Cytokine Requirements for Acute and Basal Homeostatic Proliferation of Naive and Memory CD8+ T Cells

Both naive and memory T cells undergo antigen-independent proliferation after transfer into a T cell–depleted environment (acute homeostatic proliferation), whereas only memory T cells slowly divide in a full T cell compartment (basal proliferation). We show, first, that naive and memory CD8+ T cells have different cytokine requirements for acute homeostatic proliferation. Interleukin (IL)-7 receptor(R)α–mediated signals were obligatory for proliferation of naive T cells in lymphopenic hosts, whereas IL-15 did not influence their division. Memory T cells, on the other hand, could use either IL-7Rα– or IL-15–mediated signals for acute homeostatic proliferation: their proliferation was delayed when either IL-7Rα was blocked or IL-15 removed, but only when both signals were absent was proliferation ablated. Second, the cytokine requirements for basal and acute homeostatic proliferation of CD8+ memory T cells differ, as basal division of memory T cells was blocked completely in IL-15–deficient hosts. These data suggest a possible mechanism for the dearth of memory CD8+ T cells in IL-15– and IL-15Rα–deficient mice is their impaired basal proliferation. Our results show that naive and memory T lymphocytes differ in their cytokine dependence for acute homeostatic proliferation and that memory T lymphocytes have distinct requirements for proliferation in full versus empty compartments

Construction of a biosynthetic mutant of Amycolatopsis balhimycina via module insertion in the non-ribosomal peptide synthetase BpsB using a combinatorial biosynthetic approach and its mass spectrometric characterization

Herstellung einer Biosynthesemutante von Amycolatopsis balhimycina durch Modulinsertion in die nicht-ribosomale Peptidsynthetase BpsB mittels eines kombinatorisch-biosynthetischen Ansatzes und deren massenspektro-metrische Charakterisierung Die vorliegende Arbeit beschäftigt sich mit der kombinatorischen Biosynthese bei nicht-ribosomalen Peptidsynthetasen (NRPS), die aufgrund der Komplexität der Strukturen von nicht-ribosomal synthetisierten Peptiden eine ernstzunehmende Alternative zur chemischen Synthese darstellt, um Strukturderivate zu erzeugen. Ziel dieser Arbeit war die Komplettierung der bisher bei NRPS literaturbekannten Modul-manipulationen „Modulaustausch“ und „Moduldeletion“ durch das noch ausstehende Experiment der „Modulinsertion“. Als Modellverbindung diente das von A. balhimycina synthetisierte Vancomycin-Typ Glykopeptidantibiotikum Balhimycin, welches durch sein stark modifiziertes Peptidgrundgerüst (Chlorierung, oxidative Seitenkettenzyklisierung, N-Methylierung und Glykosylierung) ein sehr komplexes Beispiel einer nicht-ribosomalen Peptidsynthese darstellt. In der vorliegenden Arbeit ist es gelungen, ein zusätzliches Modul in die NRPS BpsB von A. balhimycina über eine double crossover-Strategie einzuführen, was sowohl auf genetischer Ebene mittels PCR und Southern Blot-Hybridisierung als auch auf Proteinebene mittels Disk-SDS-PAGE und anschließende massenspektrometrische Identifizierung der Gelbande nachgewiesen werden konnte. Daran schloss sich eine eingehende Charakterisierung des Produktionsprofils dieser kombinatorischen Biosynthese-Mutante mittels HPLC-ESI-MS und -MS/MS an. Diese Analyse erbrachte den Beweis, dass das eingeführte Modul auch enzymatische Aktivität besitzt und somit das Peptidrückgrat des Balhimycins um eine Aminosäure erweitert. Neben dem Beweis der Machbarkeit einer Modulinsertion bei NRPS gewährte dieses kombinatorische Biosynthese-Experiment auch Einblicke in die Flexibilität des gesamten Biosyntheseapparates. Beispielsweise war die Chlorierung der Produkte durch die Halogenase BhaA nicht beeinträchtigt, während N-Methylierung und Glykosylierung in allen 17 nachgewiesenen Biosyntheseprodukten fehlten. Bezüglich der oxidativen Seitenkettenzyklisierungen brachte die erste P450-abhängige Monooxygenase OxyB noch eine gewisse Toleranz für das neue Substrat bzw. die veränderte NRPS auf, während OxyA und OxyC keine nachweisbare Aktivität mehr zeigten.The topic of the thesis is combinatorial biosynthesis with non-ribosomal peptide synthetases (NRPSs).Whereas module exchanges and module deletions are manipulations already known in literature, no example of a successful module insertion in a NRPS has been published. The model system for such an experiment was Amycolatopsis balhimycina producing the vancomycin-type glycopeptide antibiotic balhimycin which is characterized by diverse modifications of the peptidic backbone (chlorination, oxidative side-chain cyclization, N-methylation, glycosylation). The insertion of an additional module in the NRPS BpsB of Amycolatopsis balhimycina was obtained via a double crossover strategy and could be verified on the genetic and proteinogenic level. The subsequent mass spectrometric characterization of the production profile of this mutant showed the expected amino acid insertion in the peptidic backbone of balhimycin. This was the proof that the new module bears enzymatic activity

Orchard pollination in Capitol Reef National Park, Utah, USA. Honey bees or native bees?

Capitol Reef National Park in central Utah, USA surrounds 22 managed fruit orchards started over a century ago by Mormon pioneers. Honey bees are imported for pollination, although the area in which the Park is embedded has over 700 species of native bees, many of which are potential orchard pollinators. We studied the visitation of native bees to apple, pear, apricot, and sweet cherry over 2 years. Thirty species of bees visited the flowers but, except for pear flowers, most were uncommon compared to honey bees. Evidence that honey bees prevented native bees from foraging on orchard crop flowers was equivocal: generally, honey bee and native bee visitation rates to the flowers were not negatively correlated, nor were native bee visitation rates positively correlated with distance of orchards from honey bee hives. Conversely, competition was tentatively suggested by much larger numbers of honey bees than natives on the flowers of apples, apricots and cherry; and by the large increase of native bees on pears, where honey bee numbers were low. At least one-third of the native bee species visiting the flowers are potential pollinators, including cavity-nesting species such as Osmia lignaria propinqua, currently managed for small orchard pollination in the US, plus several fossorial species, including one rosaceous flower specialist (Andrena milwaukiensis). We suggest that gradual withdrawal of honey bees from the Park would help conserve native bee populations without decreasing orchard crop productivity, and would serve as a demonstration of the commercial value of native pollinators

The Effect of Child Benefit Policies on Fertility and Female Labor Force Participation in Canada

fertility, child benefits, time series models, cointegration,