Cold tolerance and plant growth promotion potential of Serratia marcescens strain SRM (MTCC 8708) isolated from flowers of summer squash (Cucurbita pepo)

Aim: To determine the cold tolerance and plant growth promotion potential of Serratia marcescens strain SRM (MTCC 8708). Methods and Results: Serratia marcescens strain SRM was isolated from the flowers of summer squash plants, showing no apparent symptoms of yellow vine disease. It was evaluated for growth and plant growth promotion attributes at 15 and 4°C. At 15°C, the isolate was able to solubilize 76·6 μg ml^?1 of P and produce Indole Acetic Acid, IAA (11·1 μg ml^?1). HCN and siderophore production were also detected at 15°C. The isolate retained all the plant growth promotion traits at 4°C. Seed bacterization with the isolate significantly enhanced plant biomass and nutrient uptake of wheat seedlings grown in cold temperatures. Conclusion: Serratia marcescens strain SRM is a promising cold‐tolerant isolate that can significantly influence wheat seedling growth at cold temperatures. Significance and Impact of the Study: This strain can be employed as a bioinoculant in cold temperature conditions.     

MicroRNA-126 regulates HOXA9 by binding to the homeobox

The PicTar program predicted that microRNA-126 (miR-126), miR-145, and let-7s target highly conserved sites within the Hoxa9 homeobox. There are increased nucleotide constraints in the three microRNA seed sites among Hoxa9 genes beyond that required to maintain protein identity, suggesting additional functional conservation. In preliminary experiments, forced expression of these microRNAs in Hoxa9-immortalized bone marrow cells downregulated the HOXA9 protein and caused loss of biological activity. The microRNAs were shown to target their predicted sites within the homeobox. miR-126 and Hoxa9 mRNA are coexpressed in hematopoietic stem cells and downregulated in parallel during progenitor cell differentiation; however, miR-145 is barely detectable in hematopoietic cells, and let-7s are highly expressed in bone marrow progenitors, suggesting that miR-126 may function in normal hematopoietic cells to modulate HOXA9 protein. In support of this hypothesis, expression of miR-126 alone in MLL-ENL-immortalized bone marrow cells decreased endogenous HOXA9 protein, while inhibition of endogenous miR-126 increased expression of HOXA9 in F9 cells.     

Renaturation, purification and characterization of streptokinase expressed as inclusion body in recombinant E. coli

Recombinant protein purification is facilitated using high expression systems which produce larger quantities of streptokinase protein as inclusion bodies. As the accumulation of active streptokinase is toxic to the host cells, we have optimized the conditions to achieve large amounts of streptokinase in the form of inclusion bodies. The solubility and yield of pure protein are highly dependent on various combinations of chemical additives, ionic and non-ionic detergents and salts, with solubilizing agents followed by refolding of denatured protein into active form. As the extraction of the purified streptokinase from inclusion bodies requires denaturation and a subsequent refolding step, careful balancing steps were needed to develop under different controlled conditions. Here the purified fragments of refolded proteins were screened to select the conditions that yield the active streptokinase having native conformation. The maximum specific activity of the purified streptokinase was achieved by these methods. The refolded recombinant streptokinase was analyzed by RP-HPLC showing a purity of 99%. Size exclusion chromatography profile shows that there are minimal aggregates in the active streptokinase protein and the percentage of renaturation is around 99%.     

UV-light exposed prion protein fails to form amyloid fibrils

Amyloid fibril formation involves three steps; structural perturbation, nucleation and elongation. We have investigated amyloidogenesis using prion protein as a model system and UV-light as a structural perturbant. We find that UV-exposed prion protein fails to form amyloid fibrils. Interestingly, if provided with pre-formed fibrils as seeds, UV-exposed prion protein formed amyloid fibrils albeit with slightly different morphology. Atomic force microscopy and electron microscopic studies clearly show the formation of fibrils under these conditions. Circular dichroism study shows loss in helicity in UV-exposed protein. UV-exposed prion protein fails to form amyloid fibrils. However, it remains competent for fibril extension, suggesting that UV-exposure results in loss of nucleating capability. This work opens up possibility of segregating nucleation and elongation step of amyloidogenesis, facilitating screening of new drug candidates for specifically inhibiting either of these processes. In addition, the work also highlights the importance of light-induced structural and functional alterations which are important in protein based therapeutics.     

High doses of dietary zinc induce cytokines, chemokines, and apoptosis in reproductive tissues during regression

In chickens, high levels of dietary zinc cause molting, and the reproductive system undergoes complete remodeling concomitant to feather replacement. In the present study, the expression profiles of cytokines and chemokines were investigated in the ovary and oviduct of control hens and of hens induced to molt by zinc feeding. The zinc-induced feed-intake suppression, the changes in corticosterone levels, the immune cell populations in the reproductive tract, and the apoptosis of reproductive tissues were analyzed. The expression of mRNAs for interleukin-6 (IL-6), interferon-γ (IFN-γ), the avian ortholog of mammalian IL-8 (chCXCLi2), and a chicken MIP-1β-like chemokine (chCCLi2) in the ovary and of mRNAs for IL-1β, IL-6, IFN-γ, transforming growth factor-β2, chCXCLi2, and chCCLi2 in the oviduct were upregulated significantly during zinc-induced molting. A simultaneous feed-intake reduction was observed with higher expression of cytokines and chemokines. The results of the present investigation also suggested that the upregulation of corticosterone was closely associated with the increased expression of cytokines and chemokines. An increase in apoptosis within reproductive tissue during tissue regression was also noted. We had previously observed the upregulation of these cytokines expression in an earlier study (molting by feed withdrawal). However, the pattern and the level of expression were different among these two methods. These findings indicate that cytokines might be a common mediator of tissue regression during molting induced by diverse methods, although the pattern of induction is different. Thus, a high dose of dietary zinc seems to induce reproductive regression via the upregulation of cytokines and chemokines, the suppression of feed intake, and the increase in serum corticosterone, resulting finally in the apoptosis of reproductive tissues.     

pH dependent unfolding characteristics of DLC8 dimer: Residue level details from NMR

Environment dependence of folding and unfolding of a protein is central to its function. In the same vein, knowledge of pH dependence of stability and folding/unfolding is crucial for many biophysical equilibrium and kinetic studies designed to understand protein folding mechanisms. In the present study we investigated the guanidine induced unfolding transition of dynein light chain protein (DLC8), a cargo adaptor of the dynein complex in the pH range 7-10. It is observed that while the protein remains a dimer in the entire pH range, its stability is somewhat reduced at alkaline pH. Global unfolding features monitored using fluorescence spectroscopy revealed that the unfolding transition of DLC8 at pH 7 is best described by a three-state model, whereas, that at pH 10 is best described by a two-state model. Chemical shift perturbations due to pH change provided insights into the corresponding residue level structural perturbations in the DLC8 dimer. Likewise, backbone (15)N relaxation measurements threw light on the corresponding motional changes in the dimeric protein. These observations have been rationalized on the basis of expected changes with increasing pH in the protonation states of the titratable residues on the structure of the protein. These, in turn provide an explanation for the change from three-state to two-state guanidine induced unfolding transition as the pH is increased from 7 to 10. All these results exemplify and highlight the role of environment vis-à-vis the sequence and structure of a given protein in dictating its folding/unfolding characteristics.     

Orally efficacious thrombin inhibitors with cyanofluorophenylacetamide as the P2 motif

2-Cyano-6-fluorophenylacetamide was explored as a novel P2 scaffold in the design of thrombin inhibitors. Optimization around this structural motif culminated in 14, which is a potent thrombin inhibitor (K_i = 1.2 nM) that exhibits robust efficacy in canine anticoagulation and thrombosis models upon oral administration.     

Biotransformation of Externally Added Vanillin Related Compounds by Multiple Shoot Cultures of Vanilla planifolia L

The multiple shoots and callus cultures of Vanilla planifolia obtained from the nodal explant on MS medium supplemented with 6-benzylaminopurine (BAP) 2 mg l^?1 and α-naphthalene acetic acid (NAA) 2 mg l^?1 were maintained by regular subculturing every 30 days and also cultured liquid MS medium of the same hormonal combination. Shoots were transferred to the MS basal medium for rooting. Different explants along with vanilla pods and in vitro cultures were analyzed using HPLC for the presence of vanillin and related compounds. When the amount of these compounds was determined in explants and in in vitro cultures after precursor feeding and curing process, explants showed different profile after precursor feeding and after undergoing curing process. During further investigations we have applied a novel approach for curing in vitro tissues as done for vanilla beans. Curing of in vitro shoots resulted in a significant change in the aromatic compound profile.