Comparison of uroplakin expression during urothelial carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine in rats and mice

The expression of uroplakins, the tissue-specific and differentiation- dependent membrane proteins of the urothelium, was analyzed immunohistochemically in N butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-treated rats and mice during bladder carcinogenesis. Male Fischer 344 rats were treated with 0.05% BBN in the drinking water for 10 wk and were cuthanatized at week 20 of the experiment. BBN was administered to male B6D2F1 mice; it was either provided at a rate of 0.05% in the drinking water (for 26 wk) or 5 mg BBN was administered by intragastric gavage twice weekly for 10 wk, followed by 20 wk without treatment. In rats, BBN-induced, noninvasive, low grade, papillary, transitional cell carcinoma (TCC) showed decreased uroplakin-staining of cells lining the lumen but showed increased expression in some nonluminal cells. In mice, nonpapillary, high-grade dysplasia, carcinoma in situ, and invasive carcinoma were induced. There was a marked decrease in the number of uroplakin-positive cells lining the lumen and in nonluminal cells. This occurred in normal-appearing urothelium in BBN-treated mice and in dysplasic urothelium, in carcinoma in situ, and in invasive TCC. The percentage of uroplakin-positive nonluminal cells was higher in control mice than in rats, but it was lower in the mouse than in the rat after BBN treatment. Uroplakin expression was disorderly and focal in BBN-treated urothelium in both species. These results indicate that BBN treatment changed the expression of uroplakins during bladder carcinogenesis, with differences in rats and mice being related to degree of tumor differentiation.

N-glycosylation in sugarcane

The N-linked glycosylation of secretory and membrane proteins is the most complex posttranslational modification known to occur in eukaryotic cells. It has been shown to play critical roles in modulating protein function. Although this important biological process has been extensively studied in mammals, much less is known about this biosynthetic pathway in plants. The enzymes involved in plant N-glycan biosynthesis and processing are still not well defined and the mechanism of their genetic regulation is almost completely unknown. In this paper we describe our first attempt to understand the N-linked glycosylation mechanism in a plant species by using the data generated by the Sugarcane Expressed Sequence Tag (SUCEST) project. The SUCEST database was mined for sugarcane gene products potentially involved in the N-glycosylation pathway. This approach has led to the identification and functional assignment of 90 expressed sequence tag (EST) clusters sharing significant sequence similarity with the enzymes involved in N-glycan biosynthesis and processing. The ESTs identified were also analyzed to establish their relative abundance.

Clonagem e expressão da glicoproteína transmembrana do retrovírus HTLV-1 em células de mamíferos

The retrovirus HTLV-1 is the etiological agent of the adult T-cell leukemia and HTLV-1 associated myelopathy/tropical spastic paraparesis. The proviral genome has 9,032 base pairs, showing regulatory and structural genes. The env gene encodes for the transmembrane glycoprotein gp 21. The development of methodologies for heterologous protein expression, as well as the acquisition of a cellular line that constituently expresses the recombinant, were the main goals of this work. The DNA fragment that encodes for gp 21 was amplified by nested-PCR and cloned into a pCR2.1-TOPO vector. After which, a sub-cloning was realized using the expressing vector pcDNA3.1+. The transfection of mammalian cells HEK 293 was performed transitorily and permanently. Production of the recombinant gp 21 was confirmed by flux cytometry experiments and the cell line producing protein will be used in immunogenicity assays.

Toxoplasma gondii: Comparison of a rhoptry-ELISA with IFAT and MAT for antibody detection in sera of experimentally infected pigs

Indirect ELISA and IFAT have been reported to be more sensitive and specific than agglutination tests. However, MAT is cheaper, easier than the others and does not need special equipment. The purpose of this study was to compare an enzyme linked immunosorbent assay using crude rhoptries of Toxoplasma gondii as coating wells (r-ELISA) with indirect fluorescence antibody test (IFAT) and modified agglutination test (MAT) to detect anti-T. gondii antibodies in sera of experimentally infected pigs. Ten mixed breed pigs between 6.5 and 7.5 weeks old were used. All pigs were negative for the presence of T. gondii antibodies by IFAT (titre < 16), r-ELISA (OD < 0.295) and MAT (titre < 16). Animals received 7 × 107 viable tachyzoites of the RH strain by intramuscular (IM) route at day 0. Serum samples were collected at days -6, 0, 7, 14, 21, 28, 35, 42, 50, and 57. IFAT detected anti-T. gondii antibodies earlier than r-ELISA and MAT. The average of antibody levels was higher at day 35 in IFAT (Log10 = 2.9) and in MAT (Log10 = 3.5), and at day 42 in r-ELISA (OD = 0.797). The antibody levels remained high through the 57th day after inoculation in MAT, and there was a decrease tendency in r-ELISA and IFAT. IFAT was used as gold standard and r-ELISA demonstrated a higher prevalence (73.3%), sensitivity (94.3%), negative predictive value (83.3%), and accuracy (95.6%) than MAT. Kappa agreements among tests were calculated, and the best results were shown by r-ELISA × IFAT (κ = 0.88, p < 0.001). Cross-reaction with Sarcocystis miescheriana was investigated in r-ELISA and OD mean was 0.163 ± 0.035 (n = 65). Additionally, none of the animals inoculated with Sarcocystis reacted positively in r-ELISA. Our results indicate that r-ELISA could be a good method for serological detection of T. gondii infection in pigs. © 2005 Elsevier Inc. All rights reserved.

Aspectos estruturais da membrana eritrocitária

This article describes the structures and functions of the erythrocyte membrane and its importance in transfusional medicine. The erythrocyte membrane is one of the best known membranes in terms of structure, function and genetic disorders. As any other plasma membrane, it mediates transport functions. It also provides the erythrocytes with their resilience and deformability. According to the International Society of Blood Transfusion (ISBT), more than 500 antigens are expressed in the erythrocyte membrane, and around 270 are involved in transfusion reaction cases and hemolytic diseases of the fetus and newborn. In the ISBT classification, the high frequency series is represented by antigens in more than 99% of population (high prevalence antigen). In transfusion, the absence of these antigens determines severe problems as for example, one woman without the P antigen suffered 6 repetitive miscarriages due to placental insufficiency, which was caused by an antibody formed against the absent P antigen. Some important erythrocyte membrane proteins are described here including Band 3, Glycophorins and spectrin. The most abundant integral membrane protein is Band 3 and its main function is to mediate exchange of chloride and bicarbonate anions across the plasma membrane. The second most abundant integral membrane protein in the human erythrocyte is sialoglycoprotein glycophorin A (GPA). With its high sialic acid content, GPA is the main contributor to the net negative cell-surface charge and is thus critical for minimizing cell-cell interactions and preventing red cell aggregation. Glycophorin C (GPC) is the receptor for PfEBP-2 (baebl, EBA-140), the newly identified erythrocyte binding ligand of Plasmodium falciparum. The ternary complex of spectrin, actin and 4.1R defines the nodes of the erythrocyte membrane skeletal network, and is inseparable from membrane stability when under mechanical stress. This erythrocyte membrane review is important for a better understanding of transfusion reactions, where the antibody formation against high prevalence antigens makes compatible transfusions difficult. The study of antigen diversity and biochemical characterization of different proteins will contribute to healthcare, as well as diagnosis, development of technology such as monoclonal antibody production and the therapeutic conduct of many diseases.

Clonagem e expressão da glicoproteína transmembrana do vírus linfotrópico de células T humanas em sistema procarioto

HTLV-1 is the virus that causes T cell lymphoma/leukemia in adults and a neurological disorder known as HTLV-associated myelopathy or tropical spastic paraparesis. One of the transmission means is through contaminated blood and its byproducts. Because of the risk of HTLV-associated infections, screening for HTLV was introduced for Brazilian blood donors in 1993. Most of the diagnostic kits used in the national blood banks are bought from foreign companies. Brazil does not have the technology to produce this material and there is a need to produce diagnostic systems with national technology. In this study, we show the expression of gp21/HTLV-1 in Escherichia coli and its reactivity towards monoclonal antibodies and the antibodies of infected patients. Expressing these proteins is the first step towards obtaining diagnostic kits with Brazilian biotechnology.

Presence and distribution of the endosymbiont Wolbachia among Solenopsis spp. (Hymenoptera: Formicidae) from Brazil and its evolutionary history

Wolbachia are intracellular bacteria that commonly infect arthropods. Its prevalence among ants of the genus Solenopsis is high. In the present study, the presence and distribution of these endosymbionts was examined among populations of Solenopsis spp. from Brazil. A phylogenetic analysis based on the wsp gene was conducted to infer the evolutionary history of Wolbachia infections within the populations surveyed. A high frequency of Wolbachia bacteria was observed among the genus Solenopsis, 51% of the colonies examined were infected. Incidence was higher in populations from southern Brazil. However, little genetic variability was found among different Wolbachia strains within supergroups A and B. Our findings also suggest that horizontal transmission events can occur through the social parasite S. daguerrei. © 2012 Elsevier Inc..

Equine tendonitis therapy using mesenchymal stem cells and platelet concentrates: A randomized controlled trial

Introduction. Tendon injury is a major cause of lameness and decreased performance in athletic equines. Various therapies for tendonitis have been described; however, none of these therapies results in complete tissue regeneration, and the injury recurrence rate is high even after long recovery periods involving rest and physiotherapy. Methods. A lesion was induced with collagenase gel in the superficial digital flexor tendon in the center portion of the metacarpal region of eight equines of mixed breed. After two weeks, the lesions of the animals in the treated and control groups were treated through the intralesional administration of mesenchymal stem cells derived from adipose tissue (adMSCs) suspended in platelet concentrate (PC) and with phosphate buffered saline (PBS), respectively. Serial ultrasound analyses were performed every two weeks. After 16 weeks of therapy, a biopsy was performed for histopathological, immunohistochemical and gene expression (type I collagen (COL1A1), type III collagen (COL3A1), tenascin-C (TNC), tenomodulin (TNMD), and scleraxis (SCX)) analyses. Results: Differences in the ultrasound and histopathological analyses were observed between the groups. Improved results were reported in the group treated with adMSCs suspended in PC. There was no difference in the gene expression levels observed after the different treatments. The main results observed from the histopathological evaluation of the treated group were as follows: a prevention of the progression of the lesion, a greater organization of collagen fibers, and a decreased inflammatory infiltrate. A lack of progression of the lesion area and its percentage was observed in the ultrasound image, and increased blood flow was measured by Power Doppler. Conclusions: The use of adMSCs combined with PC for the therapy of experimentally induced tendonitis prevented the progression of the tendon lesion, as observed in the ultrasound examination, and resulted in a greater organization and decreased inflammation, as observed in the histopathological evaluation. These data demonstrate the therapeutic potential of this therapy for the treatment of equine tendonitis. © 2013 Carvalho et al.; licensee BioMed Central Ltd.

Análise comparativa da expressão protéica em glândulas pós-faríngeas de saúva-limão, Atta sexdens rubropilosa Forel, 1908 (Hymenoptera: Formicidae) submetidas à suplementação lipídica

Pós-graduação em Ciências Biológicas (Biologia Celular e Molecular) - IBRC

Estrogen-Responsive Genes Overlap with Triiodothyronine-Responsive Genes in a Breast Carcinoma Cell Line

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Overexpression of UCP1 in tobacco induces mitochondrial biogenesis and amplifies a broad stress response

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expressão e localização de aquaporinas no epidídimo do gerbilo, Meriones unguiculatus, em diferentes fases do desenvolvimento pós-natal

Aquaporins (AQPs) are membrane protein channels expressed in a wide variety of cells and tissues that are involved in the transport of water. In the male excurrent duct, the movement of fluid and solutes across the epithelium is essential for establishing the proper luminal environment in which sperm mature and are stored. The initial aim of the present propose is to localize AQP 2 e 9 in epithelium of the initial segment, caput, corpus and cauda epididymidis in Mongolian gerbil during postnatal development (10, 30, 60, 120 and 300 days), observing possible changes in the pattern of expression of the AQPs along the postnatal development. The animals will be killed by inhalation of carbon dioxide. After euthanasia, the epididymis will be removed and the different regions will be isolated. The biological material obtained will be processed in accordance with the routines of histological techniques and imunnohistochemistry to study the expression and localization of the AQPs 2 and 9. The results obtained will be analyzed and photographed in conventional light microscope

Novel inorganic pyrophosphatase from soil metagenomic and family and subfamily prediction

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

MetaPPase by in silico analyses predicting the novel gene operate as H+ pumps.

The lastyears declined the discovery of compounds to use in industrial and naturaldiversity has been the best supplier for novel genes, enzymes and compounds inhigh demand by the biotechnology industry. We know immense diversity of microorganisms,yet most remains unexplored. For these reason we use the metagenômica approach toinvestigate the potential of uncultured microorganisms. With this purpose weused the metagenomic library of from Eucalyptus spp. arboretum (EAA), wedid screening to found positive clone and them was submitted to the process of shotgun,the data obtained was submitted a bioinformatics analyses. Our results showsthe hypothesis of high unexplored microbial diversity of soil are able to foundnovel genes and metagenomic approach is and allowed to isolate novel genes and insilico analyses are essential part to identify a novel Inorganicpyrophosphatase (PPase) prediction indicated the novel gene operate as H+ pumps. Thissuggests that a special feature, our work in situ will be cloning thegene expression vector for subsequent kinetic characterization and crystallization.

Effects of Yersinia enterocolitica O:3 derivatives on B lymphocyte activation in vivo

The potential sequelae of intestinal infection with Yersinia enterocolitica include reactive arthritis, erythema nodosum, Reiter's syndrome and other autoimmune diseases. The role of the immune response in the pathogenesis of these diseases has not been fully defined, but autoimmune manifestations may be a consequence of the increase in autoantibodies as a result of polyclonal B-cell activation induced by Yersinia. We investigated the effects of Y enterocolitica 0:3 derivatives on B lymphocyte activation in vivo. Groups of five specific pathogen free (SPF) Swiss mice were inoculated with bacterial cell extract, Yersinia outermembrane proteins (Yops) or lipopolysaccharide (LPS) obtained from Y enterocolitica 0:3 and their immunoglobulin-secreting spleen cells were detected by isotype-specific protein A plaque assay. The presence of specific anti-Yersinia antibodies and autoantibodies was determined in mouse sera by ELISA. In all experiments a marked increase in the number of secretory cells of different isotypes was observed as early as the third day after inoculation. IgG and IgM anti-Yersinia antibodies were detected in the sera of all inoculated mice, and autoantibodies against myosin in the sera of those inoculated with bacterial cell extract. The sera from animals stimulated with LPS reacted with myelin, actin and laminin, while the sera from mice inoculated with Yops reacted with myelin, thyroglobulin and cardiolipin. These results suggest that SPF Swiss mice inoculated with any one of the Y enterocolitica derivatives tested exhibited polyclonal activation of B lymphocytes as a result of stimulation by various bacterial components and not only LPS stimulation.