Slit: a roadblock for chemotaxis.

The immune system and nervous system display striking similarities. Fernandis and Ganju discuss yet another example where a protein (Slit) originally identified for its role in modulating axon pathfinding is able to regulate immune cell migration. Slit isoforms expressed in the nervous system interact with members of the Robo receptor family to modify movement stimulated by the secreted attractants netrins and their receptors. In leukocytes, Slit 2 interacting with Robo receptors inhibits movement stimulated by the chemokine receptor (CXCR4). Fernandis and Ganju discuss the therapeutic potential of Slit as an antiviral agent and in the treatment of inflammatory diseases, autoimmune disorders, and cancer.

Tyrosyl phosphorylation and activation of a type II phosphatidylinositol 4-kinase by p56(lck) in concanavalin A stimulated rat splenic lymphocytes.

Recent evidence suggests that concanavalin A modulates tyrosyl phosphorylation and activation of a type II PtdIns 4-kinase in rat splenic lymphocytes. However, the regulatory protein tyrosine kinase(s) remain to be elusive. The present manuscript provides evidence that a type II PtdIns 4-kinase associates with p56(lck) in Con A stimulated rat splenic lymphocytes. In vitro phosphorylation studies suggest that p56(lck) regulates phosphorylation and activation of type II PtdIns 4-kinase. Inhibition of p56(lck) activity in vivo has shown to reduce tyrosyl phosphorylation and activation of PtdIns 4-kinase by Con A. These results suggest that p56(lck) may be the physiological regulator of type II PtdIns 4-kinase.

Concanavalin A modulates tyrosine phosphorylation and activation of a type II phosphatidylinositol 4-kinase in rat splenic lymphocytes.

Activation of rat splenic lymphocytes by concanavalin A resulted in two-fold increase in Ptdlns 4-kinase activity and rapid tyrosine phosphorylation of the enzyme. The activation kinetics showed a strong correlation with tyrosine phosphorylation state of the enzyme. Characterization of the enzyme activity suggests that it is a type II PtdIns 4-kinase. Kinetic analysis of the enzyme reaction showed three-fold decrease in Km for PtdIns and two-fold increase in Vmax in Con A stimulated cells. These results suggest that a type II PtdIns 4-kinase is an integral component of the early signal transduction machinery during T-cell activation by concanavalin A and is actively regulated by protein tyrosine phosphorylation-dephosphorylation.

Protein tyrosine phosphorylation activates rat splenic type II phosphatidylinositol 4-kinase in vitro.

Regulation of phosphatidylinositol 4-kinase (PtdIns 4-kinase) by protein tyrosine phosphorylation has been indirect and the effects of phosphorylation are debatable. Rat splenic type II PtdIns 4-kinase was phosphorylated in vitro with protein tyrosine kinases from Con A stimulated splenic lymphocytes. Stoichiometric analysis showed one mole of phosphate was incorporated per mole of PtdIns 4-kinase. Tyrosine phosphorylation increased the enzyme activity by 3-fold. Kinetic analysis showed a reduction in Km for PtdIns and an increase in Vmax. Dephosphorylation with protein phosphotyrosine phosphatase abolished the activation of PtdIns 4-kinase while protein phosphatase 2A had no effect. Protein tyrosine phosphorylation and activation of PtdIns 4-kinase appear to be tissue specific.

Overexpression of G1/S cyclins and PCNA and their relationship to tyrosine phosphorylation and dephosphorylation during tumor promotion by metanil yellow and malachite green.

Metanil yellow (MY) and Malachite green (MG) are textile dyes, which, despite the ban, occur unscrupulously as food colouring agents. Accordingly they constitute a serious public health hazard and are of sufficient environmental concern. We have earlier reported that both MY and MG have tumor promoting effects on the development of hepatic preneoplastic lesions induced by N-nitrosodiethylamine in rats. In order to understand the possible mechanism(s) by which metanil yellow (MY) and malachite green (MG) promotes liver tumor development, we have studied the tyrosine phosphorylation and protein phosphatases during tumor promotion. We have also investigated the possible overexpression of G1/S cyclins and PCNA during tumor promotion by MY and MG. The present investigation indicates that enhanced tyrosine phosphorylation is associated with no change in levels of tyrosine protein phosphatases. We have also observed an increase in the expression of PCNA and G1/S cyclins during tumor promotion. These factors collectively may contribute to the abnormal cell proliferation during tumor promotion by MY and MG.

Enhanced sequential expression of G1/S cyclins during experimental epatocarcinogenesis and tyrosine phosphorylation.

It is now widely accepted that cancer development is a multistage process, starting from the original cell population and ending with a malignant tumor. However, the mechanisms involved in the progressive growth of cells from normalcy to preneoplasia, and from preneoplasia to malignancy are not clear. Because tyrosine phosphorylation and dephosphorylation reactions are known to play critical roles during normal and abnormal cellular growth, we have studied the tyrosine phosphorylation, tyrosine phosphorylated proteins, and protein phosphatases during the sequential development of liver cancer. The present investigation indicated that enhanced tyrosine phosphorylation and tyrosine phosphorylated proteins, with no change in the levels of tyrosine protein phosphatases may contribute to abnormal cellular proliferation during liver carcinogenesis. We have also seen an increase in the expression of proliferating cell nuclear antigen and G1/S cyclins during tumor development.

Purification and characterization of a type II phosphatidylinositol 4-kinase from rat spleen and comparison with a PtdIns 4-kinase from lymphocytes.

A PtdIns 4-kinase from rat spleen particulate fraction was purified to homogeneity and its molecular properties were compared with a PtdIns 4-kinase from splenic lymphocytes. The enzyme activity was solubilized from spleen particulate fraction with Triton X-100 and chromatographed sequentially on phosphocellulose, DEAE-sephacel, heparin acrylamide and hydroxyapatite columns. The purified enzyme preparation showed a 55 kDa band on SDS-PAGE with silver staining. Renaturation of the enzyme activity from SDS-PAGE showed that it comigrated with the 55 kDa protein. Characterization of the enzyme showed that it was a type II PtdIns 4-kinase. Polyclonal antibodies raised against PtdIns 4-kinase inhibited the enzyme activity in in vitro assays. Analysis of adult rat tissue particulate fractions on immunoblots showed restricted immunoreactivity among PtdIns 4-kinases. However, the immunoreactivity is conserved in lymphoid tissues from mouse to human, suggesting that lymphoid tissue has a distinct PtdIns 4-kinase. Activation of rat splenocytes with Con A showed two fold increase in PtdIns 4-kinase activity. Comparison of PtdIns 4-kinases from spleen and splenic lymphocytes showed identical chromatographic behaviour, molecular mass, immunoreactivity, K(m) values for PtdIns and inhibition by adenosine.

Lipidomic analysis of human placental syncytiotrophoblast microvesicles in adverse pregnancy outcomes.

PROBLEM: Syncytiotrophoblast microvesicles (STBM) are shed from placenta into the maternal circulation. STBM circulate in increased amounts in adverse pregnancies, e.g., preeclampsia and recurrent miscarriages (RM). Recently dysregulation of lipid metabolites has been proposed to be associated with their pathogenesis. Lipid composition of STBM in healthy and adverse pregnancies remains unknown. OBJECTIVE: To determine lipid composition of STBM and whether STBM lipid composition differs in pathologic and normal pregnancies. STUDY DESIGN: Patients with Preeclampsia (n = 6) or history of RM (n = 9) (>2 consecutive losses <20 weeks) and gestational age-matched normal pregnant controls (same number as cases) were recruited. STBM were prepared from placental explant culture supernatant. Lipid profiling of STBM was performed by mass spectrometry in combination with liquid chromatography. We quantified ∼200 lipids in STBM including (i) glycerophospholipids (phosphatidylcholine, PC; phosphatidylethanolamine, PE; phosphatidylinositol, PI; phosphatidylglycerol, PG; phosphatidylserine, PS; phosphatidic acid, PA); (ii) sphingolipids (sphingomyelin, SM; ceramide, Cer; Glucosylceramide, GluCer; ganglioside mannoside 3, GM3); (iii) free cholesterol and cholesteryl esters, CE. RESULTS: The major lipid classes in STBM were SM, Chol, PS, PC and PI, along with PA and GM3 enrichments. SM/PC ratio showed a unique reversal (3:1) compared to that normally found in human cells or plasma. Level of total PS was significantly upregulated (p < 0.005) in preeclampsia patients, while PI (p < 0.0005), PA (p < 0.005), and GM3 (p < 0.05) were significantly downregulated. Similar trends were obtained in RM. CONCLUSIONS: Differential lipid expression of STBM in preeclampsia or RM includes those that are implicated in immune response, coagulation, oxidative stress, and apoptosis.