Adenosine deaminase prefers a distinct sugar ring conformation for binding and catalysis: kinetic and structural studies

Several recent X-ray crystal structures of adenosine deaminase (ADA) in complex with various adenosine surrogates have illustrated the preferred mode of substrate binding for this enzyme. To define more specific structural details of substrate preferences for binding and catalysis, we have studied the ADA binding efficiencies and deamination kinetics of several synthetic adenosine analogues in which the furanosyl ring is biased toward a particular conformation. NMR solution studies and pseudorotational analyses were used to ascertain the preferred furanose ring puckers (P, nu(MAX)) and rotamer distributions (chi and gamma) of the nucleoside analogues. It was shown that derivatives which are biased toward a "Northern" (3'-endo, N) sugar ring pucker were deaminated up to 65-fold faster and bound more tightly to the enzyme than those that preferred a "Southern" (2'-endo, S) conformation. This behavior, however, could be modulated by other structural factors. Similarly, purine riboside inhibitors of ADA that prefer the N hemisphere were more potent inhibitors than S analogues. These binding propensities were corroborated by detailed molecular modeling studies. Docking of both N- and S-type analogues into the ADA crystal structure coordinates showed that N-type substrates formed a stable complex with ADA, whereas for S-type substrates, it was necessary for the sugar pucker to adjust to a 3'-endo (N-type) conformation to remain in the ADA substrate binding site. These data outline the intricate structural details for optimum binding in the catalytic cleft of ADA.     

Cloning and expression of isocitrate lyase from human round worm Strongyloides stercoralis

A full length cDNA (1463 bp) encoding isocitrate lyase (EC 4.1.3.1) of Strongyloides stercoralis is described. The nucleotide sequence of this insert identified a cDNA coding for the isocitrate lyase. The conceptually translated amino acid sequence of the open reading frame for S. stercoralis isocitrate lyase encodes a 450 amino acid residue protein with an apparent molecular weight of 50 kDa and a predicted pl of 6.39. The sequence is 69% A/T, reflecting a characteristic A/T codon bias of S. stercoralis. The amino acid sequence of S. stercoralis isocitrate lyase is compared with bifunctional glyoxylate cycle protein of Caenorhabditis elegans and isocitrate lyases from Chlamydomonas reinhardtii and Myxococcus xanthus. The full length cDNA of S. stercoralis was expressed in pRSET vector and bacteriophage T7 promoter based expression system. S. stercoralis lyase recombinant protein, purified via immobilized metal affinity chromatography, showed a molecular mass of 50 kDa on polyacrylamide gels. The role of isocitrate lyase in the glyoxylate cycle and energy metabolism of S. stercoralis is also discussed.     

Two simple and rapid methods for the detection of polymer-degrading enzymes on high-resolution, alkaline, cold, in situ-native (HiRACIN)-PAGE and high-resolution, in situ-inhibited native (HiRISIN)-PAGE

Two sensitive, high-resolution and exceedingly versatile methods for the detection of isoenzymes of polymer-degrading enzymes on high-resolution, alkaline, cold, in situ-native (HiRACIN)-PAGE and high-resolution in situ-inhibited, native (HiRISIN)-PAGE are described. Extracellular crude extracts containing xylanases and carboxymethylcellulases from Scopulariopsis sp. and glucoamylases from Aspergillus niger were subjected to non-denaturing PAGE containing substrates in the resolving gel. In case of HiRACIN-PAGE, the enzymes were prevented from degrading their respective substrates during run by carrying out electrophoresis at 4°C and the pH of running and resolving gel buffer systems were increased from 8.5 to 10.6. In case of HiRISIN-PAGE, adding competitive inhibitor of the enzyme, cellobiose, in the resolving gel prevents the degradation of polymer during the run. These techniques were successfully applied, for the first time, to visualize four, three and four sharp and distinct bands of xylanases, glucoamylases and CMCases, respectively.     

Ameliorative Effect of Chrysin on Adenine-Induced Chronic Kidney Disease in Rats

Chrysin (5, 7- dihydroxyflavone) is a flavonoid with several pharmacological properties that include antioxidant, anti-inflammatory and antiapoptotic activities. in this work, we investigated some effects of three graded oral doses of chrysin (10, 50 and 250 mg/kg) on kidney structure and function in rats with experimental chronic renal disease (CKD) induced by adenine (0.25% w/w in feed for 35 days), which is known to involve inflammation and oxidative stress. Using several indices in plasma, urine and kidney homogenates, adenine was found to impair kidney function as it lowered creatinine clearance and increased plasma concentrations of creatinine, urea, neutrophil gelatinase-associated lipocalin and N-Acetyl-beta-D-glucosaminidase activity. Furthermore, it raised plasma concentrations of the uremic toxin indoxyl sulfate, some inflammatory cytokines and urinary albumin concentration. Renal morphology was severely damaged and histopathological markers of inflammation and fibrosis were especially increased. In renal homogenates, antioxidant indices, including superoxide dismutase and catalase activities, total antioxidant capacity and reduced glutathione were all adversely affected. Most of these adenine – induced actions were moderately and dose -dependently mitigated by chrysin, especially at the highest dose. Chrysin did not cause any overt adverse effect on the treated rats. The results suggest that different doses of chrysin produce variable salutary effects against adenine-induced CKD in rats, and that, pending further pharmacological and toxicological studies, its usability as a possible ameliorative agent in human CKD should be considered.     

Quantification of Heavy Metals in Mining Affected Soil and Their Bioaccumulation in Native Plant Species

Several anthropogenic and natural sources are considered as the primary sources of toxic metals in the environment. The current study investigates the level of heavy metals contamination in the flora associated with serpentine soil along the Mafic and Ultramafic rocks northern-Pakistan. Soil and wild native plant species were collected from chromites mining affected areas and analyzed for heavy metals (Cr, Ni, Fe, Mn, Co, Cu and Zn) using atomic absorption spectrometer (AAS-PEA-700). The heavy metal concentrations were significantly (p < 0.01) higher in mine affected soil as compared to reference soil, however Cr and Ni exceeded maximum allowable limit (250 and 60 mg kg^?1, respectively) set by SEPA for soil. Inter-metal correlations between soil, roots and shoots showed that the sources of contamination of heavy metals were mainly associated with chromites mining. All the plant species accumulated significantly higher concentrations of heavy metals as compared to reference plant. The open dumping of mine wastes can create serious problems (food crops and drinking water contamination with heavy metals) for local community of the study area. The native wild plant species (Nepeta cataria, Impatiens bicolor royle, Tegetis minuta) growing on mining affected sites may be used for soil reclamation contaminated with heavy metals.     

Comparison of Univariate and Multivariate Models of 13C SSNMR and XRPD Techniques for Quantification of Nimodipine Polymorphs

The focus of the present investigation was to explore the use of solid-state nuclear magnetic resonance (¹³C ssNMR) and X-ray powder diffraction (XRPD) for quantification of nimodipine polymorphs (form I and form II) crystallized in a cosolvent formulation. The cosolvent formulation composed of polyethylene glycol 400, glycerin, water, and 2.5% drug, and was stored at 5°C for the drug crystallization. The ¹³C ssNMR and XRPD data of the sample matrices containing varying percentages of nimodipine form I and form II were collected. Univariate and multivariate models were developed using the data. Least square method was used for the univariate model generation. Partial least square and principle component regressions were used for the multivariate models development. The univariate models of the ¹³C ssNMR were better than the XRPD as indicated by statistical parameters such as correlation coefficient, R², root mean square error, and standard error. On the other hand, the XRPD multivariate models were better than the ¹³C ssNMR as indicated by precision and accuracy parameters. Similar values were predicted by the univariate and multivariate models for independent samples. In conclusion, the univariate and multivariate models of ¹³C ssNMR and XRPD can be used to quantitate nimodipine polymorphs.KEY WORDS: nimodipine polymorphs, X-ray powder diffraction, solid-state nuclear magnetic resonance, univariate, multivariate     

A Network Meta-Analysis of Efficacy and Evaluation of Safety of Subcutaneous Pegylated Interferon Beta-1a versus Other Injectable Therapies for the Treatment of Relapsing-Remitting Multiple Sclerosis

Subcutaneous pegylated interferon beta-1a (peginterferon beta-1a [PEG-IFN]) 125 μg every two or four weeks has been studied in relapsing-remitting multiple sclerosis (RRMS) patients in the pivotal Phase 3 ADVANCE trial. In the absence of direct comparative evidence, a network meta-analysis (NMA) was conducted to provide an indirect assessment of the relative efficacy, safety, and tolerability of PEG-IFN versus other injectable RRMS therapies. Systematic searches were conducted in MEDLINE, Embase, and the Cochrane Library, and conference proceedings from relevant annual symposia were hand-searched. Included studies were randomized controlled trials evaluating ≥1 first-line treatments including interferon beta-1a 30, 44, and 22 μg, interferon beta-1b, and glatiramer acetate in patients with RRMS. Studies were included based on a pre-specified protocol and extracted by a team of independent reviewers and information scientists, utilizing criteria from NICE and IQWiG. In line with ADVANCE findings, NMA results support that PEG-IFN every 2 weeks significantly reduced annualized relapse rate, and 3- and 6-month confirmed disability progression (CDP) versus placebo. There was numerical trend favoring PEG-IFN every 2 weeks versus other IFNs assessed for annualized relapse rate, and versus all other injectables for 3- and 6-month CDP (6-month CDP was significantly reduced versus IFN beta-1a 30 μg). The safety and tolerability profile of PEG-IFN beta-1a 125 μg every 2 weeks was consistent with that of other evaluated treatments. Study limitations for the NMA include variant definitions of relapse and other systematic differences across trials, assumptions that populations were sufficiently similar, and inability to perform NMA of adverse events. With similar efficacy compared to other RRMS treatments in terms of annualized relapse rate and 3- and 6-month CDP, a promising safety profile, and up to 93% reduction in number of injections (which may improve adherence), PEG-IFN every 2 weeks offers a valuable alternative treatment option for patients with RRMS.