Gelsolin role in microapocrine secretion.

A role of gelsolin in opening the way along the microvilli for secretory vesicles during microapocrine secretion is proposed here. Data obtained with different techniques showed that many digestive enzymes are released by microapocrine secretion in insects. Proteins that might be involved in the machinery of microapocrine secretion were selected from our transcriptomes and literature searches. The proteins were annexin, Complex actin-related proteins 2 and 3 (ARP 2/3) cofilin, fimbrin, gelsolin 1, gelsolin 2, moesin, myosin 1, myosin 6, protein disulphide isomerase 1 (PDI 1), PDI 2 and profilin. The cDNAs coding for annexin, fimbrin, gelsolin 1, myosin 1, PDI 1 and PDI 2 were cloned and their sequences deposited in GenBank. Only gelsolin 1 and myosin 1 are expressed exclusively in the midgut (semiquantitative reverse transcriptase PCR). As myosin 1 may have a structural role in microvilli, gelsolin 1 is the best guess to be involved in the secretory machinery. A truncated recombinant gelsolin 1 was used to generate antibodies with which it was shown labelling inside and around midgut cell microvilli shown in an electron microscope, reinforcing a microvillar role for gelsolin 1. Suppression of gelsolin 1 synthesis by RNA interference prevents secretory vesicles from advancing inside the microvilli, in agreement with its putative role in severing the actin filaments to free the way for the vesicles.

Situação epidemiológica da brucelose bovina no Estado do Paraná / Epidemiological situation of bovine brucellosis in the State of Paraná, Brazil

Realizou-se um estudo para caracterizar a situação epidemiológica da brucelose bovina no Estado do Paraná. O Estado foi estratificado em sete circuitos produtores ou regiões. Em cada circuito foram amostradas aleatoriamente cerca de 300 propriedades e, dentro dessas, foi escolhido de forma aleatória um número pré-estabelecido de animais, dos quais foi obtida uma amostra de sangue. No total, foram amostrados 14.857 animais, provenientes de 2.098 propriedades. Em cada propriedade amostrada foi aplicado um questionário epidemiológico para verificar o tipo de exploração e as práticas zootécnicas e sanitárias que poderiam estar associadas ao risco de infecção pela doença. O protocolo de testes utilizado foi o da triagem com o teste do antígeno acidificado tamponado e o reteste dos positivos com o teste do 2-mercaptoetanol. O rebanho foi considerado positivo se pelo menos um animal foi reagente às duas provas sorológicas. Para o Estado, os resultados de prevalências de focos e de animais infectados foram, respectivamente, de 4,0 por cento [3,2-4,8 por cento] e 1,7 por cento [1,1-2,4 por cento]. Para os circuitos, a prevalência de focos e a de animais foram, respectivamente: circuito 1, 14,7 por cento [10,9-19,2 por cento] e 2,8 por cento [1,2-4,4 por cento]; circuito 2, 8,8 por cento [5,9-12,6 por cento] e 2,4 por cento [1,0-3,8 por cento]; circuito 3, 3,4 por cento [1,6-6,1 por cento] e 0,85 por cento [0,21-1,5 por cento]; circuito 4, 2,3 por cento [0,94-4,8 por cento] e 0,83 por cento [0,02-1,6 por cento]; circuito 5, 2,3 por cento [0,94-4,7 por cento] e 1,6 por cento [0,06-3,3 por cento]; circuito 6, 0,3 por cento [0-1,9 por cento] e 0,09 por cento [0-0,27 por cento]; circuito 7, 1,0 por cento [0,21-2,9 por cento] e 2,2 por cento [0-6,0 por cento]. Os fatores de risco (odds ratio, OR) associados à condição de foco foram: compra de reprodutores (OR= 2,20 [1,42-3,40]) e prática de aluguel de pasto (OR= 2,45 [1,54-3,90]).

A study to characterize the epidemiological situation of bovine brucellosis in the State of Paraná was carried out. In this study, the State was divided into seven regions. Three hundred herds were randomly sampled in each region and a pre-established number of animals were sampled in each of these herds. A total of 1,4857 serum samples from 2,098 herds were collected. In each herd, it was applied an epidemiological questionnaire regarding herd features and also husbandry and sanitary practices that could be associated with risk of infection. The serum samples were screened for antibodies against Brucella spp. by the Rose-Bengal test (RBT), and all RBT-positive sera were re-tested by the 2-mercaptoethanol test (2-ME). A herd was considered positive if at least one animal was positive on both RBT and 2-ME tests. The prevalences of infected herds and infected animals in Paraná State were 4.0 percent [3.2-4.8 percent] and 1.7 percent [1.1-2.4 percent], respectively. In the regions, the prevalences of infected herds and animals were: region 1, 14.7 percent [10.9-19.2 percent] and 2.8 percent [1.2-4.4 percent]; region 2, 8.8 percent [5.9-12.6 percent] and 2.4 percent [1.0-3.8 percent]; region 3, 3.4 percent [1.6-6.1 percent] and 0.85 percent [0.21-1.5 percent]; region 4, 2.3 percent [0.94-4.8 percent] and 0.83 percent [0.02-1.6 percent]; region 5, 2.3 percent [0.94-4.7 percent] and 1.7 percent [0.06-3.3 percent]; region 6, 0.34 percent [0-1.9 percent] and 0.09 percent [0-0.27 percent]; and region 7, 1.0 percent [0.21-2.9 percent] and 2.2 percent [0-6,0 percent]. The risck factors (odds ratio, OR) associated with the presence of the infection were: purchase of animals for breeding (OR= 2.20 [1.42-3.40]) and pasture rental practice (OR=2.45 [1.54-3.90]).

Situação epidemiológica da brucelose bovina no Estado de Mato Grosso / Epidemiologic situation of bovine brucellosis in the State of Mato Grosso, Brazil

Caracterizou-se a brucelose bovina em Mato Grosso por meio de um estudo transversal realizado em 2003 para auxiliar na implementação do Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose. No Estado, estratificado em quatro circuitos pecuários, foram amostrados 13.684 animais, provenientes de 1.152 rebanhos. O protocolo de testes utilizado foi o da triagem com o teste do antígeno acidificado tamponado e os soros reagentes foram testados pelo 2-mercaptoetanol (ME) e concomitantemente retestados pelo teste de Rosa Bengala para resultado conclusivo. As prevalências de focos e de animais infectados foram: 41,2 por cento [38,0-44,4 por cento] e 10,2 por cento [7,4-13,1 por cento], respectivamente. Nos circuitos produtivos, as prevalências de focos foram 36,9 por cento [29,2-45,2 por cento], 27,2 por cento [22,8-32,1 por cento], 40,4 por cento [38,8-46,2 por cento] e 50,3 por cento [44,5-56,1 por cento]; e as prevalências de animais 7,9 por cento [3,0-12,9 por cento], 4,1 por cento [2,8-5,4 por cento], 8,1 por cento [5,2-11,1 por cento] e 15,3 por cento [9,2-21,3 por cento], respectivamente, para os circuitos 1, 2, 3 e 4. Os fatores de risco (odds ratio, OR) associados à condição de foco no Estado foram: exploração de gado de corte (OR= 1,8 [1,2-2,5]), exploração mista (OR=1,8 [1,2-2,7]), número de fêmeas no rebanho de 11 a 50 (OR=4,8 [1,1-20,8]), número de fêmeas no rebanho acima de 51 (OR=6,8 [1,6-29,0]), ocorrência de aborto (OR=1,7 [1,3-2,2]). A brucelose está homogeneamente distribuída no Estado, o que permite uniformidade de medidas sanitárias. Adicionalmente, sugere-se a intensificação da vacinação de fêmeas para todo o Estado.

A study to characterize bovine brucellosis in the State of Mato Grosso was carried out in 2003 in order to support the implementation of the National Program for the Control and Eradication of Bovine Brucellosis. The State was divided into four productive regions, and 13,684 bovines from 1,152 herds were sampled. The serum samples were screened for antibodies to Brucella spp. by the Rose-Bengal test (RBT) and all RBT-positive sera were re-tested by the 2-mercaptoethanol test (2-ME), and again by RBT for confirmation. The prevalence of infected herds and animals in the State of Mato Grosso were 41.2 percent [38.0-44.4 percent] and 10.2 percent [7.4-13.1 percent], respectively. In the productive regions, the prevalence of infected herds were 36.9 percent [29.2-45.2 percent], 27.2 percent [22.8-32.1 percent], 40.4 percent [38.8-46.2 percent], and 50.3 percent [44.5-56.1 percent], respectively; and the prevalence of infected animals were 7.9 percent [3.0-12.9 percent], 4.1 percent [2.8-5.4 percent], 8.1 percent [5.2-11.1 percent], and 15.3 percent [9.2-21.3 percent], respectively, for regions 1, 2, 3, and 4. The risk factors (odds ratio, OR) associated with the presence of the infection in the State were: beef cattle farming (OR=1.8 [1.2-2.5]), mixed (beef and dairy) cattle farming (OR=1.8 [1.2-2.7]), farms with 11 to 50 females (OR=4.8 [1.1-20.8]), farms with more than 51 females (OR=6.8 [1.6-29.0]), and occurrence of abortion (OR=1.7 [1.3-2.2]). Given that brucellosis is homogeneously distributed in the State, uniform sanitary measures might be adopted. As a homogeneous additional measure for the State, the intensification of female vaccination is suggested.

Sequencing of Spodoptera frugiperda midgut trehalases and demonstration of secretion of soluble trehalase by midgut columnar cells.

Both soluble (SfTre1) and membrane-bound (SfTre2) trehalases occur along the midgut of Spodoptera frugiperda larvae. Released SfTre2 was purified as a 67 kDa protein. Its K(m) (1.6 mM) and thermal stability (half life 10 min at 62 degrees C) are different from the previously isolated soluble trehalase (K(m)= 0.47 mM; 100% stable at 62 degrees C). Two cDNAs coding for S. frugiperda trehalases have been cloned using primers based on consensus sequences of trehalases and having as templates a cDNA library prepared from total polyA-containing RNA extracted from midguts. One cDNA codes for a trehalase that has a predicted transmembrane sequence and was defined as SfTre2. The other, after being cloned and expressed, results in a recombinant trehalase with a K(m) value and thermal stability like those of native soluble trehalase. This enzyme was defined as SfTre1 and, after it was used to generate antibodies, it was immunolocalized at the secretory vesicles and at the glycocalyx of columnar cells. Escherichia coli trehalase 3D structure and sequence alignment with SfTre1 support a proposal regarding the residue modulating the pKa value of the proton donor.

Azithromycin inhibits vertical transmission of Toxoplasma gondii in Calomys callosus (Rodentia: Cricetidae).

Toxoplasma gondii infection during pregnancy may cause severe consequences to the embryo. Current toxoplasmosis treatment for pregnant women is based on the administration of spiramycin or a drug combination as sulphadiazine-pyrimethamine-folinic acid (SPFA) in cases of confirmed fetal infection. However, these drugs are few tolerated and present many disadvantages due to their toxic effects to the host. The aim of this study was to evaluate the effectiveness of different treatments on the vertical transmission of T. gondii, including azithromycin, Artemisia annua infusion, spiramycin and SPFA in Calomys callosus as model of congenital toxoplasmosis. C. callosus females were perorally infected with 20 cysts of T. gondii ME49 strain at the day that a vaginal plug was observed (1st day of pregnancy - dop). Treatment with azithromycin, A. annua infusion, and spiramycin started at the 4th dop, while the treatment with SPFA started at the 14th dop. Placenta and embryonic tissues were collected for morphological and immunohistochemical analyses, mouse bioassay and PCR from the 15th to 20th dop. No morphological changes were seen in the placenta and embryonic tissues from females treated with azithromycin, spiramycin and SPFA, but embryonic atrophy was observed in animals treated with A. annua infusion. Parasites were found in the placenta and fetal (brain and liver) tissues of animals treated with SPFA, A. annua infusion and spiramycin, although the number of parasites was lower than in non-treated animals. Parasites were also observed in the placenta of animals treated with azithromycin, but not in their embryos. Bioassay and PCR results confirmed the immunohistochemical data. Also, bradyzoite immunostaining was observed only in placental and fetal tissues of animals treated with SPFA. In conclusion, the treatment with azithromycin showed to be more effective, since it was capable to inhibit the vertical transmission of T. gondii in this model of congenital toxoplasmosis.

Human immature dental pulp stem cells share key characteristic features with limbal stem cells.

OBJECTIVES: Limbal stem cells (LSC) are self-renewing, highly proliferative cells in vitro, which express a set of specific markers and in vivo have the capacity to reconstruct the entire corneal epithelium in cases of ocular surface injury. Currently, LSC transplantation is a commonly used procedure in patients with either uni- or bilateral total limbal stem cells deficiency (TLSCD). Although LSC transplantation holds great promise for patients, several problems need to be overcome. In order to find an alternative source of cells that can partially substitute LSC in cornea epithelium reconstruction, we aimed at investigating whether human immature dental pulp stem cells (hIDPSC) would present similar key characteristics as LSC and whether they could be used for corneal surface reconstruction in a rabbit TLSCD model. MATERIALS: We used hIDPSC, which co-express mesenchymal and embryonic stem cell markers and present the capacity to differentiate into derivative cells of the three germinal layers. TLSCD was induced by chemical burn in one eye of rabbits. After 30 days, the opaque tissue formed was removed by superficial keratectomy. Experimental group received undifferentiated hIDPSC, while control group only received amniotic membrane (AM). Both groups were sacrificed after 3 months. RESULTS AND CONCLUSIONS: We have demonstrated, using immunohistochemistry and reverse transcription-polymerase chain reaction, that hIDPSCs express markers in common with LSC, such as ABCG2, integrin beta1, vimentin, p63, connexin 43 and cytokeratins 3/12. They were also capable of reconstructing the eye surface after induction of unilateral TLSCD in rabbits, as shown by morphological and immunohistochemical analysis using human-specific antibodies against limbal and corneal epithelium. Our data suggest that hIDPSCs share similar characteristics with LSC and might be used as a potential alternative source of cells for corneal reconstruction.

Palatine mucosa of aging and experimentally induced diabetic aging rabbits: light-microscopic, scanning electron microscopic and transmission electron microscopic qualitative study.

BACKGROUND: Data associating palatine mucosa, microvascular architecture, aging and diabetes mellitus are few, so the aim of the present study was to examine these conditions structurally and ultrastructurally. METHODS: We used 18 female rabbits, aged about 2 years at the beginning of the experiment, distributed into 2 groups: aging diabetic and aging animals, prepared by three different methodologies: light microscopy, scanning electron microscopy and transmission electron microscopy. The samples were stained with hematoxylin-eosin for light microscopy, and for scanning electron microscopy, we used Mercox Cl-2B vascular corrosion casts. For transmission electron microscopy, the specimens were fixed and embedded in Epon 812 resin. RESULTS: Under light microscopy, we noted small amounts of underdeveloped connective papillae and significant flattened areas through the epithelium-connective tissue interface in the aging diabetic group. Larger blood vessels were deeply located in the palatine mucosa, branching off in the direction of the epithelium-connective tissue interface. Capillaries followed the interface contour. In both groups, vascular corrosion casts revealed capillaries sprouting out in disorganized rows but parallel between themselves. 'Hair-pin' capillary loops and convoluted capillary loops were noted. CONCLUSION: The aging diabetic group showed rarefying microvasculature areas with complex tortuous capillary loops. Transmission electron microscopy results showed that the aging diabetic group presented small cytoplasmatic projections directed to the vessel lumen and micropinocytic vesicle, i.e. caveolas. Epithelium-connective tissue interface, connective papillae, microangioarchitecture, and information about endothelial cells alterations were observed in the aging diabetic and aging animals.

Angioarchitectural characteristics of the anterodorsal surface of aging and young rabbit tongue: scanning electron microscopy study.

The three-dimensional angioarchitecture of young and aging rabbit tongues were studied using a vascular corrosion cast method. The vascular network of different lingual papillae were observed by scanning electron microscope (SEM). Young animals showed filiform papillae distributed all over considered area, with fungiform ones among them. In comparison to developed specimens, young rabbits demonstrated dense and higher organization of microvascular network.

Three-dimensional architecture of the connective tissue core and surface structures of the lingual papillae in the rabbit.

Three-dimensional characteristics of the epithelial cell layer and connective tissue interface of the tongue were studied using scanning electron microscopy. In this study, the fragments of tongue were fixed in modified Karnovsky's fixative solution. Subsequently, the specimens were treated with 10% NaOH solution for 4-7 days at room temperature and postfixed in 1% OsO4 in 0.1 M phosphate buffer (pH 7.4) for 2 hours at 4 degrees C. They were dehydrated through a graded ethanol series, and critical-point dried with CO2. The specimens were coated with gold and observed in a scanning electron microscope, JEOL JSM-6100. The results showed numerous papillae on the dorsal surface of the tongue divided into four groups (filiform, fungiform, foliate and vallate papillae). Filiform papillae are conically shaped; fungiform papillae have an irregular round surface; foliate papillae are oval in shape and have some parallel projections; and vallate papillae are located in the posterior part of the tongue and have a depression around the center. After the treatment with 10% NaOH solution, the original arrangements of connective papillae could be seen. This characteristic three-dimensional distribution of the collagen fiber bundles is typical for each superficial papillae depending on whether it is filiform, fungiform, foliate or vallate.