Lack of influence of the COX inhibitors metamizol and diclofenac on platelet GPIIb/IIIa and P-selectin expression in vitro

BACKGROUND: The effect of non-steroidal anti-inflammatory drugs (NSAIDs) for reduced platelet aggregation and thromboxane A2 synthesis has been well documented. However, the influence on platelet function is not fully explained. Aim of this study was to examine the influence of the COX-1 inhibiting NSAIDs, diclofenac and metamizol on platelet activation and leukocyte-platelet complexes, in vitro. Surface expression of GPIIb/IIIa and P-selectin on platelets, and the percentage of platelet-leukocyte complexes were investigated. METHODS: Whole blood was incubated with three different concentrations of diclofenac and metamizol for 5 and 30 minutes, followed by activation with TRAP-6 and ADP. Rates of GPIIb/IIIa and P-selectin expression, and the percentage of platelet-leukocyte complexes were analyzed by a flow-cytometric assay. RESULTS: There were no significant differences in the expression of GPIIb/IIIa and P-selectin, and in the formation of platelet-leukocyte complexes after activation with ADP and TRAP-6, regarding both the time of incubation and the concentrations of diclofenac and metamizol. CONCLUSIONS: Accordingly, the inhibitory effect of diclofenac and metamizol on platelet aggregation is not related to a reduced surface expression of P-selectin and GPIIb/IIIa on platelets.     

Activated protein C inhibits the release of macrophage inflammatory protein-1-alpha from THP-1 cells and from human monocytes

Several lines of evidence have implicated activated protein C (APC) to be an endogenous inhibitor of the inflammatory septic cascade. APC may exhibit direct anti-inflammatory properties, independent of its antithrombotic effects. Chemokines influence the interaction of monocytes at the endothelium during infection and sepsis and are involved in the molecular events leading to an adverse and lethal outcome of sepsis. Defining regulatory mechanisms on the monocytic release profile of the proinflammatory C-C chemokines macrophage inflammatory protein-1-alpha (MIP-1-alpha) and monocyte chemoattractant protein-1 (MCP-1) might have therapeutic implications for the treatment of sepsis. We established a monocytic cell model of inflammation by the addition of lipopolysaccharide (LPS) and examined the effect of human APC on LPS-stimulated chemokine release from the monocytic cell line THP-1. We found that human APC in supra-physiological concentrations of 2.5-10 microg/ml inhibited the LPS-induced release of the chemokines MIP-1-alpha and MCP-1, as measured by enzyme-linked immunosorbent assays (ELISA) at 6 up to 24 h. In addition to experiments on THP-1 cells, recombinant human APC in concentrations of 50 ng/ml was found to have an inhibiting effect on the release of MIP-1-alpha from freshly isolated mononuclear cells of septic patients. The ability of APC to decrease the release of the C-C chemokine MIP-1-alpha from the monocytic cell line THP-1 and from human monocytes may identify a novel immunomodulatory pathway by which APC exerts its anti-inflammatory action and may contribute to control the inflammatory response in sepsis.     

Tumor suppressor activity of profilin requires a functional actin binding site

Profilin 1 (PFN1) is a regulator of the microfilament system and is involved in various signaling pathways. It interacts with many cytoplasmic and nuclear ligands. The importance of PFN1 for human tissue differentiation has been demonstrated by the findings that human cancer cells, expressing conspicuously low PFN1 levels, adopt a nontumorigenic phenotype upon raising their PFN1 level. In the present study, we characterize the ligand binding site crucial for profilin's tumor suppressor activity. Starting with CAL51, a human breast cancer cell line highly tumorigenic in nude mice, we established stable clones that express PFN1 mutants differentially defective in ligand binding. Clones expressing PFN1 mutants with reduced binding to either poly-proline-stretch ligands or phosphatidyl-inositol-4,5-bisphosphate, but with a functional actin binding site, were normal in growth, adhesion, and anchorage dependence, with only a weak tendency to elicit tumors in nude mice, similar to controls expressing wild-type PFN1. In contrast, clones expressing a mutant with severely reduced capacity to bind actin still behaved like the parental CAL51 and were highly tumorigenic. We conclude that the actin binding site on profilin is instrumental for normal differentiation of human epithelia and the tumor suppressor function of PFN1.     

Pyrimidinone antibiotics--heterocyclic analogues with improved antibacterial spectrum

We report the synthesis and pharmacological evaluation of new derivatives of the natural dipeptide antibiotic TAN 1057 A,B containing heterocycles either in the beta-amino acid side chain or as mimics of the urea function. In the course of this program, we identified novel analogues that display activity towards a broader panel of Gram-positive bacteriae.     

The muscle ankyrin repeat proteins: CARP, ankrd2/Arpp and DARP as a family of titin filament-based stress response molecules

CARP, ankrd-2/Arpp, and DARP, are three members of a conserved gene family, referred to here as MARPs (muscle ankyrin repeat proteins). The expression of MARPs is induced upon injury and hypertrophy (CARP), stretch or denervation (ankrd2/Arpp), and during recovery following starvation (DARP), suggesting that they are involved in muscle stress response pathways. Here, we show that MARP family members contain within their ankyrin repeat region a binding site for the myofibrillar elastic protein titin. Within the myofibril, MARPs, myopalladin, and the calpain protease p94 appear to be components of a titin N2A-based signaling complex. Ultrastructural studies demonstrated that all three endogenous MARP proteins co-localize with I-band titin N2A epitopes in adult heart muscle tissues. In cultured fetal rat cardiac myocytes, passive stretch induced differential distribution patterns of CARP and DARP: staining for both proteins was increased in the nucleus and at the I-band region of myofibrils, while DARP staining also increased at intercalated discs. We speculate that the myofibrillar MARPs are regulated by stretch, and that this links titin-N2A-based myofibrillar stress/strain signals to a MARP-based regulation of muscle gene expression.     

In-field labeling of western corn rootworm adults (Coleoptera: Chrysomelidae) with rubidium

Field and laboratory studies were conducted in 2000 and 2001 to determine the feasibility of mass marking western corn rootworm adults, Diabrotica virgifera virgifera LeConte, with RbCl in the field. Results showed that application of rubidium (Rb) in solution to both the soil (1 g Rb/plant) and whorl (1 g Rb/plant) of corn plants was optimal for labeling western corn rootworm adults during larval development. Development of larvae on Rb-enriched corn with this technique did not significantly influence adult dry weight or survival. Rb was also highly mobile in the plant. Application of Rb to both the soil and the whorl resulted in median Rb concentrations in the roots (5,860 ppm) that were 150-fold greater than concentrations in untreated roots (38 ppm) 5 wk after treatment. Additionally, at least 90% of the beetles that emerged during the first 3 wk were labeled above the baseline Rb concentration (5 ppm dry weight) determined from untreated beetles. Because emergence was 72% complete at this time, a significant proportion of the population had been labeled. Results from laboratory experiments showed that labeled beetles remained distinguishable from unlabeled beetles for up to 4 d postemergence. The ability to efficiently label large numbers of beetles under field conditions and for a defined period with virtually no disruption of the population provides an unparalleled opportunity to conduct mark-recapture experiments for quantifying the short-range, intrafield movement of adult corn rootworms.     

The Murein Hydrolase of the Bacteriophage φ3626 Dual Lysis System Is Active against All Tested Clostridium perfringens Strains

Clostridium perfringens commonly occurs in food and feed, can produce an enterotoxin frequently implicated in food-borne disease, and has a substantial negative impact on the poultry industry. As a step towards new approaches for control of this organism, we investigated the cell wall lysis system of C. perfringens bacteriophage 3626, whose dual lysis gene cassette consists of a holin gene and an endolysin gene. Hol3626 has two membrane-spanning domains (MSDs) and is a group II holin. A positively charged beta turn between the two MSDs suggests that both the amino terminus and the carboxy terminus of Hol3626 might be located outside the cell membrane, a very unusual holin topology. Holin function was experimentally demonstrated by using the ability of the holin to complement a deletion of the heterologous phage λ S holin in λΔSthf. The endolysin gene ply3626 was cloned in Escherichia coli. However, protein synthesis occurred only when bacteria were supplemented with rare tRNA^Arg and tRNA^Ile genes. Formation of inclusion bodies could be avoided by drastically lowering the expression level. Amino-terminal modification by a six-histidine tag did not affect enzyme activity and enabled purification by metal chelate affinity chromatography. Ply3626 has an N-terminal amidase domain and a unique C-terminal portion, which might be responsible for the specific lytic range of the enzyme. All 48 tested strains of C. perfringens were sensitive to the murein hydrolase, whereas other clostridia and bacteria belonging to other genera were generally not affected. This highly specific activity towards C. perfringens might be useful for novel biocontrol measures in food, feed, and complex microbial communities.     

The matrix metalloproteinase 9 (mmp-9) hemopexin domain is a novel gelatin binding domain and acts as an antagonist

Matrix metalloproteinases (MMPs) are involved in the remodeling processes of the extracellular matrix and the basement membrane. Most MMPs are composed of a regulatory, a catalytic, and a hemopexin subunit. In many tumors the expression of MMP-9 correlates with local tumor growth, invasion, and metastasis. To analyze the role of the hemopexin domain in these processes, the MMP-9 hemopexin domain (MMP-9-PEX) was expressed as a glutathione S-transferase fusion protein in Escherichia coli. After proteolytic cleavage, the isolated PEX domain was purified by size exclusion chromatography. In a zymography assay, MMP-9-PEX was able to inhibit MMP-9 activity. The association and dissociation rates for the interaction of MMP-9-PEX with gelatin were determined by plasmon resonance. From the measured rate constants, the dissociation constant was calculated to be K(d) = 2,4 x 10(-8) m, demonstrating a high affinity between MMP-9-PEX and gelatin. In Boyden chamber experiments the recombinant MMP-9-PEX was able to inhibit the invasion of melanoma cells secreting high amounts of MMP-9 in a dose-dependent manner. These data demonstrate for the first time that the hemopexin domain of MMP-9 has a high affinity binding site for gelatin, and the particular recombinant domain is able to block MMP-9 activity and tumor cell invasion. Because MMP-9 plays an important role in metastasis, this antagonistic effect may be utilized to design MMP inhibition-based cancer therapy.     

F1-ATPase, the C-terminal end of subunit gamma is not required for ATP hydrolysis-driven rotation

ATP hydrolysis by the isolated F(1)-ATPase drives the rotation of the central shaft, subunit gamma, which is located within a hexagon formed by subunits (alphabeta)(3). The C-terminal end of gamma forms an alpha-helix which properly fits into the "hydrophobic bearing" provided by loops of subunits alpha and beta. This "bearing" is expected to be essential for the rotary function. We checked the importance of this contact region by successive C-terminal deletions of 3, 6, 9, 12, 15, and 18 amino acid residues (Escherichia coli F(1)-ATPase). The ATP hydrolysis activity of a load-free ensemble of F(1) with 12 residues deleted decreased to 24% of the control. EF(1) with deletions of 15 or 18 residues was inactive, probably because it failed to assemble. The average torque generated by a single molecule of EF(1) when loaded by a fluorescent actin filament was, however, unaffected by deletions of up to 12 residues, as was their rotational behavior (all samples rotated during 60 +/- 19% of the observation time). Activation energy analysis with the ensemble revealed a moderate decrease from 54 kJ/mol for EF(1) (full-length gamma) to 34 kJ/mol for EF(1)(gamma-12). These observations imply that the intactness of the C terminus of subunit gamma provides structural stability and/or routing during assembly of the enzyme, but that it is not required for the rotary action under load, proper.     

Quinones in long-lived clk-1 mutants of Caenorhabditis elegans

Ubiquinone (UQ) (coenzyme Q) is a lipophilic redox-active molecule that functions as an electron carrier in the mitochondrial electron transport chain. Electron transfer via UQ involves the formation of semiubiquinone radicals, which causes the generation of superoxide radicals upon reaction with oxygen. In the reduced form, UQ functions as a lipid-soluble antioxidant, and protects cells from lipid peroxidation. Thus, UQ is also important as a lipophilic regulator of oxidative stress. Recently, a study on long-lived clk-1 mutants of Caenorhabditis elegans demonstrated that biosynthesis of UQ is dramatically altered in mutant mitochondria. Demethoxy ubiquinone (DMQ), that accumulates in clk-1 mutants in place of UQ, may contribute to the extension of life span. Here we elucidate the possible mechanisms of life span extension in clk-1 mutants, with particular emphasis on the electrochemical property of DMQ. Recent findings on the biochemical function of CLK-1 are also discussed.