Red blood cell regulation of microvascular tone through adenosine triphosphate

The matching of blood flow with metabolic need requires a mechanism for sensing the needs of the tissue and communicating that need to the arterioles, the ultimate controllers of tissue perfusion. Despite significant strides in our understanding of blood flow regulation, the identity of the O(2) sensor has remained elusive. Recently, the red blood cell, the Hb-containing O(2) carrier, has been implicated as a potential O(2) sensor and contributor to this vascular control by virtue of its concomitant carriage of millimolar amounts of ATP, which it is able to release when exposed to a low-O(2) environment. To evaluate this possibility, we exposed perfused cerebral arterioles to low extraluminal O(2) in the absence and presence of red blood cells or 6% dextran and determined both vessel diameter and ATP in the vessel effluent. Only when the vessels were perfused with red blood cells did the vessels dilate in response to low extraluminal O(2). In addition, this response was accompanied by a significant increase in vessel effluent ATP. These findings support the hypothesis that the red blood cell itself serves a role in determining O(2) supply to tissue.     

A novel heparan sulphate with high degree of N-sulphation and high heparin cofactor-II activity from the brine shrimp Artemia franciscana

With the aid of heparinase and heparitinases from Flavobacterium heparinum and 13C and IH NMR spectroscopy it was shown that the heparan sulphate isolated from the brine shrimp Artemia franciscana exhibits structural features intermediate between those of mammalian heparins and heparan sulphates. These include an unusually high degree of N-sulphation (with corresponding very low degree of N-acetylation), a relatively high content of iduronic acid residues (both unsulphated and 2-O-sulphated) and a relatively low degree of 6-O-sulphation of the glucosamine residues. The major sequences (glucuronic acid-->N-sulphated glucosamine and glucuronic acid-->N, 6-disulphated glucosamine) are most probably arranged in blocks. Although exhibiting negligible anticlotting activity in the APTT and anti-factor Xa assays the A. franciscana heparan sulphate has a high heparin cofactor-II activity (about 1/3 that of heparin).     

Calcium-potassium selectivity: kinetic analysis of current-voltage relationships of the open, slowly activating channel in the vacuolar membrane of Vicia faba guard-cells

Single-channel current-voltage (IV ) relationships of the open, slowly activating vacuolar (SV) channel of Vicia faba L. were recorded in solutions with different activities of Ca²⁺ and K⁺, and have been analyzed for Ca²⁺/K⁺ selectivity. Two models with one binding site have been examined. A rigid-pore model with a main binding site between two energy barriers (nine free parameters) provides fair fits. Slightly better fits are obtained with an alternative, dynamic-pore model, where the selectivity filter is located between two Mitchellian ion wells of the cytoplasmic and luminal pore sections, and where the selectivity filter alternates the orientation of the binding site between the two faces of the pore (ten free parameters). Using sets of IV-relationships with only Ca²⁺ or only K⁺ as transportable substrates, both models consistently predict open-channel IV-relationships in the presence of both substrates. Fits of both models to the entire ensemble of␣data yield very similar flux-voltage characteristics for␣Ca²⁺ and for K⁺ in experimental conditions, and consistently predict such flux-voltage characteristics over physiologically relevant ranges of voltage and substrate concentrations. In a very general sense, physiological Ca⁺ fluxes through the open SV channel are predominantly inward and about 50 times smaller than K⁺ fluxes. The ions Cl⁻, OH⁻, and H⁺, do not pass the SV channel at significant rates. Kinetic details of the SV channel with respect to binding and passage of Ca²⁺ and K⁺ are discussed on the basis of the consistent results of the reaction-kinetic analysis of the experimental data by the two models. Received: 14 July 1997 / Accepted: 26 September 1997     

Oligomerization of profilins from birch, man and yeast. Profilin, a ligand for itself?

 Profilins are structurally well conserved low molecular weight (12–15 kDa) eukaryotic proteins which interact with a variety of physiological ligands: (1) cytoskeletal components, e.g., actin; (2) polyphosphoinositides, e.g., phosphatidylinositol-4,5-bisphosphate; (3) proline-rich proteins, e.g., formin homology proteins and vasodilatator-stimulated phosphoprotein. Profilins may thus link the microfilament system with signal transduction pathways. Plant profilins have recently been shown to be highly crossreactive allergens which bind to IgE antibodies of allergic patients and thus cause symptoms of type I allergy. We expressed and purified from Escherichia coli profilins from birch pollen (Betula verrucosa), humans (Homo sapiens) and yeast (Schizosaccharomyces pombe) and demonstrated that each of these profilins is able to form stable homo- and heteropolymers via disulphide bonds in vitro. Circular dichroism analysis of oxidized (polymeric) and reduced (monomeric) birch pollen profilin indicates that the two states have similar secondary structures. Using ¹²⁵I-labeled birch pollen, yeast and human profilin in overlay experiments, we showed that disulphide bond formation between profilins can be disrupted under reducing conditions, while reduced as well as oxidized profilin states bind to actin and profilin-specific antibodies. Exposure of profilin to oxidizing conditions, such as when pollen profilins are liberated on the surface of the mucosa of atopic patients, may lead to profilin polymerization and thus contribute to the sensitization capacity of profilin as an allergen. Received: 25 February 1998 / Revision accepted: 12 May 1998     

Ltx1, a mouse locus that influences the susceptibility of macrophages to cytolysis caused by intoxication with Bacillus anthracis lethal factor, maps to chromosome 11

The lethal factor (LF) toxin that is produced by Bacillus anthracis plays an important role in the pathogenesis of anthrax. LF has mononuclear phagocyte-specific intoxicating effects that are not well understood. We have identified genetic differences in inbred mouse strains that determine whether their cultured macrophages are susceptible to the cytolytic effect of LF intoxication. Our identification of resistant and susceptible mouse strains enabled us to analyse crosses between these strains and to map a single responsible gene (called Ltx1 ) to chromosome 11. Ltx1 probably influences intoxication events that occur after the delivery of LF to the cytosol, as all mouse macrophages are killed by polypeptides containing the catalytic domain of Diphtheria toxin fused to the domain of LF required for cytosolic transport. Furthermore, the susceptibility phenotype is dominant to resistance, suggesting that resistance is caused by an absence of or polymorphism in a molecule that acts jointly with, or downstream of, the activity of LF. Our mapping of Ltx1 is a crucial first step in its positional cloning, which will provide more information about the mechanism of LF intoxication.     

Stimulation of phospholipase C-beta2 by the Rho GTPases Cdc42Hs and Rac1.

Neutrophils contain a soluble guanine-nucleotidebinding protein, made up of two components with molecular masses of 23 and 26 kDa, that mediates stimulation of phospholipase C-beta2 (PLCbeta2). We have identified the two components of the stimulatory heterodimer by amino acid sequencing as a Rho GTPase and the Rho guanine nucleotide dissociation inhibitor LyGDI. Using recombinant Rho GTPases and LyGDI, we demonstrate that PLCbeta2 is stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP[S])-activated Cdc42HsxLyGDI, but not by RhoAxLyGDI. Stimulation of PLCbeta2, which was also observed for GTP[S]-activated recombinant Rac1, was independent of LyGDI, but required C-terminal processing of Cdc42Hs/Rac1. Cdc42Hs/Rac1 also stimulated PLCbeta2 in a system made up of purified recombinant proteins, suggesting that this function is mediated by direct protein-protein interaction. The Cdc42Hs mutants F37A and Y40C failed to stimulate PLCbeta2, indicating that the Cdc42Hs effector site is involved in this interaction. The results identify PLCbeta2 as a novel effector of the Rho GTPases Cdc42Hs and Rac1, and as the first mammalian effector directly regulated by both heterotrimeric and low-molecular-mass GTP-binding proteins.