Arterial acid-base status during digestion and following vascular infusion of NaHCO(3) and HCl in the South American rattlesnake, Crotalus durissus

Digestion is associated with gastric secretion that leads to an alkalinisation of the blood, termed the "alkaline tide". Numerous studies on different reptiles and amphibians show that while plasma bicarbonate concentration ([HCO(3)(-)](pl)) increases substantially during digestion, arterial pH (pHa) remains virtually unchanged, due to a concurrent rise in arterial PCO(2) (PaCO(2)) caused by a relative hypoventilation. This has led to the suggestion that postprandial amphibians and reptiles regulate pHa rather than PaCO(2). Here we characterize blood gases in the South American rattlesnake (Crotalus durissus) during digestion and following systemic infusions of NaHCO(3) and HCl in fasting animals to induce a metabolic alkalosis or acidosis in fasting animals. The magnitude of these acid-base disturbances were similar in magnitude to that mediated by digestion and exercise. Plasma [HCO(3)(-)] increased from 18.4+/-1.5 to 23.7+/-1.0 mmol L(-1) during digestion and was accompanied by a respiratory compensation where PaCO(2) increased from 13.0+/-0.7 to 19.1+/-1.4 mm Hg at 24 h. As a result, pHa decreased slightly, but were significantly below fasting levels 36 h into digestion. Infusion of NaHCO(3) (7 mmol kg(-1)) resulted in a 10 mmol L(-1) increase in plasma [HCO(3)(-)] within 1 h and was accompanied by a rapid elevation of pHa (from 7.58+/-0.01 to 7.78+/-0.02). PaCO(2), however, did not change following HCO(3)(-) infusion, which indicates a lack of respiratory compensation. Following infusion of HCl (4 mmol kg(-1)), plasma pHa decreased by 0.07 units and [HCO(3)(-)](pl) was reduced by 4.6 mmol L(-1) within the first 3 h. PaCO(2), however, was not affected and there was no evidence for respiratory compensation. Our data show that digesting rattlesnakes exhibit respiratory compensations to the alkaline tide, whereas artificially induced metabolic acid-base disturbances of same magnitude remain uncompensated. It seems difficult to envision that the central and peripheral chemoreceptors would experience different stimuli during these conditions. One explanation for the different ventilatory responses could be that digestion induces a more relaxed state with low responsiveness to ventilatory stimuli.     

Tempol diverts peroxynitrite/carbon dioxide reactivity toward albumin and cells from protein-tyrosine nitration to protein-cysteine nitrosation

Tempol has been shown to protect experimental animals from injuries associated with excessive nitric oxide production. In parallel, tempol decreased the levels of protein-3-nitrotyrosine in the injured tissues, suggesting that it interacted with nitric oxide-derived oxidants such as nitrogen dioxide and peroxynitrite. Relevantly, a few recent studies have shown that tempol catalytically diverts peroxynitrite/carbon dioxide reactivity toward phenol from nitration to nitrosation. To examine whether this shift occurs in biological environments, we studied the effects of tempol (10-100 microM) on peroxynitrite/carbon dioxide (1 mM/2 mM) reactivity toward proteins, native bovine serum albumin (BSA) (0.5-0.7 cys/mol) and reductively denatured BSA (7-19 cys/mol), and cells (J774 macrophages). Although not a true catalyst, tempol strongly inhibited protein-tyrosine nitration (70-90%) and protein-cysteine oxidation (20-50%) caused by peroxynitrite/carbon dioxide in BSA, denatured BSA, and cells while increasing protein-cysteine nitrosation (200-400%). Tempol consumption was attributed mainly to its reaction with protein-cysteinyl radicals. Most of the tempol, however, reacted with the radicals produced from peroxynitrite/carbon dioxide, that is, nitrogen dioxide and carbonate radical anion. Accordingly, tempol decreased the yields of BSA-cysteinyl and BSA-tyrosyl/tryptophanyl radicals, as well their decay products such as protein-3-nitrotyrosine. The parallel increase in protein-nitrosocysteine yields demonstrated that part of the peroxynitrite is oxidized to nitric oxide by the oxammonium cation produced from tempol oxidation by peroxynitrite/carbon dioxide-derived radicals. Protein-nitrosocysteine formation was shown to occur by radical and nonradical mechanisms in studies with a protein-cysteinyl radical trapper. These studies may contribute to the understanding of the protective effects of tempol in animal models of inflammation.     

Inhibition of intramolecular electron transfer in ascorbate oxidase by Ag+: redox state dependent binding

Intramolecular electron transfer within zucchini squash ascorbate oxidase is inhibited in a novel manner in the presence of an equimolar concentration of Ag(+). At pH 5.5 in acetate buffer reduction of the enzyme by laser flash photolytically generated 5-deazariboflavin semiquinone occurs at the Type I Cu with a rate constant of 5 x 10(8) M(-1)s(-1). Subsequent to this initial reduction step, equilibration of the reducing equivalent between the Type I Cu and the trinuclear Type II, III copper cluster (TNC) occurs with rate constant of 430 s(-1). The 41% of the reduced Type I Cu is oxidized by this intramolecular electron transfer reaction. When these reactions are performed in the presence of Ag(+) equimolar to dimeric AO, the bimolecular reduction of the enzyme by the 5-deazariboflavin semiquinone is not affected. As in the case of the native enzyme, intramolecular electron transfer between the Type I Cu and the TNC occurs, which continues until 25% of the reducing equivalent has been transferred. At that point, the reducing equivalent is observed to more slowly return to the Type I Cu, resulting a second reduction phase whose rate constant (100 s(-1)) is protein and Ag(+) concentration independent. The data suggest that partial reduction of the TNC results in Ag(+) binding to the enzyme which causes the apparent midpoint potential of the TNC as a whole to decrease thereby reversing the direction of electron flow. These results are consistent with the inhibitory effect of Ag(+) on the steady-state activity of ascorbate oxidase [S. Maritano, E. Malusa, A. Marchesini, presented at The Meeting on Metalloproteins, SERC Daresbury Laboratory, Warrington, England, 1992; A. Marchesini, XIX Convegno Nazionale SICA, Italian Society of Agricultural Chemistry, Reggio Calabria, Italy, September 2001.].     

Diagnostic utility of GLUT-1 expression in the cytologic evaluation of serous fluids

OBJECTIVE: To evaluate the extent to which adenocarcinomas in body cavity fluids express GLUT-1 in comparison to currently available markers for adenocarcinomas. STUDY DESIGN: Archival paraffin-embedded cell blocks of serous fluids from 25 cases of benign effusions containing reactive mesothelial cells and 39 cases of malignant effusions with metastatic adenocarcinoma (11 ovarian, 11 pulmonary, 9 gastrointestinal and 8 breast) were retrieved from the surgical pathology files. All cases were stained with antibodies for GLUT-1, Ber-Ep4, B72.3 and CEA. Positive staining was defined as distinct linear membrane staining for GLUT-1 and Ber-EP4, cytoplasmic staining for CEA, and cytoplasmic or membrane staining for B72.3. Strong staining in at least 10% of the tumor cells was required in order to consider the case positive for the particular marker. RESULTS: GLUT-1 was expressed in 72% (28 of 39) of cases of malignant effusions: 100% (11 of 11) from the ovary, 91% (10 of 11) from the lung, 67% (6 of 9) from the gastrointestinal tract and 12% (1 of 8) from the breast. None (0 of 25) of the benign effusions expressed GLUT-1. Malignant effusions expressed CEA in 74% (29 of 39), Ber-Ep4 in 85% (33 of 39), and B72.3 in 62% (24 of 39). Benign effusions expressed CEA in 3 cases and B72.3 in 2 cases. CONCLUSION: GLUT-1 is a useful marker that can be applied to cytologic specimens. It can be used as a reliable component of an antibody panel to distinguish reactive mesothelial cells from metastatic adenocarcinoma in particular adenocarcinomas of body cavity effusions, in particular adenocarcinomas of ovarian and pulmonary origin.     

Structural bioinformatics study of PNP from Schistosoma mansoni

The parasite Schistosoma mansoni lacks the de novo pathway for purine biosynthesis and depends on salvage pathways for its purine requirements. Schistosomiasis is endemic in 76 countries and territories and amongst the parasitic diseases ranks second after malaria in terms of social and economic impact and public health importance. The PNP is an attractive target for drug design and it has been submitted to extensive structure-based design. The atomic coordinates of the complex of human PNP with inosine were used as template for starting the modeling of PNP from S. mansoni complexed with inosine. Here we describe the model for the complex SmPNP-inosine and correlate the structure with differences in the affinity for inosine presented by human and S. mansoni PNPs.     

Interaction of shikimic acid with shikimate kinase

The crystal structure of shikimate kinase from Mycobacterium tuberculosis (MtSK) complexed with MgADP and shikimic acid (shikimate) has been determined at 2.3A resolution, clearly revealing the amino acid residues involved in shikimate binding. In MtSK, the Glu61 strictly conserved in SK forms a hydrogen bond and salt-bridge with Arg58 and assists in positioning the guanidinium group of Arg58 for shikimate binding. The carboxyl group of shikimate interacts with Arg58, Gly81, and Arg136, and hydroxyl groups with Asp34 and Gly80. The crystal structure of MtSK-MgADP-shikimate will provide crucial information for elucidation of the mechanism of SK-catalyzed reaction and for the development of a new generation of drugs against tuberculosis.     

L-Arginine modulates CD3zeta expression and T cell function in activated human T lymphocytes

Engagement of the T cell receptor (TCR) by antigen or anti-CD3 antibody results in a cycle of internalization and re-expression of the CD3zeta. Following internalization, CD3zeta is degraded and replaced by newly synthesized CD3zeta on the cell surface. Here, we provide evidence that availability of the amino acid L-arginine modulates the cycle of internalization and re-expression of CD3zeta and cause T cell dysfunction. T cells stimulated and cultured in presence of L-arginine, undergo the normal cycle of internalization and re-expression of CD3zeta. In contrast, T cells stimulated and cultured in absence of L-arginine, present a sustained down-regulation of CD3zeta preventing the normal expression of the TCR, exhibit a decreased proliferation, and a significantly diminished production of IFNgamma, IL5, and IL10, but not IL2. The replenishment of L-arginine recovers the expression of CD3zeta. The decreased expression of CD3zeta is not caused by a decreased CD3zeta mRNA, an increased CD3zeta degradation or T cell apoptosis.     

Helicobacter pylori arginase inhibits T cell proliferation and reduces the expression of the TCR zeta-chain (CD3zeta)

Helicobacter pylori infects approximately half the human population. The outcomes of the infection range from gastritis to gastric cancer and appear to be associated with the immunity to H. pylori. Patients developing nonatrophic gastritis present a Th1 response without developing protective immunity, suggesting that this bacterium may have mechanisms to evade the immune response of the host. Several H. pylori proteins can impair macrophage and T cell function in vitro through mechanisms that are poorly understood. We tested the effect of H. pylori extracts and live H. pylori on Jurkat cells and freshly isolated human normal T lymphocytes to identify possible mechanisms by which the bacteria might impair T cell function. Jurkat cells or activated T lymphocytes cultured with an H. pylori sonicate had a reduced proliferation that was not caused by T cell apoptosis or impairment in the early T cell signaling events. Instead, both the H. pylori sonicate and live H. pylori induced a decreased expression of the CD3zeta-chain of the TCR. Coculture of live H. pylori with T cells demonstrated that the wild-type strain, but not the arginase mutant rocF(-), depleted L-arginine and caused a decrease in CD3zeta expression. Furthermore, arginase inhibitors reversed these events. These results suggest that H. pylori arginase is not only important for urea production, but may also impair T cell function during infection.     

Nitric oxide of the supraoptic nucleus influences the salivary secretion, sodium renal excretion, urinary volume and arterial blood pressure induced by pilocarpine

Male Holtzman rats weighting 200-250 g were anesthetized with zoletil 50 mg/Kg (tiletamine chloridrate 125.0 mg and zolazepan chloridrate 125.0 mg) into quadriceps muscle and stainless steel cannulas were implanted into their supraoptic nucleus (SON). We investigated the effects of the injection into the supraoptic nucleus (SON) of FK 409, a nitric oxide donor, and NW-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor (NOS), on the salivary secretion, arterial blood pressure, sodium excretion and urinary volume induced by pilocarpine, which was injected into SON. The drugs were injected in 0.5 microl volume over 30-60 s. Controls was injected with a similar volume of 0.15 M NaCl. FK 409 and L-NAME were injected at doses of 20 microg/0.5 microl and 40 microg/0.5 microl respectively. The amount of saliva secretion was studied over a five-minute period after injection of pilocarpine into SON. Injection of pilocarpine (10, 20, 40, 80, 160 microg/microl) into SON produced a dose-dependent increase in salivary secretion. L-NAME was injected into SON prior to the injection of pilocarpine into SON, producing an increase in salivary secretion due to the effect of pilocarpine. FK 409 injected into SON attenuating the increase in salivary secretion induced by pilocarpine. Mean arterial pressure (MAP) increase after injections of pilocarpine into the SON. L-NAME injected into the SON prior to injection of pilocarpine into SON increased the MAP. FK 409 injected into the SON prior to pilocarpine attenuated the effect of pilocarpine on MAP. Pilocarpine (0.5 micromol/0.5 microl) injected into the SON induced an increase in sodium and urinary excretion. L-NAME injected prior to pilocarpine into the SON increased the urinary sodium excretion and urinary volume induced by pilocarpine. FK 409 injected prior to pilocarpine into the SON decreased the sodium excretion and urinary volume induced by pilocarpine. All these roles of pilocarpine depend on the release of nitric oxide into the SON. In summary the present results show: a) SON is involved in pilocarpine-induced salivation; b) that mechanism involves increase in MAP, sodium excretion and urinary volume.