The cellular response to vascular endothelial growth factors requires co-ordinated signal transduction,trafficking and proteolysis

VEGFs (vascular endothelial growth factors) are a family of conserved disulfide-linked soluble secretory glycoproteins found in higher eukaryotes. VEGFs mediate a wide range of responses in different tissues including metabolic homoeostasis, cell proliferation, migration and tubulogenesis. Such responses are initiated by VEGF binding to soluble and membrane-bound VEGFRs (VEGF receptor tyrosine kinases) and co-receptors. VEGF and receptor splice isoform diversity further enhances complexity of membrane protein assembly and function in signal transduction pathways that control multiple cellular responses. Different signal transduction pathways are simultaneously activated by VEGFR–VEGF complexes with membrane trafficking along the endosome–lysosome network further modulating signal output from multiple enzymatic events associated with such pathways. Balancing VEGFR–VEGF signal transduction with trafficking and proteolysis is essential in controlling the intensity and duration of different intracellular signalling events. Dysfunction in VEGF-regulated signal transduction is important in chronic disease states including cancer, atherosclerosis and blindness. This family of growth factors and receptors is an important model system for understanding human disease pathology and developing new therapeutics for treating such ailments.     

Pharmacological characterization of a recombinant, fluorescent somatostatin receptor agonist

Somatostatin (SST) is a peptide neurotransmitter/hormone found in several mammalian tissue types. Apart from its natural importance, labeled SST/analogues are utilized in clinical applications such as targeting/diagnosis of neuroendocrine tumors. We report on the development and characterization of a novel, recombinant, fluorescent somatostatin analogue that has potential to elucidate somatostatin-activated cell signaling. SST was genetically fused with a monomeric-red fluorescent protein (mRFP) as the fluorescent label. The attachment of SST to mRFP had no detectable effect on its fluorescent properties. This analogue's potency to activate the endogenous and transfected somatostatin receptors was characterized using assays of membrane potential and Ca(2+) mobilization and immunocytochemistry. SST-mRFP was found to be an effective somatostatin receptor agonist, able to trigger the membrane hyperpolarization, mobilization of the intracellular Ca(2+) and receptor-ligand internalization in cells expressing somatostatin receptors. This complex represents a novel optical reporter due to its red emission spectral band suitable for in vivo imaging and tracking of the somatostatin receptor signaling pathways, affording higher resolution and sensitivity than those of the state-of-the-art radiolabeling bioassays.     

Evolution of opinions on social networks in the presence of competing committed groups

Public opinion is often affected by the presence of committed groups of individuals dedicated to competing points of view. Using a model of pairwise social influence, we study how the presence of such groups within social networks affects the outcome and the speed of evolution of the overall opinion on the network. Earlier work indicated that a single committed group within a dense social network can cause the entire network to quickly adopt the group''s opinion (in times scaling logarithmically with the network size), so long as the committed group constitutes more than about of the population (with the findings being qualitatively similar for sparse networks as well). Here we study the more general case of opinion evolution when two groups committed to distinct, competing opinions and , and constituting fractions and of the total population respectively, are present in the network. We show for stylized social networks (including Erdös-Rényi random graphs and Barabási-Albert scale-free networks) that the phase diagram of this system in parameter space consists of two regions, one where two stable steady-states coexist, and the remaining where only a single stable steady-state exists. These two regions are separated by two fold-bifurcation (spinodal) lines which meet tangentially and terminate at a cusp (critical point). We provide further insights to the phase diagram and to the nature of the underlying phase transitions by investigating the model on infinite (mean-field limit), finite complete graphs and finite sparse networks. For the latter case, we also derive the scaling exponent associated with the exponential growth of switching times as a function of the distance from the critical point.     

A VE-cadherin-PAR3-α-catenin complex regulates the Golgi localization and activity of cytosolic phospholipase A(2)α in endothelial cells

Phospholipase A(2) enzymes hydrolyze phospholipids to liberate arachidonic acid for the biosynthesis of prostaglandins and leukotrienes. In the vascular endothelium, group IV phospholipase A(2)α (cPLA(2)α) enzyme activity is regulated by reversible association with the Golgi apparatus. Here we provide evidence for a plasma membrane cell adhesion complex that regulates endothelial cell confluence and simultaneously controls cPLA(2)α localization and enzymatic activity. Confluent endothelial cells display pronounced accumulation of vascular endothelial cadherin (VE-cadherin) at cell-cell junctions, and mechanical wounding of the monolayer stimulates VE-cadherin complex disassembly and cPLA(2)α release from the Golgi apparatus. VE-cadherin depletion inhibits both recruitment of cPLA(2)α to the Golgi and formation of tubules by endothelial cells. Perturbing VE-cadherin and increasing the soluble cPLA(2)α fraction also stimulated arachidonic acid and prostaglandin production. Of importance, reverse genetics shows that α-catenin and δ-catenin, but not β-catenin, regulates cPLA(2)α Golgi localization linked to cell confluence. Furthermore, cPLA(2)α Golgi localization also required partitioning defective protein 3 (PAR3) and annexin A1. Disruption of F-actin internalizes VE-cadherin and releases cPLA(2)α from the adhesion complex and Golgi apparatus. Finally, depletion of either PAR3 or α-catenin promotes cPLA(2)α-dependent endothelial tubule formation. Thus a VE-cadherin-PAR3-α-catenin adhesion complex regulates cPLA(2)α recruitment to the Golgi apparatus, with functional consequences for vascular physiology.     

Levan: A Biocompatible Homopolysaccharide Excipient for Stabilization of Peptide Drugs

Stabilisation of protein/peptide drugs against thermal denaturation is a challenging problem, especially for liquid formulations. Various polysaccharides at high concentrations have been reported to improve stability of polypeptides, probably by providing a crowded environment which retards kinetic unfolding and resultant degradation. Levan is a fructose homopolysaccharide which is finding increasing use in pharmaceutical applications, but its use for protein drug stabilization remains meagre. In this study, we used levan for stabilizing a liquid preparation of a peptide antibiotic, bacitracin. We prepared liquid formulations of bacitracin with or without levan and subjected them to storage at 25 °C. The stored samples were then analysed over 120 days for denaturation and antibacterial activity. Differential Scanning Calorimetry, Circular Dichroism and High Performance Liquid Chromatography were used for evaluating the effect of levan on thermal denaturation of bacitracin. We found that levan at 2.5% w/v significantly preserved the antibacterial activity of bacitracin for 120 days as compared to plain buffered bacitracin, even when stored at 25 °C. Also, levan at high concentrations maintained the secondary structure and increased the melting temperature (Tm) of bacitracin in solution. Levan did not form covalent interactions or strong complexation with bacitracin. Based on this study, levan appears as a promising stabilizing agent for preparing liquid formulations of protein/peptide drugs that can be stored at room temperature.     

The trans Golgi Network Is Lost from Cells Infected with African Swine Fever Virus

The cellular secretory pathway is important during the assembly and envelopment of viruses and also controls the transport of host proteins, such as cytokines and major histocompatibility proteins, that function during the elimination of viruses by the immune system. African swine fever virus (ASFV) encodes at least 26 proteins with stretches of hydrophobic amino acids suggesting entry into the secretory pathway (R. J. Yanez, J. M. Rodriguez, M. L. Nogal, L. Yuste, C. Enriquez, J. F. Rodriguez, and E. Vinuela, Virology 208:249-278, 1995). To predict how and where these potential membrane proteins function, we have studied the integrity of the secretory pathway in cells infected with ASFV. Remarkably, ASFV caused complete loss of immunofluorescence signal for the trans Golgi network (TGN) marker protein TGN46 and dispersed the AP1 TGN adapter complex. Loss of TGN46 signal was not due to degradation of TGN46, suggesting redistribution of TGN46 to other membrane compartments. ASFV markedly slowed transport of cathepsin D to lysosomes, demonstrating that loss of TGN structure correlated with loss of TGN function. ASFV shows a tropism for macrophages, and it is possible that ASFV compromises TGN function to augment the activity of viral membrane proteins or to suppress the function of host immunoregulatory proteins.     

Limnological Features of a Tropical Impoundment,Bhavanisagar Reservoir (Tamil Nadu), India

In the Bhavanisagar Reservoir a stable thermal stratification did not occur, but the thermal stability was fairly high. Oxygen deficits occurred frequently as well as complete anoxic conditions. Influx of flood waters improved the bottom oxygen conditions. This together with other factors indicates a high level of metabolic activity and productivity. Primary production rates were fairly high. There has been a change in fish population; some introduced species (like Catla and Labeo calbasu) have increased in the catches. It is felt that more fish could be exploited from this reservoir.     

Rejoining of radiation-induced single-strand breaks in deoxyribonucleic acid of Escherichia coli: effect of phenethyl alcohol.

Single-strand breaks in deoxyribonucleic acid of Escherichia coli B/r cells exposed to 20 krads of gamma radiation could be rejoined by incubation of irradiated cells in growth medium. In the presence of 0.25% phenethyl alcohol, this repair was completely inhibited although deoxyribonucleic acid and protein syntheses were suppressed only partially.