Bianca Maria Uva

Clinorotation-induced weightlessness influences the cytoskeleton of glial cells in culture

During and after spaceflight astronauts experience neurophysiological alterations. To investigate if the impairment observed might be traced back to cytomorphology, we undertook a ground based research using a random positioning machine (clinostat) as a simulation method for microgravity. The outcome of the study was represented by cytoskeletal changes occurring in cultured glial cells (C(6) line) after 15 min, 30 min, 1 h, 20 h and 32 h under simulated microgravity. Glia is fundamental for brain function and it is essential for the normal health of the entire nervous system. Our data showed that after 30 min under simulated microgravity the cytoskeleton was damaged: microfilaments (F-actin) and intermediate filaments (Vimentin, Glial Fibrillary Acidic Proteins GFAP) were highly disorganised, microtubules (alpha-tubulin) lost their radial array, the overall cellular shape was deteriorated, and the nuclei showed altered chromatin condensations and DNA fragmentation. This feature got less dramatic after 20 h of simulated microgravity when glial cells appeared to reorganise their cytoskeleton and mitotic figures were present. The research was carried out by immunohistochemistry using antibodies to alpha-tubulin, vimentin and GFAP, and cytochemical labelling of F-actin (Phalloidin-TRIC). The nuclei were stained with propidium iodide or 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). The cells were observed at the conventional and/or the confocal laser scanning microscope. Samples were also observed at the scanning electron microscope (SEM). Our data showed that in weightlessness alterations occur already visible at the scale of the single cell; if this may lead to the neurophysiological problems observed in flight is yet to be established.

Immunoreactive localization of vasoactive hormones (atrial natriuretic peptide and endothelin) in the heart of Protopterus annectens, an African lungfish

Abstract.The present study demonstrated, by immunohistochemistry and Western blotting, the presence of immunoreacting atrial natriuretic peptide (ANP) and endothelin in the heart of Protopterus annectens by both light and electron microscopy. The distribution of ANP granules was investigated. ANP granules were localised in myocytes from the atrium, ventricle and conus arteriosus; endothelin-1 (ET-1) was demonstrated in subendocardial myocytes of the atrium and the conus. No ET-1 immunoreactivity was observed in the ventricle wall. At the light-microscopical level, ET-1 appeared to occur in the endocardium, but at the electron-microscopical level no immunogold labelling was seen on the granules of the endocardial cells. It is suggested that ET-1 is produced and stored in the subendocardial cells and released into the subendocardial space to reach the ANP-producing myocytes and the endothelial cells.

Immunoreactive atrial natriuretic-like peptide in antarctic teleosts

Abstract 1. 1. The presence of an atrial natriuretic-like peptide (ANP) has been investigated in the Antarctic fish Chionodraco hamatus (Channichthydae) and Pagothenia bernacchii (Notothenidae). 2. 2. By radioimmunoassay an ANP-like peptide has been detected in plasma and heart homogenates of both white blooded ( Chionodraco ) and red blooded ( Pagothenia ) species. 3. 3. By immunohistochemistry atrial nutriuretic factor (ANF) immunoreactive cardiocytes have been demonstrated in the atria of Chionodraco and Pagothenia using Ab to rat 99–126 ANF. Ventricular immunoreactivity is absent in both species. 4. 4. ANP binding sites have been visualized by autoradiography in the kidney, gills and heart of both Pagothenia and Chionodraco . 5. 5. Our data indicate that ANP concentration is greater in the white blooded than in the red blooded fish.

Renin-angiotensin system in antarctic fishes

Abstract 1. 1. The presence of a renin-angiotensin-like system has been investigated in the Antarctic fishes Chionodraco hamatus (Fam. Channichthydae) and Pagothenia (Trematomus) bernacchii (Fam. Notothenidae). 2. 2. A renin-like activity is present in plasma and kidney of both the white blooded (Chionodraco) and the red blooded (Pagothenia) species. 3. 3. An angiotensin converting enzyme-like activity has been demonstrated in plasma, gills and kidneys of both species. The activity is inhibited by high temperature. 4. 4. From our data a renin-angiotensin-like system is present in the Antarctic fishes studied but the cascade of enzymes is active only at low temperatures.

Ion transport systems in the kidney and urinary bladder of two Antarctic teleosts, Chionodraco hamatus and Trematomus bernacchii

Abstract. Na+-K+-Cl- cotransport and Na+/K+ATPase were studied by immunohistochemistry in the kidney and urinary bladder of Trematomus bernacchii and Chionodraco hamatus. The activity was correlated to the density of mitochondria. The first segment of the renal proximal tubule was more active than the second one. In T. bernacchii and the temperate marine teleost Pagellus bogaraveo, the immunoreactivity for the antibody to cotransporters and to the α-subunit of the sodium pump was stronger than in the icefish. This difference indicates in the kidney of the icefish, a weaker secretory activity, a consequent lower osmolarity in the lumen and lower water loss, which correlates well with the need for a greater blood volume in the icefish. The epithelium of the urinary bladder in T. bernacchii, where intense immunostaining was observed, was composed of columnar cells. In C. hamatus the columnar cells, where the immunostaining was weaker, lined only a portion of the urinary bladder, the other region being composed of cuboidal cells.

Plasma aldosterone levels in the female tortoise Testudo hermanni Gmelin in different experimental conditions.

Abstract Plasma aldosterone concentration has been measured by radioimmunoassay in a tortoise species Testudo hermanni Gmelin. Control levels were very low, 8.26 ± 0.2 ng/100 ml, if compared with the determination carried out in other reptilian species with the same RIA method. A sodium-loaded diet for a prolonged period (30 days) depressed aldosterone levels but administration of a diuretic produced a significant elevation. Plasma aldosterone, sodium, and potassium concentrations were significantly correlated. Administration of a mammalian inhibitor of aldosterone (spironolactone) was not followed by a significant plasma aldosterone or electrolyte changes. These data are consistent in supporting the hypothesis that aldosterone is involved in hydromineral regulation, at least in terrestrial chelonians, with a direct action on the renal proximal tubule.

Occurrence of 7-dehydrocholesterol in the uropygial gland of domestic fowls

Histochemical studies on the uropygial gland of domestic fowls have shown the presence of sterols (among which cholesterol and its esters) in the lipidic fraction of the gland secret. beta-Glucuronidase activity beside A5 3beta- and 17beta-hydroxysteroid dehydrogenase activities suggests that uropygial gland might be involved in sterols metabolism. By thin layer chromatography cholesterol and 7-dehydrocholesterol can be separated from the uropygial extracts and these compounds can be identified in gas liquid chromatography.

GnRH immunodetection in the brain of the holocephalan fish Chimaera monstrosa L.: Correlation to oocyte maturation

Chimera monstrosa (rabbit fish) like other holocephalans is a rare, delicate deep sea fish. Owing to the difficulty of sampling individuals in good shape, there is a paucity of information available on the morphology and physiology of this species especially concerning reproduction. In holocephalans, a hypothalamus-pituitary-gonadal axis has been postulated and a GnRH molecule identical to cGnRH II has been identified. The aim of the present study was to correlate the presence of steroidogenic enzymes in the ovarian follicles with the presence of GnRH in the hypothalamus. Estrogens, the steroids that trigger the accumulation of yolk (vitellogenesis) in the oocytes are synthesized by the somatic cells of the follicle in the vitellogenic stages via a cascade of steroid dehydrogenases involving 3 beta-hydroxysteroid-dehydrogenase (3 beta-HSD; in the inner thecal layer) and aromatase cytochrome (P450; granulosa layer). Our results showed that 3 beta-HSD is present concomitant with the presence of cGnRH II in the preoptic area and in the ventral hypothalamus. Another form of immunoreactive GnRH, mGnRH is also present in the brain of C. monstrosa. It is localized in the ventral telencephalon and in the midbrain caudal diencephalon (boundary between ventral thalamus and tegmentum of the mesencephalon). This form of GnRH is probably correlated with sexual behaviour.

Effect of hypophysectomy on plasma electrolytes and adrenal mineralocorticoid secretion in the terrestrial chelonian Testudo hermanni

Na, K, ACTH, corticosterone and aldosterone plasma levels were studied in Testudo hermanni after hypophysectomy and dexamethasone administration. Hypophysectomy as well as dexamethasone treatment caused a deep decrease in the plasma levels of electrolytes, corticosterone and aldosterone when compared to sham-operated or normal controls. ACTH plasma level was markedly lower in hypophysectomized or dexamethasone-treated tortoises when compared to the controls but never reached undetectable levels. Administration of ACTH significantly increased plasma electrolytes and hormone levels in hypophysectomized and dexamethasone-treated tortoises. The present data seem to indicate that the pituitary plays a significant role on electrolyte homeostasis and on the regulation of plasma mineralocorticoids level in terrestrial chelonians.

Mitochondria-rich cells in Antarctic fish gills

Abstract We used transmission and scanning electron microscopy and an antibody to the inner mitochondrial membrane to study the presence and localisation of mitochondria-rich cells in the gills of two Antarctic fishes, Chionodraco hamatus (Channichthyidae) and Trematomus bernacchii (Nototheniidae). The general morphology of the gills in the two species was slightly different: in T. bernacchii the filament and secondary lamellae were short and thicker, and mitochondria-rich cells were less numerous than in C. hamatus. In the two species the mitochondria-rich cells, distributed in the secondary and primary epithelium, were of the same morphological type, similar to the α-chloride cells of temperate seawater teleosts. The study was carried out to compare the mitochondria-rich cells of Antarctic fishes to the chloride cells of temperate marine teleosts. Immunolocalisation, using a specific antibody to the α-subunit of Na+/K+-ATPase, was observed in numerous epithelial cells in the interlamellar epithelium and on the secondary lamellae, suggesting an active sodium chloride secretion.