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\section*{1}
Premature aging and immunodeficiency: Mulvihill-Smith syndrome?
Ohashi H, Tsukahara M, Murano I, Fujita K, Matsuura S, Fukushima Y, Kajii T
Department of Pediatrics, Yamaguchi University School of Medicine, Ube,
Japan.
Am J Med Genet Mar 1 1993 45 (5) p597-600
We report on a 30-year-old woman with premature aging, immunodeficiency, and
other abnormalities. She had many manifestations of the Mulvihill-Smith
syndrome, a disorder that has been described in 4 sporadic individuals, ranging
in age from 4 to 17 years. The common manifestations include short stature,
microcephaly, a senile face with an underdeveloped lower half, diminished
facial subcutaneous fat, multiple pigmented nevi, sensorineural hearing loss,
and a low IgG level. Our patient also had severe mental retardation,
brachydactyly, severe T cell dysfunction, and suffered from severe verruca
vulgaris and a chronic, active Epstein-Barr virus infection. The fact that her
parents were first cousins suggests autosomal recessive inheritance of her
disorder. Two alternative possibilities were considered: the disorder in the
patient represents the Mulvihill-Smith syndrome with immune deficiency as a
sign of its advanced stage, or a hitherto undescribed syndrome.
\section*{2}
MRA detection of vascular occlusion in a child with progeria.
Smith AS, Wiznitzer M, Karaman BA, Horwitz SJ, Lanzieri CF
Department of Radiology, University Hospitals of Cleveland, OH 44106.
AJNR Am J Neuroradiol Mar-Apr 1993 14 (2) p441-3
We report a case of progeria and the utility of visualizing the
cerebrovascular anatomy by using MR angiography. A 4-year-old child with
Hutchinson-Guilford syndrome developed symptoms of ischemia and MR angiography
showed bilateral occlusion of internal carotid and vertebral artery origins,
the anterior spinal artery was prominent.
\section*{3}
Age-associated damage in mitochondrial DNA in human hearts.
Hayakawa M, Sugiyama S, Hattori K, Takasawa M, Ozawa T
Department of Biomedical Chemistry, Faculty of Medicine, University of
Nagoya, Japan.
Mol Cell Biochem Feb 17 1993 119 (1-2) p95-103
Damage to mitochondrial DNA seems to be involved in the etiology of age-
associated degenerative diseases. The aim of this study is to elucidate
effects of aging on human mitochondrial DNA. 8-Hydroxy-deoxyguanosine, a
product of free radical damage to deoxyguanosine, is reported to cause random
point mutations. In human mitochondrial DNA, 8-hydroxy-deoxyguanosine
increased exponentially with age, and the population of mitochondrial DNA with
deletion increased also exponentially with age. Furthermore, a clear
correlation existed between the accumulation of 8-hydroxy-deoxyguanosine and
that of mitochondrial DNA with deletion. We also determined the effects of
aging on rat mitochondrial function together with 8-hydroxy-deoxyguanosine
content in mitochondrial DNA. The activities of complexes I and IV of the
mitochondrial electron transport chain decreased significantly in rats aged 100
weeks compared with those in rats aged 7 weeks. A concomitant increase in 8-
hydroxy-deoxyguanosine was observed in mitochondrial DNA of rats aged 100
weeks. From our results, it is concluded that the age-associated
accumulation of somatically acquired oxygen damage together with
deletions in mitochondrial DNA might be important contributors to the
deterioration of cardiac function associated with age.
\section*{4}
Phenotypic conversion of SV40-immortalized human diploid fibroblasts to
senescing cells by introduction of an antisense gene for SV40-T antigen.
Tanaka Y, Tang X, Hou DX, Gao H, Kitabayashi I, Gachelin G, Yokoyama K
Gene Bank, Tsukuba Life Science Center, RIKEN (The Institute of Physical and
Chemical Research, Ibaraki, Japan.
Cell Struct Funct Dec 1992 17 (6) p351-62
Normal human lung fibroblast diploid cells, WI-38, become senescent after a
definite number of divisions. VA-13 is a line of immortalized cells
established by transformation of WI-38 cells by SV40 virus. To determine
whether SV40 large T (SV40-T) antigen is essential for this immortalization of
WI-38 cells we introduced an antisense gene for T antigen into VA-13. Two
morphologically different types of antisense transformant (VA-AS5-8 and VA-AS37-
8) were obtained. In both antisense transformants the expression of T antigen
was reduced by more than 70% as compared to that in the parent cells. The
morphology of the antisense transformants indicated a partial conversion to the
senescent phenotype of WI-38. The relative number of cells in the S phase of
the antisense transformants was decreased as compared to that in cultures of VA-
13 and about 50% of cells were at G1/0. The doubling time of the transformants
was prolonged to close to the doubling time of WI-38. The level of expression
of retinoblastoma protein (pRB) complexed with SV40-T antigen of the antisense
transformants was significantly decreased although the level of total pRB was
much higher than that in VA-13. The pRB was present exclusively in the
underphosphorylated form. Thus, the decreased level of formation of the
complex between SV40-T and pRB or the underphosphorylation of pRB may explain
the suppression of growth of antisense transformants. Together, these results
show that an antisense gene for SV40-T antigen can efficiently block the cell
proliferation and the cell immortalization of VA-13 cells.
\section*{5}
The role of mitochondrial DNA rearrangements in aging and human diseases.
Osiewacz HD, Hermanns J
Deutsches Krebsforschungszentrum, Institut fur Virusforschung, Heidelberg,
Germany.
Aging (Milano) Dec 1992 4 (4) p273-86
Instabilities and point mutations of the high molecular weight mitochondrial
DNA (mtDNA) were shown to be correlated with various degenerative processes in
both lower eukaryotes as well as in mammals. In filamentous fungi, circular
and linear plasmids were demonstrated to be involved in mtDNA rearrangements
and in the genetic control of senescence. In addition, in these eukaryotic
microorganisms, which have proved to be ideal model systems in experimental
gerontology, a number of nuclear genes were identified controlling the
stability of the mitochondrial genome. Although the mitochondrial genome of
mammals, including humans, appears to be quite stable in comparison to other
species, mtDNA instabilities of the type described in fungi were observed in
mitochondria of patients with different mitochondrial degenerative disorders
(CPEO, KSS, Pearson syndrome, LHON, MERRF, MELAS). It was later demonstrated
that such mtDNA rearrangements appear to accumulate progressively during aging
in human subjects. These data suggest that instabilities of the mitochondrial
genome may play an important role in the control of life span not only in lower
eukaryotes, but also in humans.
\section*{6}
Genetic defects in proteoglycan biosynthesis.
Quentin-Hoffmann E, Harrach B, Robenek H, Kresse H
Institute of Physiological Chemistry and Pathobiochemistry, University of
Munster, Federal Republic of Germany.
Padiatr Padol 1993 28 (1) p37-41
An overview on the structure of proteoglycans and on genetic defects in
proteoglycan biosynthesis is given. Several patients with progeroid-like
symptoms have been shown to have abnormalities in the biosynthesis of
chondroitin/dermatan sulfate proteoglycans. A partial inactivity of
galactosyltransferase I which catalyzes the second glycosyl transfer reaction
in the assembly of glycosaminoglycan chains has been shown to represent the
primary defect in one of the patients. A diminished concentration of a
collagen-associated proteoglycan is considered to play a pathogenetic role in
the development of loose skin.
\section*{7}
G2 repair and aging: influence of donor age on chromosomal aberrations in
human lymphocytes.
Pincheira J, Gallo C, Bravo M, Navarrete MH, Lopez-Saez JF
Depto. de Biologia Celular y Genetica, Facultad de Medicina, Universidad de
Chile, Santiago.
Mutat Res Mar 1993 295 (2) p55-62
The caffeine effects on chromosomal aberration frequency and mean G2 duration
were studied in human lymphocytes in vitro from three age groups of normal
donors (I: 1-5 years old, II: 30-40 years old, III: 60-70 years old). Under
control conditions, the three age groups showed a similar frequency of
chromosomal aberrations. All three age groups exhibited a linear dose response
for aberrations with caffeine treatments. However, lymphocytes from aged
individuals (groups II and III) showed higher chromosomal aberration
frequencies and longer G2 duration than cells from young individuals
(group I). The caffeine effect in reducing G2 length was rather
similar in every age group. The reversion of caffeine effects by
adenosine or niacinamide in lymphocytes from older individuals was
higher than in cells from group I. The different caffeine effects
and G2 values between lymphocytes from old and young individuals are
most likely due to a higher number of DNA lesions reaching G2 phase
and/or a decrease of the G2 repair capability of lymphocytes from older
individuals.
\section*{8}
Different in situ hybridization patterns of mitochondrial DNA in cytochrome c
oxidase-deficient extraocular muscle fibres in the elderly.
Muller-Hocker J, Seibel P, Schneiderbanger K, Kadenbach B
Institute of Pathology, Munich University, Germany.
Virchows Arch A Pathol Anat Histopathol 1993 422 (1) p7-15
Previous studies have revealed an increase of cytochrome c oxidase-deficient
fibres/cells in the skeletal and heart muscle of humans during ageing. The
enzyme defect is due to a lack of both mitochondrial and nuclear coded enzyme
subunits. In the present investigation in situ hybridization of mitochondrial
DNA (mtDNA) has been performed on extraocular muscles of humans over 70 years
of age to show whether mutated mtDNA with the so called common deletion of
4,977 basepairs at position 8,482-13,460 of mtDNA accumulates in the cytochrome
c oxidase-deficient fibres. The cytochrome c oxidase-deficient fibres revealed
different hybridization patterns: a normal hybridization signal with three
different mtDNA probes, a reduced or lacking signal with all three probes
indicating depletion of mtDNA and a selective hybridization defect with the
probe recognizing the "common deletion" region of mtDNA as evidence of mtDNA
deletion. The results suggest that during ageing defects of cytochrome c
oxidase are associated with different molecular alterations of mtDNA. Deletion
and depletion of mtDNA are not the only nor probably the leading mechanisms
responsible for the loss of respiratory chain capacity during ageing. The
normal hybridization signal in most of the cytochrome c oxidase-deficient
fibres and the loss of mitochondrial and nuclear protein subunits indicate the
involvement of other, especially nuclear factors.
\section*{9}
A specific CCAAT-binding protein, CBP/tk, may be involved in the regulation
of thymidine kinase gene expression in human IMR-90 diploid fibroblasts during
senescence.
Pang JH, Chen KY
Department of Chemistry, Rutgers University, Piscataway, New Jersey 08855-
0939.
J Biol Chem Feb 5 1993 268 (4) p2909-16
The aging of IMR-90 human diploid fibroblasts in culture is accompanied by a
5-7 fold decrease in the level of thymidine kinase (TK) mRNA and TK activity
(Chang, Z. F., and Chen, K. Y. (1988) J. Biol. Chem. 263, 11431-11435). We
have employed a gel mobility shift analysis to investigate the molecular basis
of the age-dependent attenuation of TK gene expression. Several cis-elements
including two inverted CCAAT boxes, located at base pairs (bp) -36 and -67, and
GC-rich Sp1 binding sites have been identified in the TK promoter. A 28-bp (-
91 to -64) fragment containing the distal inverted CCAAT element was excised
from the TK promoter to examine possible differences in nuclear protein binding
between young and old IMR-90 cells. A prominent DNA-protein complex was
identified in serum-stimulated young cells by a gel mobility shift assay.
Competition analysis indicated that the binding was highly specific. The
nuclear protein responsible for the complex formation was named CBP/tk (CCAAT
Binding Protein for TK gene) since methylation interference assay showed that
the inverted CCAAT box was involved in binding. The appearance of the CBP/tk-
28-bp complex in IMR-90 cells was (i) serum-dependent, becoming prominent 12-24
h after serum stimulation, and (ii) age-dependent, prominent only in young but
not in old IMR-90 cells. Similar serum- and age-dependent complex formations
were also observed using a 67-bp fragment (-63 to +4) containing the proximal
CCAAT element and a TATA box. In contrast, the binding activities for the Sp1
sequence were the same in young and old cells and appeared to be serum-
independent. CBP/tk binding activity in nuclear extracts was abolished by heat
(60 degrees C, 5 min) or treatment with proteinase K (0.1 microgram/ml) and
sodium dodecyl sulfate (0.005%), but not by Nonidet P-40 or Triton X-100.
Treatment of nuclear extracts with alkaline phosphatase or lectins
(concanavalin A and wheat germ agglutinin) did not affect the binding activity.
Metal chelators such as 1,10-ortho-phenanthroline (0.5 mM) inhibited the CBP/tk
binding activity. Cycloheximide added to the serum-stimulated cultures at an
early or mid-G1 phase inhibited the CBP/tk binding activity. The half-life of
the serum-induced CBP/tk binding activity was estimated to be less than 1
...jeez..read the marathon abstract from the article itself!
\section*{10}
Insulin-degrading enzyme is differentially expressed and developmentally
regulated in various rat tissues.
Kuo WL, Montag AG, Rosner MR
Ben May Institute, University of Chicago, Illinois 60637.
Endocrinology Feb 1993 132 (2) p604-11
Insulin-degrading enzyme (IDE), a cytosolic metalloendoprotease, can degrade
insulin, insulin-like growth factor-II, insulin-like growth factor-I, and
transforming growth factor-alpha. While IDE has been implicated in the
cellular degradation of insulin, other physiological functions of this enzyme
are not known. To assess the possible role of IDE in cellular growth and
development, we determined the tissue and developmental distribution of the
enzyme. Rat IDE cDNA fragments and antibodies directed against human IDE were
used to probe IDE transcripts and proteins in rat tissues. The results
demonstrate that IDE transcripts are ubiquitous in rat tissues. The level of
rIDE transcripts is high in adult rat testis, tongue, and brain, moderate in
kidney, prostate, heart, muscle, liver, intestine, and skin, and low in spleen,
lung, thymus, and uterus. The sizes of the major transcripts of rIDE are 3.4
and 6.3 kilobases in all tissues analyzed, except testis. Surprisingly, the
highest level of rIDE mRNA in the adult rat was in the testis, and the major
transcripts of rIDE in this tissue were shifted in size to 3.8 and 6.7
kilobases. Immunocytochemical analysis localized the rIDE mainly in the
epithelium of prostate gland and kidney, and the cytosol of liver hepatocytes.
During rat development from 6-7 days of age to adulthood, rIDE mRNA levels
increased in brain, testis, and tongue, decreased in muscle and skin, and did
not significantly change in other tissues examined. These studies reveal
regulation of IDE or IDE-related genes in rat tissues and during rat
development, suggesting that this enzyme may have multiple functions relating
to cellular growth and development.
\section*{11}
Acrometageria: a spectrum of "premature aging" syndromes.
Greally JM, Boone LY, Lenkey SG, Wenger SL, Steele MW
Department of Pediatrics, University of Pittsburgh, PA.
Am J Med Genet Oct 1 1992 44 (3) p334-9
A child with manifestations of acrogeria and metageria, two "premature aging"
syndromes, is presented. Because of his indistinct phenotype and because the
question has been previously raised as to whether these conditions are
separate, we propose the designation of acrometageria to describe this
phenotypic continuum. As there is much in common clinically between
acrometageria and the syndrome of type III procollagen deficiency (Ehlers-
Danlos type IV), it might be presumed that a similar pathogenesis for
acrometageria exists. This possibility has been tested previously, without
demonstrating specific quantitative or qualitative deficits, but with some
indirect evidence that collagen metabolism is deranged in these patients. One
such crude indicator is the elevation of urinary hyaluronic acid levels,
demonstrated in our patient and also observed in the phenotypically distinct
Werner and Hutchinson-Gilford premature aging syndromes. On one hand, it could
be argued that this supports the concept that premature aging syndromes exist
as a biological continuum. On the other hand, it is equally valid to argue
that syndromes of premature aging are so described merely because they include
recognizable changes of normal aging and that the demonstration of an
underlying mutation in a collagen gene, for example, invalidates their study as
models of accelerated normal aging.
\section*{12}
Mitochondrial DNA mutation associated with aging and degenerative disease.
Nagley P, Mackay IR, Baumer A, Maxwell RJ, Vaillant F, Wang ZX, Zhang C,
Linnane AW
Centre for Molecular Biology and Medicine, Monash University, Clayton,
Victoria, Australia.
Ann N Y Acad Sci Dec 26 1992 673 p92-102
Previous theories of aging based on somatic mutation neglected mtDNA, which
has a high propensity for mutational error. Knowledge of yeast mtDNA mutations
and their functional effects, and of human mtDNA mutations identified in the
mitochondrial cytopathies, provides for a concept of aging based on the
cumulative effect of mutations affecting human mtDNA. An essential feature of
this concept is heteroplasmy, representing mixtures of normal and mutant mtDNA
at the cellular and mitochondrial level, resulting in a "tissue mosaic" of
focal bioenergetic deficits. Direct evidence for the concept is provided by
(i) focal loss of staining for mitochondrially encoded enzymes, such as
cytochrome c oxidase, in tissues of aged individuals (humans and rats) and (ii)
an age-related increase in deletional mutations in mtDNA demonstrable by
application of the polymerase chain reaction to DNA templates from individuals
of different ages.
\section*{13}
Oxidants are a major contributor to aging.
Ames BN, Shigenaga MK
Division of Biochemistry and Molecular Biology, University of California,
Berkeley 94720.
Ann N Y Acad Sci Nov 21 1992 663 p85-96
Very high level oxidative damage to DNA occurs during normal metabolism. In
each rat cell the steady-state level of this damage is estimated to be about
10(6) oxidative adducts, and about 10(5) new adducts are formed daily. This
endogenous DNA damage appears to be a major contributor to aging and to the
degenerative diseases associated with aging such as cancer. The oxidative
damage rate in mammalian species with a high metabolic rate, short life span,
and high age-specific cancer rate such as rats is much higher than the rate in
humans, long-lived mammals with a lower metabolic rate and a lower age-specific
cancer rate. It is argued that deficiency of micronutrients that protect
against oxidative DNA damage is a major contributor to human cancer.
Epidemiological studies, a large body of experimental evidence, and theoretical
work on the mechanisms of carcinogenesis point to mitogenesis as a major
contributor to cancer. Dividing cells compared to nondividing cells are at an
enormously increased risk for mutations in part due to the conversion of DNA
adducts to mutations. Mitogenesis also increases the probability of gene
amplification and loss of 5-methylcytosine. Dietary interventions that lower
mitogenesis, such as calorie restriction, decrease the incidence of cancer.
\section*{14}
Enzymes of DNA metabolism in a patient with the Wiedemann-Rautenstrauch
progeroid syndrome.
Mazzarello P, Verri A, Mondello C, Colombo S, Maga G, Spadari S, Focher F
Istituto di Genetica Biochimica ed Evoluzionistica, CNR Pavia, Italy.
Ann N Y Acad Sci Nov 21 1992 663 p440-1
\section*{15}
Sensitivity to genotoxic agents and immunological status in a case of
Werner's syndrome.
Monti D, Cossarizza A, Chiricolo M, Vannini P, Ciavarella A, Franceschi C
Istituto di Patologia Generale, University of Modena, Italy.
Ann N Y Acad Sci Nov 21 1992 663 p429-31
\section*{16}
Cytogenetic and DNA repair studies in a patient affected by the neonatal
progeroid syndrome.
Chessa L, Bastianon V, Del Porto G, Nardo T, Stefanini M
Dipartimento di Medicina Sperimentale, Rome, Italy.
Ann N Y Acad Sci Nov 21 1992 663 p423-5
\section*{17}
Genomic instability and aging. Studies in centenarians (successful aging) and
in patients with Down's syndrome (accelerated aging).
Franceschi C, Monti D, Scarfi MR, Zeni O, Temperani P, Emilia G, Sansoni P,
Lioi MB, Troiano L, Agnesini C, et al
Institute of General Pathology, University of Modena, Italy.
Ann N Y Acad Sci Nov 21 1992 663 p4-16
\section*{18}
Genomic heterogeneity of DNA repair. Role in aging?
Hanawalt PC, Gee P, Ho L, Hsu RK, Kane CJ
Department of Biological Sciences, Stanford University, California 94305-
5020.
Ann N Y Acad Sci Nov 21 1992 663 p17-25
The introduction and repair of DNA lesions are generally heterogeneous with
respect to different genomic domains. In particular, the repair of helix-
distorting damage, such as the cyclobutane pyrimidine dimers (CPD) induced by
ultraviolet light occurs selectively in expressed genes. This is due in large
part to the preferential repair of transcribed DNA strands, which is then
reflected in a bias toward mutagenesis from persisting lesions in
nontranscribed strands. Consequently, determination of overall genomic repair
efficiencies may not be a good indicator of cellular sensitivity to agents that
damage DNA. Although some studies suggest an age-related accumulation of
altered nucleotides in DNA, we do not know the intragenomic distribution of
those changes and whether they are relevant to the physiological aspects of
aging. Subtle changes in the pattern of preferential repair during maturation
could have profound effects on cell and tissue function. DNA repair has been
analyzed in differentiating cell systems as possible models for aging. We have
observed attenuated overall repair of CPD in differentiated rat myoblasts or
PC12 neuron-like cells. In both model systems, several expressed genes have
been shown to be repaired relatively efficiently but without strand specificity.
In another model system of human HT1080 fibroblasts differentiating in the
presence of dexamethasone, we demonstrated enhanced repair in the gene for
plasminogen activator inhibitor I whose transcription is induced and,
correspondingly, a reduced repair rate in the urokinase plasminogen activator
gene whose transcription is suppressed. We conclude that any attempted
correlation of the phenomena of aging with DNA repair should focus on the
relevant genes in the tissue of interest.
\section*{19}
Protein oxidation in aging brain.
Smith CD, Carney JM, Tatsumo T, Stadtman ER, Floyd RA, Markesbery WR
Department of Neurology, University of Kentucky Medical Center, Lexington
40536.
Ann N Y Acad Sci Nov 21 1992 663 p110-9
\section*{20}
Aging and cellular defense mechanisms.
Giacomoni PU
Department of Biology, Laboratoires de Recherche de L'OREAL, Aulnay sous
bois, France.
Ann N Y Acad Sci Nov 21 1992 663 p1-3
\section*{21}
Report on a case of Hutchinson-Gilford progeria, with special reference to
orthopedic problems.
Fernandez-Palazzi F, McLaren AT, Slowie DF
Head Service C of Orthopedics, Hospital San Juan de Dios, Caracas, Venezuela.
Eur J Pediatr Surg Dec 1992 2 (6) p378-82
Hutchinson-Gilford progeria is a very rare syndrome of premature aging and
often features many orthopedic abnormalities. This is a case report on a young
boy suffering from progeria. His orthopedic history included bilateral talus
deformities of the feet, bilateral dislocated hips, pes planus, a fractured
femur (which healed without complications), aseptic necrosis in the left
nuclear head of the femur, bilateral fixed hip flexion deformities, bone
dysplasia, osteoporosis and osteolysis.
\section*{22}
Cerebral infarction in progeria.
Wagle WA, Haller JS, Cousins JP
Department of Radiology, Albany Medical Center, NY 12208.
Pediatr Neurol Nov-Dec 1992 8 (6) p476-7
\section*{23}
Overview: mechanisms of antioxidant action on life span.
Warner HR
Biology of Aging Program, National Institute on Aging, Bethesda, MD 20892.
Toxicol Ind Health Jan-Apr 1993 9 (1-2) p151-61
\section*{24}
Age-associated oxygen damage and mutations in mitochondrial DNA in human
hearts.
Hayakawa M, Hattori K, Sugiyama S, Ozawa T
Department of Biomedical Chemistry, Faculty of Medicine, University of
Nagoya, Japan.
Biochem Biophys Res Commun Dec 15 1992 189 (2) p979-85
Some mutations in mitochondrial DNA (mtDNA) causing a number of neuromuscular
diseases are suggested to arise spontaneously during the life of an
individual. To substantiate the extent and the rate of these somatic
mutations, mtDNA specimens from post-mortem human heart muscles of subjects
in differing age groups were hydrolyzed. 8-Hydroxy-deoxyguanosine (8-OH-dG),
a hydroxyl-radical adduct of deoxyguanosine, in mtDNA, was quantitatively
determined using a micro high-performance liquid chromatography/mass
spectrometry system. In each specimen, the mtDNA with a 7.4 kilo base-pair
deletion was quantified by the kinetic polymerase chain reaction method.
In association with age, the 8-OH-dG content accumulated exponentially up to
1.5% with a correlative increase in the content of the deleted mtDNA
up to 7%. Clear correlation between the 8-OH-dG content in mtDNA and the
population of mtDNA with a deletion (r = 0.93, P < 0.01) gives insight
into the mechanism for the generation of a large deletion. These results
indicate that accumulation of somatically acquired oxygen damage together
with age-associated mutations in mtDNA which lead to bioenergetic
deficiency and the heart muscle weakness are inevitable in human life.
\section*{25}
The effect of Ninjinyoeito on Werner's syndrome skin fibroblasts.
Uchiyama Y, Nakajima S, Ohno T, Goto M, Kan M, Haruki E
Kanagawa Rehabilitation Institute, Japan.
Am J Chin Med 1992 20 (3-4) p295-305
The effect of Ninjinyoeito on three cases of Werner's syndrome fibroblasts
was investigated. In all three cases, groups which were treated with
Ninjinyoeito showed improved life-span doubling levels compared to groups which
were not treated with Ninjinyoeito. Also, all of the treated group in all
three cases showed significantly higher values in the rates of DNA synthesis
including two cases which showed significantly higher rates of protein
synthesis.
\section*{26}
Analyses of mitochondrial respiratory chain function and mitochondrial DNA
deletion in human skeletal muscle: effect of ageing.
Cooper JM, Mann VM, Schapira AH
Department of Neuroscience, Royal Free Hospital School of Medicine, London,
UK.
J Neurol Sci Nov 1992 113 (1) p91-8
The analysis of human skeletal muscle mitochondria revealed a progressive
decline in mitochondrial respiratory chain function with age. The activities
affected to the greatest extent were those of complexes I and IV which were
decreased by 59% and 47% respectively between the ages of 20-30 years and 60-90
years of age. Quantitation of the 5 kb 'common' deletion of mtDNA using PCR
revealed a progressive accumulation with age, from approximately 1 in 100,000
at 21 years to 1 in 10,000 at 56 years and 1 in 5000 at 78 years of age. The
low absolute levels of this mutation are unlikely to contribute significantly
to the observed mitochondrial dysfunction.
\section*{27}
Accumulation of deletions in human mitochondrial DNA during normal aging:
analysis by quantitative PCR.
Simonetti S, Chen X, DiMauro S, Schon EA
Department of Neurology, Columbia University, New York, NY 10032.
Biochim Biophys Acta Dec 10 1992 1180 (2) p113-22
We have developed a quantitative PCR technique to measure the amount of a
specific mitochondrial DNA deletion (delta mtDNA), the so-called 'common
deletion', in human tissues. Using this method, we estimate that there is a
10,000-fold increase in this delta mtDNA species in muscle during the course of
the normal human lifespan. The maximum amount of common deletion observed in
aged muscle was approx. 0.1%. Tissues that turn-over slowly, such as skeletal
muscle and heart, contained more delta mtDNA than more rapidly dividing
tissues, such as liver, in agreement with studies performed by others.
\section*{28}
Werner syndrome: molecular genetics and mechanistic hypotheses.
Monnat RJ Jr
Department of Pathology SM-30, University of Washington, Seattle, 98195.
Exp Gerontol Jul-Aug 1992 27 (4) p447-53
\section*{29}
Isolation and characterization of gene sequences overexpressed in Werner
syndrome fibroblasts during premature replicative senescence.
Thweatt R, Murano S, Fleischmann RD, Goldstein S
Department of Medicine, University of Arkansas for Medical Sciences, Little
Rock.
Exp Gerontol Jul-Aug 1992 27 (4) p433-40
\section*{30}
Metalloproteinase and TIMP-1 gene expression during replicative senescence.
Millis AJ, McCue HM, Kumar S, Baglioni C
Department of Biological Sciences, State University of New York, Albany
12222.
Exp Gerontol Jul-Aug 1992 27 (4) p425-8
\section*{31}
Inhibitors of DNA synthesis derived from senescent human diploid fibroblasts.
Smith JR
Huffington Center on Aging, Baylor College of Medicine, Houston, Texas 77030.
Exp Gerontol Jul-Aug 1992 27 (4) p409-12
\section*{32}
Addition of nuclear DNA content to the AMES risk-group classification for
papillary thyroid cancer.
Pasieka JL, Zedenius J, Auer G, Grimelius L, Hoog A, Lundell G, Wallin G,
Backdahl M
Department of Surgery, Karolinska Hospital and Institute, Stockholm, Sweden.
Surgery Dec 1992 112 (6) p1154-9, discussion 1159-60
BACKGROUND. The purpose of this study was to prospectively assess whether
nuclear DNA content added prognostic value to existing risk factors in patients
with papillary thyroid cancer. METHODS. Nuclear DNA content was measured both
on the fine needle aspiration material and the surgical specimen in 73 patients
with primary or recurrent papillary thyroid cancer. We modified the existing
age of patient, presence of distant metastases, extent and size of the tumor
(AMES) risk-group classification to include DNA ploidy with AMES (DAMES).
Patients with euploid tumors that were AMES low risk were considered to be
DAMES low risk, patients with euploid tumors that were AMES high risk became
intermediate risk, and patients with aneuploid tumors that were AMES high risk
became DAMES high risk. RESULTS. Forty-eight patients were in the DAMES low-
risk group. Recurrences and/or distant metastases developed in only four (8%)
of these patients. Twenty-two patients were in the DAMES intermediate-risk
group. Twelve (55%) of the intermediate-risk group had residual, recurrent, or
distant metastatic disease, with one death from cancer at 120 months. Three
patients were in the DAMES high-risk group. Distant metastases developed in
all three patients, who died within 24 months from thyroid cancer. A
statistically significant difference existed in the development of
recurrence/metastases or death from cancer in the DAMES high-risk group
compared with the other risk groups combined. CONCLUSIONS. Nuclear DNA
content adds prognostic value to the existing AMES risk-group classification.
Because DNA analysis on fine needle aspiration correlated well with the
surgical specimen DNA analysis, this modified classification can be used
perioperatively to further individualize the treatment of patients with
papillary thyroid cancer.
\section*{33}
The rate of DNA damage and aging.
Simic MG
Department of Pharmacology and Toxicology, School of Pharmacy, University of
Maryland, Baltimore 21201.
EXS 1992 62 p20-30 <---pl dont ask me which journal this is!
A new theory of aging based on the rate of DNA damage is presented, and the
relationship between the rate of oxidative DNA damage and maximum life span
(MLS) of mammalian species is explored. In humans the level of oxidative DNA
damage, as measured by urinary biomarkers, can be modulated by caloric
restriction and dietary composition. Consequently, longevity may depend not
only on the basal metabolic rate but also on dietary caloric intake and the
type of diet. The theory may provide the basis for a practical approach for
reduction of degenerative diseases in general, extension of life expectancy,
and optimization of individual lifestyles.
\section*{34}
Telomere length predicts replicative capacity of human fibroblasts.
Allsopp RC, Vaziri H, Patterson C, Goldstein S, Younglai EV, Futcher AB,
Greider CW, Harley CB
Department of Biochemistry, McMaster University, Hamilton, ON, Canada.
Proc Natl Acad Sci U S A Nov 1 1992 89 (21) p10114-8
When human fibroblasts from different donors are grown in vitro, only a small
fraction of the variation in their finite replicative capacity is explained by
the chronological age of the donor. Because we had previously shown that
telomeres, the terminal guanine-rich sequences of chromosomes, shorten
throughout the life-span of cultured cells, we wished to determine whether
variation in initial telomere length would account for the unexplained
variation in replicative capacity. Analysis of cells from 31 donors (aged 0-93
yr) indicated relatively weak correlations between proliferative ability and
donor age (m = -0.2 doubling per yr, r = -0.42, P = 0.02) and between telomeric
DNA and donor age (m = -15 base pairs per yr, r = -0.43, P = 0.02). However,
there was a striking correlation, valid over the entire age range of the
donors, between replicative capacity and initial telomere length (m = 10
doublings per kilobase pair, r = 0.76, P = 0.004), indicating that cell strains
with shorter telomeres underwent significantly fewer doublings than those with
longer telomeres. These observations suggest that telomere length is a
biomarker of somatic cell aging in humans and are consistent with a causal role
for telomere loss in this process. We also found that fibroblasts from
Hutchinson-Gilford progeria donors had short telomeres, consistent with their
reduced division potential in vitro. In contrast, telomeres from sperm DNA did
not decrease with age of the donor, suggesting that a mechanism for maintaining
telomere length, such as telomerase expression, may be active in germ-line
tissue.
\section*{35}
Age-dependent increases of DNA adducts (I-compounds) in human and rat brain
DNA.
Randerath K, Putman KL, Osterburg HH, Johnson SA, Morgan DG, Finch CE
Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030.
Mutat Res Jan 1993 295 (1) p11-8
Brain DNA from 20 humans ranging in age from neonatal to 100 years was
analyzed by the nuclease P1-enhanced version of the 32P-postlabeling assay for
bulky covalently modified nucleotides. A reproducible pattern of three 32P-
labeled spots was obtained by thin-layer chromatography followed by
autoradiography. Two of these spots increased with age (Mann-Whitney U-test, P
< 0.001, comparison of ages < or = 60 years and ages > 60 years). Thus, these
spots met the definition of I-compounds. Rat brain DNA exhibited the same two
I-spots, whose intensities also increased with animal age (1, 4, and 10
months). In humans, considerable individual variation of brain I-compound
levels was observed, especially at ages > 60 years, presumably reflecting
environmental, life-style, or genetic factors. This variation was not
noted for brain DNA of laboratory rats. Thus, human brain DNA undergoes
progressive covalent modifications with aging.
\section*{36}
Exogenous and endogenous DNA modifications as monitored by 32P-postlabeling:
relationships to cancer and aging.
Randerath K, Li D, Nath R, Randerath E
Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030.
Exp Gerontol Sep-Dec 1992 27 (5-6) p533-49
32P-postlabeling analysis, a highly sensitive method for the detection and
measurement of covalent carcinogen-DNA adducts and other DNA modifications,
does not require radioactive test substances and, therefore, can be applied to
DNA of mammals, including humans exposed to low doses of environmental or
occupational genotoxicants. The basic procedure entails the enzymatic
incorporation of 32P-label into hydrolysis products of DNA, followed by
chromatographic mapping and autoradiography of the 32P-labeled digestion
products and quantitative scintillation spectrometry. Microgram amounts of DNA
are analyzed: Thus the assay is suited for limited amount of cells or tissues.
Various versions of the assay afford different sensitivities of adduct
detection. A single aromatic or bulky/hydrophobic adduct in 10(8)-10(10)
nucleotides can be detected and measured (corresponding to 0.3-30 amol
adduct/micrograms DNA or 0.1-10 nmol adduct/mol DNA-P). In animal models, the
assay has been successfully applied to a variety of mutagenic (genotoxic) as
well as nonmutagenic carcinogens. In humans, DNA specimens from cigarette
smokers, iron foundry workers, and coke oven workers whose total aromatic
adduct levels ranged from 1 adduct in 10(6)-10(8) DNA nucleotides have been
examined by 32P-postlabeling. The assay also detects DNA modifications
Indigenous (I)-compounds that increase with age in untreated animals. I-
compound profiles and levels are highly species-, strain-, sex-, and tissue-
specific, and also depend on diet composition. Caloric restriction, a highly
efficient method for improving resistance to carcinogenesis and extending life
span, increased rather than decreased I-compound levels in various tissues of
male rats. Nonmutagenic hepatocarcinogens reduced levels of I-compounds in the
target organ. Because of the specificity of this effect, reduction of I-
compound levels appears to represent a novel biomarker for the action of
nonmutagenic carcinogens. DNA from various hepatomas was found largely devoid
of I-compounds. The results support a possible antineoplastic and antiaging
role of these DNA modifications.
\section*{37}
Characterization of the absence of an unique DNA-binding protein in senescent
but not in their young growing and nongrowing counterparts provides the means
to mark the final stage of the cellular aging process.
Wang E
Bloomfield Centre for Research in Aging, Lady Davis Institute, Jewish General
Hospital, Montreal, Quebec, Canada.
Exp Gerontol Sep-Dec 1992 27 (5-6) p503-17
In senescent fibroblasts, the incapability of cell replication is permanent,
and may involve the irreversible loss of gene expressions potentiating the
engagement in DNA replication. The initial attempt is to identify gene
products that are permanently lost in irreversibly growth-arrested cells. In
this article, we report the success in identifying a DNA-associated S6 antigen
found in the nuclei of growing and growth-arrested young cells, but not in the
nuclei of their senescent counterparts. The presence of the S6 antigen is
uniform throughout the nucleoplasm, except in the regions of the nucleoli, and
found to be associated with condensed chromosomes in mitotic cells. Treatment
with RNase does not abolish the antibody staining activity in the nuclei, while
treatment with DNase does remove the activity. Equal intensity of S6 antibody
staining is observed in transformed, growing, and contact-inhibited young
fibroblasts. Significant reduced level of S6 antibody staining activity is
found in the nuclei of senescent fibroblasts, indicating the loss of the
expression of this protein during cellular aging process. Immunoblotting assay
shows that the S6 antigen is of 50 kDa and with a possibility of a 100 kDa as a
dimeric precursor. Our results suggests that a permanent turning off of unique
gene expression is associated with the onset of senescence or terminal
differentiation, and this hypothesis is supported by the characterization of S6
antigen's absence in in vitro-aged fibroblasts.
\section*{38}
Growth hormone binding protein in Werner's syndrome.
Hattori N, Hino M, Ikekubo K, Ishihara T, Moridera K, Kurahachi H
Department of Endocrinology, Kobe City General Hospital, Japan.
Clin Endocrinol (Oxf) Apr 1992 36 (4) p351-3
OBJECTIVE GH and growth hormone binding protein in Werner's syndrome were
investigated to elucidate their relation to the short stature. DESIGN The
levels of GH binding protein and GH response to insulin-induced hypoglycaemia
were determined. GH binding protein levels and its Scatchard analysis in
Werner's syndrome were compared with those in normal subjects. PATIENTS Three
patients with Werner's syndrome (one man aged 45 years and two women aged 39
and 38 years) and 41 normal subjects (18 men and 23 women aged 39.3 +/- 5.5
years, mean +/- SD) were studied. MEASUREMENTS GH binding protein levels were
determined using an Ultrogel AcA44 minicolumn and GH levels were measured by a
highly sensitive enzyme immunoassay. RESULTS Two out of three patients with
Werner's syndrome had GH binding protein levels above the mean +/- 2SD value in
normal subjects. GH secretion was impaired in Werner's syndrome as judged by
the low GH response to insulin-induced hypoglycaemia. CONCLUSIONS Elevated GH
binding protein levels may lead to an increase in the bound form of GH, which
is probably less bioactive, resulting in growth failure in association with the
impaired GH secretion in Werner's syndrome.
\section*{39}
The role of DNA damage and its repair in the aging process.
Grossman L
Department of Biochemistry, Johns Hopkins University, School of Hygiene and
Public Health, Baltimore, MD 21205.
Aging (Milano) Sep 1992 4 (3) p252-5
\section*{40}
Effect of insulin on the proline transport activity in cultured fibroblasts
from patients with Werner syndrome.
Bannai C, Bannai S, Okuda Y, Yamashita K
Department of Endocrinology and Metabolism, Tsukuba University Medical
School, Ibaraki, Japan.
Biochem Med Metab Biol Oct 1992 48 (2) p174-6
The effect of insulin on the transport of proline has been studied in
cultured fibroblasts from normal individuals, non-insulin-dependent diabetic
patients, and patients with Werner syndrome. In fibroblasts from normal
individuals and those with diabetes mellitus, incubation with 10(-7) M insulin
resulted in more than a twofold increase in the transport rate after about 14 h
incubation. In contrast, fibroblasts from patients with Werner syndrome had a
markedly attenuated response to insulin, suggesting a defect in insulin action
on the transport of this amino acid in Werner syndrome.
\section*{41}
Senescent human fibroblasts are more sensitive to the effects of a phorbol
ester on macromolecular synthesis and growth characteristics.
Derventzi A, Rattan SI, Clark BF
Department of Chemistry, Aarhus University, Denmark.
Biochem Int Aug 1992 27 (5) p903-11
4-beta-phorbol-12-beta-myristate-13-alpha-acetate (PMA) alters cellular
growth properties by modulating gene expression in a wide variety of cell types.
Human diploid fibroblasts MRC-5 were treated with PMA at different phases of
their lifespan in vitro and the alterations of their short-term growth
characteristics and macromolecular synthesis in response to PMA were analysed.
PMA stimulates DNA and RNA synthesis in both Phase II (young) and Phase III
(senescent) MRC-5 cells. Treatment of senescent cells with various PMA
concentrations results in a greater stimulation of DNA and RNA synthesis than
that in young cells. Senescent cells are also more sensitive to the PMA-
induced alterations of growth characteristics and higher concentrations of PMA
become toxic for them. The age-related alterations of cellular responsiveness
are also apparent in the gradual loss of responsiveness to serum, observed in
parallel with the increased sensitivity to PMA. Furthermore, serum-induced
stimulation of macromolecular synthesis is inhibited by PMA. Since it is known
that serum and PMA elicit their effects via different signal transduction
pathways, our results point to suggest the differential regulation of the
signalling mechanisms during cellular ageing.
\section*{42}
Hypothesis: a damaging role in aging for reactive protein oxidation products?
Dean RT, Gebicki J, Gieseg S, Grant AJ, Simpson JA
Cell Biology Group, Heart Research Institute, Sydney, Australia.
Mutat Res Sep 1992 275 (3-6) p387-93
This paper discusses our knowledge of protein oxidation and its relationship
to aging. It also outlines new observations from our laboratories concerning
reactive species produced during protein oxidation, and proposes that these may
inflict damage on other molecules, and hence contribute to the progression of
aging. Whereas it has previously been difficult to see how relatively inert
protein oxidation products could possibly have any causal role in aging, the
detection of these novel reactive species implies a potentially significant
role.
\section*{43}
DNA damage and repair in brain: relationship to aging.
Rao KS, Loeb LA
Neurobiochemistry Laboratory, School of Life Sciences, University of
Hyderabad, India.
Mutat Res Sep 1992 275 (3-6) p317-29
The usefulness of conducting DNA damage and repair studies in a postmitotic
tissue like brain is emphasized. We review studies that use brain as a tissue
to test the validity of the DNA damage and repair hypothesis of aging. As far
as the accumulation of age dependent DNA damage is concerned, the data appear
to overwhelmingly support the hypothesis. However, attempts to demonstrate a
decline in DNA repair capacity as a function of age are conflicting and equally
divided. Possible reasons for this discrepancy are discussed. It is suggested
that assessment of the repair capacity of neurons with respect to a specific
type of damage in a specific gene might yield more definitive answers regarding
the role of DNA repair potential in the aging process and as a longevity
assurance system.
\section*{44}
Oxidative and other DNA damages as the basis of aging: a review.
Holmes GE, Bernstein C, Bernstein H
Department of Microbiology, College of Medicine, Howard University,
Washington, DC 20059.
Mutat Res Sep 1992 275 (3-6) p305-15
DNA damages occur continuously in cells of living organisms. While most of
these damages are repaired, some accumulate. In particular, there is evidence
for DNA damage accumulation in non-dividing cells of mammals. These
accumulated DNA damages probably interfere with RNA transcription. We consider
that the decline in the ability of DNA to serve as a template for gene
expression is the primary cause of aging. Oxidative DNA damages are among the
best documented and prevalent DNA damages and are likely to be a prominent
cause of aging.
\section*{45}
Reactive oxygen and DNA damage in mitochondria.
Richter C
Laboratory of Biochemistry I, Swiss Federal Institute of Technology (ETH),
Zurich.
Mutat Res Sep 1992 275 (3-6) p249-55
During the last decade the importance of reactive oxygen species as major
contributors to various types of cancer, heart diseases, cataracts, Parkinson's
and other degenerative diseases that come with age, and to natural aging has
become apparent. Mitochondria are the most important intracellular source of
reactive oxygen. Mitochondrial DNA is heavily damaged by reactive oxygen at
the bases, as indicated by the high steady-state level of 8-
hydroxydeoxyguanosine, the presence of which causes mispairing and point
mutations. Mitochondrial DNA is also oxidatively fragmented to a certain
extent. Conceivably, such fragmentation relates to deletions found in
mitochondrial DNA. Point mutations and deletions have recently been shown to
be etiologically linked to several human diseases and natural aging. Future
studies should address the causal relationship between mitochondrial
dysfunction, production of reactive oxygen species, and aging.
\section*{46}
New evidence for the insertion of mitochondrial DNA into the human genome:
significance for cancer and aging.
Shay JW, Werbin H
University of Texas Southwestern Medical Center, Department of Cell Biology
and Neuroscience, Dallas 75235-9039.
Mutat Res Sep 1992 275 (3-6) p227-35
We have observed and characterized in detail two cases of mitochondrial DNA
fragments which have inserted into the nucleus of HeLa cells. In one case
three non-sequential but contiguous regions of mitochondrial DNA with 92%
homology to human cytoplasmic mitochondrial DNA inserted into the nuclear
genome. In the second case the mitochondrial DNA sequence encoding cytochrome
c oxidase subunit III was contiguous with and 5' of exons 2 and 3 of the c-myc
oncogene and the chimeric gene was transcribed. Models are presented that
describe mechanisms for the transfer of mitochondrial DNA into the nucleus
involving fragmentation of mitochondrial DNA through aging and/or oxidative
damage, anomalous processing or escape of mitochondrial DNA and RNA fragments
from autophagic vacuoles, and insertion of mitochondrial DNA sequences, in some
instances after reverse transcription of mitochondrial RNA, into the nuclear
genome.
\section*{47}
Evidence for and against the causal involvement of mitochondrial DNA mutation
in mammalian ageing.
Bittles AH
Biomedical Sciences Division, King's College, University of London, Strand,
UK.
Mutat Res Sep 1992 275 (3-6) p217-25
Current experimental evidence on the role of mitochondrial DNA mutation in
ageing is assessed alongside reports implicating other genetic and non-genetic
causes, including inter-relationships between the mitochondrial and nuclear
genomes and their potential effect on mitochondrial structure and function.
The role of a 5-kb mtDNA deletion, identified as age-dependent in a variety of
human and other mammalian species, is specifically evaluated in the context of
its functional effect in mitotic and non-mitotic adult tissue. Downstream
effects of mitochondrial decline are considered in terms of the maintenance of
ATP production. Associated sequelae then are discussed specifically with
reference to restrictions in the supply of ribose moieties for DNA and RNA
synthesis, and to disruption of NADPH production and hence cellular anti-
oxidant defences.
\section*{48}
An update on the mitochondrial-DNA mutation hypothesis of cell aging.
Miquel J
Laboratorio de Neurogerontologia, Facultad de Medicina, San Juan-Alicante,
Spain.
Mutat Res Sep 1992 275 (3-6) p209-16
Our electron microscopic study of aging insects and mammals suggests that
metazoan senescence is linked to a gradual process of mitochondrial breakdown
(and lipofuscin accumulation) in fixed postmitotic cells. This led us to
propose in the early 1980s an oxyradical-mitochondrial DNA damage hypothesis,
according to which metazoan aging may be caused by mutation, inactivation or
loss of the mitochondrial genome (mtDNA) in irreversibly differentiated cells.
This extranuclear somatic gene mutation concept of aging is in agreement with
the fact that mtDNA synthesis takes place at the inner mitochondrial membrane
near the sites of formation of highly reactive oxygen species and their
products. Mitochondrial DNA may be unable to counteract the damage inflicted
by those by-products of respiration because, in contrast to the nuclear genome,
it lacks excision and recombination repair. Since mtDNA contains the
structural genes for 13 hydrophobic proteins of the respiratory chain and ATP
synthase as well as mitochondrial rRNAs and tRNAs, damage to this organellar
genome will decrease or prevent the 'rejuvenation' of the mitochondria through
the process of macromolecular turnover and organelle fission. Thus deprived of
the ability to regenerate their mitochondria, the fixed postmitotic cells will
sustain a decrease in the number of functional organelles, with resulting
decline in ATP production. At higher levels of biological organization, this
will lead to a loss in the bioenergetic capacity of cells, with concomitant
decreases in ATP dependent protein synthesis and specialized physiological
function, thus paving the way for age related degenerative diseases. The above
concept is supported by a wealth of recent observations confirming the genomic
instability of mitochondria and suggesting that animal and human aging is
accompanied by mtDNA deletions and other types of injury to the mitochondrial
genome. Our hypothesis of mtDNA damage is integrated with the classic concepts
of Weissman and Minot in order to provide a preliminary explanation of the
evolutionary roots of aging and reconcile the programed and stochastic views of
metazoan senescence.
\section*{49}
Mitochondrial DNA mutation and the ageing process: bioenergy and
pharmacological intervention.
Linnane AW, Zhang C, Baumer A, Nagley P
Department of Biochemistry, Monash University, Clayton, Vic., Australia.
Mutat Res Sep 1992 275 (3-6) p195-208
A comprehensive hypothesis concerning the contribution of mitochondrial DNA
(mtDNA) mutations to the human ageing process is reviewed and the implications
for cellular bioenergy loss and pharmacological therapy are considered. The
central idea is that random mutations in the population of mtDNA molecules of
each cell occur throughout life, and that this is a major contributor to the
gradual loss of cellular bioenergy capacity within tissues and organs,
associated with general senescence and diseases of ageing. An elaboration of
four major aspects of the general proposition, together with relevant
supporting data, is presented. (1) An extensive array of deletions in mtDNA of
many tissues of humans and other mammals has been observed to occur in an age-
related manner. (2) The preservation and selection of fully functional mtDNA
molecules in the female germ line cells is proposed to occur via a human mtDNA
cycle, in which selective amplification of a limited number of mtDNA templates
occurs during oocyte development. This proposal explains the endowment of
normal neonates with a mtDNA complement minimally contaminated by damaged mtDNA
molecules. The phenomena of maternal inheritance and rapid fixation of
sequence variants of mtDNA in mammals, as well as selection of cells based on
mitochondrial function, are taken into account. (3) Tissue bioenergy mosaics
result from accumulated mtDNA damage during ageing, representing different
rates of cellular bioenergy loss within individual cells of a tissue. The
random segregation of mtDNA during cell division will also further contribute
to the tissue energy mosaic. Cells unable to meet their particular bioenergy
demand will become non-functional, leading to cell death, the bioenergy
threshold is different for the various cell types in the tissues of the body.
(4) In order to bioenergetically resuscitate cells and tissues suffering from
impaired mitochondrial functions as a result of the ageing process, we propose
that redox compounds may be used therapeutically in the pharmacological
configurations of a by-pass strategy or as a redox sink therapy. The role of
these compounds is to maintain at least part of the mitochondrial respiratory
chain function (by-pass) as well as to maintain adequate levels of cellular
NAD+ (redox sink) for ATP synthesis, predominantly by the cytosolic glycolytic
pathway, with some contribution from mitochondrial oxidative phosphorylation.
\section*{50}
Association of mitochondrial DNA damage with aging and coronary
atherosclerotic heart disease.
Corral-Debrinski M, Shoffner JM, Lott MT, Wallace DC
Department of Genetics and Molecular Medicine, Emory University School of
Medicine, Atlanta, GA 30322.
Mutat Res Sep 1992 275 (3-6) p169-80
The role of somatic mitochondrial DNA (mtDNA) damage in human aging and
progressive diseases of oxidative phosphorylation (OXPHOS) was examined by
quantitating the accumulation of mtDNA deletions in normal hearts and hearts
with coronary atherosclerotic disease. In normal hearts, mtDNA deletions
appeared after 40 and subsequently accumulated with age. The common 4977
nucleotide pair (np) deletion (mtDNA4977) reached a maximum of 0.007%, with the
mtDNA7436 and mtDNA10,422 deletions appearing at the same time. In hearts
deprived of mitochondrial substrates due to coronary artery disease, the level
of the mtDNA4977 deletion was elevated 7-220-fold over age-matched controls,
with the mtDNA7436 and mtDNA10,422 deletions increasing in parallel. This
cumulative mtDNA damage was associated with a compensatory 3.5-fold induction
of nuclear OXPHOS gene mRNA and regions of ischemic hearts subjected to the
greatest work load (left ventricle) showed the greatest accumulation of mtDNA
damage and OXPHOS gene induction. These observations support the hypothesis
that mtDNA damage does accumulate with age and indicates that respiratory
stress greatly elevates mitochondrial damage.
\section*{51}
Deleterious mitochondrial DNA mutations accumulate in aging human tissues.
Arnheim N, Cortopassi G
Molecular Biology Section, University of Southern California, Los Angeles
90089-1340.
Mutat Res Sep 1992 275 (3-6) p157-67
This paper reviews the current state of knowledge of the contribution of
mitochondrial DNA (mtDNA) mutations to the phenotype of aging. Its major focus
is on the discovery of deletions of mtDNA which previously were thought to
occur only in individuals with neuromuscular disease. One particular deletion
(mtDNA4977) accumulates with age primarily in non-dividing cells such as muscle
and brain of normal individuals. The level of the deletion rises with age by
more than 1000 fold in heart and brain and to a lesser extent in other tissues.
In the brain, different regions have substantially different levels of the
deletion. High levels of accumulation of the deletion in tissues are
correlated with high oxygen consumption. We speculate that oxidative damage to
mtDNA may be 'catastrophic', mutations affecting mitochondrially encoded
polypeptides involved in electron transport could increase free radical
generation leading to more mtDNA damage.
\section*{52}
Mitochondrial DNA alterations as ageing-associated molecular events.
Wei YH
Department of Biochemistry, National Yang-Ming Medical College, Taipei,
Taiwan.
Mutat Res Sep 1992 275 (3-6) p145-55
Mitochondrial DNA (mtDNA) is a naked double-stranded circular
extrachromosomal genetic element continuously exposed to the matrix that
contains great amounts of reactive oxygen species and free radicals. The age-
dependent decline in the capability and capacity of mitochondria to dispose
these oxy-radicals will render mtDNA more vulnerable to mutations during the
ageing process. During the past 3 years, more than 10 different types of
deletions have been identified in the mtDNA of various tissues of old humans.
Some of them were found only in a certain tissue but some others appeared in
more than one organ or tissue. The 4977-bp deletion is the most prevalent and
abundant one among these deletions. Skeletal muscle is the target tissue of
most ageing-associated mtDNA deletions and has often been found to carry
multiple deletions. The onset age of the various deletions in mtDNA varies
greatly with individual and type of the deletion. The 4977-bp deletion has
been independently demonstrated to occur in the mtDNA of various tissues of the
human in the early third decade of life. However, the 7436-bp deletion was
only detected in the heart mtDNA of human subjects in their late thirties. The
others appeared only in older humans over 40 years old. No apparent sex
difference was found in the onset age of these ageing-associated mtDNA
deletions. The various ageing-associated deletions could be classified into
two groups. Most of the deletions belong to the first group, in which the 5'-
and 3'-end breakpoints of the deletion are flanked by 4-bp or longer direct
repeats. The deletion in the second group occurs less frequently and shows no
distinct repeat sequences flanking the deletion sites. These two groups of
mtDNA deletions may occur by different mechanisms. The first group is most
probably caused by internal recombination or slippage mispairing during
replication of mtDNA by the D-loop mechanism. The deleted mtDNA and the
deleted DNA fragment may be further degraded or escape from the mitochondria
and get translocated into the nucleus. The latter route has been substantiated
by many observations of inserted mtDNA sequences in the nuclear DNA. Thus, the
fragments of migrating mtDNA may change the information content and expression
level of certain nuclear genes and thereby promote the ageing process or cause
cancer. Similar ageing-associated alterations of mtDNA have also been observed
in aged animals and plants. I suggest that mtDNA deletions and other mutations
to be discovered are molecular events generally associated with the ageing
process.
\section*{53}
Role of impaired intracellular glucose metabolism in the insulin resistance
of aging.
Gumbiner B, Thorburn AW, Ditzler TM, Bulacan F, Henry RR
Department of Medicine, University of Rochester, NY.
Metabolism Oct 1992 41 (10) p1115-21
The insulin resistance of aging is characterized by both reduced glucose
uptake and impaired intracellular glucose metabolism. The aim of this study
was to determine whether impaired intracellular glucose metabolism contributes
to insulin resistance in the elderly independent of reduced glucose uptake. To
address this question, glucose uptake in non-obese elderly males was matched to
controls using the glucose clamp technique, and intracellular glucose
metabolism was assessed in vivo by indirect calorimetry and in vitro by
skeletal muscle biopsy for glycogen synthase activity. When elderly subjects
were compared with controls at an equivalent basal glucose uptake of
approximately 2.5 mg/kg fat-free mass (FFM)/min, muscle glycogen synthase
activity was similar (fractional velocity of glycogen synthase at 0.1 mmol/L
glucose-6-phosphate [FV0.1], 0.06 +/- 0.1 and 0.07 +/- 0.1), but whole-body
rates of glucose oxidation were reduced (1.36 +/- 0.12 v 1.90 +/- 0.11 mg/kg
FFM/min, P less than .05). During 40-mU/m2/min hyperinsulinemic clamps at
matched rates of glucose uptake (approximately 10.7 mg/kg FFM/min in both
groups), glycogen synthase activity was again similar (FV0.1, 0.15 +/- 0.02 and
0.14 +/- 0.02), and glucose oxidation remained reduced in the elderly (4.18 +/-
0.25 v 4.77 +/- 0.17 mg/kg FFM/min, P less than .05). Only during clamps in
the maximal range of glucose uptake (approximately 29.5 mg/kg FFM/min) was
glucose oxidation between the groups comparable (5.97 +/- 0.50 and 5.75 +/-
0.31 mg/kg FFM/min). Plasma free fatty acid (FFA) concentrations, fat
oxidation, and protein oxidation were similar under all study
conditions....blah!!...hmmm...where is ibuprofen for my fingers..!
\section*{54}
Characterization of osteoporosis in a patient with Werner's syndrome.
Rubin CD, Zerwekh JE, Reed-Gitomer BY, Pak CY
Department of Medicine, University of Texas Southwestern Medical Center,
Dallas 75235-8889.
J Am Geriatr Soc Nov 1992 40 (11) p1161-3
\section*{55}
Stimulation of DNA synthesis in senescent human cells following incubation
with plasma membranes.
Frederich KB, Phillips PD, Cristofalo VJ
Wistar Institute, Philadelphia, Pennsylvania 19104.
Exp Cell Res Oct 1992 202 (2) p386-90
DNA synthesis and mitosis were increased in mitogen-stimulated senescent WI-
38 cells following incubation with plasma membranes prepared from young or
senescent WI-38 cells, A431 cells, 3T3 cells, or NR6 cells. The percentage of
[3H]thymidine-labeled nuclei in senescent cultures was two- to fivefold greater
than that seen in controls in which cells were incubated in the absence of
membranes or in the presence of boiled membranes. The effect was trypsin
sensitive, suggesting that a protein moiety is necessary for stimulation of DNA
synthesis. As the culture age increased, basal levels of DNA synthesis, as
well as maximal stimulation of DNA synthesis following incubation with plasma
membranes, decreased. These observations are consistent with the hypothesis
that different subpopulations exist in senescing cultures and suggest a complex
pattern of inhibitory and stimulatory regulation of cell proliferation.
\section*{56}
Impaired S-phase transit of Werner syndrome cells expressed in lymphoblastoid
cell lines.
Poot M, Hoehn H, Runger TM, Martin GM
Department of Human Genetics, University of Wurzburg, Germany.
Exp Cell Res Oct 1992 202 (2) p267-73
The clinical phenotype of Werner's syndrome (WS) includes short stature,
premature cataracts, skin atrophy, osteoporosis, graying and loss of hair,
neoplasia, diabetes mellitus, and arteriosclerosis. Cultured cells from
patients with this autosomal recessive disorder exhibit chromosomal instability
and a markedly reduced replicative lifespan and growth rate. To elucidate the
cell cycle alterations associated with the growth deficit, we continuously
labeled lymphoid cell lines from five WS patients and from four healthy adult
controls with 5-bromodeoxyuridine. Bivariate Hoechst 33258/ethidium bromide
flow cytometry revealed a 2.4-h prolongation in the minimal duration of the S
phase of WS cells (P less than 0.005). Moreover, the fraction of proliferating
cells irreversibly arrested in the S phase (5.4% vs 1.4% in controls) was
significantly elevated in WS (P less than 0.001). Other cell cycle
compartments were not significantly affected in WS cell lines. As a partial
test of the hypothesis that the WS phenotype is due to a defect in DNA
topoisomerase I (topo I) or DNA topoisomerase II (topo II) we exposed lymphoid
cells from a healthy control to the topo I inhibitor camptothecin or to the
topo II inhibitor 4'-(9-acridinylamino)methanesulfon-m-anisidine. The cell
kinetic alterations elicited by these compounds differed from that exhibited by
untreated WS patients. Thus, a primary defect in topo I or II is unlikely in
WS. Our cell cycle results, however, provide important evidence that the
biochemical genetic lesion is in fact expressed in lymphoblastoid cell lines,
the most readily available cells from such subjects.
\section*{57}
Occurrence of a particular base substitution (3243 A to G) in mitochondrial
DNA of tissues of ageing humans.
Zhang C, Linnane AW, Nagley P
Department of Biochemistry, Monash University, Clayton, Victoria, Australia.
Biochem Biophys Res Commun Sep 15 1993 195 (2) p1104-10
Allele-specific polymerase chain reaction has been used to demonstrate the
occurrence, during the human ageing process, of the mutation A to G at
nucleotide position 3243 on mitochondrial DNA. An oligonucleotide with a 3'-
end specific to this mutation was designed to selectively amplify DNA bearing
this mutation under annealing temperature conditions that efficiently
discriminate between normal and mutant mitochondrial DNA templates. Seven out
of 38 adult tissues tested were found to contain this mutation, whereas the
mutation could not be detected in any of the 16 infant tissues analyzed. The
present demonstration of an age-related point mutation adds to the growing list
of mutations in human mitochondrial DNA, till now almost exclusively large
deletions, that accumulate in human tissues as a function of age. The method
described here has general application in the detection of other age-related
point mutations in human mitochondrial DNA.
\section*{58}
Genetic and molecular basis for cellular senescence.
Barrett JC, Annab LA, Futreal PA
Laboratory of Molecular Carcinogenesis, National Institute of Environmental
Health Sciences, Research Triangle Park, North Carolina 27709.
Adv Exp Med Biol 1993 330 p27-43
Normal human and rodent cells in culture exhibit a finite life span at the
end of which they exhibit morphological changes and cease proliferating, a
process termed cellular senescence or cellular aging. Many cancer cells differ
from normal cells in that they do not senesce and have an indefinite life span
in culture, suggesting that alterations relating to cellular senescence are
involved in the neoplastic evolution of tumor cells. Recent experimental
results strongly support a genetic basis for cellular senescence. Defects in
the senescence program in transformed cells can be corrected by introduction of
a specific chromosome from normal cells into the abnormal cells. Using this
approach, possible senescence genes have been mapped to specific chromosomes.
Cell cycle control genes that regulate entry into the DNA synthetic phase of
the cell cycle must be altered in senescent cells. Recent findings suggest
that phosphorylation of the retinoblastoma gene is altered in senescent cells.
It is possible, but not yet proven, that aging at the cellular level
contributes to the aging of the individual. Therefore, an understanding of
cellular senescence at the genetic and molecular levels may provide new
insights into both the cancer and aging processes.
\section*{59}
Human ovarian aging and mitochondrial DNA deletion.
Suganuma N, Kitagawa T, Nawa A, Tomoda Y
Department of Obstetrics and Gynecology, Nagoya University School of
Medicine, Japan.
Horm Res 1993 39 Suppl 1 p16-21
The functions of human ovary change dynamically around the menopausal period.
A decrease of the number of primordial follicles and an increase of fibrous
tissues are observed histologically in the aged ovary. As endocrinological
aspects at menopause, the synthesis and secretion of ovarian steroid hormones
such as estrogens and progesterone decrease, followed with the resultant
increases of pituitary gonadotropins. However, the mechanism of menopause and
ovarian aging is not well understood. Thus, to study the regulatory mechanism
of ovarian dysfunction by aging, we analyzed the accumulation of mitochondrial
DNA (mtDNA) mutation in the human ovary in women of various ages. The
amplification of a 5.5-kb region in mtDNA with polymerase chain reaction
revealed a 0.5-kb band in ovarian samples obtained from menopausal and
postmenopausal women, which means that the 5.0-kb deletion of mtDNA in ovarian
tissue starts at the menopausal period. The close relationship between the
occurrence of ovarian mtDNA deletion and the menstrual irregularity was also
observed. These observations suggest that the accumulation of the deleted
mtDNA may be a regulating factor of dysfunction of the ovary by aging.
\section*{60}
Close linkage of the gene for Werner's syndrome to ANK1 and D8S87 on the
short arm of chromosome 8.
Nakura J, Miki T, Nagano K, Kihara K, Ye L, Kamino K, Fujiwara Y, Yoshida S,
Murano S, Fukuchi K, et al
Department of Geriatric Medicine, Osaka University Medical School, Japan.
Gerontology 1993 39 Suppl 1 p11-5
Werner's syndrome (WRN) is a rare autosomal recessive disorder characterized
by the appearance of features of premature aging in a young adult. Skin
fibroblasts from WRN patient demonstrate slow growth, reduced life span in
vitro and mutator phenotype. The genetic defect in WRN is unknown. We have
studied 23 WRN patients mainly from first or second cousin marriage and have
applied homozygosity mapping to search for the WRN locus. A peak lod score of
5.58 at a recombination fraction of 0.03 was obtained with D8S87. We confirmed
that the WRN locus was located on the short arm of chromosome 8, 8p11.2-p12.
\section*{61}
De Barsy syndrome: report of a case, literature review, and elastin gene
expression studies of the skin.
Karnes PS, Shamban AT, Olsen DR, Fazio MJ, Falk RE
Children's Hospital Los Angeles, Division of Medical Genetics, Calif 90027.
Am J Med Genet Jan 1 1992 42 (1) p29-34
Several "progeroid" syndromes have now been identified. The De Barsy
syndrome is an autosomal recessive syndrome of dwarfism, mental deficiency, an
"aged" appearance at birth, abnormal elastic fibers on skin biopsy, and lax
skin, large helices, eye abnormalities, lax joints, hypotonia, and athetoid
posturing. We report one case and review 11 cases from the literature. To
understand the abnormal appearance of the elastic fibers on biopsy, we
performed elastin gene expression studies on fibroblasts cultured from our
patient's skin. Molecular hybridization studies revealed reduced elastin mRNA
steady-state levels as compared with age matched control individuals. Assuming
normal rates of mRNA translation, reduced elastin synthesis would occur.
Diminished dermal elastin content could explain the altered cutaneous
elasticity, decreased elastic fibers in the skin, and many clinical
manifestations of individuals with this condition.
\section*{61}
Werner syndrome and biological ageing: a molecular genetic hypothesis.
Thweatt R, Goldstein S
Department of Medicine, University of Arkansas for Medical Sciences and
Geriatric Research, Education, Little Rock.
Bioessays Jun 1993 15 (6) p421-6
Werner syndrome (WS) is an inherited disorder that produces somatic stunting,
premature ageing and early onset of degenerative and neoplastic diseases.
Cultured fibroblasts derived from subjects with WS are found to undergo
premature replicative senescence and thus provide a cellular model system to
study the disorder. Recently, several overexpressed gene sequences isolated
from a WS fibroblast cDNA library have been shown to possess the capacity to
inhibit DNA synthesis and disrupt many normal biochemical processes. Because a
similar constellation of genes is overexpressed in WS and senescent normal
fibroblasts, these data suggest the existence of a common molecular genetic
pathway for replicative senescence in both types of cell. We propose that the
primary defect in WS is a mutation in a gene for a trans-acting repressor
protein that reduces its binding affinity for shared regulatory regions of
several genes, including those that encode inhibitors of DNA synthesis (IDS).
The mutant WS repressor triggers a sequence of premature expression of IDS and
other genes, with resulting inhibition of DNA synthesis and early cellular
senescence, events which occur much later in normal cells.
\section*{62}
A ScaI RFLP at the E-selectin (SELE) locus in a progeria family.
Warnecke P, Weiss AS
Department of Biochemistry, University of Sydney, NSW, Australia.
Hum Mol Genet Jun 1993 2 (6) p825
\section*{62}
A mitochondrial DNA deletion in normally aging and in Alzheimer brain tissue.
Blanchard BJ, Park T, Fripp WJ, Lerman LS, Ingram VM
Department of Biology, Massachusetts Institute of Technology, Cambridge
02139.
Neuroreport Jun 1993 4 (6) p799-802
By quantitative polymerase chain reaction (PCR) of total cellular DNA, the
known 4977 bp deletion in human mitochondrial DNA (mtDNA delta 4977) was not
detected in rapidly dividing tissue such as placenta and lymphocytes, nor in
brain tissue from fetuses and in frontal cortex from two individuals 24 and 56
years old. However, in frontal cortex from individuals 71-95 years 0.13%
deleted/undeleted mtDNA was found, with no significant difference between
Alzheimer patients (0.14%) and age-matched controls (0.12%). We hypothesize
that the age-related accumulation of this deletion (and other expected
deletions) contributes to the down-regulation of adenosine triphosphate (ATP)
production in neurons and other non-dividing cells, a fundamental mechanism
common to aging and Alzheimer's disease.
\section*{63}
Accumulation of insulin-like growth factor binding protein-3 in conditioned
medium of human fibroblasts increases with chronologic age of donor and
senescence in vitro.
Goldstein S, Moerman EJ, Baxter RC
Department of Medicine, University of Arkansas for Medical Sciences, Little
Rock.
J Cell Physiol Aug 1993 156 (2) p294-302
We have found that insulin-like growth factor binding protein-3 (IGFBP-3)
accumulates to higher levels in medium conditioned by a strain of normal
fibroblasts at late passage (LP) and a strain derived from subjects with Werner
syndrome (WS) of premature aging, compared to medium conditioned by the same
normal cells at early passage (EP) (Goldstein et al., Proc. Natl. Acad. Sci.
USA, 88:9680-9684, 1991). To explore the generality of this phenomenon with
respect to chronological age of donor (in vivo aging) and LP (in vitro
senescence) we assayed IGFBP-3 in medium conditioned by 18 normal fibroblast
strains at EP and LP and two WS strains at the midpoint of their curtailed
replicative lifespans and assessed IGFBP-3 mRNA levels in cells by Northern
analysis. The lowest accumulations of IGFBP-3 were found in medium conditioned
by fetal cells with progressively increasing amounts postnatally, direct
correlations between IGFBP-3 levels and donor age were seen in EP cells 3 days
after subculture (during logarithmic growth) r = 0.80, P < 0.001, and 7 days
after subculture (at confluence) r = 0.77, P < 0.001. With two exceptions,
conditioned medium of cell strains accumulated more IGFBP-3 at LP, IGFBP-3
levels correlated with chronological age after 3 days, r = 0.50, P = 0.05, and
after 7 days, r = 0.75, P < 0.001. IGFBP-3 content of WS culture medium fell
within the range of LP normal cells. Cumulative IGFBP-3 levels were inversely
proportional to the thymidine labeling index, a measure of proliferative vigor.
With some exceptions IGFBP-3 mRNA levels were commensurate with the amount of
IGFBP-3 accumulated in the medium, suggesting that distal translational and
posttranslational mechanisms also regulate IGFBP-3 production in some strains.
The trend toward augmented IGFBP-3 output of fibroblasts as a direct function
of chronological age and in vitro senescence and as an inverse function of
proliferative vigor is consistent with the known inhibitory effect of excess
IGFBP-3 on IGF-mediated DNA synthesis and the reduced regenerative potential
that is evident during biological aging in vivo.
\section*{64}
Short DNA sequences from the cytoplasm of mouse tumor cells induce
immortalization of human lymphocytes in vitro.
Abken H, Hegger R, Butzler C, Willecke K
Abteilung Molekulargenetik, Universitat Bonn, Germany.
Proc Natl Acad Sci U S A Jul 15 1993 90 (14) p6518-22
Cytoplasts of mouse L929 and Ehrlich ascites tumor cells harbor DNA sequences
that induce unlimited proliferation ("immortalization") of human lymphocytes
after transfection in vitro. By equilibrium centrifugation of cytoplasmic
lysates in a neutral CsCl gradient, the immortalizing activity was recovered
together with extramitochondrial fractions at high salt densities (1.85-1.87
g/cm3). Unexpectedly, these fractions contain linear DNA molecules of 50-500
bp in length. In contrast, cytoplasts of primary, senescent cells (mouse
embryo fibroblasts, human lymphocytes) do not harbor DNA in the corresponding
fractions. Cytoplasmic DNA isolated from high-density fractions of mouse tumor
cells was cloned in subset libraries, and of 45 DNA sequences we identified 2
clones one from L929 cytoplasts (203 bp) and another one from the cytoplasm of
Ehrlich ascites cells (372 bp) that induce unlimited proliferation of human
lymphocytes in vitro. Immortalized lymphoid cells harbor 1-5 copies of
transfected DNA integrated into chromosomal DNA, whereas about 100 copies were
found as episomal DNA in the cytoplasmic fraction. No immortalization could be
induced by transfection of nuclear DNA randomly fragmented to 200-500 bp.
Although the cloned DNA sedimented at 1.70 g/cm3, after transient transfection
into lymphocytes, these DNA sequences form salt-stable complexes that sediment
in fractions at the same high density (1.82-1.88 g/cm3) from which they were
originally cloned. The high-density banding of these cytoplasmic DNA sequences
may be due to association with RNA and/or with (metallo-) proteins in vivo.
Since both cloned DNA sequences with immortalizing activity have stop codons
for protein translation in all possible reading frames, immortalization may be
induced by insertional inactivation or functional suppression of genes that are
needed to be expressed during cellular senescence or programmed cell death.
\section*{65}
Robust induction of AP-1 transcription factor DNA binding activity in the
hippocampus of aged rats.
Kaminska B, Kaczmarek L
Nencki Institute of Experimental Biology, Warsaw, Poland.
Neurosci Lett Apr 30 1993 153 (2) p189-91
Activation of expression of genes encoding components of AP-1 transcription
factor (c-fos, c-jun and their cognates) as well as AP-1 itself has been
repeatedly shown to coincide with long-term cellular responses. For the
proliferating cells where functional involvement of AP-1 in physiological
activity has been proved, cellular senescence as well as aging at level of the
organism results in an alteration of the AP-1. The aim of the present study
was to analyze whether there is an aging-related defect in the formation of AP-
1 transcription factor in the brain. AP-1 has been shown previously to be
inducible by several stimuli in the cells of the central nervous system,
including proconvulsant treatment of pentylenetetrazole (PTZ). The present
studies found that PTZ robustly induced AP-1 DNA binding activity, evaluated by
the electrophoretic mobility shift assay, in hippocampi of aged (2-year-old)
rats, indicating no aging-related AP-1 defect in the rat brain.
\section*{66}
A genetic analysis of the Werner syndrome region on human chromosome 8p.
Thomas W, Rubenstein M, Goto M, Drayna D
Department of Molecular Biology, Genentech, Inc., South San Francisco,
California 94080.
Genomics Jun 1993 16 (3) p685-90
Werner syndrome (WRN) is an inherited disorder that produces symptoms of
premature aging. This disease is caused by a recessive mutation that has
previously been mapped to chromosome 8p. We have now used genetic linkage
analysis to map the WRN gene relative to chromosome 8 reference loci, to screen
candidate genes, and to identify a novel dinucleotide repeat polymorphic marker
closely linked to WRN. The WRN locus was mapped relative to the marker loci,
PLAT, ANK1, D8S135, and D8S87 of the comprehensive chromosome 8 linkage map.
The heregulin (HRG) and the fibroblast growth factor receptor 1 genes (FGFR1)
have been mapped to chromosome 8p and are involved in cellular growth.
Recombination events were detected between WRN and the HRG and FGFR1 genes,
excluding them as candidates for the WRN gene. A polymorphic marker generated
in this study, WT251, is linked to WRN at a recombination fraction of 0.006,
with a lod score of 16.5.
\section*{67}
Mitochondrial DNA mutations associated with age [letter]
Ozawa T
Aging (Milano) Apr 1993 5 (2) p155-8
\section*{68}
Aging, mitochondria heterogeneity and pharmacological approach [letter]
Calvani M, Caruso G
Aging (Milano) Apr 1993 5 (2) p159-64
\section*{69}
Genomic damage and its repair in young and aging brain.
Rao KS
Neurobiochemistry Laboratory, School of Life Sciences, University of
Hyderabad, AP, India.
Mol Neurobiol Spring 1993 7 (1) p23-48
A brief review of the available information concerning age-related genomic
(DNA) damage and its repair, with special reference to brain tissue, is
presented. The usefulness of examining the validity of DNA-damage and repair
hypothesis of aging in a postmitotic cell like neuron is emphasized. The
limited number of reports that exist on brain seem to overwhelmingly support
the accumulation of DNA damage with age. However, results regarding the age-
dependent decline in DNA-repair capacity are conflicting and divided. The
possible reasons for these discrepancies are discussed in light of the
gathering evidence, including some human genetic disorders, to indicate how
complex is the DNA-repair system in higher animals. It is suggested that
assessment of repair potential of neurons with respect to a specific damage in
a specific gene might yield more definitive answers about the DNA-repair
process and its role in aging.
\section*{70}
Neuronal nicotinic receptors and their implications in ageing and
neurodegenerative disorders in mammals.
Nordberg A
Department of Pharmacology, Uppsala University, Sweden.
J Reprod Fertil Suppl 1993 46 p145-54
Recent biochemical, molecular biology and pharmacological studies have
revealed the existence of multiple functional subtypes of neuronal nicotinic
receptor in the rodent as well as in the human CNS. An important goal in this
research is to define the physiological role of these receptors and to
understand the mechanisms by which these receptors interact with other
receptors and neurotransmitters. Nicotinic receptors are detectable in the
immature rat brain. Exposition of various agents can change the expression of
receptors. Neuronal receptors undergo changes during ageing, the pattern of
which is different for various subtypes of nicotinic receptor.
Neurodegenerative disorders, such as Alzheimer's disease, are characterized by
losses in this type of receptor. An increased understanding of underlying
mechanisms leading to this disease will provide pathophysiological knowledge
and strategies for treatment. Imaging techniques such as positron emission
tomography provide a possible means of tracing the nicotinic receptor in vivo
in patients with Alzheimer's disease.
\section*{71}
Age distribution of latent herpes simplex virus 1 and varicella-zoster virus
genome in human nervous tissue.
Liedtke W, Opalka B, Zimmermann CW, Lignitz E
Department of Neurology, University of Essen, Faculty of Medicine, Germany.
J Neurol Sci May 1993 116 (1) p6-11
Latency in nervous tissue caused by herpes simplex virus 1 (HSV-1) and by
varicella-zoster virus (VZV) is an intriguing feature of herpes-virus'
neurotropism. HSV-1 and VZV latency are the causes of ophthalmic zoster and
recurrent HSV infections in the distributions of the trigeminal branches. HSV-
1 neuronal latency may play a role in the etiopathogenesis of HSV encephalitis.
We attempted to determine the prevalence and age distribution of VZV and HSV
latency. We applied nested polymerase chain reaction (PCR) assays to detect
HSV-1 and VZV genome in trigeminal ganglia and olfactory bulbs which were
obtained from 109 human corpses at forensic postmortems. HSV-1 latency was
found in 72.5% of trigeminal ganglia and in 15.5% of olfactory bulbs. VZV
latency was 63.3% in trigeminal ganglia and 1% in olfactory bulbs.
Simultaneous latency of VZV and HSV genome occurs in 48.8% of trigeminal
ganglia. The age-group specific prevalence of HSV neuronal latency increases
from 18.2% in 0-20 years to reach finally 100% in persons older than 60 years.
Age specific prevalences of VZV peaked for a first time with 82% between 21-30
years, fell to 50% for 40-50 years, and rose to 89% for 71-80 years. If the
latent trigeminal ganglion HSV-1 genome were the source of endogenously
acquired encephalitis, the peak incidence of HSV encephalitis in older subjects
correlates with our findings. Increased VZV latency prevalence in nervous
tissue of younger people without subsequent disease indicates sufficient immune
surveillance.
\section*{72}
Oxidized amino acids in lens protein with age. Measurement of o-tyrosine and
dityrosine in the aging human lens.
Wells-Knecht MC, Huggins TG, Dyer DG, Thorpe SR, Baynes JW
Department of Chemistry and Biochemistry, University of South Carolina,
Columbia 29208.
J Biol Chem Jun 15 1993 268 (17) p12348-52
The concentrations of ortho-tyrosine (o-Tyr) and dityrosine (DT) were
measured in noncataractous human lenses in order to assess the role of protein
oxidation reactions in the aging of lens proteins. The measurements were
conducted by selected ion monitoring-gas chromatography/mass spectrometry using
deuterium-labeled internal standards, which provided both high sensitivity and
specificity for the quantitation of o-Tyr and DT. Between ages 1 and 78 years,
the o-Tyr concentration in lens proteins varied from 0.3 to 0.9 mmol of o-
Tyr/mol of Phe (n = 19), while DT ranged from 1 to 3 mumol of DT/mol of Tyr (n
= 30). There were no significant changes in levels of o-Tyr with lens age.
There was a statistically significant, but only slight, increase in DT in lens
proteins with age (approximately 33% increases between ages 1 and 78, r = 0.5,
p < 0.01). At the same time, total protein fluorescence, measured at DT
wavelengths (Ex = 317 nm, Em = 407 nm), increased 11-fold between ages 1 and 78
and correlated strongly with age (r = 0.82, p < 0.0001). Although the
fluorescence maxima of lens proteins were similar to those of DT, DT accounted
for less than 1% of the DT-like fluorescence in lens protein at all ages.
These observations indicate that oxidation of Phe and Tyr plays a limited role
in the normal aging of lens proteins in vivo.
\section*{73}
Suppression of the growth factor-mediated induction of c-fos and down-
modulation of AP-1-binding activity are not required for cellular senescence.
Lucibello FC, Brusselbach S, Sewing A, Muller R
Institut fur Molekularbiologie und Tumorforschung (IMT), Philipps-Universitat
Marburg, Germany.
Oncogene Jun 1993 8 (6) p1667-72
It has been suggested that the impaired response of the c-fos gene to serum
growth factors and the concomitant loss of AP-1 activity may be a crucial step
in the process of cellular senescence. In the present study, we provide
evidence arguing against such a conclusion. Data obtained in five independent
experiments showed that both c-fos RNA and protein expression were similar in
'young' and in senescent serum-stimulated WI-38 cells, suggesting that the
previously reported suppression of c-fos induction is not an obligatory event
in the process of cellular senescence. Likewise, expression of fra-1, c-jun
and junB continued to be high in serum-stimulated senescent cells, while
induction of fosB was reduced approximately fivefold. Among all genes tested
fosB thus seems to be the most suitable marker for the detection of senescent
cells. Stimulated senescent cells showed only a approximately twofold
reduction of AP-1-binding activity, and senescent cells continuously exposed to
serum exhibited normal AP-1-binding activity. These observations support the
conclusion that a down-modulation of AP-1 is not crucial for human fibroblasts
to enter the senescent state.
\section*{74}
Decreased type VI collagen gene expression in cultured Werner's syndrome
fibroblasts.
Hatamochi A, Mori K, Takeda K, Arakawa M, Ueki H, Yoshioka H
Department of Dermatology, Kawasaki Medical School, Kurashiki, Japan.
J Invest Dermatol Jun 1993 100 (6) p771-4
Gene expression of collagens VI, I, and III in Werner's syndrome was studied
by measuring messenger RNA (mRNA) and protein production levels in four
fibroblast strains from patients with Werner's syndrome and comparing them with
age-matched healthy subjects. Levels of type VI collagen mRNA were decreased
in all Werner's syndrome fibroblast strains and the decreases were in parallel
in all three chains (alpha 1, alpha 2, and alpha 3) of type VI collagen. A
coordinate increase of the alpha 1(I) and alpha 1(III) collagen mRNA levels was
observed in three of the four Werner's syndrome fibroblast strains. However,
no qualitative abnormality of these mRNA transcripts in Werner's syndrome
fibroblasts were found by Northern blot analysis. Changes in type VI and type
I collagen mRNA correlated well with production levels of corresponding
proteins, as determined by immunologic assays. These data suggest that there
are changes in expression of multiple connective tissue constituents in
Werner's syndrome fibroblasts.
\section*{75}
Mitochondrial DNA deletions in human brain: regional variability and increase
with advanced age.
Corral-Debrinski M, Horton T, Lott MT, Shoffner JM, Beal MF, Wallace DC
Department of Genetics and Molecular Medicine, Emory University School of
Medicine, Atlanta, Georgia 30322.
Nat Genet Dec 1992 2 (4) p324-9
We have examined the role of somatic mitochondrial DNA (mtDNA) mutations in
human ageing by quantitating the accumulation of the common 4977 nucleotide
pair (np) deletion (mtDNA4977) in the cortex, putamen and cerebellum. A
significant increase in the mtDNA4977 deletion was seen in elderly individuals.
In the cortex, the deleted to total mtDNA ratio ranged from 0.00023 to 0.012 in
67-77 year old brains and up to 0.034 in subjects over 80. In the putamen, the
deletion level ranged from 0.0016 to 0.010 in 67 to 77 years old up to 0.12 in
individuals over the age of 80. The cerebellum remained relatively devoid of
mtDNA deletions. Similar changes were observed with a different 7436 np
deletion. These changes suggest that somatic mtDNA deletions might contribute
to the neurological impairment often associated with ageing.
\section*{76}
Growing old: the most common mitochondrial disease of all? [news]
Harding AE
Nat Genet Dec 1992 2 (4) p251-2
\section*{77}
Molecular Biology of Aging. Keystone Symposium. Lake Tahoe, California, March
19-25, 1993. Abstracts.
J Cell Biochem Suppl 1993 17D p139-68
\section*{78}
Deficient expression of decorin in infantile progeroid patients.
Beavan LA, Quentin-Hoffmann E, Schonherr E, Snigula F, Leroy JG, Kresse H
Institute of Physiological Chemistry and Pathobiochemistry, University of
Munster, Germany.
J Biol Chem May 5 1993 268 (13) p9856-62
Fibroblasts from young patients exhibiting clinical features of progeroidal
syndromes showed decreased biosynthesis of the small proteoglycan decorin.
Cells in culture were metabolically labeled, and proteoglycans secreted into
the medium were analyzed electrophoretically after immunoprecipitation with
antibodies raised against decorin and biglycan. Fluorograms showed regularly a
reduction to 15-30% of the normal amount of mature decorin and its core protein
after chondroitin ABC lyase treatment. The size of the glycosaminoglycan
chains was increased, but there was no obvious anomaly in the secretion
kinetics of the mature proteoglycan. In addition, the patients' fibroblasts
synthesized an increased amount of biglycan compared to control cells from
healthy donors. Northern blot analysis clearly demonstrated a reduction by 85-
94% in decorin mRNA, but biglycan mRNA was concomitantly increased, indicating
that these alterations occur at the transcriptional level of protein expression.
Transcription of decorin in fibroblasts from one of the patients was stimulated
up to 3-fold by treatment with interleukin-1 beta. No response to interleukin-
1 beta and transforming growth factor-beta was observed in the cells from
another patient. In situ hybridization of cultured cells with an antisense
decorin probe showed that decorin levels were reduced throughout the cell
population. Surprisingly, subsequent examination of cells from one of the
patients, now in mid-teenage, revealed a return to normal levels of decorin
expression compared to age-matched controls. These studies suggest that, as in
Marfan's syndrome where the primary defect concerns the fibrillin gene, reduced
decorin expression contributes to the formation of an abnormal matrix and the
pathogenesis of these disorders. They also indicate that this abnormality is
likely to represent a secondary phenomenon which leads to a fault in the
regulation of decorin gene transcription.
\section*{79}
Mutations of mitochondrial DNA support human aging [letter]
Kadenbach B, Munscher C, Muller-Hocker J
Aging (Milano) Feb 1993 5 (1) p73-5
\section*{80}
Decreased expression of the platelet-derived growth factor beta-receptor in
fibroblasts from a patient with Werner's syndrome.
Mori S, Kawano M, Kanzaki T, Morisaki N, Saito Y, Yoshida S
Department of Internal Medicine, School of Medicine, Chiba University, Japan.
Eur J Clin Invest Mar 1993 23 (3) p161-5
The study of Werner's syndrome, a premature ageing syndrome, may provide
insight into the processes of natural ageing. We investigated the reason for
the reduced growth potential of fibroblasts in Werner's syndrome which is
considered to be similar to that of fibroblasts in normal senescence. The
responses to different isoforms of platelet-derived growth factor (PDGF) of
fibroblasts from a patient with Werner's syndrome (W-cells) and from a normal
subject (control cells) were assessed by [3H]thymidine incorporation assay, a
125I-PDGF-AB binding experiment, and Northern blot analysis with a PDGF beta-
receptor specific cDNA probe. PDGF-stimulated increase in [3H]thymidine
incorporation in W-cells was much lower than that in control cells, especially
with PDGF-AB and -BB as mitogens. The specific binding of 125I-PDGF-AB to W-
cells was correspondingly lower than that to control cells. Furthermore,
Northern blot analysis demonstrated a decreased basal level, and lack of PDGF-
AB-induced up-regulation of the PDGF beta-receptor transcript in W-cells.
Decreased expression of the PDGF beta-receptor due to decrease in its synthesis
may be a causative factor of the decreased mitogenic response of W-cells to
PDGF.
\section*{81}
Audiologic and otolaryngologic findings in progeria: case report.
Hall JW 3d, Denneny JC 3d
Division of Hearing and Speech Sciences, School of Medicine, Vanderbilt
University, Nashville, Tennessee.
J Am Acad Audiol Mar 1993 4 (2) p116-21
Progeria is a rare syndrome, with an estimated incidence of 1 per 250,000
births. Although children with progeria have the appearance of premature aging
or senility, the term is misleading because reported cases of progeria have not
manifested most physical or biochemical aspects of old age. Many children with
progeria appear normal at birth and then progressively, and rather rapidly,
develop the characteristic features during early childhood. Although first
described in the 1880s, only approximately 100 cases of progeria are reported
in the international literature. The single case study of hearing in progeria,
which appeared in 1965, is limited to pure-tone and speech audiometry findings.
We report the results of otolaryngologic examination and pure-tone, speech,
immittance, and auditory brainstem response (ABR) audiometry for a 5-year-old
female with progeria. The patient had a mild-to-moderate, bilateral,
conductive hearing loss. Immittance measurements were consistent with fixation
of the ossicular chain and this was confirmed surgically. Mildly prolonged ABR
wave I-V latencies suggest possible auditory central nervous system
involvement.
\section*{82}
Larsen syndrome presenting as a familial syndrome of dwarfism, distinct
oldish facial appearance and bilateral clubfeet in mother and daughter.
Fryns JP, Lenaerts J, Van den Berghe H
Centre for Human Genetics, University of Leuven, Belgium.
Genet Couns 1993 4 (1) p43-6
\section*{82a}
Ah! How about reading the ZEROETH posting for reaching this far?!
Some contributions would be greatly appreciated!
\section*{83}
Elevated levels of glycoprotein gp200 in progeria fibroblasts.
Clark MA, Weiss AS
Department of Biochemistry, University of Sydney, N.S.W. Australia.
Mol Cell Biochem Mar 10 1993 120 (1) p51-60
The glycosylation of proteins in fibroblasts from people with the premature
ageing disease Hutchinson-Gilford Progeria Syndrome (progeria) was investigated.
Protein was prepared from fibroblast cell lines established from skin biopsy
taken from progeria patients and control donors. Glycoproteins were labelled
by the covalent attachment of the steroid hapten digoxygenin to the sugar group.
After separation of total protein by SDS-PAGE and electroblotting onto
Immobilon-PTM, glycoproteins were detected by enzyme immunoassay. We have
observed a glycoprotein of M(r) 200 kDa which is consistently present in
protein preparations from progeria fibroblasts and which is absent, or markedly
reduced, in preparations from control fibroblasts. This suggests that it may
be useful as a marker for progeria. Similar analysis of progeria lymphoblast
and control lymphoblast cultures did not show this altered pattern of
glycosylated proteins, indicating that it may be cell-type specific.
Glycoproteins were also detected by labelling fibroblasts in vitro with D-[6-
3H]glucosamine hydrochloride followed by SDS-PAGE of isolated protein and
subsequent fluorography. Profiles of glycoproteins from progeria and control
fibroblasts were consistent with those obtained from labelling of carbohydrate
groups with digoxygenin. Protease digestion of cell protein verified that the
band at M(r) 200 kDa contains a protein core. Characteristic features of
progeria primarily involve the connective tissue and include wrinkled and loose
skin, loss of soft tissue, thin limbs and stiff joints. Death of progeria
patients is usually a result of cardiovascular abnormalities. The most
consistent manifestations thus involve the connective tissue. The glycoprotein
of M(r) 200 kDa which we have observed in progeria fibroblasts in vitro could
reflect a perturbation in glycosylation which may underly the connective tissue
defects seen in progeria.
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