Partial truncation of the NH2-terminus affects physical characteristics and membrane binding, aggregation, and fusion properties of annexin A7

Annexin A7 (synexin, annexin VII), a member of the annexin family of proteins, causes aggregation of membranes in a Ca2+-dependent manner and has been suggested to promote membrane fusion during exocytosis of lung surfactant, catecholamines, and insulin. Although annexin A7 (A7) was one of the first annexin proteins described, limited studies of its physical characteristics or of structural domains affecting any of its proposed functions have been conducted. As postulated for other annexin proteins, the unique NH2-domain possibly determines the functional specificity of A7. Therefore, we evaluated the effects of segmental deletions in the NH2-terminus on several characteristics associated with the COOH-terminus of A7. The COOH-terminus contains the only tryptophan residue, and all potential trypsin sites, and the Ca2+ and phospholipid binding sites. Recombinant rat A7 and its deletion mutants were expressed using constructs based on the cDNA sequence obtained by screening a rat lung cDNA library. Ca2+ increased the tryptophan fluorescence of A7 and caused a small red shift in the emission maximum (lambdamax), which was further increased in presence of phospholipid vesicles (PLV). NH2-terminal deletions of 29, 51, and 109 residues affected the peak width of fluorescence and lambdamax, surface-exposure of tryptophan residue, and caused a smaller Ca2+-dependent red shift in lambdamax of membrane-bound protein in comparison to A7. Limited proteolysis with trypsin showed that Ca2+ increased the proteolysis of all proteins, but the deletions also affected the pattern of proteolysis. The presence of PLV protected against Ca2+-dependent increase in proteolysis of all proteins. The deletion of first 29 residues also caused decreased membrane binding, aggregation, and fusion, when compared with A7. Collectively, these results suggest that specific NH2-terminus domains can alter those properties of A7 that are normally associated with the COOH-terminus. We speculate that interactions between the NH2- and COOH-termini are required for membrane binding, and aggregation and fusion properties of annexin A7.     

The Rv0805 gene from Mycobacterium tuberculosis encodes a 3',5'-cyclic nucleotide phosphodiesterase: biochemical and mutational analysis

Mycobacterium tuberculosis is an important human pathogen and has developed sophisticated mechanisms to evade the host immune system. These could involve the use of cyclic nucleotide-dependent signaling systems, since the M. tuberculosis genome encodes a large number of functional adenylyl cyclases. Using bioinformatic approaches, we identify, clone, and biochemically characterize the Rv0805 gene product, the first cyclic nucleotide phosphodiesterase identified in M. tuberculosis and a homologue of the cAMP phosphodiesterase present in Escherichia coli (cpdA). The Rv0805 gene product, a class III phosphodiesterase, is a member of the metallophosphoesterase family, and computational modeling and mutational analyses indicate that the protein possesses interesting properties not reported earlier in this class of enzymes. Mutational analysis of critical histidine and aspartate residues predicted to be essential for metal coordination reduced catalytic activity by 90-50%, and several mutant proteins showed sigmoidal kinetics with respect to Mn in contrast to the wild-type enzyme. Mutation of an asparagine residue in the GNHD motif that is conserved throughout the metallophosphoesterase enzymes almost completely abolished catalytic activity, and these studies therefore represent the first mutational analysis of this class of phosphodiesterases. The Rv0805 protein hydrolyzes cAMP and cGMP in vitro, and overexpression in Mycobacterium smegmatis and E. coli reduces intracellular cAMP levels. The presence of an orthologue of Rv0805 in Mycobacterium leprae suggests that the Rv0805 protein could have an important role to play in regulating cAMP levels in these bacteria and adds an additional level of complexity to cyclic nucleotide signaling in this organism.     

Tyrphostins are inhibitors of guanylyl and adenylyl cyclases

Guanylyl cyclase C (GC-C), the receptor for guanylin, uroguanylin, and the heat-stable enterotoxin, regulates fluid balance in the intestine and extraintestinal tissues. The receptor has an extracellular domain, a single transmembrane spanning domain, and an intracellular domain that harbors a region homologous to protein kinases, followed by the C-terminal guanylyl cyclase domain. Adenine nucleotides can regulate the guanylyl cyclase activity of GC-C by binding to the intracellular kinase homology domain (KHD). In this study, we have tested the effect of several protein kinase inhibitors on GC-C activity and find that the tyrphostins, known to be tyrosine kinase inhibitors, could inhibit GC-C activity in vitro. Tyrphostin A25 (AG82) was the most potent inhibitor with an IC(50) of approximately 15 microM. The mechanism of inhibition was found to be noncompetitive with respect to both the substrate MnGTP and the metal cofactor. Interestingly, the activity of the catalytic domain of GC-C (lacking the KHD) expressed in insect cells was also inhibited by tyrphostin A25 with an IC(50) of approximately 5 microM. As with the full-length receptor, inhibition was found to be noncompetitive with respect to MnGTP. Inhibition was reversible, ruling out a covalent modification of the receptor. Structurally similar proteins such as the soluble guanylyl cyclase and the adenylyl cyclases were also inhibited by tyrphostin A25. Evaluation of a number of tyrphostins allowed us to identify the requirement of two vicinal hydroxyl groups in the tyrphostin for effective inhibition of cyclase activity. Therefore, our studies are the first to report that nucleotide cyclases are inhibited by tyrphostins and suggest that novel inhibitors based on the tyrphostin scaffold can be developed, which could aid in a greater understanding of nucleotide cyclase structure and function.     

Bufadienolides from Drimia robusta and Urginea epigea (Hyacinthaceae)

Two bufadienolides, 6beta-acetoxy-3beta,8beta,14beta-trihydroxy-12-oxobufa-4,20,22-trienolide and 14beta-hydroxybufa-3,5,20,22-tetraenolide were isolated from the dichloromethane extract of the bulbs of Drimia robusta and the methanol extract of the bulbs of Urginea epigea, respectively. The bulbs of Drimia robusta also yielded several known compounds, 6beta-acetoxy-3beta,8beta,12beta,14beta-tetrahydroxybufa-4,20,22-trienolide (12beta-hydroxyscillirosidin) from the dichloromethane extract and three common aromatic acids, 4-hydroxy-3-methoxybenzoic acid, 3,4-dihydroxybenzoic acid, and trans-3-(4'-hydroxyphenyl)-2-propenoic acid from the ethyl acetate extract.     

Process development in the QbD paradigm: Role of process integration in process optimization for production of biotherapeutics

Biotherapeutics have become the focus of the pharmaceutical industry due to their proven effectiveness in managing complex diseases. Downstream processes of these molecules consist of several orthogonal, high resolution unit operations designed so as to be able to separate variants having very similar physicochemical properties. Typical process development involves optimization of the individual unit operations based on Quality by Design principles in order to define the design space within which the process can deliver product that meets the predefined specifications. However, limited efforts are dedicated to understanding the interactions between the unit operations. This paper aims to showcase the importance of understanding these interactions and thereby arrive at operating conditions that are optimal for the overall process. It is demonstrated that these are not necessarily same as those obtained from optimization of the individual unit operations. Purification of Granulocyte Colony Stimulating Factor (G‐CSF), a biotherapeutic expressed in E. coli., has been used as a case study. It is evident that the suggested approach results in not only higher yield (91.5 vs. 86.4) but also improved product quality (% RP‐HPLC purity of 98.3 vs. 97.5) and process robustness. We think that this paper is very relevant to the present times when the biotech industry is in the midst of implementing Quality by Design towards process development. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:355–362, 2016     

Cell wall remodelling involving galactomannan de-branching influence <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of <Emphasis Type="Italic">Coffea canephora</Emphasis> somatic embryos

Direct somatic embryogenesis is favoured over indirect methods for the in vitro propagation of Coffea canephora, as the frequency of somaclonal variation is usually reduced. Ethylene action inhibitors improve the tissue culture response and thus silver nitrate (AgNO₃) is used for direct somatic embryogenesis in coffee. It was observed that silver thiosulphate (STS) that is a more potent ethylene action inhibitor, induced a much robust response in C. canephora cotyledonary leaf explants with 7.49?±?0.57 and 7.08?±?0.12 embryos/explant at 60 and 80 µM AgNO₃, respectively compared to 3.3?±?0.18 embryos/explant at 40 µM AgNO₃. Transient transformation indicated that STS improved the transformation potential of embryos by enhancing Agrobacterium tumefaciens adherence to surfaces. In vitro adherence assays demonstrated that the cell wall material from STS-derived embryos provide a better substratum for adherence of Agrobacterium. Furthermore, blocking this substratum with anti-mannan hybridoma supernatant negatively effects the adherence. The presence of galactose and mannose residues in the decomposed cellulose fraction of STS treated somatic embryos are indicative of de-branching and re-modelling of galactomannan in response to ethylene inhibition. Genes of mannan biosynthesis, degradation and de-branching enzyme were affected to different extents in embryos derived in AgNO₃ and STS containing somatic embryogenesis medium. The results indicate that ethylene-mediated cell wall galactomannan remodelling is vital for improving the transgenic potential in coffee.     

Novel,quinone-thiosemicarbazone hybrid (QTSCHY) non-platinum antitumor agents: inhibition of DNA biosynthesis in P388 lymphocytic cells by coordinatively unsaturated copper(II) and iron(III) complexes of naphthoquinone thiosemicarbazones

Coordinately unsaturated Cu(II) and Fe(III) complexes of the stoichiometry [Cu(L)Cl] and [Fe(L)Cl₂], where L=tridentate anion of 2-hydroxy-1,4-naphthoquinone 1-thiosemicarbazone (2HNQTSC) and its 3-methyl derivative (3M2HNQTSC), were screenedin vitro against P388 lymphocytic leukemia cells. Copper complexes were found to be more effective inhibitors of DNA synthesis than analogous Fe(III) compounds. The inhibitory activities are suggested to be related to Cu(II)–Cu(I) redox couple or nitrogen adduct formation.     

Geochemistry Shapes Bacterial Communities and their Metabolic Potentials in Tertiary Coalbed

Culture-dependent and independent approaches were used to understand the microbiota thriving in tertiary coalbed, located in Jammu and Kashmir, India. We observed changes in physicochemical properties of the surface sediment (CM1) and coalbed (CM2) which detailed the influence of environmental factors on the structure and capabilities of bacterial communities. A total of 316 bacterial isolates representing 35 genera were isolated. We noted comparable difference in uncultivable bacterial communities which revealed the predominance of Proteobacteria in both the study sites. Moreover, we observed differential abundance of phyla Actinobacteria (49.6%), Firmicutes (4.2%), and Bacteroidetes (0.8%) in CM1, whereas Actinobacteria (11%), Firmicutes (37.8%), and Bacteroidetes (2.3%) in CM2. Additionally, functional imputations using PICRUSt depicted ～30% higher assemblage of major gene families in CM1 in comparison to CM2. Bacterial communities residing at CM1 were predominantly involved in methane oxidation, whereas CM2 communities found to play a vital process of conversion of coal to biogenic-methane enabling microbes to survive under constraints of high sulfur content, salt precipitation, and low nutrients and also provide clues to understand the potential of methanogenesis.