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91.
LeDay AM Awe SO Kulkarni K Harris LC Opere C Dash A Ohia SE 《Neurochemical research》2004,29(4):811-818
In the present study, we investigated the effect of naturally occurring and synthetic peroxides on K+-depolarization-evoked release of [3H]D-aspartate from bovine isolated retinae. Furthermore, effect of peroxides on endogenous glutamate concentrations were measured by HPLC in bovine neural retinae and vitreous humor of eyes treated with hydrogen peroxide (H2O2) ex vivo. Both naturally occurring H2O2 (1-100 microM) and synthetic (cumene hydroperoxide, cuOOH; 1-100 microM) peroxides caused a concentration-dependent inhibition of K+-evoked [3H]D-aspartate release without affecting basal tritium efflux. The antioxidant, trolox (2 mM) prevented the inhibition of evoked [3H]D-aspartate overflow elicited by both H2O2 (30 microM) and cuOOH (10 microM). Inhibition of catalase by 3-amino-triazole (3- AT 100 mM) enhanced an inhibitory effect of a low concentration of H2O2 (1 microM) but antagonized the effect of H2O2 (30 microM) on K+-induced [3H]D-aspartate release. In ex vivo experiments, exogenously applied H2O2 (1-100 microM) also caused a concentration-related decrease in glutamate levels in the bovine retina. We conclude that peroxides can inhibit K+-evoked release of [3H]D-aspartate and also decrease endogenous glutamate concentrations in the bovine retina. 相似文献
92.
93.
Multicenter evaluation of reverse line blot assay for detection of drug resistance in Mycobacterium tuberculosis clinical isolates 总被引:7,自引:0,他引:7
Mokrousov I Bhanu NV Suffys PN Kadival GV Yap SF Cho SN Jordaan AM Narvskaya O Singh UB Gomes HM Lee H Kulkarni SP Lim KC Khan BK van Soolingen D Victor TC Schouls LM 《Journal of microbiological methods》2004,57(3):323-335
A multicenter study was conducted with the objective to evaluate a reverse line blot (RLB) assay to detect resistance to rifampin (RIF), isoniazid (INH), streptomycin (STR), and ethambutol (EMB) in clinical isolates of Mycobacterium tuberculosis. Oligonucleotides specific for wild type and mutant (drug resistance linked) alleles of the selected codons in the genes rpoB, inhA, ahpC, rpsL, rrs, embB, were immobilized on a nylon membrane. The RLB assay conditions were optimized following analysis of DNA samples with known sequences of the targeted genes. For validation of the method at different geographical locations, the membranes were sent to seven laboratories in six countries representing the regions with high burdens of multudrug-resistant tuberculosis. The reproducibility of the assay for detection of rpoB genotypes was initially evaluated on a blinded set of twenty reference DNA samples with known allele types and overall concordant results were obtained. Further mutation analysis was performed by each laboratory on the local strains. Upon RLB analysis of 315 clinical isolates from different countries, 132 (85.2%) of 155 RIF-resistant and 28 (51.0%) of 55 EMB-resistant isolates were correctly identified, showing applicability of the assay when targeting the rpoB hot-spot region and embB306. Mutations in the inhA and ahpC promoter regions, conferring resistance to INH, were successfully identified in respectively 16.9% and 13.2% of INH-resistant strains. Likewise, mutations in rrs513 and rpsL88 that confer resistance to STR were identified in respectively 15.1% and 10.7% of STR-resistant strains. It should be mentioned that mutation analysis of the above targets usually requires rather costly DNA sequencing to which the proposed RLB assay presents rapid and inexpensive alternative. Furthermore, the proposed method requires the same simple equipment as that used for spoligotyping and permits simultaneous analysis of up to 40 samples. This technique is a first attempt to combine different targets in a single assay for prediction of antituberculosis drugs resistance. It is open to further development as it allows easy incorporation of new probes for detection of mutations in other genes associated with resistance to second-line (e.g., fluoroquinolones) and new antituberculosis compounds. 相似文献
94.
Tandon VK Kumar M Awasthi AK Saxena HO Goswamy GK 《Bioorganic & medicinal chemistry letters》2004,14(12):3177-3180
The synthesis and pharmacological evaluation of substituted oximino-ethers 1 and 2 of naphth[1,2-b]- and naphth[2,1-b]-oxepin-5-ones (4 and 8) were carried out. The hypotensive activity of oximino-ethers 1 and 2 was evaluated on anaesthetized cats. The results indicated that 1c caused a fall of 80 mm/Hg for >100' at a dose of 5mg/kg iv in anaesthetized cats. 相似文献
95.
Jin J Smith FD Stark C Wells CD Fawcett JP Kulkarni S Metalnikov P O'Donnell P Taylor P Taylor L Zougman A Woodgett JR Langeberg LK Scott JD Pawson T 《Current biology : CB》2004,14(16):1436-1450
BACKGROUND: 14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine. RESULTS: We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo. CONCLUSION: 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3. 相似文献
96.
Gore MA Karmalkar RN Kulkarni MG 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2004,804(1):211-221
Molecularly imprinted polymers are being increasingly investigated as selective sorbents. For the recovery of cholesterol from aqueous media, the utility of the molecularly imprinted polymers has been limited by modest capacities and selectivities, especially when compared with alternative adsorbents reported for the binding of bile acids [Macromolecules 34 (2001) 1548]. This paper describes the use of cholesterol conjugated monomers and cross-linkers, which bind to the template cholesterol molecule by hydrophobic interactions. This leads to enhanced capacities and selectivities during the recovery of cholesterol from aqueous media. The templating effect is clearly seen in the enhanced capacity and selectivity in the retention of cholesterol vis-a-vis stigmasterol and testosterone. 相似文献
97.
Ramakrishna NV Vishwottam KN Puran S Koteshwara M Manoj S Santosh M Chidambara J Wishu S Sumatha B 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2004,809(2):243-249
A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantitation of tadalafil (I) in human plasma, a new selective, reversible phosphodiesterase 5 inhibitor. The analyte and internal standard (sildenafil, II) were extracted by liquid-liquid extraction with diethyl ether/dichloromethane (70/30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on reverse phase Xterra MS C18 column with a mobile phase of 10mM ammonium formate/acetonitrile (10/90, v/v, pH adjusted to 3.0 with formic acid). The protonate of analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 390.4 --> 268.0 and m/z 475.5 --> 58.3 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 10-1000 ng/mL for tadalafil in human plasma. The lower limit of quantitation was 10 ng/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 1.2 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies. 相似文献
98.
Schenck HA Lenkowski PW Choudhury-Mukherjee I Ko SH Stables JP Patel MK Brown ML 《Bioorganic & medicinal chemistry》2004,12(5):979-993
Themisone, also known as Atrolactamide, was found, in the 1950s, to be a very potent anticonvulsant. It was hypothesized that the -CF(3) substitution would maintain the anticonvulsant activity. Anticonvulsant testing of our novel compounds by the National Institute of Health's Anticonvulsant Screening Project of the Antiepileptic Drug Discovery Program identified analogue 1, 3,3,3-trifluoro-2-hydroxy-2-phenyl-propionamide, to have potent anticonvulsant activity (MES ED(50) of 9.9 mg/kg, ScMET ED(50) of 34 mg/kg and TD(50) of 100 mg/kg). Therefore, a diverse range of analogues were synthesized utilizing multiple synthetic pathways to explore the structure-activity relationship. Patch clamp electrophysiology experiments demonstrate that compound 1 is an effective T-type calcium channel blocker. Altogether, these results suggest these compounds as a class of orally available anticonvulsants. 相似文献
99.
Quibell M Benn A Flinn N Monk T Ramjee M Wang Y Watts J 《Bioorganic & medicinal chemistry》2004,12(21):5689-5710
A stereoselective synthesis of (3aS,6aR)-tetrahydrofuro[3,2-b]pyrrol-3-ones and (3aS,7aR)-hexahydrofuro[3,2-b]pyridine-3-ones has been developed through Fmoc protected scaffolds 12 and 13. A key design element within these novel bicyclic scaffolds, in particular the 5,5-fused system, was the inherent stability of the cis-fused geometry in comparison to that of the corresponding trans-fused. Since the bridgehead stereocentre situated beta to the ketone was of a fixed and stable configuration, the fact that cis ring fusion is both kinetically and thermodynamically stable with respect to trans ring fusion provides chiral stability to the bridgehead stereocentre that is situated alpha to the ketone. To exemplify this principle, building blocks 12 and 13 were designed, prepared and utilised in a solid phase combinatorial synthesis of peptidomimetic inhibitors 10, 45a-e, 11 and 46. Both series were chirally stable with 5,5-series 10 and 45a-e exhibiting potent in vitro activity against a range of CAC1 cysteinyl proteinases. Compound 10, a potent and selective inhibitor of cathepsin K, possessed good primary DMPK properties along with promising activity in an in vitro cell-based human osteoclast assay of bone resorption. 相似文献
100.
UvrA protein is a major component of ABC endonuclease complex involved in nucleotide excision repair (NER) mechanism. Although NER system is best characterized in Escherichia coli, not much information is available in Haemophilus influenzae. However, based on amino acid homology, uvrA ORF has been identified on H. influenzae genome [gene identification No. HI0249, Science 269 (1995) 496]. H. influenzae Rd uvrA ORF was cloned and overexpressed in E. coli. The expressed UvrA protein was purified using a two-step column chromatography protocol to a single band of expected molecular weight (104 kDa) and characterized for its ATPase and DNA binding activity. In addition, when H. influenzae uvrA was introduced in E. coli uvrA mutant strain AB1886, its UV resistance was restored to near wild type level. 相似文献