Possible role of ILK-affixin complex in integrin-cytoskeleton linkage during platelet aggregation

Integrin-mediated adhesion induces the formation of focal adhesions that link the extracellular matrix and intracellular actin cytoskeletal networks. We previously showed that integrin-linked kinase (ILK), which can interact with beta1 and beta3 integrins, and its interacting protein, affixin, play an essential role in the initial assembly of focal adhesion structures and actin stress fibers. Although the relevant structures are also observed in integrin alphaIIbbeta3 in platelets, the precise underlying molecular mechanism remains unclarified. Here, we found that ILK stably forms a complex with ss-affixin in platelets. Thrombin stimulation induces their association with integrin beta3, which is followed by their incorporation into the Triton-insoluble membrane-cytoskeletal fraction. During the course of thrombin-induced platelet aggregation, ILK activity was enhanced within 90s to 2.1-fold of the basal level, independent of phosphatidylinositol 3-kinase. Taken together with the observation that the treatment with an anti-integrin beta3 antibody stimulates ILK activity without inducing platelet aggregation, these results suggest that the outside-in signaling induced by fibrinogen binding to integrin enhances ILK activity and results in the initial phase to reorganize the actin cytoskeleton.     

Light-harvesting complex II pigments and proteins in association with Cbr, a homolog of higher-plant early light-inducible proteins in the unicellular green alga Dunaliella

 Like higher plants, unicellular green algae of the genus Dunaliella respond to light stress by enhanced de-epoxidation of violaxanthin and accumulation of Cbr, a protein homologous to early light-inducible proteins (Elips) in plants. Earlier studies indicated that Cbr was associated with the light-harvesting complex of photosystem II (LHCII) and suggested it acted as a zeaxanthin-binding protein and fulfilled a photo-protective function (Levy et al. 1993, J. Biol. Chem. 268: 20892–20896). To characterize the protein-pigment subcomplexes containing Cbr in greater detail than attained so far, thylakoid membranes from Dunaliella salina grown in high light or normal light were solubilized with dodecyl maltoside and fractionated by isoelectric-focusing. Analysis of the resolved LHCII subcomplexes indicated preferred associations among the four LHCIIb polypeptides and between them and Cbr: subcomplexes including Cbr contained one or two of the more acidic of the four LHCIIb polypeptides as well as large amounts of lutein and zeaxanthin relative to chlorophyll a/b. After sucrose gradient centrifugation, Cbr free of LHCIIb polypeptides was detected together with released pigments; this Cbr possibly originated in subcomplexes dissociated in the course of the analysis. These results agree with the conclusion that Cbr is part of the network of LHCIIb protein-pigment complexes and suggest that the role played by Cbr involves the organization and/or stabilization of assemblies highly enriched in zeaxanthin and lutein. Such assemblies may function to protect PSII from photodamage due to overexcitation. Received: 6 August 1999 / Accepted: 23 November 1999     

Evolutionary Rate and Genetic Heterogeneity of Human T-Cell Lymphotropic Virus Type II (HTLV-II) Using Isolates from European Injecting Drug Users

Seven new Italian and two new British HTLV-II isolates were obtained from injecting drug users and the entire long terminal repeat (LTR) region was sequenced. Restriction analysis showed that all the Italian isolates are of the IIb subtype, whereas the British isolates are of the IIa subtype. To understand whether the further differentiation of each two principal HTLV-II subtypes in several subgroups could be statistically supported by phylogenetic analysis, the neighbor-joining, parsimony, and maximum likelihood methods were used. The separation between IIa and IIb is very well supported by all three methods. At least two phylogenetic subgroups exist within the HTLV-IIa and at least three within the HTLV-IIb subtype. In the present analysis, no statistical support was obtained for additional phylogroups. Two particular subgroups seem interesting because they include all European and North American injecting drug user strains within the IIa and IIb subtypes, respectively. These data confirm that European HTLV-II infection among drug users is probably derived from North America. They also suggest that though a certain differentiation by restriction analysis in different subgroups is possible, carefully interpreted phylogenetic analyses remain necessary. Using the likelihood ratio test, a molecular clock for the drug user strains was calibrated. A fixation rate between 1.08 × 10⁻⁴ and 2.7 × 10⁻⁵ nucleotide substitutions per site per year was calculated for the IIa and IIb injecting drug user strains. This is the lowest fixation rate so far reported for RNA viruses, including for HIV, which typically range between 10⁻² and 10⁻⁴.     

From a peptide lead to an orally active peptidomimetic fibrinogen receptor antagonist

Antagonists of the platelet fibrinogen receptor (GP IIb/IIIa receptor) are expected to be a new promising class of antithrombotic agents. The binding of fibrinogen to the fibrinogen receptor depends on an Arg-Gly-Asp-Ser (RGDS) tetrapeptide recognition motif. Structural modifications of the RGDS lead have led to the discovery of a non-peptide RGD mimetic GP IIb/IIIa antagonist 20 (S 1197). Compound 20 inhibits dose-dependently and reversibly human platelet aggregation. Modeling studies based on structure–activity data revealed the following structural features of the drug as important for receptor binding: the amidino group, the carboxylate group, hydrophobic substitutions at the carboxyl-terminus and at the side chain carrying the positive charge, the carboxyl-terminal NH group of the -amino acid as a hydrogen bond donor and one oxygen atom of the hydantoin as a hydrogen bond acceptor. The ethyl ester prodrug of 20 (S 5740) is an orally active antithrombotic agent which has the potential to be used to treat and prevent thrombotic diseases in humans.     

From a peptide lead to an orally active peptidomimetic fibrinogen receptor antagonist

Summary Antagonists of the platelet fibrinogen receptor (GP IIb/IIIa receptor) are expected to be a new promising class of antithrombotic agents. The binding of fibrinogen to the fibrinogen receptor depends on an Arg-Gly-Asp-Ser (RGDS) tetrapeptide recognition motif. Structural modifications of the RGDS lead have led to the discovery of a non-peptide RGD mimetic GP IIb/IIIa antagonist20 (S 1197). Compound20 inhibits dose-dependently and reversibly human platelet aggregation. Modeling studies based on structure-activity data revealed the following structural features of the drug as important for receptor binding: the amidino group, the carboxylate group, hydrophobic substitutions at the carboxyl-terminus and at the side chain carrying the positive charge, the carboxyl-terminal NH group of the β-amino acid as a hydrogen bond donor and one oxygen atom of the hydantoin as a hydrogen bond acceptor. The ethyl ester prodrug of20 (S 5740) is an orally active antithrombotic agent which has the potential to be used to treat and prevent thrombotic diseases in humans.     

Inhibition of Platelet Aggregation of a Mutant Proinsulin Chimera Engineered by Introduction of a Native Lys-Gly-Asp-containing Sequence

An eight amino acid sequence, CAKGDWNC, from disintegrin barbourin, was introduced into an inactive human proinsulin molecule between the B28 and A2 sites to construct a chimeric, anti-thrombosis recombinant protein. The constructed Lys-Gly-Asp (KGD)-proinsulin gene was expressed in Escherichia coli and then purified. The KGD-proinsulin chimera protein inhibits human platelet aggregation, induced by ADP, with an IC₅₀ value (molar concentration causing 50% inhibition of platelet aggregation) of 830 nM and demonstrates also specific affinity to glycoprotein IIb/IIIa receptor. Its insulin receptor binding activity remaines as low as 0.04% with native insulin as a control.     

Glycolytic fast‐twitch muscle fiber restoration counters adverse age‐related changes in body composition and metabolism

Aging is associated with the development of insulin resistance, increased adiposity, and accumulation of ectopic lipid deposits in tissues and organs. Starting in mid‐life there is a progressive decline in lean muscle mass associated with the preferential loss of glycolytic, fast‐twitch myofibers. However, it is not known to what extent muscle loss and metabolic dysfunction are causally related or whether they are independent epiphenomena of the aging process. Here, we utilized a skeletal‐muscle‐specific, conditional transgenic mouse expressing a constitutively active form of Akt1 to examine the consequences of glycolytic, fast‐twitch muscle growth in young vs. middle‐aged animals fed standard low‐fat chow diets. Activation of the Akt1 transgene led to selective skeletal muscle hypertrophy, reversing the loss of lean muscle mass observed upon aging. The Akt1‐mediated increase in muscle mass led to reductions in fat mass and hepatic steatosis in older animals, and corrected age‐associated impairments in glucose metabolism. These results indicate that the loss of lean muscle mass is a significant contributor to the development of age‐related metabolic dysfunction and that interventions that preserve or restore fast/glycolytic muscle may delay the onset of metabolic disease.     

New peptidomimetics in the chemistry of fibrinogen receptor antagonists

Summary RGD-peptidomimetics are currently being investigated as a class of potential antithrombotics that antagonize the fibrinogen receptor, GP IIb/IIIa, on the surface of platelets. These mimetics are expected to have decisive advantages-such as higher activity and specificity, oral bioavailability and longer duration of action-over known antithrombotics. For further optimization in this respect, novel peptidomimetic GP IIb/IIIa antagonists with an oxazolidinonemethyl central building block were synthesized. This building block proved to be very versatile as an anchor for structurally different C-termini and was the starting point for highly efficient and orally active compounds.     

Interactions between a soil fungus,Trichoderma harzianum,and IIb metals—adsorption to mycelium and production of complexing metabolites

Fungi are capable of accumulating metals and, in soil, such accumulation may influence metal speciation and transport. The interactions between a common soil fungus, Trichoderma harzianum, and IIb elements were studied in the present investigation. The accumulation of the metals zinc, cadmium and mercury by starved and non-starved mycelium at different pH was determined by a batch technique using radioactive tracers; uptake of the metals was found to be large, with respective distribution coefficients of about 10^3.5, 10^2.5 and 10^4.0 for zinc, cadmium and mercury, respectively. Metal accumulation by a starved system was largely independent of pH in the range 3–9, where in a non-starved system an increased accumulation of zinc (at 10^– m) was observed at low pH (3–5). Potentiometric titrations performed on the two systems revealed significant differences in acid capacities, i.e. values close to zero for the starved system and 500–800 meq kg^– for the non-starved system. The maximum metal uptake was at least 50 mmol kg^– at pH 6.5 (calculated from adsorption isotherms). The present findings suggests that in the non-starved system a metabolite is produced and then released when the pH is within a certain range.