Savignin, a potent thrombin inhibitor isolated from the salivary glands of the tick Ornithodoros savignyi (Acari: Argasidae).

A thrombin (E.C. 3.4.21.5) inhibitor, savignin, was isolated from the salivary glands of Ornithodoros savignyi by a combination of size exclusion, anion-exchange, and reversed-phase chromatography. The inhibitor has a molecular mass of 12,430.4 Da as determined by electrospray mass spectrometry. The behavior of savignin during anion-exchange chromatography indicated that it has an acidic pI. The available N-terminal sequence (residues 1-11) differed from that of ornithodorin with only one residue. Savignin inhibits thrombin-induced platelet aggregation, but has no effect on ADP- or collagen-induced aggregation. Kinetic studies indicated that savignin is a competitive, slow-, tight-binding inhibitor of alpha-thrombin (K(i) = 4.89 +/- 1.39 pM). Tight-binding kinetics showed that the inhibitor has a lower affinity for gamma-thrombin (K(i) = 22.3 +/- 5.9 nM). Plasmin, factor Xa, and trypsin are not inhibited by savignin.     

Vitrification of carnation in vitro: Changes in cell wall mechanical properties,cellulose and lignin content

Vitrification of internodes of carnation was brought about by culturing in liquid medium. Cell wall extensibility of these internodes was kinetically followed in comparison to that of normal plants using the constant stress method. Liquid culture induced increased immediate and total deformation capacities of the walls from the second day. Measurements indicated that these deformation capacities involved plastic properties rather than elastic ones. These changes were paralleled by decreased relative levels of cellulose and lignin.     

Microelectrode studies of potential difference responses to changes in stromal K+ in bullfrog cornea

The effects of changing stromal K+ were studied using microelectrodes in an in vitro preparation of frog cornea. The intracellular potential (V0) responded in two opposite ways under short-circuit conditions: (1) depolarization (normal response) when stromal K+ was increased from 4 to 20 or to 79 mM, about 30 mV per 10-fold K+ concn. change; (2) a hyperpolarization (anomalous response) of 10 mV maximum when stromal K+ was increased from 0 to 4 mM. The increase from 4 to 20 or 79 mM decreased or even reversed the short-circuit current (Isc). The transepithelial conductance (gt) increased when K+ was increased to 79 mM but no change occurred in the apical membrane fractional resistance (fRo). Increase of stromal K+ from 0 to 4 mM increased Isc and minimally changed gt and fRo. Ouabain (10(-3) M) abolished the anomalous responses, that is, the increases in V0 and Isc when stromal K+ was increased from 0 to 4 mM. These results are interpreted in terms of two K+ conductive pathways in the basolateral membrane of the corneal epithelium, a Nernstian conductance and an electrogenic (Na+ + K+)-ATPase pump transporting more Na+ than K+ ions per cycle. The normal or anomalous potential difference responses to changes in stromal K+ appear to depend on the relative resistance of the two pathways at the time stromal K+ is changed.     

Microelectrode studies of amphotericin B on Na+ and K+ conductance in bullfrog cornea

Addition of 10(-5) M amphotericin B to the tear solution of an in vitro preparation of the frog cornea increased the transepithelial conductance, gt, and decreased the apical membrane fractional resistance, f(R0), in the presence or absence of tear Na+ and Cl-. In the presence of tear Na+ and Cl-, amphotericin B increased the short-circuit current, Isc, from 3.9 to 8.8 microA.cm-2 and changed the intracellular potential, V0, from -48.5 to -17.9 mV probably due to a higher increase in the Na+ than in the K+ conductance. In the absence of tear Na+ and Cl-, amphotericin B decreased Isc from 5.5 to about 0 microA.cm-2 due to K+ (and possibly Na+) flux from cell to tear and changed V0 from -35.4 to -63.6 mV due to the increase in conductance of both ions. Increase in the tear K+ from 4 to 79 mM (in exchange for choline), in the presence of amphotericin B and absence of tear Na+ and Cl-, decreased f(R0) from 0.09 to 0.06, increased gt from 0.23 to 0.31 mS, increased Isc from 0.63 to 7.3 microA.cm-2, and changed V0 from -65.5 to -17.3 mV due to the change in EK in the presence of a high conductance in the tear membrane. Similar effects were observed with an increase of tear Na+. Results support the concept that the Na+ conductance opened by amphotericin B in the apical membrane is greater than the K+ conductance. Previously observed transepithelial effects of the ionophore may be explained mostly on the basis of its effect on the apical membrane.     

Testicular expression of PC4 in the rat: molecular diversity of a novel germ cell-specific Kex2/subtilisin-like proprotein convertase.

The rat cDNA sequence of PC4 (rPC4), representing a new member of the Kex2/subtilisin-like proprotein convertases, demonstrated the presence of at least three rPC4 mRNAs resulting in the production of rPC4-A (654 amino acids), rPC4-B (619 amino acids), and rPC4-C (609 amino acids) with different C-terminal sequences. Analogous to rat PC4, three cDNAs were also found for the mouse PC4. The observed molecular diversity of PC4 mRNA possibly results from the differential splicing and/or exon skipping of the parent gene. PC4 mRNA, with a major form at 2.8 kilobases, was highly abundant in the rat testis but could not be detected by Northern analysis in any other tissues including the central nervous system and peripheral tissues. Testicular cell separation studies combined with Northern analysis indicate the high expression levels of PC4 in germ cells but not in Leydig, Sertoli, or peritubular cells. In situ hybridization histochemistry confirms the site of PC4 gene expression as the pachytene spermatocytes and the round spermatids but not in the elongating spermatids. We also demonstrate the colocalization of PC4 with proenkephalin in testicular germ cells by in situ hybridization. A study of the ontogeny of PC4 indicated that PC4 mRNA was first expressed postnatally between days 19 and 22, coinciding with the first stages of spermiogenesis. The stage-specific expression of PC4 in testis indicates its potential role in the developmental maturation of germ cells and that this convertase may play a specific physiological function in reproduction.     

Action du CCC et de l'Amo 1618 sur la germination,la croissance et les activités AIA-oxydasique,peroxydasique, catalasique in vitro et in vivo de la racine de la Lentille

Summary Amo 1618 inhibits germination and root growth of Lentil seedlings in the dark and in the light, with some symptoms of toxicity; CCC has no effect.Both CCC and Amo 1618 inhibit the catalase activity of a lentil root extract.Increasing concentrations of Amo 1618 progressively increase the activity of peroxidase and IAA-oxidase in vivo; the catalase activity remains unchanged.The effect of Amo 1618 on root growth can thus be explained by a diminished auxin level mediated by an increased auxin catabolism.The effect of Amo 1618 and that of kinetin on root growth and enzymes are parallet. Gibberellic acid has an opposite effect on auxin catabolism.

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Une partie de ce travail a fait l'objet du mémoire de Licence de J. L. et a été réalisée au Laboratoire de Biochimie végétale de l'Institut de Botanique de Liège.