Characterization of hemocytes from the marine amphipod Parhyale hawaiensis (Dana 1853): Setting the basis for immunotoxicological studies

Hemocytes are circulating blood cells that play a crucial function in amphipods and other crustacean immune systems. The hemocytes of the marine tropical amphipod Parhyale hawaiensis have been used for the evaluation of DNA damage and micronuclei, but they have not been characterized in the scientific literature. The aim of this study was to describe the hemolymph cells of P. hawaiensis and study their phagocytotic activity. Basic dyes were used to differentiate the cell types and the presence of lipids. The total hemocyte counts (THCs) and the proportion and sizes of the hemocyte types were determined. Hemolymph was exposed to Escherichia coli for verification of the presence of phagocytosis. Three cell types, all containing lipids, were identified in P. hawaiensis: granulocytes (oval shape, 13.4 × 7.6 μm), semi-granulocytes (oval shape, 14.1 × 7.2 μm), and hyalinocytes (round shape, 9.6 × 7.2 μm). Those three cell types were found in different percentages in males (64.8%, 31.1%, and 4.2%) and females (70.1%, 28.2%, and 1.7%). THCs for males were 9007 ± 3800 cells per individual and 4695 ± 1892 cells per individual for females. The cells of E. coli were phagocytized by the hemocytes. Our findings increased the knowledge of hemocytes in P. hawaiensis and is a step forward in using hemocyte-based immune responses as an endpoint in ecotoxicology.     

The effect of multiplicity of infection on recombination values in bacteriophageT 4 D

Zusammenfassung In Massenlysaten und Einzelwürfen vonT 4-Kreuzungen steigt der Prozentsatz der Rekombinanten mit steigender Infektionsmultiplizität an, und zwar sowohl zwischen gekoppelten als auch zwischen ungekoppelten Genen. Dieser Befund stimmt mitTrautners (1960) Ergebnissen beiT 1-Kreuzungen überein. Der scheinbarte Widerspruch zuEpsteins (1958) früheren Resultaten beiT 4-Kreuzungen läßt sich erklären, da verschiedene Wirtsbakterien verwendet wurden.Es wird diskutiert, ob dieser Effekt durch die Annahme erklärt werden kann, daß die infizierenden Phagegenome bei der Rekombination brechen und daß die Bruchstellen zufallsgemäß über die Genome einer Phagenpopulation verteilt sind. Die beschriebenen Versuche schließen jedochTrautners Interpretation nicht aus, daß in der infizierten Bakterienzelle eine Topographie existiert, d. h., daß die verschiedenen Genotypen während der Vermehrung nach Einzelinfektion nicht so vollständig durchmischt werden wie nach Mehrfachinfektion.

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Identification of anticoagulant activities in the salivary glands of the soft tick,Ornithodoros savignyi

Salivary gland extracts of the sand tampan, Ornithodoros savignyi, prolonged the activated partial thromboplastin time (APTT) and prothrombin time (PT) significantly in a concentration-dependent manner. There was also a pronounced inhibition of human activated factor Xa (fXa) by salivary gland extracts. The salivary gland extracts inhibited chromogenic assays specific for both fXa and thrombin. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the salivary gland proteins followed by elution of specific areas or bands from a polyvinylidene difluoride (PVDF)-membrane, showed that various anticoagulant factors are present when screened by means of the APTT assay. The most active component was associated with a band of M _r of 14 kDa. Partial purification of this component was achieved using isoelectric focusing (IEF) and size-exclusion highperformance liquid chromatography (HPLC).     

Respiratory muscle reserve in rats during heavy exercise

Gosselin, Luc E., David Megirian, Joshua Rodman, DonnaMueller, and Gaspar A. Farkas. Respiratory muscle reserve in ratsduring heavy exercise. J. Appl.Physiol. 83(4): 1405-1409, 1997.The extent towhich the respiratory pump muscles limit maximal aerobic capacity inquadrupeds is not entirely clear. To examine the effect of reducedrespiratory muscle reserve on aerobic capacity, whole bodypeak oxygen consumption(O_{2 peak}) wasmeasured in healthy Sprague-Dawley rats before and after Sham,unilateral, or bilateral hemidiaphragm denervation (Dnv) surgery.O_{2 peak} wasdetermined by using a graded treadmill running test.Hemidiaphragm paralysis was verified after testing byrecording the absence of electromyographic activity duringinspiration. Before surgery, O_{2 peak} averaged 86, 87, and 92 ml · kg¹ · min¹for the Sham, unilateral, and bilateral Dnv groups, respectively. Twoweeks after surgery, there was no significant change inO_{2 peak} foreither the Sham or unilateral Dnv group. However,O_{2 peak} decreased~19% in the bilateral Dnv group 2 wk after surgery. These findingsstrongly suggest that the pulmonary system in rats is designed suchthat during heavy exercise, the remaining respiratory pump muscles areable to compensate for the loss of one hemidiaphragm, but not of both.

     

Peroxidases in Acetabularia: their possible role in development

Abstract. Crude enzymatic extracts from Acetabularia exhibit very low peroxidase activity after a lag period. Starch gel electrophoresis of extracts from growing algae shows a single, extremely anodic band. Extracts of small, slow-growing or cap-bearing algae, which do not grow any more, do not exhibit any peroxidase band. Cytochemical staining with benzidine reveals changes in both the quantity and distribution of peroxidase along the polarized Acetabularia cell. The homogenous staining of small algae becomes distributed along a negative apico-basal gradient when the algae initiate their rapid growth phase. This polarized pattern is repeated on the hair whorls. A similar developmental sequence directs cap growth, with an initial intense staining reaction of the primordium, which later leaves only the corona inferior stained blue. Finally, the Acetabularia cell remains slightly blue at the edges of the rhizoidal out-growths and cap rays. Crude extracts of Acetabularia induce a lag in standard horseradish peroxidase (HRP) activity. The inhibitor is always present in small and growing algae; it is sometimes absent or less active in cap-bearing algae. In no case does it change the kinetics of the HRP reaction with guaïacol. The lag is completely suppressed by pretreatment with either H₂O₂ or ascorbate oxidase. The changes in peroxidase activity, correlated with developmental stage and according to a polarized gradient, suggest that the enzyme could be involved in some way in the control of morphogenesis in Acetabularia . An inhibitor of peroxidase activity, which disappears as the cap matures, might, in turn, exert a regulatory function.     

Comparison of isolated peanut agglutinin receptor glycoproteins from human,bovine and porcine erythrocyte membranes

Affinity chromatography has been used to isolate and compare the peanut agglutinin receptors from neuraminidase-treated human, bovine and porcine erythrocyte membranes. Passage of Triton X-100-solubilised membrane material through either Sepharose- or acrylamide-based affinity columns resulted in the reversible binding of receptor molecules to the immobilised lectin. Elution with 0.2M galactose released specifically bound glycoprotein fractions, the composition and molecular weights of which were determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate.Carbohydrate analysis by gas chromatography identified these bound glycoprotein fractions as the major sources of the $\text{O-}\text{glycosidic-linked}$ disaccharide $\text{galactosyl}\text{-β-(1 → 3)-N-}\text{acetylgalactosamine}$ in these membranes. It is suggested that these isolated fractions represent a discrete population of glycoproteins within the membranes studied, which possess both $\text{O-}\text{glycosidic}$- and $\text{N-}\text{glycosidic-linked}$ carbohydrates.     

Vegetative multiplication of sugarbeet through in vitro culture of inflorescence pieces

In vitro vegetative multiplication of sugarbeet was obtained by culturing of inflorescence explants. Subapical segments or 5-mm-long tips from nine varieties developed axillary shoots (up to 50 per tip) on a medium containing indolebutyric acid (IBA) and benzylaminopurine (BAP). Zeatin was ineffective as cytokinin. Gibberellic acid (GA₃) enhanced the process. Such vegetative shoots were subsequently isolated and were each allowed to develop up to 20 supplementary axillary shoots on a multiplication medium containing IBA, BAP, and naphthaleneacetic acid (NAA). Rooting of shoots was obtained in the absence of growth regulators and plants were established.     

Peroxidases of high molecular weight identified as the membrane peroxidases in lentils]

Peroxidases extracted from lentil roots are separated in two peaks by gel chromatography on Sephadex G-100 or on Bio Gel A-5 M. On both resins, the first peak of extremely large molecular weight is demonstrated to be an association of some peroxidases with microsomes. These enzymes can be detached from membranes by NaCl. Starch gel electrophoresis shows that isoperoxidases associated electrostatically to microsomes are basic peroxidases apparently not different from those of the soluble fraction.     

Inhibition of ileal brush-border chloride conductance by specific antibody

Summary Antibody raised in mice was used in attempting to identify proteins responsible for the conductive chloride transport that can be measured in porcine ileal brush border membrane vesicles. Ileal brush-border membrane vesicle protein from pig was separated into five different molecular mass fractions by preparative SDS polyacrylamide disc gel electrophoresis. Separated protein fractions were used to immunize mice. Antibody was screened for reactivity with antigen by Western blotting, and for effects on conductive chloride transport in ileal brush border membrane vesicles. Immunization with brush-border protein from fraction I proteins (>110 kDa) produced polyclonal antisera which specifically inhibited the conductive component of chloride uptake by ileal brush border vesicle preparations. Western blotting of the antigen showed the presence of several protein species of molecular mass >100 kDa that were recognized by immune serum. Spleen cells from a mouse producing antiserum that inhibited conductive chloride transport were fused with a myeloma cell line. The resulting hybridoma colonies produced antibody that reacted with at least seven distinct protein bands by Western blot assay and inhibited chloride conductance in brush-border membrane vesicles.