Genetic assessment of environmental features that influence deer dispersal: implications for prion-infected populations

The landscape can influence host dispersal and density, which in turn, affect infectious disease transmission, spread, and persistence. Understanding how the landscape influences wildlife dispersal and pathogen epidemiology can enhance the efficacy of disease management in natural populations. We applied landscape genetics to examine relationships among landscape variables, dispersal of white-tailed deer hosts and transmission/spread of chronic wasting disease (CWD), a fatal prion encephalopathy. Our focus was on quantifying movements and population structure of host deer in infected areas as a means of predicting the spread of this pathology and promoting its adaptive management. We analyzed microsatellite genotypes of CWD-infected and uninfected deer from two disease foci (Southern Wisconsin, Northern Illinois). We quantified gene flow and population structure using F _ST, assignment tests, and spatial autocorrelation analyses. Gene flow estimates were then contrasted against a suite of landscape variables that potentially mediate deer dispersal. Forest fragmentation and grassland connectivity promoted deer movements while rivers, agricultural fields and large urbanized areas impeded movement. Landscape variables, deer dispersal, and disease transmission covaried significantly and positively in our analyses. Habitats with elevated host gene flow supported the concept of dispersal-mediated CWD transmission by reflecting a concomitant, rapid CWD expansion. Large, interrelated social groups isolated by movement barriers overlapped disease foci, suggesting that philopatry exacerbated CWD transmission. Our results promote adaptive management of CWD by predicting patterns of its spread and identifying habitats at risk for invasion. Further, our landscape genetics approach underscores the significance of topography and host behavior in wildlife disease transmission.     

Cosubstrate-induced dynamics of D-3-hydroxybutyrate dehydrogenase from Pseudomonas putida

D-3-Hydroxybutyrate dehydrogenase from Pseudomonas putida belongs to the family of short-chain dehydrogenases/reductases. We have determined X-ray structures of the D-3-hydroxybutyrate dehydrogenase from Pseudomonas putida, which was recombinantly expressed in Escherichia coli, in three different crystal forms to resolutions between 1.9 and 2.1 A. The so-called substrate-binding loop (residues 187-210) was partially disordered in several subunits, in both the presence and absence of NAD(+). However, in two subunits, this loop was completely defined in an open conformation in the apoenzyme and in a closed conformation in the complex structure with NAD(+). Structural comparisons indicated that the loop moves as a rigid body by about 46 degrees . However, the two small alpha-helices (alphaFG1 and alphaFG2) of the loop also re-orientated slightly during the conformational change. Probably, the interactions of Val185, Thr187 and Leu189 with the cosubstrate induced the conformational change. A model of the binding mode of the substrate D-3-hydroxybutyrate indicated that the loop in the closed conformation, as a result of NAD(+) binding, is positioned competent for catalysis. Gln193 is the only residue of the substrate-binding loop that interacts directly with the substrate. A translation, libration and screw (TLS) analysis of the rigid body movement of the loop in the crystal showed significant librational displacements, describing the coordinated movement of the substrate-binding loop in the crystal. NAD(+) binding increased the flexibility of the substrate-binding loop and shifted the equilibrium between the open and closed forms towards the closed form. The finding that all NAD(+) -bound subunits are present in the closed form and all NAD(+) -free subunits in the open form indicates that the loop closure is induced by cosubstrate binding alone. This mechanism may contribute to the sequential binding of cosubstrate followed by substrate.     

Human T-lymphocytes--clinical and methodological studies on the detection of Fc-IgM- and Fc-IgG-receptor bearing T-lymphocytes (Tm and Tg)

Several methodical aspects for determination of T lymphocytes with Fc receptors for IgM (TM) and IgG (TG) were studied including separation technique of T cells with E-rosetting, culture conditions of T cells for determination of TM and the rosetting of TM and TG with EA complexes. The bests results were obtained by stabilization of E-rosettes with human serum albumin, after separation of E-rosette forming cells lysis of sheep erythrocytes with save hypotonic shock, culturing of T cells in medium containing 20% AB Serum. Furthermore it was shown the possibility using EA complexes produced with not purifieded IgG or IgM anti-ox-red-blood cells antisera without lost of specifity for TM and TG. The percentage of TM and TG in peripheral blood of thirty healthy persons as well as monitoring TM and TG in three cases was investigated.     

The role of alpha-amidated neuropeptides in hydroid development--LWamides and metamorphosis in Hydractinia echinata

Peptides are increasingly attracting attention as primary signals in the control of development. Even though a large number of peptides have been characterized in cnidarians, little experimental evidence addresses their endogenous role. The life cycle of Hydractinia echinata includes metamorphosis from planula larva into the adult stage of the polyp. This process of stage conversion includes internal signalling, which controls cell cycle activity, cell differentiation, cell death and proportion-controlled morphogenesis. LWamide peptides are considered to be part of the control system. We implemented methods to silence gene activity by dsRNAi in Hydractinia and show a substantial knock-down of LWamide gene activity. In addition, LWamide function was knocked-out pharmacologically by targeting the biosynthesis of amidated peptides and thus preventing functional LWamides. Here we show that extinction of bioactive LWamides from planulae causes loss of metamorphosis competence, a deficiency which can be rescued by synthetic LWamide peptides. Thus, it is shown that LWamides are indispensable and act by conveying outer metamorphosis stimuli to target cells within the animal. Considering non-availability of genetic analysis and the so-far limited success in expressing transgenes in hydroids, gene functions are difficult to analyse in hydroids. The approach as outlined here is suitable for functional analysis of genes encoding amidated peptides in hydroids.     

Biosynthesis of a nonphysiological sialic acid in different rat organs, using N-propanoyl-D-hexosamines as precursors.

In this study it could be shown that in rat the normally occurring N-acetyl neuraminic acid can be modified in its N-acyl moiety by in vivo administration of the chemically synthesized N-propanoyl precursors, N-propanoyl-D-glucosamine or N-propanoyl-D-mannosamine. It could be shown that each of these nonphysiological amino sugar analogues was incorporated into both membrane and serum glycoproteins. After treatment of rats with radiolabeled N-[acyl-1-14C]D-mannosamine, radioactivity could be removed from serum glycoprotein fractions by incubation with neuraminidase from Clostridium perfringens or from Arthrobacter ureafaciens. Mild acid hydrolysis removed 98% of the radioactivity after in vivo labeling with N-[acetyl-1-14C]D-mannosamine and 86% after labeling with N-[propanoyl-1-14C]D-mannosamine. Chromatographic analysis yielded two compounds, i.e. N-acetyl neuraminic acid and N-propanoyl neuraminic acid, the latter being identified by gas liquid chromatography/mass spectrometry studies. Measurement of protein-bound radioactivity in different rat organs revealed a different organotropy of the natural and the nonphysiological neuraminic acid precursor. Of the glucosamine derivatives, N-acetyl-D-glucosamine showed the higher rate of uptake and incorporation in most organs (except in the submandibulary gland), and especially in kidney cortex and Morris hepatoma 7777. Natural and the unphysiological mannosamine derivatives were incorporated at similar rates, except in liver, where N-acetyl-D-mannosamine was taken up and metabolized more effectively. This finding indicates that it is possible to modify the acyl group of N-acetyl neuraminic acid in vivo by the introduction of an N-propanoyl group and possibly other homologous N-acyl groups. This procedure may provide a tool for a further characterization of the biological function of sialic acids.     

Microencapsulation of hybridomas by cellulose sulfate-polydimethyldiallylammonium chloride procedure

Two hybridoma cell lines of human origin and two of murine origin were encapsulated using the cellulose sulfate-polydimethyldiallylammonium chloride procedure. All lines could be cultivated in capsules, but the time of growth and product synthesis were limited. The membranes formed were impermeable to albumin and transferrin over a period of 240 minutes. Therefore, the stop of cell proliferation is assumed to be caused by the lack of at least these two proteins. The cell densities inside the capsules reached a maximum of 4.1 × 10⁶/ml (human line) and 3.9 × 10⁷/ml (murine line). The maximum product concentration in the capsules measured by IgG- and IgM-ELISA was 0.17 mg/ml (human line) and 7 mg/ml (murine line). The calculated corresponding retention was 93% (human IgM) and 52% (murine IgG). The characterization of the product showed that the immunoglobulin was not intact and might have been destroyed during harvest. Using the same cell lines the method was compared with the alginate-PLL procedure originally developed by LIM and SUN [1].     

Immunoelectron cytochemical localization of motilin and substance P in rabbit bile duct enterochromaffin (EC) cells

Summary Using an immunoreactive technique the two peptides, motilin and Substance P, have been localized at the ultrastructural level in enterochromaffin (EC) cells. Motilin occurs in cells containing a mixed population of biconcave and round secretory granules whereas Substance P is found in cells with exclusively round granules. These observations confirm the existence of at least two functionally and morphologically different types of EC cell in rabbit bile duct, both of which contain 5-hydroxytryptamine. Classification of the endocrine cells of the gut on a purely morphological basis is clearly impossible, however.On leave from the Department of Pathology, University of Basle, in receipt of a grant from the Swiss Academy of Medical Sciences     

The chromatin structure of the lysozyme GAS41 origin of DNA replication changes during the cell cycle

We used a rapid and simple protocol using lysolecithin for mapping HS sites in vivo. The protocol is based on partial digestion with DNase I of exponentially growing cells following permeabilization by short treatment with lysolecithin. Using this protocol, we analyzed the chromatin structure of the region surrounding two overlapping elements, an origin of bidirectional DNA replication and the GAS41 promoter, in chicken myelomonocytic HD11 cells arrested in G0, G1 and S phases as well as at the G1/S border. The results show that the chromatin of this region became more nuclease sensitive when cells were arrested in G1 phase and that this change in chromatin structure was reversible after the cells began to enter S phase.