2-Aminoethyl Dihydrogen Phosphate (2-AEH2P) Associated with Cell Metabolism-Modulating Drugs Presents a Synergistic and Pro-Apoptotic Effect in an In Vitro Model of the Ascitic Ehrlich Tumor.

The progression and maintenance of cancer characteristics are associated with cellular components linked to the tumor and non-cellular components with pro-tumoral properties. Pharmacological association with antagonists of the cellular components of the tumor, such as anti- and pro-apoptotic drugs, represents a novel adjuvant strategy. In this study, the antiproliferative, pro-apoptotic, and pharmacological effects of the combination of monophosphoester 2-AEH2P with Simvastatin, Coenzyme Q10, the chemotherapeutic drug paclitaxel, and colony-stimulating factor (GM-CSF) were evaluated. Tests were conducted to determine cytotoxic activity using the MTT method, cell cycle phases, and fragmented DNA by flow cytometry, mitochondrial membrane potential, expression of cell markers Bcl2, TNF-α/DR-4, Cytochrome c, caspase 3, and P53, and analysis of drug combination profiles using Synergy Finder 2.0 Software. The results showed a synergistic effect among the combinations, compared to individual treatments with the monophosphoester and other drugs. In addition, there was modulation of marker expression, indicating a pro-apoptotic and immunomodulatory effect of 2-AEH2P. Pharmacological analysis revealed that tumor cells treated with GM-CSF + 2-AEH2P exhibited a synergistic effect, while groups of tumor cells treated with paclitaxel, Coenzyme Q10, and Simvastatin showed additive effects. Furthermore, treatment with the paclitaxel + 2-AEH2P combination (12 h) resulted in a significant reduction in mitochondrial membrane potential. Pharmacological combinations for normal cells did not exhibit deleterious effects compared to mammary carcinomatosis tumor (EAT) cells.

2-Aminoethyl dihydrogen phosphate (2-AEH2P) Associated with cell metabolism-modulating drugs presents a synergistic and pro-apoptotic effect in an in vitro model of the ascitic ehrlich tumor

The progression and maintenance of cancer characteristics are associated with cellular components linked to the tumor and non-cellular components with pro-tumoral properties. Pharmacological association with antagonists of the cellular components of the tumor, such as anti- and pro-apoptotic drugs, represents a novel adjuvant strategy. In this study, the antiproliferative, pro-apoptotic, and pharmacological effects of the combination of monophosphoester 2-AEH2P with Simvastatin, Coenzyme Q10, the chemotherapeutic drug paclitaxel, and colony-stimulating factor (GM-CSF) were evaluated. Tests were conducted to determine cytotoxic activity using the MTT method, cell cycle phases, and fragmented DNA by flow cytometry, mitochondrial membrane potential, expression of cell markers Bcl2, TNF-α/DR-4, Cytochrome c, caspase 3, and P53, and analysis of drug combination profiles using Synergy Finder 2.0 Software. The results showed a synergistic effect among the combinations, compared to individual treatments with the monophosphoester and other drugs. In addition, there was modulation of marker expression, indicating a pro-apoptotic and immunomodulatory effect of 2-AEH2P. Pharmacological analysis revealed that tumor cells treated with GM-CSF + 2-AEH2P exhibited a synergistic effect, while groups of tumor cells treated with paclitaxel, Coenzyme Q10, and Simvastatin showed additive effects. Furthermore, treatment with the paclitaxel + 2-AEH2P combination (12 h) resulted in a significant reduction in mitochondrial membrane potential. Pharmacological combinations for normal cells did not exhibit deleterious effects compared to mammary carcinomatosis tumor (EAT) cells.

Structural and Ultrastructural Changes in the Tongue of mdx Mice.

The mdx mouse is an experimental model of Duchenne muscular dystrophy, a genetic disorder characterized by progressive muscular degeneration which affects the oral cavity musculature, and promotes difficulty in swallowing. This study aimed to describe morphological, structural, and ultrastructural changes in the tongue mucosa and musculature of mdx mice. Forty six-month-old mice were divided into two groups: Control C57bl/10 (n = 20) and mdx C57bl/10mdx (n = 20). The tongue was dissected and analyzed with light microscopy, scanning electron microscopy, and transmission electron microscopy techniques. Our results showed conical and triangular filiform, fungiform, foliate, and vallate papillae, and their connective tissue cores. The epithelium layers identified were corneum, granulosum, spinosum, and basale. The mdx group had a thicker epithelium. Lamina propria was composed of reddish and greenish collagen. In mdx, collagen was present in the musculature of the tongue's body and in the muscular tissue between mucous and serous glands of the caudal region. Musculature was also characterized by a shorter length of sarcoplasmic invaginations, myocytolysis in mitochondrial groupings, and inflammatory focus. In conclusion, the tongue of 6-month-old mdx mice had morphology, structure, and ultrastructure revealed, showing higher wear of filiform papillae indirect reflex from the muscular degeneration process.

Microstructural morphology of dermal and oral denticles of the sharpnose sevengill shark Heptranchias perlo (Elasmobranchii: Hexanchidae), a deep-water species.

The dermal denticles are among the unique morphological adaptations of sharks, which have been acquired throughout their long evolutionary process of more than 400 million years. Species-specific morphological characteristics of these structures has been applied specially as tools for functional and taxonomic (family-level) studies. Nevertheless, few studies have explored the diversity of denticle structure in different around the body and oral cavity. In the present study, we described the morphological differences observed in skin and oral cavity of sharpnose sevengill shark Heptranchias perlo, using scanning electron microscopy. Our findings demonstrate substantial variation in morphological structure of the denticles of the body and oral cavity. Overall, the dermal denticles observed across body surface were overlapped, tricuspid, with the central cuspid being more pronounced, pointed, and triangular in shape compared with lateral ones. Unlike, the denticles on the tip of the nose had a smooth crown, with rounded edges, being compact, and overlapped. The oral denticles were found in the ventral and dorsal region of the oral cavity. They also were tricuspid, but with differences in arrangement and ridges. These results suggest a strict functional relationship with the morphological characteristics observed. Such morphological diversity body-region-dependent highlights the need for comparative studies that include oral denticles, since this structure has an important functional role in sharks and can be found in fossil and recent records.

Microscopic aspects of electrosensory system on the partially euryhaline lesser guitarfish

ABSTRACT The electrosensory system on elasmobranchs consists of subcutaneous electroreceptor organs known as ampullae of Lorenzini. The present study investigated the ampullae of Lorenzini morphology of the lesser guitarfish Zapteryx brevirostris, using light microscopy and scanning electron microscopy. The pore number found in the ventral skin surface is much higher than that found in the dorsal portion, characteristic of species that inhabit the euphotic zone. Under light microscopy it was possible to observe that the wall canal consists of a single layer of squamous epithelial cells. The canal features distal expansion, where the ampullae are located with up to six alveoli. The sensory epithelium of ampullae is composed by cubic cells, with oval nucleus, restricted to the interior of the alveoli. With analysis the clusters under scanning electron microscopy, it was possible to observe the structure and the random arrangement of individual ampullae, canals and nerves. The distribution of dorsal and ventral pores and ampullae in Z. brevirostris resembled those of the same family. The number of alveoli per ampullae was similar to that found in euryhaline elasmobranchs species, suggesting that the morphological organization in Z. brevirostris is linked to its possible evolutionary transitory position among batoids.

Morphogenesis of the rhea (Rhea americana) respiratory system in different embryonic and foetal stages / Morfogênese do sistema respiratório da ema (Rhea americana) em diferentes estágios embrionários e fetais

The rhea (Rhea americana) is an important wild species that has been highlighted in national and international livestock. This research aims to analyse embryo-foetal development in different phases of the respiratory system of rheas. Twenty-three embryos and foetuses were euthanized, fixed and dissected. Fragments of the respiratory system, including the nasal cavity, larynx, trachea, syrinx, bronchi and lungs, were collected and processed for studies using light and scanning electron microscopy. The nasal cavity presented cubic epithelium in the early stages of development. The larynx exhibited typical respiratory epithelium between 27 and 31 days. The trachea showed early formation of hyaline cartilage after 15 days. Syrinx in the mucous membrane of 18-day foetuses consisted of ciliated epithelium in the bronchial region. The main bronchi had ciliated epithelium with goblet cells in the syringeal region. In the lung, the parabronchial stage presented numerous parabronchi between 15 and 21 days. This study allowed the identification of normal events that occur during the development of the rhea respiratory system, an important model that has not previously been described. The information generated here will be useful for the diagnosis of pathologies that affect this organic system, aimed at improving captive production systems.(AU)

A ema (Rhea americana) representa importante espécie silvestre que vem se destacando na pecuaria nacional e internacional. Esta pesquisa objetiva analisar o desenvolvimento embrionário-fetal, em diferentes fases, do sistema respiratório de emas. Vinte e três embriões e fetos foram eutanasiados, fixados e dissecados. Fragmentos do sistema respiratório: cavidade nasal, laringe, traqueia, siringe, brônquios e pulmões, foram coletados e processados para estudos por meio de microscopia de luz e microscopia eletrônica de varredura. A cavidade nasal apresentou, nas primeiras fases de desenvolvimento, epitélio estratificado cúbico. A laringe exibiu epitélio respiratório típico entre 27 e 31 dias. A traqueia aos 15 dias apresentou início de formação da cartilagem hialina. Na siringe a túnica mucosa de fetos de 18 dias e formada por epitélio estratificado ciliado na região bronquial. Os brônquios principais apresentavam epitélio estratificado ciliado com células caliciformes na região siringeal. No pulmão, o estágio parabronquial apresentou numerosos parabrônquios entre 15 a 21 dias. Este estudo permitiu a identificação de eventos normais que ocorrem durante o desenvolvimento do sistema respiratório de emas, importante modelo ainda não descrito. As informações geradas serão úteis para o diagnóstico de patologias que acometem este sistema orgânico, visando a melhoria dos sistemas de produção em cativeiro.(AU)

Microscopic aspects of electrosensory system on the partially euryhaline lesser guitarfish.

The electrosensory system on elasmobranchs consists of subcutaneous electroreceptor organs known as ampullae of Lorenzini. The present study investigated the ampullae of Lorenzini morphology of the lesser guitarfish Zapteryx brevirostris, using light microscopy and scanning electron microscopy. The pore number found in the ventral skin surface is much higher than that found in the dorsal portion, characteristic of species that inhabit the euphotic zone. Under light microscopy it was possible to observe that the wall canal consists of a single layer of squamous epithelial cells. The canal features distal expansion, where the ampullae are located with up to six alveoli. The sensory epithelium of ampullae is composed by cubic cells, with oval nucleus, restricted to the interior of the alveoli. With analysis the clusters under scanning electron microscopy, it was possible to observe the structure and the random arrangement of individual ampullae, canals and nerves. The distribution of dorsal and ventral pores and ampullae in Z. brevirostris resembled those of the same family. The number of alveoli per ampullae was similar to that found in euryhaline elasmobranchs species, suggesting that the morphological organization in Z. brevirostris is linked to its possible evolutionary transitory position among batoids.

A preliminary investigation into the morphology of oral papillae and denticles of blue sharks (Prionace glauca) with inferences about its functional significance across life stages.

Sensory organs in elasmobranchs (sharks, skates, rays) detect and respond to a different set of biotic and/or abiotic stimuli, through sight, smell, taste, hearing, mechanoreception and electroreception. Although gustation is crucial for survival and essential for growth, mobility, and maintenance of neural activity and the proper functioning of the immune system, comparatively little is known about this sensory system in elasmobranchs. Here we present a preliminary investigation into the structural and dimensional characteristics of the oral papillae and denticles found in the oropharyngeal cavity of the blue shark (Prionace glauca) during embryonic development through adulthood. Samples were obtained from the dorsal and ventral surface of the oropharyngeal cavity collected from embryos at different development stages as well as from adults. Our results suggest that development of papillae occurs early in ontogeny, before the formation of the oral denticles. The diameter of oral papillae gradually increases during development, starting from 25 µm in stage I embryos, to 110 µm in stage IV embryos and 272-300 µm in adults. Embryos exhibit papillae at early developmental stages, suggesting that these structures may be important during early in life. The highest density of papillae was observed in the maxillary and mandibular valve regions, possibly related to the ability to identify, capture and process prey. The oral denticles were observed only in the final embryonic stage as well as in adults. Accordingly, we suggest that oral denticles likely aid in ram ventilation (through reducing the hydrodynamic drag), to protect papillae from injury during prey consumption and assist in the retention and consumption of prey (through adhesion), since these processes are only necessary after birth.

Ultrastructural analysis of the spermatogenesis in the guinea pig (Cavia porcellus) / Análise ultraestrutural da espermatogênese do porquinho-da-Índia (Cavia porcellus)

Understanding of the reproductive function is essential for both, the establishment of appropriate management systems, and for the use of new species as animal models. In this study, we used light and electron microscopy to characterize the sexual development stages of the guinea pig (Cavia porcellus) in specimens of 30, 45 and 90 days of age. We observed the differentiation of spermatocytes only through transmission electron microscopy in the leptotene, zygotene and pachytene phases of meiosis, in 30-day-old animals. During puberty, there was differentiation of the germinative epithelium and formation of the acrosome. Spermatozoa, however, were not detected. Thus, we could infer that puberty happens after 45 days of age. Sexual maturity was evident in 90-day-old specimens. Our results showed that changes in the testicular germinative epithelium during the postnatal sexual development in guinea pig led to morphological changes, including the ones related to the development of Leydig and Sertoli cells, which are directly related to puberty. In this work, we provide new morphological subsidies for a better understanding of reproductive parameters of this species, enabling its use as an animal model in the field of the reproductive biology.(AU)

A função reprodutiva é um fator de vital compreensão tanto para o estabelecimento de sistemas apropriados de manejo, quanto para o uso de novas espécies como modelos animais. Neste estudo através da microscopia de luz e eletrônica caracterizou-se a fase de desenvolvimento sexual do porquinho-da-Índia (Cavia porcellus) em espécimes de 30, 45 e 90 dias de desenvolvimento. Nos animais de 30 dias, a diferenciação dos espermatócitos foi visualizada somente na microscopia eletrônica de transmissão em leptóteno, zigóteno e paquíteno. Durante a puberdade, houve diferenciação do epitélio germinativo, formação do acrossoma, porém não foram evidenciados espermatozóides, assim, infere-se que a puberdade acontece a partir dos 45 dias de idade. A maturidade sexual foi evidente aos 90 dias de idade. Nossos resultados mostraram que ao longo do desenvolvimento sexual pós-natal do porquinho-da-Índia, mudanças no epitélio germinativo testicular levam há alterações morfológicas, inclusive com relação ao desenvolvimento das células de Sertoli e de Leydig, as quais estão diretamente relacionadas com a puberdade. Assim, novos subsídios morfológicos são fornecidos para um melhor entendimento dos parâmetros reprodutivos desta espécie, a fim de viabilizar sua utilização como modelo animal no campo da biologia reprodutiva.(AU)

Morphological analysis of the oviduct, oviducal gland and isthmus of the blue shark Prionace glauca (Linnaeus, 1758) (Elasmobranchii: Carcharhiniformes)

Oviducal gland present in elasmobranchs is correlated to the organism's reproductive strategy, and its functions are to produce mucus, to form the egg's tertiary envelope and to store sperm. The gland contains four zones: club, papillary,baffle and terminal. The structures of the oviduct, oviducal gland and isthmus of blue shark Prionace glauca were described using macroscopic, light microscopy and scanning electron microscopy techniques. The epithelium of the oviduct and isthmus is folded and is a simple, columnar, ciliated lining epithelium with glandular cells. In the oviducal gland, the lining tissues in the four zones are similar to the oviduct and isthmus lining. The terminal zone shows the presence of sperm in the lumen of the secretory tubules, which remains stored even in the absence of recent copulation. Here, these organs were studied and their connections in an attempt to elucidate the mechanisms of reproduction in the blue shark, showing the three-dimensional aspects, thus adding morphological information important for the understanding of the structure and functioning of these organs of fundamental importance in the life of the majority of elasmobranchs.

A glândula oviducal presente nos elasmobrânquios está correlacionada com a estratégia reprodutiva, cuja função é de produzir o muco, formar o envelope terciário do ovo e armazenar espermatozóides. A glândula contém quatro zonas: club, papilar, baffle e terminal. Foi descrita a estrutura do oviduto, glândula oviducal e istmo do tubarão-azul, Prionace glauca , pelas técnicas macroscópica, microscópica de luz e eletrônica de varredura. Foi observado que no oviduto e istmo o epitélio é pregueado de revestimento simples, colunar, ciliado com células glandulares. Na glândula oviducal o tecido de revestimento é semelhante ao oviduto e istmo nas quatro zonas. Na zona terminal observou-se a presença de espermatozoides no lúmen dos túbulos secretores que permanecem estocados mesmo na ausência de copula recente. Aqui, estudaram-se esses órgãos e suas conexões na tentativa de elucidar os mecanismos da reprodução no tubarão azul, apresentando os aspectos tridimensionais, desta forma agregando informações morfológicas importantes para o entendimento da estrutura e funcionamento desses órgãos de fundamental importância na vida da maioria dos elasmobrânquios.

Ultraestrutura comparativa da língua do sagui-de-tufo-preto (Callithrix penicillata) e do bugio-preto (Alouatta caraya) em diferentes faixas etárias / Comparative ultrastructure of the tongue of black-tufted-ear-marmoset (Callithrix penicillata) and black-howler-monkey (Alouatta caraya) in different age groups

Para a análise e descrição comparativa da morfologia da língua e de suas papilas, bem como a distribuição destas, foram utilizados três animais de faixas etárias distintas (filhote, jovem, adulto), de duas espécies de primatas, Callithrix penicillata (sagui-de-tufo-preto) e Alouatta caraya (bugio-preto), ambas sendo observadas através da microscopia eletrônica de varredura. Os animais da espécie Callithrix penicillata eram procedentes de um criadouro comercial de animais selvagens em Atibaia/SP e vieram a óbito por causas naturais, e os da espécie Alouatta caraya eram provenientes do acervo do Laboratório de Anatomia Macroscópica da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo (FMVZ-USP). Com o material analisado e a técnica empregada pôde-se constatar que as línguas dos saguis-de-tufo-preto e dos bugios-pretos analisadas apresentavam papilas linguais similares às descritas para outras espécies de primatas. As línguas possuíam diferenças morfológicas, principalmente para alguns tipos de papilas linguais, em detrimento do avançar da idade dos animais. De forma geral, foram observados quatro tipos de papilas linguais, sendo estas: filiforme, fungiforme, valada e folhada. As línguas dos bugio-pretos apresentavam características de animais com dietas herbívoras. Possuindo desta forma papilas filiformes com formatos variados (coroa, lança, multifilamentar), de acordo com as regiões da língua, e também robustas papilas, principalmente na proeminência lingual. Já as línguas dos saguis-de-tufo-preto possuíam características de animais onívoros. Com papilas filiformes em formato de coroa, grandes quantidades de papilas fungiformes e desenvolvidas papilas folhadas. Observou-se que as modificações das papilas linguais que ocorrem nos animais após o nascimento apresentaram correlação com a mudança nos tipos de alimentos consumidos. Sendo presumível então concluir que as dietas possuem relação direta para com as alterações morfológicas e estruturais das papilas linguais nos animais ora aqui analisados.

For the comparative analysis and description of the morphology of the tongue and taste buds as well as the distribution of these we have used three animals of different age groups (puppy, young adult), of two species of primate, Callithrix penicillata (black-tufted-ear-marmoset) and Alouatta caraya (black-howler-monkey), both being observed by scanning electron microscopy. The animals of the species Callithrix penicillata were from a commercial breeding of wild animals in Atibaia-SP and they had died of natural causes, and the specie Alouatta caraya were from the collection of Macroscopic Anatomy Laboratory of Faculty of Veterinary Medicine and Animal Science University of São Paulo (FMVZ-USP). With the material analyzed and the technique employed could be seen that the tongue of black-tufted-ear-marmosets and black-howler-monkeys had examined lingual papillae similar to those described for other primate species. The tongue has morphological differences, especially for some types of lingual papillae, at the expense of advancing age of the animals. Overall, we found four types of lingual papillae, which are: filiform, fungiform, vallate and litter. The tongues of the black-howler-monkey had characteristics of animals with herbivorous diets. Possessing this way with the filiform papillae varied formats (crown, spear, multifilament), according to the regions of the tongue, and also robust taste, especially in the lingual prominence. Since the tongues of the black-tufted-ear-marmosets owned features of omnivorous animals. With filiform-shaped crown, large amounts of fungiform papillae and foliate papillae developed. It was observed that the changes of tongue papillae that occur in animals after birth correlated with the change in the types of foods consumed. Being suspected then conclude that diets have direct relation to the morphological and structural of the lingual papillae now here in the animals analyzed.

Development of bovine embryos derived from reproductive techniques.

Assisted reproduction techniques have improved agricultural breeding in the bovine. However, important development steps may differ from the situation in vivo and there is a high mortality rate during the first trimester of gestation. To better understand these events, we investigated the development of embryos and fetal membranes following fixed-time AI (FTAI), IVF and nuclear transfer (NT). The onset of yolk-sac development was not normal in cloned embryos. Later steps differed from conditions in vivo in all three groups; the yolk-sac was yellowish and juxtaposed with the amniotic membrane. Vascularisation of the chorioallantoic membrane was relatively late and low in NT gestations, but normal in the others. The overall development of the embryos was normal, as indicated by morphology and regression analysis of growth rate. However, NT conceptuses were significantly smaller, with the livers in some embryos occupying the abdominal cavity and others exhibiting heart abnormalities. In conclusion, the yolk-sac and the cardiovascular system seem to be vulnerable to morphogenetic alterations. Future studies will focus on gene expression and early vascularisation processes to investigate whether these changes may be responsible for the high incidence of intrauterine mortality, especially in clones.

Patterns of cell proliferation and apoptosis by topographic region in normal Bos taurus vs. Bos indicus crossbreeds bovine placentae during pregnancy.

BACKGROUND: Placental and fetal growth requires high rates of cellular turnover and differentiation, which contributes to conceptus development. The trophoblast has unique properties and a wide range of metabolic, endocrine and angiogenic functions, but the proliferative profile of the bovine placenta characterized by flow cytometry analysis and its role in fetal development are currently uncharacterized. Complete understanding of placental apoptotic and proliferative rates may be relevant to development, especially if related to the pathogenesis of pregnancy losses and placental abnormalities. METHODS: In this study, the proliferation activity and apoptosis in different regions of normal bovine placenta (central and boundary regions of placentomes, placentomal fusion, microplacentomes, and interplacentomal regions), from distinct gestation periods (Days 70 to 290 of pregnancy), were analyzed by flow cytometry. RESULTS: Our results indicated that microplacentomes presented a lower number of apoptotic cells throughout pregnancy, with a higher proliferative activity by the end of gestation, suggesting that such structures do not contribute significantly to normal of placental functions and conceptus development during pregnancy. The placentome edges revealed a higher number of apoptotic cells from Day 170 on, which suggests that placentome detachment may well initiate in this region. CONCLUSION: Variations involving proliferation and apoptotic rates may influence placental maturation and detachment, compromising placental functions and leading to fetal stress, abnormalities in development and abortion, as frequently seen in bovine pregnancies from in vitro fertilization and cloning procedures. Our findings describing the pattern of cell proliferation and apoptosis in normal bovine pregnancies may be useful for unraveling some of the developmental deviations seen in nature and after in vitro embryo manipulations.