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Biomedical subjects

Q M Chen

Publications and source records attributed to Q M Chen.

At least 19 recordsLinked to original sources
PubMed2005

The requirements and mechanism for capsid assembly and budding of bovine foamy virus.

Little is known about assembly of non-primate foamy virus (FV) such as bovine foamy virus (BFV). To help determine the requirements for assembly of BFV, we constructed BFV-Gag expression plasmids containing all or part of the gag gene, with or without modification by addition of myristate (Myr). Each construct was transfected alone, and with pFenv, into Sf-9 insect cells. The results showed that only the entire Gag could transit through nucleus, which is required for BFV viral assembly in the cytoplasm. Unlike other retroviruses (but like primate foamy viruses), BFV requires the coexpression of the Env protein for viral particle budding. In the case of BFV, this occurs at the plasma membrane rather than the endoplasmic reticulum (ER), due to lack of a functional ER retrieval signal (ERRS). The results also showed that addition of a Myr-membrane targeting signal to the C-terminus of Gag could restore the budding from plasma membrane, implying that Myr-membrane targeting signal could substitute for Env protein in budding.

Animals↗
PubMed2004

Photochemical degradation of 1,3-dinitrobenzene in aqueous solution in the presence of hydrogen peroxide.

A study on the destruction of 1,3-dinitrobenzene (1,3-DNB) in aqueous solution was carried out under ultraviolet (UV) irradiation alone and UV irradiation in the presence of hydrogen peroxide (H2O2). The combination of UV and H2O2 is significantly effective in degrading 1,3-DNB in terms of initial reaction rate and the mineralization of organic carbons. The photodegradation process can be influenced in certain extent by increasing the content of H2O2 and the acidity of reaction matrices. It was found that a variety of phenolic intermediates and inorganic acid were formed via hydroxyl radicals attacking the parent compound. The UV/H2O2 oxidation of 1,3-DNB was characterized by pseudo-zero order reaction for the degradation of 1,3-DNB with a 20 times enhanced rate constant of 1.36 x 10(-7) Ms(-1) and the initial rate constant was dependent on the initial concentration of 1,3-DNB.

Chromatography, High Pressure Liquid↗
PubMed2001

Uncoupling the senescent phenotype from telomere shortening in hydrogen peroxide-treated fibroblasts.

Normal human cells have a limited replicative potential and inevitably reach replicative senescence in culture. Replicatively senescent cells show multiple molecular changes, some of which are related to the irreversible growth arrest in culture, whereas others resemble the changes occurring during the process of aging in vivo. Telomeres shorten as a result of cell replication and are thought to serve as a replicometer for senescence. Recent studies show that young cells can be induced to develop features of senescence prematurely by damaging agents, chromatin remodeling, and overexpression of ras or the E2F1 gene. Accelerated telomere shortening is thought to be a mechanism of premature senescence in some models. In this work, we test whether the acquisition of a senescent phenotype after mild-dose hydrogen peroxide (H(2)O(2)) exposure requires telomere shortening. Treating young HDFs with 150 microM H(2)O(2) once or 75 microM H(2)O(2) twice in 2 weeks causes long-term growth arrest, an enlarged morphology, activation of senescence-associated beta-galactosidase, and elevated expression of collagenase and clusterin mRNAs. No significant telomere shortening was observed with H(2)O(2) at doses ranging from 50 to 200 microM. Weekly treatment with 75 microM H(2)O(2) also failed to induce significant telomere shortening. Failure of telomere shortening correlated with an inability to elevate p16 protein or mRNA in H(2)O(2)-treated cells. In contrast, p21 mRNA was elevated over 40-fold and remained at this level for at least 2 weeks after a pulse treatment of H(2)O(2). The role of cell cycle checkpoints centered on p21 in premature senescence induced by H(2)O(2) is discussed here.

Blotting, Northern↗
PubMed2001

Enteric nervous system, interstitial cells of cajal, and smooth muscle vacuolization in segmental dilatation of jejunum.

BACKGROUND/PURPOSE: The etiology of congenital segmental dilatation (CSD) of bowel remains elusive. Intestitial cell of Cajal plays a role in the pace making of the intestine. Its abnormality has been documented in a variety of conditions of abnormal intestinal motility. The current study attempts to evaluate the roles of intestitial cells of Cajal, enteric nervous system, and smooth muscle in segmental dilatation of small bowel. METHODS: Resected specimen from a neonate with segmental dilatation of jejunum was stained with H&E, Alcian blue, Periodic Acid-Schiff (PAS), and immunostained with S100, Ret, MAP5, and c-kit antibodies using the standard immunohistochemical process. RESULTS: The immunostaining of S100, Ret, MAP5 and c-kit of CSD specimen were positive. Localized vacuolization was, however, detected in the circular smooth muscle of the jejunum. The Alcian blue and PAS staining of the vacuolization were negative. CONCLUSIONS: CSD shows no abnormality in the enteric nervous system and pace makers. Localized vacuolization suggests myopathy to be a contributing factor to the disease. J Pediatr Surg 36:930-935.

Dilatation, Pathologic↗
PubMed2001

Molecular mechanisms of cardiac hypertrophy induced by toxicants.

Cardiac hypertrophy is an end point of chronic cardiac toxicity from a number of toxicants. Doxorubicin, cocaine, acetaldehyde, monocrotaline, and azide are examples of these toxicants, which may induce hypertrophy by increasing oxidants, circulating levels of catecholamines, and hemodynamic load or by inducing hypoxia. We summarize here the major signal transduction pathways and common changes in gene expression found with the classical hypertrophy inducers angiotensin II, endothelin 1, and catecholamines. Activation of G-proteins, calcium signaling, phosphoinositide 3-kinase (PI3K), certain family members of protein kinase Cs (PKCs), and three branches of mitogenactivated protein kinases (MAPKs), i.e. extracellular signal-regulated kinases (ERKs), p38, and c-Jun N-terminal kinases (JNKs), are important for developing a hypertrophic phenotype in cardiomyocytes. Characteristic changes of gene expression in hypertrophy include the elevated transcription of atrial natriuretic factor (ANF), beta-myosin heavy chain (beta MHC), skeletal alpha-actin (SkA), certain variants of integrins and perhaps tubulin genes, and reduced expression of the sarcoplasmic reticulum proteins phospholamban and sarco(endo)plasmic reticulum Ca2+-ATPase 2 alpha (SERCA2 alpha), and of the ryanodine receptors. Although which toxicants induce these molecular changes remains to be tested, increasing lines of evidence support that oxidants play a central role in cardiac hypertrophy. Oxidants activate small G-proteins, calcium signaling, PI3K, PKCs, and MAPKs. Oxidants cause cardiomyocytes to enlarge in vitro. Recent developments in transgenic, genomic, and proteomic technologies will provide needed tools to reveal the mechanism of chronic cardiac toxicity at the cellular and molecular levels.

Animals↗
PubMed2000

Subcytotoxic H2O2 stress triggers a release of transforming growth factor-beta 1, which induces biomarkers of cellular senescence of human diploid fibroblasts.

Stress-induced premature senescence (SIPS) is induced 3 days after exposure of human diploid fibroblasts to subcytotoxic oxidative stress with H(2)O(2), with appearance of several biomarkers of replicative senescence. In this work, we show that transforming growth factor-beta1 (TGF-beta1) regulates the induction of several of these biomarkers in SIPS: cellular morphology, senescence-associated beta-galactosidase activity, increase in the steady-state level of fibronectin, apolipoprotein J, osteonectin, and SM22 mRNA. Indeed, the neutralization of TGF-beta1 or its receptor (TGF-beta RII) using specific antibodies decreases sharply the percentage of cells positive for the senescent-associated beta-galactosidase activity and displaying a senescent morphology. In the presence of each of these antibodies, the steady-state level of fibronectin, osteonectin, apolipoprotein J, and SM22 mRNA is no more increased at 72 h after stress. Results obtained on fibroblasts retrovirally transfected with the human papillomavirus E7 cDNA suggest that retinoblastoma protein (Rb) regulates the expression of TGF-beta1 in stressful conditions, leading to SIPS and overexpression of these four genes.

Cells, Cultured↗
PubMed2000

Apoptosis or senescence-like growth arrest: influence of cell-cycle position, p53, p21 and bax in H2O2 response of normal human fibroblasts.

Early-passage human diploid fibroblasts (HDFs) undergo senescence-like growth arrest in response to sublethal concentrations of H(2)O(2) [Chen and Ames (1994) Proc. Natl. Acad. Sci. USA. 95, 4130-4134]. We determine here whether H(2)O(2) can cause apoptosis in HDFs and the molecular changes that differ between apoptosis and senescence-like growth arrest. When exponentially growing early-passage IMR-90 cells were treated for 2 h with 50-200 microM (or 0.25-1 pmol/cell) H(2)O(2), a fraction of cells detached at 16-32 h after the treatment. The cells remaining attached were growth-arrested and developed features of senescence in 1 week. The detached cells showed caspase-3 activation and typical morphological changes associated with apoptosis. Caspase-3 activation was H(2)O(2) dose-dependent and preceded nuclear condensation or plasma membrane leakage. Apoptotic cells were mainly distributed in the S-phase of the cell cycle, while growth-arrested cells exhibited predominantly G1- and G2/M-phase distributions. H(2)O(2) pretreatment induced G1 arrest and prohibited induction of apoptosis by a subsequent H(2)O(2) challenge. The p53 protein showed an average 6.1-fold elevation in apoptotic cells and a 3.5-fold elevation in growth-arrested cells. Reduction of p53 levels with human papillomavirus E6 protein prohibited the activation of caspase-3 and decreased the proportion of apoptotic cells. Growth-arrested cells had elevated p21, while p21 was absent in apoptotic cells. Bcl-2 was elevated in both growth-arrested and apoptotic cells. Finally, although the overall level of bax did not change in growth-arrested or apoptotic cells, the solubility of bax protein increased in apoptotic cells. Our data suggest that in contrast with growth-arrested cells, apoptotic cells show an S-phase cell cycle distribution, a higher degree of p53 elevation, an absence of p21 protein and increased solubility of bax protein.

Apoptosis↗
PubMed2000

Linking gene expression to mechanisms of toxicity.

Activation of gene expression is one of the earliest cellular responses to toxicity. However, our understanding of the biological and biochemical signals that activate these toxicant-responsive genes as well as the consequences of gene activation to survival of the organism remains sketchy. In this article, strategies that can be used to link changes in gene expression to biochemical mechanisms of toxicity are addressed using the hsp70 and grp78 genes as examples. The data indicate that activation of hsp70 is linked to changes in thiol-disulfide redox perturbations while grp78 activation may be caused by loss of calcium from the endoplasmic reticulum. Each gene is part of a discrete feedback regulated signaling pathway designed to protect cells against the toxic signals that activate gene expression.

Animals↗
PubMed2000

Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast.

We tested the long-term effects of sublethal oxidative stresses on replicative senescence. WI-38 human diploid fibroblasts (HDFs) at early cumulative population doublings (CPDs) were exposed to five stresses with 30 microM tert-butylhydroperoxide (t-BHP). After at least 2 d of recovery, the cells developed biomarkers of replicative senescence: loss of replicative potential, increase in senescence-associated beta-galactosidase activity, overexpression of p21(Waf-1/SDI-1/Cip1), and inability to hyperphosphorylate pRb. The level of mRNAs overexpressed in senescent WI-38 or IMR-90 HDFs increased after five stresses with 30 microM t-BHP or a single stress under 450 microM H(2)O(2). These corresponding genes include fibronectin, osteonectin, alpha1(I)-procollagen, apolipoprotein J, SM22, SS9, and GTP-alpha binding protein. The common 4977 bp mitochondrial DNA deletion was detected in WI-38 HDFs at late CPDs and at early CPDs after t-BHP stresses. In conclusion, sublethal oxidative stresses lead HDFs to a state close to replicative senescence.

Biomarkers↗
PubMed2000

Hydrogen peroxide dose dependent induction of cell death or hypertrophy in cardiomyocytes.

Cardiomyocyte hypertrophy and cell death are often observed in the end stages of heart failure. The triggers of these two cellular processes are not known under most pathological conditions. Oxidants are by-products of aerobic metabolism. The level of oxidants increases as a result of ischemic reperfusion. Using H9C2 and primary cultured neonatal rat cardiomyocytes, we found that a 2-h pulse treatment with H(2)O(2) at 250 microM or lower caused activation of DEVD sequence specific caspases. The activity of DEVD-ase peaked with 200 microM H(2)O(2) at 24 h. While a fraction of the cells detached and showed nuclear condensation, the majority of the cells (>55%) survived the treatment and appeared to enlarge when cultured for 5 days. These cells showed increases in cell surface area, cell volume, and protein content. With 200 microM H(2)O(2), treated cells appeared to be six times bigger in volume and contained three times more protein per cell than untreated cells. The enlarged cells showed enhanced actin stress fibers and disrupted myofibrils. Our data indicate that while H(2)O(2) can cause cell death, the surviving cardiomyocytes undergo hypertrophy.

Actins↗
PubMed2000

Cell cycle regulation in H(2)O(2)-induced premature senescence of human diploid fibroblasts and regulatory control exerted by the papilloma virus E6 and E7 proteins.

Many biomarkers of replicative senescence appear in stress-induced premature senescence (SIPS) of human diploid fibroblasts (HDFs). The mRNA level of key cell cycle regulators was studied in H(2)O(2)-induced premature senescence of HDFs expressing or not the papillomavirus E6 and E7 proteins, which enhanced, respectively, the proteolysis of p53 and Rb. The CdKI's p21(waf-1) and p16(Ink-4a) were found overexpressed in H(2)O(2)-induced premature senescence, while p19(Ink-4d)and p27(Kip-1) were repressed. The results obtained in E6 HDFs suggest that p21(waf-1) and p16(Ink-4a) overexpressions are p53-independent, while p27(Kip-1) and p19(Ink-4d) down-regulations are p53-dependent.E6 regulated Rb, p130, p53 and p16(Ink-4a) mRNA level in non-stressing conditions, and regulated p130, p107, p53, p19(Ink-4d), p27(Kip-1) mRNA level in SIPS. SIPS modified the E6-mediated regulatory control on p107, p16(Ink-4a), p19(Ink-4d) and p27(Kip-1) mRNA level, when compared to normal conditions.E7 regulated the mRNA level of all the genes studied, in all conditions, suggesting that the Rb family or other E7-interacting proteins might modify the expression of these genes. SIPS modified strongly the E7-mediated regulatory control on p107, p16(Ink-4a), p19(Ink-4d), p27(Kip-1), p21(Waf-1) and Rb mRNA level, when compared to normal conditions. Further work is ongoing to test whether this E7-mediated regulatory control takes place through interactions with Rb or other E7-interacting proteins.

Cell Cycle↗
PubMed2000

Measurements of hydrogen peroxide induced premature senescence: senescence-associated beta-galactosidase and DNA synthesis index in human diploid fibroblasts with down-regulated p53 or Rb.

Human diploid fibroblasts (HDFs) inevitably undergo replicative senescence in culture after serial passages. Recent work indicates that early passage HDFs undergo irreversible growth arrest and develop features of senescence after being treated with oxidants and other agents. Senescence is usually measured by a decrease in DNA synthesis index and an increase in the activity of senescence-associated beta-galactosidase (SA beta-gal). We compared these two measurements here and found that IMR-90 HDFs lost the ability to synthesize DNA immediately but did not activate SA beta-gal until 4 days after the treatment with 75 microM or 0.75 pmol/cell H2O2. Expression of human papillomaviral E6 or/and E7 genes results in reduction of p53 or/and Rb protein levels and increases in ED50 for DNA synthesis inhibition or SA beta-gal expression. A small fraction of wild type and E7 expressing cells could not synthesize DNA and did not express SA beta-gal one week following the treatment with H2O2 at doses lower than 150 microM or 1.5 pmol/cell. The dose response curve of SA beta-gal activation overlapped with that of DNA synthesis inhibition in E6 and E6E7 expressing cells. The results indicate that the expression of SA beta-gal correlates with inability of DNA synthesis in the majority of wild type, E6, E7 or E6E7 expressing cells one week after H2O2 treatment.

Cell Line↗
PubMed2000

Replicative senescence and oxidant-induced premature senescence. Beyond the control of cell cycle checkpoints.

Normal human diploid fibroblasts (HDFs) undergo replicative senescence inevitably in tissue culture after a certain number of cell divisions. A number of molecular changes observed in replicative senescent cells occur in somatic cells during the process of aging. Genetic studies on replicative senescence indicate the control of tumor suppression mechanisms. Despite the significance of replicative senescence in aging and cancer, little is known about the central cause of the complex changes observed in replicative senescent cells. The interest in the phenomenon has intensified in recent years, since damaging agents, certain oncogenes and tumor suppressor genes have been found to induce features of senescence in early passage young HDFs or in immortalized tumor cells. The reported features of senescence are summarized here in order to clarify the concept of replicative senescence or premature senescence. The experimental results of extending the replicative life span by reducing ambient oxygen tension or by N-tert-butyl-alpha-phenylnitrone (PBN) argue a role of oxidative damage in replicative senescence. By inducing premature senescence with a pulse treatment of H2O2, we can study the role of the cell cycle checkpoint proteins p53, p21, p16 and Rb in gaining each feature of senescence. Although p53 and Rb control G1 arrest and Rb appears to control cell enlargement, activation of the senescent associate beta-galactosidase, loss of cell replication and multiple molecular changes observed in premature senescent or replicative senescent cells are likely controlled by mechanisms beyond the cell cycle checkpoints.

Animals↗
PubMed2000

Involvement of Rb family proteins, focal adhesion proteins and protein synthesis in senescent morphogenesis induced by hydrogen peroxide.

Early passage human diploid fibroblasts develop senescent morphology prematurely within a week after a 2-hour pulse treatment with low or mild dose H(2)O(2). We test here the role of cell cycle checkpoints, cytoskeletal proteins and de novo protein synthesis in senescent morphogenesis following H(2)O(2) treatment. H(2)O(2) treatment causes transient elevation of p53 protein and prolonged inhibition of Rb hyperphosphorylation. Expression of human papillomaviral E6 gene prevented elevation of p53 but did not affect senescent morphogenesis. Expression of human papillomaviral E7 gene reduced the level of Rb protein and prevented induction of senescent morphology by H(2)O(2). The mutants of the E7 gene, in which the Rb family protein binding site was destroyed, could not reduce Rb protein or prevent H(2)O(2) from inducing senescent morphology. Senescent-like cells showed enhanced actin stress fibers. In untreated cells, vinculin and paxillin preferentially distributed along the edge of the cells. In contrast, vinculin and paxillin distributed randomly and sporadically throughout senescent-like cells. E7 expression prevented enhancement of actin filament formation and redistribution of vinculin or paxillin. Neither wild-type nor E7 cells showed changes in the protein level of actin, vinculin or paxillin measured by western blot after H(2)O(2) treatment. Finally, depletion of methionine in the culture medium after H(2)O(2) treatment prevented senescent morphogenesis without affecting dephosphorylation of Rb protein. Our results suggest that senescent morphology likely develops by a program involving activated Rb family proteins, enhancement of actin stress fibers, redistribution of focal adhesion proteins and de novo protein synthesis.

Actins↗
PubMed2000

[Construction of B. thuringiensis shuttle vector and expression of the cry1C gene].

We have constructed the E. coli-Bt shuttle vector pHV-1 by cloning the replicon (approximately 1.6 kb) of Bt ken-Ag and the aphI gene of pUC4K into pUC19. The rate of plasmid maintenance is more than 80% after 100 generations in E. coli, whereas 80% after 40 generations in Bti 4Q8. We have also constructed pHV-cry1C through cloning the alpha-amylase promoter from B. licheniformis and the cry1C gene from Bt 9510 into pHV-1 and introduced it into Bti 4Q8 by means of electroporation. Under the microscope, we can see that there is no crystal in Bti 4Q8, however, there are many rhomboid crystals in Bti 4Q8 (pHV-cry1C), which are smaller than those of Bt 9510. The bioassay result of Bti 4Q8 (pHV-cry1C) demonstrates that the expressed crystal protein is insecticidally active against Spodoptera exigue.

Animals↗
PubMed1998

Molecular analysis of H2O2-induced senescent-like growth arrest in normal human fibroblasts: p53 and Rb control G1 arrest but not cell replication.

Human diploid fibroblasts lose the capacity to proliferate and enter a state termed replicative senescence after a finite number of cell divisions in culture. When treated with sub-lethal concentrations of H2O2, pre-senescent human fibroblasts enter long-term growth arrest resembling replicative senescence. To understand the molecular basis for the H2O2-induced growth arrest, we determined the cell cycle distribution, levels of p53 tumour suppressor and p21 cyclin-dependent kinase inhibitor proteins, and the status of Rb phosphorylation in H2O2-treated cells. A 2-h pulse of H2O2 arrested the growth of IMR-90 fetal lung fibroblasts for at least 15 days. The arrested cells showed a G1 DNA content. The level of p53 protein increased 2- to 3-fold within 1.5 h after H2O2 exposure but returned to the control level by 48 h. The induction of p53 protein was dose dependent, beginning at 50-75 microM and reaching a maximum at 100-250 microM. The induction of p53 did not appear to correlate with the level of DNA damage as measured by the formation of 8-oxo-2'-deoxyguanosine in DNA. The level of p21 protein increased about 18 h after H2O2 exposure and remained elevated for at least 21 days. During this period, Rb remained underphosphorylated. The induction of p53 by H2O2 was abolished by the iron chelator deferoxamine and the protein synthesis inhibitor cycloheximide. The human papillomavirus protein E6, when introduced into the cells, abolished the induction of p53, reduced the induction of p21 to a minimal level and allowed Rb phosphorylation and entry of the cells into S-phase. The human papillomavirus protein E7 reduced the overall level of Rb and also abolished H2O2-induced G1 arrest. Inactivating G1 arrest by E6, E7 or both did not restore the replicative ability of H2O2-treated cells. Thus H2O2-treated cells show a transient elevation of p53, high level of p21, lack of Rb phosphorylation, G1 arrest and inability to replicate when G1 arrest is inactivated.

Cell Division↗
PubMed1998

[Effect of changes of amino acids of N-terminal region of the mature protein on secretion of alpha-amylase in B. subtilis].

The mutant plasmid pAmy413C, in which G takes the place of A at the 271 position of alpha-amylase gene on the pAmy413 from B. licheniformis, was constructed by site-direct mutagenesis. At the N-terminus of the mature alpha-amylase, amino acid +2Asn was substituted by +3Asp in the wild type protein. Then, the alpha-amylase output of the mutant plasmid pAmy413C in B. subtilis was 2.02-2.57 times higher than that of the wild type pAmy413C in the same strain. The amino acid sequencing at the N-terminus of the matural alpha-amylase revealsed that the recognition site of signal peptidase I moved one amino acid upstream, from Ala-(+2)Asn to AlaAla-(+3) Asp. That is, the +2Asn of the wild type was changed to the +3Asp of the mutant. The secondary structural analysis showed that a 14-cycle structure formed in the alpha-amylase mRNA when the free energy was -51.7 kcal. In this case, the mutant is identical with the wild type. The difference between them is that G at 271 position is no longer paired with U at 211 position, hence, a G-overhang is formed. The secondary structural analysis of protein showed that one amino acid diminished in the turn structure of amino acid at 33-37 position, and this very amino acid is involed in an alpha-helix structure. In short, all the changes mentioned above in conformation and charged amino acids contribute to the increase in the protein secretion in B. subtilis.

Amino Acid Sequence↗