Search PubMed⌕ Search

Biomedical subjects

C Chen

Publications and source records attributed to C Chen.

At least 865 records · Page 48Linked to original sources
PubMed1995

Age-dependent changes in response of rat prostatic tissues to isoproterenol and forskolin: changes with sexual maturation in function of G proteins.

Developmental changes in the responses of rat ventral prostate to isoproterenol (IPR) and forskolin (F) were studied in relation to the function of beta-adrenoceptor-adenylate cyclase system. The response of adenylate cyclase in the tissues to IPR at 10(-7)M and above was steadily enhanced after birth and reached a maximum at 12 weeks, followed by a decrease with age. In contrast, the response of the enzyme to F at 10(-7)M and above was highest at 2 weeks, but thereafter decreased. The changes in the response of the enzyme to IPR coincided with changes in the beta-adrenoceptor density and the binding ability of GTP binding proteins (G proteins) to GTP. The ADP-ribosylation of inhibitory G proteins (Gi proteins) catalyzed by pertussis toxin (IAP) decreased 70% in the tissues from 4 to 8 weeks, and then maintained this level. On the other hand, the ADP-ribosylation of stimulatory G proteins (Gs proteins) catalyzed by cholera toxin (CT) increased only 20% in the tissues from 2 to 4 weeks. Thus, the ratio of ADP-ribosylation of Gs to that of Gi significantly increased from 4 weeks, reaching a maximum at 12 weeks, but thereafter decreased gradually with age. These changes paralleled those in the function of G proteins and the response of the enzyme to IPR. It is suggested that the rapid and marked decrease in apparent level of Gi proteins in the rat ventral prostate after 4 weeks may have a key role in controlling the function of the beta-adrenoceptor-adenylate cyclase system in the tissues.

Adenosine Diphosphate Ribose↗
PubMed1995

Enhancement and destruction of antibody function by somatic mutation: unequal occurrence is controlled by V gene combinatorial associations.

We examined the positive and negative effects of somatic mutation on antibody function using saturation mutagenesis in vitro to mimic the potential of the in vivo process to diversify antibodies. Identical mutations were introduced into the second complementarity determining region of two anti-phosphocholine antibodies, T15 and D16, which share the same germline VH gene sequence. T15 predominates in primary responses and does not undergo affinity maturation. D16 is representative of antibodies that co-dominate in memory responses and do undergo affinity maturation. We previously reported that > 50% of T15 mutants had decreased antigen binding capacity. To test if this high frequency of binding loss was unique to T15 or a consequence of random point mutations applicable to other combining sites, we analyzed the same mutations in D16. We show that D16 suffers a similar loss of function, indicating an equally high potential for B-cell wastage. However, only D16 displayed the capacity for somatic mutation to improve antigen binding, which should enhance its persistence in memory responses. Mutation of residues contacting the haptenic group, as determined by molecular modeling, did not improve binding. Instead, productive mutations occurred in residues that either contacted carrier protein or were distant from the antigen binding site, possibly increasing binding site flexibility through long-range effects. Targeting such residues for mutation should aid in the rational design of improved antibodies.

Amino Acid Sequence↗
PubMed1995

The third extracellular loop of the mu opioid receptor is important for agonist selectivity.

To investigate the interaction between the mu opioid receptor and its ligands, we compared the binding of mu-selective ligands to two mu/kappa chimeric opioid receptors and to mu and kappa receptors. The two chimeras were constructed from cloned rat mu and kappa receptors in which a segment from the middle of the third intracellular loop to the C terminus was exchanged. When this portion of the kappa receptor was replaced by that of the mu receptor, affinities of mu selective agonists, DAMGO (Tyr-D-Ala-Gly-NMePhe-Gly-ol), PL017 (Tyr-Pro-NMePhe-D-Pro-NH2), sufentanil, and morphine, were greatly increased as compared to those for the kappa receptor. Conversely, when this region of the mu receptor was substituted by that of the kappa receptor, affinities for these agonists were substantially decreased as compared with those of the mu receptor. Unlike selective agonists, the mu-selective antagonist, CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-penicillamine-Thr-NH2), displayed a low affinity for both chimeric receptors, similar to that of the kappa receptor. Thus, the region from the middle of the third intracellular loop to the C terminus of the mu receptor is important for the binding of selective agonists. Conversely, the determinants for selective binding of the antagonist CTAP reside in a more extended region of the receptor.

Amino Acid Sequence↗
PubMed1995

Dopamine causes stimulation of protein kinase C in rat renal proximal tubules by activating dopamine D1 receptors.

Although it is suggested that in the renal proximal tubules, dopamine D1 receptor activation causes inhibition of Na+/K+ATPase via a phospholipase C and protein kinase C coupled pathway, the direct stimulation of protein kinase C by dopamine has not been reported. The present study was designed to examine the effects of dopamine and selective dopamine D1 receptor and dopamine D2 receptor agonists on protein kinase C activity. The renal proximal tubule suspensions were obtained from male Sprague-Dawley rats. The tubules were incubated separately with dopamine and fenoldopam in the presence or absence of dopamine D1 receptor antagonist, SCH 23390 ([(R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3- benzazepine]). The protein kinase C activity was measured by using a kinase target peptide, conjugated to a fluorescent molecule in water. The amino acid sequence of this peptide is, Proline-Leucine-Serine-Arginine-Threonine-Leucine-Serine-Valine-Alanine- Alanine-Lysine(PKSRTLSVAAK). We found that dopamine and fenoldopam [6-chloro-2,3,4,5-tetrahydro-1-(4-hydroxyphenyl)-1H-3-benzazepine-7,8-di ol] produced concentration-dependent increases in protein kinase C activity, which was blocked by SCH 23390. However, the dopamine D2 receptor agonist, bromocriptine [(5' alpha)-2-bromo-12'-hydroxy-2'-(1-methyl-ethyl)-5'-(2-methylpropyl)erg o- taman-3',6',18-trione] failed to stimulate protein kinase C activity at all the concentrations tested. These results provide direct evidence that dopamine stimulates protein kinase C activity via activation of dopamine D1 receptors.

Animals↗
PubMed1995

The Drosophila insulin receptor contains a novel carboxyl-terminal extension likely to play an important role in signal transduction.

The nucleic acid and deduced amino acid sequence of the Drosophila insulin receptor homologue (dir) has been determined. The coding sequence of dir is contained within 10 exons spanning less than 8 kilobase pairs of genomic DNA. The deduced amino acid sequence of the dir encodes a protein of 2148 amino acids, larger than the human insulin receptor due to amino- and carboxyl-terminal extensions. The overall level of amino acid identity between the DIR and human insulin and insulin-like growth factor-I receptors is 32.5 and 33.3%, respectively. Higher levels of identity are found in exon 2 (45 and 43%, respectively) and in the beta subunit (50 and 48%, respectively), and the positions of most cysteine residues in the alpha subunit cysteine-rich domain are conserved. A novel, 400-amino acid, carboxyl-terminal extension contains 9 tyrosine residues, four of which are present in YXXM or YXXL motifs, suggesting that they function as binding sites for SH2 domain-containing signaling proteins. The presence of multiple putative SH2 domain binding sites in the DIR represents a significant difference from its mammalian homologues and suggests that, unlike the human insulin and insulin-like growth factor-I receptors, the DIR forms stable complexes with signaling molecules as part of its signal transduction mechanism.

Amino Acid Sequence↗
PubMed1995

Nitric oxide donors enhanced Ca2+ currents and blocked noradrenaline-induced Ca2+ current inhibition in rat sympathetic neurons.

1. The effects of NO donors on Ca2+ channel currents and noradrenaline (NA)-induced Ca2+ current inhibition were investigated in superior cervical ganglion (SCG) neurons using the whole-cell patch-clamp technique. 2. A 500 microM concentration of the NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP), enhanced Ca2+ current amplitude after either extracellular or intracellular application. The magnitude of Ca2+ current enhancement induced by NO donors was greater after intracellular application than after extracellular application. 3. Intracellular application of 1 mM guanosine 3',5'-cyclic monophosphate (cGMP) or 100 microM M&B 22948 (2-O-propoxyphenyl-8-azapurine-6-one), a cGMP phosphodiesterase inhibitor, or extracellular application of 1 mM 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) also increased the amplitude of Ca2+ currents thus mimicking the effect of the NO donors on Ca2+ channels. In contrast, pretreatment with Methylene Blue (100 microM) decreased the SNP (500 microM)-induced enhancement of Ca2+ currents. 4. Intracellular application of 500 microM SNP and SNAP, 100 microM M&B 22948 or 1 mM cGMP, or extracellular application of 200 microM 8-Br-cGMP reduced the magnitude of Ca2+ current inhibition induced by 5 microM NA. In addition, 500 microM SNP prevented the NA-induced shift of tail current activation curves to more depolarized potentials. 5. Internal dialysis with 500 microM SNP and SNAP or 1 mM cGMP, or extracellular application of 200 microM 8-Br-cGMP, reduced Ca2+ current facilitation produced by a depolarizing conditioning pulse both in the absence and presence of 5 microM NA. 6. The results suggest that NO donors induce enhancement of Ca2+ currents and block NA-induced Ca2+ current inhibition of SCG neurons via stimulation of cGMP formation.

Animals↗
PubMed1995

Cigarette smoke extract inhibits oxidative modification of low density lipoprotein.

Oxidative modification of low density lipoprotein (LDL) by cigarette smoke has been suggested in several recent studies. To characterize possible modification of LDL by cigarette smoke extract (CSE), we incubated LDL with CSE either in the presence or absence of the chemical pro-oxidants, cupric chloride or 2,2'-azo-bis(2-amidinopropane) hydrochloride (AAPH). Surprisingly, CSE inhibited oxidative modification of LDL induced by either copper or AAPH. Under such oxidant stress conditions, CSE inhibited formation of thiobarbituric acid-reactive substances and also inhibited the increased agarose gel electrophoretic mobility of LDL in a dose-response manner. In addition, CSE prevented degradation of phosphatidylcholine to lysophosphatidylcholine and also fragmentation of the apolipoprotein B-100 moiety of LDL. Finally, CSE inhibited loss of immunoreactivity of the treated LDL with a murine monoclonal antibody against human apolipoprotein B-100. On the other hand, at higher concentrations, CSE per se was still able to cause structural changes in LDL. After incubation with CSE for 24 h, LDL showed a slight increase in agarose gel electrophoretic mobility, a slight loss of immunoreactivity with monoclonal antibody, and a marked increase in protein carbonyl formation. Lipid peroxidation did not appear to be involved in the modification of LDL caused by CSE. It is suggested that reactive aldehydes present in cigarette smoke may cause direct chemical modification of LDL. Furthermore, the free radical-scavenging potential of the tar fraction of cigarette smoke may be responsible for the apparent antioxidant properties of CSE against LDL oxidation.

Electrophoresis, Agar Gel↗
PubMed1995

The site and stage of anti-DNA B-cell deletion.

Antibodies to DNA and nucleoproteins are found in sera of individuals with systemic autoimmune disease. In the population (and in the autoimmune mouse strain MRL/lpr) there is a great variety of such antinuclear antibodies, but individuals with systemic lupus erythematosus or single MRL mice express a subset only of the antinuclear specificities found in the population. These observations have been interpreted to mean that these antibodies arise by immunization. The oligoclonal nature of the autoantibody response and the evidence of selection acting on somatically mutated autoantibodies favour this interpretation. Specific activation of autoantibodies in disease implies either that autoantibodies are regulated in non-diseased individuals or that autoantigen availability is variable. The former has been demonstrated in anti-DNA transgenic mice. In normal mice, transgene-encoded antibodies against double-stranded (ds) DNA are not expressed in serum or on B cells. Here we describe modified anti-dsDNA transgenic mice which allow us to study the site and developmental stage at which such B-cell regulation occurs. This model shows that in normal mice B cells expressing anti-DNA specificity are deleted in the bone marrow at a pre-B to immature B transitional stage.

Animals↗
PubMed1995

Alpha 1-adrenoceptor subtypes mediating stimulation of Na+,K(+)-ATPase activity in rat renal proximal tubules.

Although both alpha 1A- and alpha 1B-adrenoceptors are present in renal proximal tubules, the involvement of these receptor subtypes in the stimulation of Na+,K(+)-ATPase activity is not known. This study was undertaken to delineate the receptor subtype(s) involved in alpha 1-adrenoceptor-mediated increase in Na+,K(+)-ATPase activity and to identify the cellular signaling mechanisms such as stimulation of inositol triphosphate formation (IP3) and protein kinase C activation in this phenomenon. It was found that norepinephrine-induced increase in Na+,K(+)-ATPase activity was attenuated by prazosin, but not by rauwolscine, indicating the involvement of alpha 1-adrenoceptors. Furthermore, this response was selectively inhibited by the alpha 1B-adrenoceptor inactivator, chloroethylclonidine (100 microM), but not by the alpha 1A-adrenoceptor antagonist, WB4101 (0.01 microM). We examined whether these effects on Na+,K(+)-ATPase activity are mediated via the activation of IP3 and protein kinase C. Phenylephrine-induced increase in IP3 levels was abolished by prazosin, and significantly inhibited by WB4101, but not by chloroethylclonidine. Similarly, phenylephrine-induced activation of protein kinase C was sensitive to blockade by WB4101, but not by chloroethylclonidine. These results suggest that whereas both alpha 1A- and alpha 1B-adrenoceptors are present in proximal tubules, alpha 1B-adrenoceptors are involved in stimulating Na+,K(+)-ATPase activity and alpha 1A-adrenoceptors are predominantly linked to renal tubular IP3 production and protein kinase C activation. Therefore, it appears that norepinephrine-induced stimulation of Na+,K(+)-ATPase activity does not involve phospholipase-C-coupled protein kinase C pathway.

Adrenergic alpha-1 Receptor Agonists↗
PubMed1995

Inhibition of lecithin: cholesterol acyltransferase activity in human blood plasma by cigarette smoke extract and reactive aldehydes.

Cigarette smoking is a risk factor for atherosclerosis. It is conceivable that reactive chemical components in cigarette smoke may adversely affect reverse cholesterol transport at the level of lecithin: cholesterol acyltransferase (LCAT) and promote atherogenesis. Hence, the effect of cigarette smoke extract (CSE) on the activity of LCAT in human plasma was studied. When incubated with plasma, CSE caused both concentration- and time-dependent losses of LCAT activity. Addition of glutathione, but not ascorbate, to plasma prevented loss of LCAT activity caused by CSE. Incubation of plasma with some reactive aldehydes known to be present in cigarette smoke also inhibited LCAT activity. Among five aldehydes tested, acrolein was the strongest inhibitor of LCAT, with complete enzyme inhibition occurring at 1 mM. Acetaldehyde was the weakest inhibitor of LCAT, with 85% enzyme inhibition at 50 mM. Hexanal, formaldehyde, and malondialdehyde completely inhibited LCAT activity at 10, 50, and 50 mM, respectively. When plasma was incubated with 1 mM acrolein in the presence of 2.5 mM glutathione or dihydrolipoic acid, 100 and 57% of LCAT activity, respectively, remained after incubation. This finding suggest that reactive aldehydes may form adducts with certain free sulfhydryl groups functioning in the active site of LCAT to inhibit enzyme activity. It is concluded that reactive aldehydes are at least partially responsible for the reduction in LCAT activity in plasma treated with CSE.

Adult↗
PubMed1995

Comparison of arbitrarily primed polymerase chain reaction and ribotyping for subtyping Actinobacillus actinomycetemcomitans.

This study investigated the compatibility of arbitrarily primed polymerase chain reaction (AP-PCR) and ribotyping in the characterization of Actinobacillus actinomycetemcomitans , a major pathogen in the mixed anaerobic microflora of human periodontitis. AP-PCR was performed directly on lysed bacterial colonies using a random-sequence 10-base oligonucleotide primer. Ribotyping was carried out by using purified bacterial chromosomal DNA digested with BglI. DNA fragments were separated electrophoretically, blotted onto a nylon membrane and hybridized with the plasmid pKK3535 containing the rRNA operon of Escherichia coli. The two genetic methods were evaluated on isolates from single individuals and from family members. Twelve AP-PCR types and 47 ribotypes were distinguished among 76 A. actinomycetemcomitans isolates of different serotypes. AP-PCR typing and ribotyping gave compatible results in 18 of 20 comparisons. Although AP-PCR detected less genetic heterogeneity in A. actinomycetemcomitans than ribotyping, the rapid and relatively simple AP-PCR technique seems to be sufficiently discriminative to be used in large scale epidemiological studies which preclude the application of the more laborious ribotyping technique.

Journal Article↗
PubMed1995

Potentiation by enalaprilat of fenoldopam-evoked natriuresis is due to blockade of intrarenal production of angiotensin-II in rats.

We have previously shown that the natriuretic response to DA-1 receptor agonist fenoldopam is markedly potentiated by angiotensin converting enzyme inhibitor captopril. Since inhibition of angiotensin converting enzyme can lead to decreased production of angiotensin-II and increased levels of kinins (e.g., bradykinin), it is likely that both of these mechanisms might be involved in this phenomenon. However, it is not known whether and to what degree the accumulation of kinins contributes to the overall potentiation of natriuretic response to fenoldopam seen during angiotensin converting enzyme inhibition. In the present study, we have examined the effect of angiotensin converting enzyme inhibitor enalaprilat and angiotensin-II receptor antagonist losartan as well as bradykinin-2 receptor antagonist HOE 140 on fenoldopam-induced natriuresis. Intravenous infusion of fenoldopam (1 microgram/kg/min) for 30 min produced significant increases in urine output and urinary sodium excretion without causing any changes in glomerular filtration rate, renal blood flow and mean arterial blood pressure, a phenomenon suggestive of a direct tubular site of action. In animals treated with either the angiotensin converting enzyme inhibitor enalaprilat or angiotensin-II receptor antagonist losartan, the diuretic and natriuretic effects of fenoldopam were potentiated to a similar degree. Whereas no significant changes in glomerular filtration rate occurred when fenoldopam alone was given to control rats, in animals treated with either enalaprilat or losartan, fenoldopam produced a modest but significant increase in glomerular filtration rate. In a separate group of animals, the effects of bradykinin-2 receptor antagonist HOE 140 on potentiation of fenoldopam-induced natriuresis by enalaprilat was examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Antagonists↗
PubMed1995

Modulation of Na+ channel inactivation by the beta 1 subunit: a deletion analysis.

Na+ currents recorded from Xenopus oocytes expressing the Na+ channel alpha subunit alone inactivate with two exponential components. The slow component predominates in monomeric channels, while co-expression with the beta 1 subunit favors the fast component. Macropatch recordings show that the relative rates of these components are much greater than previously estimated from two-electrode measurements (approximately 30-fold vs approximately 5-fold). A re-assessment of steady-state inactivation, h infinity (V), shows that there is no depolarized shift of the slow component, provided a sufficiently long prepulse duration and repetition interval are used to achieve steady-state entry and recovery from inactivation, respectively. Deletion mutagenesis of the beta 1 subunit was used to define which regions of the subunit are required to modulate inactivation kinetics. The carboxy tail, comprising the entire predicted intracellular domain, can be deleted without a loss of activity; whereas small deletions in the extracellular amino domain or the signal peptide totally disrupt function.

Amino Acid Sequence↗
PubMed1995

Effect of peroxyl radicals on lecithin/cholesterol acyltransferase activity in human plasma.

The objective of this study was to determine whether subjecting human plasma to oxidant stress reduces the activity of lecithin/cholesterol acyltransferase (LCAT, EC 2.3.1.43). Plasma was incubated for 4 h with 2.25-45 mM of 2,2'-azobis(2-amidinopropane)HCl (AAPH), a source of peroxyl radicals. A time- and concentration-dependent reduction of LCAT activity occurred, relative to control samples incubated in the absence of AAPH. Reduction of LCAT activity was disproportionate to elevation of thiobarbituric acid-reactive substances (TBARS) in the plasma. Added ascorbate was able to significantly prevent reduction of LCAT activity, but this effect was unrelated to blockage of TBARS formation by the antioxidant. The results suggest that LCAT activity can be down-modulated by oxidant stress, but not necessarily by lipid peroxidation.

Amidines↗
PubMed1995

Intracardiac ultrasonographic assessment of atrial septal defect area: in vitro validation and technical considerations.

Assessment of atrial septal defect (ASD) size and shape is important for planning and guiding its transcatheter occlusion and can potentially be achieved by intracardiac ultrasonography (ICUS). ICUS accuracy, however, must first be established against stable standards and technical imaging requirements defined. We therefore used 10, 20, and 30 MHz ICUS catheters to examine 17 ASDs that were 0.16 to 6.7 cm2 in area and were surgically created in excised ovine hearts with 10, 20, and 30 MHz ICUS catheters. ASD shape and area by ICUS were compared with direct video images of the actual ASD. In all instances minimal area by ICUS pullback agreed well with actual values (y = 1.04x + 0.2, SEE = 0.23 cm2, r = 0.99) and corresponded well with defect shapes. The maximum angle between ultrasonography beam and septal plane allowing for complete ASD visualization was 20 degrees. The angle depended on transducer frequency and septal thickness. This new technique has potential value for the accurate assessment of ASD shape and size and may be especially useful in the setting of transcatheter occlusion.

Animals↗