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PubMed1991

Child behaviours as stressors: replicating and extending the use of the CARS as a measure of stress: a research note.

Autistic children represent a source of stress for their parents, but the most appropriate way to measure this stress is not clear. Two studies in the literature compared parents' and professionals' ratings of symptom severity, and had parents rate the stressfulness of each symptom. The present study sought to replicate past research, and to extend it by validating this method of measuring parental stress compared to more traditional measures. Results indicated that mothers, fathers, and professionals agreed on the severity of child behaviours. Mothers found these behaviours significantly more stressful than fathers. The stress related to specific child behaviours was significantly correlated with more general measures of parental stress.

Adaptation, Psychological↗

PubMed2005

Role of Nbs1 in the activation of the Atm kinase revealed in humanized mouse models.

Nijmegen breakage syndrome (NBS) is a chromosomal fragility disorder that shares clinical and cellular features with ataxia telangiectasia. Here we demonstrate that Nbs1-null B cells are defective in the activation of ataxia-telangiectasia-mutated (Atm) in response to ionizing radiation, whereas ataxia-telangiectasia- and Rad3-related (Atr)-dependent signalling and Atm activation in response to ultraviolet light, inhibitors of DNA replication, or hypotonic stress are intact. Expression of the main human NBS allele rescues the lethality of Nbs1-/- mice, but leads to immunodeficiency, cancer predisposition, a defect in meiotic progression in females and cell-cycle checkpoint defects that are associated with a partial reduction in Atm activity. The Mre11 interaction domain of Nbs1 is essential for viability, whereas the Forkhead-associated (FHA) domain is required for T-cell and oocyte development and efficient DNA damage signalling. Reconstitution of Nbs1 knockout mice with various mutant isoforms demonstrates the biological impact of impaired Nbs1 function at the cellular and organismal level.

ATP-Binding Cassette Transporters↗

PubMedDate not supplied

DNA topoisomerase I: customs officer at the border between DNA and RNA worlds?

DNA topoisomerase I is required for the normal development of multicellular organisms, probably because it plays a role in controlling gene activity, in addition to its function in relieving tortional stress during DNA replication and transcription. The discovery of DNA topoisomerase I as a specific kinase that phosphorylates serine-arginine rich (SR) splicing factors may provide new insights into their precise function in regulating gene expression. It is clear that the splicing factors phosphorylated by DNA topoisomerase I can modulate gene expression by changing the splicing pattern of structural genes. Studies of the splicing mechanism suggest that the phosphorylation of serine residues of SR proteins contribute to their activity. As this phosphorylation can be accomplished by several kinases, it remains to be determined whether phosphorylation by DNA topoisomerase I protein kinase is the limiting step in regulating this process. The availability of specific inhibitors of DNA topoisomerase I, structurally related to the alkaloid camptothecin, have made it possible to address this question experimentally. These inhibitors, which hold great promise as antineoplastic drugs, lead to specific inhibition of SR protein phosphorylation in cultured cells. This observation will hopefully lead to improved understanding of the mechanism by which these drugs act at cellular level.

Animals↗

PubMed1995

Peroxisome proliferation: current mechanisms relating to nongenotoxic carcinogenesis.

A wide variety of chemicals have been shown to produce liver enlargement, peroxisome proliferation and induction of peroxisomal and microsomal fatty acid-oxidising enzyme activities in rats and mice. Some peroxisome proliferators have also been shown to increase the incidence of liver tumours in these species. Rodent peroxisome proliferators are not considered to be genotoxic agents. Proposed mechanisms of liver tumour formation include induction of sustained oxidative stress, enhanced cell replication, promotion of spontaneous preneoplastic lesions and inhibition of apoptosis. Marked species differences in the effects of peroxisome proliferators have been observed in both in vitro and in vivo studies. Key issues concerning the risk assessment to humans of exposure to rodent peroxisome proliferators are discussed.

Animals↗

PubMed2006

Polo-like kinase-1 controls proteasome-dependent degradation of Claspin during checkpoint recovery.

DNA-damage checkpoints maintain genomic integrity by mediating a cell-cycle delay in response to genotoxic stress or stalled replication forks. In response to damage, the checkpoint kinase ATR phosphorylates and activates its effector kinase Chk1 in a process that critically depends on Claspin . However, it is not known how exactly this kinase cascade is silenced. Here we demonstrate that the abundance of Claspin is regulated through proteasomal degradation. In response to DNA damage, Claspin is transiently stabilized, and its expression depends on Chk1 kinase activity. In addition, we show that Claspin is degraded upon mitotic entry, a process that depends on the beta-TrCP-SCF ubiquitin ligase and Polo-like kinase-1 (Plk1). We demonstrate that Claspin interacts with both beta-TrCP and Plk1 and that inactivation of these components or the beta-TrCP recognition motif in Claspin prevents its mitotic degradation. Interestingly, expression of a nondegradable Claspin mutant inhibits recovery from a DNA-damage-induced checkpoint arrest. Thus, we conclude that Claspin levels are tightly regulated, both during unperturbed cell cycles and after DNA damage. Moreover, our data demonstrate that the degradation of Claspin at the onset of mitosis is an essential step for the recovery of a cell from a DNA-damage-induced cell-cycle arrest.

Adaptor Proteins, Signal Transducing↗

PubMed2001

A novel copper-regulated promoter system for expression of heterologous proteins in Schizosaccharomyces pombe.

The increasing use of the fission yeast Schizosaccharomyces pombe as a model organism for elucidating the mechanisms of critical biological processes such as cell-cycle control, DNA replication, and stress-mediated signal transduction has fostered the development and utilization of expression systems for gene function analysis. Using the promoter of the ctr4(+) copper transporter gene from S. pombe, we created a series of vectors, named pctr4(+)-X, which regulate the expression of heterologous genes as a function of copper availability. In this system, the addition of copper ions at levels that are non-toxic to yeast cells represses gene expression, while copper deprivation strongly induces gene expression. Conveniently, changes of growth medium or carbon sources are not required to shut down or induce gene expression. The Cu-starvation-mediated inducible expression system is rapid, producing heterologous proteins within 3 h, with sustained expression of proteins that persists for several hours. The pctr4(+)-X expression vectors harbor unique restriction sites constructed in-frame to DNA sequences encoding for epitope tags, which facilitate the detection or purification of the heterologous proteins using commercially available antibodies and affinity columns. Furthermore, the pctr4(+)-X copper-regulatable protein expression vectors have been constructed with three different selectable markers, offering more versatility for studying gene function in fission yeast.

Cation Transport Proteins↗

PubMed1998

Fission yeast cut mutations revisited: control of anaphase.

Studies of anaphase are approaching a golden age. Several different disciplines have contributed immensely to advances in our understanding of cell-cycle control and chromosome and spindle dynamics during mitosis. This article describes control of anaphase based on results obtained from Schizosaccharomyces pombe cut (cell untimely torn) mutants. These temperature-sensitive mutants were isolated by selection for uncoordinated mitosis with aberrant sister-chromatid separation and post-anaphase events. Characterization of some of the cut gene products has led to identification of novel molecular events related to chromosome condensation, sister-chromatid separation, anaphase-promoting proteolysis, fatty-acid metabolism, and cell-cycle arrest induced by stress or a replication block.

Anaphase↗

PubMed1999

Apoptosis, Bcl-2, and proliferating cell nuclear antigen in the failing human heart: observations made after implantation of left ventricular assist device.

BACKGROUND: Heart failure is characterized by progressive left ventricular remodeling, a complex process that results from cell growth and cell death. The quantitative contribution of apoptotic cells toward left ventricular remodeling has varied widely in tissue removed from cardiomyopathic hearts. Apoptosis has been responsive to angiotensin-converting enzyme inhibition in experimental heart failure, but the dynamics and responsiveness to chronic left ventricular unloading have not been studied. METHODS AND RESULTS: We studied 8 patients with severe heart failure before and after chronic left ventricular unloading with a left ventricular assist device (LVAD). Tissue from the left ventricular apex removed at the time of LVAD implantation was examined for apoptosis using the technique of terminal deoxynucleotidyl transferase deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) in 10 patients. These same hearts explanted at the time of cardiac transplantation were then examined for apoptosis after patients had been on the LVAD for 99 +/- 20 (SEM) days. An additional 10 patients with equally severe heart failure who underwent heart transplantation without the use of an LVAD served as controls. Eight hearts obtained at autopsy approximately 6 hours after death from patients who died of non-cardiovascular disease causes served as non-heart failure controls. Additionally, 6 hearts were examined by immunohistochemistry for the antiapoptotic protein, Bcl-2, and for the repair and/or proliferation marker, proliferating cell nuclear antigen (PCNA), before and after LVAD. Apoptosis was not detected in the tissue sections from the hearts of 8 patients at the time of LVAD implantation. Only 1 of these patients had limited apoptosis (< 1 apoptotic cell/1,000 myocytes) after LVAD insertion. Three of 10 patients with severe heart failure who did not receive an LVAD but underwent transplantation showed limited apoptosis (< 1 apoptotic cell/1,000 myocytes). Likewise, none of the control hearts from patients who died of noncardiovascular disease manifested apoptosis. Six of 6 patients overexpressed Bcl-2 at the time of LVAD insertion. In all these patients, Bcl-2 returned to negligible levels after chronic unloading of the heart. Likewise, PCNA was abundantly expressed in 5 of 6 failing hearts at the time of LVAD implantation and was reduced in 4 of 5 hearts after chronic unloading by LVAD. CONCLUSION: Apoptosis is a rare or inconsistent finding in the failing human heart. Overexpression of such indicators of cellular stress and DNA replication and/or repair as Bcl-2 and PNCA in heart failure may be altered by optimizing left ventricular loading conditions by such mechanical devices as the LVAD.

Adult↗

PubMed1997

Achieving transcriptional specificity with NF-kappa B.

Nuclear Factor-Kappa B (NF-kappa B) was first identified by Sen and Baltimore (1986, Cell 46, 705-716) as a constitutively active transcription factor binding the kappa light chain immunoglobulin enhancer in B cells. Shortly afterwards, the same researchers found NF-kappa B to be present in other cell types in an inactive cytoplasmic form which upon cellular stimulation could be induced to translocate to the nucleus and bind DNA. Subsequently, it has been demonstrated that NF-kappa B performs a critical role as a regulator of the immune system, the response to stress, apoptosis, viral replication and is involved in many diseases, leading to it becoming one of the most intensively studied transcription factors of the last decade. The pivotal role played by NF-kappa B is illustrated not only by the great diversity of genes that it regulates, but also by the large variety of stimuli leading to its activation. This article will address how NF-kappa B, a ubiquitously expressed transcription factor composed of dimers formed from five subunits, differentially regulates the expression of such a diverse array of genes with different functions, in different cell types and at different times. Recent research indicates that this behavioral diversity arises from a delicately balanced network of protein: protein interactions: NF-kappa B activity is determined not only through its regulated nuclear localization but is also dependent on the cellular context in which it is found.

Animals↗

PubMed2026

Biomimetic Hydrogels with Nucleus Pulposus-like Viscoelasticity and ECM Peptides for Discogenic Differentiation of Stem Cells.

Intervertebral disc (IVD) degeneration is a leading cause of low back pain (LBP), primarily originating in the nucleus pulposus (NP). Regenerative strategies combining mesenchymal stem cells (MSCs) with biomaterials offer great potential for NP repair by replenishing cells and restoring extracellular matrix (ECM). However, key translational challenges remain, including limited stem cell differentiation, poor cell survival in the harsh degenerative niche, and insufficient biomaterial support. While matrix viscoelasticity has been shown to influence adipose-derived stem cell (ASC) discogenic differentiation, its interplay with cell-adhesive ligands for IVD regeneration remains unclear. Moreover, most current hydrogels fail to replicate the ultrafast stress relaxation properties of native non-degenerative human NP tissue. Here, we developed viscoelastic ECM peptide-functionalized hydrogels (VEPH), specifically designed to mimic healthy human NP biomechanics and promote ASC differentiation for NP regeneration. We biochemically conjugated NP ECM-derived adhesive peptides (IKVAV, hA5G26, CHAD) through maleimide-thiol click chemistry, achieving hydrogels with significantly faster stress relaxation (∼25 s) compared to conventional viscoelastic alginate hydrogels (>100 s). Our results demonstrated that VEPH supported >95% ASC viability and robust metabolic activity over 21 days in 3D culture. Notably, the IKVAV-functionalized hydrogel significantly enhanced ASC cell-matrix interactions, upregulated NP marker expression (KRT18, HIF-1α, ITGA3, and CD24), and promoted type-II collagen secretion, indicating an NP-committed cell fate. Our findings highlight the synergistic roles of matrix viscoelasticity and NP-specific biochemical cues in directing ASC discogenic differentiation and advancing novel biomaterial design for IVD regeneration.

cell-adhesive peptides↗

PubMed2005

YB-1 represses AP1-dependent gene transactivation and interacts with an AP-1 DNA sequence.

Involvement of the AP-1 (activator protein-1) transcription factor has been demonstrated previously in the regulation of cell proliferation and cell-cycle progression, in the control of cell migration, invasion and metastasis, and in signal transduction, stress responsiveness, DNA replication and DNA repair. YB-1 (Y-box-binding protein-1) has also been implicated in many of these processes. However, the mechanism by which YB-1 mediates these processes is poorly understood. In the present study, we report that overexpression of a transfected gene encoding YB-1 in human HeLa cervical carcinoma cells significantly represses the transactivation of a minimal AP-1 reporter construct in response to the tumour promoter PMA. YB-1 also represses mRNA expression and PMA-induced promoter transactivation of the endogenous AP-1 target gene encoding matrix metalloproteinase-12 (metalloelastase). YB-1 transrepression of both the minimal and matrix metalloproteinase-12 promoter reporter constructs is dependent on the AP-1 sequence. To identify new nuclear proteins that bind specifically to the AP-1 DNA-binding site, we devised a DNA-affinity-chromatography-based assay termed NAPSTER (nucleotide-affinity preincubation specificity test of recognition) and discovered a 49 kDa protein from human cancer cells that binds in a sequence-specific manner to the AP-1 DNA sequence. By tandem MS fragmentation sequencing analyses we determined that p49 is a YB-1. Immunoblotting of the NAPSTER-purified p49 protein using anti-YB-1 antibodies confirmed YB-1 binding to the AP-1 DNA sequence, as did gel mobility-supershift assays using YB-1 antibodies. This is the first report of YB-1 transrepression and interaction at the AP-1 DNA-binding site.

Base Sequence↗

PubMed2004

Human nucleotide excision repair efficiently removes chromium-DNA phosphate adducts and protects cells against chromate toxicity.

Intracellular reduction of carcinogenic Cr(VI) leads to the extensive formation of Cr(III)-DNA phosphate adducts. Repair mechanisms for chromium and other DNA phosphate-based adducts are currently unknown in human cells. We found that nucleotide excision repair (NER)-proficient human cells rapidly removed chromium-DNA adducts, with an average t((1/2)) of 7.1 h, whereas NER-deficient XP-A, XP-C, and XP-F cells were severely compromised in their ability to repair chromium-DNA lesions. Activation of NER in Cr(VI)-treated human fibroblasts or lung epithelial H460 cells was manifested by XPC-dependent binding of the XPA protein to the nuclear matrix, which was also observed in UV light-treated (but not oxidant-stressed) cells. Intracellular replication of chromium-modified plasmids demonstrated increased mutagenicity of binary Cr(III)-DNA and ternary cysteine-Cr(III)-DNA adducts in cells with inactive NER. NER deficiency created by the loss of XPA in fibroblasts or by knockdown of this protein by stable expression of small interfering RNA in H460 cells increased apoptosis and clonogenic death by Cr(VI), providing genetic evidence for the role of monofunctional chromium-DNA adducts in the toxic effects of this metal. The rate of NER of chromium-DNA adducts under saturating conditions was calculated to be approximately 50,000 lesions/min/cell. Because chromium-DNA adducts cause only small changes in the DNA helix, rapid repair of these modifications in human cells indicates that the presence of major structural distortions in DNA is not required for the efficient detection of the damaged sites by NER proteins in vivo.

Cell Line↗

PubMed1995

The role of antioxidants in gastric carcinogenesis.

The epidemiologic evidence for a role of antioxidants in inhibiting carcinogenesis in humans is briefly summarized. Mechanistic pathways of protection by antioxidants are illustrated with the prevailing model of gastric carcinogenesis in humans. In this model, ascorbic acid may be involved at the early stage of chronic gastritis; its secretion into the stomach is inhibited by infection with Helicobacter pylori, a major cause of gastritis. Ascorbic acid may also play a role in blocking the nitrosation of amines and related compounds in the gastric microenvironment. Carotenoids are also involved in the chain of gastric cancer causation and apparently play an important role in preventing mutations in the advanced stages of the process. The gastric cancer model supports the notion that invasive cancer is the result of insufficient control of oxidative stress applied to replicating cells over a prolonged period of time.

Antioxidants↗

PubMed1995

Isolation of mutants of Saccharomyces cerevisiae requiring DNA topoisomerase I.

Despite evidence that DNA topoisomerase I is required to relieve torsional stress during DNA replication and transcription, yeast strains with a top1 null mutation are viable and display no gross defects in DNA or RNA synthesis, possibly because other proteins provide overlapping functions. We isolated mutants whose inviablility or growth defect is relieved when TOP1 is expressed [trf mutants (topoisomerase one-requiring function)]. The TRF genes define at least four complementation groups. TRF3 is allelic to TOP2. TRF1 is allelic to HPR1, previously shown to be homologous to TOP1 over two short regions. TRF4 encodes a novel 584-amino acid protein with homology to the N-terminus of Saccharomyces cerevisiae topo I. Like top1 mutants, trf4 mutants have elevated rDNA recombination and fail to shut off RNA polymerase II transcription in stationary phase. trf4 null mutants are cs for viability, display reduced expression of GAL1 and Cell Cycle Box UAS::LacZ fusions, and are inviable in combination with trfI null mutants, indicating that both proteins may share a common function with DNA topoisomerase I. The existence of multiple TRF complementation groups suggests that not all biological functions of topo I can be carried out by topo II.

Alleles↗

PubMed2006

Regulation of mitotic function of Chk1 through phosphorylation at novel sites by cyclin-dependent kinase 1 (Cdk1).

Chk1 is phosphorylated at Ser317 and Ser345 by ATR in response to stalled replication and genotoxic stresses. This Chk1 activation is thought to play critical roles in the prevention of premature mitosis. However, the behavior of Chk1 in mitosis remains largely unknown. Here we reported that Chk1 was phosphorylated in mitosis. The reduction of this phosphorylation was observed at the metaphase-anaphase transition. Two-dimensional phosphopeptide mapping revealed that Chk1 phosphorylation sites in vivo were completely overlapped with the in vitro sites by cyclin-dependent protein kinase (Cdk) 1 or by p38 MAP kinase. Ser286 and Ser301 were identified as novel phosphorylation sites on Chk1. Treatment with Cdk inhibitor butyrolactone I induced the reduction of Chk1-S301 phosphorylation, although treatment with p38-specific inhibitor SB203580 or siRNA did not. In addition, ionizing radiation (IR) or ultraviolet (UV) light did not induce Chk1 phosphorylation at Ser317 and Ser345 in nocodazole-arrested mitotic cells. These observations imply the regulation of mitotic Chk1 function through Chk1 phosphorylation at novel sites by Cdk1.

Amino Acid Sequence↗

PubMed2006

Reduced expression levels of the senescence biomarker clusterin/apolipoprotein j in lymphocytes from healthy centenarians.

Clusterin/apolipoprotein J (CLU) is a conserved, ubiquitously expressed secreted glycoprotein that has been implicated in several physiological processes and was found to accumulate in many severe physiological disturbances. We have previously shown that the CLU gene and protein are upregulated during replicative senescence, stress-induced premature senescence, in vivo aging, and in several age-related diseases. In this study we have examined the CLU gene relationship to human longevity. We recruited and further analyzed 96 blood samples from Italian and Greek healthy donors of different ages, including 49 centenarians. We found that although the CLU gene expression levels increase during aging, in the centenarians' samples CLU levels were lower than those found in old donors. We then investigated the possible existence of a genetic polymorphism related to longevity at the CLU structural locus. A neutral noncoding sequence variant was detected 35 nucleotides upstream from exon 6, which does not correlate, however, with the age of the donor. We conclude that CLU gene accumulation during in vivo aging does not directly relate to chronological age, but rather indicates increased levels of organismal stress due to a progressive failure of homeostasis and/or to prolonged exposure to a stressful environment.

Adult↗

PubMedDate not supplied

ERM proteins and Cdk5 in cellular senescence.

Cellular senescence is a tumor-suppressive process instigated by proliferation in the absence of telomere replication, by cellular stresses such as oncogene activation, or by activation of the retinoblastoma tumor suppressor protein, pRb. This process is characterized by an irreversible cell cycle exit, a unique morphology, and expression of senescence-associated-beta-galactosidase (SA-beta-gal). Despite the potential biological importance of cellular senescence, little is known of the mechanisms leading to the senescent phenotype. We have recently discovered that expression of active pRb induces expression and altered localization of the ERM family member ezrin, an actin-binding protein involved in membrane-cytoskeletal signaling. pRb expression results in the stimulation of cdk5-mediated phosphorylation of ezrin with subsequent membrane association and induction of cell shape changes, linking pRb activity to cytoskeletal regulation in senescent cells. Cdk5 activity increases in senescing cells and is required for expression of SA-beta-gal and for actin polymerization accompanying acquisition of the senescent morphology. These results begin to illuminate the mechanisms underlying induction of senescence and the senescent shape change and describe new pathways that may contribute to the ability of senescent cells to influence tumor growth.

Cell Cycle↗

PubMed2006

Claspin: timing the cell cycle arrest when the genome is damaged.

DNA damage checkpoints maintain genomic integrity by delaying cell cycle progression in response to genotoxic stress and stalled replication forks. One central pathway in the checkpoint response is the ATR-Chk1 pathway, in which, upon DNA damage, ATR phosphorylates and activates the effector kinase Chk1. This process depends on the adaptor protein Claspin that bridges ATR and Chk1. Once the damage is repaired, this pathway must somehow be switched off to allow the cell to continue the cell division process, an event known as checkpoint recovery. Polo-like kinase 1 (Plk1) plays a central role during checkpoint recovery. Interestingly, the Xenopus homologue of Plk1, Plx1, is able to bind and phosphorylate Claspin, releasing it from DNA and thereby contributing to Chk1 inactivation. Moreover, it was recently demonstrated that Claspin levels are controlled by proteasomal degradation, and this is regulated by Plk1. Importantly, Plk1-mediated proteosomal degradation of Claspin appears to be essential for check-point recovery. Here we review these recent findings and discuss the mechanisms of checkpoint regulation by Claspin.

Adaptor Proteins, Signal Transducing↗