Computational kinetic studies of pyruvate metabolism in Carboxydothermus hydrogenoformans Z-2901 for improved hydrogen production

Hydrogen is considered as a renewable energy source and it is also regarded as future fuel. Currently, hydrogen production through a biotechnological approach is a research priority. Hydrogenogens, a microbial species, are of significant interest to researchers because of their ability to produce biological hydrogen. Carboxydothermus hydrogenoformans Z-2901 is one among the hydrogenogens that can grow anaerobically by utilizing pyruvate as a carbon source, and can produce molecular hydrogen. In the present study, we performed an in silico kinetic simulation using the available Kyoto Encyclopedia of Genes and Genomes (KEGG) model and reconstructed pyruvate metabolism in C. hydrogenoformans Z-2901. During this metabolism, dissimilation of pyruvate leads to the formation of energy co-factors, such as ATP and NAD⁺/ NADH, and the level of these co-factors influences the specific growth rate of organism and hydrogen production. Our strategy for improving hydrogen production involves maximizing the ATP and NAD⁺ yield by modification of kinetic properties and adding new reactions in pyruvate metabolism through metabolic pathway reconstruction. Moreover, the influence of phosphoenol pyruvate carboxylase and pyruvate dehydrogenase enzyme concentration on cofactor productions was also simulated. The theoretical molar yield of ATP and NAD⁺ were obtained as 2.32 and 1.83 mM, respectively, from 1 mM/mg of phosphoenol pyruvate (PEP) utilization. A higher yield of ATP is achieved when the PEP level reaches 5 mM/mg. This work also suggests that PEP can be considered as an alternative substrate. In conclusion, the simulation results reported in this paper can be applied to design and evaluate strategies of strain construction for optimal hydrogen yield in C. hydrogenoformans.     

Osteopontin stimulates cell motility and nuclear factor kappaB-mediated secretion of urokinase type plasminogen activator through phosphatidylinositol 3-kinase/Akt signaling pathways in breast cancer cells

We have recently reported that osteopontin (OPN) induces nuclear factor kappaB (NFkappaB)-mediated promatrix metalloproteinase-2 activation through IkappaBalpha/IKK signaling pathways and that curcumin (diferulolylmethane) down-regulates these pathways (Philip, S., and Kundu, G. C. (2003) J. Biol. Chem. 278, 14487-14497). However, the molecular mechanism by which upstream kinases regulate the OPN-induced NFkappaB activation and urokinase type plasminogen activator (uPA) secretion in human breast cancer cells is not well defined. Here we report that OPN induces the phosphatidylinositol 3'-kinase (PI 3'-kinase) activity and phosphorylation of Akt in highly invasive MDA-MB-231 and low invasive MCF-7 cells. The OPN-induced Akt phosphorylation was inhibited when cells were transfected with a dominant negative mutant of the p85 domain of PI 3-kinase (Deltap85) and enhanced when cells were transfected with an activated form of PI 3-kinase (p110CAAX), indicating that PI 3'-kinase is involved in Akt phosphorylation. OPN enhances the interaction between IkappaBalpha kinase (IKK) and phosphorylated Akt. OPN also induces NFkappaB activation through phosphorylation and degradation of IkappaBalpha by inducing the IKK activity. However, both pharmacological (wortmannin and LY294002) and genetic (Deltap85) inhibitors of PI 3'-kinase inhibited OPN-induced Akt phosphorylation, IKK activity, and NFkappaB activation through phosphorylation and degradation of IkappaBalpha. OPN also enhances uPA secretion, cell motility, and extracellular matrix invasion. Furthermore, cells transfected with Deltap85 or the super-repressor form of IkappaBalpha suppressed the OPN-induced uPA secretion and cell motility, whereas cells transfected with p110CAAX enhanced these effects. Pretreatment of cells with PI 3-kinase inhibitors or NFkappaB inhibitory peptide (SN-50) reduced the OPN-induced uPA secretion, cell motility, and invasion. To our knowledge, this is first report that OPN induces NFkappaB activity and uPA secretion by activating PI 3'-kinase/Akt/IKK-mediated signaling pathways and further demonstrates a functional molecular link between OPN-induced PI 3'-kinase-dependent Akt phosphorylation and NFkappaB-mediated uPA secretion, and all of these ultimately control the motility of breast cancer cells.     

Dynamic antagonism between ETR-3 and PTB regulates cell type-specific alternative splicing

Inclusion of cardiac troponin T (cTNT) exon 5 in embryonic muscle requires conserved flanking intronic elements (MSEs). ETR-3, a member of the CELF family, binds U/G motifs in two MSEs and directly activates exon inclusion in vitro. Binding and activation by ETR-3 are directly antagonized by polypyrimidine tract binding protein (PTB). We use dominant-negative mutants to demonstrate that endogenous CELF and PTB activities are required for MSE-dependent activation and repression in muscle and nonmuscle cells, respectively. Combined use of CELF and PTB dominant-negative mutants provides an in vivo demonstration that antagonistic splicing activities exist within the same cells. We conclude that cell-specific regulation results from the dominance of one among actively competing regulatory states rather than modulation of a nonregulated default state.     

Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion

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Structure and Function of the Hypertension Variant A486V of G Protein-coupled Receptor Kinase 4

G-protein-coupled receptor (GPCR) kinases (GRKs) bind to and phosphorylate GPCRs, initiating the process of GPCR desensitization and internalization. GRK4 is implicated in the regulation of blood pressure, and three GRK4 polymorphisms (R65L, A142V, and A486V) are associated with hypertension. Here, we describe the 2.6 Å structure of human GRK4α A486V crystallized in the presence of 5′-adenylyl β,γ-imidodiphosphate. The structure of GRK4α is similar to other GRKs, although slight differences exist within the RGS homology (RH) bundle subdomain, substrate-binding site, and kinase C-tail. The RH bundle subdomain and kinase C-terminal lobe form a strikingly acidic surface, whereas the kinase N-terminal lobe and RH terminal subdomain surfaces are much more basic. In this respect, GRK4α is more similar to GRK2 than GRK6. A fully ordered kinase C-tail reveals interactions linking the C-tail with important determinants of kinase activity, including the αB helix, αD helix, and the P-loop. Autophosphorylation of wild-type GRK4α is required for full kinase activity, as indicated by a lag in phosphorylation of a peptide from the dopamine D₁ receptor without ATP preincubation. In contrast, this lag is not observed in GRK4α A486V. Phosphopeptide mapping by mass spectrometry indicates an increased rate of autophosphorylation of a number of residues in GRK4α A486V relative to wild-type GRK4α, including Ser-485 in the kinase C-tail.     

Adult murine skeletal muscle contains cells that can differentiate into beating cardiomyocytes in vitro

It has long been held as scientific fact that soon after birth, cardiomyocytes cease dividing, thus explaining the limited restoration of cardiac function after a heart attack. Recent demonstrations of cardiac myocyte differentiation observed in vitro or after in vivo transplantation of adult stem cells from blood, fat, skeletal muscle, or heart have challenged this view. Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion. We now show a novel population of nonsatellite cells in adult murine skeletal muscle that progress under standard primary cell-culture conditions to autonomously beating cardiomyocytes. Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials. Within 2 d after tail-vein injection of these marked cells into a mouse model of acute infarction, the marked cells are visible in the heart. By 6 d they begin to differentiate without fusing to recipient cardiac cells. Three months later, the tagged cells are visible as striated heart muscle restricted to the region of the cardiac infarct.     

Template‐free protein structure prediction and quality assessment with an all‐atom free‐energy model

Biophysical forcefields have contributed less than originally anticipated to recent progress in protein structure prediction. Here, we have investigated the selectivity of a recently developed all‐atom free‐energy forcefield for protein structure prediction and quality assessment (QA). Using a heuristic method, but excluding homology, we generated decoy‐sets for all targets of the CASP7 protein structure prediction assessment with <150 amino acids. The decoys in each set were then ranked by energy in short relaxation simulations and the best low‐energy cluster was submitted as a prediction. For four of nine template‐free targets, this approach generated high‐ranking predictions within the top 10 models submitted in CASP7 for the respective targets. For these targets, our de‐novo predictions had an average GDT_S score of 42.81, significantly above the average of all groups. The refinement protocol has difficulty for oligomeric targets and when no near‐native decoys are generated in the decoy library. For targets with high‐quality decoy sets the refinement approach was highly selective. Motivated by this observation, we rescored all server submissions up to 200 amino acids using a similar refinement protocol, but using no clustering, in a QA exercise. We found an excellent correlation between the best server models and those with the lowest energy in the forcefield. The free‐energy refinement protocol may thus be an efficient tool for relative QA and protein structure prediction. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

MicroRNA profiles in Sorghum exposed to individual drought or heat or their combination

Sorghum is largely grown for food, fodder and for biofuel production in semi-arid regions where the drought or high temperature or their combination co-occur. Plant microRNAs (miRNAs) are integral to the gene regulatory networks that control almost all biological processes including adaptation to stress conditions. Thus far, plant miRNA profiles under separate drought or heat stresses have been reported but not under combined drought and heat. In this study, we report miRNA profiles in leaves of sorghum exposed to individual drought or heat or their combination. Approximately 29 conserved miRNA families represented by 80 individual miRNAs, 26 families represented by 47 members of less conserved or sorghum-specific miRNA families as well as 8 novel miRNA families have been identified. Of these, 25 miRNAs were found to be differentially regulated in response to stress treatments. The comparative profiling revealed that the miRNA regulation was stronger under heat or combination of heat and drought compared to the drought alone. Furthermore, using degradome sequencing, 48 genes were confirmed as targets for the miRNAs in sorghum. Overall, this study provides a framework for understanding of the miRNA-guided gene regulations under combined stresses.