Direct enantioseparation of some β-adrenergic blocking agents using impregnated thin-layer chromatography

The resolution of (±)-atenolol, (±)-propranolol and (±)-metoprolol into their enantiomers was achieved by TLC on silica-gel plates impregnated with optically pure

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-lysine (0.5%) and

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-arginine (0.5%) as the chiral selectors. In all cases, different combinations of acetonitrile–methanol solvent systems were found to be successful in resolving these compounds. Spots were detected using iodine vapour. The detection limit for both (±)-atenolol and (±)-propranolol was 2.6 μg and for (±)-metoprolol, it was 0.26 μg.     

Studies on the maintenance and expression of cloned DNA fragments in the nuclear genome of the green alga Chlamydomonas Reinhardtii

Using a biolistic device built here and based on the principle of the device described by Klein et al. (1987). we have reproducibly obtained transformants of Chlamydomonas reinhardtii . The reproducibility of the method has allowed us to examine the maintenance and expression of cloned DNA fragments introduced into C. Reinhardtii .     

BALT development and augmentation of hyperoxic lung injury in mice deficient in NQO1 and NQO2

NAD(P)H/NRH:quinone oxidoreductases (NQO1 and NQO2) protect against oxidative stress and neoplasia. Cross-breeding of NQO1-/- with NQO2-/- mice generated double-knockout (DKO) mice. DKO mice were born normal yet showed myelogenous hyperplasia as observed in single-knockout mice. DKO mice also showed bronchial-associated lymphoid tissue (BALT) that increased in number and size with age. BALT was absent in wild-type and single-knockout mice. Further analysis demonstrated infiltration of neutrophils and macrophages in BALT and significant increases in the serum cytokines TNFalpha, IL-6, and IL-1beta and increased expression of iNOS and higher nitric oxide in lung macrophages. The development of BALT in DKO mice presumably led to the release of cytokines and higher lung macrophage activation, because histologically spleen, thymus, and blood cultures and urine analysis showed absence of infection. Additionally, the DKO mice upon exposure to hyperoxia demonstrated severe intra-alveolar edema and perivascular inflammation and massive infiltration with neutrophils, compared with wild-type mice. These results suggest that NQO1 and NQO2 combined protect mice against lung inflammation, BALT, and hyperoxic lung injury.     

Suproxide dismutase in human testis preparations

A protein fraction from human testis was structurally investigated. The main component of the fraction reported to contain inhibin-like activity was purified and analyzed by tryptic digestion. The peptides obtained identified the protein as an enzyme, superoxide dismutase, previously known to be present in seminal plasma. The results show that superoxide dismutase is a major enzyme, also of testicular material. They further demonstrate the importance of using pure fractions, and controls such as checks with structural analysis or synthetic peptides, in the work of elucidating the nature of inhibin and other hormonal peptides.     

Dosimetric impact of Acuros XB on cervix radiotherapy using RapidArc technique: a dosimetric study

BackgroundAcuros XB (AXB) may predict better rectal toxicities and treatment outcomes in cervix carcinoma. The aim of the study was to quantify the potential impact of AXB computations on the cervix radiotherapy using the RapidArc (RA ) technique as compared to anisotropic analytical algorithm (AA) computations.Materials and methodsA cohort of 30 patients previously cared for cervix carcinoma (stages II–IIIB) was selected for the present analysis. The RA plans were computed using AA and AXB dose computation engines under identical beam setup and MLC pattern.ResultsThere was no significant (p > 0.05) difference in D_95% and D_98% to the planning target volume (PTV); moreover, a significant (p < 0.05) rise was noticed for mean dose to the PTV (0.26%), D_50% (0.26%), D_2% (0.80%) and V_110% (44.24%) for AXB computation as compared to AA computations. Further, AXB estimated a significantly (p < 0.05) lower value for maximum and minimum dose to the PTV. Additionally, there was a significant (p < 0.05) reduction observed in mean dose to organs at risk (OARs) for AXB computation as compared to AA, though the reduction in mean dose was non-significant (p > 0.05) for the rectum. The maximum difference observed was 4.78% for the rectum V_50Gy, 1.72%, 1.15% in mean dose and 2.22%, 1.48% in D_2% of the left femur and right femur, respectively, between AA and AXB dose estimations.ConclusionFor similar target coverage, there were significant differences observed between the AAA and AXB computations. AA underestimates the V_50Gy of the rectum and overestimates the mean dose and D_2% for femoral heads as compared to AXB. Therefore, the use of AXB in the case of cervix carcinoma may predict better rectal toxicities and treatment outcomes in cervix carcinoma using the RA technique.     

Cytotoxic activity of nucleoside diphosphate kinase secreted from Mycobacterium tuberculosis.

Pathogenicity of Mycobacterium tuberculosis is closely related to its ability to survive and replicate in the hostile environment of macrophages. For some pathogenic bacteria, secretion of ATP-utilizing enzymes into the extracellular environment aids in pathogen survival via P2Z receptor-mediated, ATP-induced death of infected macrophages. A component of these enzymes is nucleoside diphosphate kinase (Ndk). The ndk gene was cloned from M. tuberculosis H37Rv and expressed in Escherichia coli. Ndk was secreted into the culture medium by M. tuberculosis, as determined by enzymatic activity and Western blotting. Purified Ndk enhanced ATP-induced macrophage cell death, as assayed by the release of [14C]adenine. A catalytic mutant of Ndk failed to enhance ATP-induced macrophage cell death, and periodate-oxidized ATP (oATP), an irreversible inhibitor of P2Z receptor, blocked ATP/Ndk-induced cell death. Purified Ndk was also found to be autophosphorylated with broad specificity for all nucleotides. Conversion of His117-->Gln, which is part of the nucleotide-binding site, abolished autophosphorylation. Purified Ndk also showed GTPase activity. Collectively, these results indicate that secreted Ndk of M. tuberculosis acts as a cytotoxic factor for macrophages, which may help in dissemination of the bacilli and evasion of the immune system.     

Characterization of rat brain microsomal acyl-coenzyme A ligases: different enzymes for the synthesis of palmitoyl-coenzyme A and lignoceroyl-coenzyme A

Palmitic acid solubilized with Triton WR-1339 was converted to palmitoyl-CoA by microsomal membranes but lignoceric acid solubilized with Triton WR-1339 was not an effective substrate even though the detergent dispersed the same amount of these fatty acids and was also not inhibitory to the enzyme [I. Singh, R. P. Singh, A. Bhushan, and A. K. Singh (1985) Arch. Biochem. Biophys. 236, 418-426]. This observation suggested that palmitoyl-CoA and lignoceroyl-CoA may be synthesized by two different enzymes. We have solubilized the acyl-CoA ligase activities for palmitic and lignoceric acid of rat brain microsomal membranes with Triton X-100 and resolved them into three separate peaks (fractions) by hydroxylapatite chromatography. Fraction A (palmitoyl-CoA ligase) had high specific activity for palmitic acid and Fraction C (lignoceroyl-CoA ligase) for lignoceric acid. Specific activity of palmitoyl-CoA ligase for palmitic acid was six times higher than in Fraction C and specific activity of lignoceroyl-CoA ligase for lignoceric acid was four times higher than in Fraction A. At higher concentrations of Triton X-100 (0.5%), lignoceroyl-CoA ligase loses activity whereas palmitoyl-CoA ligase does not. Lignoceroyl-CoA ligase lost 60% of activity at 0.6% Triton X-100. Palmitoyl-CoA ligase (T1/2 of 4.5 min) is more stable at 40 degrees C than lignoceroyl-CoA ligase (T1/2 of 1.5 min). The pH optimum of palmitoyl-CoA ligase was 7.7 and that of lignoceroyl-CoA ligase was 8.4. Similar to our results with intact membranes, palmitic acid solubilized with Triton WR-1339 was converted to palmitoyl-CoA by palmitoyl-CoA ligase whereas lignoceric acid when solubilized with Triton WR-1339 was not able to act as substrate for lignoceroyl-CoA ligase. Since solubilized enzyme activities for synthesis of palmitoyl-CoA and lignoceroyl-CoA from microsomal membranes can be resolved into different fractions by column chromatography and demonstrate different properties, we suggest that in microsomal membranes palmitoyl-CoA and lignoceroyl-CoA are synthesized by two different enzymes.     

Biotransformation of CL-20 by a dehydrogenase enzyme from Clostridium sp. EDB2

In a previous study, a marine isolate Clostridium sp. EDB2 degraded 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20) under anaerobic conditions (Bhushan B, Halasz A, Thiboutot S, Ampleman G, Hawari J (2004c) Chemotaxis-mediated biodegradation of cyclic nitramine explosives RDX, HMX, and CL-20 by Clostridium sp. EDB2. Biochem Biophys Res Commun 316:816–821); however, the enzyme responsible for CL-20 degradation was not known. In the present study, we isolated and purified an enzyme, from strain EDB2, responsible for CL-20 degradation. The enzyme was membrane-associated and NADH-dependent and had a molecular weight of 56 kDa (with SDS-PAGE). N-terminal amino acid sequence of enzyme revealed that it belonged to dehydrogenase class of enzymes. The purified enzyme degraded CL-20 at a rate of 18.5 nmol/h mg protein under anaerobic conditions. Carbon and nitrogen mass balance of the products were 100 and 64%, respectively. In LC–MS–MS studies, we detected three different initial metabolites from CL-20, i.e., mono-nitroso derivative, denitrohydrogenated product, and double-denitrated isomers with molecular weight of 422, 393, and 346 Da, corresponding to presumed empirical formulas of C₆H₆N₁₂O₁₁, C₆H₇N₁₁O₁₀, and C₆H₆N₁₀O₈, respectively. Identity of all the three metabolites were confirmed by using ring-labeled [¹⁵N]CL-20 and the nitro-group-labeled [¹⁵NO₂]CL-20. Taken together, the above data suggested that the enzyme degraded CL-20 via three different routes: Route A, via two single electron transfers necessary to release two nitro-groups from CL-20 to produce two double-denitrated isomers; Route B, via a hydride transfer necessary to produce a denitrohydrogenated product; and Route C, via transfer of two redox equivalents to CL-20 necessary to produce a mono-nitroso derivative of CL-20. This is the first biochemical study which showed that CL-20 degradation can be initiated via more than one pathway.     

Stereo-specificity for pro-(R) hydrogen of NAD(P)H during enzyme-catalyzed hydride transfer to CL-20

A dehydrogenase from Clostridium sp. EDB2 and a diaphorase from Clostridium kluyveri were reacted with CL-20 to gain insights into the enzyme-catalyzed hydride transfer to CL-20, and the enzyme's stereo-specificity for either pro-R or pro-S hydrogens of NAD(P)H. Both enzymes biotransformed CL-20 at rates of 18.5 and 24nmol/h/mg protein, using NADH and NADPH as hydride-source, respectively, to produce a N-denitrohydrogenated product with a molecular weight of 393Da. In enzyme kinetics studies using reduced deuterated pyridine nucleotides, we found a kinetic deuterium isotopic effect of 2-fold on CL-20 biotransformation rate using dehydrogenase enzyme against (R)NADD as a hydride-source compared to either (S)NADD or NADH. Whereas, in case of diaphorase, the kinetic deuterium isotopic effect of about 1.5-fold was observed on CL-20 biotransformation rate using (R)NADPD as hydride-source. In a comparative study with LC-MS, using deuterated and non-deuterated NAD(P)H, we found a positive mass-shift of 1Da in the N-denitrohydrogenated product suggesting the involvement of a deuteride (D(-)) transfer from NAD(P)D. The present study thus revealed that both dehydrogenase and diaphorase enzymes from the two Clostridium species catalyzed a hydride transfer to CL-20 and showed stereo-specificity for pro-R hydrogen of NAD(P)H.