Determination of glucose and ethanol in bioethanol production by near infrared spectroscopy and chemometrics.

The concentrations of glucose and ethanol in substrates from bioethanol processes have been modeled by near infrared (NIR) spectroscopy data. NIR spectra were acquired in the wavelength range of 1100-2300 nm by means of a transflectance probe for measurements in liquid samples. For building of regression models a genetic algorithm has been applied for variable selection, and partial least-squares (PLS) regression for creation of linear models. A realistic estimation of the prediction performance of the models was obtained by a repeated double cross-validation (rdCV). Reduced data sets with only 15 variables showed improved prediction qualities, in comparison with models containing 235 variables, particularly for the determination of the ethanol concentration in distillation residues (stillages). The squared correlation coefficient, R(2), between the concentrations obtained by HPLC analysis and the concentrations derived from NIR data (using 15 selected wavelengths, test set samples) was 0.999 for ethanol in stillage, and 0.977 for glucose in mash. The standard deviation of prediction errors, SEP, obtained from test set samples was 0.6 g L(-1) for ethanol (2% of the mean ethanol concentration), and 2.0 g L(-1) for glucose (9.6% of the mean glucose concentration).

Aspergillus fumigatus conidial pigment and cAMP signal transduction: significance for virulence.

The conidial pigment of Aspergillus fumigatus contains 1,8-dihydroxynaphthalene (DHN)-like pentaketide melanin. It plays a major role in the protection of the fungus against immune effector cells; for example, it is able to scavenge reactive oxygen species generated by alveolar macrophages and neutrophiles. The polyketide synthase PKSP (ALB1) is a key-enzyme of the biosynthesis pathway; its structural gene is part of a gene cluster. Furthermore, the presence of a functional pksP (albl) gene in A. fumigatus conidia is associated with an inhibition of phagolysosome fusion in human monocyte-derived macrophages. Moreover, the analysis of mutants that are defective in elements of the cAMP signaling pathway found that they are almost avirulent in an optimized low dose murine inhalation model. Taken together, our results indicate that the cAMP/PKA signal transduction pathway is required for A. fumigatus pathogenicity. In addition, we showed that the expression of the pksP gene is, at least in part, controlled by the cAMP/ PKA signal transduction pathway. Currently, we hypothesize that pentaketide melanin is important for defence against ROS. However, besides its contribution to the biosynthesis of DHN-like melanin, PKSP also appears to be involved in the formation of another compound which is immunosuppressive.

cAMP signaling in Aspergillus fumigatus is involved in the regulation of the virulence gene pksP and in defense against killing by macrophages.

Aspergillus fumigatus is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease and resulting in high mortality. In order to determine the importance of the cAMP signaling pathway for virulence, three genes encoding putative elements of the pathway have been cloned and characterized: the adenylate cyclase gene acyA, and gpaA and gpaB, both of which encode alpha subunits of heterotrimeric G proteins. The acyA and gpaB genes were each deleted in A. fumigatus. Both mutants showed reduced conidiation, with the deltaacyA mutant producing very few conidia. The growth rate of the deltaacyA mutant was also reduced, in contrast to that of the deltagpaB mutant. Addition of 10 mM dibutyryl-cAMP to the culture medium completely restored the wild-type phenotype in both mutant strains. To study the influence of GPAB on the expression of the gene pksP, which encodes a virulence factor that is involved in pathogenicity, a pksPp-lacZ gene fusion was generated and integrated as a single copy at the pyrG gene locus of both the parental strain and the deltagpaB mutant strain. The deltagpaB mutant showed reduced expression of the pksPp-lacZ reporter gene relative to that in the parental strain. In mycelia of both the parental strain and the deltagpaB mutant pksPp-lacZ expression was increased when isobutyl-methyl-xanthine, an inhibitor of intracellular phosphodiesterases, was added to the medium. The survival rate of conidia after ingestion by human monocyte-derived macrophages was also determined. The killing rate for conidia from deltaacyA and deltagpaB strains was significantly higher than that for wild-type conidia. Taken together, these findings suggest that cAMP triggers a system that protects A. fumigatus from the effects of immune effector cells of the host.

The effect of ciclotropium on human heart rate.

Ciclotropium is a recently developed parasympathicolytic agent. Plasma concentration and heart rate increase (the most prominent anticholinergic effects) were measured in 12 healthy subjects before, during and after a 15-min intravenous infusion of 10 mg ciclotropium. The effect was described by using either a linear or a nonlinear (Emax) effect model linked to a linear three-compartment kinetic model via an effect compartment. Maximum heart rate increase was 33 (10) beats.min-1, and half-value duration of effect was 41 (9) min. Total plasma clearance was 0.51 (0.13) l.min-1, and mean terminal elimination half-life was 12(4) h, whereas the equilibration half-lives of drug removal from the effect compartment ranged from 2 to 14 min.

Studies on the metabolic clearance of ciclotropium to alpha-phenylciclopentylacetic acid using a new enantiospecific metabolite assay.

Ester hydrolysis represents an important biotransformation pathway for various parasympatholytic agents. Cleavage of the ciclotropium ester bond results in the formation of alpha-phenylciclopentylacetic acid (PCA). The relevance of this metabolic route for ciclotropium bromide (HIT-PCE, CAS 85166-20-7) including its stereochemical aspects was studied in a preliminary pharmacokinetic study. An enantiospecific assay for biological material was developed that is based on chiral derivatization of PCA with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDAC) and the primary amine S-FLOPA, a chiral coupling component for carboxylic acids derived from S-flunoxaprofen, followed by HPLC resolution. R-(--)-Ibuprofen was used as internal standard. From plasma or urine PCA can be extracted into n-hexane/ethanol (9:1) at pH 4 under addition of sodium chloride. Derivatization with EDAC/FLOPA was performed under addition of 1-hydroxybenzotriazole in anhydrous dichloromethane that contained trace amounts of pyridine (ambient temperature; 2 h reaction time). The chromatographic separation was performed on a silica gel stationary phase (Zorbax Sil) using n-hexane-chloroform-ethanol (100:10:1, by vol.) as mobile phase (flow rate, 2 ml/min; fluorescence-detection, 305/355 nm; elution order of the derivatives, (-) before (+)). Limit of quantification was 1.0 ng/ml for plasma and 10 ng/ml for urine. In the pharmacokinetic study in two healthy volunteers who received a single i.v. dose of 10 mg ciclotropium race-mate the PCA concentrations in plasma were below the detection limit, but approx. 1.5% of the administered dose were excreted into urine as the respective glucuronides.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the quaternary compound ciclotropium in human biological material after hydrolysis and derivatization with the fluorophor flunoxaprofen chloride.

The quantitative determination of the quaternary spasmolytic compound ciclotropium and its metabolite N-isopropyltropinium is described for human plasma and urine. The analytical procedure consists of ion-pair extraction from biological material, alkaline hydrolysis, subsequent derivatization with the fluorophor flunoxaprofen chloride and separation by high-performance liquid chromatography on a reversed-phase column with fluorimetric monitoring. The detection limits of 0.5 ng/ml in plasma and 10 ng/ml in urine at signal-to-noise ratios higher than 3 permit the determination of pharmacokinetic parameters after therapeutic doses.