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Biomedical subjects

M Chen

Publications and source records attributed to M Chen.

At least 577 records · Page 32Linked to original sources
PubMed1996

[Kidney reinforcing and yang supporting action of cistanche deserticola Y. C. Ma before and after preparation].

The weights of seminal vesicle and prostate gland of castrated young rats were significantly increased by administration of alcoholsoluble extract from decoction of Cistanche deserticola. The weights of testes, seminal vesicle and prostate gland in mice and rats were also increased by the extract. The phagocytic function of intra-abdominal macrophage in mice was activated by decoction of Cistanche deserticola. The results showed no statistical differences between crude and prepared drugs. The maximum oral tolerance for mouse was 40 g/kg.

Animals↗
PubMed1995

Aplysia CREB2 represses long-term facilitation: relief of repression converts transient facilitation into long-term functional and structural change.

The switch from short- to long-term facilitation induced by behavioral sensitization in Aplysia involves CREB-like proteins, as well as the immediate-early gene ApC/EBP. Using the bZIP domain of ApC/EBP in a two-hybrid system, we have cloned ApCREB2, a transcription factor constitutively expressed in sensory neurons that resembles human CREB2 and mouse ATF4. ApCREB2 represses ApCREB1-mediated transcription in F9 cells. Injection of anti-ApCREB2 antibodies into Aplysia sensory neurons causes a single pulse of serotonin (5-HT), which induces only short-term facilitation lasting minutes, to evoke facilitation lasting more than 1 day. This facilitation has the properties of long-term facilitation: it requires transcription and translation, induces the growth of new synaptic connections, and occludes further facilitation by five pulses of 5-HT.

Activating Transcription Factor 2↗
PubMed1995

Cell type-dependent regulation of the activity of the negative regulatory element of the hepatitis B virus core promoter.

The Hepatitis B virus core promoter regulates the expression of the core protein, the precore protein, and the viral DNA polymerase. This promoter is transactivated by HNF4, a liver-enriched transcription factor, through an HNF4 binding site located upstream of the core promoter. The transactivation activity of HNF4 on the core promoter is antagonized by a negative regulatory element (NRE) located upstream of the HNF4 binding site. While the NRE can effectively antagonize HNF4 to suppress the core promoter in HeLa cervical carcinoma cells, it has only a marginal suppressing activity on the core promoter in Huh7 hepatoma cells. By performing deletion-mapping experiments, we have found that the NRE contains at least three independent subregions named NRE alpha, NRE beta, and NRE gamma. Each of these three subregions possesses a weak suppressing activity, but together they generate a strong synergistic suppressing effect on the core promoter. The NRE gamma subregion is active in both HeLa and Huh7 cells and is bound by a protein factor slightly less than 130 kDa in molecular mass. The NRE alpha and NRE beta subregions are active in HeLa cells but not in Huh7 cells. Thus, the marginal suppressing effect of the NRE observed in Huh7 cells was mostly due to the activity of the NRE gamma subregion. No clear protein factor binding sites could be identified in the NRE alpha and NRE beta subregions when the HeLa nuclear extract was used for the DNaseI-footprinting analysis, indicating weak or no protein association with these two subregions in this cell type. However, extensive protein factor binding sites could be identified throughout the sequences of these two subregions when the Huh7 nuclear extract was used for the analysis. These results indicate that a different set of protein factors binds to the NRE alpha and NRE beta subregions in Huh7 cells and may account for the inactivity of these two subregions in this cell type. Thus, our results indicate that the cell type-dependent activity of the NRE is due to differential regulation of the activities of the NRE alpha and NRE beta subregions by the cell types. This regulation is most likely mediated by cell type-dependent protein factors.

Base Sequence↗
PubMed1995

Atherosclerosis alters the composition, structure and function of arterial smooth muscle cell plasma membranes.

The object of this study was to examine changes in plasma membranes of arterial smooth muscle (ASM) during atherogenesis obtained from cholesterol-fed (2%) rabbits. A microsomal fraction highly enriched with plasma membrane markers was prepared by subcellular organelle fractionation from ASM freshly isolated from the thoracic aorta. The membranes were analyzed for unesterified (free) cholesterol (FC) content, membrane bilayer structural parameters (X-ray diffraction), phospholipid (PL) composition, and Na+/K(+)-ATPase activity and kinetics. Following 8 weeks on diet, membrane FC content increased 67.1%. Small angle X-ray diffraction demonstrated an increase in membrane hydrocarbon core electron density and an increase in overall lipid bilayer width (56-62 A). This increase in bilayer width was highly correlated with the membrane FC content (r = 0.992). Both membrane FC content And bilayer width independently correlated with time on cholesterol diet. The phospholipid profile of the membrane revealed a 16.4% increase in phosphatidylcholine (PC), 19.3% decrease in phosphatidylethanolamine (PE) and 62.8% increase in sphingomyelin (SM) content with no change in total PL content. Na+/K(+)-ATPase activity was decreased 52.2% (P < 0.005), and [3H]ouabain binding kinetics demonstrated a 27.6% decrease in maximum binding sites (Bmax) (P < 0.01) while the dissociation constant (Kd) remained unaltered. Membranes obtained from control ASM cells enriched with FC in culture demonstrated changes similar to those in atherosclerotic ASM membranes including an increase in membrane FC content, an increase in bilayer width, and a decrease in Na+/K(+)-ATPase activity with decreased ouabain Bmax. These data demonstrate marked compositional, structural and functional changes in ASM cell membrane characteristics in dietary atherosclerosis. These changes were highly correlated with cholesterol accumulation in the plasma membrane bilayer and were observed before the appearance of visible lesions. We suggest that these membrane defects may be linked with early atherogenesis.

Animals↗
PubMed1995

Tethered ligand library for discovery of peptide agonists.

We exploited the mechanism underlying thrombin receptor activation to develop a novel screening method to identify peptide agonists. The thrombin receptor is activated by limited proteolysis of its amino-terminal exodomain. Thrombin cleaves this domain to unmask a new amino terminus, which then functions as a tethered peptide agonist, binding intramolecularly to the body of the receptor to trigger signaling. The thrombin receptor's amino-terminal exodomain can also donate the tethered agonist intermolecularly to activate nearby thrombin receptors. We utilized this ability by co-expressing a "tethered ligand library," which displayed the thrombin receptor's amino-terminal exodomain bearing random pentapeptides in place of the native tethered ligand together with target receptors in Xenopus oocytes. Clones that conferred thrombin-dependent signaling by intermolecular ligation of the target receptor were isolated by sib selection. Agonists for the thrombin receptor itself (GFIYF) and for the formyl peptide receptor (MMWLL) were identified. Surprisingly, the latter agonist was quite active at the formyl peptide receptor even without N-formylation, and its formylated form, fMMWLL, was more potent than the classical formyl peptide receptor agonist fMLF. In addition to identifying novel peptide agonists for targets of pharmacological interest, this method might be used to discover agonists for orphan receptors. It also suggests a possible evolutionary path from peptide to protease-activated receptors.

Amino Acid Sequence↗
PubMed1995

Blockage of the early events of mitogenic signaling by interferon-gamma in macrophages in response to colony-stimulating factor-1.

Inhibition of cell proliferation is an important biologic function of interferons (IFNs), which has been exploited in therapeutic treatment of certain hematologic malignancies. However, the molecular mechanism was not clear. We have recently shown that IFNs (alpha/beta and gamma) inhibit protein kinase C (PKC)-dependent (such as PDGF and phorbol ester) but not PKC-independent (such as epidermal growth factor) activation of Raf-1 and mitogen-activated protein kinases (MAPK/ERKs) in fibroblasts (Xu et al, Mol Cell Biol 14:8018, 1994), suggesting a novel mechanism by which IFNs execute their antiproliferative function. Monocytes/macrophages are primary targets in vivo for IFN-gamma, the major activity of macrophage-activating factor. In the present study, mechanism of IFN-gamma-induced antiproliferative action in macrophages in response to colony-stimulating factor-1 (CSF-1) has been investigated. Our results show that antiproliferative effect of IFN-gamma overrode mitogenic effect of CSF-1 and phorbol ester, as measured by early gene expression, DNA synthesis and cell proliferation. Although activation, phosphorylation, and turnover of the CSF-1 receptor and CSF-1-induced increase in diacylglycerol production remained normal, IFN-gamma blocked CSF-1-stimulated activation of mitogen-activated protein kinases, Raf-1 kinase, increase in GTP-bound Ras and tyrosine phosphorylation, and activation of protein kinase C delta (PKC-delta). PKC-delta was required for CSF-1-induced mitogenic signaling and a primary target for IFN-gamma-induced inhibition. Interestingly, although phorbol myristate acetate stimulated Ras activation, PKC-delta did not appear to be an upstream activator of Ras. These studies clearly indicated that IFN-gamma specifically inhibits PKC-delta activation, resulting in blockage of the early events of mitogenesis in macrophages in response to CSF-1.

Animals↗
PubMed1995

Mechanisms of thrombin receptor agonist specificity. Chimeric receptors and complementary mutations identify an agonist recognition site.

Identification of the docking interactions by which peptide agonists activate their receptors is critical for understanding signal transduction at the molecular level. The human and Xenopus thrombin receptors respond selectively to their respective hexapeptide agonists, SFLLRN and TFRIFD. A systematic analysis of human/Xenopus thrombin receptor chimeras revealed that just two human-for-Xenopus amino acid substitutions, Phe for Asn87 in the Xenopus receptor's amino-terminal exodomain and Glu for Leu260 in the second extracellular loop, conferred human receptor-like specificity to the Xenopus receptor. This observation prompted complementation studies to test the possibility that Arg5a in the human agonist peptide might normally interact with Glu260 in the human receptor. The mutant agonist peptide SFLLEN was a poor agonist at the wild type human receptor but an effective agonist at a mutant human receptor in which Glu260 was converted to Arg. An "arginine scan" of the receptor's extracellular surface revealed additional complementary mutations in the vicinity of position 260 and weak complementation at position 87 but not elsewhere in the receptor. Strikingly, a double alanine substitution that removed negative charge from the Glu260 region of the human receptor also effectively complemented the SFLLEN agonist. The functional complementation achieved with single Arg substitutions was thus due at least in part to neutralization of a negatively charged surface on the receptor and not necessarily to introduction of a new salt bridge. By contrast, charge neutralization did not account for the gain of responsiveness to SFLLRN seen in the human/Xenopus receptor chimeras. Thus two independent approaches, chimeric receptors and arginine scanning for complementary mutations, identified the Glu260 region and to a lesser degree Phe87 as important determinants of agonist specificity. These extracellular sites promote receptor responsiveness to the "correct" agonist and inhibit responsiveness to an "incorrect" agonist. They may participate directly in agonist binding or regulate agonist access to a nearby docking site.

Amino Acid Sequence↗
PubMed1995

Platelets are the primary source of amyloid beta-peptide in human blood.

The main component of Alzheimer's disease (AD) amyloid deposits is amyloid beta-peptide (A beta), a fragment of the larger amyloid precursor protein (APP). The cellular source of A beta is not known, but a circulatory origin has been postulated. We studied human blood from healthy individuals and found that platelets account for almost 90% of the total anti-A beta immunoreactivity detected in whole blood. Using reverse-phase HPLC, we identified a platelet peptide which corresponds to A beta by three criteria: (a) it shares a retention time with the synthetic A beta 1-40 peptide in two consecutive HPLC tests; (b) it interacts with two anti-A beta antibodies in separate ELISAs; and, (c) its partial N-terminal amino acid sequence closely matches that of A beta. The detection of this peptide in platelets indicates that, aside from the well-known non-amyloidogenic (secretory) pathway, the processing of APP in platelets from healthy individuals also involves an amyloidogenic pathway. These findings are consistent with the view that platelets are one of the major sources of A beta in the circulation.

Alzheimer Disease↗
PubMed1995

Differences in inhibition of chromosome separation and G2 arrest by DNA topoisomerase II inhibitors merbarone and VM-26.

Merbarone, a novel DNA topoisomerase II (topo II) inhibitor, differs from teniposide (VM-26) in that it inhibits topo II activities without stabilizing topo II-DNA covalent complexes. Thus, while the cellular effects of VM-26 are the consequences of inhibition of topo II catalytic activities and generation of topo II-mediated DNA damage, those of merbarone may be due to inactivation of topo II catalytic function. To address the issues of mechanisms of cell cycle effects and pharmacological actions of these two topo II inhibitors in mammalian cells, we used synchronized cultures of HeLa cells to study the effects of these drugs on cell cycle processes where topo II is essential (e.g., chromosome separation) or possibly involved (e.g., G2 arrest, DNA replication). We found that both drugs inhibited chromosome separation and cell division without preventing cells from exiting mitosis. Both drugs caused S-phase retardation G2 arrest, and phase-specific cytotoxicity in that they are more toxic to S, M, and G2 cells than G0/G1 cells. However, merbarone produced the above effects in convergent dosages that were within one to five times its 90% inhibitory cytotoxic concentration, whereas the concentrations of VM-26 to cause quantitatively similar effects were quite divergent. VM-26 is 50-100-fold more efficient in causing G2 arrest than in inhibiting chromosome separation. Furthermore, at concentrations showing similar levels of S-phase suppression, VM-26 caused significant DNA breaks, while merbarone had no such effect. Our data suggest that the effects of merbarone and VM-26 during mitosis are most likely due to inhibition of topo II function. We conclude that while G2 arrest by VM-26 is related to topo II-mediated DNA damage and its sequelae, G2 arrest by merbarone likely results from different mechanisms.

Cell Division↗
PubMed1995

Suppression of Bcl-2 messenger RNA production may mediate apoptosis after ionizing radiation, tumor necrosis factor alpha, and ceramide.

Recent studies have proposed that tumor necrosis factor alpha (TNF-alpha) and ionizing radiation induce apoptosis by activating hydrolysis of sphingomyelin to ceramide. Bcl-2 and a related gene, Bcl-X, inhibit several forms of apoptosis. Herein, we report that internucleosomal DNA fragmentation, characteristic of apoptosis and induced by ionizing radiation, is accompanied by concomitant decreases in Bcl-2 and Bcl-X mRNA levels in HL-60 and U-937 human leukemia cells. Apoptotic DNA fragmentation after exposure to TNF-alpha and C2-ceramide was also associated with down-regulation of Bcl-2 mRNA in HL-60 and U-937 cells, while Bcl-X mRNA production was unaffected. These results suggest that modulation of Bcl-2 gene expression may be a target for ceramide-mediated apoptosis following exposure to ionizing radiation and TNF-alpha. Changes in Bcl-2 expression may be the basis for the interactive killing observed between radiation and TNF-alpha in some human and tumor cells.

Apoptosis↗
PubMed1995

[An experimental investigation on preventive measures of skin ulcers induced by extravascular mitomycin C].

In order to make an investigation on skin ulcer induced by Mitomycin C extravasation, the preventive measures were undergone by the following ex-periment. A subcutaneous ulcerative model induced by 0.1mg Mitomycin C (MMC) extravasation in the leg of BALA/C mice was established. On the model subject, 8 pharmacologic adjuvants or physical modulation were adopted. It is preferential to choose local injection with 0.1 ml 99% dimethyl sulfoxide (DMSO), or with additional smearing DMSO on the affected skin. 10 days later, the ulcerative areas with DMSO treatment were significantly decreased in contrast with 0.1 ml 0.9% NaCI treatment (P < 0.001). The other preferential treatment were in turn by the injection of 0.1 ml 5% NaHCO3 (P < 0.001); 0.1 ml 1% procainamidi hydrochloridum (including 0.4% dexame-thasonum) (P < 0.001) or smearing with 0.1 ml 90% DMSO (including 10% Vit E) (P < 0.01). Delayed application of DMSO after MMC extravasation 30 min had been proven the limited effects on the prevention of ulcer (P < 0.05). Neither cooling of skin or local injection of sodium thiosulfate reduced MMC ulceration (P > 0.05). The mechanisms of prevention of MMC extravascular ulcer by DMSO or 5% NaHCO3 were briefly discussed.

Animals↗
PubMed1995

Key role of a CCAAT element in regulating hepatitis B virus surface protein expression.

Two separate promoters, the upstream preS1 and the downstream S promoters, give rise to transcripts encoding three forms of the hepatitis B virus surface protein. Overproduction of large surface protein because of increased preS1 transcripts leads to a block in secretion of all forms of the surface protein and of virion particles. We show here that a CCAAT element in the S promoter not only increases the amount of S transcripts, but also decreases the amount of preS1 transcripts by up to fivefold. Consequently, mutations in this element cause intracellular accumulation of surface proteins because of the secretory block. Therefore, this CCAAT element appears to be critical for maintaining the high ratio of S versus preS1 transcripts that is necessary for the viral life cycle.

Base Sequence↗
PubMed1995

Assignment of the 36.5-kDa (RFC5), 37-kDa (RFC4), 38-kDa (RFC3), and 40-kDa (RFC2) subunit genes of human replication factor C to chromosome bands 12q24.2-q24.3, 3q27, 13q12.3-q13, and 7q11.23.

Replication factor C is a multimeric primer-recognition protein consisting of five subunits (p145, p40, p38, p37, and p36.5) and is essential for the processive elongation of DNA chains catalyzed by DNA polymerase delta or epsilon in human cells. We have mapped the locations on human chromosomes of the genes coding for the four smaller subunits [p36.5 (RFC5), p37 (RFC4), p38 (RFC3), and p40 (RFC2)] using both PCR amplification from DNAs of a panel of somatic hybrids and fluorescence in situ hybridization to bands 12q24.2-q24.3, 3q27, 13q12.3-q13, and 7q11.23, respectively.

Base Sequence↗
PubMed1995

Vaccination against Lyme disease caused by diverse Borrelia burgdorferi.

Diversity and mutations in the genes for outer surface proteins (Osps) A and B of Borrelia burgdorferi sensu lato (B. burgdorferi), the spirochetal agent of Lyme disease, suggests that a monovalent OspA or OspB vaccine may not provide protection against antigenically variable naturally occurring B. burgdorferi. We now show that OspA or OspB immunizations protect mice from tick-borne infection with heterogeneous B. burgdorferi from different geographic regions. This result is in distinct contrast to in vitro killing analyses and in vivo protection studies using syringe injections of B. burgdorferi as the challenge inoculum. Evaluations of vaccine efficacy against Lyme disease and other vector-borne infections should use the natural mode of transmission and not be predicated on classification systems or assays that do not rely upon the vector to transmit infection.

Animals↗
PubMed1995

Evaluation of a multiple peptide assay for typing of antibodies to the hepatitis C virus: relation to genomic typing by the polymerase chain reaction.

A panel of 16 type-specific synthetic peptides corresponding to variable antigenic regions within the hepatitis C virus (HCV) core, nonstructural 4 (NS4), and NS5 proteins was synthesised. The peptide panel was used to develop an enzyme immunoassay (EIA) for the detection of antibodies directed to HCV type 1 (genotypes I/1a and II/1b), type 2 (genotypes III/2a and IV/2b), and type 3 (genotype V/3). The peptides corresponded to residues 68-81 of the HCV core (types 1, 2, and 3), residues 1692-1705 and 1710-1728 of HCV NS4 (types 1a, 1b, 2a, 2b, and 3), and residues 2303-2319 of HCV NS5 (types 1a, 1b, 2a, and 2b). The 16-peptide panel was evaluated using human sera from 46 carriers of HCV, which were genotyped in parallel by the polymerase chain reaction (PCR) using primers specific for types I, II, III, IV, and V of HCV core. Of the 46 carriers, 14 (30%) were infected by HCV genotype I, 7 (15%) by genotype II, 16 (35%) by HCV genotype IV, and 6 (13%) by HCV of genotype V. Two carriers had double infections of types I and II, and the HCV strain of one carrier could not be genotyped. Using the serotyping system, 40 (89%) out of the 45 genotyped carriers were found to contain type-specific antibodies corresponding to the genotypes identified by PCR. In 5 of the 23 carriers infected by genotypes I and/or II, antibodies specific for HCV type 1 could not be detected, whereas all 16 carriers infected by genotype IV were serologically typed as type 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗