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Biomedical subjects

H Cheng

Publications and source records attributed to H Cheng.

At least 289 records · Page 16Linked to original sources
PubMed1996

Monocyte chemoattractant protein 1 and interleukin-8 production in mononuclear cells stimulated by oral microorganisms.

Chemokines are a family of low-molecular-weight proinflammatory cytokines that stimulate recruitment of leukocytes. The chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) are relatively specific chemoattractants for neutrophils and monocytes, respectively. Chemokine expression contributes to the presence of different leukocyte populations observed in normal and pathologic states. In the present studies, peripheral blood mononuclear cells (PBMC) were stimulated by microbes (Candida albicans, Streptococcus mutans, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans) selected based upon their importance as oral pathogens. IL-8 and MCP-1 gene expression and protein release were determined by Northern blot (RNA blot) analysis and enzyme-linked immunosorbent assay. C. albicans, P. gingivalis, and A. actinomycetemcomitans induced high levels of production of both MCP-1 and IL-8. S. mutans was a strong inducer of MCP-1, but it did not stimulate significant production of IL-8. C. albicans, S. mutans, and A. actinomycetemcomitans were 500 to 5,000 times more potent than P. gingivalis in terms of MCP-1 production. In general, the microbe-to-PBMC ratios required for maximum gene expression of MCP-1 were lower than those for IL-8. However, for actual protein release of MCP-1 versus IL-8, differences in the effects of various microbe concentrations were observed only for A. actinomycetemcomitans. These results demonstrate that different oral pathogens induce specific dose-dependent patterns of chemokine gene expression and release. Such patterns may help explain the immunopathology of oral infections, particularly with regard to inflammatory leukocyte recruitment.

Aggregatibacter actinomycetemcomitans↗
PubMed1996

Calcium sparks and [Ca2+]i waves in cardiac myocytes.

Local elevations in intracellular calcium ("Ca2+ sparks") in heart muscle are elementary sarcoplasmic reticulum (SR) Ca(2+)-release events. Ca2+ sparks occur at a low rate in quiescent cells but can also be evoked by electrical stimulation of the cell to produce the cell-wide Ca2+ transient. In this study we investigate how Ca2+ sparks are related to propagating waves of elevated cytosolic Ca2+ induced by "Ca2+ overload." Single ventricular myocytes from rat were loaded with the Ca(2+)-sensitive indicator fluo 3 and imaged with a confocal microscope. After extracellular Ca2+ concentration was increased from 1 to 10 mM to produce Ca2+ overload, the frequency of spontaneous Ca2+ sparks, which occur at the t tubule/SR junction, increased approximately 4-fold, whereas the spark amplitude and spatial size increased 4.1-and 1.7-fold, respectively. In addition, a spectrum of larger subcellular events, including propagating Ca2+ waves, was observed. Ca2+ sparks were seen to occur at the majority (65%) of the sites of wave initiation. For slowly propagating Ca2+ waves, discrete Ca(2+)-release events, similar to Ca2+ sparks, were detected in the wave front. These Ca2+ sparks appeared to recruit other sparks along the wave front so that the wave progressed in a saltatory manner. We conclude that Ca2+ sparks are elementary events that can explain both the initiation and propagation of Ca2+ waves. In addition, we show that Ca2+ waves and electrically evoked Ca2+ transients have the same time course and interact with each other in a manner that is consistent with both phenomena having the same underlying mechanism(s). These results suggest that SR Ca2+ release during Ca2+ waves, like that during normal excitation-contraction coupling, results from the spatial and temporal summation of Ca2+ sparks.

Animals↗
PubMed1996

Relation between the sarcolemmal Ca2+ current and Ca2+ sparks and local control theories for cardiac excitation-contraction coupling.

Ca2+ sparks, the elementary events underlying excitation-contraction (E-C) coupling, occur when sarcoplasmic reticulum (SR) Ca2+ release channels open. They are activated locally by Ca2+ influx through sarcolemmal (SL) Ca2+ channels. By measuring the probability of spark occurrence under conditions in which their probability of occurrence is low, we address two important questions raised by our recent work: (1) When a Ca2+ spark is triggered, how many SL Ca2+ channels (at a minimum) contribute to its activation? (2) What is the relation between the subcellular local [Ca2+], produced by the opening of SL Ca2+ channels and the consequent SR Ca2+ release? By comparing the voltage dependence of Ca2+ sparks in rat ventricular myocytes with the Ca2+ current, we show that the opening of a single SL Ca2+ channel can trigger a Ca2+ spark. Furthermore, we deduce that the probability of SR Ca2+ release depends of the square of the local [Ca2+]i produced by SL Ca2+ channel openings. These results are discussed with respect to the properties of Ca2+-induced Ca2+-release (CICR) and the local control theory of excitation-contraction coupling.

Animals↗
PubMed1996

Identification of classical, novel, and atypical protein kinase C isoenzymes in the bovine parathyroid.

Because phospholipid metabolism leading to the activation of protein kinase C (PKC) may play a key regulatory role in the degradation and secretion of PTH, we examined parathyroid cell fractions for the presence of various PKC isoenzymes. Hydroxylapatite chromatography identified the classical PKCs, alpha and beta, but not gamma in parathyroid cell extracts. Western blot analysis confirmed the presence of PKC alpha and beta in these extracts. Of the so-called novel PKCs, Western blot analysis revealed the presence of only one isoenzyme, novel PKC epsilon in parathyroid cell soluble extracts. Western blot analysis using an antibody to the C-terminus of the atypical isoenzyme, PKC zeta, identified a protein of lower molecular weight in addition to PKC zeta. This lower molecular weight protein presumably represents PKC lambda, which shares a high degree of C-terminal sequence similarity with PKC zeta. These findings suggest the possibility that members of all three groups of the PKC family are present and may play a regulatory role in the bovine parathyroid cell.

Animals↗
PubMed1996

Differential distribution of lumican and fibromodulin in tooth cementum.

The objectives of this study were to isolate and characterize the major proteoglycans of tooth cementum in relation to the tissue's mineralization. Cementum was collected from the root apex region of bovine molars and pulverized. It was first extracted with 6M guanidine-HCI, pH 7.4 (G-extract, mineral-unassociated) and then demineralized and extracted with 0.5M EDTA (E-extract, mineral-associated). Both extracts were applied to anion exchange and then molecular sieve chromatography to isolate proteoglycans. The fractions collected were assayed for chondroitin-(CS) and keratan sulfate (KS) containing proteoglycans using the monoclonal antibodies 2-B-6 and 5-D-4, respectively. It was found that the KS was the major glycosaminoglycan and was enriched in the G-extract fraction. The major KS fraction was then applied to 7.5% SDS-PAGE. The major broad band (69 kDa) was 5-D-4 positive in Western blot analysis and separated into two bands (46 kDa and 50 kDa) after treatment with keratanase II and endo-beta-galactosidase. These two proteins were transfered to PVDF membrane and analyzed for amino acid sequence. The results showed the major band (46 kDa) to be lumican and the minor (50 kDa) fibromodulin. In addition, based on the immunohistochemical study using a number of mono- and polyclonal antibodies including 5-D-4, anti-lumican core protein as well as anti-fibromodulin core protein antibodies, the KSPGs were found to be located almost exclusively in nonmineralized portions of cementum such as precementum and the pericementocyte area. These biochemical as well as immunohistochemical data suggest that the major KSPGs of cementum, lumican and fibromodulin, have a specific tissue distribution and may have regulatory roles in cementum mineralization.

Amino Acid Sequence↗
PubMed1996

Identification of human liver cytochrome P450 isoforms involved in the in vitro metabolism of cyclobenzaprine.

Cyclobenzaprine (Flexeril) is a muscle relaxant, possessing a tricyclic structure. Numerous therapeutic agents containing this structure are known to be metabolized by polymorphic cytochrome P4502D6. The aim of this study was to determine if cytochrome P4502D6 and other isoforms are involved in the metabolism of cyclobenzaprine in human liver microsomes. Selective cytochrome P450 inhibitors for CYP1A1/2 (furafylline and 7,8-benzoflavone) and CYP3A4 (troleandomycin, gestodene, and ketoconazole) inhibited the formation of desmethylcyclobenzaprine, a major metabolite of cyclobenzaprine, in human liver microsomes. Antibodies directed against CYP1A1/2 and CYP3A4 inhibited the demethylation reaction whereas anti-human CYP2C9/10, CYP2C19, and CYP2E1 antibodies did not show any inhibitory effects. When a panel of microsomes prepared from human B-lymphoblastoid cells that expressed specific human cytochrome P450 isoforms were used, only microsomes containing cytochromes P4501A2, 2D6, and 3A4 catalyzed N-demethylation. In addition, demethylation catalyzed by these recombinant cytochromes P450 can be completely inhibited with selective inhibitors at concentrations as low as 1 to 20 microM. Interestingly, cyclobenzaprine N-demethylation was significantly correlated with caffeine 3-demethylation (1A2) and testosterone 6 beta-hydroxylation (3A4) but not with dextromethorphan O-demethylation (2D6) in human liver microsomes. To further determine the involvement of cytochrome P4502D6 in cyclobenzaprine metabolism, liver microsomes from a human that lacked CYP2D6 enzyme activities was included in this study. The data showed that cyclobenzaprine N-demethylation still occurred in the incubation with this microsome. These results suggested that cytochrome P4502D6 plays only a minor role in cyclobenzaprine N-demethylation whereas 3A4 and 1A2 are primarily responsible for cyclobenzaprine metabolism in human liver microsomes. Due to the minimum involvement of CYP2D6 in the vitro metabolism of cyclobenzaprine, the polymorphism of cytochrome P4502D6 in man should not be of muci concern in the clinical use of cyclobenzaprine.

Adult↗
PubMed1996

Age-of-onset related HLA-DQA1 genetic heterogeneity of insulin-dependent diabetes mellitus.

OBJECTIVE: To clarify whether there is an association of insulin-dependent diabetes mellitus (IDDM) susceptibility with HLA-DQA1 locus in adult-onset IDDM. PATIENTS AND METHODS: Possible heterogeneity in immunogenetic aspects was investigated by comparing the findings on relationship between HLA-DQA1 52 Arg(+) and IDDM predisposition in three groups with different ages of IDDM onset (group A 14 years, group B 15-30 years, group C > 31 years). RESULTS: The frequency of HLA-DQA1 52 Arg(+) in group A (87.5%) was higher than that in control (53.9%, P < 0.05) and in group B and C (P < 0.05). The frequency of HLA-DQA1 52 Arg(+)/Arg(+) phenotype in group A (75%) was higher than that in group C (23.1%, P < 0.05). CONCLUSION: The contribution of HLA-DQA1 52 arginine to IDDM susceptibility is heterogeneous. Further study is necessary to clarify the immunogenetic entity of IDDM.

Adult↗
PubMed1996

[The role of thoracoscopy in the diagnosis and treatment of lung cancer].

Using video-assisted thoracoscopic techniques in 32 lung cancer patients over the past 3 years, we have performed a vriety of procedures previously accomplished by "open" techniques. This group of patients included stage I (7 patients), stage II-IIIa (10), stage IV (15). Our procedures include wedge resections of lung tumor using endoscopic mechnical stapling devicesin 7 patients; pulmonary lobectomy in 7; lung cancer staging in 7; pleural biopsies and pleurodesis in 11. There were no mortality and no serious complications associated with the procedure. Our experience indicates on expanded role of thoracoscopy in the diagnosis and treatment of lung cancer.

Adult↗
PubMed1996

[Expression of human brain myelin basic protein gene cDNA in Escherichia coli].

A 612bp full-length cDNA sequence coding for 21.5 kd myelin basic protein (MBP) was amplified from human brain cDNA library by polymerase chain reaction, cloned into vector pGEM-3Zf(+) with blunt-end. The 561bp cloned fragment was recovered from recombinant pGEMP after BamHI digestion, and then inserted into the BamHI site downstream of the glutathione-S-transferase gene of the expression vector pGEX-5T. The recombinant vector p5TBP was transformed into E. coli JM 109 and positive colonies were incubated in LB medium containing 1 mmol/L IPTG for six hours at 37 degrees C. A new polypeptide band with an apparent molecular weight 49 kd was detected in transformed cell lysates by SDS-PAGE. Western blotting analysis confirmed that this fusion protein reacted specifically with rabbit antibodies to human MBP. The expression level of MBP in this system was about 50 mg/L of bacterial culture estimate by immuno-dot blot, ELISA and absorbance scanning of SDS-PAGE.

Cloning, Molecular↗
PubMed1995

A novel endoprotease responsible for the specific cleavage of transducin gamma subunit.

Isoprenylated/methylated heterotrimeric G proteins play important roles in a large number of signal transduction processes. While the enzymology of isoprenylated/methylated protein biosynthesis is well understood, nothing is known about how these proteins are degraded. In this article, a novel endoproteolytic activity has been identified from bovine retina and is shown specifically to remove the glycylfarnesylcysteine moiety from the carboxyl terminus of T gamma. When tested in a GTP binding assay, freshly prepared proteolyzed T beta gamma was unable to catalyze the binding of guanosine 5'-(gamma-thio)triphosphate (GTP-gamma-S) to T alpha in the presence of detergent solubilized rhodopsin. The optimum pH for this proteolytic activity is approximately 6, and the pH profile corresponds to an enzyme having pKa's of 4.4 +/- 0.1 and 7.7 +/- 0.1 for its active site residues. After analyzing a series of protease inhibitors, we found E-64, a specific thiol protease inhibitor, to be the most effective irreversible inhibitor of this enzyme, suggesting that the endoprotease might be a thiol protease. Affinity labeling studies using biotinylated affinity labeling probes have identified a 35 kDa protein as a candidate for the endoprotease.

Amino Acid Sequence↗
PubMed1995

Relaxation of arterial smooth muscle by calcium sparks.

Local increases in intracellular calcium ion concentration ([Ca2+]i) resulting from activation of the ryanodine-sensitive calcium-release channel in the sarcoplasmic reticulum (SR) of smooth muscle cause arterial dilation. Ryanodine-sensitive, spontaneous local increases in [Ca2+]i (Ca2+ sparks) from the SR were observed just under the surface membrane of single smooth muscle cells from myogenic cerebral arteries. Ryanodine and thapsigargin inhibited Ca2+ sparks and Ca(2+)-dependent potassium (KCa) currents, suggesting that Ca2+ sparks activate KCa channels. Furthermore, KCa channels activated by Ca2+ sparks appeared to hyperpolarize and dilate pressurized myogenic arteries because ryanodine and thapsigargin depolarized and constricted these arteries to an extent similar to that produced by blockers of KCa channels. Ca2+ sparks indirectly cause vasodilation through activation of KCa channels, but have little direct effect on spatially averaged [Ca2+]i, which regulates contraction.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Site-specific mutagenesis demonstrates that the structural requirements for efficient electron transfer in Anabaena ferredoxin and flavodoxin are highly dependent on the reaction partner: kinetic studies with photosystem I, ferredoxin:NADP+ reductase, and cytochrome c.

Electron transfer reactions involving site-specific mutants of Anabaena ferredoxin (Fd) and flavodoxin (Fld) modified at surface residues close to the prosthetic groups, with photoexcited P700 in spinach photosystem I (PSI) particles, ferredoxin:NADP+ reductase (FNR), and horse cytochrome c (cytc), have been investigated by laser flash photolysis and stopped-flow spectrophotometry. Nonconservative mutations in Fd at F65 and E94, which have been shown to result in very large inhibitions of electron transfer to FNR, were found to yield wild-type behavior in reactions with PSI and cytc. In general, the effects of Fd mutagenesis on the PSI reactions were considerably smaller than those observed for the FNR reaction. In the case of Fld, mutagenesis was found to have only small effects on both the FNR and PSI reactions, although the specific sites whose mutation caused changes in electron transfer properties differed for the two systems. In contrast, several of the Fld mutants showed appreciably larger effects on the nonphysiological reaction with cytc. We conclude from these studies that the structural requirements for efficient electron transfer involving the Fd and Fld molecules differ, depending upon the reactant with which these redox proteins interact. This is consistent with the multiple roles that these proteins have in vivo in biological electron transfer and implies that different conserved residues in these proteins have evolved to satisfy varying requirements of particular reaction partners.

Anabaena↗
PubMed1995

The control of calcium release in heart muscle.

The control of calcium release from intracellular stores (the sarcoplasmic reticulum) in cardiac muscle was examined with the use of a confocal microscope and voltage clamp techniques. Depolarization evoked graded calcium release by altering the extent of spatial and temporal summation of elementary calcium release events called "calcium sparks." These evoked sparks were triggered by local L-type calcium channel currents in a stochastic manner, were similar at different potentials, and resembled spontaneous calcium sparks. Once triggered, the calcium release from the sarcoplasmic reticulum during a calcium spark was independent of the duration of the triggering calcium influx. These results were used to develop a unifying model for cardiac excitation-contraction coupling that explains the large (but paradoxically stable) amplification of the trigger calcium influx by a combination of digital and analog behavior.

Animals↗