Sodium translocation by the iminoglycinuria associated imino transporter (SLC6A20)

The system IMINO transporter plays an essential role in the transport of proline and hydroxyproline in the intestine and kidney. Its molecular correlate has been identified and named SIT1 or IMINO (SLC6A20). Initial characterization of the transporter showed it to be Na⁺ and Cl^?-dependent, but the stoichiometry remained unresolved. Using homology modeling along the structure of the bacterial leucine transporter LeuT, we identified two highly conserved Na⁺-binding sites and a putative Cl^?-binding site. Mutation of all residues in the two proposed Na⁺-binding sites revealed that most of them were essential for uptake and completely inactivated the transporter. However, mutants A22V (Na⁺-binding site 1) and mutants S20A, S20G, S20G/G405S (Na⁺-binding site 2) were partially active and characterized further. Flux studies suggested that mutations of Na⁺-binding site 1 caused a decrease of the Na⁺-K_0.5, whereas mutations of site 2 increased the K_0.5. Mutation of Na⁺-binding site 1 also changed the ion selectivity of the IMINO transporter. IMINO actively translocates ³⁶Cl^? demonstrating that the proposed chloride binding site is used in the transporter. Accumulation experiments and flux measurements at different holding potentials showed that the transporter can work as a 2Na⁺/1Cl^?-proline cotransporter. The proposed homology model allows to study mutations in IMINO associated with iminoglycinuria.     

The quantification of the haemagglutinin content of influenza whole virus and Tween-ether split vaccines

Monovalent whole virus and Tween-ether split vaccines prepared from influenza A/Bangkok, A/Brazil and B/Singapore were assayed for haemagglutinin content using single radial immunodiffusion (SRID), quantitative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunization of guinea pigs. When SRID was performed with split vaccines, haemagglutinin values were consistently recorded which were in the range of 50 to 25% of the values obtained before disruption of virions. When, however, disruption was conducted in the presence of excess detergent, thus preventing aggregate formation of solubilized haemagglutinin, test values comparable with those of whole virus vaccines were obtained. In agreement with these results, immunization experiments revealed that whole virus and corresponding split vaccines exhibited comparable immunogenicity in guinea pigs. Additionally it could be calculated from SDS-PAGE and densitometer tracings, obtained by scanning the gels after staining with either Coomassie blue or FITC-Con A, that 90 to 95% of whole virus HA2 was recovered in Tween-ether split vaccines. On the basis of these findings we conclude that precise quantification of Tween-ether split vaccines is not possible by the SRID test alone. As aggregate formation of solubilized haemagglutinin occurs, we suggest that either a physico-chemical method including a disaggregation procedure, such as SDS treatment, or immunological evaluation of the original whole virus preparation before disruption of virions should be applied as an additional criterion for quantification of influenza Tween-ether split vaccines.     

Solutions to a degenerate system of parabolic equations from marine biology

Summary A system of parabolic and ordinary differential equations u _t = a ² u _xx + F(u, v, w), v _t = a ² v _xx + G(u, v, w),w _x = – k(u)w is studied which has been proposed by Radach and Maier-Reimer for the dynamics of phytoplankton and nutrient in dependence of light intensity. It is shown that there is a unique solution to this system satisfying given initial and boundary conditions. The solution depends continuously on the data. For specific nonlinearities F, G, and k bounds for the solutions are given.     

Examining olfactory and visual cues governing host-specificity of a weed biological control candidate species to refine pre-release risk assessment

In weed biological control programs, pre-release host-specificity testing relies traditionally on no-choice and choice feeding, oviposition, and development tests. Rarely have they included detailed examination of behavioral responses to olfactory and visual cues of biological control candidates, although a better understanding of the mechanisms underlying host recognition may explain potential discrepancies between choice and no-choice tests, and/or between tests conducted in the lab versus field conditions. We investigated how the seed-feeding weevil, Mogulones borraginis, distinguishes its host plant, Cynoglossum officinale, from three native confamilial non-target species in North America. In behavioral bioassays, M. borraginis responded to olfactory and visual cues individually and, to an even greater extent, to both plant cue modalities when offered simultaneously. In tests with the combined cues, M. borraginis was attracted to C. officinale but responded with indifference or was repelled by non-target plants. In electrophysiological experiments, we identified that M. borraginis responded to ten volatile compounds and four wavelengths of lights from inflorescences of C. officinale. We propose that studies of responses to multimodal plant cues can advance our understanding of how biocontrol candidate species discriminate among host plants and closely related non-target species, thereby increasing the accuracy of environmental safety assessments pre-release.     

A structural analysis of the interaction between ncd tail and tubulin protofilaments

ncd is a minus-end directed, kinesin-like motor, which binds to microtubules with its motor domain and its cargo domain as well. Typical of retrograde motors, the motor domain of ncd locates to the C-terminal end of the polypeptide chain, and hence, the cargo domain constitutes the N-terminal region. To date, several studies have investigated the interaction properties of the motor domain with microtubules, but very few structural data are available about the tail itself or its interaction with microtubules as cargo. Here, we applied cryo-electron microscopy and helical 3D image reconstruction to 15 protofilament microtubules decorated with an ncd tail fragment (N-terminal residues 83-187, named NT6). In our study, the ncd tail shows a behaviour resembling filamentous MAPs such as tau protein, exhibiting a highly flexible structure with no large globular domains. NT6 binds to four different sites on the outer side of microtubules within the proximity of the kinesin motor-binding site. Two of these sites locate within the groove between two neighbouring protofilaments, and appear as strong binding sites, while the other two sites, located at the outer rim, appear to play a secondary role. In addition, the ncd tail fragment induces the formation of large protofilament sheets, suggesting a tail-induced modification of lateral protofilament contacts.     

Untersuchungen zum Bekämpfungsrichtwert bei der Haferblattlaus,Rhopalosiphum padi (L.), an Sommergerste

In unseren Untersuchungen prüften wir den Einfluß einer Saatgutbehandlung des Maises mit Imidacloprid (Gaucho) sowohl auf die Übertragung des persistenten barley yellow dwarf virus (BYDV) als auch auf die nichtpersistenten Potyviren maize dwarf mosaic virus (MDMV) und sugarcane mosaic virus (SCMV). Dazu wurden Freiland‐ und Gewächshausversuche sowie Experimente in einer Klimakammer angestellt. Für die Virusübertragungen nutzten wir die Blattlausarten Rhopalosiphum padi L. und weniger häufig Sitobion avenae F. Im Vergleich zu S. avenae wanderte R. padi schneller von den behandelten Pflanzen ab. In Gewächshausversuchen zeigte sich, daß das SCMV nach einer Saatgutbehandlung in deutlich geringerem Umfang übertragen wurde, wenn die Inokulation bis 3 Wochen nach dem Auslegen erfolgte. Inokulationen von älteren Pflanzen ließen keine insektizide Wirkung erkennen. Ebenso ergaben Freilandversuche keinen eindeutigen Zusammenhang zwischen Saatgutbehandlung und Virusbefall. Im Falle des BYDV konnte bei einer Inokulation bis 9 Tage nach dem Auslegen durch einen Imidacloprid‐Einsatz eine signifikante Reduzierung der Übertragung erreicht werden. Spätere Inokulationen verliefen in der Regel negativ. Die Ergebnisse des Feldversuches 1992 bestätigten die im Gewächshaus gewonnenen Resultate. Im Freiland war die natürliche Besiedlung des Maises durch Blattläuse in den Parzellen mit behandeltem Saatgut deutlich vermindert. Hinzu kam eine zeitliche Verzögerung des Massenwechselgeschehens um ca. 2 Wochen.

Für eine abschließende Beurteilung von Imidacloprid sind weiterführende Untersuchungen sowohl zum Saugverhalten der Blattläuse als auch zu seiner Wirkung auf die Virusübertragung unter Einbeziehung verschiedener Sorten erforderlich.     

Intestinal peptidases form functional complexes with the neutral amino acid transporter B(0)AT1

The brush-border membrane of the small intestine and kidney proximal tubule are the major sites for the absorption and re-absorption of nutrients in the body respectively. Transport of amino acids is mediated through the action of numerous secondary active transporters. In the mouse, neutral amino acids are transported by B(0)AT1 [broad neutral ((0)) amino acid transporter 1; SLC6A19 (solute carrier family 6 member 19)] in the intestine and by B(0)AT1 and B(0)AT3 (SLC6A18) in the kidney. Immunoprecipitation and Blue native electrophoresis of intestinal brush-border membrane proteins revealed that B(0)AT1 forms complexes with two peptidases, APN (aminopeptidase N/CD13) and ACE2 (angiotensin-converting enzyme 2). Physiological characterization of B(0)AT1 expressed together with these peptidases in Xenopus laevis oocytes revealed that APN increased the substrate affinity of the transporter up to 2.5-fold and also increased its surface expression (V(max)). Peptide competition experiments, in silico modelling and site-directed mutagenesis of APN suggest that the catalytic site of the peptidase is involved in the observed changes of B(0)AT1 apparent substrate affinity, possibly by increasing the local substrate concentration. These results provide evidence for the existence of B(0)AT1-containing digestive complexes in the brush-border membrane, interacting differentially with various peptidases, and responding to the dynamic needs of nutrient absorption in the intestine and kidney.