Search PubMed⌕ Search

Biomedical subjects

J Barnett

Publications and source records attributed to J Barnett.

At least 55 records · Page 3Linked to original sources
PubMed1995

High-level production from a baculovirus expression system and biochemical characterization of human GMP synthetase.

GMP synthetase, a key enzyme in the de novo synthesis of guanine nucleotides, is a potential target for immunosuppression and anticancer chemotherapy. In order to closely examine the catalytic mechanism and active-site topography of this enzyme, large amounts of pure protein are needed. Catalytically active human GMP synthetase was expressed in a baculovirus system. A high-level production system has been established from which the yield of pure protein is routinely more than 50 mg/10 liters of cell culture. The recombinant enzyme was purified to homogeneity and characterized. Like native GMP synthetase, the recombinant enzyme was resolved into two forms by ion-exchange chromatography. The two forms are both monomers and they differ in their isoelectric points. There is no evidence that these forms are in equilibrium or interconvertible. Protein sequence analysis reveals that both forms are blocked at the amino-terminus and they are essentially identical in sequence. Since they can be produced by a cDNA with a single open reading frame, we believe that they represent post-translational modification variants. The recombinant GMP synthetase is not distinguishable from the native enzyme in terms of chromatographic profiles, subunit composition, molecular weight, and kinetic properties. The inhibition constants and the modes of inhibition toward decoyinine, a selective inhibitor of GMP synthetase, are also the same as the native enzyme. The high-level production of active enzyme is invaluable to the determination of the three-dimensional structure and the discovery of potent and selective drug candidates.

Animals↗
PubMed1995

Evaluation of the objectivity of an infection control audit tool.

A national study involving 22 districts was carried out during 1993 to evaluate an infection control audit tool. The tool was found to be objective and can be used by infection control teams to monitor practices and assess the impact of training programmes. It can also be used to provide purchasers with a measurable standard in relation to infection control quality issues. In total, 440 wards were audited. A few districts had problems in meeting some of the standards, particularly on sharps and waste. A subgroup of the West Midlands Infection Control Nurses Association was able to adapt the audit tool for specialized areas including general practice, nursing homes and dentistry.

Evaluation Studies as Topic↗
PubMed1995

Conversion of human 15-lipoxygenase to an efficient 12-lipoxygenase: the side-chain geometry of amino acids 417 and 418 determine positional specificity.

Positional specificity determinants of human 15-lipoxygenase were examined by site-directed mutagenesis and by kinetic analysis of the wild-type and variant enzymes. By comparing conserved differences among sequences of 12- and 15-lipoxygenases, a small region responsible for functional differences between 12- and 15-lipoxygenases has been identified. Furthermore, the replacement of only two amino acids in 15-lipoxygenase (at 417 and 418 in the primary sequence) by those found in certain 12-lipoxygenases results in an enzyme that has activity similar to 12-lipoxygenase. An examination of the activity of nine variants of lipoxygenase demonstrated that the amino acid side-chain bulk and geometry of residues 417 and 418 are the key components of the positional specificity determinant of 15-lipoxygenase. Overexpression of a variant (containing valines at positions 417 and 418) that performs predominantly 12-lipoxygenation was achieved in a baculo-virus-insect cell culture system. This variant was purified to > 90% homogeneity and its kinetics were compared with the wild-type 15-lipoxygenase. The variant enzyme has no change in its apparent KM for arachidonic acid and a minor (3-fold) change in its Vmax. For linoleic acid, the variant has no change in its KM and a 10-fold reduction in its Vmax, as expected for an enzyme performing predominantly 12-lipoxygenation. The results are consistent with a model in which two amino acids of 15-lipoxygenase (isoleucine 417 and methionine 418) constitute a structural element which contributes to the regiospecificity of the enzyme. Replacement of these amino acids with those found in certain 12-lipoxygenases results in an enzyme which can bind arachidonic acid in a catalytic register that prefers 12-lipoxygenation.

Arachidonate 12-Lipoxygenase↗
PubMed1995

Effects of Vitamin E on susceptibility of low-density lipoprotein and low-density lipoprotein subfractions to oxidation and on protein glycation in NIDDM.

OBJECTIVE: To evaluate the effect of vitamin E supplementation on the susceptibility of low-density lipoprotein (LDL) and LDL subfractions to oxidation and on protein glycation in non-insulin-dependent diabetes mellitus (NIDDM). RESEARCH DESIGN AND METHODS: Twenty-one men with NIDDM (HbA1c = 6-10%), ages 50-70, were randomly assigned to either 1,600 IU/day of vitamin E or placebo for 10 weeks after a 4-week placebo period. LDL and LDL subfractions were isolated after 4 weeks of placebo and after 6 and 10 weeks of therapy. Susceptibility of LDL to copper-mediated oxidation was measured by conjugated diene formation (lag time) and formation of thiobarbituric acid-reactive substances (TBARS). Fasting serum glucose, mean weekly blood glucose, HbA1c, and glycated plasma protein concentrations were also determined at these time points. RESULTS: Vitamin E content in plasma and LDL increased 4.0- and 3.7-fold, respectively, in the vitamin E-treated group. Vitamin E decreased the susceptibility of LDL to oxidation in comparison with placebo (lag time, 243 +/- 46 vs. 151 +/- 22 min, P < 0.01; 3 h TBARS, 24 +/- 12 vs. 66 +/- 18 nmol malondialdehyde/mg LDL, P < 0.05). Vitamin E content also increased significantly in both buoyant and dense LDL subfractions, and their oxidation was dramatically reduced. The lag time of LDL oxidation correlated well with the content of vitamin E in both LDL and its subfractions (r = 0.69-0.92). Glycemic indexes did not change significantly in either group during the study. Protein glycation, including glycated hemoglobin, glycated albumin, glycated total plasma proteins, and glycated LDL were unchanged in the vitamin E group. CONCLUSIONS: Supplementation of vitamin E in NIDDM leads to enrichment of LDL and LDL subfractions and reduced susceptibility to oxidation. Despite a greater percentage increase in vitamin E content in small dense LDL, it remained substantially more susceptible to oxidation than was buoyant LDL. This suggests that dense, LDL may gain less protection against oxidation from antioxidant supplementation than does larger, more buoyant LDL. In contrast to previous reports, vitamin E supplementation did not reduce glycation of intracellular or plasma proteins.

Aged↗
PubMed1994

Purification, characterization and selective inhibition of human prostaglandin G/H synthase 1 and 2 expressed in the baculovirus system.

Human prostaglandin G/H synthase 1 and 2 were expressed in the baculovirus expression system and purified to high levels. Both enzymes were glycosylated. PGHS-1 appeared to be homogeneous by SDS-PAGE analysis but two closely migrating bands were detected in PGHS-2 preparation which were evidently due to heterogeneity in glycosylation. The amino-acid sequence of the N-termini of both isoforms indicated that the signal sequences were efficiently cleaved by the insect cells. The recombinant human PGHS-1 and PGHS-2 possessed both cyclooxygenase and peroxidase activities. Both had high affinities for arachidonate as substrate and underwent self-inactivation during catalysis. The recombinant isoforms were not pharmacologically identical, since some NSAIDs were selective inhibitors of either PGHS-1 or PGHS-2. This is the first report of high levels of expression and purification of human PGHS isoforms. The recombinant enzymes are invaluable in developing potent PGHS-2 selective inhibitors that may be efficacious anti-inflammatory drugs with no or low levels of toxicity.

Amino Acid Sequence↗
PubMed1994

Production, purification, and characterization of human matrilysin (PUMP) from recombinant Chinese hamster ovary cells.

A process for semicontinuous production of matrilysin zymogen secreted from recombinant Chinese hamster ovary (CHO) cells was developed. The zymogen was purified to apparent homogeneity by sequential ion-exchange and metal chelation chromatography. These processes were scaled-up to purify gram quantities of the zymogen. The N-terminus of the secreted zymogen from the recombinant cells was the same as the observed sequence of the zymogen from natural sources. Furthermore, activation and autocatalysis of the recombinant zymogen resulted in a form with an N-terminus identical to that of the corresponding native enzyme. The three C-terminal amino acids of both the recombinant zymogen and the corresponding smaller activated form were missing. Activated matrilysin was shown to have activity against a synthetic peptide substrate. The large quantities of matrilysin that can be produced and purified from the recombinant CHO cells will be useful in determination of the structure of matrilysin.

Amino Acid Sequence↗
PubMed1994

A test of interpersonal theory of depression in children and adolescents using a projective technique.

Using a projective technique among child and adolescent psychiatric inpatients, we tested several hypotheses derived from Coyne's (1976b) interpersonal theory of depression. First, we predicted that depression would be associated with rejection, even controlling for the effects of aggression. Depression and rejection were associated only when aggression effects were covaried. Second, we hypothesized that the depression-rejection relation would display symptom specificity. Consistent with this hypothesis, we found that depression, but not anxiety, was related to rejection. Third, we assessed whether an index of interpersonal style--reliance on others--moderated the depression-rejection relation. Results suggested that depression and overreliance were both associated with high rejection scores, whereas the combination of low depression and low reliance on others was associated with low rejection scores. We found that results held across age groups. The implications of our findings for the interpersonal theory of depression in youngsters are discussed.

Adolescent↗
PubMed1993

Overexpression, purification and characterization of human recombinant 15-lipoxygenase.

Human 15-lipoxygenase was expressed to high levels (approx. 20% of cellular protein) in a baculovirus/insect cell expression system. Catalytically active enzyme was readily purified (90-95% pure) from cytosolic fractions by anion-exchange chromatography on a Mono Q column with approx. 95% recovery of enzymatic activity. Routinely, a yield of 25-50 mg of pure enzyme per L of culture and a specific activity of 7.1-21 mumol 13-hydroxyoctadecadienoic acid (13-HODE)/mg.min (turnover rate of 8.4-25 s-1) were obtained. Both the specific activity and the enzyme's iron content was significantly increased by the addition of ferrous ions to either the purified enzyme or to the insect cell culture medium during production. An isoelectric point of 5.85 was determined and the N-terminal amino acid sequence was found to be identical to that predicted from the cDNA. The purified recombinant enzyme exhibits a dual positional specificity with arachidonic acid (formation of 15S- and 12S-hydroxyeicosatetraenoic acid (12S-HETE) in a ratio of 12:1). Double oxygenation products 14R,15S- and various 8,15-DiHETE isomers were also identified. With linoleic acid as substrate, a pH-optimum of 7.0 and a KM of 3 microM were determined. The enzyme undergoes suicidal inactivation during fatty acid oxygenation, is sensitive to standard lipoxygenase inhibitors, and oxygenates phospholipids, cholesterol esters, biomembranes and human low-density lipoprotein. Contrary to prior studies on the rabbit enzyme, no glycosylation was detected.

Animals↗
PubMed1993

Oxygenation of lipoproteins by mammalian lipoxygenases.

Oxidative modification converts low-density lipoprotein (LDL) into its atherogenic form and appears to be a necessary precondition for LDL uptake by macrophages during foam cell formation. Cellular lipoxygenases have been implicated in this process. We studied the interaction of purified mammalian lipoxygenases with human LDL in vitro and found that the arachidonate 15-lipoxygenases of rabbit and man are capable of oxygenating lipoproteins as indicated by oxygen uptake and by the formation of thiobarbituric-acid-reactive substances. Furthermore, oxygenated polyenoic fatty acids, such as 13-hydro(pero)xy-9Z,11E-octadecadienoic acid and 15-hydro(pero)xy-5,8,11,13(Z,Z,Z,E)-eicosatetraenoic acid were detected in the lipid compartment of various lipoproteins classes after lipoxygenase treatment. More than 90% of the oxygenated polyenoic fatty acids were found in the ester-lipid fraction, particularly in the cholesterol esters, whereas only small amounts of free hydro(pero)xy polyenoic fatty acids were detected. Lipoxygenase-catalyzed oxygenation of LDL is not restricted to the lipid compartment but also leads to a cooxidative modification of the apoproteins as indicated by changes in the electrophoretic mobility and by the formation of carbonyl derivatives of amino acid side chains. The possible biological significance of lipoxygenase-induced oxidative modification of lipoproteins in the pathogenesis of atherosclerosis is discussed.

Amino Acid Sequence↗
PubMed1993

Fed-batch culture of insect cells: a method to increase the yield of recombinant human nerve growth factor (rhNGF) in the baculovirus expression system.

A fed-batch method was developed which increased the density of insect cells (Spodoptera frugiperda, Sf-9 cells) in suspension culture and the feeding of nutrients improved the yield of a recombinant protein produced by a baculovirus expression system. Analysis of spent medium samples indicated that depletions of glucose and glutamine correlated with the retardation of cell growth. Feeding of a mixture of nutrients consisting of glucose, glutamine, yeastolate and lipids solution restored the growth rate. In fed-batch culture, cell density was increased from 3 x 10(6) cells per ml to 1.2 x 10(7) cells per ml and the increased cell density enhanced the yield of the desired recombinant product, in this case, human nerve growth factor (rhNGF). The optimal conditions for the production of rhNGF were also defined by selecting the appropriate viral multiplicity of infection (MOI). At a cell density of 5 x 10(6) ml-1, a MOI of 0.05 (plaque forming units per cell) gave the highest yield of rhNGF in culture fluid 3 d post-infection. The yield of rhNGF was 20 mg l-1. The fed-batch method was scaled up to 12 l stirred bioreactor.

Animals↗
PubMed1993

A clinical infection control audit programme: evaluation of an audit tool used by infection control nurses to monitor standards and assess effective staff training.

In today's competitive market infection control teams are under increasing pressure from purchasing authorities to provide hard data on quality issues. The initial aim of this study was to determine whether a modified form of the audit system used to monitor infection control standards in Derbyshire could be used effectively across three district health authorities. Infection control nurses in each of the study districts have different approaches to training with only Stafford using a link nurse system. Our additional aim therefore was to examine the relationship between the knowledge base of nursing staff and their ward audit score, and to determine whether wards with link nurses achieved significantly higher scores. To establish this a questionnaire was devised and completed by three different grades of staff on each ward at the time of the audit. These results were fed back to the wards and to managers by the infection control nurse, and an action plan given with a review date where applicable. Results of the study indicated that the audit tool could be used effectively in any district but would need modifying for psychiatric departments. The value of education in improving standards of infection control was clearly demonstrated. An important finding in Stafford was that wards with link nurses obtained significantly higher scores. The value of using such a clinically based audit will be discussed.

England↗
PubMed1993

Immunocytochemical evidence that the beta-protein precursor is an integral component of neurofibrillary tangles of Alzheimer's disease.

Amyloid beta (A beta) immunoreactivity has been demonstrated in all extracellular neurofibrillary tangles (E-NFT) and most intraneuronal neurofibrillary tangles (I-NFT). We undertook this immunocytochemical study to understand the relationship between A beta immunoreactivity localized in NFT and beta-protein precursor (beta PP). We found epitopes of amino-, mid-, and carboxyl-terminal domains of beta PP in I-NFT and the majority of E-NFT. NFT retained beta PP after ionic detergent extraction, demonstrating that beta PP is an integral component of NFT. Finding beta PP in regions of A beta immunoreactivity raises the possibility that beta PP or its fragments associate with amyloid, and that the stability of A beta is responsible for its dominance in amyloid deposits.

Aged↗
PubMed1992

Regulated expression of the human acetylated low density lipoprotein receptor gene and isolation of promoter sequences.

The acetylated low density lipoprotein (AcLDL) receptor is expressed on tissue macrophages after their differentiation from monocyte precursors and has been proposed to play a role in the generation of foam cells in atherosclerotic lesions. In the present studies, THP-1 human monocytic leukemia cells were used to investigate mechanisms responsible for expression of the AcLDL receptor gene after treatment with phorbol 12-myristate 13-acetate (TPA). TPA-dependent accumulation of AcLDL receptor mRNA was not detected until after a lag phase of 12 hr and was blocked by concurrent treatment with cycloheximide. In addition, the TPA-dependent induction of AcLDL receptor activity and mRNA levels was inhibited by retinoic acid and dexamethasone treatment. Isolation and sequence analysis of the promoter regions for the human and bovine AcLDL receptor genes indicated high sequence similarity. Binding sites for AP-1 proteins or other known transcription factors were not conserved between the two species, suggesting that novel factors are required for AcLDL receptor expression.

Base Sequence↗
PubMed1991

Extracorporeal lithotripsy. An important adjunct in the nonoperative management of retained or recurrent bile duct stones.

Retained or recurrent bile duct stones can be successfully removed in up to 80% to 85% of patients with the use of percutaneous or endoscopic techniques. However, problems related to difficult biliary access, large stones, and biliary strictures may decrease the success rate of this approach. We evaluated the safety and efficacy of extracorporeal shock-wave lithotripsy (ESWL) in 16 patients with complicated biliary stones treated prospectively over a 24-month period. Successful stone fragmentation was achieved in 15 patients (94%) using a Dornier HM3 lithotripter (average of 2290 shocks at 22 kV). Three patients (19%) required a second ESWL treatment. Biliary clearance of stone fragments was spontaneous in seven (43%) of the patients and required additional treatment in eight (57%) of the patients. Complications from ESWL were minor and included transient hematuria and ecchymoses at the skin entry site. Extracorporeal shock-wave lithotripsy failed in one patient (6%) with a biliary stricture and surgery was required. At hospital discharge, all patients were asymptomatic and stone free. Treatment with ESWL appears to be a safe and effective adjunct for selected patients with complex biliary stone disease.

Adult↗
PubMed1991

Physicochemical characterization of recombinant human nerve growth factor produced in insect cells with a baculovirus vector.

Recombinant human nerve growth factor (rhNGF) secreted by insect cells was purified by ion-exchange and reversed-phase chromatography to near homogeneity. The N-terminus of the secreted molecule was analogous to that of mouse salivary gland NGF. In its native conformation, the insect cell produced rhNGF molecules were homodimers consisting of 120 amino acid polypeptide chains. Mature rhNGF was found not to be significantly glycosylated (less than 0.08 mol of N-acetylglucosamine/mol of protein). The rhNGF was homogeneous with regard to molecular weight and amino acid sequence. Isoelectric focusing resolved the rhNGF into one major and one minor component. Because rhNGF from insect cells can be obtained in large quantities, purified to near homogeneity, and is similar to natural NGF with regard to physicochemical properties and biological activity, it is suitable for further evaluation in animal models as a therapeutic molecule for neurodegenerative diseases such as Alzheimer's disease.

Amino Acid Sequence↗
PubMed1990

High-density lipoprotein inhibits the oxidative modification of low-density lipoprotein.

Oxidatively modified low-density lipoprotein (LDL), generated as a result of incubation of LDL with specific cells (e.g., endothelial cells, EC) or redox metals like copper, has been suggested to be an atherogenic form of LDL. Epidemiological evidence suggests that higher concentrations of plasma high-density lipoprotein (HDL) are protective against the disease. The effect of HDL on the generation of the oxidatively modified LDL is described in the current study. Incubation of HDL with endothelial cells, or with copper, produced much lower amounts of thiobarbituric acid-reactive products (TBARS) as compared to incubations that contained LDL at equal protein concentrations. Such incubations also did not result in an enhanced degradation of the incubated HDL by macrophages in contrast to similarly incubated LDL. On the other hand, inclusion of HDL in the incubations that contained labeled LDL had a profound inhibitory effect on the subsequent degradation of the incubated LDL by the macrophages while having no effect on the generation of TBARS or the formation of conjugated dienes. This inhibition was not due to the modification of HDL as suggested by the following findings. (A) There was no enhanced macrophage degradation of the HDL incubated with EC or copper alone, together with LDL, despite an increased generation of TBARS. (B) HDL with the lysine groups blocked (acetyl HDL, malondialdehyde (MDA) HDL) was still able to prevent the modification of LDL and (C) acetyl HDL and MDA-HDL competed poorly for the degradation of oxidatively modified LDL. It is suggested that HDL may play a protective role in atherogenesis by preventing the generation of an oxidatively modified LDL. The mechanism of action of HDL may involve exchange of lipid peroxidation products between the lipoproteins.

Animals↗