Biomedical subjects

M Liang

Publications and source records attributed to M Liang.

At least 55 records · Page 3Linked to original sources

PubMed2000

Mechanisms underlying induction of heme oxygenase-1 by nitric oxide in renal tubular epithelial cells.

We examined whether nitric oxide-generating agents influence expression of heme oxygenase-1 (HO-1) in renal proximal tubular epithelial cells, LLC-PK(1) cells, and the mechanisms underlying any such effects. In sublytic amounts, the nitric oxide donor sodium nitroprusside induced HO-1 mRNA and protein and HO activity in a dose-dependent and time-dependent fashion; this induction was specific for nitric oxide since the nitric oxide scavenger carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide significantly reduced such induction. The induction of HO activity by sodium nitroprusside, or by another nitric oxide donor, spermine NONOate, was markedly reduced by the iron chelator deferoxamine. Two different thiol-containing agents, N-acetylcysteine and dithiothreitol, blunted such induction of HO by nitric oxide. Downstream products of nitric oxide, such as peroxynitrite or cGMP, were not involved in inducing HO. In higher concentrations (millimolar amounts), sodium nitroprusside induced appreciable cytotoxicity as assessed by lactate dehydrogenase (LDH) release and lipid peroxidation, and both of these effects were markedly reduced by deferoxamine. Inhibition of HO did not affect the cytotoxic effects (measured by LDH release) of sodium nitroprusside. We thus provide the novel description of the induction of HO-1 in renal proximal tubular epithelial cells exposed to nitric oxide donors and provide the first demonstration in kidney-derived cells for the involvement of a redox-based mechanism in such expression. We also demonstrate that, in LLC-PK(1) cells exposed to nitric oxide donors, chelatable iron is involved in eliciting the HO-1 response observed at lower concentrations of these donors, and in mediating the cytotoxic effects of these donors when present in higher concentrations.

Animals↗

PubMed2000

[Human Fab antibodies derived from phage display library neutralize hepatitis A virus in vitro].

OBJECTIVE: Development of recombinant human monoclonal antibody to hepatitis A virus as a emergent measure for prevention of hepatitis A virus infection. METHODS: Human neutralizing monoclonal antibody Fab fragments to HAV have been developed by using phage display technique. The heavy and light chains of human IgG Fab genes were amplified from a HAV patient in convalescent stage. The combinatorial phage antibody library was established by inserting both heavy and light chains of Fab genes into phage mid-vector pComb3 and followed by help phage infection after 4 rounds of panning with purified HAV as coated antigen. RESULTS: The human Fab fragments to HAV were selected and expressed in bacteria. CONCLUSIONS: The specific binding of Fab antibodies to HAV were demonstrated by their reaction with HAV antigen in ELISA. These results provide the basis for further development of a neutralizing recombinant human whole IgG molecule and hold promise for future use in the prophylaxis of HAV infection.

Antibodies, Monoclonal↗

PubMed2000

[The partial nucleotide sequence of L segment of Hantavirus strain].

OBJECTIVE: To study the nucleotide sequence of L segment of Hantavirus strain A9. METHODS: The partial L segment cDNA of A9 strain was amplified by PCR, the PCR product was sequenced. RESULTS: The nucleotide sequence of partial L segment of A9 strain was provided, the sequence was A, T rich, similar to other Hantaviruses, and similar to both M, S segments. Compared with nucleotide sequences from other Hantaviruses, it revealed a higher homology to HTN virus strain 76-118,83.5% at nucleotide level and 96.7% at deduced amino level. SNV, PUU and Tula viruses shared the lower homology. A9 and 76-118 were at same cluster in phylogenetic analysis, close to SEO virus but not to PUU, SNV, Tula viruses. Comparing of partial nucleotide sequence of strain A9 with 76-118, some conserved regions were found, but also some regions of A9 were more conserved to other viruses not 76-118. It suggested that when design the primers for L segments amplification, more than one type of Hantavirus sequence should he concerned. Like the homology from M and S segments of Hantaviruses, the viruses isolated from same host animal showed high homology. CONCLUSIONS: Nucleotide sequence of L segment of Hantavirus strain A9 revealed homology between 85.2% -70.8% to different types of Hantavirus and shared a higher homology of 85.2% to strain 76-118.

Animals↗

PubMed2000

[Generation of monoclonal antibody Fab fragments to parathyroid hormone related protein by phage display technology].

OBJECTIVE: To develop monoclonal antibody Fab fragments using display technique. METHODS: The mouse IgG Fab genes of heavy and light chains were amplified from spleen cells of a parathyroid hormone-related protein (PTHrP) immunized mouse. The combinatorial phage antibody library was prepared by inserting both heavy and light chain Fab genes into phagemid vector pComb3 and followed by infection of helper phage. The library was selected by purified recombinant PTHrP. RESULTS: The combinatorial phage antibody library was constructed successfully and the specific mouse Fabs to PThrP were selected and expressed in E. coli. CONCLUSIONS: The selected specific mouse Fabs can recognize PTHrP with high specificity.

Animals↗

PubMed2000

[Detection of IgG antibody to hepatitis G virus by ELISA].

OBJECTIVE: To study the relation of HGV IgG antibody (anti- HGV IgG) to various viral infections, ALT and HGV RNA. METHODS: To detect anti-HGV IgG from 315 persons infected with various viruses hepatitis (A - E) and 117 healthy blood donors by ELISA. The detection of ALT of these peoples, and detect HGV RNA of the samples of positive anti-HGV IgG by RT-PCR were also carried out. RESULTS: The positive rate of anti-HGV IgG of 117 blood donors was 3.42% (4/117), and that of the people infected by non A- E, HAV, HBV, HCV, HEV, HBV and HCV, HBV and HDV, HBV and HEV were 5.95%(5/84), 0(0/3), 3.75% (3/80), 17. 28%(14/81), 0(0/15), 33.33%(5/15), 3.33%(1/30), 16.66%(1/6), respectively. The relativity of anti-HGV IgG and HGV RNA was 30.30% (10/33), and 33.33% of ALT level in the antibody positive samples were elevated. CONCLUSIONS: HGV may infect people alone, or coinfect with HBV or /and HCV, but is highly relative to HCV, and the coinfection of HGV with HCV may increase the degree of liver damage.

Alanine Transaminase↗

PubMed1999

Synthesis of NAADP and cADPR in mitochondria.

Here we investigated whether cADPR and NAADP are synthesized in mitochondria. We found that ADPR-cyclase activity is present in mitochondria. In addition, we describe for the first time synthesis of NAADP in this intracellular organelle. ADPR-cyclase activities (V(MAX)) and NAADP synthesis in mitochondria were about 4-fold lower than that in plasma membranes. Otherwise, ADPR-cyclases in mitochondria and in plasma membranes have similar catalytic properties in terms of apparent K(m) for the substrate NGD and K(i) values for inhibition by dithiotreitol, beta-NAD, and nicotinamide. ADPR-cyclase in plasma membranes and to a lesser degree mitochondrial enzyme, was inhibited by Zn(2+) and Cu(2+); ADPR-cyclase from mitochondria was more stable upon thermal inactivation. CD38 antigen, determined by Western blot, was well-expressed in plasma membranes but was far less so (17-fold less) in mitochondria. The major difference between ADPR-cyclase activity in mitochondria and plasma membranes is that mitochondrial cyclase activity was increased by incubation with nonionic detergents. Conversely, the incubation with phosphatidylinositol-specific phosphodiesterase C (PI-PLC) released ADPR-cyclase activity from plasma membranes, but not from mitochondria. We conclude that ADPR-cyclase in mitochondria and in plasma membranes are both multifunctional enzymes with similar catalytic properties; however, the two ADPR-cyclases differ in the mode of anchoring to the membrane: by glycosylphosphoinositol anchor in plasma membranes and by hydrophobic interactions in mitochondria. In addition, synthesis of NAADP can also be found in intracellular organelles via mitochondria. We propose that independent mitochondrial cADPR and NAADP systems may have an intracrine signaling function that is not dependent on direct input by extracellular hormonal stimuli, but rather responds to changes of intermediary cellular metabolism.

ADP-ribosyl Cyclase↗

PubMed1999

Discovery of novel non-peptide CCR1 receptor antagonists.

Ligands for the CCR1 receptor (MIP-1alpha and RANTES) have been implicated in a number of chronic inflammatory diseases, most notably multiple sclerosis and rheumatoid arthritis. Because these ligands share a common receptor, CCR1, we sought to discover antagonists for this receptor as an approach to treating these disorders. A novel series of 4-hydroxypiperidines has been discovered by high throughput screening (HTS) which potently inhibits the binding of MIP-1alpha and RANTES to the recombinant human CCR1 chemokine receptor. The structure-activity relationships of various segments of this template are described as the initial HTS lead 1 was optimized synthetically to the highly potent receptor antagonist 6s. This compound has been shown to have at least 200-fold selectivity for inhibition of CCR1 over other human 7-TM receptors, including other chemokine receptors. In addition, data obtained from in vitro functional assays demonstrate the functional antagonism of compound 6s and structurally related analogues against the CCR1 receptor in a concentration dependent manner. The discovery and optimization of potent and selective CCR1 receptor antagonists represented by compound 6s potentially represent a novel approach to the treatment of chronic inflammatory diseases.

Anti-Inflammatory Agents↗

PubMed1999

Nitric oxide enhances paracellular permeability of opossum kidney cells.

BACKGROUND: Nitric oxide (NO) has been shown to be a paracrine/autocrine regulator of proximal tubular transport. In this study, we investigated the effect of NO on the paracellular permeability of opossum kidney (OK) cells, a proximal tubule cell line that possesses a leaky paracellular pathway resembling that of the in vivo proximal tubule. METHODS: Paracellular permeability of OK cells cultured on permeable supports was measured as the apparent paracellular permeability coefficient (Papp) for 3[H]-D-mannitol. Changes in cell viability, cellular adenosine triphosphate (ATP) content, cGMP levels, and lipid peroxidation were assessed. RESULTS: Incubation with 2 mM sodium nitroprusside (SNP), an NO donor, for 24 hours significantly enhanced the Papp of OK cell sheets by 30.6 +/- 7.0% (N = 8, P < 0.05). This effect was largely blunted by hemoglobin, a NO scavenger. Cell viability was not compromised. This effect of SNP was concomitant with a moderate reduction of cellular ATP content, an increase in lipid peroxidation, and an increase in cellular cGMP levels. The antioxidant superoxide dismutase (SOD) significantly attenuated the effect of SNP on cellular ATP content and blunted the increase in Papp caused by SNP. A soluble guanylate cyclase inhibitor did not affect the effect of SNP on the Papp. CONCLUSIONS: NO enhances the paracellular permeability of OK cells possibly via mechanisms involving decreases in cellular ATP content.

Adenosine Triphosphate↗

PubMed1999

Nitric oxide reduces the molecular activity of Na+,K+-ATPase in opossum kidney cells.

BACKGROUND: Nitric oxide (NO) directly inhibits fluid and solute reabsorption in the proximal tubule. In the present study, we investigated the effect of NO on the Na+, K+-ATPase of opossum kidney (OK) cells, a proximal tubule cell line, and its mechanisms. METHODS: Na+,K+-ATPase activity in the membrane fraction of OK cells was measured as the ouabain-sensitive ATP hydrolytic activity. The enzyme unit number on intact cells was measured by ouabain-binding assay. RESULTS: Incubation with 0.5 mM sodium nitroprusside (SNP), a NO donor, for two hours inhibited the catalytic activity of the membrane-associated Na+,K+-ATPase in OK cells to 65.5 +/- 9.7% of control (N = 6, P < 0.05 vs. control). This effect of SNP was concentration- and time-dependent. The NO scavenger hemoglobin blunted, while another NO donor spermine NONOate (5 microM) mimicked this effect of SNP. At all concentrations and time points tested, SNP did not alter the molecular number of Na+,K+-ATPase on intact OK cells, indicating that NO inhibited the molecular activity of Na+,K+-ATPase. The soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ), blunted the inhibitory effect of SNP on the Na+,K+-ATPase activity. An exogenous cGMP analog similarly inhibited the Na+,K+-ATPase activity. Neither lipid soluble antioxidants vitamin E/probucol or thiol group compound DL-dithiothreitol (DTT) altered the inhibitory effect of SNP on the Na+,K+-ATPase activity. CONCLUSIONS: NO inhibited the molecular activity of the Na+,K+-ATPase of the OK proximal tubule cell line probably via cGMP-dependent mechanisms.

Animals↗

PubMed1999

The paracellular permeability of opossum kidney cells, a proximal tubule cell line.

UNLABELLED: The paracellular permeability of opossum kidney cells, a proximal tubule cell line. BACKGROUND: The regulation of the unusually leaky paracellular pathway of the proximal tubule is poorly understood partially because of the lack of an appropriate in vitro cell model. In this study, we determined whether the paracellular permeability of opossum kidney (OK) cells would resemble that of the in vivo proximal tubule epithelium. METHODS: The parental and subclonal OK cells and, for comparison, LLC-PK1 cells were cultured on permeable Transwell supports. The apparent paracellular permeability coefficient (Papp) for the extracellular marker 3H-mannitol was determined. RESULTS: The Papp of OK cell sheets (12.17 x10-6 cm/sec) was remarkably close to the previously reported Papp of rat proximal tubules. The Papp of LLC-PK1 cells, another proximal tubule cell line, however, was approximately 20-fold lower than that of both OK cells and the in vivo proximal tubule. Phorbol 12-myristate 13-acetate, a protein kinase C activator, enhanced the Papp of OK cell sheets. The characteristic response of paracellular permeability to Ca2+ switch was demonstrated in OK cell sheets. Slight variations of Papp among several OK subclones were observed. Basal to apical Papp was uniformly higher than apical to basal Papp, independent of cell subtype. This rectification was attenuated by inhibition of active transport. CONCLUSIONS: OK cell sheets cultured on Transwell supports possess a leaky paracellular pathway resembling that of the proximal tubule epithelium in vivo.

Amiloride↗

PubMed1999

Nitric oxide activates PKCalpha and inhibits Na+-K+-ATPase in opossum kidney cells.

Nitric oxide (NO) reduces the molecular activity of Na+-K+-ATPase in opossum kidney (OK) cells, a proximal tubule cell line. In the present study, we investigated the cellular mechanisms for the inhibitory effect of NO on Na+-K+-ATPase. Sodium nitroprusside (SNP), a NO donor, inhibited Na+-K+-ATPase in OK cells, but not in LLC-PK1 cells, another proximal tubule cell line. Similarly, phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, inhibited Na+-K+-ATPase in OK, but not in LLC-PK1, cells. PKC inhibitors staurosporine or calphostin C, but not the protein kinase G inhibitor KT-5823, abolished the inhibitory effect of NO on Na+-K+-ATPase in OK cells. Immunoblotting demonstrated that treatment with NO donors caused significant translocation of PKCalpha from cytosolic to particulate fractions in OK, but not in LLC-PK1, cells. Furthermore, the translocation of PKCalpha in OK cells was attenuated by either the phospholipase C inhibitor U-73122 or the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. U-73122 also blunted the inhibitory effect of SNP on Na+-K+-ATPase in OK cells. The phospholipase A2 inhibitor AACOCF3 did not blunt the inhibitory effect of SNP on Na+-K+-ATPase in OK cells. AACOCF3 alone, however, also decreased Na+-K+-ATPase activity in OK cells. In conclusion, our results demonstrate that NO activates PKCalpha in OK, but not in LLC-PK1, cells. The activation of PKCalpha in OK cells by NO is associated with inhibition of Na+-K+-ATPase.

Alkaloids↗

PubMed1999

[Determination of 6-methoxy-2-naphthylacetic acid, a major metabolite of nabumetone, in human plasma by HPLC].

This paper reports a sensitive and rapid method for determining 6-methoxy-2-naphthylacetic acid (MNA), a major metabolite of nabumetone in human plasma using naproxen as the internal standard. High performance liquid chromatograph model 680 (Waters, USA) with a variable wavelength UV detector and reversed-phase YWG-C18 column (10 microns, 250 x 4.6 mm) was used. After the addition of acetate buffer(pH3.0), the plasma sample was extracted with methylene chloride. The mobile phase of methanol-pH3.0, 0.02 mol/L acetate buffer(74:26) was pumped at 1.0 ml/min through the column. The detector at 0.01AUFS was set at 270 nm. The retention times for MNA and naproxen were 3.98 min and 4.73 min respectively. Standard curve was linear in the concentration range of 0.5 to 64 mg/L. The detection limit in serum was 0.02 mg/L. Extraction recovery was 88%-94%; method recovery 96%-102%; withinday RSD less than 3.5%; inter-day RSD less than 5%.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1999

[Simultaneous determination of norepinephrine and epinephrime in plasma by using high performance liquid chromatograph].

A sensitive and rapid method was reported for the determination of nerepinephrine (NE) and epinephrine (E) in plasma by using high-performance liquid chromatography with an electrochemical detector (HPLC-EC) and YWG-C18 column(5u). 3,4-Dihydroxybenzylamine(DHBA) was used as an internal standard. The mobile phase of 0.05 mol/L actate buffer(pH3.0)-methanol-0.05 mol/L EDTA-Na2(78:20:2) with of 200 mg/L SDS solution was pumped through the column with a flow rate of 1.0 ml/min. The lengths of retention time for NE, E and DHBA(IS) was 7.3 min, 8.3 min and 10.4 min, respectively. The standard curve was linear within the concentration range of 31.25 to 4000 ng/L for NE and E (NE: r = 0.9999 E: r = 0.9996). The within day RSD (%) were less than 1.87% for NE and less than 2.07% for E, inter-day RSD(%) were less than 8.92% for NE and less than 9.65% for E respectively. The average extraction recovery(%) for NE and E were 93.5% +/- 5.50% and 91.3% +/- 1.77% respectively. This method has been applied to the diagnosis of pheochromocytoma and clinical research.

Adrenal Gland Neoplasms↗

PubMed1999

[Study on chromatographic peak identification by means of peak shape analysis].

A new method for chromatographic peak identification by means of peak shape analysis is presented in this paper. Directly measurable parameters, i.e. peak width and asymmetry factor are first utilized for peak shape analysis. It is showed from theoretical and experimental results that for a component having almost the same retention time with the standard, an overlapped peak can be obtained by injecting the mixture of analyte and standard. In comparing the peak shape of overlapped peak with the standard one, the component can be identified whether it is the same with the standard or not.

Chromatography↗

PubMed1998

Differential effect of pH upon cyclic-ADP-ribose and nicotinate-adenine dinucleotide phosphate-induced Ca2+ release systems.

We investigated the pH dependence and the effects of thimerosal and dithiothreitol (DTT) upon the Ca2+ release induced by cADP-ribose (cADPR) and nicotinate-adenine dinucleotide phosphate (NAADP) in sea urchin egg homogenates. Both Ca2+ release triggered by cADPR and the binding of [3H]cADPR to sea urchin egg homogenates were decreased by alkalization of the assay media from pH 7.2 to 8.9. In contrast, NAADP-triggered Ca2+ release was not influenced by changes in pH. The Ca2+ release induced by cADPR was potentiated by thimerosal and inhibited by DTT, but neither thimerosal nor DTT had any effect upon the Ca2+ release induced by NAADP. We conclude that cADPR-sensitive Ca2+-release mechanisms are dependent on pH of the assay media and are sensitive to thiol group modification. On the other hand, these functional properties are not shared by NAADP-regulated Ca2+ channels.

Adenosine Diphosphate Ribose↗

PubMed1998

Identification and characterization of small molecule functional antagonists of the CCR1 chemokine receptor.

The CC chemokines macrophage inflammatory protein-1alpha (MIP-1alpha) and RANTES (regulated on activation normal T cell expressed) have been implicated in rheumatoid arthritis and multiple sclerosis. Since their effects are mediated through the CCR1 chemokine receptor, we set up a small molecule CCR1 antagonist program to search for inhibitors. Through high capacity screening we discovered a number of 4-hydroxypiperidine compounds with CCR1 antagonist activity and report their synthesis and in vitro pharmacology here. Scatchard analysis of the competition binding data revealed that the compounds had Ki values ranging from 40 to 4000 nM. The pharmacological profile of the most potent member of this series, compound 1 (2-2-diphenyl-5-(4-chlorophenyl)piperidin-lyl)valeronitri te), was further evaluated. Compound 1 showed concentration-dependent inhibition of MIP-1alpha-induced extracellular acidification and Ca2+ mobilization demonstrating functional antagonism. When given alone, the compound did not elicit any responses, indicating the absence of intrinsic agonist activity. Compound 1 inhibited MIP-1alpha- and RANTES-induced migration in peripheral blood mononuclear cells in a dose-responsive manner. Selectivity testing against a panel of seven transmembrane domain receptors indicated that compound 1 is inactive on a number of receptors at concentrations up to 10 microM. This is the first description of CCR1 receptor antagonists that may be useful in the treatment of chronic inflammatory diseases involving MIP-1alpha, RANTES, and CCR1.

Arthritis, Rheumatoid↗

PubMed1998

Identification and characterization of the CXCR4 chemokine receptor in human T cell lines: ligand binding, biological activity, and HIV-1 infectivity.

The CXCR4 chemokine receptor has been shown to respond to the C-X-C chemokine stromal-derived factor (SDF-1) and has recently been shown to be an important coreceptor for HIV-1 infection. In the present paper we have tested a number of human lymphocyte cell lines, including Jurkat, HUT78, CEM, and Sup-T1 for the presence of CXCR4 receptors. We found that these T cell lines bind SDF-1alpha and SDF-1beta with high affinity. The CXCR4 Ab 12G5 inhibited both SDF-1 binding and HIV-1LAI-mediated fusion of CEM. Scatchard analysis revealed the presence of approximately 150,000 SDF-1alpha-binding sites per cell with a Kd between 5 and 10 nM. Cross-competition experiments using unlabeled SDF-1alpha and SDF-1beta revealed that both chemokines are equally capable of displacing their radiolabeled counterparts. Internalization studies with [125]I-SDF-1alpha revealed that Jurkat cells internalized greater than 90% of the ligand by 2 h at 37 degrees C. SDF-1alpha was also chemotactic for Jurkat cells and caused an increase in the rate of extracellular acidification that was half-maximal at 18 nM SDF-1alpha and could be inhibited by pretreatment with the SDF-1 proteins, pertussis toxin, or the Ab 12G5. Finally, SDF-1alpha also caused an increase in the cytosolic Ca2+ concentration in Sup-T1 cells that was abolished by preincubating the cells with pertussis toxin or PMA and inhibited by the Ab 12G5. This molecular characterization of CXCR4 receptors should prove useful in clarifying receptor interaction with SDF-1 proteins and with HIV-1 glycoprotein, with the ultimate aim of targeting the viral interaction for therapeutic intervention.

Binding Sites↗

PubMed1998

Nucleotide sequence and phylogenetic analysis of the medium (M) genomic RNA segments of three hantaviruses isolated in China.

The medium (M) genome segment of hantaviruses (family Bunyaviridae) encodes the two virion glycoproteins. G1 and G2, as a precursor protein in the complementary sense RNA. We determined the nucleotide sequences of the M genome segments of three Chinese hantavirus isolates, a Hantaan-type (HTN) virus designated A9 and two Seoul-type (SEO) viruses designated L99 and HB55, and compared them to those of other HTN or SEO viruses isolated in Eastern Asia. The M segment of A9 is 3616 nucleotides in length and shows 99.5% identity at the nucleotide level and 99.1% identity at the amino acid level to that of the Chinese HTN isolate HV114. The M segments of L99 and HB55 are 3652 nucleotides in length, one nucleotide longer than the M segments of other sequenced SEO isolates such as SEO 80-39, SR-11, and Biken-1. The Chinese SEO isolates showed 95% nucleotide sequence identity and 99% amino acid sequence identity to SEO 80-39. We also sequenced a 736 nucleotides region of the M genome segment of another Chinese SEO isolate, R22, which revealed errors in the published data. Phylogenetic analysis of the available sequences indicated that both the Chinese HTN- and SEO-type viruses form lineages distinct from those of the isolates from other parts of Eastern Asia.

Amino Acid Sequence↗