Influence of putrescine on anthocyanin production in callus cultures of <Emphasis Type="Italic">Daucus carota</Emphasis> mediated through calcium ATPase

The influence of Putrescine (Put) on the growth and elicitation of anthocyanin in callus cultures of Daucus carota var. Nantes scarlet was investigated through the use of α-DL-difluoromethylarginine (DFMA), the polyamine (PA) biosynthetic inhibitor. It was observed that the addition of Put (0.05 mM) resulted in enhancement of growth and anthocyanin content. The anthocyanin content was found to be enhanced by 1.68 fold on the 21^st day as compared to the untreated controls. The PA inhibitor was found to result in lowering of the growth and the anthocyanin accumulation, which could be partially restored by the addition of Put in combination with this inhibitor. The levels of Ca²⁺ ATPase were also found to be elevated in treatment with Put suggesting the involvement of calcium in the elicitation of anthocyanin. The endogenous titres of PAs and the ethylene production under these treatments were also studied. The treatment with DFMA resulted in lower levels of endogenous PAs and higher levels of ethylene. Lowering of ethylene by putrescine treatment shows that PA treatment also inhibited ethylene formation, which would also imply that endogenous ethylene does not influence anthocyanin production in carrot callus cultures.     

Ion pairs in non-redundant protein structures

Ion pairs contribute to several functions including the activity of catalytic triads, fusion of viral membranes, stability in thermophilic proteins and solvent-protein interactions. Furthermore, they have the ability to affect the stability of protein structures and are also a part of the forces that act to hold monomers together. This paper deals with the possible ion pair combinations and networks in 25% and 90% non-redundant protein chains. Different types of ion pairs present in various secondary structural elements are analysed. The ion pairs existing between different subunits of multisubunit protein structures are also computed and the results of various analyses are presented in detail. The protein structures used in the analysis are solved using X-ray crystallography, whose resolution is better than or equal to 1.5 A and R-factor better than or equal to 20%. This study can, therefore, be useful for analyses of many protein functions. It also provides insights into the better understanding of the architecture of protein structure.     

Computation of non-covalent interactions in TNF proteins and interleukins

The roles played by the non-covalent interactions have been investigated for a set of six TNF proteins and nine Interleukins. The stabilizing residues have been identified by a consensus approach using the concepts of available surface area, medium and long-range interactions and conservation of amino acid residues. The cation-pi interactions have been computed based on a geometric approach such as distance and energy criteria. We identified an average of 1 energetically significant cation-pi interactions in every 94 residues in TNF proteins and 1 in every 62 residues in Interleukins. In TNF proteins, the cationic groups Lys preferred to be in helix while Arg preferred to be in strand regions while in Interleukins the Arg residues preferred to be in helix and Lys preferred to be in strand regions. From the available surface area calculations, we found that, almost all the cation and pi residues in TNF proteins and Interleukins were either in buried or partially buried regions and none of them in the exposed regions. Medium and long-range interactions were predominant in both TNF proteins and Interleukins. It was observed that the percentage of stabilizing centers were more in TNF proteins as compared to the Interleukins, while the percentage of conserved residues were more in Interleukins than in TNF proteins. In the stabilizing residues Lys was observed to be a stabilizing residue in both TNF proteins and Interleukins. Among the aromatic group, Phe was seen to be a stabilizing residue in both TNF and Interleukins. We suggest that this study on the computation of cation-pi interactions in TNF proteins and Interleukins would be very helpful in further understanding the structure, stability and functional similarity of these proteins.     

Arginine metabolic pathways determine its therapeutic benefit in experimental heatstroke: Role of Th1/Th2 cytokine balance

We have demonstrated that therapeutic administration of L-arginine (L-arg) (120 mg/kg) at +2 h of whole body hyperthermia (WBH) could rescue the mice from heatstroke-induced death. Studies were undertaken to elucidate the role of L-arg in the immunomodulation of the heat-stressed mice. Administration of L-arginine (L-arg), (120 mg/kg, i.p.), at +2 h of WBH, rescued the mice from heat-induced death and reduced the hypothermia. At +4 and +24 h of WBH, levels of IL-1beta, IFN-gamma, nitrite, TNF-alpha, IL-4, TGF-beta1, inducible form of nitric oxide synthase (iNOS), and corticosterone significantly increased compared to the sham group. The elevated levels of Th(1) cytokines, namely TNF-alpha, IL-1beta, IFN-gamma, nitrite, and iNOS, decreased significantly both at +4 and +24 h of WBH, following L-arg administration. However, L-arg administration did not reduce the increased levels of Th(2) cytokines, namely IL-4 and TGF-beta1, in WBH mice at +4 h of WBH. L-arg administration significantly increased the levels of Th(2) cytokines at +24 h of WBH, compared to the saline-treated WBH mice. L-arg administration significantly increased both the splenic and hepatic arginase activity at +4 and +24 h of WBH compared to the saline-treated WBH mice. L-NAME treatment at +2 h of WBH and anti-TGF-beta antibody treatment at 0 h of WBH significantly increased the mortality compared to the saline-treated WBH mice. Altered liver histopathology was attenuated following the administration of L-arg at +2 h of WBH. These results suggest that therapeutic administration of L-arg at appropriate concentration and time attenuates the acute inflammatory response, leading to the rescue of mice from heatstroke.     

Discovery of novel 5-(ethyl or hydroxymethyl) analogs of 2'-'up' fluoro (or hydroxyl) pyrimidine nucleosides as a new class of Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium avium inhibitors

Discovery of novel antimycobacterial compounds that work on distinctive targets and by diverse mechanisms of action is urgently required for the treatment of mycobacterial infections due to the emerging global health threat of tuberculosis. We have identified a new class of 5-ethyl or hydroxy (or methoxy) methyl-substituted pyrimidine nucleosides as potent inhibitors of Mycobacterium bovis, Mycobacterium tuberculosis (H37Ra, H37Rv) and Mycobacterium avium. A series of 2'-'up' fluoro (or hydroxy) nucleosides (1, 2, 4-6, 9, 10, 13, 16, 18, 21, 24) was synthesized and evaluated for antimycobacterial activity. Among 2'-fluorinated compounds, 1-(3-bromo-2,3-dideoxy-2-fluoro-β-d-arabinofuranosyl)-5-ethyluracil (13) exhibited promising activity against M. bovis and Mtb alone, and showed synergism when combined with isoniazid. The most active compound emerging from these studies, 1-(β-d-arabinofuranosyl)-4-thio-5-hydroxymethyluracil (21) inhibited Mtb (H37Ra) (MIC(50)=0.5 μg/mL) and M. bovis (MIC(50)=0.5 μg/mL) at low concentrations, and was ten times more potent against Mtb (H37Ra) than cycloserine (MIC(50)=5.0 μg/mL), a second line drug. It also showed an additive effect when combined with isoniazid. Compound 21 retained sensitivity against a rifampicin-resistant (H37Rv) strain of Mtb (MIC(50)=1 μg/mL) at concentrations similar to that for a rifampicin-sensitive (H37Rv) strain, suggesting that it has no cross-resistance to a first-line anti-TB drug. In addition, the replication of M. avium was also inhibited by 21 (MIC(50)=10 μg/mL). No cellular toxicity of 13 or 21 was observed up to the highest concentration tested (CC(50)>100 μg/mL). These observations offer promise for a new drug treatment regimen to augment and complement the current chemotherapy of TB.     

Production and characterization of pharmacologically active recombinant human phosphodiesterase 4B in Dictyostelium discoideum

Phosphodiesterase 4B (PDE4B) is an important therapeutic target for asthma and chronic obstructive pulmonary disease. To identify PDE4 subtype-specific compounds using high-throughput assays, full-length recombinant PDE4 proteins are needed in bulk quantity. In the present study, full-length human PDE4B2 was expressed in the cellular slime mould Dictyostelium discoideum (Dd). A cell density of 2 x 10(7) cells/mL was obtained and up to 1 mg/L recombinant PDE4B2 was purified through Ni-NTA affinity chromatography. The expressed protein was soluble and its activity was comparable to PDE4B2 protein expressed in mammalian cells (K(m)=1.7 microM). The functional significance of the Dd expression system is supported by the demonstration that, in concert with proteins expressed in mammalian systems, there are no major changes in the affinity for PDE4B2 inhibitors and substrates. These findings thus provide the first evidence that Dd can be utilized for the expression and purification of functionally active full-length human PDE4B2 in large amounts required for high-throughput screening of pharmacologically active compounds against this therapeutic target.