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C Dash

Publications and source records attributed to C Dash.

9 recordsLinked to original sources
PubMed2001

Structural and mechanistic insight into the inhibition of aspartic proteases by a slow-tight binding inhibitor from an extremophilic Bacillus sp.: correlation of the kinetic parameters with the inhibitor induced conformational changes.

We present here the first report of a hydrophilic peptidic inhibitor, ATBI, from an extremophilic Bacillus sp. exhibiting a two-step inhibition mechanism against the aspartic proteases, pepsin and F-prot from Aspergillus saitoi. Kinetic analysis shows that these proteases are competitively inhibited by ATBI. The progress curves are time-dependent and consistent with slow-tight binding inhibition: E + I right arrow over left arrow (k(3), k(4)) EI right arrow over left arrow (k(5), k(6)) EI. The K(i) values for the first reversible complex (EI) of ATBI with pepsin and F-prot were (17 +/- 0.5) x 10(-9) M and (3.2 +/- 0.6) x 10(-6) M, whereas the overall inhibition constant K(i) values were (55 +/- 0.5) x 10(-12) M and (5.2 +/- 0.6) x 10(-8) M, respectively. The rate constant k(5) revealed a faster isomerization of EI for F-prot [(2.3 +/- 0.4) x 10(-3) s(-1)] than pepsin [(7.7 +/- 0.3) x 10(-4) s(-1)]. However, ATBI dissociated from the tight enzyme-inhibitor complex (EI) of F-prot faster [(3.8 +/- 0.5) x 10(-5) s(-1)] than pepsin [(2.5 +/- 0.4) x 10(-6) s(-1)]. Comparative analysis of the kinetic parameters with pepstatin, the known inhibitor of pepsin, revealed a higher value of k(5)/k(6) for ATBI. The binding of the inhibitor with the aspartic proteases and the subsequent conformational changes induced were monitored by exploiting the intrinsic tryptophanyl fluorescence. The rate constants derived from the fluorescence data were in agreement with those obtained from the kinetic analysis; therefore, the induced conformational changes were correlated to the isomerization of EI to EI. Chemical modification of the Asp or Glu by WRK and Lys residues by TNBS abolished the antiproteolytic activity and revealed the involvement of two carboxyl groups and one amine group of ATBI in the enzymatic inactivation.

Aspartic Acid Endopeptidases↗
PubMed2001

Novel bifunctional inhibitor of xylanase and aspartic protease: implications for inhibition of fungal growth.

A novel bifunctional inhibitor (ATBI) from an extremophilic Bacillus sp. exhibiting an activity against phytopathogenic fungi, including Alternaria, Aspergillus, Curvularia, Colletotricum, Fusarium, and Phomopsis species, and the saprophytic fungus Trichoderma sp. has been investigated. The 50% inhibitory concentrations of ATBI ranged from 0.30 to 5.9 microg/ml, whereas the MIC varied from 0.60 to 3.5 microg/ml for the fungal growth inhibition. The negative charge and the absence of periodic secondary structure in ATBI suggested an alternative mechanism for fungal growth inhibition. Rescue of fungal growth inhibition by the hydrolytic products of xylanase and aspartic protease indicated the involvement of these enzymes in cellular growth. The chemical modification of Asp or Glu or Lys residues of ATBI by 2,4,6-trinitrobenzenesulfonic acid and Woodward's reagent K, respectively, abolished its antifungal activity. In addition, ATBI also inhibited xylanase and aspartic protease competitively, with K(i) values 1.75 and 3.25 microM, respectively. Our discovery led us to envisage a paradigm shift in the concept of fungal growth inhibition for the role of antixylanolytic activity. Here we report for the first time a novel class of antifungal peptide, exhibiting bifunctional inhibitory activity.

Alternaria↗
PubMed2000

Interactions of a novel inhibitor from an extremophilic Bacillus sp. with HIV-1 protease: implications for the mechanism of inactivation.

The active site cleft of the HIV-1 protease (PR) is bound by two identical conformationally mobile loops known as flaps, which are important for substrate binding and catalysis. The present article reports, for the first time, an HIV-1 PR inhibitor, ATBI, from an extremophilic Bacillus sp. The inhibitor is found to be a hydrophilic peptide with Mr of 1147, and an amino acid sequence of Ala-Gly-Lys-Lys-Asp-Asp-Asp-Asp-Pro-Pro-Glu. Sequence homology exhibited no similarity with the reported peptidic inhibitors of HIV-1 PR. Investigation of the kinetics of the enzyme-inhibitor interactions revealed that ATBI is a noncompetitive and tight binding inhibitor with the IC(50) and K(i) values 18.0 and 17.8 nm, respectively. The binding of the inhibitor with the enzyme and the subsequent induction of the localized conformational changes in the flap region of the HIV-1 PR were monitored by exploiting the intrinsic fluorescence of the surface exposed Trp-42 residues, which are present at the proximity of the flaps. We have demonstrated by fluorescence and circular dichroism studies that ATBI binds in the active site of the HIV-1 PR and thereby leads to the inactivation of the enzyme. Based on our results, we propose that the inactivation is due to the reorganization of the flaps impairing its flexibility leading toward inaccessibility of the substrate to the active site of the enzyme.

Amino Acid Sequence↗
PubMed2000

Fabrication, characterization, and enzymatic activity of encapsulated fungal protease--fatty lipid biocomposite films.

Encapsulation of an aspartic protease from the fungus Aspergillus saitoi (F-prot) in thermally evaporated fatty acid films by a simple beaker-based immersion technique under enzyme-friendly conditions is described. The approach is based on diffusion of the enzyme from aqueous solution, driven primarily by attractive electrostatic interaction between charged groups on the enzyme surface and ionized lipid molecules in the film. The encapsulated enzyme molecules could be "pumped out" of the biocomposite film into solution by modulating the electrostatic interaction between the enzyme and fatty acid molecules via solution pH variation. The kinetics of F-prot diffusion into the acid films was followed using quartz crystal microgravimetry measurements while the secondary and tertiary structure of the enzyme in the lipid matrix was studied using Fourier transform infrared (FT-IR) and fluorescence spectroscopies. FT-IR and fluorescence measurements indicated little perturbation to the native structure of the enzyme. A chemical analysis of the F-prot-fatty acid biocomposite film was also performed using X-ray photoelectron spectroscopy. The encapsulated F-prot molecules showed catalytic activity (as estimated by reaction with hemoglobin) comparable to free enzyme molecules in solution, indicating facile access of biological analytes/reactants in solution to the enzyme molecules. The advantages/disadvantages of this approach vis-à-vis methods currently used for encapsulation of biomolecules are briefly discussed.

Aspergillus↗
PubMed1998

Ribavirin and interferon alfa-2b in chronic hepatitis C: assessment of possible pharmacokinetic and pharmacodynamic interactions.

AIMS: The primary objective of this study was to determine whether pharmacokinetic interactions occurred between interferon alpha-2b (IFN) and ribavirin in patients with chronic hepatitis C infections. Additionally this study assessed the single and multiple-dose pharmacokinetics of ribavirin and IFN, and compared the safety, tolerability and antiviral pharmacodynamics of IFN plus ribavirin compared with either drug alone. METHODS: In this open label parallel group study, patients with chronic hepatitis C were randomized to receive IFN 3 million IU thrice weekly s.c. alone, ribavirin 600 mg twice daily p.o. alone or both drugs in combination over 6 weeks. Single and multiple dose pharmacokinetics and indices of antiviral pharmacodynamics were assessed during weeks 1 and 6, along with safety assessments during the study. RESULTS: The range of mean ribavirin terminal phase half-lives after single doses was 44-49 h. Comparison of week 1 and week 6 AUC(0,12h) values showed accumulation in plasma of approximately 6-fold. The range of mean washout half-lives after week 6 was 274-298 h, reflecting release of ribavirin from deep compartment stores. The range of single and multiple dose IFN terminal phase half-lives was 5-7 h. IFN demonstrated an increase in bioavailability (approximately 2-fold) upon multiple dose administration. Ribavirin and IFN pharmacokinetic parameters for combined ribavirin and IFN were similar to those during monotherapy with either compound, although the power of this study to detect differences was low. Serum HCV-RNA titers and ALT concentrations were reduced by IFN alone, ribavirin alone reduced ALT concentrations only, and combined IFN plus ribavirin produced numerically greater falls in both measurements than either treatment alone. Serum concentrations of neopterin and activity of 2',5'-oligoadenylate synthetase (2'5'-OAS) were increased by IFN alone and in combination with ribavirin, whereas serum 2'5'-OAS activity was decreased and neopterin concentrations unaltered by ribavirin monotherapy. IFN and ribavirin monotherapy produced characteristic changes in safety laboratory tests (IFN--reductions in white cells, neutrophils and platelets; ribavirin--reduced haemoglobin) and characteristic adverse event profiles (IFN--headache, flu-like symptoms, fatigue, anorexia, nausea, myalgia, and insomnia; ribavirin--headache, fatigue, myalgia, and pruritus). There was no additive effect of combination therapy on safety laboratory tests or reported adverse events. All changes were fully reversible upon treatment cessation. CONCLUSIONS: There was no evidence of pharmacokinetic interactions between IFN and ribavirin in this study. There were numerical trends indicating that the combination of IFN and ribavirin reduced titers of HCV-RNA to a greater extent than did either treatment alone, and the safety profile of combination therapy was similar to those of both monotherapy treatments.

Adolescent↗
PubMed1996

Changes in cardiovascular risk factors by combined pharmacological and nonpharmacological strategies: the main results of the CELL Study.

OBJECTIVE: The objectives of the Cost Effectiveness of Lipid Lowering (CELL) study were twofold: (i) to evaluate the effect on overall cardiovascular risk of two types of health care advice ("usual' and "intensive') given in primary care, with or without pharmacological medication, with the target being to attain a moderate decrease in cholesterol; (ii) to evaluate the ritual of daily medication on compliance with the health care advice. DESIGN: A prospective, double-blind, randomized, controlled trial of 18 months' duration. SETTING: The study was carried out in 32 health centres (out of a total of approximately 850) in Sweden. SUBJECTS: In all, 681 subjects, aged 30-59 years, were randomized. They had at least two cardiovascular risk factors in addition to moderate primary hyperlipidaemia (total cholesterol of at least 6.50 mmol L-1 on three occasions measured by Reflotron triglycerides less than 4.0 mmol L-1 and an LDL:HDL cholesterol ratio of more than 4.0). Most (87%) of the subjects were males; 626 subjects (92%) completed the 18-month follow-up. INTERVENTION: Half the subjects were randomized to 'intensive advice' given in group sessions led by doctors and nurses in primary care. The other half received 'usual advice'. In each of the two advice groups, one-third received an active lipid-lowering drug (pravastatin), one-third placebo, and one-third no drug at all. The tablets were titrated to achieve a 15% reduction in cholesterol. MAIN OUTCOME MEASURES: Changes in the overall Framingham risk score, and the development of adverse events in each group. RESULTS: The change in Framingham risk score was significantly reduced only in subjects taking lipid-lowering medication (together with intensive advice -0.13; 95% CI-0.20, -0.06, and together with usual advice -0.16; 95% CI -0.23, -0.09). The other subjects receiving intensive advice tended to fare better than those on usual advice. Lifestyle was not influenced significantly over the study period. The ritual of daily medication did not affect the outcome. CONCLUSION: As expected, lipid-lowering medication reduced serum cholesterol as well as overall cardiovascular risk in subjects with several risk factors for cardiovascular disease. There was no additive effect of intensive advice to these subjects. However, there was a meagre but significant effect of intensive advice in subjects not receiving active lipid-lowering drugs. One explanation for this difference may be that those on active lipid-lowering medication who had substantial drops in cholesterol might have felt less inclined to change their lifestyle compared with those on other treatment regimens who had less successful drops in cholesterol. There was no benefit from the ritual of taking daily medication.

Adult↗
PubMed1995

The impact of health care advice given in primary care on cardiovascular risk. CELL Study Group.

OBJECTIVE: To evaluate the additional benefit of "intensive" health care advice through six group sessions, compared with the advice usually offered to subjects with multiple risk factors for cardiovascular disease. DESIGN: Prospective, randomised controlled clinical study lasting 18 months. SETTING: 681 subjects aged 30-59 years, with at least two cardiovascular risk factors in addition to moderately high lipid concentrations: total cholesterol > or = 6.5 mmol/l on three occasions, triglycerides < 4.0 mmol/l, and ratio of low density lipoprotein cholesterol to high density lipoprotein cholesterol > 4.0. Most (577) of the subjects were men. MAIN OUTCOME MEASURE: Percentage reduction in total cholesterol concentration (target 15%); quantification of the differences between the two types of health care advice (intensive v usual) for the Framingham cardiovascular risk and for individual risk factors. RESULTS: In the group receiving intensive health care advice total cholesterol concentration decreased by 0.15 mmol/l more (95% confidence interval 0.04 to 0.26) than in the group receiving usual advice. The overall Framingham risk dropped by 0.068 more (0.014 to 0.095) in the group receiving intensive advice, and most of the risk factors showed a greater change in a favourable direction in this group than in the group receiving usual advice, but the differences were seldom significant. The results from questionnaires completed at the group sessions showed that the subjects improved their lifestyle and diet. CONCLUSION: Limited additional benefit was gained from being in the group receiving the intensive health care advice. It is difficult to make an important impact on cardiovascular risk in primary care by using only the practice staff. Better methods of communicating the messages need to be devised.

Adult↗
PubMedDate not supplied

On the preparation, characterization, and enzymatic activity of fungal protease-gold colloid bioconjugates.

We present herein details pertaining to the preparation of bioconjugates of colloidal gold with aspartic protease from the fungus Aspergillus saitoi (F-prot) and their characterization and enzymatic activity. Simple mixing of the colloidal gold and protein solutions under protein-friendly conditions (pH = 3) followed by centrifugation (to remove uncomplexed gold nanoparticles and protein molecules) results in the formation of the fungal protease-gold nanoparticle conjugates. The protein-gold nanoparticle bioconjugate was redispersed in buffer solution and indicated the formation of efficient bioconjugates with intact native protein structures. The bioconjugates in solution were characterized by UV-vis spectroscopy, fluorescence spectroscopy, and biocatalytic activity measurements while drop-dried bioconjugate films on Si (111) substrates were characterized by scanning electron microscopy (SEM), energy dispersive analysis of X-rays (EDAX), and X-ray diffraction (XRD) measurements. Microscopy images do show some aggregate formation, but the intactness of the native structure of the enzyme in the bioconjugate material was verified by fluorescence and biocatalytic activity measurements. The enzyme retains substantial biocatalytic activity in the bioconjugate material and was comparable to that of free enzyme in solution.

Aspartic Acid Endopeptidases↗