Modulation of guinea pig intrinsic cardiac neurons by prostaglandins

Activation of cardiac mast cells has been shown to alter parasympathetic neuronal function via the activation of histamine receptors. The present study examined the ability of prostaglandins to alter the activity of guinea pig intracardiac neurons. Intracellular voltage recordings from whole mounts of the cardiac plexus showed that antigen-mediated mast cell degranulation produces an attenuation of the afterhyperpolarization (AHP), which was prevented by the phospholipase A2 inhibitor 5,8,11,14-eicosatetraynoic acid. Exogenous application of either PGD2 or PGE2 produced a biphasic change in the membrane potential and an inhibition of both AHP amplitude and duration. Examination of prostanoid receptors using bath perfusions (1 microM PGE2 and PGD2), specific agonists (BW245C, sulprostone, and butaprost), and antagonists (AH6809 and SC19220) found evidence for both the PGE2-specific EP2 and EP3 receptors, but not for EP1 or the PGD2-specific prostanoid (DP) receptors. Sulprostone was able to mimic the PGE2 responses in some cells, but not in all PGE2-sensitive cells. Butaprost was able to mimic the PG-induced hyperpolarization in some cells, but did not alter the AHP. Inhibition of specific potassium channels with either TEA, charybdotoxin, or apamin showed that neither TEA nor charybdotoxin could prevent the PGE2-induced AHP attenuation. Apamin alone inhibited AHP duration, with PGs having no further effect in these cells. These results demonstrate that guinea pig intracardiac neurons can be modulated by PG, most likely through either EP2, EP3, or potentially EP4 receptors, and this response is due, at least in part, to a reduction in small-conductance KCa currents.     

Influences of base excision repair defects on the lethality and mutagenicity induced by Me-lex,a sequence-selective N3-adenine methylating agent

Due to its minor groove selectivity, Me-lex preferentially generates N3-methyladenine (3-MeA) adducts in double-stranded DNA. We undertook a genetic approach in yeast to establish the influence of base excision repair (BER) defects on the processing of Me-lex lesions on plasmid DNA that harbors the p53 cDNA as target. We constructed a panel of isogenic strains containing a reporter gene to test p53 function and the following gene deletions: deltamag1, deltaapn1apn2, and deltaapn1apn2mag1. When compared with the wild-type strain, a decrease in survival was observed in deltamag1, deltaapn1apn2, and deltaapn1apn2mag1. The Me-lex-induced mutation frequency increased in the following order: wild type < deltamag1< deltaapn1apn2 = deltaapn1apn2mag1. A total of 77 mutants (23 in wild type, 31 in deltamag1, and 23 in deltaapn1apn2) were sequenced. Eighty-one independent mutations (24 in wild type, 34 in deltamag1, and 23 in deltaapn1apn2) were detected. The majority of base pair substitutions were AT-targeted in all strains (14/23, 61% in wild type; 20/34, 59%, in deltamag1; and 14/23, 61%, in deltaapn1apn2). The Mag1 deletion was associated with a significant decrease of GC > AT transitions when compared with both the wild-type and the AP endonuclease mutants. This is the first time that the impact of Mag1 and/or AP endonuclease defects on the mutational spectra caused by 3-MeA has been determined. The results suggest that 3-MeA is critical for Me-lex cytotoxicity and that its mutagenicity is slightly elevated in the absence of Mag1 glycosylase activity but significantly higher in the absence of AP endonuclease activity.     

Sensory lateralization in pain subjective perception for noxious heat stimulus

The aim of the present study was to identify and characterize hemispheric lateralization for pain intensity perception. A sample of 351 healthy volunteers was tested by the immersion of the right hand for 10 s followed by the same test for the left hand (RL group; n = 199) or in a random sequence (RND group; n = 152) into a water bath (48 degrees C, 15 s). Pain intensity was self-reported by the Visual Analogue Scale (VAS). The motor hemispherical Lateralization Index (LI) was obtained by the Edinburgh Inventory. Gender, hand skin fold, interstimulus time and menstrual cycle data in case of female subjects were recorded. The sample, 60.7% females and 39.3% males, 20.4 +/- 0.18 (mean +/- SEM) years old, showed 92.1% right-handed subjects. Left hand VAS was significantly higher than right hand VAS for RL (7.24 +/- 1.31 vs 6.74 +/- 1.52; p < 0.01) and RND (7.24 +/- 0.82 vs 6.73 +/- 1.25; p < 0.01) both for right- and left-handed subjects. A low but significant correlation for VAS scores and LI was found (r = 0.14; p < 0.05 or r = 0.18; p < 0.05, for left or right hand, respectively). Skin fold was statistically similar in both hands (p > 0.05) being highly correlated with each other (r = 0.68; p < 0.05). Pain subjective perception was not correlated to interstimulus time (r = -0.01; p > 0.05). Females showed significantly higher values than males for both left and right hand VAS scores. Periovulatory phase VAS value was significantly higher than luteal phase VAS only for the right hand test (7.57 +/- 0.20 vs 6.47 +/- 0.33; p < 0.01). The results of the present study suggest a lateralization of pain intensity perception to the right hemisphere not correlated with the motor hemispheric lateralization.     

Automated subcellular localization and quantification of protein expression in tissue microarrays

The recent development of tissue microarrays-composed of hundreds of tissue sections from different tumors arrayed on a single glass slide-facilitates rapid evaluation of large-scale outcome studies. Realization of this potential depends on the ability to rapidly and precisely quantify the protein expression within each tissue spot. We have developed a set of algorithms that allow the rapid, automated, continuous and quantitative analysis of tissue microarrays, including the separation of tumor from stromal elements and the sub-cellular localization of signals. Validation studies using estrogen receptor in breast carcinoma show that automated analysis matches or exceeds the results of conventional pathologist-based scoring. Automated analysis and sub-cellular localization of beta-catenin in colon cancer identifies two novel, prognostically significant tumor subsets, not detected by traditional pathologist-based scoring. Development of automated analysis technology empowers tissue microarrays for use in discovery-type experiments (more typical of cDNA microarrays), with the added advantage of inclusion of long-term demographic and patient outcome information.     

Different modulation by live or killed Helicobacter pylori on cytokine production from peripheral blood mononuclear cells

The mechanisms by which H. pylori colonizes and persists within the gastric mucosa are poorly understood. The induction and maintenance of gastric inflammation appear to depend on the complex interaction between a number of cytokines and chemokines. The gastric immune response observed "in vivo", during H. pylori infection, is characterized by a polarization of Th1 cell type that seems to be responsible for gastric pathology. The purpose of this study was to test the direct effect of H. pylori (live or gentamicin-killed) on human PBMC in order to evaluate the "in vitro" Th1-Th2 balance by monitoring IL-18, IFNgamma and IL-10 production. This study demonstrates for the first time that "in vitro" pretreatment with gentamicin-killed H. pylori of PBMC, followed by infection with live bacteria, downregulates the production of inflammatory cytokines such as IL-18 and IFNgamma Our results provide a possible strategy to restore the immunological disorders determined by H. pylori infection.     

Expression in E. coli and purification of recombinant fragments of wild type and mutant human prion protein

Prion diseases are fatal neurodegenerative disorders of the CNS of men and animals, characterized by spongiform degeneration of the CNS, astrogliosis and deposition of amyloid into the brain.The conversion of a cellular glycoprotein (the prion protein, PrP(C)) into an altered isoform (the prion scrapie, PrP(Sc)), which accumulates within the brain tissue by virtue of its resistance to the intracellular catabolism, is currently believed to represent the etiologic agent responsible for these diseases.Synthetic or recombinant polypeptides are commonly used to elucidate the mechanism of proteins involved in neurodegenerative diseases. Here we describe a procedure, which allows the synthesis and purification in its native folding, of the human prion protein fragment 90-231, corresponding to the protease resistant core of PrP(Sc). We synthesized the polypeptides 90-231 of both the wild type and the E200K mutant isoforms of PrP. Using a gluthatione S-transferase (GST) fusion protein approach, milligram amounts of polypeptides were obtained after expression in E. coli. The recovery of the purified fusion protein was monitored following the evaluation of the GST activity. The PrP fragment was released from the fusion protein immobilized on a glutathione-coupled agarose resin by direct cleavage with thrombin.The recombinant protein was identified by comassie stained acrylamide gel and by immunoblotting employing a monoclonal anti-PrP antibody. The peptide purified by gel filtration chromatography showed mainly an alpha-helix structure, as analysed by circular dichroism (CD) and an intact disulfide bridge. The same procedure was also successfully employed to synthesize and purify the E200K mutant PrP fragment.     

Differential induction of TNFalpha and IL-18 in human peripheral blood mononuclear cells infected with Leishmania major or Leishmania donovani

Several cytokines are involved in the host response to Leishmania. However, the role played by cytokines during infection with different species of Leishmania is not univocal. In this work, the production of tumor necrosis factor alpha (TNFalpha) and interleukin 18 (IL-18) during interaction of human phagocytes with Leishmania major or L. donovani was comparatively investigated. Peripheral blood mononuclear cells (PBMC) and monocytes from healthy donors were used. The release of TNFalpha and IL-18 during infection of cells with different species of Leishmania "in vitro" was evaluated. L. donovani induced in both PBMC and monocytes significantly more TNFalpha and IL-18 with respect to L. major. The amounts of TNFalpha released by PBMC were always significantly higher than those released by monocytes of the same donors.     

Treatment of PBMC with killed Helicobacter pylori subverts the environment of inflammatory cytokines

It is well known that inflammation induced by Helicobacter pylori is characterized by the local production of cytokines and chemokines. In the present study, we analyse the kinetics of MCP-1, IL-12 and IL-4 induction during the interaction of peripheral blood mononuclear cells with killed and/or live H. pylori. Our results demonstrate that live H. pylori does not induce IL-4 release whereas it stimulates MCP-1 and IL-12 production. In addition, the neutralization of IL-12 with monoclonal antibodies determines a lower MCP-1 release. These data demonstrate that MCP-1 production is in part supported by IL-12 induced by live H. pylori. On the contrary, killed H. pylori stimulates the IL-4 but not MCP-1 and IL-12 production. The combined treatment with killed and live H. pylori upregulates the IL-4 production and at the same time downregulates IL-12 and MCP-1 production.