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PubMed1996

Dopamine D2 receptor (DRD2) gene and susceptibility to posttraumatic stress disorder: a study and replication.

Subjects on an addiction treatment unit who had been exposed to severe combat conditions in Vietnam were screened for posttraumatic stress disorder (PTSD). Of 24 with PTSD, 58.3% carried the D2A1 allele. Of the remaining eight who did not meet PTSD criteria, 12.5% carried the D2A1 allele (p = 0.04). In a replication study of 13 with PTSD, 61.5% carried the D2A1 allele. Of the remaining 11 who did not meet criteria for PTSD, 0% carried the D2A1 allele (p = 0.002). For the combined group 59.5% of those with PTSD carried the D2A1 allele versus 5.3% of those who did not have PTSD (p = 0.0001). These results suggest that a DRD2 variant in linkage disequilibrium with the D2A1 allele confers an increased risk to PTSD, and the absence of the variant confers a relative resistance to PTSD.

Adult↗
PubMed2006

Nuclear localization of the Saccharomyces cerevisiae ribonucleotide reductase small subunit requires a karyopherin and a WD40 repeat protein.

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides and is an essential enzyme for DNA replication and repair. Cells have evolved intricate mechanisms to regulate RNR activity to ensure high fidelity of DNA replication during normal cell-cycle progression and of DNA repair upon genotoxic stress. The RNR holoenzyme is composed of a large subunit R1 (alpha, oligomeric state unknown) and a small subunit R2 (beta(2)). R1 binds substrates and allosteric effectors; R2 contains a diferric-tyrosyl radical [(Fe)(2)-Y.] cofactor that is required for catalysis. In Saccharomyces cerevisiae, R1 is predominantly localized in the cytoplasm, whereas R2, which is a heterodimer (betabeta'), is predominantly in the nucleus. When cells encounter DNA damage or stress during replication, betabeta' is redistributed from the nucleus to the cytoplasm in a checkpoint-dependent manner, resulting in the colocalization of R1 and R2. We have identified two proteins that have an important role in betabeta' nuclear localization: the importin beta homolog Kap122 and the WD40 repeat protein Wtm1. Deletion of either WTM1 or KAP122 leads to loss of betabeta' nuclear localization. Wtm1 and its paralog Wtm2 are both nuclear proteins that are in the same protein complex with betabeta'. Wtm1 also interacts with Kap122 in vivo and requires Kap122 for its nuclear localization. Our results suggest that Wtm1 acts either as an adaptor to facilitate nuclear import of betabeta' by Kap122 or as an anchor to retain betabeta' in the nucleus.

Catalysis↗
PubMed2006

Glucose-induced replicative senescence in mesenchymal stem cells.

Mesenchymal stem cells (MSCs) show great promise for use in a variety of cell-based therapies. Because isolated primary mesenchymal stem cells are low in numbers, in vitro expansion is necessary. However, the expansion potential is limited and in vitro aging leads to loss of multipotency and replicative senescence. Stress induced by culture conditions is likely to be a major cause of replicative senescence and reduced multipotency of MSC and optimization of culture conditions might be able to reduce this. Caloric restriction (CR) is the only established method to delay aging and extend lifespan. In vitro caloric restriction experiments are rare, but have demonstrated beneficial effects. Therefore, we investigated the effect of culture medium glucose concentration on the proliferative and differentiation potential of mesenchymal stem cells. Reduction in glucose concentrations led to decreased apoptosis and an increased rate of MSC proliferation and increased the number and size of fibroblastic colonies in the colony-forming unit assay.

Animals↗
PubMedDate not supplied

Replication and validation of anticipated turnover model for urban registered nurses.

Replication of the Anticipated Turnover Model Among Nurses for urban registered nurses provided substantial support for the stability and generalizability of the theoretical model. In both the original and replication studies, causal modeling was used to test the theoretical model predicting job satisfaction, anticipated turnover, and actual turnover. The replication study included 385 full-time nurses in two public and two private urban hospitals. The replication validated, with younger, more educated staff, the major findings from the original study. Group cohesion and job satisfaction effectively predicted anticipated turnover in the replication and the original study. Anticipated turnover was a good predictor of actual turnover, with discriminant analysis yielding 73.2% successful predictions in the replication study and 76.2% in the original study. Job satisfaction effectively buffered job stress. The replication also substantiated the position that job satisfaction strategies need to be targeted specifically to the types of clinical services.

Adult↗
PubMed2005

The human stress-activated protein kin17 belongs to the multiprotein DNA replication complex and associates in vivo with mammalian replication origins.

The human stress-activated protein kin17 accumulates in the nuclei of proliferating cells with predominant colocalization with sites of active DNA replication. The distribution of kin17 protein is in equilibrium between chromatin-DNA and the nuclear matrix. An increased association with nonchromatin nuclear structure is observed in S-phase cells. We demonstrated here that kin17 protein strongly associates in vivo with DNA fragments containing replication origins in both human HeLa and monkey CV-1 cells. This association was 10-fold higher than that observed with nonorigin control DNA fragments in exponentially growing cells. In addition, the association of kin17 protein to DNA fragments containing replication origins was also analyzed as a function of the cell cycle. High binding of kin17 protein was found at the G(1)/S border and throughout the S phase and was negligible in both G(0) and M phases. Specific monoclonal antibodies against kin17 protein induced a threefold inhibition of in vitro DNA replication of a plasmid containing a minimal replication origin that could be partially restored by the addition of recombinant kin17 protein. Immunoelectron microscopy confirmed the colocalization of kin17 protein with replication proteins like RPA, PCNA, and DNA polymerase alpha. A two-step chromatographic fractionation of nuclear extracts from HeLa cells revealed that kin17 protein localized in vivo in distinct protein complexes of high molecular weight. We found that kin17 protein purified within an approximately 600-kDa protein complex able to support in vitro DNA replication by means of two different biochemical methods designed to isolate replication complexes. In addition, the reduced in vitro DNA replication activity of the multiprotein replication complex after immunodepletion for kin17 protein highlighted for a direct role in DNA replication at the origins.

Animals↗
PubMed1997

A replicated prospective investigation of life stress, coping, and depressive symptoms in multiple sclerosis.

Life stress and coping responses jointly contribute to psychological adjustment in many chronic illness populations, but their significance in multiple sclerosis (MS) has not been extensively investigated. Physical disability, cognitive status, negative life stress, coping strategies, and depressive symptoms were prospectively assessed in 27 adults with definite or probable MS. Of the original subjects, 22 provided two additional assessments at 6-month intervals. After accounting for cognitive status and physical disability, life stress was positively correlated with current as well as future depressive symptoms; the prospective relationship was replicated within the second pair of prospective data waves. Escape avoidance was the only coping strategy that added to the prediction of future mood symptoms, but this was not replicated. Results suggest that MS-related depressive symptoms are a function of prior disease-related impairment, life stress, and possibly escape avoidance coping.

Activities of Daily Living↗
PubMed2002

Stress-induced premature senescence and replicative senescence are different phenotypes, proteomic evidence.

In this paper, we illustrate how a proteomic analysis can be useful to approach complex biological problems, in this case the concept of stress-induced premature senescence (SIPS). According to the stochastic theories of ageing, damage that accumulate with time in the cellular components are responsible for cellular ageing. As a corollary, some sort of premature senescence should appear if the damage level is artificially increased due to the presence of stressing agents at subcytotoxic level. It has been shown, in several different models, that at a long-term after subcytotoxic stresses of many different natures, human diploid fibroblasts (HDFs) display biomarkers of replicative senescence (RS), which led to the concept of SIPS as compared to telomere-dependent RS. We compared RS and SIPS of HDFs by proteome analysis. SIPS was induced by two very different stressors: tert-butyhydroperoxide or ethanol. First, only a part of the protein expression changes observed in RS were also observed in SIPS. Second, HDFs in SIPS show changes specific either to the long-term effects of t-BHP or ethanol or independent of the nature of the stress. These changes have been termed "molecular scars" of subcytotoxic stresses. This work is also an excellent opportunity to discuss on important methodological issue in proteomics: the absolute requirement to start from reliable and reproducible models, which was the case in this study. We also focus on the data handling and statistical analysis allowing to use two-dimensional gel electrophoresis patterns in a semi-quantitative analysis.

Aging, Premature↗
PubMed2023

DNA binding and RAD51 engagement by the BRCA2 C-terminus orchestrate DNA repair and replication fork preservation.

The tumor suppressor BRCA2 participates in DNA double-strand break repair by RAD51-dependent homologous recombination and protects stressed DNA replication forks from nucleolytic attack. We demonstrate that the C-terminal Recombinase Binding (CTRB) region of BRCA2, encoded by gene exon 27, harbors a DNA binding activity. CTRB alone stimulates the DNA strand exchange activity of RAD51 and permits the utilization of RPA-coated ssDNA by RAD51 for strand exchange. Moreover, CTRB functionally synergizes with the Oligonucleotide Binding fold containing DNA binding domain and BRC4 repeat of BRCA2 in RPA-RAD51 exchange on ssDNA. Importantly, we show that the DNA binding and RAD51 interaction attributes of the CTRB are crucial for homologous recombination and protection of replication forks against MRE11-mediated attrition. Our findings shed light on the role of the CTRB region in genome repair, reveal remarkable functional plasticity of BRCA2, and help explain why deletion of Brca2 exon 27 impacts upon embryonic lethality.

DNA Replication↗
PubMed2003

Inhibition of bacterial DNA replication by zinc mobilization during nitrosative stress.

Phagocytic cells inhibit the growth of intracellular pathogens by producing nitric oxide (NO). NO causes cell filamentation, induction of the SOS response, and DNA replication arrest in the Gram-negative bacterium Salmonella enterica. NO also induces double-stranded chromosomal breaks in replication-arrested Salmonella lacking a functional RecBCD exonuclease. This DNA damage depends on actions of additional DNA repair proteins, the RecG helicase, and RuvC endonuclease. Introduction of a recG mutation restores both resistance to NO and the ability of an attenuated recBC mutant Salmonella strain to cause lethal infection in mice, demonstrating that bacterial DNA replication is inhibited during host-pathogen interactions. Inhibition of DNA replication during nitrosative stress is invariably accompanied by zinc mobilization, implicating DNA-binding zinc metalloproteins as critical targets of NO-related antimicrobial activity.

Animals↗
PubMed2006

New functions for Y family polymerases.

The Y family of DNA polymerases plays crucial roles in carrying out translesion synthesis past damaged bases in DNA. Several recent papers suggest that they might have other roles as well in gene conversion, in nucleotide excision repair (NER), and in DNA replication under stressed conditions.

Animals↗
PubMed1990

Students' self-ratings of stress in medical school: a replication across 20 months.

We replicated the essential results of a prior study on the capacity of the BAROMAS scales to reflect stress in medical school as perceived by students. As before, subjective stress was high at the start of medical school, and when facing the exams prerequisite to entry into clinical clerkships. On most measures, stress was lowest when the second year began (i.e. after having passed the first). Once again, most test-retest reliabilities (significant rs ranged from 0.24 to 0.66 for confidence ratings at 12- and 20-months after entry) were moderate.

Adaptation, Psychological↗
PubMed2002

Replicative aging, telomeres, and oxidative stress.

Aging is a very complex phenomenon, both in vivo and in vitro. Free radicals and oxidative stress have been suggested for a long time to be involved in or even to be causal for the aging process. Telomeres are special structures at the end of chromosomes. They shorten during each round of replication and this has been characterized as a mitotic counting mechanism. Our experiments show that the rate of telomere shortening in vitro is modulated by oxidative stress as well as by differences in antioxidative defence capacity between cell strains. In vivo we found a strong correlation between short telomeres in blood lymphocytes and the incidence of vascular dementia. These data suggest that parameters that characterise replicative senescence in vitro offer potential for understanding of, and intervention into, the aging process in vivo.

Aging↗
PubMed2003

Re-adaptation of the colonic mucosa following protracted stress in rats.

BACKGROUND: We previously found that DNA synthesis of the colonic mucosa rises under protracted physical stress. At 8 stress weeks DNA values regained those of untreated animals, and was regarded as DNA adaptation to stress. AIMS: To assess whether the 8 weeks' DNA adaptation to stress could be maintained following stress-free intervals, ending with a stress stimulus. METHODS: Sixty rats were transported to the stress laboratory. Thirty were water plunged and 30 sham handled once a day, 5 days a week, for 8 weeks. After 8 stress weeks, groups of five animals were allowed to rest for 1, 2, 3 and 4 weeks, ending with single water plunging or sham handling. Five additional rats were untreated. All 65 animals received an i.p. injection of 3H-thymidine before sacrifice. The ratio radioactive DNA/total DNA reflected the DNA synthesis of the colonic mucosa. RESULTS: Following DNA adaptation at 8 stress weeks, DNA replication in the right colon of the stressed rats significantly increased at 9, 10 and 11 weeks (P < 0.05), but at 12 weeks it had decreased to 8-week values. CONCLUSIONS: DNA replication in the right colon of the stressed rats did not prevail: it increased considerably in the following weeks, but at 12 weeks (only 4 weeks later), re-adaptation to DNA synthesis had occurred. The review of the literature indicates that this is the first report in which, following adaptation to protracted stress, a re-adaptation phase to DNA replication is demonstrated in the colonic mucosa of experimental animals.

Adaptation, Physiological↗
PubMed2000

Transportation of goats: effects on physiological stress responses and live weight loss.

The management of food animals prior to slaughter influences both profitability and animal well-being. This experiment was conducted as a split-unit design to determine live weight shrink and stress responses in goats due to differences in stocking density during transportation and holding. A total of 150 Spanish does were transported on two different days (replicate) and held overnight (18 h) without feed in low- (LD) or high-density (HD) groups. On each day, 75 does were transported 2.5 h with floor spaces of .18 m2 and .37 m2/animal in LD (25 does) and HD (50 does) groups, respectively. The average temperatures in the trailer during transportation were 34.6 and 35 degrees C, respectively, on d 1 and 2. All animals were blood-sampled before loading (PRELOAD) and four does from each treatment were sampled immediately after loading (POSTLOAD). Animals were blood-sampled in holding pens either at 0, 1, 2, 3, 4, or 18 h after transportation (time) to assess the time course (n = 8 does per time per replicate) of stress responses. Individual animals were weighed just before loading onto a trailer and after overnight holding to assess shrinkage. Treatment or treatment x time did not have a significant effect on any of the dependent variables studied. There were significant effects of time (P < .01) on plasma cortisol, glucose, and urea nitrogen (PUN) concentrations. Time also had significant effects (P < .01) on plasma creatine kinase (CK) activity, differential leukocyte counts (neutrophils, lymphocytes, monocytes, and eosinophils), and ratio of neutrophils to lymphocytes (N:L). However, plasma leptin concentrations were not influenced by time. Cortisol concentrations increased at POSTLOAD sampling, peaked at 0 h, and decreased thereafter before spiking again at 18 h of holding. The PUN was higher at 18 h than at other time periods studied. Plasma glucose concentrations increased and remained at higher levels at 0, 1, and 2 h and began decreasing at 3 h, reaching PRELOAD levels at 18 h. Plasma CK kinase activity peaked at approximately 2 h after transportation. The N:L ratio was higher at all time periods after transportation than prior to starting the journey, indicating a prolonged effect of transportation stress on the immune system. The mean (+/- SE) shrinkage losses were 10.2 +/- .68 and 9.8 +/- .68 in HD and LD treatment groups, respectively. The results indicate that the stress responses of goats due to transportation begin decreasing within 3 h after transportation. However, prolonged holding periods without feed may increase stress responses and bring about metabolic changes.

Animals↗
PubMed2006

The budding yeast protein Chl1p is required to preserve genome integrity upon DNA damage in S-phase.

The budding yeast protein, Chl1p, is required for sister-chromatid cohesion, transcriptional silencing, rDNA recombination and aging. In this work, we show that Chl1p is also required for viability when DNA replication is stressed, either due to mutations or if cells are treated with genotoxic agents like methylmethane sulfonate (MMS) and ultraviolet (UV) rays. The chl1 mutation caused synthetic growth defects with mutations in DNA replication genes. At semi-permissive temperatures, the double mutants grew poorly, were less viable and showed nuclear fragmentation. They were, however, not limited in their bulk DNA synthesis. When chl1 cells were treated with relatively low levels of MMS in S-phase, they lost viability. The S-phase DNA damage checkpoint pathway, however, remained active in these cells. Agarose gel electrophoresis of genomic DNA isolated from wild-type and chl1 cells, after recovery from MMS treatment, suggested that the wild-type was more proficient in the repair of DNA damage than the mutant. Our work suggests that Chl1p is required for genome integrity when cells suffer endogenously or exogenously induced DNA damage.

Alkylating Agents↗
PubMed1981

The dynamic response of vascular endothelial cells to fluid shear stress.

We have developed an in-vitro system for studying the dynamic response of vascular endothelial cells to controlled levels of fluid shear stress. Cultured monolayers of bovine aortic endothelial cells are placed in a cone-plate apparatus that produces a uniform fluid shear stress on replicate samples. Subconfluent endothelial cultures continuously exposed to 1-5 dynes/cm2 shear proliferate at a rate comparable to that of static cultures and reach the same saturation density (congruent to 1.0-1.5 X 10(5) cells/cm2). When exposed to a laminar shear stress of 5-10 dynes/cm2, confluent monolayers undergo a time-dependent change in cell shape from polygonal to ellipsoidal and become uniformly oriented with flow. Regeneration of linear "wounds" in confluent monolayer appears to be influenced by the direction of the applied force. Preliminary studies indicate that certain endothelial cell functions, including fluid endocytosis, cytoskeletal assembly and nonthrombogenic surface properties, also are sensitive to shear stress. These observations suggest that fluid mechanical forces can directly influence endothelial cell structure and function. Modulation of endothelial behavior by fluid shear stresses may be relevant to normal vessel wall physiology, as well as the pathogenesis of vascular diseases, such as atherosclerosis.

Animals↗
PubMed2006

Altered hippocampal gene expression 2 days after general anesthesia in rats.

We profiled changes in gene expression in the hippocampus 2 days after a 4 h general anesthetic with isoflurane and nitrous oxide. Eighteen month old Fisher 344 rats were anesthetized for 4 h with 1.2% isoflurane and 70% nitrous oxide (N=9) whereas control rats breathed 30% oxygen for 4 h (N=9). Rats were sacrificed 48 h later and RNA extracted from the hippocampus for gene expression profiling. Three gene arrays were used per group, with samples prepared by pooling RNA from three rats. Differentially expressed genes were analyzed based on a weighted error statistical model. Microarray results for 6 differentially expressed genes were verified with reverse transcriptase polymerase chain reaction. Compared to unanesthetized controls, 297 genes were differentially expressed 2 days following anesthesia (P<0.05). Of these, 113 are named genes; 64% were up-regulated and 36% were down-regulated. The majority of differentially expressed genes are implicated in cell stress and replication, signal transduction, transcription, protein biosynthesis, cell structure, and metabolism. The correlation between fold changes on array and reverse transcriptase polymerase chain reaction was good (R2=0.85) for the 6 genes examined with both methods. These results demonstrate that in rats general anesthesia is associated with persistent changes in hippocampal gene expression, suggesting that recovery of the brain from anesthesia is considerably slower than generally recognized.

Anesthetics, Inhalation↗
PubMedDate not supplied

The design and fabrication of three-chamber microscale cell culture analog devices with integrated dissolved oxygen sensors.

Whole animal testing is an essential part in evaluating the toxicological and pharmacological profiles of chemicals and pharmaceuticals, but these experiments are expensive and cumbersome. A cell culture analog (CCA) system, when used in conjunction with a physiologically based pharmacokinetic (PBPK) model, provides an in vitro supplement to animal studies and the possibility of a human surrogate for predicting human response in clinical trials. A PBPK model mathematically simulates animal metabolism by modeling the absorption, distribution, metabolism, and elimination kinetics of a chemical in interconnected tissue compartments. A CCA uses mammalian cells cultured in interconnected chambers to physically represent the corresponding PBPK. These compartments are connected by recirculating tissue culture medium that acts as a blood surrogate. The purpose of this article is to describe the design and basic operation of the microscale manifestation of such a system. Microscale CCAs offer the potential for inexpensive, relatively high throughput evaluation of chemicals while minimizing demand for reagents and cells. Using microfabrication technology, a three-chamber ("lung"-"liver"-"other") microscale cell culture analog (microCCA) device was fabricated on a 1 in. (2.54 cm) square silicon chip. With a design flow rate of 1.76 microL/min, this microCCA device achieves approximate physiological liquid-to-cell ratio and hydrodynamic shear stress while replicating the liquid residence time parameters in the PBPK model. A dissolved oxygen sensor based on collision quenching of a fluorescent ruthenium complex by oxygen molecules was integrated into the system, demonstrating the potential to integrate real-time sensors into such devices.

Animals↗