Estudo retrospectivo de ovariossalpingo-histerectomia em cadelas e gatas atendidas em Hospital Veterinário Escola no período de um ano / Retrospective ovariosalpingohisterectomy study in bitches and queens assisted at a Veterinary School Hospital during one year

Realizou-se um estudo retrospectivo das indicações de ovariossalpingo-histerectomia - eletiva e terapêutica - no período de um ano. Foram analisados 193 prontuários de cadelas e gatas atendidas em Hospital Veterinário Escola, entre março de 2010 e março de 2011, levando em consideração a espécie, a idade e o uso ou não de anticoncepcional. Constatou-se que a demanda de OSH com caráter terapêutico (78,8%) é consideravelmente mais alta que a eletiva (21,2%). Observou-se que as anormalidades reprodutivas detectadas com maior frequência foram piometra (53,36%) e complicações obstétricas (25,38%) e que o uso de anticoncepcional foi o fator relevante para o delineamento desses quadros.

An ovariosalpingohisterectomy procedure indication - elective and therapeutic - retrospective study was conducted during one year. Record files from 193 bitches and queens assisted at a Veterinary School Hospital between March 2010 and March 2011 were analyzed, considering species, age and contraceptive use. It was possible to conclude that the therapeutic OSH demand (78.8%) is remarkably superior to the elective OSH request (21.2%). It was also observed that the most frequently detected reproductive abnormalities were pyometra (53.36%) and gestational complications (25.38%), and also that the use of contraceptives is a relevant factor for those events.

Ultrastructural and functional analysis of secretory goblet cells in the midgut of the lepidopteran Anticarsia gemmatalis.

Defoliation caused by Anticarsia gemmatalis larvae affects the commercial production of the soybean. Although regulation of the digestion of soybean components has become part of the suggested strategy to overcome problems caused by Anticarsia larvae, few studies have focused on the morphological and cellular aspects of Anticarsia intestinal tissue. We have therefore further analyzed the morphology and ultrastructure of the midgut of 5th instar larvae of A. gemmatalis. Dissected midgut was subjected to chemical or cryo-fixation and then to several descriptive and analytical techniques associated with both light and electron microscopy in order to correlate anatomical and physiological aspects of this organ. Histological analysis revealed typical anatomy composed of a cell layer limited by a peritrophic membrane. The identified lepidoptera-specific goblet cells were shown to contain several mitochondria inside microvilli of the goblet cell cavity and a vacuolar H(+)-ATPase possibly coupled to a K(+)-pumping system. Columnar cells were present and exhibited microvilli dispersed along the apical region that also presented secretory characteristics. We additionally found evidence for the secretion of polyphosphate (PolyP) into the midgut, a result corroborating previous reports suggesting an excretion route from the goblet cell cavity toward the luminal space. Thus, our results suggest that the Anticarsia midgut not only possesses several typical lepidopteran features but also presents some unique aspects such as the presence of a tubular network and PolyP-containing apocrine secretions, plus an apparent route for the release of cellular debris by the goblet cells.

New insights into the in situ microscopic visualization and quantification of inorganic polyphosphate stores by 4',6-diamidino-2-phenylindole (DAPI)-staining.

Inorganic polyphosphate (PolyP) is a biological polymer that plays important roles in the cell physiology of both prokaryotic and eukaryotic organisms. Among the available methods for PolyP localization and quantification, a 4',6-diamidino-2-phenylindole(DAPI)-based assay has been used for visualization of PolyP-rich organelles. Due to differences in DAPI permeability to different compartments and/or PolyP retention after fixation, a general protocol for DAPI-PolyP staining has not yet been established. Here, we tested different protocols for DAPI-PolyP detection in a range of samples with different levels of DAPI permeability, including subcellular fractions, free-living cells and cryosections of fixed tissues. Subcellular fractions of PolyP-rich organelles yielded DAPI-PolyP fluorescence, although those with a complex external layer usually required longer incubation times, previous aldehyde fixation and/or detergent permeabilization. DAPI-PolyP was also detected in cryosections of OCT-embedded tissues analyzed by multi-photon microscopy. In addition, a semi-quantitative fluorimetric analysis of DAPI-stained fractions showed PolyP mobilization in a similar fashion to what has been demonstrated with the use of enzyme-based quantitative protocols. Taken together, our results support the use of DAPI for both PolyP visualization and quantification, although specific steps are suggested as a general guideline for DAPI-PolyP staining in biological samples with different degrees of DAPI and PolyP permeability.

Inorganic polyphosphates are stored in spherites within the midgut of Anticarsia gemmatalis and play a role in copper detoxification.

Inorganic polyphosphates (PolyP) are widespread molecules that have been shown to play a role in metal detoxification and heavy-metal tolerance. In the present report, we investigated the functional role of spherites as PolyP-metal binding stores in epithelial cells of the midgut of Anticarsia gemmatalis, a lepidopteran pest of soybean. PolyP stores were detected by DAPI staining and indirect immunohistochemistry as vesicles distributed in columnar cells and around goblet cell cavities. These PolyP vesicles were identified as spherites by their elemental profile in cell lysates that were partially modulated by P- or V-ATPases. PolyP levels along the midgut were detected using a recombinant exopolyphosphatase assay. When copper was added in the diet of larva, copper detection in spherites by X-ray microanalysis correlated with an increase in the relative phosphorous X-ray signal and with an increase in PolyP levels in epithelia cell lysate. Transmission electron microscopy of chemically fixed or cryofixed and freeze substituted tissues confirmed a preferential localization of spherites around the goblet cell cavity. Taken together, these results suggest that spherites store high levels of PolyP that are modulated during metal uptake and detoxification. The similarity between PolyP granules and spherites herein described also suggest that PolyP is one of the main phosphorous source of spherites found in different biological models. This suggests physiological roles played by spherites in the midgut of arthropods and mechanisms involved in heavy metal resistance among different insect genera.

Inorganic polyphosphate inhibits an aspartic protease-like activity in the eggs of Rhodnius prolixus (Stahl) and impairs yolk mobilization in vitro.

Inorganic polyphosphate (poly P) is a polymer of phosphate residues that has been shown to act as modulator of some vertebrate cathepsins. In the egg yolk granules of Rhodnius prolixus, a cathepsin D is the main protease involved in yolk mobilization and is dependent on an activation by acid phosphatases. In this study, we showed a possible role of poly P stored inside yolk granules on the inhibition of cathepsin D and arrest of yolk mobilization during early embryogenesis of these insects. Enzymatic assays detected poly P stores inside the eggs of R. prolixus. We observed that micromolar poly P concentrations inhibited cathepsin D proteolytic activity using both synthetic peptides and homogenates of egg yolk as substrates. Poly P was a substrate for Rhodnius acid phosphatase and also a strong competitive inhibitor of a pNPPase activity. Fusion events have been suggested as important steps towards acid phosphatase transport to yolk granules. We observed that poly P levels in those compartments were reduced after in vitro fusion assays and that the remaining poly P did not have the same cathepsin D inhibition activity after fusion. Our results are consistent with the hypothesis that poly P is a cathepsin D inhibitor and a substrate for acid phosphatase inside yolk granules. It is possible that, once activated, acid phosphatase might degrade poly P, allowing cathepsin D to initiate yolk proteolysis. We, therefore, suggest that degradation of poly P might represent a new step toward yolk mobilization during embryogenesis of R. prolixus.

Polyphosphate polymers during early embryogenesis of Periplaneta americana.

Inorganic polyphosphates (PolyP) are linear polymers of phosphate (Pi) residues linked by high-energy phosphoanhydride bonds. Despite a wide distribution, their role during insect embryogenesis has not been examined so far. In this study, we show the mobilization of PolyP polymers during the embryogenesis of the cockroach Periplaneta americana. PolyP was detected by enzymatic and fluorimetric assays and found to accumulate in two main sizes by agarose gel electrophoresis. Confocal microscopy showed their presence in small vesicles. In addition, X-ray microanalysis of small vesicles showed considerable amounts of calcium, sodium and magnesium, suggesting an association of PolyP with these elements. Variations of the free Ca+2, Pi and PolyP levels were observed during the first days of embryogenesis. Our results are consistent with the hypothesis that phosphate ions modulate PolyP variation and that PolyP hydrolysis result in increasing free Ca+2 levels. This is the first investigation of PolyP metabolism during embryogenesis of an insect and might shed light on the mechanisms involving Pi storage and homeostasis during this period. We suggest that PolyP, mainly stored in small vesicles, might be involved in the functional control of Ca+2 and Pi homeostasis during early embryogenesis of P. Americana.

Characterization of a tyrosine phosphatase activity in the oogenesis of Periplaneta americana.

In this work, phosphatase activity was characterized in the ovary and the haemolymph of Periplaneta americana. The optimum pH for these activities was 4.0, and a temperature of 44 degrees C was ideal for the maximal enzyme activity. The phosphatase activities were inhibited by NaF, sodium tartrate, Pi, sodium orthovanadate, and ammonium molybdate. The ovarian phosphatase activity at pH 4.0 was almost exclusive against phosphotyrosine, with little or no effect on the residues of phosphoserine or phosphothreonine. These results indicate that this phosphatase activity is due to the presence of an acid tyrosine phosphatase. The phosphatase activities of acid extracts from P. americana ovaries (OEX) and an acid extract from P. americana haemolymph (HEX) were analyzed in non-denaturant gel electrophoresis using an analog substrate beta-naphtyl phosphate. The gel revealed two bands with phosphatase activity in the ovary and one band in the haemolymph; these bands were excised and submitted to a 10% SDS-PAGE showing a single 70-kDa polypeptide in both samples. Histochemistry of the ovary with alpha-naphtyl phosphate for localization of acid phosphatase activity showed mainly labeling associated to the oocyte peripheral vesicles, basal lamina, and between follicle cells. Electron microscopy analysis showed that acid phosphatase was localized in small peripheral vesicles in the oocyte, but not inside yolk granules. The possible role of this phosphatase during oogenesis and embryogenesis is also discussed in this article.

The role of lipoxygenase products on the endocytosis of yolk proteins in insects: participation of cAMP.

The participation of eicosanoids and second messengers in the regulation of endocytosis by the ovaries was investigated using the uptake of Rhodnius heme binding protein (RHBP) as an experimental model. The rate of RHBP uptake decreased up to 40% in the presence of BWA4C and NDGA, 5 and 12-lipoxygenase inhibitors, respectively, suggesting the involvement of lipoxygenase products in endocytosis regulation. Addition of Leukotriene B4 (LTB(4); one product of the 5 lipoxygenase pathway) increased in vitro the uptake of RHBP by 30%. The content of cAMP in the Rhodnius' ovaries were monitored after treatment with different eicosanoids and inhibitors of eicosanoids synthesis. The amount of cAMP decreased in the presence of indomethacin (by 50%), while treatment with PGE(2) induced an increase of 85% of this messenger in the ovaries. The presence of LTB(4) in the medium inhibited in 60% the content of cAMP in the ovaries, while BWA4C induced a 100% increase of this messenger in the ovaries. Addition of 1 microM DBcAMP in the medium resulted in a 30% decrease in the rate of RHBP uptake. Taken together, these data show that cyclooxygenase and lipoxygenase products participate in the control of protein internalization by modulation of cAMP levels.

A new model for proton pumping in animal cells: the role of pyrophosphate.

The H+-PPase activity was characterized in membrane fractions of ovary and eggs of Rhodnius prolixus. This activity is totally dependent on Mg2+, independent of K+ and strongly inhibited by NaF, IDP and Ca2+. The membrane proteins of eggs were analyzed by western blot using antibodies to the H+-PPase from Arabidopsis thaliana. The immunostain was associated with a single 65-kDa polypeptide. This polypeptide was immunolocalized in yolk granule membranes by optical and transmission electron microscopy. We describe the acidification of yolk granules in the presence of PPi and ATP. This acidification is inhibited in the presence of NAF, Ca2+ and antibodies against H+-PPase. These data show for the first time in animal cells that acidification of yolk granules involves an H+-PPase as well as H+-ATPase.

Synthesis of vitellogenin by the follicle cells of Rhodnius prolixus.

The synthesis and secretion of vitellogenin by the ovary of Rhodnius prolixus was investigated. Using whole ovary or epithelial cells isolated from follicles of different sizes, it is shown that the follicle cells are a site of synthesis for this protein in the ovary. The ovaries or follicle cells were incubated in vitro with [(35)S]-methionine or (32)Pi and the secretion of newly synthesized ovarian vitellogenin (O-Vg) was estimated by the radioactivity associated with the immunoprecipitate or acid-precipitate proteins in the culture medium. The radioactive O-Vg was analyzed by SDS-PAGE followed by autoradiography or after elution from a DEAE-Toyopearl column. The presence of O-Vg inside the follicle cells was detected by immunofluorescence and immunogold labels. Both methods revealed strong labeling inside the follicle cells. While the capacity for total protein synthesis by the follicle cells was maximal during the early phase of vitellogenesis (in small follicles), the synthesis of O-Vg reached its peak during the late phase of oocyte growth, just before formation of the chorion. A possible role for ovarian vitellogenin in Rhodnius and its relationship with Vg synthesis by the fat body is discussed.

A heme-binding protein from hemolymph and oocytes of the blood-sucking insect, Rhodnius prolixus. Isolation and characterization.

A heme-binding protein has been isolated and characterized from both the hemolymph and oocytes of the blood-sucking insect, Rhodnius prolixus. The protein from both sources is identical in most aspects studied. The Rhodnius heme-binding protein (RHBP) is composed of a single 15-kDa polypeptide chain coiled in a highly alpha-helical structure which binds non-covalently one heme/polypeptide chain. This RHBP is not produced by limited degradation of hemoglobin from the vertebrate host, since specific polyclonal antibodies against it do not cross-react with rabbit hemoglobin, and since it differs from hemoglobin in having a distinct amino-acid composition and NH2-terminal sequence. The spectrum of the dithionite-reduced protein has peaks at 426, 530, and 559 nm and resembles that of a b-type cytochrome. RHBP from hemolymph is not saturated with heme and promptly binds heme added to the solution. The oocyte protein, on the other hand, is fully saturated and is not capable of binding additional heme.

Variant of congenital dyserythropoietic anemia.

Nonspherocytic hemolytic anemia was diagnosed in a 34-year-old man with jaundice since childhood. Splenectomy at the age of 8 had no influence on the anemia. Bronze diabetes was diagnosed at age 31, presumably due to hemosiderosis and secondary hemochromatosis. Iron chelation was unsuccessful in controlling iron overload, but phlebotomies proved effective without aggravating the anemia. We believe the anemia represents a variant of congenital dyserythropoietic anemia, type I.

Proliferation and differentiation of human myeloid leukemic cells in immunodeficient mice: electron microscopy and cytochemistry.

To study the influence of a biologic environment on cultured human leukemia cells, KG-1, KG-1a, and HL-60 cells were inoculated subcutaneously into newborn nude mice. The cells developed myelosarcomas at the site of inoculation and in lungs and kidneys. KG-1 and HL-60 myelosarcomas were successfully passaged through adult nude mice, whereas KG-1a tumors proliferated only after transplantation into newborn hosts. The human nature of the cells forming myelosarcomas in mice was assessed by chromosomal analyses and detection of cross-reactivity with an antibody to the human leukemia cell line K562. We undertook electron microscopic and cytochemical examinations of the cells proliferating in vitro and in the mice. The granules of KG-1 cells in vivo did not react for acid phosphatase, as observed in vitro, and the HL-60 cells proliferating in mice lost the perinuclear myeloperoxidase (MPO) demonstrated in cultured cells. Although the influence of an in vivo selection of cell subpopulations cannot be ruled out, the enzymatic changes are compatible with induced cell differentiation. Conclusive evidence of differentiation in vivo was observed in the KG-1a cell subline. The undifferentiated KG-1a blasts developed cytoplasmic granules and synthesized MPO during proliferation in vivo. These observations indicate that human leukemia cells from established cell lines proliferate in nude mice and may acquire new differentiated properties in response to the in vivo environment.

Local and metastatic growth and in vivo differentiation of human myeloid leukemia cells transplanted in nude mice.

Cells from the established human myeloid cell lines KG-1, KG-1a, and HL-60, transplanted subcutaneously (sc) into nude mice, developed discrete tumors (myelosarcomas). These myelosarcomas had a host's age-associated pattern of growth identical to that of experimental tumors produced by sc transplantation of cells derived from malignant solid neoplasias. Thus, leukemia cells yielded either localized myelosarcomas at the site of inoculation or a disseminated neoplastic growth after inoculation in adult (more than 4 weeks old) or newborn (1-3 days old) nude mice, respectively. Human myeloid leukemia cells proliferating in the nude mice preserved the human karyotype and a surface antigenic determinant and did not influence the hematopoietic tissues of the host. The KG-1 and HL-60 cell lines consistently attained varying degrees of differentiation along the myeloid series in vitro, and these features were maintained during proliferation in the mice. Furthermore, cells of the variant subline KG-1a, which had a blastic morphology, developed signs of differentiation that were not seen in culture. The presence of readily identifiable markers, such as cytoplasmic granules containing myeloperoxidase, in the cell lines tested makes these models particularly useful for studying the influence of a biological environment on cell differentiation and its influence on tumor growth. These experimental systems are also suitable for investigating the mechanism(s) of metastases and for in vivo experimental therapeutic trials.

Proliferation of human malignant hematopoietic cells in immunodeficient mice: suppression by antibody to pluripotent K-562 leukemia cells involves direct cytolysis and effector cells.

Six human hematopoetic cell lines were successfully heterotransplanted into athymic (nude) and asplenic-athymic (lasat) neonatal mice. The tumors arising from leukemia and lymphoma cells could then be serially transplanted into adult nude mice. Seven days after the fourth serial mouse passage, each mouse was treated with goat immune gamma globulin against K-562 cells. One control group was treated similarly, but with nonimmune (normal) gamma globulin, while another control group was not treated. The goat gamma globulin was not toxic for nude and lasat mice, and the immune, but not the normal, gamma globulin suppressed local subcutaneous growth of myelosarcomas, lymphosarcomas, and Burkitt lymphoma cells. On the other hand, the growth of lung, breast, and prostatic carcinomas and a melanoma of human origin were not altered by the immune gamma globulin. Since suppression of cell growth occurred equally well in decomplemented mice, a complement-mediated cytotoxicity apparently cannot be considered as responsible for the abrogation. The Fab fragment of the immunoglobulin did not suppress the growth of the myelosarcomas. We conclude that antibody suppression of the in vivo proliferation was specific for malignant hematopoietic cells and that the Fc portion of IgG is necessary for in vivo cytolysis of leukemia cells. The most probable mechanisms are direct antibody cytolysis and antibody-dependent macrophage-mediated cytotoxicity.

Absence of morphological, chromosomal and antigenic changes in the K-562 cell line growing as localized or disseminated tumours in nude mice.

Transplantation of K-562 cells into adult and newborn nude mice led to the development of localized s.c. and disseminated myelosarcomas, respectively. This age-associated, changing pattern of in vivo proliferation of K-562 cells derived from a single aliquot was consistently repeated throughout sequential passages. The only variable in this experimental system was the age of the recipient mice. Not only did the mice have an identical genetic background, but also the transplanted K-562 cells were derived from a single culture passage. As shown by cytological and histological examinations, the characteristic morphology and percentage composition of the subpopulations of the K-562 cell line were preserved in successive in vitro and in vivo passages. The K-562 cells had no prevailing phenotypic traits which could be associated with the growth either in the s.c. tissue or in the viscera. Furthermore, the cells maintained the human karyotype, including their typical chromosomal abnormalities and antigenic determinants, as demonstrated by the binding of a specific antibody, throughout all passages. Our results demonstrate that heterotransplanted K-562 cells may change their behaviour in vivo without undergoing modifications associated with different types of growth. These findings would indicate that the ability of neoplastic cells to proliferate in various environments (metastases) is not the consequence of predetermined cellular characteristics but is functionally conditioned.

Arrest and extravasation of neoplastic cells. An electron microscopy study of serial sections at sequential stages.

The morphological aspects of the arrest and extravasation of malignant cells of human origin (K-562 cell line) in the lungs of athymic (nude) and asplenic-athymic (lasat) mice were studied by electron microscopy examination of serial sections. The specimens were obtained at sequential stages after the sc inoculation into newborn mice of 10(7) malignant cells. K-562 cells (10(5)) were also injected iv into control groups of nude and lasat mice to assess the influence of the route of inoculation on the in vivo behavior of K-562 cells. Our results demonstrated that K-562 cells were arrested and proliferated within the pulmonary capillaries without the participation of platelets or fibrin. The neoplastic cells extravasated by attrition and penetration of the endothelium (rather than by diapedesis) and continued to proliferate in the interstitial tissue of the lung, developing into neoplastic nodules. Following iv injection, K-562 cells induced the formation of platelet-tumor cell aggregates within the pulmonary capillaries. However, under these conditions, the neoplastic cells did not adhere to the endothelium nor did they proliferate or extravasate. These aggregates were flushed out by the circulation, restoring the permeability of the capillaries.

Malaria no municipio de Humaita, Estado do Amazonas. XIV - Inquerito coprologico em habitantes da regiao em relacao as taxas de hemoglobina. / Malaria in the municipality of Humaita, State of Amazonas. XIV - Coprological survey in inhabitants of the region in relation to hemoglobin levels

Em outubro de 1981, foram estudados 213 individuos do Municipio de Humaita, Estado do Amazonas, dos quais 91 eram habitantes de localidades situadas ao longo da calha do Rio Madeira e 122 do Bairro da Olaria, na zona urbana. Todos foram submetidos a inquerito clinico, epidemiologico e parasitologico de fezes pelas tecnicas de BAERMANN, FAUST & col. e HOFFMAN, PONS & JANER. De 65 habitantes da zona urbana e de 43 do Rio Madeira, foi feita determinacao da hemoglobina. De 25 habitantes da zona urbana e de 16 Rio Madeira, foi feita determinacao hemoglobina A2 pela cromatografia em microcoluna de D.E.A.E. celulose Os resultados revelaram que houve maior proporcao de infestacao parasitaria unica ou multipla entre os habitantes do Rio Madeira. Estes achados pode ser explicado pela diferenca das condicoes de higiene locais, pois, os habitantes do Rio Madeira nao dispoe de qualquer elemento de saneamento basico. Essas diferencas, contudo, nao influiram nos niveis de hemoglobina e de hemoglobina A2 que se mostraram semelhantes nos dois grupos