Cervical relaxation for non-surgical uterus access in Santa Inês ewes / Relaxamento cervical para acesso uterino não cirúrgico em ovelhas Santa Inês

The present study was composed by two experiments aiming to develop a cervical dilation technique for non-surgical access to Santa Inês ewe's uterus. In Experiment 1, thirty ewes underwent four epidural treatments. The three experimental treatments used 2.0mg/kg ketamine. The group receiving this drug alone was denominated KG, whereas other group had ketamine associated with 0.1mg/kg morphine (KM) and KX a third group had ketamine associated with 0.05mg/kg xylazine (KX). Control treatment was 1mL/7.5kg saline solution epidurally (CON). Cervical dilation was evaluated in both experiments by attempting to pass a metal rod through the cervix. In Experiment 2, three different hormonal protocols for cervical dilation were tested in thirty ewes. Epidural anesthesia with 2.0mg/kg ketamine was the control treatment (KG) and was combined with hormonal treatments: Misoprostol (MI); Oxytocin + Estradiol (OE); Misoprostol + Oxytocin + Estradiol (MOE). In Experiment 1 transposition rate was not different among groups. In Experiment 2, OE presented the highest rate (90%) while MOE presented 86.2%, MI 68.9% and CON 62.1%. The study developed a pharmacological protocol that increased cervical transposition making the non-surgical access to the uterus feasible in Santa Inês ewes.(AU)

O presente estudo teve como objetivo desenvolver uma técnica para acesso não cirúrgico ao útero de ovelhas Santa Inês e foi realizado em duas etapas. No experimento 1, 30 ovelhas foram submetidas a quatro tratamentos epidurais. Os três tratamentos teste usaram 2,0mg/kg de cetamina. O grupo que recebeu apenas cetamina foi denominado KG, enquanto no segundo grupo a cetamina foi combinada com morfina (0,1mg/kg - KM) e um terceiro grupo recebeu cetamina associada com xilazina 0,05mg/kg - KX). No grupo controle (CON), usou-se solução salina (1mL/7,5kg de peso). A dilatação cervical foi avaliada em ambos os experimentos pela tentativa de transposição cervical com uma haste metálica. No experimento 2, três protocolos hormonais de dilatação cervical foram testados em 30 ovelhas. Anestesia epidural com 2,0mg/kg de cetamina foi o tratamento controle (CON), combinada com tratamentos hormonais nos grupos: misoprostol (MI); ocitocina + estradiol (OE); misoprostol + ocitocina + estradiol (MOE). No experimento 1, a taxa de transposição não variou entre os grupos. No experimento 2, OE teve a maior taxa (90%), enquanto MOE apresentou 86,2%, MIi 68,9% e CON 62,1%. O estudo desenvolveu um protocolo farmacológico que aumentou a taxa de transposição cervical, tornando o acesso uterino não cirúrgico viável em ovelhas Santa Inês.(AU)

Mast cell tryptase, a still enigmatic enzyme.

Tryptases constitute a subfamily of trypsin-like proteinases, stored in the mast cell secretory granules of all mammalian organisms. These enzymes are released along with other mediators into the extracellular medium upon mast cell activation/degranulation. Among the trypsin-like enzymes, tryptases are unique: they are present as active enzymes in the mast cell granules, but display activity only extracellularly, and have a specificity which is much more restricted than trypsin. Tryptases are mostly tetrameric, and in only few organisms (not in humans) are they inhibited by endogenous inhibitors in vitro. The enzymatic and molecular properties of tryptases are far better characterized that any of their plausible biological functions. On the basis of its structural and functional features it could be predicted that tryptase would not degrade a large number of proteins in vivo due to low accessibility to the tetramer central pore where the active sites face inwards. Although their biological function has not yet been clarified, tryptases seem to be involved in a number of mast cell-mediated allergic and inflammatory diseases. In particular, the involvement of tryptase in asthma, an inflammatory disease of the airways often caused by allergy, has been proposed. Here we review the present knowledge on the structure-function relationship of tryptases from different organisms, with special emphasis on human enzymes, and on their role in a variety of pathophysiological processes.

Determinação da concentração alveolar mínima do isoflurano em cães com estímulo elétrico ou mecânico

O artigo não apresenta resumo.

Minimum alveolar concentration of halothane in capuchin-monkeys (Cebus apella)

The article has no abstract.

Concentração alveolar mínima do isoflurano associado ou não ao óxido nitroso durante a maturação pós-natal em cães. Resultados preliminares

O artigo não apresenta resumo.

Lovastatin-induced apoptosis in thyroid cells: involvement of cytochrome c and lamin B.

OBJECTIVE: The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, lovastatin, induces apoptosis in the thyroid cell line TAD-2 and in proliferating normal human thyroid cells in culture, through a p53-independent mechanism involving caspase-3-like proteases. The combination of lovastatin with other anti-neoplastic drugs potentiates chemotherapy of tumors. This drug has been suggested for the chemotherapy of tumors and is potentially useful in the treatment of thyroid proliferative diseases. Based on this premise, we analyzed in more detail the role of some molecular effectors and the role of the caspase family proteases in the lovastatin-induced apoptotic pathway in TAD-2 cells. METHODS: TAD-2 cells were treated with lovastatin to induce apoptosis, and expression of p53, Bc1-2, Bcl-XL and Bax was analyzed by Western blot. Caspase activation was evaluated by the assay of enzymatic activity with chromogenic peptides and Western blot. Nuclear, cytosolic and mitochondrial fractions were prepared by differential centrifugation and the presence of cytochrome c and lamin B was evaluated by Western blot. RESULTS: p53, Bc1-2, Bcl-XL and Bax protein expression were unchanged during apoptosis. Cytochrome c was released from mitochondria into the cytosol, a pivotal event in the activation of caspase-3. Caspase-3 and -6 but not caspase-2 were activated, and proteolysis of PARP and lamin B, a caspase-6 substrate located in the inner nuclear membrane, was demonstrated by Western blot. The nuclear localization of lamin B was also inhibited by lovastatin. CONCLUSIONS: These data demonstrate that, in TAD-2 thyroid cells, lovastatin induces lamin B proteolysis and inhibits its nuclear localization and induces cytochrome c release from mitochondria into the cytosol.

Cytochrome c reconstituted from two peptide fragments displays native-like redox properties.

Recombination of two fragments of horse cytochrome c (the heme-containing N-fragment, residues 1-56, and the C-fragment, residues 57-104), which are substantially unstructured at neutral pH, gives rise to a 1:1 fragment complex with a compact conformation, in which the alpha helical structure and the native Met80-Fe(III) axial bond are recovered. With respect to the native protein, the ferric complex shows a less rigid atomic packing and a decreased stability [Delta(DeltaG(o))D = 14.7 kJ.mol(-1)], ascribed to perturbations involving the Trp59 microenvironment and, to a lower extent, the heme pocket region. The redox potential, E1/2 = 234 +/- 5 mV vs. normal hydrogen electrode at 25 degrees C, is close to that of the intact protein, consistent with recovery of the native Met80-heme Fe(III) axial bond. Furthermore, the fragment complex shows reactivity similar to intact cytochrome c, in the reaction with cytochrome c oxidase. We conclude that the absence in the complex of some native cross-links and interlocked packing important for protein rigidity and stability is not as relevant for maintaining the native redox properties of the protein, provided that some structural requirements (i.e. recovering of the native-like alpha helical structure) are fulfilled and coordination of Met80 to the heme-iron is restored.

Structure of the Fe-heme in the hemodimeric hemoglobin from Scapharca inaequivalvis and in the T721 mutant: an X-ray absorption spectroscopic study at low temperature.

The Fe site structure in the recombinant wild-type and T721 mutant of the cooperative homodimeric hemoglobin (HbI) of the mollusc Scapharca itnaequivalvis has been investigated by measuring the Fe K-edge X-ray absorption near edge structure (XANES) spectra of their oxy, deoxy and carbonmonoxy derivatives, and the cryogenic photoproducts of the carbonmonoxy derivatives at T = 12 K. According to our results, the Fe site geometry in T72I HbI-CO is quite similar to that of human carbonmonoxy hemoglobin (HbA-CO), while in native HbI-CO it seems intermediate between that of HbA-CO and sperm whale MbCO. The XANES spectra of oxy and deoxy derivatives are similar to the homologous spectra of human HbA, except for T72I HbI, for which the absorption edge is blue-shifted (about + 1 eV) towards the spectrum of the oxy form. XANES spectra of the cryogenic photoproducts of HbA-CO (HbA*), HbI-CO (HbI*) and mutant HbI-CO (T72I HbI*) were acquired under continuous illumination at 12 K. The Fe-heme structures of the three photoproducts are similar; however, while in the case of HbA* and HbI* the data indicate incomplete structural relaxation of the Fe-heme towards its deoxy-like (T) form, the relaxation in T72I HbI* is almost completely towards the proposed "high affinity" Fe-heme structure of T72I HbI. This evidence suggests that minor tertiary restraints affect the Fe-heme dynamics of T72I HbI, corresponding to a reduction of the energy necessary for the T --> R structural transition, which can contribute to the observed dramatic enhancement in oxygen affinity of this hemoprotein, and the decreased cooperativity.

Selective inhibition of human mast cell tryptase by gabexate mesylate, an antiproteinase drug.

Gabexate mesylate is a non-antigenic synthetic inhibitor of trypsin-like serine proteinases that is therapeutically used in the treatment of pancreatitis and disseminated intravascular coagulation and as a regional anticoagulant for hemodialysis. Considering the structural similarity between gabexate mesylate and arginine-based inhibitors of trypsin-like serine proteinases, the effect of gabexate mesylate on human and bovine mast cell tryptase action was investigated. Values of the inhibition constant (K(i)) for gabexate mesylate binding to human and bovine tryptase were 3.4 x 10(-9) M and 1.8 x 10(-7) M (at pH 7.4 and 37.0 degrees ), respectively. Furthermore, gabexate mesylate inhibited the fibrinogenolytic activity of human tryptase. On the basis of the available x-ray crystal structure of human tryptase, the possible binding mode of gabexate mesylate to human and bovine tryptase was analyzed. Human tryptase inhibition by gabexate mesylate may account for the reported prevention of inflammation, erosion, and ulceration of skin and mucosae.

Amiodarone induces cytochrome c release and apoptosis through an iodine-independent mechanism.

Amiodarone (AMD) is one of the most effective antiarrhythmic drugs available. However, its use is often limited by side-effects, mainly hypo- or hyperthyroidism. As AMD displays direct toxic effect on different cell types, we investigated the cytotoxic effect of AMD and its main metabolite, desethylamiodarone (DEA), in thyroid (TAD-2) and nonthyroid (HeLa) cell lines. Both AMD and DEA displayed a dose-dependent toxicity in TAD-2 and HeLa cells, although DEA was more effective. Both TAD-2 and HeLa cells underwent apoptosis, as evidenced by plasma membrane phosphatidylserine exposure and DNA fragmentation. Inhibition of protein synthesis with cycloheximide and inhibition of endogenous peroxidase activity with propylthiouracil did not affect this AMD- and DEA-induced apoptosis in TAD-2 cells. Western blot analysis did not display variations in the expression of p53, Bcl-2, Bcl-XL, and Bax proteins during the treatment with AMD and DEA. Generation of reactive oxygen species, investigated by flow cytometry with dichlorofluorescein diacetate, did not show the production of free radicals during drug treatment. Furthermore, Western blot analysis of cytosolic and mitochondrial fractions prepared from AMD-treated cells demonstrated that AMD induces the release of cytochrome c into the cytosol from the mitochondria. These data indicate that AMD induces cytochrome c release from mitochondria, triggering apoptosis through an iodine-independent mechanism, and that this process is not mediated by modulation of p53, Bcl-2, Bcl-XL, or Bax protein expression and does not involve the generation of free radicals.

Scapharca inaequivalvis A and B miniglobin genes: promoter activity of the 5' flanking regions and in vivo transcription.

Globin genes of the bivalve mollusk Scapharca inaequivalvis have the two intron/three exon organization typical of vertebrate and many invertebrate globins, with introns in highly conserved positions. Sequence studies on the A and B globin genes of the mollusk tetrameric hemoglobin gave evidence for the existence of 'minigenes' spanning part of the first and second intron, in-frame with the heme binding domain encoded by the central exon. Putative promoter and regulatory sequences flanking these minigenes were identified in the 3' regions of intron I. Here we report cloning and functional analysis of these regions ( approximately 400bp) and their promoter activity, which was assessed in K562 cells by transient transfection, was established. Moreover, in vitro reverse transcriptase-polymerase chain reaction (RT-PCR) on total cytoplasmatic RNA demonstrated that the A and B minigenes are transcriptionally active in the erythrocytes of S. inaequivalvis. Thus, the present results lead to the conclusion that the present-day organization of the globin genes of S. inaequivalvis tetrameric hemoglobin is still reminiscent of an ancestral globin gene before exon shuffling.

The heme-containing N-fragment (residues 1-56) of cytochrome c is a bis-histidine functional system.

The structural and redox properties of a heme-containing fragment (1-56 residues) of cytochrome c have been investigated by spectroscopic (circular dichroism, electronic absorption, and EPR) and voltammetric techniques. The results indicate that the N-fragment lacks ordered secondary structure and has two histidines axially bound to the heme-iron (the native His18 and a misligated His26 or His33). Despite the absence of ordered secondary structure, the peptide chain shields the heme group from solvent, as shown by (i) the pK(a) of protonation of the nonnative histidine ligand (5.18 +/- 0.05), lower than that of the bis-histidine guanidine-unfolded cytochrome c (5.58 +/- 0.05), and (ii) the redox potential, E(o) = 0 +/- 5 mV versus NHE, close to that of bis-histidine cytochrome c mutants but less negative than that of bis-histidine complexes of microperoxidase with short peptides. The electroactive N-fragment may be taken as a "minichrome c" model, with interesting potential for application to biosensor technology; further, the system provides useful information for a deeper understanding of cytochrome c folding and structural/functional organization.

Human mast cells take up and hydrolyze anandamide under the control of 5-lipoxygenase and do not express cannabinoid receptors.

Human mast cells (HMC-1) take up anandamide (arachidonoyl-ethanolamide, AEA) with a saturable process (K(m)=200+/-20 nM, V(max)=25+/-3 pmol min(-1) mg protein(-1)), enhanced two-fold over control by nitric oxide-donors. Internalized AEA was hydrolyzed by a fatty acid amide hydrolase (FAAH), whose activity became measurable only in the presence of 5-lipoxygenase, but not cyclooxygenase, inhibitors. FAAH (K(m)=5.0+/-0.5 microM, V(max)=160+/-15 pmol min(-1) mg protein(-1)) was competitively inhibited by palmitoylethanolamide. HMC-1 cells did not display a functional cannabinoid receptor on their surface and neither AEA nor palmitoylethanolamide affected tryptase release from these cells.

Iodide excess induces apoptosis in thyroid cells through a p53-independent mechanism involving oxidative stress.

Thyroid toxicity of iodide excess has been demonstrated in animals fed with an iodide-rich diet; in vitro iodide is cytotoxic, inhibits cell growth, and induces morphological changes in thyroid cells of some species. In this study, we investigated the effect of iodide excess in an immortalized thyroid cell line (TAD-2) in primary cultures of human thyroid cells and in cells of nonthyroid origin. Iodide displayed a dose-dependent cytotoxicity in both TAD-2 and primary thyroid cells, although at different concentrations, whereas it had no effect on cells of nonthyroid origin. Thyroid cells treated with iodide excess underwent apoptosis, as evidenced by morphological changes, plasma membrane phosphatidylserine exposure, and DNA fragmentation. Apoptosis was unaffected by protein synthesis inhibition, whereas inhibition of peroxidase enzymatic activity by propylthiouracil completely blocked iodide cytotoxicity. During KI treatment, reactive oxygen species were produced, and lipid peroxide levels increased markedly. Inhibition of endogenous p53 activity did not affect the sensitivity of TAD-2 cells to iodide, and Western blot analysis demonstrated that p53, Bcl-2, Bcl-XL, and Bax protein expression did not change when cells were treated with iodide. These data indicate that excess molecular iodide, generated by oxidation of ionic iodine by endogenous peroxidases, induces apoptosis in thyroid cells through a mechanism involving generation of free radicals. This type of apoptosis is p53 independent, does not require protein synthesis, and is not induced by modulation of Bcl-2, Bcl-XL, or Bax protein expression.

Kinetic and thermodynamic analysis of leech-derived tryptase inhibitor interaction with bovine tryptase and bovine trypsin.

The interaction of leech-derived tryptase inhibitor (LDTI) with bovine liver capsule tryptase (BLCT) and bovine trypsin has been studied using both thermodynamic and kinetic approaches. Several differences were detected: (i) the equilibrium affinity of LDTI for BLCT (Ka = 8.9 x 10(5) M(-1)) is about 600-fold lower than that for bovine trypsin (Ka = 5.1 x 10(8) M(-1)); (ii) LDTI behaves as a purely non-competitive inhibitor of BLCT, while it is a purely competitive inhibitor of bovine trypsin. These functional data are compared with those previously reported for the LDTI binding to human tryptase, where tight inhibition occurs at two of the four active sites of the tetramer (Ka = 7.1 x 10(8) M(-1)). Amino acid sequence alignment of BLCT, human betaII-tryptase and bovine trypsin allows us to infer some possible structural basis for the observed functional differences.

Functional modulation of the Thr72-->Ile mutant from Scapharca inaequivalvis homodimeric hemoglobin.

The pH and temperature dependence of both the kinetic and thermodynamic properties of the Thr72-->Ile mutant of Scapharca inaequivalvis homodimeric hemoglobin were investigated between pH 2 and 10 and between 8 degrees C and 36 degrees C, in comparison with the wild-type recombinant protein. Results demonstrate pH-independent O2-binding properties, at least between pH 5 and 10, with the higher affinity of the mutant being related to a less negative entropy change. This observation may relate to a variation in the number of water molecules involved in the intersubunit communication. Furthermore, the kinetic properties of ligand association and dissociation seem to be in keeping with possible structural alterations of water molecules at the subunit interface occurring in the Thr72-->Ile mutant as well as with amino acid residues involved in the modulation of reactivity and cooperativity at the level of (1) the proximal side of the heme pocket and of (2) the heme propionates bridging the two subunits.

The exon/intron organization of the globin gene of Scapharca inaequivalvis homodimeric hemoglobin: unusual intron homology with other bivalve mollusc globin genes.

In this study, we have investigated the positions of introns in the globin gene of Scapharca inaequivalvis homodimeric hemoglobin. We found the three exon/two intron organization typical of vertebrate globin genes, with the two introns in highly conserved positions, as it occurs in the A and B globin genes of the tetrameric hemoglobin from the same organism, confirming the absence of the so-called 'central intron' found in the globin genes of plants and of some invertebrates. We identified two homodimeric globin genes (3207 and 2723 bp) that differ only with respect to the size of the first intron. Sequence analysis of the two first introns (1668 and 1364 bp) has revealed that they are highly homologous, except for a 569- and 296-bp insertion in each intron I. Interestingly, the two first introns contain regions with an unusually high identity (approximately 80%) with regions of the first intron of the congeneric clam Anadara trapezia and the related clam Barbatia reveana globin genes, suggesting that these uncoding regions may have played a regulatory role that has subsequently been lost during the course of the evolution.

Mutational destabilization of the critical interface water cluster in Scapharca dimeric hemoglobin: structural basis for altered allosteric activity.

A cluster of interface ordered water molecules has been proposed to act as a key mediator of intersubunit communication in the homodimeric hemoglobin of Scapharca inaequivalvis. Mutations of Thr72 to Val and Ile, which lack the hydroxyl group to hydrogen bond the deoxy interface water molecules, result in sharply altered functional properties. We have determined the high resolution (1.6-1. 8 A) crystal structures of these two mutants in both the deoxygenated and CO-liganded states. These structures show minimal protein structural changes relative to the same native derivatives, despite greater than 40-fold increases in oxygen affinity. In the deoxy state of both mutants two water molecules at the periphery of the water cluster are lost, and the remaining cluster water molecules are destabilized. The CO-liganded structures show key differences between the two mutants including a more optimal interface packing involving Ile72 that acts to stabilize its high affinity (R) state. This additional stabilization allows rationalization of its lowered cooperativity within the context of a two-state model. These studies support a key role of ordered water in cooperative functioning and illustrate how subtle structural alterations can result in significantly altered functional properties in an allosteric molecule.