Structural and metabolic correlation for Bacillus megaterium ACBT03 in response to colchicine biotransformation

This study aims to evaluate the effects of colchicine on metabolic and structural changes in Bacillus megaterium ACBT03, enduring colchicine bioconversion. Electron microscopy examination of cells adapted to different concentrations of colchicine for its bioconversion to pharmacologically active 3-demethylated colchicine, endowed changes in cell shape, decreased cell wall and plasma membrane thickness. In line with microscopic studies, lipid and membrane protein contents were drastically reduced in bacterial cells adapted to higher concentrations of colchicine and resulting into decrease in cell membrane thickness. More numbers of polyhydroxybutyrate (PHB) rich inclusion bodies were found inside the colchicine adapted cells and presence of higher amount of PHB, a carbon source for generation of redox potential, indicates that it might be responsible for activation of P450 BM-3 enzyme and plays significant role in colchicine demethylation. The presence of dense ribosome like bodies in colchicine adapted cells showed higher biosynthesis of P450 BM-3. Reduction in cell wall and cell membrane thickness, presence of more inclusion bodies and ribosome like masses in colchicine adapted cells were some of the key interlinked phenomena responsible for colchicine bioconversion. This is the first study which reports that colchicine demethylation process severely affects the structural and metabolic functions of the bacteria.     

A non-ionic surfactant reduces the induction time and enhances expression levels of bubaline somatotropin in Pichia pastoris

This study describes a simple approach for enhanced secretory expression of bubaline somatotropin (BbST) in the methylotropic yeast Pichia pastoris. A Mut^s Pichia transformant carrying multi-copy, non-codon optimized BbST cDNA sequence, expressed and secreted the recombinant protein into the culture medium to a level of 25 % of the total proteins in the culture supernatant, after 120 h of induction. Inclusion of polysorbate-80 in the inducing medium resulted in a significant improvement in the BbST expression (up to 45 % of the total culture supernatant proteins) with concomitant reduction in the induction time to 48 h. The amount of BbST obtained was 148 mg/L, which was around fivefold higher than that obtained without the surfactant. BbST was purified to near homogeneity by FPLC on Q-sepharose FF anion-exchange column. Protein authenticity was judged by SDS-PAGE and western blot analyses. A bioassay based on proliferation of Nb2 rat lymphoma cell lines confirmed that the purified, recombinant BbST is biologically active. Use of polysorbate-80 in combination with methanol, during the induction phase, is likely to have general applicability in lowering the induction time and enhancing the secretory expression of other commercially important proteins in Mut^s strains of P. pastoris.     

Lifting the Mask: Identification of New Small Molecule Inhibitors of Uropathogenic Escherichia coli Group 2 Capsule Biogenesis

Uropathogenic Escherichia coli (UPEC) is the leading cause of community-acquired urinary tract infections (UTIs), with over 100 million UTIs occurring annually throughout the world. Increasing antimicrobial resistance among UPEC limits ambulatory care options, delays effective treatment, and may increase overall morbidity and mortality from complications such as urosepsis. The polysaccharide capsules of UPEC are an attractive target a therapeutic, based on their importance in defense against the host immune responses; however, the large number of antigenic types has limited their incorporation into vaccine development. The objective of this study was to identify small-molecule inhibitors of UPEC capsule biogenesis. A large-scale screening effort entailing 338,740 compounds was conducted in a cell-based, phenotypic screen for inhibition of capsule biogenesis in UPEC. The primary and concentration-response assays yielded 29 putative inhibitors of capsule biogenesis, of which 6 were selected for further studies. Secondary confirmatory assays identified two highly active agents, named DU003 and DU011, with 50% inhibitory concentrations of 1.0 µM and 0.69 µM, respectively. Confirmatory assays for capsular antigen and biochemical measurement of capsular sugars verified the inhibitory action of both compounds and demonstrated minimal toxicity and off-target effects. Serum sensitivity assays demonstrated that both compounds produced significant bacterial death upon exposure to active human serum. DU011 administration in mice provided near complete protection against a lethal systemic infection with the prototypic UPEC K1 isolate UTI89. This work has provided a conceptually new class of molecules to combat UPEC infection, and future studies will establish the molecular basis for their action along with efficacy in UTI and other UPEC infections.     

Agrobacterium-Mediated Transformation of Tomato with rolB Gene Results in Enhancement of Fruit Quality and Foliar Resistance against Fungal Pathogens

Tomato (Solanum lycopersicum L.) is the second most important cultivated crop next to potato, worldwide. Tomato serves as an important source of antioxidants in human diet. Alternaria solani and Fusarium oxysporum cause early blight and vascular wilt of tomato, respectively, resulting in severe crop losses. The foremost objective of the present study was to generate transgenic tomato plants with rolB gene and evaluate its effect on plant morphology, nutritional contents, yield and resistance against fungal infection. Tomato cv. Rio Grande was transformed via Agrobacterium tumefaciens harbouring rolB gene of Agrobacterium rhizogenes. rolB. Biochemical analyses showed considerable improvement in nutritional quality of transgenic tomato fruits as indicated by 62% increase in lycopene content, 225% in ascorbic acid content, 58% in total phenolics and 26% in free radical scavenging activity. Furthermore, rolB gene significantly improved the defence response of leaves of transgenic plants against two pathogenic fungal strains A. solani and F. oxysporum. Contrarily, transformed plants exhibited altered morphology and reduced fruit yield. In conclusion, rolB gene from A. rhizogenes can be used to generate transgenic tomato with increased nutritional contents of fruits as well as improved foliar tolerance against fungal pathogens.     

Induction of Apoptosis and Antiproliferative Activity of Naringenin in Human Epidermoid Carcinoma Cell through ROS Generation and Cell Cycle Arrest

A natural predominant flavanone naringenin, especially abundant in citrus fruits, has a wide range of pharmacological activities. The search for antiproliferative agents that reduce skin carcinoma is a task of great importance. The objective of this study was to analyze the anti-proliferative and apoptotic mechanism of naringenin using MTT assay, DNA fragmentation, nuclear condensation, change in mitochondrial membrane potential, cell cycle kinetics and caspase-3 as biomarkers and to investigate the ability to induce reactive oxygen species (ROS) initiating apoptotic cascade in human epidermoid carcinoma A431 cells. Results showed that naringenin exposure significantly reduced the cell viability of A431 cells (p<0.01) with a concomitant increase in nuclear condensation and DNA fragmentation in a dose dependent manner. The intracellular ROS generation assay showed statistically significant (p<0.001) dose-related increment in ROS production for naringenin. It also caused naringenin-mediated epidermoid carcinoma apoptosis by inducing mitochondrial depolarization. Cell cycle study showed that naringenin induced cell cycle arrest in G₀/G₁ phase of cell cycle and caspase-3 analysis revealed a dose dependent increment in caspase-3 activity which led to cell apoptosis. This study confirms the efficacy of naringenin that lead to cell death in epidermoid carcinoma cells via inducing ROS generation, mitochondrial depolarization, nuclear condensation, DNA fragmentation, cell cycle arrest in G₀/G₁ phase and caspase-3 activation.     

Habitat Fragmentation and Species Extirpation in Freshwater Ecosystems; Causes of Range Decline of the Indus River Dolphin (Platanista gangetica minor)

Habitat fragmentation of freshwater ecosystems is increasing rapidly, however the understanding of extinction debt and species decline in riverine habitat fragments lags behind that in other ecosystems. The mighty rivers that drain the Himalaya - the Ganges, Brahmaputra, Indus, Mekong and Yangtze - are amongst the world’s most biodiverse freshwater ecosystems. Many hundreds of dams have been constructed, are under construction, or are planned on these rivers and large hydrological changes and losses of biodiversity have occurred and are expected to continue. This study examines the causes of range decline of the Indus dolphin, which inhabits one of the world’s most modified rivers, to demonstrate how we may expect other vertebrate populations to respond as planned dams and water developments come into operation. The historical range of the Indus dolphin has been fragmented into 17 river sections by diversion dams; dolphin sighting and interview surveys show that river dolphins have been extirpated from ten river sections, they persist in 6, and are of unknown status in one section. Seven potential factors influencing the temporal and spatial pattern of decline were considered in three regression model sets. Low dry-season river discharge, due to water abstraction at irrigation barrages, was the principal factor that explained the dolphin’s range decline, influencing 1) the spatial pattern of persistence, 2) the temporal pattern of subpopulation extirpation, and 3) the speed of extirpation after habitat fragmentation. Dolphins were more likely to persist in the core of the former range because water diversions are concentrated near the range periphery. Habitat fragmentation and degradation of the habitat were inextricably intertwined and in combination caused the catastrophic decline of the Indus dolphin.     

A Bub1–Mad1 interaction targets the Mad1–Mad2 complex to unattached kinetochores to initiate the spindle checkpoint

Recruitment of Mad1–Mad2 complexes to unattached kinetochores is a central event in spindle checkpoint signaling. Despite its importance, the mechanism that recruits Mad1–Mad2 to kinetochores is unclear. In this paper, we show that MAD-1 interacts with BUB-1 in Caenorhabditis elegans. Mutagenesis identified specific residues in a segment of the MAD-1 coiled coil that mediate the BUB-1 interaction. In addition to unattached kinetochores, MAD-1 localized between separating meiotic chromosomes and to the nuclear periphery. Mutations in the MAD-1 coiled coil that selectively disrupt interaction with BUB-1 eliminated MAD-1 localization to unattached kinetochores and between meiotic chromosomes, both of which require BUB-1, and abrogated checkpoint signaling. The identified MAD-1 coiled-coil segment interacted with a C-terminal region of BUB-1 that contains its kinase domain, and mutations in this region prevented MAD-1 kinetochore targeting independently of kinase activity. These results delineate an interaction between BUB-1 and MAD-1 that targets MAD-1–MAD-2 complexes to kinetochores and is essential for spindle checkpoint signaling.     

Protein secretion systems in Fusobacterium nucleatum: Genomic identification of Type 4 piliation and complete Type V pathways brings new insight into mechanisms of pathogenesis

Recent genomic analyses of the two sequenced strains F. nucleatum subsp. nucleatum ATCC 25586 and F. nucleatum subsp. vincentii ATCC 49256 suggested that the major protein secretion systems were absent. However, such a paucity of protein secretion systems is incongruous with F. nucleatum pathogenesis. Moreover, the presence of one or more such systems has been described for every other Gram-negative organism sequenced to date. In this investigation, the question of protein secretion in F. nucleatum was revisited. In the current study, the absence in F. nucleatum of a twin-arginine translocation system (TC #2.A.64.), a Type III secretion system (TC #3.A.6.), a Type IV secretion system (TC #3.A.7.) and a chaperone/usher pathway (TC #1.B.11.) was confirmed. However, contrary to previous findings, our investigations indicated that a Type I protein secretion system was also absent from F. nucleatum. In contrast, members of the holin family (TC #1.E) and the machinery required for a Type 4 piliation/fimbriation system (TC #3.A.15.2.) were identified using a variety of bioinformatic tools. Furthermore, a complete range of proteins resembling members of the Type V secretion pathway, i.e., the Type Va (autotransporter; TC #1.B.12.), Type Vb (two-partner secretion system; TC #1.B.20.) and Type Vc (YadA-like trimeric autotransporter; TC #1.B.42.), was found. This work provides new insight into the protein secretion and virulence mechanisms of F. nucleatum.     

Polyiodine units in starch–iodine complex: INDO CI study of spectra and comparison with experiments

The starch–iodine blue complex formation does not involve negatively charged iodine species like I, I, or I; rather, neutral iodine units are involved. The heat of reaction is determined to be about ?110 kJ for every mole of I-I unit in the amylose helix, which suggests that the dissociation of I₂ (binding energy 149 kJ/mol) does not take place during the complex formation. Quantum mechanical (INDO CI) calculations indicate that the linear as well as nonlinear polyiodine units, I₆, with interiodine distance of 3.0 Å are responsible for characteristic absorbance bands of the starch–iodine complex. Based on our previous article [(1989) J. Polym. Sci. A 27 , 4161] and the present studies we identify (C₆H₁₀O₅)_16.5I₆ to be the polymeric unit responsible for the characteristic blue color of the complex.