The evaluation and prognosis of the psychophysiological status of a human operator

In experiments on 56 healthy subjects (18-20 years old) the quality of their activity was determined during compensatory watching the mark at complicating regimes of work. Depending on the difficulty of the task five groups of subjects were singled out with optimum working capacity in one of four working conditions: normal, ordinary and strenuous work, model of stress situation. It is established that the change of the number of significant correlative connections between main parameters of psychophysiological state of man-operator reflects the condition of his functional systems. On the basis of computation of total range of organization values of both R-R intervals of the ECG and duration of expiration, the success of the man-operator work in complex conditions of activity is predicted.     

Fine Mapping the Spatial Distribution and Concentration of Unlabeled Drugs within Tissue Micro-Compartments Using Imaging Mass Spectrometry

Readouts that define the physiological distributions of drugs in tissues are an unmet challenge and at best imprecise, but are needed in order to understand both the pharmacokinetic and pharmacodynamic properties associated with efficacy. Here we demonstrate that it is feasible to follow the in vivo transport of unlabeled drugs within specific organ and tissue compartments on a platform that applies MALDI imaging mass spectrometry to tissue sections characterized with high definition histology. We have tracked and quantified the distribution of an inhaled reference compound, tiotropium, within the lungs of dosed rats, using systematic point by point MS and MS/MS sampling at 200 µm intervals. By comparing drug ion distribution patterns in adjacent tissue sections, we observed that within 15 min following exposure, tiotropium parent MS ions (mass-to-charge; m/z 392.1) and fragmented daughter MS/MS ions (m/z 170.1 and 152.1) were dispersed in a concentration gradient (80 fmol-5 pmol) away from the central airways into the lung parenchyma and pleura. These drug levels agreed well with amounts detected in lung compartments by chemical extraction. Moreover, the simultaneous global definition of molecular ion signatures localized within 2-D tissue space provides accurate assignment of ion identities within histological landmarks, providing context to dynamic biological processes occurring at sites of drug presence. Our results highlight an important emerging technology allowing specific high resolution identification of unlabeled drugs at sites of in vivo uptake and retention.     

Deoxyadenosine/deoxycytidine kinase from Bacillus subtilis. Purification, characterization, and physiological function

Three different deoxyribonucleoside kinases with specificities toward thymidine, deoxyguanosine, and deoxyadenosine/deoxycytidine, respectively, are identified in Bacillus subtilis. The deoxyadenosin/deoxycytidine kinase is purified 950-fold employing blue Sepharose CL-6B column chromatography. The two deoxyribonucleoside kinase activities copurify and are present in the same band after polyacrylamide gel electrophoresis. The molecular weight is determined by gel filtration to be 47,000. Cytidine, adenosine, arabinosylcytosine, and arabinosyladenine are substrates for the enzyme. The activities toward these substrates are less than 20% of the activities obtained with deoxyadenosin and deoxycytidine. The deoxycytidine and deoxyadenosine saturation curves are hyperbolic with Km values for both nucleosides around 5 microM. The maximal velocities for the two deoxyribonucleosides are nearly identical with GTP as phosphate donor. GTP is the best donor showing hyperbolic saturation curves and Km values around 150 microM depending on the deoxyribonucleoside concentration. dATP and dCTP are inhibitors when GTP is the phosphate donor. They may both act as phosphate donors themselves. A divalent metal ion is required, Mg2+ giving the highest activity. A spontaneous mutant, selected as resistant to 5-fluorodeoxycytidine, lacks both deoxycytidine and deoxyadenosine kinase activity, while it retains normal activities toward deoxyguanosine, deoxyuridine, and thymidine.     

Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.     

Biorefining of wood: combined production of ethanol and xylanase from waste fiber sludge

The possibility to utilize fiber sludge, waste fibers from pulp mills and lignocellulose-based biorefineries, for combined production of liquid biofuel and biocatalysts was investigated. Without pretreatment, fiber sludge was hydrolyzed enzymatically to monosaccharides, mainly glucose and xylose. In the first of two sequential fermentation steps, the fiber sludge hydrolysate was fermented to cellulosic ethanol with the yeast Saccharomyces cerevisiae. Although the final ethanol yields were similar, the ethanol productivity after 9.5?h was 3.3?g/l/h for the fiber sludge hydrolysate compared with only 2.2?g/l/h for a reference fermentation with similar sugar content. In the second fermentation step, the spent fiber sludge hydrolysate (the stillage obtained after distillation) was used as growth medium for recombinant Aspergillus niger expressing the xylanase-encoding Trichoderma reesei (Hypocrea jecorina) xyn2 gene. The xylanase activity obtained with the spent fiber sludge hydrolysate (8,500?nkat/ml) was higher than that obtained in a standard medium with similar monosaccharide content (1,400?nkat/ml). Analyses based on deglycosylation with N-glycosidase?F suggest that the main part of the recombinant xylanase was unglycosylated and had molecular mass of 20.7?kDa, while a minor part had N-linked glycosylation and molecular mass of 23.6?kDa. Chemical analyses of the growth medium showed that important carbon sources in the spent fiber sludge hydrolysate included xylose, small aliphatic acids, and oligosaccharides. The results show the potential of converting waste fiber sludge to liquid biofuel and enzymes as coproducts in lignocellulose-based biorefineries.     

An adaptor strategy to subclone entire cDNA libraries as single insert recombinants

The current methods for subcloning entire cDNA libraries usually result in a significant portion of recombinants containing multiple inserts, since in most instances the inserts derived from the library to be subcloned are released as a bulk of self-ligatable DNA fragments. By use of an adaptor strategy, a method is presented to confer noncompatible ends to primarily self-ligatable inserts, resulting in efficient subcloning of entire libraries as single insert recombinants.     

Characterization of epithelial cell islets in primary monolayer cultures of human breast carcinomas by the tetrazolium reaction for glucose 6-phosphate dehydrogenase

Epithelial cell islets in primary monolayer cultures of human breast biopsies were characterized by combined immuno-, enzyme- and DNA cytochemistry as well as by analysis of attachment-, spread- and growth patterns. For cultivation we used explants from reduction mammoplasties, benign lesions, primary carcinomas and metastases. Milk fat globule membrane antigen (MFGM-A) was detected with a monoclonal antibody, and the tetrazolium reaction for glucose 6-phosphate dehydrogenase (G6PDH) as well as DNA content of the cultured cells were quantified. Spreading and growth of individual islets were studied by image analysis. Fibroblast-like cells did not express MFGM-A, and whereas epithelial (MFGM-A positive) cell islets of normal and benign origin showed cells with no or low G6PDH reaction, respectively, the majority of epithelial cell islets from 11 out of 21 carcinomas showed strong reaction. Cell islets with strong G6PDH reaction were sometimes hyperdiploid. Moreover, whereas cell islets with no or low reaction from both benign lesions and carcinomas readily attached and spread in a serum-free medium and showed population doubling times of 30 to 110 h, cell islets with strong reaction from carcinomas and metastatic lesions required serum for attachment and their growth rate was too low to be determined.