Properties of lipoamide dehydrogenase and thioredoxin reductase from Escherichia coli altered by site-directed mutagenesis.
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Binding Sites↗
Biomedical subjects
N Allison
Publications and source records attributed to N Allison.
Overexpression and mutagenesis of the lipoamide dehydrogenase of Escherichia coli.
A 'split-gene' technique for the overexpression and mutagenesis of the gene encoding the lipoamide dehydrogenase of Escherichia coli was developed in order to overcome the instability problems encountered when attempting to mutate the intact gene. The lipoamide dehydrogenase gene, lpd, was dissected into two fragments which were separately subcloned into M13 vectors for mutagenesis in vitro followed by reconstitution in the pJLA504 expression vector under the transcriptional control of the lambda PR and lambda PL promoters and a temperature-sensitive lambda repressor. After thermo-induction, E. coli cells transformed with the plasmid carrying the reconstituted lpd gene contained 4-5 times more lipoamide dehydrogenase activity than is normally found in the wild-type organism. The strategy was used to engineer a Glu-188----Asp replacement in lipoamide dehydrogenase, and this generated an enzyme with markedly different kinetic properties.
Cloning, Molecular↗
Diagnosis of Aelurostrongylus abstrusus and Dirofilaria immitis infections in cats from a human shelter.
During a 6-month period, 108 sexually mature stray cats were euthanatized at a humane shelter in Alabama. Aelurostrongylus abstrusus was identified in 20 of the cats (18.5%) by Baermann fecal examination, necropsy, or histologic examination. The Baermann fecal examination was a more sensitive test for aelurostrongylosis than was necropsy or histologic examination. Thoracic radiography was useful in identifying infected cats, but was a less sensitive and more nonspecific method of detection than was the fecal examination. Changes in CBC and serum protein concentrations were not helpful identifying cats with aelurostrongylosis. Six of 17 (35%) heartworm-free, lungworm-infected cats had antibodies to Dirofilaria immitis, as determined by an ELISA method, but none had circulating D immitis antigen, as determined by an ELISA method. Three (2.8%) cats had D immitis. Two of the heartworm-infected cats had no detectable antemortem radiographic or clinicopathologic evidence of the infection. Results of ELISA were positive for antibodies to adult heartworms in 2 cats and for circulating heartworm antigen in 2 cats. One cat that had only one heartworm had no detectable circulating heartworm antigen.
Alabama↗
Bacillus piliformis infection in an adult dog.
Bacillus piliformis infection (Tyzzer's disease) was diagnosed in a 7-year-old spayed dog that had icterus, hepatosplenomegaly, and polyuria. Hematology revealed regenerative anemia, leukocytosis, lymphopenia, and thrombocytopenia. Serum chemical analyses indicated hypocalcemia, high alkaline phosphatase activity, hypoalbuminemia, and hyperglobulinemia. At necropsy, the liver was stippled with gray-white focal lesions. Microscopically, the liver lesions were necrotic and inflammatory. Warthin-Starry-stained sections revealed rod-shaped bacteria in crisscrossing patterns characteristic of B piliformis. This dog was considerably older than dogs previously reported to have Tyzzer's disease and had a concurrent systemic hyphomycosis, suggesting it had been immunocompromised.
Age Factors↗
Chordoma in two ferrets.
Chordomas, characterized by lobules of vacuolated cells lying in a mucinous matrix, are described in 2 ferrets. This is the first report of this rare tumour of notochordal remnant origin in the ferret.
Animals↗
Comparison of mandelate dehydrogenases from various strains of Acinetobacter calcoaceticus: similarity of natural and 'evolved' forms.
In previous work it had been shown that Acinetobacter calcoaceticus wild-type strain NCIB 8250 had only an L-mandelate deydrogenase but it could give rise to mutants that contained an evolved D-mandelate dehydrogenase; conversely, wild-type strain EBF 65/65 had only a D-mandelate dehydrogenase but gave rise to mutants that possessed an evolved L-mandelate dehydrogenase. Several other wild-type strains of A. calcoaceticus have now been shown to grow on both enantiomers of mandelate. In every case the L-mandelate dehydrogenases were found to be much more heat-stable and insensitive to inhibition by p-chloromercuribenzoate than were the D-mandelate dehydrogenases when measured in bacterial extracts. All the D-mandelate dehydrogenases in the wild-type strains were inactivated to about the same extent by an antiserum that had been raised in a rabbit against an evolved D-mandelate dehydrogenase. An evolved D-mandelate deydrogenase (from a mutant strain derived from strain NCIB 8250) and an original D-mandelate dehydrogenase (from a mutant strain derived from strain EBF 65/65) were purified to homogeneity by the same procedure and were indistinguishable as judged by immunological cross-reactivity of the native and the sodium-dodecyl-sulphate-denatured enzymes, solubility in cholate, net charge at pH 7.5, pI value, salting-out properties, Mr value, apparent K(m) value for D-mandelate, heat-stability and sensitivity to p-chloromercuribenzoate. The most likely explanation for the appearance of evolved mandelate dehydrogenases in strains of A. calcoaceticus is that cryptic genes become expressed.
Acinetobacter↗
Purification and properties of L-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus.
L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a 'wall + membrane' fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus.
Acinetobacter↗
Delirium related to tiaprofenic acid.
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Aged↗
The effect of acetaminophen on the disposition of fenoldopam: competition for sulfation.
Both fenoldopam and acetaminophen undergo conjugation with sulfate in humans. The present study was undertaken to determine if a metabolic interaction exists between these two compounds in humans. Twelve healthy male volunteers participated in a single-dose crossover study with 100 mg fenoldopam (capsule) alone or in combination with 1000 mg acetaminophen. The effects of chronic acetaminophen dosing (1000 mg three times/day for 7 days) on a single 100 mg tablet of fenoldopam were studied in a second crossover study in seven additional volunteers. Concomitant dosing with fenoldopam with acetaminophen resulted in increases in peak fenoldopam plasma concentration and AUC after both single (+32% and +50%, respectively) and chronic (+73% and +66%, respectively) acetaminophen dosing. Decreases in peak plasma concentrations and AUC for fenoldopam's sulfated metabolites were seen in both studies. These findings indicate a metabolic basis for the interaction between fenoldopam and acetaminophen, presumably through a competition for inorganic sulfate.
Acetaminophen↗
Nasal rhinosporidiosis in two dogs.
Nasal rhinosporidiosis was diagnosed in 2 dogs. Cytologic criteria for diagnosis were the presence of 5- to 10-microns endospores and 50- to 1,000-microns sporangia. These findings made it easy to differentiate rhinosporidiosis from the more common nasal mycoses such as cryptococcosis. Treatment is principally surgical, but medical management can be performed if the lesion is inoperable or recurs despite multiple surgeries. Dapsone is one drug that has been used in such patients.
Animals↗
Temelastine, a new H1-receptor antagonist.
Temelastine is a selective, competitive histamine H1-receptor antagonist which does not penetrate the central nervous system. The effect of varying doses of temelastine was compared in a randomized, double-blind, controlled study by measuring the inhibition of cutaneous histamine wheals. In twelve subjects single oral doses of 50, 100 and 200 mg of temelastine produced dose-dependent reductions in wheal areas. The inhibition of wheal size was maximal by 2 hr after dosing and was present at 8 hr. At 2 hr the 50, 100, and 200 mg doses reduced the wheal size by 53, 64, and 78%, respectively. Chlorpheniramine, 4 mg, reduced wheal size by 32% at the same period. The ability of temelastine to antagonize the histamine-induced skin reaction over 20 hr was evaluated in a second randomized, double-blind study. Eight subjects participated. Temelastine, 100 mg, produced reductions of 64, 49, 56 and 51% in histamine wheal area at 8, 12, 16 and 20 hr, respectively. Plasma concentrations at these times were 4.04, 2.77, 1.88, and 1.44 mumol/l, respectively. These data suggest that blood levels as low as 1.44 mumol/l may be sufficient to produce an antihistaminic effect, and that daily or twice daily dosing with 100 mg may be adequate to control allergic symptoms.
Adult↗
Membrane-bound lactate dehydrogenases and mandelate dehydrogenases of Acinetobacter calcoaceticus. Purification and properties.
Procedures were developed for the optimal solubilization of D-lactate dehydrogenase, D-mandelate dehydrogenase, L-lactate dehydrogenase and L-mandelate dehydrogenase from wall + membrane fractions of Acinetobacter calcoaceticus. D-Lactate dehydrogenase and D-mandelate dehydrogenase were co-eluted on gel filtration, as were L-lactate dehydrogenase and L-mandelate dehydrogenase. All four enzymes could be separated by ion-exchange chromatography. D-Lactate dehydrogenase and D-mandelate dehydrogenase were purified by cholate extraction, (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography and chromatofocusing. The properties of D-lactate dehydrogenase and D-mandelate dehydrogenase were similar in several respects: they had relative molecular masses of 62 800 and 59 700 respectively, pI values of 5.8 and 5.5, considerable sensitivity to p-chloromercuribenzoate, little or no inhibition by chelating agents, and similar responses to pH. Both enzymes appeared to contain non-covalently bound FAD as cofactor.
Acinetobacter↗
Membrane-bound lactate dehydrogenases and mandelate dehydrogenases of Acinetobacter calcoaceticus. Location and regulation of expression.
Acinetobacter calcoaceticus possesses an L(+)-lactate dehydrogenase and a D(-)-lactate dehydrogenase. Results of experiments in which enzyme activities were measured after growth of bacteria in different media indicated that the two enzymes were co-ordinately induced by either enantiomer of lactate but not by pyruvate, and repressed by succinate or L-glutamate. The two lactate dehydrogenases have very similar properties to L(+)-mandelate dehydrogenase and D(-)-mandelate dehydrogenase. All four enzymes are NAD(P)-independent and were found to be integral components of the cytoplasmic membrane. The enzymes could be solubilized in active form by detergents; Triton X-100 or Lubrol PX were particularly effective D(-)-Lactate dehydrogenase and D(-)-mandelate dehydrogenase could be selectively solubilized by the ionic detergents cholate, deoxycholate and sodium dodecyl sulphate.
Acinetobacter↗