Renal glutamine utilization: glycylglycine stimulation of gamma-glutamyltransferase

Glycylglycine stimulation of renal glutamine utilization was studied on the homogenate, subcellular and purified enzyme level. The results clearly establish the existence of two glutamine utilizing pathways, the mitochondrial dependent L-glutamine amidohydrolase (PDG) and a second, extramitochondrial pathway. In contrast to the mitochondrial pathway which produces stoichiometric amounts of ammonia and glutamate, this second pathway hydrolyzes glutamine to produce ammonia and transfers the gamma-glutamyl moiety, producing gamma-glutamyl peptides. In the crude systems, containing cyclotransferase, the gamma-glutamyl moiety appears mainly as 5-oxoproline; however, in the enzyme preparation, purified 112-fold, gamma-glutamyl peptides (transpeptidation) and a small amount of glutamate (hydrolysis) appear. D-Glutamine was also hydrolyzed, in contrast to the stereospecific PDG, but at less than one-half the rate of the L-isomer. The molecular weight of this extramitochondrial D- and L-glutamine utilizing enzyme was estimated by gel filtration on a Sephadex G-200 column and found to be approximately 70 000. Based on product formation, molecular weight estimation and copurification with the activity responsible for p-nitroanilide release from gamma-glutamyl-p-nitroanilide, we conclude that this reaction is catalyzed by gamma-glutamyltranspeptidase. Glycylglycine stimulated this enzyme to produce more ammonia while decreasing the appearance of glutamate; in contrast, the mitochondrial glutaminase was unaffected by glycylglycine. This extramitochondrial glutamine utilizing pathway can make a significant contribution to in vivo renal ammoniagenesis.     

Identification of Heterotrophic Nanoflagellates by Restriction Fragment Length Polymorphism Analysis of Small Subunit Ribosomal DNA

Thirty clones derived from twenty isolates of heterotrophic nanoflagellates originating from a variety of marine and freshwater environments were examined by restriction fragment length polymorphism analysis of small subunit ribosomal RNA genes amplified by the polymerase chain reaction (riboprinting). The data were compared with light and electron microscopical identification of the isolates. On morphological criteria, sixteen of the thirty clones belonged to the genus Paraphysomonas De Saedeleer, seven to the genus Spumella Cienkowski, four to the genus Pteridomonas Penard and three to the genus Cafeteria Fenchel and Patterson. Among these taxa, eleven ribotypes were detected by analysis with the restriction enzymes Hinf I, Hae III, Sau3A I, and Msp I. Differentiation of nanoflagellate taxa by the riboprinting method supported taxonomic classification based on morphology at the generic and species level. The utility of the method for discriminating the 'naked' flagellates and for confirming the identity of polymorphic forms among species of Paraphysomonas is demonstrated.     

Pharmacophore feature prediction and molecular docking approach to identify novel anti‐HCV protease inhibitors

Discovering a potential drug for HCV treatment is a challenging task in the field of drug research. This study initiates with computational screening and modeling of promising ligand molecules. The foremost modeling method involves the identification of novel compound and its molecular interaction based on pharmacophore features. A total of 197 HCV compounds for NS3/4A protein target were screened for our study. The pharmacophore models were generated using PHASE module implemented in Schrodinger suite. The pharmacophore features include one hydrogen bond acceptor, one hydrogen bond donor, and three hydrophobic sites. As a result, based on mentioned hypothesis the model ADHHH.159 corresponds to the CID 59533233. Furthermore, docking was performed using maestro for all the 197 compounds. Among these, the CID 59533313 and 59533233 possess the best binding energy of ?11.75 and ?10.40 kcal/mol, respectively. The interactions studies indicated that the CID complexed with the NS3/4A protein possess better binding affinity with the other compounds. Further the compounds were subjected to calculate the ADME properties. Therefore, it can be concluded that these two compounds could be a potential alternative drug for the development of HCV.     

Characterization of beta-endorphin in human pituitary by fast atom bombardment mass spectrometry of trypsin-generated fragments

A novel mass spectrometric method possessing a high level of structural specificity is described for characterization in biological fluids and tissues of endogenous beta-endorphin of the human amino acid sequence (beta h-EP). The method is based upon purification of tissue extracts by an RP-HPLC gradient, followed by trypsinolysis of that particular HPLC fraction corresponding to the elution time of synthetic beta h-EP. The tryptic digest of that endogenous beta h-EP fraction was purified further by a second RP-HPLC gradient. A unique tryptic fragment selected from the second gradient was analyzed by fast atom bombardment mass spectrometry and B/E linked-field scan MS/MS techniques to provide molecular weight and amino acid sequence-determining fragment ion information, respectively, of that fragment. Collectively, these independent analytical methodologies provided unequivocal structure evidence for the presence of endogenous beta h-EP in human pituitary. The method was established first by utilizing synthetic beta h-EP to optimize experimental parameters, and then applied to the analysis of beta h-EP in post-mortem human pituitary extracts. The suitability of the present method for semi-quantitation of tissue extracts is also demonstrated. The corresponding detection limit of the synthetic beta h-EP was 90 fmol, and human pituitary contained 1.5 pmol of beta h-EP mg-1 protein. The method can be extended readily to the analysis of beta-endorphin derived from other species and tissues.     

Methionine-enkephalin peptides in human teeth

The presence of the free opioid pentapeptide methionine enkephalin (ME) and of ME-containing peptide(s) was established firmly in decalcified, depulped human teeth by using a combination of methods including RP-HPLC, radioimmunoassay, radioreceptorassay, trypsin, carboxypeptidase B, fast atom bombardment mass spectrometry, and MS/MS methodology. Positive structural identification of ME was made with mass spectrometry. Those data demonstrate the presence of the preproenkephalinergic A system in the human trigeminal sensory termini.     

Adaptation of γ-glutamyltransferase to acidosis

This study established the following points: (1) the rat kidney contains a mitochondrial (PDG) andm extramitochondrial glutamine utilizing enzymes; (2) this extramitochondrial activity exhibits a molecular weight of 70,000 identical with γ-glutamyltranspeptidase (γ-GTP) and is capable of hydrolyzing both isomers of glutamine; (3) this pathway adapts to metabolic acidosis consistent with a significant contribution to in vivo renal ammoniagenesis.     

Response of Pusa Seedless Grape to 4-CPA, Kinetin, Uracil and GA

Response of Pusa Seedless, a cultivar of Vitis vinifera to 4-chlorophenoxyacetic acid (4-CPA), kinetin, uracil and gibberellic acid (GA) separately and in combinations was tested. Dipping the clusters in GA at 50–100 mg/1 at 2–3 days after full bloom significantly increased the bunch and berry weight of Pusa Seedless Grape. 4-CPA and uracil treatments increased the bunch and berry weight to some extent but not as effectively as GA treatments and kinetin had no effect. GA proved to be the best in increasing the berry weight of this cultivar. A synergistic effect was observed when 4-CPA was combined with GA at 50 mg/1 but combinations of 4-CPA with higher concentrations of GA did not show such synergistic effect. In general, GA alone and especially in combination with 4-CPA lowered the quality of the berries. Uracil on the other hand, improved the quality when applied alone and in combination with GA.     

Callus induction and whole plant regeneration in elite Indian maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) inbreds

Callus induction and regeneration ability of five elite maize inbred lines, CM 111, CM 117, CM 124, CM 125 and CM 300 were investigated using 14-day-old immature embryos as explants. Genotype, medium, source of auxin and their concentrations influenced induction of callus. Explants grown on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid at 1 mg l⁻¹ showed the highest frequency of callusing. Among all the media tested, explants grown on N6 medium gave the highest frequency of organogenic callus. Moreover, N6 supplemented with Dicamba promoted higher callus response in terms of both frequency of induction as well as quality, compared to N6 medium with 2,4-D. N6 supplemented with 2 mg l⁻¹ Dicamba induced the highest frequency of organogenic callus. Among the five genotypes tested, CM 124, CM 125, and CM 300 gave the best callus. Explants of both CM 124 and CM 300 incubated on MS medium supplemented with 1 mg l⁻¹ benzyladenine and 0.5 mg l⁻¹ indole acetic acid promoted the highest frequency of shoot induction. Though CM 124 induced higher percentage of shoot formation than CM 300, the mean number of developed shoots per explant was higher for CM 300. The highest frequency of root formation was observed when shoots were grown on MS medium supplemented with 2 mg l⁻¹ naphathalene acetic acid. Percentage of regenerated plants ranged from 54 to 66.     

4-1BB-mediated amelioration of experimental autoimmune uveoretinitis is caused by indoleamine 2,3-dioxygenase-dependent mechanisms

Interphotoreceptor retinoid binding protein (IRBP)-induced experimental autoimmune uveoretinitis (EAU) is a CD4+ T cell-mediated autoimmune disease. Development of EAU is inhibited by treatment with an agonistic anti-4-1BB mAb. Even established EAU was alleviated by anti-4-1BB mAb. However, inhibition of 4-1BB/4-1BB ligand (4-1BBL) interaction does not suppress the development of EAU. It appears that cross-linking of 4-1BB evokes an active antigen-specific suppression mechanism rather than merely blocking 4-1BB/4-1BBL interaction. We found that administration of anti-4-1BB mAb induced massive clonal expansion of CD11c+CD8+ T cells that produced IFN-gamma, resulting in accumulation of a high level of indoleamine 2,3-dioxygenase (IDO) in CD11c+ dendritic cells. 4-1BB-mediated suppression of EAU was reversed by the pharmacological IDO inhibitor, 1-methyl-tryptophan (1-MT). These studies demonstrate that suppression of EAU results from antigen-driven, 4-1BB-mediated expansion of novel CD11c+CD8+ T cells that suppress antigen-specific CD4+ T cells via an IDO-dependent mechanism.