Endotoxin transduces Ca2+ signaling via platelet-activating factor receptor.

Lipopolysaccharide (LPS) is a pathogenic substance causing severe multiple organ failures and high mortality. Although several LPS binding proteins have been identified, the molecular mechanism underlying the LPS signaling pathway still remains obscure. We have found that the LPS-induced Ca2+ increase in platelets and platelet aggregation is blocked by selective platelet-activating factor (PAF) receptor antagonists, thus suggesting a cross-talk between LPS and the PAF receptor. Next, we confirmed this hypothesis using the cloned PAF receptors [(1991) Nature 349, 342-346; (1991) J. Biol. Chem. 266, 20400-20405] expressed in Xenopus oocytes and Chinese hamster ovary (CHO) cells. In both systems, cells responded to LPS only when PAF receptors were expressed, and specific PAF binding was successfully displaced and reversibly dissociated by LPS. PAF receptor activation by LPS may represent a novel important pathway in the pathogenesis of circulatory collapse and systemic thrombosis caused by endotoxin.

Micro-trap phosphorylation assay of mitogen-activated protein (MAP) kinases to detect their activation by lipopolysaccharides.

We designed a microplate-based assay method for mitogen-activated protein (MAP) kinase. Using anion-exchanger resin, MAP kinases from murine macrophages were partially purified in 96-well plates. The activities of these purified enzymes correlated well with those detected in heretofore used assays. The micro-trap phosphorylation assay has advantages over conventional methods (immunoprecipitation, Western blotting for the detection of mobility shift, or kinase detection assay in myelin basic protein (MBP)-containing gel), in terms of sensitivity, economy and rapid execution for hundreds of samples. Using micro-trap phosphorylation assay, it was demonstrated that MAP kinase activities in macrophages were persistently increased by lipopolysaccharide (LPS) stimulation, and this activation was inhibited by polymyxin B or tyrosine kinase inhibitors. This method is expected to give a wide range of application, such as determining effects of drug inhibitors or antisense oligonucleotides on MAP kinases, or measuring the various protein kinases after specificity controls were done.

Future molecular approaches to the diagnosis and treatment of glomerular disease.

Current diagnoses and treatment decisions for renal disease are made based upon a combination of clinical and pathological determinations. With the advances in both biochemical and molecular biological techniques, identifying the underlying biochemical and genetic changes that may have initiated and/or contributed to the disease is possible. We describe here technologies that may lead to significant changes in renal disease diagnosis, characterization, treatment, and potentially prevention. For example, differential display techniques and DNA gene chip arrays show the changes in mRNA expression patterns and can potentially identify previously unknown genes and reveal new roles for previously known genes in renal disease. The generation of the single nucleotide polymorphisms (SNP) genomic map will facilitate genetic screening that may identify a gene or combination of genes that produce enhanced disease susceptibility. Combining genomic analysis with epidemiological studies may identify environmental factors that contribute to renal disease onset in genetically susceptible individuals. A number of novel therapies are already on the horizon. These include reagents that abrogate the function of specific cytokines, chemokines, and effector cells. With the list of renal disease genes in hand, their role in renal physiology and pathophysiology can be determined, which should lead to the discovery of pharmacological intervention directed at those genes and their products that play a role in the pathogenesis of renal disease.

PAF-induced MAPK activation is inhibited by wortmannin in neutrophils and macrophages.

In the present study we examined the mechanism by which PAF activates MAPK in native cells such as guinea-pig neutrophils and P388D1 macrophage-like cells. We found that PAF activates MAPK through two distinct pathways. One calcium-dependent pathway that likely involves cPKC, and another calcium-independent but wortmannin-sensitive pathway. Using molecular biological methods we are presently examining whether hetrodimeric (p85/p110) type PI 3-kinase is the actual target of wortmannin involved in PAF mediated activation of MAPK.

Cloning and characterization of a murine platelet-activating factor receptor gene.

A murine gene encoding a platelet-activating factor receptor (PAFR) was cloned. The gene was mapped to a distal region of chromosome 4 both by fluorescence in situ hybridization and by molecular linkage analysis. Northern blot analysis showed a high expression of the PAFR message in peritoneal macrophages. When C3H/HeN macrophages were treated with bacterial lipopolysaccharide (LPS) or synthetic lipid A, the PAFR gene expression was induced. Bacterial LPS, but not lipid A, induced the level of PAFR mRNA in LPS-unresponsive C3H/HeJ macrophages. These induction patterns were parallel to those of tumor necrosis factor-alpha mRNA. Thus, the PAFR in macrophages plays important roles in LPS-induced pathologies.

Spontaneous tool use by wild capuchin monkeys (Cebus libidinosus) in the Cerrado.

Among primates, only chimpanzees and orang-utans are credited with customary tool use in nature. Among monkeys, capuchins stand out with respect to the number of accounts of tool use. However, the majority of capuchin tool use observations reported in nature is anecdotal or idiosyncratic. In this report, we documented the stone pounding of dry fruits (Hymenea courbaril and Acrocomia aculeata) in two wild free-ranging groups of Cebus libidinosus in the Brasilia National Park, a preserved area representative of the Cerradobiome of Central Brazil. In 2004, we noted 2 episodes at which 4 monkeys used stones to crack open nuts. In 2005, we recorded 5 pounding episodes involving 2 different monkeys. Observations of tool use over the course of 2 consecutive years by some individuals, as well as other indirect evidence, indicate that this behaviour could be habitual in the studied groups. We propose that the probability of the emergence of the use of pounding stones as tools may be dependent on the ecological variables that influence the degree of terrestriality and extractive foraging and the complex interaction of these factors.

Microplate chromatography assay for acetyl-CoA: lysoplatelet-activating factor acetyltransferase.

Acetyl-CoA:lysoplatelet-activating factor (1-O-alkyl-sn-glycero-3-phosphocholine) acetyltransferase (lysoPAF-AT) (EC 2.3.1.67) is a key enzyme in the biosynthesis of platelet-activating factor (PAF) and has been shown to be activated by various extracellular stimuli. A novel method to determine the enzyme activity is described here, which enables 96 simultaneous assays in a standard 96-well microplate format. The assay is based on the quantification of the incorporation of [3H]acetyl-CoA into PAF in the presence of lysoPAF. The radioactive products are separated from the substrate with a 96-well-formatted chromatography device using a Multiscreen plate (Millipore) prefilled with octyl-silica gel. As little as 1 mg octyl-silica gel was sufficient for the efficient recovery of the radioactive product, resulting in the very low background and thus high sensitivity. The enzyme activity could be measured directly with whole cell lysates from various cells cultured in 96-well microplate scale. This tailor-made microplate chromatography separation step is readily applicable for other kinds of enzyme assays.

Prostaglandin F2 alpha stimulates formation of p21ras-GTP complex and mitogen-activated protein kinase in NIH-3T3 cells via Gq-protein-coupled pathway.

Prostaglandin (PG) F2 alpha activated mitogen-activated protein (MAP) kinase and MAP kinase kinase in NIH-3T3 cells by a mechanism that was completely inhibited by protein kinase inhibitors, staurosporine (20 nM) or H-7 (20 microM), but was insensitive to pretreatment with islet-activating protein (100 ng/ml; 24 h) or 12-O-tetradecanoylphorbol 13-acetate (2.5 microM; 24 h). PGF2 alpha stimulation also led to a significant increase in Ras.GTP complex. Transfection of a cDNA encoding a constitutively active mutant of Gq alpha-subunit (Q209L) mimicked PGF2 alpha-induced MAP kinase activation, increase in Ras.GTP complex, and DNA synthesis in these cells, suggesting that activation of Gq mediates the PGF2 alpha-activation of Ras-MAP kinase pathway and mitogenesis in NIH-3T3 cells. These data provide a new insight into regulatory mechanisms of Ras-MAP kinase pathway through heterotrimeric G-protein-mediated pathways.

A murine platelet-activating factor receptor gene: cloning, chromosomal localization and up-regulation of expression by lipopolysaccharide in peritoneal resident macrophages.

A murine gene encoding a platelet-activating factor receptor (PAFR) was cloned. The gene was mapped to a region of the D2.2 band of chromosome 4 both by fluorescence in situ hybridization and by molecular linkage analysis. Northern blot analysis showed a high expression of the PAFR message in peritoneal macrophages. When C3H/HeN macrophages were treated with bacterial lipopolysaccharide (LPS) or synthetic lipid A, the PAFR gene expression was induced. Bacterial LPS, but not lipid A, induced the level of PAFR mRNA in LPS unresponsive C3H/HeJ macrophages. These induction patterns were parallel to those of tumor necrosis factor-alpha mRNA. Thus the PAFR in macrophages is important in LPS-induced pathologies.

On the mechanism of cytosolic phospholipase A2 activation in CHO cells carrying somatostatin receptor: wortmannin-sensitive pathway to activate mitogen-activated protein kinase.

We examined the mechanism of arachidonate release induced by somatostatin-14 (SS14) in CHO-K1 cells overexpressing rat hippocampal somatostatin receptor SSTR4. SSTR4 couples to pertussis toxin (PTX)-sensitive G-protein in CHO cells and does not lead to phosphoinositides breakdown or intracellular calcium ([Ca2+]i) mobilization (Bito et al.: J. Biol. Chem. 269, 12722-12730, 1994). SSTR4 activated mitogen-activated protein (MAP) kinase and induced the phosphorylation of 85kDa cytosolic phospholipase A2 (cPLA2), in a PTX-sensitive manner. Furthermore, activations of both MAP kinase and cPLA2 were inhibited by treatment with wortmannin, at almost identical IC50 values. Thus, SSTR4 appears to stimulate MAP kinase and cPLA2 in a Gi-dependent, and through a wortmannin-sensitive pathway. We also showed that stimulation with SS14, in combination with calcium-ionophore, strongly enhanced arachidonate release from these cells.

Wortmannin inhibits mitogen-activated protein kinase activation induced by platelet-activating factor in guinea pig neutrophils.

Stimulation of guinea pig neutrophils with platelet-activating factor (PAF) caused a rapid and transient activation of mitogen-activated protein kinase (MAPK). Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, partially (approximately 50%) inhibited PAF-induced MAPK activation. Half-maximal inhibition was observed with 200-300 nM wortmannin, while it did not inhibit phorbol ester-induced MAPK activation. Neutrophils preloaded with 1,2-bis-(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM) failed to raise cytosolic Ca2+ concentrations toward PAF, while they still responded to PAF with a 40-50% activation of MAPK. However, when cells were treated with BAPTA/AM and wortmannin in combination, the MAPK activation was completely inhibited. These results suggest that PAF activates MAPK through two distinct pathways in guinea pig neutrophils, one Ca(2+)-dependent, and the other Ca(2+)-independent but wortmannin-sensitive.

Two distinct signal transduction pathways for the activation of guinea-pig macrophages and neutrophils by endotoxin.

Lipopolysaccharide (LPS, endotoxin) is a major component of the outer membrane of gram-negative bacteria. Although it interacts with many types of cells and is linked to numerous events associated with sepsis and endotoxic shock, the mechanisms underlying these actions are poorly understood. We found that Ca-signaling induced by endotoxin in guinea-pig neutrophils and macrophages is caused by cross-recognition of LPS with platelet activating factor (PAF) receptors. However, the synthesis of tumor necrosis factor-alpha or the priming effect of O2- production was not affected by PAF antagonists. Thus, at least two distinct pathways are involved in the actions of LPS, one via the PAF receptor, while the other is independent of a PAF receptor and Ca-signaling.

Integrin beta2 (CD18)-mediated cell proliferation of HEL cells on a hematopoietic-supportive bone marrow stromal cell line, HESS-5 cells.

Cellular interactions between hematopoietic cells and stromal cells play important roles in the proliferation and differentiation of hematopoietic cells. The proliferation of a human erythroleukemia cell line, HEL cells, which can differentiate into macrophage- and megakaryocyte-like cells, and erythroid precursors was dramatically induced on coculture with a hematopoietic-supportive stromal cell line, HESS-5 cells, which can support long-term hematopoiesis in vitro without fetal bovine serum. HEL cells proliferated when they were cocultured with but not without direct cell contact. Because the coculture supernatants with direct cell contact and cytokines such as interleukins and growth factors did not exhibit growth-stimulating activity toward HEL cells, it was suggested that some molecule that has growth-stimulating activity exists on the surface of the cells. Extracellular matrix components such as fibronectin, laminin, vitronectin, and collagen did not affect the proliferation of HEL cells. An anti-CD18 monoclonal antibody, which recognizes the common beta chain of the beta2 integrin subfamily, induced dramatic proliferation of HEL cells. Moreover, the proliferation of HEL cells was inhibited by an antisense oligonucleotide of CD18 mRNA. As judged from these observations, the proliferation of HEL cells was mediated by CD18 molecules expressed on HEL cells. On the contrary, the common counter-receptor of the beta2 integrin subfamily, intercellular adhesion molecule-1, which is expressed on CHO-K1 cells, did not stimulate the growth of HEL cells. It is known that other counter molecules of the beta2 integrin subfamily, such as complement C3bi and fibrinogen, are not produced by stromal cells. These findings suggest that the proliferation of HEL cells may be induced through an interaction between a novel molecule of the beta2 integrin subfamily on HEL cells and the counter-receptor on HESS-5 cells. The beta2 integrin subfamily may regulate the growth of hematopoietic cells in hematopoiesis in vivo and/or cause the abnormal growth of leukemia cells.

JTE-607, a novel inflammatory cytokine synthesis inhibitor without immunosuppression, protects from endotoxin shock in mice.

OBJECTIVE AND DESIGN: We investigated the effect of a novel N-benzoyl-L-phenylalanine derivative compound (JTE-607) on production of various cytokines and other immune responses in vitro and on endotoxin shock in vivo. MATERIALS AND METHODS: Human, monkey, rabbit, mouse and rat peripheral blood mononuclear cells (PBMCs), and human fibroblasts, umbilical vein endothelial cells (HUVEC), mesangial cells and T cells were used in vitro. Endotoxin shock was induced by lipopolysaccharide (LPS) in Corynebacterium parvum (C. parvum) sensitized male C57BL/6 mice in vivo. RESULTS: JTE-607 inhibited inflammatory cytokine production, including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-8 and IL-10, from LPS-stimulated human PBMCs, with IC50 values of 11, 5.9, 8.8, 7.3 and 9.1 nM, respectively. The inhibitory effects of JTE-607 were also seen in mRNA expression of those cytokines. The potency of JTE-607 on cytokine production from PBMCs of other species, and from other human cells were much lower than that on human PBMCs. JTE-607 did not affect either LPS-stimulated microbead phagocytosis or reactive oxygen species production at 1 microM in human PBMCs but slightly suppressed expression of major histocompatibility complex class II antigen at 1 microM, although it was 100-fold less active than it was as a cytokine inhibitor. JTE-607 (0.3-10 mg/kg, i.v.) showed dose dependent inhibition of mortality after LPS challenge in C. parvum sensitized mice in accordance with a decrease of plasma TNF-alpha. CONCLUSIONS: These results suggest that JTE-607 is a multiple cytokine inhibitor specific for human PBMCs. This compound may be useful for the treatment of various cytokine mediated diseases such as septic shock without causing immunosuppression.

Wortmannin inhibits mitogen-activated protein kinase activation by platelet-activating factor through a mechanism independent of p85/p110-type phosphatidylinositol 3-kinase.

We have shown previously that wortmannin partially inhibits mitogen-activated protein kinase (MAPK) activated by platelet-activating factor (PAF) in guinea pig neutrophils (Ferby, M. I., Waga, I., Sakanaka, C., Kume, K., and Shimizu, T. (1994) J. Biol. Chem. 269, 30485-30488). To identify whether p85-dependent phosphatidylinositol 3-kinase is a target molecule of wortmannin in this inhibitory process, we established a murine macrophage cell line (P388D1), inducibly expressing a dominant-negative p85, delta p85. Upon induction of delta p85 by isopropyl-beta-D-thiogalactopyranoside, PAF still induced unaltered activation of MAPK, which was inhibited completely by wortmannin and 1,2-bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester in an additive manner. Thus, PAF activates MAPK in P388D1 cells via two distinct pathways, one calcium-dependent and another calcium-independent, but wortmannin-sensitive. The inhibition of calcium-independent activation of MAPK by wortmannin does not involve p85-dependent phosphatidylinositol 3-kinase.

Probing the dark energy with quasar clustering.

We show through Monte Carlo simulations that the Alcock-Paczynski test, as applied to quasar clustering, is a powerful tool to probe the cosmological density and equation of state parameters Omega(m0), Omega(x0), and w. By taking into account the effect of peculiar velocities upon the correlation function we obtain for the Two-Degree Field QSO Redshift Survey the predicted confidence contours for the cosmological constant (w = -1) and spatially flat (Omega(m0)+Omega(x0) = 1) cases. For w = -1, the test is especially sensitive to the difference Omega(m0)-Omega(Lambda0), thus being ideal to combine with cosmic microwave background results. For the flat case, it is competitive with future supernova and galaxy number count tests, besides being complementary to them.

Rapid activation of MAP kinase by estrogen in the bone cell line.

We examined the effect of estrogen on mitogen-activated protein kinase (MAPK) in osteoblastic cells. Rat ROS 17/2.8 cells were exposed to 17beta-estradiol (E2) and MAPK activity in the cells was measured by an in vitro phosphorylation assay. E2 treatment caused a rapid and transient MAPK activation within 5 min. Insulin-like growth factor-I, which acts via their membrane receptors, caused a similar effect, but it required 10 min to reach the maximum level. Western blot analyses with anti-MAPK and anti-phosphotyrosine antibodies demonstrated that the E2 activation of MAPK was accompanied by phosphorylation of the enzyme. The concentration range (10 nM-1 pM) of E2 needed for this MAPK activation was less than that (1 microM-0.1 nM) needed for the transcriptional activation via the nuclear estrogen receptor (ER). These data provide the first evidence of MAPK activation by E2 through phosphorylation, which may be mediated through a putative plasma membrane receptor in the cultured bone cells.

Bronchial hyperreactivity, increased endotoxin lethality and melanocytic tumorigenesis in transgenic mice overexpressing platelet-activating factor receptor.

Although platelet-activating factor (PAF) has been shown to exert pleiotropic effects on isolated cells or tissues, controversy still exists as to whether it plays significant pathophysiological roles in vivo. To answer this question, we established transgenic mice over-expressing a guinea-pig PAF receptor (PAFR). The transgenic mice showed a bronchial hyperreactivity to methacholine and an increased mortality when exposed to bacterial endotoxin. An aberrant melanogenesis and proliferative abnormalities in the skin were also observed in the transgenic mice, some of which spontaneously bore melanocytic tumors in the dermis after aging. Thus, PAFR transgenic mice proved to be a useful model for studying the basic pathophysiology of bronchial asthma and endotoxin-induced death, and screening of therapeutics for these disorders. Furthermore, our findings provide new insights regarding the role of PAF in the morphogenesis of dermal tissues as well as the mitogenic activity of PAF and PAFR in vivo.

Functional coupling of adenosine A2a receptor to inhibition of the mitogen-activated protein kinase cascade in Chinese hamster ovary cells.

Activation of Gs-coupled receptors enhances the increase in cyclic AMP mediated by adenylate cyclases. As it has been shown that cyclic AMP inhibits the epidermal growth factor-activated mitogen-activated protein kinase (MAPK) signalling pathway, stimulation of Gs-coupled receptors may lead to the inhibition of MAPK activation. To investigate the effect of a Gs-coupled receptor on the MAPK cascade, we cloned the adenosine (Ado) A2a receptor from a guinea-pig leucocyte cDNA library, and established Chinese hamster ovary (CHO) cells stably expressing the receptor (CHOAdoA2R). The [3H]5'-N-ethylcarbamoyladenosine (NECA) binding characteristics (Kd = 91.0 +/- 5.4 nM, Bmax = 707 +/- 11 fmol/mg of protein, n = 3) and NECA-induced cyclic AMP production indicate that the cloned Ado A2a receptor was functionally expressed in the cells. In CHO cells, thrombin induced intracellular Ca2+ increase and MAPK activation through the intrinsic G-coupled receptor. In CHOAdoA2R cells, NECA partially inhibited thrombin-elicited MAPK activation. When combining NECA-treatment with 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA-AM) loading, a nearly complete inhibition of the MAPK activation occurred. Forskolin also partially inhibited the MAPK activation and synergized with BAPTA-AM, suggesting that partial inhibition of MAPK activation by NECA results from cyclic AMP production via Ado A2a receptor activation. The same synergism of MAPK inhibition between wortmannin and BAPTA-AM was observed, but not between wortmannin and NECA. These results suggest that cyclic AMP production through Ado A2a receptor inhibits thrombin-elicited MAPK activation by a Ca(2+)-independent/wortmannin-sensitive pathway in CHO cells.