Versatile regulation of neuronal nitric oxide synthase by specific regions of its C-terminal tail

The C-terminal tail (CT) of neuronal nitric oxide synthase (nNOS) is a regulatory element that suppresses nNOS activities in the absence of bound calmodulin (CaM). A crystal structure of the nNOS reductase domain (nNOSr) (Garcin, E. D., Bruns, C. M., Lloyd, S. J., Hosfield, D. J., Tiso, M., Gachhui, R., Stuehr, D. J., Tainer, J. A., and Getzoff, E. D. (2004) J. Biol. Chem. 279, 37918-37927) revealed how the first half of the CT interacts with nNOSr and thus provided a template for detailed studies. We generated truncation mutants in nNOS and nNOSr to test the importance of 3 different regions of the CT. Eliminating the terminal half of the CT (all residues from Ile1413 to Ser1429), which is invisible in the crystal structure, had almost no impact on NADP+ release, flavin reduction, flavin autoxidation, heme reduction, reductase activity, or NO synthesis activity, but did prevent an increase in FMN shielding that normally occurs in response to NADPH binding. Additional removal of the CT alpha-helix (residues 1401 to 1412) significantly increased the NADP+ release rate, flavin autoxidation, and NADPH oxidase activity, and caused hyper-deshielding of the FMN cofactor. These effects were associated with increased reductase activity and slightly diminished heme reduction and NO synthesis. Further removal of residues downstream from Gly1396 (a full CT truncation) amplified the aforementioned effects and in addition altered NADP+ interaction with FAD, relieved the kinetic suppression on flavin reduction, and further diminished heme reduction and NO synthesis. Our results reveal that the CT exerts both multifaceted and regiospecific effects on catalytic activities and related behaviors, and thus provide new insights into mechanisms that regulate nNOS catalysis.     

Optimization of a binding fragment targeting the “enlarged methionine pocket” leads to potent Trypanosoma brucei methionyl-tRNA synthetase inhibitors

Potent inhibitors of Trypanosoma brucei methionyl-tRNA synthetase were previously designed using a structure-guided approach. Compounds 1 and 2 were the most active compounds in the cyclic and linear linker series, respectively. To further improve cellular potency, SAR investigation of a binding fragment targeting the “enlarged methionine pocket” (EMP) was performed. The optimization led to the identification of a 6,8-dichloro-tetrahydroquinoline ring as a favorable fragment to bind the EMP. Replacement of 3,5-dichloro-benzyl group (the EMP binding fragment) of inhibitor 2 using this tetrahydroquinoline fragment resulted in compound 13, that exhibited an EC₅₀ of 4 nM.     

Effect of volatile fatty acids on Shigella dysenteriae during anaerobic digestion of human night soil

Thein vitro toxic effect of different volatile fatty acids (VFA) on Shigella dysenteriae was studied in pure culture. Volatile fatty acids viz., acetate, propionate, butyrate, valerate, caproate and heptanoate, exerted pH dependent toxic effect on the pathogen, with minimum inhibitory concentration in the range of 10–3000 mg l⁻¹. The effect of high levels of VFA on S. dysenteriae was studied during anaerobic digestion of human night soil in an experimental digester with VFA level ≅ 9000 mg l⁻¹ and pH ≅ 6.5. Another digester, with VFA level ≅ 700 mg l⁻¹ and pH 7.4, served as the control. In the experimental digester, S. dysenteriae was completely eliminated within 18 days. In the control digester, a four-log reduction in pathogen count was achieved however the pathogen was not completely eliminated. T ₉₀ values for the experimental and control digesters were 2.2 and 3.7 days respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

MicroRNA‐206 induces G1 arrest in melanoma by inhibition of CDK4 and Cyclin D

Expression profiling of microRNAs in melanoma lesional skin biopsies compared with normal donor skin biopsies, as well as melanoma cell lines compared with normal melanocytes, revealed that hsa‐miR‐206 was down‐regulated in melanoma (?75.4‐fold, P = 1.7 × 10^?4). MiR‐206 has been implicated in a large number of cancers, including breast, lung, colorectal, ovarian, and prostate cancers; however, its role in tumor development remains largely unknown, its biologic function is poorly characterized, and its targets affecting cancer cells are largely unknown. MiR‐206 reduced growth and migration/invasion of multiple melanoma cell lines. Bioinformatics identified cell cycle genes CDK2, CDK4, Cyclin C, and Cyclin D1 as strong candidate targets. Western blots and 3′UTR reporter gene assays revealed that miR‐206 inhibited translation of CDK4, Cyclin D1, and Cyclin C. Additionally, hsa‐miR‐206 transfection induced G1 arrest in multiple melanoma cell lines. These observations support hsa‐miR‐206 as a tumor suppressor in melanoma and identify Cyclin C, Cyclin D1, and CDK4 as miR‐206 targets.     

Synthesis of potential antiprogestogens.

Acylated derivatives of 17 alpha-hydroxyprogesterone were prepared in order to test the hypothesis that dialkylamino alkyl moieties have the effect of transforming progestogens into antiprogestogens. This approach has been successful with certain estrogens. Compounds with other functional groups were synthesized to determine whether these might exert binding influence outside the area occupied by progesterone itself. The compounds were tested for competitive affinity against tritiated progesterone and receptor from rabbit uterus cytosol. The low affinity of all derivatives makes it unlikely that they would be active as antiprogestational agents.     

Novel application of quantitative immunoassays for screening seed globulins of cowpea varieties

Using antibodies raised in rabbits, radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) are standardized for cowpea (var. Pusa Barsati) seed globulins. The RIA, when used to screen three stages of seed development, reveals that maximum globulins are detected at 28 days after flowering. When three different varieties of cowpea are assayed for their globulin content by RIA and ELISA, it is observed that the Bold Grain cowpea has the highest amount of related globulin as compared to two other varieties, namely Pusa Phalgun and Asparagus Bean.NCL Communication No. 3821.     

Characterization of Calmodulin-Free Murine Inducible Nitric-Oxide Synthase

Nitric-Oxide Synthase (NOS), that produces the biological signal molecule Nitric-Oxide (NO), exists in three different isoforms called, neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). All NOS isoforms require post-translational interaction with the calcium-binding protein, calmodulin (CaM) for manifesting their catalytic activity. However, CaM has been suggested to control the translational assembly of the enzyme as well, particularly in helping its inducible isoform, iNOS assume a stable, heme-replete, dimeric and active form. Expression of recombinant murine iNOS in E.coli in the absence of CaM has been previously shown to give extremely poor yield of the enzyme which was claimed to be absolutely heme-free, devoid of flavins, completely monomeric and catalytically inactive when compared to the heme-replete, active, dimeric iNOS, generated through co-expression with CaM. In contrast, we found that although iNOS expressed without CaM does produce significantly low amounts of the CaM-free enzyme, the iNOS thus produced, is not completely devoid of heme and is neither entirely monomeric nor absolutely bereft of catalytic activity as reported before. In fact, iNOS synthesized in the absence of CaM undergoes compromised heme incorporation resulting in extremely poor dimerization and activity compared to its counterpart co-expressed with CaM. Moreover, such CaM-free iNOS has similar flavin content and reductase activity as iNOS co-expressed with CaM, suggesting that CaM may not be as much required for the functional assembly of the iNOS reductase domain as its oxygenase domain. LC-MS/MS-based peptide mapping of the CaM-free iNOS confirmed that it had the same full-length sequence as the CaM-replete iNOS. Isothermal calorimetric measurements also revealed high affinity for CaM binding in the CaM-free iNOS and thus the possible presence of a CaM-binding domain. Thus CaM is essential but not indispensible for the assembly of iNOS and such CaM-free iNOS may help in elucidating the role of CaM on iNOS catalysis.     

A Review on Bioactive Porcine Peptide,Protegrin-1

International Journal of Peptide Research and Therapeutics - Multi drug resistance is a major problem of the twenty first century. In order to combat this issue, there is an urgent need in the...     

Distinct States of Methionyl-tRNA Synthetase Indicate Inhibitor Binding by Conformational Selection

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Highlights? Complexes of MetRS from T. brucei with its substrate, intermediate, and inhibitors ? In crystals, substrate-bound MetRS undergoes extensive changes with inhibitor binding ? The inhibitor-bound conformation is surprisingly similar to the ligand-free state ? This indicates inhibitor binding occurs via conformational selection not by induced fit