Influence of nebivolol and metoprolol on inflammatory mediators in human coronary endothelial or smooth muscle cells. Effects on neointima formation after balloon denudation in carotid arteries of rats treated with nebivolol.

OBJECTIVE AND BACKGROUND: Inflammation plays a critical role in all stages of atherogenesis. Proliferating vascular smooth muscle cells (SMC) and endothelial cells (EC) enhancing the inflammatory response, both contribute to the progression of atherosclerosis. Anti-proliferative, anti-inflammatory and anti-oxidative therapy seems to be a promising therapeutic strategy. The aim of this study was to assess the anti-proliferative and anti-inflammatory effect of the beta-blocker nebivolol in comparison to metoprolol in vitro and to find out whether nebivolol inhibits neointima formation in vivo. METHODS AND RESULTS: Real-time-RT-PCR revealed a decrease in VCAM-1, ICAM-1, PDGF-B, E-selectin and P-selectin mRNA expression in human coronary artery EC and SMC incubated with nebivolol for 72 hours while metoprolol did not have this effect. Nebivolol reduced MCP-1 and PDGF-BB protein in the culture supernatant of SMC and EC, respectively. Sprague-Dawley rats were treated with nebivolol for 0 or 35 days before and 28 days after carotid balloon injury. Immunohistological analyses showed that pre-treatment with nebivolol was associated with a decreased number of SMC layers and macrophages and an increased lumen area at the site of the arterial injury. The intima area was reduced by 43% after pre-treatment. CONCLUSION: We found that nebivolol reduced the expression of proinflammatory genes in endothelial cells and vascular smooth muscle cells in vitro whereas metoprolol did not. In vivo, nebivolol inhibited neointima formation by reducing SMC proliferation and macrophage accumulation.     

Production and characterization of an iminoimidazolidine specific monoclonal antibody using para-aminoclonidine as antigen.

Para-aminoclonidine coupled to hemocyanin was used to produce mouse monoclonal antibodies directed against clonidine. The properties of one of these, called mFE7, secreted by a clone of hybrid myeloma, are described. This antibody displayed total crossreactivity with imidazolidines and no crossreactivity at all with catecholamines or other known naturally occurring substances tested. A liquid phase radioimmunoassay permitted the detection of immunoreactivity in human brain extracts. The mFE7 antibody could be useful for immunopurifying the endogenous ligand of Imidazolines Preferring Receptors (IPR) which are catecholamines insensitive.     

Dynamic changes in plasma membrane properties of semliki forest virus infected cells related to cell fusion

The mechanism of the processes leading to membrane fusion is as yet unknown. In this report we demonstrate that changes in membrane potential and potassium fluxes correlate with Semliki Forest virus induced cell-cell fusion at mildly acidic pH. The changes observed occur only at pH's below 6.2 corresponding to values required to trigger the fusion process. A possible role of these alterations of the plasma membrane related to membrane fusion phenomena is discussed.     

Synthesis and evaluation of inhibitors of cytochrome P450 3A (CYP3A) for pharmacokinetic enhancement of drugs

The HIV protease inhibitor ritonavir (RTV) is also a potent inhibitor of the metabolizing enzyme cytochrome P450 3A (CYP3A) and is clinically useful in HIV therapy in its ability to enhance human plasma levels of other HIV protease inhibitors (PIs). A novel series of CYP3A inhibitors was designed around the structural elements of RTV believed to be important to CYP3A inhibition, with general design features being the attachment of groups that mimic the P2–P3 segment of RTV to a soluble core. Several analogs were found to strongly enhance plasma levels of lopinavir (LPV), including 8, which compares favorably with RTV in the same model. Interestingly, an inverse correlation between in vitro inhibition of CYP3A and elevation of LPV was observed. The compounds described in this study may be useful for enhancing the pharmacokinetics of drugs that are metabolized by CYP3A.     

Lipoprotein from the osmoregulated ABC transport system OpuA of Bacillus subtilis: purification of the glycine betaine binding protein and characterization of a functional lipidless mutant.

The OpuA transport system of Bacillus subtilis functions as a high-affinity uptake system for the osmoprotectant glycine betaine. It is a member of the ABC transporter superfamily and consists of an ATPase (OpuAA), an integral membrane protein (OpuAB), and a hydrophilic polypeptide (OpuAC) that shows the signature sequence of lipoproteins (B. Kempf and E. Bremer, J. Biol. Chem. 270:16701-16713, 1995). The OpuAC protein might thus serve as an extracellular substrate binding protein anchored in the cytoplasmic membrane via a lipid modification at an amino-terminal cysteine residue. A malE-opuAC hybrid gene was constructed and used to purify a lipidless OpuAC protein. The purified protein bound radiolabeled glycine betaine avidly and exhibited a KD of 6 microM for this ligand, demonstrating that OpuAC indeed functions as the substrate binding protein for the B. subtilis OpuA system. We have selectively expressed the opuAC gene under T7 phi10 control in Escherichia coli and have demonstrated through its metabolic labeling with [3H]palmitic acid that OpuAC is a lipoprotein. A mutant expressing an OpuAC protein in which the amino-terminal cysteine residue was changed to an alanine (OpuAC-3) was constructed by oligonucleotide site-directed mutagenesis. The OpuAC-3 protein was not acylated by [3H]palmitic acid, and part of it was secreted into the periplasmic space of E. coli, where it could be released from the cells by cold osmotic shock. The opuAC-3 mutation was recombined into an otherwise wild-type opuA operon in the chromosome of B. subtilis. Unexpectedly, this mutant OpuAC system still functioned efficiently for glycine betaine acquisition in vivo under high-osmolarity growth conditions. In addition, the mutant OpuA transporter exhibited kinetic parameters similar to that of the wild-type system. Our data suggest that the lipidless OpuAC-3 protein is held in the cytoplasmic membrane of B. subtilis via its uncleaved hydrophobic signal peptide.     

Rapid detection of carbapenem resistance in Acinetobacter baumannii using matrix-assisted laser desorption ionization-time of flight mass spectrometry

Rapid detection of carbapenem-resistant Acinetobacter baumannii strains is critical and will benefit patient care by optimizing antibiotic therapies and preventing outbreaks. Herein we describe the development and successful application of a mass spectrometry profile generated by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) that utilized the imipenem antibiotic for the detection of carbapenem resistance in a large series of A. baumannii clinical isolates from France and Algeria. A total of 106 A. baumannii strains including 63 well-characterized carbapenemase-producing and 43 non-carbapenemase-producing strains, as well as 43 control strains (7 carbapenem-resistant and 36 carbapenem-sensitive strains) were studied. After an incubation of bacteria with imipenem for up to 4 h, the mixture was centrifuged and the supernatant analyzed by MALDI-TOF MS. The presence and absence of peaks representing imipenem and its natural metabolite was analyzed. The result was interpreted as positive for carbapenemase production if the specific peak for imipenem at 300.0 m/z disappeared during the incubation time and if the peak of the natural metabolite at 254.0 m/z increased as measured by the area under the curves leading to a ratio between the peak for imipenem and its metabolite being <0.5. This assay, which was applied to the large series of A. baumannii clinical isolates, showed a sensitivity of 100.0% and a specificity of 100.0%. Our study is the first to demonstrate that this quick and simple assay can be used as a routine tool as a point-of-care method for the identification of A. baumannii carbapenemase-producers in an effort to prevent outbreaks and the spread of uncontrollable superbugs.     

Cytoplasmic trafficking of minute virus of mice: low-pH requirement,routing to late endosomes,and proteasome interaction

The cytoplasmic trafficking of the prototype strain of minute virus of mice (MVMp) was investigated by analyzing and quantifying the effect of drugs that reduce or abolish specific cellular functions on the accumulation of viral macromolecules. With this strategy, it was found that a low endosomal pH is required for the infection, since bafilomycin A(1) and chloroquine, two pH-interfering drugs, were similarly active against MVMp. Disruption of the endosomal network by brefeldin A interfered with MVMp infection, indicating that viral particles are routed farther than the early endocytic compartment. Pulse experiments with endosome-interfering drugs showed that the bulk of MVMp particles remained in the endosomal compartment for several hours before its release to the cytosol. Drugs that block the activity of the proteasome by different mechanisms, such as MG132, lactacystin, and epoxomicin, all strongly blocked MVMp infection. Pulse experiments with the proteasome inhibitor MG132 indicated that MVMp interacts with cellular proteasomes after endosomal escape. The chymotrypsin-like but not the trypsin-like activity of the proteasome is required for the infection, since the chymotrypsin inhibitors N-tosyl-L-phenylalanine chloromethyl ketone and aclarubicin were both effective in blocking MVMp infection. However, the trypsin inhibitor Nalpha-p-tosyl-L-lysine chloromethyl ketone had no effect. These results suggest that the ubiquitin-proteasome pathway plays an essential role in the MVMp life cycle, probably assisting at the stages of capsid disassembly and/or nuclear translocation.     

Influence of dissolved O on the fermentative production of gallidermin by Staphylococcus gallinarum

Decreased O supply during the fermentative production of gallidermin by Staphylococcus gallinarum decreased biomass formation by 65% compared to that obtained with optimal oxygen supply. However the antibiotic, gallidermin, increased by more than 50% at the same time. This effect was used in a process strategy, that allows biomass formation under oxygen saturation first and then switches to a prolonged production phase after a carefully directed shift to oxygen limitation.