Mizue Imoto Egami

Cytochemical aspects of the peripheral blood cells of Oreochromis (Tilapia) niloticus. (Linnaeus, 1758) (Cichlidae, Teleostei): part II

Morphologicaly, seven types of cells were identified in the blood of Oreochromis niloticus: erythrocytes, thrombocytes, neutrophils, eosinophils, basophils, lymphocytes and monocytes. Glycogen was present not only in the cytoplasm of neutrophils and thrombocytes but also in some lymphocytes and monocytes. The positive reaction for myeloperoxidase and Sudan black was observed in neutrophils and eosinophils. The bromphenol blue method was strongly positive for erythrocytes and eosinophils.

Identification and Characterization of a Sialidase Released by the Salivary Gland of the Hematophagous Insect Triatoma infestans

Sialidases (EC 3.2.1.18) are commonly found in viruses, bacteria, fungi, protozoa, and vertebrates, but not in invertebrates. We have previously reported the presence of a new sialidase activity in the gut of exclusively hematophagous insects of the Triatoma genus, which transmit Chagas’ disease (Amino, R., Acosta, A., Morita, O. M., Chioccola, V. L. P., and Schenkman, S. (1995) Glycobiology 5, 625–631). Here we show that this sialidase is present in the salivary gland ofTriatoma infestans, and it is released with the saliva during the insect bite. The sialidase was purified to homogeneity (>5000 times) to a specific activity of more than 20 units/mg. It elutes from a gel filtration column with a volume corresponding to the size of 33 kDa, and it migrates as a single 26-kDa band in SDS-polyacrylamide gel electrophoresis, which is unusually smaller when compared with other known sialidases. T. infestanssialidase hydrolyzes preferentially α2→3-linked sialic acids at pH 4–8, with maximal activity between pH 5.5 and 6.5, which is compatible with the optimal pH of secreted sialidases. The sialidase is competitively inhibited by 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid (K i = 0.075 mm) and differently from many sialidases, with exception of Salmonella typhimuriumsialidase, it is inhibited competitively by HEPES (K i = 15 mm). The fact that T. infestans sialidase is released with the saliva and can hydrolyze sialyl-LewisX blood groups, which are the ligands for selectins, suggests that it might have a role in the blood feeding.

Cytochemical, immunocytochemical and ultrastructural observations on leukocytes and thrombocytes of fat snook (Centropomus parallelus)

The cytochemical, immunocytochemical and ultrastructural characteristics of leukocytes and thrombocytes in the peripheral blood of the fat snook (Centropomus paralellus) - a fish occurring in Brazil - were investigated. The cytochemical methods were performed to demonstrate four enzymatic reactions - o-toluidine-hydrogen peroxide, naphtol AS-MX phosphate, naphtol AS-BI phosphate and alpha-naphtil acetate to detect myeloperoxidase (MPO), alkaline phosphatase (ALP), acid phosphatase (ACP) and non-specific esterase (α-NAE), respectively - and two non-enzymatic ones - Periodic-Acid Schiff (PAS) and Sudan black B (SBB) to detect the occurrence of glycogen and phospholipids, respectively. Immunocytochemical method utilizing polyclonal rabbit antibody against mammal metalloproteinases (MMPs) 2 and 9 were done. Standard method for Electron Microscopy (EM) was applied for the ultrastructural study. The cytochemical reactions were positive in neutrophils for MPO, ACP, α-NAE, glycogen and phospholipids; in lymphocytes for ACP and α-NAE; in monocytes for ACP and α-NAE and in thrombocytes for ACP, α-NAE and glycogen. Only neutrophils were positive for MMPs 2 and 9, and none of the cells studied were positive for ALP. Ultrastructurally: 1) neutrophil showed a spherical shape with a spherical, indented or lobulated euchromatic nucleus, and cytoplasm containing granules of varied sizes and mitochondria of varied shapes and sizes. The nucleus/cytoplasm relation and the size of granules suggest neutrophil maturation in peripheral blood; 2) lymphocytes showed partially heterochromatic nucleus and minimal cytoplasm; 3) monocytes had long cytoplasmic projections, an indented nucleus, evident nucleolus and cytoplasm with granules of varied sizes and vacuoles; 4) thrombocytes were predominantly elliptical or roughly spherical in shape, had a partially heterochromatic nucleus and cytoplasm containing electron-dense granules, intricate canalicular system and vacuoles occasionally holding phagocytic material.

ASPECTOS MORFOLÓGICOS Y CITOQUÍMICOS DE LAS CÉLULAS SANGUÍNEAS DE Salminus maxillosus Valenciennes, 1840 (CHARACIFORMES, CHARACIDAE)

Las caracteristicas morfologicas de sangre periferica del pez Salminus maxillosus fueron descritas usando microscopia de luz. Para demostrar el comportamiento citoquimico de las celulas sanguineas del pez en estudio, fueron aplicados metodos especificos para la deteccion de cuatro tipos basicos de componentes macromoleculares, de naturaleza quimica diferente: carbohidratos, lipidos, proteinas enzimaticas y estructurales. Fueron observados 6 tipos de celulas: eritrocitos, trombocitos, monocitos, linfocitos, neutrofilos de los tipos I y II y eosinofilos. Los trombocitos y los neutrofilos de los tipos I y II presentaron glucogeno en el citoplasma. Los neutrofilos del tipo II presentan granulos sudanofilos y mieloperoxidasa. Ademas presentan, reaccion difusa intensa para proteinas en el citoplasma. Los eosinofilos muestran reaccion intensa en sus granulos.

Comparative study on hematological parameters of farmed matrinxã, Brycon amazonicus Spix and Agassiz, 1829 (Characidae: Bryconinae) with others Bryconinae species

The aim of this study was not only to determine the red blood cells parameters, thrombocyte and leukocyte counts in farmed Brycon amazonicus (matrinxa), to compare these parameters among Bryconinae species from literature, and also to investigate the presence of special granulocytic cells in these fish. The results of the blood cells parameters here established for farmed B. amazonicus, a species of great economic importance in Brazilian aquaculture, could help a better understanding of the blood features in natural populations of this Amazon species. Blood parameters varied between Bryconinae species investigated, mainly the red blood cell counts, hemoglobin, hematocrit and mean corpuscular volume (MCV). The presence of the blood granulocytes, neutrophils and heterophils in matrinxa suggest that both leukocytes can be a characteristic for Bryconinae family. Furthermore, it indicates that the existence of special granulocytic cells in the blood of Bryconinae species from literature is an artifact, and this was herein discussed.

Aspectos morfológicos e citoquímicos dos glóbulos sangüíneos de Caiman crocodilus yacare (Daudin, 1802) (Reptilia, Crocodilia)

Samples of peripheral blood were collected from 10 healthy Caiman crocodilus yacare and stained with Leishman, for morphological analysis, or submitted to the following cytochemical methods: PAS, Sudan black B, o-toluidine and mercury bromophenol blue. Seven types of cells were identified: erythrocytes, thrombocytes, heterophils, eosinophils, basophils, lymphocytes and azurofilic monocytes. Erythrocytes were elliptical and positive for bromophenol blue. Some mitotic figures were noted. Spindle-shaped thrombocytes showed large nucleus with striking indentation. The cytoplasm was attenuated at one or both poles into knob-like projections containing glycogen. Heterophils revealed round eccentric nuclei and cytoplasm containing glycogen and many polymorphic granules. These granules showed partial positive reaction for Sudan and activity for myeloperoxidase. Eosinophils showed eccentrically located lenticular-shaped nuclei and numerous round granules stained positive with myeloperoxidase and Sudan. Basophils revealed a round central nuclei and cytoplasm with few basophilic round granules. Lymphocytes appeared as polygonal or irregular cells with thin cytoplasm forming bleb-like protrusions. Azurophilic monocytes were round with eccentrically located nuclei and basophilic cytoplasm containing numerous azurophilic granules.

Rudimentary Eyes of Squamate Fossorial Reptiles (Amphisbaenia and Serpentes)

The rudimentary characteristic of the eyes of fossorial animals raises some questions regarding its evolution and functionality. Would these eyes result from atrophy or from stagnated development? How would its visual function work? Anatomical investigations of these organs are the fundamental preamble to answer those questions, which are still little explored by the literature. In this article we have studied anatomical aspects of the eyes of three species of fossorial reptiles, within the suborder Amphisbaena (Amphisbaena alba, Amphisbaena mertensi, Leposternon infraorbitale), as well as a species within the ophidian suborder (Typhlops brongersmianus). The minuscule eyes (1–2 mm diameter) were visualized through a scale, a translucent area which corresponds to the spectacle. This spectacle is a thinner and transparent scale, covering a conjunctival sac. The retrobulbar space was filled with the harderian gland. The eyes of Typhlops presented an oval shape, whereas Amphisbaena specimens presented cup‐shaped eyes. In Amphisbaenian sclera is comprised of cartilage, while the thin sclera of Typhlops consists of connective tissue and striated muscle fibers. The retina presented all the typical layers found in vertebrates, regardless the species. The characteristics involved in the fossil adaptation of these species include: reduced size of the eyeball, rudimentary cornea, absence of the anterior chamber, presence of a complex iris‐ciliary body, and lens with amorphous nucleate cells. The analysis of the eye morphology of these animals suggests that there might be a specific function concerning light perception. Anat Rec, 2010.

Ativação da proteína TGFbetaI latente em pulmão irradiado in vivo

PURPOSE: assess the latent and active TGFb1 localization in the lung, whether or not radiation induces latent TGFb1 activation, and the distribution of collagen fibers in the irradiated lung. METHODS: Thirty two C57BL mice were randomly assigned in two groups: GI (non irradiated animals) and GII (irradiated animals). The mice from GII received a single whole ¾ body radiation dose of 7Gy, using a 60Co source at a dose rate of 0.97 Gy/min. They were sacrificed by cervical dislocation at 1, 14, 30 and 90 days after radiation. RESULTS: The irradiated lungs showed: 1) vascular congestion and thickness of the alveolar septa 30 days and more intense 90 days after irradiation; 2) significant increase of collagen deposition in all time periods after irradiation; 3) weak latent TGFb1 activation 1 day and strong activation 14 days after irradiation in the bronchi and alveoli. Our results suggest that some bronchial and alveolar cells may have a role in the complex process of radiation-induced lung fibrosis acting as cellular sources of active TGFb.

Phosphorylation and cytoplasmic localization of MAPK p38 during apoptosis signaling in bone marrow granulocytes of mice irradiated in vivo and the role of amifostine in reducing these effects

We studied p38 phosphorylation and its intracellular localization during p53 and Puma (a p53 upregulated modulator of apoptosis) apoptotic signaling pathway in bone marrow granulocytes in mice irradiated in vivo and the role of the radioprotector amifostine in ameliorating these responses. Sixty-four C57BL mice were randomly assigned in two non-irradiated (Ami-/rad- and Ami+/rad-) and two irradiated (Ami-/rad+ and Ami+/rad+) groups. Animals received 400mg/kg of amifostine i.p. 30 min prior to a single whole body radiation dose of 7Gy. The experiments were performed using immunohistochemistry for caspase-3, cleaved caspase-3, p53, p-p53 (Ser 15), Puma, p38 and p-p38 (Thr 180/Tyr 182) protein expression. In addition transmission electron microscopy was used for ultrastructural characterization of apoptosis. Data showed that: (i) amifostine significantly reduced the number of apoptotic cells, (ii) p-p53 and Puma proteins were strongly immunostained in granulocytes after irradiation (Ami-/rad+), (iii) amifostine decreased the immunostaining of the proteins (Ami+/rad+), (iv) p38 was immunolocalized in physiological conditions in the nucleus and cytoplasm of granulocytes and neither radiation nor amifostine changed the protein immunostaining or its subcellular distribution, but influenced its activation, (v) radiation-induced p38 phosphorylation and its cytoplasmic accumulation during apoptosis signaling in granulocytes after whole body high radiation dose and amifostine markedly reduced these effects.

ASPECTOS ULTRAESTRUTURALES DE TROMBOCITOS, EOSINOFILOS Y HETEROFILOS DE Caiman crocodilus yacare (Daudin, 1802) (Reptilia, Crocodilia)

Fueron utilizados 10 ejemplares de Caiman crocodilus yacare provenientes del municipio de Caceres (Estado de Mato Grosso do Sul, Brasil), a cada uno de los cuales les fueron extraidos 5ml de sangre periferica. Las muestras obtenidas fueron utilizadas para analisis ultraestructurales, referentes a trombocitos, eosinofilos y heterofilos. Los trombocitos son de forma eliptica, poseen citoplasma abundante solo en los polos y su nucleo presenta surcos profundos o hendiduras acentuadas, mostrando algunos poros en su envoltura. Su citoplasma contiene abundantes tubulos y vesiculas electrolucidas del sistema canalicular. Son observados vacuolas con cuerpos densos y microtubulos. Los heterofilos poseen formato aproximadamente esferico y nucleo excentrico o periferico. Su citoplasma esta repleto de granulos polimorficos de formas de huso, en baqueta, oval o esferica, con superficie, en general, irregular. Los eosinofilos son esfericos y de nucleo lenticular excentrico o periferico. El citoplasma contiene granulos esfericos u ovales. Cabe destacar la presencia de granulos citoplasmaticos voluminosos con diferencias en la electrodensidad, de superficie uniforme, pudiendo contener una estructura interna heterogenea. En heterofilos y eosinofilos, el nucleo presenta heterocromatina, eucromatina, nucleolo y la envoltura nuclear es porosa