Microtubule-associated protein 2 (MAP2)-immunoreactive neurons in the retina of Bufo marinus: colocalisation with tyrosine hydroxylase and serotonin in amacrine cells

Summary Neuron populations in the retina of the toad, Bufo marinus, were labelled with a monoclonal antibody raised against microtubule-associated protein 2 (MAP2). A subpopulation of cones, probably corresponding to the blue-sensitive small single cones, large diameter amacrine cells in the most proximal row of the inner nuclear layer and some large ganglion cells in the ganglion cell layer were labelled. Double labelling experiments were carried out to establish the colocalisation of MAP2 with known putative transmitter substances of the anuran amacrine cells. MAP2 was colocalised in a subpopulation of serotonin-immunoreactive and in all tyrosine hydroxylase-immunoreactive amacrine cells. The results indicate, that the MAP2 content in the neurons of the anuran retina can be correlated with other well-defined neurochemical and/or physiological properties.On leave from Department of Zoology, Attlia József University, Szeged, Hungary     

Appearance of an intra-acrosomal antigen during the terminal step of spermiogenesis in the rat

Summary In a survey of sperm antigens in the rat, a new intra-acrosomal antigen was found using a monoclonal antibody MC41 raised against rat epididymal spermatozoa. The MC41 was immunoglobulin G₁ and recognized spermatozoa from rat, mouse and hamster. Indirect immunofluorescence with MC41 specifically stained the crescent region of the anterior acrosome of the sperm head. Immuno-gold electron microscopy demonstrated that the antigen was localized within the acrosomal matrix. Immunoblot study showed that MC41 recognized a band of approximately 165000 dalton in the extract of rat sperm from the cauda epididymidis. Immunohistochemistry with MC41 demonstrated that the antigen was first detected in approximately step-2 spermatids, and distributed over the entire cytoplasmic region of spermatids from step 2 to early step 19. The head region became strongly stained in late step-19 spermatids and then in mature spermatozoa. Distinct immunostaining was not found in the developing acrosome of spermatids throughout spermiogenesis. These results suggest that the MC41 antigen is a unique intra-acrosomal antigen which is accumulated into the acrosome during the terminal step of spermiogenesis.     

Design of liposome to improve encapsulation efficiency of gelonin and its effect on immunoreactivity and ribosome inactivating property

Gelonin, purified from the seeds of Gelonium multiflorum, using cation-exchange and gel-filtration chromatography was characterised for its purity, homogeneity and molecular weight by reverse-phase HPLC (RP-HPLC) and SDS-PAGE analysis. The HPLC purified gelonin was used for entrapment studies in the liposomes. Liposomes were prepared by reverse phase evaporation (REV) technique using three different types of lipid composition in the same molar ratio. The method resulted in 75–80% entrapment efficiency of gelonin in the liposomes. Entrapped and unentrapped gelonin was characterized for physico-chemical, immunochemical and biological properties. The immunoreactivity of entrapped gelonin was fully preserved but the ribosome-inactivating property was slightly inhibited. The method involved mild conditions, highly reproducible and the liposomes produced appeared to be stable for several months. It has important implications in the development of cell type specific cytotoxic agents where a chemical cross-linking is involved which significantly inhibits both immunoreactivity and ribosome-inactivating ability of the toxin.     

Phosphorylation of atrial natriuretic factor R1 receptor by serine/threonine protein kinases: evidences for receptor regulation

The 130 kDa atrial natriuretic factor receptor (ANF-R₁) purified from bovine adrenal zona glomerulosa is phosphorylated in vitro by serine/threonine protein kinases such as cAMP-, cGMP-dependent and protein kinase C. This phosphorylation is independent of the presence of ANF (99–126) and there is no detectable intrinsic kinase activity associated with the ANF-R₁ receptor or with its activated form. In bovine adrenal zona glomerulosa cells, TPA (phorbol ester) induces a marked inhibition of the ANF-stimulated cGMP accumulation as well as of the membrane ANF-sensitive guanylate cyclase catalytic activity without any change in the binding capacity or affinity for ¹²⁵I-ANF. However, we have demonstrated a significant ³²P incorporation in the ANF-R₁ receptor of the TPA-treated cells. The effect of TPA on the zona glomerulosa ANF-R₁ receptors was abolished by calphostin C, a specific protein kinase C inhibitor. Altered ANF actions due to blunted response of guanylate cyclase to ANF could be a consequence of the ANF receptor phosphorylation by excessive activity of protein kinase C and might be involved in the pathogenesis of hypertension.Abbreviations ANF Atrial Natriuretic Factor - ANF-R₁ Atrial Natriuretic Factor Receptor, subtype 1 - ATP Adenosine Triphosphate - CaCl₂ Calcium Chloride - cAMP Adenosine cyclic 3,5-Monophosphate acid - cGMP Guanosine cyclic 35-Monophosphate acid - EDC 1-Ethyl-3-[3-Dimethylaminopropyl] Carbodiimide - EDTA Ethylenediaminetetraacetic Acid - GTP Guanosine Triphosphate - IBMX 3-isobutyl-1-methylxanthine - kDa Kilodaltons - MgCl₂ Magnesium Chloride - MgAC Magnesium Acetate - NaCl Sodium Chloride - PAGE Polyacrylamide Gel Electrophoresis - PKA cAMP-dependent protein kinase - PKG cGMP-dependent Protein Kinase - PKC Calcium/Phospholipid-dependent Protein Kinase - RIA Radioimmunoassay - SDS Sodium Dodecyl Sulfate - SHR Spontaneously Hypertensive Rat - Tris HCl Tris (Hydroxymethyl) aminomethane Hydrochloride - TPA 12-O-Tetradecanoyl-Phorbol-13-Acetate     

Effects of phorbol esters on insulin receptor function and insulin action in hepatocytes: evidence for heterogeneity

We investigated the effect of phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator on insulin receptors and insulin action in freshly isolated and primary cultures of rat hepatocytes. PMA (1 x 10^–7 M) did not alter insulin receptor numbers or affinity either acutely or chronically but within 60 minute inactivated insulin stimulated tyrosine kinase of the insulin receptor. PKC activation inhibitied insulin (1 x 10^–7M) stimulation of glycogen and lipid synthesis with a decrease or no change in basal glycogenesis and lipogenesis respectively. However, PKC activation did not alter insulin stimulated or basal amino acid transport even though PCK activation inhibited insulin stimulation of the insulin. receptor tyrosine kinase. Thus, within one tissue, PKC activation has differential effect on insulin action depending on which pathway is examined. Furthermore, insulin stimulation of the insulin receptor tyrosine kinase may not be a necessary step for all insulin signaling pathways.     

Calcium-binding protein regucalcin increases calcium-independent proteolytic activity in rat liver cytosol

The effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was investigated. The Ca²⁺-requiring proteinase required 5–10 µM Ca²⁺ for maximal activity in the presence of a protein substrate (globin). The proteinase activity was markedly elevated by the addition of regucalcin (0.25–2.0 µM) in the absence or presence of Ca²⁺ (5.0 µM) added. The effect of regucalcin, however, was the greater in the absence of Ca²⁺ than that in the presence. The pronounced effect of regucalcin on the proteinase activity was also seen in the presence of 1.0 mM EGTA with or without Ca²⁺ (5.0 µM). In the absence of Ca²⁺, the regucalcin-increased proteinase activity was clearly inhibited by the presence of anti-regucalcin antiserum (diluted to 240-fold), leupeptin (20 and 200 µg/ml), and heavy metals (25 µM cadmium or 25 µM zinc), although the inhibition was not complete at the concentration used. The present findings suggest that regucalcin increases proteolytic activity in rat liver cytosol, and that regucalcin may activate Ca²⁺-independent neutral cysteinyl-proteinase.     

Plasma volume, osmolarity, total protein and electrolytes during treadmill running and cycle ergometer exercise

While haemoconcentration due to loss of plasma volume is well established during cycling, the existence of similar changes during running remains contentious. This study compared the changes in plasma volume and associated blood indices during 60 min of running and cycling at the same relative intensity (approximately 65% VO2max), with all changes referenced to blood indices obtained after 30 min seated at rest on a cycle ergometer. Plasma osmolarity increased similarly with both forms of exercise but was less than predicted for water loss alone, such that there was a net loss of sodium during exercise and of potassium postexercise, with essentially no loss of protein. Plasma volume decreased similarly (approximately 6.5%) in both exercise trials, but while that with cycling was initiated by exercise itself and was essentially maximal within 5 min, the reduction in plasma volume in the running trial was induced by adopting the upright posture and was complete before exercise began. These data would indicate that different mechanisms are responsible for the changes in plasma volume induced by running and cycling, while the similarity of change would suggest that there is a lower limit to any reduction in plasma volume, regardless of mechanism. Furthermore, the observation that the changes in plasma volume were complete before or early in exercise, would imply that oral water ingestion during prolonged exercise, which is essential for thermoregulation, may be more concerned with homeostasis of extravascular water rather than plasma volume.     

Analysis of Con A-binding glycoproteins in synaptosomal membranes

Concanavalin A (Con A)-binding proteins obtained from solubilized synaptosomal membranes of bovine brain were analyzed by two-dimensional electrophoresis (2DE), and were identified by peroxidase conjugated Con A (Con A-peroxidase staining), after transfer from 2DE gel to nitrocellulose paper. The Con A-binding proteins were resolved up to 40 spots, ranging in isoelectric points (pI) from 4.5 to 8.0 and molecular weight (MW) from 10 kDa to 120 kDa. Most of the Con A-binding proteins were streaked across a pH gradient and/or exhibited as multiple spots, indicating broad charge and molecular weight heterogeneity. The presence of protein groups that showed high affinities for Con A were revealed. Most interesting group (named GP51), which consisted of seven spots separated horizontally in charge heterogeneity (pI5.85-7.5) with MW 51kDa, was characterized by its binding to an immobilized protein A gel. This implies that GP51 is related to immunoglobulins and/or GP51 may be a new member of the immunoglobulin supergene family.     

Antigenic heterogeneity of strains of Saccharomyces cerevisiae and Candida albicans recognised by serum antibodies from patients with Crohn''s disease

Abstract Vitronectin, a serum and extracellular matrix protein involved in immunological reactions, interacts with Helicobacter pylori strains. Of the 20 H. pylori strains tested three strains bound more than 50% of the vitronectin added, five strains bound 25–40%, nine strains bound 10–20% and three strains bound 5–8% vitronectin. Two strains, one with high- and one with low-binding properties, were selected for further characterization of ¹²⁵I-vitronectin binding. Binding to the urea-activated ¹²⁵I-labelled vitronectin was fast, saturable and reversible when an excess of unlabelled vitronectin was added to the bacteria with bound ¹²⁵I-vitronectin. The binding was heat- and protease-sensitive, suggesting that the binding was mediated by bacterial cell-surface proteins. Since components such as fetuin and orosomucoid but not asialofetuin inhibited the binding, sialic-acid specific proteins, related to H. pylori sialic-acid specific haemagglutinins, were probably involved.