Jorge Luiz Pesquero

Effect of a High-Intensity Exercise Training on the Metabolism and Function of Macrophages and Lymphocytes of Walker 256 Tumor–Bearing Rats

Epidemiologic studies suggest that moderately intense training promotes augmented immune function, whereas strenuous exercise can cause immunosupression. Because the combat of cancer requires high immune function, high-intensity exercise could negatively affect the host organism; however, despite the epidemiologic data, there is a lack of experimental evidence to show that high-intensity training is harmful to the immune system. Therefore, we tested the influence of high-intensity treadmill training (10 weeks, 5 days/week, 30 mins/day, 85% VO2max) on immune system function and tumor development in Walker 256 tumor–bearing Wistar rats. The metabolism of glucose and glutamine in lymphocytes and macrophages was assessed, in addition to some functional parameters such as hydrogen peroxide production, phagocytosis, and lymphocyte proliferative responses. The metabolism of Walker 256 cells was also investigated. Results demonstrated that high-intensity training increased the life span of tumor-bearing rats, promoted a reduction in tumor mass, and prevented indicators of cachexia. Several changes, such as a reduction in body weight and food intake and activation of glutamine metabolism in macrophages and lymphocytes induced by the presence of Walker 256 tumor, were prevented by high intensity training. The reduction in tumor growth was associated with an impairment of tumor cell glucose and glutamine metabolism. These data suggest that high-intensity exercise training may be a viable strategy against tumors.

Role of the kinin B1 receptor in insulin homeostasis and pancreatic islet function.

Abstract Kinins are potent vasoactive peptides generated in blood and tissues by the kallikrein serine proteases. Two distinct kinin receptors have been described, one constitutive (subtype B2) and one inducible (subtype B1), and many physiological functions have been attributed to these receptors, including glucose homeostasis and control of vascular permeability. In this study we show that mice lacking the kinin B1 receptor (B1 -/- mice) have lower fasting plasma glucose concentrations but exhibit higher glycemia after feeding when compared to wild-type mice. B1 -/- mice also present pancreas abnormalities, characterized by fewer pancreatic islets and lower insulin content, which leads to hypoinsulinemia and reduced insulin release after a glucose load. Nevertheless, an insulin tolerance test indicated higher sensitivity in B1 -/- mice. In line with this phenotype, pancreatic vascular permeability was shown to be reduced in B1 receptor-ablated mice. The B1 agonist desArg9bradykinin injected intravenously can induce the release of insulin into serum, and this effect was not observed in the B1 -/- mice or in isolated islets. Our data demonstrate the importance of the kinin B1 receptor in the control of pancreatic vascular homeostasis and insulin release, highlighting a new role for this receptor in the pathogenesis of diabetes and related diseases.

An improved method to distinguish Entamoeba histolytica and Entamoeba dispar

A 482 base pair gene fragment from samples of amoebae E. histolytica and E. dispar was amplified by PCR. The amplification products of fragments from the 2 species of amoebae presented differences in mobility in non-denaturing polyacrylamide gel, probably due to sequence-dependent conformational alterations in the DNA fragments. The method described here permits E. histolytica and E. dispar to be distinguished with greater sensitivity and rapidity.

Purification and partial characterization of kininogenase activity from Schistosoma mansoni adult worms

An enzyme presenting kallikrein-like activity (designated sK1) was purified from the supernatant of Schistosoma mansoni adult worm homogenate. The enzyme cleaves bradykinin from purified rat plasma kininogen. Activity was optimal at pH 9.0 and the enzyme showed amidolytic activity, since it hydrolysed the kallikrein synthetic substrate D-Pro-Phe-Arg-p-nitroanilide. The activity of sK1 upon rat plasma kininogen was strongly inhibited by the serine proteinase inhibitors phenylmethanesulfonyl fluoride, aprotinin or soybean trypsin inhibitor, but not by ethylenediaminetetraacetic acid or sodium tetrathionate. The molecular mass of sK1, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was 66 kDa and the pI value, estimated by analytical chromatofocusing, was 4.2. Physical and chemical properties suggest that sK1 is a serine proteinase of the kallikrein family. Evidence is presented which suggests that sK1 is a component of the tegumental surface of the parasite and the levels of its activity in the male adult worm are approximately 21 times higher than those in the female adult worm. The intravenous injection of 3 micrograms of sK1 into an anaesthetized rat induced a drastic reduction in the arterial blood pressure of the animal. This effect lasted for about 1 min, and was followed by a progressive recovery of the arterial pressure. Neither bradycardia nor cardiac arrhythmias were noticed, suggesting a peripheral vasodilation effect. The presence of sK1 on the surface of adult male worms could play an important role in the wandering capacity of coupled worms into the visceral vasculature of the host.

A single step duplex PCR to distinguish Entamoeba histolytica from Entamoeba dispar

In this study, a single-step duplex polymerase chain reaction procedure was developed for rapid, specific and sensitive identification of Entamoeba histolytica and for its diagnostic differentiation from E. dispar. Specific oligonucleotide primers were combined for the amplification of a cysteine proteinase 5 gene target sequence of 242 bp, present only in E. histolytica. Additionally, another oligonucleotide primer pair for both the E. histolytica and E. dispar actin gene target of 300 bp was designed to amplify only from amoebae DNA. The PCR developed was specific and efficiently identified and differentiated these parasites from each other in either cultured parasites or from stool material.

Molecular structure and alternative splicing of the human carboxypeptidase M gene.

Abstract Using RACE technology the 5 and 3 ends of human carboxypeptidase M (CPM) mRNA were determined and found to be divergent from the published sequence. With these results the complete structure of the human CPM gene was established based on the human genome sequence in the GenBank database. The gene was shown to contain 9 exons comprising at least 75 kb of genomic sequence. A novel first exon of 30 bp was identified and an upstream promoter sequence containing several transcription factor binding sites was found by computer analysis. Furthermore, the ATG starting codon was detected defining an open reading frame of 1329 bp that codes for a protein of 443 residues. Additionally, the polyadenylation site was discovered, determining a 3 noncoding region of 2000 nucleotides. The exonintron boundaries diverged substantially compared to those of the other basic carboxypeptidases, CPD, CPE, CPN, and AEBP1. Cloning and sequencing of RTPCR products from different tissues revealed alternative splicing of exons 3 and 5, which results in the generation of four different mRNA isoforms. RNA extracted from tumor tissues contained more CPM mRNA than control tissue, suggesting an upregulation of CPM expression in tumors and raising the question of the role of this enzyme in cancer.

Diagnóstico de resistência do Mycobacterium tuberculosis à rifampicina utilizando-se da reação em cadeia da polimerase

A resistencia do Mycobacterium tuberculosis aos tuberculostaticos tem surgido como grande ameaca a Saude Publica. A resistencia a rifampicina pode ser considerada como um marcador para a multi-resistencia a farmacos e tem sido atribuida a mudancas estruturais da RNA polimerase, produto de expressao do gene rpoB. Os pacientes portadores dessas cepas tem baixa perspectiva frente ao tratamento. Os testes convencionais de sensibilidade aos farmacos realizados em cultura do Mtb requerem varias semanas para o crescimento. Por este motivo, a Reacao em Cadeia da Polimerase (PCR), metodo de baixo custo e que pode reduzir o tempo para o diagnostico, representa alternativa viavel e promissora. Neste artigo estao descritos os metodos mais comumente empregados na deteccao de mutantes resistentes a rifampicina baseados na PCR, como analise de Polimorfismo Conformacional de Fita Simples (Single-Strand Conformation Polymorphism, SSCP), PCR Heteroduplex e INNO-LIPA. Recentemente, padronizou-se a tecnica de PCR em baixa estringencia, usando um unico iniciador (Low Stringency Single Specific Primer, LSSP), que se mostrou um metodo rapido e sensivel na deteccao de mutacoes no gene rpoB.

A new method for bovine pepsinogen purification preparation of a specific antibody

Bovine pepsinogen was purified from abomasum by ammonium sulphate precipitation and ionic exchange chromatography on DEAE-cellulose and Mono Q columns. Purified pepsinogen was shown to be homogeneous by analytical electrophoresis, having an estimated molecular mass of 46,000 Daltons. The isoelectric point, determined by analytical chromatofocusing was 4.6. Using this pepsinogen preparation to immunize rabbits, a specific antiserum of high titer was obtained.

High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterization

Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein(-1) min(-1)). This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes.

TRPM8 Channel Activation Induced by Monoterpenoid Rotundifolone Underlies Mesenteric Artery Relaxation.

In this study, our aims were to investigate transient receptor potential melastatin-8 channels (TRPM8) involvement in rotundifolone induced relaxation in the mesenteric artery and to increase the understanding of the role of these thermosensitive TRP channels in vascular tissue. Thus, message and protein levels of TRPM8 were measured by semi-quantitative PCR and western blotting in superior mesenteric arteries from 12 week-old Spague-Dawley (SD) rats. Isometric tension recordings evaluated the relaxant response in mesenteric rings were also performed. Additionally, the intracellular Ca2+ changes in mesenteric artery myocytes were measured using confocal microscopy. Using PCR and western blotting, both TRPM8 channel mRNA and protein expression was measured in SD rat mesenteric artery. Rotundifolone and menthol induced relaxation in the isolated superior mesenteric artery from SD rats and improved the relaxant response induced by cool temperatures. Also, this monoterpene induced an increase in transient intracellular Ca2+. These responses were significantly attenuated by pretreatment with capsazepine or BCTC, both TRPM8 channels blockers. The response induced by rotundifolone was not significantly attenuated by ruthenium red, a non-selective TRP channels blocker, or following capsaicin-mediated desensitization of TRPV1. Our findings suggest that rotundifolone induces relaxation by activating TRPM8 channels in rat superior mesenteric artery, more selectively than menthol, the classic TRPM8 agonist, and TRPM8 channels participates in vasodilatory pathways in isolated rat mesenteric arteries.