A simple and rapid liquid chromatography method for simultaneous determination of zidovudine and nevirapine in plasma

We describe a simple, fast, isocratic, reversed-phase high performance liquid chromatographic method for simultaneous determination of plasma zidovudine and nevirapine with UV detection at 260 nm. The method involves liquid-liquid extraction with ethyl acetate and using 3-isobutyl 1-methyl xanthine as internal standard. The system requires a C(18) column (150 mm x 4.6 mm I.D.) and a mobile phase composed of potassium dihydrogen phosphate (15 mM; pH 7.5) and acetonitrile in the ratio of 80:20 (v/v). The assay was linear from 0.025 to 10.0 microg/ml for zidovudine and 0.05 to 10.0 microg/ml for nevirapine. The intra- and inter-day variations were less than 10% for both the drugs. The method was specific and sensitive enough to allow quantification of zidovudine and nevirapine in concentrations observed clinically. The average recoveries of zidovudine and nevirapine from plasma were 95 and 94%, respectively. The method was applied to a pharmacokinetic study in HIV-infected patients who were receiving antiretroviral treatment with zidovudine and nevirapine containing regimens. The method spans the blood concentration range of clinical interest. Due to its simplicity, the assay can be used for pharmacokinetic studies and therapeutic drug monitoring in patients taking a combination treatment of zidovudine and nevirapine.     

Simple and rapid liquid chromatography method for determination of efavirenz in plasma

A simple and rapid high performance liquid chromatographic method for determination of efavirenz in human plasma was developed. The method involved extraction of sample with ethyl acetate and analysis using a reversed-phase C(18) column (150 mm) with UV detection. The assay was linear from 0.0625 to 10.0 microg/ml. The method was specific for efavirenz estimation and the drug was stable in plasma up to one month at -20 degrees C. The average recovery of efavirenz from plasma was 101%. Due to its simplicity, the assay can be used for pharmacokinetic studies and therapeutic drug monitoring of efavirenz.     

Water-assisted dual mode cofactor recognition by HhaI DNA methyltransferase.

Energetically competent binary recognition of the cofactor S-adenosyl-L-methionine (AdoMet) and the product S-adenosyl-L-homocysteine (AdoHcy) by the DNA (cytosine C-5) methyltransferase (M.HhaI) is demonstrated herein. Titration calorimetry reveals a dual mode, involving a primary dominant exothermic reaction followed by a weaker endothermic one, for the recognition of AdoMet and AdoHcy by M.HhaI. Conservation of the bimodal recognition in W41I and W41Y mutants of M.HhaI excludes the cation-pi interaction between the methylsulfonium group of AdoMet and the pi face of the Trp(41) indole ring from a role in its origin. Small magnitude of temperature-independent heat capacity changes upon AdoMet or AdoHcy binding by M.HhaI preclude appreciable conformational alterations in the reacting species. Coupled osmotic-calorimetric analyses of AdoMet and AdoHcy binding by M.HhaI indicate that a net uptake of nearly eight and 10 water molecules, respectively, assists their primary recognition. A change in water activity at constant temperature and pH is sufficient to engender and conserve enthalpy-entropy compensation, consistent with a true osmotic effect. The results implicate solvent reorganization in providing the major contribution to the origin of this isoequilibrium phenomenon in AdoMet and AdoHcy recognition by M.HhaI. The observations provide unequivocal evidence for the binding of AdoMet as well as AdoHcy to M.HhaI in solution state. Isotope partitioning analysis and preincubation studies favor a random mechanism for M.HhaI-catalyzed reaction. Taken together, the results clearly resolve the issue of cofactor recognition by free M.HhaI, specifically in the absence of DNA, leading to the formation of an energetically and catalytically competent binary complex.     

CARd-3D: Carbon Distribution in 3D Structure Program for Globular Proteins

Spatial arrangement of carbon in protein structure is analyzed here. Particularly, the carbon fractions around individual atoms are compared. It is hoped that it follows the principle of 31.45% carbon around individual atoms. The results reveal that globular protein''s atoms follow this principle. A comparative study on monomer versus dimer reveal that carbon is better distributed in dimeric form than in its monomeric form. Similar study on solid versus liquid structures reveals that the liquid (NMR) structure has better carbon distribution over the corresponding solid (X-Ray) structure. The carbon fraction distributions in fiber and toxin protein are compared. Fiber proteins follow the principle of carbon fraction distribution. At the same time it has another broad spectrum of carbon distribution than in globular proteins. The toxin protein follows an abnormal carbon fraction distribution. The carbon fraction distribution plays an important role in deciding the structure and shape of proteins. It is hoped to help in understanding the protein folding and function.     

The FAK–Arp2/3 interaction promotes leading edge advance and haptosensing by coupling nascent adhesions to lamellipodia actin

Cell migration is initiated in response to biochemical or physical cues in the environment that promote actin-mediated lamellipodial protrusion followed by the formation of nascent integrin adhesions (NAs) within the protrusion to drive leading edge advance. Although FAK is known to be required for cell migration through effects on focal adhesions, its role in NA formation and lamellipodial dynamics is unclear. Live-cell microscopy of FAK^−/− cells with expression of phosphorylation deficient or a FERM-domain mutant deficient in Arp2/3 binding revealed a requirement for FAK in promoting the dense formation, transient stabilization, and timely turnover of NA within lamellipodia to couple actin-driven protrusion to adhesion and advance of the leading edge. Phosphorylation on Y397 of FAK promotes dense NA formation but is dispensable for transient NA stabilization and leading edge advance. In contrast, transient NA stabilization and advance of the cell edge requires FAK–Arp2/3 interaction, which promotes Arp2/3 localization to NA and reduces FAK activity. Haptosensing of extracellular matrix (ECM) concentration during migration requires the interaction between FAK and Arp2/3, whereas FAK phosphorylation modulates mechanosensing of ECM stiffness during spreading. Taken together, our results show that mechanistically separable functions of FAK in NA are required for cells to distinguish distinct properties of their environment during migration.     

Continuous Biosorption of Pb,Cu, and Cd by Phanerochaete chrysosporium in a Packed Column Reactor

The dynamic removal of lead, copper and cadmium in a single component system by Phanerochaete chrysosporium was studied in packed columns. The packed columns consisted of biomass of P. chrysosporium immobilized on polyurethane foam cubes. The performances of packed columns were described through the concept of breakthrough and the values of column parameters predicted as a function of bed depth. The column biosorption data were evaluated in terms of maximum (equilibrium) capacity of the column, the amount of metal loading and the yield of the process. The maximum capacities for lead, copper and cadmium were 70.7, 43.7 and 70.8 mg, respectively, and their yields were 39.2, 40.6 and 41%, respectively. The kinetic and mass transfer aspects of the dynamic removal of the three metals were studied using three mathematical models commonly used to describe the column performance in adsorption processes. Column studies showed good agreement between the experimental data and the simulated breakthrough curves obtained with Adams-Bohart or the Wolborska model and the Clark model. While the initial segment of the breakthrough curve was defined by the Adams-Bohart and Wolborska models, the whole breakthrough curve was well predicted by the Clark model for all the three metals studied.     

An interdependent network of functional enhancers regulates transcription and EZH2 loading at the INK4a/ARF locus

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