K.M. Gothandam

OsPPR1, a pentatricopeptide repeat protein of rice is essential for the chloroplast biogenesis

In this paper, we report a novel pentatricopeptide repeat (PPR) protein gene in rice. PPR, a characteristic repeat motif consisted of tandem 35 amino acids, has been found in various biological systems including plant. Sequence analysis revealed that the gene designated OsPPR1 consisted of an open reading frame of 2433 nucleotides encoding 810 amino acids that include 11 PPR motifs. Blast search result indicated that the gene did not align with any of the characterized PPR genes in plant. The OsPPR1 gene was found to contain a putative chloroplast transit peptide in the N-terminal region, suggesting that the gene product targets to the chloroplast. Southern blot hybridization indicated that the OsPPR1 is the member of a gene family within the rice genome. Expression analysis and immunoblot analysis suggested that the OsPPR1 was accumulated mainly in rice leaf. Antisense transgenic strategy was used to suppress the expression of OsPPR1 and the resulted transgenic rice showed the typical phenotypes of chlorophyll-deficient mutants; albinism and lethality. Cytological observation using microscopy revealed that the antisense transgenic plant contained a significant defect in the chloroplast development. Taken together, the results suggest that the OsPPR1 is a nuclear gene of rice, encoding the PPR protein that might play a role in the chloroplast biogenesis. This is the first report on the PPR protein required for the chloroplast biogenesis in rice.

BcXTH1, a Brassica campestris homologue of Arabidopsis XTH9, is associated with cell expansion

Xyloglucan endotransglucosylase/hydrolases (XTHs) are a group of the enzymes that are responsible for reorganization of the cellulose–xyloglucan framework by catalyzing cleavage and religation of the xyloglucan chains in the plant cell wall. In this study, we report the isolation and characterization of a XTH gene from a pistil cDNA library of Brassica campestris. Sequence analysis of the gene, designated BcXTH1, revealed that it is homologous to the XTH9 gene of Arabidopsis. The highly conserved domain (DEIDFEFLG) found among all XTHs was also present in BcXTH1 but with the two amino acid substitutions (NEFDFEFLG) also found in Arabidopsis XTH9. These results suggest that BcXTH1 is the B. campestris homologue of XTH9. Expression analysis of BcXTH1 revealed that it was expressed in most of the plant organs. In situ hybridization showed that the gene is highly expressed in the floral primodia, especially in the epidermal cell layer. Southern blot analysis indicated that the BcXTH1 gene exists as a multi-copy gene in the B. campestris genome. The function of the BcXTH1 gene was deduced from using an overexpression strategy in Arabidopsis. Interestingly, the transgenic plants showed a pronounced cell expansion phenotype. Immunoelectron microscopy shows that BcXTH1 is localized almost exclusively to the cell wall, supporting our conclusion that it participates in the regulation of cell expansion in B. campestris.

Ultrastructural study of rice tapetum under low-temperature stress

The development of male reproductive organs in rice is very sensitive to various environmental stresses. For example, exposing plants to low temperatures during the heading stage leads to a reduction in grain yield. Here, we grew rice under normal conditions and also at three different temperatures -- 16, 18, and 20°C. Treatment at a low temperature significantly decreased pollen viability and grain production. Cytological observations of the anther showed that the tapetum was the most sensitive to low-temperature stress, resulting in male sterility due to functional loss of the tissue. Detailed observations by transmission electron microscopy suggested that this abnormality was restricted primarily to the ER structures. The endoplasmic reticulum, a highly vulnerable sub-cellular organelle, showed two typical morphological aberrations, one in its pattern of arrangement, the other in the formation of ER bodies. Of our three experimental chilling temperatures, the most severe abnormalities were observed in tapetal cells exposed to 16°C.

Hepatoprotective effect of polyphenols rich methanolic extract of Amorphophallus commutatus var. wayanadensis against CCl4 induced hepatic injury in swiss albino mice.

The present study is to evaluate the hepatoprotective and antioxidant activity of methanolic extract of Amorphophallus commutatus var. wayanadensis against carbon tetrachloride induced hepatotoxicity in mice models. Hepatic injury was induced by injecting 0.2% CCl4 in olive oil intra peritoneally on 15th day of drug administration. Hepatoprotective activity was evaluated by estimating the levels of serum markers like alanine aminotransferase (ALT) and aspartate aminotransferase (AST), alkaline phosphatase (ALP), bilirubin and histopathological studies. Antioxidant potential of the extract was estimated by measuring the levels of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione-s-transferase (GST), glutathione peroxidase (GPx) and total reduced glutathione in the liver samples. Histopathological and biochemical results elicited the methanolic extract of A. commutatus has significant hepatoprotective activity comparable to the standard silymarin. The extract also showed dose dependent increase of antioxidant enzymes in CCl4 induced hepatotoxicity models. The methanolic extract of A. commutatus showed significant hepatoprotective and antioxidant activity which might be attributed due to the polyphenolic compounds present in the extract.

Immunomodulatory activity of methanolic extract of Amorphophallus commutatus var. wayanadensis under normal and cyclophosphamide induced immunosuppressive conditions in mice models.

The present study investigates the immunomodulatory activity of methanolic extract of Amorphophallus commutatus var. wayanadensis (MEAC) under normal and cyclophosphamide induced immunosuppressive conditions in Swiss albino mice models. The splenocyte proliferation assay was performed to study in-vitro immunomodulatory activity of MEAC, where sheep RBC (SRBC) was used to induce immune responses in the experimental animals. The in-vivo immunomodulatory activity was evaluated by humoral antibody titer, quantification of plaque forming cells, qualitative hemolysis, delayed type hypersensitivity assay, phagocytic index and neutrophil adhesion assays. The chemoprotective effect of MEAC was determined against cyclophosphamide induced immunosuppression in mice models. MEAC exhibited significant mitogenic and co-mitogenic activity on Con-A, PHA and LPS stimulated splenocytes isolated from mouse spleen in a dose dependent manner. Furthermore, MEAC also elicited significant immunomodulatory activity with enhanced activation of humoral immune response along with a suppressive effect on cell mediated immune response. Hematological and histopathological analysis revealed the protective effect of MEAC against CP induced immunosuppression. The significant immunomodulatory activity of MEAC observed in the current study could be due to the fatty acids and phytosterols present in the extract.

Cytotoxic Effect on Cancerous Cell Lines by Biologically Synthesized Silver Nanoparticles

The biosynthesis of nanoparticles has been proposed as an environmental friendly and cost effective alternative to chemical and physical methods. Silver nanoparticles are biologically synthesized and characterized were used in the study. The invitro cytotoxic effect of biologically synthesized silver nanoparticles against MCF-7 cancer cell lines were assessed. The cytotoxic effects of the silver nanoparticles could significantly inhibited MCF-7 cancer cell lines proliferation in a time and concentration-dependent manner by MTT assay. Acridine orange, ethidium bromide (AO/EB) dual staining, caspase-3 and DNA fragmentation assays were carried out using various concentrations of silver nanoparticles ranging from 1 to 100 μg/mL. At 100 μg/mL concentration, the silver nanoparticles exhibited significant cytotoxic effects and the apoptotic features were confirmed through caspase-3 activation and DNA fragmentation assays. Western blot analysis has revealed that nanoparticle was able to induce cytochrome c release from the mitochondria, which was initiated by the inhibition of Bcl-2 and activation of Bax. Thus, the results of the present study indicate that biologically synthesized silver nanoparticles might be used to treat breast cancer. The present studies suggest that these nanoparticles could be a new potential adjuvant chemotherapeutic and chemo preventive agent against cytotoxic cells. However, it necessitates clinical studies to ascertain their potential as anticancer agents.

Residue centrality in alpha helical polytopic transmembrane protein structures.

Transmembrane proteins serve as receptors, transporters or as enzymes. They mediate a broad range of fundamental cellular activities including signal transduction, cell trafficking and photosynthesis. In this study, we analyzed the significance of central residues in the polytopic transmembrane proteins. Each protein is represented as an undirected graph, where residues represent nodes and inter-residue interactions as the edges. Residue centrality was calculated by removing the nodes and its corresponding edges from the protein contact network. Results revealed that 80% of the predicted central residues had normalized conservation values below the mean since they were slowly evolving conserved sites. We also found that 56% of amino acids were interacting with the ligand molecules and metal ions. Predicted central residues in the polytopic transmembrane proteins were found to account for 84% of binding and active site amino acids. From mutation sensitivity analysis, it was observed that 89% of central residues had deleterious mutations whose probabilities were greater than their mean value. Interestingly, we find that z-score values of each amino acid positively correlate with the conservation scores and also with the degrees of each node. Results show that 87% of central residues are hub residues.

Systematic functional analysis and application of a cold-active serine protease from a novel Chryseobacterium sp.

Psychrotolerant bacteria isolated from natural and artificially cold environments were screened for synthesis of cold-active protease. The strain IMDY showing the highest protease production at 5°C was selected and phylogenetic analysis revealed that IMDY as novel bacterium with Chryseobacterium soli(T) as its nearest neighbor. Classical optimization enhanced the protease production from 18U/mg to 26U/mg and the enzyme was found to be active at low temperature, activity enhanced by CaCl2, inhibited by PMSF, stable against NaCl, and its activity retained in the presence of surfactants, organic solvents and detergents. On testing, the meat tenderization, myofibril fragmentation, pH, and TBA values were favorable in IMDY-protease treated meat compared to control. SDS profiling and SEM analysis also showed tenderization in meat samples. Hence, this study proposes to consider the cold-active protease from Chryseobacterium sp. IMDY as a pertinent candidate to develop potential applications in food processing industry.

Astaxanthin from psychrotrophic Sphingomonas faeni exhibits antagonism against food-spoilage bacteria at low temperatures.

Food production and processing industry holds a perpetual relationship with microorganisms and their by-products. In the present study, we aimed to identify beneficial cold-adapted bacteria devoid of any food spoilage properties and study their antagonism against common food-borne pathogens at low temperature conditions. Ten isolates were obtained on selective isolation at 5 °C, which were spread across genera Pseudomonas, Sphingomonas, Psychrobacter, Leuconostoc, Rhodococcus, and Arthrobacter. Methanol extracts of strains were found to contain several bioactive metabolites. Among the studied isolates, methanol extracts of S. faeni ISY and Rhodococcus fascians CS4 were found to show antagonism against growth of Escherichia coli, Proteus mirabilis, Enterobacter aerogenes, Listeria monocytogenes and Vibrio fischeri at refrigeration temperatures. Characterization of the abundant yellow pigment in methanol extracts of S. faeni ISY through UV-Vis spectrophotometry, high performance liquid chromatography (HPLC) and mass spectrometry (LC-MS) revealed the presence of astaxanthin, which, owing to its presence in very large amounts and evidenced to be responsible for antagonistic activity of the solvent extract.

Accumulation of phytoene, a colorless carotenoid by inhibition of phytoene desaturase (PDS) gene in Dunaliella salina V-101

The aim of this work was to study the accumulation of phytoene in Dunaliella salina V-101 by down-regulating its phytoene desaturase (PDS) gene expression using RNA interference and Antisense technology. RNAi and antisense constructs were introduced into the Dunaliella cells by Agrobacterium-mediated transformation. Among thirty-two transformants, six showed positive down-regulation of PDS expression with RNAi construct and five positive transformants were obtained using antisense construct. Characterization of PDS suppression was carried out using semi-quantitative RT-PCR and quantitative determination of phytoene as well as other carotenoids by HPLC. Both the RNAi and antisense lines showed a significant decrease in the expression levels of phytoene desaturase and carotenoid content compared to wild type cells. The RNAi line #5 showed maximum Phytoene content (108.34±22.34µg/100mg DCW) compared to other transgenic lines. These phytoene-accumulating phenotypes exhibited slower growth rates and were found to be sensitive to high light conditions.