Nitric oxide synthase in the spinal cord of the frog, Xenopus laevis

The expression of nitric oxide synthase was investigated in the spinal cord of the South African clawed frog by NADPH diaphorase histochemistry and immunohistochemistry. The dorsal field contained many strongly positive neurons and a dense plexus of processes. Only few nitric oxide synthase-positive cells occurred in the lateral and central field. Motoneurons were negative. A dense accumulation of stained neurons was located dorsal and dorsomedial to the motoneurons. The white matter harbored many positive fibers. These were most abundant in the dorsal funiculus, and obviously consist of nonprimary projections to the brainstem. These results suggest that nitric oxide represents a widely used messenger molecule in the frog spinal cord, in particular with respect to the processing of sensory information.     

Purification and properties of the catalytic domain of human 3-hydroxy-3-methylglutaryl-CoA reductase expressed in Escherichia coli

Three fragments of the cDNA encoding human 3-hydroxy-3-methylglutaryl-CoA reductase, all incorporating the majority of the catalytic domain of the protein, were subcloned into Escherichia coli expression vectors containing the pL promoter. The two larger expressed fragments (58 and 52 kDa) were soluble and had enzymatic activity, while the smallest (48 kDa) was insoluble. The two active fragments were purified by a combination of conventional techniques and affinity chromatography. A number of properties of the two enzymes were compared including specific activity, kinetic parameters, relative solubility, and cold lability. The 52-kDa enzyme was observed to change from a dimeric to monomeric form and to lose activity at 4 degrees C. In contrast, the 58-kDa enzyme was found to be much less cold labile, and was dimeric at both 20 and 4 degrees C. In order to resolve the number of subunits required to form an active site, the number of inhibitor binding sites for a known inhibitor was determined to be one per subunit in the 58-kDa enzyme.     

Vipera lebetina venom contains two disintegrins inhibiting laminin-binding beta1 integrins

To explain the myotoxic effects of snake venoms, we searched for inhibitors of alpha7beta1 integrin, the major laminin-binding integrin in skeletal muscle. We discovered two inhibitors in the venom of Vipera lebetina. One of them, lebein-1 (known as lebein), has already been proposed to be a disintegrin because of its RGD-containing primary sequence. The other, lebein-2, is a novel protein that also interacts firmly with alpha3beta1, alpha6beta1, and alpha7beta1 integrins, but not with the collagen-binding alpha1beta1 and alpha2beta1 integrins. Ligand binding of laminin-recognizing beta1 integrins was efficiently blocked by both lebein-1 and lebein-2. In cell attachment assays, lebein-1 and lebein-2 inhibited myoblast attachment not only to laminin, but also to fibronectin. However, neither lebein-1 nor lebein-2 interacted with alpha7beta1 integrin in an RGD-dependent manner, similar to the interaction of the laminin with alpha7beta1 integrin. Identical divalent cation dependence of integrin binding to laminin and to either of the two inhibitors and their mutually exclusive binding suggest that both lebein-1 and lebein-2 interact with the ligand-binding site of laminin-binding beta1 integrins by mimicking the yet unknown integrin-binding structure of laminins. Like lebein-1, lebein-2 is a soluble heterodimeric disintegrin of low molecular mass. Together with membrane-bound ADAM-2 and ADAM-9, the two inhibitors seem to form a small group of disintegrins that can bind to laminin-binding beta1 integrins. Because of their inhibitory capability both in vitro and in vivo, lebein-1 and lebein-2 may be valuable tools in influencing laminin-induced, integrin-mediated cell functions such as cell anchorage, migration, and mechanical force transduction on laminin-rich basement membranes.     

The cytokinesis gene KEULE encodes a Sec1 protein that binds the syntaxin KNOLLE

KEULE is required for cytokinesis in Arabidopsis thaliana. We have positionally cloned the KEULE gene and shown that it encodes a Sec1 protein. KEULE is expressed throughout the plant, yet appears enriched in dividing tissues. Cytokinesis-defective mutant sectors were observed in all somatic tissues upon transformation of wild-type plants with a KEULE-green fluorescent protein gene fusion, suggesting that KEULE is required not only during embryogenesis, but at all stages of the plant's life cycle. KEULE is characteristic of a Sec1 protein in that it appears to exist in two forms: soluble or peripherally associated with membranes. More importantly, KEULE binds the cytokinesis-specific syntaxin KNOLLE. Sec1 proteins are key regulators of vesicle trafficking, capable of integrating a large number of intra- and/or intercellular signals. As a cytokinesis-related Sec1 protein, KEULE appears to represent a novel link between cell cycle progression and the membrane fusion apparatus.     

Nck and phosphatidylinositol 4,5-bisphosphate synergistically activate actin polymerization through the N-WASP-Arp2/3 pathway

The Wiskott-Aldrich syndrome protein (WASP) and its relative neural WASP (N-WASP) regulate the nucleation of actin filaments through their interaction with the Arp2/3 complex and are regulated in turn by binding to GTP-bound Cdc42 and phosphatidylinositol 4,5-bisphosphate. The Nck Src homology (SH) 2/3 adaptor binds via its SH3 domains to a proline-rich region on WASP and N-WASP and has been implicated in recruitment of these proteins to sites of tyrosine phosphorylation. We show here that Nck SH3 domains dramatically stimulate the rate of nucleation of actin filaments by purified N-WASP in the presence of Arp2/3 in vitro. All three Nck SH3 domains are required for maximal activation. Nck-stimulated actin nucleation by N-WASP.Arp2/3 complexes is further stimulated by phosphatidylinositol 4,5-bisphosphate, but not by GTP-Cdc42, suggesting that Nck and Cdc42 activate N-WASP by redundant mechanisms. These results suggest the existence of an Nck-dependent, Cdc42-independent mechanism to induce actin polymerization at tyrosine-phosphorylated Nck binding sites.     

Evidence for synergistic anion binding to iron in ovotransferrin complexes from resonance Raman and extended X-ray absorption fine structure analysis

The 2,3-dihydroxybenzoate and thioglycolate complexes of iron(III)-ovotransferrin have been studied with resonance Raman and extended x-ray absorption fine structure spectroscopies, respectively, to obtain evidence for the coordination of the synergistic anion to the iron center. The dihydroxybenzoate complex exhibits resonance-enhanced Raman vibrations arising from both the endogenous tyrosinates and the added dihydroxybenzoate. A comparison of the extended x-ray absorption fine structure spectra of the carbonate and thioglycolate complexes shows a large feature at about 1.95 A assigned to Fe-(O,N) interactions. The latter complex exhibits an added feature at 2.32 A assigned to an Fe-S interaction. These experiments demonstrate that the Lewis base functions in the synergistic anions coordinate to the iron in ovotransferrin.     

Use of [3H] Spiperone for Labelling Dopaminergic and Serotonergic Receptors in Bovine Caudate Nucleus

Abstract— [³H]Spiperone binding has been used to study neurotransmitter receptors in bovine caudate nucleus in displacement and saturation binding experiments. Displacement curves for several antagonists are biphasic and can be analysed into contributions from dopaminergic and serotonergic sites. Antagonist binding at each class of sites follows the simple mass action equations for binding at a homogeneous set of sites (slope factors close to unity). Agonist displacement curves also indicate complex behaviour, but agonist binding to the dopaminergic sites alone exhibits heterogeneous properties (slope factors less than unity). Saturation binding experiments have been conducted on each class of site, defining dopaminergic binding of [³H]spiperone as that binding displaced by 0.1 m m -dopamine and serotonergic binding as that displaced by 0.3 μ m -mianserin. In each case, a single class of binding sites was detected: the binding parameters derived in this way have been used to calculate the proportions of the two classes of binding site observed in displacement experiments. Good agreement was obtained between calculated and observed values.     

Influence of Nematode Parasitism,Body Size,Temperature, and Diel Period on the Flight Capacity of <Emphasis Type="Italic">Sirex noctilio</Emphasis> F. (Hymenoptera: Siricidae)

Sirex noctilio F. (Hymenoptera: Siricidae) is a woodwasp of pine trees that has recently invaded and established in North American forests. Although S. noctilio has had a limited impact in North America to date, there is some concern that it could have a significant impact on pine plantations, especially in the southeastern U.S.A. Moreover, there are few data on the flight capacity of male S. noctilio. We found no association between parasitism by D. siricidicola and whether or not S. noctilio initiated flight on the flight mill. Male wasps that were parasitized by nematodes were heavier than non-parasitized males, but there was no significant difference in mass between parasitized and non-parasitized females. We also examined the flight capacity of male and female S. noctilio in relation to nematode parasitism, body mass, temperature (for only males), and diel period. Body mass, temperature, and diel period affected flight in S. noctilio such that wasps were generally observed to fly faster, farther, and more frequently if they were heavier, flying at warmer temperatures, and flying during the photoperiod. The fact that nematode-parasitized male wasps were found to fly farther than the non-parasitized males is consistent with the hypothesis that nematode parasitism does not negatively affect the flight capacity of S. noctilio.     

Phylogenetic relationships of the European lacertid genera Archaeolacerta and Iberolacerta and their relationships to some other 'Archaeolacertae' (sensu lato) from Near East, derived from mitochondrial DNA sequences

Parts of the mitochondrial genes coding for 12SrRNA and 16SrRNA (together about 960 bp) were sequenced for all Mediterranean species of 'Mountain lizards' of the genera Archaeolacerta ( sensu lato ) and Iberolacerta . All subspecies of the Iberian species Iberolacerta cyreni and I. monticola were included in this study. In addition, samples of Apathya cappadocica and Darevskia rudis were analysed to elucidate the relationships of the European 'Mountain lizards' to their possible relatives in the Near East. Maximum parsimony and neighbour joining analyses lead to the following major conclusions: (i) the monophyly of the genus Iberolacerta is very well supported; (ii) Archaeolacerta bedriagae (the type species of the genus) is most basal with respect to the ingroup taxa. If we accept Iberolacerta as a genus, Archaeolacerta becames paraphyletic. Therefore, we propose to restrict Archaeolacerta to the type species and to treat A. mosorensis and A. oxycephala provisionally as members of the collective genus Lacerta ; (iii) within the genus Iberolacerta three groups were found: a Pyrenean group, an Iberian group and I. horvathi . The relationships among these groups remain unresolved; and (iv) the Peña de Francia lizards, described originally as a subspecies of I. cyreni , are in fact more closely related to I. monticola .