Flow cytometric determination of basophils in whole blood with n-propyl Astra Blue iodide

Within the past year, it has become apparent, in connection with its use on automatic flow cytometers, that the quality of commercially available Alcian Blue has significantly declined. A homologous series of alkylated (C1-C7) Astra Blue quaternary ammonium halides was prepared, characterized, and evaluated for the detection of basophils in whole blood. On the Technicon H6000 flow cytometer, the resolution of the basophil cluster from the main population of unstained white blood cells was found to depend on the chain length of the quaternizing alkyl group. Optimal basophil resolution was observed for the n-propyl derivative. Correlation of the new method vs Alcian Blue as the reference on the H6000 was expressed as follows: %Baso (Astra Blue) = 0.89% Baso (Alcian Blue) + 0.12% for 180 fresh whole blood samples. Within-run precision at a basophil differential count of 0.73% was characterized by SD = 0.11, identical to that obtained for Alcian Blue. Aqueous solutions of n-propyl Astra Blue iodide, in contrast to Alcian Blue, are thermally stable. Heating the reagent for 1 h at 100 degrees C did not alter solubility or cytochemical behavior. In contrast, parallel treatment of Alcian Blue yielded insoluble material by hydrolysis of the isothiouronium groups. The reagent for basophil detection comprises n-propyl Astra Blue iodide, lanthanum chloride, sodium chloride, Tween 20, and cetylpyridinium chloride. The Astra Blue derivatives were characterized by uv-vis, ir, percentage halide, paper chromatography, and 13C NMR.     

Fine Mapping the Spatial Distribution and Concentration of Unlabeled Drugs within Tissue Micro-Compartments Using Imaging Mass Spectrometry

Readouts that define the physiological distributions of drugs in tissues are an unmet challenge and at best imprecise, but are needed in order to understand both the pharmacokinetic and pharmacodynamic properties associated with efficacy. Here we demonstrate that it is feasible to follow the in vivo transport of unlabeled drugs within specific organ and tissue compartments on a platform that applies MALDI imaging mass spectrometry to tissue sections characterized with high definition histology. We have tracked and quantified the distribution of an inhaled reference compound, tiotropium, within the lungs of dosed rats, using systematic point by point MS and MS/MS sampling at 200 µm intervals. By comparing drug ion distribution patterns in adjacent tissue sections, we observed that within 15 min following exposure, tiotropium parent MS ions (mass-to-charge; m/z 392.1) and fragmented daughter MS/MS ions (m/z 170.1 and 152.1) were dispersed in a concentration gradient (80 fmol-5 pmol) away from the central airways into the lung parenchyma and pleura. These drug levels agreed well with amounts detected in lung compartments by chemical extraction. Moreover, the simultaneous global definition of molecular ion signatures localized within 2-D tissue space provides accurate assignment of ion identities within histological landmarks, providing context to dynamic biological processes occurring at sites of drug presence. Our results highlight an important emerging technology allowing specific high resolution identification of unlabeled drugs at sites of in vivo uptake and retention.     

Identification and design of a novel series of MGAT2 inhibitors

[Acyl CoA]monoacylglycerol acyltransferase 2 (MGAT2) is of interest as a target for therapeutic treatment of diabetes, obesity and other diseases which together constitute the metabolic syndrome. In this Letter we report our discovery and optimisation of a novel series of MGAT2 inhibitors. The development of the SAR of the series and a detailed discussion around some key parameters monitored and addressed during the lead generation phase will be given. The in vivo results from an oral lipid tolerance test (OLTT) using the MGAT2 inhibitor (S)-10, shows a significant reduction (68% inhibition relative to na?ve, p <0.01) in plasma triacylglycerol (TAG) concentration.     

The E3/19K protein of adenovirus type 2 binds to the domains of histocompatibility antigens required for CTL recognition

The E3/19K protein of human adenovirus type 2 binds to HLA class I antigens and blocks their terminal glycosylation and cell surface expression. The nature of this interaction is non-covalent and involves neither disulfide bridges between the two molecules nor their carbohydrates. The murine H-2 Kd antigen associates with the E3/19K protein in a similar fashion to human HLA antigens whereas the allelic product H-2 Kk does not. Hybrid genes between the Kd and Kk alleles were constructed and their products were expressed in embryonic kidney cells together with the E3/19K protein. This allowed us to identify the alpha 1 and alpha 2 domains as the essential structures of the histocompatibility antigens for binding the viral protein. Interestingly, these domains are also crucial for T cell recognition. The implications for the evolution of adenoviruses and their ability to cause persistent infections are discussed.     

Host plant recognition in monophagous weevils: specificity in feeding responses of Ceutorhynchus constrictus and the variable effect of sinigrin

Host plant relations of the monophagous weevil Ceutorhynchus constrictus Marsh. (Coleoptera: Curculionidae: Ceutorhynchinae) feeding on garlic mustard, Alliaria petiolata (Bieb.) Cavara & Grande (Cruciferae) were studied in the laboratory. Most other crucifers were rejected in choice tests using garlic mustard as a reference plant, but Brassica nigra, Sinapis alba and Thlaspi arvense were as acceptable as the host plant. Flowering plants of Descurainia sophia were acceptable while young plants of this species were not. The most important feeding stimulants in extracts of garlic mustard were uncharged, water soluble compounds. The most abundant glucosinolate in garlic mustard, sinigrin, was a feeding stimulant, too. However, the feeding stimulatory activity of sinigrin was only expressed in the presence of still unidentified uncharged compounds from garlic mustard leaves. Host plant relations in monophagous crucifer-feeding insects is discussed in relation to the distinctness of glucosinolate patterns found in their host plants.

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Zusammenfassung Ceutorhynchus constrictus Marsh. (Coleoptera: Chrysomelidae: Ceutorhynchinae) ist ein monophager Rüsselkäfer, der an Knoblauchhederich frisst. Das Wirtswahl-Verhalten dieses Käfers ist im Labor untersucht worden. Die meisten Crucifiren waren im Wahlversuche nicht akzeptiert, wenn Knoblauchhederich als Vergleichspflanze vorhanden war. Von Brassica nigra, Sinapis alba, und Thlaspi arvense wurden im Vergleich gleiche Mengen verzehrt wie von der Wirtspflanze. Blühende Descurainia sophia Pflanzen wurden, im Gegensatz zu Jungpflanzen der gleichen Art, angenommen. Die wichtichsten Phagostimulanten in Extrakten von Knoblauchhederich-Blättern waren ungeladene, wasserlösliche Substanzen. Das häufigste Glukosinolat im Knoblauchhederich, Sinigrin, war auch ein Phagostimulant. Doch war die phagostimulierende Wirkung von Sinigrin nur in Kombinationen mit noch nicht identifizierten, ungeladenen Substanzen aus Knoblauchhederich-Blätter nachweisbar. Wirtspfanzen-Beziehungen von monophagen Insekten werden diskutiert im Zusammenhang mit der Eigenart des Glukosinolat-Inhaltes ihrer Wirtspflanzen.

     

The effect of glucosinolates on responses of young Phyllotreta nemorum larvae to non-host plants

All recorded host plants of Phyllotreta nemorum L. (Coleoptera: Chrysomelidae) contain glucosinolates and belong to the plant families Brassicaceae (Cruciferae), Resedaceae and Capparaceae. The acceptability of 56 plant species from 28 other plant families (non-hosts) for young larvae has been studied in the laboratory. None of these species were fully acceptable for initiations of leaf mines when intact untreated leaves were presented, and only one species, Malva silvestris L. (Malvaceae), was partially acceptable. The acceptability of some species increased when leaf discs were presented instead of intact leaves; but the highest percentages of mine initiations occurred in leaf discs treated with the glucosinolate, sinigrin. A stimulatory effect of sinigrin could be demonstrated in experiments with 7 plant species: Papaver dubium L., Papaver rhoeas L., Fumaria officinalis L., Malva silvestris L., Pisum sativum L., Campanula latifolia L. and Lactuca sativa L. The majority of species remained unacceptable even after treatment with glucosinolates.The main causes for these differences between plant species are supposed to be differences in contents of deterrents and/or other stimulants for mine initiation. These possibilities are discussed in relation to the content of allelochemicals in acceptable plants and the position of these plants in botanical classifications.

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Zusammenfassung Phyllotreta nemorum L. ist ein oligophager Erdfloh, der an Cruciferen und anderen Glukosinolat-haltigen Pflanzenarten gebunden ist. Die Imagines fressen Löcher in die Blätter und die Weibchen legen ihre Eier in den Boden. Die Larven sind Blattminierer. Nach dem Schlüpfen im Boden klettern sie an die Pflanzen hoch, und die Einbohrung und der Anfang der Minierung erfolgt in eines der unteren Blätter der Wirtspflanze.Die Wirkung von Glukosinolaten auf die Einbohrung von Junglarven in Pflanzenarten, die keine natürliche Inhalt von Glukosinolaten haben, ist in Laborexperimenten untersucht worden. 56 Pflanzenarten aus 28 Familien wurden präsentiert teils als unbehandelte Blätter und teils als Glukosinolatbehandelte Blattscheiben. Unbehandelte Blätter von nicht-Glukosinolathaltigen Arten waren immer unbefriedigend für die Larven. Nur in eine Art, Malva silvestris L. war die Frequenz der Einbohrung ein bisschen höher als 10%. Eine signifikante Erhöherung der Anzahl eingebohrten Larven nach der Sinigrin-behandlung erfolgte in 7 Pflanzenarten: Papaver dubium L., P. rhoeas L., Fumaria officinalis L., Malva silvestris L., Pisum sativum L., Campanula latifolia L. und Lactuca sativa L. Doch blieben die meisten Pflanzenarten (84%) auch nach der Sinigrin-Behandlung unbesiedelt.Pflanzenarten, die nach der Sinigrin-Behandlung nicht besiedelt werden enthalten vielleicht frasshemmende Stoffe, oder ihnen fehlen noch weitere Frass-stimulierende Stoffe. Diese Möglichkeiten werden diskutiert in Zusammenhang mit den Inhalt von Allelochemikalien in besiedelten Pflanzenarten und mit ihrer taxonomischer Position.

     

Augmented analgesic effects of L-tryptophan combined with low current transcranial electrostimulation

The analgesic effects of low current transcranial electrostimulation are both naloxone and pCPA-reversible, suggesting that they may be mediated in part by endogenous opioid and serotonergic activity. The present experiments indicate that pretreatment with the serotonin precursor L-tryptophan results in an increased analgesic effect of electrostimulation as measured by the 50 degrees C wet tail flick test in the rat. Rats receiving both L-tryptophan and electrostimulation displayed significantly more analgesia than rats receiving electrostimulation and injection vehicle alone, rats receiving drug and sham stimulation or rats receiving vehicle and sham stimulation.     

Augmented analgesic effects of enkephalinase inhibitors combined with transcranial electrostimulation

The analgesic effects of very low current transcranial electrostimulation are naloxone-reversible and thus presumably mediated by endogenous opioid activity. The present experiments indicate that blocking enkephalinase activity by i.c.v. thiorphan or i.p. acetorphan results in an increased analgesic effect of electrostimulation as measured by the 50 degrees C wet tail flick test. In the case of each drug, rats receiving both drug and electrostimulation displayed significantly more analgesia than rats receiving electrostimulation and injection vehicle alone, rats receiving drug and sham stimulation or rats receiving vehicle and sham stimulation.     

Recolonization following past persecution questions the importance of persistent snow cover as a range limiting factor for wolverines

Globally, climate is changing rapidly, which causes shifts in many species' distributions, stressing the need to understand their response to changing environmental conditions to inform conservation and management. Northern latitudes are expected to experience strongest changes in climate, with milder winters and decreasing snow cover. The wolverine (Gulo gulo) is a circumpolar, threatened carnivore distributed in northern tundra, boreal, and subboreal habitats. Previous studies have suggested that wolverine distribution and reproduction are constrained by a strong association with persistent spring snow cover. We assess this hypothesis by relating spatial distribution of 1589 reproductive events, a fitness-related proxy for female reproduction and survival, to snow cover over two decades. Wolverine distribution has increased and number of reproductive events increased 20 times in areas lacking spring snow cover during our study period, despite low monitoring effort where snow is sparse. Thus, the relationship between reproductive events and persistent spring snow cover weakened during this period. These findings show that wolverine reproductive success and hence distribution are less dependent on spring snow cover than expected. This has important implications for projections of future habitat availability, and thus distribution, of this threatened species. Our study also illustrates how past persecution, or other factors, that have restricted species distribution to remote areas can mask actual effects of environmental parameters, whose importance reveals when populations expand beyond previously restricted ranges. Overwhelming evidence shows that climate change is affecting many species and ecological processes, but forecasting potential consequences on a given species requires longitudinal data to revisit hypotheses and reassess the direction and magnitude of climate effects with new data. This is especially important for conservation-oriented management of species inhabiting dynamic systems where environmental factors and human activities interact, a common scenario for many species in different ecosystems around the globe.