Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils

Stimulation of human neutrophils with f-met-leu-phe, platelet-activating factor, or leukotriene B4 resulted in an increase in [Ca2+]i. The [Ca2+]i rise was greater in the presence than absence of external Ca2+; the component that was dependent on external Ca2+ was blocked by Ni2+, or could be reconstituted by addition of external Ca2+ following discharge of the internal Ca2+ store. These measurements of [Ca2+]i responses provide only indirect evidence for agonist-stimulated Ca2+ entry, and here we have used an alternative approach to demonstrate directly agonist-stimulated divalent cation entry. In the presence of extracellular Mn2+, f-met-leu-phe, leukotriene B4, and platelet-activating factor stimulate a quench in fluorescence of fura-2-loaded human neutrophils. This quench was due to stimulated Mn2+ influx and was blocked by Ni2+. When Mn2+ was added in the continued presence of agonist, after discharge of the internal store of Ca2+, a stimulated quench was seen; this result shows that an elevated [Ca2+]i is not needed for the stimulation of Mn2+ entry. Depolarization by high [K+] or addition of the L-type Ca2+ channel agonist, BAY-R-5417, had little or no effect on either [Ca2+]i or Mn2+ entry. These results show that agonists stimulate divalent cation entry (Ca2+ or Mn2+) by a mechanism independent of changes in [Ca2+]i and unrelated to voltage-dependent Ca2+ channels.     

Functional comparison of hemoglobin purified by different methods and their biophysical implications

Hemoglobin (Hb) that is purified from red blood cells (RBCs) is commonly subjected to harsh processing conditions, such as high temperatures and extensive column separation, which may damage the Hb by altering the heme prosthetic group and/or the Hb protein structure. In this study, bovine and human Hb purified by tangential flow filtration (TFF) was compared to commercial preparations of human Hb (Hemosol, Inc., Toronto, Canada) and bovine Hb (Biopure, Inc., Cambridge, MA). Purified Hbs were characterized by measuring their overall purity (SDS–PAGE, SEC, and ESI‐MS), susceptibility to oxidation (k_ox), responses to physiological conditions (pH, [Cl^?], [IHP], and T), and ligand binding kinetics (O₂, NO, and CO). All Hbs evaluated possessed comparable biophysical properties, however, a small amount of catalase was detected in the TFF‐purified Hbs that reduced the rate of autoxidation. Mass changes observed by mass spectrometry suggest that structural alterations may be introduced into Hbs that are purified by extensive chromatographic separations. These results demonstrate that TFF is a suitable process for the purification of Hb from RBCs with a quality equivalent to that of commercial Hb preparations that employ more extensive purification strategies. This work also shows that TFF can yield highly pure Hb which can be used for Hb‐based O₂ carrier synthesis. Biotechnol. Bioeng. 2010; 106: 76–85. © 2010 Wiley Periodicals, Inc.     

Environmental temperature modulates olfactory reception in Drosophila melanogaster

Sensory systems, including the olfactory system, are able to adapt to changing environmental conditions. In nature, changes in temperature modify the volatility and concentration of odorants in the air. If the olfactory system does not adapt to these changes, it could relay wrong information about the distance to or direction of odor sources. Recent behavioral studies in Drosophila melanogaster showed olfactory acclimation to temperature. In this report, we investigated if temperature affects olfaction at the level of the receptors themselves. With this aim, we performed electroantennograms (EAGs) and single sensillum recordings (SSRs) to measure the response to several odorants in flies that had been submitted to temperature treatments. In response to all tested odorants, the amplitude of the EAGs increased in flies that had been exposed to a higher temperature and decreased after cold treatment, revealing that at least part of the reported change in olfactory perception happens at reception level. SSRs of odorant stimulated basiconic sensilla ab2 and ab3 showed some changes in the number of spikes after heat or cold treatment. However, the number and shape of spontaneous action potentials were unaffected, suggesting that the observed changes related specifically to the olfactory function of the neurons.     

Larval and juvenile Pacific herring Clupea pallasii are not susceptible to infectious hematopoietic necrosis under laboratory conditions

Infectious hematopoietic necrosis (IHN) leads to periodic epidemics among certain wild and farmed fish species of the Northeast (NE) Pacific. The source of the IHN virus (IHNV) that initiates these outbreaks remains unknown; however, a leading hypothesis involves viral persistence in marine host species such as Pacific herring Clupea pallasii. Under laboratory conditions we exposed specific pathogen-free (SPF) larval and juvenile Pacific herring to 10(3) to 10(4) plaque-forming units (pfu) of IHNV ml(-1) by waterborne immersion. Cumulative mortalities among exposed groups were not significantly different from those of negative control groups. After waterborne exposure, IHNV was transiently recovered from the tissues of larvae but absent in tissues of juveniles. Additionally, no evidence of viral shedding was detected in the tank water containing exposed juveniles. After intraperitoneal (IP) injection of IHNV in juvenile herring with 10(3) pfu, IHNV was recovered from the tissues of sub-sampled individuals for only the first 5 d post-exposure. The lack of susceptibility to overt disease and transient levels of IHNV in the tissues of exposed fish indicate that Pacific herring do not likely serve a major epizootiological role in perpetuation of IHNV among free-ranging sockeye salmon Oncorhynchus nerka and farmed Atlantic salmon Salmo salar in the NE Pacific.     

The stability of the ternary interferon-receptor complex rather than the affinity to the individual subunits dictates differential biological activities

Type I interferons (IFNs) signal for their diverse biological effects by binding a common receptor on target cells, composed of the two transmembrane IFNAR1 and IFNAR2 proteins. We have previously differentially enhanced the antiproliferative activity of IFN by increasing the weak binding affinity of IFN to IFNAR1. In this study, we further explored the affinity interdependencies between the two receptor subunits and the role of IFNAR1 in differential IFN activity. For this purpose, we generated a panel of mutations targeting the IFNAR2 binding site on the background of the IFNalpha2 YNS mutant, which increases the affinity to IFNAR1 by 60-fold, resulting in IFNAR2-to-IFNAR1 binding affinity ratios ranging from 1000:1 to 1:1000. Both the antiproliferative and antiviral potencies of the interferon mutants clearly correlated to the in situ binding IC(50) values, independently of the relative contributions of the individual receptors, thus relating to the integral lifetime of the complex. However, the antiproliferative potency correlated throughout the entire range of affinities, as well as with prolonged IFNAR1 receptor down-regulation, whereas the antiviral potency reached a maximum at binding affinities equivalent to that of wild-type IFNalpha2. Our data suggest that (i) the specific activity of interferon is related to the ternary complex binding affinity and not to affinity toward individual receptor components and (ii) although the antiviral pathway is strongly dependent on pSTAT1 activity, the cytostatic effect requires additional mechanisms that may involve IFNAR1 down-regulation. This differential interferon response is ultimately mediated through distinct gene expression profiling.     

Influence of oxygen tension on myocardial performance. Evaluation by tissue Doppler imaging

Background

Endothelial function in hypercholesterolemic rabbits is usually evaluated ex vivo on isolated aortic rings. In vivo evaluation requires invasive imaging procedures that cannot be repeated serially.

Aim

We evaluated a non-invasive ultrasound technique to assess early endothelial function in rabbits and compare data with ex vivo measurements.

Methods

Twenty-four rabbits (fed with a cholesterol diet (0.5%) for 2 to 8 weeks) were given progressive infusions of acetylcholine (0.05–0.5 μg/kg/min) and their endothelial function was assessed in vivo by transcutaneous vascular ultrasound of the abdominal aorta. Ex vivo endothelial function was evaluated on isolated aortic rings and compared to in vivo data.

Results

Significant endothelial dysfunction was demonstrated in hypercholesterolemic animals as early as 2 weeks after beginning the cholesterol diet (aortic cross-sectional area variation: -2.9% vs. +4% for controls, p < 0.05). Unexpectedly, response to acetylcholine at 8 weeks was more variable. Endothelial function improved in 5 rabbits while 2 rabbits regained a normal endothelial function. These data corroborated well with ex vivo results.

Conclusion

Endothelial function can be evaluated non-invasively in vivo by transcutaneous vascular ultrasound of the abdominal aorta in the rabbit and results correlate well with ex vivo data.     

Vancomycin derivative with damaged D-Ala-D-Ala binding cleft binds to cross-linked peptidoglycan in the cell wall of Staphylococcus aureus

Des-N-methylleucyl-4-(4-fluorophenyl)benzyl-vancomycin (DFPBV) retains activity against vancomycin-resistant pathogens despite its damaged d-Ala-d-Ala binding cleft. Using solid-state nuclear magnetic resonance (NMR), a DFPBV binding site in the cell walls of whole cells of Staphylococcus aureus has been identified. The cell walls were labeled with d-[1-(13)C]alanine, [1-(13)C]glycine, and l-[epsilon-(15)N]lysine. Internuclear distances from (19)F of the DFPBV to the (13)C and (15)N labels of the cell-wall peptidoglycan were determined by rotational-echo double-resonance (REDOR) NMR. The (13)C{(19)F} and (15)N{(19)F} REDOR spectra show that, in situ, DFPBV binds to the peptidoglycan as a monomer with its vancosamine hydrophobic side chain positioned near a pentaglycyl bridge. This result suggests that the antimicrobial activity of other vancosamine-modified glycopeptides depends upon both d-Ala-d-Ala stem-terminus recognition (primary binding site) and stem-bridge recognition (secondary binding site).     

Early tetrapodomorph biogeography: Controlling for fossil record bias in macroevolutionary analyses

The fossil record provides direct empirical data for understanding macroevolutionary patterns and processes. Inherent biases in the fossil record are well known to confound analyses of this data. Sampling bias proxies have been used as covariates in regression models to test for such biases. Proxies, such as formation count, are associated with paleobiodiversity, but are insufficient for explaining species dispersal owing to a lack of geographic context. Here, we develop a sampling bias proxy that incorporates geographic information and test it with a case study on early tetrapodomorph biogeography. We use recently-developed Bayesian phylogeographic models and a new supertree of early tetrapodomorphs to estimate dispersal rates and ancestral habitat locations. We find strong evidence that geographic sampling bias explains supposed radiations in dispersal rate (potential adaptive radiations). Our study highlights the necessity of accounting for geographic sampling bias in macroevolutionary and phylogenetic analyses and provides an approach to test for its effect.