The alpha 7 nicotinic receptor agonist PHA-543613 hydrochloride inhibits <i>Porphyromonas gingivalis</i>-induced expression of interleukin-8 by oral keratinocytes

Objective: The alpha 7 nicotinic receptor (α7nAChR) is expressed by oral keratinocytes. α7nAChR activation mediates anti-inflammatory responses. The objective of this study was to determine if α7nAChR activation inhibited pathogen-induced interleukin-8 (IL-8) expression by oral keratinocytes.&lt;p&gt;&lt;/p&gt; Materials and methods: Periodontal tissue expression of α7nAChR was determined by real-time PCR. OKF6/TERT-2 oral keratinocytes were exposed to &lt;i&gt;Porphyromonas gingivalis&lt;/i&gt; in the presence and absence of a α7nAChR agonist (PHA-543613 hydrochloride) alone or after pre-exposure to a specific α7nAChR antagonist (α-bungarotoxin). Interleukin-8 (IL-8) expression was measured by ELISA and real-time PCR. Phosphorylation of the NF-κB p65 subunit was determined using an NF-κB p65 profiler assay and STAT-3 activation by STAT-3 in-cell ELISA. The release of ACh from oral keratinocytes in response to &lt;i&gt;P. gingivalis&lt;/i&gt; lipopolysaccharide was determined using a GeneBLAzer M3 CHO-K1-blacell reporter assay.&lt;p&gt;&lt;/p&gt; Results: Expression of α7nAChR mRNA was elevated in diseased periodontal tissue. PHA-543613 hydrochloride inhibited &lt;i&gt;P. Gingivalis&lt;/i&gt;-induced expression of IL-8 at the transcriptional level. This effect was abolished when cells were pre-exposed to a specific α7nAChR antagonist, α-bungarotoxin. PHA-543613 hydrochloride downregulated NF-κB signalling through reduced phosphorylation of the NF-κB p65-subunit. In addition, PHA-543613 hydrochloride promoted STAT-3 signalling by maintenance of phosphorylation. Furthermore, oral keratinocytes upregulated ACh release in response to &lt;i&gt;P. Gingivalis&lt;/i&gt; lipopolysaccharide.&lt;p&gt;&lt;/p&gt; Conclusion: These data suggest that α7nAChR plays a role in regulating the innate immune responses of oral keratinocytes.&lt;p&gt;&lt;/p&gt

Sphingosine Kinase-1 Is Required for Toll Mediated β-Defensin 2 Induction in Human Oral Keratinocytes

Host defense against invading pathogens is triggered by various receptors including toll-like receptors (TLRs). Activation of TLRs is a pivotal step in the initiation of innate, inflammatory, and antimicrobial defense mechanisms. Human beta-defensin 2 (HBD-2) is a cationic antimicrobial peptide secreted upon gram-negative bacterial perturbation in many cells. Stimulation of various TLRs has been shown to induce HBD-2 in oral keratinocytes, yet the underlying cellular mechanisms of this induction are poorly understood.Here we demonstrate that HBD-2 induction is mediated by the Sphingosine kinase-1 (Sphk-1) and augmented by the inhibition of Glycogen Synthase Kinase-3beta (GSK-3beta) via the Phosphoinositide 3-kinase (PI3K) dependent pathway. HBD-2 secretion was dose dependently inhibited by a pharmacological inhibitor of Sphk-1. Interestingly, inhibition of GSK-3beta by SB 216763 or by RNA interference, augmented HBD-2 induction. Overexpression of Sphk-1 with concomitant inhibition of GSK-3beta enhanced the induction of beta-defensin-2 in oral keratinocytes. Ectopic expression of constitutively active GSK-3beta (S9A) abrogated HBD-2 whereas kinase inactive GSK-3beta (R85A) induced higher amounts of HBD-2.These data implicate Sphk-1 in HBD-2 regulation in oral keratinocytes which also involves the activation of PI3K, AKT, GSK-3beta and ERK 1/2. Thus we reveal the intricate relationship and pathways of toll-signaling molecules regulating HBD-2 which may have therapeutic potential

Glycogen Synthase Kinase-3 regulates multiple myeloma cell growth and bortezomib-induced cell death

BACKGROUND: Glycogen Synthase Kinase-3 (GSK-3) \u3b1 and \u3b2 are two serine-threonine kinases controlling insulin, Wnt/\u3b2-catenin, NF-\u3baB signaling and other cancer-associated transduction pathways. Recent evidence suggests that GSK-3 could function as growth-promoting kinases, especially in malignant cells. In this study, we have investigated GSK-3\u3b1 and GSK-3\u3b2 function in multiple myeloma (MM). METHODS: GSK-3 \u3b1 and \u3b2 expression and cellular localization were investigated by Western blot (WB) and immunofluorescence analysis in a panel of MM cell lines and in freshly isolated plasma cells from patients. MM cell growth, viability and sensitivity to bortezomib was assessed upon treatment with GSK-3 specific inhibitors or transfection with siRNAs against GSK-3 \u3b1 and \u3b2 isoforms. Survival signaling pathways were studied with WB analysis. RESULTS: GSK-3\u3b1 and GSK-3\u3b2 were differently expressed and phosphorylated in MM cells. Inhibition of GSK-3 with the ATP-competitive, small chemical compounds SB216763 and SB415286 caused MM cell growth arrest and apoptosis through the activation of the intrinsic pathway. Importantly, the two inhibitors augmented the bortezomib-induced MM cell cytotoxicity. RNA interference experiments showed that the two GSK-3 isoforms have distinct roles: GSK-3\u3b2 knock down decreased MM cell viability, while GSK-3\u3b1 knock down was associated with a higher rate of bortezomib-induced cytotoxicity. GSK-3 inhibition caused accumulation of \u3b2-catenin and nuclear phospho-ERK1, 2. Moreover, GSK-3 inhibition and GSK-3\u3b1 knockdown enhanced bortezomib-induced AKT and MCL-1 protein degradation. Interestingly, bortezomib caused a reduction of GSK-3 serine phosphorylation and its nuclear accumulation with a mechanism that resulted partly dependent on GSK-3 itself. CONCLUSIONS: These data suggest that in MM cells GSK-3\u3b1 and \u3b2 i) play distinct roles in cell survival and ii) modulate the sensitivity to proteasome inhibitors

A conductometeric study of the complex formation between copper (I) perchlorate and some organic ligands in acetonitrile

1079-1083Copper (I) perchlorate solutions of different concentrations in acetonitrile have been conductometerically titrated with solutions of thiourea (TU), N,N-dimethylthiourea (DMTU), N-phenylthiourea (NPTU), N, N-diphenylthioarea (DPTU), 1,10-phenanthroline (PHEN), 2,2' -bipyridyl (BPY) and triethylphosphite (TEP) at 298 K. The plots of conductivity versus moles of ligand added show significant decrease in conductivity followed by a region of almost constant conductivity. The fall of conductivity in the cases of thiourea and substituted thioureas is linear while in all the other cases it is non-linear. From these plots knowledge about the stoichiometry of the complexes formed between copper (I) perchlorate and various ligands has been derived. A complex of stoichiometry 1:2 is observed between copper (I) perchlorate and 1, 10-phenanthroline and 2, 2' -bipyridyl and 1:4 complex with triethylphosphite. Copper (I) perchlorate, however, forms complexes with thiourea and substituted thioureas, the stoichiometry of which strongly depends upon the concentrations of copper (I) perchlorate as well as of the ligands used for the titration. With N-phenylthiourea and N,N-diphenylthiourea, the complex formed has copper (I) perchlorate to ligand ratio as 1:2 under the present conditions employed, while with thiourea and N,N-dimethylthiourea, the stoichiometry of the complexes formed between copper (I) perchlorate and ligands is either 1:2 or 1:3 or 1:4 depending upon the conditions

Shear relaxation times of some binary liquid systems and electrolyte solutions

45-48Ultrasonic velocities (u), densities (p) and viscosities (Tl) of LiCIO4. NaCIO4, Mg(CIO4)2 and AI(CIO4)3 have been measured in water in the concentration range 0.02 to 4.0 mol kg-I at 298 K. The isentropic compressibilities (Ks) of various salts have been calculated and used to evaluate shear relaxation times (&tau;s). Shear relaxation times of CuClO4 and NaClO4 in cyanobenzene, cyanomethane and pyridine at 298, 308 and 318 K as well as of a large number of binary liquid systems at 298 K have also been calculated by using ultrasonic velocity and viscosity data from our previous studies. In binary liquid mixtures the relaxation time shows a .strong dependence on solvent composition and in electrolyte solutions on salt concentration. For AI(ClO4)3. Mg(ClO4)2 and LiClO4, the relaxation time increases significantly while for NaCIO4 it decreases slightly with the increase in salt concentration. At a given concentration, the relaxation time varies in the order: Al(ClO4)3&gt; Mg(CIO4)2&gt; LiClO4. &gt; NaCIO4. Both for CUClO4 and NaClO4 in cyanobenzene, cyanomethane and pyridine. the shear relaxation time also increases significantly with the increase in salt concentration. With the exception ofCuClO4 in cyanomethane, the relaxation time decreases with the increase in the temperature. The relaxation times for both these salts are in the order: cyanobenzene &gt; pyridine&gt; cyanomethane

Transport studies and enzyme assays in mice infected with human Giardia lamblia

It is well established that Giardia infection causes malabsorption. However, the precise mechanism of such a malabsorptipn is not known. To investigate this, transport studies, using the tissue accumulation technique, were carried out in mice infected with G. lamblia obtained from human stools. There was a significant fall in the transport of D-glucose, L-alanine and glycine in the infected animals compared with the controls. Kinetics of the D-glucose and glycine transport system were examined by measuring the tissue uptake in the presence of different concentrations of the substrate. For glucose, the affinity constant (Km) for the transport site was the same (4.37mM) in normal and infected animals but the maximal transport rate (V max) was considerably reduced in infected animals (158.7 μ moles/hr/g tissue) compared with (357.1 μ moles/hr/g tissue) in controls. Results with glycine were similar; the Km was similar in control and infected animals (5.7 mM) whereas the V max was reduced in infected animals (27.02 µ moles/hr/g tissue) compared with controls (45.5 μ moles/hr/g tissue). Analysis of the intestinal enzymes showed a significant decrease in the levels of brush border sucrase, lactase and alkaline phosphatase in infected animals; the cellular enzymes, LDH, GOT and GPT remained unaffected. The observed aberrations in the transport functions and brush border enzymes suggest that G. lamblia causes malabsorption by damaging the epithelial membrane of the enterocyte