Detection of somatic mutations of the PIG-A gene in Brazilian patients with paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal syndrome characterized by intravascular hemolysis mediated by complement, thrombotic events and alterations in hematopoiesis. Basically, the molecular events which underlie the complexity of the syndrome consist of the absence of the glycosylphosphatidylinositol (GPI) anchor as a consequence of somatic mutations in the PIG-A gene, located on the X chromosome. The GPI group is responsible for the attachment of many proteins to the cytoplasmic membrane. Two of them, CD55 and CD59, have a major role in the inhibition of the action of complement on the cellular membrane of blood cells. The absence of GPI biosynthesis can lead to PNH. Since mutations in the PIG-A gene are always present in patients with PNH, the aim of this study was to characterize the mutations in the PIG-A gene in Brazilian patients. The analysis of the PIG-A gene was performed using DNA samples derived from bone marrow and peripheral blood. Conformation-sensitive gel electrophoresis was used for screening the mutation and sequencing methods were used to identify the mutations. Molecular analysis permitted the identification of three point mutations in three patients: one G->A transition in the 5' portion of the second intron, one T->A substitution in the second base of codon 430 (Leu430->stop), and one deletion deltaA in the third base of codon 63. This study represents the first description of mutations in the PIG-A gene in a Brazilian population.

Short-term evaluation of non-absorbable microgranular hydroxyapatite infiltration in the guinea pig subepidermal abdominal region

Non-absorbable microgranular hydroxyapatite was infiltrated into the subepidermal abdominal region of guinea pigs in order to assess the possibility of using this material to correct deficiencies in orbital volume. Microgranular hydroxyapatite (2.0 ml) was subepidermally infiltrated into the abdominal region of 20 guinea pigs. The animals were divided into four experimental groups of 5 animals each, which were killed 7 (G1), 15 (G2), 30 (G3) and 60 (G4) days after infiltration. The area and the largest and smallest diameters of the nodules formed by infiltration were evaluated at the site of infiltration and histological examination was performed. The mean granuloma area was similar in all groups. Histopathological examination showed that the material remained isolated from surrounding tissues by a pseudocapsule that became denser throughout the experiment. A host reaction started with young fibroblastic tissue that evolved to dense tissue until cartilaginous tissue was formed in G4, progressively advancing towards the center of the granuloma from G1 to G4. Non-absorbable microgranular hydroxyapatite is an inert material that was well tolerated by the animals studied, with maintenance of the infiltrated volume, and may perhaps be useful to fill anophthalmic cavities.

Standardization of an isolated pig heart preparation with parabiotic circulation: methodological considerations

In the present study we standardized an experimental model of parabiotic circulation of isolated pig heart. The isolated heart was perfused with arterial blood from a second animal as support and submitted to regional ischemia for 30 min, followed by total ischemia for 90 min and reperfusion for 90 min. Parameters for measurement of ventricular performance using different indices measured directly or indirectly from intraventricular pressure were defined as: maximum peak pressure, final diastolic pressure, pressure developed, first derivative of maximum pressure (dP/dt max), first derivative of minimum pressure (dP/dt min), systolic stress of the left ventricle (sigmas), and maximum elastance of the left ventricle. Isolated hearts subjected to regional and global ischemia presented significant worsening of all measured parameters. Less discriminative parameters were dP/dt max and dP/dt min. Elastance was the most sensitive parameter during the reperfusion period, demonstrating an early loss of ventricular function during reperfusion. The model proved to be stable and reproducible and permitted the study of several variables in the isolated heart, such as ischemia and reperfusion phenomena, the effects of different drugs, surgical interventions, etc. The model introduces an advantage over the classical models which use crystalloid solutions as perfusate, because parabiotic circulation mimics heart surgery with extracorporeal circulation.

Inotropic effects of extracts of Psidium guajava L. (guava) leaves on the guinea pig atrium

Many pharmacological effects have been ascribed to extracts of Psidium guajava L. (guava) leaves. However, in spite of its widespread use in Brazilian folk medicine and a reasonable number of scientific reports about it, we could not find any study dealing with its action on the mammalian myocardium. In the present study, by measuring isometric force, we observed that the crude extract of P. guajava (water-alcohol extract obtained by macerating dry leaves) depresses the guinea pig atrial contractility in a concentration-dependent fashion (N = 8 hearts, 15 trials). The compound with cardiac activity was concentrated by extraction in a Soxhlet apparatus using 17 M glacial acetic acid after removing the less polar fractions (hexane, chloroform, acetone, ethanol and methanol), suggesting that this compound is a highly polar substance. In the isolated guinea pig left atrium the acetic acid fraction (10-800 mg/l) of P. guajava 1) reversibly decreased myocardial force in a concentration-dependent fashion (EC50 = 0.07g/l, N = 5 hearts, 9 trials, P<0.05), 2) increased the atrial relaxation time measured at 20% of the force amplitude up to 35% (91 ± 15 to 123 ± 30 ms, N = 3 hearts, 6 trials, P<0.05), 3) abolished the positive staircase effect (Bowditch phenomenon) in a concentration-dependent fashion suggesting a decrease of the cellular inward calcium current (N = 4 hearts, 8 trials, P<0.05), and 4) its inotropic effect was abolished by cholinergic receptor blockade with 1.5 mM atropine sulfate, indicating a cholinergic involvement in the mechanism of action of the extract (N = 7 hearts, 15 trials, P<0.05). The acetic acid extract was 20 times more potent than crude extract (EC50 = 1.4 g/l). The results showed that extracts from P. guajava leaves depress myocardial inotropism.

Cholinergic-opioidergic interaction in the central amygdala induces antinociception in the guinea pig

Several studies have demonstrated the involvement of the central nucleus of the amygdala (CEA) in the modulation of defensive behavior and in antinociceptive regulation. In a previous study, we demonstrated the existence of a cholinergic-opioidergic interaction in the CEA, modulating the defensive response of tonic immobility in guinea pigs. In the present study, we investigated a similar interaction in the CEA, but now involved in the regulation of the nociceptive response. Microinjection of carbachol (2.7 nmol) and morphine (2.2 nmol) into the CEA promoted antinociception up to 45 min after microinjection in guinea pigs as determined by a decrease in the vocalization index in the vocalization test. This test consists of the application of a peripheral noxious stimulus (electric shock into the subcutaneous region of the thigh) that provokes the emission of a vocalization response by the animal. Furthermore, the present results demonstrated that the antinociceptive effect of carbachol (2.7 nmol; N = 10) was blocked by previous administration of atropine (0.7 nmol; N = 7) or naloxone (1.3 nmol; N = 7) into the same site. In addition, the decrease in the vocalization index induced by the microinjection of morphine (2.2 nmol; N = 9) into the CEA was prevented by pretreatment with naloxone (1.3 nmol; N = 11). All sites of injection were confirmed by histology. These results indicate the involvement of the cholinergic and opioidergic systems of the CEA in the modulation of antinociception in guinea pigs. In addition, the present study suggests that cholinergic transmission may activate the release of endorphins/enkephalins from interneurons of the CEA, resulting in antinociception.

Estradiol and testosterone concentrations in follicular fluid as criteria to discriminate between mature and immature oocytes

The objective of the present study was to examine the association between follicular fluid (FF) steroid concentration and oocyte maturity and fertilization rates. Seventeen infertile patients were submitted to ovulation induction with urinary human follicle-stimulating hormone, human menopausal gonadotropin and human chorionic gonadotropin (hCG). A total of 107 follicles were aspirated after hCG administration, the oocytes were analyzed for maturity and 81 of them were incubated and inseminated in vitro. Progesterone, estradiol (E2), estrone, androstenedione, and testosterone were measured in the FF. E2 and testosterone levels were significantly higher in FF containing immature oocytes (median = 618.2 and 16 ng/ml, respectively) than in FF containing mature oocytes (median = 368 and 5.7 ng/ml, respectively; P < 0.05). Progesterone, androstenedione and estrone levels were not significantly different between mature and immature oocytes. The application of the receiver-operating characteristic curve statistical approach to determine the best cut-off point for the discrimination between mature and immature oocytes indicated levels of 505.8 ng/ml for E2 (81.0% sensitivity and 81.8% specificity) and of 10.4 ng/ml for testosterone (90.9% sensitivity and 82.4% specificity). Follicular diameter was associated negatively with E2 and testosterone levels in FF. There was a significant increase in progesterone/testosterone, progesterone/E2 and E2/testosterone ratios in FF containing mature oocytes, suggesting a reduction in conversion of C21 to C19, but not in aromatase activity. The overall fertility rate was 61% but there was no correlation between the steroid levels or their ratios and the fertilization rates. E2 and testosterone levels in FF may be used as a predictive parameter of oocyte maturity, but not for the in vitro fertilization rate.

Inotropic effect of Citrus sinensis (L.) Osbeck leaf extracts on the guinea pig atrium

The objective of the present investigation was to determine the contractile effect of crude and acetone leaf extracts of Citrus sinensis (L.) Osb. on mammalian myocardium. Crude leaf extracts have been used in folk medicine to treat neurological disorders. Some flavonoids isolated from this plant presented a positive inotropic effect on myocardium. This motivated us to test the extracts on the atria of guinea pigs of both sexes (300-500 g) and surprisingly we observed inotropic depression instead of an increase in force. The maximum effect of the crude extract was 79.4 ± 8.1% of the control force amplitude (N = 5 hearts, 10 trials, 27 ± 0.1ºC, stimulus: 2 Hz, 400 V, 0.5 ms). The EC50 for crude, ethanol, acetic, aqueous, and acetone extracts was 300, 300, 600, 1000, and 140 µg/ml, respectively, with a Hill constant of 1.8, 2.0, 2.5, 2.0, and 1.4, respectively. Blockade of cholinergic, beta-adrenergic, or opioid membrane receptors with 1.5 µM atropine sulfate, 1 µM propranolol, and 10 µM naloxone, respectively, did not change the effect of the crude extract. The acetone extract abolished the Bowditch positive staircase phenomenon (N = 5 hearts, 10 trials, 27 ± 0.1ºC), suggesting a possible reduction of the calcium inward current, and also promoted the so-called Woodworth phenomenon. The effect was concentration-dependent and indicated the existence of another inhibitory contractile mechanism such as the simultaneous activation of some of the membrane potassium channels reducing the myocardial action potential duration and further decreasing the cellular calcium entry.

Negative inotropic and chronotropic effects on the guinea pig atrium of extracts obtained from Averrhoa carambola L. leaves

It has been reported that star fruit can lead to a fatal outcome in uremic patients. The intoxication syndrome consists of hiccups, mental confusion, dizziness, and vomiting. On the other hand, folk medicine uses teas and infusions of carambola leaves to treat headache, vomiting, cough, insomnia, and diabetes. This motivated us to determine if Averrhoa carambola can act on the contractility and automaticity of the guinea pig heart. We measured the atrial isometric force in stimulated left atria and determined the chronotropic changes in spontaneously beating right atria. The carambola leaf extracts (1.5 mg/ml) abolished the contractile force in a concentration-dependent manner. Among the crude, methanolic, ethanolic, aqueous, and acetic extracts, the aqueous one was the most potent (EC50 = 520 ± 94 µg/ml; flavonoids and tannins are the main constituents; Na+ and K+ contents in 1.0 mg/ml of aqueous extract were 0.12 ± 0.016 and 1.19 ± 0.15 mM, respectively). The aqueous extract abolished the positive Bowditch staircase phenomenon and reduced the inotropic response to CaCl2 (0.17-8.22 mM), events that are dependent on the cellular Ca2+ inward current. The adrenergic, muscarinic or opioid membrane receptors do not seem to participate in the mechanism of action of the cardioactive substance(s). In spontaneously beating atria, the aqueous extract promoted a negative chronotropic effect that was antagonized by 0.1 µM isoproterenol bitartrate. With this agonist, the EC50 of the aqueous extract increased from 133 ± 58 to 650 ± 100 µg/ml. These data regarding the effect of A. carambola on guinea pig atrial contractility and automaticity indicate an L-type Ca2+ channel blockade.

Purification, partial characterization and role in lipid transport to developing oocytes of a novel lipophorin from the cowpea weevil, Callosobruchus maculatus

Lipid transport in arthropods is achieved by highly specialized lipoproteins, which resemble those described in vertebrate blood. Here we describe purification and characterization of the lipid-apolipoprotein complex, lipophorin (Lp), from adults and larvae of the cowpea weevil Callosobruchus maculatus. We also describe the Lp-mediated lipid transfer to developing oocytes. Lps were isolated from homogenates of C. maculatus larvae and adults by potassio bromide gradient and characterized with respect to physicochemical properties and lipid content. The weevil Lp (465 kDa) and larval Lp (585 kDa), with hydrated densities of 1.22 and 1.14 g/mL, contained 34 and 56% lipids and 9 and 7% carbohydrates, respectively. In both Lps, mannose was the predominant monosaccharide detected by paper chromatography. SDS-PAGE revealed two apolipoproteins in each Lp with molecular masses of 225 kDa (apolipoprotein-I) and 79 kDa (apolipoprotein-II). The lipids were extracted and analyzed by thin-layer chromatography. The major phospholipids found were phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine in adult Lp, and phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in larval Lp. Hydrocarbons, fatty acids and triacylglycerol were the major neutral lipids found in both Lps. Lps labeled in the protein moiety with radioactive iodine (125I-iodine) or in the lipid moiety with fluorescent lipids revealed direct evidence of endocytic uptake of Lps in live oocytes of C. maculatus.

Effect of supplementation of green tea polyphenols on the developmental competence of bovine oocytes in vitro

The objective of the present study was to examine the effect of green tea polyphenols (GTPs) supplementation during in vitro maturation, in vitro fertilization, and in vitro culture on the developmental competence of bovine oocytes. Cumulus-oocyte complexes aspirated from the ovaries were matured in vitro (38.5ºC for 24 h) and fertilized (38.5ºC for 15-18 h) and embryos were cultured (38.5ºC for 192 h) in a defined conditioned medium with or without GTPs supplementation. The GTPs used in the present study contained 99% catechin derivatives, with the major components being 50% (-)-epigallocatechin gallate, 22% (-)-epicatechin gallate, 18% (-)-epigallocatechin, and 10% (-)-epicatechin. Four replicate trials were done for each type of experiment. GTPs supplementation (15 µM) of the maturation medium led to a significant increase in the rate of blastocyst formation (34.0 vs 21.4%, P < 0.05). However, the rate of blastocyst formation was not improved when higher GTPs concentrations (20 or 25 µM) were added to the in vitro maturation medium. During in vitro fertilization, supplementation with higher GTPs concentrations (20 or 25 µM) significantly reduced the rate of blastocyst formation (P < 0.05). Supplementation of the culture medium with 15 µM GTPs improved the rate of blastocyst formation, while higher GTPs concentrations (25 µM) significantly reduced embryo development (P < 0.05). In conclusion, these results demonstrate that supplementation with GTPs at low concentration (15 µM) during in vitro maturation and in vitro culture improved the developmental competence of bovine oocytes.

Illustration - Drawing of Pig while Blindfolded, 23 July 1900

The illustration of a pig is part of an autograph book collection of Ethelwyn Wetherald. Her collection consists of drawings of pigs her guests create while blindfolded. The signature of each guest accompanies the illustration. This illustration was done by Margaret H. Dorland in the year 1900.

Regulation of cholesterol intake by the corpus luteum

Studies were funded by Colegio de Postgraduados, México. CONACyT, México. SRE, México. Ministère de l’Éducation du Québec, University of Montreal and an Operating Grant to B.D. Murphy from the Canadian Institutes of Health Research.

The effect of Nystatin on the inner ear : an experimental guinea pig study

Objectifs: Le Nystatin est un antibiotique efficace pour le traitement d’otomycose. Bien que sa sécurité au niveau de l’oreille externe soit bien établie, son utilisation n’est pas recommandée lorsqu’il y a une perforation tympanique. L’objectif de cette étude est d’évaluer le potentiel ototoxique du Nystatin lorsque celui-ci est appliqué directement au niveau de l’oreille moyenne. Méthodes: Nous avons fait une étude expérimentale avec 18 cochons d’Indes de souche Hartley que nous avons divisés en deux groupes. En exposant l’oreille moyenne de chaque animal au Nystatin (groupe I) ou à la néomycine (groupe II) et chaque oreille controlatérale à une solution physiologique (NaCl), la fonction auditive a été évaluée avec un test de potentiels évoqués auditif du tronc cérébral avant et après les injections. Une étude par microscopie électronique a permis une comparaison histologique de l’état des cellules ciliées cochléaires entre les 2 groupes. Résultats: Les pertes auditives moyennes du groupe « Nystatin » étaient de 13.0 dB et comparables aux pertes moyennes observées dans les oreilles ayant été injectées avec du NaCl (4.0 dB dans le groupe I et 15.1 dB dans le groupe II). Le groupe de contrôle « néomycine » a subi une perte auditive moyenne de 39.3 dB, ce qui représente une différence cliniquement et statistiquement significative (p<0.001). L’étude histologique avec une microscopie à balayage électronique a démontré une conservation de l’architecture des cellules ciliées cochléaires dans les groupe Nystatin et NaCl. La néomycine a causé une destruction marquée de ces structures. Conclusions: Le Nystatin ne provoque pas d’atteinte auditive ni de destruction des cellules ciliées externes après injection directe dans l’oreille moyenne chez le cochon d’Inde.

Impact of neonatal total parenteral nutrition and early glucose-enriched diet on glucose metabolism and physical phenotypes in Guinea Pig

Les oxydants infusés avec la nutrition parentéral (NP) néonatale induisent une modification du métabolisme des lipides et du glucose, donnant lieu à l’âge adulte à un phénotype de carence énergétique (faible poids, baisse de l’activité physique). L’hypothèse qu’une diète précoce riche en glucose prévient ces symptômes plus tard dans la vie, fut évalué chez le cobaye par un ANOVA en plan factoriel complet à deux facteurs (p < 0:05) : NP du jour 3 à 7, suivit d’une nourriture régulière (chow) (NP+) vs. chow à partir du 3ième jour (NP-), combiné avec une eau de consommation enrichie en glucose (G+) ou non (G-) à partir de la 3ième semaine. Les paramètres suivant ont été mesurés à l’âge de 9 semaine: taux de croissance, activité physique, activité de phosphofructokinase-1 et glucokinase (GK), niveau hépatique de glucose-6-phosphate (G6P), glycogène, pyruvate et potentiel redox du glutathion, poids du foie, glycémie, tolérance au glucose, concentrations hépatiques et plasmatiques en triacylglycérides (TG) et cholestérol. Le groupe G+ (vs. G-) avait un taux de croissance plus bas, une activité de GK et une concentration en G6P plus élevée, et un potentiel redox plus bas (moins oxydé). Le niveau plasmatique de TG était moins élevé dans le groupe NP+ (vs. NP-). Les traitements n’eurent aucun effet sur les autres paramètres. Ces résultats suggèrent qu’indépendamment de la NP, une alimentation riche en glucose stimule la glycolyse et déplace l’état redox vers un statut plus réduit, mais ne surmonte pas les effets de la NP sur le phénotype physique de carence énergétique.

Potential pathogenicity and antimicrobial resistance of Escherichia coli from pig and poultry feces on-farm and carcasses at the abattoir in Vietnam

E. coli avec potentiel zoonotique pourrait éclore dans les réservoirs porcins et avicoles. Cette étude consiste à examiner la présence de souches E. coli porteuses de gènes virulents associés aux STEC (E. coli producteurs de Shiga-toxines), EPEC (E. coli entéropathogène), et ExPEC (E. coli pathogène extra-intestinal) chez les porcs et volailles élevés au Vietnam. Des prélèvements d’excréments et de carcasses ont été effectués dans des fermes et abattoirs porcins et avicoles sélectionnés où les animaux ont été suivis de l’élevage à l’abattage. Un total de 13,1% des souches, toutes sources confondues, ont été catégorisées comme potentiellement contaminées par ExPEC, possédant un ou plusieurs gènes de virulence iucD, tsh, papC et cnf. Peu d’isolats d’autres pathotypes ont été observés. Tous les gènes de virulence ExPEC, à l’exception de cnf, ont été identifiés plus fréquemment dans les isolats de fèces et carcasses avicoles que dans les isolats porcins. Même constatation pour le groupe du phylogénétique D. Une multirésistance aux médicaments a été régulièrement observée chez les deux isolats ExPEC. Les isolats de fèces de volailles ont souvent été associés à une résistance à l’acide nalidixique et à la ciprofloxacine (P<0.05), de même qu’au gène blaTEM, alors que les gènes qnr et aac(6’)-Ib ont peu été rencontrés des deux côtés. Cette étude démontre que les isolats ExPEC avicoles sont potentiellement plus pathogèniques que ceux porcins et que les isolats ExPEC de carcasses porcines et avicoles peuvent provenir de leurs excréments par la contamination associée au processus d'abattage. Ainsi, la volaille, particulièrement, serait un facteur de transmission de souches ExPEC zoonotiques.