Sphingosine 1-phosphate-induced cell rounding and neurite retraction are mediated by the G protein-coupled receptor H218

Sphingosine 1-phosphate (SPP) is a lipid second messenger that also acts as a first messenger through the G protein-coupled receptor Edg-1. Here we show that SPP also binds to the related receptors H218 and Edg-3 with high affinity and specificity. SPP and sphinganine 1-phosphate bind to these receptors, whereas neither sphingosylphosphorylcholine nor lysophosphatidic acid compete with SPP for binding to either receptor. Transfection of HEK293 cells with H218 or edg-3, but not edg-1, induces rounded cell morphology in the presence of serum, which contains high levels of SPP. SPP treatment of cells overexpressing H218 cultured in delipidated serum causes cell rounding. A similar but less dramatic effect was observed in cells overexpressing Edg-3 but not with Edg-1. Cell rounding was correlated with apoptotic cell death, probably as a result of loss of attachment. Nerve growth factor-induced neuritogenesis in PC12 cells was inhibited by overexpression of H218 and to a lesser extent Edg-3. SPP treatment rapidly enhanced neurite retraction in PC12 cells overexpressing Edg-1, Edg-3, or H218. Thus, H218, and possibly Edg-3, may be the cell surface receptors responsible for cell rounding and neurite retraction induced by SPP. Moreover, the identification of these two additional SPP receptors indicates that a family of highly specific receptors exists that mediate different responses to SPP.     

Exogenous sphingosine 1-phosphate induces neurite retraction possibly through a cell surface receptor in PC12 cells

Alterations in thyroid function tests are very common in patients with NTI. Multiple, complex, and incompletely understood mechanisms are involved in these abnormalities. Knowledge of these abnormalities is necessary to avoid errors in the diagnosis of thyroid disease. Measurement of serum TSH, free T4, and free T3 levels by direct equilibrium dialysis/RIA methods probably yield most useful (accurate) information in the setting of NTI. Patients with low free T4 by these methods and normal or low TSH have secondary hypothyroidism. This may be due to NTI per se, drugs administered for treatment of NTI, or associated pituitary or hypothalamic disease; the latter consideration may require evaluation of cortisol reserve, PRL, and/or gonadotropins. A serum TSH level above 20-25 microU/mL probably reflects primary hypothyroidism; accompanying findings of goiter, low free T4, and positive antithyroid antibodies help establish the diagnosis. An elevated serum concentration of rT3 argues against hypothyroidism. Studies have demonstrated no discernible benefit of treatment of NTI patients with T4. Some studies have shown a few benefits of treatment with T3 in selected cases, but much more needs to be learned. There is no evidence of harm by treatment of NTI patients with up to replacement doses of T3. As some NTI patients may indeed be hypothyroid, the term ESS should be replaced with NTIS.     

Aberrant tau phosphorylation and neurite retraction during NGF deprivation in PC12 cells

OBJECTIVE: To investigate the final results of cases with preoperative diagnoses of thyroid follicular neoplasms by fine needle aspiration cytology (FNAC). STUDY DESIGN: A retrospective review of 6,499 patients who received thyroid ultrasonography with FNAC at Chang Gung Memorial Hospital. Among 6,499 patients, 209 (3.2%) were diagnosed by FNAC as having follicular neoplasms, of which 84 received surgical treatment. Eighty-two of the 84 cases had a frozen section prepared during the operation. RESULTS: Thyroid malignancy was confirmed histopathologically in 164 cases. Among 84 thyroid follicular neoplasm patients, 21 cases were diagnosed as malignant tumors, including papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma and Hürthle's cell carcinoma. Ultrasonography on these 21 patients revealed that 16 cases (76.2%) had low echo density in the thyroid nodule. The percentage was statistically significantly different from that in benign cases, 23.8% (P < .05). A higher incidence of malignancy was found in males, but the data did not reach statistical significance (P = .0586). CONCLUSION: Most of the follicular neoplasms revealed by FNAC were benign lesions. Low echo density on ultrasonography and male sex carried a higher risk of malignancy.     

Modulation of cell surface fibronectin assembly sites by lysophosphatidic acid

Lysophosphatidic acid is a product of activated platelets and has diverse actions on cells. We have characterized the effect of lysophosphatidic acid on cell-mediated binding and assembly of fibronectin, an extracellular matrix protein. Serum made from whole blood, but neither platelet-poor plasma nor serum made from platelet-poor plasma, caused enhanced binding of fibronectin to cultured fibroblastic cells. The ability of whole blood serum to enhance binding of fibronectin was abolished by phospholipase B. These results indicate that lysophosphatidic acid derived from platelets is the principal component in whole blood serum that is active in the fibronectin binding assay. 1-oleoyl lysophosphatidic acid, 20-200 nM, was as active as 0.1-0.2% whole blood serum. The stimulatory effect of lysophosphatidic acid on the binding of fibronectin or the amino-terminal 70-kD fragment of fibronectin was rapid, sustained, and lost upon removal of lysophosphatidic acid. The stimulatory effect on binding could not be duplicated by bradykinin, platelet-activating factor, bombesin, or a peptide agonist of the thrombin receptor. Enhanced binding of the 70-kD fragment was due to increases in both the number and affinity of binding sites. Enhanced binding and assembly of fibronectin correlated with changes in cell shape and actin-containing cytoskeleton. The binding sites for fibronectin on lysophosphatidic acid-stimulated cells, as assessed by fluorescence, video, and scanning electron microscopy, were on areas of cell membrane containing numerous filopodia that extended between cells or between cells and substratum. These observations suggest that lysophosphatidic acid functions as a powerful and specific modulator of cell shape and early matrix assembly during wound healing.     

Restoration of beta1A integrins is required for lysophosphatidic acid-induced migration of beta1-null mouse fibroblastic cells

Cells lacking the beta1 integrin subunit or expressing beta1A with certain cytoplasmic mutations have poor directed cell migration to platelet-derived growth factor or epidermal growth factor, ligands of receptor tyrosine kinases (Sakai, T., Zhang, Q., F?ssler, R., and Mosher, D. F. (1998) J. Cell Biol. 141, 527-538). We investigated the effect of expression of beta1A integrins on lysophosphatidic acid (LPA)-induced migration of fibroblastic cells derived from beta1-null mouse embryonic stem cells. These cells expressed edg-2, a G-protein-linked receptor for LPA, as well as the related edg-1 receptor. Cells expressing wild type beta1A demonstrated enhanced cell migration across filters coated with gelatin or adhesive proteins in response to LPA, whereas beta1-deficient cells lacked LPA-induced cell migratory ability. Checkerboard analyses indicated that LPA causes both chemotaxis and chemokinesis of beta1-replete cells. Cells expressing beta1A with mutations of prolines or tyrosines in conserved cytoplasmic NPXY motifs, threonine in the inter-motif sequence, or a critical aspartic acid in the extracellular domain had low migratory responses to LPA. These findings indicate that active beta1A integrin is required for cell migration induced by LPA and that the cytoplasmic domain of ligated beta1A interacts with pathways that are common to both receptor tyrosine kinase and G-protein-linked receptor signaling.     

Excess formation of lysophosphatidic acid with age inhibits myristic acid-induced superoxide anion generation in intact human neutrophils

AIM: To examine whether the elevation of endogenous NG, NG-dimethylarginine (DMA) content is related to lipid peroxidation in the high lipid-fed rabbit. METHODS: In high lipid diet-fed rabbits, concentrations of serum cholesterol, triglyceride, malondialdehyde (MDA), and DMA were measured, and endothelium-dependent relaxation to acetylcholine (ACh) was tested. RESULTS: After 6-wk on a high lipid-diet, the levels of serum total cholesterol, triglyceride, MDA, and DMA were increased vs those in control group (MDA was 2.88 +/- s 0.20 vs 1.54 +/- 0.13 nmol.L-1, P < 0.01 and DMA was 1.51 +/- 0.07 vs 0.75 +/- 0.13 mumol.L-1, P < 0.01), while the endothelium-dependent vasodilation in the isolated thoracic aorta was impaired (the maximal response to ACh was 45.59 +/- 3.10 vs 76.93 +/- 5.68%). Supplementation with vitamin E decreased MDA and DMA content and improved the endothelium-dependent vasodilation. CONCLUSIONS: An increase in serum concentration of DMA may be secondary to the elevation of lipid peroxides in the hyperlipidemic rabbit.     

Retinoic acid-induced HL-60 cell differentiation is augmented by copper supplementation

The objective of this study was to determine how copper influences the ability of HL-60 cells to differentiate into cells of the granulocytic lineage. We hypothesized that granulopoiesis requires copper because copper-deficient humans become neutropenic. Differentiation of HL-60 cells along the granulocytic lineage with retinoic acid was enhanced by copper. The results showed a greater number of cells were more differentiated when copper was added to the medium for 96 h. The respiratory burst activity of retinoic acid-induced cells was increased by copper supplementation, but intracellular superoxide anion generation was not affected. Supplementation with copper resulted in more cell-associated copper in both noninduced and induced cells; however, the induced cells accumulated three times more copper than the noninduced cells. Even though the amount of copper associated with retinoic acid-treated cells was greater than in untreated cells, the activity of a copper-requiring enzyme, copper/zinc superoxide dismutase, was significantly lower. Copper supplementation increased the activity of this enzyme in both retinoic acid-treated and untreated cells. Cytochrome c oxidase activity was not affected by retinoic acid treatment or by copper supplementation. Copper seems to play a specific role during the early stages of granulocyte differentiation.     

Inhibition of natural killer cell cytotoxicity by cell growth-related molecules

Certain MHC class I molecules on target cells are known to inhibit the cytotoxic action of NK cells. By using monoclonal antibody (mAb) Cho-1, we have found inhibitory non-MHC class I cell surface molecules that are noncovalently-associated with 200 kDa and 40 kDa antigens. Poly I-C-induced rat NK cells were not cytotoxic to rat fetus-derived fibroblast WFB cell line. In contrast, NK cells were cytotoxic to H-ras oncogene-induced transformants of WFB, W14 and W31. FACS analysis indicated that mAb Cho-1 reacts with WFB, but not with W14 and W31 cells. Thus, this antigen may disappear concomitantly with cell growth and transformation. Cho-1 antigens were also expressed on other NK-resistant lines, such as mouse BALB3T3 fibroblast, EL-4 lymphoma and human fibroblast HEPM. However, they were not expressed on NK-sensitive mouse YAC-1 and H-ras transformant (Brash) of BALB3T3 cells. Furthermore, treatment of target cells with IFN-gamma clearly induced the cell surface expression of Cho-1 antigens, and conferred a resistance to NK cytolysis on target cells. These data strongly suggest that Cho-1 antigen expression may correlate with target cell susceptibility to NK cells. Indeed, treatment of NK-resistant WFB as well as HEPM cells with F(ab')2 fragments of mAb Cho-1 resulted in the acquisition of susceptibility to NK cytolysis. Cho-1 antigens may be novel molecules that regulate the NK resistance of cells.     

Inhibition of muscarinic receptor-stimulated glial cell proliferation by ethanol

Acetylcholine and other muscarinic agonists stimulate the proliferation of rat cortical astrocytes and 132 1N1 human astrocytoma cells by activating muscarinic m3 cholinergic receptors. Ethanol was a potent inhibitor of carbachol-stimulated proliferation, measured by [3H]thymidine incorporation, with an IC50 of 10 mM. On the other hand, basal and serum-stimulated proliferation of astrocytes and astrocytoma cells was inhibited by ethanol with lower potency (IC50 = 200-250 mM). Concentration-response experiments with carbachol, in the presence of 10 mM ethanol, suggested that inhibition of proliferation by the alcohol was of the noncompetitive type. Experiments with acetaldehyde and with the alcohol dehydrogenase inhibitor 4-methylpyrazole suggested that the inhibitory effect of alcohol was due to ethanol itself and not to its metabolite acetaldehyde. Proliferation of astrocytoma cells induced by carbachol and the inhibitory effects of ethanol were also confirmed by flow cytometry using the 5-bromodeoxyuridine-Hoechst 33258 method. Ethanol (10 mM) had no effect on proliferation induced by 50 micrograms/ml insulin and 100 ng/ml platelet-derived growth factor BB; on the other hand, the mitogenic effect of 1 mM histamine, 100 U/ml interleukin-1, and 100 ng/ml 12-O-tetradecanoylphorbol 13-acetate were inhibited by approximately 50%. These results indicate that proliferation of glial cells induced by muscarinic agonists is especially sensitive to the inhibitory effect of ethanol. This action of ethanol may be relevant to its developmental neurotoxicity, particularly microencephaly, which is one of the common features of the fetal alcohol syndrome.     

Tenascin-C inhibits beta1 integrin-dependent cell adhesion and neurite outgrowth on fibronectin by a disialoganglioside-mediated signaling mechanism

We have previously shown that the extracellular matrix molecule tenascin-C inhibits fibronectin-mediated cell adhesion and neurite outgrowth by an interaction with a cellular RGD-independent receptor which interferes with the adhesion and neurite outgrowth promoting activities of the fibronectin receptor(s). Here we demonstrate that the inhibitory effect of tenascin-C on beta1integrin-dependent cell adhesion and neurite outgrowth is mediated by the interaction of the protein with membrane-associated disialogangliosides, which interferes with protein kinase C-related signaling pathways. First, in substratum mixtures with fibronectin, an RGD sequence-containing fragment of the molecule or synthetic peptide, tenascin-C inhibited cell adhesion and spreading by a disialoganglioside-dependent, sialidase-sensitive mechanism leading to an inhibition of protein kinase C. Second, the interaction of intact or trypsinized, i.e., cell surface glycoprotein-free, cells with immobilized tenascin-C was strongly inhibited by gangliosides or antibodies to gangliosides and tenascin-C. Third, preincubation of immobilized tenascin-C with soluble disialogangliosides resulted in a delayed cell detachment as a function of time. Similar to tenascin-C, immobilized antibody to GD2 (3F8) or sphingosine, a protein kinase C inhibitor, strongly inhibited RGD-dependent cell spreading. Finally, the degree of tenascin-C-induced inhibition of cell adhesion was proportional to the degree of disialoganglioside levels of expression by different cells suggesting the relevance of such mechanism in modulating integrin-mediated cell-matrix interactions during pattern formation or tumor progression.     

Inhibition of peripheral blood mononuclear cell proliferation by cardiac glycosides

We prospectively studied the epidemiologic, clinic signs and outcome of bacterial meningitis in 125 children who were admitted into a PICU (Pediatric Intensive Care Unit) of 11 hospitals of Spain and whose meningitis was diagnosed between May 1994 and April 1995. RESULTS: The median age of the children was 3.55 +/- 3.32 years (range 1 month to 16.5 yrs). Eighty-eight were bacterial meningitis, probably bacterial 30 and aseptic 7. The most frequently isolated organisms were N. meningitidis (52), H. influenza type b (17) and S. pneumoniae (8). Twenty-five percent of N. meningitidis had C serotype. Incidence rate of each germen was depending of age. All patients diagnosed of H. influenza type b meningitis were less than 3 years old. H. influenza type b and meningococcus had similar incidence rate during the first year of life (27% versus 31%). During the first three years of life H. influenza type b produced one third of bacterial meningitis. A mortality rate of 5.6% (seven patients: 3 S. pneumoniae, 1 N. meningitidis, 1 H. influenza type b and 2 unknown germen) was observed. Patients who die had lower Glasgow coma score (p = 0.034) and seizures (p = 0.001) at admission. At discharge of PICU, 9 survivors (7.2%) had sequelae: mental retardation in 7 patients and hearing loss in two. One third of patients needed hemodynamic support and a 15% of them ventilatory support. CONCLUSIONS: Age is an important epidemiological factor in the etiology of pediatric acute meningitis. H. influenza type b and N. meningitidis had similar incidence rate during the first year of life. S. pneumoniae had the highest mortality rate (37.5%). The presence of coma and seizures at admission were associated with mortality.     

Inhibition of smooth muscle cell proliferation and neointimal growth by low-anticoagulant heparin

Low-anticoagulant heparin (LA-heparin) obtained by affinity chromatography on antithrombin III Sepharose inhibits the proliferation of cultured arterial smooth muscle cells in an in vitro bioassay system as effectively as standard heparin. A growth inhibition of smooth muscle cells of about 60% is achieved when LA-heparin or heparin is added to the culture medium to a concentration of 50 micrograms/ml. In normolipemic rats LA-heparin suppresses the formation of neointimal thickenings and stenosis after balloon catheter-induced deendothelialization of the carotid artery. In terms of mass a dose of 5 mg/kg body weight/d given subcutaneously twice daily one week before and 2 weeks after balloon injury the cross sectional area of the neointima is reduced to 36% as compared with the nontreated control group (100%). This 64% reduction is statistically highly significant (p < 0.001). After treatment with 0.5 mg LA-heparin/kg/d the reduction of the neointima was 11% (p < 0.05). At a dose of 5 mg/kg body weight single or repeated administrations of LA-heparin caused only a small and transient increase in activated partial thromboplastin time values. The results show that subcutaneous administration of LA-heparin very effectively prevents smooth muscle cell proliferation and balloon catheter-induced neointimal growth. The well tolerated systemic application of this chemically non-modified LA-heparin might justify clinical trials for prevention of restenosis after percutaneous transluminal coronary angioplasty or other invasive cardiovascular interventions without complications of bleeding.     

Inhibition of glomerular mesangial cell proliferation and extracellular matrix deposition by halofuginone

Mesangial cell proliferation, increased deposition of collagen, and expansion of the mesangial extracellular matrix (ECM) are key features in the development of mesangioproliferative diseases. Halofuginone, a low molecular weight anti-coccidial quinoazolinone derivative, inhibits collagen type alpha 1(I) gene expression and synthesis. We investigated the effect of halofuginone on both normal and SV40 transformed mesangial cell proliferation, collagen synthesis, and ECM deposition. Proliferation of both cell types was almost completely inhibited in the presence of 50 ng/ml halofuginone. The cells were arrested in the late G1 phase of the cell cycle and resumed their normal growth rate following removal of the compound from the culture medium. The antiproliferative effect of halofuginone was associated with inhibition of tyrosine phosphorylation of cellular proteins. Similar results were obtained whether the mesangial cells were seeded on regular tissue culture plastic or in close contact with a naturally produced ECM resembling their local environment in vivo. Halofuginone also inhibited synthesis and deposition of ECM by mesangial cells as indicated by a substantial reduction in 14C-glycine and Na2(35)SO4 incorporation into the ECM, and by the inhibition of collagen type I synthesis and gene expression. It is proposed that by inhibiting collagen type I synthesis and matrix deposition, halofuginone exerts a potent antiproliferative effect that may be applied to inhibit mesangial cell proliferation and matrix expansion in a variety of chronic progressive glomerular diseases.     

Requirement for Rho-mediated myosin light chain phosphorylation in thrombin-stimulated cell rounding and its dissociation from mitogenesis

Thrombin treatment causes a dose-dependent rounding of 1321N1 astrocytoma cells. This cytoskeletal response is rapid, peaking 2 h after thrombin stimulation, and reverses by 50% after 24 h. The thrombin receptor peptide SFLLRNP also induces cell rounding, whereas other G protein-linked receptor agonists such as carbachol, lysophosphatidic acid, or bradykinin fail to do so. Results of studies using pharmacological inhibitors do not support a requirement for phosphatidylinositol 3-kinase, mitogen-activated protein kinase, or Ca2+ mobilization in this response. Inhibition of protein kinase C or tyrosine kinase produces minimal blockade. Pertussis toxin treatment is also without effect. However, thrombin-induced rounding is fully blocked by the C3 toxin from Clostridium botulinum, which specifically ADP-ribosylates and inactivates the small G protein Rho. Thrombin also leads to a rapid, 2.4-fold increase in 32P incorporation into myosin light chain while carbachol does not. Myosin phosphorylation, like cell rounding is inhibited by inactivation of Rho with C3 exoenzyme, suggesting that myosin phosphorylation is necessary for this cytoskeletal response. This is supported by the observation that thrombin-induced rounding is also blocked by the myosin light chain kinase inhibitor KT5926. However, treatment with KT5926 fails to inhibit mitogenesis. Thus, cell rounding is not prerequisite to thrombin-induced DNA synthesis. We conclude that stimulation of the heterotrimeric G protein-coupled thrombin receptor in 1321N1 cells activates Rho-dependent pathways for both DNA synthesis and cell rounding, the cytoskeletal response being mediated in part through increases in myosin phosphorylation.     

Inhibition of tumor cell adhesion and metastasis

Since the establishment of an experimental metastasis model using some tumor metastatic cell lines, progress has been made in basic studies on tumor metastasis. In addition, the complex mechanisms of tumor metastasis have been elucidated by the cellular and molecular studies. Here we focus our attention on the relationship between tumor metastasis and cellular adhesiveness, a factor which is a critical determinant in tumor progression and metastasis. The roles of cell adhesive molecules in the metastatic process are also described. This type of study is invaluable in understanding the molecular and regulatory mechanisms of tumor metastasis. We also report the antimetastatic effect of some polypeptides of fibronectin in the extracellular matrices, poly (RGD) and recombinant fusion peptide containing cell- and heparin-binding domain of fibronectin (CH-271) in regulating the mechanism of tumor cell adhesion.     

Gelsolin and functionally similar actin-binding proteins are regulated by lysophosphatidic acid

An extensive survey was carried out for compounds capable of regulating actin-binding proteins in a manner similar to phosphatidylinositol 4,5 bisphosphate (PI 4,5-P2). For this purpose we developed a sensitive assay involving release of radioactively phosphorylated actin from the fragminP-actin complex. We found that the structurally simplest lysophospholipid, lysophosphatidic acid (LPA), dissociated the complex between fragminP and actin, whereas other lysophospholipids or sphingosine-1-phosphate were inactive. Furthermore, LPA inhibited the F-actin severing activity of human gelsolin, purified from plasma or as recombinant protein, mouse adseverin and Physarum fragminP. Dissociation of actin-containing complexes by LPA analyzed by gelfiltration indicated that LPA is active as a monomer, in contrast to PI 4,5-P2. We further show that binding of LPA to these actin-regulatory proteins promotes their phosphorylation by pp60(c-src). A PI 4,5-P2-binding peptide counteracted the effects mediated by LPA, suggesting that LPA binds to the same target region in these actin-binding proteins. When both LPA and PI 4,5-P2 were used in combination we found that LPA reduced the threshold concentration at which PI 4,5-P2 was active. Significantly, LPA promoted the release of gelsolin from barbed actin filaments in octylglucoside-permeabilized human platelets. These results suggest that lysophosphatidic acid could act as an intracellular modulator of actin-binding proteins. Our findings can also explain agonist-induced changes in the actin cytoskeleton that are not mediated by polyphosphoinositides.     

Growth of glycine picrate crystal by conventional and SR method

Single crystals of an organic nonlinear optical (NLO) material, glycine picrate (DGP) has been grown from mixed solution by using the novel uniaxial crystal growth method of Sankaranarayanan and Ramasamy (SR) and conventional method. The novel method allows the crystal to grow in one specified axis with well developed faces. The grown crystals have been subjected to crystal structure studies and characterized by single crystal X-ray diffraction, Powder X-ray diffraction (PXRD), UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), Thermogravimetric/differential thermal analysis (TG/DTA), Vickers microhardness, dielectric measurements, etching and High-resolution x-ray diffraction (HRXRD). The NLO efficiency of the crystal has been confirmed by using the Kurtz powder technique. The present study indicates that the SR method is superior to conventional method for the growth of DGP single crystals.