[Is monotherapy with ß-lactam antibiotics still up to date? New aspects for treatment of severe infections]. / Sind Monotherapien mit ß-Laktamantibiotika noch zeitgemäss?

Mortality of sepsis is still high. Crucial for therapeutic response are the early start of treatment as well as the choice of antibiotics or antibiotic combinations. ß-lactam antibiotics with bactericidal mode of action are often recommended in guidelines. But this antibiotic class can trigger the immune system to a maximum by releasing cell wall components or exotoxins. This may lead to a worsening of the patient's clinical situation. In contrast, antibiotics with bacteriostatic action often inhibit bacterial protein synthesis with decrease of production of virulence factors and minimize release of cell wall components. The purpose of this review is to summarise the significance of some bacteriostatic antibiotics and to discuss whether a combination of bactericidal and bacteriostatic agents may improve the course of the illness.

Evidence for orthologous S-locus-related I genes in several genera of Brassicaceae.

In the Brassica genus, self-incompatibility (SI) is considered to be controlled by the combined action of several highly polymorphic genes located at the S-locus. These genes, including the S-Locus Gene (SLG), and the S-Receptor Kinase (SRK) are all members of the complex multigenic S-family. The S-Locus Related I gene (SLR1) is a member of the S-family, but is not involved in SI control since it is not linked to the S-locus and is essentially monomorphic. Here we confirm or demonstrate the occurrence of SLR1 as highly diverged but not very polymorphic genes in several genera of the Brassicaceae family (Arahidopsis, Brassica, Hirschfeldia, Raphanus, Sinapis). They show similar expression patterns with respect to location (stigmatic papillae), developmental stage (before and during anthesis) and transcript size (1.6 kb). In addition, they are assumed to be involved in the same biological function (late pollen adhesion). These features suggest that the pollen adhesion function might have evolved towards self-pollen recognition through duplication of SLR1 and recruitment of a protein kinase gene.

Metabolism and excretion of tolcapone, a novel inhibitor of catechol-O-methyltransferase.

AIMS: To investigate the rate of excretion and routes of metabolism of tolcapone, a novel inhibitor of catechol-O-methyltransferase (COMT). METHODS: Six healthy male volunteers were given 200 mg [14C]-tolcapone (approximately 50 muCi) orally. To assess excretion balance and to identify metabolites, urine and faeces were collected before administration and until radioactivity fell below 75 d min-1 ml-1 (urine) and 100 d min-1 mg-1 (faeces). Blood samples were collected frequently before and after administration to determine plasma radioactivity, to identify tolcapone metabolites and to measure plasma tolcapone and its methylated derivative 3-O-methyltolcapone (3-OMT). RESULTS: The mean proportion of the dose excreted in urine was 57.3% and in faeces 40.5%. Excretion was almost complete (more than 95%) in all participants after 9 days. The major early metabolite present in plasma was the 3-O-beta, d-glucuronide conjugate, which was detectable within 2 h after dosing. The major late metabolite in plasma was 3-OMT. The 3-O-beta, d-glucuronide was also the most abundant metabolite in urine and faeces, accounting for 27% and 33%, respectively, of the total radioactivity excreted by these routes and for 26% of the original dose. Reduction of the nitro moiety yields an amine derivative, detected in both urine and faeces, with subsequent modifications, such as acetylation of the amine group and conjugation with glucuronic acid or sulphate, or both. Oxidative reactions due to cytochrome P450 enzymes are of small significance, as is 3-O-methylation by COMT. CONCLUSIONS: Tolcapone is almost completely metabolized and excreted in urine and faeces (only 0.5% of tolcapone was excreted unchanged); glucuronidation is the most important route of metabolism. The relatively long duration of excretion is caused by the long half-life of 3-OMT.

Determination of the catechol-O-methyltransferase inhibitor tolcapone and three of its metabolites in animal and human plasma and urine by reversed-phase high-performance liquid chromatography with ultraviolet detection.

Reversed-phase HPLC procedures were developed for the determination of tolcapone (Ro 40-7592) and its metabolites Ro 40-7591, Ro 61-1448, and Ro 47-1669 in plasma and in urine samples. One of the procedures for plasma involved the determination of tolcapone and its metabolite Ro 40-7591 and the other, the determination of the two other metabolites. The urine assay enabled the simultaneous determination of tolcapone and all metabolites in one run. Sample preparation in plasma involved protein precipitation with acetonitrile. Urine was simply diluted. The compounds of interest were monitored in the UV at 270 nm. The limits of quantification were 0.05 microg/ml for each compound (plasma assay) and 0.2 microg/ml for the urine assay. The mean inter-assay precisions (C.V.) were < or = 6% (plasma assay) and < or = 8% (urine assay). The procedures were successfully applied to the sample analysis of animal pharmacokinetic (rat, dog, mouse, rabbit and cynomolgus monkey) and clinical pharmacology studies.

Pollen-stigma adhesion in Brassica spp involves SLG and SLR1 glycoproteins.

The adhesion of pollen grains to the stigma is the first step of pollination in flowering plants. During this step, stigmas discriminate between pollen grains that can and cannot be permitted to effect fertilization. This selection is operated by various constituents of the cell walls of both partners. Several genes structurally related to the self-incompatibility system that prevents self-pollination in Brassica spp are known to target their products into the stigma cell wall. We proposed previously that one of these genes, the one encoding the S locus glycoprotein (SLG)-like receptor 1 (SLR1), which is coexpressed with that encoding SLG, may participate in pollen-stigma adhesion. Here, we exploit a biomechanical assay to measure the pollen adhesion force and show that it is reduced both by transgenic suppression of SLR1 expression and by pretreatment of wild-type stigmas with anti-SLR1 antibodies, anti-SLG antibodies, or pollen coat-protein extracts. Our results indicate a common adhesive function for the SLR1 and SLG proteins in the pollination process.

Pharmacokinetics and pharmacodynamics after oral and intravenous administration of tolcapone, a novel adjunct to Parkinson's disease therapy.

OBJECTIVE: To evaluate fully the pharmacokinetics and pharmacodynamics of tolcapone, a novel inhibitor of catechol-O-methyltransferase (COMT), after oral and intravenous administration. METHODS: Sixteen healthy male volunteers were given tolcapone in single doses of 200 mg orally and 50 mg intravenously, separated by a washout period of 7 days or more, in a single-center, open-label, randomized, cross-over study. Pharmacokinetic parameters were calculated using both compartmental and non-compartmental methods; pharmacodynamics were evaluated from erythrocyte COMT activity. RESULTS: After an initial lag time of 0.5 h, tolcapone was rapidly absorbed (peak plasma concentrations were reached within approximately 2 h) following either zero- or first-order absorption kinetics. The absolute bioavailability of an oral dose was approximately 60%. The volume of distribution was approximately 9 1, and the total clearance was approximately 71.h-l, resulting in a mean plasma half-life of 1.8 h. The degree of erythrocyte COMT inhibition was closely related to tolcapone plasma concentration; a rebound in COMT activity was observed after tolcapone withdrawal. Both oral and intravenous tolcapone were well tolerated. DISCUSSION: Because of its relatively low systemic clearance, tolcapone exhibits only a small degree of first-pass metabolism and a relatively good oral bioavailability, which explains the higher plasma concentrations and stronger COMT inhibition observed with tolcapone compared with entacapone, another COMT inhibitor. The pharmacokinetic and pharmacodynamic profile of tolcapone obtained in this study underlines the potential of the agent to be used as an adjunct to levodopa in the treatment of Parkinson's disease.

Effect of liver impairment on the pharmacokinetics of tolcapone and its metabolites.

OBJECTIVE: To assess the effect of liver impairment on the pharmacokinetics of tolcapone and to derive appropriate dose recommendations for patients with this disease who are undergoing treatment for Parkinson's disease. STUDY DESIGN: In an open, two-way crossover study, 16 patients with moderate liver disease (eight with cirrhotic and eight with noncirrhotic liver disease) and eight healthy subjects received an oral dose of 200 mg tolcapone and an intravenous dose of 50 mg tolcapone on separate occasions. The concentrations of total and unbound tolcapone and its three major metabolites (tolcapone glucuronide, carboxylic acid, and 3-O-methyl metabolite) were assessed in plasma and urine. RESULTS: On the basis of total drug concentration, the differences in tolcapone pharmacokinetics between the groups were small. However, lower clearance and volume of distribution of unbound drug were found among patients with cirrhosis than among control subjects. Plasma concentration of the pharmacologically inactive glucuronide metabolite was increased among patients with cirrhosis. CONCLUSIONS: Half of the recommended dosage of tolcapone should be administered to patients with cirrhosis of the liver to maintain the target steady-state concentration of unbound drug and to avoid accumulation of tolcapone glucuronide. Our data did not indicate a requirement for dosage adjustment in the presence of moderate chronic hepatitis.

Animal pharmacokinetics and interspecies scaling of Ro 25-6833 and related (lactamylvinyl)cephalosporins.

From a series of six (lactamylvinyl)cephalosporins, candidates for clinical evaluation were selected on the basis of their kinetic profile in animals and predicted pharmacokinetics in man. Exploratory pharmacokinetic studies with Ro 25-6833 and five related cephalosporins were performed following intravenous administration to rats, dogs, and cynomolgus monkeys. All compounds were characterized by a high protein binding in rat, monkey, and human plasma (unbound fraction < or = 5%), whereas in dog plasma, protein binding was markedly lower. Accordingly, for most compounds, clearance was highest in dogs, and lowest in monkeys. Comparison of the renal clearance of unbound drug with creatinine clearance suggests a renal elimination of Ro 25-6833 by glomerular filtration in both rats and dogs (urinary excretion in monkey was not determined due to drug instability in monkey urine). All other compounds showed different renal excretion mechanisms in rats and dogs, thus making the validity of allometric scaling questionable. Unbound clearances in man were predicted by allometric scaling (Ro 25-6833 only) and by a correlation analysis of cephalosporin pharmacokinetics in monkey and man. Limitations of both methods are discussed. When Ro 25-6833 was later studied in man, the predicted pharmacokinetic data in man from both approaches were found to be in good agreement with the observed values.

Integrated pharmacokinetics and pharmacodynamics of Ro 48-8684, a new benzodiazepine, in comparison with midazolam during first administration to healthy male subjects.

AIMS: This study aimed to investigate the pharmacodynamics and pharmacokinetics of ascending doses of Ro 48-8684, compared with midazolam, in healthy subjects during first administration to man. METHODS: The study was double-blind and five-way crossover (three ascending doses, placebo, fixed midazolam dose), performed in two groups of five males. Ro 48-8684 was infused in doses of 0.1-0.3-1 mg in the first group, and 1-3-10 mg in the second, with different infusion rates (expressed as mg min(-1)) among doses. Midazolam was infused at 0.1 mg(-1) kg. Infusions were stopped after 20 min or if sedation became too strong for proper performance of saccadic eye movements. Pharmacokinetics and pharmacodynamics and their relationships were evaluated as described in the companion article. RESULTS: Ro 48-8684 caused dose-dependent sedation. No serious adverse events occurred. The volume of distribution and clearance of Ro 48-8684 were larger than of midazolam (337+/-114 vs 50+/-121 and 2.4+/-0.5 vs 0.47+/-0.11 l min(-1), resp). The recovery of saccadic eye movements from equal levels of sedation was on average almost half an hour faster for Ro 48-8684 than for midazolam, with considerable interindividual differences (range 2, 55 min). The doses of Ro 48-8684 leading to the same clinical endpoint as midazolam were comparable, but the corresponding predicted effect compartment concentrations of Ro 48-8684 were on average 2.6 times lower (range 1.5, 4.9 times). The slope of the linear concentration-effect-relationship for saccadic peak velocity was on average 2.2 times steeper for 10 mg Ro 48-8684 than for midazolam (range 1.3, 3.3). The slope decreased on average 4.4-fold (range 1.6, 7.3 times), with doses of Ro 48-8684 increasing from 1 to 10 mg. The metabolite Ro 61-2466 had a longer half-life than the parent compound Ro 48-8684. The influence of this metabolite during prolonged administration should be further investigated. CONCLUSIONS: These results show that Ro 48-8684 has a considerably shorter duration of action than midazolam. There may be a reduction of sensitivity to Ro 48-8684 with repeated administration of rising doses due to as yet undetermined factors.

Pollen-Stigma Adhesion in Kale Is Not Dependent on the Self-(In)Compatibility Genotype.

The adhesion of pollen on the stigmas of flowering plants is a critical step for the success of reproduction in angiosperms, long considered to present some specificity in terms of self-incompatibility. We carried out quantitative measurements of the pollen-stigma adhesion (expressed in Newtons) in kale (Brassica oleracea), using the flotation force of Archimedes exerted by dense sucrose solutions (50%, w/v) to release pollen grains fixed on the surface of stigmas. We demonstrate that pollen adhesion varies with the genotypes of the plants used as partners, but increases with time in all cases for about 30 to 60 min after pollination. There is no correlation with the self- or cross-status of the pollinations, nor with the self-compatible or -incompatible genotypes of the parents. Only late events of pollination, after the germination or arrest of the pollen tube, depend on compatibility type. Biochemical and physiological dissection of pollen-stigma adhesion points to major components of this interaction: among male components, the pollen coating, eliminated by delipidation (or modified by mutation in the case of the cer mutants of the related species Arabidopsis thaliana), plays a major role in adhesion; the genetic background of the pollen parent is also of some importance. On the female side, the developmental stage of the stigma and the protein constituents of the stigmatic pellicle are critical for pollen capture. The SLG and SLR1 proteins are not involved in the initial stages of pollen adhesion on the stigma but one or both may be involved in the later stages.

Pharmacokinetics of retinoids in women after meal consumption or vitamin A supplementation.

These studies were conducted to evaluate the pharmacokinetics of several retinoids after meal consumption or vitamin A supplementation to establish a reference for future assessment of teratogenic risks of retinoid therapeutic agents. In the first study, 36 healthy young female volunteers consumed single meals containing vitamin A amounts ranging from 1,305 to 169,474 IU. In the second study, 24 other female volunteers took vitamin A supplements at a dose level of 5,000, 10,000, or 25,000 IU/day for 60 days. Plasma concentrations of tretinoin, isotretinoin, 4-oxo-tretinoin, and 4-oxo-isotretinoin in samples collected during the studies were analyzed using a high-performance liquid chromatography method with ultraviolet detection. Pharmacokinetic parameters for the retinoids were calculated using model-independent methods. Plasma concentrations of tretinoin were not altered by meal consumption or vitamin A supplementation. Plasma levels of 4-oxo-tretinoin were below the assay detection limit (0.3 ng/mL) in the majority of samples collected throughout the studies. Linear relationships between dose and maximum concentration (Cmax) and dose and area under the concentration-time curve (AUC) for isotretinoin and 4-oxo-isotretinoin were derived from data from the meal study. For the most bioavailable formulation used in the supplement study, daily ingestion of 5,000 IU of vitamin A caused increases of 141 +/- 53% and 171 +/- 77% from baseline in the 24-hour AUCs of isotretinoin and 4-oxo-isotretinoin, respectively. Dose-related increases in systemic exposure to retinoids were observed after ingestion of vitamin A by means of a meal or a supplement. Findings from these studies can be used as a basis for future safety evaluations of retinoid compounds.

Characterization of the S locus genes, SLG and SRK, of the Brassica S3 haplotype: identification of a membrane-localized protein encoded by the S locus receptor kinase gene.

The S locus, which controls the self-incompatibility response in Brassica, has been shown to contain at least two genes. SLG encodes a secreted S locus glycoprotein whilst SRK encodes a putative S locus receptor kinase. SRK has been shown potentially to encode a functional kinase and genetic evidence indicates that this gene is essential for the self-incompatibility response. Here the characterization of the SRK and SLG genes of a Brassica line homozygous for the S3 haplotype is described. A 120 kDa glycoprotein was identified in stigmas and several lines of evidence indicated that this protein is encoded by the SRK3 gene. First, the 120 kDa glycoprotein was recognized by antibodies raised against peptides based on the SRK3 gene sequence. Secondly, this protein is polymorphic and, in an F2 population segregating for the S3 haplotype, was expressed only in plants possessing the S3 haplotype. Thirdly, the 120 kDa protein was expressed specifically in stigmas. Finally, the 120 kDa protein was only extracted from stigmas in the presence of detergent indicating that it is anchored in the membrane. SRK has been predicted to encode a transmembrane glycoprotein based on the deduced amino acid sequence. Located on the membrane, SRK is in a position to interface between an extracellular recognition event between pollen and pistil and an intracellular signal transduction pathway which initiates the self-incompatibility response.

Expression level of the SLG gene is not correlated with the self-incompatibility phenotype in the class II S haplotypes of Brassica oleracea.

In Brassica, the S-locus glycoprotein (SLG) gene has been strongly implicated in the self-incompatibility reaction. Several alleles of this locus have been sequenced, and accordingly grouped as class I (corresponding to dominant S-alleles) and class II (recessive). We recently showed that a self-compatible (Sc) line of Brassica oleracea expressed a class II-like SLG (SLG-Sc) gene. Here, we report that the SLG-Sc glycoprotein is electrophoretically and immunochemically very similar to the recessive SLG-S15 glycoprotein, and is similarly expressed in stigmatic papillae. Moreover, by seed yield analysis, we observed that both alleles are associated with a self-compatibility response, in contrast with the other known recessive S haplotypes (S2 and S5). By genomic DNA blot analysis, we show the existence of molecular homologies between the Sc and S15 haplotypes, but demonstrate that they are not identical. On the other hand, we also report that the S2 haplotype expresses very low amounts of SLG glycoproteins, although it exhibits a self-incompatible phenotype. These results strongly question the precise role of the SLG gene in the molecular mechanisms that control the self-incompatibility reaction of Brassica.

Isolation and microinjection of active sperm nuclei into egg cells and central cells of isolated maize embryo sacs.

Artificial fertilisation was attempted in maize by microinjecting sperm nuclei into the egg cell or central cell of isolated embryo sacs. A protocol for isolation of nuclei from pollen grains was developed and a pure fraction of sperm nuclei was obtained after centrifugation on a Percoll gradient. The in vitro transcriptional activity of the nuclei was tested by incorporation of radioactive UTP into RNA. The level of labelled nucleotide incorporation increased and reached a maximum after between 30 and 40 min in the incubation medium. The embryo sacs were enzymatically isolated and their viability determined by observation of cytoplasmic streaming in the female cells. The embryo sacs were immobilised by embedding in low-melting-point agarose and a single male nucleus was injected with a bevelled microcapillary. The presence of the injected nucleus in the egg or central cell was demonstrated using a cytological approach. This paper presents an alternative method for studying the intimate processes of fertilisation in plants.

Tissue printing and its applications in self-incompatibility studies.

In this study, the tissue printing technique has been used to rapidly localize in female tissues the presence of specific mRNA representing the products (or some of the products) of the self-incompatibility S-locus gene(s). The methodology, initially developed for Brassica oleracea (sporophytic self-incompatibility) has been successfully employed on Solanum chacoense (gametophytic self-incompatibility). In the Brassica system tissue printing has allowed rapid discrimination between S alleles belonging to class 1 (dominant types) vs. class 2 (recessive types), and thus parallels findings obtained by restriction analyses. In the Solanum system the level of the S-RNase messages was analysed by scanning laser densitometry, and it was found that the message levels of the allele S14 declined faster than those coming from S13 in mature flowers.

Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea.

In cruciferous plants, self-pollination is prevented by the action of genes situated at the self-incompatibility locus or S-locus. The self-incompatibility reaction is associated with expression of stigma glycoproteins encoded by the S-locus glycoprotein (SLG) gene. Only a few cases of self-compatible plants derived from self-incompatible lines in the crucifer Brassica have been reported. In these cases, self-compatibility was generally ascribed to the action of single genes unlinked to the S-locus. In contrast, we report here a line of Brassica oleracea var acephala with a self-compatible phenotype linked to the S-locus. By means of both biochemical and immunochemical analyses, we showed that this self-compatible (Sc) line nonetheless possesses stigmatic SLGs (SLG-Sc) that are expressed with a similar spatial and temporal pattern to that described for the SLGs of self-incompatible Brassica plants. Moreover, the SLG-Sc products segregate with the self-compatibility phenotype in F2 progeny, suggesting that changes at the S-locus may be responsible for the occurrence of the self-compatibility character. A cDNA clone encoding the SLG-Sc product was isolated, and the deduced amino acid sequence showed this glycoprotein to be highly homologous to the pollen recessive S2 allele glycoprotein. Hence, self-compatibility in this Brassica Sc line correlates with the expression of a pollen recessive-like S allele in the stigma.

Pharmacodynamics and pharmacokinetics of carprofen, a non-steroidal anti-inflammatory drug, in healthy cows and cows with Escherichia coli endotoxin-induced mastitis.

The pharmacodynamics of carprofen and its pharmacokinetics in plasma and milk of healthy cows and cows with endotoxin-induced mastitis were studied after a single intravenous dose of 0.7 mg/kg body weight. Carprofen was administered to five clinically healthy cows and to the same cows 3 weeks later, 2 h after intramammary infusion of endotoxin. Mastitis developed in all endotoxin-infused quarters. The pharmacokinetic characteristics of carprofen in healthy cows were a small volume of distribution (0.09 l/kg), a relatively low systemic clearance (2.4 ml/h kg), and a long elimination half-life (30.7 h). In the mastitic cows, systemic clearance (1.4 ml/h kg) was significantly lower (P less than 0.01), and elimination half-life (43.0 h) was significantly longer (P less than 0.01) than in the normal animals. Concentrations of carprofen in milk from healthy quarters were below the limit of detection for the assay (0.022 micrograms/ml). In milk from mastitic quarters, concentrations of carprofen increased up to 0.164 micrograms/ml during the first 12 h after induction of mastitis, but were less than 0.022 micrograms/ml at 24 to 48 h. Compared with the untreated mastitic controls, carprofen treatment significantly reduced heart rate (P less than 0.01), rectal temperature (P less than 0.001), quarter swelling (P less than 0.01) and other parameters measured. Local and systemic adverse reactions to carprofen were not observed.

PCR detection of transcripts homologous to the self-incompatibility gene in anthers ofBrassica.

The polymerase chain reaction (PCR) is particularly well suited for the detection of rare sequences. Taking advantage of the recent isolation of sequences associated with stigma self-incompatibility inBrassica oleracea, we used PCR amplifications with primers synthesized to the S6 cDNA sequence, to demonstrate the presence of mRNA homologous to stigmaS-locus gene (SLG) in anthers during early microsporogenesis. In addition, otherS-locus-related (SLR) sequences were shown to be transcribed in sexual as well as in vegetative tissues (roots, leaves), suggesting that the SLG family might be involved not only in pollen-stigma recognition, but more generally in various forms of plant cell signalling processes. This information corroborates the recent discovery of a cDNA-deduced protein kinase from maize roots, whose extracellular receptor displays high homology withBrassica S-locus-specific glycoproteins.