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Biomedical subjects

M Y Liu

Publications and source records attributed to M Y Liu.

At least 109 records · Page 6Linked to original sources
PubMed1993

Use of bacterially-expressed antigen for detection of antibodies to the EBV-specific deoxyribonuclease in sera from patients with nasopharyngeal carcinoma.

A cDNA clone, BG9, corresponding to the open reading frame BGLF5 of Epstein-Barr virus (EBV) DNase was inserted into an E. coli expression vector, pET3a, to generate a recombinant plasmid, pDNase 5. High level of expression of a DNase activity was detected in the E. coli transformed with pDNase 5 following induction with IPTG. The enzyme activity was purified using DEAE-cellulose, phosphocellulose and DNA-cellulose column chromatography. The purified protein appeared to be nearly homogeneous in SDS-PAGE using Coomassie blue staining. The requirement for divalent cations and optimum pH as well as inhibitory concentrations of ionic strength and polyamines for the purified enzyme activity were determined and seemed to be very similar to those of the enzyme activity purified from an EBV producing lymphoblastoid cell line. Using the purified enzyme as an antigen and anti-IgA as the secondary antibody, 82% (64/78) and 91% (71/78) of sera from patients with nasopharyngeal carcinoma (NPC) were shown to be positive by dot immunobinding assay and ELISA, respectively. The results suggest that purified E. coli expressed EBV DNase may be useful for preparing specific test for large scale screening of patients with NPC.

Antibodies, Viral↗
PubMed1993

Identification and molecular characterization of csrA, a pleiotropic gene from Escherichia coli that affects glycogen biosynthesis, gluconeogenesis, cell size, and surface properties.

Current evidence suggests that a few global regulatory factors mediate many of the extensive changes in gene expression that occur as Escherichia coli enters the stationary phase. One of the metabolic pathways that is transcriptionally activated in the stationary phase is the pathway for biosynthesis of glycogen. To identify factors that regulate glycogen biosynthesis in trans, a collection of transposon mutants was generated and screened for mutations which independently increase or decrease glycogen levels and the expression of a plasmid-encoded glgC'-lacZ fusion. The glycogen excess mutation TR1-5 was found to be pleiotropic. It led to increased expression of the genes glgC (ADPglucose pyrophosphorylase) and glgB (glycogen branching enzyme), which are representative of two glycogen synthesis operons, and the gluconeogenic gene pckA (phosphoenolpyruvate carboxykinase), and it exhibited effects on cell size and surface (adherence) properties. The mutated gene was designated csrA for carbon storage regulator. Its effect on glycogen biosynthesis was mediated independently of cyclic AMP (cAMP), the cAMP receptor protein, and guanosine 3'-bisphosphate 5'-bisphosphate (ppGpp), which are positive regulators of glgC expression. A plasmid clone of the native csrA gene strongly inhibited glycogen accumulation and affected the ability of cells to utilize certain carbon sources for growth. Nucleotide sequence analysis, complementation experiments, and in vitro expression studies indicated that csrA encodes a 61-amino-acid polypeptide that inhibits glycogen biosynthesis. Computer-assisted data base searches failed to identify genes or proteins that are homologous with csrA or its gene product.

Amino Acid Sequence↗
PubMed1993

Analysis of Epstein-Barr virus BamHI F DNA fragment in nude mouse-passaged nasopharyngeal carcinoma tissues.

Two nude mouse-passaged nasopharyngeal carcinoma (NPC) tissues, NPC-295 and NPC-306, were different in ability to transform cord blood lymphocytes and contained Epstein-Barr virus (EBV) genomes with difference in BamHI F DNA fragment. Four clones containing DNA sequences of the BamHI F fragment (54,853-62,249) were obtained from genomic libraries of NPC-295 and NPC-306 and their partial restriction enzyme maps and sequences were determined. The restriction enzyme maps of EBV DNA at the BamHI F fragment in NPC-295 and NPC-306 appeared to be similar to that of EBV B95-8 strain. However, 8 nucleotide differences were revealed between NPC-295 and the B95-8 strain when 566 nucleotides (55,405-55,970) were compared, and 15 out of 677 nucleotides analyzed (55,410-56,086) were different between NPC-306 and B95-8.

Animals↗
PubMed1993

[Total intravenous anesthesia with O2 mask in transthoracic endoscopic sympathectomy for palmar hyperhidrosis].

Using non-invasive monitors (such as blood pressure, O2 saturation, end-tidal CO2 pressure monitors and electrocardiograms), the application of total intravenous anesthesia with O2 mask in transthoracic endoscopic sympathectomy was evaluated. A total of 198 palmar hyperhidrosis patients were studied. In the study, the durations of surgery were 5-15 min (mean 8 min) with anesthesia duration of 8-20 min (mean 12 min), respectively. The cardiovascular system was quite stable during operation, with systolic blood pressures of 90-165 mmHg (mean 132 mmHg), diastolic blood pressures of 50-106 mmHg (mean 92 mmHg) and heart rates of 56-130 beats/min (mean 104 beats/min). The respiratory function was maintained within a safety range with O2 saturation of 95-100% (mean 99%) and end-tidal CO2 pressure of 32-40 mmHg (mean 36 mmHg). After surgery, the major discomfort was chest pain (132/198), but this could be improved with intravenous analgesics. Patients' consciousness returned within 1-3 min. After ensuring their general condition was stable, patients were discharged within 24 hr. Preliminary study showed that total intravenous anesthesia plus muscle relaxant, with O2 mask and assisted ventilation, could serve as a safe and simple anesthesia for palmar hyperhidrosis patients who received transthoracic endoscopic sympathectomy.

Adolescent↗
PubMed1992

Two homologs of the Drosophila polarity gene frizzled (fz) are widely expressed in mammalian tissues.

The frizzled (fz) locus of Drosophila encodes a protein (Fz) with a seven-transmembrane-domain profile characteristic of G-protein-coupled receptors. In Drosophila, genetic evidence suggests that Fz functions to transmit and transduce polarity signals in epidermal cells during hair and bristle development. We have isolated from a UMR 106 rat osteosarcoma cell library a cDNA (fz-1) encoding a predicted 641-residue protein (Fz-1) with 46% homology with Drosophila Fz. We also identified a second cDNA (fz-2) encoding a protein (Fz-2) of 570 amino acids that is 80% homologous with Fz-1, with divergence most evident in the extracellular domains. Southern blots of rat genomic DNA indicated that fz-1 and fz-2 represent distinct genes. Northern analysis revealed the presence of a single fz-1 mRNA (4.7 kilobases) and two fz-2 mRNAs (2.5 and 4.5 kilobases) in rat tissues. The fz-1 and fz-2 genes are widely expressed in rat tissues with the highest steady-state levels of mRNA in kidney, liver, heart, uterus, and ovary. fz-1 and -2 mRNA levels were greater in neonatal than in corresponding adult tissues. Treatment of UMR 106 cells with bone resorbing agents including parathyroid hormone, epidermal growth factor, and 1,25-dihydroxyvitamin D3 produced increases in fz-1 and -2 mRNA levels. We suggest that hormonal induction of Fz proteins in osteoblasts serves to promote intercellular signaling required for functional responses such as increased bone resorption. Fz-1 and Fz-2 may represent products of a gene family whose members serve as transducers or intercellular transmitters of signals required for normal morphogenesis and/or differentiated function in diverse tissues.

Amino Acid Sequence↗
PubMed1992

Stimulation of macrophage colony-stimulating factor synthesis by interleukin-1.

Interleukin-1 (IL-1), which plays an important role in the inflammatory response, was found to induce colony-stimulating factor-1 (CSF-1) expression in the MIA PaCa-2 cells. IL-1-induced CSF-1 production was markedly suppressed (70%) by pertussis toxin. This inhibition by pertussis toxin was reversed by benzamide, an inhibitor of ADP-ribosylation reactions. Similarly, IL-1-induced CSF-1 production was inhibited by cholera toxin and this inhibition was reversed by an arginine analog, p-methoxy-benzylaminodecamethylene guanidine sulfate. Dibutyryl-cAMP as well as other cAMP elevating agents such as theophylline and forskolin also suppressed IL-1-induced CSF-1 production, suggesting that cAMP concentrations inversely regulate the biosynthesis of CSF-1. Measurement of cAMP concentration indicated that IL-1 treatment of MIA PaCa-2 cells did not change the cAMP level. IL-1-induced CSF-1 production was not suppressed by the protein kinase C (PKC) inhibitor, H7, under conditions in which 12-O-tetradecanoylphorbol-13-acetate-induced CSF-1 production was completely abolished. These data suggest that IL-1-induced CSF-1 production is not mediated via the activation of PKC. Analysis of oncogene c-fos and c-jun expression has shown the enhancement of expression of both protooncogenes prior to CSF-1, suggesting that the expression of these two oncogenes may be the mechanism which triggers CSF-1 gene expression.

Bucladesine↗
PubMed1992

NMR and EPR studies on a monoheme cytochrome c550 isolated from Bacillus halodenitrificans.

A c-type monoheme ferricytochrome c550 (9.6 kDa) was isolated from cells of Bacillus halodenitrificans sp.nov., grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme-methionine coordination. The midpoint redox potential was determined at several pH values by visible spectroscopy. The redox potential at pH 7.6 is 138 mV. When studied by 1H-NMR spectroscopy as a function of pH, the spectrum shows a pH dependence with pKa values of 6.0 and 11.0. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c550. The first pKa is probably associated with protonation of the propionate groups. The second pKa value introduces a larger effect in the 1H-NMR spectrum and is probably due to the ionisation of the axial histidine. Studies of temperature variation of the 1H-NMR spectra for both the ferrous and ferri forms of the cytochrome were performed. Heme meso protons, the heme methyl groups, the thioether protons, two protons from a propionate and the methylene protons from the axial methionine were identified in the reduced form. The heme methyl resonances of the ferri form were also assigned. EPR spectroscopy was also used to probe the ferric heme environment. A signal at gmax approximately 3.5 at pH 7.5 was observed indicating an almost axial heme environment. At higher pH values the signal at gmax approximately 3.5 converts mainly to a signal at g approximately 2.96. The pKa associated with this change is around 11.3. The N-terminal sequence of this cytochrome was determined and compared with known amino acid sequences of other cytochromes.

Amino Acid Sequence↗
PubMed1992

Direct spectroscopic evidence for the presence of a 6Fe cluster in an iron-sulfur protein isolated from Desulfovibrio desulfuricans (ATCC 27774)

A novel iron-sulfur protein was purified from the extract of Desulfovibrio desulfuricans (ATCC 27774) to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a monomer of 57 kDa molecular mass. It contains comparable amounts of iron and inorganic labile sulfur atoms and exhibits an optical spectrum typical of iron-sulfur proteins with maxima at 400, 305, and 280 nm. Mössbauer data of the as-isolated protein show two spectral components, a paramagnetic and a diamagnetic, of equal intensity. Detailed analysis of the paramagnetic component reveals six distinct antiferromagnetically coupled iron sites, providing direct spectroscopic evidence for the presence of a 6Fe cluster in this newly purified protein. One of the iron sites exhibits parameters (delta EQ = 2.67 +/- 0.03 mm/s and delta = 1.09 +/- 0.02 mm/s at 140 K) typical for high spin ferrous ion; the observed large isomer shift indicates an iron environment that is distinct from the tetrahedral sulfur coordination commonly observed for the iron atoms in iron-sulfur clusters and is consistent with a penta- or hexacoordination containing N and/or O ligands. The other five iron sites are most probably high spin ferric. Three of them show parameters characteristic for tetrahedral sulfur coordination. In correlation with the EPR spectrum of the as-purified protein which shows a resonance signal at g = 15.3 and a group of signals between g = 9.8 and 5.4, this 6Fe cluster is assigned to an unusual spin state of 9/2 with zero field splitting parameters D = -1.3 cm-1 and E/D = 0.062. Other EPR signals attributable to minor impurities are also observed at the g = 4.3 and 2.0 regions. The diamagnetic Mössbauer component represents a second iron cluster, which, upon reduction with dithionite, displays an intense S = 1/2 EPR signal with g values at 2.00, 1.83, and 1.31. In addition, an EPR signal of the S = 3/2 type is also observed for the dithionite-reduced protein.

Amino Acid Sequence↗
PubMed1992

Regulation of mitochondrial respiration by controlling the permeability of the outer membrane through the mitochondrial channel, VDAC.

Mitochondrial functions depend not only on the properties of the particular enzyme systems, but also on the continual flux of metabolites between the cytoplasm and mitochondrial spaces. We report the results of experiments that strongly indicate that a soluble mitochondrial protein can regulate mitochondrial respiration by reducing the permeability of the outer membrane. This protein is known as the VDAC modulator because it induces the outer mitochondrial membrane channel, VDAC, to close. When added to intact mitochondria, the modulator reduces the ADP-stimulated respiration. This inhibition can be prevented by damaging the outer membrane prior to modulator addition. Another mitochondrial activity, adenylate kinase, is reduced by 40% by the addition of the VDAC modulator to intact mitochondria. Again, damaging the outer membrane removed the modulator effect. Dextran sulfate, an artificial polyanion that acts on VDAC channels in a similar way to the VDAC modulator, has the same effects on intact mitochondria. The findings correlate well with observations of the actions of the VDAC modulator on reconstituted VDAC channels, in which the modulator induces the channel to enter a very low conductive state. The ability of a mitochondrial protein to regulate mitochondrial activities by reducing the permeability of the outer membrane further fuels the hypothesis that this membrane participates in the overall regulation of mitochondrial functions.

Adenylate Kinase↗
PubMed1992

Detection of human herpesvirus-6 DNA by polymerase chain reaction in serum or plasma.

Human herpesvirus-6 (HHV-6) is a newly identified human pathogen. Currently clinicians rely mainly on blood lymphocyte culture and serological tests to diagnose HHV-6 infection. The polymerase chain reaction (PCR) was carried out on the plasma or sera of patients to determine the value of PCR in the diagnosis of HHV-6 infection. A total of 30 patients entered the study; 10 were experiencing acute HHV-6 infections and 20 were healthy and served as controls. HHV-6 DNA was detected by PCR in the serum or plasma of the 10 cases with acute HHV-6 infections. All 20 controls had no HHV-6 DNA in their sera. The time for serum to become PCR-positive coincided with the appearance of IgG HHV-6 antibody. The relatively late presence of HHV-6 DNA in serum might result from late lysis of infected cells by immune responses. It is concluded that detection of HHV-6 DNA by PCR in the serum is a valuable tool for the diagnosis of acute and/or active viral infection.

Adult↗
PubMed1992

Use of antigen expressed in bacteria for detection of EBV-specific thymidine kinase antibodies in sera from patients with nasopharyngeal carcinoma.

Two cDNA clones covering the N- and C-terminal portions of the EBV BXLF1 open reading frame were selected from a cDNA library derived from P3HR1 cells. The two clones were ligated, the N-terminal untranslated region truncated, and the product inserted into an E. coli expression vector, pET3CP*. The fusion protein was expressed under control of the T7 phage phi 10 gene promoter and shown to possess thymidine kinase activity. The protein was then used as an antigen to detect antibody reactivities in serum samples of nasopharyngeal carcinoma patients and healthy blood donors. Using a 1:400 dilution of serum samples in Western blot analyses, it was possible to differentiate the reactivities of serum IgA of NPC patients and healthy donors. The prevalence of positive reactivity to EBV TK in NPC was around 84%. The test was compared to others used for early diagnosis of NPC and was able to detect some patients who were negative in those tests.

Antibodies, Viral↗
PubMed1992

A soluble mitochondrial protein increases the voltage dependence of the mitochondrial channel, VDAC.

A soluble protein isolated from mitochondria has been found to modulate the voltage-dependent properties of the mitochondrial outer membrane channel, VDAC. This protein, called the VDAC modulator, was first found in Neurospora crassa and then discovered in species from other eukaryotic kingdoms. The modulator-containing fraction (at a crude protein concentration of 20 micrograms/ml) increases the voltage dependence of VDAC channels over 2-3-fold. At higher protein concentrations (50-100 micrograms/ml), some channels seem to remain in a closed state or be blocked while others display the higher voltage dependence and are able to close at low membrane potentials. By increasing the steepness of the voltage-dependent properties of VDAC channels, this modulator may serve as an amplifier in vivo to increase the sensitivity of the channels in response to changes in the cell's microenvironment, and consequently, regulate the metabolic flux across the outer mitochondrial membrane by controlling the gating of VDAC channels.

Electrophysiology↗
PubMed1992

Cloning and expression of a cDNA encoding the Epstein-Barr virus thymidine kinase gene.

A clone of the Epstein-Barr virus (EBV) thymidine kinase (TK) gene was derived from a cDNA library of P3HR1 cells. The gene product was expressed as a fusion protein in a procaryotic system by using T7 RNA polymerase. The recombinant TK showed a molecular mass of 67 kDa and was biologically active. Antiserum raised in mice immunized with partially purified TK recognized an antigen present in EBV-superinfected Raji cells using an indirect immunofluorescence assay.

Animals↗
PubMed1992

Prevalence of antibody to Epstein-Barr virus DNase in children.

Antibodies to Epstein-Barr-virus-specific DNase in children under 15 years old from Taipei, Taiwan were determined by an enzyme neutralization test. Serum was taken as positive for the anti-DNase antibody when 1 ml of serum could neutralize > or = 2 units of the DNase activity. Among the 1,292 sera tested, 5.6% of the children were shown to have anti-EBV DNase antibodies, a result similar to that for adults. Maternal antibodies against the EBV DNase were found to decrease very rapidly 2 months after the child's birth, and new antibody appeared upon natural EBV infection.

Adolescent↗
PubMed1992

Induction of human monocyte cell line U937 differentiation and CSF-1 production by phorbol ester.

The monoblast-like human histiocytic lymphoma cell line U937 can be induced by phorbol 12-myristate 13-acetate (PMA) at concentrations of 8-16 ng/ml to undergo differentiation. The induced cells show growth inhibition, morphological changes to monocyte-macrophage-like cells, and increases in nitroblue tetrazolium (NBT) and nonspecific esterase activities. The expression of surface markers MO1 and MO2 for macrophages is also elevated. The induced differentiation of U937 cells is accompanied by the appearance of colony-stimulating activity in the culture medium as assayed on mouse bone marrow cells. Gel filtration chromatography of the conditioned media shows a peak of colony-stimulating factor (CSF) activity with an apparent molecular weight of approximately 100 kd. Morphological analysis of the colonies reveals a predominant monocyte-macrophage population. This CSF activity can be neutralized by anti-human colony-stimulating factor 1 (CSF-1) antibody, and Western blot analysis shows a CSF-1 band. All these results strongly indicate that CSF produced by induced U937 cells is CSF-1. Northern blot analysis of RNA isolated from U937 cells demonstrated that CSF-1 gene expression started after PMA induction for 3 h and declined after 18 h. Protooncogenes c-fos and c-jun were expressed after half an hour of incubation with PMA.

Blotting, Northern↗
PubMed1991

The primary structure of rubrerythrin, a protein with inorganic pyrophosphatase activity from Desulfovibrio vulgaris. Comparison with hemerythrin and rubredoxin.

The complete polypeptide chain of rubrerythrin from the sulfate reducing bacterium Desulfovibrio vulgaris, strain Hildenborough NCIB 8303, was found by protein chemical techniques to consist of 191 residues and to have the amino acid sequence [sequence: see text] The C-terminal part of the protein (position 153----191) shows the typical sequence features of rubredoxin, a protein with a nonheme iron center also present in the same and other Desulfovibrio species. Based on the known three-dimensional structure of D. desulfuricans rubredoxin, we propose that the C-terminal part of rubrerythrin is folded in a similar way and suggest that the deletion of the extra 10 residues is compatible with the same basic rubredoxin-fold. After characterization of the C-terminal region, and in contrast to what could be expected from previously published spectroscopic analyses, the N-terminal region 1-152 of rubrerythrin appears to have no sequence similarity with the eukaryotic protein hemerythrin which is known to contain a binuclear iron center bound by 5 histidine ligands. However, the N-terminal region of rubrerythrin does contain 5 histidine residues but they are differently spaced along the peptide chain. We suggest that at least one of the 3 histidine residues located in the rubredoxin-like center of rubrerythrin may be liganded to one iron atom of the hemerythrin-like center. This paper is the first sequence report of a protein with pyrophosphatase activity although the physiological substrate for the rubrerythrin may be not inorganic pyrophosphate.

Amino Acid Sequence↗
PubMed1991

Calcium is required for the reduction of sulfite from hydrogen in a reconstituted electron transfer chain from the sulfate reducing bacterium, Desulfovibrio gigas.

Calcium is found a strong stimulator of sulfite reduction from hydrogen. A coupling protein of molecular weight 65,000 can be isolated from Desulfovibrio gigas. It functions in a reconstituted electron transfer chain between hydrogenase and sulfite reductase. Its N-terminal sequence shows high homologies with calcium or magnesium binding sites from other calcium-binding proteins.

Amino Acid Sequence↗