Imunolocalização de receptores de leptina no ovário de preás (Galea spixii Wagler, 1831) / Immunolocalization of leptin receptor of Spix's Yellow-toothed Cavy (Galea spixii Wagler, 1831) ovary

A leptina, uma citocina produzida pelas células adiposas, é alvo da comunidade científica por acreditarem que ela apresente impacto sobre a reprodução dos animais promovendo a puberdade, foliculogênese e oogênese, ciclo estral e auxiliando na fecundação. A compreensão dos mecanismos que controlam a atividade reprodutiva de preás (Galea spixii) possui papel relevante para a preservação da espécie. Desta forma, o presente trabalho propôs analisar a imunolocalização dos receptores de leptina (Ob-R) no ovário de preás. Coletaram-se os ovários de 20 fêmeas adultas, não prenhes e saudáveis. As amostras foram fixadas em paraformaldeído a 4% em tampão fosfato, incluídas em parafina e seccionadas para a realização de imunohistoquímica (IHC). As secções foram fotomicrografadas e avaliadas quanto à intensidade da reação. Observou-se forte imunorreação no oócito e nas células da teca, moderada nas células do estroma ovariano e nas células luteínicas grandes e fracamente coradas nas células da granulosa, endoteliais, perivasculares e células luteínicas pequenas. Quando comparado a expressão de receptores ao longo do desenvolvimento folicular foi observado que o oócito e as células da teca se mantiveram com expressão na mesma intensidade. Entretanto, as células da granulosa apresentaram forte marcação nos estádios pré-antrais enquanto que nos folículos antrais apresentou fraca intensidade. Concluímos que em ovários de Galea spixii existe a presença de Ob-R nas principais estruturas do ovário sugerindo que este hormônio desempenhe papel fundamental na reprodução desta espécie.

Leptin, a cytokine produced by adipose cells, is the target of the scientific community for believing that it has an impact on the reproduction of the animals promoting puberty, folliculogenesis and oogenesis, estrous cycle and aiding in fertilization. The understanding of the mechanisms controlling the reproductive activity of Spix's Yellow-toothed Cavy (Galea spixii) plays a relevant role in the preservation of the species. Thus, the present study proposed to analyze the immunolocalization of leptin receptors (Ob-R) in the ovary of cavies. Ovaries from 20 adult, non-pregnant, healthy females were collected. The samples were fixed in 4% phosphate buffered paraformaldehyde, embedded in paraffin and sectioned for immunohistochemistry. The sections were photomicrographs and intensity of the reaction was measured. Strong immunoreaction was observed in oocyte and theca cells, moderate in ovarian stromal cells and large luteal cells and weak stained in granulosa, endothelial, perivascular and small luteal cells. When compared to receptor expression along follicular development it was observed that the oocyte and the theca cells remained with expression at the same intensity. However, the granulosa cells presented strong stained in the preantral stages, whereas in the antral follicles it presented low intensity. We conclude that in the ovaries of Galea spixii there is the presence of Ob-R in the main structures of the ovary sugesting that this hormone plays a fundamental role in the reproduction of this species.

Caracterização das proteínas caveolinas -1 e -2 na placenta de conceptos bovinos clonados transgênicos / Caracterization of caveolin -1 and -2 proteins in cloned and transgenic placenta of cattle

A utilização da transgenia com a proteína fluorescente verde (GFP) como marcador de células de origem fetal nas placentas de clones bovinos servirá de modelo inédito para estudo morfofisiológico e imunológico da interação materno-fetal, visto que possibilitará o seu mapeamento, diferenciando as células fetais das maternas. Tal modelo terá aplicação direta, principalmente porque estes são animais que apresentam problemas em relação ao seu desenvolvimento. Com o auxílio deste modelo, pretende-se verificar o transporte de substâncias entre a mãe e o feto via endocitose, pela imunolocalização das proteínas chamadas de caveolinas. Para tanto foram utilizados 06 bovinos clonados e 30 bovinos de inseminação artificial (IA) com idade até 90 dias de gestação, os quais tiveram seu desenvolvimento interrompido mediante abate humanitário das receptoras e ovariosalpingohisterectomia, com posterior recuperação do útero gestante. Foram coletados os placentônios e o cório. Uma parte das amostras foi recortada e fixada, por imersão, em solução de parafolmaldeído a 4% ou formoldeído a 10% em tampão fosfato de sódio (PBS) a 0,1M pH 7.4, solução de Zamboni (4% de paraformoldeído, 15% de ácido pícrico, em tampão fosfato de sódio a 0,1M pH 7.4), metacarn (60% de metanol, 30% de clorofórmio, e 10% de ácido acético glacial), para verificação da morfologia e realização de imuno-histoquímica para as proteínas caveolinas -1 e -2 (CAV -1 e CAV-2)...

The transgenic application of green fluorescent protein (GFP) as fetal cell marker on cattle cloned placenta could provide an exclusive model for studying the morphologic and immunologic maternal-fetal interactions, providing information about its mapping, distinguishing the fetal from maternal cells. This model will have direct application, mainly because these animals present problems during its development. With this model's support, we intend to verify the substances transport between mother and fetus during endocytosis, through the immunolocalization of protein named caveolae. For these, we used 06 cloned bovine and 30 cattle samples of artificial insemination (AI) with 90 days of pregnancy, which had been their development interrupted by humanitarian slaughter of the recipient and recovery of the pregnant uterus. We collected the placentome and the chorion. A part of the samples was cut and fixed, by immersion, on a solution containing 4% of parafomaldehyde or 10% of formaldehyde on a sodium phosphate buffer (PBS), at 0,1M pH 7.4, Zamboni solution (4% of paraformaldehyde, 15% of picric acid, on sodium phosphate buffer 0,1M pH 7.4), metacarn (60% of metanol, 30% of chloroform, and 10% glacial acetic acid), for morphologic and immunohistochemistry verification for caveolinas proteins -1 and -2 (CAV -1 and CAV- 2). The caveolins -1 were found in fetal and maternal villi, but its strongest staining was observed in the endometrial stroma. The caveolins -2 had positive staining in trophoblast and chorioallantoic membrane, and specifically in giant trophoblastic binucleated cell. Therefore the results were compared between cloned cattle and from AI or natural mating, for assisting on detection of the reason of many placental alterations, embryonic losses, spontaneous abortion, post-natal mortality and large offspring syndrome on laboratory-manipulated animals. The result suggests that the proteins caveolins -1 and -2 (CAV-1 and CAV-2)...

Ultrastructural immunolocalization of beta-defensin-27 in granulocytes of the dermis and wound epidermis of lizard suggests they contribute to the anti-microbial skin barrier / 대한해부학회지

The high resistance to infections in lizard wounds suggests that these reptiles possess effective antimicrobial peptides in their tissues. The present immunocytochemical study shows the cellular localization of beta-defensin 27 in tail tissues and in the blood, a defensin previously identified in the lizard Anolis carolinensis through biomolecular methods. Beta-defensin-27 immunoreactivity is only observed in some large granules mainly contained in heterophilic granulocytes that are sparse within the dermis of the skin or in the isolated blood. This peptide is absent in other cell types of the skin, in keratinocytes and in subdermal muscle tissue of the tail in normal conditions. Pre-corneous keratinocytes of the regenerating tail epidermis are unlabeled or show a weak labeling for the peptide only in sparse cytoplasmic areas or in the extracellular spaces among corneocytes of the wound and regenerating epidermis. The study suggests that beta-defensin 27 is normally stored in granulocytes present in the blood or in connective tissues while in the epidermis keratinocytes do not show the presence of this peptide unless these cells are stimulated from injury to produce and likely release beta-defensins.

Preparation of Rabbit Polyclonal Antibody against ELF3 and Its Immunolocalization in Mice / 华中科技大学学报(医学版)

Objective To prepare and identify rabbit polyclonal antibody against embryonic liver fordrin 3(ELF3),and investigate the distribution of ELF3 in mice tissue.Methods ELF3 specific N-terminal peptide was synthesized,and conjugated to Keyhole limpet hemocyanin(KLH)as immunogen.The ELF3-KLH complex was injected into rabbits subcutaneously,and then ELF3 antibody was purified using affinity chromatography.The titer of the antibody was evaluated by ELISA.The specificity of antibody against ELF3 and immunolocalization of ELF3 were evaluated by using Western blot and immunohistochemistry.Results Rabbit polyclonal antibody against ELF3 was prepared by the immunization of ELF3-KLH complex.ELISA and Western blot results showed the antibody against ELF3 had high titer and specificity.Western blot and immunohistochemical studies demonstrated ELF3 was expressed in the mouse heart,liver,brain and kidney tissue,particular on the cell membrane.Conclusion The preparation of polyclonal antibody against ELF3 was successful due to its high titer and specificity;ELF3 was expressed in the mice heart,liver,and kidney,particular on the cell membrane.It will provide an excellent tool for further study on the ELF3 function.

Molecular Cloning and Characterization of Two Hommologues of Yeast Sir2 Gene in the Parasitic Protist Trichomonas vaginalis / 热带医学杂志

Objective To screen cell growth and senescence-related genes of the parasitic pmtist Trichomonas vaginalis,we launched an EST program and isolated two cDNA clones from a T.vaginalis cDNA library,which showed high homology in deduced amino acid sequences to yeast Sir2 and designated as TvSir2 and TvSir2-like.Method The cDNA sequence of TvSIR2 had a length of 1034 base pairs (bp) with an open reading frame of 915 bp,and TvSIR2-like,1214 bp with an open reading frame of 1116 bp.Result The two deduced amino acid sequences shared all the three conserved cole domains with yeast Sir2 and its homologues,suggesting that the two clones were Sir2 homologues. A cDNA fragment from each cDNA clone was subvloned into the expression vector pET-41a.The expression of the fusion proteins in E.coli BL21 stains was induced by isopropylthio-β-D-galactoside (IPTG).Two anti-sera were prepared by immunizing two guinea pigs with the purified fusion proteins, Western-blot analysis demonstrated that each anti-serum reacted with the corresponding recombinant protein and detected a clear band (TvSir2,34 000 Mr;TvSir2-like,42 000 Mr)in protein extracts of the protist.Immunofluolescence techniques showed that TvSir2 and TvSir2-like proteins were both localized in the legions of perinuelear (ER) and Golgi complex.Conclusion Our data suggest that TvSir2 and TvSir2-like were two members of Sir2 family.Their biological functions in the protist would be further studied.

Preparation of porcine LIF polyclonal antibody and its immunolocalization in pig / 吉林大学学报(医学版)

Objective To construct the expression plasmid of porcine leukemia inhibitory factor(LIF) in the prokaryotic system,and purify the expressed recombinant porcine LIF protein as antigen to immune rabbit for preparation of rabbit anti-pig LIF polyclonal antibody,and immunolocalize of LIF in pigs.Methods The cDNA fragment encoding mature porcine LIF protein was amplified by PCR,then cloned into the prokaryotic expression vector PET-31b at restriction sites NdeⅠand XhoⅠ.After transformed into E.coli BL21 for expression,the recombinant porcine LIF protein was purifed for preparation of anti-pig LIF polyclonal antibody as immunogen.Western blotting and ELISA were used to test the specificity and titer of the purifed IgG.Immunostaining was used to assay the expression of LIF in pigs.Results The constructed expression plasmid was identified by DNA sequencing and restriction enzyme digestion.The expression of the recombinant porcine LIF protein was performed exactly in E.coli BL21.By immuning the rabbit,the polyclonal antibody was successfully prepared.The results of ELISA and Western blotting showed that the antibody had high titer(1∶10000) and specificity.By immunostaining,LIF protein was faintly expressed in porcine myocardial cell,splenic cord and monolayer covering epithelium cell and pulmonary fibroblast.Conclusion The prepared polyclonal antibody has high titer and specificity;LIF has faint positive immunoreactivity in the normal porcine,myocardial cell,splenic cord and monolayer covering epithelium cell and pulmonary fibroblast.

Excretory bladder: the source of cysteine proteases in Paragonimus westermani metacercariae

The cysteine proteases of Paragonimus westermani metacercariae are involved in metacercarial excystment, host immune modulation, and possibly in tissue penetration. In order to clarify the origin of the enzymes, 28 and 27 kDa cysteine proteases in metacercarial excretory-secretory products were purified through the FPLC system using Mono Q column chromatography. The polyclonal antibodies to the enzymes were produced in BALB/c mice. Immunolocalization studies revealed that both cysteine proteases were distributed at the linings of excretory bladder and excretory concretions of the metacercariae. It was suggested that the excretory epithelium of P. westermani undertake the secretory function of metacercarial cysteine proteases, in addition to its role as a route for eliminating waste products.