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Biomedical subjects

C Fu

Publications and source records attributed to C Fu.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1994

An experimental model for study of Candida survival and transmission in human volunteers.

In order to determine the potential for cross-transmission of Candida spp. between health-care workers and patients, the survival of clinical isolates of five species of Candida on the palms of human volunteers was tested. One hundred microliters of a McFarland 1.0 density suspension (5 x 10(5) cfu) from an overnight culture of Candida albicans, Candida krusei, Candida parapsilosis, Candida tropicalis and Candida glabrata was used as inoculum. The degree of hydrophobicity of the different Candida species was also tested and did not influence the survival. The half-lives were brief, being 9.5, 12.4, 7.4, 12.8, 9.6 min for Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, and Candida tropicalis, respectively, but at 45 min 2.6 x 10(3) to 3 x 10(4) organisms remained on the hands. Survival of Candida albicans for as long as 24 h on inanimate surfaces was observed. Transmission from one hand to a second hand occurred in 69% of the experiments and from the first to a third hand in 38%. Transmission to and from inanimate surfaces was successful in most of the experiments (90%). This experimental model aids in the biological study of Candida spp. and suggests some of the potential mechanisms of transmission.

Candida↗
PubMed1994

The FeSII protein of Azotobacter vinelandii is not essential for aerobic nitrogen fixation, but confers significant protection to oxygen-mediated inactivation of nitrogenase in vitro and in vivo.

The FeSII protein of Azotobacter vinelandii has been proposed to mediate the 'conformational protection' of the molybdenum-dependent nitrogenase components against oxygen inactivation. We have cloned and characterized the structural gene for the FeSII protein (the fesII locus). Hybridization studies did not reveal the presence of fesII-like genes in a number of diverse species of well-studied nitrogen-fixing bacteria, with the exception of Azotobacter chroococcum. The fesII locus is transcriptionally expressed during both nitrogen fixing and non-nitrogen fixing conditions, although the level of its message is upregulated by approximately 2.5-fold during nitrogen fixation. The promoter region was identified by primer extension analysis, and is similar to other sigma 70-type promoters. Mutants devoid of the FeSII protein were constructed. These mutants possessed growth characteristics on a variety of carbon substrates during non-diazotrophic as well as diazotrophic growth that were essentially indistinguishable from the wild-type strain. Nevertheless, the nitrogenase activity in cell-free extracts is significantly more sensitive to irreversible oxygen inactivation in the mutants as compared with the wild type. When treated with 250 mM NaCl (a condition known to dissociate FeSII from nitrogenase components), the wild-type and mutant extracts were equally hypersensitive to oxygen inactivation. Upon energy starvation, conditions in which 'respiratory protection' is inoperable, the MoFe and Fe proteins of nitrogenase are degraded much more rapidly in vivo in the deletion mutants, compared to the wild type. Strains relying on either the vanadium or the 'iron-only' alternative nitrogenases exhibited similar growth rates irrespective of the presence or absence of the FeSII protein, and the in vitro inactivation of the vanadium nitrogenase components was not affected by the lack of the FeSII protein. All in all, these results are consistent with a model whereby 'respiratory protection' is the major physiological mechanism responsible for the protection of all three nitrogenases during energy-supplemented growth. Upon energy starvation, however, 'conformational protection', mediated by the FeSII protein is capable of temporarily protecting the conventional molybdenum nitrogenase components from inactivation and subsequent degradation.

Aerobiosis↗
PubMed1994

Sequences and characterization of hupU and hupV genes of Bradyrhizobium japonicum encoding a possible nickel-sensing complex involved in hydrogenase expression.

A 2.7-kb DNA fragment of Bradyrhizobium japonicum previously shown to be involved in hydrogenase expression has been sequenced. The area is located just upstream of the hupSLCDF operon and was found to contain two open reading frames, designated hupU and hupV; these encode proteins of 35.4 and 51.8 kDa, respectively. These proteins are homologous to Rhodobacter capsulatus HupU, a possible repressor of hydrogenase expression in that organism. B. japonicum HupU is 54% identical to the N terminus of R. capsulatus HupU, and HupV is 50% identical to the C terminus of R. capsulatus HupU. HupU and HupV also show homology to the [Ni-Fe] hydrogenase small and large subunits, respectively. Notably, HupV contains the probable nickel-binding sites RxCGxC and DPCxxCxxH, which are located in the N- and C-terminal portions, respectively, of the large subunit of hydrogenases. Hydrogenase activity assays, immunological assays for hydrogenase subunits, and beta-galactosidase assays on mutant strain JHCS2 (lacking a portion of HupV) were all indicative that HupV is necessary for transcriptional activation of hydrogenase. A physiological role as a possible nickel- or other environmental (i.e., oxygen or hydrogen)-sensing complex is proposed for HupU and HupV.

Amino Acid Sequence↗
PubMed1994

Omeprazole potentiates atracurium and succinylcholine paralysis in vivo in rats.

We examined the effect of proton pump inhibitor omeprazole on neuromuscular paralysis induced with either nondepolarizing or depolarizing neuromuscular blocking drugs in anesthetized and mechanically ventilated rats. Neuromuscular paralysis, as judged by tibialis anterior muscle twitch tension in response to sciatic nerve stimulation, was maintained at about 50% with intravenous (i.v.) bolus and infusion regimens of either atracurium or succinylcholine. Omeprazole, 0.5, 1, and 10 mg/kg i.v., was then administered at 10-min intervals while the infusion of the neuromuscular blocker was continued. Omeprazole at all three doses increased the steady-state neuromuscular paralysis produced with either atracurium (pre-omeprazole versus final post-omeprazole paralysis; mean +/- SE, n = 6, 53.0% +/- 2.3% vs 80.0% +/- 5.3%) or succinylcholine (50.8% +/- 1.5% vs 86.4% +/- 5.1%). Omeprazole, 0.5, 1.0, and 10 mg/kg i.v., given directly and without any neuromuscular blocker, produced approximately 5% depression of the muscle twitch response. Omeprazole, i.v. at human therapeutic doses, alters neuromuscular function and enhances the action of both atracurium and succinylcholine in vivo in rats.

Animals↗
PubMed1994

[Dynamic study on relationship between serum SEAIC level and hepatic pathological changes in mice infected with Schistosoma japonicum].

Using purified rabbit polyclonal antibodies to SEA, avidin-biotin system and capture ELISA technique, we observed the dynamic changes in the level of the circulating soluble egg antigen-antibody complex (SEAIC) in murine sera at various weeks post infection. Simultaneously, the diameter and area of liver egg granuloma were measured by using profile analytical technique. Serum SEAIC was first detected 4 weeks post infection (p.i.), reaching peak level at 6-7th week, and then gradually dropped, and maintained at moderately high level till the end of the observation (12 weeks p.i.). Schistosome eggs appeared in liver tissue at 4 weeks p.i. No egg granuloma could be found until 6 weeks p.i. The peak of the average diameter and area of liver egg granuloma was noted at 7 weeks p.i., then dropped gradually. Its dynamic changes were consistent with that of the serum SEAIC level. It is therefore suggested that the serum SEAIC level could be a reference index reflecting the extent of the pathological changes of the liver. Moreover, SEAIC might play an important role in the pathogenesis of schistosomiasis japonica.

Animals↗
PubMed1993

Monoclonal antibodies for structure-function studies of (R)-3-hydroxybutyrate dehydrogenase, a lipid-dependent membrane-bound enzyme.

Monoclonal antibodies (mAbs) have been used to study structure-function relationships of (R)-3-hydroxybutyrate dehydrogenase (BDH) (EC 1.1.1.30), a lipid-requiring mitochondrial membrane enzyme with an absolute and specific requirement for phosphatidylcholine (PC) for enzymic activity. The purified enzyme (apoBDH, devoid of phospholipid and thereby inactive) can be re-activated with preformed phospholipid vesicles containing PC or by short-chain soluble PC. Five of six mAbs cross-react with BDH from bovine heart and rat liver, including two mAbs to conformational epitopes. One mAb was found to be specific for the C-terminal sequence of BDH and served to: (1) map endopeptidase cleavage and epitope sites on BDH; and (2) demonstrate that the C-terminus is essential for the activity of BDH. Carboxypeptidase cleavage of only a few (< or = 14) C-terminal amino acids from apoBDH (as detected by the loss of C-terminal epitope for mAb 3-10A) prevents activation by either bilayer or soluble PC. Further, for BDH in bilayers containing PC, the C-terminus is protected from carboxy-peptidase cleavage, whereas in bilayers devoid of PC the C-terminus is cleaved, and subsequent activation by PC is precluded. We conclude that: (1) the C-terminus of BDH is essential for enzymic activity, consistent with the prediction, from primary sequence analysis, that the PC-binding site is in the C-terminal domain of BDH; and (2) the allosteric activation of BDH by PC in bilayers protects the C-terminus from carboxypeptidase cleavage, indicative of a PC-induced conformational change in the enzyme.

Animals↗
PubMed1993

Rapid and efficient selection of recombinant site-directed mutants of Bradyrhizobium japonicum by colony hybridization.

Due to the high incidence of spontaneous antibiotic resistance and slow growth of Bradyrhizobium japonicum strains, screening for site-directed mutants is cumbersome and time-consuming. A rapid method for selection of recombinant site-directed mutants of B. japonicum was developed. A kanamycin (Km) and a spectinomycin (Sp) cassette were each used to replace DNA fragments in the chromosome by homologous recombination. The primary new features of this method involve a simple plate selection for the antibiotic (Km or Sp) resistant mutants, then colony streaking, and lysis for DNA hybridization on a nitrocellulose filter enabling direct identification of the recombinant site-directed mutants. This method has permitted us to quickly and easily identify a large number of positive recombinant mutants from a large number of individual colonies. The procedure eliminates the need to first isolate genomic DNA from each mutant for Southern hybridization. All of the tested site-directed mutants from this method were confirmed to exhibit the expected mutant phenotype.

DNA, Bacterial↗
PubMed1993

Activation of a nonselective cation channel by swelling in atrial cells.

Cell swelling has been shown to increase the permeability of the plasma membrane to ions such as K+, Na+, Ca2+ or Cl- in many types of cells. In cardiac cells, swelling has been reported to increase Cl- conductance, but whether cation-selective currents are activated by swelling is not known. Low Cl- or Cl(-)-free solutions were used to study the presence of such currents. Lowering the osmolarity of the extracellular medium from 299 to 219 mOsm resulted in cell swelling and concurrent activation of a cation-selective whole-cell current. When cell-attached patches were formed on swollen cells, opening of bursting single channel currents were observed in 18% of the patches studied. Ion substitution experiments indicated that the channel discriminated poorly among monovalent cations, and was impermeable to Cl-. The channel was permeable to Ca2+. In symmetrical 140 mM K+, the current-voltage relation was linear with a single channel conductance of 36 +/- 3 pS. Depolarization increased channel open probability. Interestingly, depending on the membrane patch studied, application of negative pressure to the pipette caused either an increase or a decrease in the open probability of the channel already activated by swelling. Thus, the sensitivity to tension of the swelling-activated channel was different from those of previously reported stretch-activated channels. These findings suggest that nonselective cation channels exist in rat atrial cells and may be involved in swelling-induced changes in cell function.

Animals↗
PubMed1993

Immunocytochemical study with anti-muscle actin antibody (HHF35) on myocardial ischaemia and reperfusion injury in rats.

Myocardial ischaemia and reperfusion injury in 16 anaesthetized Sprague-Dawley rats, eight of which were pretreated with morphine (5 mg/kg, intraperitoneally) to prevent arrhythmias, were studied immunocytochemically with anti-muscle actin specific monoclonal antibody (HHF35). Eight cases of simple ischaemia and eight cases of sham-operated rats were used as controls. With HHF35 ABC immunocytochemical method, the left ventricular myocardium in the reperfusion group (without morphine) showed large areas of staining loss, but no loss of staining was seen in controls. No significant changes were seen with H&E stain in all hearts. Compared with HBFP, acridine orange (AO), eosin-fluorescence, HHF35 ABC staining best demonstrated myocardial reperfusion injury. The results indicate that the degree of myocardial damage may be related to the arrhythmias.

Animals↗
PubMed1993

A genetic region downstream of the hydrogenase structural genes of Bradyrhizobium japonicum that is required for hydrogenase processing.

Deletion of a 2.9-kb chromosomal EcoRI fragment of DNA located 2.2 kb downstream from the end of the hydrogenase structural genes resulted in the complete loss of hydrogenase activity. The normal 65- and 35-kDa hydrogenase subunits were absent in the deletion mutants. Instead, two peptides of 66.5 and 41 kDa were identified in the mutants by use of anti-hydrogenase subunit-specific antibody. A hydrogenase structural gene mutant did not synthesize either the normal hydrogenase subunits or the larger peptides. Hydrogenase activity in the deletion mutants was complemented to near wild-type levels by plasmid pCF1, containing a 6.5-kb BglII fragment, and the 65- and 35-kDa hydrogenase subunits were also recovered in the mutants containing pCF1.

Cosmids↗
PubMed1992

Misreading of the argI message in Escherichia coli.

It has previously been shown that either phenylalanine codon, UUU or UUC, could be misread as leucine during phenylalanine starvation, if the codons encoded residue 8 of the Escherichia coli argI gene product, ornithine transcarbamylase (OTC). However, no leucine misincorporation was detected when either of these same codons encoded residue 3. Here we report that leucine misincorporation can be directed by a UUU codon for residue 3 of OTC during phenylalanine starvation, if the argI gene has been mutated so that the codon preceding the UUU has been changed from the rarely used glycine codon GGG to the more commonly used GGC.

Amino Acid Sequence↗