Infectious agents are not necessary for murine atherogenesis.

Recent work has revealed correlations between bacterial or viral infections and atherosclerotic disease. One particular bacterium, Chlamydia pneumoniae, has been observed at high frequency in human atherosclerotic lesions, prompting the hypothesis that infectious agents may be necessary for the initiation or progression of atherosclerosis. To determine if responses to gram-negative bacteria are necessary for atherogenesis, we first bred atherosclerosis-prone apolipoprotein (apo) E(-/)- (deficient) mice with animals incapable of responding to bacterial lipopolysaccharide. Atherogenesis was unaffected in doubly deficient animals. We further tested the role of infectious agents by creating a colony of germ-free apo E(-/)- mice. These animals are free of all microbial agents (bacterial, viral, and fungal). Atherosclerosis in germ-free animals was not measurably different from that in animals raised with ambient levels of microbial challenge. These studies show that infection is not necessary for murine atherosclerosis and that, unlike peptic ulcer, Koch's postulates cannot be fulfilled for any infectious agent in atherosclerosis.

Inhibition of dermal MRSA colonization by microalgal micro- and nanoparticles.

The aim of this study was to investigate the prevention of the dermal colonization of methicillin-resistant Staphylococcus aureus (MRSA) strains by the application of micro- and nanoparticles called Maresometrade mark. Maresometrade mark were prepared from selected microalgae by a novel emulsion technique. They contain lipids and all other components of the microalgae in an encapsulated form. It could be shown that Maresometrade mark prepared from a cyanobacterial strain of the order Nostocales (Bio33-Maresometrade mark) were able to inhibit the dermal colonization of different MRSA strains (North German Epidemic Strain, Col, N315) and even of the vancomycin-resistant strain MU50 in the models 'mouse ear' and 'cow udder teat'. Pretreatment of the skin with Maresometrade mark reduced the number of attached MRSA by 3-4 log units in comparison to the control. We assume that a prophylactic skin care with Maresometrade mark could complete the multibarrier anti-infectious strategy against MRSA.

Development and application of a scintillation proximity assay (SPA) for identification of selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1.

Pre-receptor metabolism of glucocorticoids by the 11beta-hydroxysteroid dehydrogenase (11betaHSD) enzymes has been implicated in the etiology of the metabolic syndrome. Recent studies have shown that alterations in the activity of the type 1 isozyme can affect many aspects of the disease. This paper describes the optimization and application of a high-throughput scintillation proximity assay (SPA) developed to identify selective specific inhibitors of 11betaHSD1. Microsomes containing 11betaHSD1 were incubated in the presence of NADPH and [3H]cortisone, and the product, [3H]cortisol, was specifically detected in the mixture by a monoclonal antibody coupled to protein A-coated SPA beads with greater than 2 log higher affinity for cortisol than cortisone. Dimethyl sulfoxide and NADPH co-substrate additions were optimized for 11betaHSD1 reductase activity. Titrated test compound, when introduced into the optimized assay, reproducibly inhibited the enzyme and yielded consistent IC50 data in either 96- or 384-well format. An 11betaHSD2 counterscreen was performed by incubating 11betaHSD2 microsomes with [3H]cortisol and NAD+ and monitoring substrate disappearance.

Simvastatin has anti-inflammatory and antiatherosclerotic activities independent of plasma cholesterol lowering.

Inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, such as simvastatin, lower circulating cholesterol levels and prevent myocardial infarction. Several studies have shown an unexpected effect of HMG-CoA reductase inhibitors on inflammation. Here, we confirm that simvastatin is anti-inflammatory by using a classic model of inflammation: carrageenan-induced foot pad edema. Simvastatin administered orally to mice 1 hour before carrageenan injection significantly reduced the extent of edema. Simvastatin was comparable to indomethacin in this model. To determine whether the anti-inflammatory activity of simvastatin might affect atherogenesis, simvastatin was tested in mice deficient in apoE. Mice were dosed daily for 6 weeks with simvastatin (100 mg/kg body wt). Simvastatin did not alter plasma lipids. Atherosclerosis was quantified through the measurement of aortic cholesterol content. Aortas from control mice (n=20) contained 56+/-4 nmol total cholesterol/mg wet wt tissue, 38+/-2 nmol free cholesterol/mg, and 17+/-2 nmol cholesteryl ester/mg. Simvastatin (n=22) significantly (P<0.02) decreased these 3 parameters by 23%, 19%, and 34%, respectively. Histology of the atherosclerotic lesions showed that simvastatin did not dramatically alter lesion morphology. These data support the hypothesis that simvastatin has antiatherosclerotic activity beyond its plasma cholesterol-lowering activity.

Induction of 11beta-hydroxysteroid dehydrogenase type 1 but not -2 in human aortic smooth muscle cells by inflammatory stimuli.

The 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes catalyze the interconversion of active glucocorticoids (GC) with their inert metabolites, thereby regulating the functional activity of GC. While 11beta-HSD type 1 (11beta-HSD1) activates GC from their 11-keto metabolites, 11beta-HSD type 2 (11beta-HSD2) inactivates GC. Here we report that both of these enzymes are expressed in human aortic smooth muscle cells (SMC), and that 11beta-HSD1 is more abundant and is differentially regulated relative to 11beta-HSD2. Stimulation of SMC with IL-1beta or TNFalpha led to a time- and dose-dependent increase of mRNA levels for 11beta-HSD1, while 11beta-HSD2 mRNA levels decreased. Parallel enzyme activity studies showed increased conversion of 3H-cortisone to 3H-cortisol but not 3H-cortisol to 3H-cortisone, demonstrating 11beta-HSD1 in SMC acts primarily as a reductase. A similar increase of 11beta-HSD1 mRNA expression was also found in human bronchial SMC upon stimulation, indicating the regulatory effect is not limited to vascular smooth muscle. Additional parallel studies revealed a similar pattern of induction for 11beta-HSD1 and monocyte chemoattractant protein-1, a well-defined proinflammatory molecule. These data suggest 11beta-HSD1 may play an important role in regulating inflammatory responses in the artery wall and lung.

Cyanobacteria--a potential source of new biologically active substances.

Hydrophilic and lipophilic extracts of twelve cyanobacterial strains, isolated from fresh and brackish water, and two waterblooms, collected during the summer from the Baltic Sea, were investigated for their antibiotic activities against seven microorganisms. No inhibitory effects were found against the three Gram-negative bacteria Escherichia coli, Proteus mirabilis and Serratia marcescens and the yeast Candida maltosa. Of all cyanobacterial samples, extracts from seven species inhibited the growth of at least one of the Gram-positive bacteria Micrococcus flavus, Staphylococcus aureus and Bacillus subtilis. M. flavus proved to be the most sensitive bacterium in the agar diffusion test system. In particular, the hexane and dichlormethane extracts showed antimicrobial effects. But only one water extract, prepared from material of a natural waterbloom, was found to be active.

[Detection of muscarinic receptors on cultivated endothelial cells]. / Zum Nachweis muscarinerger Receptoren an kultivierten Endothelzellen.

In order to contribute to the verification of the hypothesis of Furchgott et al. that the effect of acetylcholine on blood vessels is mediated by factors produced by endothelial cells, the authors tried to provide evidence for the existence of muscarinic receptors on these cells. Using cultivated cells of the endothelium of calve aortas, a specific saturable binding of the potent muscarinic antagonist 3H-Quinuclidinyl benzilate and therewith the existence of muscarinic receptors on the cells could be demonstrated. Long time cultivation leads to the disappearance of the receptors.

Biochemical and pharmacological investigations of selected cyanobacteria.

Cyanobacteria are a very old group of prokaryotic organisms that produce a variety of secondary metabolites with antibiotic, algicide, cytotoxic, immunosuppressive and enzyme inhibiting activities. In the last decades structures of pure compounds have been determined as phenols, peptides, alkaloids or terpenoids (Falch, 1996). Screening of lipophilic and hydrophilic extracts from cultured cyanobacteria or waterbloom material, isolated from German lakes and the Baltic sea for antiviral, antibiotic, immunomodulating and enzyme inhibiting activity in different in vitro systems revealed strains with interesting effects. These strains were cultivated in 45 litre photobioreactors to produce enough biomass for bioassay-guided isolation of the active substances. First results characterising active substances are reported.

[Cultivated aortic cells--demonstration of muscarinic receptors and reaction behavior in the presence of acetylcholine]. / Kultivierte Aortenzellen--Nachweis muscarinerger Rezeptoren und Reaktionsverhalten in Gegenwart von Acetylcholin.

Using the muscarinic ligand 3H-quinuclidinyl benzilate the presence of muscarinic receptors on cells isolated from aortas of newborn rats and cultivated for 6 d could be shown. The number of receptors per cell was estimated with 17,400 on cells of confluent and with 87,100 on cells of nonconfluent cultures. Incubation of cells with increasing concentrations of acetylcholine in the range of 10(-13)-10(-7) mol.l-1 resulted in the reaction of growing numbers of cells following the all-or-nothing-law. Physiological concentrations of acetylcholine lead to contractions, showing that dilatations of blood vessels after application of acetylcholine in situ are caused by reaction products of endothelial cells, being absent in the cultures.

[Investigations of the immunomodulatory effect of cyanobacterial extracts]. / Untersuchungen zur immunmodulatorischen Wirkung von Extrakten aus Cyanobakterien.

Resulting from the knowledge that cyanobacteria (blue-green algae) are able to produce pharmacologically active substances the aqueous extracts from several cyanobacteria species and strains (Microcystis aeruginosa, Synechocystis aquatilis, Oscillatoria redekei, Anabaena flos-aque, Aphanizomenon flos-aquae, Oscillatoria rubescens, Oscillatoria tenuis) were tested for their immunomodulating activity. Extracts from Oscillatoria redekei 051, Oscillatoria tenuis 01 and Synechocystis aquatilis 428 caused an immunosuppression. They inhibited not only the incorporation of 3H-thymidine into mitogen stimulated lymphocytes but reduced also the number of plaque-forming cells of mice as shown by hemolysis-plaque-assay. Only extracts from Oscillatoria redekei 051 did not show any cytotoxic effects in lymphocyte cytotoxic test. This may be an evidence for a specific action on the proliferation of lymphocytes.

Complex formation of the spinach chloroplast psbA mRNA 5' untranslated region with proteins is dependent on the RNA structure.

RNA-protein interactions are part of many regulatory pathways in gene expression. In chloroplasts of higher plants and of green algae, gene regulation by posttranscriptional processes such as differential regulation of mRNA stability and control of translation plays a major role during chloroplast development and light-dependent protein expression. Regulation here is mediated by interactions of RNA-binding proteins with the respective mRNAs. In this work, structural requirements for protein-RNA complex formation between the 5' untranslated region of the spinach psbA mRNA (encoding the D1 protein of photosystem II) and stromal proteins are analyzed. For this, a combination of temperature gradient gel electrophoresis and gel shift analysis is employed to study several variants of the psbA 5' untranslated region. Supported by theoretical interpretation of the data and analysis of the structures by chemical probing, we show that a certain structure of the RNA is necessary for protein complex formation. Already very subtle structural changes within the RNA interfere with binding and thereby with the biological activity of the mRNA.

Cyanobacteria a potential source of antiviral substances against influenza virus.

Aqueous and methanolic extracts of cultured cyanobacteria of several genera, Microcystis, Nodularia, Oscillatoria, Scytonema, Lyngbya and Calothrix were evaluated for their in vitro antiviral activity against influenza A virus in Madin Darby canine kidney cells. None of the methanolic extracts showed cytotoxic effects. The inhibitory concentration (IC(50)) of antiviral activity ranged between 20.0 micro g to 79.0 micro g extract/ml. The most active extract in this screening derived from genus Microcystis. The further analysis of methanolic extracts of cultured strains of genus Microcystis revealed a remarkable antiviral activity against influenza A virus for M. aeruginosa, M. ichthyoblabe and M. wesenbergii. The observed antiviral activity was associated with protease inhibitory activity of approximately 90% and suggest that protease inhibitory activity may be responsible for reducing virus replication. These results show that cyanobacteria are able to produce compounds with biological activity that may be of potential clinical interest.

[Anesthesia in cats using tiletamine/zolazepam in minimal doses]. / Zur Anästhesie bei der Katze mit Tiletamin/Zolazepam in Minimaldosierung.

The object of this study was to evaluate minimal dose anesthesia with Tiletamine/Zolazepam for castration, dental treatments and other minor surgical procedures in cats. The study included 264 cats treated either at the Department of Veterinary Surgery, Ludwig-Maximilians University of Munich or under private practice conditions, in a small animal clinic in Hamburg (Germany). The drug dose needed for anesthesia for a 10 minute surgical procedure was calculated in each case using a formula. Side effects that occurred with doses recommended by the manufacturer could not be eliminated by decreasing the drug dose, but could be reduced considerably in severity and duration. Tiletamine/Zolazepam was found to be a useful drug for short anesthesia in cats at an average dose of 4.2 mg/kg.

Enzymatically inactive macrophage migration inhibitory factor inhibits monocyte chemotaxis and random migration.

Macrophage migration inhibitory factor (MIF) is a cytokine that was first described as an inhibitor of the random migration of monocytes and macrophages and has since been proposed to have a number of immune and catalytic functions. One of the functions assigned to MIF is that of a tautomerase that interconverts the enol and keto forms of phenylpyruvate and (p-hydroxyphenyl)pyruvate and converts D-dopachrome, a stereoisomer of naturally occurring L-dopachrome, to 5,6-dihydroxyindole-2-carboxylic acid. The physiological significance of the MIF enzymatic activity is unclear. The three-dimensional structure of MIF is strikingly similar to that of two microbial enzymes (4-oxalocrotonate tautomerase and 5-carboxymethyl-2-hydroxymuconate isomerase) that otherwise share little sequence identity with MIF. MIF and these two enzymes have an invariant N-terminal proline that serves as a catalytic base. Here we report a new biological function for MIF, as an inhibitor of monocyte chemoattractant protein 1- (MCP-1-) induced chemotaxis of human peripheral blood monocytes. We find that MIF inhibition of chemotaxis does not occur at the level of the CC chemokine receptor for MCP-1, CCR2, since MIF does not alter the binding of (125)I-MCP-1 to monocytes. The role of MIF enzymatic activity in inhibition of monocyte chemotaxis and random migration was studied with two MIF mutants in which the N-terminal proline was replaced with either a serine or a phenylalanine. Both mutants remain capable of inhibiting monocyte chemotaxis and random migration despite significantly reduced or no phenylpyruvate tautomerase activity. These data suggest that this enzymatic activity of MIF does not play a role in its migration inhibiting properties.

ApoE(-/-) mice develop atherosclerosis in the absence of complement component C5.

Previous studies have suggested that the terminal complex of complement may contribute to the pathogenesis of atherosclerosis. C5b-9 complexes colocalize with the extracellular lipid in the aortic intima of hypercholesterolemic rabbits, and C6-deficient rabbits develop less atherosclerosis than controls. To test the role of complement in atherosclerosis in a different animal model, C5 deficient (C5def) mice were cross-bred with atherosclerosis susceptible apoE(-/-) mice, generating mice deficient in both apoE and C5 and control apoE(-/-) mice. Progeny were typed for C5 titer and serum cholesterol levels. Both male and female mice were fed a high fat diet from weaning until 22 weeks of age. At that time there were no significant differences in plasma cholesterol or triglycerides between apoE(-/-) control and apoE(-/-)/C5def groups. Morphometric analysis of the aortic root lesions gave mean (+/-SEM) lesion areas for male apoE(-/-) and apoE(-/-)/C5def mice of 468,176 +/- 21,982 and 375,182 +/- 53,089 microm(2), respectively (n = 10 each, P value = 0.123). In female apoE(-/-) mice (n = 5), the mean lesion area was 591,981 +/- 53,242 microm(2), compared to 618,578 +/- 83,457 microm(2) for female apoE(-/-)/C5def mice (n = 10) (P value = 0.835). Thus neither male nor female mice showed a significant change in lesion area when C5 was not present. In contrast to the case in the hypercholesterolemic rabbit, activation of the terminal complex of complement does not play a major role in the development of atherosclerosis in apoE(-/-) mice.