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Molecules to Medicine With Mtor Translating Critical
Pathways of the Mammalian Target of Rapamycin into
Novel Therapeutic Strategies 1st Edition Kenneth Maiese
Md Digital Instant Download
Author(s): Kenneth Maiese MD
ISBN(s): 9780128027332, 0128027339
Edition: 1
File Details: PDF, 30.02 MB
Year: 2016
Language: english
MOLECULES TO MEDICINE WITH mTOR
MOLECULES
TO MEDICINE
WITH mTOR
Translating Critical Pathways into
Novel Therapeutic Strategies
Edited by
KENNETH MAIESE
Cellular and Molecular Signaling, Newark, NJ, USA
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List of Contributors
Michelle M. Adams Interdisciplinary Graduate Program in Frédéric Calon Neurosciences Axis, Centre de recherche
Neuroscience, Bilkent University, Ankara, Turkey; du CHU de Québec, Québec, QC, Canada; Faculty of
Department of Psychology, Bilkent University, Ankara, Pharmacy, Université Laval, Québec, QC, Canada
Turkey Mar Castellanos Department of Neurology, University
Garrett R. Ainslie Section of Experimental and Systems Hospital A Coruña, A Coruña, Spain
Pharmacology, College of Pharmacy, Washington State Sarmishtha Chatterjee Environmental Toxicology
University, Spokane, WA, USA Laboratory, Department of Zoology, Centre for Advanced
António Francisco Ambrósio Laboratory of Pharmacology Studies, Visva-Bharati (A Central University),
and Experimental Therapeutics, Institute for Biomedical Santiniketan, India
Imaging and Life Sciences (IBILI), Faculty of Medicine, Rita Citraro Science of Health Department, School of
University of Coimbra, Coimbra, Portugal; Center for Medicine, University of Catanzaro, Italy
Neuroscience and Cell Biology, Institute for Biomedical
Andrew Constanti Department of Pharmacology, UCL
Imaging and Life Sciences (CNC-IBILI) Consortium,
School of Pharmacy, London, United Kingdom
University of Coimbra, Coimbra, Portugal
Antonietta Coppola Epilepsy Centre; Department of
Camila O. Arent Laboratório de Neurociências, Programa
Neuroscience, Reproductive and Odontostomatological
de Pós-Graduação em Ciências da Saúde, Unidade
Sciences, Federico II University, Naples, Italy
Acadêmica de Ciências da Saúde, Universidade do
Extremo Sul Catarinense, Criciúma, SC, Brazil Anindita Das Pauley Heart Center, Division of Cardiology,
Department of Internal Medicine, Virginia
Athira A.P. Department of Biochemistry, University of
Commonwealth University Medical Center, Richmond,
Kerala, Thiruvananthapuram, Kerala, India
VA, USA
Keith Baar Department of Neurobiology, Physiology and Giovambattista De Sarro Science of Health Department,
Behavior, Functional Molecular Biology Lab, University of School of Medicine, University of Catanzaro, Italy
California Davis, Davis, CA, USA
Bernard Fauconneau EA3808 Molecular Targets and
Fusun Doldur-Balli Department of Molecular Biology and Therapeutics of Alzheimer’s Disease, Poitiers University
Genetics, Bilkent University, Ankara, Turkey
Hospital, University of Poitiers, Poitiers, France
Fuller W. Bazer Center for Animal Biotechnology and Rosa Fernandes Laboratory of Pharmacology and
Genomics, Texas A&M University, College Station, TX, Experimental Therapeutics, Institute for Biomedical
USA; Department of Animal Science, Texas A&M Imaging and Life Sciences (IBILI), Faculty of Medicine,
University, College Station, TX, USA University of Coimbra, Coimbra, Portugal; Center for
Jacob T. Beckley Department of Neurology, University of Neuroscience and Cell Biology, Institute for Biomedical
California, San Francisco, CA, USA Imaging and Life Sciences (CNC-IBILI) Research Unit,
Shelley Bhattacharya Environmental Toxicology University of Coimbra, Coimbra, Portugal
Laboratory, Department of Zoology, Centre for Advanced Arnaud Francois Neurosciences Axis, Centre de recherche
Studies, Visva-Bharati (A Central University), du CHU de Québec, Québec, QC, Canada; Faculty of
Santiniketan, India Pharmacy, Université Laval, Québec, QC, Canada
Agnès Rioux Bilan EA3808 Molecular Targets and K. Michael Gibson Section of Experimental and Systems
Therapeutics of Alzheimer’s Disease, Poitiers University Pharmacology, College of Pharmacy, Washington State
Hospital, University of Poitiers, Poitiers, France University, Spokane, WA, USA
Binu S. Department of Biochemistry, University of Kerala, Maria Grazia Giovannini Department of Health Sciences,
Thiruvananthapuram, Kerala, India Section of Clinical Pharmacology and Oncology,
Ergul Dilan Celebi-Birand Interdisciplinary Graduate University of Florence, Florence, Italy
Program in Neuroscience, Bilkent University, Ankara, Connie G. Glasgow Cardiovascular and Pulmonary
Turkey Branch, National Heart, Lung, and Blood Institute,
Zhiyou Cai Department of Neurology, Renmin Hospital, National Institutes of Health, Bethesda, MD, USA
Hubei University of Medicine, Shiyan, Hubei Province, Carme Gubern Cerebrovascular Research Group,
People’s Republic of China Biomedical Research Institute (IdiBGi), Girona, Spain
xi
xii LIST OF CONTRIBUTORS
Michael N. Hall Biozentrum, University of Basel, Basel, Joel Moss Cardiovascular and Pulmonary Branch, National
Switzerland Heart, Lung, and Blood Institute, National Institutes of
David C. Hughes Department of Neurobiology, Physiology Health, Bethesda, MD, USA
and Behavior, Functional Molecular Biology Lab, Chayan Munshi Environmental Toxicology Laboratory,
University of California Davis, Davis, CA, USA Department of Zoology, Centre for Advanced Studies,
Ken-ichi Inoue Second Department of Surgery, Dokkyo Visva-Bharati (A Central University), Santiniketan, India
Medical University, Mibu, Japan Akihiro Nakamura Department of Genetics and
Thierry Janet EA3808 Molecular Targets and Therapeutics Development, Krembil Research Institute, University
of Alzheimer’s Disease, Poitiers University Hospital, Health Network, Toronto, Ontario, Canada
University of Poitiers, Poitiers, France Pierluigi Navarra Institute of Pharmacology, Catholic
University Medical School, Rome, Italy
Elisabet Kadar Department of Biology, University of
Girona, Girona, Spain Marc Paccalin EA3808 Molecular Targets and Therapeutics of
Alzheimer’s Disease, Poitiers University Hospital, University
Mohit Kapoor Department of Genetics and Development,
of Poitiers, Poitiers, France; Geriatrics Department, Poitiers
Krembil Research Institute, University Health Network,
University Hospital, Poitiers, France; Centre Mémoire de
Toronto, Ontario, Canada; Department of Surgery,
Ressources et de Recherche, France; INSERM CIC-P 1402,
University of Toronto, Toronto, Ontario, Canada; Arthritis
Poitiers University Hospital, Poitiers, France
Program, University Health Network, Toronto, Ontario,
Canada Gustavo Pacheco-Rodriguez Cardiovascular and Pulmonary
Branch, National Heart, Lung, and Blood Institute, National
Elif Tugce Karoglu Interdisciplinary Graduate Program in
Institutes of Health, Bethesda, MD, USA
Neuroscience, Bilkent University, Ankara, Turkey
Guylène Page EA3808 Molecular Targets and Therapeutics
Hiroshi Kato Division of Rheumatology, Department of
of Alzheimer’s Disease, Poitiers University Hospital,
Medicine, State University of New York Upstate Medical
University of Poitiers, Poitiers, France
University, Syracuse, New York, NY, USA
Filipe Palavra Laboratory of Pharmacology and
Goran B.G. Klintmalm Texas A&M University
Experimental Therapeutics, Institute for Biomedical
College of Medicine, Dallas, TX, USA; Simmons
Imaging and Life Sciences (IBILI), Faculty of Medicine,
Transplant Institute, Department of Surgery, Transplant
University of Coimbra, Coimbra, Portugal; Center for
Surgery, Baylor University Medical Center, Dallas,
Neuroscience and Cell Biology, Institute for Biomedical
TX, USA
Imaging and Life Sciences (CNC-IBILI) Consortium,
Keiichi Kubota Second Department of Surgery, Dokkyo University of Coimbra, Coimbra, Portugal
Medical University, Mibu, Japan
Andras Perl Division of Rheumatology, Department of
Rakesh C. Kukreja Pauley Heart Center, Division of Medicine, State University of New York Upstate Medical
Cardiology, Department of Internal Medicine, Virginia University, Syracuse, New York, NY, USA
Commonwealth University Medical Center, Richmond, Meagan R. Pitcher Center for Translational Psychiatry,
VA, USA Department of Psychiatry and Behavioral Sciences,
Daniele Lana Department of Health Sciences, Section of Medical School, The University of Texas Health Science
Clinical Pharmacology and Oncology, University of Center at Houston, Houston, TX, USA
Florence, Florence, Italy João Quevedo Center for Translational Psychiatry,
Antonio Leo Science of Health Department, School of Department of Psychiatry and Behavioral Sciences, Medical
Medicine, University of Catanzaro, Italy School, The University of Texas Health Science Center at
Lucia Lisi Institute of Pharmacology, Catholic University Houston, Houston, TX, USA; Laboratório de Neurociências,
Medical School, Rome, Italy Programa de Pós-Graduação em Ciências da Saúde,
Unidade Acadêmica de Ciências da Saúde, Universidade do
Xiangwei Liu Shanghai Institute of Cardiovascular Diseases, Extremo Sul Catarinense, Criciúma, SC, Brazil
Zhongshan Hospital, Fudan University, Shanghai, PR
China; Center for Cardiovascular Research and Alternative Flávio Reis Laboratory of Pharmacology and Experimental
Medicine, School of Pharmacy, University of Wyoming Therapeutics, Institute for Biomedical Imaging and Life
College of Health Sciences, Laramie, WY, USA Sciences (IBILI), Faculty of Medicine, University of
Coimbra, Coimbra, Portugal; Center for Neuroscience and
Kenneth Maiese Cellular and Molecular Signaling, Cell Biology, Institute for Biomedical Imaging and Life
Newark, NJ, USA Sciences (CNC.IBILI) Consortium, University of Coimbra,
Francesco Massari Medical Oncology, Azienda Ospedaliera Coimbra, Portugal
Universitaria Integrata, University of Verona, Verona, Jun Ren Shanghai Institute of Cardiovascular Diseases,
Italy Zhongshan Hospital, Fudan University, Shanghai, PR
Greg J. McKenna Simmons Transplant Institute, Baylor China; Center for Cardiovascular Research and
University Medical Center, Dallas, TX, USA; Texas A&M Alternative Medicine, School of Pharmacy, University of
University College of Medicine, Dallas, TX, USA Wyoming College of Health Sciences, Laramie, WY, USA
LIST OF CONTRIBUTORS xiii
Gislaine Z. Réus Center for Translational Psychiatry, Sudhakaran P.R. Inter-University Centre for Genomics and
Department of Psychiatry and Behavioral Sciences, Gene Technology, University of Kerala, Thiruvananthapuram,
Medical School, The University of Texas Health Science Kerala, India; Department of Biochemistry, University
Center at Houston, Houston, TX, USA; Laboratório de of Kerala, Thiruvananthapuram, Kerala, India; Department of
Neurociências, Programa de Pós-Graduação em Ciências Computational Biology and Bioinformatics, University of
da Saúde, Unidade Acadêmica de Ciências da Saúde, Kerala, Thiruvananthapuram, Kerala, India
Universidade do Extremo Sul Catarinense, Criciúma, Marta M. Swierczynska Biozentrum, University of Basel,
SC, Brazil Basel, Switzerland
Dorit Ron Department of Neurology, University of Julie Verite EA3808 Molecular Targets and Therapeutics of
California, San Francisco, CA, USA Alzheimer’s Disease, Poitiers University Hospital,
University of Poitiers, Poitiers, France
Cinzia Dello Russo Institute of Pharmacology, Catholic
University Medical School, Rome, Italy Kara R. Vogel Section of Experimental and Systems
Pharmacology, College of Pharmacy, Washington State
Emilio Russo Science of Health Department, School of University, Spokane, WA, USA
Medicine, University of Catanzaro, Italy
Marita A. Wallace Department of Neurobiology,
Matteo Santoni Clinica di Oncologia Medica, AOU Physiology and Behavior, Functional Molecular Biology
Ospedali Riuniti, Polytechnic University of the Marche Lab, University of California Davis, Davis, CA, USA
Region, Ancona, Italy Xiaoqiu Wang Center for Animal Biotechnology and
Genomics, Texas A&M University, College Station, TX,
Norisuke Shibuya Second Department of Surgery, Dokkyo
USA; Department of Animal Science, Texas A&M
Medical University, Mibu, Japan
University, College Station, TX, USA
Soumya S.J. Inter-University Centre for Genomics and Guoyao Wu Center for Animal Biotechnology and
Gene Technology, University of Kerala, Genomics, Texas A&M University, College Station, TX,
Thiruvananthapuram, Kerala, India USA; Department of Animal Science, Texas A&M
Wendy K. Steagall Cardiovascular and Pulmonary University, College Station, TX, USA
Branch, National Heart, Lung, and Blood Liang-Jun Yan Department of Pharmaceutical Sciences,
Institute, National Institutes of Health, Bethesda, UNT System College of Pharmacy, University of North
MD, USA Texas Health Science Center, Fort Worth, TX, USA
About the Editor
Kenneth Maiese is an internationally recognized received outstanding investigator awards, was named a
physician researcher, healthcare executive, and editor Johnson & Johnson Distinguished Investigator, received
with broad research, clinical, and leadership experience the Albrecht Fleckenstein Memorial Award for
in both academia and industry. He was born and raised Distinguished Achievement in Basic Research, was
in New Jersey and was the Valedictorian of his high elected to America’s Top Physicians and The Best of
school class at Pennsauken High School. He graduated U.S. Physicians, and his highly cited work has received
from the University of Pennsylvania Summa cum the distinction of “High Impact Research and Potential
Laude with Distinction and was a Teagle Scholar, Public Health Benefit” by the National Institutes of
Grupe Scholar, and Joseph Collins Scholar at Weill Health. He has been fortunate to have benefited from
Medical College of Cornell University. Dr Maiese sub- continuous funding from sources that include the
sequently trained as a physician-scientist at Cornell, the American Diabetes Association, the American Heart
National Institutes of Health (NIH), and as a senior Association, the Bugher Foundation, Johnson and
executive at the Harvard School of Public Health. He Johnson Focused Giving Award, and the National
has extensive experience in academic medicine, health- Institutes of Health. His service on Executive Councils
care delivery, and drug development, holding positions for Graduate Schools has fostered innovative graduate
as tenured Professor and Chair, and Chief of Service of student training programs and he was elected as board
the Department of Neurology and Neurosciences of and advisor member to develop and execute inaugural
Rutgers University, Global Head of Translational MD/PhD Degree Programs and new Translational
Medicine and External Innovation, Board Member of Medicine Programs. He chairs national grant commit-
the Cancer Institute of New Jersey, Steering Committee tees and is a chartered panel member or consultant for
Member for the Foundation for the National Institutes numerous national and international foundations as
of Health, tenured Professor in Neurology, Anatomy & well as multiple study sections and special emphasis
Cell Biology, Molecular Medicine, the Barbara Ann panels for the National Institutes of Health. Dr Maiese
Karmanos Cancer Institute, and the National Institute is frequently honored as the chairperson and/or the
of Health Center at Wayne State University, and plenary speaker for a number of international sympo-
Founding Editor and Editor-in-Chief of multiple inter- siums, organizations, and presentations to federal
national journals. Early in his career, Dr Maiese policy-makers.
xv
Preface
Throughout the world, the age of the population is The costs of caring for individuals with DM are
increasing at a significant rate. In developed countries extremely high. In the United States (US) alone, the
over the course of the last half-century, the number of costs of care for individuals with DM in 2012 equaled
individuals that are over the age of 65 has approxi- 8915 US dollars per person and almost 17% of the
mately doubled. This increase in life expectancy and country’s gross domestic product. Although early
age has been accompanied by a 1% decrease in the diagnosis of individuals with DM may be critical to
age-adjusted death rate over the years 2000 through prevent the progression of this disease, the presence of
2011. Developing nations also share in the increased impaired glucose tolerance in the young raises addi-
life expectancy of the world’s population. In these tional concerns for the future development of DM.
countries, the number of elderly people will rise from Obesity, now increasing in incidence, is another risk
5% of the population to approximately 10% of the pop- factor for the development of DM since it leads to
ulation over future decades. cellular oxidative stress, insulin resistance, and lipid-
This positive news for the increased lifespan of indi- induced impairment of pancreatic β cells.
viduals in developed nations as well as developing DM can affect multiple systems of the body and
countries is somewhat buffered by the parallel rise in can impact cardiovascular disease, neurodegenerative
non-communicable diseases (NCDs). Improvements in disorders, the musculoskeletal system, cancer, and
healthcare have fostered increased longevity of the immune function. Acute and chronic neurodegenera-
global population. For example, of the five leading tive disorders lead to disability and death in more
causes of death that are cardiac disease, cancer, chronic than 30 million individuals worldwide. It is predicted
lower respiratory disease, stroke, and traumatic acci- that the number of individuals with dementia will
dents, stroke is no longer ranked as the third leading continue to grow, doubling every 20 years, and reach
cause of death. Multiple factors most likely have led to almost 115 million individuals by 2050. Most of the
this lower rank for stroke that involve heightened pub- increase is expected to occur in developing countries,
lic awareness for the rapid treatment of stroke, a with Alzheimer’s disease being considered a signifi-
reduction in tobacco consumption, and improved care cant public health concern. In regards to costs, care
for disorders involving diabetes mellitus (DM), hyper- for those suffering from dementia equals more than
tension, and low-density lipoprotein cholesterol. 600 billion US dollars annually. With chronic neurode-
Active treatment with recombinant tissue plasminogen generative disorders such as Parkinson’s disease,
activator has also led to a reduction in mortality and approximately 50,000 new cases are present in the US
morbidity in stroke patients. Yet, therapeutic treat- alone each year and this number is predicted to dou-
ments to resolve disability and death from stroke con- ble by 2030. During this same period, other disorders
tinue to remain limited for the majority of patients. such as epilepsy are predicted to affect over 50 million
Overall, a rise in the prevalence of NCDs continues people and peripheral nerve disease is estimated to be
with the advancing age of the world’s population. present in almost 300 million individuals. With cancer,
According to World Health Organization (WHO) almost 50% of cancer survivors are 70 years or older,
records, greater than 60% of the annual 57 million indicating the increased prevalence with advancing
global deaths are due to NCDs. Furthermore, almost age. In contrast, 5% of individuals with cancer are
80% of these NCDs occur in low- and middle-income younger than 40 years of age. The number of cancer
countries. Disorders such as DM will continue to grow survivors will continue to grow and reach at least 19
according to WHO estimates, such that DM will be the million by 2024 in the US alone. For cardiovascular
seventh leading cause of death by the year 2030. As of disease, this disorder remains the leading cause
2013, approximately 350 million individuals are esti- of death and increases with aging, but can easily affect
mated to suffer from DM and millions of individuals all ages of the world’s population. Elevated choles-
are estimated to be currently undiagnosed with DM. terol and hypertension are significant risk factors for
xvii
xviii PREFACE
cardiovascular disease and contribute to approxi- cellular biology and new therapeutic strategies with
mately 13% of all deaths. mTOR that involve the maintenance of proliferation of
In light of the limited courses of action to treat stem cells, conceptus development, implantation and
many NCDs that currently have only symptomatic or placentation, myogenesis, cartilage homeostasis, and
a handful of therapeutic options, development of novel cell survival during xenobiotic cell injury are pre-
therapeutic strategies to address the growing preva- sented. Section II extends these topics with the discus-
lence of NCDs with the accompanying advancing age sion of mTOR in the nervous system and the role of
of the global population is considered to be highly mTOR in congenital disease. In Section III, higher cog-
warranted to fill this void. The mechanistic target of nitive function and aging are explored with the exami-
rapamycin (mTOR) is one such strategic pathway that nation of the role of mTOR in memory formation as
has gained high visibility as a critical target for multi- well as cognitive impairment, aging processes of the
ple disease entities. mTOR is also known as the mam- brain, depression, addiction, and behavior modifica-
malian target of rapamycin and the FK506-binding tion. The cardiovascular system is discussed in
protein 12-rapamycin complex-associated protein 1. Section IV, highlighting the role of mTOR in nutrient
mTOR is a 289-kDa serine-threonine protein kinase, sensing, new vessel growth, cardiac ischemic disease,
present throughout the body, and oversees multiple and the maintenance of cardiac function during meta-
functions that involve gene transcription, protein for- bolic disorders. In Section V, the vital role of mTOR in
mation, oxidative stress, stem cell development, cell adaptive and innate immune function, organ trans-
metabolism, cell survival, cell senescence, immunity, plantation, inflammatory pathways in the nervous sys-
aging, tissue regeneration and repair, cancer, and path- tem, and glial function are presented. Section VI
ways of programmed cell death that include autop- further expands the analysis of mTOR in the endocrine
hagy and apoptosis. system and provides scrutiny of the influence of
Molecules to Medicine with mTOR: Translating Critical mTOR in endocrine disorders, metabolic disease, dia-
Pathways into Novel Therapeutic Strategies is a unique betogenesis, and integrated pathways that involve
resource that employs a translational medicine both renal and nervous system impairment. The last
approach to present novel work and new insights for segment of the book, Section VII, provides a vital per-
the role of mTOR during normal physiology, in dis- spective for the role of mTOR as a proliferative agent
ease states, and for the development and refinement of to foster tumor cell growth in a variety of settings and
therapeutic strategies applicable to multiple systems of to affect cancer cell metabolic pathways. Molecules to
the body. Prominent internationally recognized experts Medicine with mTOR: Translating Critical Pathways into
explore the complexity of mTOR signaling and the crit- Novel Therapeutic Strategies provides an innovative
ical need for therapeutic targeting of this pathway, but examination of the pivotal function mTOR holds in the
maintain a clear and insightful presentation of clinical human body and highlights the compelling need to
and basic research perspectives for a broad audience critically consider mTOR signaling pathways in the
that encompasses healthcare providers, scientists, drug translation of cellular biology to effective and safe clin-
developers, and students. ical treatments for the growing prevalence of multiple
Molecules to Medicine with mTOR: Translating Critical disorders that mirror the increasing age and lifespan
Pathways into Novel Therapeutic Strategies begins with of the global population.
the presentation of mTOR in cellular development,
proliferation, and survival in Section I. Topics of Kenneth Maiese, Editor
C H A P T E R
1
Novel Stem Cell Strategies with mTOR
Kenneth Maiese
Cellular and Molecular Signaling, Newark, NJ, USA
1.1 LIFE EXPECTANCY AND THE of death (Table 1.1) [6,7]. Other NCDs, such as diabetes
SIGNIFICANCE OF GLOBAL mellitus (DM), can also contribute to acute neurodegen-
NONCOMMUNICABLE DISEASES erative diseases that include cerebrovascular disease
[8,9]. Stroke leads to multiple complications that affect
In developed nations, the age of the population has both the livelihood and minimal daily function of an
significantly risen. Life expectancy is approaching individual [10,11]. As a leading cause of death [4],
almost 80 years for all individuals and has been stroke affects approximately 15 million individuals
marked by a 1% decrease in the age-adjusted death every year. In the United States, approximately 800,000
rate from the years 2000 through 2011 [1]. The number strokes occur per year at an annual cost of 75 billion US
of individuals over the age of 65 also has doubled dollars [6]. It is of interest to note that stroke is no lon-
during the prior 50 years [2]. If one examines large ger ranked as the third leading cause of death. Factors
developing nations, such as China and India, it is related to improved management of hypertension and
expected that the proportion of elderly individuals low-density lipoprotein cholesterol disorders, reduction
will increase from the present levels of approximately in tobacco consumption, heightened public education
5% to almost 10% over the next several decades. and awareness for the need for rapid treatment of cere-
Improvements in treatment resources and preventive brovascular disorders, and more focused management
care have contributed to the increased lifespan of the on metabolic disorders such as DM may have contrib-
global population. uted to this lower ranking for stroke [6,7]. In addition,
However, accompanied with the increase in life treatment with recombinant tissue plasminogen activa-
expectancy has been a rise in chronic disorders and tor has led to a reduction in mortality and morbidity in
noncommunicable diseases (NCDs; Table 1.1). Through patients presenting with stroke [12,13]. Yet, overall ther-
data obtained by the World Health Organization, apeutic strategies for patients presenting with stroke
greater than 60% of the 57 million annual global deaths remain limited for the majority of patients. Treatment
result from NCDs. Approximately 80% of these NCDs with recombinant tissue plasminogen activator is appli-
occur in low- and middle-income countries [3]. cable to only a subgroup of patients that requires a
NCDs affect approximately 30% of the population under narrow therapeutic window.
60 in low- and middle-income countries. In high-income Together, acute and chronic neurodegenerative
countries, 13% of the population under 60 is affected. disorders lead to disability and death in more than 30
The five leading causes of death are cardiac disease, million individuals worldwide annually (Table 1.1) [14].
cancer, chronic lower respiratory disease, stroke, and Acute neurodegenerative disorders can place a severe
traumatic accidents [4]. Elevated cholesterol and hyper- burden on the world’s population [6,15]. Acute
tension are significant risk factors for cardiovascular traumatic brain injury (TBI) leads to severe neurological
disease. For example, hypertension contributes to disability [16,17]. A twofold detrimental effect on
approximately 13% of all deaths [5]. the clinical outcome of patients can occur with TBI.
Disorders such as elevated cholesterol and hyperten- TBI can result in acute injury to the nervous system
sion can also lead to an increase in acute neurodegener- as well as subsequent chronic impairment [18 20].
ative disorders such as stroke, the fourth leading cause In the United States, approximately 50,000 individuals
Molecules to Medicine with mTOR

DOI: http://dx.doi.org/10.1016/B978-0-12-802733-2.00020-7 3 © 2016 Elsevier Inc. All rights reserved.
4 1. NOVEL STEM CELL STRATEGIES WITH mTOR
TABLE 1.1 Global Noncommunicable Diseases die every year as a result of TBI and more than 100,000
individuals must live with chronic disability [21].
NCD Clinical presentation
During severe trauma, approximately 50% of indivi-
Neurodegenerative Acute and chronic neurodegenerative duals eventually die.
disorders disorders result in disability and death in over With the aging population, a rise in the incidence of
30 million individuals worldwide
chronic neurodegenerative disorders is also expected to
TBI results in acute injury to the nervous ensue [22,23]. One such example is Alzheimer’s disease
system as well as subsequent chronic (AD). The familial cases of AD consist of less than 2%
impairment
of all presentations [24] and usually occur prior to age
More than 5 million individuals have AD and 55 [25]. Familial cases of AD occur as an autosomal
3.5 million receive treatment at an annual cost dominant form of a mutated amyloid precursor protein
of 4 billion US dollars
(APP) gene as well as mutations in the presenilin 1 or 2
Almost 4% of individuals over age 60 suffer genes [26]. Familial AD is present in approximately
from PD globally and this is expected to 200 families worldwide and results from variable
double by the year 2030
single-gene mutations on chromosomes 1, 14, and 21.
As the fourth leading cause of death, stroke Mutations on chromosome 1 lead to altered presenilin 2,
affects approximately 15 million individuals mutations on chromosome 14 result in altered
every year at an annual cost of 75 billion US
dollars
presenilin 1, and mutations on chromosome 21 lead to
altered APP. In contrast, 10% of the global population
In the year 2030, epilepsy is predicted to affect over the age of 65 is affected with sporadic AD [27].
over 50 million people and neuropathies are
estimated to afflict almost 300 million
At present, more than 5 million individuals are diag-
individuals nosed with AD and 3.5 million are under treatment at
an annual cost of almost 4 billion US dollars. It is esti-
Cardiovascular As the leading cause of death, cardiac disease
disease affects all ages in the global population
mated that the number of those suffering from AD will
increase significantly and rise to more than 30 million
Elevated cholesterol and hypertension are individuals over the next 15 20 years [14,27,28]. If one
significant risk factors for cardiovascular disease
considers other chronic progressive neurodegenerative
Hypertension contributes to approximately disorders such as Parkinson’s disease (PD) [29,30],
13% of all deaths and results in acute almost 50,000 new cases present in the United States
neurodegenerative disorders such as stroke
alone each year. Approximately 1 4% of individuals
Diabetes mellitus DM affects all systems of the body and leads to over age 60 suffer from PD globally and this number of
pathology in the cardiovascular system, affected individuals may double by the year 2030.
nervous system, immune system, and
musculoskeletal system
By the year 2030, epilepsy is predicted to affect over
50 million people, neuropathies are estimated to afflict
DM is increasing throughout the world and almost 300 million individuals, and neurological injuries
approximately 350 million individuals are
currently afflicted with this disorder
may change the lives of 243 million individuals [31].
However, similar to acute neurodegenerative disorders,
Over 8 million individuals may be the availability of treatments that can prevent the initia-
undiagnosed for metabolic disease
tion or block the progression of chronic neurodegenera-
Overall care for patients with DM consumes tive disorders is limited.
17% of the gross domestic product in the DM also is a significant NCD for the world’s
United States
population (Table 1.1) [32]. The incidence of DM is
Cancer The most common cancers among male increasing throughout the world and approximately
survivors are prostate, colon and rectal, and 350 million individuals currently have DM [33 37].
melanoma
Another 8 million individuals are believed to suffer
The most common cancers among female from metabolic disorders and are currently undiag-
survivors are breast, uterine corpus, and colon nosed [38 40]. The costs to care for individuals with
and rectal
DM are high. Approximately 9000 US dollars are spent
Approximately one half of cancer survivors are in the United States for each individual with DM
at least 70 years or older. Approximately 5% of per year and overall care for patients with DM con-
individuals with cancer are younger than 40
years of age
sumes 17% of the gross domestic product [41]. Early
diagnosis and proper care of individuals with DM
In the United States, it is estimated that the can impact care for this disease by modulating
number of cancer survivors will increase to 19
million individuals by the year 2024
epigenetic changes in age-related genes involved
with DM and other degenerative disorders [42 46].
I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

1.2 mTOR STRUCTURE AND SIGNALING 5
Impaired glucose tolerance in the young raises TABLE 1.2 mTOR Structure and Signaling
additional concerns for the risk of developing DM [34].
mTOR Highlights
Obesity is another risk factor for DM [33 37]. Obesity
and excess body fat can precipitate pancreatic β-cell Structure and mTOR is a 289-kDa serine/threonine protein
injury [47], cellular inflammation [8], impaired growth identification kinase that is encoded by a single gene
FRAP1
factor release [48 51], changes in protein tyrosine
phosphatase signaling [37,52], oxidative stress [35,53], TOR was identified through the use of
and insulin resistance [8,32,54,55]. rapamycin-resistant TOR mutants in S.
cerevisiae. The genes TOR1 and TOR2 yield
DM can involve any system of the body [8,9]. two protein isoforms in yeast, known as
DM leads to vascular disease resulting in endothelial TOR1 and TOR2
cell senescence [56], injury to endothelial cells
mTOR is a central component of the protein
[51,57 62], cardiovascular disease [63 71], platelet complexes mTORC1 and mTORC2
dysfunction [72,73], atherosclerosis [11,74 76], loss of
endothelial progenitor cells [52,77 81], dysfunctional mTORC1 consists of Raptor, the proline-rich
Akt substrate 40 kDa (PRAS40), Deptor, and
maintenance and mobilization of endothelial progeni- mLST8/GβL
tor cells [79,80], and impaired angiogenesis [62,69,82].
DM also can have negative effects on the immune mTORC2 consists of Rictor, mLST8, Deptor,
the mSIN1, and the protein observed with
system [33,70,83 88], renal function [89 93], hepatic Rictor-1 (Protor-1)
metabolism [35,54,94 98], and musculoskeletal integrity
[53,74,99 101]. DM affects all components of the central Post-translational Post-translational phosphorylation of mTOR
phosphorylation occurs through multiple avenues
and peripheral nervous systems. DM can lead to retinal
disease [39,102 104], peripheral nerve disorders The C-terminal domain with sequence
[95,105], memory loss [106 108], psychiatric disorders homology to the catalytic domain of the
PI 3-K family has several phosphorylation
[109,110], stroke [22,46,72,76,111,112], dementia such as sites that regulate mTOR
AD [50,106,113,114], and impairment of neuronal lon-
gevity [50]. Within the HEAT domain, serine1261
can be phosphorylated in mTORC1
Interestingly, tight glucose control does not always and mTORC2 by insulin signaling through
result in the resolution of complications from DM the PI 3-K pathway to increase mTOR
[34,115]. Use of diet control treatments may be effective activity
to prevent hyperglycemia events, but can potentially Cellular signaling Rapamycin blocks mTORC1 activity by
decrease organ mass through processes that involve binding to immunophilin FKBP12 to prevent
autophagy [97]. These observations point to the need the phosphorylation of mTOR
for additional strategies for the treatment of DM and Chronic administration of rapamycin can
its complications. also inhibit mTORC2 activity
PRAS40 inhibits mTOR activity and blocks
binding of mTORC1 to Raptor
1.2 mTOR STRUCTURE AND SIGNALING
Deptor blocks mTORC1 activity by binding
to the FAT domain of mTOR
It is clear that NCDs are becoming a growing concern
for the global population given the concurrent rise in mLST8 promotes mTOR kinase activity
longevity and life expectancy. Increased lifespan is through p70S6K and eIF4E-4EBP1 that bind
to Raptor
accompanied by a greater exposure to several NCDs that
involve multiple systems of the body as well as pro- mTOR signaling relies upon the pathways
cesses closely tied to cellular metabolism. Given the need of PI 3-K, Akt, and AMPK
for novel treatments to effectively treat NCDs that cur- In contrast to the inhibition of
rently have limited therapeutic options, the mechanistic mTORC1, mTORC2 is activated by
target of rapamycin (mTOR) is increasingly being recog- the TSC1/TSC2
nized as a critical target for drug discovery development Akt, protein kinase B; AMPK, AMP activated protein kinase; eIF4E-4EBP1,
against NCDs that involve cardiovascular disease, eukaryotic initiation factor 4E-binding protein 1; FAT domain,
FKBP12-rapamycin-associated protein (FRAP), ataxia-telangiectasia
neurological disorders, metabolic disease, and cancer. (ATM), and the transactivation/transformation domain-associated protein
mTOR is also known as the mammalian target domain; HEAT, Huntingtin, Elongation factor 3, a subunit of protein
of rapamycin and the FK-506-binding protein phosphatase-2A, and TOR1; mTOR, mechanistic target of rapamycin;
mTORC1, mTOR complex 1; mTORC2, mTOR complex 2; PI 3-K,
12-rapamycin complex-associated protein 1 (Table 1.2). phosphoinositide 3-kinase; p70S6K, p70 ribosomal S6 kinase; TOR, target
It is a 289-kDa serine/threonine protein kinase of rapamycin.

that is encoded by a single gene, FRAP1 [116,117]. and allows it to bind to its complex constituents [126].
Initially, the target of rapamycin (TOR) was identified Rheb phosphorylates Raptor residue serine863 and other
through the use of rapamycin-resistant TOR mutants residues that include serine859, serine855, serine877,
in Saccharomyces cerevisiae with the genes TOR1 and serine696, and threonine706. mTORC1 activity is limited
TOR2 that yield two protein isoforms in yeast if serine863 remains unphosphorylated [127]. Once
known as Tor1 and Tor2 [118]. The carboxyterminal mTOR is active, mTOR also can modulate Raptor activ-
domain of mTOR has four domains for catalytic ity that can be blocked by rapamycin [127]. PRAS40
activity that include FAT, FKBP12-rapamycin-binding inhibits mTOR activity and can prevent the binding of
domain (FRB), the catalytic PI3/PI4-related kinase mTORC1 to Raptor [128]. Phosphorylation of PRAS40
domain, and FATC [116]. The FAT domain, which con- by protein kinase B (Akt) frees PRAS40 from Raptor
sists of FKBP12-rapamycin-associated protein (FRAP), and allows PRAS40 to be sequestered by the cyto-
ataxia-telangiectasia (ATM), and the transactivation/ plasmic docking protein 14-3-3 to activate mTORC1
transformation domain-associated protein domain, [128 132]. Deptor inhibits mTORC1 activity by binding
resides adjacent to the FRB that allows association to the FAT domain of mTOR. Without Deptor, the
between mTOR and FKBP12 when bound to rapamy- activity of Akt, mTORC1, and mTORC2 are enhanced
cin. The PI3/PI4-related kinase domain and the [133]. In contrast to PRAS40 and Deptor, mLST8 pro-
small FATC domain follow these two domains. The motes mTOR kinase activity through p70 ribosomal S6
N-terminal portion of mTOR contains at least a 20 kinase (p70S6K) and the eukaryotic initiation factor 4E
HEAT (Huntingtin, Elongation factor 3, a subunit of (eIF4E)-binding protein 1 (4EBP1) that bind to Raptor
Protein phosphatase-2A, and TOR1) repeat. This area [134]. PRAS40 can block mTORC1 activity by prevent-
promotes binding with the regulatory proteins Raptor ing the association of p70S6K and 4EBP1 with Raptor
(regulatory-associated protein of mTOR) and Rictor [14,128]. In addition, mLST8 promotes mTOR kinase
(rapamycin-insensitive companion of mTOR) [119]. activity and is known to control insulin signaling
Post-translational phosphorylation of mTOR can through the transcription factor FoxO3 [135], be neces-
occur through a number of processes. The C-terminal sary for Akt and protein kinase C-α (PKCα) phosphor-
domain with sequence homology to the catalytic ylation [135], and is required for the association
domain of the phosphoinositide 3-kinase (PI 3-K) between Rictor and mTOR [135].
family [120] contains several phosphorylation sites that mTOR signaling is closely associated with the
regulate mTOR. Within the HEAT domain, serine1261 pathways of PI 3-K, Akt, and AMP activated protein
can be phosphorylated in mTOR complex 1 (mTORC1) kinase (AMPK) (Table 1.2) [64,136]. Akt regulates the
and mTOR complex 2 (mTORC2) by insulin signaling activity of the hamartin (tuberous sclerosis 1)/tuberin
through the PI 3-K pathway to increase mTOR activity (tuberous sclerosis 2) (TSC1/TSC2) complex, an
[120]. Phosphorylation of serine1261 also results in inhibitor of mTORC1 [137 140]. Although several
mTOR serine2481 autophosphorylation [120]. regulatory phosphorylation sites are known to exist for
mTOR is a central component of the protein TSC1, control of the TSC1/TSC2 complex is primarily
complexes mTORC1 and mTORC2 (Figure 1.1) mediated though the phosphorylation of TSC2 by Akt,
[27,29,121,122]. Rapamycin is a macrolide antibiotic extracellular signal-regulated kinases, activating pro-
derived from Streptomyces hygroscopicus that inhibits tein p90 ribosomal S6 kinase 1, AMPK, and glycogen
both TOR activity and mTOR activity [123]. mTORC1 synthase kinase-3β. TSC2 functions as a GTPase-
is more sensitive to the inhibitory effects of rapamycin activating protein (GAP) converting a small G protein
than mTORC2 [124]. Rapamycin blocks mTORC1 Rheb (Rheb-GTP) to the inactive GDP-bound form
activity by binding to immunophilin FK-506-binding (Rheb-GDP). Once Rheb-GTP is active, Rheb-GTP
protein 12 (FKBP12) that attaches to FRB at the associates with Raptor to regulate the binding of
C-terminal of mTOR to prevent the phosphorylation of 4EBP1 to mTORC1 and increase mTORC1 activity
mTOR [125]. Chronic administration of rapamycin can [141]. Akt phosphorylates TSC2 on multiple sites that
inhibit mTORC2 activity that may occur from the leads to the destabilization of TSC2 and disruption of
disruption of the assembly of mTORC2 [124]. its interaction with TSC1. Phosphorylation of TSC2 at
mTORC1 consists of Raptor, the proline-rich Akt serine939, serine981, and threonine1462 results in the
substrate 40 kDa (PRAS40), Deptor (DEP domain- binding of TSC2 to protein 14-3-3, disruption of the
containing mTOR interacting protein), and mLST8/ TSC1/TSC2 complex, and subsequent activation
GβL (mammalian lethal with Sec13 protein 8, abbrevi- of Rheb and mTORC1 [142]. During some paradigms
ated as mLST8 or G protein beta subunit-like (GβL)) of cellular protection, a limited activity of TSC2,
(Table 1.2) [116]. Phosphorylation of Raptor through as well as AMPK [143], appears necessary since
several pathways that can include the protein Ras complete knockdown of TSC2 can prevent cellular
homolog enriched in brain (Rheb) activates mTORC1 protection [144].

1.2 mTOR STRUCTURE AND SIGNALING 7
Oxidative stress via neurodegeneration,

cardiovascular disease,
cancer, or diabetes mellitus
Cellular membrane
PI 3-K
Akt AMPK
TSC1/TSC2
mTORC1 mTORC2
Raptor PRAS40 Rictor Protor-1
mTORC
mTORC
Deptor mLST8 mSIN1
mLST8
Deptor
Wnt, WISp1,
SIRT1, forkhead
transcription factors,
& growth factors
Stem cell
Stem cell development, pluripotency,
apoptosis & autophagy &
differentiation
Stem cell
maintenance & senescence
FIGURE 1.1 mTOR oversees stem cell development, pluripotency, differentiation, maintenance, and programmed cell death in clinical dis-
ease. Multiple disease entities that involve neurodegenerative disorders, cardiovascular disease, DM, and cancer can be the result of
oxidative-stress-mediated cell injury. Through oxidative stress and the generation of ROS, the pathways of mTOR and PI 3-K, protein kinase B
(Akt), AMPK, and the TSC1/TSC2 complex become significant determinants of stem cell survival. mTOR is a vital component of mTORC1
and mTORC2. mTORC1 consists of Raptor, the proline-rich Akt substrate 40 kDa (PRAS40), Deptor, and mLST8/GβL. mTORC2 is composed
of Rictor, mLST8, Deptor, the mSIN1, and the protein observed with Rictor-1 (Protor-1). Intimately tied to these pathways are Wnt, WISP1,
SIRT1, forkhead transcription factors, and growth factors. Ultimately, mTOR signaling governs stem cell apoptosis, autophagy, maintenance,
cell senescence, development, pluripotency, and differentiation.
AMPK also controls the activity of the TSC1/TSC2 conversion of nicotinamide to nicotinamide mononu-
complex and blocks mTORC1 function. AMPK cleotide [85], increases nicotinamide adenine dinucleo-
phosphorylates TSC2 to increase GAP activity to turn tide (NAD1) levels, decreases levels of the SIRT1
Rheb-GTP into Rheb-GDP and thus inhibits the inhibitor nicotinamide, and promotes SIRT1 transcrip-
activity of mTORC1 [145]. AMPK also controls TSC1/ tion [9,148,149]. SIRT1 expression in combination with
TSC2 activity through RTP801 (REDD1/product of the AMPK activation promotes the induction of autophagy
Ddit4 gene) [146]. AMPK activity can increase REDD1 that can protect endothelial cells exposed to oxidized
expression during hypoxia to suppress mTORC1 low-density lipoproteins [150]. SIRT1 may function
activity by releasing TSC2 from its inhibitory binding similar to AMPK as an inhibitor of mTOR to block its
to protein 14-3-3 [146]. In addition, AMPK modulates activity [35].
the sirtuin silent mating type information regulation 2 In regards to mTORC2, mTORC2 consists of
homolog 1 (S. cerevisiae) (SIRT1) activity that can Rictor, mLST8, Deptor, the mammalian stress-
be vital for stem cell survival and proliferation [22]. activated protein kinase interacting protein (mSIN1),
AMPK increases the cellular NAD1/NADH ratio that and the protein observed with Rictor-1 (Protor-1)
leads to deacetylation of the SIRT1 targets peroxisome [6,119,151 154]. Rictor [155] and mSIN1 [156] can
proliferator-activated receptor-gamma coactivator 1 activate Akt at serine473 and facilitate threonine308
and the forkhead transcription factors FoxO1 [68] phosphorylation by phosphoinositide-dependent kinase
and FoxO3a [147]. AMPK increases nicotinamide 1 to promote cell survival [14,151,156]. The kinase
phosphoribosyltransferase activity that catalyzes the domain of mTOR has been shown to phosphorylate

mSIN1, preventing the lysosomal degradation of As a result of this aerobic production for ATP, ROS
mSIN1 [157]. Protor-1 is a Rictor-binding subunit of are generated. At reduced levels, production of
mTORC2 and can activate serum and glucocorticoid ROS may be important for tolerance against hypoxia
induced protein kinase 1 (SGK1), since loss of Protor-1 and ischemia. Moderate levels of ROS also may be
reduces the hydrophobic motif phosphorylation of required for the regulation of inflammatory cell activa-
SGK1 and the substrate N-Myc down-regulated gene 1 tion [168]. However, increased levels of ROS can lead
in the kidney [158]. mTORC2 also phosphorylates to cellular injury and result in mitochondrial and other
and activates SGK1, a member of the protein kinase A/ organelle injury, DNA damage, protein misfolding,
protein kinase G/protein kinase C family of protein and neuronal synaptic dysfunction [17,22,164,169,170].
kinases [159]. mTORC2 controls cytoskeleton remodel- Protective pathways serve to insulate against damage
ing through PKCα and oversees cell migration through from ROS and involve vitamins B, C, D, and K
the Rac guanine nucleotide exchange factors P-Rex1 [76,85,171 173], glutathione peroxidase [173,174],
and P-Rex2 and through Rho signaling [160]. In and superoxide dismutase [163,169,175 182].
contrast to the inhibition of mTORC1, mTORC2 is
activated by the TSC1/TSC2 complex through the
N-terminal region of TSC2 and the C-terminal region of 1.4 mTOR, STEM CELL SURVIVAL,
Rictor [161]. Absence of the TSC1/TSC2 complex leads APOPTOSIS, AND AUTOPHAGY
to the loss of mTORC2 kinase activity in vitro [161].
Closely associated with oxidative stress cell injury
are the pathways of programmed cell death that
1.3 mTOR AND OXIDATIVE CELL STRESS involve apoptosis and autophagy (Figure 1.1)
[183 186]. Apoptosis has an early phase that involves
mTOR has a critical role in cell signaling pathways the loss of plasma membrane lipid asymmetry and a
and regulates multiple cellular processes that include later phase that leads to genomic DNA degradation
cellular proliferation, cellular senescence, metabolism, [187 189]. The loss of asymmetry of membrane
gene transcription, cellular survival, and cellular death phosphatidylserine (PS) distribution is an early compo-
(Figure 1.1). Targeting mTOR and its signaling nent of apoptosis that can be reversible [32,81,190].
pathways that control stem cell maintenance may However, if not reversed, cell death ensues and geno-
prove essential for the development of new strategies mic DNA degradation prevails [187,188,191 195].
for NCDs. Stem cells have the potential for treating During the later phase of apoptotic cell injury, cellular
multiple disorders. As a result, the ability of mTOR to DNA is destroyed, which is usually not a reversible
oversee processes of cell injury and cell death becomes process [39,179,196,197]. Externalization of PS residues
a vital consideration in the development of stem-cell- and the onset of genomic DNA degradation are the
mediated therapies. result of a series of activation of nucleases and
Oxidative stress can markedly impact cellular proteases that occur during apoptosis [148,198]. The
survival and longevity [11,53,162 167]. During early phase of apoptosis with membrane PS externali-
oxidative stress, reactive oxygen species (ROS) are zation activates inflammatory cells to engulf and
generated that include nitrogen-based free radical remove injured cells [191,193,199,200]. Inhibition of
species such as nitric oxide and peroxynitrite as well membrane PS externalization is necessary to prevent
as superoxide free radicals, hydrogen peroxide, and the loss of functional cells that are temporarily injured
singlet oxygen [163]. Mitochondria also lead to the and expressing membrane PS residues.
generation of ROS. Mitochondria produce adenosine Under most circumstances, activation of mTOR and
triphosphate (ATP) through the oxidation of glucose, its related pathways of PI 3-K and Akt can prevent
pyruvate, and NAD1 that are present in the cytosol. apoptotic cell death in stem cells. Loss of mTOR
NAD1 and flavin adenine dinucleotide (FAD) are activity, such as with rapamycin, leads to endothelial
reduced to NADH and FADH2 in the tricarboxylic progenitor cell apoptotic death. These detrimental
acid cycle. This redox energy from NADH and effects during mTOR diminished activity may be
FADH2 is transferred to oxygen through the electron related to inhibition of growth factor signaling [201].
transport chain. Protons are then transferred from Growth factors, such as erythropoietin (EPO) [49,202],
complexes I, III, and IV in the inner membrane to the are protective against apoptosis through mTOR
intermembrane space with a subsequent proton gradi- activity against multiple insults. These include sepsis-
ent that is formed across the inner membrane. associated encephalopathy [203], oxidative stress [129],
Complex V (ATP synthase) accumulates the energy cerebral microglial injury [204], and beta-amyloid (Aβ)
from this gradient to produce ATP from adenosine toxicity [205]. EPO relies upon mTOR for cell
diphosphate and inorganic phosphate (Pi). development, differentiation, and survival [11,35].

1.4 mTOR, STEM CELL SURVIVAL, APOPTOSIS, AND AUTOPHAGY 9
EPO requires mTOR for the differentiation of neural autophagy employs cytosolic chaperones for the
precursor cells to achieve a neuronal phenotype [206] transport of cytoplasmic components across lysosomal
and for the protection of retinal progenitor cells from membranes [14].
oxidative stress [207]. EPO controls mTOR and its The proteins TOR and mTOR are directly tied with
downstream signaling pathways that involve PRAS40 genes that control autophagy [27,29,225]. At least 33
to increase neuronal survival during oxygen-glucose autophagic related genes (Atg) have been identified in
deprivation [129]. EPO, through mTOR and Wnt sig- yeast that can affect multiple disorders including DM,
naling, maintains microglial survival during oxidative vascular disease, cancer, and neurodegenerative disor-
stress [204]. EPO can block Aβ toxicity through Wnt ders [10,185,222,223,226 230]. Of these autophagic
signaling and mTOR pathways and prevent caspase related genes, Atg1 and Atg13 (also known as Apg13)
activation and apoptosis [205]. During hypoxia- are associated with PI 3-K, Akt, and the TOR
reoxygenation stress, EPO increases mTOR activity to pathways. Dephosphorylation of Atg13 that can occur
protect hippocampus-derived neuronal cells [208]. In during starvation leads to the activation of Atg1 and
the mTOR pathway, AMPK may also affect the biolog- the induction of autophagy. Phosphorylation of Atg13
ical function of EPO. The ability of EPO to oversee through a TOR-dependent pathway releases Atg13
neuroinflammation may be linked to AMPK activity from Atg1 and reduces Atg1 activity. In mammals, the
[209]. EPO may require a specific level of AMPK activ- homologs of Atg1 are UNC-51 like kinase 1 (ULK1)
ity to alleviate detrimental effects of oxidative stress and ULK2 [29]. Mammalian Atg13 binds to ULK1,
[210]. In addition, the concentration and activity of ULK2, and FIP200 (focal adhesion kinase family inter-
EPO may influence the protective actions of mTOR acting protein of 200 kDa) to activate ULKs, foster
and signaling pathways associated with AMPK. High phosphorylation of FIP200 by ULKs, and lead to the
concentrations of EPO may promote cellular damage activation of autophagy [231]. mTOR activity blocks
and lessen the activity of mTOR [211]. the induction of autophagy by phosphorylating Atg13
Other growth factors in addition to EPO also and ULKs to inhibit the ULK Atg13 FIP200
rely upon mTOR to maintain stem cell integrity. In complex.
experimental models with mice, the growth factors Under some conditions, autophagy serves as a vital
epidermal growth factor (EGF) and fibroblast growth component to promote stem cell survival that involves
factor (FGF) are protective of stem cells and neurons both SIRT1 and mTOR pathways. In embryonic stem
[212 216]. EGF and FGF use mTOR to maintain the cells, SIRT1 is protective [232] and appears to have
proliferation of neural stem and progenitor cells [217]. an inverse relationship with mTOR [22]. SIRT1 can
EGF requires PI 3-K, Akt, and mTOR pathways depress mTOR-mediated pathways as well as promote
to block cell injury during metabolic stress [218] and to autophagy to preserve the integrity of human embry-
prevent memory impairment [219]. Brain-derived onic stem cells exposed to oxidative stress [233]. SIRT1
neurotrophic factor (BDNF) uses mTOR activation for can foster inhibition of mTOR signaling to promote
memory consolidation [220]. Yet, under some condi- neuronal growth [234]. SIRT1 activity is necessary to
tions, growth factor protection that is dependent upon promote autophagy to maintain proteostasis, produce
autophagy may require blockade of mTOR activity. energy during nutrient deprivation, and maintain mus-
Cortical neurons are protected by BDNF through the cle stem cell activation [235]. In endothelial cells
induction of autophagy and the inhibition of mTOR exposed to oxidized low-density lipoproteins that can
during oxygen deprivation [221]. lead to atherosclerosis, SIRT1 up-regulation in combi-
Autophagy is another pathway of programmed cell nation with AMPK activity leads to autophagy that is
death that is dependent upon mTOR signaling to necessary for cellular protection [150].
influence stem cell survival. Autophagy is a process that However, activation of autophagy during mTOR
recycles components in the cell cytoplasm to remove inhibition can also lead to stem cell demise in some cir-
nonfunctional organelles for disposal and tissue remo- cumstances. Autophagy may foster cellular senescence
deling [15,185,222,223]. Autophagy has classifications [236]. Endothelial progenitor cells that regenerate
that include microautophagy, chaperone-mediated vascular endothelium become dysfunctional with the
autophagy, and macroautophagy [32]. Macroautophagy activation of autophagy during exposure to elevated
is the primary category of autophagy. This process con- glucose [80].
sists of the sequestration of cytoplasmic proteins and Wnt signaling pathways with mTOR can also modu-
organelles into autophagosomes that combine with lyso- late programmed cell death to play a role in stem cell
somes for degradation and recycling [32,66,80,223,224]. survival. Wnt proteins are cysteine-rich glycosylated
Microautophagy results in the invagination of proteins that regulate stem cell proliferation and tumor
lysosomal membranes for the sequestration and diges- cell growth [237 243]. During the activation of autop-
tion of cytoplasmic components. Chaperone-mediated hagy, breast cancer stem cells have been shown to

succumb to apoptosis and the inhibition of Wnt signal- activity leads to mesenchymal stem cell senescence
ing [228]. In addition, growth factors, such as EPO, [258]. Loss of mTOR activity can result in cell pluripo-
may require Wnt signaling for the preservation of mes- tency, cell proliferation, and inhibition of mesoderm
enchymal stem cells [244] and to limit “proapoptotic” and endoderm activities in embryonic stem cells [259].
forkhead transcription factor activity [245,246]. In contrast, activation of mTOR and its downstream
A downstream target in the Wnt pathway is Wnt1 signal transduction pathways can lead to cell differenti-
inducible signaling pathway protein 1 (WISP1), which ation. Increased mTOR and p70S6K activity results in
is also known as CCN4 [9]. WISP1 is a member of the embryonic stem cell differentiation [260].
six secreted extracellular matrix-associated CCN family
of proteins that are involved in cellular survival
and stem cell proliferation [247]. WISP1 is present in 1.6 mTOR, STEM CELLS, AND METABOLIC
the brain, heart, kidney, lung, pancreas, placenta, DISEASE
epithelium, ovaries, small intestine, and spleen [247].
WISP1 is closely tied to mTOR and can significantly mTOR pathways are critical components for insulin
influence stem cell survival and proliferation. WISP1 signaling (Figure 1.1) [64]. mTOR inhibition, such as
can control induced pluripotent stem cell reprogram- with rapamycin administration, results in reduced
ming [248,249]. WISP1 is also one of several genes β-cell function and mass, insulin resistance, decreased
that are overexpressed during pancreatic regeneration insulin secretion, and DM [261]. Blockade of mTOR
[250] and can support vascular regeneration during activity with rapamycin also reduces food intake and
saphenous vein crush injury [251]. However, WISP1 prevents fat-diet-induced obesity in mice. However,
oversees cellular senescence [252] and does not appear rapamycin administration can impair glucose uptake
to foster excessive cellular proliferation under circum- through the blockade of insulin-generated Akt activa-
stances involving aging vascular cells [253]. WISP1 can tion and alteration in the translocation of glucose
also be differentially regulated in stem cells. WISP1 transporters to the plasma membrane as has been
expression is increased during stem cell migration [254] demonstrated in skeletal muscle [99]. Furthermore,
and is repressed during hepatic differentiation in loss of p70S6K activity results in hypoinsulinemia,
adipose-derived stem cells [238]. WISP1 also relies insulin insensitivity to glucose secretion, glucose intol-
upon pathways of mTOR to prevent injury to cells erance, and decreased pancreatic β-cell size [262].
[6,29]. WISP1 activates mTOR, inhibits PRAS40 [131], Activation of the mTOR pathways p70S6K and 4EBP1
and limits TSC2 activity [144] to increase microglial cell in pancreatic β-cells in mice leads to improved insulin
survival during oxidative stress and Aβ toxicity. WISP1 secretion and resistance to β-cell streptozotocin toxicity
also controls the post-translational phosphorylation of and obesity [263].
AMPK [35,64,136,255]. WISP1 regulates AMPK activa- Yet, activation of mTOR does not always lead to
tion by differentially decreasing phosphorylation of improved cellular metabolism and may require limited
TSC2 at serine1387, a target of AMPK, and increasing activity through the inhibition of this pathway. mTOR
phosphorylation of TSC2 at threone1462, a target of Akt can function in a negative feedback loop and produce
[144]. This enables WISP1 to provide a minimal but not glucose intolerance by inhibiting the insulin receptor
excessive level of TSC2 and AMPK activity to promote substrate 1 (IRS-1). mTOR signaling through the
cellular survival during toxic insults [144]. TSC1/TSC2 complex can inactivate IRS-1 and phos-
phorylate p70S6K to block IRS-1 activity [264]. With
this pathway and mTOR activation, mTOR leads to
1.5 mTOR, STEM CELL PROLIFERATION, inhibitory phosphorylation of IRS-1, impaired Akt
AND STEM CELL DIFFERENTIATION signaling, and insulin resistance in studies with high-
fat-fed obese rats [265]. Consumption of high-fat diets
mTOR can oversee the proliferation of stem cells in can also activate the renin angiotensin aldosterone
multiple systems of the body (Figure 1.1) [29]. system with increased circulating angiotensin II that
Embryonic stem cell proliferation is decreased during activates mTOR and p70S6K to phosphorylate IRS-1
the deletion of the C-terminal six amino acids of mTOR and foster insulin resistance [266].
that leads to inhibition of kinase activity [256]. Deletion Additional studies also suggest that limited activity
of the mTOR gene results in limited trophoblast of mTOR may be beneficial during DM. In studies
growth, faulty implantation, and the inability to that have examined caloric restriction in male mice,
establish embryonic stem cells [257]. mTOR can also genes with the greatest statistical change after
control undifferentiated stem cell growth in human caloric restriction involved those associated with
embryonic stem cells as well as lead to activation of sirtuin activation and mTOR inhibition [267]. SIRT1
cell differentiation. Under some conditions, mTOR activation can prevent endothelial senescence during

1.8 mTOR, STEM CELLS, AND THE VASCULAR SYSTEM 11
hyperglycemia [268], limit endothelial atherosclerotic necessary for the expression of the neuronal phenotype
lesions during elevated lipid states [269], and prevent of postmortem neuronal precursors [206].
endothelial cell apoptosis during experimental DM Growth factors that employ mTOR can also direct
[193,270]. SIRT1 and mTOR signaling appear to have stem cell development. EPO promotes Wnt signaling
an inverse relationship in the control of cellular to maintain the survival of mesenchymal stem cells
metabolism. Hepatic SIRT1 deficiency yields hepatic [244]. EPO also requires mTOR to regulate bone
glucose overproduction, hyperglycemia, initiation of homeostasis with osteoblastogenesis and osteoclasto-
oxidative stress, and inhibition of the gene encoding genesis [281]. Differentiation of neural precursor cells
Rictor that results in impaired mTORC2 and Akt that may be used for the treatment of neurodegenera-
signaling [271]. SIRT1 can function as a negative tive disorders is dependent upon both EPO and
regulator of unfolded protein response signaling and mTOR [206].
inhibit mTOR in DM to attenuate hepatic steatosis, Importantly, the degree of mTOR activity may affect
ameliorate insulin resistance, and restore glucose different populations of stem cells. Inhibition of mTOR
homeostasis [272]. During experimental DM models activity can result in stem cell differentiation into
with high glucose exposure to mesangial cells, astrocytic cells [217]. Combined Akt and mTORC1
activation of SIRT1 with mTOR inhibition is required inhibition can influence stem cell proliferation and has
to promote mesangial cell proliferation [273]. SIRT1 been shown to lead to reduced neuronal stem cell
is necessary for the protection of endothelial progeni- self-renewal and earlier neuronal and astroglial differ-
tor cells during high glucose exposure [274] and entiation [282]. Non-neuronal neighboring cells may
decreased SIRT1 levels have been observed in represent another factor to influence the growth
endothelial progenitor cells in patients with DM [78]. of neuronal stem cells. Endothelial cells can foster
SIRT1 also has been shown to foster angiogenesis for mTOR activity and lead to the expansion of long-term
vascular repair mechanisms during DM [275]. SIRT1 glioblastoma stem-like cells [283].
may provide progenitor cell protection through the
maintenance of mitochondrial pathways. Autophagy
and mitochondrial impairment occur in endothelial
progenitor cells during exposure to elevated glucose 1.8 mTOR, STEM CELLS, AND THE
exposure, suggesting a mechanism for dysfunction of VASCULAR SYSTEM
endothelial progenitor cells during DM [80]. SIRT1
has been shown to prevent mitochondrial depolariza- Protein kinase signaling plays an important role in
tion, cytochrome c release, and Bad, caspase 1, and stem cell development in the cardiovascular system
caspase 3 activation [193,233,270,276]. (Figure 1.1). In particular, mTOR is one of several
components important for the proliferation of human
embryonic stem-cell-derived cardiomyocytes [284].
mTOR can regulate the proliferation of hematopoietic
1.7 mTOR, STEM CELLS, AND THE stem cells and progenitor cells [285]. Loss of mTOR
NERVOUS SYSTEM results in endothelial cell death [201]. Similar to neural
stem cells that are dependent upon growth factors
In the nervous system, a loss of mTORC1 activity in and mTOR for survival, failed endothelial progenitor
neural stem cells results in reduced lineage expansion, cell development may be the result of decreased
blocked differentiation, and failed neuronal production growth factor signaling and loss of mTOR activity
(Figure 1.1) [277]. mTOR is necessary for insulin- [201]. Insulin-like growth factor relies upon mTOR
induced neuronal differentiation in neuronal progeni- and Akt for the development of cardiac progenitor
tor cells [278]. mTOR also governs the timing and cells [286]. mTOR may be required for endothelial
control of neurogenesis. Inhibition of mTOR through progenitor cells to initiate angiogenesis that can
the RTP801/REDD1 pathway delays neuronal differen- subsequently offer neuroprotection during cerebral
tiation. Over time, the expression of RTP801/REDD1 is ischemia [287]. In some studies, mTOR activity has
altered with maturation. Levels of RTP801/REDD1 in been demonstrated to have a dual role. mTOR not
newborn and mature neurons become diminished and only leads to stem cell proliferation, but also promotes
mTOR activity is increased to promote the maturation apoptotic cell death, suggesting that a defined degree
of neurons [279]. Interestingly, with aging, the loss of of mTOR activity is necessary to maximize stem cell
mTOR activity may affect neural stem cell prolifera- viability [288]. For the maintenance of hematopoietic
tion. mTOR signaling in the aged brain is reduced and stem cells with blocked differentiation, reduction in
is accompanied by a reduction in the proliferation of mTOR signaling with decreased phosphorylation of
active neural stem cells [280]. mTOR activity is also p70S6K is required [289].

1.9 mTOR, STEM CELLS, the proliferation of cancer [314 316] and blood brain
AND TUMORIGENESIS barrier injury [317]. In addition, downstream pathways
of Wnt, such as WISP1, can promote distant metastatic
Strongly associated with proliferative pathways, disease [318]. Variants of WISP1 are aggressive in
mTOR also may lead to detrimental effects and promoting cell growth [319]. In contrast, nonvariant
promote tumor cell growth through cancer stem WISP1 expression can block tumor cell invasion, motil-
cell proliferation. mTOR activity is associated with ity, and metastatic disease [320]. Under these scenarios
neurofibromatosis type 1, tuberous sclerosis, of tumor cell growth with Wnt signaling, mTOR
Lhermitte Duclos disease, and glioblastoma multi- activity may represent a “biological checkpoint” that
forme [29]. As a result, the US Food and Drug can limit excessive cell growth that is promoted by
Administration (FDA) has approved rapamycin (siroli- Wnt signaling. For example, mTOR activation
mus) and several rapamycin derivative compounds, maintains cell senescence and prevents tumor cell
known as “rapalogs,” that inhibit mTOR for the growth that is normally fostered by Wnt [321].
treatment of subependymal giant cell astrocytoma
associated with tuberous sclerosis, renal cancer, and
neuroendocrine pancreatic tumors [116,139]. 1.10 FUTURE CONSIDERATIONS
In regards to stem cell proliferation that can foster
tumor growth, neuronal activity that promotes The 289-kDa serine/threonine protein kinase mTOR
high-grade glioma proliferation in neural precursor oversees stem cell development, maintenance, prolifer-
and oligodendroglial precursor cells has recently been ation, and differentiation in multiple systems of the
linked to mTOR activity (Figure 1.1) [290,291]. body. In human embryonic stem cells, mTOR controls
Blockade of mTOR activity can prevent the conversion undifferentiated stem cell growth as well as the
of astrocytoma cells to oligodendroglioma cells that processes that lead to stem cell differentiation. During
foster the development of glioblastoma multiforme metabolic disorders such as DM, activation of mTOR
[292]. Inhibition of mTOR signaling may limit the pop- may be necessary for proper pancreatic β-cell function
ulation of cancer stem cells that can lead to disease and insulin sensitivity. However, a limited level of
recurrence and therapeutic resistance [293]. For mTOR activity with SIRT1 activation is required
example, activation of mTOR has been correlated with to restore glucose homeostasis, foster mesangial
chemotherapy resistance of breast cancer cells with cell proliferation, promote endothelial progenitor cell
stem cell characteristics [294]. Furthermore, in naso- number, and allow for vascular repair mechanisms
pharyngeal carcinoma, cancer stem cell properties are during DM. In the nervous system, mTOR activity is
reduced and invasion potential is restrained with the necessary for lineage expansion, cell differentiation,
inhibition of mTOR signaling [295]. and neuronal cell production. During aging, mTOR
However, the role of mTOR during tumor growth is activity is diminished and leads to a reduction in the
complex since mTOR signaling is linked to other proliferation of active neural stem cells. In the
proliferative pathways. An example of this involves cardiovascular system, mTOR regulates proliferation
the Wnt signaling pathway. During injury paradigms, of hematopoietic stem cells, cardiac progenitor cells,
Wnt signaling can be cytoprotective [239,241,296 298]. and endothelial progenitor cells that are necessary for
Activation of Wnt pathways can block inflam- angiogenesis. mTOR also plays a prominent role in
matory cell loss during neurodegenerative disorders tumorigenesis through cancer stem cell maintenance,
[187,192,205,299], limit cerebral ischemia [300,301], proliferation, and invasion. The function of mTOR in
protect cells during experimental DM [57,58,302], cancer is complex, since at times mTOR can serve
foster neurogenesis [303] and stem cell differentiation to block tumor cell growth.
[304], and promote wound healing [305]. Growth In the development of stem cell strategies for
factors also require Wnt to prevent cerebral endothelial NCDs, several considerations for mTOR need to be
cell injury [58], limit apoptosis during forkhead addressed to move forward. First, what are the
transcription factor activation [57,306], preserve mesen- pathways of programmed cell death that would be
chymal stem cells [244], maintain immune cells in the most conducive to promote stem cell maintenance and
nervous system [204], and block Aβ toxicity in cerebral proliferation? Under a number of conditions, mTOR
microglia [205]. However, Wnt signaling may lead to activation can prevent apoptotic cell death and may
glioma proliferation [307,308], metastatic disease assist growth factor protection of stem cells through
[309 312], and malignant melanoma [313]. Prolonged the blockade of apoptosis. However, some populations
exposure of growth factors such as EPO that rely upon of stem cells, such as cortical neurons, use autophagic
Wnt signaling can have detrimental effects including pathways for cell protection that inhibit mTOR.

REFERENCES 13
In combination with SIRT1 activation and inhibition of apoptotic cell death, implying that a defined degree of
mTOR, autophagy can also preserve human embryonic mTOR activity is necessary to maximize stem cell
stem cells exposed to oxidative stress and promote neu- viability. As a proliferative agent, mTOR can also
ronal cell growth. However, the pathways of autophagy promote tumor cell growth through cancer stem cells.
associated with mTOR are not straightforward. mTOR inhibition may be necessary to block cancer
Activation of autophagy with mTOR inhibition can stem cell growth that can lead to disease recurrence
also result in stem cell demise. Autophagy may lead to and therapeutic resistance. However, mTOR holds a
cellular senescence and result in endothelial progeni- complicated role in tumor cell growth, since it is linked
tor cell dysfunction. Further necessary analysis may to other proliferative cell pathways necessitating
involve the investigation of the role of pathways such careful governance of the cellular growth properties
as Wnt signaling and WISP1 that also employ mTOR for mTOR. mTOR may function as a “biological check-
signaling and can promote stem cell survival, but point” by maintaining cell senescence and blocking
under some circumstances do not promote excessive tumor cell growth that is normally fostered through
cellular proliferation. proliferative pathways such as Wnt signaling.
Another challenge for targeting mTOR involves
the ability to direct stem cell differentiation. How
can mTOR activity be finely controlled and balanced Acknowledgments
to oversee stem cell differentiation and maintain This research was supported by the following grants to Kenneth
pluripotency? Absent or limited mTOR activity leads Maiese: American Diabetes Association, American Heart Association,
to cell pluripotency and cell proliferation. In contrast, NIH NIEHS, NIH NIA, NIH NINDS, and NIH ARRA.
activation of mTOR leads to stem cell differentiation,
stem cell senescence, and potentially stem cell
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C H A P T E R
2
mTOR: The Master Regulator of Conceptus
Development in Response to Uterine
Histotroph During Pregnancy in Ungulates
Xiaoqiu Wang1,2, Guoyao Wu1,2 and Fuller W. Bazer1,2
1
Center for Animal Biotechnology and Genomics, Texas A&M University, College Station, TX, USA 2Department of
Animal Science, Texas A&M University, College Station, TX, USA
2.1 INTRODUCTION and growth factors) into the uterine lumen that are collec-
tively known as histotroph, which is required for growth
In all mammalian species, the greatest constraint to and development of the conceptus during the periods of
reproductive performance is embryonic mortality, implantation and placentation. Animal studies have
which, in most cases, claims 20 40% of embryos [1]. shown that an inappropriate nutrient supply reduces
Of these embryonic losses, two-thirds occurs during utero placental blood flows and stunts fetal growth,
the peri-implantation period of pregnancy due to fac- resulting in intrauterine growth restriction (IUGR)
tors that include asynchrony between conceptus [6 8,10 12]. Successful establishment and maintenance
(embryo/fetus and its associated extraembryonic of pregnancy requires appropriate development of the
membranes) and uterine signals that regulate con- conceptus for pregnancy recognition signaling [e.g., inter-
ceptus elongation (ungulates), expansion (horse), or feron tau (IFNT) in ruminants, estrogen in pigs, prolactin,
invasion (rodents and primates) and uterine receptiv- placental lactogen or prolactin-like hormones in rodents,
ity to implantation, resulting in defects in conceptus and chorionic gonadotrophin in primates] required for
development and implantation [1,2]. Even for those maintenance of the corpus luteum (CL) that secretes pro-
conceptuses who survive and are eventually born, gesterone to maintain pregnancy [1,2]. Progesterone is
inappropriate hormonal regulation, metabolism, and required for an intrauterine environment that supports
cell signaling such as via nutrient-sensing pathways implantation, placentation, and uterine functions essen-
most likely will predispose them to fetal origins of tial for birth of healthy offspring [1,2]. There is growing
adult disease, the phenomenon known as fetal pro- evidence that the mechanistic target of rapamycin
gramming [3 5]. Namely, alterations in fetal nutrition (mTOR) cell signaling pathway is the master regulator of
and endocrine status may result in developmental cell growth intensively involved in conceptus develop-
adaptations that permanently change the structure, ment, implantation, and placentation in response to com-
physiology, and metabolism of the offspring [6 8], ponents of uterine histotroph during pregnancy.
thereby predisposing individuals to metabolic, endo- Therefore, this review focuses on the relationship
crine, and cardiovascular disease in adult life [3,9]. between selected components of uterine histotroph and
During pregnancy, the uterine epithelia secrete or mTOR as related to development of conceptuses during
selectively transport molecules (e.g., nutrients, enzymes, preangncy, particularly in ungulates.

24 2. mTOR: THE MASTER REGULATOR OF CONCEPTUS DEVELOPMENT
2.2 DEVELOPMENTAL EVENTS the conceptus migrates and undergoes orientation with-
OF UNGULATE CONCEPTUS out definitive cellular contacts between conceptus Tr
and uterine LE, and initiates pregnancy recognition sig-
Despite differences in duration of the preimplanta- naling. IFNT is the pregnancy recognition signal pro-
tion period, as well as type of implantation (noninva- duced and secreted by ruminant conceptuses, whereas
sive vs invasive) and placentation (epitheliochorial in estrogen, placental lactogen or prolactin-like hormones,
pigs, synepitheliochorial in ruminants; endotheliochor- and chorionic gonadotrophin are the pregnancy recog-
ial in rodents; and hemochorial in primates), the early nition signals in pigs, rodents, and primates, respec-
stages of embryonic development and phases of blasto- tively [24 28]. Apposition is phase 3 wherein the
cyst implantation are common across mammalian spe- conceptus Tr associates closely with uterine LE for
cies [13 15]. For early development of a zygote unstable adhesion and, particularly in ungulates, devel-
(a fertilized ovum), successive cleavage events during ops finger-like villi or papillae which extend into the
stage 1 results in formation of a morula (32- to 64-cell superficial ducts of the uterine glands for subsequent
embryo). Compaction is stage 2 of development stable adhesion and to absorb histotroph. Phase 4 is the
wherein the morula forms a blastocyst characterized by adhesion phase during which the Tr becomes firmly
two distinct cell populations, that is, inner cell mass adhered to uterine LE and, in ungulates, superficial
(ICM) which develops into the embryo/fetus, and tro- glandular epithelium (sGE). In sheep and other rumi-
phectoderm (Tr) which gives rise to the placenta. nants, this is the period of interdigitation of conceptus
Briefly, the innermost cells of the morula develop gap Tr and uterine LE in both caruncular and intercaruncu-
junctions that increase intercellular communication and lar areas of the endometrium in preparation for devel-
coordination, whereas the outer cells of the morula opment of cotyledons on the chorion and caruncles on
begin compaction by forming cell cell adhesions the uterine endometrium to form placentomes [23,29].
known as tight junctions that allow them to become Also, mononuclear Tr cells differentiate into trophoblast
polarized and differentiated for transport and accumu- giant binucleate cells that produce placental lactogen,
lation of water and nutrients in a central cavity called progesterone, and other molecules. In pigs, this is the
the blastocele [16 20]. The blastocele is surrounded by period of initiation of the epitheliochorial type of pla-
Tr cells while the ICM is localized to a single pole of the centation in which two apposed cell layers (Tr and uter-
blastocyst. For instance, in sheep, the blastocyst at this ine LE) form an anatomical interaction involving
stage contains approximately 3000 blastomeres, is interdigitated microvilli and increased folding of the
150 200 μm in diameter and is located within the uter- endometrium [29,30]. Phase 5 is unique to species with
ine lumen [17,19]. invasive implantation whereby the conceptus (rodents
Within the uterine lumen, the events of blastocyst and primates), instead of undergoing expansion or elon-
development prior to implantation are divided into five gation, invades through uterine LE, stroma, and even
phases (see Figure 2.1), that include: (1) shedding of the maternal endothelial cells to achieve close contact
zona pellucida (ZP); (2) precontact and blastocyst orien- between Tr and maternal blood in order to increase
tation; (3) apposition of Tr and uterine luminal epithe- nutrient and gas exchange.
lium (LE); (4) adhesion of Tr to uterine LE; and (5) With respect to sheep and pigs, the conceptus
invasion of the blastocyst into the uterine endometrium undergoes a rapid transition from spherical (0.4 mm
which is unique to species such as rodents and primates in diameter at days 10 11) to tubular (1.0 mm in
that have an invasive implantation. Invasive implanta- diameter 3 33 mm in length at days 12 13) to filamen-
tion involves migration of the blastocyst through the tous forms (68 190 mm in length between days 14 and
uterine LE and into the uterine stroma which then 16 of pregnancy in sheep; and 700 1000 mm in length
becomes decidualized [21 23]. Briefly, during phase 1 between days 14 and 16 of pregnancy in pigs) [1,26].
the blastocyst continues to grow by mitotic divisions This process is highly correlated with the composition
and the blastocele increases in volume and pressure. of histotroph. In fact, it is during this period of mor-
The ZP breaks down in response to proteolytic enzymes phological and functional transition in development
from the Tr, and the blastocyst (ICM and Tr) becomes that 30 40% of ungulate conceptuses die as many fail
free to expand further within the uterine lumen. to elongate and achieve sufficient contact between con-
Thereafter, the floating blastocyst, now termed the conceptus Tr and uterine LE/sGE for uptake of nutrients
ceptus, undergoes expansion (horse), elongation from a and other components of histotroph, resulting in inap-
spherical to tubular and filamentous form (ungulates), propriate outside-in or inside-out signal transduction,
or remains spherical prior to implantation (rodents and and failure of maternal recognition of pregnancy
primates). During phase 2, the preattachment period, signaling.

2.3 ROLE OF mTOR AS A MASTER REGULATOR OF CELL GROWTH 25
FIGURE 2.1 The phases of blastocyst implantation in mammals.
2.3 ROLE OF mTOR AS A MASTER lipid, and nucleotide synthesis [43,44], ribosome and lyso-
REGULATOR OF CELL GROWTH some biosynthesis, expression of metabolism-regulated
genes, autophagy, and cytoskeletal reorganization [45].
The mTOR, also known as FK506 binding protein Unlike Saccharomyces cerevisiae that encodes two different
12-rapamycin associated protein 1 is a serine/threonine TOR proteins (TOR1 and TOR2) [46], most eukaryotes
protein kinase that regulates a variety of biological pro- and all mammals have only one gene that encodes TOR/
cesses in response to multiple environmental cues, includ- mTOR. However, all eukaryotes have two TOR/mTOR-
ing, but not restricted to nutrients, growth factors, containing complexes, that is, the MTOR complex 1
hormones, as well as diverse forms of stress [31 33]. (mTORC1) and the mTOR complex 2 (mTORC2) (see
mTOR was first identified and named mammalian target Figure 2.2) [44,47,48]. In mammals, mTOR regulates
of rapamycin in 1994 [34,35] as the mammalian homolog protein synthesis and, therefore, cell proliferation via
of TOR discovered during genetic studies of yeast. mTORC1, which consists of mTOR itself and other
However, the early studies related to mTOR/TOR started components, including: regulatory-associated protein
several decades ago with the compound rapamycin, of mTOR (RAPTOR), mammalian lethal with SEC13
which is a potent antifungal macrolide originally isolated protein 8 (MLST8), proline-rich Akt/PKB substrate
from the soils of Rapa Nui, commonly known as Easter 40 kDa (PRAS40), and DEP domain-containing mTOR-
Island [36]. This compound stirred up clinical and interacting protein (DEPTOR) [31,45,49 51]. mTORC1
research interests due to its antiproliferative and immuno- regulates cell proliferation in part by phosphorylating
suppressive properties in not only prokaryotes, but also ribosomal protein S6 kinase 1 (RPS6K1) and the eIF-
eukaryotes [37 41]. mTOR, the direct target of 4E-binding protein 1 (EIF4EBP1), known regulators
rapamycin [34,35,42], controls many biological processes, of protein synthesis. mTOR also controls cell survival
including cell proliferation via regulation of protein, and spatial aspects of growth, such as cytoskeletal

FIGURE 2.2 Model for induction of cell sig-

naling for proliferation, migration, adhesion, and
cytoskeletal remodeling of conceptuses via
mTORC1 and mTORC2 signaling cascade.
AKT1, proto-oncogenic protein kinase 1; FAK,
focal adhesion kinase; PDK1, phosphoinositide-
dependent protein kinase 1; mTOR, mechanistic
target of rapamycin; RAPTOR, regulatory-
associated protein of mTOR; RICTOR,
rapamycin-insensitive companion of mTOR;
IGF2, insulin-like growth factor 2; type I IGF2,
type I insulin-like growth factor receptor;
ILK, integrin-linked kinases; IRS1, insulin
receptor substrate 1; PKC, protein kinase C; SGK,
serum/glucocorticoid-regulated kinase; MLST8,
mammalian lethal with SEC13 protein 8;
PRAS40, proline-rich Akt/PKB substrate 40 kDa;
DEPTOR, DEP domain-containing mTOR-
interacting protein; MSIN1, mammalian stress-
activated MAP kinase interacting protein 1;
PROTOR, protein observed with RICTOR;
NCK2, noncatalytic region of tyrosine kinase,
beta; NO, nitric oxide; NOS3, nitric oxide
synthase 3; ODC1, ornithine decarboxylase;
PI3K, phosphatidylinositol 3-kinase; LIMS1, LIM
and senescent cell antigen-like domains 1; S6K,
S6 kinase; SPP1, secreted phosphoprotein 1.
organization and Akt/PKB phosphorylation [52], through epithelia, particularly uterine sGE and mid- (mGE) to
mTORC2 that contains mTOR itself and the following deep- (dGE) glandular epithelia into the uterine lumen.
components: rapamycin-insensitive companion of mTOR Histotroph contains molecules that include nutrient
(RICTOR), mammalian stress-activated MAP kinase inter- transport proteins, ions, mitogens, cytokines, lympho-
acting protein 1 (MSIN1), MLST8, DEPTOR, and protein kines, enzymes, hormones, growth factors, adhesion
observed with RICTOR (PROTOR) [51,53 55]. RICTOR, proteins, proteases and protease inhibitors, amino acids,
MSIN1, MLST8, and mTOR are the essential core compo- glucose, fructose, vitamins, and other substances
nents of the complexes required for maintaining struc- [2,57,58]. During pregnancy, the composition of histo-
tural integrity. However, DEPTOR and PROTOR are not troph is essential to the development of the conceptuses
required for mTORC2 activity, but function as regulatory in pigs, ruminants, rodents, and primates, regardless of
proteins whereby DEPTOR seems to negatively regulate differences in type of implantation and placentation. For
mTORC2, whereas the function of PROTOR is unclear example, in primates that have invasive implantation,
[56]. mTORC2 controls actin cytoskeleton organization production of histotroph by uterine GE is a primary
and Akt/PKB phosphorylation [52], which further regu- source of nutrition for conceptus development during at
lates cell migration, growth, survival, and metabolism. least the first trimester of pregnancy, before hemato-
trophic nutrition is fully established [59].
Studies with the uterine gland knockout (UGKO)
2.4 HISTOTROPH AND THE mTOR CELL ewe substantiated the essential role for histotroph from
SIGNALING PATHWAY DURING uterine glands to regulate luteolysis during normal
CONCEPTUS DEVELOPMENT estrous cycles, early conceptus development and initia-
tion of pregnancy recognition signaling [60 62]. This
In mammalian species, histotroph includes secretions UGKO ewe model is produced by the administration of
produced and/or selectively transported by uterine a synthetic, nonmetabolizable progestin to neonatal ewe

2.4 HISTOTROPH AND THE mTOR CELL SIGNALING PATHWAY DURING CONCEPTUS DEVELOPMENT 27
lambs during the critical period of endometrial gland system for arginine includes three members, CAT1,
morphogenesis from birth to postnatal day 56 [63]. This CAT2, and CAT3, which are encoded by members of
early exposure to a progestin permanently ablates the solute carrier family 7 (SLC7) genes known as
development of uterine GE without apparent defects on SLC7A1, SLC7A2, and SLC7A3 genes, respectively.
development of the myometrium or other Müllerian- During the peri-implantation period of pregnancy in
duct-derived structures in the female reproductive tract sheep, expression of both SLC7A1 and SLC7A2 mRNAs
or function of the hypothalamic pituitary ovarian is enhanced in uterine LE and sGE so that more
axis [63,64]. Consequently, the adult UGKO ewes are maternal-derived arginine is transported into uterine
unable to experience normal estrous cycles or support lumen, whereas only SLC7A1 mRNA increases in Tr
conceptus elongation due to the absence of histotroph and extraembryonic endoderm (En) of conceptuses for
from uterine glands. The UGKO ewes also fail as surro- transport of arginine into those tissues [73]. Moreover,
gates to accommodate hatched blastocysts transferred SLC7A3 mRNA is constitutively and weakly expressed
from normal synchronous ewes, in terms of normal in uterine LE, sGE, and conceptus Tr and En [73].
elongation and pregnancy recognition signaling [62]. Therefore, an in utero morpholino antisense oligonucle-
However, uterine histotroph may not be critical for otide (MAO) loss-of-function approach was used to
early embryonic development from the zygote to knockdown translation of SLC7A1 mRNA, the major
hatched blastocyst stage as large spherical and some- arginine transporter in ovine conceptus Tr [73], thereby
times early tubular conceptuses can be found in the successfully depriving the conceptus of arginine and
uterine flushings of bred UGKO ewes [61,62]. resulting in retarded development of conceptuses and a
Uterine flushings from UGKO ewes on day 14 con- significant decrease in IFNT production [79].
tain many components such as galectin-15, glycosylated In vitro studies with an established ovine trophecto-
cell adhesion molecule 1 (GLYCAM1), and secreted derm (oTr1) cell line isolated from day 15 ovine con-
phophoprotein 1 (SPP1, also known as osteopontin), but ceptuses demonstrated that arginine induces both
other unidentified components are absent or reduced proliferation and migration of oTr cells through
significantly which affects the fertility of these ewes stimulation of the tuberous sclerosis 2 (TSC2)-mTOR-
[61,65,66]. mTOR, as the master regulator of cell growth, RPS6K-RPS6 signaling cascade [80 82] and mTORC2-
seems to be heavily involved in such important biologi- mediated cytoskeletal reorganization (X. Wang, G. Wu,
cal processes. In the following sections, the relationships G.A. Johnson, and F.W. Bazer, unpublished results),
between identified components in histotroph and respectively. Consistent with activation of these cell
mTOR cell signaling pathways are summarized. signaling molecules, arginine increases protein synthe-
sis and reduces protein degradation in oTr1 cells [81].
Further studies with oTr1 cells using inhibitors of NOS
such as L-NG-nitroarginine methyl ester and ODC1
2.4.1 Arginine and mTOR difluoromethylornithine, as well as donors of NO such
Arginine is a conditionally essential amino acid for as S-nitrosothiol and 1-[N-(2-Aminoethyl)-N-(2-ammo-
adult mammals, including sheep and pigs [67,68]. It is nioethyl)amino]diazen-1-ium-1,2-diolate and polya-
one of the most abundant amino acids deposited in fetal mines (putrescine) revealed that activation of the
tissue proteins. Concentrations of arginine in histotroph TSC2-mTORC1 cell signaling pathway is induced by
increase by 8 13-fold during the peri-implantation NO and polyamines synthesized from arginine, but
period of pregnancy [3,69 71]. Besides being a building also by arginine itself [82]. Therefore, arginine per se is
block for protein synthesis, arginine is the common sub- a growth factor that cooperatively stimulates prolifera-
strate for production of nitric oxide (NO) via NO tion of oTr cells via the TSC2-mTORC1 cell signaling
synthases (NOSs) and for the biosynthesis of polyamines pathway [82].
via arginase followed by ornithine decarboxylase 1
(ODC1) [11,72 75]. In addition, supplementation of the
diet with arginine increases embryonic and conceptus
2.4.2 Nitric Oxide and mTOR
survival and growth rate of conceptuses in gilts, sheep,
and rats [76,77]. This evidence suggests that the abun- NO, a product of arginine catabolism, is an impor-
dance of arginine available via histotroph or hemato- tant cell signaling gas molecule involved in many
trophic exchange at the utero placental interface is physiological processes in mammals. It is generated
important for fetal growth and development during from conversion of arginine to citrulline by eNOS
pregnancy. (NOS3) and/or iNOS (NOS2) in Tr cells and activates
Arginine transport into cells is mediated primarily guanylate cyclase to produce cyclic guanosine mono-
by the Na1-independent system y1 for cationic amino phosphate that stimulates migration of Tr cells perhaps
acids [78]. The cationic amino acid transporter (CAT) by modifying the extracellular matrix (ECM), inducing

vasodilation of maternal blood vessels [83], or regulat- polyamines via agmatinase (AGMAT) [103,105]. We
ing cellular energy metabolism [84]. During elongation discovered that the alternative ADC/AGMAT pathway
and implantation of ovine conceptuses, NOS3 is the is functional in ovine conceptuses for polyamine biosyn-
major isoform that converts arginine to NO and citrul- thesis and compensates for loss of ODC1 activity follow-
line [72]. Therefore, in vivo knockdown of NOS3 ing in vivo MAO knockdown of translation of ODC1
mRNA reduces NO production by conceptus Tr suffi- mRNA, thereby maintaining the healthy phenotype of
ciently to retard development of the elongating con- conceptuses. Interestingly, not all of the conceptuses
ceptuses; however, the conceptuses produce normal that lost ODC1 activity were able to compensate via
amounts of IFNT [85]. Furthermore, results of in vitro activation of the alternative ADC/AGMAT pathway in
studies revealed that NO stimulates proliferation of order to maintain physiological levels of polyamines. In
oTr1 cells via activation of the TSC2-mTORC1 cell sig- fact, for MAO-ODC1 ovine conceptuses with a defi-
naling cascade, that is, increased abundance of phos- ciency in polyamines, conceptus development including
phorylated TSC2, mTOR, RPS6K, and EIF4EBP1 elongation was completely retarded. Therefore, the
[81,82]. However, NO is not required for IFNT produc- majority of polyamine synthesis may normally be via
tion by oTr1 cells [82], which is consistent with results the conventional ODC1-dependent pathway; however,
of in vivo knockdown of NOS3. the ADC/AGMAT-dependent pathway is, at least in
During the peri-implantation period of ovine con- sheep, a complimentary or compensatory pathway for
ceptus development, there is a significant increase in production of polyamines for supporting survival and
expression of secreted phosphoprotein 1 (SPP1, also development of conceptuses. In addition, activation of
known as osteopontin) by uterine GE [86] and NO such a compensatory pathway may be associated with
induces expression of SPP1 that increases cell adhesion genotype of conceptus, perhaps sexual dimorphism or
and invasion in cultured cells [87,88]. Moreover, polymorphism, but that has not been investigated.
hepatocyte-growth factor-induced motility of human Cell signaling pathways induced by polyamines
trophoblast cells is activated by NO signaling through include tyrosine- and mitogen-activated protein kinase
phosphatidylinositol bisphosphate-3 kinase (PI3K), (MAPK) and proto-oncognes, c-myc, c-jun, and c-fos
serine/threonine kinase (AKT), and mTOR [89,90]. [90]. Of interest in reproductive tissues, polyamines
Expression of NOS2 is highest in peri-implantation also activate mTORC1 cell signaling to stimulate pro-
mouse blastocysts [91]. There are increases in NOS3 tein synthesis and increase proliferation of both por-
and NOS2 activities in ovine placentomes between cine and ovine Tr cells [82,106]. Furthermore,
days 30 and 60 of gestation that are sustained to day polyamines increase production of IFNT, the preg-
140 of gestation to increase placental NO synthesis that nancy recognition signal in ruminants, via activation
is coordinated with increases in placental vascular of mTOR cell signaling in oTr1 cells [82]. In rodents,
growth and utero placental blood flows [91]. polyamines also stimulate Tr cell motility via activa-
tion of mTOR activity, which allows blastocysts to
adhere to uterine LE and undergo superficial implan-
tation [107]. Knockout of the ODC1 gene in mice is not
2.4.3 Polyamines and mTOR lethal until the gastrulation stage of embryogenesis
Polyamines (putrescine, spermidine, and spermine) [108]. There is a requirement for polyamines later in
are products of arginine catabolism required for DNA embryogenesis as ODC1-null embryos at the late mor-
regulation and protein synthesis [92 94], scavenging ula to early blastocyst stages do not survive in vitro
reactive oxygen species [95], cell proliferation [81,96,97], due to apoptotic cell loss in the ICM, but this condition
and differentiation of tissues [96,98], thereby sup- can be rescued by providing putrescine (a precursor of
porting placental development and embryogenesis in spermidine and spermine) in drinking water of the
mammals [89,90,99 101]. Depletion of cellular polya- dam up to the early implantation stage, but not
mines prevents translation of mRNAs and growth of beyond that stage of pregnancy [108].
mammalian cells [102]. During the peri-implantation
period of pregnancy in ungulates, increases in
expression of ODC1 [72], as well as production of
intracellular polyamines, are highly correlated with
2.4.4 Leucine and mTOR
elongation and implantation of conceptuses [103]. Unlike arginine, leucine is not synthesized de novo in
ODC1 is the key enzyme for classical de novo biosyn- any animal cells. However, like arginine, leucine is
thesis of polyamines from arginine, proline, and orni- highly abundant in histotroph during pregnancy and it
thine [104]; however, it is also known that arginine is an important signaling molecule for conceptus devel-
can be converted to agmatine via arginine decarbox- opment. In mice, leucine is required for expanded blas-
ylase (ADC) and agmatine can then be converted to tocysts to exhibit motility and outgrowth of Tr, which is

2.4 HISTOTROPH AND THE mTOR CELL SIGNALING PATHWAY DURING CONCEPTUS DEVELOPMENT 29
essential for implantation [107,109 111]. Of note, leu- abundant hexose sugar in fetal fluids of cetaceans and
cine is the first amino acid that was found to activate ungulates [128 131]. In ewes, for example, the concen-
mTOR in mammalian cells, and this mechanism helps tration of fructose is between 11.1 and 33.3 mM in allan-
explain the initial observation in the early 1970s that toic fluid during pregnancy, whereas the maximum
leucine stimulates protein synthesis and inhibits prote- concentration of glucose is only 1.1 mM [125]. Fructose
olysis in skeletal muscle of rats. It is now known that is also present, but as a relatively minor sugar com-
leucine induces expression of genes such as insulin-like pared with glucose, in fetal blood and fetal fluids of
growth factor 2 (IGF2) [112 114]. Previous studies with humans and other mammals (e.g., dog, cat, guinea pig,
leucine and amino acid transporters suggest that uptake rabbit, rat, and ferret) [126,132,133]. In general, high
of leucine is via transporters, such as SLC3A1, and that concentrations of fructose are found in the fetal fluids
leucine stimulates mTOR cell signaling to induce blasto- of mammals having epitheliochorial and synepithelio-
cyst motility and differentiation [107,115 117]. Leucine chorial placentae [126], such as pigs and sheep, which
may also function to drive arginine transport as a are invaluable animal models for studying IUGR in
counter transporter for uptake of arginine via the Na1- humans [134 136]. The placentae of ungulates contain
dependent system SLC3A1. little or no glycogen. In contrast, rodents and humans
with endotheliochorial and hemochorial placentae,
respectively, metabolize glucose primarily via glycolysis
2.4.5 Glutamine and mTOR to form pyruvate and lactate. In all mammals, rates of
placental glucose utilization via the pentose cycle may
Glutamine is a major physiological precursor of orni-
be particularly high to support DNA synthesis and anti-
thine and arginine, and an essential substrate for the
oxidative reactions.
synthesis of purine and pyrimidine nucleotides for cell
Studies with pregnant ewes have demonstrated that:
division, amino sugars, and nicotinamide adenine dinu-
(1) injection of glucose into ewes causes a rapid increase
cleotide in mammals [70,71,118 120]. Also, glutamine
in glucose followed by a protracted increase in fructose
serves as an energy source for rapidly dividing cells. In
in fetal blood; (2) the placenta is the site of conversion
sheep, the maternal to fetal flux of glutamine is the
of glucose to fructose; (3) production of fructose by the
greatest among all amino acids, particularly between
placenta is independent of concentrations of glucose in
days 13 and 16 of pregnancy when conceptuses are
fetal blood; and (4) glucose can move from blood of the
undergoing rapid elongation [69]. Transfer of glutamine
conceptus to maternal blood, whereas fructose derived
from ewes to their fetuses is also greatest among all
from glucose in the conceptus is not transported into
amino acids measured during mid-gestation [69,121].
maternal blood [130,137 139]. Studies with pigs con-
Physiological levels of both leucine and glutamine
firmed that the placenta is the site of conversion of glu-
induce cell proliferation via stimulation of mTOR
cose to fructose [127]. However, the role of fructose
and RPS6K activities [122]. Interestingly, the actions
during pregnancy in ungulates is largely ignored
of glutamine require the presence of physio-
because fructose is not metabolized via the glycolytic
logical concentrations of glucose or fructose [a pre-
pathway or the Krebs cycle as an energy source.
cursor of fructose-6-phosphate (fructose-6-P) and thus
Proposed roles of fructose are associated with pen-
glucosamine-6-phosphate (GlcN-6-P)], supporting the
tose cycle and hexosamine biosynthetic pathways.
view that the hexosamine pathway plays a cell signaling
Studies with fetal pigs and lambs showed that
role in conceptus growth and development [123]. Further 14
C-labeled fructose could be metabolized for synthesis
study demonstrated that glutamine induces proliferation
of ribose sugars (precursor for nucleic acids) and gen-
of oTr1 cells via hexosamine-mediated activation of the
eration of reducing equivalents in the form of nicotin-
TSC2-mTOR cell signaling pathway (X. Wang, G. Wu,
amide adenine dinucleotide phosphate, which is
and F.W. Bazer, unpublished results). Moreover, gluta-
related to redox status, as well as synthesis of lipids
mine is required to facilitate the uptake of leucine,
[140,141]. However, whether this is quantitatively and
thereby leading to activation of mTORC1 [124].
physiologically significant is unclear, as the purity of
the labeled fructose was not verified in the early stud-
ies. In porcine placentae obtained on days 25 and 60 of
2.4.6 Fructose and mTOR gestation, and in porcine Tr cells, metabolism of fruc-
During pregnancy, placentae of cetaceans (e.g., tose (1, 5, and 30 mM) via the pentose cycle, as
whales and porpoises) and ungulates (e.g., pigs and assessed using [1-14C]fructose and [6-14C]fructose, was
ruminants) are fructogenic as glucose that is not metab- negligible, and there was no detectable production of
olized via glycolysis, pentose cycle, and glycogenesis is pyruvate or lactate from fructose (G. Lin, X. Wang,
converted into fructose by conceptus Tr cells and stored F.W. Bazer, and G. Wu, unpublished data). Recently,
in allantoic fluid [125 127]. In fact, fructose is the most we discovered that fructose is actively involved in

stimulating cell proliferation and mRNA translation

via activation of mTOR cell signaling, and synthesis of
glycosaminoglycans, especially hyaluronic acid, via the
hexosamine metabolic pathway [123]. Specifically,
fructose stimulates phosphorylation of the down-
stream effectors of mTOR, that is, RPS6K1 and
EIF4EBP1, as well as the upstream regulators, that is,
PI3K and AKT [123], which are involved with nutrient
and energy sensing and protein synthesis and respon-
siveness to insulin, growth factors, serum, phospha-
tidic acid, amino acids, and oxidative stress [43].
Once transported into cells, fructose as well as glucose
can be metabolized to fructose-6-P, which is further uti-
lized for synthesis of GlcN-6-P via glutamine:fructose-6-
phosphate transaminase 1 (GFPT1). Inhibition of GFPT1
activity by azaserine abrogates mTOR activation as well
as proliferation of pTr cells [123]. Uridine diphosphate
N-acetylglucosamine (UDP-GlcNAc), the product of
GlcN-6-P, is associated with intracellular signaling as a
substrate for O-linked β-N-acetylglucosamine transferase
(OGT), nuclear pore formation and nuclear signaling
and the glucose-sensing mechanism, as well as insulin
sensitivity of cells [142]. UDP-GlcNAc is the donor
substrate for glycosylation of proteins and lipids. Unlike
N-linked glycosylation, protein O-GlcNAcylation is an
O-linked glycosylation involving attachment of GlcNAc
to serine/threonine residues catalyzed by OGT without
further extension of GlcNAc, whose removal is catalyzed
by O-GlcNAcase (OGA). O-GlcNAcylation is dynamic FIGURE 2.3 Schematic diagram of the GFPT1-OGT-mediated
and reciprocal to phosphorylation at the same or Akt-TSC2-mTOR signaling cascade affected by glucose and fruc-
adjacent serine/threonine residues, and often mutually tose in oTr1 cells. Fructose stimulates GFPT1 for hexosamine biosyn-
inhibitory; however, recent results have also shown the thesis to provide UDP-GlcNAc for O-GlcNAcylation, thereby activating
existence of cooperativeness [143,144]. We further Akt-TSC2-mTOR signaling cascade for stimulation of oTr1 cell prolifera-
tion and growth. Akt, proto-oncogenic protein kinase Akt; TSC2, tuber-
discovered the functional role of fructose to induce ous sclerosis 2; mTOR, mechanistic target of rapamycin; RPS6, ribosomal
proliferation and adhesion of oTr1 cells via activation of protein S6; RPS6K, ribosomal protein S6K; EIF4EBP1, eukaryotic transla-
AKT-TSC2-mTOR signaling cascade (X. Wang, K.A. tion initiation factor 4E-binding protein 1; GlcN-6-P, glucosamine-6-
Dunlap, G. Wu, and F.W. Bazer, unpublished results). phosphate; GFPT1, glutamine-fructose-6-phosphate transaminase 1;
The phosphorylation for activation of this cascade is OGT, O-linked N-acetylglucosamine transferase; OGA, O-GlcNAcase;
UDP-GlcNAc, UDP-N-acetylglucosamine.
mediated by O-GlcNAcylation from UDP-GlcNAc, the
end product of hexosamine biosynthesis (see Figure 2.3).
These results indicate the functional role of fructose in
promoting embryonic/fetal growth and development peri-implantation period of pregnancy, attachment of Tr
during pregnancy, and also provide insights into under- to uterine LE is facilitated by a mosaic of interactions
standing of the relationship between excessive fructose between integrins and ECM proteins, particularly SPP1,
intake and metabolic disorders in human medicine. that contribute to stable adhesion of Tr to uterine LE for
implantation [152 154]. In most mammals studied, secre-
tion of SPP1 by uterine GE increases significantly during
pregnancy [86]. For example, in sheep, implanting con-
2.4.7 SPP1 and mTOR ceptuses secrete IFNT to prolong the lifespan of CL which
SPP1 is a multifunctional ECM protein that binds to secretes progesterone to induce SPP1 synthesis and secre-
cell surface integrin receptors via its Arg-Gly-Asp amino tion from uterine GE into the uterine lumen. Using our
acid sequence to regulate cell proliferation, migration, oTr1 cell line, we demonstrated that SPP1 binds to αvβ3
adhesion, differentiation, survival, and immune functions and αvβ5 integrin heterodimers to initiate focal adhesion
in many physiological systems [86,145 151]. During the assembly, a prerequisite for adhesion and migration of

REFERENCES 31
cells via activation of: (1) P70S6K via crosstalk between nutrient-sensing cell signaling pathways, including
mTOR and MAPK pathways; (2) mTOR, PI3K, MAPK3/ mTOR, the master regulator of cell growth (prolifer-
MAPK1 (Erk1/2), and MAPK14 (p38) signaling to stimu- ative growth via mTORC1 and spatial growth via
late cell migration; and (3) focal adhesion assembly and mTORC2), for growth, development, and survival of
myosin II motor activity to induce adhesion of Tr cells conceptuses, as well as for optimization of culture
[86,146 148,151]. These cell signaling pathways, acting in media for in vitro studies of conceptus development.
concert, mediate adhesion, migration, and cytoskeletal Given the importance of the peri-implantation period of
remodeling of Tr cells required for expansion and elonga- pregnancy in setting the stage for implantation and pla-
tion of conceptuses and attachment to uterine LE for centation, the effects of select components of histotroph
implantation. However, SPP1 alone fails to stimulate pro- on conceptus development have long-term conse-
liferation of oTr1 cells. Interestingly, further investigations quences for the health and wellbeing of the fetus and
related to individual and combined effects of arginine into its adulthood, the developmental origins of health
and recombinant SPP1 have shown their multiple cooper- and disease concept (DOHAD). Although mechanisms
ative effects on adhesion, migration, and proliferation of responsible for differential growth and development of
oTr1 cells [155]. At physiological concentrations, arginine the conceptus resulting in DOHAD phenomena remain
(0.2 mM) significantly increased oTr1 cell proliferation, unclear, they likely involve epigenetic events involving
but SPP1 had no effect. However, the combination of argi- methylation of DNA as well as histone modifications.
nine and SPP1 significantly increased cell proliferation Moreover, for assisted reproductive technologies used
through activation of PDK1-AKT-TSC2-mTORC1 cell sig- in human medicine and animal agriculture, gametes
naling. Arginine is the driving force for cell proliferation and embryos may be prone to epigenetic alterations
and SPP1, by its ability to increase cell spreading and trig- when exposed to specific culture media with an
ger focal adhesion assembly, facilitates cell proliferation imbalanced mixture of nutrients during in vitro proce-
and cytoskeletal reorganization. The synergistic effects of dures. Therefore, future research on the effects of
the combination of arginine and SPP1 on cell adhesion selected components of histotroph on conceptus devel-
and migration were achieved via mTORC2-mediated opment, as well as relationships between histotroph-
cytoskeleton reorganization, including microtubule induced mTOR regulation and epigenetic alterations
α-tubulin, microfilament F-actin, and intermediate fila- during conceptus development, is of great signifi-
ment cytokeratin (X. Wang, G.A. Johnson, G. Wu, and cance for human medicine and animal agriculture.
F.W. Bazer, unpublished results). These results suggest Understanding such relationships between cell signal-
that SPP1 together with arginine, activate mTORC1 and ing pathways and developmental events is critical for
mTORC2 in Tr cells to allow blastocysts to make the criti- enhancing conceptus development, implantation, and
cal transition from spherical to tubular and filamentous placentation, thereby increasing the probability for
conceptuses, which is a prerequisite for signaling preg- birth of healthy offspring.
nancy recognition, implantation, and placentation
required for a successful outcome of pregnancy in ungu-
lates and other mammals. Acknowledgments
Research in our laboratories was supported by National Research
Initiative Competitive Grants from the Animal Reproduction Program
2.5 SUMMARY (2008-35203-19120, 2009-35206-05211, 2011-67015-20067, and 2011-67015-
20028) and Animal Growth & Nutrient Utilization Program (2008-
35206-18764) of the USDA National Institute of Food and Agriculture,
Histotroph includes molecules secreted and/or and Texas A&M AgriLife Research (H-8200). The important contribu-
transported into the uterine lumen and then into the tions of our graduate students and colleagues in this research are grate-
fetal placental vascular system to support key events fully acknowledged.
involved in the development and survival of concep-
tuses during pregnancy in mammals. The increase in
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C H A P T E R
3
mTORC1 in the Control of Myogenesis
and Adult Skeletal Muscle Mass
Marita A. Wallace, David C. Hughes and Keith Baar
Department of Neurobiology, Physiology and Behavior, Functional Molecular Biology Lab,
University of California Davis, Davis, CA, USA
3.1 SKELETAL MUSCLE— sarcomere. It is this alignment that gives skeletal muscle
AN INTRODUCTION its striated appearance.
Skeletal muscle cells are composed of a heteroge-
Skeletal muscle is the most abundant tissue in the neous collection of muscle fiber types and this allows
human body, comprising about 40% of total body for the wide variety of capabilities and functions that
mass [1]. The primary functions of skeletal muscle this organ displays. In human skeletal muscle the three
include the control of movement, posture, and breath- fiber types are referred to as type I, type IIa, and type
ing. Beyond these mechanical roles skeletal muscle also IIx and are named after the respective myosin heavy-
controls whole-body metabolism [1]. Skeletal muscle is chain (MHC) isoform they contain [2]. In rodent
comprised of bundles of muscle fibers (fascicles), skeletal muscle, another fast fiber type exists, type IIb;
nerves, and blood vessels, which are enclosed in a however, this protein is not expressed in human skele-
connective tissue sheath called the epimysium. Each tal muscle. Each muscle fiber type is characterized not
fascicle is surrounded by another connective tissue only by specific contractile properties, but by unique
sheath called the perimysium, whereas each muscle metabolic properties as well. Due to their slow contrac-
fiber within the fascicle is tightly packed and separated tion speed, low fatigability, high mitochondrial content
from other fibers by the endomyosium. Muscle fibers and oxidative capacity, type I fibers are often called
are long cylindrical and multinucleated cells that lie slow oxidative fibers. These fibers are adapted for
parallel to each other. The nuclei of muscle fibers are endurance exercise and are very rare in mice. In con-
located against the inside of the plasma membrane in trast, type IIx fibers are called fast glycolytic fibers
muscle that is called the sarcolemma. Centrally located since they are adapted for short and powerful activi-
within the muscle fibers are the myofibrils, which are ties due to their fast contraction speed, low mitochon-
long bundles of contractile and regulatory proteins, drial content, and high glycolytic capacity. Type IIa
myofilaments. The myofibrils are surrounded by the fibers, or fast oxidative fibers, express fast MHC but
sarcoplasm, which contains important organelles such still have a high oxidative capacity. In fact, in human
as the mitochondria (oxidative metabolism), sarcoplas- mixed muscles type IIa fibers can have more mito-
mic reticulum (calcium storage for muscle contraction), chondria than type I fibers [3].
and the transverse tubules (pathway for action poten- Skeletal muscle is a highly adaptive tissue that can
tials). The apparatus that contracts the muscle cell alter its size and biochemical makeup in response to
consists of two types of myofilaments; a thick filament changes in its environment [4]. Skeletal muscle size
containing the motor protein myosin and a thin and function are tightly regulated by the balance
filament composed mainly of the protein actin. Myosin between the processes controlling muscle protein syn-
and actin filaments are organized longitudinally in thesis (MPS) and degradation. When adult skeletal
the smallest contractile unit of skeletal muscle, the muscle grows, it does so by increasing the size of each

38 3. mTORC1 IN THE CONTROL OF MYOGENESIS AND ADULT SKELETAL MUSCLE MASS
cell much more than the number of cells [5]. This stimulation of its upstream activator Rheb (the ras
growth is positively influenced by increased loading homolog enriched in brain). In both cases, this is
(resistance exercise) and changes in nutrition and hor- achieved by the movement of the tuberous sclerosis
mone levels. On the other hand, adult muscle loss complex 2 (TSC2) away from Rheb; even though the
(atrophy) or wasting is observed in disease states, dis- upstream kinase responsible for phosphorylating and
use, and aging [6,7]. Importantly, muscle atrophy is a moving TSC2 is different [13]. Amino acids (AAs), on
strong predictor of morbidity and mortality in cardio- the other hand, activate mTORC1 by moving mTOR to
vascular, musculoskeletal, nervous, renal, and respira- Rheb. Since AAs and resistance exercise activate
tory diseases, as well as cancer [6,8 10]. Independent mTORC1 in different ways, they have an additive
of disease, muscle loss occurs as we age (sarcopenia), effect when they are combined [14]. The control of skel-
reducing functional independence and quality of life etal muscle growth/hypertrophy via regulation of pro-
as well as increasing the risk of falls and fractures [11]. tein synthesis is the best-known downstream process
As in disease states, muscle strength is one of the best regulated by mTORC1 activation in skeletal muscle.
predictors of longevity with aging [12]. Therefore, the mTORC1 regulates protein synthesis by controlling
maintenance of skeletal muscle mass is essential for the translational activity and capacity of muscle by
improving longevity as well as the quality of life. governing the activity and number of ribosomes,
Extensive research over the past 15 years has both of which are important for muscle growth [15].
improved our understanding of the molecular signal- mTOR, primarily through its role in the mTORC1
ing pathways involved in controlling skeletal muscle complex, plays an essential role in regulating adult
development, growth, and maintenance, as well as muscle hypertrophy. However, mTORC1 also plays
those involved in muscle loss and disease. As in all an important role in embryonic muscle development
cell types, the activity of the mechanistic (or mamma- and regeneration. The following sections will high-
lian) target of rapamycin (mTOR) signaling pathway light the current knowledge of the role of mTORC1
plays an important role in these processes in skeletal in the development of muscle from the embryo
muscle and will be the focus of this chapter. through to muscle growth and the maintenance of
healthy muscle in adult life.
3.2 THE mTOR SIGNALING PATHWAY

IN SKELETAL MUSCLE 3.3 SKELETAL MUSCLE MYOGENESIS—
AN OVERVIEW
mTOR is a serine/threonine (Ser/Thr) kinase that
senses mechanical stress, hormones, and cellular nutri- Myogenesis, the process of generating skeletal mus-
ents and in turn regulates a wide array of cellular cle, occurs during embryonic development and is reca-
processes including cell growth (proliferation and pitulated in adult skeletal muscle following severe
hypertrophy), metabolism, and autophagy. The mTOR injury. During embryonic muscle development, skele-
kinase is part of two distinctive complexes, mTOR tal muscle precursor cells travel from the somite to the
complex 1 (mTORC1) and 2 (mTORC2), which are limb buds to form the early myoblasts [16]. The migra-
generally referred to as being rapamyacin-sensitive tion of these cells and formation of myoblasts are
and rapamyacin-insensitive, respectively. The mTOR dependent upon the expression of the tyrosine kinase
complexes both contain: (1) the mTOR subunit; (2) receptor (RTK), c-met, which is in turn regulated by
the DEP domain containing mTOR-interacting protein the transcription factor, paired box protein 3 (Pax-3)
(DEPTOR); (3) the mammalian lethal with sec-13 [17]. Following the formation of myoblasts, these early
protein 8 [mLST8; also known as the G-protein beta muscle cells are stimulated to grow and divide (prolif-
subunit-like protein (GβL)]; and (4) the Tti1/Tel2 com- erate) by several cell cycle regulatory factors. Myoblast
plex. Unique to mTORC1 is the regulatory-associated proliferation is dependent on the expression of muscle
protein of mammalian target of rapamycin (raptor) and regulatory factors (MRFs): myogenic determination
the proline-rich Akt substrate 40 kDa (PRAS40). In con- protein 1 (MyoD) and myogenic factor 5 (Myf-5)
trast, mTORC2 contains the rapamycin-insensitive com- [18 21]. Once sufficient myoblasts have formed, they
panion of mTOR (rictor), protein observed with rictor 1 exit the cell cycle and begin to fuse. Myoblast fusion
and 2 (protor1/2), and mammalian stress-activated map occurs in two distinct phases. During the initial phase,
kinase-interacting protein 1 (mSIN1). The unique pro- embryonic myoblasts fuse to form primary (nascent)
teins in each complex serve to localize mTOR within myotubes [22]. The formation of these primary myo-
the cell, and identify downstream targets. tubes requires myogenin and the myocyte enhancer
In skeletal muscle, growth factors and mechanical factor 2 and ends when the myotubes become inner-
load (resistance exercise) activate mTORC1 through vated and begin contracting [23 27]. At the onset of

3.3 SKELETAL MUSCLE MYOGENESIS—AN OVERVIEW 39
contraction, fetal myoblasts fuse to each other or with multiple processes during muscle differentiation. Since
the primary myotubes to form the secondary fibers then, mTOR has been shown to regulate myogenesis at
that predominate in mature skeletal muscle [22]. MRF4 two main stages and these are controlled in both a
(also known as Myf-6) and the nuclear factor 1X [28] kinase-independent and -dependent manner. In C2C12
are required during secondary myogenesis, with MRF4 cells, myoblasts fuse to form nascent myotubes when
becoming the prominent MRF expressed in adult skel- expressing a kinase-inactive mTOR (KI-mTOR); how-
etal muscle [29 31]. Following the completion of myo- ever, further maturation does not occur [52]. Similarly,
genesis, muscle fiber number is largely set varying in mice expressing RR/KI-mTOR, primary myofiber
only B5% throughout adult life [5]. formation occurs during regeneration even in the pres-
Myogenesis in adult skeletal muscle (regeneration ence of rapamycin. However, in both cases further
after injury) depends on the activation of muscle- maturation of these fibers is arrested by rapamycin. In
specific stem cells called satellite cells that have the contrast, muscle regeneration occurs normally in the
potential to differentiate into new fibers [32]. This type presence of rapamycin in the RR-mTOR mice [49].
of myogenesis occurs when mature muscle is injured These data suggest that initial fusion of myoblasts
and satellite cells are activated, proliferate, and fuse to requires mTOR, but not its kinase activity, whereas
each other or existing fibers to repair the tissue maturation of muscle fibers requires an active form of
and reestablish homeostasis. In adult muscle, satellite mTORC1 (see Figure 3.1). What is known about each
cells lie outside the sarcolemma but inside the basal of these processes will be further discussed below.
(external) lamina [33 37]. These skeletal muscle pre-
cursor cells can be identified due to the expression of a 3.3.1.1 Kinase-Independent mTOR Regulation
number of distinctive genetic markers such as Pax-3 of Myogenesis
and Pax-7 [38,39]. Following muscle damage, there is Recent work suggests that insulin-like growth factor
an accumulation of leukocytes and macrophages, 2 (IGF-2) is the key intermediary of kinase-independent
which aid in the removal of cellular debris [40,41]. mTOR signaling during myoblast fusion. In this
Macrophages also play an important role in muscle scenario, mTORC1 relocalization by AAs [53,54] drives
regeneration through the production of proinflamma- IGF-2 transcription in both C2C12 myoblasts [54] and in
tory cytokines, such as interleukin-1 (IL-1), interleukin- the early stage of skeletal muscle regeneration in mice
6 (IL-6), and tumor necrosis factor-α, which are [49]. There are two mechanisms by which mTOR con-
involved in satellite cell activation [42]. Upon activa- trols the transcription of IGF-2 in a kinase-independent
tion, satellite cells re-enter the cell cycle, proliferate to manner, first by activating a muscle-specific enhancer
fill the wound site, then differentiate and fuse with the [54] and second, by reducing the post-transcriptional
damaged fibers or form new fibers [43]. inhibition of IGF-2 by microRNA-125b [55]. The result-
ing increased expression of IGF-2 activates the phospha-
tidylinositide 3-kinase (PI3K)/Akt pathway [54,56],
which is essential for fusion. Whether there are other
3.3.1 The Role of mTOR in Myogenesis ways that fusion is controlled by the mTOR kinase-
mTOR is an essential component of the myogenic independent mechanism remains to be determined.
process both in vivo and in vitro. Rapamyacin inhibits
muscle cell differentiation in vitro in rat L6 [44] and 3.3.1.2 Kinase-Dependent mTOR Regulation
mouse C2C12 myoblasts [45 47], as well as inhibiting of Myogenesis
skeletal muscle regeneration in rodents [48,49]. To The final stage of fusion and further maturation of
demonstrate the specific requirement of mTORC1, a myotubes/myofibers is mediated by secreted proteins,
rapamycin-resistant mutant of mTOR (RR-mTOR) has which are regulated in an mTOR kinase-dependent
been used both in C2C12 myoblasts and mice. In the manner. Late in fusion, follistatin is secreted from
presence of rapamycin, muscle cells expressing the myocytes and is a powerful stimulator of muscle
RR-mTOR mutant were able to differentiate and whole cell differentiation and maturation [57,58]. The expres-
muscles regenerated normally, indicating that the sion of follistatin is negatively regulated by histone dea-
effect of rapamycin was specific to its ability to inhibit cetylases (HDACs) and positively regulated by the
mTOR [47,49,50]. Complete knockout of mTOR in activation of the nitric oxide/cGMP signaling [59 61].
mouse muscle results in premature death from a The kinase activity of mTOR enhances production of
severe myopathy, decreased mitochondrial biogenesis, follistatin by regulating the expression of miR-1 in a
and reduced oxidative capacity [51]. MyoD-dependent manner [62]. The mTOR-dependent
Erbay and Chen [47] were the first to demonstrate rise in miR-1 suppresses HDAC4, resulting in enhanced
that rapamycin inhibited myogenesis at multiple transcription of follistatin [62]. The ability of mTOR to
stages, suggesting that mTORC1 was required for control microRNAs might not be limited to mIR-1 [63].

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enemmän seuraa sinulle, mutta minulla oli vähän sanottavaa —
ehdotettavaa — ja arvelin, että ehkä — —"
John keskeytti hänet sydämellisesti torjuen, ja vanhan miehen

ryppyiset silmät värähtelivät.
"Minulle tuottaa sanomatonta iloa, rakas poikaseni, että olet

jättänyt veljeskunnan, ja minä otaksun, että aiot liittyä kirkkoon."
John aikoi suorittaa täydellisen papintutkinnon ja sitten jatkaa
työtään pappina.
"Niin juuri, niinpä juuri" — pitkät kapeat sormet naputtivat pöytää

— "ja minä tahtoisin jollakin tavalla auttaa sinua."
Juoden vettä viinilasistaan alkoi pääministeri sitten hitaasti selitellä

tuumaansa matalalla, virallisella äänellä. Oli eräs piispanistuin
avoinna. "Se oli siirtomaissa — Colombon piispan-istuin. Tulot olivat
pienet, tuhat seitsemänsataa puntaa vain, työ ei ollut helppoa, ja
hiippakuntaan kuului kahdeksankymmentä pappia. Paitsi sitä
siirtomaan piispanistuin ei ole aina portaana kotimaiseen virkaan,
mutta kumminkin —"
John keskeytti hänet taas: "Te olette niin kovin ystävällinen, setä,
mutta minä en halua muuta kuin elää elämäni köyhänä pappina vain
etäällä maailman ja kirkon huomiopiiristä."
"Colombo on epäilemättä kyllin etäällä, poikani."
"Mutta minä en ymmärrä, miksi minun tulisi jättää Lontoo."
Pääministeri katseli häntä vakavasti ja tarkkaan kuin mies, joka on

tottunut tunkeutumaan toisen ihmisen ajatusten ja tunteitten perille.
"Miksikä — miksi sinä siis —"
"Miksikä luovuin luostarista, setä? Siksi että minä huomasin

luostarijärjestelmän perustuvan väärään käsitykseen kristikunnasta.
Sitä on koeteltu melkein yhdeksäntoista vuosisataa, mutta se ei ole
onnistunut. Munkkilaisuuden teoria on se, että Kristus kuoli
vapahtaakseen meidän lihallisen luontomme ja ettei meillä ole muuta
tehtävää kuin uskoa ja rukoilla. Mutta siinä ei ole kylliksi, että Kristus
kuoli kerran. Hänen täytyy kuolla alati — joka päivä — ja jokaisessa
meistä. Jumala kutsuu meitä tällä aikakaudella toteuttamaan
evankeliumia uudella yhteiskunnallisella tavalla, taikka oikeammin
palaamaan takaisin vanhaan käsitykseen."
"Mitenkä siis?"
"Esittämään Kristusta käytännöllisessä elämässä elävänä Mestarina

ja Kuninkaana ja esimerkkinä sekä toteuttamaan kristillisyyttä oman
aikakautemme elämässä."
Pääministeri ei ollut kääntänyt katsettaan hänestä. "Mitähän tuo

tarkoittaa?" kysyi hän itseltään, mutta samalla hän hymyili vain
selittämätöntä hymyään ja rupesi puhelemaan iloisesti. Jos tuo
uskonto soveltui yksityiselle, soveltui se myöskin valtiolle. Mutta
kuvittelepa ministeristöä, joka johtaisi valtion asioita seuraamalla
noita viehättäviä periaatteita, ettei pidä "huolehtia huomispäivästä"
ja että pitää "rakastaa vihollisiaan" ja "kääntää hänelle toinenkin
poski", ja "myydä kaikki sekä jakaa köyhille."
John alkoi puolustautua. Jos kristinusko ei voinut olla ainoana

auktoriteettina kansakuntaa hallittaessa, niin oli siihen syynä meidän
uskomme heikkous ja se, että luotamme liian paljon valtiomiesten
laatimiin koneellisiin lakeihin ja liian vähän Kristuksen laatimiin
siveellisiin lakeihin. "Kristuksen elämä korkeimpana päämääränä ja
esimerkkinä merkitsee joko jotain taikka ei mitään. Jos se merkitsee
jotain, niin koettakaamme seurata sitä. Ellei se merkitse mitään, niin
viskatkaamme herran tähden se syrjään häikäilemättömänä,
petollisena ja moitittavana narripelinä."
Pääministeri kysyi yhä itseltään: "Missä on avain tuohon?" ja

katseli Johnia aivan kuin probleemia, joka oli ratkaistava, mutta siitä
huolimatta hän yhä vielä hymyili ja puheli iloisesti. Totta on, että me
lausuimme rukouksen ja vannoimme valan käsi raamatulla
parlamentissa, mutta ajatteliko kukaan hetkeäkään, että meidän on
aikomus sulkea valtiomme Jeesuksen valtio-opin viehättävän
romanttisuuden huomaan? Mitä taasen kirkkoon tulee, on se
perustettu parlamentin säädöksille, valtio on sen ylläpitäjä ja
perustaja, sen päänä on hallitsija, sen papit ovat siviilivirkamiehiä,
jotka käyvät kunniatervehdyksillä, liikkuvat vastaanottosalien
nurkissa ja kuluttavat ovikoputinta n:o 10:ssä Downin kadun varrella.
Ja mitä Kristuksen lakeihin tulee, selittää niitä tässä maassa
valtioneuvosto, ja ne ovat hallitusdepartementin suoranaisen
valvonnan alaisina. Mutta olihan toiselta puolen vallan viatonta
taikauskoa, että me muka olemme kristitty valtio. Se helpotti
rahvaan pitämistä aisoissa, ja jos John arveli, että —
Pääministeri vaikeni.
"Sano minulle, poikaseni", — hän kosketti Johnin käsivartta, —

"aiotko itse elää… sanalla sanoen… no niin, seuraamalla Kristuksen
elämän esimerkkiä?"
"Siinä määrin kuin heikko, turhamainen ja syntinen luontoni sallii,

setä."
"Ja miten aiot toteuttaa tuota uutta käsitystäsi kristillisyydestä?"
"Minun kokeeni olisi yhteiskunnallista laatua ja etupäässä se tulisi

koskemaan naisia." John oli kuumissaan, ja hänen kasvonsa
punehtuivat.
Pääministeri vilkaisi häneen salaa pöydän poikki, pyyhkäisi laihalla

kädellään otsaansa ja ajatteli: "Vai sillä kannalla asiat ovat!" Mutta
John oli vaipunut syvälle aineeseensa eikä huomannut mitään.
Naisen nykyinen asema oli sietämätön. Naisen menestymisestä ja
varsinkin työtä tekevän naisen menestyksestä riippuu koko
yhteiskunnan onni. Mutta millainen on heidän asemansa nyt?
Ajatelkaapa — ajatelkaapa, kuinka riippuvaisia he ovat miehestä,
ajatelkaapa heidän kiusauksiaan, heidän palkkaansa, heidän
rangaistustaan! Puolitoista penceä on naisen keskimääräinen
tuntipalkka Englannissa — kaiken tämän rikkauden keskellä,
komeuden sydämessä! Ja heillä on aina avoinna tie, jolla pääsevät
helposti ja houkuttelevasti pakenemaan köyhyyttä! Turmio odottaa
heitä, se viittailee heille, se houkuttelee heitä tanssisaleissa,
varieteesaleissa ja rikkaiden, itsekästen miesten luona.
"Ei yksi miljoonasta miehestä voisi käydä semmoisen kiirastulen

läpi turmeltumatta. Mutta mitä me teemme — mitä kirkko tekee
noitten uljaiden olentojen hyväksi, joiden siveydestä ja sankariudesta
kansan onni riippuu? Jos he lankeavat, heittää se heidät syrjään eikä
heillä ole mitään muuta valittavana kuin kadut, rikos, köyhäinhuone
tai itsemurha. Ja kuitenkin kirkko vihkii samat miehet, jotka juuri
ovat heidän viettelijöitään, sieviin, suojattuihin kodin lemmikkeihin,
joiden rikkauden, säädyn tai kauneuden tähden köyhät tyttöraukat
on työnnetty syrjään. Voi, setä, kulkiessani pitkin Regent-katua
päiväiseen aikaan olen vihainen, mutta kulkiessani samaa katua yöllä
olen häpeissäni. Ja kuinka äärettömän yksin sellainen työtätekevä
tyttö on, joka tahtoo elää puhdasta elämää Lontoossa — ja missä
äärettömässä henkisessä yksinäisyydessä hän elää!"
Johnin ääni sortui, mutta pääministeri tuskin kuuli häntä enää.

Ponnistamalla ankarasti ajatustaan hän vihdoin pääsi asiasta
selvyyteen: hänen oma nuoruutensa palasi Johnin puheessa hänen
eteensä, ja hän tunsi syvää sääliä.
"Suvaitseeko teidän ylhäisyytenne juoda kahvia kirjastossa?" kysyi
palvelija hänen kyynäspäänsä takaa.
"Kirjastossa", vastasi pääministeri ja tarttuen taas Johnin

käsivarteen hän palasi toiseen huoneeseen.
Valkea paloi nyt uunissa, ja pienellä pöydällä oli lampun vieressä

kirja, jonka keskipalkoille oli pistetty hopeainen paperiveitsi
kirjanmerkiksi.
"Kuinka sinä muistutat äitiäsi joskus, John! Tuo on juuri kuin

hänen äänensä!"
Kahden tunnin perästä hän saattoi John Stormin pitkin pitkää

käytävää eteiseen. Hänen kalpeat kasvonsa näyttivät lempeiltä, ja
hänen syvä äänensä värähteli hiukan. "Hyvästi, rakas poikaseni, ja
muista nyt, että rahasi ovat täällä sinua odottamassa. Kunnes sinun
kristillis-sosiaalinen valtiosi on perustettu olet sinä vain sosialismin
puoltaja ja voit huoleti käyttää omaa omaisuuttasi. Jos sinun
kristillisyytesi on samaa kuin ensimmäisen vuosisadan, täytyy sen
jollakin tavoin pysyä elossa yhdeksännellätoistakin vuosisadalla,
ymmärräthän. Sinä et voi elää tyhjällä etkä lentää ilman siipiä. Minä
olen utelias näkemään, kuinka paljon nykyinen sivistyskanta sallii
lähestymistä kristittyyn ihanteeseen. Minä en tiedä, mitä sinun
uskolliset ystäväsi ja ihmisten suuri lauma ajattelee sinusta — he
pitävät kaiketi sinua hulluna tai ainakin heikkona ja lapsellisena,
ehkäpä myöskin helposti saavutetun kuuluisuuden hakijana. — Mutta
hyvää yötä nyt, ja Jumala siunatkoon sinua, olitpa sitten kirkossasi
tai työssä vihamiehen vainiolla."
John oli punakka ja kiihoittunut. Hän oli kertonut tuumistaan,

toiveistaan, otaksumisistaan. Jumala pitäisi huolta hänestä siinä
suhteessa, kuten kaikissa muissakin, ja Jumalan papin tulisi olla
köyhä Jumalan tähden. — Sillä välin kaksi samettipukuun puettua
palvelijaa odotti häntä ovella. Toinen ojensi hänelle hänen hattunsa,
toinen kepin ja hansikkaat.
Pääministeri astui takaisin hiljaista käytävää myöten säännöllisin

askelin ja kädet selän takana. Palattuaan kirjastoon otti hän kirjansa
ja koetti lukea. Se oli novelli, mutta hän ei voinut nyt kiintyä toisten
ihmisten elämän tapahtumiin. Yhä uudelleen ja uudelleen hän sanoi
itsekseen: "Poikaraukka! Löytäneekö hän hänet?"
Hän oli näet luonut itselleen varman, liikuttavan käsityksen asiasta

— hän uskoi, että John Stormin rakkaus Jumalaan oli rakkautta
naiseen, ja tuo nainen oli langennut ja turmioon joutunut.
Paria viikkoa myöhemmin John kirjoitti Glorylle:
"Vihdoinkin olen päässyt hyvään alkuun, Glory! Olen

suorittanut täydellisen papintutkinnon ja saanut piispan
'lupakirjan papintoimiin' sekä löytänyt kirkon itselleni. Se on
Pyhän Maria Magdalenan kirkko Crown-kadulla Sohossa, jota
kaupunginosaa jo kolmesataa vuotta on nimitetty 'Vihamiehen
vainioksi' ja joka vieläkin hyvin ansaitsee tuon nimen. Se on
vanha rakennus, jossa on sallittu pitää kirkonmenoja, vaikka
se ei ole kirkoksi vihitty. Ennen se oli kreikkalaisten tai
italialaisten tai ranskalaisten tai muitten maanpakolaisten
turvapaikkana, mutta se on ollut jo kauan autiona ja vallan
epäkunnossa. Sen nykyiset omistajat, eräs kreikkalainen
yhtiö, ovat muuttaneet Sohosta Cityyn, ja koska he ovat liian
köyhiä (minun isäntinäni) korjaamaan rakennusta, on minun
täytynyt pyytää rahaa sitä tarkoitusta varten sedältäni,
pääministeriltä. Mutta rahat ovat kaikesta päättäen minun
omiani, sillä setä on vaatinut isältäni kymmenentuhatta
puntaa äitini myötäjäisiä minun käytettäväkseni ja hän on
myöskin saanut ne. Ja nyt minä aion tuhlata kaksituhatta
puntaa kirkkoni korjaamiseksi, huolimatta pääministerin ja
rouva Callenderin vastustuksesta. Setä sanoo minua
'turkkilais-kreikkalaiseksi pastoriksi', ja rouva Callender on
keksinyt, että minä olen 'nuori sentimentaalinen itkusuu ja
narrimainen tuhlari'. Epäilemättä minä olen kaikkea, mitä hän
sanoo, ja vielä paljoa enemmänkin maailman viisauden
kannalta nähden — mutta mitäpä siitä.
En malttanut odottaa työmiehiä, ennenkuin aloitin puuhat.

Ensimmäinen jumalanpalvelus oli viime sunnuntaina. Ei
mitään urkuria, ei kuoroa, ei lukkaria ja tuskin mitään
seurakuntaakaan. Ei muita kuin kirkon puhdistaja, hyvä,
yksinkertainen vanha sielu nimeltä Pincher, hänen poikansa,
entinen juoppo ja panttilainaaja, sekä vielä toinenkin
kääntynyt, eräs klubin viinuri. Siitä huolimatta minä toimitin
koko jumalanpalveluksen, aamu- ja iltapalveluksen,
rukoukset, psalmit ja saarnan. Jumalalle tulee sitä suurempi
kunnia.
Olen aloittanut uuden ristiretkeni naisten auttamiseksi ja

kulkenut Sohon katuja ristiin rastiin kulkueineni, johon kuuluu
kokonaista kolme henkeä, ensiksikin panttilainaajani, kantaen
lippua (valkoinen risti, johon on tähdätty jos jonkinlaisia
heittoaseita), sitten viinurini, joka kantaa pientä harmoniota
ja on tuttavain kesken tunnettu nimellä 'posetiivi-ukko', ja
lopuksi minä itse, puettuna kauhtanaan. Viimeksimainittu
henkilö näkyy jo olevan hyvin populaari ilmiö ja tunnetaan
'miehenä, jolla on yllään musta alushame'. Yöllä meitä on
seurannut paljoa suurempi kulkue, väkeä, joka sanoo itseään
'Skellingtoneiksi', mutta; jota muut sanovat 'skeleteiksi', s.o.
joukko huonoja naisia ja tappelujunkkareita, jotka elävät
petoelämää tässä tahraisessa, viheliäisessä, unohdetussa
sopukassa. Jumalan kiitos, näitten pahantekijäin asuinsijat
alkavat olla vaarassa, ja he tietävät sen!
Kirkkoni takana on synkkä, epäterveellinen puistokäytävä,

jonka nimi on Crook Lane. Siellä on meidän pappilamme, joka
nykyään vielä on vuokralaisten hallussa, kellarikerroksessa
viinakauppa. Niin pian kuin heidän vuokrakontrahtinsa
saadaan rikotuksi, aion muuttaa huoneiston työnaisten
klubiksi. Miksi en sitä tekisi? Entisinä aikoina tuli kirkko
kansan luo, tulkoon se niin nytkin! Täällä me olemme keskellä
perkeleen, synnin ja rikoksen valtakunnan mahtavaa
linnoitusta. Muukalaisia klubeja, kasinoja, tanssikouluja ja
pelihuoneita on kaikkialla ympärillämme. Mitä meidän on
tehtävä? Sulkeutua telkien taakse (kuten luostarissa) ja
pysytellä erillämme kaikesta kosketuksesta ja
saastaisuudesta? Taivas varjelkoon! Menkäämme alas noihin
siveellisesti sairaisiin luoliin ja puhdistakaamme ne.
Työnaisraukat Sohossa tarvitsevat sunnuntainsa: antakaamme
se heille. He pyytävät musiikkia ja soittoa: antakaamme sitä
heille. He tahtovat tanssia: antakaamme heille sitäkin.
Jumalan tähden antakaamme sitä heille kirkoissamme, sillä
muutoin perkele antaa sitä heille helvetissään!
Tietysti minulle kohta huudetaan ja ulvotaan. Muutamat

hyvät ihmiset varmaan tulevat sanomaan, että olen intoilija
tai kavala keinottelija, kun taasen papilliset paikanonkijat,
jotka rakastavat Egyptin lihapatoja ja joitten katseet ovat
suuntautuneet tämän maailman ja kirkon valtaistuimiin,
tulevat tuomitsemaan minun 'maallisuuttani' ja selittämään,
että minä 'yllytän publikaanien ja syntisten paheita.' Mutta
mitäpä siitä, jos Jumala sitten sallii hedelmien näkyä. Jos
yksikin kalpeakasvoinen, yksinäinen tyttö muuttuu terveeksi
ja onnellisen näköiseksi, tai jos yksi ainoakin iloinen nuori
nainen on saatu pelastetuksi kadotuksen partaalta, on siinä
suurempi uskonnon hedelmä kuin mitä monikaan heistä on
saanut nähdä useiden vuosien kuluessa.
Heti kun työmiehet ovat lopettaneet työnsä kirkossa, aion

ruveta pitämään jumalanpalveluksia joka päivä ja annan
kirkon olla aina avoinna. Asun vielä rouva Callenderin luona,
kuten näette, mutta minä en ota vastaan mitään kutsuja,
paitsi papillisiin toimiin, ja kumminkin on aikani nyt jo niin
täpärällä, että tuskin joudan henkeäni vetämään. Pyhän Maria
Magdalenan kirkko ei tuota mitään tuloja tai tuskin mitään —
juuri sen verran, että se riittää vahtimestarin palkaksi —
mutta minun ei sovi valittaa sitä seikkaa, sillä juuri siksi olen
saanutkin kirkkoni, koska ei kukaan muu ole siitä välittänyt
sellaisissa olosuhteissa. Minä olin ruvennut katsomaan
luostarissa elettyä aikaani turhaan kulutetuksi, mutta Jumala
ymmärsi sen asian paremmin. Se auttaa minua elämään
köyhyyden ja puhtauden elämää vapaana maailmasta.
Tervehdykseni isoisälle ja neideille! Oi kuinka toivoisin, että

te olisitte kanssani taistelun tulessa! Joskus uneksin, että
olettekin, ja kuvittelen näkeväni teidät noitten iloisten, kukilla
ja höyhenillä koristautuvien nuorten olentojen keskellä —
niistä tulee vielä hyviä kristityitä! Omituista kyllä tuntui
minusta sinä päivänä, jolloin läksitte saarelle, kuin joka tunti,
joka vei teidät etäämmälle, olisi tuonut teidät lähemmäksi
minua. Tervehdykseni!"
VII.
Glenfaba, "Saaripahanen."
Oi, armollinen ja laupias ystävä, vihdoinkin olette muistanut

"yksinäistä olentoraukkaa", joka elää elävänä kuolleessa maassa! Jos
minulla olisi ollenkaan sitä sappea, jota oikeaan katkeruuteen
tarvitaan, niin lähettäisin teidän myöhästyneen kirjeenne takaisin ja
kirjoittaisin "tuntematon" teidän "Hyvän Glorynne" poikki, koska
näillä mailla ei enää ole ketään, jolla olisi oikeutta tuohon
arveluttavaan nimitykseen. Mutta oi, voi! Ei ole ajat niinkuin oli
ennen, ja häpeän kyynelin minä tunnustan, että joka ikinen kirje
Lontoosta on minulle kuin enkelin kuiskaus, joka ilman läpi
hengäistään korvaani.
Kun varmasti uskon teidän vaatimuksenne menevän niin pitkälle,

että minun olisi pitänyt kirjoittaa ja kertoa tulostani, ja koska minä
tiedän, että mies on syntynyt turhamaiseksi yhtä varmaan kuin
kipinät lentävät ylöspäin, niin olen jo useamman kuin yhden kerran
tarttunut kynään kirjoittaakseni. Mutta tässä saari-ilmassa on jotain
niin nukuttavaa, että kaikki tähänastiset päätökseni ovat olleet
kuolleena syntyneitä lapsukaisia, jotka ovat hengittäneet, mutta
eivät ole huutaneet.
Tietäkää siis, että matkani tänne tapahtui tavallista kyytiä, eikä
minulle sattunut mitään sen kummempia onnettomuuksia kuin
muulloinkaan, jolloin olen tehnyt mitä minun ei olisi pitänyt tehdä ja
jättänyt tekemättä mitä minun olisi tullut tehdä. Edellisistä
mainittakoon tässä tapauksessa esimerkkinä useat itkupuuskat, jotka
antoivat aihetta eräälle samassa vaunussa matkustavalle
naisveijarille osoittaa osanottoaan — jälkimmäisistä olkoon mainittu
se, että otin esille pikku laukkuni, mikä teki mahdolliseksi tuolle
kohteliaalle olennolle vetää taskustani minun koruompeluilla
koristettu nenäliinani ja viedä se mukanaan.
Minä ennätin hyvissä ajoin laivaan Liverpoolissa, ja se oli kuten

tavallista ahdettu täyteen Lancashiren poikia ja tyttöjä, joissa
rakastuminen on yhtä tarttuvaa kuin pussitauti. Koska minulla sattui
olemaan mieli mustana, jakun muistin, minkä hurjan kiihkon
ajamana olin kerran purjehtinut tänne, niin aloin huomata, ettei
kyynelvarastoni vielä ollut läheskään loppuun kulutettu, mutta
onneksi kiintyi huomioni rakastettavaan pojannallikkaan, joka
kaikessa hiljaisuudessa oli kiertänyt käsivartensa naapurinsa
vyötäisille ja katseli tuontuostakin ympärilleen niin viattomana kuin
ei vasen käsi lainkaan tietäisi, mitä oikea käsi tekee.
Mutta me olimme tuskin ennättäneet ulapalle, kun ilman voimien

hallitsija alkoi kadehtia noitten miekkosten onnea, ja jos olisitte
nähnyt, millainen ilma meillä oli seuraavien neljän tunnin aikana,
olisitte varmaan myöntänyt, että pirulla oli sormensa pelissä. Suuri
laine vyöryi perän yli, ja matkustajat vyöryivät kannen alle
hoiperrellen ja kirkuen kuin rotat olutpanimossa, ja me kiidimme
kotkan siivillä aivan niinkuin Israelin lapset ennen muinoin lensivät
ulos Egyptistä. Koska minun omat merijalkani olivat ennättäneet
käydä hiukan epävarmoiksi, arvelin soveliaaksi mennä alas minäkin,
koira kainalossani, ja koska löysin tyhjän penkin naisten salongissa,
rupesin siihen nukkumaan enkä herännyt ennenkuin olimme jo
saapuneet ristiaallokkoihin aivan saaren edustalle. Kesken kaikkia
oihkimisia ja voihkimisia ja muita ääniä kuulin erään pahoinvoivan
matkustajan kysyvän siistijättäreltä kyynelten sortamalla äänellä:
"Onko mitään toivoa enää?"
Juna saapui Peeliin juuri auringon laskiessa jylhien, vanhojen

linnanmuurien taakse, ja kun taas näin tuon rakkaan pikku
kaupungin, herahti puoli kyyneltä silmistäni ja minut valtasi hurja
halu hypätä ulos ja juosta sitä vastaan. Isoisä oli junalla
"Cæsareineen" ja kiesseineen, ja kun minä olin lopettanut
suutelemiseni ja hän oli lopettanut uhkimisensa ja puhkimisensa
ynnä kaikenlaiset ihmettelevät hassutukset, aivan kuin minä olisin
ollut kuningatar Viktoria ja Ranskan keisarinna yhdistettynä yhdeksi
henkilöksi, — niin, kaiken tuon perästä minä olisin voinut purskahtaa
itkuun nähdäkseni, kuinka pieneksi ja heikoksi isoisä oli käynyt sen
jälkeen kun läksin pois. Me emme päässeet heti lähtemään, sillä
viattomassa ilossaan minun kotiintulostani hän tervehti kaikkia
ihmisiä ja kaikki ihmiset tervehtivät häntä, ja torilla tuo rakas vanha
fariseus puhalsi torveensa niin usein saadakseen ihmisten huomion
kiinnitetyksi meihin, etten uskonut meidän enää mitenkään sinä
iltana saapuvan Glenfabaan. Kun vihdoin viimein pääsimme perille,
odottivat tätöset portilla, ja silloin isoisä taas alkoi päivitellä: "Eikö
hän ole kasvanut pitkäksi? Katsokaa nyt häntä! Eikö hän nyt ole
komea!" Ja sitten tädit rupesivat vanhaan tapaansa myöntelemään:
"Jaa, jaa! Jaha! Niin, niin! Niinpä kyllä!" Kun sitten vihdoinkin olin
suudellut kaikkia toistakymmentä kertaa, olin punasilmäinen kuin
teiri.
Sitten mentiin sisälle, ja ensimmäisten viiden minuutin kuluessa en
voinut käsittää, mikä kumma oli muuttanut tuon vanhan tutun
paikan, kun se yhtäkkiä näytti niin pieneltä ja vähäpätöiseltä. Tuntui
aivan siltä kuin huoneitten seinät olisivat olleet harmonikan palkeita,
joita joku äkkiä puristi kokoon. Mutta tuossa naksutti porstuan iso
kello, ja tuossa kehräsi sir Thomas Traddles vanhalla paikallaan
uunin matolla. Tuossa olivat myöskin samat lautaset kaapin päällä, ja
lieden yläpuolella riippui sama Afrikan kartta, johon oli punaisella
musteella merkitty se paikka, missä isäni kuoli.
Kuu kimalteli meren pinnalla sinä yönä, kun menin levolle, ja kun
heräsin aamulla, paistoi aurinko aalloilla. Varis lensi vaakkuen
ikkunani ohi, ja minä kuulin isoisän hyräilevän itsekseen puutarhan
käytävällä. Ja pitkän Lontoossa-oloni jälkeen sekä edellisen päivän
rautatiematkan perästä tuntui nyt kuin olisin nukkunut uneen
myrskyssä vyöryvillä aalloilla ja herännyt jossakin tyynessä
satamassa.
Siis minä olen palannut takaisin Glenfabaan, vanhaan pikku

kamariini, entiseen pikku vuoteeseeni, ja kaikki on samanlaista kuin
ennen muinoin. Minä alan uskoa, että te menemällä luostariin
ennätitte vain hiukan minun edelleni kuolemisessa pois tästä
maailmasta. Olihan täällä ennen edes muutamia ihmisiä, mutta nyt
ei ole jälellä muuta kuin koira. Kaikki nuoret ovat menneet "muille
maille", ja kaikki vanhat — "taivas tiesi minne." Joskus kuuluu
vuorilta lampaitten määkymistä, joskus lokkien kirkumista ilmasta, ja
joskus pitävät koko maailman harakat kokousta jalavassa
ruohokentällä. Me emme ajattele huomispäivää, mitä me syömme
taikka millä me itsemme vaatetamme, ja sunnuntaisin me menemme
kirkkoon niinkuin Israelin lapset erämaassa, puettuina vaatteisiin,
jotka ovat pysyneet Vanhenematta, vaikka niitä on käytetty
neljäkymmentä vuotta. Viikon muina päivinä me katselemme, kuinka
kärpäset lyövät tyhmät päänsä kattoon, ja kuuntelemme sirkkain
laulua ruohikossa ja nukumme mehiläisten suristessa ja heräämme
vanhan Neiluksen aasin huutaessa hii-haa ja meidän ikämme on
seitsemänkymmentä vuotta ja jos… oo-hoi (haukotus).
Tietysti se on hirveän kiittämätöntä, että minä puhelen näin, sillä

tämä rakas paikka on aivan kylliksi kaunis häiritäkseen
kuvitelmiamme paratiisista ja tämä aamu on raitis kuin kaste
ruohikossa. Leivoset visertelevät ilmassa, virta kohisee tuolla alhaalla
ja pilvet leijailevat kuin vaahtokiehkurat meren yli. Ja mitä taaskin
tulee noihin kolmeen vanhaan ihmislapseen, jotka rakastavat minua
paljoa enemmän kuin ikinä ansaitsen, häpeän koko sielustani
kuullessani, kuinka he pohtivat, mitä herkkuja he keksivät minulle
syötäväksi ja mitä ihmeellisiä hauskuuksia he voisivat saada aikaan,
ettei minulle tulisi ikävä ja etten kaipaisi komeutta, johon olen
tottunut. "Kuulkaa, minä pelkään, että Glory ei välitäkään
herajuustosta", kuulin isoisän kuiskaavan Anna-tädille eräänä
aamuna, ja puoli tuntia myöhemmin hän torui Rakel-tätiä siitä, että
täti muka kiusasi minua liiaksi tulemaan köyhäinkeittolaan keittoa
jakelemaan.
He pitelevät minua kuin pientä paitaressua, ja tietysti minä lapsen

tavalla palkitsen hallitsemalla koko taloa. Mutta ei, voi, voi!
Ikipäiviksi menneet ovat ne ajat, jolloin saatoin elää vesivellillä ja olla
onnellinen reikätuolissa. Niin, niin, muutos on minussa itsessäni eikä
heissä tai vanhassa kodissa, ja mitä hyötyä olisi kellon viisarin
työntämisestä taaksepäin, kun aurinko kumminkin itsepäisesti kulkee
kulkuaan eteenpäin. Minä voisin ehkä vieläkin olla onnellinen
Glenfabassa, jos vain saisin takaisin ne päivät, jolloin puutarhan puut
olivat minun voimistelutelineinäni ja minulla oli tapana kiikkua oksalla
laulellen kuin lintunen, taikka jolloin johdin kylän poikia varastamaan
omenoita meidän omasta puutarhastamme — uljas teko, jonka
vuoksi piispa Anna singahutti minua sekä muita alaisiaan vastaan
mitä ankarimman rangaistusjulistuksensa — käyttäen siinä
muistaakseni omaa tohveliaan vertauskuvana. Mutta minä en voi
enää päivät pitkät juosta avojaloin märässä hiekassa rannalla, missä
suolainen vaahto räiskähtää kasvoihini, ja yksikolmattavuotias
neitonen on hyvin harvoin hippasilla tai rosvosilla kymmenvuotisten
kylätyttöjen kanssa aukeilla paikoilla.
Luonnollisena seurauksena kaikesta tästä on, että minun entiset

seikkailuni ovat nyt supistuneet ratsastusmatkoiksi pikku Cæsarin
selässä. Mutta Cæsar on käynyt niin vanhaksi ja lihavaksi ja se
huojuu kuin vanha ankka, joten ratsastaminen sillä on samanlaista
kuin matruusina ponnisteleminen kiikkuvalla laivalla. Minä siis
pakotan itseni istumaan ompeluseuroissa ja hankaamaan
riimukirjoituksia isoisälle, pesemään maitokehloja Anna-tädille ja
alistumaan semmoisiinkin pyhiin ajanviettoihin kuin säännölliset
kirkossakäynnit ovat, jolloin saan ihailla vanhaa Neilusta hänen
kantaessaan haavia ympäri kirkkoa seurakunnan veisatessa "sun
'valos' meille paistaa suo" — "valon" on Neiluksen käytännöllinen
järki nähtävästi käsittänyt synonyymiksi "hopea" sanalle, joka nyt on
paistava kolmen pencen kappaleissa.
Mutta oi, voi, voi! Minä olen kumminkin susi lammaslaumassa tällä
saarella. Rakkaat vanhat lapsukaiset eivät vielä ole sanoneet
sanaakaan minun siirtymisestäni vihamiehen leiriin (s.o. perkeleen ja
varieteeteatterien), ja jos vain puhe sattuu joskus hiukan
luiskahtamaan siihen suuntaan, niin syntyy heti kuolonhiljaisuus
ympäristössäni. Tämä ei kuitenkaan ole mitään siihen kamalaan
kauhuun verraten, joka valtaa jokaisen, kun vieraat, jotka käyvät
Glenfabassa, kyselevät millä minä itseni elätän Lontoossa. Tuskin
tiedän pitäisikö itkeä vai nauraa tuolle helpotuksen huokaukselle,
joka puhkeaa tätösien sydämestä, kun minä pääsen pujottelemaan
karien läpi valehtelematta. Mutta ankeriasta ei voi piilottaa säkkiin, ja
minä olen varma siitä, että totuus vielä jonakin päivänä pujahtaa
esiin. Eilispäivänä viimeksi, kun kävin Rakel-tädin kanssa köyhiä
tervehtimässä, narrasi eräs kunnon mies, joka pitää ravintolaa
kalastajille, tädin ja minut kapakkansa vierashuoneeseen katsomaan
"Glorian" kuvaa, jonka hän oli leikannut eräästä kuvalehdestä ja
kiinnittänyt nuppineuloilla kamarin seinään "koska se oli niin kovasti
minun näköiseni." Oi, voi, voi, voi! Minä olisin todellakin tahtonut
ilmoittaa asian oikean laidan heti paikalla siinä pahan maineeni
näkyväisen todistuksen edessä, mutta huomatessani pikku tätiraukan
kauhistuneen katseen rupesin lavertelemaan kuin myllyn ratas ja
kiitän Jumalaa, että loput Peelin asukkaista eivät ole kuin muut
ihmiset eivätkä myöskään niinkuin tämä publikaani. [Sama sana
englanninkielessä merkitsee ravintoloitsijaa ja publikaania. Suoment.
muist.]
Minä olen myöskin saanut sanomalehtiä, joita ystäväni Rosa on

minulle lähettänyt, ja niin kauan kuin uutisten onkijat kertoivat
minun hyvistä ja pahoista töistäni valehdellen hellästi kuin
hautakirjoitus minun katoamisestani Lontoosta, leikkasin lehdet
palasiksi ja poltin ne. Mutta kun he sitten unohtivat minut ja
rupesivat kertomaan muitten ihmisten menestyksestä, valitsin
Neiluksen paperikorikseni sillä nimenomaisella suostumuksella, että
sanomalehdet annettaisiin kalastajille, jotka juuri olivat palanneet
Kinsalesta. Vähän väliä hän kertoi minulle, että ne "kiertävät ympäri,
neiti, kiertävät ympäri", uskotellen siten minulle, että niiden
kiertokulku oli laajin maailmassa. Ja se olikin kyllä tavallaan totta,
vaikka tuo vanha veijari jätti sanomatta, että ne kiersivät ympäri
paperitehtaassa muuttuen vähitellen paperimassaksi.
Mutta hohhoo! En minä tarvitse mitään sanomalehtiä muistaakseni

Lontoota! Kuten pyhällä Paavalilla on minullakin paholainen, joka
takoo minua nyrkeillään, ja aina kirkkaina päivinä, kun silmä kantaa
kauas, tuo sama paholainen pakottaa minut kiipeämään Slieu
Whallinin [vuori Man-saarella. Tekijän muist.] harjalle ja istumaan
majakan viereen tuntikausiksi sekä tuijottamaan Englantiin päin
nähdäkseni siitä vilahduksen. Silloin on minussa sama tunne kuin
Lotin vaimossa hänen katsoessaan taaksensa vanhaan kotiinsa, ja
sitten astun alas sydän raskaana ja suussani kyynelten maku, aivan
kuin minäkin olisin muuttunut suolapatsaaksi. Rakas vanha Lontoo!
Mutta kaiketi se kulkee vanhaa kulkuaan liikevirtoineen ja
tungoksineen ja vaeltavine kaupittelijoineen, jotka kirkuen tarjoavat
tavaroitaan, ja kaikki on entisellään, vaikkei Glory olekaan siellä!
Kello puoli 11 illalla. — Minun piti keskeyttää kirjoitukseni

aamupäivällä, sillä isoisä sairastui äkkiä ja sai pyörtymiskohtauksen.
Tietysti lähetimme heti hakemaan lääkäriä, mutta ennenkuin hän
saapui, oli isoisä toipunut. Tohtori katsoi siksi meihin niin rikkiviisaan
näköisenä, kuin isoisä olisi ollut kuva-arvoitus, joka olisi selitettävä
ennen hänen ensi käyntiään, ja sitten hän kirjoitti meille nimensä
(vallan ihmeellisiä harakanvarpaita) sekä läksi pois. Sen jälkeen on
isoisää hoitanut paljoa puheliaampi tohtori, jota isoisä nimittää
"Glenfaban kukaksi" (minä näet olen se), ja kun olin laverrellut
vanhalle lapselleni koko päivän sekä pelannut sakkia hänen kanssaan
illalla, meni hän levolle nauraen, ja minä palasin omaan huoneeseeni
lopettamaan kirjettäni kevyemmällä mielellä. — Viimeisen puolen
tunnin aikana ovat revontulet leimunneet pohjoisella taivaankannella,
ja minä olen ajatellut, että ne ovat kaiketi myrskynmerkkejä
taivaassa aivan samoin kuin rakeet ja sumu sekä vihurinpuuskat ovat
myrskyn ennustajia maan päällä. Mutta revontulet alkavat jo hälvetä,
ja jälellä on vain sysimusta yö, jonka kuluessa Knockaloen sekä
Ballamoarin koirat ulvoen lähettävät sanomia toisilleen.
Oi, armias! Vasta puoli yksitoista! Kuinka omituista on ajatella, että

nyt, kun me täällä maalla juuri olemme menossa nukkumaan ja
kaikki on haudan hiljaista ympärillämme, nyt vasta liike alkaa
Lontoossa! Maatessani vuoteellani ja koettaessani nukkua näen taas
nuo laajat puistot, joitten keskellä on jonkun muistopatsas, ja
teatterin ovella kimaltelee lyhtyjä, ja koko tuo ihmeellinen
pääkaupunki uhkuu eloa! Huh! Tuntuu aivan kuin olisin oma aaveeni,
joka kulkee yläkerran huoneissa ja pimeissä käytävissä kuunnellen
musiikin kaikua ja tanssin humua alakerran tanssisalista.
Mutta, oi armahin Glory! (Sillä tavalla minä vielä voin aivan syystä
huudahtaa). Mitä huolia voisi iloisella olla — ja minä olen aina
iloinen! — Kuinka teidän koiranne voi? Minun rakkini kirjoittaisi
kirjeen sille, mutta rakkiraukan sydän on särkymäisillään, ja jos vain
tällaista asiain menoa jatkuu, täytyy sen kohta ryhtyä laatimaan
testamenttiaan. Parast'aikaa se makaa vuoteeni jalkopäässä miettien
syviä asioita ja käskien minua kertomaan teille, että koetettuaan yhä
uudelleen ja uudelleen päästä kokemasta Glenfaban home rulea
(koska hän ei ole noita luonteita, joita kärsimykset vain jalostavat,
kuten esim. hänen emäntäänsä) pysyy hän nykyään elävien joukossa
vain "oman tahdonlujuutensa avulla" tässä talossa, joka on täynnä
vanhojapiikoja ja jossa ei ole mitään muuta jahdattavaa kuin vanha
kissa; ja sitä todellakaan ei maksa vaivaa ajaa, sillä tuolla kurjalla ei
ole edes häntääkään. Tietysti hän (nimittäin rakki) koettaa elää
tahratonna (kuten eräs toinenkin) tässä matoisessa maailmassa,
joka on niin täynnä viettelyksiä, mutta hän tunnustaa kumminkin
rehellisesti, että pieni annos "pirullisuutta" silloin tällöin auttaisi
häntä katselemaan elämää hurskaammalta kannalta.
Minä toivotan teille "onnea ja lykkyä tykö" uuteen puuhaanne,

mutta jos aiotte ruveta naisen ritariksi tässä maailmassa —
puolustamaan hänen hyviä ja pahoja puoliaan — niin ei teidän pitäisi
olla niin äärettömän ankara moraalissanne. Minä kuulin täällä
kertomuksen eräästä kauniista nuoresta pastorista, joka alituiseen
saarnatuolissaan toisti: "Rakastatko minua?" kunnes eräs neitonen
seurasi häntä sakaristoon tunnustaen, että kyllä hän (neitonen)
rakasti pastoria. — Vakavasti puhuen on teidän aikeenne suuri ja
jalo, joten minun melkein tekisi mieli rakastaa teitä sen tähden. Jos
miehet tahtovat, että naisten pitää olla hyviä, niin he ovat hyviä, sillä
naiset tanssivat miesten nuottien mukaan, kuten he ovat tehneet
Aatamin ajoista saakka — minä en nyt ollenkaan unosia mainitun
herran kiusausta enkä myöskään hänen puolustustaan: "Vaimo,
jonka annoit minulle", mikä osoittaa, ettei hän ollut, herra paratkoon,
kovinkaan ihailtava aviomies, vaikka tosin toiselta puolen täytyy
myöntää, ettei hänellä myöskään ollut hyvin suurta valitsemisen
varaa hakiessaan rouvaa itselleen.
Terveiseni rakkaalle vanhalle Lontoolle! Joskus tekisi mieleni

riistäytyä irti ja lähteä itse viemään terveisiäni. Ehkä tekisinkin sen,
ellei Rosa kirjoittaisi minulle, että hän tahtoisi tulla viettämään
kesälomaansa Peeliin. Enkö ole kertonut teille Rosasta? Hän on se
naiskirjailija, jolle herra Drake esitteli minut. Mutta
'Nukkumaan sä houkkio,
Nukkumatti kutsuu jo!'
Glory.
J.K. — Tärkeätä. — Lontoosta lähdettyäni on minua koko ajan
kiusannut muisto Polly-raukan lapsesta. Hän antoi sen hoidettavaksi
rouva Jupelle, josta kerroin teille, ja tämä henkilö vei sen jollekin
toiselle. Äidin eläessä minä en voinut sekaantua asiaan, mutta nyt
minä en saa tuota orporaukkaa mielestäni. Mikähän hänen
kohtalokseen lienee tullut? Toivottavasti hän on Jeshurunin tavoin
lihonut ja oppinut potkimaan, mutta kumminkin te menettelisitte
miehen ja pappismiehen tavoin, jos ottaisitte minun laiminlyödyt
velvollisuuteni niskoillenne ja menisitte katsomaan, onko tuolla
lapsiraukalla ketään koko maailmassa, joka rakastaisi sitä. Rouva
Jupen osoite on 5 A Little Turnstile, Holbornin ja Lincoln's Inn
Fields'in välillä.
VIII.
John Storm sai Gloryn kirjeen lauantai-aamuna ja saman päivän

iltana hän läksi hakemaan rouva Jupea. Hänen asuntoaan ei ollut
helppo löytää, ja kun John vihdoinkin keksi sen, tunsi hän tuskaa
ajatellessaan, että Glory oli asunut tuossa likaisessa luolassa.
Tullessaan pienen tupakkapuodin ovelle kuuli hän kimeän äänen
sisällä huutavan: "Lapsella ei ole mitään hätää, ja koska te ette voi
maksaa mitään, ei teidän myöskään tarvitse tulla tänne
rettelöimään." Samassa nuori nainen tuli John Stormia vastaan
kynnyksellä. Se oli pieni, hento olento, kuin kukkanen, jonka sade on
murtanut. John tunsi hänet samaksi tytöksi, joka oli kanniskellut
lasta Crook Lanella sinä päivänä, jolloin John ensi kerran kävi
Sohossa. Tyttö itki nyt ja peittääkseen turvonneita silmiään painoi
hän päänsä alas kulkiessaan nuoren papin ohi, mutta tämä huomasi
hänen virttyneet hatunnauhansa ja likaisen olkihattunsa.
Keski-ikäinen nainen tiskin takana kumarsi kunnioittavasti papin

pukuun puetulle herralle, ja pikkuinen tyttö sisähuoneen kynnyksellä
vilkaisi häneen salaa, pää kallellaan.
"Isä Storm, varmaankin. Astukaa sisään ja istukaa, herra pastori!

Vahdi sinä puotia, Booboo. Minun mieheni on kertonut minulle teistä.
'Sinä tuntisit hänet heti paikalla, Lyydi', sanoo hän aina. Hän ei ole
vielä palannut kotiin klubista, mutta hän voi tulla millä hetkellä
tahansa."
John Storm oli istuutunut pieneen, pimeään vierashuoneeseen

katsellen ympärilleen ja muistellen Glorya.
"Ei sillä ole väliä, minulla onkin asiaa teille, rouva Jupe", vastasi
pappi, ja samassa rouva Jupen paksut posket, jotka tähän saakka
olivat olleet yhtenä ainoana hymynä, jäykistyivät ja silmistä välähti
pelokas ilme.
"Mitä on tapahtunut?" kysyi hän vetäen puodin oveni kiinni.
"Ei mitään, toivoakseni, hyvä rouva", sanoi John selittäen asiansa.
Rouva Jupe kuunteli tarkkaan ja näkyi miettivän itsekseen, kuka

oli papin lähettänyt.
"Nuori äitiraukka on nyt kuollut, kuten ehkä tiedätte, ja —"
"Mutta isä ei ole kuollut", tiuskaisi vaimo kiivaasti, "ja, anteeksi

herra pastori, mutta jos hän tahtoo tietää, missä lapsi on, voi hän
tulla itse, eikä lähettää toisia puhumaan puolestaan!"
"Jos lapsi voi hyvin ja on hyvässä hoidossa, hyvä rouva —"
"Se on hyvässä hoidossa ja se on semmoisen henkilön luona,

johon minä luotan."
"Mitä teillä siis on salattavaa? Sanokaa minulle, missä se on ja —"
"E-hei, en ollenkaan! Jos se on hänen lapsensa ja hän tahtoo sen

itselleen, niin maksakoon hän myös siitä ja sen hoidosta tähän
päivään saakka. Nuo keikarit ovat aina niin valmiit lähettämään
pappeja ajamaan heidän kirjavia asioitaan."
"Jos otaksutte, että olen täällä puhumassa isän puolesta,

erehdytte kokonaan, sen voin vakuuttaa teille."
"Oh, todellako? Vai niin!"
Asiat olivat ennättäneet näin pitkälle, kun ovi aukeni ja herra Jupe
astui sisään. Hattu lensi miehen päästä kunnioittavaan
tervehdykseen, mutta huomatessaan pilven vaimonsa otsalla
keskeytti mies tervehdyshommansa. Rouvan esiliinan nurkka kohosi
silmille heti paikalla.
"Mikä nyt on hätänä?" kysyi mies. John Storm koetti selittää asian,
mutta eukko piti parhaana jatkaa itkuaan.
"Se on nyt sillä lailla, isä-Storm, että minun vaimoni pitää niin
hirveästi lapsista, ja hänen sydämensä pakahtuu jos —"
Oliko tuo mies hullu vai ulkokullattu?
"Herra Jupe", sanoi John nousten ylös, "pelkään, että teidän

rouvanne on menetellyt laittomasti ja epärehellisesti —"
"So, soh, herra pastori", sanoi mies pudistellen päätään. "Minä

kunnioitan herra pastori John Stormia paljon, mutta minä kunnioitan
vielä paljoa enemmän rouva Lyydi Jupea, ja mitä laittomaan sekä
epärehelliseen menettelyyn tulee, niin —"
"Älä välitä hänestä, Henry", sanoi vaimo, itkien nyt ääneensä.

"Ja älä sinäkään pane sitä niin pahaksesi, Lyydi", sanoi mies, ja
sitten he katsoivat toisiinsa sillä tavoin, että sopi otaksua heidän
kohta syleilevän toisiaan.
John Storm ei voinut tätä enää kestää. Mennessään alas kapeata

katua hän tunsi katkeruutta ajatellessaan, että Gloryn oli täytynyt
elää kuukausia tuon petturin luona. Samassa joku koski hänen
käsivarteensa pimeässä. Se oli sama tyttö, jonka hän oli nähnyt
mennessään tupakkapuotiin. Tyttö itki yhä vielä.
"Minä satuin näkemään teidät Crook Lanella, herra pastori, sinä

päivänä, jolloin minun lapseni ristittiin, ja nyt minä jäin odottamaan,
koska arvelin, että te ehkä voisitte auttaa minua."
"Tulkaa tätä tietä", sanoi John, ja kulkien Johnin rinnalla Lincoln's

Inn Fieldsin paljaan muurin vartta kertoi tyttö tarinansa. Hän asui
John Stormin pappilassa kirkon; takana. Koska hänen täytyi työllään
elättää itsensä, oli hän vastannut erääseen sunnuntailehdessä
olleeseen ilmoitukseen, ja siten rouva Jupe oli tullut ottaneeksi
hänen lapsensa hoitoonsa. Oli kyllä totta, että hän (tyttö) oli
luopunut kaikista oikeuksistaan lapseen, mutta hän ei malttanut olla
menemättä katsomaan sitä — se oli niin suloinen ja herttainen.
Sitten hän sai selville, että rouva Jupe oli antanut sen jollekin
toiselle. Hän (tyttö) ei olisi ikinä enää saanut kuulla lapsestaan, ellei
hänen "ystävänsä" olisi sattumalta löytänyt sitä eräästä talosta
Westminsteristä. Se oli kamala paikka, jonne miehet menivät korttia
pelaamaan. Sen paikan isäntä oli juuri päässyt vapaaksi
kahdeksantoista kuukauden vankeudesta, ja miehensä ollessa
vankilassa oli tuon talon emäntä ruvennut hoitamaan kasvattilapsia.
Se oli vallan hirveä nainen, ja isäntä oli myöskin kauhea ihminen, ja
molemmat löivät ja pieksivät lapsiraukkoja ihan julmasti. Naapurit
kuulivat alituiseen lyöntejä ja valitusta, joten se heidän mielestään
oli ilmeinen häpeä. Tyttö olisi tahtonut ottaa lapsensa pois sieltä,
mutta vaimo ei antanut sitä, koska kolmen viikon vuokra oli
maksamatta eikä tytöllä ollut rahaa sitä maksaa.
"Voisitteko viedä minut tuohon taloon?"
"Kyllä, herra pastori."
"Tulkaa sitten kirkkoon huomenna jumalanpalveluksen jälkeen."
Tytön itkettyneet kasvot loistivat kuin kevätauringon paiste. "Ja te

autatte siis minua saamaan takaisin pikku tyttöni? Voi, kuinka hyvä
te olette! Jokainen sanoo, että nyt me todellakin olemme saaneet
isän…" Tyttö vaikeni äkkiä ja jatkoi sitten tyynemmin: "Ehkä joku
lainaa minulle villahuivin, johon voin kääriä hänet. Pantiksi kuuluu
kelpaavan vaikka mikä, ja minä voisin pantata sen sievän valkoisen
hameen, jonka ostin tytölleni… Voi, en milloinkaan enää päästä
pienoistani luotani, en milloinkaan!"
"Mikä teidän nimenne on, tyttöseni?"
"Agatha Jones", vastasi tyttö.
Kello oli jo yksitoista sunnuntai-iltana ennenkuin he pääsivät

lähtemään asialleen. Sillä välin Aggie oli esiintynyt kahdessa
muukalaisklubissa ja John Storm oli johtanut kulkueensa kerran
Crown-kadun toisesta päästä toiseen, jolloin eräs "skeleteistä" oli
heittänyt häntä kivellä. Siitä huolimatta pappi ei ollut antanut
heittäjää poliisin huostaan. — John ja Aggie seisahtuivat puiston
nurkkaukseen, ja nuori pappi meni rouva Pincherin luo pyytämään
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Molecules to Medicine with mTOR

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

Oxidative stress via neurodegeneration,

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

Molecules to Medicine with mTOR

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

FIGURE 2.1 The phases of blastocyst implantation in mammals.

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

FIGURE 2.2 Model for induction of cell sig-

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

stimulating cell proliferation and mRNA translation

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

Molecules to Medicine with mTOR

3.2 THE mTOR SIGNALING PATHWAY

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

I. mTOR IN CELLULAR DEVELOPMENT, PROLIFERATION, AND SURVIVAL

John Storm tunkeutui joukon läpi, ja hänen mustapukuinen

"Mistä on kysymys?" tiuskaisi hän. "Eikö kukaan tahdo puhua?"

Silloin tuli kerskaileva miehen kuvatus aution kirkon vieressä

"Ristiäisistä. Pari viinan kulausta ei haittaisi, hyvä mies, vai mitä?"