Analysis of mice deficient in late endosomal SNARE proteins VAMP8/endobrevin and Vti1b

Bei der Fusion von Membranen kommt es zu einem synchronisierten Zusammenspiel von verschiedenen Proteinen. SNAREs spielen dabei eine zentrale Rolle. SNARE-Proteine, die sowohl auf der Vesikel-, als auch auf der Zielmembran verankert sind, interagieren dabei in eine coil-coiled-Struktur miteinander und bilden somit den Core-Komplex aus. Vamp8 ("Vesicle associated-membrane-protein-8), auch als Endobrevin bezeichnet, ist ein säugerspezifisches R-SNARE, das an zwei Komplexen beteiligt ist. Zum einen trägt es zur Fusion der späten Endosomen in Zusammenspiel mit Syntaxin8, Syntaxin7 und Vti1b bei. Zweitens bildet Vamp8 zusammen mit SNAP23 und Syntaxin4 einen Komplex, welcher die Exozytose der zymogenen Granular in dem Acinarzellen des Pankreas vermittelt.Ziel dieser Arbeit war die Charakterisierung der physiologischen Funktion von Vamp8 unter Verwendung entsprechender Knockout-Mäusen. Vamp 8 -/- Mäuse entwickeln sich bis zur Geburt normal. In einem Alter von 10 bis 12 Tagen sterben jedoch ein Drittel der Tiere. Diese Mäuse weisen einen fortschreitenden Gewichtsverlust auf, der erstmals ab einem Alter von 8-9 Tagen zu beobachten ist. Die Tiere können in zwei Gruppen eingeteilt werden: Zur Gruppe 1 zählen die Mäuse, die sich später, trotz des geringeren Gewichtes, zu normalen adulten Mäusen entwickeln. In Gruppe 2 kommt es bei den Tieren innerhalb von wenigen Tagen zu einem dramatischen Verlauf des Gewichtsverlustes, der zum Tod führt. Kennzeichen dieser nicht lebensfähigen Vamp8-/- Mäuse ist ein nur halb so großes Körpergewicht gegenüber den Wurfgeschwistern und ein extrem kleiner Thymus Dessen Medulla ist reduziert und eine deutliche Abgrenzung zum Cortex ist nicht zu erkennen. Die sich entwickelnden Thymozyten, einschließlich der CD4-CD8- DN1-4 und CD4/CD8 Population, zeigen eine massive Störungen in der Reifung. Der Thymus der nicht überlebensfähigen Vamp8-/- Tiere weist eine hohe Anzahl an bereits toten Zellen auf, während die Thmyozyten der kleinen, aber gesunden Knockout-Mäuse sensitiver gegenüber apoptotischen Stimuli in vitro reagieren. Es konnte gezeigt werden, dass sich hämatopoetische Vorläuferzellen aus dem Knochenmark der kleinen und kranken Knockout-Mäusen bei Transplantation in RAG2-/-γc-/- Tiere zu funktionsfähigen T-und B-Lymphozyten entwickeln. Dies ist ein Hinweis darauf, dass ein Defekt in den Stromazellen vorliegt, während die thymozytischen Vorläuferzellen nicht beeinflusst werden. Unabhängig davon weisen Vamp8 doppelt-defiziente Mäuse keine Schäden innerhalb der Exozytose, des endosomalen Transports, Phagozytose und der lysosomalen Degradation auf. Dies gibt wiederum einen Hinweis darauf, dass möglicher weise Vamp8 keine essentielle Rolle während der späten Endosomen-Fusionsereignisse spielt

Standardization of protocol for in vitro multiplication of rose (Rosa × hybrida) cv. Happiness

An efficient protocol for in vitro multiplication of Rosa × hybrida L. cv. Happiness was standardized using axillary bud segments. Out of different pre treatments for explants, the highest explant survival (80.25%) was obtained with T1 pre-treatment comprising 0.2% Carbendazim + 0.2% Mancozeb-45 +150 mg/l 8-HQC for 4 hragitation on a horizontal shaker (200 rpm). Sucrose concentration of 30g/l in the medium was found to be optimum for in vitro shoot multiplication. Murashige and Skoog (MS) medium supplemented with 2.5 mg/l BAP + 5.0 mg/l kinetin + 0.1 mg/l NAA + 0.5 mg/l GA3 was found most effective for culture establishment, however, MS medium comprising 2.5 mg/l BAP + 2.5 mg/l kinetin + 0.1mg/l NAA+ 0.5mg/l GA3 along with 40 mg/l adenine sulphate was found to be better for shoot proliferation with highest number of micro shoots (7.10 shoots/explant). Rooting of micro shoots was induced on half strength MS basal medium supplemented with NAA (0.5 mg/l) and IBA (0.5 mg/l) rooting growth regulators. The regenerated plantlets were efficiently hardened in glass jars filled with coco peat + vermiculite + perlite (2:1:1) moistened with half strength MS medium salts and covered with polypropylene lids, thereafter, plants were successfully transferred to the glasshouse with good survival

Genetic diversity analysis of chrysanthemum (Chrysanthemum grandiflorum) cultivars using RAPD markers

Identification and characterization of new cultivars is essential to meet DUS testing, address IPR issues and their utilization and conservation. Traditionally, morphological markers were used for germplasm characterization as being simple and irreplaceable. A total of 57 RAPD primers were screened and out of these 22 primers which gave sufficient amplification were selected for the study. Out of 207 bands generated, 175 were polymorphic with a polymorphism percentage of 84.54%. The polymorphism percentage ranged between 60% (OPA04) to 100% (OPA17, OPB-06, OPX14, OPX-19). The primer OPA08 generated least numbers of bands (4) and OPA06 generated maximum number of bands (17). The Rp values ranged between 0.82 to 8.18 for RAPD primers OPY-4 and OPX16, respectively with a mean value of 3.94 and the correlation between Rp and number of cultivars identified by each primer was fairly high (0.96). The primers OPX16, OPY6 and OPA04 distinguished higher number of cultivars of 49, 46 and 37 respectively. The UPGMA dendrogram indicated that the cultivars Maghi Orange and Maghi Yellow were sharing the maximum similarity (89%) and were close to Maghi White at a similarity level of 86%. The other cultivars sharing higher similarity levels were Flirt with Yellow Gold, Pink Cloud with Korean Small and Sadbhawna with Jubilee. RAPD proved to be useful for the characterization of the genotypes for their efficient utilization, management and IPR protection

Determination of genetic variation for vegetative and floral traits in African marigold (Tagetes erecta)

Twenty-one genotypes of African marigold were (Tagetes erecta L.) evaluated for 11 growth and flowering related traits to study their genetic parameters such as variability, heritability, genetic (GCV), phenotypic (PCV) coefficient of variation and correlation and path coefficient analysis. Analysis of variance for all the traits showed significant differences among genotypes for all the growth and flowering related traits. High range in mean performance has been observed for traits, viz. plant height (64.00-106.67 cm), plant spread (49.33-72.00 cm), flower diameter (3.77- 6.17 cm), days required for flowering (78.67-99.33 days), number of secondary branches (22.13-37.47) and flower duration (26.00-44.83 days). Higher genotypic and phenotypic coefficient of variation was observed for traits such as fresh flower weight per plant, flower fresh weight per 10 flowers, number of flowers per plant, stem girth, flowering duration, etc. The high value (> 90%) of heritability was observed for all traits except plant height, plant spread and stem girth. The genetic advance was found ranged from 1.23 for flower diameter to 288.69 for fresh flower weight per plant. High values of genetic advance as per cent of mean was recorded for number of flowers (59.79%) followed by fresh flower weight per plant (59.32%) and flower fresh weight per 10 flowers (58.09%). Fresh flower weight per plant is significantly and positively correlated both at genotypic and phenotypic level for plant spread, flower fresh weight per flower, number of flowers per plant and flower diameter. Path coefficient analysis at genotypic level revealed that the number of primary branches per plant contributed highest and has significantly positive direct effect on fresh flower weight per plant followed by number of flowers per plant, flower diameter, flower fresh weight per flower, days required for flowering and stem girth. The different genotypes were identified to be performing differently for different quantitative traits. Hence, those genotypes with superior traits could be involved in the hybridization programme for assembling of desirable traits in a single genotype

The Exocytosis of Lytic Granules Is Impaired in Vti1b- or Vamp8-Deficient CTL Leading to a Reduced Cytotoxic Activity following Antigen-Specific Activation

Dressel R, Elsner L, Novota P, Kanwar N, Fischer von Mollard G. The Exocytosis of Lytic Granules Is Impaired in Vti1b- or Vamp8-Deficient CTL Leading to a Reduced Cytotoxic Activity following Antigen-Specific Activation. JOURNAL OF IMMUNOLOGY. 2010;185(2):1005-1014

Effect of polymer microstructure on the docetaxel release and stability of polyurethane formulation

Abstract not availableMohsin Shaikh, Namita Roy Choudhury, Robert Knott, Jagat Rakesh Kanwar, Sanjay Gar

<span style="font-size:11.0pt;font-family: "Times New Roman";mso-fareast-font-family:"Times New Roman";mso-bidi-font-family: Mangal;mso-ansi-language:EN-GB;mso-fareast-language:EN-US;mso-bidi-language: HI" lang="EN-GB">Molecular fingerprinting and assessment of genetic diversity in rose (<i style="mso-bidi-font-style:normal">Rosa </i>× <i style="mso-bidi-font-style: normal">hybrida</i>)</span>

518-524Rose (Rosa × hybrida) is economically important floricultural crop and represents a major product for commercial floriculture market as well as oil industry. Understanding its genetic diversity is important for crop improvement programmes, while DNA fingerprinting for accurate identification of clones, cultivars and varieties. In the present investigation, genetic variability among 32 rose cultivars were analysed by using 20 RAPD and 10 ISSR markers, where a total of 306 bands were detected with a range of 6 to 18 bands per marker. ISSR markers were found to be more useful since the average polymorphic information content<span style="mso-bidi-font-weight: bold"> (PIC), resolving power of primer (RP) and marker index (MI) was better compared to that of RAPD markers. <span style="mso-bidi-font-weight: bold">UPGMA-cluster-analysis based on genetic distance coefficients ranged 0.44 to 0.91, clearly distinguished all the genotypes<span style="mso-bidi-font-style: italic">, including Suryodaya and Suryakiran, which are morphologically almost identical. Thus the molecular fingerprinting data of rose cultivars generated in the present study would be helpful for the varietal identification and conservation. Moreover, results of diversity analysis suggested that the cultivated rose varieties display a high level of genetic variability despite the fact that the morphological and physiological characters were found to be less polymorphic. </span

SHP-1 Phosphatase Is a Critical Regulator in Preventing Natural Killer Cell Self-Killing

<div><p>Balance of signals generated from the engaged activating and inhibitory surface receptors regulates mature NK cell activities. The inhibitory receptors signal through immunoreceptor tyrosine based inhibitory motifs (ITIM), and recruit phosphatases such as SHP-1 to inhibit NK cell activation. To directly examine the importance of SHP-1 in regulating activities and cell fate of mature NK cells, we used our established lentiviral-based engineering protocol to knock down the SHP-1 protein expression in primary C57BL/6NCrl cells. Gene silencing of the SHP-1 in primary NK cells abrogated the ability of ITIM-containing NK inhibitory receptors to suppress the activation signals induced by NK1.1 activating receptors. We followed the fates of stably transduced SHP-1 silenced primary NK cells over a longer period of time in IL-2 containing cultures. We observed an impaired IL-2 induced proliferation in the SHP-1 knockdown NK cells. More interestingly, these “de-regulated” SHP-1 knockdown NK cells mediated specific self-killing in a real-time live cell microscopic imaging system we developed to study NK cell cytotoxicity in vitro. Selective target recognition of the SHP-1 knockdown NK cells revealed also possible involvement of the SHP-1 phosphatase in regulating other NK functions in mature NK cells.</p> </div

Specific recognition of the SHP-1 gene knockdown NK cells as targets.

<p>SHP-1 shRNA transduced and puromycin selected purified NK cells mixed with green CMFDA-labelled normal C57BL/6NCrl NK cells at 1∶1 ratio, resuspended in Hanks Buffered Salt Solution (HBSS) with 10% FCS and 50 U/ml IL-2 containing 7AAD. Cells were imaged every 25 seconds for up to 5 hours using 10X magnification objective on a Zeiss Observer 710 station. The images in the figure were taken from the live cell imaging videos (Video S7) at different time points. Red box in insert A indicated real cell conjugate formation between two unlabelled NK cells. These conjugated cells can be tracked 46 minutes later (red box, insert B) to show subsequent cytolysis as the dying cells took up the 7-AAD dye. Orange box indicated transient aggregates that disintegrated at later time points, and showed no evidence of cytolysis. At late time-points (such as 150 minutes of imaging), more apoptotic cells (red) were observed in the unlabelled SHP-1 knockdown NK cells (Blue box).</p