EFFECT OF FERMENTATION PARAMETERS ON BIO-ALCOHOLS PRODUCTION FROM GLYCEROL USING IMMOBILIZED Clostridium pasteurianum: AN OPTIMIZATION STUDY

This article addresses the issue of effect of fermentation parameters for conversion of glycerol (in both pure and crude form) into three value-added products, namely, ethanol, butanol, and 1,3-propanediol (1,3-PDO), by immobilized Clostridium pasteurianum and thereby addresses the statistical optimization of this process. The analysis of effect of different process parameters such as agitation rate, fermentation temperature, medium pH, and initial glycerol concentration indicated that medium pH was the most critical factor for total alcohols production in case of pure glycerol as fermentation substrate. On the other hand, initial glycerol concentration was the most significant factor for fermentation with crude glycerol. An interesting observation was that the optimized set of fermentation parameters was found to be independent of the type of glycerol (either pure or crude) used. At optimum conditions of agitation rate (200 rpm), initial glycerol concentration (25 g/L), fermentation temperature (30°C), and medium pH (7.0), the total alcohols production was almost equal in anaerobic shake flasks and 2-L bioreactor. This essentially means that at optimum process parameters, the scale of operation does not affect the output of the process. The immobilized cells could be reused for multiple cycles for both pure and crude glycerol fermentation.     

Identification of a single and non-essential cysteine residue in dextransucrase of Leuconostoc mesenteroides NRRL B-512F

Amino acid analysis of purified dextransucrase (sucrose: 1,6-α-D-glucan 6-α-D-glucosyltransferase EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F was carried out. The enzyme is virtually devoid of cysteine residue there being only one cysteine residue in the whole enzyme molecule comprising over 1500 amino acid residues. The enzyme is rich in acidic amino acid residues. The number of amino acid residues was calculated based on the molecular weight of 188,000 (Goyal and Katiyar 1994). Amino sugars were not found, implying that the enzyme is not a glycoprotein. It has been shown earlier that the cysteine residue in dextransucrase is not essential for enzyme activity (Goyal and Katiyar 1998). The presence of only one cysteine residue per enzyme molecule illustrates that its tertiary structure is solely dependent on other types of non-covalent interactions such as hydrogen bonding, ionic and nonpolar hydrophobic interactions.     

Development and characterization of a new Bombyx mori cell line for protein expression

A Bombyx mori continuous cell line, designated DZNU-Bm-17, was established from larval ovaries. The cells were initially grown in MGM-448 insect cell culture medium supplemented with 10% fetal bovine serum and 3% heat inactivated B. mori hemolymph at 25 ± 1 °C and later adapted gradually to TNM-FH medium. Partially adhered refractive cells were the predominant cell type in the culture. The cells took about 1055 days to complete 100 passages in TNM-FH medium. The population doubling time of the cell line was about 30–34 h at 25 ± 1 °C. The cell population was largely diploid, but a few triploids and tetraploids were also observed. DNA profiles using simple sequence repeat loci established the differences between the DZNU-Bm-1, Bm-5, DZNU-Bm-12, DZNU-Bm-17, and BmN cell lines. The cell line was susceptible to budded virus of B. mori nucleopolyhedrovirus (BmNPV), and 85–92% of the cells harbored BmNPV with an average of 15 occlusion bodies/infected cell. The cells expressed the luciferase and green fluorescent proteins using the BmNPV bacmid vector. We suggest the usefulness of the DZNU-Bm-17 cell line for BmNPV-based baculoviral expression studies.     

Targeting RET to induce medullary thyroid cancer cell apoptosis: an antagonistic interplay between PI3K/Akt and p38MAPK/caspase-8 pathways

Mutations in REarranged during Transfection (RET) receptor tyrosine, followed by the oncogenic activation of RET kinase is responsible for the development of medullary thyroid carcinoma (MTC) that responds poorly to conventional chemotherapy. Targeting RET, therefore, might be useful in tailoring surveillance of MTC patients. Here we showed that theaflavins, the bioactive components of black tea, successfully induced apoptosis in human MTC cell line, TT, by inversely modulating two molecular pathways: (i) stalling PI3K/Akt/Bad pathway that resulted in mitochondrial transmembrane potential (MTP) loss, cytochrome-c release and activation of the executioner caspases-9 and -3, and (ii) upholding p38MAPK/caspase-8/caspase-3 pathway via inhibition of Ras/Raf/ERK. Over-expression of either constitutively active myristoylated-Akt-cDNA (Myr-Akt-cDNA) or dominant-negative-caspase-8-cDNA (Dn-caspase-8-cDNA) partially blocked theaflavin-induced apoptosis, while co-transfection of Myr-Akt-cDNA and Dn-caspase-8-cDNA completely eradicated the effect of theaflavins thereby negating the possibility of existence of other pathways. A search for the upstream signaling revealed that theaflavin-induced disruption of lipid raft caused interference in anchorage of RET in lipid raft that in turn stalled phosphorylation of Ras and PI3Kinase. In such anti-survival cellular micro-environment, pro-apoptotic signals were triggered to culminate into programmed death of MTC cell. These findings not only unveil a hitherto unexplained mechanism underlying theaflavin-induced MTC death, but also validate RET as a promising and potential target for MTC therapy.     

S-Adenosyl-Homocysteine Is a Weakly Bound Inhibitor for a Flaviviral Methyltransferase

The methyltransferase enzyme (MTase), which catalyzes the transfer of a methyl group from S-adenosyl-methionine (AdoMet) to viral RNA, and generates S-adenosyl-homocysteine (AdoHcy) as a by-product, is essential for the life cycle of many significant human pathogen flaviviruses. Here we investigated inhibition of the flavivirus MTase by several AdoHcy-derivatives. Unexpectedly we found that AdoHcy itself barely inhibits the flavivirus MTase activities, even at high concentrations. AdoHcy was also shown to not inhibit virus growth in cell-culture. Binding studies confirmed that AdoHcy has a much lower binding affinity for the MTase than either the AdoMet co-factor, or the natural AdoMet analog inhibitor sinefungin (SIN). While AdoMet is a positively charged molecule, SIN is similar to AdoHcy in being uncharged, and only has an additional amine group that can make extra electrostatic contacts with the MTase. Molecular Mechanics Poisson-Boltzmann Sovation Area analysis on AdoHcy and SIN binding to the MTase suggests that the stronger binding of SIN may not be directly due to interactions of this amine group, but due to distributed differences in SIN binding resulting from its presence. The results suggest that better MTase inhibitors could be designed by using SIN as a scaffold rather than AdoHcy.     

Structure guided inhibitor designing of CDK2 and discovery of potential leads against cancer

On the basis of stereo specific information obtained from crystal structures of CDK2, indole and chromene analogues were designed by suitably substituting the pharmacophores on their moiety and docked with target protein for calculating binding affinities. The binding affinities are represented in glide score. (5E)-5-[(1-methyl-1H-indol-3-yl)methylidene]-2,4,6-trioxotetrahydro-2H-pyrimidin-1-ide (I₁), (5E)-5-(1H-indol-3-ylmethylidene)-2,4,6-trioxotetrahydro-2H-pyrimidin-1-ide (I₂) and 2-amino-4-(4-methyl phenyl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (C₉) were selected for synthesis and biological testing based on vital interactions. (5E)-5-(1H-indol-3-ylmethylidene)-2,4,6-trioxotetrahydro-2H-pyrimidin-1-ide(I₂) and 2-amino-4-(4-methyl phenyl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (C₉) were proved to be active against MCF-7 and HeLa cell lines.     

Structural insights into the interaction of human S100B and basic fibroblast growth factor (FGF2): Effects on FGFR1 receptor signaling

S100B is a calcium sensing protein belonging to the S100 protein family with intracellular and extracellular roles. It is one of the EF hand homodimeric proteins, which is known to interact with various protein targets to regulate varied biological functions. Extracellular S100B has been recently reported to interact with FGF2 in a RAGE-independent manner. However, the recognition mechanism of S100B–FGF2 interaction at the molecular level remains unclear. In this study, the critical residues on S100B–FGF2 interface were mapped by combined information derived from NMR spectroscopy and site directed mutagenesis experiments. Utilizing NMR titration data, we generated the structural models of S100B–FGF2 complex from the computational docking program, HADDOCK which were further proved stable during 15 ns unrestrained molecular dynamics (MD) simulations. Isothermal titration calorimetry studies indicated S100B interaction with FGF2 is an entropically favored process implying dominant role of hydrophobic contacts at the protein–protein interface. Residue level information of S100B interaction with FGF2 was useful to understand the varied target recognition ability of S100B and further explained its role in effecting extracellular signaling diversity. Mechanistic insights into the S100B–FGF2 complex interface and cell-based assay studies involving mutants led us to conclude the novel role of S100B in FGF2 mediated FGFR1 receptor inactivation.     

Computational modeling of novel inhibitory peptides targeting proteoglycan like region of carbonic anhydrase IX and in vitro validation in HeLa cells

Abstract

Carbonic anhydrase IX (CAIX) is a tumour-associated, hypoxia-induced, membrane-bound metallo-enzyme which catalyzes the reversible hydration of carbon dioxide (CO₂) to bicarbonate (HCO₃^?) and proton (H⁺) ions. Over expression of CAIX is observed in cancers of colon, lung, kidney, breast, etc. CAIX plays a vital role in maintaining favourable intracellular pH for tumour cell growth and extracellular acidification which in-turn leads to drug resistance and spread of factors influencing tumour invasion. The N-terminal proteoglycan (PG) – like fragment of CAIX is unique to this isoform and is considered as potential druggable hotspot. Recently, M75 monoclonal antibody targeting the LPGEEDLPG epitope of PG like region has been proposed to reduce cellular adhesion in cancer cells. LPGEEDLPG fragment in complex with M75 has been crystallized and it serves as a strong base for development of peptide inhibitors based on interacting interfaces. Thus, in this study, an in-depth analysis of intermolecular interactions in LPGEEDLPG-M75 complex was carried out by implementing extensive molecular dynamics simulations, binding free energy calculations so as to infer the major determinant fragments of M75 that can be used as peptide inhibitors targeting PG region. Based on these analyses, 3 peptides (Pep1, Pep2 and Pep3) were synthesized and validated by in vitro assays involving cytotoxicity assessment, CAIX inhibition analysis through Direct and Indirect functional assays, and inhibition of Cell adhesion in HeLa cells. The results reveal Pep1 to be a promising inhibitor as it could efficiently modulate CAIX mediated pH homeostasis and cell adhesion in cancer cells.

Communicated by Ramaswamy H. Sarma     

Promising Polyphenols in Parkinson’s Disease Therapeutics

Neurochemical Research - Parkinson’s disease (PD) is a slow progressive, second most common neurodegenerative disease characterized by the loss of dopaminergic neurons from the nigrostriatal...     

Signaling role of nitric oxide in the induction of plant defense by exogenous application of abiotic inducers

The objective of this work was to search out the probable molecule behind the activation of broad spectrum resistance during abiotic elicitors such as arachidonic acid, cupric chloride, chitosan, isonicotinic acid and salicylic acid mediated induced systemic resistance (ISR) in Raphanus sativus L. The elicitor compounds were sprayed on the radish leaves of healthy plant and after 24 h incubation a significant increase of β-1,3 glucanase, peroxidase, polyphenol oxidase and phenolics as well as a remarkable increase of nitric oxide (NO), a probable potent defense-signaling molecule in plant, was observed. Furthermore, treatment of the host with NO donor, sodium nitroprusside, also induced the same defense molecules. The results suggests that NO might be the signaling molecule during abiotic elicitor mediated ISR induction in the host system.