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Biomedical subjects

J Zhao

Publications and source records attributed to J Zhao.

At least 685 records · Page 38Linked to original sources
PubMed1993

In vitro expression of the alpha-smooth muscle actin isoform by rat lung mesenchymal cells: regulation by culture condition and transforming growth factor-beta.

alpha-Smooth muscle actin (alpha SM actin)-containing cells recently have been demonstrated in intraalveolar lesions in both rat and human tissues following lung injury. In order to develop model systems for the study of such cells, we examined cultured lung cell lines for this phenotype. The adult rat lung fibroblast-like "RL" cell lines were found to express alpha SM actin mRNA and protein and to organize this actin into stress fiber-like structures. Immunocytochemical staining of subclones of the RL87 line demonstrated the presence in the cultures of at least four cell phenotypes, one that fails to express alpha SM actin and three distinct morphologic types that do express alpha SM actin. The proportion of cellular actin that is the alpha-isoform was modulated by the culture conditions. RL cells growing at low density expressed minimal alpha SM actin. On reaching confluent densities, however, alpha SM actin increased to at least 20% of the total actin content. This effect, combined with the observation that the most immunoreactive cells were those that displayed overlapping cell processes in culture, suggests that cell-cell contact may be involved in actin isoform regulation in these cells. Similar to the response of some smooth muscle cell lines, alpha SM actin expression in RL cells also was promoted by conditions, e.g., maintenance in low serum medium, which minimize cell division. alpha SM actin expression was modulated in RL cells by the growth factor transforming growth factor-beta. Addition of this cytokine to growing cells substantially elevated the proportion of alpha SM actin protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1993

[A study on P21(ras) in human lung cancer and body fluid of cancer patients].

The McAb of anti-P21(ras) has been used to screen 9 cases of human lung cancer tissues, 18 cases of samples and 2 cases of hydrothorax samples of lung cancer patients by means of PAGE-immunoblots. The expression of ras gene was found in 8 cases of human lung cancer among which the expression of 4 cases was higher than that of normal control. P21(ras) has not been detected in serum and hydrothorax samples of cancer patients. The present study indicated that there was P21(ras) in human lung cancer and normal control and the expression level of ras gene in lung cancer was related to the differentiation of cancer.

Adenocarcinoma↗
PubMed1992

[The biotransformation of NSAIDs: a common elimination site and drug interactions].

Many NSAIDs are eliminated predominantly through hepatic biotransformation in man. We have studied, in human hepatic microsomes, the role of specific cytochrome P450 isozymes in the formation of the major metabolites of oxicam (piroxicam and tenoxicam), phenylacetic (diclofenac) and propionic acid (ibuprofen) derivatives. A common isozyme (P450TB, CYP2C subfamily) controls the major elimination pathway of these NSAIDs. We have also determined, in two in vitro models of P450TB, the affinity for this isozyme of NSAIDs from other chemical classes (acetylsalicylic acid, mefenamic acid and indomethacin). All NSAIDs tested displayed a high affinity (3-300 microM) for cytochrome P450TB. Cytochrome P450TB plays a major role in the elimination of several NSAIDs with different chemical structures. NSAIDs are substrates as well as potential inhibitors of cytochrome P450TB. Their elimination can therefore be reduced by concomitant administration of known inhibitors of P450TB (antifungals, antibacterial sulfonamides, calcium channel blockers).

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1992

Effects of exercise training and diabetes on cardiac myosin heavy chain composition.

This study determined whether the beneficial effects of exercise training on the diabetic heart previously observed are associated with alterations in ventricular myosin heavy chain (MHC) isoform composition. Diabetes was induced in rats by i.v. streptozotocin. Trained rats were run on a treadmill for 60 min/day, 27 m/min, 10% grade. After 10 wks, ventricular MHC isoenzyme protein composition was analyzed for MHC composition using gel electrophoresis. alpha-MHC and beta-MHC mRNA were determined by Northern and slot blot hybridization techniques. Both protein and mRNA analyses indicated that sedentary control rats exhibited a predominance of alpha-MHC. Sedentary diabetics exhibited a shift to beta-MHC. Exercise trained diabetic rats showed a predominance of beta-MHC. The results indicate that treadmill exercise training of diabetic rat does not prevent the diabetes-induced shift in MHC composition towards the beta-MHC isoform, thus it is unlikely that the beneficial effects of exercise training on the diabetic heart, previously shown, are due to a normalization of the myosin isoform composition.

Animals↗
PubMed1992

Protection of the ischemic diabetic heart by L-propionylcarnitine therapy.

Diabetics suffer from an increased incidence of myocardial infarction and are less likely to survive an ischemic insult. Since L-propionylcarnitine (LPC) has been shown to protect against ischemic/reperfusion injury, we hypothesized that LPC may be of even greater benefit to the diabetic heart. Diabetes was induced by i.v. streptozotocin, 60 mg/kg; duration: 12 wks. The chronic effect of LPC was determined by daily i.p. injections (100 mg/kg) for 8 wks. The acute effects of LPC were determined by adding it to the perfusion medium (5 mM) of control and diabetic hearts. Initial cardiac contractile performance of isolated perfused working hearts was assessed by varying left atrial filling pressure. Hearts were then subjected to 90 min of low flow global ischemia followed by 30 min reperfusion. Chronic LPC treatment had no effect on initial cardiac performance in either control or diabetic hearts. Acute addition of LPC to the perfusion medium enhanced pump performance of control hearts, but had no effect in diabetic hearts. Both acute and chronic LPC significantly improved the ability of control and diabetic hearts to recover cardiac contractile performance after ischemia and reperfusion, however, chronic treatment was more effective in diabetic hearts.

Animals↗
PubMed1992

Regulation of glycoprotein IIb-IIIa receptor function studied with platelets permeabilized by the pore-forming complement proteins C5b-9.

Recent evidence suggests that the cytoplasmic domains of platelet glycoprotein (GP) IIb-IIIa are involved in the agonist-initiated transformation of this integrin into a receptor for fibrinogen. To identify intracellular reactions that regulate the receptor function of GP IIb-IIIa, membrane-impermeable agonists and antagonists were introduced into the platelet by permeabilizing the plasma membrane with the pore-forming complement proteins C5b-9. Platelet responses were then analyzed by flow cytometry. Non-lytic concentrations of C5b-9 caused permeabilization of the platelet plasma membrane, as determined by uptake of a water-soluble fluorescent tracer dye. The complement pores were large enough to permit the entry of fluorescein isothiocyanate (FITC)-labeled oligopeptides in a size-dependent manner. Under conditions of low external Ca2+, C5b-9 treatment per se did not activate GP IIb-IIIa, as measured by binding of the activation-dependent antibody FITC-PAC1. However, FITC-PAC1 binding to C5b-9-permeabilized platelets was stimulated by a thrombin receptor agonist acting at the cell surface and by guanosine 5'-O-(thiotriphosphate), a membrane-impermeable activator of G proteins. Permeabilization also permitted the entry of cyclic AMP and the peptide, RFARKGALRQKNV, a pseudo-substrate inhibitor of protein kinase C. Each of these inhibited agonist-induced FITC-PAC1 binding to permeabilized platelets but not to intact platelets. Agonist-induced GP IIb-IIIa activation in permeabilized platelets was also inhibited by tyrphostin-23, a protein tyrosine kinase inhibitor. Thus, C5b-9 can be used to permeabilize the plasma membrane to permit the selective entry of small peptides and other bioactive compounds into permeabilized platelets. Results obtained with these platelets indicate that GP IIb-IIIa receptor function is regulated by a network of signaling reactions involving G proteins, serine/threonine kinases, and tyrosine kinases.

Amino Acid Sequence↗
PubMed1992

Phycocyanin alpha-subunit phycocyanobilin lyase.

Phycobiliproteins, unlike other light-harvesting proteins involved in photosynthesis, bear covalently attached chromophores. The bilin chromophores are attached through thioether bonds to cysteine residues. The cyanobacterium Synechococcus sp. PCC 7002 has eight distinct bilin attachment sites on seven polypeptides, all of which carry the same chromophore, phycocyanobilin. When two genes in the phycocyanin operon of this organism, cpcE and cpcF, are inactivated by insertion, together or separately, the surprising result is elimination of correct bilin attachment at only one site, that on the alpha subunit of phycocyanin. We have overproduced CpcE and CpcF in Escherichia coli. In vitro, these proteins catalyze the attachment of phycocyanobilin to the alpha subunit of apophycocyanin at the appropriate site, alpha-Cys-84, to form the correct adduct. CpcE and CpcF also efficiently catalyze the reverse reaction, in which the bilin from holo-alpha subunit is transferred either to the apo-alpha subunit of the same C-phycocyanin or to the apo-alpha subunit of a heterologous C-phycocyanin. The forward and reverse reactions each require both CpcE and CpcF and are specific for the alpha-Cys-84 position. Phycocyanobilin is the immediate precursor of the protein-bound bilin.

Bile Pigments↗
PubMed1992

Liquid sample injection using an atmospheric pressure direct current glow discharge ionization source.

An atmospheric pressure DC glow discharge in helium has been used as an ionization source for organic samples introduced by liquid injection into atmospheric pressure ionization mass spectrometry (API/MS). The glow source operates typically in the range up to 1 mA of current at less than 1 kV, although the source can be operated up to a discharge current of 10 mA. Even at the high current used in this work, the protonated molecule, MH+, is observed with little or no fragmentation for many of the samples studied. The detection limits achieved for API glow discharge detection are typically in the low femtomole region for small organic molecules including small biological neurotransmitters, drugs, pesticides, phenylthiohydantoin-substituted amino acids, and explosives. A detection limit of approximately 2 pg has been achieved for tyramine with linear quantitation over at least 3 orders of magnitude. The sensitivity in these experiments has been further improved by optimization of the skimmer-interface system and the liquid injection/nebulization design.

Amino Acid Sequence↗
PubMed1992

Site-directed conversion of a cysteine to aspartate leads to the assembly of a [3Fe-4S] cluster in PsaC of photosystem I. The photoreduction of FA is independent of FB.

The terminal electron acceptors FA and FB exist as two [4Fe-4S] clusters located on the 8.9-kDa PsaC protein in photosystem I. We have used site-directed mutagenesis to produce a complementary pair of mutant PsaC proteins in which specific cysteine ligands to the [4Fe-4S] clusters were changed to aspartic acid residues. The mutant proteins, denoted C14D and C51D, were overproduced in Escherichia coli; the iron-sulfur clusters were inserted in vitro; and the reconstituted proteins were rebound to the P700-FX core of Synechococcus sp. PCC 6301 in the presence of the PsaD protein. In complexes reconstituted with C51D a rhombic ESR spectrum with g-values of 2.063, 1.934, and 1.879 in the reduced state identifies the intact [4Fe-4S] cluster as FB, while an intense axial spectrum with g-values of 2.020 and 1.997 in the oxidized state identifies the altered cluster in the aspartate site as a [3Fe-4S] cluster. The [3Fe-4S] cluster corresponding to FA can be reduced chemically with dithionite and photochemically by illumination at room temperature but is not reduced by illumination at 15 K. With reconstituted C14D a rhombic ESR spectrum with g-values of 2.043, 1.942, and 1.853 in the reduced state identified the unaltered [4Fe-4S] cluster as FA, while a complex spectrum with a gz-value of 2.194 and an asymmetric gx,y set of resonances between 2.092 and 1.999 indicates an altered cluster of unknown identity in the site containing the aspartate ligand. The ESR signals arising from the altered cluster corresponding to FB are not diminished by illumination at either room temperature or 15 K.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartic Acid↗
PubMed1992

Contribution of the N-linked carbohydrate of erythrocyte antigen CD59 to its complement-inhibitory activity.

The contribution of N-linked carbohydrate to the complement-inhibitory function of the human erythrocyte membrane glycoprotein, CD59, was investigated. Amino acid sequence analysis of tryptic peptides labeled with [3H]borohydride revealed an N-linked carbohydrate moiety at the Asn18 residue. No O-linked carbohydrate was detected, as judged by the failure of asialo-CD59 to bind peanut agglutinin and by its resistance to digestion by O-glycanase. The apparent molecular mass of CD59 was reduced from 18-20 to 14 kDa upon complete digestion with N-glycanase, with no detectable proteolysis. N-glycanase digestion of CD59 was associated with an 88 +/- 4% loss of the complement-inhibitory activity of the protein, as assessed by its capacity to protect chicken erythrocytes from lysis by the human C5b-9 proteins. By contrast, no change in function was observed after digestion of CD59 with neuraminidase, under conditions that removed greater than 60% of [3H]sialic acid residues. Despite loss of functional activity after N-glycanase digestion, we detected no change in the capacity of the deglycosylated CD59 to incorporate into erythrocyte membranes or to bind specifically and with species selectivity to the C8 and C9 components of the membrane attack complex. In order to alter the branched-chain structure of the N-linked carbohydrate of CD59 without enzymatic digestion, Chinese hamster ovary (CHO) cells transfected with cDNA for human CD59 were grown in the alpha-mannosidase inhibitor, 1-deoxymannojirimycin, resulting in conversion of approximately 70% of the membrane glycoprotein to a high mannose. When grown in the presence of 1-deoxymannojirimycin, the C5b-9-inhibitory activity of CD59 expressed on the surface of the transfected CHO cells was reduced by an amount comparable to that observed for the N-glycanase digested protein. Taken together, these data suggest that normal glycosylation of Asn18 in CD59 is required for the normal expression of its complement-inhibitory activity on membrane surfaces, although these N-linked sugar residues do not contribute to CD59's affinity for the C8 and C9 components of the C5b-9 complex.

Animals↗
PubMed1992

On the nature of origins of DNA replication in eukaryotes.

Chromosomal origins of DNA replication in higher eukaryotes differ significantly from those of E. coli (oriC) and the tumor virus, SV40 (ori sequence). Initiation events appear to occur throughout broad zones rather than at specific origin sequences. Analysis of four chromosomal origin regions reveals that they share common modular sequence elements. These include DNA unwinding elements, pyrimidine tracts that may serve as strong DNA polymerase-primase start sites, scaffold associated regions, transcriptional regulatory sequences, and, possibly, initiator protein binding sites and inherently destabilized regions. Based on the novel organization of chromosomal origin regions, we propose a model for initiation of DNA replication in higher eukaryotes. Unwinding of duplex DNA during initiation may be uncoupled, both temporally and spatially, from DNA synthesis, resulting in transient single-stranded intermediates that function in lieu of conventional replication forks during chromosomal DNA replication. DNA synthesis begins subsequently at multiple sites within the unwound regions rather than at specific origin sequences.

Animals↗
PubMed1992

Reversible dissociation and unfolding of the dimeric protein thymidylate synthase.

Conditions for in vitro unfolding and refolding of dimeric thymidylate synthase from Lactobacillus casei were found. Ultraviolet difference and circular dichroism spectra showed that the enzyme was completely unfolded at concentrations of urea over 5.5 M. As measured by restoration of enzyme activity, refolding was accomplished when 0.5 M potassium chloride was included in the refolding mixture. Recombination of subunits from catalytically inactive mutant homodimers to form an active hybrid dimer was achieved under these unfolding-refolding conditions, demonstrating a monomer to dimer association step.

Circular Dichroism↗
PubMed1992

Dystrophin and a dystrophin-related protein in intrafusal muscle fibers, and neuromuscular and myotendinous junctions.

To determine whether or not and how dystrophin exists in neuromuscular junctions (NMJs) and myotendinous junctions (MTJs), we studied the mid-belly and peripheral portions of control and mdx muscles, immunohistochemically and immunoelectrophoretically, using six kinds of polyclonal antibodies, and an antibody against a dystrophin-related protein (DRP). In controls these regions and the polar region of intrafusal muscle fibers showed a rather clearer immunohistochemical dystrophin reaction than those of extrafusal muscle fibers with all antibodies used. In the muscles of mdx mice NMJs only showed a positive dystrophin reaction with the c-terminal antibody, that is, no reaction with the other five antibodies, and MTJs in mdx showed a positive reaction with the c-terminal antibody and a faint to negative reaction with the other five antibodies. In biopsied human muscles NMJs and MTJs also showed a clear reaction with all ten antibodies, i.e., six polyclonal and four monoclonal ones. Although an immunohistochemical DRP reaction was clearly seen at NMJs, only a faint or no reaction was seen on MTJs and on intrafusal muscle fibers in both mouse and human materials. Western blot analysis of control mouse muscle for dystrophin showed a clearer band for the peripheral portion, which contains many MTJs, than for the mid-belly portion. These data suggest that dystrophin really exists on MTJs, and that dystrophin and DRP exist on NMJs in mouse and human muscles.

Animals↗
PubMed1992

Dystrophin isoforms and/or cross-reactive proteins on neurons and glial cells in control and mdx central nervous systems.

We studied the central nervous system (CNS) of control mice in comparison with that of mdx mice, immunohistochemically and immunoelectrophoretically, using 5 kinds of polyclonal antibodies against dystrophin (DMDP-II, 60-kDa, 30-kDa, P-20 and DMDP-IV) to determine whether or not and, if so, how dystrophin exists in the central nervous system. A positive dystrophin reaction was seen on the neurons and glial cells in both control and mdx tissue, without any immunohistochemical difference. In control mice, Western blot analysis showed two relatively clear bands corresponding to 400-kDa, with all 4 antibodies used (60-kDa, 30-kDa, P-20 and DMDP-IV), and 280-kDa, with 3 of them, the exception being 30-kDa, and 2 other faint bands corresponding to larger M(r) than 400-kDa, with 3 of them, the exception being P-20, respectively. In the mdx CNS, the 400-kDa band was absent, the other 3 bands being seen. The results suggest that dystrophin really exists in the control CNS, and some dystrophin isoforms or cross-reactive proteins exist on the neurons and glial cells in mdx as well as control mice. The localization of dystrophin in CNS also suggests its physiological function in the conduction system rather than a mechanical one, and a defect of it in CNS is a possible cause of the mental retardation in Duchenne muscular dystrophy.

Animals↗
PubMed1992

In vitro oxidation of oxicam NSAIDS by a human liver cytochrome P450.

The nature of the enzyme(s) catalyzing the major metabolic pathway (5'-hydroxylation) of oxicam NSAIDs was investigated in subcellular preparations of human liver tissue. Microsomal, but not cytosolic, fractions catalyzed the 5'-hydroxylation of tenoxicam. This reaction required NADPH and was inhibited by various nonselective P450 inhibitors (CO, SKF-525A, ketoconazole), but not by the peroxidase inhibitor NaN3. Tenoxicam 5'-hydroxylation exhibited simple Michaelis-menten kinetics compatible with catalysis by a single enzyme, but it strongly inhibited its own oxidation at concentrations higher than 100-150 microM. Piroxicam competitively inhibited tenoxicam 5'-hydroxylation and, conversely, tenoxicam competitively inhibited piroxicam 5'-hydroxylation. Tenoxicam 5'-hydroxylation kinetics were similar in microsomes from one poor and five extensive metabolizers of debrisoquin (CYP2D6). Dextromethorphan (CYP2D6 prototype substrate) and midazolam (CYP3A prototype substrate) had no influence on tenoxicam 5'-hydroxylation, whereas mephenytoin, tolbutamide and sulfaphenazole (Ki = 0.1 microM) inhibited it. This indicates that the 5'-hydroxylation of both piroxicam and tenoxicam is predominantly catalyzed by at least one cytochrome P450 isozyme of the CYP2C subfamily.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1992

Effects of dietary fish oil on myocardial ischemic/reperfusion injury of Wistar Kyoto and stroke-prone spontaneously hypertensive rats.

The purpose of the present study was to determine whether 15 weeks of dietary fish oil supplementation to Wistar Kyoto (WKY) and stroke-prone spontaneously hypertensive rats (SP-SHR) would alter cardiac contractile performance. Isolated perfused working hearts were used to determine the effects of varying left atrial filling pressures on cardiac pump output and pressure development. In addition, the ability of these hearts to recover cardiac output after a 60-minute period of low-flow ischemia followed by 30 minutes of reperfusion was assessed. Myocardial high-energy phosphate levels and long-chain acylcarnitine content were measured. Fish oil supplementation had no effect on indirect tail-cuff systolic blood pressure of WKY rats, but produced a small decrease in systolic blood pressure of SP-SHR. In response to varying left atrial filling pressures, hearts from WKY rats and SP-SHR exhibited no major differences in aortic flow, left intraventricular diastolic pressure, systolic pressure, positive and negative rate of pressure change per unit of time (dP/dt), or relaxation time; however, coronary flow values were lower in SP-SHR groups relative to respective WKY groups. Fish oil supplementation had no effect on these cardiac parameters. When these hearts were subjected to ischemia and reperfusion, the recovery of cardiac contractile performance was significantly improved in fish oil-fed WKY and SP-SHR groups, compared with their corn oil-fed counterparts. There were no differences in the recovery of the above cardiac hemodynamic parameters between corn oil-fed WKY and SP-SHR. These results provide further support to the hypothesis that dietary fish oil can significantly reduce the mortality risk from cardiovascular disease.

Animals↗
PubMed1992

Altered expression of adenovirus 12 DNA-binding protein but not DNA polymerase during abortive infection of hamster cells.

Replication of human adenovirus type 12 DNA is blocked in abortively infected baby hamster kidney cells. The activity and accumulation of adenovirus 12 DNA polymerase is equivalent in infected hamster and human cell extracts. However, the accumulation of adenovirus type 12 DNA-binding protein is approximately 120-fold lower in extracts from infected hamster cells when compared to infected permissive human cells. This difference in accumulation is not due to replication of viral DNA during productive infection, since this difference is observed in the presence of hydroxyurea. The DNA-binding protein from infected hamster cells retains the ability to bind denatured DNA-cellulose. An adenovirus 5 early region 1 transformed hamster cell line competent to complement the adenovirus 12 DNA replication defect also stimulates accumulation of the DNA-binding protein even when the cells are treated with hydroxyurea. Thus, the reduced expression of the viral DNA-binding protein may play a role in the mechanism of abortive infection of hamster cells by adenovirus 12.

Adenovirus Early Proteins↗