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Biomedical subjects

G M Martin

Publications and source records attributed to G M Martin.

At least 19 recordsLinked to original sources
PubMed2000

Overexpression of wild-type and nuclear-targeted catalase modulates resistance to oxidative stress but does not alter spontaneous mutant frequencies at APRT.

Animal cells generate hydrogen peroxide as a byproduct of energy metabolism. In the presence of reduced metals H(2)O(2) can decompose to a highly reactive hydroxyl radical that attacks essentially all organic molecules, including DNA. We wished to determine if overexpression of catalase and/or the targeting of the enzyme to the nucleus could protect cells from oxidative stress and reduce the frequency of mutation. Wild-type human catalase, which localizes to peroxisomes, and a modified construct, which targets catalase to the nucleus, were overexpressed in a murine line of embryonic carcinoma cells (P19). Both constructs enhanced the resistance of the cells to hydrogen peroxide, but sensitized them to bleomycin. Overexpression of wild-type catalase protected cells against paraquat, while nuclear targeting sensitized them to this agent. Expression of neither construct significantly altered spontaneous mutant frequencies at the endogenous murine adenosine phosphoribosyl transferase (APRT) locus; however, nuclear-targeted catalase prevented an increase in mutant frequency after H(2)O(2) treatment. These results suggest that endogenous levels of hydrogen peroxide may not generate DNA damage in vivo, or that such damage may be efficiently repaired in murine embryonic carcinoma cells.

Adenine Phosphoribosyltransferase↗
PubMed2000

Broadly altered expression of the mRNA isoforms of FE65, a facilitator of beta amyloidogenesis, in Alzheimer cerebellum and other brain regions.

FE65 is a key "adapter" protein that links a multiprotein complex to an intracellular domain of beta-amyloid precursor protein (betaPP). Its overexpression modulates the trafficking of betaPP and facilitates the generation of beta-amyloid (Abeta). FE65 is predominantly expressed in brain tissues. An exon 9-inclusive isoform is exclusively expressed in neurons, and an exon 9-exclusive isoform is only expressed in non-neuronal cells. We quantitated the two isoforms in middle temporal cortex, middle frontal cortex, cerebellar cortex and caudate nucleus of 17 Alzheimer disease (AD) patients, 12 normal controls and 9 non-AD neurodegenerative disease controls by reverse transcription-competitive polymerase chain reaction (RT-cPCR). Expression of the two isoforms was significantly and differentially altered, with a 30-57% decrease in levels of the neuronal form (P < 0.05-0.002) and a 73-135% increase in levels of non-neuronal form (P < 0.02-0.001), in the temporal and frontal cortex of AD brains. These alterations presumably reflect advanced neurodegenerative processes of these regions. Surprisingly, expression of both isoforms was significantly up-regulated by 42-66% in the cerebellar cortex and caudate nucleus of AD brains when compared to normal brains (P < 0.05-0.005). Diffuse Abeta-positive plaques were observed in the cerebellum of these AD subjects but not in the normal controls. Selective up-regulation of only the FE65 neuronal isoform was seen in the cerebellar cortex in association with other neurodegenerative diseases (largely Parkinson's disease). Because FE65 modulates trafficking of betaPP toward the production of Abeta, the up-regulation of FE65 in AD cerebellum may be relevant to the genesis of diffuse plaques. Thus, early biochemical alterations in AD, not complicated by advanced pathology, may be beneficially investigated in the less-affected regions of the brain, such as the cerebellum.

Alzheimer Disease↗
PubMed2000

Molecular mechanisms of late life dementias.

A brief overview of the molecular pathology of dementia of the Alzheimer type (DAT), frontotemporal dementias (FTD), and Lewy body dementias (LBD) is preceded by a discussion of the evolutionary biological basis for the types of gene action responsible for the emergence of late life dementias. The beta amyloid cascade theory of the pathogenesis of DAT still predominates, but possible upstream events are being explored. Some familial forms of FTD have been shown to result from dominant mutations in the microtubular associated protein tau. A key element in pathogenesis is a shift in the ratios of various isoforms. Rare forms of Parkinson disease have been associated with dominant mutations in alpha synuclein, a protein of probable importance for synaptic plasticity. Aberrations in the metabolism of this protein (which is found in Lewy body fibrillar material) may therefore be of importance to the genesis of some LBD cases.

Aging↗
PubMed2000

Multiple points of entry into a circular enclosure prevent place learning despite normal vestibular orientation and cue arrays: evidence for map resetting.

This study identified sources of map orientation critical for successful spatial problem solving by rats of a plus maze embedded in water. Disorientation slowed, but it did not prevent acquisition of goal location. Use of a circular enclosure with multiple points of entry prevented reliable goal location. A single entry point enabled the rats to locate a fixed goal. A cue array within the enclosure was ineffective in providing orientation. These data suggest that stable map orientation can be derived from entry location when enclosure geometry is uniformative, but is not readily taken from cue arrays. They further suggest that map orientation is reset when rats enter an enclosure.

Animals↗
PubMed2000

Cellular Werner phenotypes in mice expressing a putative dominant-negative human WRN gene.

Mutations at the Werner helicase locus (WRN) are responsible for the Werner syndrome (WS). WS patients prematurely develop an aged appearance and various age-related disorders. We have generated transgenic mice expressing human WRN with a putative dominant-negative mutation (K577M-WRN). Primary tail fibroblast cultures from K577M-WRN mice showed three characteristics of WS cells: hypersensitivity to 4-nitroquinoline-1-oxide (4NQO), reduced replicative potential, and reduced expression of the endogenous WRN protein. These data suggest that K577M-WRN mice may provide a novel mouse model for the WS.

4-Nitroquinoline-1-oxide↗
PubMed2000

Some new directions for research on the biology of aging.

A highly selective, eclectic, and personal view of new directions and new opportunities for research on the biology of aging is briefly outlined. Some concern is raised regarding the present emphasis on the use of centenarians for the definition of genetic loci responsible for unusually robust retention of structure and function. More progress is likely to be made were we to focus on the genetic basis for "elite" aging in middle-aged subjects examined for very specific phenotypes, as these are likely to be far less polygenic. Descriptive gerontology is entering a renaissance, given such new clinical tools as functional MRI and basic science tools such as functional genomics and proteomics. Advances in genomics should expedite answers to such questions as why some avian species have exceptionally long lifespans despite unusual loads of oxidative stress. One hopes to see renewed mechanistic studies, using such tools, at the systems levels. New methodologies are permitting the evaluation of stochastic alterations in gene structure and function in postreplicative cells. The exciting work on molecular misreading should prompt us to reexplore the Orgel hypothesis as it applies to such cell types. Epigenetic shifts in gene expression that occur in association with sexual maturation and the cessation of growth may have deleterious consequences late in the life course. It will therefore be important for gerontologists to investigate the molecular biology of pubescence. Finally, our community should investigate the impact of environmental "gerontogens," agents that accelerate specific processes of aging and specific senescent phenotypes.

Aging↗
PubMed1999

Alternatively spliced isoforms of FE65 serve as neuron-specific and non-neuronal markers.

FE65 is predominantly expressed in brain and is especially rich in the regions with the highest densities of neurons. The FE65 protein binds to an intracellular domain of the beta-amyloid precursor protein (betaPP) and may modulate the production of beta-amyloid peptide (AP). One of FE65 exons, a mini-exon (exon 9, 6 bp), is alternatively spliced, giving rise to two isoforms varying only in 6 base pairs. We quantitated the two isoforms by a sensitive reverse transcription-competitive polymerase chain reaction technique, and characterized their expressions in various tissues and cell cultures, and the kinetics of expression of the two isoforms in P19 embryonal carcinoma cell lines during neuronal differentiation. Our results show that the exon 9-inclusive (E9) form, the more abundant form in brain, was exclusively expressed in neurons, while the exon 9-exclusive (DeltaE9) form was widely expressed in all non-neuronal cells, but was not expressed in differentiated neurons. When P19 cells were differentiated to neurons, expression of FE65 was significantly up regulated ( approximately 30-fold) and the splicing pattern of the FE65 pre-mRNA was switched from the DeltaE9 pattern to the E9 form. Based upon their distinctive expression patterns, these two isoforms may serve as neuronal and non-neuronal markers, and determination of their ratios may have applications in neuropathological diagnosis.

Aged↗
PubMed1999

Neurotrophic factors [activity-dependent neurotrophic factor (ADNF) and basic fibroblast growth factor (bFGF)] interrupt excitotoxic neurodegenerative cascades promoted by a PS1 mutation.

Although an excitotoxic mechanism of neuronal injury has been proposed to play a role in chronic neurodegenerative disorders such as Alzheimer's disease, and neurotrophic factors have been put forward as potential therapeutic agents, direct evidence is lacking. Taking advantage of the fact that mutations in the presenilin-1 (PS1) gene are causally linked to many cases of early-onset inherited Alzheimer's disease, we generated PS1 mutant knock-in mice and directly tested the excitotoxic and neurotrophic hypotheses of Alzheimer's disease. Primary hippocampal neurons from PS1 mutant knock-in mice exhibited increased production of amyloid beta-peptide 42/43 and increased vulnerability to excitotoxicity, which occurred in a gene dosage-dependent manner. Neurons expressing mutant PS1 exhibited enhanced calcium responses to glutamate and increased oxyradical production and mitochondrial dysfunction. Pretreatment with either basic fibroblast growth factor or activity-dependent neurotrophic factor protected neurons expressing mutant PS1 against excitotoxicity. Both basic fibroblast growth factor and activity-dependent neurotrophic factor stabilized intracellular calcium levels and abrogated the increased oxyradical production and mitochondrial dysfunction otherwise caused by the PS1 mutation. Our data indicate that neurotrophic factors can interrupt excitotoxic neurodegenerative cascades promoted by PS1 mutations.

Alzheimer Disease↗
PubMed1999

Polymorphisms at the Werner locus: I. Newly identified polymorphisms, ethnic variability of 1367Cys/Arg, and its stability in a population of Finnish centenarians.

The Werner syndrome gene (WRN) encodes a novel helicase of 1,432 amino acids. Homozygous mutations, all of which result in the truncation of the protein, lead to Werner syndrome. However, little is known about the role of WRN in "normal" aging. We have identified four missense polymorphisms and four conservative polymorphsims in WRN gene. A single study showed that a polymorphism at amino acid 1367 Cys(TTG)/ Arg(CTG) is associated with a variation in risk of myocardial infarction among a Japanese population. The 1367 Cys/Arg polymorphism was examined during aging in three different populations: Finnish, Mexican, and North American. The frequencies of 1367 Cys were higher than those of 1367 Arg in all the populations examined, though the frequencies varied among populations. The frequency of the 1367 Arg allele, thought to be protective against myocardial infarction in a Japanese population, was approximately three times higher in the North American and Finnish adult populations. When newborns and centenarians were compared within the Finnish population, no differences were observed in the proportions of 1367 Cys/Arg across age groups. Within the Finnish population, we confirmed a significant decrease of the APOE epsilon2 allele and an increase in the epsilon4 allele in newborn infants compared with centenarians. Thus, unlike the APOE polymorphism, there is no evidence of an association of this WRN polymorphism with longevity.

Adult↗
PubMed1999

Open field motor patterns and object marking, but not object sniffing, are altered by ibotenate lesions of the hippocampus.

Objectmarking and object sniffing were assessed in an open field during six 5-min trials in unoperated Long-Evans rats and rats with ibotenate lesions of the hippocampus and/or neocortex. Object marking was higher in hippocampally lesioned rats than in unoperated rats. Object marking did not differ between neocortically lesioned rats and unoperated rats. Object sniffing durations and visits did not differ between unoperated and hippocampally lesioned rats nor between unoperated and neocortically lesioned rats. A new object elicited longer sniffing by both unoperated and hippocampally lesioned rats. Neocortically lesioned rats did not show this effect. There were no effects of the new object on marking. Computerized tracing of open field paths revealed a smaller perimeter track for hippocampally lesioned rats than for unoperated rats. This difference reflected distinct ambulatory patterns. Hippocampally lesioned rats made stereotyped hind-limb pivots at each corner, while normal rats used forelimb pivots or reared and reoriented adjacent to the wall. Rearing was lower in hippocampally lesioned rats, and higher in neocortically lesioned rats, than in unoperated rats. These data indicate that investigative object behavior (sniffing) is resistant to the effects of hippocampal damage, while object-elicited marking and motoric output may be profoundly altered. The data on sniffing suggest either that (1) the noticeability of the objects used elicited investigative behaviors in hippocampally damaged rats comparable to those of novelty-induced exploration in normal rats or (2) object exploration is not used to create a spatial map, and, hence, not disturbed by hippocampal loss. Object marking may require spatial locale information to be exercised normally, or may index the mediation of an olfactory-modulated behavioral pattern through the hippocampal system.

Animals↗
PubMed1999

Werner helicase expression in human fetal and adult aortas.

Werner syndrome is a human progeroid syndrome caused by mutations at the Werner helicase locus (WRN). Progeroid features and diseases associated with aging (including arteriosclerosis) do not become apparent until after puberty. We entertained two alternative hypotheses to explain the post-pubertal onset: 1) WRN expression is induced at the time of puberty, its earlier functions being satisfied by another member of that family of helicases; and 2) it is expressed at all ages, but the phenotype of deficiency becomes apparent only after puberty. We report initial experiments consistent with the second hypothesis. Steady-state levels of WRN mRNA in aortic tissues were determined by semiquantitative reverse transcription-polymerase chain reaction. WRN mRNA was detectable as early as 49 days of gestation (the earliest available material). There was no statistically significant change in these levels between fetal and adult tissues. The presence of the WRN protein in fetal aorta was confirmed by Western analysis. This rules out the possibility that Werner syndrome phenotypes manifest after the puberty because of peripubertal induction of WRN expression.

Adult↗
PubMed1999

Increased vulnerability of hippocampal neurons to excitotoxic necrosis in presenilin-1 mutant knock-in mice.

Excitotoxicity, a form of neuronal injury in which excessive activation of glutamate receptors results in cellular calcium overload, has been implicated in the pathogenesis of Alzheimer disease (AD), although direct evidence is lacking. Mutations in the presenilin-1 (PS1) gene on chromosome 14 are causally linked to many cases of early-onset inherited AD (refs. 5,6). We generated PS1 mutant mice (PS1M146VKI) that express the PS1 M146V targeted allele at normal physiological levels. Although PS1M146VKI mice have no overt mutant phenotype, they are hypersensitive to seizure-induced synaptic degeneration and necrotic neuronal death in the hippocampus. Cultured hippocampal neurons from PS1M146VKI mice have increased vulnerability to death induced by glutamate, which is correlated with perturbed calcium homeostasis, increased oxidative stress and mitochondrial dysfunction. Agents that suppress calcium influx or release and antioxidants protect neurons against the excitotoxic action of the PS1 mutation. These findings establish a direct link between a genetic defect that causes AD and excitotoxic neuronal degeneration, and indicate new avenues for therapeutic intervention in AD patients.

Animals↗
PubMed1999

Increased vulnerability of hippocampal neurons from presenilin-1 mutant knock-in mice to amyloid beta-peptide toxicity: central roles of superoxide production and caspase activation.

Many cases of early-onset inherited Alzheimer's disease (AD) are caused by mutations in the presenilin-1 (PS1) gene. Overexpression of PS1 mutations in cultured PC12 cells increases their vulnerability to apoptosis-induced trophic factor withdrawal and oxidative insults. We now report that primary hippocampal neurons from PS1 mutant knock-in mice, which express the human PS1M146V mutation at normal levels, exhibit increased vulnerability to amyloid beta-peptide toxicity. The endangering action of mutant PS1 was associated with increased superoxide production, mitochondrial membrane depolarization, and caspase activation. The peroxynitrite-scavenging antioxidant uric acid and the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone protected hippocampal neurons expressing mutant PS1 against cell death induced by amyloid beta-peptide. Increased oxidative stress may contribute to the pathogenic action of PS1 mutations, and antioxidants may counteract the adverse property of such AD-linked mutations.

Alzheimer Disease↗
PubMed1999

The Werner syndrome protein is involved in RNA polymerase II transcription.

Werner syndrome (WS) is a human progeroid syndrome characterized by the early onset of a large number of clinical features associated with the normal aging process. The complex molecular and cellular phenotypes of WS involve characteristic features of genomic instability and accelerated replicative senescence. The gene involved (WRN) was recently cloned, and its gene product (WRNp) was biochemically characterized as a helicase. Helicases play important roles in a variety of DNA transactions, including DNA replication, transcription, repair, and recombination. We have assessed the role of the WRN gene in transcription by analyzing the efficiency of basal transcription in WS lymphoblastoid cell lines that carry homozygous WRN mutations. Transcription was measured in permeabilized cells by [3H]UTP incorporation and in vitro by using a plasmid template containing the RNA polymerase II (RNA pol II)-dependent adenovirus major late promoter. With both of these approaches, we find that the transcription efficiency in different WS cell lines is reduced to 40-60% of the transcription in cells from normal individuals. This defect can be complemented by the addition of normal cell extracts to the chromatin of WS cells. Addition of purified wild-type WRNp but not mutated WRNp to the in vitro transcription assay markedly stimulates RNA pol II-dependent transcription carried out by nuclear extracts. A nonhelicase domain (a direct repeat of 27 amino acids) also appears to have a role in transcription enhancement, as revealed by a yeast hybrid-protein reporter assay. This is further supported by the lack of stimulation of transcription when mutant WRNp lacking this domain was added to the in vitro assay. We have thus used several approaches to show a role for WRNp in RNA pol II transcription, possibly as a transcriptional activator. A deficit in either global or regional transcription in WS cells may be a primary molecular defect responsible for the WS clinical phenotype.

Amino Acid Sequence↗
PubMed1998

Werner helicase is localized to transcriptionally active nucleoli of cycling cells.

Mutations at the Werner helicase locus (WRN) are responsible for the Werner syndrome (WS), a "caricature of aging." We have localized the Werner protein (WRNp) to the nucleoli of replicating mammalian cells, where its appearance is associated with transcriptional activity. A dramatic reduction of the nucleolar signal and of [3H]uridine incorporation occurred when cultures were made quiescent or were exposed to 4-nitroquinoline-1-oxide (4NQO), to which WS cells are particularly susceptible. Total cellular levels of WRNp, however, did not change, and virtually all WRNp was in the nuclear fractions, consistent with translocation to the nucleoplasm and/or masking of the epitopes. The 4NQO-induced altered state of WRNp was prevented by Na3VO4, but not by okadaic acid, suggesting that WRNp localization/function is partially regulated by kinases/phosphatases for Tyr substrates on WRNp or interacting proteins. The repression of rDNA transcription by 4NQO was not reversed by Na3VO4. We suggest that physiological states and genotoxic agents modulate the interaction of WRNp with rDNA, consistent with a role of WRNp in rDNA transcription.

4-Nitroquinoline-1-oxide↗
PubMed1998

Structure and function of the human Werner syndrome gene promoter: evidence for transcriptional modulation.

The Werner syndrome (WS) is an autosomal recessive segmental progeroid syndrome caused by mutations in a novel member ( WRN ) of the RecQ family of helicases. Somatic WS cells are hypermutable and have elongated S phases, suggesting possible defects in DNA replication and/or repair. As an initial approach to the investigation of how this locus might be responsive to DNA damage, we determined the structure of the human WRN promoter. The WRN promoter region has two transcription initiation sites and exhibits several features characteristic of so-called constitutive promoters, including the absence of TATA and CAAT boxes. A luciferase reporter assay revealed that the upstream promoter was used 2-10-fold less frequently than the downstream promoter, the variation being a function of cell type. The activity of the WRN promoter was dramatically reduced in cells from WS patients. The reduction of activity was not seen in three other promoters tested, including one TATA-less promoter and one TATA-containing promoter. This is consistent with the presence of a positive regulatory mechanism of WRN expression.

Base Sequence↗