Differential inhibition of the Fas- and granule-mediated cytolysis pathways by the orthopoxvirus cytokine response modifier A/SPI-2 and SPI-1 protein.

Cytotoxic T lymphocytes are important effectors of antiviral immunity, and they induce target cell death either by secretion of cytoplasmic granules containing perforin and granzymes or by signaling through the Fas cell surface antigen. Although it is not known whether the granule-mediated and Fas-mediated cytolytic mechanisms share common components, proteinase activity has been implicated as an important feature of both pathways. The orthopoxviruses cowpox virus and rabbitpox virus each encode three members of the serpin family of proteinase inhibitors, designated SPI-1, SPI-2, and SPI-3. Of these, SPI-2 (also referred to as cytokine response modifier A in cowpox virus) has been shown to inhibit the proteolytic activity of both members of the interleukin 1 beta converting enzyme family and granzyme B. We report here that cells infected with cowpox or rabbitpox viruses exhibit resistance to cytolysis by either cytolytic mechanism. Whereas mutation of the cytokine response modifier A/SPI-2 gene was necessary to relieve inhibition of Fasmediated cytolysis, in some cell types mutation of SPI-1, in addition to cytokine response modifier A/SPI-2, was necessary to completely abrogate inhibition. In contrast, viral inhibition of granule-mediated killing was unaffected by mutation of cytokine response modifier A/SPI-2 alone, and it was relieved only when both the cytokine response modifier A/SPI-2 and SPI-1 genes were inactivated. These results suggest that an interleukin 1 beta converting enzyme-like enzymatic activity is involved in both killing mechanisms and indicate that two viral proteins, SPI-1 and cytokine response modifier A/SPI-2, are necessary to inhibit both cytolysis pathways.

Polycystin, the polycystic kidney disease 1 protein, is expressed by epithelial cells in fetal, adult, and polycystic kidney.

Polycystic kidney disease 1 (PKD1) is the major locus of the common genetic disorder autosomal dominant polycystic kidney disease. We have studied PKD1 mRNA, with an RNase protection assay, and found widespread expression in adult tissue, with high levels in brain and moderate signal in kidney. Expression of the PKD1 protein, polycystin, was assessed in kidney using monoclonal antibodies to a recombinant protein containing the C terminus of the molecule. In fetal and adult kidney, staining is restricted to epithelial cells. Expression in the developing nephron is most prominent in mature tubules, with lesser staining in Bowman's capsule and the proximal ureteric bud. In the nephrogenic zone, detectable signal was observed in comma- and S-shaped bodies as well as the distal branches of the ureteric bud. By contrast, uninduced mesenchyme and glomerular tufts showed no staining. In later fetal (>20 weeks) and adult kidney, strong staining persists in cortical tubules with moderate staining detected in the loops of Henle and collecting ducts. These results suggest that polycystin's major role is in the maintenance of renal epithelial differentiation and organization from early fetal life. Interestingly, polycystin expression, monitored at the mRNA level and by immunohistochemistry, appears higher in cystic epithelia, indicating that the disease does not result from complete loss of the protein.

Insertional mutagenesis and marker rescue in a protozoan parasite: cloning of the uracil phosphoribosyltransferase locus from Toxoplasma gondii.

Nonhomologous integration vectors have been used to demonstrate the feasibility of insertional mutagenesis in haploid tachyzoites of the protozoan parasite Toxoplasma gondii. Mutant clones resistant to 5-fluorouracil were identified at a frequency of approximately 10(-6) (approximately 2 x 10(-5) of the stable transformants). Four independent mutants were isolated, all of which were shown to lack uracil phosphoribosyl-transferase (UPRT) activity and harbor transgenes integrated at closely linked loci, suggesting inactivation of the UPRT-encoding gene. Genomic DNA flanking the insertion point (along with the integrated vector) was readily recovered by bacterial transformation with restriction-digested, self-ligated total genomic DNA. Screening of genomic libraries with the recovered fragment identified sequences exhibiting high homology to known UPRT-encoding genes from other species, and cDNA clones were isolated that contain a single open reading frame predicted to encode the 244-amino acid enzyme. Homologous recombination vectors were exploited to create genetic knock-outs at the UPRT locus, which are deficient in enzyme activity but can be complemented by transient transformation with wild-type sequences--formally confirming identification of the functional UPRT gene. Mapping of transgene insertion points indicates that multiple independent mutants arose from integration at distinct sites within the UPRT gene, suggesting that nonhomologous integration is sufficiently random to permit tagging of the entire parasite genome in a single transformation.

Infecção concomitante experimental de Rattus norvegicus por Toxocara canis e Toxoplasma gondii: estudo comportamental e histopatológico

A hipótese de \"manipulação comportamental\" supõe que um parasito pode alterar o comportamento de seu hospedeiro visando aumentar a probabilidade de completar seu ciclo evolutivo. Tais alterações aumentariam a taxa de transmissão hospedeirohospedeiro, assegurando ao parasito ou a seus propágulos o encontro de novo hospedeiro. A possibilidade de infecções parasitárias provocarem mudanças comportamentais em seus hospedeiros e a elevada frequência com que o acometimento de seres humanos por larvas de Toxocara e cistos de Toxoplasma ocorre, têm chamado à atenção de pesquisadores interessados no estudo das relações hospedeiro-parasita. Na infecção por Toxoplasma gondii e Toxocara canis, cistos e larvas estão presentes em diversos locais anatômicos incluindo musculatura, coração, pulmões, olhos e cérebro. A presença de parasitos no cérebro dá oportunidade de manipulação do comportamento do hospedeiro. Entretanto, não se sabe qual ou quais mecanismos estão envolvidos no processo de manipulação do comportamento. Os objetivos do presente estudo foram verificar alterações na ansiedade, medo, memória e aprendizagem de Rattus norvegicus experimentalmente infectados por Toxocara canis e/ou Toxoplasma gondii em dois períodos após infecção, bem como a localização das larvas e cistos e presença de placas beta amiloide ( A) na região do hipocampo no tecido cerebral desses roedores corado pela técnica de Hematoxilina e Eosina (HE), e Vermelho de Congo, respectivamente. Foram utilizadas 40 exemplares fêmeas da espécie Rattus norvegicus, com seis a oito semanas. Os animais foram divididos em quatro grupos: Toxocara - 10 ratos infectados com 300 ovos de Toxocara canis, Toxoplasma -10 ratos infectados com 10 cistos de Toxoplasma gondii, Infecção dupla - 10 ratos infectados com 300 ovos de Toxocara canis e 10 cistos de Toxoplasma gondii, e controle - 10 ratos sem infecção. Nos dias 40, 41, 70 e 71 após a infecção, os animais dos grupos infectados e controle foram submetidos à avaliação no Labirinto em Cruz Elevado e Campo aberto. Aos 120 após infecção foi feita avaliação da memória, aprendizado e aversão a urina de gato dos animais no Labirinto de Barnes. No final das análises comportamentais os animais foram levados a eutanásia para retirada do cérebro e confecção dos cortes histológicos preparados em HE e Vermelho de Congo. Os resultados mostraram efeito ansiolítico para ambas as infecções, principalmente para Toxoplasma gondii. Não houve comprometimento da memória e aprendizado no LB, porém os animais infectados por Toxocara canis ou Toxoplasma gondii apresentaram menor tempo para encontrar a toca com urina e entrar nela. A leitura dos cortes histológicos corados com HE mostraram larvas de Toxocara canis e cistos de Toxoplasma gondii em regiões do sistema nervoso central dos animais relacionadas com memória e aprendizado. As lâminas coradas com Vermelho de Congo apresentaram placas beta amiloides ( A) em metade dos animais infectados por Toxoplasma gondii. Conclui-se que a infecção por ambos os parasitos apresenta efeito ansiolítico quando ocorre infecção única. Quando a Infecção ocorre concomitantemente há modulação no comportamento. Além disso, ratas infectadas com infecção única apresentam-se menos aversivas à urina de gatos.

Characterization of the human sulfate anion transporter (hsat-1) protein and gene (SAT1; SLC26A1)

Sulfate plays an essential role during growth, development, bone/cartilage formation, and cellular metabolism. In this study, we have isolated the human sulfate anion transporter cDNA (hsat-1; SCL26A1) and gene (SAT1), determined its protein function in Xenopus oocytes and characterized SAT1 promoter activity in mammalian renal cell lines. hsat-1 encodes a protein of 75 kDa, with 12 putative transmembrane domains, that induces sulfate, chloride, and oxalate transport in Xenopus oocytes. hsat-1 mRNA is expressed most abundantly in the kidney and liver, with lower levels in the pancreas, testis, brain, small intestine, colon, and lung. The SAT1 gene is comprised of four exons stretching 6 kb in length, with an alternative splice site formed from an optional exon. SAT1 5' flanking region led to promoter activity in renal OK and LLC-PK1 cells. Using SAT1 5' flanking region truncations, the first 135 bp was shown to be sufficient for basal promoter activity. Mutation of the activator protein-1 (AP-1) site at position 252 in the SAT1 promoter led to loss of transcriptional activity, suggesting its requirement for SAT1 basal expression. This study represents the first functional characterization of the human SAT1 gene and protein encoded by the anion transporter hsat-1.

Receptor for activated C Kinase 1 protein binds to and activates the human estrogen receptor α

The classical concept of estrogen receptor (ER) activation is that steroid passes the cell membrane, binds to its specific protein receptor in the cell's cytoplasm and the steroid-receptor complex travels to the nucleus where it activates responsive genes. This basic idea has been challenged by results of experiments demonstrating insulin-like growth factor 1 (IGF-1) activation of the ER in the complete absence of estrogen suggesting at least one other mechanism of ER activation not involving steroid. One explanation is that activation of the cell surface IGF-1 receptor leads to synthesis of an intracellular protein(s) able to bind to and stimulate the ER. Based on results using the two-hybrid system, coimmunoprecipitation and transfection-luciferase assays, we herein show that one of these proteins could well be receptor for activated C kinase 1 (RACK-1). Using the human ER type α (ER-α) as bait, a cloned complementary deoxyribonucleic acid (cDNA) library from IGF-1 treated human breast cancer MCF-7 cells was screened for ER-α - protein interactions. Many positive clones were obtained which contained the RACK-1 cDNA sequence. Coimmunoprecipitation of in-vitro translation products of the ER-α and RACK-1 confirmed the interaction between the two proteins. Transfection studies using the estrogen response element spliced to a luciferase reporter gene revealed that constitutive RACK-1 expression was able to powerfully stimulate ER-α activity under estrogen-free conditions. This effect could be enhanced by 17β-estradiol (E2) and blocked by tamoxifen, an E2 antagonist. These results show that RACK-1 is able to activate the ER-α in the absence of E2, although together with the latter, enhanced effects occur. Since RACK-1 gene expression is stimulated by IGF-1, it is distinctly possible that RACK-1 is the mediator of the stimulatory effects of IGF-1 on ER-α. © 2014 JMS.

Az európai uniós támogatások hatékonysága és hatásossága közötti különbség a ROP 1.2 intézkedésnél (The difference between the efficiency and effectiveness of using the EU-subsidies at Regional Operational Program)

A szerző cikkében megpróbál rávilágítani annak fontosságára, hogy az Európai Unióból érkező fejlesztési támogatások hasznosulása és a támogatási rendszer hatékonysága között jelentős különbség van. Ha nem tudjuk hatékonyan és hatásosan felhasználni az Unióból érkező pénzügyi eszközöket, akkor a cél: a kohézió, a konvergencia az Európai Unió régi tagállamainak fejlettségi szintjéhez, jólétéhez még nehezebben és lassabban érhető el. Az uniós támogatások hatékonysága alapvetően a rendelkezésre álló pénz lekötésének, lehívásának arányából, illetve a lekötött, lehívott és esetleg visszafizetett összeg arányából állapítható meg (kvantitatív megközelítés). A támogatások felhasználásának hatékonyságánál bonyolultabb, jóval összetettebb megközelítést igényel a hatásosság fogalma. A felhasználás hatásosságát a projektszinten a támogatás hatására megtermelt hozzáadott értékkel, programszinten a GDP hozzáadott növekedéssel lehet kifejezni. A cikk alapvetően a ROP 1.2 "Turisztikai fogadóképesség javítása" pályázati kiírás nyertes projektjeinél végzett kutatás eredményein keresztül kívánja szemléltetni a projektszintű vagy mikro hatásosság fontosságára (kvalitatív megközelítés). _________ The author tries to highlight the importance the difference between the efficiency and effectiveness of using the EU-subsidies. If Hungary cannot use the financial means of the EU efficiently and effectively, than the goal: cohesion and convergence to the level of the old, developed countries of the EU will be much harder and slowly. The efficiency of the EU-subsidies can be measured by the ratio of the amount of money obliged and the amount of money spent, and by the amount of money withdrawn by the Commission, which is actually lost (quantitative approach). The effectiveness of EU-subsidies needs a much more complicated and complex approach, than the efficiency. The effectiveness of usage on project level can be measured by the "added value" of the project, on program level by the added GDP growth or employment. The article is analysing basically the results of a survey made among the winner project of the application ROP 1.2 (Regional Operational Program 1.2) Improving the capacity for tourism (project level or micro effectiveness – qualitative approach).

Mecanismos protetores induzidos pelo tratamento prévio com antígeno solúvel de taquizoítas (STAg) no intestino delgado de camundongos BALB/c e C57BL/6 infectados oralmente por Toxoplasma gondii

T. gondii can infect the gut mucosa by direct invasion of epithelial cells in the small intestine and these cells may respond directly to infection promoting a local immune response. C57BL/6 mice orally infected with a high parasitic load of T.gondii are highly susceptible, presenting a lethal ileitis. Recently, it was demonstrated that pretreatment with STAg protects C57BL/6 mice against intestinal pathology in oral T. gondii infection. To investigate the mechanisms induced by STAg in the small intestine in oral T.gondii infection, BALB/c and C57BL/6 mice were treated with STAg 48 hours before oral infection with 30 ME-49 cysts and sacrificed at 8 days of infection. Previous treatment with STAg were able of decrease parasitism and pathology in peripheral organs of BALB/c and C57BL/6 mice and induced a increase in amounts of goblet cells, IgA positive cells, Paneth cells and expression of cryptidin in the small intestine of both lineages of mice, moreover BALB/c mice presented higher amount of these cells comparing with C57BL/6 mice. The results suggests that STAg is able of promoting protective mechanisms in both lineages of mice, although these protection is more evidenced in BALB/c mice, and these mechanisms could be in part mediated by increase in goblet, Paneth and local secretion of IgA in the small intestine of mice orally infected with T.gondii.

Utilização de peptídeos sintéticos derivados de moléculas imunodominantes de Toxoplasma gondii para diagnóstico sorológico da toxoplasmose em ovinos e galinhas caipiras

Chapter I - The obligate intracellular parasite Toxoplasma gondii is the causative agent of toxoplasmosis, a disease that affects humans and generates economic losses in farm animals. When prevention fails disease refers to the diagnosis and subsequent treatment if the individual is diagnosed as positive. Therefore, the development of new accurate diagnostic tools for detecting T. gondii infection is a need in particular to determine the environmental source of infection which can result in more appropriate public health policies against different routes of infection and prevent potential damage that toxoplasmosis can cause when animals are infected. Chapter II - The domestic chicken (Gallus gallus domesticus) are considered epidemiological sentinels, still representing a major source of recombinant strains when predated by cats, it is common to find them with multiple infections. We evaluate the diagnostic potential of six synthetic peptides (SAG2Y, MIC1, M2AP, GRA10, ROP2 and ROP7) predicted in silico from tachyzoites immunodominant markers of T. gondii in samples from naturally infected chickens, comparing synthetic peptides with antigen total soluble (STAg). In general, our results demonstrated that reactivity rates and positivity for these peptides are similar to the STAg, and the ROP7 peptide and the combination of peptides MIC1+ROP2 have significant sensitivity, confirming them as potential diagnostic tools for the diagnosis of toxoplasmosis in chickens. Chapter III - Sheep (Ovis aries) are commonly infected with Toxoplasma gondii due to his eating habits. Infection in pregnant sheep can have serious consequences such as embryonic death, fetal resorption, mummification, and neonatal death. One concern regarding the infection in these animals is that the meat can be a source of contamination to humans and other carnivores. Therefore perform accurate diagnosis in these animals is of fundamental importance. In the present study we evaluated the potential of new synthetic peptides as a diagnostic tool. Synthetic peptides (SAG2Y, SRS52A, MIC14, GRA4, GRA10 and GRA15) were predicted in silico from immunodominant proteins of T. gondii. We determine the levels of IgG antibodies using sera obtained from two farms in the city of Uberlândia. Analyzing the results together, the peptide combination GRA10+GRA15 (Accuracy = 0,82) showed better characteristics compared with the other mixtures. This preparation could be better analyzed with an antigenic mixture potential use in the diagnosis of toxoplasmosis in sheep and other species.

Enrofloxacina e toltrazuril reduzem a infecção por Toxoplasma gondii em células trofoblásticas humanas e em explantes de vilos placentários humanos de terceiro trimestre

The classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic targets is indispensable to minimize the undesirable effects and improve the control of the infection. Previous studies demonstrated that enrofloxacin and toltrazuril were able to control the infection triggered by Neospora caninum and Toxoplasma gondii. Therefore, the aim of this present study was evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii proliferation in human trophoblast cells (BeWo lineage) and in human villous explants from third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine or association (sulfadiazine + pyrimethamine) in other to verify their viability by MTT or LDH assay, respectively. Next, BeWo cells were infected with T. gondii RH (2F1 clone) or ME49 strain, whereas villous were infected only with RH strain (2F1 clone), after, both cells and villous were treated or not with the same antibiotics and analyzed to T. gondii intracellular proliferation by beta-galactosidase assay (for RH strain) or blue toluidine staining (for ME49 strain). ELISA was performed in the supernatant to evaluate the cytokine profile. Enrofloxacin and toltrazuril did not change strongly the viability in cells and villous. Furthermore, the drugs decreased the parasite intracellular proliferation regardless T. gondii strain in BeWo cells and villous explants when compared to untreated and infected conditions. In BeWo cells infected by RH, enrofloxacin induced high levels of IL-6 low levels of MIF, while both cytokines were upregulated by enrofloxacin and toltrazuril in BeWo cells infected by ME49 strain. Additionally, in villous explantes, enrofloxacin induced high MIF production. Thus, enrofloxacin and toltrazuril were able to control the parasitism in BeWo cells and villous explants, and probably it occurs by modulation of immune response in these cells or tissues and direct action on parasite, but future experiments are necessary to verify this hypothesis.

Biochemical Characterization of Human Exonuclease 1 Protein-protein Interactions

Human Exonuclease 1 (Exo1) plays important roles in numerous DNA metabolic/repair pathways including DNA mismatch repair, DNA double strand break repair, Okazaki fragment maturation etc. The nuclease activity of Exo1 is tightly regulated in vivo. The regulation of Exo1 in different pathways is achieved by interactions with different protein partners. The focus of this dissertation will be on characterization of Exo1 interactions with traditional protein partners and providing experimental evidences for new Exo1 interactions.

Molecular cloning, biochemical assays, collaborative nuclear magnetic resonance and X-ray crystallography have been employed to study Exo1 interactions with protein partners. This work contains: (i) the experimental evidence for new Exo1 interactions, and (ii) the detailed characterization of Exo1 interactions with PCNA, MLH1 and MutSα/β.

Taken together, the research progress presented in this dissertation further advances our understanding of traditional Exo1 interaction network and probably provides new insights to new functions and new regulations of Exo1.

Determinación de la prevalencia serológica de brucella abortus, toxoplasma gondii, sarcocystis spp y los factores de riesgo asociados en estudiantes de medicina veterinaria de la Universidad de la Salle

Tesis (Médico Veterinario). -- Universidad de La Salle. Facultad de Ciencias Agropecuarias. Programa de Medicina Veterinaria, 2013

Pathogenicity and phenotypic sulfadiazine resistance of Toxoplasma gondii isolates obtained from livestock in northeastern Brazil

Toxoplasma gondii is the causative protozoan agent of toxoplasmosis, which is a common infection that is widely distributed worldwide. Studies revealed stronger clonal strains in North America and Europe and genetic diversity in South American strains. Our study aimed to differentiate the pathogenicity and sulfadiazine resistance of three T. gondii isolates obtained from livestock intended for human consumption. The cytopathic effects of the T. gondii isolates were evaluated. The pathogenicity was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using a CS3 marker and in a rodent model in vivo. Phenotypic sulfadiazine resistance was measured using a kinetic curve of drug activity in Swiss mice. IgM and IgG were measured by ELISA, and the dihydropteroate synthase (DHPS) gene sequence was analysed. The cytopathic effects and the PCR-RFLP profiles from chickens indicated a different infection source. The Ck3 isolate displayed more cytopathic effects in vitro than the Ck2 and ME49 strains. Additionally, the Ck2 isolate induced a differential humoral immune response compared to ME49. The Ck3 and Pg1 isolates, but not the Ck2 isolate, showed sulfadiazine resistance in the sensitivity assay. We did not find any DHPS gene polymorphisms in the mouse samples. These atypical pathogenicity and sulfadiazine resistance profiles were not previously reported and served as a warning to local health authorities.

Ocorrência da infecção por Toxoplasma gondii e avaliação da imunização em caprinos do sertão do cabugi, Rio Grande do Norte

Toxoplasmosis is one zoonosis caused by Toxoplasma gondii protozoan. Goats, amongst the production animals, are one of the species most susceptible to this parasite, being one them main involved agents in ovine and goat abortions, determining great economic losses and implications for public health, since the presence it parasite in the products of goat origin, consist in one of the main sources of infection for the man. In this study 244 blood samples in 8 farms situated in 4 cities from the Sertão do Cabugi region, Rio Grande do Norte State, northeast of Brazil and, tested by ELISA assay. The results had shown a prevalence of 47.13% for anti- T. gondii antibodies and a significant association between positivity and variable evaluated as age, locality and property. The IgG avidity assay evaluated in 115 positive samples was carried to discriminate acute and chronic infection. Twelve samples (10.4%) had presented antibodies of low avidity while 103 (89.6%) presented high avidity antibodies; indicating that most of the animals was precocious exposure to the parasite. Significant difference was verified only for the variable sex. We also evaluate the capacity of recombinant adenoviruses codifying SAG1, SAG2, SAG3 and CMV in inducing activation of specific immune response in goat. These 109 animals received 109 pfu of the AdSAG1, AdSAG2, AdSAG3, AdCMV or PBS in vaccine protocol with 3 immunizations. Serum samples of the each animal, before and after mmunization, had been submitted to the ELISA. The results demonstrate that the immunizations had induced the production of IgG antibodies specific against T. gondii proteins

Serological evidence of Toxoplasma gondii in hunted wild boar from Portugal

Toxoplasmosis is an important parasitic zoonosis with a worldwide distribution, being the parasitic disease with the highest occurrence in Europe. Wild boar has an important role in the epidemiological cycle of Toxoplasma gondii as an intermediate host, that can potentially infect humans when the meat is consumed raw or undercooked. The purpose of this work was to determine the presence of antibodies to T. gondii in serum of hunted wild boar. During the hunting season 2011/2012, sera samples were collected from 97 wild boar and tested for IgG antibodies to T. gondii, using the modified agglutination test. Twenty out of the 97 wild boar (20.6%) were seropositive for T. gondii IgG antibodies. Multivariate logistic regression analysis showed that males and older animals were associated with T. gondii seropositivity. These results show that T. gondii has an important presence in wild boar population from Portugal, suggesting a potential zoonotic risk for humans when wild boar meat or meat products are consumed raw or undercooked.