Structure and function analysis of CaMdr1p, a major facilitator superfamily antifungal efflux transporter protein of Candida albicans: identification of amino acid residues critical for drug/H+ transport

We have cloned and overexpressed multidrug transporter CaMdr1p as a green fluorescent protein-tagged protein to show its capability to extrude drug substrates. The drug extrusion was sensitive to pH and energy inhibitors and displayed selective substrate specificity. CaMdr1p has a unique and conserved antiporter motif, also called motif C [G(X6)G(X3)GP(X2)GP(X2)G], in its transmembrane segment 5 (TMS 5). Alanine scanning of all the amino acids of the TMS 5 by site-directed mutagenesis highlighted the importance of the motif, as well as that of other residues of TMS 5, in drug transport. The mutant variants of TMS 5 were placed in four different categories. The first category had four residues, G244, G251, G255, and G259, which are part of the conserved motif C, and their substitution with alanine resulted in increased sensitivity to drugs and displayed impaired efflux of drugs. Interestingly, first category mutants, when replaced with leucine, resulted in more dramatic loss of drug resistance and efflux. Notwithstanding the location in the core motif, the second category included residues which are part of the motif, such as P260, and those which were not part of the motif, such as L245, W248, P256, and F262, whose substitution with alanine resulted in a severe loss of drug resistance and efflux. The third category included G263, which is a part of motif C, but unlike other conserved glycines, its replacement with alanine or leucine showed no change in the phenotype. The replacement of the remaining 11 residues of the fourth category did not result in any change. The putative helical wheel projection showed clustering of functionally critical residues to one side and thus suggests an asymmetric nature of TMS 5.     

Conserved cysteine variants of metagenomic derived polygalacturonase concurrently shift its optima at acidic pH and enhanced thermostability: structural and functional analysis

To study the effect of conserved cysteins on biochemical properties of a previously cloned metagenomic polygalacturonase (PecJKR01), single point variants A42C, M283C, and double variants M283C + F24C, M283C + A42C were constructed. Mutations resulted in shifting the pH toward lower range and enhanced thermostability. The mutants were optimally active at pH 5.0 as compared to pH 7.0 for wild type. Point variants demonstrated slightly higher enzyme activity at 60^o C than that of the wild type. In addition, the A42C/M283C + A42C variants displayed nearly 28–40% enhanced thermostability, while M283C + 24C was least thermostable among all variants/ wild type. Cys (pKa 8.18) possibly interfered in the ionization state resulting in change in pH optima of variants. Structure function analysis suggested that the increased activity in A42C could be due to van der Waals interactions in S···Ar with Phe29 and formation of an additional hydrogen bond between Cys42-S....HN-Ala31. Higher thermostability and decreased enzymatic activity of M283C might be attributed to the incorporation of additional disulfide linkage between Cys283 S=S Cys255 and decreased cavity size. Overall cysteine at position 42 was most promising in shifting the optimum pH toward lower range as well as for thermostability of enzyme.     

Effect of lithium on thyroidal 131iodine uptake, its clearance, and circulating levels of triiodothyronine and thyroxine in lead-treated rats

The influence of lead acetate (50 mg per kg body weight) on the 131iodine (131I) biokinetics (uptake and retention) in rat thyroid and serum levels of triiodothyronine (T3) as well as thyroxine (T4) was evaluated as a function of time and in combination with lithium treatment. The 2-h and 24-h uptake of 131I in the thyroid was stimulated significantly by lead treatment. The 24-h uptake showed a maximum stimulation after 4 months of lead treatment. Lithium supplementation, however, showed the opposite effect by reducing the iodine uptake whereby the maximum decrease was noticed after 2 months of treatment. Further, simultaneous lead and lithium treatment resulted in an even more pronounced increase of 2-h 131I uptake with a maximum after 3 months. However, the 24-h uptake after 3 months and 4 months of treatment did not differ significantly from the lead treated reference groups. The thyroidal biological half-life of 131I (Tbiol) was found to have clearly increased following the lead/lithium treatment. Interestingly, the combined lead/lithium treatment applied for 4 months caused a further growth of Tbiol, thus reflecting an increased retention of 131I. A maximum increase of Tbiol was seen after 2 months of combined treatment. A progressive decline of the circulating T3 and T4 levels following lead or lithium treatment was noticed and was more pronounced after combined treatment.     

Resistance to oxidative stress in a freshwater fish Channa punctatus after exposure to inorganic arsenic

The biochemical toxicity of arsenic trioxide (As^III) in a freshwater edible fish Channa punctatus has been studied on exposures ranging from 7 to 90 d. The arsenic concentration increased exponentially in liver, kidney, gills, and muscles of fish up to 60 d of exposure to arsenic. However, arsenic concentration in these tissues declined at 90 d of exposure. This relationship between period of exposure and concentration of arsenic in selected tissues suggests an adaptive response of fish to arsenic. Furthermore, exposure to arsenic-induced lipid peroxidation in these organs increased initially at 7 d of exposure; however, it decreased up to 60 d of exposure but increased again at 90 d of treatment. Values of reduced glutathione (GSH) reflected the observations of lipid peroxidation. The role of GSH in this adaptive response has been discussed.     

Role of zinc in regulating the levels of hepatic elements following nickel toxicity in rats

This study was designed to determine the protective effects of zinc on the hepatotoxicity induced by nickel in rats. Female Sprague-Dawley (SD) rats received either nickel sulfate alone in the dose of 800 mg/L nickel in drinking water, zinc sulfate alone in the dose of 227 mg/L zinc in drinking water, and nickel plus zinc or drinking water alone for a total duration of 8 wk. The effects of different treatments were studied on activities of rat liver marker enzymes like alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferases (AST) and on the status of essential elements in rat liver. The study revealed a significant increase in the activities of enzymes ALP and ALT in rats subjected to nickel treatment. Interestingly, zinc supplementation to rats treated with nickel brought back the raised activities of these enzymes to within normal limits. Further, the levels of elements in liver that include zinc, copper, selenium, and potassium were found to be significantly suppressed following nickel treatment, whereas the levels of iron and sulfur were elevated. However, zinc treatment alone did not cause any appreciable change in the concentration of these elements. To the contrary, when zinc was given to nickel-treated rats, the concentrations of zinc, copper, potassium, and phosphorus were not significantly different from that of normal controls, whereas the levels of iron, selenium, and sulfur were improved in comparison to nickel-treated rats but were not within the normal limits. The present study concludes that zinc has the ability to maintain the levels of hepatic elements and has bearing in regulating the liver functions by maintaining the activities of marker enzymes in conditions of nickel toxicity.     

Mass transfer studies of cell permeabilization and recovery of alkaline phosphatase from Escherichia coli by reverse micellar solutions

Transfer of alkaline phosphatase (AP) directly from Escherichia coli cells into reverse micellar solutions (RMS) was studied by varying the water content, pH, and ionic strength of RMS. Prior to the mass transfer studies, the optimum conditions for the activity of alkaline phosphatase in reverse micellar solutions were determined. The maximum enzyme activity could be detected at higher pH and water content, indicating the ionization of p-nitrophenol to be crucial in the enzyme activity. The transfer of AP from E. coli followed a two-step process with most of the recovery being achieved in the first 3-10 min beyond which it slowed. A model suggesting separate locations for the enzyme in the cell wall has been proposed.     

Evaluation of mucoadhesive properties of chitosan microspheres prepared by different methods

The mucoadhesive properties of chitosan microspheres prepared by different method were evaluated by studying the interaction between mucin and microspheres in aqueous solution. The interaction was determined by the measurement of mucin adsorbed on the microspheres. A strong interaction between chitosan microspheres and mucin was detected. The intensity of the interaction was dependent upon the method of preparation of chitosan microspheres and the amount of mucin added. The extent of mucus adsorption was proportional to the absolute values of the positive zeta potential of chitosan microspheres. The zeta potential in turn was found to be dependent upon the method of preparation of microspheres. The adsorption of type III mucin (1% sialic acid content) was interpreted using Freundlich or Langmuir adsorption isotherms. The values ofr ² were greater for Langmuir isotherm as compared with Freundlich isotherm. The adsorption of a suspension of chitosan microspheres in the rat small intestine indicated that chitosan microspheres prepared by tripolyphosphate cross-linking and emulsification ionotropic gelation can be used as an excellent mucoadhesive delivery system. The microspheres prepared by glutaraldehyde and thermal cross-linking showed good stability in HC1 as compared with microspheres prepared by tripolyphosphate and emulsification ionotropic gelation.     

Sodium channel modulating activity in a delta-conotoxin from an Indian marine snail

A 26 residue peptide (Am 2766) with the sequence CKQAGESCDIFSQNCCVG-TCAFICIE-NH(2) has been isolated and purified from the venom of the molluscivorous snail, Conus amadis, collected off the southeastern coast of India. Chemical modification and mass spectrometric studies establish that Am 2766 has three disulfide bridges. C-terminal amidation has been demonstrated by mass measurements on the C-terminal fragments obtained by proteolysis. Sequence alignments establish that Am 2766 belongs to the delta-conotoxin family. Am 2766 inhibits the decay of the sodium current in brain rNav1.2a voltage-gated Na(+) channel, stably expressed in Chinese hamster ovary cells. Unlike delta-conotoxins have previously been isolated from molluscivorous snails, Am 2766 inhibits inactivation of mammalian sodium channels.