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S L Wong

Publications and source records attributed to S L Wong.

At least 19 recordsLinked to original sources
PubMed1992

Sorbitol dehydrogenase from Bacillus subtilis. Purification, characterization, and gene cloning.

Cloning of the sorbitol dehydrogenase gene (gutB) from Bacillus subtilis offers an excellent system for studying zinc binding, substrate specificity, and catalytic mechanism of this enzyme through protein engineering. As a first step to clone gutB, B. subtilis sorbitol dehydrogenase has been purified to homogeneity and characterized. It is a tetrameric enzyme with a molecular mass of 38 kDa for each subunit. Atomic absorption analysis shows the presence of 1 mol of zinc atom/subunit. Substrate specificity and stereospecificity of the enzyme toward C-2 and C-4 of hexitols were established. Sequence of the first 31 amino acids was determined, and a set of oligonucleotide probes was designed for gene cloning. A positive clone carrying a 5-kilobase pair HindIII insert was isolated and sequenced. Sequence alignment indicated that the deduced amino acid sequence of B. subtilis sorbitol dehydrogenase shows 36% identity in sequence with the liver sorbitol dehydrogenase from sheep, rat, and human. In reference to the sequence of alcohol dehydrogenase, two potential zinc binding sites were identified. Sequence information related to the structure-function relationships of the enzyme is discussed.

Amino Acid Sequence↗
PubMed1992

Production of stable anti-digoxin Fv in Escherichia coli.

We have created a bacterial expression-export system and have used it to express (14 mg l-1) the variable region fragment (Fv) of an anti-digoxin antibody (26-10) in Escherichia coli. The expression-export plasmid contains a T7 promoter and the E. coli signal sequences ompA [Movva et al., J. biol. Chem. 255, 27-29 (1980)] and phoA [Inouye et al., J. Bacteriol. 149, 434-439 (1982)] fused to heavy chain (VH) and light chain (VL) variable region sequences to generate an artificial cistron. The 26-10 Fv protein made using this system was soluble, unlike many other expression systems which produce insoluble proteins in the form of inclusion bodies. The 26-10 VH and VL proteins were cleaved at their mature N-termini and exported into the bacterial periplasm where they could be easily extracted and affinity purified on ouabain-Sepharose. 26-10 Fv bound to digoxin with similar affinity and specificity as the whole 26-10 antibody (Ka for Fv, 1.3 x 10(9) M-1, Ka for IgG, 7 x 10(9) M-1). 26-10 Fv appears to be remarkably stable in comparison with other Fv fragments. The half-life for chain dissociation of 26-10 Fv was 48 hr compared to the reported 1.5 hr half-life of McPC603 Fv. We present the proton NMR spectra of the 26-10 Fv as preliminary evidence that this expression-export system can be used to facilitate the analysis of the solution structure of 26-10 Fv by NMR.

Amino Acid Sequence↗
PubMed1992

Analysis of zidovudine distribution to specific regions in rabbit brain using microdialysis.

The distribution of zidovudine (3'-azido-3'-deoxythymidine; AZT) into two regions of rabbit brain was investigated in crossover using microdialysis. Six rabbits had guide cannulas surgically implanted in the lateral ventricle and thalamus by stereotaxic placement. After recovery, microdialysis probes were positioned and i.v. bolus doses of 5, 10, 20, and 30 mg/kg were administered to each animal over a period of 2 weeks. Blood was drawn via a marginal ear vein catheter for 8 hr. Brain dialysate was collected at 3 microliters/min from ventricle and thalamus dialysis probes every 10 min. Simulated cerebrospinal fluid (CSF), to which 3'-azido-2',3'-dideoxyuridine (AZdU) was added, was used as perfusate. AZdU loss, which was measured during simultaneous retrodialysis, served as a marker for in vivo recovery of AZT. AZT concentrations in plasma, as well as in ventricle and thalamus dialysate, were determined using a sensitive HPLC assay, and AZdU was simultaneously analyzed in the dialysates. Calculation of in vivo recovery of AZT was based on loss of AZdU from the perfusate during retrodialysis and was used to estimate the concentration of drug at both sites in the brain. In vitro loss of AZdU and recovery of AZT showed good agreement, demonstrating a bivariate regression slope of 0.99. The half-lives and AUCs (normalized to dose) achieved in the plasma, ventricle, or thalamus were not significantly different for the four doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Competitive inhibition of zidovudine clearance by probenecid during continuous coadministration.

The pharmacokinetics of zidovudine in the rabbit were studied during coadministration of probenecid at two infusion rates. Each animal (n = 6) served as its own control during an initial 8-hr infusion of zidovudine. In the second 8-hr infusion period, probenecid was coadministered with zidovudine. Urine samples were collected by bladder flush hourly for 19 hr. Plasma samples were taken at the midpoint of the urine collection interval and at predetermined intervals for 3 hr postinfusion. Plasma concentrations of zidovudine reached steady state during control periods but showed incomplete attainment of steady state during the infusions of probenecid at the higher rate. Total and renal clearance of zidovudine were reduced by 24.0 +/- 4.0 and 20.7 +/- 15%, respectively, during low-dose probenecid treatment and 48.9 +/- 7.4 and 55.7 +/- 3.4%, respectively, with high-dose probenecid treatment. Plasma probenecid concentrations during low-dose and high-dose infusion were 56.9 +/- 12 and 248 +/- 42 micrograms/ml. Postinfusion data showed that the zidovudine terminal half-life during high-dose probenecid treatment was longer than that with low-dose probenecid treatment (58.2 +/- 4.6 vs 39.0 +/- 9.1 min). The volume of distribution of zidovudine also decreased (1.76 +/- 0.27 vs. 1.10 +/- 0.095 L/kg) as a result of probenecid coadministration. The results are consistent with competitive inhibition of renal and nonrenal clearances. A drug interaction model relating zidovudine clearances to plasma probenecid concentrations was derived. Michaelis-type constants for probenecid inhibition of zidovudine renal and nonrenal clearances were 73 and 55 micrograms/ml, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Site-directed mutagenesis of the hydrogenase signal peptide consensus box prevents export of a beta-lactamase fusion protein.

A secretion vector, pVN1, expressing the [NiFe] hydrogenase signal peptide of Desulfovibrio vulgaris Hildenborough fused to beta-lactamase from Escherichia coli was constructed in order to study the unusual characteristics of hydrogenase signal peptides, which share a strictly conserved sequence, the consensus box: R-R-X-F-X-K. Although the hydrogenase signal peptide-beta-lactamase fusion protein was processed much more slowly than the fusion of beta-lactamase with its own signal peptide, the system mimicked several features expected for hydrogenase biosynthesis in E. coli, including increased export under anaerobic conditions. Site-directed mutagenesis of R(-28), the first arginine residue of the consensus box, to a glutamate completely inhibited export and processing of the fusion protein. The same mutation of R(-33), located outside the consensus box, had almost no effect. The data indicate a specific role for the consensus box sequence in the export mechanism for hydrogenase.

Amino Acid Sequence↗
PubMed1992

The role of the pro-sequence in the processing and secretion of the thermolysin-like neutral protease from Bacillus cereus.

The Bacillus cereus cnp gene coding for the thermolysin-like neutral protease (TNP) has been cloned, sequenced, and expressed in Bacillus subtilis. The protease is first produced as a pre-pro-protein (M(r) = 61,000); the pro-peptide is approximately two-thirds of the size of the mature protein. The pro-sequence has been compared with those of six other TNPs, and significant homologies have been found. Additionally, the TNP pro-sequences are shown to be homologous to the pro-sequence of Pseudomonas aeruginosa elastase. A mutant has been constructed from cnp, in which 23 amino acids upstream from the pro-protein processing site have been deleted. This region has no homologous analogue in any of the other TNP pro-sequences. The deletion results in a delay of six to eight hours in detection of active protease in the growth medium, as well as a 75% decrease in maximum protease production. N-terminal analysis of the mutant mature protein demonstrates that the processing site is unaltered by the pro-sequence deletion. The deletion must, therefore, modulate the kinetics of processing and/or secretion of the pro-protein.

Amino Acid Sequence↗
PubMed1992

Cloning and characterization of the groESL operon from Bacillus subtilis.

The sequence of the 10 N-terminal amino acids of a Bacillus subtilis protein that cross-reacts with antibody to Escherichia coli GroEL was used to design a set of degenerate oligonucleotide probes. These probes identified a clone which carries almost the entire groESL operon from a B. subtilis subgenomic library. By chromosomal walking, an additional fragment carrying the 3' end of groESL and its flanking sequence was isolated. Sequence analysis revealed two open reading frames (ORFs) in the cloned DNA. The upstream ORF encodes a 10-kDa protein which has 47% amino acid identity with E. coli GroES. The downstream ORF encodes a 58-kDa protein which is 62% identical to E. coli GroEL. A 2.1-kb groESL mRNA from B. subtilis was detected independently by Northern (RNA) blot analyses with a groES- and a groEL-specific probe. This demonstrated that groES and groEL are in an operon. The groESL promoter was located by using a promoter-probing plasmid, and the apparent transcription start site was mapped by primer extension analysis. The same promoter is utilized under normal and heat shock conditions. This promoter has the same features as a typical sigma A promoter. A strain in which the groESL operon was under the control of the sucrose-inducible sacB promoter was created. With this strain, it was possible to show that both groES and groEL are essential genes under both normal and heat shock conditions.

Amino Acid Sequence↗
PubMed1992

Influence of socioeconomic status on cardiovascular diseases in Hong Kong.

STUDY OBJECTIVE: The aim was to explore the relationships between five socioeconomic variables and three main cardiovascular diseases (ischaemic heart disease, hypertensive disease, and cerebrovascular disease) in Hong Kong. DESIGN: This cross sectional study used data from the 1986 by-census and registered death data for 1985 to 1987 in Hong Kong. For each of 24 districts, the correlation coefficients between log standardised mortality ratios for the three cardiovascular diseases and the percentages of professional and administrative workers, production and agricultural workers, persons aged 15 and over having tertiary education, households with higher income, and people living in private residential blocks were calculated. Besides simple linear regression and correlation, factor analysis was used to produce a new single surrogate measure summarising the five most useful variables in 24 sets of districts. SETTING: The whole Hong Kong area (population approximately 5.5 million) was divided into 24 districts, which were the study units. MEASUREMENTS AND MAIN RESULTS: For ischaemic heart disease in men, the correlation coefficients of the log standardised mortality ratios with the five socioeconomic variables as well as with the factor score were all statistically significant. For women, statistical significance was obtained in only two of five socioeconomic variables. No such trends were obtained for the other two cardiovascular diseases for either men or women. CONCLUSIONS: The study suggests that in Hong Kong in recent years, a higher level of socioeconomic status is associated with higher risk of death from ischaemic heart disease; but this association is not present for hypertensive disease and cerebrovascular disease.

Adolescent↗
PubMed1991

High-performance liquid chromatographic determination of 2',3'-didehydro-3'-deoxythymidine (d4T) in human and rabbit plasma and urine and its application to pharmacokinetic studies in the rabbit.

A rapid and sensitive liquid chromatographic assay for 2',3'-didehydro-3'-deoxythymidine (d4T) in plasma and urine is described. This assay uses thymidine oxetane (TO), a synthetic precursor of d4T, as internal standard. Sample preparation involves a simple extraction of plasma or urine with 5% isopropyl alcohol in methylene chloride. The method is specific and sensitive, allowing a linear response over a 2000-fold range of concentrations in human plasma (5 ng/ml to 10 micrograms/ml) and urine (50 ng/ml to 100 micrograms/ml). This assay, developed for human plasma and urine, is also applicable to rabbit samples with minor modification. Intravenous bolus doses of 10 mg/kg d4T to rabbits showed that the plasma concentration-time profile followed a biexponential decay. Estimates of the distribution and elimination half-lives were 6.7 +/- 0.9 and 51 +/- 6 min, respectively. The total-body and renal clearances were 23.4 +/- 3.6 and 8.82 +/- 3.9 ml/min.kg, respectively. That the renal clearance exceeds the glomerular filtration rate in the rabbit suggests that d4T is actively secreted in the renal tubule. The fraction excreted unchanged in the urine was 36 +/- 8%. Similar results were obtained in the same rabbits at steady state during constant-rate intravenous infusion. Noncompartmental analysis estimates of the MRT and Vdss were 46 +/- 5 min and 1.08 +/- 0.13 L/kg, respectively.

Animals↗
PubMed1991

Cloning and characterization of a pair of novel genes that regulate production of extracellular enzymes in Bacillus subtilis.

Two novel Bacillus subtilis genes that regulate the production of several extracellular enzymes were clones and characterized. These two genes are organized as part of an operon. When cloned in a multicopy plasmid, the first gene (tenA, transcription enhancement) stimulates alkaline protease production at the transcriptional level. The second gene (tenI) exerts an opposite effect to reduce alkaline protease production. The production of neutral protease, levansucrase, and alkaline protease can be stimulated up to 11- to 55-fold. Thus, tenA is a new member of the deg (regulatory genes for degradative enzymes) family in B. subtilis. A functional degS product is required to observe the stimulatory effect from tenA. Between the promoter and the ribosome-binding site of tenA, there exists a terminatorlike structure. Deletion of this structure doubles the expression of tenA. Neither tenA nor tenI is essential for cell growth and the production of extracellular enzymes. However, inactivation of these genes causes a delay in sporulation. This operon is located close to tre on the genetic linkage map. The overall organization of this operon and its relationship with other known regulatory factors in the deg family are discussed.

Amino Acid Sequence↗
PubMed1991

Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases.

We describe the development of an expression-secretion system in Bacillus subtilis to improve the quality and quantity of the secreted foreign proteins. This system consists of a strain (WB600) deficient in six extracellular proteases and a set of sacB-based expression vectors. With the inactivation of all six chromosomal genes encoding neutral protease A, subtilisin, extracellular protease, metalloprotease, bacillopeptidase F, and neutral protease B, WB600 showed only 0.32% of the wild-type extracellular protease activity. No residual protease activity could be detected when WB600 was cultured in the presence of 2 mM phenylmethylsulfonyl fluoride. By using TEM beta-lactamase as a model, we showed that WB600 can significantly improve the stability of the secreted enzyme. To further increase the production level we constructed an expression cassette carrying sacY, a sacB-specific regulatory gene. This gene was placed under the control of a strong, constitutively expressed promoter, P43. With this cassette in the expression vector, an 18-fold enhancement in beta-lactamase production was observed. An artificial operon, P43-sacY-degQ, was also constructed. However, only a partial additive enhancement effect (24-fold enhancement) was observed. Although degQ can stimulate the production of beta-lactamase in the system, its ability to increase the residual extracellular protease activity from WB600 limits its application. The use of the P43-sacY cassette and WB600 would be a better combination for producing intact foreign proteins in high yield.

Bacillus subtilis↗
PubMed1991

Cloning and expression of a novel protease gene encoding an extracellular neutral protease from Bacillus subtilis.

We have cloned from Bacillus subtilis a novel protease gene (nprB) encoding a neutral protease by using a shotgun cloning approach. The gene product was determined to have a molecular mass of 60 kDa. It has a typical signal peptide-like sequence at the N-terminal region. The expression of nprB can be stimulated by using a B. subtilis strain, WB30, carrying a sacU(h)h mutation. Expression of this protease gene results in production of a 37-kDa protease in the culture medium. The first five amino acid residues from the N terminus of the mature protease were determined to be Ala-Ala-Gly-Thr-Gly. This indicates that the protease is synthesized in a preproenzyme form. The purified protease has a pH optimum of around 6.6, and its activity can be inhibited by EDTA, 1,10-phenanthroline (a zinc-specific chelator), and dithiothreitol. It retained 65% of its activity after treatment at 65 degrees C for 20 min. Sequence comparison indicates that the mature form of this protease has 66% homology with the two thermostable neutral proteases from B. thermoproteolyticus and B. stearothermophilus. It also shares 65, 61, and 56% homology with the thermolabile neutral proteases from B. cereus, B. amyloliquefaciens, and B. subtilis, respectively. The zinc-binding site and the catalytic residues are all conserved among these proteases. Sequence homology extends into the "propeptide" region. The nprB gene was mapped between metC and glyB and was not required for growth or sporulation.

Amino Acid Sequence↗
PubMed1990

Cloning, genetic organization, and characterization of a structural gene encoding bacillopeptidase F from Bacillus subtilis.

Bacillopeptidase F is an extracellular serine protease that is expressed at the beginning of the stationary phase. To study its structure, regulation of expression, and physiological roles, we have cloned and characterized the structural gene (bpf) encoding this protease from Bacillus subtilis. DNA sequence analysis suggests this protease is synthesized as a preproenzyme (Mr = 92,000). Through processing at both the NH2 and COOH termini, it is gradually converted into various forms with molecular mass ranging from 80 to 48 kDa. Shortening the 3' end of bpf demonstrates that at least 290 amino acid residues from the COOH-terminus of bacillopeptidase F are not required for either catalytic activity or secretion. Bacillopeptidase F exhibits sequence similarity with several serine proteases. Its gene is found immediately downstream from the fts operon which was mapped at 135 degrees on the B. subtilis genetic linkage map. Inactivation of the chromosomal copy of bpf shows no effect on cell growth and sporulation. A triple protease-deficient strain (WB300 with the structural genes for bacillopeptidase F and two other major proteases inactivated) was constructed to serve as a better expression host for the production and secretion of foreign proteins.

Amino Acid Sequence↗
PubMed1989

Development of an inducible and enhancible expression and secretion system in Bacillus subtilis.

A set of inducible secretion vectors has been developed in Bacillus subtilis based on the regulatory region and the signal peptide sequence of the sacB gene encoding an extracellular enzyme, levansucrase. The expression of the inserted foreign gene (bla) encoding TEM beta-lactamase (Bla), can be induced by the addition of sucrose to the medium. Either the installation of a sacQ expression cassette into the same secretion vector, or the use of a sacUh two-protease-deficient strain (WB30), can significantly enhance expression of the bla gene. However, the combined use of the sacQ-containing secretion vector and the WB30 strain results in no further increase in Bla activity. During development of the secretion vector, the nucleotide sequence around the signal peptidase cleavage site has been redesigned, so that unique restriction sites were installed to facilitate the insertion of foreign genes.

Amino Acid Sequence↗
PubMed1989

Repeated prevalence surveys for monitoring effectiveness of hospital infection control.

In a 1400-bedded teaching hospital single-day prevalence surveys of hospital infection were done every six months for 3 years. The prevalence of community-acquired infection remained constant; but, after the introduction of a general infection-control policy, the prevalence of hospital-acquired infection (HAI) fell linearly from 10.5% in the second survey to 5.6% in the last. After the introduction of a specific urinary catheter care policy, the prevalence of hospital-acquired urinary tract infection (HAUTI) fell from 3.2% in the first four surveys to 2.0% in the last three. These differences persisted when the results were adjusted by logistic regression for patient risk factors, which varied between surveys: the declines for HAI and HAUTI were then 9.9% to 6.0% and 2.9% to 2.2% respectively. Infection control policies, therefore, can have substantial impact on the prevalence of HAI, and their effectiveness can readily be measured by repeated prevalence surveys.

Chi-Square Distribution↗
PubMed1989

Bacillus subtilis subtilisin gene (aprE) is expressed from a sigma A (sigma 43) promoter in vitro and in vivo.

In vitro studies demonstrated that the Bacillus subtilis subtilisin gene (aprE) could be transcribed by RNA polymerase holoenzyme reconstituted from core and sigma A factor obtained from vegetative cells. Upstream deletions (from -45) reduced the amount of transcription from the promoter. A deletion downstream of the promoter that overlapped a putative downstream minor promoter did not affect transcription from the sigma A promoter, which indicated that the putative downstream promoter is not utilized in vivo. S1 nuclease mapping studies showed that there was a low level of transcription from the subtilisin promoter during the growth phase and that the site of transcription initiation was the same during log and stationary phases. We conclude from these findings that there is only one promoter for the subtilisin gene and that it can be transcribed by the sigma A form of RNA polymerase in vitro.

Bacillus subtilis↗
PubMed1989

Developmental patterns of antioxidant defense mechanisms in human erythrocytes.

To obtain a profile of erythrocyte antioxidant defense potential during late fetal development, we studied selected antioxidant parameters in blood samples from 65 neonates with birth wt between 520 and 4210 g and from 12 healthy adults. Erythrocyte superoxide dismutase activity did not change significantly with maturation and no significant differences were observed among preterm infants grouped in increasing birth wt categories, term neonates, and adults. Erythrocyte catalase and glutathione peroxidase, as well as plasma vitamin E levels, showed highly significant positive correlations (p less than 0.001) with increasing fetal wt and gestational age; by term, CAT activity reached a level similar to the adult control group, but glutathione peroxidase activity, as well as plasma vitamin E levels, were markedly lower in all the preterm and in the term groups than in adults (p less than 0.01). Erythrocyte glutathione S-transferase activity showed a negative correlation with increasing gestational age (p less than 0.01) and the adult values were considerably lower than any of the neonatal levels (p less than 0.001). The role of glutathione S-transferase in erythrocyte metabolism remains obscure. Maturational changes in the activity of the red cell enzymes that were studied and in the plasma vitamin E level were apparent from about 31-36 wk of gestation, suggesting that the stimulation for these changes may have commenced from about 28-31 wk.

Adult↗