Bactericidal activity of human eosinophilic granulocytes against Escherichia coli

Eosinophils participate in allergic inflammation and may have roles in the bodys defense against helminthic infestation. Even under noninflammatory conditions, eosinophils are present in the mucosa of the large intestine, where large numbers of gram-negative bacteria reside. Therefore, roles for eosinophils in host defenses against bacterial invasion are possible. In a system for bacterial viable counts, the bactericidal activity of eosinophils and the contribution of different cellular antibacterial systems against Escherichia coli were investigated. Eosinophils showed a rapid and efficient killing of E. coli under aerobic conditions, whereas under anaerobic conditions bacterial killing decreased dramatically. In addition, diphenylene iodonium chloride (DPI), an inhibitor of the NADPH oxidase and thereby of superoxide production, also significantly inhibited bacterial killing. The inhibitor of nitric oxide (NO) production L-N5-(1-iminoethyl)-ornithine dihydrochloride did not affect the killing efficiency, suggesting that NO or derivatives thereof are of minor importance under the experimental conditions used. To investigate the involvement of superoxide and eosinophil peroxidase (EPO) in bacterial killing, EPO was blocked by azide. The rate of E. coli killing decreased significantly in the presence of azide, whereas addition of DPI did not further decrease the killing, suggesting that superoxide acts in conjunction with EPO. Bactericidal activity was seen in eosinophil extracts containing granule proteins, indicating that oxygen-independent killing may be of importance as well. The findings suggest that eosinophils can participate in host defense against gram-negative bacterial invasion and that oxygen-dependent killing, i.e., superoxide acting in conjunction with EPO, may be the most important bactericidal effector function of these cells

Membrane-Lipid Therapy in Operation: The HSP Co-Inducer BGP-15 Activates Stress Signal Transduction Pathways by Remodeling Plasma Membrane Rafts

Aging and pathophysiological conditions are linked to membrane changes which modulate membrane-controlled molecular switches, causing dysregulated heat shock protein (HSP) expression. HSP co-inducer hydroxylamines such as BGP-15 provide advanced therapeutic candidates for many diseases since they preferentially affect stressed cells and are unlikely have major side effects. In the present study in vitro molecular dynamic simulation, experiments with lipid monolayers and in vivo ultrasensitive fluorescence microscopy showed that BGP-15 alters the organization of cholesterol-rich membrane domains. Imaging of nanoscopic long-lived platforms using the raft marker glycosylphosphatidylinositol-anchored monomeric green fluorescent protein diffusing in the live Chinese hamster ovary (CHO) cell plasma membrane demonstrated that BGP-15 prevents the transient structural disintegration of rafts induced by fever-type heat stress. Moreover, BGP-15 was able to remodel cholesterol-enriched lipid platforms reminiscent of those observed earlier following non-lethal heat priming or membrane stress, and were shown to be obligate for the generation and transmission of stress signals. BGP-15 activation of HSP expression in B16-F10 mouse melanoma cells involves the Rac1 signaling cascade in accordance with the previous observation that cholesterol affects the targeting of Rac1 to membranes. Finally, in a human embryonic kidney cell line we demonstrate that BGP-15 is able to inhibit the rapid heat shock factor 1 (HSF1) acetylation monitored during the early phase of heat stress, thereby promoting a prolonged duration of HSF1 binding to heat shock elements. Taken together, our results indicate that BGP-15 has the potential to become a new class of pharmaceuticals for use in ‘membrane-lipid therapy’ to combat many various protein-misfolding diseases associated with aging

A Genome Scan for Positive Selection in Thoroughbred Horses

Thoroughbred horses have been selected for exceptional racing performance resulting in system-wide structural and functional adaptations contributing to elite athletic phenotypes. Because selection has been recent and intense in a closed population that stems from a small number of founder animals Thoroughbreds represent a unique population within which to identify genomic contributions to exercise-related traits. Employing a population genetics-based hitchhiking mapping approach we performed a genome scan using 394 autosomal and X chromosome microsatellite loci and identified positively selected loci in the extreme tail-ends of the empirical distributions for (1) deviations from expected heterozygosity (Ewens-Watterson test) in Thoroughbred (n = 112) and (2) global differentiation among four geographically diverse horse populations (FST). We found positively selected genomic regions in Thoroughbred enriched for phosphoinositide-mediated signalling (3.2-fold enrichment; P<0.01), insulin receptor signalling (5.0-fold enrichment; P<0.01) and lipid transport (2.2-fold enrichment; P<0.05) genes. We found a significant overrepresentation of sarcoglycan complex (11.1-fold enrichment; P<0.05) and focal adhesion pathway (1.9-fold enrichment; P<0.01) genes highlighting the role for muscle strength and integrity in the Thoroughbred athletic phenotype. We report for the first time candidate athletic-performance genes within regions targeted by selection in Thoroughbred horses that are principally responsible for fatty acid oxidation, increased insulin sensitivity and muscle strength: ACSS1 (acyl-CoA synthetase short-chain family member 1), ACTA1 (actin, alpha 1, skeletal muscle), ACTN2 (actinin, alpha 2), ADHFE1 (alcohol dehydrogenase, iron containing, 1), MTFR1 (mitochondrial fission regulator 1), PDK4 (pyruvate dehydrogenase kinase, isozyme 4) and TNC (tenascin C). Understanding the genetic basis for exercise adaptation will be crucial for the identification of genes within the complex molecular networks underlying obesity and its consequential pathologies, such as type 2 diabetes. Therefore, we propose Thoroughbred as a novel in vivo large animal model for understanding molecular protection against metabolic disease

The effect of powder blend and tablet structure on drug release mechanisms of hydrophobic starch acetate matrix tablets

This study investigates the release mechanism of a hydrophilic drug (caffeine) from hydrophobic matrix tablets composed of starch acetate. Different particle size fractions of starch acetate were mixed with caffeine (22% V/V) to obtain various mixture organisations in the powder, as 14 well as in the final tablet. The organisation of powder mixtures was calculated by the carrier payload of starch acetate particles, while the pore size distributions in tablets were measured by mercury intrusion porosimetry. A carrier payload below I indicated the existence of a free starch acetate particle surface, while numbers greater than 1 pointed to a complete occupation of the starch acetate particle surface area by caffeine particles. The carrier payload calculations gave a good prediction for the existence of a starch acetate matrix in the tablet structures. Caffeine matrices in tablets compressed from the mixtures could be detected by mercury intrusion porosimetry measurements. The existence of different matrices, as well as different pore networks, determined the physical changes of the tablets and the release mechanism of caffeine during dissolution tests. When a tablet contained only a caffeine matrix, rapid tablet disintegration and immediate release of the total amount of caffeine occurred. A single matrix of starch acetate resulted in tablets that remained intact, although cracks were formed. The co-existence of matrices of both materials created surface erosion of the tablet. The caffeine release profiles of tablets that remained intact or showed erosion were fitted by an equation containing both diffusional and relaxational factors to describe the effect of tablet porosity on drug release. (c) 2005 Elsevier B.V. All rights reserved

Pharmacological and molecular studies on the interaction of varenicline with different nicotinic acetylcholine receptor subtypes. Potential mechanism underlying partial agonism at human 42 and 34 subtypes

In order to determine the structural components underlying differences in affinity, potency, and selectivity of varenicline for several human (h) nicotinic acetylcholine receptors (AChRs), functional and structural experiments were performed. The Ca2+ influx results established that: (a) varenicline activates (EC50s in μM) AChR subtypes with the following rank sequence: hα7 (0.18 ± 0.02) > hα4β4 (0.37 ± 0.08) > hα4β2 (1.30 ± 0.18) > hα3β4 (6.4 ± 1.2) >>> hα1β1γδ (>100); (b) varenicline binds to AChR subtypes with the following affinity order (Kis): hα4β2 (0.090 ± 0.007 nM) ~ hα4β4 (0.121 ± 0.004 nM) > hα3β4 (13 ± 1 nM) > hα7 (77 ± 6 nM) >>> Tα1β1γδ (116 ± 8 μM). The molecular docking results indicating that more hydrogen bond interactions are apparent for 4-containing AChRs in comparison to other AChRs may explain the observed higher affinity; and that (c) varenicline is a full agonist at hα7 (101%) and hα4β4 (93%), and a partial agonist at hα4β2 (20%) and hα3β4 (45%), relative to ()-epibatidine. The allosteric sites found at the extracellular domain (EXD) of h34 and h42 AChRs could explain the partial agonistic activity of varenicline on these AChR subtypes. Molecular dynamics simulations show that the interaction of varenicline to each allosteric site decreases the capping of Loop C at the hα4β2 AChR, suggesting that these allosteric interactions limit the initial step in the gating process. In conclusion, we propose that in addition to hα4β2 AChRs, hα4β4 AChRs can be considered as potential targets for the clinical activity of varenicline, and that the allosteric interactions at the h34- and h42-EXDs are alternative mechanisms underlying partial agonism at these AChRs