Characterization of two 3-hydroxybutyrate dehydrogenases in poly(3-hydroxybutyrate)-degradable bacterium, Ralstonia pickettii T1.

Two D-(-)-3-hydroxybutyrate (3HB) dehydrogenases, BDH1 and BDH2, were isolated and purified from a poly(3-hydroxybutyrate) (PHB)-degradable bacterium, Ralstonia pickettii T1. BDH1 activity increased in R. pickettii T1 cells grown on several organic acids as a carbon source but not on 3HB, whereas BDH2 activity markedly increased in the same cells grown on 3HB or PHB. To examine their biochemical properties, bdh1 and bdh2 were cloned and overexpressed in Escherichia coli, and their purified products were characterized. The kinetic parameters indicate that BDH1 is more suitable for converting acetoacetate to 3HB than BDH2, whereas BDH2 is more efficient for the reverse reaction than BDH1. Thus, R. pickettii T1 contains two BDHs with different biochemical properties and physiological roles: BDH1 for cell growth on organic acids other than 3HB and BDH2 for cell growth on 3HB or PHB.

Development of a commercial amperometric biosensor electrode for the ketone D-3-hydroxybutyrate.

Representatives of the common classes of quinoid NADH redox mediator, including Meldola Blue (MB) 3, 4-methyl-1,2-benzoquinone (4-MBQ) 4, 1-methoxy phenazine methosulphate (1-MeO-PMS) 5 and 2,6-dichloroindophenol (DCIP) 6, are shown to inhibit the NAD-dependent enzyme D-3-hydroxybutyrate dehydrogenase (HBDH), severely limiting their utility in the construction of a stable biosensor electrode for the ketone body D-3-hydroxybutyrate (3-OHB). It is proposed that these mediators bind covalently to important thiol groups in the enzyme. This mode of inhibition is overcome through the use of mediators such as 1,10-phenanthroline quinone (1,10-PQ) 7, which avoid 1,4-nucleophilic addition with enzyme amino acid residues such as Cys. As a result, 1,10-PQ 7 was selected for incorporation in a biosensor electrode for 3-OHB. The resulting MediSense Optiumtrade mark beta-Ketone electrode is stable (

The content and distribution of troponin I, troponin T, myoglobin, and alpha-hydroxybutyric acid dehydrogenase in the human heart.

We studied the content and distribution of heart-specific markers troponin I and troponin T in relation to conventional non-heart specific myoglobin and alpha-hydroxybutyric acid dehydrogenase (HBD) in the hearts of 34 patients who died of various causes. Tissue was obtained from the right and left ventricles, the interventricular septum, and the right and left atria. We found significant differences in the contents expressed per gram wet weight tissue in the right and left ventricles for troponin I (by 1 of the 2 methods used), troponin T, myoglobin, and HBD and no differences per gram of protein. The biochemical contents per gram wet weight tissue and per gram protein were significantly lower in the right and left atria for all studied markers compared with the right and left ventricles. No significant differences were found in biochemical contents between the right and left atria. These findings imply that estimation of myocardial damage through cardiac markers levels in serum depends on the site of injury (atrium or ventricle). Comparison of myocardial injury among individuals using marker levels in serum is not reliable because of the varied ranges of markers in tissue contents.

Three enzymes newly identified from the genus Eimeria and two more newly identified from E. maxima, leading to the discovery of some aliphatic acids with activity against coccidia of the domesticated fowl.

Nine enzymes were detected in sporulated oocysts of Eimeria tenella and E. maxima, parasites of the domesticated fowl (Gallus gallus). Three enzymes, hydroxybutyrate dehydrogenase, alanine aminotransferase and gamma-glutamyltransferase, all identified for the first time in Eimeria of fowl, occurred both in E. tenella and in E. maxima. The remaining enzymes assayed had previously been found in various Eimeria species of fowl, although creatine kinase and glutamate dehydrogenase were hitherto unknown from E. maxima. The three enzymes newly recorded from Eimeria of fowl are of interest as potential genetic markers, and also as potential chemotherapeutic targets. The discovery of hydroxybutyrate dehydrogenase led to the demonstration of anticoccidial activity by some aliphatic acids. The paper also includes a list of the enzymes detected in Eimeria of fowl in previous studies.

Enantioselective depletion of mitochondrial glutathione concentrations by (S)- and (R)-3-hydroxy-4-pentenoate.

(R,S)-3-Hydroxy-4-pentenoate rapidly and selectively depletes the mitochondrial glutathione pool in rat hepatocytes, but shows little cytotoxicity and does not induce mitochondrial dysfunction [Shan, X., et al. (1993) Chem. Res. Toxicol. 6, 75-81]. The objective of the present studies was to investigate the 3-hydroxybutanoate dehydrogenase-dependent oxidation of (R)- and (S)-3-hydroxy-4-pentenoate and the enantioselectivity of 3-hydroxy-4-pentenoate-induced depletion of mitochondrial glutathione concentrations in isolated rat liver mitochondria and hepatocytes. (S)-3-Hydroxy-4-pentenoate, but not (R)-3-hydroxy-4-pentenoate, was a substrate for 3-hydroxybutanoate dehydrogenase. Incubation of rat liver mitochondria or hepatocytes with (S)-3-hydroxy-4-pentenoate resulted in a time- and concentration-dependent depletion of mitochondrial glutathione concentrations, whereas (R)-3-hydroxy-4-pentenoate produced little depletion. These results show that (S)-3-hydroxy-4-pentenoate is a substrate for 3-hydroxy-butanoate dehydrogenase and is converted to the Michael acceptor 3-oxo-4-pentenoate, which reacts with glutathione and thereby depletes the mitochondrial glutathione pool. (S)-3-Hydroxy-4-pentenoate may find use in the study of mitochondrial glutathione homeostasis and the role of mitochondrial glutathione in cellular protection.

Identification and typing of pyogenic streptococci by enzyme electrophoretic polymorphism.

Polyacrylamide-agarose gel electrophoresis was used to study polymorphism of lactate dehydrogenase (LDH), nucleoside phosphorylase (NSP), phosphoglucose isomerase (PGI), hydroxybutyrate dehydrogenase (HBD), adenylate kinase (ADK) and esterases of 44 strains of Streptococcus pyogenes, 25 group G streptococcal strains, 11 "S. equisimilis" strains, seven S. dysgalactiae strains, four S. canis strains, three S. equi strains and seven S. zooepidemicus strains. Analysis of LDH, NSP, PGI, HBD and ADK provided valuable interspecies differentiation, by showing that four groups of strains corresponded to the four known DNA homology groups. Esterases showed greater intraspecies variation than the other enzymes. The combined analysis of the six enzymes indicated 31 zymotypes among S. pyogenes, 14 in group G streptococci and 11 in "S. equisimilis" strains. This was shown to be an effective technique for typing pyogenic streptococci.

Combined enzyme histochemical and ultrastructural study on cryostat sections of pig heart to detect early reperfusion damage after ischaemia.

In cardiac surgery, recognition of peroperative myocardial infarction may improve patient selection for prolonged circulatory support. The value of enzyme histochemistry to discriminate between reversible and irreversible myocardial damage at short periods of reperfusion was studied in an in vivo model of regional ischaemia in pig hearts. The left anterior descending coronary artery (LADCA) was clamped for 45 min followed by 2 h reperfusion (group 1, n = 3). Post-mortem heart tissue showed markedly decreased activities of lactate dehydrogenase (LDH) and beta-hydroxybutyrate dehydrogenase (BDH) as demonstrated in cryostat sections, accompanied by massive leakage of LDH in the venous effluent. The depleted areas showed irreversible cell damage based on the presence of flocculent densities in mitochondria. In group 2 (n = 6), LADCA flow was reduced to 40 per cent of the base-line value followed by 2 h reperfusion. Heart tissue showed normal LDH and BDH activities, except for some cells surrounding blood vessels, which activity was minimally decreased. Flocculent densities in mitochondria were never observed. We conclude that enzyme histochemistry of LDH and BDH activity on cryostat sections is a useful tool for detecting irreversible myocardial cell damage as early as 2 h reperfusion after ischaemia of the pig heart. The technique has potential applications in the detection of peroperative infarction in human biopsies.

Production and characterization of monoclonal antibodies to bovine brain succinic semialdehyde reductase.

Monoclonal antibodies against bovine brain succinic semialdehyde reductase were produced and characterized. A total of nine monoclonal antibodies recognizing different epitopes of the enzyme were obtained, of which two inhibited the enzyme activity and three stained cytosol of rat spinal cord neurons as observed by indirect immunofluorescence microscopy. When unfractionated total proteins of bovine brain homogenate were separated by gel electrophoresis and immunoblotted, the antibodies specifically recognized a single protein band of 34 kDa, which comigrates with purified bovine succinic semialdehyde reductase. Using the antisuccinic semialdehyde reductase antibodies as probes, we investigated the cross-reactivities of brain succinic semialdehyde reductases from some mammalian and an avian species. The immunoreactive bands on western blots appeared to be the same in molecular mass--34 kDa--in all animal species tested, including humans. The result indicates that brain succinic semialdehyde reductase is distinct from other aldehyde reductases and that mammalian brains contain only one succinic semialdehyde reductase. Moreover, the enzymes among the species are immunologically very similar, although some properties of the enzymes reported previously were different from one another.

[Aspects of the enzymatic evaluation in Plasmodium falciparum malaria infection]. / Aspects du bilan enzymatique au cours de l'infection palustre à Plasmodium falciparum.

To try to find effects of malaria on clinical serum activity of certain enzymes, 3 groups of infants--malarial, asymptomatic carrier and normal controls--have been designed. Parasitologic data have been compared with serum concentration of lactate dehydrogenase (LDH), Hydroxybutyrate dehydrogenase (HBDH) alanine aminotransferase (ALT), gamma glutamyl transferase (GGT) and 5'nucleotidase (5'Nu). Results show that only LDH and HBDH are significantly increased. Respective coefficients of correlation r = 0.32 (p < 0.05) and r = 0.39 (p < 0.01) show that increasing in LDH and HBDH are linked to malarial parasite density. LDH and HBDH increasing might therefore constitute a marker of malaria.

Comparison of magnetic resonance imaging studies with enzymatic indexes of myocardial necrosis for quantification of myocardial infarct size.

To evaluate the potential of gadolinium-diethylene triamine pentaacetic acid (DTPA)-enhanced magnetic resonance imaging (MRI) in the quantification of infarct size in patients with a first acute myocardial infarction, 24 patients with a first acute myocardial infarction were studied by electrocardiographic gated MRI at a mean of 4.3 days after the acute event. Multislice, single-phase, T1-weighted, spin-echo MRI in the true short-axis plane was performed 20 minutes after intravenous injection of gadolinium-DTPA (0.15 mmol/kg of body weight). Circumscript myocardial regions of increased signal intensity on gadolinium-DTPA-enhanced images were considered to be infarcted. Infarct size (in g) was determined using Simpson's rule, and was compared with that based on cumulative release of alpha-hydroxybutyrate dehydrogenase activity in plasma and with peak creatine kinase-MB level in plasma. Infarct size quantified with MRI correlated well with "enzymatic" infarct size (in g equivalents) (y = 0.99 x + 0.71; r = 0.93; p = 0.0001) and peak creatine kinase-MB levels (r = 0.72; p = 0.002). It is concluded that gadolinium-DTPA-enhanced MRI enables accurate quantification of infarct size in patients with a first acute myocardial infarction.