Differentiation of Melipona quadrifasciata L. (Hymenoptera, Apidae, Meliponini) subspecies using cytochrome b PCR-RFLP patterns

Melipona quadrifasciata quadrifasciata and M. quadrifasciata anthidioides are subspecies of M. quadrifasciata, a stingless bee species common in coastal Brazil. These subspecies are discriminated by the yellow stripe pattern of the abdominal tergites. We found Vsp I restriction patterns in the cytochrome b region closely associated to each subspecies in 155 M. quadrifasciata colonies of different geographical origin. This mitochondrial DNA molecular marker facilitates diagnosis of M. quadrifasciata subspecies matrilines and can be used to establish their natural distribution and identify hybrid colonies.

Cirurgia de catarata: características e opiniões de pacientes com visão mono versus binocular

OBJETIVO: Verificar em dois grupos de pacientes com visão monocular (grupo 1) e com visão binocular (grupo 2), a serem submetidos à cirurgia de catarata num hospital universitário, opiniões em relação ao problema ocular, à qualidade da visão e à cirurgia de catarata. MÉTODOS: Foi realizado estudo transversal e comparativo, de forma consecutiva, por meio de questionário estruturado, aplicado por entrevista a pacientes, elaborado a partir de estudo exploratório e medidas acuidade visual e causa da perda visual. RESULTADOS: A amostra foi constituída por 96 indivíduos do grupo 1 (50,0% homens; 50,0% mulheres, com idade entre 41 e 91 anos, média 69,3 anos ± 10,4 anos) e 110, do grupo 2 (40,9% homens; 59,1% mulheres, com idade entre 40 e 89 anos, média 68,2 anos ± 10,2 anos). A maioria dos indivíduos de ambos os grupos apresentava baixa escolaridade. Não houve diferença estatisticamente significante entre os grupos em relação ao sexo (p=0,191), à idade (p=0,702) e à escolaridade (p=0,245). Não exerciam atividade laboral 95,8% dos indivíduos do grupo 1 e 83,6%, do grupo 2 (p=0,005) e 30,4% do grupo 1 mencionaram não ter possibilidade de trabalhar por causa da deficiência visual. Observou-se acuidade visual do olho a ser operado menor que 0,05 em 40,6% (grupo 1) e 33,6% (grupo 2), entre 0,25 e 0,05. Quase a totalidade dos indivíduos de ambos os grupos afirmou ter dificuldade para realização das atividades de vida diária e qualificou como insuficiente a respectiva acuidade visual; 71,9% dos entrevistados do grupo 1 e 71,6%, do grupo 2 mencionaram saber a causa da visão fraca; desses, 87,1% do grupo 1 e 83,3% do grupo 2 referiram a catarata como causa da baixa acuidade visual. CONCLUSÃO: Os indivíduos de ambos os grupos tiveram acesso à cirurgia de catarata com acuidade visual menor do que a idealmente indicada; os pacientes com visão monocular apresentaram acuidade visual significativamente menor em relação aos com visão binocular; a maioria dos entrevistados de ambos os grupos referiu dificuldades para realizar atividades cotidianas como consequência da baixa visão; muitos indivíduos de ambos os grupos desconheciam a causa da dificuldade visual ou a atribuíram a outra causa que não a catarata.

Induction of Heme Oxygenase-1 Can Halt and Even Reverse Renal Tubule-Interstitial Fibrosis

Background: The tubule-interstitial fibrosis is the hallmark of progressive renal disease and is strongly associated with inflammation of this compartment. Heme-oxygenase-1 (HO-1) is a cytoprotective molecule that has been shown to be beneficial in various models of renal injury. However, the role of HO-1 in reversing an established renal scar has not yet been addressed. Aim: We explored the ability of HO-1 to halt and reverse the establishment of fibrosis in an experimental model of chronic renal disease. Methods: Sprague-Dawley male rats were subjected to unilateral ureteral obstruction (UUO) and divided into two groups: non-treated and Hemin-treated. To study the prevention of fibrosis, animals were pre-treated with Hemin at days -2 and -1 prior to UUO. To investigate whether HO-1 could reverse established fibrosis, Hemin therapy was given at days 6 and 7 post-surgery. After 7 and/or 14 days, animals were sacrificed and blood, urine and kidney tissue samples were collected for analyses. Renal function was determined by assessing the serum creatinine, inulin clearance, proteinuria/creatininuria ratio and extent of albuminuria. Arterial blood pressure was measured and fibrosis was quantified by Picrosirius staining. Gene and protein expression of pro-inflammatory and pro-fibrotic molecules, as well as HO-1 were performed. Results: Pre-treatment with Hemin upregulated HO-1 expression and significantly reduced proteinuria, albuminuria, inflammation and pro-fibrotic protein and gene expressions in animals subjected to UUO. Interestingly, the delayed treatment with Hemin was also able to reduce renal dysfunction and to decrease the expression of pro-inflammatory molecules, all in association with significantly reduced levels of fibrosis-related molecules and collagen deposition. Finally, TGF-beta protein production was significantly lower in Hemin-treated animals. Conclusion: Treatment with Hemin was able both to prevent the progression of fibrosis and to reverse an established renal scar. Modulation of inflammation appears to be the major mechanism behind HO-1 cytoprotection.

Spectroscopic, structural, and functional characterization of the alternative low-spin state of horse heart cytochrome c

The alternative low-spin states of Fe3+ and Fe2+ cytochrome c induced by SDS or AOT/hexane reverse micelles exhibited the heme group in a less rhombic symmetry and were characterized by electron paramagnetic resonance, UV-visible, CD, magnetic CD, fluorescence, and Raman resonance. Consistent with the replacement of Met 80 by another strong field ligand at the sixth heme iron coordination position, Fe3+ ALSScytc exhibited 1-nm Soret band blue shift and e enhancement accompanied by disappearance of the 695-nm charge transfer band. The Raman resonance, CD, and magnetic CD spectra of Fe3+ and Fe2+ ALSScytc exhibited significant changes suggestive of alterations in the heme iron microenvironment and conformation and should not be assigned to unfold because the Trp(59) fluorescence remained quenched by the neighboring heme group. ALSScytc was obtained with His(33) and His(26) carboxyethoxylated horse cytochrome c and with tuna cytochrome c (His(33) replaced by Asn) pointing out Lys(79) as the probable heme iron ligand. Fe3+ ALSScytc retained the capacity to cleave tert-butylhydroperoxide and to be reduced by dithiothreitol and diphenylacetaldehyde but not by ascorbate. Compatible with a more open heme crevice, ALSScytc exhibited a redox potential similar to 200 mV lower than the wild-type protein (1220 mV) and was more susceptible to the attack of free radicals.

Ethanol-induced vasoconstriction is mediated via redox-sensitive cyclo-oxygenase-dependent mechanisms

The present study investigated the role of ROS (reactive oxygen species) and COX (cyclooxygenase) in ethanol-induced contraction and elevation of [Ca(2+)](i) (intracellular [Ca(2+)]). Vascular reactivity experiments, using standard muscle bath procedures, showed that ethanol (1-800 mmol/l) induced contraction in endothelium-intact (EC(50): 306 +/- 34 mmol/l) and endothelium-denuded (EC(50): 180 +/- 40 mmol/l) rat aortic rings. Endothelial removal enhanced ethanol-induced contraction. Preincubation of intact rings with L-NAME [N(G)-nitro-L-arginine methyl ester; non-selective NOS (NO synthase) inhibitor, 100 mu mol/l], 7-nitroindazole [selective nNOS (neuronal NOS) inhibitor, 100 mu mol/l], oxyhaemoglobin (NO scavenger, 10 mu mol/l) and ODQ (selective inhibitor of guanylate cyclase enzyme, 1 mu mol/l) increased ethanol-induced contraction. Tiron [O(2)(-) (superoxide anion) scavenger, 1 mmol/l] and catalase (H(2)O(2) scavenger, 300 units/ml) reduced ethanol-induced contraction to a similar extent in both endothelium-intact and denuded rings. Similarly, indomethacin (non-selective COX inhibitor, 10 mu mol/l), SC560 (selective COX- I inhibitor, 1 mu mol/l), AH6809 [PGF(2 alpha) (prostaglandin F(2 alpha))] receptor antagonist, 10 mu mol/l] or SQ29584 [PGH(2)(prostaglandin H(2))/TXA(2) (thromboxane A(2)) receptor antagonist, 3 mu mol/l] inhibited ethanol-induced contraction in aortic rings with and without intact endothelium. In cultured aortic VSMCs (vascular smooth muscle cells), ethanol stimulated generation of O(2)(-) and H(2)O(2). Ethanol induced a transient increase in [Ca(2+)](i), which was significantly inhibited in VSMCs pre-exposed to tiron or indomethacin. Our data suggest that ethanol induces vasoconstriction via redox-sensitive and COX-dependent pathways, probably through direct effects on ROS production and Ca(2+) signalling. These findings identify putative molecular mechanisms whereby ethanol, at high concentrations, influences vascular reactivity. Whether similar phenomena occur in vivo at lower concentrations of ethanol remains unclear.

Dehydromonocrotaline induces cyclosporine A-insensitive mitochondrial permeability transition/cytochrome c release

Monocrotaline (MCT) is a pyrrolizidine alkaloid present in plants of the genus Crotalaria that causes cytotoxicity and genotoxicity in animals and humans. It is well established that the toxicity of MCT results from its hepatic bioactivation to dehydromonocrotaline (DHM), an alkylating agent, but the exact mechanism of action remains unknown. In a previous study, we demonstrated DHM`s inhibition of mitochondrial NADH-dehydrogenase activity at micromolar concentrations, which is an effect associated with a significant reduction in ATP synthesis. As a follow-up study, we have evaluated the ability of DHM to induce mitochondrial permeability transition (MPT) and its associated processes in isolated rat liver mitochondria. In the presence of 10 mu M Ca(2+), DHM (50-250 mu M) elicited MPT in a concentration-dependent, but cyclosporine A-independent manner, as assessed by mitochondrial swelling, which is associated with mitochondrial Ca(2+) efflux and cytochrome c release. DHM (50-250 mu M) did not cause hydrogen peroxide accumulation but did deplete endogenous glutathione and NAD(P)H, while oxidizing protein thiol groups. These results potentially indicate the involvement of mitochondria, via apoptosis, in the well-documented cytotoxicity of monocrotaline. (C) 2009 Elsevier Ltd. All rights reserved.

Tetra-crowned porphyrin as P450 biomimetic model for carbamazepine oxidation

A substituted porphyrin bearing four crown ether units, H(2)(TCP), was synthesized from the reaction between (5,10,15,20-tetra(o-aminophenyl) porphyrin) and the acyl derivative of the ether (4-carboxy-18-crown-6). The free-base porphyrin was characterized by C, N, and H elemental analysis; UV-vis and IR spectroscopies; and (1)H NMR. The corresponding ironporphyrin, Fe(TCP)Cl, was obtained via iron insertion into H(2)(TCP). Fe(TCP)Cl was employed as catalyst for carbamazepine (CBZ) oxidation by iodosylbenzene (PhIO), 3-chloroperoxybenzoic acid (m-CPBA) or sodium hypochlorite (NaOCl), in methanol or in a biphasic water/dichloroethane system. The crowned ironporphyrin proved to be a highly efficient and selective catalyst for CBZ epoxidation even in the biphasic dichloroethane /H(2)O system, with no need for an additional phase transfer agent.

The common p450 oxidoreductase variant A503V is not a modifier gene for 21-hydroxylase deficiency

Context: 21-hydroxylase deficiency (21OHD) is a common genetic disorder caused by mutations in the CYP21A2 gene, which encodes the adrenal 21-hydroxylase, microsomal P450c21. CYP21A2 gene mutations generally correlate well with impaired P450c21 enzymatic activity and the clinical findings in 21OHD, but occasional discrepancies between genotype and phenotype suggest the effects of modifier genes. Mutations in P450 oxidoreductase (POR), the protein that transfers electrons from reduced nicotinamide adenine dinucleotide phosphate to all microsomal P450s, can ameliorate the 21OHD phenotype and, therefore, could be a modifier gene. Objectives: We sought to identify POR variants in patients with 21OHD having discordant phenotype and genotype, and to evaluate their effect on 21-hydroxylase activity. Patients and Methods: We determined the CYP21A2 genotypes of 313 Brazilian patients with 21OHD and correlated the genotype and phenotype. The POR gene was sequenced in 17 patients with discordant genotype and phenotype. Wild-type and A503V POR, and P450c21 were expressed in bacteria and reconstituted in vitro. Activities were assayed by conversion of [C-14] progesterone to deoxycorticosterone and [H-3]17-hydroxyprogesterone to 11-deoxycortisol, and assessed by thin layer chromatography and phosphorimaging. Results: The A503V POR variant was found in 10 of 30 alleles, the same ratio as in the normal population. There were no significant differences in Michaelis constant, maximum velocity and maximum velocity/Michaelis constant of 21-hydroxylase activity supported by wild-type and A503V POR. Conclusion: The only POR missense polymorphism found in atypical 21OHD patients was A503V. Although A503V reduces P450c17 enzymatic activity, it does not influence P450c21 activity, indicating that POR A503V does not modify the 21OHD phenotype.

Molecular basis of infantile reversible cytochrome c oxidase deficiency myopathy

Childhood-onset mitochondrial encephalomyopathies are usually severe, relentlessly progressive conditions that have a fatal outcome. However, a puzzling infantile disorder, long known as `benign cytochrome c oxidase deficiency myopathy` is an exception because it shows spontaneous recovery if infants survive the first months of life. Current investigations cannot distinguish those with a good prognosis from those with terminal disease, making it very difficult to decide when to continue intensive supportive care. Here we define the principal molecular basis of the disorder by identifying a maternally inherited, homoplasmic m.14674T > C mt-tRNA(Glu) mutation in 17 patients from 12 families. Our results provide functional evidence for the pathogenicity of the mutation and show that tissue-specific mechanisms downstream of tRNA(Glu) may explain the spontaneous recovery. This study provides the rationale for a simple genetic test to identify infants with mitochondrial myopathy and good prognosis.

DIVERGENT ROLE OF HEME OXYGENASE INHIBITION IN THE PATHOGENESIS OF SEPSIS

The reduction of neutrophil migration to an infectious focus is associated with a high mortality in severe sepsis. Previously, we showed that heme oxygenase (HO) products downregulate neutrophil recruitment in a noninfectious inflammatory model. The present study was designed to determine the role of HO in sepsis induced by cecal ligation and puncture (CLP) model. We demonstrated that pretreatment, but not the combination of pretreatment plus posttreatment with zinc protoporphyrin IX (ZnPP IX), an HO inhibitor, prevented the reduction of CXCR2 on circulating neutrophils and the failure of intraperitoneal neutrophil migration to the site of infection. Consequently, bacterial dissemination, systemic inflammatory response, and organ injury were prevented. In addition, pretreatment with the HO inhibitor avoided hypotension and consequently increased survival. Moreover, in mice subjected to severe CLP, the pretreatment, but not the combination of pretreatment plus posttreatment with ZnPP IX, prevented the increase of plasmatic free heme observed in nontreated severe CLP. The administration of exogenous hemin to mice subjected to moderate sepsis consistently increased the mortality rate. Furthermore, hemin resulted in a reduction of neutrophil migration both in vivo and in vitro. Altogether, our results demonstrated that pretreatment with the HO inhibitor prevents the pathological findings in severe CLP. However, the combination of pretreatment plus posttreatment with ZnPP IX enhances sepsis severity because of an increase in circulating levels of heme, which is deleterious to the host tissues and also inhibits neutrophil migration.

Gastroprotective effect of heme-oxygenase 1/biliverdin/CO pathway in ethanol-induced gastric damage in mice

Our objective was to evaluate the role of heme-oxygenase 1 (HO-1)/biliverdin/CO pathway in gastric defense against ethanol-induced gastric damage in mice. Mice were pre-treated with saline, hemin (HO-1 inducer), biliverdin (HO-1 product), dimanganese decacarbonyl (DMDC, CO donor) or zinc protoporphyrin IX (ZnPP IX, HO-1 antagonist). Another group received soluble guanylate cyclase (sGC) inhibitor (ODQ) 30 min before hemin, biliverdin or DMDC. After 30 min, gastric damage was induced by ethanol. After one hour, rats were sacrificed. Gastric lesions were measured using a computer planimetry program, and gastric corpus pieces were assayed for malonylaldehyde (MDA), glutathione (GSH) or bilirubin. HO-1 expression was determined after saline or ethanol administration by polymerase chain reaction (PCR) or immunohistochemistry. Ethanol (25% or 50%) induced gastric damage, increased MDA levels and reduced GSH in the gastric tissue. Ethanol 50% increased HO-1 mRNA transcripts, HO-1 immunoreactivity, and bilirubin concentration in gastric mucosa. Pre-treatment with hemin reduced gastric damage and MDA formation and increased GSH concentration in the gastric mucosa. ZnPP IX amplified the ethanol-induced gastric lesion, increased MDA formation and decreased GSH concentration in gastric mucosa. Biliverdin and DMDC reduced gastric damage and MDA formation and increased GSH concentration in the gastric tissue. ODQ completely abolished the DMDC protective gastric effect However, effects of hemin or biliverdin did not change with ODQ treatment. Our results suggest that HO-1/biliverdin/CO pathway plays a protective role against ethanol-induced gastric damage through mechanisms that can be dependent (CO) or independent (biliverdin) of sGC activation. (C) 2010 Elsevier B.V. All rights reserved.

Association of drug metabolism gene polymorphisms with toxicities, graft-versus-host disease and survival after HLA-identical sibling hematopoietic stem cell transplantation for patients with leukemia

Individual differences in drug efficacy or toxicity can be influenced by genetic factors. We investigated whether polymorphisms of pharmacogenes that interfere with metabolism of drugs used in conditioning regimen and graft-versus-host disease (GvHD) prophylaxis could be associated with outcomes after HLA-identical hematopoietic stem cell transplantation (HSCT). Pharmacogenes and their polymorphisms were studied in 107 donors and patients with leukemia receiving HSCT. Candidate genes were: P450 cytochrome family (CYP2B6), glutathione-S-transferase family (GST), multidrug-resistance gene, methylenetetrahydrofolate reductase (MTHFR) and vitamin D receptor (VDR). The end points studied were oral mucositis (OM), hemorrhagic cystitis (HC), toxicity and venoocclusive disease of the liver (VOD), GvHD, transplantation-related mortality (TRM) and survival. Multivariate analyses, using death as a competing event, were performed adjusting for clinical factors. Among other clinical and genetic factors, polymorphisms of CYP2B6 genes that interfere with cyclophosphamide metabolism were associated with OM (recipient CYP2B6*4; P=0.0067), HC (recipient CYP2B6*2; P=0.03) and VOD (donor CYP2B6*6; P=0.03). Recipient MTHFR polymorphisms (C677T) were associated with acute GvHD (P=0.03), and recipient VDR TaqI with TRM and overall survival (P=0.006 and P=0.04, respectively). Genetic factors that interfere with drug metabolisms are associated with treatment-related toxicities, GvHD and survival after HLA-identical HSCT in patients with leukemia and should be investigated prospectively.

Role of the spinal cord heme oxygenase-carbon monoxide-cGMP pathway in the nociceptive response of rats

The aim of the present study was to investigate the role of the spinal cord heme oxygenase (HO)-carbon monoxide (CO)-soluble guanylate cyclase (sGC)-cGMP pathway in nociceptive response of rats to the formalin experimental nociceptive model. Animals were handled and adapted to the experimental environment for a few days before the formalin test was applied. For the formalin test 50 mu l of a 1% formalin solution was injected subcutaneously in the dorsal surface of the right hind paw. Following injections, animals were observed for I h and flinching behavior was measured as the nociceptive response. Thirty min before the test, rats were pretreated with intrathecal injections with the HO inhibitor, zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) or heme-lysinate, which is known to induce the HO pathway. Control animals were treated with vehicles. We observed a significant increase in nociceptive response of rats treated with ZnDPBG, and a drastic reduction of flinching nociceptive behavioral response in the heme-lysinate treated animals. Furthermore, the HO pathway seems to act via cGMP, since methylene blue (a sGC inhibitor) prevented the reduction of flinching nociceptive behavioral response caused by heme-lysinate. These findings strongly indicate that the HO pathway plays a spinal antinociceptive role during the formalin test, acting via cGMP. (C) 2007 Elsevier B.V. All rights reserved.

Involvement of the heme oxygenase-carbon monoxide-cGMP pathway in the nociception induced by acute painful stimulus in rats

Heme oxygenase-carbon monoxide-cGMP (HO-CO-cGMP) pathway has been reported to be involved in peripheral and spinal modulation of inflammatory pain. However, the involvement of this pathway in the modulation of acute painful stimulus in the absence of inflammation remains unknown. Thus, we evaluated the involvement of the HO-CO-cGMP pathway in nociception by means the of analgesia index (AI) in the tail flick test. Rats underwent surgery for implantation of unilateral guide cannula directed toward the lateral ventricle and after the recovery period (5-7 days) were subjected to the measures of baseline tail flick test Animals were divided into groups to assess the effect of intracerebroventricular administration (i.c.v.) of the following compounds: ZnDPBG (HO inhibitor) or vehicle (Na(2)CO(3)), heme-lysinate (substrate overload) or vehicle (L-lysine), or the selective inhibitor of soluble guanilate cyclase ODQ or vehicle (DMSO 1%) following the administration of heme-lysinate or vehicle. Heme overload increased AI, indicating an antinociceptive role of the pathway. This response was attenuated by i.c.v. pretreatment with the HO inhibitor ZnDPBG. In addition, this effect was dependent on cGMP activity, since the pretreatment with ODQ blocked the increase in the AI. Because CO produces most of its actions via cGMP, these data strongly imply that CO is the HO product involved in the antinociceptive response. This modulation seems to be phasic rather than tonic, since i.c.v. treatment with ZnDPBG or ODQ did not alter the AI. Therefore, we provide evidence consistent with the notion that HO-CO-cGMP pathway plays a key phasic antinociceptive role modulating noninflammatory acute pain. (C) 2011 Elsevier B.V. All rights reserved.