Morphological and quantitative study of ganglionated plexus of Calomys callosus trachea

Calomys callosus is a wild, native forest rodent found in South America. In Brazil, this species has been reported to harbour the parasitic protozoan Trypanosoma cruzi. The ganglionated plexus of this species was studied using whole-mount preparations of trachea that were stained using histological and histochemical methods. The histological methods were used to determine the position of the ganglia with respect to the trachea muscle and to determine the presence of elastic and collagen fibers. The histochemical method of NADH-diaphorase was used for morphometric evaluations of the plexus. The tracheal plexus lies exclusively over the muscular part of the organ, dorsal to the muscle itself. It varies in pattern and extent between animals. The average number of neurons was 279 and the cellular profile area ranged from 38.37 mu m(2) to 805.89 mu m(2). Acetylcholinesterase (AChE) histochemistry verified that both ganglia and single neurons lie along nerve trunks and are reciprocally interconnected with the plexus. Intensely AChE-reactive neurons were found to be intermingled with poorly reactive ones. Two longitudinal AChE-positive nerve trunks were also observed and there was a diverse number of ganglia along the intricate network of nerves interconnecting the trunks. A ganglion capsule of collagen and elastic fibers surrounding the neurons was observed. Under polarized light, the capsule appeared to be formed by Type I collagen fibers. (C) 2008 Elsevier B.V. All rights reserved.

Early weaning accelerates the differentiation of mucous neck cells in rat gastric mucosa: Possible role of TGF alpha/EGFR

The development of the gastric mucosa is controlled by hormones, growth factors and feeding behavior. Early weaning (EW), which means the abrupt interruption of suckling, increases proliferation and differentiation in the rat gastric epithelium. Transforming growth factor alpha(TGF alpha) is secreted in the stomach, binds to the epidermal growth factor receptor( EGFR) and may control cell proliferation, differentiation and migration. Here, we investigated the influence of suckling-weaning transition on the differentiation of mucous neck cells in the stomach and its association to the expression of TGF alpha and EGFR. Fifteen-day-old Wistar rats were divided into two groups: suckling( control), in which pups were kept with the dam, and early weaning( EW), in which rats were separated from their mother and fed with hydrated powdered chow. TGF alpha and EGFR levels were increased at 18 days in EW animals compared to control ones (p<0.05). Histochemical reactions with Periodic Acid-Schiff reagent+Alcian Blue or Bandeiraea simplicifolia II lectin were used to stain the mucous neck cells and showed an increase in this cell population throughout EW, which was more pronounced at 17 days when compared to suckling pups (p<0.05). These morphological results were confirmed by RT-PCR for mucin 6. The levels of mucin 6 mRNA were higher in EW animals from the 16th to the 18th day(1-3 days post-weaning) when compared to the respective control group. Inhibition of EGFR through AG1478 administration to EW animals prevented the expansion of mucous neck cell population induced by EW (p<0.05). Therefore, early weaning up regulated TGF alpha/EGFR expression and induced differentiation of mucous neck cells. Moreover, we showed that EGFR takes part in the maturation of this cell population. We conclude that regular suckling-weaning transition is crucial to guarantee the development of the gastric mucosa. (C) 2009 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved.

Immunocytochemical identification of adenohypophyseal cells in the pirarucu (Arapaima gigas), an Amazonian basal teleost

The adenohypophysis (AH) of juvenile pirarucu (Arapaima gigas), a representative species of the Osteoglossomorpha (bonytongue fishes, one of the oldest living groups of the teleosts), was studied using histochemical and immunocytochemical methods. The AH is comprised of the pars distalis (PD), without a clear distinction between rostral pars distalis (RPD) and proximal pars distalis (PPD), and the pars intermedia (PI). The neurohypophysis (NH) is positioned on top of the PD and penetrates and branches into the PI. In the most rostral dorsal portion of the PD, adrenocorticotropic cells and fusiform gonadotropic cells were found. In the central PD, scarce prolactin-producing cells and growth-hormone-producing cells were located mainly in the dorsal part, whereas round gonadotropic cells were abundant in the ventral portion of this region. Human thyrotropin immunoreactive cells were not found in the entire AH. In the PI, melanotropic, some adrenocorticotropic, and somatolactin-producing cells were located intermingled surrounding the neurohypophyseal branches. Our results showed that the A. gigas pituitary has some basal characteristics between the ancient Actinopterygii and the more derived teleosts.

Improved skeletal muscle oxidative enzyme activity and restoration of PGC-1α and PPARß/δ gene expression upon rosiglitazone treatment in obese patients with type 2 diabetes mellitus

Objective: To examine whether rosiglitazone alters gene expression of some key genes involved in mitochondrial biogenesis and oxidative capacity in skeletal muscle of type 2 diabetic patients, and whether this is associated with alterations in skeletal muscle oxidative capacity and lipid content.

Design: Skeletal muscle gene expression, mitochondrial protein content, oxidative capacity and lipid accumulation were measured in muscle biopsies obtained from diabetic patients, before and after 8 weeks of rosiglitazone treatment, and matched controls. Furthermore, whole-body insulin sensitivity and substrate utilization were assessed.

Subjects: Ten obese type 2 diabetic patients and 10 obese normoglycemic controls matched for age and BMI.

Methods: Gene expression and mitochondrial protein content of complexes I–V of the respiratory chain were measured by quantitative polymerase chain reaction and Western blotting, respectively. Histochemical staining was used to quantify lipid accumulation and complex II succinate dehydrogenase (SDH) activity. Insulin sensitivity and substrate utilization were measured during a hyperinsulinemic–euglycemic clamp with indirect calorimetry.

Results: Skeletal-muscle mRNA of PGC-1a and PPARb/d – but not of other genes involved in glucose, fat and oxidative metabolism – was significantly lower in diabetic patients (Po0.01). Rosiglitazone significantly increased PGC-1a (B2.2-fold, Po0.01) and PPARb/d (B2.6-fold, Po0.01), in parallel with an increase in insulin sensitivity, SDH activity and metabolic flexibility (Po0.01). Surprisingly, none of the measured mitochondrial proteins was reduced in type 2 diabetic patients, nor affected by rosiglitazone treatment. No alterations were seen in muscular fat accumulation upon treatment.

Conclusion: These results suggest that the insulin-sensitizing effect of rosiglitazone may involve an effect on muscular oxidative capacity, via PGC-1a and PPARb/d, independent of mitochondrial protein content and/or changes in intramyocellular lipid.

Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult zebrafish.

The zebrafish is a useful model organism for developmental and genetic studies. The morphology and function of zebrafish myeloid cells were characterized. Adult zebrafish contain 2 distinct granulocytes, a heterophil and a rarer eosinophil, both of which circulate and are generated in the kidney, the adult hematopoietic organ. Heterophils show strong histochemical myeloperoxidasic activity, although weaker peroxidase activity was observed under some conditions in eosinophils and erythrocytes. Embryonic zebrafish have circulating immature heterophils by 48 hours after fertilization (hpf). A zebrafish myeloperoxidase homologue (myeloid-specific peroxidase; mpx) was isolated. Phylogenetic analysis suggested it represented a gene ancestral to the mammalian myeloperoxidase gene family. It was expressed in adult granulocytes and in embryos from 18 hpf, first diffusely in the axial intermediate cell mass and then discretely in a dispersed cell population. Comparison of hemoglobinized cell distribution, mpx gene expression, and myeloperoxidase histochemistry in wild-type and mutant embryos confirmed that the latter reliably identified a population of myeloid cells. Studies in embryos after tail transection demonstrated that mpx- and peroxidase-expressing cells were mobile and localized to a site of inflammation, indicating functional capability of these embryonic granulocytes. Embryonic macrophages removed carbon particles from the circulation by phagocytosis. Collectively, these observations have demonstrated the early onset of zebrafish granulopoiesis, have proved that granulocytes circulate by 48 hpf, and have demonstrated the functional activity of embryonic granulocytes and macrophages. These observations will facilitate the application of this genetically tractable organism to the study of myelopoiesis.

ATP7B expression in human breast epithelial cells is mediated by lactational hormones

A role for the copper transporter, ATP7B, in secretion of copper from the human breast into milk has previously not been reported, although it is known that the murine ortholog of ATP7B facilitates copper secretion in the mouse mammary gland. We show here that ATP7B is expressed in luminal epithelial cells in both the resting and lactating human breast, where it has a perinuclear localization in resting epithelial cells and a diffuse location in lactating tissue. ATP7B protein was present in a different subset of vesicles from those containing milk proteins and did not overlap with Menkes ATPase, ATP-7A, except in the perinuclear region of cells. In the cultured human mammary line, PMC42-LA, treatment with lactational hormones induced a redistribution of ATP7B from a perinuclear region to a region adjacent, but not coincident with, the apical plasma membrane. Trafficking of ATP7B was copper dependent, suggesting that the hormone-induced redistribution of ATP7A was mediated through an increase in intracellular copper. Radioactive copper (⁶⁴Cu) studies using polarized PMC42-LA cells that overexpressed mAtp7B protein showed that this transporter facilitates copper efflux from the apical surface of the cells. In summary, our results are consistent with an important function of ATP7B in the secretion of copper from the human mammary gland.

Abscisic acid regulation of plant defence responses during pathogen attack

The plant hormone, abscisic acid (ABA), has previously been shown to have an impact on the resistance or susceptibility of plants to pathogens. In this thesis, it was shown that ABA had a regulatory effect on an extensive array of plant defence responses in three different plant and pathogen interaction combinations as well as following the application of an abiotic elicitor. In unique studies using ABA deficient mutants of Arabidopsis, exogenous ABA addition or ABA biosynthesis inhibitor application and simulated drought stress, ABA was shown to have a profound effect on the outcome of interactions between plants and pathogens of differing lifestyles and from different kingdoms. The systems used included a model plant and an important agricultural species: Arabidopsis thaliana (Arabidopsis) and Peronospora parasitica (a biotrophic Oomycete pathogen), Arabidopsis and Pseudomonas syringae pathovar tomato (a biotrophic bacterial pathogen) and an unrelated plant species, soybean (Glycine max) and Phytophthora sojae (a hemibiotrophic Oomycete pathogen), Generally, a higher than basal endogenous ABA concentration within plant tissues at the time of avirulent pathogen inoculation, caused an interaction shift towards what phenotypically resembled susceptibility. Conversely, a lower than basal endogenous ABA concentration in plants inoculated with a virulent pathogen caused a shift towards resistance. An extensive suppressive effect of ABA on defence responses was revealed by a range of techniques that included histochemical, biochemical and molecular approaches. A universal effect of ABA on suppression or induction of the phenylpropanoid pathway via regulation of the key entry point gene, phenylalanine ammonia-lyase (PAL), when stimulated by biotic or abiotic elicitors was shown. ABA also influenced a wide variety of other defence-related components such as: the development of a hypersensitive response (HR), the accumulation of the reactive oxyden species, hydrogen peroxide and the cell wall strengthening compounds lignin and callose, accumulation of SA and the phytoalexin, glyceollin and the transcription of the SA-dependent pathogenesis- related gene (PR-1). The near genome-wide microarray gene expression analysis of an ABA induced susceptible interaction also revealed an yet unprecedented insight into the great diversity of defence responses that were influenced by ABA that included: disease resistance like proteins, antimicrobial proteins as well as phenylpropanoid and tryptophan pathway enzymes. Subtle differences were found in the number and type of defence responses that were regulated by ABA in each type of plant and pathogen interaction that was studied. This thesis has clearly identified in plant/pathogen interactions previously unknown and important roles for ABA in the regulation of many defence responses.

Defence mechanisms of a resistant monocot model to phytophthora cinnamomi

The plant pathogen Phytophthora cinnamomi causes devastating disease in natural and agricultural systems worldwide. While some plants can survive, little is known about the underlying mechanisms of resistance. This research used histochemical and genome-wide analysis to identify key cellular and molecular defence mechanisms within the resistant plant Zea mays.

Double label autoradiography : an improvement

A convenient method for double-label autoradiography is described that uses an aqueous mountant, Gelutol (polyvinyl alcohol), which keeps the gelatin spacer in the final autoradiograph permanently swollen to a thickness of around 18 microns in contrast to its 5 microns thickness during exposure of the autoradiograph. This greatly improves optical discrimination between upper and lower layers without the loss of sensitivity or resolution that would result if a 18 microns spacer were used during exposure.

Transformação genética em cevada por bombardeamento de partículas

A presente Tese de Doutorado objetivou: (1) definir um método eficiente de transformação genética, por bombardeamento de partículas, para a obtenção de plantas transgênicas de cultivares brasileiras de cevada e (2) identificar gene(s) codificante(s) de quitinase(s) potencialmente capaz(es) de conferir resistência ao fungo patogênico de cevada Bipolaris sorokiniana. Culturas de calos obtidos a partir de escutelos imaturos das cultivares Brasileiras de cevada MN-599 e MN-698 (Cia. de Bebidas das Américas, AMBEV) foram bombardeadas com partículas de tungstênio e avaliadas quanto à expressão do gene repórter gusA através de ensaios histoquímicos de GUS e quanto ao efeito dos bombardeamentos na indução estruturas embriogênicas e regeneração de plantas. As condições de biobalística analisadas incluíram a região promotora regulando a expressão de gusA, tipo e pressão de gás hélio de dois aparelhos de bombardeamento, distância de migração das partículas, número de tiros e a realização de pré e pós-tratamento osmótico dos tecidos-alvo. No presente trabalho foram obtidos um número bastante alto de pontos azuis por calo, a indução de calos embriogênicos e embriões somáticos em uma freqüência de até 58,3% e a regeneração de 60 plantas, sendo 43 de calos bombardeados. As melhores condições observadas foram o promotor e primeiro íntron do gene Adh de milho (plasmídeo pNGI), o aparelho de bombardeamento “ Particle Inflow Gun” (PIG) utilizando-se a distância de migração de partículas de 14,8 cm, dois tiros disparados por placa e a realização de tratamento osmótico dos explantes com 0,2 M de manitol e 0,2 M de sorbitol 4-5 horas antes e 17-19 horas depois dos bombardeamentos. Das 43 plantas obtidas de calos bombardeadas, 3 apresentaram atividade de GUS na base das suas folhas. A utilização de primers sintéticos definidos a partir de genes de quitinases descritos na literatura em PCRs resultou na amplificação de dois fragmentos de aproximadamente 700 e 500 pb a partir de DNA total das cvs. MN-599 e MN-698 de cevada e um fragmento, com aproximadamente 500 pb, a partir do DNA total do isolado A4c de Trichoderma sp. Estes fragmentos foram purificados dos géis de agarose e diretamente seqüenciados de forma manual e automática. Os fragmentos de 700 e 500 pb amplificados do genoma da cultivar MN-599 foram identificados como genes de quitinases de cevada e o fragmento de 500 pb do isolado A4c de Trichoderma sp. não apresentou homologia com seqüências conhecidas de quitinases depositadas no EMBL/GenBank. A utilização de novos pares de primers, representando seqüências conservadas de quitinases do fungo Metarhizium anisopliae, resultou na amplificação de 3 fragmentos a partir do DNA total do isolado A4b de Trichoderma sp., que estão sendo purificados para realização de seqüenciamento.

Astrocytic and microglial response and histopathological changes in the brain of horses with experimental chronic Trypanosoma evansi infection

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

Preliminary study on age- and sex-dependent alterations in the composition of skeletal muscle fibers of Brasileiro de Hipismo horses

The aim of this work was to characterize the distribution of myofibers in the gluteus medius muscle of inactive horses of the Brasileiro de Hipismo (BH) breed at different ages by means of histochemical analyses, according to sex and depth of the biopsy. A total of 78 inactive horses (9 castrated males, 35 stallions, and 34 females) of the BH breed, aged 1 to 4 years, were used. A percutaneous muscle biopsy was obtained with a 6.0-mm Bergstrom-type needle, which allowed the removal of muscle fragments at depths of 20 and 60 mm. Myofiber types were determined based on myofibrillar adenosine triphosphatase (mATPase) and nicotinamide dinucleotide tetrazolium reductase (NADH-TR) techniques. Morphometry of the fibers was determined based on cross-sectional area (CSA), mean frequency (F), and relative cross-sectional area (RCSA). The current study demonstrated that BH horses 3 and 4 years of age show a greater percentage of, and area occupied by, type IIA fibers and lower percentage of type IIX fibers in the gluteus medius muscle compared with horses 1 and 2 years of age. No difference was found between sexes in the frequency of and area occupied by the different fiber types at any of the depths and ages studied. In this study, females showed a greater CSA for all fibers in comparison with males, at 1 year of age. The results of the current study indicate that the gluteus medius muscle of inactive BH horses shows modifications in its structural and biochemical composition during the growth of the animals, leading to a better oxidative capacity.

Light exercise causes adaptive changes in muscles of young Brasileiro de Hipismo horses

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

Composição de fibras musculares esqueléticas de eqüinos jovens da raça Brasileiro de Hipismo

The aim of this study was to typify the skeletal striated fibers of the gluteus medius muscle of young Brasileiro de Hipismo (BH) horses by means of histochemical analysis with m-ATPase and NADH-TR according to the sex and the biopsy depth. It was observed that the frequency (F;%) and the relative cross sectional area (RCSA;%) of the fibers type IIX were greater than the fibers type IIA, which F and RCSA were greater than the fibers type I. The comparison between sex and muscles depht, showed no significant difference in F and RCSA in the three types of fibers. The results of morphometry showed that the gluteus medius muscle has greater glycolitic metabolism and anaerobic capacity because of the presence of large proportion of type IIX fibers. This may be justified by the genetic influence of Thoroughbred in the formation of Brasileiro de Hipismo breed.

Immunohistochemical characterization of mononuclear cells and MHC II expression in the brain of horses with experimental chronic Trypanosoma evansi infection

Este estudo objetivou caracterizar a resposta imune celular no sistema nervoso central (SNC) de eqüinos com infecção crônica experimental por Trypanosoma evansi. Para este propósito, foram utilizados os métodos histoquímicos (HE) e imunoistoquímicos do complexo avidina-biotina peroxidase (ABC). O fenótipo do infiltrado celular foi caracterizado com o auxílio de anticorpos anti - CD3, para linfócitos T e antiBLA36 para linfócitos B. Os macrófagos foram marcados com anticorpo antiantígenos da linhagem mielóide/histiócitos (Clone Mac387). A lesão no sistema nervoso central (SNC) dos eqüinos infectados com T. evansi foi caracterizada como meningoencefalite e meningomielite não supurativa. A gravidade das lesões variou em diferentes segmentos do SNC, refletindo distribuição irregular das alterações vasculares. A distribuição de células T e B e antígenos do complexo maior de histocompatibilidade classe II foram avaliados dentro do SNC de eqüinos cronicamente infectados com T. evansi. O infiltrado perivascular e meníngeo eram constituídos predominantemente por células T e B. Macrófagos foram raramente visualizados. T.evansi não foi identificado no parênquima do SNC dos eqüinos.