KCl evokes contraction of airway smooth muscle via activation of RhoA and Rho-kinase

Airway smooth muscle (ASM) cells express voltage-dependent Ca2+ channels, primarily of the L-subtype. These may play a role in excitation-contraction coupling of ASM, although other signaling pathways may also contribute: one of these includes Rho and its downstream effector molecule Rho-associated kinase (ROCK). Although voltage-dependent Ca2+ influx and Rho/ROCK signaling have traditionally been viewed as entirely separate pathways, recent evidence in vascular smooth muscle suggest differently. In this study, we monitored contractile activity (muscle baths) in bronchial and/or tracheal preparations from the pig, cow, and human, and further examined Rho and ROCK activities (Western blots and kinase assays) and cytosolic levels of Ca2+ (fluo 4-based fluorimetry) in porcine tracheal myocytes. KCl evoked substantial contractions that were suppressed in tracheal preparations by removal of external Ca2+ or using the selective L-type Ca2+ channel blocker nifedipine; porcine bronchial preparations were much less sensitive, and bovine bronchi were essentially unaffected by 1 microM nifedipine. Surprisingly, KCl-evoked contractions were also highly sensitive to two structurally different ROCK inhibitors: Y-27632 and HA-1077. Furthermore, the inhibitory effects of nifedipine and of the ROCK inhibitors were not additive. KCl also caused marked stimulation of Rho and ROCK activities, and both these changes were suppressed by nifedipine or by removal of external Ca2+. KCl-induced elevation of [Ca2+]i was not affected by Y-27632 but was reversed by NiCl2 or by BAPTA-AM. We conclude that KCl acts in part through stimulation of Rho and ROCK, possibly secondary to voltage-dependent Ca2+ influx.     

Surface modifications created by using engineered hydrophobins

Hydrophobins are small (ca. 100 amino acids) secreted fungal proteins that are characterized by the presence of eight conserved cysteine residues and by a typical hydropathy pattern. Class I hydrophobins self-assemble at hydrophilic-hydrophobic interfaces into highly insoluble amphipathic membranes, thereby changing the nature of surfaces. Hydrophobic surfaces become hydrophilic, while hydrophilic surfaces become hydrophobic. To see whether surface properties of assembled hydrophobins can be changed, 25 N-terminal residues of the mature SC3 hydrophobin were deleted (TrSC3). In addition, the cell-binding domain of fibronectin (RGD) was fused to the N terminus of mature SC3 (RGD-SC3) and TrSC3 (RGD-TrSC3). Self-assembly and surface activity were not affected by these modifications. However, physiochemical properties at the hydrophilic side of the assembled hydrophobin did change. This was demonstrated by a change in wettability and by enhanced growth of fibroblasts on Teflon-coated with RGD-SC3, TrSC3, or RGD-TrSC3 compared to bare Teflon or Teflon coated with SC3. Thus, engineered hydrophobins can be used to functionalize surfaces.     

Propionate formation by Opitutus terrae in pure culture and in mixed culture with a hydrogenotrophic methanogen and implications for carbon fluxes in anoxic rice paddy soil

Propionate-forming bacteria seem to be abundant in anoxic rice paddy soil, but biogeochemical investigations show that propionate is not a correspondingly important intermediate in carbon flux in this system. Mixed cultures of Opitutus terrae strain PB90-1, a representative propionate-producing bacterium from rice paddy soil, and the hydrogenotrophic Methanospirillum hungatei strain SK maintained hydrogen partial pressures similar to those in the soil. The associated shift away from propionate formation observed in these cultures helps to reconcile the disparity between microbiological and biogeochemical studies.     

Divergent role for TNF-alpha in IFN-gamma-induced killing of Toxoplasma gondii and Salmonella typhimurium contributes to selective susceptibility of patients with partial IFN-gamma receptor 1 deficiency

Patients with defects in IFN-gamma- or IL-12-mediated immunity are susceptible to infections with Salmonella and non-tuberculous mycobacteria, but rarely suffer from infections with other intracellular pathogens such as Toxoplasma gondii. Here we describe macrophage and T cell function in eight individuals with partial IFN-gamma receptor 1 (IFN-gammaR1) deficiency due to a mutation that results in elevated cell surface expression of a truncated IFN-gammaR1 receptor that lacks the intracellular domain. We show that various effector mechanisms dependent on IFN-gammaR signaling are affected to different extents. Whereas TNF-alpha production was normally up-regulated in response to IFN-gamma, IL-12 production and CD64 up-regulation were strongly reduced, and IFN-gamma-mediated killing of the intracellular pathogens Salmonella typhimurium and T. gondii was completely abrogated in patient's macrophages. Since these patients suffer selectively from infections with non-tuberculous mycobacteria and Salmonella, but not T. gondii, despite sero-immunity in six of eight patients, which indicates previous contact with this pathogen, we next studied the role of TNF-alpha as a possible immune compensatory mechanism. IFN-gamma-induced killing of T. gondii appeared to be partially mediated by TNF-alpha, and addition of TNF-alpha could compensate for the abrogated killing of T. gondii in the patient's macrophages. In contrast, IFN-gamma-mediated killing of S. typhimurium appeared to be independent of TNF-alpha. We propose that the divergent role of TNF-alpha in IFN-gamma-induced killing of T. gondii and S. typhimurium may at least partially explain the highly selective susceptibility of patients.     

Biotic ligand model development predicting Zn toxicity to the alga Pseudokirchneriella subcapitata: possibilities and limitations

Biotic ligand models have been developed for various metals (e.g. Cu, Ag, Zn) and different aquatic species. These models incorporate the effect of physico-chemical water characteristics (major cations, pH, dissolved organic carbon) on the bioavailability and toxicity of the metal. In this study, the individual effects of calcium, magnesium, potassium, sodium and pH on zinc toxicity to the green alga Pseudokirchneriella subcapitata (formerly and better known as Selenastrum capricornutum and Raphidocelis subcapitata) were investigated. Stability constants for binding to algal cells (K(BL)) were derived for those cations affecting zinc toxicity, using the mathematical approach proposed by De Schamphelaere and Janssen [Environ. Sci. Technol. 63, (2002) 48-54]. Potassium proved to be the only cation tested that did not alter zinc toxicity to algae significantly. Log (K(BL)) values for Ca, Mg and Na, derived at pH 7.5, were 3.2, 3.9 and 2.8, respectively. Toxicity tests performed at different pH values (5.5-8.0) indicated that competition between H(+) and Zn(2+) reduces zinc toxicity. However, the observed relationship between (H(+)) and the 72h-EbC(50) [expressed as microM (Zn(2+))] is not linear and suggests that pH affects the physiology of the biotic ligand. Although, in general, our findings seem to suggest that zinc toxicity to algae can be modelled as a function of key water characteristics, the results also demonstrate that the part of the conventional BLM-hypothesis-i.e. that the binding characteristics of the biotic ligand are independent of the test medium characteristics-is not valid for algae. The observed pH-dependent change of stability constants should therefore be further investigated and incorporated in future BL-modelling efforts with algae.     

Receptors and signaling pathway underlying relaxations to isoprostanes in canine and porcine airway smooth muscle

Using muscle bath techniques, we examined the inhibitory activities of several E- and F-ring isoprostanes in canine and porcine airway smooth muscle. 8-Isoprostaglandin E1 and 8-isoprostaglandin E2 (8-iso PGE2) reversed cholinergic tone in a concentration-dependent manner, whereas the F-ring isoprostanes were ineffective. Desensitization with 8-iso-PGE2 and PGE2 implicated isoprostane activity at the PGE2 receptor (EP). We found that the inhibitory E-ring isoprostane responses were significantly augmented by rolipram (a type IV phosphodiesterase inhibitor), while 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (a guanylate cyclase inhibitor) had no effect, suggesting a role for cAMP in isoprostane-mediated relaxations. 8-Iso-PGE2 did not reverse KCl tone, suggesting that voltage-dependent Ca2+ influx and myosin light chain kinase are not suppressed by isoprostanes. Patch-clamp studies showed marked suppression of K+ currents by 8-iso-PGE2. We conclude that E-ring isoprostanes exert PGE2 receptor-directed, cAMP-dependent relaxations in canine and porcine airway smooth muscle. This activity is not dependent on K+ channel activation or the direct inhibition of voltage-operated Ca2+ influx or myosin light chain kinase.     

Measuring principles of frictional coefficients in cartilaginous tissues and its substitutes

The frictional properties of cartilaginous tissues, such as the hydraulic permeability, the electro-osmotic permeability, the diffusion coefficients of various ions and solutes, and the electrical conductance, are vital data to characterise the extracellular environment in which chondrocytes reside. This paper analyses one-dimensional measurement principles of these coefficients. Particular attention is given to the deformation dependence of them and the highly deformable nature of the tissues. A suggested strategy is the combination of a diffusion experiment using radiotracer methods, an electro-osmotic flow experiment and an electro-osmotic pressure experiment at low electric current.     

Specific granules of human eosinophils have lysosomal characteristics: presence of lysosome-associated membrane proteins and acidification upon cellular activation

Eosinophils possess characteristic specific granules. Their content may be important during host defense but it can also cause damage after release at sites of inflammation. We investigated possible lysosomal characteristics of these granules. Lysosome-associated membrane protein (LAMP)-1 and 2, were detected by Western blot, subcellular fractionation, and immunoelectron microscopy (IEM) and were localized to the membrane of specific granules and in vesicles of the cytoplasm, separate from secretory vesicles. No binding of mannose 6-phosphate receptor to proteins of specific granules could be detected, indicating that they are dephosphorylated and mature. Cellular activation by interleukin-5 caused acidification of specific granules, as detected by pH-dependent probes. The acidification was inhibited by concanamycin A (inhibitor of vacuolar H(+)-ATPase). Activation of eosinophils by serum-treated zymosan (STZ) caused degranulation into STZ-containing phagosomes and incorporation of LAMPs to their membranes. In conclusion, specific granules of eosinophils can be regarded as specialized primary lysosomes, a feature that may be important for their function and integrity.     

Chromosomal breakpoint mapping by arrayCGH using flow-sorted chromosomes

Despite the recent completion of the human genome project, the mapping of disease-related chromosomal translocation breakpoints and genes has remained laborious. Here, we describe a novel and rapid procedure to map such translocation breakpoints using flow-sorted chromosomes in combination with array-based comparative genomic hybridization (arrayCGH). To test the feasibility of this approach, we used a t(12;15)(q13;q25)-positive cell line with known breakpoint positions as a model. The derivative 12 chromosomes were flow-sorted, labeled, and hybridized to a genome-wide array containing 3648 well-characterized human genomic clones. The exact locations of the breakpoints on both chromosome 12 and 15 could be determined in a single hybridization experiment. In addition, we have tested the minimal amount of material necessary to perform these experiments and show that it is possible to obtain highly reliable profiles using as little as 10,000 flow-sorted chromosomes.