Search PubMed⌕ Search

Biomedical subjects

J F Shaw

Publications and source records attributed to J F Shaw.

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

Site-directed mutagenesis of a novel serine arylesterase from Vibrio mimicus identifies residues essential for catalysis.

Site-directed mutagenesis (SDM) of an arylesterase (the arylesterase) from Vibrio mimicus revealed that residues S29, H153, and D96 constituted a catalytic triad. The use of a serine residue for ester hydrolysis by the arylesterase proves that the enzyme is a novel serine arylesterase. SDM also showed that D28 was necessary for the esterase activity; to our knowledge it is the first time that a residue immediately preceding the active-site serine in esterases was shown biochemically to possess such a property. The results further suggest that D28 plays a role in substrate-binding. Residue 31 was firmly shown to participate in the binding of N-acetyl-D, L-phenylalanine beta-naphthyl ester (NAPNE), an artificial substrate for chymotrypsin. The S31G enzyme showed a 4 fold decrease in the Km for NAPNE over that of wild type enzyme, proving residue 31 is important for substrate-specificity. A mechanism for binding and catalysis of esters by the arylesterase is proposed, which includes the unique role of S31 for aromatic (hydrophobic) acyl-binding. The biochemical properties of the arylesterase suggest that the enzyme stands out as a member of a distinct subfamily within a recently proposed, lipolytic enzyme family.

Amino Acid Sequence↗
PubMed1995

Vibrio mimicus arylesterase has thioesterase and chymotrypsin-like activity.

A Vibrio mimicus serine arylesterase and an Escherichia coli thioesterase/serine protease share 49.4% amino acid identity. The arylesterase has thioesterase activity for benzoyl-CoA and chymotrypsin-like activity for N-carbobenzoxy-L-phenylalanine p-nitrophenyl ester (NBPNPE). The gene encoding the V. mimicus enzyme is designated etpA. Substituting Ser31 of the V. mimicus enzyme with a glycine or an alanine altered its activity. In comparison with wild type enzyme, the S31A enzyme showed a 5-fold increase and 57% decrease in the catalytic efficiency for benzoyl-CoA and NBPNPE, respectively, and the S31G enzyme showed a 3.6-fold increase and 43% decrease in the catalytic efficiency for benzoyl-CoA and NBPNPE, respectively. For the two mutant enzymes an 8-fold decrease and a 6- to 7-fold increase in Km were seen for benzoyl-CoA and NBPNPE, respectively. The mutagenesis results prove that residue 31 plays an important role in the substrate-specificity.

Acyl Coenzyme A↗
PubMed1995

Crystallization and preliminary X-ray crystallographic analysis of arylesterase from Vibrio mimicus.

Single crystals of arylesterase (EC 3.1.1.2) from Vibrio mimicus have been obtained from ammonium sulfate as a precipitant at room temperature for 2 months. The present crystals diffract up to 2.2 A resolution and belong to monoclinic space group P2(1). The cell dimensions are a = 55.65(1) A, b = 53.46(1) A, c = 65.79(1) A, and beta = 106.54(1) degrees. There are two molecules of molecular weight 22 kDa in an asymmetric unit with a solvent content of 43%.

Ammonium Sulfate↗
PubMed1995

Subunit interaction enhances enzyme activity and stability of sweet potato cytosolic Cu/Zn-superoxide dismutase purified by a His-tagged recombinant protein method.

The coding region of copper/zinc-superoxide dismutase (Cu/Zn-SOD) cDNA from sweet potato, Ipomoea batatas (L.) Lam. cv. Tainong 57, was introduced into an expression vector, pET-20b(+). The Cu/Zn-SOD purified by His-tagged technique showed two active forms (dimer and monomer). The amount of proteins of dimer and monomer appeared to be equal, but the activity of dimeric form was seven times higher than that of monomeric form. The enzyme was dissociated into monomer by imidazole buffer above 1.0 M, acidic pH (below 3.0), or SDS (above 1%). The enzyme is quite stable. The enzyme activity is not affected at 85 degrees C for 20 min, in alkali pH 11.2, or in 0.1 M EDTA and also quite resistant to proteolytic attack. Dimer is more stable than monomer. The thermal inactivation rate constant kd calculated for the monomer at 85 degrees C was 0.029 min-1 and the half-life for inactivation was about 28 min. In contrast, there is no significant change of dimer activity after 40 min at 85 degrees C. The enzyme dimer and monomer retained 83% and 58% of original activity, respectively, after 3 h incubation with trypsin at 37 degrees C, while those retained 100% and 31% of original activity with chymotrypsin under the same condition. These results suggest subunit interaction might change the enzyme conformation and greatly improve the catalytic activity and stability of the enzyme. It is also possible that the intersubunit contacts stabilize a particular optimal conformation of the protein or the dimeric structure enhances catalytic activity by increasing the electrostatic steering of substrate into the active site.

Base Sequence↗
PubMed1994

Nucleotide sequence of a novel arylesterase gene from Vibro mimicus and characterization of the enzyme expressed in Escherichia coli.

A gene coding for an arylesterase of Vibrio mimicus was cloned. Sequence determination reveals that the esterase gene has an open reading frame of 600 nucleotides which encodes a protein of M(r) 22,300. The deduced amino acid sequence contain a pentapeptide GDSLS (residues 27-31), which was also found in the phospholipid-cholesterol acyltransferase from Aeromonas hydrophila. Substitution of Ser-29 by alanine or cysteine in the cloned gene abolished the esterase activity in the tributyrin plate assay. On the other hand, the activity was not lost when Ser-31 was changed to alanine. The cloned gene was expressed in Escherichia coli, and the protein purified by a four-step procedure. The purified protein migrated on SDS/PAGE as a single band with an apparent M(r) of 22,100. This enzyme favoured the hydrolysis of several arylesters and was classified as an arylesterase (EC 3.1.1.2). N-Terminal analysis showed that Ser-20 was the first amino acid of the mature secreted protein, suggesting that the N-terminal 19 hydrophobic amino acids served as a signal peptide.

Amino Acid Sequence↗
PubMed1993

Cloning and characterization of a cDNA encoding the cytosolic copper/zinc-superoxide dismutase from sweet potato tuberous root.

A full-length cDNA clone encoding a putative copper/zinc-superoxide dismutase (SOD) of sweet potato, Ipomoea batatas (L.) Lam. cv Tainong 57, was isolated from a cDNA library constructed in lambda gt10 from tuber root mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete open reading frame coding for 152 amino acid residues. The deduced amino acid sequence showed higher homology (78-86%) with the sequence of the cytosolic SOD than that of the chloroplast SOD from other plant species. The residues required for coordinating copper and zinc are conserved as they are among all reported Cu/Zn-SOD sequences. In addition, it lacks recognizable plastic or mitochondrial targeting sequences. These data suggest that the isolated sweet potato clone encodes a cytosolic Cu/Zn-SOD.

Amino Acid Sequence↗
PubMed1992

Ultrasound assessment of the problem kidney transplant: a surgical prospective.

Ultrasound investigation contributes to the assessment of transplanted kidneys. Ureteric obstruction and peri-transplant fluid collections are shown satisfactorily, but the differentiation of graft rejection from cyclosporin A nephrotoxicity and continuing acute tubular necrosis is less straightforward. Doppler ultrasound is used to detect changes in graft blood circulation, but the results of such assessment vary between centers, and further improvements are required to improve the accuracy of diagnosis of causes of graft impairment by noninvasive methods.

Cyclosporine↗
PubMed1992

Prevalence of serum antibodies against lymphocytic choriomeningitis virus in selected populations from two U.S. cities.

An ELISA was developed for measuring serum antibodies against the arenavirus lymphocytic choriomeningitis virus (LCMV) and a closely related isolate termed callitrichid hepatitis virus (CHV). The ELISA was used to test sera from healthy adults and from hepatitis patients. In Birmingham, Alabama, the seropositivity rate for healthy black women was 5.1% (7/138), and the rate for patients with all types of hepatitis or cirrhosis was 4.3% (2/46). In San Antonio, Texas, the seropositivity rate among a clinical series of patients with non-A, non-B hepatitis was 0 (0/20), and the rate among persons rejected from blood donation because of high serum alanine aminotransferase levels was 2.4% (2/82). These results indicate that infection with LCMV or CHV is common in Birmingham but that infection is not associated with hepatitis.

Adult↗
PubMed1991

Immobilized enzyme system for determination of sialic acid in serum or urine.

An immobilized enzyme system has been developed and employed to determine the concentration of sialic acid (N-acetylneuraminic acid) in human serum and urine. Two enzyme pairs, neuramindiase-Neu-5-Ac lyase and pyruvate oxidase-peroxidase, have been respectively co-immobilized onto 1,12-aminododecane-agarose with glutaraldehyde. The relative specific activity of the co-immobilized neuraminidase and Neu-5-Ac lyase were 60% and 78%, and those of pyruvate oxidase and peroxidase were 50% and 95% of the corresponding soluble enzymes, respectively. The optimal reaction pH at 37 degrees C for each of the co-immobilized enzymes was about one pH unit higher than that of the corresponding soluble enzyme. The optimal reaction temperature of each enzyme was increased as a result of immobilization. The thermal stability at 45 degrees C of the immobilized neuraminidase, Neu-5-Ac lyase, pyruvate oxidase, and peroxidase were increased 80-, 83-, 115-, and 147-fold, respectively. Km and Vm of each immobilized and co-immobilized enzyme have also been determined. The system provided a convenient and rapid method to determine the concentration of total sialic acid without pretreatment of the sample. The results correlated satisfactorily with those obtained by using a soluble enzyme system. The co-immobilized enzymes were stable for at least 1 year of 500 tests when used repeatedly. The system is thus a reproducible and reliable novel assay method for sialic acid in the serum or urine sample.

Enzymes, Immobilized↗
PubMed1990

Single dose cefotaxime plus metronidazole versus three dose cefuroxime plus metronidazole as prophylaxis against wound infection in colorectal surgery: multicentre prospective randomised study.

OBJECTIVE: To establish whether a single preoperative dose of cefotaxime plus metronidazole was as effective as a standard three dose regimen of cefuroxime plus metronidazole in preventing wound infection after colorectal surgery. DESIGN: Prospective randomised allocation to one of two prophylactic antibiotic regimens in a parallel group trial. Group sequential analyses of each 250 patients were performed. SETTING: 14 District general and teaching hospitals. PATIENTS: 1018 Adults having colorectal operations were randomised, of whom 943 were evaluated. Demographic features, conditions requiring surgery, and operative procedures were similar in the two groups. Most patients had surgery for carcinoma of the colon or rectum. INTERVENTIONS: Group 1 received cefotaxime 1 g intravenously plus metronidazole 500 mg intravenously preoperatively. Group 2 received cefuroxime 1.5 g intravenously plus metronidazole 500 mg intravenously preoperatively, followed by cefuroxime 750 mg intravenously plus metronidazole 500 mg intravenously eight hours and 16 hours postoperatively. MAIN OUTCOME MEASURES: Development of surgical wound infection (as evidenced by the presence of pus), death, or discharge from hospital. RESULTS: Wound condition was scored on a five point scale on alternate days until discharge or for up to 20 days postoperatively. Wound infection rates were: group 1, 32/453 (7.1%; 95% confidence interval 4.7% to 9.4%); group 2, 33/454 (7.3%; 95% confidence interval 4.9% to 9.6%). Death rates (group 1: 26/470 (5.5%); group 2: 31/471 (6.6%], the incidence of postoperative complications, the median duration of hospital stay (12 days), and antibiotic tolerance were all similar in the two groups. Pooled data from groups 1 and 2 showed that wound infections were more frequent when minor faecal contamination had occurred at operation and when the duration of operation exceeded 90 minutes (greater than 90 min 11.2% of cases; less than 90 min 4.8%) and were associated with an extended hospital stay. CONCLUSIONS: A single preoperative dose of cefotaxime plus metronidazole is an efficacious as a three dose regimen of cefuroxime plus metronidazole in preventing wound infection after colorectal surgery and has practical advantages in eliminating the need for postoperative antibiotics.

Adolescent↗
PubMed1990

Survey of the use of thrombolysis for acute limb ischaemia in the UK and Ireland.

A total of 156 vascular surgeons from the UK and Ireland responded to a postal questionnaire on the use of thrombolysis in acute limb ischaemia. Almost half had used thrombolysis in the previous 12 months. Low dose intra-arterial streptokinase (89 per cent) was the regimen most frequently used, but 33 per cent had used intravenous streptokinase. Detailed results for 103 patients (limb salvage rate 60 per cent, amputation rate 35 per cent, mortality rate 5 per cent) were similar to those of previously published reports. Complications in most series were infrequent, but in the entire survey there were six bleeding-related deaths and one death from anaphylaxis. A total of 67 per cent of surgeons with working experience indicated that selected patients with acute arterial thromboses were most suitable for thrombolysis, particularly if there was no neurological deficit and no run-off for surgical bypass. Distal or late emboli and graft occlusions were regarded as appropriate by some surgeons. This study demonstrated that many vascular surgeons currently use thrombolysis, although sparingly and in selected cases. Patients with acute limb ischaemia should be managed by experienced vascular surgeons who are aware of the new therapeutic alternatives, an ideal supported by almost three-quarters of respondents to this survey.

Acute Disease↗
PubMed1989

Pleomorphic xanthoastrocytoma. Report of four cases.

Four cases of pleomorphic xanthoastrocytoma (PXA), a low-grade leptomeningeal glioma with a reported favorable prognosis affecting young patients, are reported together with a discussion and review of management and prognosis. A literature review has confirmed a favorable prognosis in at least 50% of patients with this disorder. Seventeen of 35 reported patients are still alive and often seizure-free for a mean period of 7.4 years (range 2 to 18 years) after diagnosis. Five patients have died within 2 years and four between 9 and 25 years after diagnosis of PXA. In some cases in which death followed shortly after diagnosis, there may have been histological confusion between PXA and a malignant glioma with heavily lipidized tumor cells. Nonetheless, transformation of PXA into a malignant astrocytoma or glioblastoma with eventual death may occur many years after initial diagnosis. From the currently reported cases it does not appear possible on clinicopathological grounds to predict which patients will have a favorable prognosis. Optimal management of PXA seems to be primary surgical resection with later surgery for residual or recurrent tumor. The role of radiotherapy in the management of PXA is at this time uncertain.

Adolescent↗
PubMed1988

Interference between coinoculated viroids.

Experiments were carried out to seek evidence of an interaction between two viroid RNAs introduced to tomato plants in the same inoculum. At the level of symptom expression, the severe isolate of potato spindle tuber viroid (PSTV) dominated the mild isolate. Seventy-five percent of the plants inoculated with a 100-fold excess of the mild isolate developed unattenuated symptoms of severe disease. Other experiments revealed that infectious RNA molecules transcribed from cloned DNA templates containing PSTV sequences reduced the level of hop stunt viroid (HSV) RNA present in nucleic acid extracts of plants which had been inoculated with a mixture of dimeric plus-strand transcripts of these two viroids. Plants inoculated with dual transcripts--containing two copies of PSTV linked to two copies of HSV--developed characteristic symptoms of severe PSTV. Dot hybridization demonstrated that only PSTV replicated to detectable levels in these plants. A likely interpretation of these results is that the HSV portion of the dual transcripts failed to replicate because of interference from PSTV. These results raise questions about how the process of viroid replication is related to symptom expression, and lead to suggested models for the effect of viroid-like RNAs in cells under both normal and pathogenic circumstances.

Plant Diseases↗
PubMed1988

Cranial diabetes insipidus secondary to arrested hydrocephalus.

An unusual case is described linking cranial diabetes insipidus with longstanding arrested hydrocephalus. The latter was demonstrated by computed tomographic (CT) and nuclear magnetic resonance (NMR) scans and cerebrospinal fluid pressure measurements. The increasing use of CT and NMR scans may result in this association of cranial diabetes and hydrocephalus being better defined.

Diabetes Insipidus↗
PubMed1988

Role of a separate subcutaneous cerebro-spinal fluid reservoir in the management of hydrocephalus.

A retrospective survey has been carried out on 56 children with shunted hydrocephalus either with a primary idiopathic cause or as a result of a low spina bifida complex. In all 56 children, a separate reservoir has been inserted at some stage in the management of their hydrocephalus. There was no mortality. Morbidity was not increased from CSF infection or shunt blockage. There was less chance of the initial shunt blocking and there was a lesser incidence of visual and schooling handicap. Double cortical puncture did not result in an increased incidence of hemiplegia or epilepsy. We conclude that a separate reservoir greatly eases the management of these children and does not cause significant increased morbidity.

Adolescent↗