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B R Thomas

Publications and source records attributed to B R Thomas.

At least 37 records · Page 2Linked to original sources
PubMed1998

Three cis-elements required for rice alpha-amylase Amy3D expression during sugar starvation.

Expression of alpha-amylase genes during seedling development plays a key role in production of sugar from the starch stored in the cereal seed. Rice alpha-amylase Amy3D promoter/GUS constructs in transgenic rice cell lines were studied to identify cis elements in the promoter of this metabolite-regulated gene. Three sequences having the greatest effects on Amy3D gene expression included the amylase element (TATCCAT), the CGACG element, and a G box-related element (CTACGTGGCCA). These promoter cis elements are needed for high-level expression of Amy3D under conditions of sugar starvation. The involvement of G box cis-elements in environmental stress responses suggest a link between the nutrient stress and the environmental stress responses of the plant.

Base Sequence↗
PubMed1996

Heterogeneity determination and purification of commercial hen egg-white lysozyme.

Hen egg-white lysozyme (HEWL) is widely used as a model protein, although its purity has not been adequately characterized by modern biochemical techniques. We have identified and quantified the protein heterogeneities in three commercial HEWL preparations by sodium dodecyl sulfate polyacrylamide gel electrophoresis with enhanced silver staining, reversed-phase fast protein liquid chromatography (FPLC) and immunoblotting with comparison to authentic protein standards. Depending on the source, the contaminating proteins totalled 1-6%(w/w) and consisted of ovotransferrin, ovalbumin, HEWL dimers, and polypeptides with approximate M(r) of 39 and 18 kDa. Furthermore, we have obtained gram quantities of electrophoretically homogeneous [> 99.9%(w/w)] HEWL by single-step semi-preparative scale cation-exchange FPLC with a yield of about 50%. Parallel studies of crystal growth kinetics, salt repartitioning and crystal perfection with this highly purified material showed fourfold increases in the growth-step velocities and significant enhancement in the structural homogeneity of HEWL crystals.

Journal Article↗
PubMed1996

Repartitioning of NaCl and protein impurities in lysozyme crystallization.

Nonuniform precipitant and impurity incorporation in protein crystals can cause lattice strain and, thus, possibly decrease the X-ray diffraction resolution. To address this issue, a series of crystallization experiments were carried out, in which initial supersaturation, NaCl concentration, protein purity level and crystallized fraction were varied. Lysozyme and protein impurities, as well as sodium and chloride were independently determined in the initial solution, supernatant and crystals. The segregation coefficients for Na(+) and Cl(-) were found to be independent of supersaturation and NaCl concentration, and decreased with crystallized fraction/crystal size. Numerical evaluation of the extensive body of data, based on a nucleation-growth-repartitioning model, suggests a core of approximately 40 micro m in which salt is incorporated in much greater concentrations than during later growth. Small crystals containing higher amounts of incorporated NaCl also had higher protein impurity contents. This suggests that the excess salt is associated with the protein impurities in the core. X-ray topography revealed strain fields in the center of the crystals comparable in size to the inferred core. The growth rates of crystals smaller than 30-40 micro m in size were consistently 1.5-2 times lower than those of larger crystals, presumably due to higher chemical potentials in the core.

Journal Article↗
PubMed1995

Characterization of rice alpha-amylase isozymes expressed by Saccharomyces cerevisiae.

Two rice alpha-amylase isozymes, AmylA and Amy3D, were produced by secretion from genetically engineered strains of Saccharomyces cerevisiae. They have distinct differences in enzymatic characteristics that can be related to the physiology of the germinating rice seed. The rice isozymes were purified with immunoaffinity chromatography. The pH optima for Amy3D (pH optimum 5.5) and Amy1A (pH optimum 4.2) correlate with the pH of the endosperm tissue at the times in rice seedling development when these isozymes are produced. Amy3D showed 10-14 times higher reactivity to oligosaccharides than Amy1A. Amy1A, on the other hand, showed higher reactivity to soluble starch and starch granules than Amy3D. These results suggest that the isozyme Amy3D, which is expressed at an early stage of germination, produces sugars from soluble starch during the early stage of seed germination and that the isozyme Amy1A works to initiate hydrolysis of the starch granules.

Carbohydrate Metabolism↗
PubMed1994

Validated micellar electrokinetic capillary chromatography method for quality control of the drug substances hydrochlorothiazide and chlorothiazide.

A stability-indicating, quality control analysis method was developed and validated for the diuretic drug substances hydrochlorothiazide (HCTZ) and chlorothiazide (CTZ). Micellar electrokinetic capillary chromatography employing the anionic detergent sodium dodecyl sulfate at 30 mM in 20 mM sodium borate buffer pH 9.5 was utilized to separate and quantify the active drug substance HCTZ from CTZ and the common impurity, 4-amino-6-chloro-1,3-benzenedisulfonamide (DSA). A 100 microns I.D. uncoated fused-silica capillary was necessary to provide the sensitivity, i.e. 1 microgram/ml, for quantification of the DSA impurity. In this study, the linearity, precision, selectivity, accuracy, reproducibility and limit of quantitation for the method were investigated for HCTZ, CTZ and DSA. As the first validation of a drug substance method by capillary electrophoresis in this laboratory, unusual care was taken to insure reliability and ruggedness with multiple instruments, capillaries and analysts. Precision and reproducibility in the range of 1% R.S.D. was achieved by controlling subtle injection factors. These included minimizing the time in which the capillary ends were not immersed in buffer or sample during the injection process and minimizing the number of assays for each anode or inlet buffer vial. Stacking induced by differences in ionic strength between sample and capillary buffer was reduced by using a sample buffer concentration similar to that of the capillary buffer. Although stacking accomplished by using lower sample buffer concentrations increased sensitivity, reproducibility was decreased. Achievement of the 1% R.S.D. precision level means that many quality control assays for drugs with good absorbance characteristics can be validated with HPLC reproducibility and CE efficiency. These micellar electrokinetic capillary chromatography methods conform to the USA and European Pharmacopoeial validation guidelines.

Chlorothiazide↗
PubMed1994

Mixed ion pair liquid chromatography method for the simultaneous assay of ascorbic acid, caffeine, chlorpheniramine maleate, dextromethorphan HBr monohydrate and paracetamol in Frenadol sachets.

The five active drug substances and two of the excipients present in Frenadol, a cold medication, were separated. The active drug components dextromethorphan HBr monohydrate, ascorbic acid, caffeine, paracetamol and chlorpheniramine maleate were quantitatively assayed by a mixed ion pair LC method. The excipients separated were citric acid and maleic acid. The HPLC assay included dual-wavelength detection to simultaneously quantify the large concentration of paracetamol and the much lower concentration of chlorpheniramine and dextromethorphan. Both tetrabutylammonium hydrogen sulphate (TBA) and pentane sulphonic acid (PSA) were necessary for resolution of the seven compounds. The TBA was necessary to lessen peak tailing for dextromethorphan and chlorpheniramine, to retain ascorbic acid and to shorten assay time. The pentane sulphonic acid enhanced peak shape for dextromethorphan and chlorpheniramine. The assay of the active drug substances was validated for use in quality control applications. Validation studies demonstrated that the procedure was accurate, linear, precise, reproducible and rugged. The method conformed to both USP and EC validation guidelines.

Acetaminophen↗
PubMed1993

Metabolic regulation of alpha-amylase gene expression in transgenic cell cultures of rice (Oryza sativa L.).

Expression of two genes in the alpha-amylase gene family is controlled by metabolic regulation in rice cultured cells. The levels of RAmy3D and RAmy3E mRNAs in rice cultured cells are inversely related to the concentration of sugar in the culture medium. Other genes in the rice alpha-amylase gene family have little or no expression in cultured cells; these expression levels are not controlled by metabolic regulation. A RAmy3D promoter/GUS gene fusion was metabolically regulated in the transgenic rice cell line 3DG, just as the endogenous RAmy3D gene is regulated. An assay of GUS enzyme activity in 3DG cells demonstrated that RAmy3D/GUS expression is repressed when sugar is present in the culture medium and induced when sugar is removed from the medium. The 942 bp fragment of the RAmy3D promoter that was linked to the coding region of the GUS reporter gene thus contains all of the regulatory sequences necessary for metabolic regulation of the gene.

Base Sequence↗
PubMed1993

Molecular characterization of duplicate cytosolic phosphoglucose isomerase genes in Clarkia and comparison to the single gene in Arabidopsis.

The nucleotide sequence of PgiC1-a which encodes a cytosolic isozymes of phosphoglucose isomerase (PGIC; EC 5.3.1.9) in Clarkia lewisii, a wildflower native to California, is described and compared to the previously published sequence of the duplicate PgiC2-a from the same genome. Both genes have the same structure of 23 exons and 22 introns located in identical positions, and they encode proteins of 569 amino acids. Exon and inferred protein sequences of the two genes are 96.4% and 97.2% identical, respectively. Intron sequences are 88.2% identical. The high nucleotide similarity of the two genes is consistent with previous genetic and biosystematic findings that suggest the duplication arose within Clarkia. A partial sequence of PgiC2-b was also obtained. It is 99.5% identical to PgiC2-a in exons and 99.7% in introns. The nucleotide sequence of the single PgiC from Arabidopsis thaliana was also determined for comparison to the Clarkia genes. The A. thaliana PgiC has 21 introns located at positions identical to those in Clarkia PgiC1 and PgiC2, but lacks the intron that divides Clarkia exons 21 and 22. The A. thaliana PGIC protein is shorter, with 560 amino acids, and differs by about 17% from the Clarkia PGICs. The PgiC in A. thaliana was mapped to a site 20 cM from restriction fragment length polymorphism marker 331 on chromosome 5.

Amino Acid Sequence↗
PubMed1992

Affinity purification of functional receptors for Escherichia coli heat-stable enterotoxin from rat intestine.

Active receptors for Escherichia coli heat-stable enterotoxin (ST) were partially purified by ligand-affinity chromatography. The affinity column was prepared by coupling ST to biotin derivatized with an extended N-hydroxysuccinylated spacer arm prior to binding to monomeric avidin immobilized on agarose. Detergent extracts of rat intestinal mucosa membranes were quantitatively depleted of ST binding activity when chromatographed on this affinity matrix. Biotinylated ST-receptor complexes were eluted from affinity columns with 2 mM biotin and these complexes quantitatively dissociated with bile salts. Using this technique, functional ST receptors were purified maximally about 2000-fold, with about 3% of the total activity in crude extracts recovered in these purified preparations. Analysis of affinity-purified preparations by polyacrylamide gel electrophoresis and silver staining demonstrated a major protein subunit of 74 kDa. Affinity cross-linking of these preparations to 125I-ST demonstrated specific labeling predominantly of the 74-kDa subunit. In addition, lower amounts of labeled ST were incorporated into subunits of 164 and 45 kDa, confirming the heterogeneous nature of ST receptors. Purified receptors bound ST in a concentration-dependent fashion, with an IC50 of 10(-9) M. These studies demonstrate that ligand-affinity chromatography can be employed to purify ST receptors. The availability of purified receptors will facilitate further studies of mechanisms underlying ST-induced intestinal secretion.

Animals↗
PubMed1992

Molecular analysis of the plant gene encoding cytosolic phosphoglucose isomerase.

The gene encoding a cytosolic isozyme of phosphoglucose isomerase (PGI, EC 5.3.1.9) was isolated from Clarkia lewisii, a wild flower native to California, and the structure and sequence of the entire coding region determined. PGI catalyzes an essential step in glycolysis and carbohydrate biosynthesis in plants. Spanning about 6 kb, the gene has 23 exons and 22 introns, the highest number yet reported in plants. The exons range in size from 43 to 156 nt and encode a protein of 569 amino acids. The protein is about 44-46% identical to the inferred protein sequences of pig, Escherichia coli and Saccharomyces cerevisiae. All of the introns are bordered with the consensus 5'-GT...AG-3' dinucleotides. The longest intron includes a large stem-loop structure bounded by a perfect 9 nt direct repeat. We cloned the PGI gene from a genomic library prepared from a single plant of known PGI genotype. The locus and allele of the clone were identified by matching restriction fragments to fragments from genetically defined genomic DNAs by Southern hybridization.

Amino Acid Sequence↗
PubMed1991

Liver lead burden in hunter-killed ducks from the Coeur d'Alene River Valley of northern Idaho.

Liver lead burden was evaluated in ducks from an area in northern Idaho contaminated with mining waste and lead shot. Hunter-killed ducks with lead shot in their gizzard had a mean liver lead concentration of 15.18 +/- 2.51 micrograms/g wet-weight (N = 23) and ducks without lead shot had a mean liver lead concentration of 4.21 +/- 0.47 micrograms/g wet-weight (N = 85). The most hazardous source of lead for waterfowl in this area has been thought to be mining waste-contaminated soils and sediments. Three sediment samples had a mean concentration of 4,520 micrograms lead/g wet-weight.

Animals↗
PubMed1990

A high-performance liquid chromatography method for determination of 4-aminopyridine in tissues and urine.

An analytical method was developed to measure 4-aminopyridine in tissues and urine to determine appropriate diagnostic samples in acute poisoning cases. Tissues from rats dosed with 4-aminopyridine were extracted with methylene chloride. Extracts were analyzed by high-performance liquid chromatography using an isocratic solvent system of acetonitrile and aqueous solution (15/85 v/v) consisting of 0.015 M sodium salt of l-heptane-sulfonic acid, 0.002 M tetramethylammonium bromide, and 0.01 M sodium dihydrogen phosphate adjusted to pH 3.0 with phosphoric acid. We concluded that suitable diagnostic samples for acute poisoning cases include stomach contents, kidney, liver, and urine.

4-Aminopyridine↗
PubMed1989

Cefotaxime and desacetylcefotaxime pharmacokinetics in very low birth weight neonates.

The single-dose pharmacokinetics of cefotaxime (CTX) and desacetylcefotaxime (dCTX) after a 50.0 mg/kg intravenous dose were evaluated in 18 very low birth weight neonates (13 male; 1015.6 +/- 349.8 gm; 28.4 +/- 2.4 weeks gestational age) during the first week of life. Microanalytic high-performance liquid chromatography was used to quantitate both CTX and dCTX from serum. A two-compartment open model best characterized the disposition of CTX during a 24-hour post-dose period. The disposition of dCTX was adequately characterized by a one-compartment model. The elimination half-life, apparent steady-state volume of distribution, and total body clearance of CTX (mean +/- SEM) were 4.44 hours, 0.461 +/- 0.027 L/kg, and 0.074 +/- 0.003 L/hr/kg, respectively. Peak concentrations (mean +/- SD) of dCTX (17.96 +/- 5.54 mg/L) occurred at 0.6 to 8.3 hours (5.9 +/- 1.9 hours) after CTX administration, and the apparent elimination half-life of dCTX was 9.36 hours. Comparison of CTX and dCTX pharmacokinetic parameters between very low birth weight neonates who weighed less than 1000 gm (n = 9; 703.3 +/- 46.6 gm; 27.0 +/- 0.8 weeks gestational age) and greater than or equal to 1000 gm (n = 9; 1328.8 +/- 48.6 gm; 29.8 +/- 0.5 weeks gestational age) revealed no significant differences, but significant linear correlations were found between gestational age and weight versus CTX half-life and total body clearance. Because of the prolonged clearance of both CTX and dCTX in the very low birth weight neonate, a CTX dose of 50 mg/kg every 24 hours may provide effective serum concentrations for susceptible infections outside the central nervous system.

Cefotaxime↗
PubMed1989

Renal function in freshwater and chronically saline-stressed male and female Pekin ducks.

Hematocrit (Hct) and plasma ionic and osmotic concentrations were measured in Pekin Ducks, Anas platyrhynchos, drinking freshwater (FW), during rapid acclimation to sea water (RA), and after 6 mo exposure to 180-mM NaCl (SW). Glomerular filtration rate (GFR) and extracellular fluid volume (ECFV) were measured in FW and SW ducks. Estimated renal plasma flow (ERPF) was measured in SW ducks. During RA, Hct and plasma concentration increased significantly (P less than .05) when drinking water sodium concentration ([Na]dw) was 300 mM; body mass decreased (P less than .01) when [Na]dw was 375 mM. The Hct and plasma ionic or osmotic concentrations of FW and SW ducks were the same, except that female SW ducks had lower (P less than .05) plasma chloride concentrations than FW female ducks. Relative to body mass, the FW female duck ECFV (26.88%) was larger (P less than .01) than that of the male (17.50%). The ECFV was not affected by SW in either sex. Mean (+/- SE) GFR determined using inulin and polyethylene glycol (PEG) in the same five FW ducks, were the same, 4.4 +/- .7 and 4.7 +/- .6 mL(min.kg)-1, respectively. In female FW ducks, the GFR (PEG) was 5.00 +/- .30 mL(min.kg)-1, significantly (P less than .001) greater than that of the male, which was 3.07 +/- .21 mL(min.kg)-1. Female and male SW duck GFR were 3.96 +/- .57 and 2.60 +/- .37 mL(min.kg)-1, respectively, but the decrease in GFR was significant (P less than .05) only for females. The GFR were the same, relative to kidney mass, in both sexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Physiological↗
PubMed1987

Isolation and identification of 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3, a new metabolite of 1,25-dihydroxyvitamin D3 produced in rat kidney.

A new metabolite of vitamin D3 was produced in vitro by perfusing rat kidneys with 1,25-dihydroxyvitamin D3 (4 X 10(-6) M). It was isolated and purified from the lipid extract of the kidney perfusate by high-performance liquid chromatography. By means of ultraviolet absorption spectrophotometry, mass spectrometry, chemical derivatization, and chemical synthesis, the new metabolite was identified as 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3. Along with the new metabolite, three other previously identified metabolites, namely, 1,24,25-trihydroxyvitamin D3, 1,25-dihydroxy-24-oxovitamin D3, and 1,23,25-trihydroxy-24-oxovitamin D3, were also isolated. The new metabolite was also formed when 1,23,25-trihydroxy-24-oxovitamin D3 was used as the substrate. Thus, the new metabolite fits into the following metabolic pathway: 1,25-dihydroxyvitamin D3----1,24(R),25-trihydroxyvitamin D3----1,25-dihydroxy-24-oxovitamin D3----1,23,25-trihydroxy-24-oxovitamin D3----1,23-dihydroxy-24,25,26,27-tetranorvitamin D3. Further, we used 1 alpha,25-dihydroxy[1 beta-3H]vitamin D3 in the kidney perfusion system and demonstrated 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 as the major further metabolite of 1,25-dihydroxyvitamin D3, circulating in the final perfusate when kidneys were perfused with 1,25-dihydroxyvitamin D3 (6 X 10(-10) M) for 4 h. The biological activity of 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 (C-3 alcohol) and its metabolic relationship to 1-hydroxy-23-carboxy-24,25,26,27-tetranorvitamin D3 (calcitroic acid or C-23 acid), the other previously identified side-chain cleavage metabolite of 1,25-dihydroxyvitamin D3, are unknown and are presently undergoing investigation.

Animals↗
PubMed1985

Effect of Chilling Temperatures upon Cell Cultures of Tomato.

The effect of chilling temperatures upon cell cultures of tomato (Lycopersicon esculentum Mill cv ;VF36,' and cv ;VFNT Cherry,' and L. hirsutum Humb. & Bonpl.) was tested. Doubling times for L. esculentum were 2 to 3 days at 28 degrees C, and 3 to 8 days at 12 degrees C. No growth was observed at 8 degrees C, indicating an abrupt limit to growth between 8 and 12 degrees C. Fluorescein diacetate staining indicated that 80 to 90% of the cells were alive when cells were maintained at 8 degrees C for up to 2 weeks. When cultures kept at 8 degrees C for up to 30 days were transferred to 28 degrees C, growth resumed quickly, and at a rate virtually identical to that for unchilled cells. Similar results were found for cells maintained at 0 degrees C, and for cells of ;VFNT Cherry' and of L. hirsutum. Under certain conditions, cultures slowly doubled in fresh weight and cell volume at 8 or 9 degrees C but additional growth at 8 degrees C did not occur, nor could growth be maintained by subculture at 8 or 9 degrees C. The results are contrary to reports that cell cultures of tomato die when exposed to temperatures below 10 degrees C for 1 or 2 weeks. Our observations indicate that chilling temperatures quickly inhibit growth of tomato cells, but do not kill them.

Journal Article↗
PubMed1981

On the origin of biological systems and the role of poly-nucleotides -- the initiation of evolution, the structural basis of the genetic code and the mechanism of protein biosynthesis.

There is much information on the nature and function of biological systems in the structures of the small molecules that affect them and in the biosynthetic and other reactions in which they are involved. Combining this with biochemical and polynucleotide sequence information allows us to derive explanations for a number of biological problems that earlier were unclear or even quite obscure. Thus polynucleotide systems give a good account of the origin of life, of the genetic code and of the function of the ribosomal mechanism of protein biosynthesis. It is apparent that polynucleotides play a more important role than has been fully appreciated.

Biological Evolution↗