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C Fu

Publications and source records attributed to C Fu.

At least 37 records · Page 2Linked to original sources
PubMed1996

Hydrogen uptake hydrogenase in Helicobacter pylori.

The peptic ulcer-causing bacterium Helicobacter pylori was found to contain an H2-uptake hydrogenase activity coupled to whole cell (aerobic) respiration. The activity was localized to membranes which functioned in the H2-oxidizing direction with a variety of artificial and physiological electron acceptors of positive redox potential. Immunoblotting of H. pylori membrane components with anti (B. japonicum) hydrogenase large and small subunit-specific antisera identified H. pylori hydrogenase peptides of approximately 65 and 26 kDa respectively, and H. pylori genomic DNA fragments hybridizing to the (B. japonicum) hydrogenase structural genes were identified. The membrane-bound activity was subject to anaerobic activation, like many NiFe hydrogenases. Difference absorption spectral studies revealed absorption peaks characteristic of b and c-type cytochromes, as well as of a bd-type terminal oxidase in the H. pylori H2-oxidizing membrane-associated respiratory chain.

Aerobiosis↗
PubMed1996

Ca2+ waves in lung capillary endothelium.

Although cytosolic Ca2+ importantly regulates organ function, lung microvascular [Ca2+]i regulation remains poorly understood because of the lack of direct in situ quantification. In the present study, we report the first endothelial [Ca2+]i quantification by the fura 2 method in microscopically imaged venular capillaries of the isolated blood-perfused rat lung. Sequential images indicated the presence of intercellular Ca2+ waves that spontaneously originated from pacemaker endothelial cells and then spread for short distances along the capillary wall, inducing synchronous endothelial [Ca2+]i oscillations. Fast Fourier analyses of the oscillations revealed a dominant wave component with an amplitude of 37 nmol/L, frequency of 0.4 min-1, and velocity of 5 microns/s. The intracellular Ca2+ wave was unaffected by blood flow stoppage or by infusions of Ca(2+)-containing or Ca(2+)-free dextran. Inhibition of the wave by thapsigargin in Ca(2+)-free dextran and by the gap junction uncoupler, heptanol, indicated that it was generated by endosomal Ca2+ release in the pacemaker cell and was propagated by gap junctional communication. In the presence of histamine, enhancement of the wave accounted for a significant component of the coordinated [Ca2+]i increase in the capillary segment. No intercellular Ca2+ waves were evident in adjoining alveolar epithelial cells. Our findings indicate a novel mechanism of [Ca2+]i regulation in the lung capillary under both resting and stimulated conditions. Pacemaker-induced Ca2+ waves, generated intracellularly by unknown initiating mechanisms, communicated to adjoining cells to determine [Ca2+]i profiles in short interbranch segments of capillary walls.

Animals↗
PubMed1995

Soluble ligands of the alpha v beta 3 integrin mediate enhanced tyrosine phosphorylation of multiple proteins in adherent bovine pulmonary artery endothelial cells.

Binding of substrate-bound extracellular matrix proteins to cell surface integrins results in a variety of cellular responses including adhesion, cytoskeletal reorganization, and gene expression. We have previously shown that addition of soluble SC5b-9, the complement-vitronectin complex, resulted in an RGD-dependent increase in lung venular hydraulic conductivity (Ishikawa, S., Tsukada, H., and Bhattacharya, J. (1993) J. Clin. Invest. 91, 103-109). To identify specific integrin(s) and signal transduction pathways that are responsive to soluble vitronectin-containing ligands, we exposed confluent bovine pulmonary artery cells to purified soluble human mono- or multimeric vitronectin, or SC5b-9, and determined the extent of endothelial cell protein tyrosine phosphorylation. Monomeric vitronectin (Vn) did not induce enhanced protein tyrosine phosphorylation. However, multimeric Vn and SC5b-9 elicited time- and concentration-dependent increases in tyrosine phosphorylation of numerous proteins. Antiserum against vitronectin, RGD peptides, and monoclonal and polyclonal antibodies against the alpha v beta 3 integrin blocked the vitronectin- or SC5b-9-induced enhanced accumulation of tyrosine phosphoproteins, while antibodies against beta 1 integrins and the alpha v beta 5 integrin did not. Clustering of the alpha v beta 3 integrin using monoclonal antibody LM609 caused a pattern of enhanced tyrosine phosphorylation similar to that caused by multimeric Vn and SC5b-9, suggesting that aggregation of alpha v beta 3 was critical for signaling. Among the proteins that underwent enhanced tyrosine phosphorylation in response to vitronectin were the cytoskeletal proteins paxillin, cortactin, and ezrin, as well as the SH2 domain-containing protein Shc, and p125FAK. We conclude that ligation of the alpha v beta 3 integrin by soluble ligands promotes enhanced phosphorylation of several proteins implicated in tyrosine kinase signaling and suggest that this pathway may be important in inflammatory states which are accompanied by accumulation of SC5b-9.

Animals↗
PubMed1995

HypB protein of Bradyrhizobium japonicum is a metal-binding GTPase capable of binding 18 divalent nickel ions per dimer.

Bradyrhizobium japonicum hypB encodes a protein containing an extremely histidine-rich region (24 histidine residues within a 39-amino-acid stretch) and guanine nucleotide-binding domains. The product of the hypB gene was overexpressed in Escherichia coli and purified by Ni(2+)-charged metal chelate affinity chromatography (MCAC) in a single step. In SDS/PAGE, HypB migrated at 38 kDa--slightly larger than the calculated molecular mass (32.8 kDa). Purified HypB has GTPase activity with a kcat of 0.18 min-1 and a Km for GTP of 7 microM, and it has dGTPase activity as well. HypB exists as a dimer of molecular mass 78 kDa in native solution as determined by fast protein liquid chromatography on Superose 12. It binds 9.0 +/- 0.14 divalent nickel ions per monomer (18 Ni2+ per dimer) with a Kd of 2.3 microM; it also binds Zn2+, Cu2+, Co2+, Cd2+, and Mn2+. In-frame deletion of the histidine-rich region (deletion of 38 amino acids including 23 histidine residues) resulted in a truncated HypB that did not bind to the MCAC column, whereas in-frame deletion of 14 amino acids including 8 histidine residues within HypB resulted in a truncated HypB that still bound to the column. The results indicate that the histidine residues within the histidine-rich region of HypB are involved in metal binding.

Amino Acid Sequence↗
PubMed1995

Structure and function of the mouse insulin-like growth factor binding protein 5 gene promoter.

The actions of insulin-like growth factors I and II (IGF-I and -II) are modulated by interactions with one or more of a family of secreted IGF binding proteins (IGFBPs). IGFBP-5, the most conserved of the six known IGFBPs, is a 252-amino-acid protein that has been shown both to potentiate and inhibit IGF action. In previous studies, we have cloned and characterized the mouse IGFBP-5 gene and demonstrated that it is expressed in a hierarchical pattern in different adult mouse tissues and during rodent embryonic development. In this report, we describe the initial analysis of the IGFBP-5 gene promoter. By transient gene transfer studies, we show the orientation-specific activity of DNA fragments containing from 31 to 4,100 bp from the 5'-flanking region of the mouse IGFBP-5 gene in directing expression of the heterologous reporter gene luciferase in Hep G2 cells. DNA fragments with only 156 bp of 5'-flanking sequence mediated over 60% of maximal promoter activity, and a segment containing the TATA box and the first 120 bp of exon 1 still conferred some promoter function. Within the highly active 156-bp region, we identified a 37-bp segment from -70 to -34 that exhibited specific binding in DNase I footprinting and gel-mobility shift experiments with Hep G2 nuclear protein extracts. The footprinted region, which is almost completely conserved in the rat and human IGFBP-5 genes, was responsible for at least 70% of the activity of the intact promoter, as evidenced by the deleterious consequences of small internal deletions within this sequence on promoter function.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Different properties of the atrial G protein-gated K+ channels activated by extracellular ATP and adenosine.

Extracellular ATP (ATPo) and adenosine activate G protein-gated inwardly rectifying K+ currents in atrial cells. Earlier studies have suggested that the two agonists may use separate pathways to activate the K+ current. Therefore, we examined whether the K+ channels activated by the two agonists have different properties under identical ionic conditions. In cell-attached patches, K+ channels activated by 100 microM ATP in the pipette had a single-channel conductance and mean open time of 32.0 +/- 0.2 pS and 0.5 +/- 0.1 ms, respectively, compared with 31.3 +/- 0.3 pS and 0.9 +/- 0.1 ms for the K+ channels activated by adenosine (140 mM KCl). With ATPo as the agonist, the K+ channel activity in cell-attached patches was approximately threefold lower than that in inside-out patches with 100 microM GTP in the bath. Applying ATP to the cytoplasmic side of the membrane (ATPi) produced a biphasic concentration-dependent effect on channel activity: an increase at low [mean affinity constant (K0.5) = 190 microM] and a decrease at high (K0.5 = 1.3 mM) concentrations. In contrast, with adenosine as the agonist, K+ channel activity in cell-attached patches was approximately fourfold greater than that in inside-out patches with 100 microM GTP in the bath. In inside-out patches, ATPi only augmented the K+ channel activity (K0.5 = 32 microM). These results show that although both ATPo and adenosine activate kinetically similar K+ channels in atrial cells, the channels are regulated differently by intracellular nucleotides.

Adenosine↗
PubMed1995

Ligation of endothelial alpha v beta 3 integrin increases capillary hydraulic conductivity of rat lung.

Complement-mediated pulmonary edema results from increases in lung capillary hydraulic conductivity (Lp), possibly by receptor-mediated mechanisms. We considered the Lp effects of vitronectin and the vitronectin-containing complement complex SC5b-9, which ligate the integrin alpha v beta 3. Vitronectin, SC5b-9, and SC5b-9-enriched zymosan-activated serum all rapidly increased Lp, as determined by the split-drop technique in single lung capillaries of rat lung. The Lp increases were inhibited by a monospecific (LM609) and a polyclonal (R838) antibody against the alpha v beta 3 integrin but not by an irrelevant monoclonal antibody isotype matched with LM609, by a monoclonal antibody against the alpha v beta 5 integrin, or by preimmune rabbit serum. Vitronectin monomers failed to increase Lp. The tyrosine kinase blockers genistein and methyl 2,5-dihydroxycinnamate caused significant concentration-dependent inhibitions of Lp increases due to vitronectin and zymosan-activated serum. By contrast, the protein kinase C blocker calphostin C had no major effect. We conclude that (1) multivalent ligation of the luminally located alpha v beta 3 integrin of lung capillary endothelium increases transcapillary liquid flux, and (2) the dominant signal transduction pathway for this effect occurs through tyrosine kinase activation.

Animals↗
PubMed1995

Effects of TNF alone or in combination with chemotherapeutic agents on human ovarian cancers in vitro and in nude mice.

Using the tetrazolium (MTT) assay, we examined the cytotoxicities of recombinant human tumor necrosis factor (rhTNF) and five chemotherapeutic agents, namely CTX, 5-FU, VCR, DDP and KSM, on human ovarian cancer cell lines OVCAR3 and CAOV3. The results showed that the cytotoxicities of rhTNF at concentrations of 50-50,000 U/ml on OVCAR3 cell line and CAOV3 cell line exposed to rhTNF for 24 hours were from 14.2% +/- 6.8% to 67.2% +/- 3.0% and from 8.2% +/- 4.3% to 60.9% +/- 1.3%, respectively. The cytotoxicities of all five chemotherapeutic agents tested on the two cell lines were much lower than that of rhTNF. We also studied the combined antitumor potential of rhTNF with the five chemotherapeutic agents and the results showed that there were various degrees of synergism in cytotoxicities of rhTNF in combination with DDP or KSM on the two cell lines. Based on experiments in vitro, the in vivo antitumor activities of rhTNF, both alone and in combination with KSM, were examined in OVCAR3 cancer transplanted in nude mice. The results showed a considerable antitumor effect of rhTNF when it was used alone and a marked synergistic effect when it was used in combination with KSM on the xenograft tumors.

Animals↗
PubMed1995

[Effect of praziquantel treatment on serum circulating soluble egg antigen-antibody complex (SEAIC) level in mice infected with Schistosoma japonicum].

Employing Capture ELISA, we determined the level of the circulating soluble egg antigen-antibody complex (SEAIC) in sera from mice infected with Schistosoma japonicum for 7 weeks and then treated the infected mice with praziquantel (100 mg/kg body weight). It was noted that the serum SEAIC level in treated mice dropped gradually after treatment. There was a significant decrease in 13 out of 15 mice in serum SEAIC levels at five weeks after treatment, OD value dropped from 0.358 +/- 0.074 to 0.165 +/- 0.049 (P < 0.01). The serum SEAIC level in 10 mice declined to disappear (OD < 0.097) by the 7th week after treatment. By contrast, no significant changes in serum SEAIC levels were found in the non-treated mice, the serum SEAIC level in the control group maintained moderately high (OD = 0.266 +/- 0.076) till the end of the observation.

Animals↗
PubMed1995

A study of immunoactivity of retinal S-antigen in retinoblastoma.

PURPOSES: To study retinal S-antigen expression in human retinoblastoma and assess if there is a correlation between S-antigen immunoactivity and degree of retinoblastoma cell differentiations. METHODS: Ten cases of Chinese retinoblastoma parafin-embedded tissues were applied for this study. A strain of monoclonal antibody,MabA9C6, which defines an epitope in S-antigen retained in fixed-tissue sections, was used to study S-antigen expression in 10 cases of retinoblastomas. S-antigen was localized by the biotinavidin indirect immunoperoxidase technique and purified MabA9C6 ascites fluid was used with 1:100 dilution. The whole procedure could be finished within a few hours. RESULTS: The S-antigen immunoactivity was observed in different patterns: the "normal" photoreceptor elements incorporated in 3 cases of growing tumors; 3 of 4 Fleurettes and E-W rosettes; and scattered tumor cells in 50% of the cases. CONCLUSIONS: The result suggests that the expression of S-antigen in retinoblastoma may be used to assess the degree of tumor differentiation as another tumor marker in retinoblastoma.

Antigens↗
PubMed1994

Cloning and sequence of the Xenopus laevis homologue of the midkine cDNA.

The Xenopus laevis (Xl) homologue of the midkine-encoding gene (MK) has been isolated and sequenced from a X. laevis cDNA library made from the head region of stage-22 embryo. The nucleotide and deduced amino-acid sequences show a high degree of conservation with the avian and mammalian MK and pleiotrophin (PTN) genes and gene products. Highly conserved domains may indicate important regions for the function of the MK and PTN proteins.

Amino Acid Sequence↗
PubMed1994

Organization of the hydrogenase gene cluster from Bradyrhizobium japonicum: sequences and analysis of five more hydrogenase-related genes.

Previously, the deletion of a 2.9-kb chromosomal EcoRI fragment of DNA located 2.2 kb downstream from the end of the Bradyrhizobium japonicum hydrogenase structural genes caused lack of normal-sized hydrogenase (Hup) subunits and complete loss of Hup activity. It was suggested that this region encodes one or more genes required for Hup processing. Sequencing of a 3322-bp XcmI fragment of DNA covering this 2.9-kb EcoRI fragment within the hup gene cluster revealed the presence of five open reading frames (ORFs) designated hupG, hupH, hupI, hupJ and hupK, encoding polypeptides with calculated molecular masses of 15.8, 30.7, 7.6, 18.1 and 38 kDa, respectively. Based on deduced amino acid (aa) sequences, all five products of the hupGHIJK genes showed significant homology with other genes' products in several H2-utilizing bacteria. Of particular interest are HupG and HupI. HupG showed 70% similarity (28% identity) to the HyaE of the Escherichia coli hydrogenase-1 operon which was demonstrated to be involved in the processing of hydrogenase-1. HupI showed strong identity to rubredoxin and rubredoxin-like proteins from many other bacteria. The latter proteins contain two 'C-X-X-C' motifs, which may serve as iron ligands for non-heme iron proteins involved as intermediate electron carriers or in the assembly process for Fe-S (or NiFe-S) clusters.

Amino Acid Sequence↗
PubMed1994

The immunocytochemical demonstration of a relative lack of nerve fibres in the atrioventricular node and bundle of His in the sudden infant death syndrome (SIDS).

Whilst examining the variation with age of the nerve fibre content of the cardiac conduction system (CCS), using an immunocytochemical approach, it became evident that in two sudden infant death syndrome (SIDS) cases there was a selective lack of S100 positive nerve fibres in the atrioventricular (AV) node and His bundle. In the present study therefore, the examination of CCS with S100 was extended to a further five SIDS cases and three cases of sudden explained death. Also, in addition to S100--which selectively marks Schwann cells associated with both myelinated and non-myelinated nerves--PGP 9.5 (protein gene product) was used to reveal the presence of nerve axonal elements associated with the CCS. The results showed a uniform presence of S100 and PGP 9.5 positive nerve fibres in the sinoatrial (SA) node, the AV node and His bundle tissue of all three control cases. In contrast, five out of seven SIDS cases showed a uniform lack of staining with these markers in the AV node and His bundle tissue, whilst in the two remaining cases it was present in greatly diminished amounts. Staining in the SA node, although present in all seven cases, was reduced when compared with the control cases. This is the first time the CCS of SIDS cases has been studied with immunocytochemical markers of nerve elements. The overall results taken in conjunction with the epidemiology of SIDS suggest that the lack of AV node and His bundle innervation most probably reflects a delay in the development or maturation of the nerve elements of the CCS, similar to that noted for other parts of the central and peripheral nervous systems in SIDS.

Atrioventricular Node↗
PubMed1994

A ribosomal frameshifting error during translation of the argI mRNA of Escherichia coli.

Using fusions between the Escherichia coli genes argI and lacZ, it has been demonstrated that ribosomal frameshifting occurs at a frequency of between 3% and 16% within the argI mRNA, soon after the initiation codon. The frameshift involves a phenylalanyl-tRNA shifting into the +1 frame at the sequence UUU-U/C. The shift does not occur if the in-frame phenylalanine codon UUU is replaced by UUC. The level of frameshifting is higher in dense cultures and is not dependent on phenylalanine starvation. In the wild-type argI gene this frameshifting event would be an error, leading to a truncated, non-functional protein. Therefore, it is unlike the numerous examples of required frameshifting events that have been described in other genes.

Amino Acid Sequence↗
PubMed1994

Bacterial genes involved in incorporation of nickel into a hydrogenase enzyme.

Nickel is an essential component of all H2-uptake hydrogenases. A fragment of DNA that complements a H2-uptake-deficient but nickel-cured mutant strain (JHK7) of Bradyrhizobium japonicum was isolated and sequenced. This 4.5-kb DNA fragment contains four open reading frames designated as ORF1, hupN, hupO, and hupP, which encode polypeptides with predicted masses of 17, 40, 19, and 63.5 kDa, respectively. The last three open reading frames (hupNOP) are most likely organized as an operon with a putative sigma 54-type promoter. Based on its hydropathy profile, HupN is predicted to be a transmembrane protein. It has 56% identity to the previously described HoxN (high-affinity nickel transport protein) of Alcaligenes eutrophus. A subclone (pJF23) containing the hupNOP genes excluding ORF1 completely complemented (in trans) strain JHK7 for hydrogenase activity in low nickel conditions. pJF26 containing only a functional hupN complemented the hydrogenase activity of mutant strain JHK7 to 30-55% of the wild-type level. Mutant strain JHK70, with a chromosomal deletion in hupP but with an intact hupNO, showed greater activities than pJF26-complemented JHK7 but still had lower activities than the wild type at all nickel levels tested. pJF25, containing the entire hupO and hupP, but without hupN (a portion of hupN was deleted), did not complement hydrogenase activity of mutant strain JHK7. The results suggest that the products of the hupNOP operon are all involved in nickel incorporation/metabolism into the hydrogenase apoprotein. Based on (previous) nickel transport studies of strain JHK7, the hupNOP genes appear not to be involved in nickel transport by whole cells. The deleterious effects on hydrogenase expression are most pronounced by lack of the HupN product.

Amino Acid Sequence↗
PubMed1994

Sequence and characterization of three genes within the hydrogenase gene cluster of Bradyrhizobium japonicum.

A 2.0-kb DNA fragment downstream from the hydrogenase-encoding structural genes within the hydrogenase gene cluster of Bradyrhizobium japonicum was sequenced. Analysis of the nucleotide (nt) sequence revealed three open reading frames (ORFs), designated hupC, hupD and hupF, which encode polypeptides of 28, 21 and 10.7 kDa, respectively. Based on analysis of the nt sequence and physiological studies, hupSL (hydrogenase structural genes) and hupCDF are organized as a single transcriptional unit. Plasmid pRY12 carrying hupSL genes did not complement (restore) hydrogenase activity of the hupSL deletion mutant strain (JHCS2), whereas the activity of the mutant was considerably restored by pLD22 harboring the entire hydrogenase operon (hupSLCDF genes). Western blots revealed a very low level of hydrogenase protein in JHCS2 containing pRY12. The results suggest that the products of the hupCDF genes may be involved in either stabilizing the hydrogenase peptides (i.e., from degradation) or in post-translational regulation of hydrogenase production. The products of hupC and hupD were successfully expressed in Escherichia coli by a phage T7 promoter system, although the apparent sizes of the gene products were slightly larger than those calculated from the deduced amino-acid sequences.

Amino Acid Sequence↗
PubMed1994

Nucleotide sequences of two hydrogenase-related genes (hypA and hypB) from Bradyrhizobium japonicum, one of which (hypB) encodes an extremely histidine-rich region and guanine nucleotide-binding domains.

Sequencing of a 1359-bp (NruI-AccI) DNA fragment located approximately 5.2 kb downstream from the end of the hydrogenase structural genes of Bradyrhizobium japonicum revealed two open reading frames designated hypA and hypB, encoding polypeptides with predicted molecular masses of 12.3 and 32.8 kDa, respectively. Both hypA and hypB showed strong homology with other genes in hydrogenase-containing bacteria. Two 'C-X-X-C' motifs were contained in the deduced amino acid sequence of hypA, a motif that is present in all known products homologous to HypA. The deduced product of hypB contains an area remarkably rich in histidine residues at the N-terminus (24 histidines within a 39 amino acid stretch). The deduced HypB also contains GTP-binding domains. We postulate that the product of hypB is involved in nickel binding and accumulation, and may utilize energy (GTP) to mobilize nickel for its subsequent incorporation into hydrogenase.

Amino Acid Sequence↗