Actinoalloteichus spitiensis sp. nov., a novel actinobacterium isolated from a cold desert of the Indian Himalayas.

An actinobacterial strain, RMV-1378T, isolated from a cold desert of the Indian Himalayas, was subjected to polyphasic taxonomic characterization. The strain formed branching, non-fragmenting vegetative hyphae and did not produce diffusible pigments. Neither aerial mycelium nor spore formation was observed. The G+C content of the DNA was 72.0 mol%. The strain had chemotaxonomic characteristics typical of the genus Actinoalloteichus and was closely related (99.3 % 16S rRNA gene sequence similarity) to Actinoalloteichus cyanogriseus, currently the only Actinoalloteichus species with a validly published name. However, the results of DNA-DNA hybridization experiments showed 51.9 % relatedness with the type strain of A. cyanogriseus. On the basis of the above data and the physiological and biochemical distinctiveness of RMV-1378T (=MTCC 6194T=JCM 12472T=DSM 44848T), this strain should be classified as the type strain of a novel species of Actinoalloteichus, for which the name Actinoalloteichus spitiensis sp. nov. is proposed.

Cloning and characterization of aspartate-beta-semialdehyde dehydrogenase from Mycobacterium tuberculosis H37 Rv.

AIMS: To clone and characterize the aspartate-beta-semialdehyde dehydrogenase of Mycobacterium tuberculosis H37Rv. METHODS AND RESULTS: The asd gene of M. tuberculosis H37Rv was cloned in pGEM-T Easy vector, subcloned in expression vector pQE30 having a T5 promoter, and overexpressed in Escherichia coli. The ASD enzyme was expressed to levels of 40% but was found to be inactive. Functional ASD was obtained by altering induction and growth conditions and the enzyme was purified to near homogeneity using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The K(m) and V(max) values for the three substrates L-ASA, NADP and Pi, the turnover number and specific activity of the enzyme were determined. CONCLUSIONS: Functional ASD enzyme of M. tuberculosis was obtained by gene cloning and protein purification using affinity chromatography. The K(cat) and specific activity of the enzyme were 8.49 s(-1) and 13.4 micromol min(-1) microg(-1) respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: The ASD enzyme is a validated drug target. We characterized this enzyme from M. tuberculosis and future work would focus on deducing the three-dimensional structure of the enzyme and design of inhibitors, which could be used as drugs against TB.

Chaperone-assisted overexpression of an active D-carbamoylase from Agrobacterium tumefaciens AM 10.

The N-carbamoyl-D-amino acid amidohydrolase (D-carbamoylase) gene (dcb) from Agrobacterium tumefaciens AM 10 was cloned by polymerase chain reaction in plasmid pET28a and was overexpressed in Escherichia coli JM109 (DE3). However, almost 80% of the enzyme remained trapped in inclusion bodies. To facilitate the expression of the properly folded active enzyme, the chaperones GroEL/ES were coexpressed in plasmid pKY206. This resulted in a 43-fold increase in active enzyme production compared to the wild-type strain. The histidyl-tagged D-carbamoylase was purified by a single step nickel-affinity chromatography to a specific activity of 9.5 U/mg protein.

Purification and characterization of two extracellular alkaline proteases from a newly isolated obligate alkalophilic Bacillus sphaericus.

Two novel extracellular serine proteases were purified to homogeneity from the cell-free culture filtrate of an obligate alkalophilic Bacillus sphaericus by a combination of ultrafiltration, ammonium sulfate precipitation and chromatographic methods. The enzymes showed similar substrate specificities, but differed in hydrophobicity and molecular mass. Protease A was a monomeric protease with a relative molecular mass (M(r)) of 28.7 kDa, whereas protease B, with a M(r) of 68.0 kDa, apparently consisted of smaller subunits. The purified protease A had a specific activity on hemoglobin of 5.1 U/mg protein compared to 40.9 U/mg protein in the case of protease B. Both proteases were most active on SAAPF-pNa, a substrate for chymotrypsin-like serine proteases. However, the K(m) values of these two proteases on SAAPF-pNa were higher than that for alpha-chymotrypsin, indicating a lower affinity of proteases A and B for this substrate compared to chymotrypsin. Unlike other Bacillus serine proteases, neither protease A nor B stained with Coomasie blue R-250, even with loading of a large amount of protein, and they stained poorly with the silver staining method. However, NH(2)-terminal amino acid sequencing of protease B revealed a high similarity with subtilisin Carlsberg (67% homology). Almost total inhibition of both proteases by PMSF, but very little/no inhibition by trypsin and chymotrypsin inhibitors (TPCK and TLCK) or thiol reagents (PCMB and iodoacetic acid), further supported the view that the enzyme belonged to the serine protease family.

Two-step purification of d(-)-specific carbamoylase from Agrobacterium tumefaciens AM 10.

A simple, economical and rapid affinity chromatography procedure with red dye as a ligand has been described for the two-step purification of a relatively thermostable d(-)-carbamoylase from the cell-free extract of Agrobacterium tumefaciens AM 10. The enzyme was purified 232-fold to homogeneity with a recovery of 30% in the presence of 2 mM dithiothreitol. The specific activity of the enzyme was 7.88 U/mg protein. The enzyme is a dimer with a native molecular mass of 67 kDa and a subunit relative molecular mass of 38 kDa. The isoelectric point of the enzyme was found to be 5.83. The K(m) values for N-carbamoyl-dl-methionine and N-carbamoyl-d-phenylglycine were 3.84 and 5.0 mM, respectively.

Rapid degradation of ferulic acid via 4-vinylguaiacol and vanillin by a newly isolated strain of bacillus coagulans.

A new strain Bacillus coagulans BK07 was isolated from decomposed wood-bark, based on its ability to grow on ferulic acid as a sole carbon source. This strain rapidly decarboxylated ferulic acid to 4-vinylguaiacol, which was immediately converted to vanillin and then oxidized to vanillic acid. Vanillic acid was further demethylated to protocatechuic acid. Above 95% substrate degradation was obtained within 7 h of growth on ferulic acid medium, which is the shortest period of time reported to date. The major degradation products, was isolated and identified by thin-layer chromatography, high performance liquid chromatography and 1H-nuclear magnetic resonance spectroscopy were 4-vinylguaiacol, vanillin, vanillic acid and protocatechuic acid.

A thermostable D-hydantoinase isolated from a mesophilic Bacillus sp.AR9.

A thermostable hydantoinase has been characterized from a mesophilic Bacillus sp.AR9. The hydantoinase produced by this Bacillus sp.AR9 is strictly D-specific and is constitutively produced with high yields (4500 U/ml) in this strain. The enzyme is not only alkalo- and thermostable but has a pH and temperature optimum of 9.5 and 65 degrees C, respectively, which is advantageous for the bioconversion of DL-5-monosubstituted-hydantoin derivatives. The enzyme has a half life of 80 minutes at 70 degrees C and loses only 33% of its activity in 4 hr at 60 degrees C. The enzyme has a broad substrate specificity with a maximum of 100% with hydantoin and about 26% with dihydrouracil. Co+ + ions enhance the activity of the enzyme by more than 60%.

Novel assay for screening rifamycin B-producing mutants.

A simple method that allows easy identification of rifamycin B-producing strains is described. This method involves the use of an enzyme, rifamycin oxidase, which converts inactive rifamycin B to active rifamycin S. In this method, colonies to be tested are grown in pairs. The two colonies are then transferred to two plates seeded with a sensitive strain of Staphylococcus aureus, one plate of which contains the enzyme rifamycin oxidase. All paired colonies which show a larger inhibition zone diameter on the enzyme-containing plate are identified as rifamycin B producers.

Production of laccase by Curvularia sp.

A Curvularia sp. isolated from soil was found to contain laccase activity toward guaiacol as substrate. The organism produced an extracellular laccase in a medium containing yeast extract, peptone and dextrose. Initial medium pH 4.0 and cultivation temperature 30 degrees C were found to be most suitable for maximum enzyme production. The optimum pH and temperature for laccase activity were found to be 5.2 and 50 degrees C, respectively. Under optimum conditions, the enzyme had a Km (guaiacol) of 0.75 mmol/L and a V of 1.50 CU min-1 ml-1. Some divalent metal ions inhibited laccase activity at very low concentrations.

Comparative analysis of glycosylated hemoglobins by ion-exchange column chromatography and high-performance liquid chromatography in patients with hemoglobinopathies.

Cation-exchange chromatography gives falsely decreased values for glycosylated hemoglobins (GHbs) in patients with abnormal hemoglobins (Hbs) such as S, D, G, C, E, and O. This decrease is thought to be proportional to the percentage of abnormal Hb present. In the authors' study, cation-exchange column chromatographic GHb values on 84 nondiabetic patients heterozygous for Hb S, 28 AS diabetic patients, and 23 nondiabetic patients heterozygous for Hb C were calculated to account for the percentage of abnormal Hb, and the resulting values were compared with the GHb values obtained by high-performance liquid chromatography (HPLC). There existed a good correlation between the calculated GHb and the GHb obtained by HPLC (r = 0.92 for AS and r = 0.94 for AC). In patients with elevated Hb F, chromatographic GHb values are falsely high. In such cases, correction can be made by subtracting the Hb F value from the observed GHb. In laboratories where cation exchange chromatography is used, accurate determination of GHb can be made by adjusting observed values for portion of the abnormal Hb present.

Monitoring of drug therapy in epileptic children.

Twenty children who were receiving phenytoin either alone or in combination with phenobarbitone, were monitored for their plasma steady state levels. A wide interindividual variation was observed in steady state plasma levels. Among children who were receiving only phenytoin, nearly 50% had drug levels above 10 micrograms/ml. It was observed that children with drug levels above 10 micrograms/ml exhibited good therapeutic response. Two children who were presented with acute toxicity showed drug levels above 40 micrograms/ml. Children receiving combination of phenytoin and phenobarbitone showed extreme degree of variation in drug levels which was reflective of drug interaction. In view of the wide interindividual variation in level of phenytoin and the unpredictability of interaction with other anticonvulsants, the monitoring of drug levels become mandatory.

Monitoring of phenobarbitone in epileptic children.

Seventy-five children with different nutritional status, who were receiving phenobarbitone for treatment of various seizure disorders, were monitored for their plasma steady state level, therapeutic efficacy and toxicity. A wide inter individual variation in the steady state level was observed. About 10 percent of them had subtherapeutic level, while nearly 30 percent of them had potentially toxic levels, the remaining were within therapeutic range of 10-25 micrograms/ml. Poor compliance was found to be an important contributing factor for the variations in the level. Steady state levels were significantly higher in children with grade II protein energy malnutrition (PEM) than normally nourished children. A good correlation existed between plasma drug level and therapeutic response in nearly 60 percent of the children. Clinical toxicity was observed in nearly two thirds of the children, sedation and behavioral problems being the most common. The importance of monitoring the drug level is discussed for the proper management of epileptic children.

Serum ferritin levels in hematologic malignant neoplasms.

Data from 90 patients with a variety of hematologic malignant neoplasms were studied to determine the relation between changes in serum ferritin concentration and the clinical status of the patients. Patients with Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, blastic crisis of chronic myelocytic leukemia, acute myeloblastic leukemia and acute lymphoblastic leukemia were found to have significantly elevated serum ferritin levels. Further study of serum ferritin concentration in certain hematologic malignant neoplasms might provide a valuable insight into the role of serum ferritin determination in the diagnosis and follow-up of patients with malignant diseases.

Hemophiliac with hemolytic anemia resulting from factor VIII concentrate.

Bilateral herniorrhaphy was successfully performed on a group AB hemophiliac with cirrhosis of the liver. Adequate hemostasis was maintained with infusions of commercial factor VIII concentrates and fresh frozen plasma. An anti-A antibody mediated hemolytic reaction occurred in the postoperative period. Hemolysis subsided after the cessation of commercial factor VIII infusions. The risk of such hemolytic reactions could be eliminated through the use of group-specific cryoprecipitated factor VIII.