Requirement for PLC gamma 2 in IL-3 and GM-CSF-Stimulated MEK/ERK Phosphorylation in Murine and Human Hematopoietic Stem/Progenitor Cells

Even though the involvement of intracellular Ca(2+) (Ca(i)(2+)) in hematopoiesis has been previously demonstrated, the relationship between Ca(i)(2+) signaling and cytokine-induced intracellular pathways remains poorly understood. Herein, the molecular mechanisms integrating Ca(2+) signaling with the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in primary murine and human hematopoietic stem/progenitor cells stimulated by IL-3 and GM-CSF were studied. Our results demonstrated that IL-3 and GM-CSF stimulation induced increased inositol 1,4,5-trisphosphate (IP(3)) levels and Ca(i)(2+) release in murine and human hematopoietic stem/ progenitor cells. In addition, Ca(i)(2+) signaling inhibitors, such as inositol 1,4,5-trisphosphate receptor antagonist (2-APB), PKC inhibitor (GF109203), and CaMKII inhibitor (KN-62), blocked phosphorylation of MEK activated by IL-3 and GM-CSF, suggesting the participation of Ca(2+)-dependent kinases in MEK activation. In addition, we identify phospholipase C gamma 2 (PLC gamma 2) as a PLC gamma responsible for the induction of Ca(2+) release by IL-3 and GM-CSF in hematopoietic stem/progenitor cells. Furthermore, the PLCg inhibitor U73122 significantly reduced the numbers of granulocyte-macrophage colony-forming units after cytokine stimulation. Similar results were obtained in both murine and human hematopoietic stem/progenitor cells. Taken together, these data indicate a role for PLC gamma 2 and Ca(2+) signaling through the modulation of MEK in both murine and human hematopoietic stem/ progenitor cells. J. Cell. Physiol. 226: 1780-1792, 2011. (C) 2010 Wiley-Liss, Inc.

Adenosine modulates alpha2-adrenergic receptors through a phospholipase C pathway in brainstem cell culture of rats

Adenosine acts in the nucleus tractus solitarii (NTS), one of the main brain sites related to cardiovascular control. In the present study we show that A(1) adenosine receptor (A(1R)) activation promotes an increase on alpha(2)-adrenoceptor (Alpha(2R)) binding in brainstem cell culture from newborn rats. We investigated the intracellular cascade involved in such modulatory process using different intracellular signaling molecule inhibitors as well as calcium chelators. Phospholipase C, protein kinase Ca(2+)-dependent, IP(3) receptor and intracellular calcium were shown to participate in A(1R)/Alpha(2R) interaction. In conclusion, this result might be important to understand the role of adenosine within the NTS regarding autonomic cardiovascular control. (C) 2009 Elsevier B.V. All rights reserved.

Role of phospholipase C and protein kinase C in Aspergillus nidulans during growth on pectin or glucose: Effects on germination and duplication cycle

The effects of PLC and Pkc inhibitors on Aspergillus nidulans depend on the carbon source. PLC inhibitors Spm and C48/80 delayed the first nuclear division in cultures growing on glucose, but stimulated it in media supplemented with pectin. Less intense were these effects on the mutant transformed with PLC-A gene rupture (AP27). Neomycin also delayed the germination in cultures growing on glucose or pectin; however, on glucose, the nuclear division was inhibited whereas in pectin it was stimulated. These effects were minor in AP27. The effects of Ro-31-8425 and BIM (both Pkc inhibitors) were also opposite for cultures growing on glucose or pectin. On glucose cultures of both strains BIM delayed germination and the first nuclear division, whereas on pectin both parameters were stimulated. Opposite effects were also detected when the cultures were growing on glucose or pectin in the presence of Ro-31-8425.

Phosphatidylinositol phospholipase C mediates carbon sensing and vegetative nuclear duplication rates in Aspergillus nidulans

In this work, we disrupted one of three putative phosphatidylinositol phospholipase C genes of Aspergillus nidulans and studied its effect on carbon source sensing linked to vegetative mitotic nuclear division. We showed that glucose does not affect nuclear division rates during early vegetative conidial germination (6-7 h) in either the wild type or the plcA-deficient mutant. Only after 8 h of cultivation on glucose did the mutant strain present some decrease in nuclear duplication. However, decreased nuclear division rates were observed in the wild type when cultivated in media amended with polypectate, whereas our plcA-deficient mutant did not show slow nuclear duplication rates when grown on this carbon source, even though it requires induction and secretion of multiple pectinolytic enzymes to be metabolized. Thus, plcA appears to be directly linked to high-molecular-weight carbon source sensing.

Metabotropic glutamate receptors transduce signals for neurite outgrowth after binding of the prion protein to laminin gamma 1 chain

The prion protein (PrP(C)) is highly expressed in the nervous system, and its abnormal conformer is associated with prion diseases. PrP(C) is anchored to cell membranes by glycosylphosphatidylinositol, and transmembrane proteins are likely required for PrP(C)-mediated intracellular signaling. Binding of laminin (Ln) to PrP(C) modulates neuronal plasticity and memory. We addressed signaling pathways triggered by PrP(C)-Ln interaction in order to identify transmembrane proteins involved in the transduction of PrP(C)-Ln signals. The Ln gamma 1-chain peptide, which contains the Ln binding site for PrP(C), induced neuritogenesis through activation of phospholipase C (PLC), Ca(2+) mobilization from intracellular stores, and protein kinase C and extracellular signal-regulated kinase (ERK1/2) activation in primary cultures of neurons from wild-type, but not PrP(C)-null mice. Phage display, coimmunoprecipitation, and colocalization experiments showed that group I metabotropic glutamate receptors (mGluR1/5) associate with PrP(C). Expression of either mGluR1 or mGluR5 in HEK293 cells reconstituted the signaling pathways mediated by PrP(C)-Ln gamma 1 peptide interaction. Specific inhibitors of these receptors impaired PrP(C)-Ln gamma 1 peptide-induced signaling and neuritogenesis. These data show that group I mGluRs are involved in the transduction of cellular signals triggered by PrP(C)-Ln, and they support the notion that PrP(C) participates in the assembly of multiprotein complexes with physiological functions on neurons.-Beraldo, F. H., Arantes, C. P., Santos, T. G., Machado, C. F., Roffe, M., Hajj, G. N., Lee, K. S., Magalhaes, A. C., Caetano, F. A., Mancini, G. L., Lopes, M. H., Americo, T. A., Magdesian, M. H., Ferguson, S. S. G., Linden, R., Prado, M. A. M., Martins, V. R. Metabotropic glutamate receptors trans-duce signals for neurite outgrowth after binding of the prion protein to laminin gamma 1 chain. FASEB J. 25, 265-279 (2011). www.fasebj.org

Invasion of Ureaplasma diversum in Hep-2 cells

Background: Understanding mollicutes is challenging due to their variety and relationship with host cells. Invasion has explained issues related to their opportunistic role. Few studies have been done on the Ureaplasma diversum mollicute, which is detected in healthy or diseased bovine. The invasion in Hep-2 cells of four clinical isolates and two reference strains of their ureaplasma was studied by Confocal Laser Scanning Microscopy and gentamicin invasion assay. Results: The isolates and strains used were detected inside the cells after infection of one minute without difference in the arrangement for adhesion and invasion. The adhesion was scattered throughout the cells, and after three hours, the invasion of the ureaplasmas surrounded the nuclear region but were not observed inside the nuclei. The gentamicin invasion assay detected that 1% of the ATCC strains were inside the infected Hep-2 cells in contrast to 10% to the clinical isolates. A high level of phospholipase C activity was also detected in all studied ureaplasma. Conclusions: The results presented herein will help better understand U. diversum infections, aswell as cellular attachment and virulence.

Crystal and molecular structures of dichloro((Z)-2-chloro-2-phenylvinyl)(organo)tellurium(IV) for organo = n-butyl and phenyl

In each of the title compounds, R[Ph(Cl)C=(H)C]TeCl(2), R = nBu (1) and Ph (2), the primary geometry about the Te(IV) atom is a pseudo-trigonal-bipyramidal arrangement, with two Cl atoms in apical positions, and the lone pair of electrons and C atoms in the equatorial plane. As the Te(IV) is involved in two, an intra- and an inter-molecular, Te center dot center dot center dot Cl interactions the coordination geometry might be considered as a Psi-pentagonal bipyramid in each case. In addition, in (2) there is a hint of a Te center dot center dot center dot pi interaction (Te center dot center dot center dot C = 3.911(3) A). The key feature in the crystal structure of both compounds is the formation of supramolecular chains mediated by Te center dot center dot center dot Cl contacts. (1): C(12)H(15)Cl(3)Te, triclinic, P (1) over bar, a = 5.9471 (11), b = 10.7826(22), c = 11.7983(19) angstrom, alpha = 75.416(12), beta = 78.868(13), gamma = 80.902(14)degrees, V = 713.6(2) angstrom(3), Z = 2, R(1) = 0.021; (2): C14HIIC13Te, orthorhombic, Pcab, a=7.7189(10), b=17.415(2), c=21.568(3)angstrom, V = 2899.3(6) angstrom(3), Z = 8, R(1) = 0.027.

Guanosine promotes B16F10 melanoma cell differentiation through PKC-ERK 1/2 pathway

Malignant melanoma is one of the most lethal cancers. Nowadays, several anti-melanoma therapies have been employed. However, the poor prognosis and/or the increased toxicity of those treatments clearly demonstrate the requirement of searching for new drugs or novel combined chemotherapeutic protocols, contemplating both effectiveness and low toxicity. Guanosine (Guo) has been used in combination with acriflavina to potentiate the latter`s antitumor activity, through still unknown mechanisms. Here, we show that Guo induces B16F10 melanoma cell differentiation, attested by growth arrest, dendrite-like outgrowth and increased melanogenesis, and also reduced motility. A sustained ERK 1/2 phosphorylation was observed after Guo treatment and ERK inhibition led to blockage of dendritogenesis. Intracellular cyclic AMP was not involved in ERK activation, since its levels remained unchanged. Protein kinase C (PKC), in contrast to phospholipase C (PLC), inhibition completely prevented ERK activation. While the classical melanoma differentiation agent forskolin activates cAMP-PKA-Raf-MEK-ERK pathway in B16F10 cells, here we suggest that a cAMP-independent, PKC-ERK axis is involved in Guo-induced B16F10 differentiation. Altogether, our results show that Guo acts as a differentiating agent, with cytostatic rather than cytotoxic properties, leading to a decreased melanoma malignancy. Thus, we propose that Guo may be envisaged in combination with lower doses of conventional anti-melanoma drugs, in an attempt to prevent or diminish their adverse effects. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

Direct measurement of intrinsic atomic scale magnetostriction

Using differential x-ray absorption spectroscopy (DiffXAS) we have measured and quantified the intrinsic, atomic-scale magnetostriction of Fe(81)Ga(19). By exploiting the chemical selectivity of DiffXAS, the Fe and Ga local environments have been assessed individually. The enhanced magnetostriction induced by the addition of Ga to Fe was found to originate from the Ga environment, where lambda(gamma,2)(approximate to (3/2)lambda(100)) is 390 +/- 40 ppm. In this environment, < 001 > Ga-Ga pair defects were found to exist, which mediate the magnetostriction by inducing large strains in the surrounding Ga-Fe bonds. For the first time, intrinsic, chemically selective magnetostrictive strain has been measured and quantified at the atomic level, allowing true comparison with theory.

Prognostic index expression of cyclin-D1, cerbB-2 and VEGF: metastases vs corresponding primary cancers and metastatic vs non-metastatic adenocarcinomas

The prognostic relevance of different molecular markers in lung cancer is a crucial issue still worth investigating, and the specimens collected and analyzed represent a valuable source of material. Cyclin-D1, c-erbB-2 and vascular endothelial growth factor (VEGF) have shown to be promising as prognosticators in human cancer. In this study, we sought to examine the importance of Cyclin-D1, c-erbB-2 and VEGF, and to study the quantitative relationship among these factors and disease progression in metastases vs corresponding primary cancer, and metastatic vs non metastatic cancers. Material and Methods: We used immunohistochemistry and morphometric analysis to evaluate the amount of tumour staining for Cyclin-D1, c-erbB-2 and VEGF in 52 patients with surgically excised ademocarcinoma of the lung, and the outcome for our study was survival time until death from hematogenic metastases. Results: Metastasis presented lower c-erbB-2 expression than corresponding primary cancers (p=0.02). Cyclin-D1 and VEGF expression were also lower in metastases than in corresponding primary cancers, but this difference did not achieve statistical significance. Non-metastatic cancers also presented significantly lower Cyclin-D1 and c-erbB-2 expression than metastatic cancers (p<0.01 and p<0.01, respectively). Equally significant was the difference between higher c-erbB-2 expression by metastatic cancers compared to non-metastatic cancers (p=0.02). Considering survival in Kaplan-Maier analysis, Cyclin-D1 (p=0.04), c-erbB-2 (p=0.04) and VEGF (p<0.01) were important predictors of survival in metastatic cancers.

Continuous extraction of alpha-toxin from a fermented broth of Clostridium perfringens Type A in perforated rotating disc contactor using aqueous two-phase PEG-phosphate system

A 2(3-1) factorial experimental design was used to evaluate the performance of a perforated rotating disc contactor to extract alpha-toxin from the fermented broth of Clostridium perfringens Type A by aqueous two-phase system of polyethylene glycol-phosphate salts. The influence of three independent variables, specifically the dispersed phase flowrate, the continuous phase flowrate and the disc rotational speed, was investigated on the hold up, the mass transfer coefficient, the separation efficiency and the purification factor, taken as the response variables. The optimum dispersed phase flowrate was 3.0 mL/min for all these responses. Besides, maximum values of hold up (0.80), separation efficiency (0. 10) and purification factor (2.4) were obtained at this flowrate using the lowest disc rotational speed (35 rpm), while the optimum mass transfer coefficient (0. 165 h(-1)) was achieved at the highest agitation level (140 rpm). The results of this study demonstrated that the dispersed phase flowrate strongly influenced the performance of PRDC, in that both the mass transfer coefficient and hold up increased with this parameter. (c) 2007 Elsevier B. V. All rights reserved.

Chronic methionine load-induced hyperhomocysteinemia impairs the relaxation induced by bradykinin in the isolated rat carotid

This study investigates the effects of chronic methionine intake on bradykinin (BK)-relaxation. Vascular reactivity experiments were performed on carotid rings from male Wistar rats. Treatment with methionine (0.1, 1 or 2 g kg(-1) per day) for 8 and 16 weeks, but not for 2 and 4 weeks, reduced the relaxation induced by BK. Indomethacin, a non-selective cyclooxygenase (COX) inhibitor, and SQ29548, a selective thromboxane A(2) (TXA(2))/prostaglandin H(2) (PGH(2)) receptor antagonist prevented the reduction in BK-relaxation observed in the carotid from methionine-treated rats. Conversely, AH6809, a selective prostaglandin F(2 alpha) (PGF(2 alpha)) receptor antagonist did not alter BK-relaxation in the carotid from methionine-treated rats. The nitric oxide synthase (NOS) inhibitors L-NAME, L-NNA and 7-nitroindazole reduced the relaxation induced by BK in carotids from control and methionine-treated rats. In summary, we found that chronic methionine intake impairs the endothelium-dependent relaxation induced by BK and this effect is due to an increased production of endothelial vasoconstrictor prostanoids (possibly TXA(2)) that counteracts the relaxant action displayed by the peptide.

Bradykinin regulates calpain and proinflammatory signaling through TRPM7-sensitive pathways in vascular smooth muscle cells

Yogi A, Callera GE, Tostes R, Touyz RM. Bradykinin regulates calpain and proinflammatory signaling through TRPM7-sensitive pathways in vascular smooth muscle cells. Am J Physiol Regul Integr Comp Physiol 296: R201-R207, 2009. First published September 17, 2008; doi: 10.1152/ajpregu.90602.2008.-Transient receptor potential melastatin-7 (TRPM7) channels have recently been identified to be regulated by vasoactive agents acting through G protein-coupled receptors in vascular smooth muscle cells (VSMC). However, downstream targets and functional responses remain unclear. We investigated the subcellular localization of TRPM7 in VSMCs and questioned the role of TRPM7 in proinflammatory signaling by bradykinin. VSMCs from Wistar-Kyoto rats were studied. Cell fractionation by sucrose gradient and differential centrifugation demonstrated that in bradykinin-stimulated cells, TRPM7 localized in fractions corresponding to caveolae. Immunofluorescence confocal microscopy revealed that TRPM7 distributes along the cell membrane, that it has a reticular-type intracellular distribution, and that it colocalizes with flotillin-2, a marker of lipid rafts. Bradykinin increased expression of calpain, a TRPM7 target, and stimulated its cytosol/membrane translocation, an effect blocked by 2-APB (TRPM7 inhibitor) and U-73122 (phospholipase C inhibitor), but not by chelerythrine (PKC inhibitor). Expression of proinflammatory mediators VCAM-1 and cyclooxygenase-2 (COX-2) was time-dependently increased by bradykinin. This effect was blocked by Hoe-140 (B(2) receptor blocker) and 2-APB. Our data demonstrate that in bradykinin-stimulated VSMCs: 1) TRPM7 is upregulated, 2) TRPM7 associates with cholesterol-rich microdomains, and 3) calpain and proinflammatory mediators VCAM-1 and COX2 are regulated, in part, via TRPM7- and phospholipase C-dependent pathways through B2 receptors. These findings identify a novel signaling pathway for bradykinin, which involves TRPM7. Such phenomena may play a role in bradykinin/B(2) receptor-mediated inflammatory responses in vascular cells.

Compound 48/80 induces endothelium-dependent and histamine release-independent relaxation in rabbit aorta

Compound 48/80 (C48/80) is a synthetic condensation product of N-methyl-p-methoxyphenethyl am me with formaldehyde and is an experimental drug used since the 1950s to induce anaphylactic shock through histamine release. This study was carried out to further elucidate the mechanism by which this drug induces nitric oxide (NO) release. Our specific goals were: (a) to verify if C48/80`s relaxation occurs through the stimulation of histamine receptors; (b) to evaluate the endothelium-dependent relaxation induced by C48/80; (c) to identify NO as the endothelium-relaxing factor released by C48/80; (d) to identify the NO synthase (NOS) responsible for NO release; and (e) to verify if the relaxation induced by C48/80 is calcium and cyclic guanidine monophosphate (cGMP) dependent. Rabbit aorta segments, with and without endothelium, were suspended in organ chambers (25 ml) filled with Krebs solution maintained at 37 degrees C, bubbled with 95% O-2/5% CO2 (pH 7.4). Phenylephrine was used to contract the segments. Other protocol drugs included H-1- and H-2-receptor antagonists, cyclooxygenase, NOS, guanylyl cyclase and phospholipase C (PLC) inhibitors. Endothelium-dependent relaxation induced by C48/80 was also studied in calcium-free Krebs solution associated with a calcium chelator. In summary, our investigation demonstrated that the C48/80 vasodilating action: (a) does not depend on H-1 and H-2 histamine receptors; (b) is NO endothelium-dependent; (c) is dependent on the endothelial constitutive NOS (NOS-3) isoform activation; (d) is cGMP-dependent; and that NOS-3 activation by C48/80: (a) is independent of PLC up to 25 mu g/ml and (b) is partially dependent of this lipase in higher doses. (C) 2007 Elsevier Inc. All rights reserved.

Antiobesity Effects of yerba mate Extract (Ilex paraguariensis) in High-fat Diet-induced Obese Mice

Because the potential of yerba mate (Ilex paraguariensis) has been suggested in the management of obesity, the aim of the present study was to evaluate the effects of yerba mate extract on weight loss, obesity-related biochemical parameters, and the regulation of adipose tissue gene expression in high-fat diet-induced obesity in mice. Thirty animals were randomly assigned to three groups. The mice were introduced to standard or high-fat diets. After 12 weeks on a high-fat diet, mice were randomly assigned according to the treatment (water or yerba mate extract 1.0 g/-kg). After treatment intervention, plasma concentrations of total cholesterol, high-density lipoprotein cholesterol, triglyceride, low-density lipoprotein (LDL) cholesterol, and glucose were evaluated. Adipose tissue was examined to determine the mRNA levels of several genes such as tumor necrosis factor-alpha (TNF-alpha), leptin, interleukin-6 (IL-6), C-C motif chemokine ligand-2 (CCL2), CCL receptor-2 (CCR2), angiotensinogen, plasminogen activator inhibitor-1 (PAI-1), adiponectin, resistin, peroxisome proliferator-activated receptor-gamma(2) (PPAR-gamma(2)), uncoupling protein-1 (UCP1), and PPAR-gamma coactivator-1 alpha (PGC-1 alpha). The F4/80 levels were determined by immunoblotting. We found that obese mice treated with yerba mate exhibited marked attenuation of weight gain, adiposity, a decrease in epididymal fat-pad weight, and restoration of the serum levels of cholesterol, triglycerides, LDL cholesterol, and glucose. The gene and protein expression levels were directly regulated by the high-fat diet. After treatment with yerba mate extract, we observed a recovery of the expression levels. In conclusion, our data show that yerba mate extract has potent antiobesity activity in vivo. Additionally, we observed that the treatment had a modulatory effect on the expression of several genes related to obesity.