Dieldrin initiates apoptosis in rat thymocytes

In vitro incubation for 6 hr to pesticide dieldrin resulted in a dose-dependent decrease of cell viability comparable to that of dexamethasone. In vivo experiments also demonstrated that dieldrin administration induced a dose-dependent thymic atrophy which appeared to be mediated by endogenous corticosteroids. Agarose gel electrophorosis analysis, revealed the generation of typical apoptotic oligosomal DNA fragmentation in presence of dieldrin. However, in response to high concentrations of pesticide, cells seemed to undergo necrosis pathway. Thus, it may be concluded that dieldrin induced apoptosis in rat thymocytes.     

Glycogen resynthesis in the absence of food ingestion during recovery from moderate or high intensity physical activity: novel insights from rat and human studies

The finding that during recovery from high intensity exercise, rats have the capacity to replenish their muscle glycogen stores even in the absence of food intake has provided us with an experimental model of choice to explore further this process. Our objective here is to share those questions arising from research carried out by others and ourselves on rats and humans that are likely to be of interest to comparative biochemists/physiologists. On the basis of our findings and those of others, it is proposed that across vertebrate species: (1). the capacity of muscles to replenish their glycogen stores from endogenous carbon sources is dependent on the type of physical activity and animal species; (2). lactate and amino acids are the major endogenous carbon sources mobilized for the resynthesis of muscle glycogen during recovery from exercise, their relative contributions depending on the duration of recovery and type of exercise; (3). the relative contributions of lactate glyconeogenesis and hepatic/renal gluconeogenesis to muscle glycogen synthesis is species- and muscle fiber-dependent; and (4). glycogen synthase and phosphorylase play an important role in the control of the rate of glycogen synthesis post-exercise, with the role of glucose transport being species-dependent.     

Interaction of C-terminal loop 13 of sodium-glucose cotransporter SGLT1 with lipid bilayers

We have previously shown that C-terminal loop 13 of SGLT1 acts as a major binding domain for the aglucon residues of d-glucose transport inhibitors, phlorizin (Raja, M. M., Tyagi, N. K., and Kinne, R. K. H. (2003) Phlorizin Recognition in a C-terminal Fragment of SGLT1 Studied by Tryptophan Scanning and Affinity Labeling, J. Biol. Chem. 278, 49154-49163) and alkyl glucosides (Raja, M. M., Kipp, H., and Kinne, R. K. H. (2004) C-Terminus Loop 13 of Na(+) Glucose Cotransporter SGLT1 Contains a Binding Site for Alkyl Glucosides, Biochemistry 43, 10944-10951). Topology of this loop with regard to the membrane lipids is hitherto a point of debate. Here we report on in vitro incorporation studies using fluorescence of Trp mutants of loop 13 to determine the position of various parts of the loop with the lipid bilayer. Six single Trp mutants were prepared as described in previous studies (Raja et al., 2003) and subsequently incorporated into DOPC:DOPG (60:40% molar ratio) lipid vesicles. Upon addition of the phospholipids only one mutant, R601W, exhibited no change in the fluorescence intensities, position of maxima, or acrylamide accessibility. Mutants Q581W, E621W, and L630W exhibited the most pronounced blue shifts (3-6 nm) and protection against acrylamide, suggesting a position of these segments within the lipid bilayer. This assumption was confirmed by the result that the fluorescence of only these mutants was quenched by doxyl spin membrane embedded labels in the 5- or 12-positions of the acyl side chain of phospholipids. The other parts of the peptide appear to remain outside of the lipid vesicles. Trp-591 and Trp-611 showed, although to a different extent, increase in fluorescence, blue shift of maxima, and decrease in acrylamide accessibility but no interaction with the spin-labeled phospholipids. This suggests changes in the conformation of the peptide itself. These conformation changes are probably induced by the interaction of an adjacent lysine rich region of the peptide with the negatively charged DOPG, since in the absence of this lipid no incorporation of loop 13 into the bilayer is observed. Trypsin cleavage experiments of loop 13 in proteoliposomes yield a peptide containing amino acid residues 603 to 614, confirming that this part of the loop is accessible at the extravesicular face of the membranes. The studies show that at least in the in vitro system the part of loop 13 essential for the interaction with the transport inhibitors is located extracellularly, making a similar arrangement in the intact SGLT1 probable.     

Cooperative regulation of p70S6 kinase by receptor tyrosine kinases and G protein-coupled receptors augments airway smooth muscle growth

We have previously demonstrated that concomitant activation of receptor tyrosine kinases and certain G protein-coupled receptors (GPCRs) can promote a synergistic increase in the rate of airway smooth muscle cell (ASM) proliferation. Here we clarify the role of p70S6 kinase (p70S6K) as an integrator of receptor tyrosine kinase and GPCR signaling that augments ASM DNA synthesis by demonstrating that specific p70S6K phosphorylation sites receive distinct regulatory input from GPCRs that promotes sustained kinase activity critical to mitogenesis. Prolonged stimulation of ASM cells with EGF and thrombin induced a greater than additive effect in levels of p70S6K phosphorylated at residue T389, whereas a significant but more modest increase in the level of T229 and T421/S424 phosphorylation was also observed. The augmenting effects of thrombin could be dissociated from p42/p44 MAPK activation, as selective inhibition of thrombin-stimulated p42/p44 failed to alter the profile of cooperative p70S6K T389 phosphorylation, p70S6K kinase activity, or ASM [(3)H]thymidine incorporation. Thrombin stimulated a sustained increase in the level of Akt phosphorylation and also augmented EGF-stimulated Akt phosphorylation. The cooperative effects of thrombin on Akt/p70S6K phosphorylation and [(3)H]thymidine incorporation were all attenuated by heterologous expression of Gbetagamma sequestrants. These data suggest that PI3K-dependent T389/T229 phosphorylation is limiting in late-phase p70S6K activation by EGF and contributes to the cooperative effect of GPCRs on p70S6K activity and cell growth.     

Effects of protein-protein interaction in ultrafiltration based fractionation processes

This paper discusses the use of pulsed sample injection ultrafiltration (UF) for investigating protein-protein interaction, particularly its effect on protein transmission through UF membranes. Several binary protein mixtures were investigated; the proteins in each mixture being selected such that one of the proteins in the pair would be preferentially transmitted while the other would be either totally or substantially retained. The "retained" protein either decreased or increased or did not affect the sieving coefficient of the "transmitted" protein, this depending the type of protein-protein interaction, that is, associative, repulsive, or neutral. The type of protein-protein interaction depended on the particular protein pair under investigation as well as on the operating conditions used (pH and salt concentration). The magnitude of either decrease or increase in transmission of a preferentially transmitted protein due to the presence of a retained protein was found to be independent of the manner in which the proteins were injected into the system, that is, simultaneous or sequential. These magnitudes however correlated well with the ratio of the two proteins present in the feed.     

Senescence and hyperspectral reflectance of cotton leaves exposed to ultraviolet-B radiation and carbon dioxide

The objectives of this study were to determine the effects of UV-B radiation and atmospheric carbon dioxide concentrations ([CO(2)]) on leaf senescence of cotton by measuring leaf photosynthesis and chlorophyll content and to identify changes in leaf hyperspectral reflectance occurring due to senescence and UV-B radiation. Plants were grown in controlled-environment growth chambers at two [CO(2)] (360 and 720 micro mol mol(-1)) and three levels of UV-B radiation (0, 7.7 and 15.1 kJ m(-2) day(-1)). Photosynthesis, chlorophyll, carotenoids and phenolic compounds along with leaf hyperspectral reflectance were measured on three leaves aged 12, 21 and 30 days in each of the treatments. No interaction was detected between [CO(2)] and UV-B for any of the measured parameters. Significant interactions were observed between UV-B and leaf age for photosynthesis and stomatal conductance. Elevated [CO(2)] enhanced leaf photosynthesis by 32%. On exposure to 0, 7.7 and 15.1 kJ of UV-B, the photosynthetic rates of 30-day-old leaves compared with 12-day-old leaves were reduced by 52, 76 and 86%, respectively. Chlorophyll pigments were not affected by leaf age at UV-B radiation of 0 and 7.7 kJ, but UV-B of 15.1 kJ reduced the chlorophylls by 20, 60 and 80% in 12, 21 and 30-day-old leaves, respectively. The hyperspectral reflectance between 726 and 1142 nm showed interaction for UV-B radiation and leaf age. In cotton, leaf photosynthesis can be used as an indicator of leaf senescence, as it is more sensitive than photosynthetic pigments on exposure to UV-B radiation. This study revealed that, cotton leaves senesced early on exposure to UV-B radiation as indicated by leaf photosynthesis, and leaf hyperspectral reflectance can be used to detect changes caused by UV-B and leaf ageing.     

Nicotinic Receptor Inhibition by Tetraponera Ant Alkaloids

1. Tetraponerines are a group of alkaloids occurring in the venoms of ants belonging to the genus Tetraponera. Eight compounds had been isolated and their structures elucidated, but their mechanisms of action had not yet been reported. We have studied the actions of several of these tetraponerines on vertebrate neuromuscular, ganglionic, and brain nicotinic acetylcholine receptors (nAChRs) using a variety of techniques including muscle contracture, cultured cell functional assays, neuronal patch clamping, and radioligand binding methods. 2. Potency for inhibition of the frog muscle carbachol-elicited contracture increased as the carbon 9 side chain alkyl group was increased in length to 10-12 carbons, then decreased when the chain was 18-carbons long. Potency differences between T-7 and T-8, which differ only in the stereochemistry of the carbon pentyl side chain were rather small. Quaternization of either N atom in a T-8 analog bearing a 10-carbon length alkyl substituent did not greatly affect potency for inhibition of the muscle response; thus the ionized form is an active form of this tetraponerine. 3. T-7 inhibited the nicotine-stimulated efflux of 86Rb from cultured PC12 cells, which primarily express alpha3-beta4 ganglionic type nicotinic receptors. T-8 blockade of BTX-sensitive and insensitive neuronal nAChRs, as studied by patchclamp recordings from cultured rat brain neurons, was also consistent with a noncompetitive type of inhibition. 4. T-7 displaced binding of the nAChR ion channel binding ligand thienylcyclophenidyl (TCP), an analog of PCP, to Torpedo neuromuscular type receptors. The affinity of the TCP binding site for T-7 did not depend upon the desensitization state of the receptor. 5. We conclude that the tetraponerines act at a site on nAChRs different from the ACh binding site which is probably located within the ion channel.     

SNP2CAPS: a SNP and INDEL analysis tool for CAPS marker development

With the influx of various SNP genotyping assays in recent years, there has been a need for an assay that is robust, yet cost effective, and could be performed using standard gel-based procedures. In this context, CAPS markers have been shown to meet these criteria. However, converting SNPs to CAPS markers can be a difficult process if done manually. In order to address this problem, we describe a computer program, SNP2CAPS, that facilitates the computational conversion of SNP markers into CAPS markers. 413 multiple aligned sequences derived from barley ESTs were analysed for the presence of polymorphisms in 235 distinct restriction sites. 282 (90%) of 314 alignments that contain sequence variation due to SNPs and InDels revealed at least one polymorphic restriction site. After reducing the number of restriction enzymes from 235 to 10, 31% of the polymorphic sites could still be detected. In order to demonstrate the usefulness of this tool for marker development, we experimentally validated some of the results predicted by SNP2CAPS.     

Cloning, expression, and purification of the 27 kDa (MPT51, Rv3803c) protein of Mycobacterium tuberculosis

A limited number of proteins of Mycobacterium tuberculosis have been characterized so far for their use as potential candidates for diagnosis and vaccine studies. This study was aimed at cloning, expression, and purification of a 27 kDa protein (otherwise known as the MPT51 or Rv3803c protein) of M. tuberculosis. The Rv3803c gene was PCR amplified using primers that contain specific restriction sites. The amplified product was inserted initially into pTOPO and then sub-cloned into pET15b and pET24d vectors, such that the recombinant protein is predicted to contain an N-terminal or a C-terminal histidine tag, respectively. The recombinant plasmids were introduced into Escherichia coli BL21 (DE3) and the recombinant proteins were purified from the cytosolic fractions of the E. coli sonicates by nickel-NTA chromatography. The purity, molecular mass, and the conformation of the proteins were determined by high performance liquid chromatography (HPLC), matrix assisted laser desorption-ionization-time-of-flight (MALDI-TOF), and circular dichroism (CD) studies, respectively. The purified proteins were found to be immunogenic and useful for immunodiagnostic studies of tuberculosis by enzyme linked immunosorbent assay (ELISA), with a sensitivity of 71% and specificity of 95%.