Clonality of Vibrio anguillarum strains isolated from fish from the Scandinavian countries, Sweden, Finland and Denmark

In order to investigate whether outbreaks of vibriosis in the Baltic region were caused by the spread of certain pathogenic clones, 291 Vibrio anguillarum isolates from Finland (n = 156), Sweden (n = 88) and Denmark (n = 47) were studied with respect to serogroup, ribotype, plasmid content, and biochemical phenotypes as expressed with the PhenePlate (PhP) typing system. For comparison, 54 V. anguillarum serogroup O1 from other countries worldwide were included. Most isolates from Finland, Sweden and Denmark belonged to serogroup O1 (255), followed by O2 (30). Four Finnish isolates cross-reacted strongly with antisera against two new serogroups VaNT2 and VaNT4, whereas two strains were non-typeable. The serogroup O1 isolates displayed ten different ribotype patterns, whereas the other strains were considerably more diverse with respect to ribotypes. Most of the O1 isolates carried the 67 kb virulence plasmid and a group of Finnish isolates, in addition, carried an 86 kb plasmid. Additional plasmids with molecular weights of 63, 76, 135 or 260-290 kb were found in single O1 isolates. With few exceptions, strains of serogroup O2 either had no plasmids or carried one or two small plasmids. PhenePlate typing revealed considerable diversity within the species, serogroup O1 being the most homogeneous. A few PhP types were dominant, whereas other types were observed only in one to four isolates. The prevalence of the different types changed significantly from one year to another but in Finland, one clonal lineage became increasingly important from 1992 (20% of isolates) to 1996 (80%). Remaining clones were mostly restricted to specific geographic areas. By cluster analysis, it was demonstrated that most of the isolates from Finland, Sweden and Denmark belonged to two clusters, and most of the strains from Southern Europe fell into two other, distinct clusters. Most isolates from the UK, North America, Chile and Tasmania grouped together in a distinct cluster. For the typing of V. anguillarum, O-serotyping should be the primary method. For isolates belonging to serogroups other than O1, plasmid profiling in combination with ribotyping gives a very good discrimination between strains, whereas for serogroup O1, another method is required. It is concluded that PhP typing is a tool that provides a good discrimination between O1 isolates.     

Recombinant avidin and avidin-fusion proteins

Both chicken egg-white avidin and its bacterial relative streptavidin are well known for their extraordinary high affinity with biotin (Kd approximately 10(-15) M). They are widely used as tools in a number of affinity-based separations, in diagnostic assays and in a variety of other applications. These methods have collectively become known as (strept)avidin-biotin technology. Biotin can easily and effectively be attached to different molecules, termed binders and probes, without destroying their biological activity. The exceptional stability of the avidin-biotin complex and the wide range of commercially available reagents explain the popularity of this system. In order by genetic engineering to modify the unwanted properties of avidin and to further expand the existing avidin-biotin technology, production systems for recombinant avidin and avidin-fusion proteins have been established. This review article presents an overview of the current status of these systems. Future trends in the production and applications of recombinant avidin and avidin-fusion proteins are also discussed.     

Xylose utilisation: cloning and characterisation of the Xylose reductase from Candida tenuis

Xylose reductases catalyse the initial reaction in the xylose utilisation pathway, the NAD(P)H+H+ dependent reduction of xylose to xylitol. In this work, the xylose reductase gene from Candida tenuis CBS 4435 was cloned and successfully expressed in E. coli. From the purified and partially sequenced protein primers were deduced for PCR. The fragment obtained was used for Southern blot analysis and screening of a subgenomic library. The clone containing the open reading frame was sequenced; the gene consisted of 969 nucleotides coding for a 322 amino acids protein with a molecular mass of 36 kDa. Putative regulatory signals were identified with the help of a Saccharomyces cerevisiae regulatory sequence database. In order to express the xylose reductase in E. coli, the gene was placed under positive and negative control. At low temperatures, the xylose reductase was expressed in soluble and active form up to about 10% of the soluble protein; with rising temperatures formation of visible inclusion bodies occurred. In refolding experiments we were able to recover the major portion of xylose reductase activity from the pellet fraction.     

Über den submikroskopischen Bau des Grundhäutchens der Hirnkapillaren

Zusammenfassung Auf Grund der polarisationsoptischen Analyse besitzt das homogene Kapillar-Grundhäutchen eine geordnete Feinstruktur. Es ist ein Eiweiß-Lipoidsystem, in dem die Proteinkomponente als Träger- oder Gerüstsubstanz mengenmäßig überwiegt. Die Proteine zeigen teilweise eine fibrilläre Struktur mit geringer mizellarer Orientierung. In der Fläche der Membran haben die Fibrillen keine bevorzugte Verlaufsrichtung, sie liegen statistisch ungeordnet. Zu der Membranoberfläche verlaufen sie jedoch annähernd parallel, so daß eine folienartige Textur resultiert. In dieses Proteingerüst sind Lipoidmolekeln in radiärer Richtung orientiert eingelagert. Nach einer Näherungsrechnung dürfte es sich um eine bimolekulare Lamelle handeln, wahrscheinlich ist, daß die Lipoide inselförmig in das Proteingerüst eingesprengt sind. Die gefundene Struktur wird in Beziehung zu den Befunden neuerer Arbeiten über die Blut-Hirnschranke und die Blut-Liquorschranke gesetzt. Hierbei wird die Bedeutung der Membranlipoide für die Regulierung des Durchtritts lipoidlöslicher Stoffe, diejenige der Proteinlamellen für die Regulierung der Durchlässigkeit für Wasser und wasserlösliche Stoffe erörtert. Die Endothelmembran, d. h. das Grundhäutchen der Kapillare wird als das morphologische Substrat der Schrankenfunktion angesehen.Die Untersuchung wurde mit dankenswerter Unterstützung der Deutschen Forschungsgemeinschaft ausgeführt.     

A hierarchical Bayesian model to predict the duration of immunity to Haemophilus influenzae type b

A hierarchical Bayesian regression model is fitted to longitudinal data on Haemophilus influenzae type b (Hib) serum antibodies. To estimate the decline rate of the antibody concentration, the model accommodates the possibility of unobserved subclinical infections with Hib bacteria that cause increasing concentrations during the study period. The computations rely on Markov chain Monte Carlo simulation of the joint posterior distribution of the model parameters. The model is used to predict the duration of immunity to subclinical Hib infection and to a serious invasive Hib disease.     

Effect of cationic lipids in the formation of asymmetries in supported bilayers

We have studied the formation of a supported bilayer containing both cationic and zwitterionic lipids by fusion of small unilamellar vesicles (SUV) onto the solid surface at low salt conditions using a combination of attenuated total reflection infrared (ATR-IR) and deuterium NMR spectroscopy with microcalorimetry. The data suggest that a significant cationic lipid asymmetry between the outer (distal) and the inner (proximal) monolayer of a supported bilayer results under conditions of prolonged incubation times of the solid support with the SUV coating solution. For a SUV composition of DPPC/DHDAB (4:1) we observed an enrichment of the cationic component in the proximal monolayer of up to 200% compared to the distal monolayer after 12 h incubation. It is suggested that the electrostatic potential arising from the solid surface is the driving force for the creation of this asymmetry by means of directed flip-flop between the monolayers and/or by temporary fusion between SUV from the bulk with the supported bilayer.     

Biophysics of ceramide signaling: interaction with proteins and phase transition of membranes

Ceramides have been implied in intracellular signal transduction systems regulating cellular differentiation, activation, survival and apoptosis and thus appear capable of changing the life style of virtually any cell type. Ceramide belongs to the group of sphingosine-based lipid second messenger molecules that are critically involved in the regulation of diverse cellular responses to exogenous stimuli. The emerging picture suggests that coupling of ceramide to specific signaling cascades is both stimulus and cell-type specific and depends on the subcellular topology of its production. However, little is understood about the molecular mode of ceramide action. In particular, in lieu of a defined ceramide binding motif it is not clear how ceramide would directly interact with putative target signaling proteins. This article proposes two modes of ceramide action. First, a protruding alkyl chain of ceramide may interact with a hydrophobic cavity of a signaling protein providing a lipid anchor to attach proteins to membranes. Second, the generation of ceramide generally increases the volume of hydrocarbon chains within the lipid bilayer thereby enhancing its propensity of to form a hexagonal II phase (Hex II). Besides the generation of a hydrophobic interaction site for proteins local hexagonal phase II formation can also change the membrane fluidity and permeability, which may impinge on membrane fusion processes, solubilization of detergent-resistant signaling rafts, or membrane receptor internalization. Thus, ceramide production by sphingomyelinases (SMase) can play a pivotal signaling role through direct interaction with signaling proteins or through facilitating the formation and trafficking of signal transduction complexes.