Src homology 3 binding sites in the P2Y2 nucleotide receptor interact with Src and regulate activities of Src, proline-rich tyrosine kinase 2, and growth factor receptors

Many G protein-coupled receptors activate growth factor receptors, although the mechanisms controlling this transactivation are unclear. We have identified two proline-rich, SH3 binding sites (PXXP) in the carboxyl-terminal tail of the human P2Y(2) nucleotide receptor that directly associate with the tyrosine kinase Src in protein binding assays. Furthermore, Src co-precipitated with the P2Y(2) receptor in 1321N1 astrocytoma cells stimulated with the P2Y(2) receptor agonist UTP. A mutant P2Y(2) receptor lacking the PXXP motifs was found to stimulate calcium mobilization and serine/threonine phosphorylation of the Erk1/2 mitogen-activated protein kinases, like the wild-type receptor, but was defective in its ability to stimulate tyrosine phosphorylation of Src and Src-dependent tyrosine phosphorylation of the proline-rich tyrosine kinase 2, epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor. Dual immunofluorescence labeling of the P2Y(2) receptor and the EGFR indicated that UTP caused an increase in the co-localization of these receptors in the plasma membrane that was prevented by the Src inhibitor PP2. Together, these data suggest that agonist-induced binding of Src to the SH3 binding sites in the P2Y(2) receptor facilitates Src activation, which recruits the EGFR into a protein complex with the P2Y(2) receptor and allows Src to efficiently phosphorylate the EGFR.     

Mechanisms of P2X7 receptor-mediated ERK1/2 phosphorylation in human astrocytoma cells

Astrocytes are involved in normal andpathological brain functions, where they become activated and undergoreactive gliosis. Astrocytes have been shown to respond toextracellular nucleotides via the activation of P2 receptors, either Gprotein-coupled P2Y receptors or P2X receptors that are ligand-gatedion channels. In this study, we have examined the manner in whichactivation of the P2X₇ nucleotide receptor, anextracellular ATP-gated ion channel expressed in astrocytes, can leadto the phosphorylation of ERK1/2. Results showed that theP2X₇ receptor agonist2',3'-O-(4-benzoyl)benzoyl-ATP induced ERK1/2phosphorylation in human astrocytoma cells overexpressing therecombinant rat P2X₇ receptor (rP2X₇-R), aresponse that was inhibited by the P2X₇ receptorantagonist, oxidized ATP. Other results suggest thatrP2X₇-R-mediated ERK1/2 phosphorylation was linked to thephosphorylation of the proline-rich/Ca²⁺-activated tyrosinekinase Pyk2, c-Src, phosphatidylinositol 3'-kinase, and proteinkinase C activities and was dependent on the presence ofextracellular Ca²⁺. These results support the hypothesisthat the P2X₇ receptor and its signaling pathways play arole in astrocyte-mediated inflammation and neurodegenerative disease.

     

The P2Y2 nucleotide receptor mediates UTP-induced vascular cell adhesion molecule-1 expression in coronary artery endothelial cells

P2Y2 receptor up-regulation and activation induces intimal hyperplasia and monocyte/macrophage infiltration in the collared rabbit carotid artery model of vascular injury, suggesting a potential role for P2Y2 receptors in monocyte recruitment by vascular endothelium. In this study, we addressed the hypothesis that activation of P2Y2 receptors by extracellular nucleotides modulates the expression of adhesion molecules on vascular endothelial cells that are important for monocyte recruitment. Results indicated that the equipotent P2Y2 receptor agonists UTP or ATP (1-100 microm) stimulated the expression of vascular cell adhesion molecule-1 (VCAM-1) in human coronary artery endothelial cells (HCAEC) in a time- and dose-dependent manner. P2Y2 antisense oligonucleotides inhibited VCAM-1 expression induced by UTP but not by tumor necrosis factor-alpha. Furthermore, UTP induced VCAM-1 expression in human 1321N1 astrocytoma cell transfectants expressing the recombinant P2Y2 receptor, whereas vector-transfected control cells did not respond to UTP. The effect of UTP on VCAM-1 expression in HCAEC was prevented by depletion of intracellular calcium stores with thapsigargin or by inhibition of p38 mitogen-activated protein kinase or Rho kinase, but was not affected by inhibitors of the mitogen-activated protein/extracellular signal-regulated kinase pathway (i.e. MEK1/2). Consistent with a role for VCAM-1 in the recruitment of monocytes, UTP or ATP increased the adherence of monocytic U937 cells to HCAEC, an effect that was inhibited by anti-VCAM-1 antibodies. These findings suggest a novel role for the P2Y2 receptor in the p38- and Rho kinase-dependent expression of VCAM-1 that mediates the recruitment of monocytes by vascular endothelium associated with the development of atherosclerosis.     

Multiple Allelism of the net Gene in Drosophila melanogaster and Drosophila simulans

Thenetgene mutations are known to cause abnormal pattern of veining in all wing regions except for the first posterior cells. In natural populations of Drosophila melanogaster, the net alleles were identified, which differ in phenotypic expression from standard mutations. The mutants net-extra-analis from a population Belokurikha-2000 have only a single additional vein in the third posterior cell. A line from Chernobyl-1986 population have another nontypical allele net ^Ch86 and shows a lower degree of abnormalities than that usually observed. About 10% of these flies have an additional vein fragment in the first posterior cell. In both males and females ofD. simulans population Tashkent -2001, which exhibit net ^ST91 mutation, a net of additional veins is formed as a specific additional fragment in the first posterior cell. The pattern of veining conferred by alleles net-extra-analis and net ^Ch86 is altered to a lesser extent; these alleles are dominant with respect to alleles net ^2-45 and net ^ST91, which cause more abnormalities. The heterozygotes for alleles net ^ST9 and net ^Ch86 and for Df(2) net ⁶² deletion have an additional fragment in the first posterior cell and show similarly strong deviations from normal wing vein pattern. The naturalnet alleles correspond, presumably, to different molecular gene defects involved into uncertain local interactions with numerous modifying factors and other genes that specify the wing vein pattern.     

Regulation of Fab1 phosphatidylinositol 3-phosphate 5-kinase pathway by Vac7 protein and Fig4, a polyphosphoinositide phosphatase family member

The Saccharomyces cerevisiae FAB1 gene encodes the sole phosphatidylinositol 3-phosphate [PtdIns(3)P] 5-kinase responsible for synthesis of the polyphosphoinositide PtdIns(3,5)P(2). VAC7 encodes a 128-kDa transmembrane protein that localizes to vacuolar membranes. Both vac7 and fab1 null mutants have dramatically enlarged vacuoles and cannot grow at elevated temperatures. Additionally, vac7Delta mutants have nearly undetectable levels of PtdIns(3,5)P(2), suggesting that Vac7 functions to regulate Fab1 kinase activity. To test this hypothesis, we isolated a fab1 mutant allele that bypasses the requirement for Vac7 in PtdIns(3,5)P(2) production. Expression of this fab1 allele in vac7Delta mutant cells suppresses the temperature sensitivity, vacuolar morphology, and PtdIns(3,5)P(2) defects normally exhibited by vac7Delta mutants. We also identified a mutant allele of FIG4, whose gene product contains a Sac1 polyphosphoinositide phosphatase domain, which suppresses vac7Delta mutant phenotypes. Deletion of FIG4 in vac7Delta mutant cells suppresses the temperature sensitivity and vacuolar morphology defects, and dramatically restores PtdIns(3,5)P(2) levels. These results suggest that generation of PtdIns(3,5)P(2) by the Fab1 lipid kinase is regulated by Vac7, whereas turnover of PtdIns(3,5)P(2) is mediated in part by the Sac1 polyphosphoinositide phosphatase family member Fig4.     

On the role of protein phosphorylation in the ATP-dependent permeabilization of transformed cells

Incubation of transformed mouse fibroblasts with external ATP in alkaline medium low in divalent cations causes an increase in the permeability of the plasma membrane to nucleotides and other small molecules. Previous suggestions that the phosphorylation of a 44,000 dalton membrane protein is involved in this permeabilization process have been pursued. Fractionation of cells that had been incubated with [γ-³²P] ATP revealed that the labeled 44K phosphoprotein was found in both the membrane and mitochondrial fractions. Incubation of fractions isolated from unlabeled cells with [γ-³²P] ATP resulted in substantial formation of ³²P-44K in the mitochondrial fraction and less incorporation in the membrane fraction. The 44,000 dalton protein was identified as the α-subunit of mitochondrial pyruvate dehydrogenase by partial proteolytic mapping and immunological cross-reactivity with antibodies prepared against bovine pyruvate dehydrogenase. The phosphorylation of this protein in whole cells by externally added ATP is suppressed by inclusion in the incubation medium of carboxyatractyloside (CAT) and EDTA. These substances have no effect on ATP-dependent permeabilization, indicating that the phosphorylation of pyruvate dehydrogenase is not involved in this process.     

Modeling brewers' yeast flocculation

Flocculation of yeast cells occurs during the fermentation of beer. Partway through the fermentation the cells become flocculent and start to form flocs. If the environmental conditions, such as medium composition and fluid velocities in the tank, are optimal, the flocs will grow in size large enough to settle. After settling of the main part of the yeast the green beer is left, containing only a small amount of yeast necessary for rest conversions during the next process step, the lagering. The physical process of flocculation is a dynamic equilibrium of floc formation and floc breakup resulting in a bimodal size distribution containing single cells and flocs. The floc size distribution and the single cell amount were measured under the different conditions that occur during full scale fermentation. Influences on flocculation such as floc strength, specific power input, and total number of yeast cells in suspension were studied. A flocculation model was developed, and the measured data used for validation. Yeast floc formation can be described with the collision theory assuming a constant collision efficiency. The breakup of flocs appears to occur mainly via two mechanisms, the splitting of flocs and the erosion of yeast cells from the floc surface. The splitting rate determines the average floc size and the erosion rate determines the number of single cells. Regarding the size of the flocs with respect to the scale of turbulence, only the viscous subrange needs to be considered. With the model, the floc size distribution and the number of single cells can be predicted at a certain point during the fermentation. For this, the bond strength between the cells, the fractal dimension of the yeast, the specific power input in the tank and the number of yeast cells that are in suspension in the tank have to be known. Copyright 1998 John Wiley & Sons, Inc.     

In Vitro Studies on the Biosynthesis of the Surface Glycoprotein of Trypanosoma congolense1,2

Tritiated leucine, glucosamine, mannose, and galactose were incorporated into the variant specific surface glycoprotein (VSG) of Trypanosoma congolense in vitro. The uptake of the precursors is shown by SDS-polyacrylamide electrophoresis and fluorography, by assay of the radioactivity in immunoprecipitates obtained with specific antisera, and by the isolation of the labeled antigens by affinity chromatography on concanavalin A-sepharose and isoelectric focusing. The in vitro labeled VSG exhibits the same degree of microheterogeneity as that observed in the VSG isolated from trypanosomes grown in animals. Analysis of the incorporated sugars after hydrolysis of the glycoprotein showed that glucosamine and mannose were utilized in biosynthesis of the carbohydrate moiety directly whereas galactose was converted possibly to other intermediates before being incorporated into the antigen. Tunicamycin completely prevented the incorporation of the radiolabeled sugars into the surface glycoprotein. The unglycosylated VSG with a molecular weight of 47 kDa had completely lost its size heterogeneity.     

Role of heterotrophic nutrition in growth of the alga Scenedesmus obliquus in high-rate oxidation ponds

The green alga Scenedesmus obliquus readily adapted to heterotrophic growth in the dark, utilizing glucose as the sole carbon source. Heterotrophic cells differed significantly from photoautotrophic cells with respect to several physiological properties such as the rate of photoassimilation of CO2, rate of incorporation of glucose, and chlorophyll a concentration. Oxidation pond cells shared features common to both photoautotrophic and heterotrophic cells. Approximately 15 percent of oxidation pond algal carbon was derived from glucose assimilated directly without first being oxidized by bacteria. Bacteria seem to play a minor role in biological oxygen demand reduction in high-rate oxidation ponds, and their role is probably confined to degradation of biopolymers, thus producing substrates for algal consumption.     

Active Ca2+ transport by vesicles reconstituted from triton X-100-solubilized pigeon erythrocyte membrane

Pigeon erythrocyte membrane was solubilized partially, but relatively unselectively by Triton X-100. Vesicles were reconstituted from mixtures of Triton-solubilized membrane and lipid (phosphatidylcholine plus phosphatidylethanolamine plus cholesterol) by addition of bovine high-density lipoprotein. This efficiently removed the Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electropherograms of reconstituted vesicles showed band patterns resembling those of the original membrane. The reconstituted vesicles showed ATP-dependent active accumulation of ⁴⁵Ca²⁺. ATP-dependent ⁴⁵Ca²⁺ uptake by the reconstituted vesicles resembled the corresponding activity of the original membrane vesicles; in both preparations the Ca²⁺ uptake rate depended on the square of the Ca²⁺ concentration and had similar $\text{[Ca}{}^{\mathrm{2+}}\text{]}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values, 0.16 μM and 0.18 μM, respectively.