Characterization of an adenylate cyclase gene (cyaB) deletion mutant of Corynebacterium glutamicum ATCC 13032

Genome analysis of C. glutamicum ATCC 13032 has showed one putative adenylate cyclase gene, cyaB (cg0375) which encodes membrane protein belonging to class III adenylate cyclases. To characterize the function of cyaB, a deletion mutant was constructed, and the mutant showed decreased level of intracellular cyclic AMP compared to that of wild-type. Interestingly, the cyaB mutant displayed growth defect on acetate medium, and this effect was reversed by complementation with cyaB gene. Similarly, it showed growth defect on glucose-acetate mixture minimal medium, and the utilization of glucose was retarded in the presence of acetate. The deletion mutant retained the activity of glyoxylate bypass enzymes. Additionally, the mutant could grow on ethanol but not on propionate medium. The data obtained from this study suggests that adenylate cyclase plays an essential role in the acetate metabolism of C. glutamicum, even though detailed regulatory mechanisms involving cAMP are not yet clearly defined. The observation that glyoxylate bypass enzymes are derepressed in cyaB mutant indicates the involvement of cAMP in the repression of aceB and aceA.     

Structure and rearrangements in the carboxy-terminal region of SpIH channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels regulate the spontaneous firing activity and electrical excitability of many cardiac and neuronal cells. The modulation of HCN channel opening by the direct binding of cAMP underlies many physiological processes such as the autonomic regulation of the heart rate. Here we use a combination of X-ray crystallography and electrophysiology to study the allosteric mechanism for cAMP modulation of HCN channels. SpIH is an invertebrate HCN channel that is activated fully by cAMP, but only partially by cGMP. We exploited the partial agonist action of cGMP on SpIH to reveal the molecular mechanism for cGMP specificity of many cyclic nucleotide-regulated enzymes. Our results also elaborate a mechanism for the allosteric conformational change in the cyclic nucleotide-binding domain and a mechanism for partial agonist action. These mechanisms will likely extend to other cyclic nucleotide-regulated channels and enzymes as well.     

COX-2 expression in cystic kidneys is dependent on dietary n-3 fatty acid composition

Dietary n-3 fatty acids generally attenuate elevated cyclooxygenase-2 (COX-2) levels in disease states. However, models of renal cystic disease (RCD) exhibit reduced renal COX-2 expression. Therefore, the in vivo regulation of COX-2 expression by dietary n-3 fatty acids was examined. In archived tissues from dietary studies, COX-2 protein and gene expression was up-regulated in diseased pcy mouse and Han:SPRD-cy rat kidneys when given diets containing eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA), but not those containing -linolenic acid (ALA), compared to control diets with linoleic acid (LA). The presence of disease was necessary to elicit these effects as COX-2 expression was unaltered by diet in normal kidneys. The effects were specific for COX-2, since COX-1 levels were unaltered by these dietary manipulations in either model. Thus, in RCD, diets containing EPA and DHA but not ALA appear to specifically up-regulate renal COX-2 gene and protein levels in vivo.     

In vivo humoral and cellular reactions, and fate of injected bacteria Aeromonas hydrophila in freshwater prawn Macrobrachium rosenbergii

Live non-opsonized and opsonized Aeromonas hydrophila were injected into juveniles of freshwater prawn Macrobrachium rosenbergii to study the cells involved in phagocytosis, distribution of bacteria, cellular reactions and clearance of both forms of bacteria from the system. The bacteria were rapidly distributed to various tissues viz., gills, heart, hepatopancreas within 1h, and the tissues revealed haemocytic nodule formation after 3 h of injection. There was rapid clearance of both the forms of bacteria from the circulation. However, clearance efficiency was significantly (P < 0.05) faster in the case of opsonized bacteria at 12 h after injection. Similarly, the nodule formation, that was prominent in cardiac musculature, was rapidly eliminated from the tissues of the group injected with opsonized bacteria as compared to non-opsonized bacteria injected group, thus confirming the existence of opsonic factors in haemolymph of this prawn. In another experiment, various dose levels of bacteria were injected intramuscularly into prawns and haemolymph was collected after 1, 6, 24, 72 h and 7 days of injection to study various immune parameters. Although, no major alterations in the total and differential haemocyte counts were observed in bacteria injected prawns compared to control, there was a significant decline in phenoloxidase activity in the highest dose bacteria injected group at the earlier phase and a rise in agglutinin levels at the later phase of the experimental period in the higher dose groups.     

Production of thermostable pectinase and xylanase for their potential application in bleaching of kraft pulp

A very high level of alkalophilic and thermostable pectinase and xylanase has been produced from newly isolated strains of Bacillus subtilis and Bacillus pumilus respectively. Enzyme production for pectinase was carried out under SSF using combinations of cheap agricultural residues while xylanase was produced under submerged fermentation using wheat bran as substrate to minimize the cost of production of these enzymes Among the various substrates tested, the highest yield of pectinase production was observed by using combination of WB + CW (6592 U/g of dry substrate) supplemented with 4% yeast extract when incubated at 37 °C for 72 h using deionized water of pH 7.0 as moistening agent. The biobleaching effect of these cellulase free enzymes on kraft pulp was determined. Both xylanase and pectinase showed stability over a broad range of pH from 6 to 10 and temperature from 55 to 70 °C. The bleaching efficiency of the pectinase and xylanase on kraft pulp was maximum after 150 min at 60 °C using enzyme dosage of 5 IU/ml of each enzyme at 10% pulp consistency with about 16% reduction in kappa number and 84% reduction in permanganate number. Enzyme treated pulp when subjected to CDED₁D₂ steps, 25% reduction in chlorine consumption and upto 19% reduction in consumption of chlorine dioxide was observed for obtaining the same %ISO brightness. Also an increase of 22 and 84% in whiteness and fluorescence respectively and a decrease of approximately 19% in the yellowness of the biotreated pulp were observed by pretreatment of the pulp with our enzymatic mixture.     

Measuring the Binding Stoichiometry of HIV-1 Gag to Very-Low-Density Oligonucleotide Surfaces Using Surface Plasmon Resonance Spectroscopy

The interaction of the HIV Gag polyprotein with nucleic acid is a critical step in the assembly of viral particles. The Gag polyprotein is composed of the matrix (MA), capsid (CA), and nucleocapsid (NC) domains. The NC domain is required for nucleic acid interactions, and the CA domain is required for Gag-Gag interactions. Previously, we have investigated the binding of the NC protein to d(TG)(n) oligonucleotides using surface plasmon resonance (SPR) spectroscopy. We found a single NC protein is able to bind to more than one immobilized oligonucleotide, provided that the oligonucleotides are close enough together. As NC is believed to be the nucleic acid binding domain of Gag, we might expect Gag to show the same complex behavior. We wished to analyze the stoichiometry of Gag binding to oligonucleotides without this complication due to tertiary complex formation. We have therefore analyzed Gag binding to extremely low oligonucleotide density on SPR chips. Such low densities of oligonucleotides are difficult to accurately quantitate. We have determined by Fourier transform ion cyclotron (FTICR) mass spectrometry that four molecules of NC bind to d(TG)(10) (a 20-base oligonucleotide). We developed a method of calibrating low-density surfaces using NC calibration injections. Knowing the maximal response and the stoichiometry of binding, we can precisely determine the amount of oligonucleotide immobilized at these very-low-density surfaces (<1 Response Unit). Using this approach, we have measured the binding of Gag to d(TG)(10). Gag binds to a 20-mer with a stoichiometry of greater than 4. This suggests that once Gag is bound to the immobilized oligonucleotide, additional Gag molecules can bind to this complex.     

A systems biology approach to model neural stem cell regulation by notch, shh, wnt, and EGF signaling pathways

The Notch, Sonic Hedgehog (Shh), Wnt, and EGF pathways have long been known to influence cell fate specification in the developing nervous system. Here we attempted to evaluate the contemporary knowledge about neural stem cell differentiation promoted by various drug-based regulations through a systems biology approach. Our model showed the phenomenon of DAPT-mediated antagonism of Enhancer of split [E(spl)] genes and enhancement of Shh target genes by a SAG agonist that were effectively demonstrated computationally and were consistent with experimental studies. However, in the case of model simulation of Wnt and EGF pathways, the model network did not supply any concurrent results with experimental data despite the fact that drugs were added at the appropriate positions. This paves insight into the potential of crosstalks between pathways considered in our study. Therefore, we manually developed a map of signaling crosstalk, which included the species connected by representatives from Notch, Shh, Wnt, and EGF pathways and highlighted the regulation of a single target gene, Hes-1, based on drug-induced simulations. These simulations provided results that matched with experimental studies. Therefore, these signaling crosstalk models complement as a tool toward the discovery of novel regulatory processes involved in neural stem cell maintenance, proliferation, and differentiation during mammalian central nervous system development. To our knowledge, this is the first report of a simple crosstalk map that highlights the differential regulation of neural stem cell differentiation and underscores the flow of positive and negative regulatory signals modulated by drugs.     

Evaluation of macrocyclic Grb2 SH2 domain-binding peptide mimetics prepared by ring-closing metathesis of C-terminal allylglycines with an N-terminal beta-vinyl-substituted phosphotyrosyl mimetic

Preferential binding of ligands to Grb2 SH2 domains in beta-bend conformations has made peptide cyclization a logical means of effecting affinity enhancement. This is based on the concept that constraint of open-chain sequences to bend geometries may reduce entropy penalties of binding. The current study extends this approach by undertaking ring-closing metathesis (RCM) macrocyclization between i and i+3 residues through a process involving allylglycines and beta-vinyl-functionalized residues. Ring closure in this fashion results in minimal macrocyclic tetrapeptide mimetics. The predominant effects of such macrocyclization on Grb2 SH2 domain binding affinity were increases in rates of association (from 7- to 16-fold) relative to an open-chain congener, while decreases in dissociation rates were less pronounced (approximately 2-fold). The significant increases in association rates were consistent with pre-ordering of solution conformations to near those required for binding. Data from NMR experiments and molecular modeling simulations were used to interpret the binding results. An understanding of the conformational consequences of such i to i+3 ring closure may facilitate its application to other systems where bend geometries are desired.     

Tissue- and gene-specific recruitment of steroid receptor coactivator-3 by thyroid hormone receptor during development

Numerous coactivators that bind nuclear hormone receptors have been isolated and characterized in vitro. Relatively few studies have addressed the developmental roles of these cofactors in vivo. By using the total dependence of amphibian metamorphosis on thyroid hormone (T3) as a model, we have investigated the role of steroid receptor coactivator 3 (SRC3) in gene activation by thyroid hormone receptor (TR) in vivo. First, expression analysis showed that SRC3 was expressed in all tadpole organs analyzed. In addition, during natural as well as T3-induced metamorphosis, SRC3 was up-regulated in both the tail and intestine, two organs that undergo extensive transformations during metamorphosis and the focus of the current study. We then performed chromatin immunoprecipitation assays to investigate whether SRC3 is recruited to endogenous T3 target genes in vivo in developing tadpoles. Surprisingly, we found that SRC3 was recruited in a gene- and tissue-dependent manner to target genes by TR, both upon T3 treatment of premetamorphic tadpoles and during natural metamorphosis. In particular, in the tail, SRC3 was not recruited in a T3-dependent manner to the target TRbetaA promoter, suggesting either no recruitment or constitutive association. Finally, by using transgenic tadpoles expressing a dominant negative SRC3 (F-dnSRC3), we demonstrated that F-dnSRC3 was recruited in a T3-dependent manner in both the intestine and tail, blocking the recruitment of endogenous coactivators and histone acetylation. These results suggest that SRC3 is utilized in a gene- and tissue-specific manner by TR during development.