PubMed Health⌕ Search

Biomedical subjects

J B Edwards

Publications and source records attributed to J B Edwards.

At least 19 recordsLinked to original sources
PubMed1992

Cloning of two different 5' untranslated exons of bovine acidic fibroblast growth factor by the single strand ligation to single-stranded cDNA methodology.

In an attempt to characterize the 5' UTR of the aFGF mRNAs we used the new anchored PCR methodology, single strand ligation to ss-cDNAs (SLIC). In bovine brain and retina, two kinds of aFGF cDNA clones were isolated. They contained two alternative exons located 34 bp upstream to the translation initiation codon ATG. Taking into account the number of clones specific for each exon, the two mRNAs are expressed with the same ratio in both tissues. One of these bovine 5' UTR exons (136 bp) showed 81% identity to a human 5' UTR exon, the second one (323 bp) was 70% identical to the second human 5' UTR exon with a central region of 90 nucleotides showing 41% identity. The conservation of the splicing positions for these 5' UTR alternate exons in both bovine and human species, suggests that the overall structure of the aFGF gene is conserved in mammals. Furthermore, the conservation of the nucleotide sequences and of the localization of these 5' UTR exons suggests that these non-coding regions may be involved in the control of aFGF gene expression.

Amino Acid Sequence↗
PubMed1992

Promoter elements of the rat choline acetyltransferase gene allowing nerve growth factor inducibility in transfected primary cultured cells.

Choline acetyltransferase, the enzyme responsible for the synthesis of acetylcholine, provides a convenient index for cholinergic neurons. Using a previously identified rat cDNA clone, we have isolated several corresponding genomic clones and have characterized a 1,902-bp fragment that contains part of the first noncoding exon as well as promoter sequences. The promoter activity of this fragment was tested, taking advantage of the recently developed lipopolyamine-mediated DNA transfer method, which allows transfection of primary neurons. The 1,902-bp sequence drives the expression of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene in a culture of dissociated cells prepared from the septal area of fetal (embryonic day 17) rats, a structure rich in cholinergic neurons. Moreover, addition of nerve growth factor to the culture increases CAT expression by approximately 56-fold, indicating that our DNA fragment contains sequences required for NGF induction. In addition, it contains consensus sequences for various transcription factors, including those of the basic helix-loop-helix family. Finally, experiments to characterize the transcription start site are presented.

Animals↗
PubMed1992

Optimized spectrophotometric determination of aldehyde dehydrogenase activity in erythrocytes.

We describe a reliable and sensitive semiautomated spectrophotometric assay of aldehyde dehydrogenase (ALDH; EC 1.2.1.3) activity in erythrocytes. The hemolysate can be stabilized with sucrose, and the technique involves only microliters of hemolysate on a centrifugal analyzer. The use of microcolumns to remove interfering hemoglobin is avoided, and reproducibility of the assay has been improved by manipulating the inherent lactate dehydrogenase activity of erythrocytes by adding lactate and oxalate to the reaction mixture. These modifications have decreased the analytical imprecision of the assay, allowing a better appraisal of aldehyde dehydrogenase activity in erythrocytes as a biological marker of excess alcohol consumption. Erythrocytic ALDH activity was significantly less in 40 alcoholics than in 145 teetotallers (median activity 128 vs 219 mU/g of hemoglobin, respectively; P = 0.0001), indicating the potential of this assay as a useful marker of excess alcohol consumption.

Acetaldehyde↗
PubMed1991

Oligodeoxyribonucleotide ligation to single-stranded cDNAs: a new tool for cloning 5' ends of mRNAs and for constructing cDNA libraries by in vitro amplification.

Cloning full length cDNAs is a difficult task especially if mRNAs are not abundant or if tissue is only available in limited amounts. Current strategies are based on in vitro amplification of cDNAs after adding a homopolymeric tail at the 3' end of the ss-cDNA. Since subsequent amplification steps yield unspecific amplified DNA mostly due to non-specific annealing of the reverse primer containing a homopolymeric tail, we have devised a new strategy based on the ligation of single-stranded oligodeoxyribonucleotide to the 3' end of single-stranded cDNAs. The efficiency of the strategy was assessed by analyzing the 5' ends of the rat pineal gland tryptophan hydroxylase messenger. The 5' end of the least abundant messenger (0.005% of total mRNAs) could be cloned without selection. Sixty percent of the analyzed clones correspond to TPH. This technique revealed a 5-nt stretch not apparent using dG tailing strategy. The potentiality of the method for generating cDNAs libraries was tested with 10(4) PC12 cells. In this library, the abundance of tyrosine hydroxylase clones (0.03%) correlated well with the abundance of the corresponding messenger, showing that no major distortion was introduced into the construction of the library.

Animals↗
PubMed1988

Ultralow volume application of organophosphate concentrate in grain terminals: a new occupational health hazard.

The introduction of ultralow volume (ULV) application of the organophosphate pesticide Fenitrothion in grain terminals presents a risk to workers of skin contact with concentrate. Blood testing, by the Ellman method, of a group of five grain terminal workers working on grain treatment showed a lowering of mean red blood cell cholinesterase (RBC ChE) activity to 23 units/gm Hb (normal value 28-40) with a range of 16-29. The probable cause was identified as percutaneous absorption of Fenitrothion concentrate by workers using ungloved hands to clean blocked drip feed nozzles. Modification of work practices was followed by a rise of mean RBC ChE to 33.6 units/gm Hb (range 32-36) during the following grain treatment season. RBC ChE activity measured during the intervening winter season--that is, a non-exposure period--showed a mean of 33.3 units/gm Hb (range 23-40).

Agricultural Workers' Diseases↗
PubMed1983

Human prostatic acid phosphatase: properties of the native enzyme, and the enzyme-antibody complex.

Acid phosphatase purified from human prostatic tissue was shown to be homogeneous by polyacrylamide gel electrophoresis and N-terminal amino acid analysis. However, isoelectric focusing revealed a large number of isoenzymes which were reduced to four by digestion with neuraminidase. It is suggested that the patterns observed are due to differences in bound carbohydrate attached to the same protein backbone. Antiserum to the purified enzyme was produced in rabbits and reacted with the enzyme to form an enzymatically active complex of large molecular weight. This complex is more stable at high temperatures than the native enzyme. Kinetic analysis of both the enzyme and the enzyme-antibody complex demonstrated that the binding of the antibody caused no significant change to the active site of the enzyme.

Acid Phosphatase↗
PubMed1983

Preparation and characterization of a human serum matrix suitable for quality control or reference materials.

We describe the preparation of a human serum based matrix suitable for inclusion in long-term control materials. The procedure entails the removal of cells, fibrin and lipoproteins from blood to produce a clear, stable material which has been stored frozen at -70 degrees C for up to 14 mth without any apparent deterioration. When incorporated into a quality control material, storage at 4 degrees and -20 degrees C may cause a change in several unstable constituents, but the material remains optically clear. A noticeable reduction in lipoprotein as a result of the preparative procedure is probably responsible for the continued clarity of the matrix. The nature of the matrix has been examined by polyacrylamide gel electrophoresis and does not reveal any unusual protein bands, whereas commercially available materials when similarly examined often reveal protein bands not present in normal serum. The matrix has been used to prepare control material for the Chemical Pathology Quality Assurance Programme Group of the Royal College of Pathologists of Australasia/Australian Association of Clinical Biochemists.

Blood Chemical Analysis↗
PubMed1983

Urinary oxalate determination by use of immobilized oxalate oxidase in a continuous-flow system.

In this procedure, oxalate oxidase (EC 1.2.3.4) immobilized in a continuous-flow system is used to determine oxalate in urine. The hydrogen peroxide formed from oxalate is detected by use of a color reaction with peroxidase (EC 1.11.1.7), 3-methyl-2-benzothiazoline hydrazine, and N,N-dimethylalanine. However, urine contains an oxalate oxidase inhibitor, which cannot be removed by heating, ion-exchange resins, or cellulose columns. This makes it necessary to precipitate the oxalate before assay. The overall assay system is accurate (oxalate recovery, 95.9%), sensitive (less than or equal to 5 mumol/L), precise (within-batch CV less than 1.25%, between-batch CV less than 5%), and relatively rapid (60 samples per working day). The assay system has better accuracy than an established chemical method and a gas-chromatographic method, and is considerably less arduous than and correlates well (r = 0.94) with a modified chemical method. The reference interval for urinary oxalate excretion is 0.16-0.56 mmol per day (n = 97). Only nonphysiological concentrations of ascorbate interfere with the assay, by increasing the oxalate result in the overall assay, presumably by post-micturition formation of oxalate from ascorbate in the urine samples.

Adult↗
PubMed1982

Models for the metabolic production of oxalate from xylitol in humans: a role for fructokinase and aldolase.

It has been proposed previously that oxalate precursors may be formed in the transketolase reaction during the metabolism of xylitol. It is shown in this paper that fructokinase and aldolase, purified from human liver, provide an alternative model in that, in coupled sequence, they produce glycolaldehyde, an oxalate precursor, from D-xylulose via D-xylulose 1-phosphate; D-fructose does not give rise to glycolaldehyde. It is concluded that metabolic pathways based on a combination of the transketolase, fructokinase and aldolase reactions can account for the production of glucose, lactate, tetronates (C-threonic and D-erythronic acids) and oxalate (precursors) during the metabolism of xylitol administered parenterally.

Fructokinases↗
PubMed1982

A mechanism for the thiamin-sparing action of dietary xylitol in the rat.

The changes induced by dietary xylitol in the gastrointestinal tract of the rat were investigated in relation to the phenomenon of vitamin-sparing. Within 18 days of consuming a synthetic diet, deficient in thiamin, riboflavin and pyridoxine, rats ceased to grow and began to lose weight rapidly. If xylitol was then included in the diet (10% w/w), the effect of the vitamin-deficient diet on growth was reversed. Moreover, within 3 days of the rats ingesting xylitol, the metabolism of this sugar polyol by the caecal microflora was increased 17-fold and the caecal concentrations of thiamin and thiamin pyrophosphate were increased 5-fold. Increases were also observed in the caecal size, the weight of the caecal contents, and the weight of the caecal wall. In contrast to the rapid changes observed within the caecum, liver thiamin pyrophosphate levels did not rise until 6-12 days after the feeding of xylitol, at which time the rats had begun to gain weight. The caecal contents were shown to contain facultative bacteria which have the ability to metabolise and grow on xylitol and which can, at the same time, synthesise thiamin. Species of the genera Klebsiella, Serratia and Micrococcus which have this ability were isolated from the caecal contents of rats. It is assumed that coprophagy is the means by which the thiamin and other vitamins synthesised by enteral bacteria become available to the host, although some absorption from the caecum cannot be excluded.

Animals↗