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.     

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.     

Structural basis for differential receptor binding of cholera and Escherichia coli heat-labile toxins: influence of heterologous amino acid substitutions in the cholera B-subunit

The closely related B-subunits of cholera toxin (CTB) and Escherichia coli heat-labile enterotoxin (LTB) both bind strongly to GM1 ganglioside receptors but LTB can also bind to additional glycolipids and glycoproteins. A number of mutant CT B-subunits were generated by substituting CTB amino acids with those at the corresponding positions in LTB. These were used to investigate the influence of specific residues on receptor-binding specificity. A mutated CTB protein containing the first 25 residues of LTB in combination with LTB residues at positions 94 and 95, bound to the same extent as native LTB to both delipidized rabbit intestinal cell membranes, complex glycosphingolipids (polyglycosylceramides) and neolactotetraosylceramide, but not to non-GM1 intestinal glycosphingolipids. In contrast, when LTB amino acid substitutions in the 1–25 region were combined with those in the 75–83 region, a binding as strong as that of LTB to intestinal glycosphingolipids was observed. In addition, a mutant LTB with a single Gly-33→Asp substitution that completely lacked affinity for both GM1 and non-GM1 glycosphingolipids could still bind to receptors in the intestinal cell membranes and to polyglycosylceramides. We conclude that the extra, non-GM1 receptors for LTB consist of both sialylated and non-sialylated glycoconjugates, and that the binding to either class of receptors is influenced by different amino acid residues within the protein.     

Galanin attenuates retention of one-trial reward learning.

Galanin is a neuropeptide that coexists with acetylcholine in the septohippocampal pathway. Galanin appears to have a negative modulating influence on cholinergic transmission, suggesting that it might interfere with memory formation on a one-trial discriminative reward learning task. The apparatus was a starburst maze with five radiating alleys, one an ascending baited alley. The subjects were 38 two to three month old Sprague-Dawley rats cannulated in the body of the lateral ventricles and deprived to 80% of initial weight. Ten rats were infused i.c.v. over six mins. with 8 micrograms galanin in 24 microliters saline and 10 with saline alone. Twenty mins. after completion of infusion, each rat was placed in the maze and observed under "blind" conditions for number of errors (blind alleys entered) and latency to reach reward. Each rat's speed score was 100 sec./latency. One day later, each rat was retested in the maze. Each rat's retention scores were its decrease in errors and increase in speed between the single training trial and the retention trial. Galanin infused rats showed significantly less retention by both measures. In a second experiment, either the same dose of galanin or saline alone was infused 20 mins. before the retention trial. There was no significant effect, suggesting that galanin may interfere with memory formation rather than memory retrieval or task performance.     

Site factors are more important than management for indicator species in semi-natural grasslands in southern Sweden

Plant Ecology - Management of semi-natural grasslands is essential to retain the characteristic diversity of flora and fauna found in these habitats. To maintain, restore or recreate favourable...