Direct Electron Microscopy Study on the Morphological Diversity of Bacteriophage Populations in Lake Plußsee

Direct electron microscopy of bacteriophages adsorbed to a carbon film without prior enrichment by specific host strains or concentration by physical or chemical methods was used to study the morphological diversity of natural bacteriophage assemblages in a North German lake. All samples contained a mixture of morphologically different tailed viruses, which were regarded as bacteriophages. Most of them had isometric heads and long noncontractile tails, belonging to morphotype B1 (Siphoviridae). In addition, members of morphotypes A1 (Myoviridae), B2 (Siphoviridae with elongated heads), and C1 (Podoviridae) were present in lower numbers. Only one cubic virus was detected, while no filamentous or pleomorphic phages were found. Up to 11 different phages per sample, and a total of 39 phages when all samples were considered together, could be distinguished by morphological criteria. The total number of phages was estimated to be on the order of 108/ml

Arkeologisk undersøkelse av bosetningsspor fra eldre bronsealder, romertid og folkevandringstid på Mosterøy

Oppdragsgiver: Statens vegvesen, region vestArkeologisk museum, Universitetet i Stavanger gjennomførte en arkeologisk undersøkelse etter kulturminnelovens § 8 og 10 i tidsrommet 25.07.2016 – 02.11. 2016 av 4 automatisk fredete lokaliteter på Mosterøy, Rennesøy kommune. Undersøkelsesområdet lå på sørsiden av Mosterøyveien (Fv 561) mellom gårdene Vold og Kåda. Undersøkelsen omfattet følgende arkeologiske kulturminner i kulturminnedatabasen «Askeladden»: ID 170565, ID 170445, ID 170569 og ID 170572. En rekke kulturminner fra forskjellige perioder ble oppdaget og dokumentert. De viktigste av disse var: • Et toskipet hus fra eldre bronsealder på lokalitet ID170565 3 • Deler av minst et treskipet langhus fra romertid med tilhørende strukturer på lokalitet ID 170445 • Velbevarte rester av et treskipet langhus fra yngre romertid / folkevandringstid med bevarte gulvlag og komplekse strukturer i tilknytning til huset på lokalitet ID 170569 • Tekniske anlegg eller groper med spor av handverk og metallbearbeidelse fra middelalder og eldre jernalder på lokalitet ID 17056

Etablierung einer Methode zum Nachweis der Assoziation von Legionella sp. mit A / A PCR-method for the detection of the association between Legionella sp. and Amoeba sp.

Abstract With two pairs of primers for the amplification of the MIP- (macrophage infectivity potentiator) and the SS rDNA-fragment, it was possible to establish a DNA extraction and a PCR method for the detection of Legionella sp. in water-samples and, after cultivation, in Amoeba sp.. Therefore, water-samples from a warmwater-system in a hospital were taken. In all samples, legionellae were detected by the PCR method and identified by cultivation and a direct immunfluorescence-method as L. pneumophila (serogroup 1). Legionellae and amoebae of the same water sample were cocultured. Legionellae were also adherent at the outer-membrane. To separate the amoebae from the legionellae, the amoebae were sedimented selectively by centrifugation at 200 x g. This washing procedure had to be repeated seven times in order to eliminate the extraamoebale legionellae for sure. After DNA-extraction of water samples and heat treatment of the intraamoebale legionellae respectively, the amplification was performed with the MIP- and 5S rDNA-primers. In 14 of 16 cocultivations growth of legionellae was found. This result and the detection of legionellae and amoebae in the same water samples suggest that an infection of amoebae may also take place in the watersystem of the hospital. This is important for the disinfection as a procedure to eliminate legionellae, since intraamoebale bacteria are more resistant to environmental manipulation. Because in two of the cocultivations no growth of legionellae in amoebae was found, it can be assumed that only specific subtypes of legionellae can infect amoebae species. Zusammenfassung Zum Nachweis von Legionelia sp. aus Wasserproben sowie von intrazellulären Legionellen nach Kokultur mit Amoeba sp. konnte ein DNA-Extraktionsverfahren und eine Po- lymerasekeftenreaktion (PCR)-Methode mit Hilfe der Primerpaare für ein Fragment des 5S rDNA-Gcns und ein Fragment des iVllP (macrophage infectivity potentiator)-Gens entwickelt werden. Als Untersuchungsmaterial dienten Proben aus dem Warmwasserleitungssystem eines Krankenhauses. Zunächst wurden mit Hilfe eines DNA-Extraktionsverfahrcns und der PCR-Methode in allen Wasserproben Legionellen nachgewiesen und mittels direkter Immunfluoreszenz als Serogruppe 1 identifiziert. Anschließend wurden Legionellen und Amöben, die aus derselben Wasserprobe isoliert wurden, kokultiviert. Amöben ließen sich nach dieser Kokultivierung durch Zentrifugation bei 200 X g selektiv sedimentieren und dadurch von extrazellulären Legionellen befreien. Dieser Waschvorgang mußte mindestens siebenmal durchgeführt werden, um sicher alle freien, bzw. außen an den Amöben haftenden Legionellen zu entfernen. Nach Extraktion der DNA bzw. LIitzebehandlung der intraamöbalen Bakterien erfolgte der Nachweis durch Amplifizierung legionellenspezifischer 5S rDNA- und MIP-Fragmente. Von 16 angelegten Kokulturcn konnte in 14 ein intraamöbales Wachstum der Legionellen gezeigt werden. Dieses Resultat in Kombination mit dem simultanen Vorkommen von Legionellen und Amöben in den Wasserproben deutet darauf hin, daß eine Infektion von Amöben mit Legionellen auch im Warmwassersystem des beprobten Krankenhauses stattfindet. Dies wäre für die Durchführung von Sanierungsmaßnahmen von Bedeutung, da in- traamöbale Legionellen gegen Desinfektionsverfahren relativ wirksam geschützt sind. Die Beobachtung, daß nicht bei allen Kokulturen ein intraamöbales Vorkommen der Legionellen gezeigt werden konnte, weist darauf hin, daß nur einige Legionellen-Serogruppen bzw. -Subtypen und/oder Amöbenspezies eine Assoziation eingehen bzw. Unterschiede in der Infektion von Amöben zwischen virulenten und avirulenten Legionellen bestehen

Neuropeptide Y (NPY) cleaving enzymes : structural and functional homologs of dipeptidyl peptidase 4

N-terminal truncation of NPY has important physiological consequences, because the truncated peptides lose their capability to activate the Y1-receptor. The sources of N-terminally truncated NPY and related peptides are unknown and several proline specific peptidases may be involved. First, we therefore provide an overview on the peptidases, belonging to structural and functional homologues of dipeptidyl peptidase 4 (DP4) as well as aminopeptidase P (APP) and thus, represent potential candidates of NPY cleavage in vivo. Second, applying selective inhibitors against DP4, DP8/9 and DP2, respectively, the enzymatic distribution was analyzed in brain extracts from wild type and DP4 deficient F344 rat substrains and human plasma samples in activity studies as well as by matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF)-mass spectrometry. Third, co-transfection of Cos-1 cells with Dpp4 and Npy followed by confocal lasermicroscopy illustrated that hNPY-dsRed1-N1 was transported in large dense core vesicles towards the membrane while rDP4-GFP-C1 was transported primarily in different vesicles thereby providing no clear evidence for co-localization of NPY and DP4. Nevertheless, the review and experimental results of activity and mass spectrometry studies support the notion that at least five peptidases (DP4, DP8, DP9, XPNPEP1, XPNPEP2) are potentially involved in NPY cleavage while the serine protease DP4 (CD26) could be the principal peptidase involved in the N-terminal truncation of NPY. However, DP8 and DP9 are also capable of cleaving NPY, whereas no cleavage could be demonstrated for DP2

Proteolytic degradation of neuropeptide Y (NPY) from head to toe: Identification of novel NPY-cleaving peptidases and potential drug interactions in CNS and Periphery.

The bioactivity of neuropeptide Y (NPY) is either N-terminally modulated with respect to receptor-selectivity by dipeptidyl-peptidase 4 (DP4)-like enzymes or proteolytic degraded by neprilysin or meprins, thereby abrogating signal transduction. However, neither the subcellular nor the compartmental differentiation of these regulatory mechanisms is fully understood. Using mass spectrometry, selective inhibitors and histochemistry, studies across various cell types, body fluids and tissues revealed that most frequently DP4-like enzymes, aminopeptidases P (AmpP), secreted meprin-A (Mep-A) and cathepsin D (CTSD) rapidly hydrolyze NPY, depending on the cell type and tissue under study. Novel degradation of NPY by cathepsins B, D, L, G, S and tissue kallikrein could also be identified. Expression of DP4, CTSD, and Mep-A at the median eminence indicates that the bioactivity of NPY is regulated by peptidases at the interphase between the periphery and the CNS. Detailed ex vivo studies on human sera and CSF samples recognized CTSD as the major NPY-cleaving enzyme in the CSF, whereas an additional C-terminal truncation by angiotensin-converting enzyme (ACE) could be detected in serum. The latter finding hints to potential drug interaction between antidiabetic DP4 inhibitors and anti-hypertensive ACE inhibitors, while it ablates suspected hypertensive side-effects of only antidiabetic DP4-inhibitors application. This article is protected by copyright. All rights reserved

Phenotyping of congenic dipeptidyl peptidase 4 (DP4) deficient Dark Agouti (DA) rats suggests involvement of DP4 in neuro-, endocrine, and immune functions

Background: Treatment of diabetes type 2 using chronic pharmacological inhibition of dipeptidyl peptidase 4 (DP4) still requires an in-depth analysis of models for chronic DP4 deficiency, because adverse reactions induced by some DP4 inhibitors have been described. Methods: In the present study, a novel congenic rat model of DP4 deficiency on a “DP4-high” DA rat genetic background was generated (DA.F344-Dpp4 m/SvH rats) and comprehensively phenotyped. Results: Similar to chronic pharmacological inhibition of DP4, DP4 deficient rats exhibited a phenotype involving reduced diet-induced body weight gain and improved glucose tolerance associated with increased levels of glucagon-like peptide-1 (GLP-1) and bound leptin as well as decreased aminotransferases and triglycerides. Additionally, DA.F344-Dpp4 m/SvH rats showed anxiolytic-like and reduced stress-like responses, a phenomenon presently not targeted by DP4 inhibitors. However, several immune alterations, such as differential leukocyte subset composition at baseline, blunted natural killer cell and T-cell functions, and altered cytokine levels were observed. Conclusions: While this animal model confirms a critical role of DP4 in GLP-1-dependent glucose regulation, genetically induced chronic DP4 deficiency apparently also affects stress-regulatory and immune-regulatory systems, indicating that the use of chronic DP4 inhibitors might have the potential to interfere with central nervous system and immune functions in vivo