Production of thermostable pullulanase by Clostridium thermosulfurogenes SV2 in solid-state fermentation: optimization of enzyme leaching conditions using response surface methodology

The optimization of parameters for the effective leaching of thermostable pullulanase from Clostridium thermosulfurogenes SV2-fermented bran was carried out using response surface methodology based on the central composite rotatable design. The design contains a total of 54 experimental trials with the first 32 organized in a fractional factorial design and experimental trials from 33-40 and 51-54 involving the replication of the central points. The design was employed by selecting solvent to wheat bran ratio (S/BB), process temperature, solvent pH, shaking (RPM) and contact time (h) as model factors. Among the five independent variables studied, the S/BB, solvent pH and shaking were found to be significant. S/BB ratio of 9.0, 200 RPM shaking and solvent pH 6.0 were identified as optimum for the leaching of thermostable pullulanase from the strain SV2-fermented bran.     

Analysis of propagation of <Emphasis Type="Italic">Bacopa monnieri</Emphasis> (L.) from hairy roots,elicitation and Bacoside A contents of Ri transformed plants

Agrobacterium rhizogenes mediated transformation has been experimented in leaf explants of the memory herb Bacopa monnieri in order to assess the regeneration potential of hairy roots (HR) followed by the elicitation of transformed plants for increased Bacoside A production. Out of the four strains tested, A4 and MTCC 532 derived HR exhibited regrowth in MS basal medium while MTCC 2364 derived HR showed regeneration in MS medium supplemented with suitable phyto hormones. R1000 derived HR possessed no regeneration potential. Comparable to A4, MTCC 532 derived HR displayed maximum regrowth frequency of about 85.71 ± 1.84 % with an increase in biomass to threefold. Therefore, five HR plant lines (MTCC 532 derived) were generated and maintained in MS basal liquid medium in which HR3 topped the others in producing a huge biomass of about 67.09 ± 0.66 g FW. PCR amplification and southern hybridization analysis of rol A gene (280 bp) has been performed in order to confirm the transformation process. Moreover, HR3 plant line has accumulated highest total phenolic content of about 165.68 ± 0.82 mg GAE/g DW and highest total flavonoid content of about 497.78 ± 0.57 mg QRE/g DW when compared to other lines and untransformed controls. In addition, HR3 plant extract showed 85.58 ± 0.14 % of DPPH (2, 2-diphenyl-1-picryl hydrazyl) inhibition displaying its reliable anti oxidant potential. Further on elicitation with 10 mg/L chitosan for 2 weeks, HR3 has produced 5.83 % of Bacoside A which is fivefold and threefold increased production when compared to untransformed and transformed unelicited controls respectively. This is the first report on eliciting HR plants for increased metabolite accumulation in B. monnieri.     

Delta12-prostaglandin D2 is a potent and selective CRTH2 receptor agonist and causes activation of human eosinophils and Th2 lymphocytes

Prostaglandin D2 (PGD2) is a lipid mediator produced by mast cells, macrophages and Th2 lymphocytes and has been detected in high concentrations in the airways of asthmatic patients. There are two receptors for PGD2, namely the D prostanoid (DP) receptor and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). The proinflammatory effects of PGD2 leading to recruitment of eosinophils and Th2 lymphocytes into inflamed tissues is thought to be predominantly due to action on CRTH2. Several PGD2 metabolites have been described as potent and selective agonists for CRTH2. In this study we have characterized the activity of delta12-PGD2, a product of PGD2 isomerization by albumin. Delta12-PGD2 induced calcium mobilization in CHO cells expressing human CRTH2 receptor, with efficacy and potency similar to those of PGD2. These effects were blocked by the TP/CRTH2 antagonist ramatroban. delta12-PGD2 bound to CRTH2 receptor with a pKi of 7.63, and a 55-fold selectivity for CRTH2 compared to DP. In Th2 lymphocytes, delta12-PGD2 induced calcium mobilization with high potency and an efficacy similar to that of PGD2. delta12-PGD2 also caused activation of eosinophils as measured by shape change. Taken together, these results show that delta12-PGD2 is a potent and selective agonist for CRTH2 receptor and can cause activation of eosinophils and Th2 lymphocytes. These data also confirm the selective effect of other PGD2 metabolites on CRTH2 and illustrate how the metabolism of PGD2 may influence the pattern of leukocyte infiltration at sites of allergic inflammation.     

Antizyme (AZ) regulates intestinal cell growth independent of polyamines

Since antizyme (AZ) is known to inhibit cell proliferation and to increase apoptosis, the question arises as to whether these effects occur independently of polyamines. Intestinal epithelial cells (IEC-6) were grown in control medium and medium containing 5 mM difluoromethylornithine (DFMO) to inhibit ODC, DFMO + 5 µM spermidine (SPD), DFMO + 5 µM spermine (SPM), or DFMO + 10 µM putrescine (PUT) for 4 days and various parameters of growth were measured along with AZ levels. Cell counts were significantly decreased and mean doubling times were significantly increased by DFMO. Putrescine restored growth in the presence of DFMO. However, both SPD and SPM when added with DFMO caused a much greater inhibition of growth than did DFMO alone, and both of these polyamines caused a dramatic increase in AZ. The addition of SPD or SPM to media containing DFMO + PUT significantly inhibited growth and caused a significant increase in AZ. IEC-6 cells transfected with AZ-siRNA grew more than twice as rapidly as either control cells or those incubated with DFMO, indicating that removal of AZ increases growth in cells in which polyamine synthesis is inhibited as well as in control cells. In a separate experiment, the addition of SPD increased AZ levels and inhibited growth of cells incubated with DFMO by 50 %. The addition of 10 mM asparagine (ASN) prevented the increase in AZ and restored growth to control levels. These results show that cell growth in the presence or absence of ODC activity and in the presence or absence of polyamines depends only on the levels of AZ. Therefore, the effects of AZ on cell growth are independent of polyamines.