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PubMed1994

Re-examination of arousing and de-arousing effects of cigarette smoking.

An attempt was made to replicate arousing and de-arousing effects of cigarette smoking originally reported by Golding and Mangan. Changes in electrodermal activity, heart rate, and magnitude in the EEG alpha (7.5-11.5 Hz) and beta (13.5-20 Hz) bands were measured under conditions of both mild sensory isolation as well as stress induced by loud white-noise. Under both conditions, real smoking as well as sham smoking an unlit cigarette increased beta activity and the skin potential response. In contrast, only real smoking produced a significant increase in heart rate, decrease in alpha activity, and increase in skin potential. De-arousing effects reported by Golding and Mangan for white-noise stress were not replicated.

Adult↗
PubMed2005

The effect of heel-pad thickness and loading protocol on measured heel-pad stiffness and a standardized protocol for inter-subject comparability.

BACKGROUND: Heel-pad stiffness is an important parameter in clinical assessments of the lower limb and is usually quantified by the slope of the force-deformation curve. However, the data produced is affected by the geometry of the heel, thus making inferences about tissue behaviour difficult. METHOD: With the use of finite element analysis the aim of this study is to explore the possibility of expressing heel-pad stiffness in terms of stress-strain data. An axisymmetric, non-linear and time-dependent representation of the heel was created. The material model, incorporating non-linearity and viscoelasticity, was based on a series of experiments involving healthy, cadaveric specimens and loading at different loading rates (0, 175 and 350 mm/s). The conditions of an in vivo study were then replicated and stress-strain data of the model were compared. Good agreement was achieved (error <5%) at higher strains (>0.2). Probe diameter, loading rate and heel-pad thickness were then varied and heel-pad stiffness, expressed in terms of both force-deformation and stress-strain characteristics, reported. FINDINGS: In terms of the force-deformation characteristics, thin heels are consistently stiffer than thick heels. In terms of stress-strain characteristics, thicker heels are stiffer than thin heels using small probes whereas thinner heels are stiffer than thick heels using large probes. It was possible to predict stress-strain data of the heel-pad that are least-dependent of heel-pad thickness using large probes and slow-rising loads. INTERPRETATION: It is suggested that stress-strain curves derived from large probes under slow loads would provide the most robust and standardized measure of heel-pad stiffness.

Biomechanical Phenomena↗
PubMed2003

Relief of oxidative stress by ascorbic acid delays cellular senescence of normal human and Werner syndrome fibroblast cells.

Primary human cells have a definite life span and enter into cellular senescence before ceasing cell growth. Oxidative stress produced by aerobic metabolism has been shown to accelerate cellular senescence. Here, we demonstrated that ascorbic acid, used as an antioxygenic reagent, delayed cellular senescence in a continuous culture of normal human embryonic cells, human adult skin fibroblast cells, and Werner syndrome (WS) cells. The results using human embryonic cells showed that treatment with ascorbic acid phospholic ester magnesium salt (APM) decreased the level of oxidative stress, and extended the replicative life span. The effect of APM to extend the replicative life span was also shown in normal human adult cells and WS cells. To understand the mechanism of extension of cellular life span, we determined the telomere lengths of human embryonic cells, both with and without APM treatment, and demonstrated that APM treatment reduced the rate of telomere shortening. The present results indicate that constitutive oxidative stress plays a role in determining the replicative life span and that suppression of oxidative stress by an antioxidative agent, APM, extends the replicative life span by reducing the rate of telomere shortening.

Antioxidants↗
PubMedDate not supplied

Normal brain aging: a commentary.

Brain gerontology encounters problems that, analogically, are similar with those encountered in biogerontology in general. These problems are namely interactions between aging per se, stress response and bioenergetics. These interaction exist for instance as concerns cross-talks between stress response- and senescence-specific signaling pathways. Analogies with replicative senescence and stress-induced premature senescence are drawn.

Aging↗
PubMed2006

Mammalian TIMELESS and Tipin are evolutionarily conserved replication fork-associated factors.

The function of the mammalian TIMELESS protein (TIM) has been enigmatic. TIM is essential for early embryonic development, but little is known regarding its biochemical and cellular function. Although identified based on similarity to a Drosophila circadian clock factor, it also shares similarity with a second family of proteins that is more widely conserved throughout eukaryotes. Members of this second protein family in yeast (S.c. Tof1p, S.p. Swi1p) have been implicated in DNA synthesis, S-phase-dependent checkpoint activation and chromosome cohesion, three processes coordinated at the level of the replication fork complex. The present work demonstrates that mammalian TIM and its constitutive binding partner, Tipin (ortholog of S.c. Csm3p, S.p. Swi3p), are replisome-associated proteins. Both proteins associate with components of the endogenous replication fork complex, and are present at BrdU-positive DNA replication sites. Knock-down of TIM also compromises DNA replication efficiency. Further, the direct binding of the TIM-Tipin complex to the 34 kDa subunit of replication protein A provides a biochemical explanation for the potential coupling role of these proteins. Like TIM, Tipin is also involved in the molecular mechanism of UV-dependent checkpoint activation and cell growth arrest. Tipin additionally associates with peroxiredoxin2 and appears to be involved in checkpoint responses to H(2)O(2), a role recently described for yeast versions of TIM and Tipin. Together, this work establishes TIM and Tipin as functional orthologs of their replisome-associated yeast counterparts capable of coordinating replication with genotoxic stress responses, and distinguishes mammalian TIM from the circadian-specific paralogs from which it was originally identified.

Animals↗
PubMed1987

Influence of drug therapy on myocardial hypertrophy in left ventricular failure.

The terminal phases of volume-overload heart failure are characterised by eccentric ventricular hypertrophy. Relatively little attention has been directed to exploring the mechanisms involved in this development. The increase in diastolic wall stress leads to replication of the sarcomeres in series. The hypertrophied myocytes has a defective contractile ability that is due to an intrinsic depression of contractility; this is restored when failure is reversed. The development of appropriate hypertrophy is dependent upon adequacy of the coronary blood supply to the failing myocytes. Reduction of preload and afterload can be expected to be more effective in reducing systolic wall stress, and hence myocardial oxygen demands, in eccentric hypertrophy than in the nonfailing heart with concentric hypertrophy. However, there is no information available on the influence of anti-heart-failure drugs on the myocardial hypertrophy associated with severe volume-overload failure. The presence of such hypertrophy carries an ominous prognosis and may be associated with the high incidence of arrhythmic sudden deaths. The detection of myocardial hypertrophy in the patient with failure emphasises the urgency of adequate reduction of the elevated preload and afterload without impairment of the coronary perfusion gradient.

Cardiomegaly↗
PubMed2006

Simultaneous proteomic profiling of four different growth states of human fibroblasts, using amine-reactive isobaric tagging reagents and tandem mass spectrometry.

In general, permanent growth arrest due to exhaustive cell replication can be induced prematurely by either stress or overexpression of selected oncogenes. In an attempt to examine key proteins involved in achieving premature senescence, and how they differ from those in serially passaged, replicatively exhausted cells, we used a novel proteomic profiling approach, isobaric tagging for relative and absolute quantitation (iTRAQ), to perform simultaneous four-way comparison of replicatively senescent fibroblasts, oxidatively stressed prematurely senescent fibroblasts, and their young replicating and quiescent counterparts. Two hundred and forty proteins were identified and quantified simultaneously; data analysis reveals: (1) groups of proteins whose expressions are uniformly either up- or down-regulated in all three growth arrest states; (2) signature proteins which may serve as candidate proteomic markers to differentiate the quiescent state from permanent growth arrest by either exhaustive replication or stress induction and (3) that while oxidative stress-induced, prematurely senescent fibroblasts morphologically resemble their replicatively exhausted counterparts, they exhibit different protein expression patterns. Results from simultaneous proteomic profiling were validated by Western blotting for selected proteins: collagen type I, HSP90 and vimentin. In conclusion, this report shows that iTRAQ proteomic profiling is a powerful technique for globally mapping protein signatures for different culture growth states.

Amino Acid Sequence↗
PubMed2003

Human CLK2 links cell cycle progression, apoptosis, and telomere length regulation.

Mutations in the clk-2 gene of the nematode Caenorhabditis elegans affect organismal features such as development, behavior, reproduction, and aging as well as cellular features such as the cell cycle, apoptosis, the DNA replication checkpoint, and telomere length. clk-2 encodes a novel protein (CLK-2) with a unique homologue in each of the sequenced eukaryotic genomes. We have studied the human homologue of CLK-2 (hCLK2) to determine whether it affects the same set of cellular features as CLK-2. We find that overexpression of hCLK2 decreases cell cycle length and that inhibition of hCLK2 expression arrests the cell cycle reversibly. Overexpression of hCLK2, however, renders the cell hypersensitive to apoptosis triggered by oxidative stress or DNA replication block and gradually increases telomere length. The evolutionary conservation of the pattern of cellular functions affected by CLK-2 suggests that the function of hCLK2 in humans might also affect the same organismal features as in worms, including life span. Surprisingly, we find that hCLK2 is present in all cellular compartments and exists as a membrane-associated as well as a soluble form.

Apoptosis↗
PubMed1988

Fragile X expression in thymidine-prototrophic and auxotrophic human-mouse somatic cell hybrids under low and high thymidylate stress conditions.

Expression of the fragile X site fra(X)(q27.3) was studied in thymidine-prototrophic and auxotrophic human-mouse somatic cell hybrids. In these cells, low thymidylate stress, achieved by 5-fluoro-2'-deoxyuridine (FdU) treatment and by limiting the exogenous supply of thymidine (dT), induced fragile X expression. High thymidylate stress, produced by supplying excess amounts of dT, was also effective in inducing fragile X expression, even in a hybrid clone that retained a fragile X chromosome as the only human chromosome; addition of deoxycytidine (dC) completely abolished this effect. In contrast, 5-bromo-2'-deoxyuridine (BrdU) did not induce fragile X expression. Cell-cycle analysis of BrdU-deprived thymidine-auxotrophic hybrid cells indicated that one round of DNA replication under thymidylate stress conditions is sufficient for fragile X expression. Our results suggest that the expression is an intrinsic property of the fragile site itself, which is believed to be composed of replicon clusters with pyrimidine-rich DNA sequence(s).

Animals↗
PubMed1990

Eukaryotic DNA replication. Enzymes and proteins acting at the fork.

A complex network of interacting proteins and enzymes is required for DNA replication. Much of our present understanding is derived from studies of the bacterium Escherichia coli and its bacteriophages T4 and T7. These results served as a guideline for the search and the purification of analogous proteins in eukaryotes. model systems for replication, such as the simian virus 40 DNA, lead the way. Generally, DNA replication follows a multistep enzymatic pathway. Separation of the double-helical DNA is performed by DNA helicases. Synthesis of the two daughter strands is conducted by two different DNA polymerases: the leading strand is replicated continuously by DNA polymerase delta and the lagging strand discontinuously in small pieces by DNA polymerase alpha. The latter is complexed to DNA primase, an enzyme in charge of frequent RNA primer syntheses on the lagging strand. Both DNA polymerases require several auxiliary proteins. They appear to make the DNA polymerases processive and to coordinate their functional tasks at the replication fork. 3'----5'-exonuclease, mostly part of the DNA polymerase delta polypeptide, can perform proof-reading by excising incorrectly base-paired nucleotides. The short DNA pieces of the lagging strand, called Okazaki fragments, are processed to a long DNA chain by the combined action of RNase H and 5'----3'-exonuclease, removing the RNA primers, DNA polymerase alpha or beta, filling the gap, and DNA ligase, sealing DNA pieces by phosphodiester bond formation. Torsional stress during DNA replication is released by DNA topoisomerases. In contrast to prokaryotes, DNA replication in eukaryotes not only has to create two identical daughter strands but also must conserve higher-order structures like chromatin.

Chromatin↗
PubMed2006

Mpt5p, a stress tolerance- and lifespan-promoting PUF protein in Saccharomyces cerevisiae, acts upstream of the cell wall integrity pathway.

Pumilio family (PUF) proteins affect specific genes by binding to, and inhibiting the translation or stability of, their transcripts. The PUF domain is required and sufficient for this function. One Saccharomyces cerevisiae PUF protein, Mpt5p (also called Puf5p or Uth4p), promotes stress tolerance and replicative life span (the maximum number of doublings a mother cell can undergo before entering into senescence) by an unknown mechanism thought to partly overlap with, but to be independent of, the cell wall integrity (CWI) pathway. Here, we found that mpt5Delta mutants also display a short chronological life span (the time cells stay alive in saturated cultures in synthetic medium), a defect that is suppressed by activation of CWI signaling. We found that Mpt5p is an upstream activator of the CWI pathway: mpt5Delta mutants display the appropriate phenotypes and genetic interactions, display low basal activity of the pathway, and are defective in activation of the pathway upon thermal stress. A set of mRNAs that specifically bind to Mpt5p was recently reported. One such putative target, LRG1, encodes a GTPase-activating protein for Rho1p that directly links Mpt5p to CWI signaling: Lrg1p inhibits CWI signaling, LRG1 mRNA contains a consensus Mpt5p-binding site in its putative 3' untranslated region, loss of Lrg1p suppresses the temperature sensitivity and CWI signaling defects of mpt5Delta mutants, and LRG1 mRNA abundance is inhibited by Mpt5p. We conclude that Mpt5p is required for normal replicative and chronological life spans and that the CWI pathway is a key and direct downstream target of this PUF protein.

Adaptation, Physiological↗
PubMed1997

Gene expression of glucokinase regulatory protein in regenerating rat liver.

The activity and messenger RNA (mRNA) levels of glucokinase, and the concentration and mRNA levels of its regulatory protein, were analyzed during liver regeneration. The activity of glucokinase and the concentration of its regulatory protein decreased to 30% and 50%, respectively, after liver resection, remaining low after 1 week. No significant variations in the level of these proteins were found in sham-operated animals. The regulatory protein/glucokinase molar ratio increased during the replicative phase, to a maximum at 48 hours. The mRNA levels of glucokinase and of its regulatory protein decreased rapidly after partial hepatectomy to minimum values at 6 hours (15%) and at 12 hours (4%), respectively, returning to normal values at 24 hours and 168 hours, respectively. Sham-operated animals showed a similar decrease in mRNA levels during the prereplicative phase of liver regeneration, suggesting that the initial effects observed in the gene expression of these proteins were due to surgical stress. During the replicative phase, a specific inhibition of the regulatory protein's gene expression was observed in the regenerating liver. A decrease in the content of regulatory protein and the glucokinase activity, and an increase in the molar ratio of these two proteins correlate with the observed decrease in glycolytic flux, providing further evidence that the phosphorylation of glucose is a control point in the glycolytic/gluconeogenic flux during liver regeneration.

Animals↗
PubMed2004

Expression profiles of p53 and p66shc during oxidative stress-induced senescence in fetal bovine fibroblasts.

Somatic cells undergo a permanent cell cycle arrest, called cellular senescence, after a limited number of cell divisions in vitro. Both the tumor suppressor protein p53 and the stress-response protein p66(shc) are suggested to regulate the molecular events associated with senescence. This study was undertaken to investigate the effect of different oxygen tensions and oxidative stress on cell longevity and to establish the role of p53 and p66(shc) in cells undergoing senescence. As a model of cellular senescence, primary fetal bovine fibroblasts were cultured in either 20% O(2) or 5% O(2) atmospheres until senescence was reached. Fibroblasts cultured under 20% O(2) tension underwent senescence after 30 population doublings (PD), whereas fibroblasts cultured under 5% O(2) tension did not exhibit signs of senescence. Oxidative stress, as measured by protein carbonyl content, was significantly elevated in senescent cells compared to their younger counterparts and to fibroblasts cultured under 5% O(2) at the same PD. p53 mRNA gradually decreased in 20% O(2) cultured fibroblasts until senescence was reached, whereas p53 protein levels were significantly increased as well as p53 phosphorylation on serine 20, suggesting that p53 might be stabilized by posttranslational modifications during senescence. Senescence was also associated with high levels of p66(shc) mRNA and protein levels, while the levels remained low and stable in dividing fibroblasts under 5% O(2) atmosphere. Taken together, our results show an effect of oxidative stress on the replicative life span of fetal bovine fibroblasts as well as an involvement of p53, serine 20-p53 phosphorylation and p66(shc) in senescence.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Peroxisome proliferators: their biological and toxicological effects.

One of the most rapidly developing areas of organelle biology, which has a major involvement in biochemical pharmacology, is the research into the peroxisomal function. There is a large group of compounds that are capable of inducing liver enlargement, proliferation of peroxisomal structures, and induction of peroxisomal and extraperoxisomal fatty acid-oxidizing enzymes in rodent liver, called peroxisome proliferators. This list includes hypolipidemic drugs, analgesics, uricosuric drugs, environmental pollutants, phthalates, etc. Some peroxisome proliferators have also been shown to increase the incidence of liver tumors. This review describes the characteristics of peroxisome proliferation in rodent liver and gives examples of different classes of chemicals that produce this effect. Mechanisms of initiation of peroxisome proliferation in rodent hepatocytes, including peroxisome proliferator-activated receptors, are also described. Rodent peroxisome proliferators are not considered to be genotoxic agents. Proposed mechanisms of liver tumor formation include induction of sustained oxidative stress, enhanced cell replication, promotion of spontaneous preneoplastic lesions, and inhibition of apoptosis. In addition, the absence of effects of peroxisome proliferators on peroxisome proliferator-associated parameters supports the hypothesis that human liver cells are refractory to peroxisome proliferator-induced hepatic carcinogenesis.

Animals↗
PubMed2006

Herpes simplex virus infection, with particular reference to the progression and complications of primary herpetic gingivostomatitis.

Primary herpetic gingivostomatitis (PHGS) represents the clinically apparent pattern of primary herpes simplex virus (HSV) infection, since the vast majority of other primary infections are symptomless. PHGS is caused predominantly by HSV-1 and affects mainly children. Prodromal symptoms, such as fever, anorexia, irritability, malaise and headache, may occur in advance of disease. The disease presents as numerous pin-head vesicles, which rupture rapidly to form painful irregular ulcerations covered by yellow-grey membranes. Sub-mandibular lymphadenitis, halitosis and refusal to drink are usual concomitant findings. Following resolution of the lesions, the virus travels through the nerve endings to the nerve cells serving the affected area, whereupon it enters a latent state. When the host becomes stressed, the virus replicates and migrates in skin, mucosae and, in rare instances, the central nervous system. A range of morbidities, or even mortality, may then occur, i.e., recurrent HSV infections, which are directly or indirectly associated with PHGS. These pathological entities range from the innocuous herpes labialis to life-threatening meningoencephalitis.

Disease Progression↗
PubMed1997

Amino Acid Effects on Post-Stress Ulcers: Relationship to Brain Serotonin, 5-HIAA and Norepinephrine.

To replicate and extend results of earlier studies on amino acid effects on post-stress ulcers, rats were subjected to i.p. injections of (a) saline, (b) tryptophan, (c) tyrosine + valine or(d) tryptophan + tyrosine + valine, either 30 minutes before or immediately after one hour of water restraint stress. Gastric lesions, brain norepinephrine, serotonin and 5-hydroxyindoleacetic acid (5-HIAA) were examined after one hour of poststress rest. We hypothesised that post-stress lesions could be aggravated by central noradrenergic hypoactivity and serotonergic hyperactivity during the post-stress period. Other studies have indicated that tyrosine + valine reduces central serotonergic activity, while additional tryptophan blocks this effect. We therefore expected post-stress lesions to be reduced in tyrosine + valine but not in tryptophan + tyrosine + valine treated animals. Although these expectations were met tentatively in animals injected prior to stress, thus replicating tyrosine + valine effects we had observed earlier, opposite results were found in animals treated post-stress. The brain analyses indicate that the data cannot be explained by a norepinephrine/serotonin imbalance hypothesis. The time dependency of the effects underlines the need for caution in clinical applications of these amino acid treatments.

Journal Article↗
PubMed2001

p53-dependent induction of p21(Cip1/WAF1/Sdi1) protects against oxygen-induced toxicity.

The beneficial effects of supplemental oxygen delivered to patients suffering from acute respiratory distress is offset by its reduction to genotoxic reactive oxygen species (ROS) that inhibit proliferation and kill pulmonary cells. Cells respond to oxygen-induced damage by expressing the tumor suppressor p53 and the cyclin-dependent kinase inhibitor p21(Cip1/WAF1/Sdi1) (p21), which limits proliferation by blocking entry into S phase. Since preventing DNA synthesis during genotoxic stress may enhance survival, the current study examines whether hyperoxia induces p21 through a p53-dependent pathway and whether p21 protects cells from the toxic effects of oxygen. HCT116 colon carcinoma cells and clonal lines lacking p53 or p21were used in this study because they allow direct cytotoxic comparisons between isogenic cells, without complications arising from unknown genetic differences between nonhomologous cell lines. Hyperoxia (95% O2, 5% CO2) increased p53 abundance, phosphorylation of p53 on serine 15, and p21 mRNA and protein in parental HCT116 cells that ceased proliferation. In contrast, p21 was not detected in either p53- or p21-deficient HCT116 cells, which exited the G1 compartment and were arrested in S and G2/M phases during hyperoxia. Trypan blue-dye exclusion revealed that induction of p21 markedly enhanced survival during exposure and colony survival assays showed that p21 enhanced the ability to resume proliferation during recovery in room air. The observation that p53-dependent induction of p21 prevents exit from G1 and promotes survival during hyperoxia is consistent with the importance of limiting DNA replication during genotoxic stress caused by oxygen exposure.

Blotting, Western↗
PubMed2004

Replication-incompetent virions of Japanese encephalitis virus trigger neuronal cell death by oxidative stress in a culture system.

It has been shown that replication of the Japanese encephalitis virus (JEV) can trigger infected cells to undergo apoptosis. In the present study, it is further demonstrated that replication-incompetent virions of JEV, obtained by short-wavelength ultraviolet (UV) irradiation, could also induce host-cell death. It was found that UV-inactivated JEV (UV-JEV) caused cell death in neuronal cells such as mouse neuroblastoma N18 and human neuronal NT-2 cells, but not in non-neuronal baby hamster kidney BHK-21 fibroblast or human cervical HeLa cells. Only actively growing, but not growth-arrested, cells were susceptible to the cytotoxic effects of UV-JEV. Killing of UV-JEV-infected N18 cells could be antagonized by co-infection with live, infectious JEV, suggesting that virions of UV-JEV might engage an as-yet-unidentified receptor-mediated death-signalling pathway. Characteristically, mitochondrial alterations were evident in UV-JEV-infected N18 cells, as revealed by electron microscopy and a loss of membrane potential. N18 cells infected by UV-JEV induced generation of reactive oxygen species (ROS) as well as the activation of nuclear factor kappa B (NF-kappaB), and the addition of anti-oxidants or specific NF-kappaB inhibitors to the media greatly reduced the cytotoxicity of UV-JEV. Together, the results presented here suggest that replication-incompetent UV-JEV damages actively growing neuronal cells through a ROS-mediated pathway.

Animals↗