Involvement of Ras in Bruton's tyrosine kinase-mediated JNK activation

Defects in Bruton's tyrosine kinase (Btk) result in B cell immunodeficiencies in humans and mice. Recent studies showed that Btk is required for maximal activation of JNK, a family of stress-activated protein kinases, induced by several extracellular stimuli including interleukin (IL)-3. On the other hand, IL-3-induced JNK activation is dependent on Ras. In the present study we have investigated whether Ras is involved in Btk-mediated JNK activation in BaF3 mouse pro-B cells. Overexpression of wild-type Btk protein in these cells enhanced JNK activation upon IL-3 stimulation, whereas expression of kinase-dead Btk partially suppressed JNK activation. Induced expression of the dominant negative Ras(N17) in the cells overexpressing wild-type Btk suppressed JNK activation. Importantly, overexpression of Btk enhanced the level of the GTP-bound, active form of Ras in response to IL-3 stimulation. Btk overexpression also increased the Shc-Grb2 association induced by IL-3 stimulation. Expression of either N17Ras or V12Ras did not impose any effects on Btk kinase activity. These data collectively indicate that Ras plays a role of an intermediary signaling protein in Btk-mediated JNK activation induced by the IL-3 signaling pathway.     

Prevalence of mood disorders and utility of the PRIME-MD in patients undergoing radiation therapy

PURPOSE: To validate a short, structured interview procedure that allows practicing oncologists to quickly and reliably identify mood disorders in their patients, and to estimate the prevalence and types of mood disorders in a radiation therapy patient setting, noting relationships between mood disorders and patient characteristics. METHODS: Consecutive, eligible adult patients from the practices of two radiation oncologists were administered the Primary Care Evaluation of Mental Disorders (PRIME-MD) by the treating physician. A subset of these patients was also evaluated with the SCID, administered by trained mental health care personnel. Agreement between the two instruments was examined using the kappa statistic. Prevalence of mood disorders was determined from the PRIME-MD. The significance of relationships between patient characteristics and mood disorders was examined by chi-square and ANOVA analysis, and subsequently by multivariate logistic regression analysis. RESULTS: One hundred twenty-two patients were studied. Fifty-three of these were administered the SCID. Agreement between the two instruments was very good (kappa = 0.70). A diagnosis of a depressive or anxiety disorder by the PRIME-MD was made in 59 of the 122 patients (48%, 95% confidence interval = 39%, 58%). Multivariate analysis showed that a diagnosis of a depressive mood disorder was significantly related to pain intensity and prior history of depression. CONCLUSION: We have demonstrated the validity and feasibility of the PRIME-MD administered by oncologists in making diagnoses of mood disorders. The prevalence of mood disorders in our set of patients undergoing a course of RT was nearly 50%. Future studies should describe the natural history of these disorders, and determine optimal intervention strategies.     

Elucidating the Molecular Origins of Solute/Solvent Friction

This paper describes experimental studies of the friction a solute molecule experiences in a solvent. More specifically, this work focuses on the dielectric friction needed to describe the rotational diffusion of medium-sized molecules (displaced volume of a few hundred cubic angstroms) in polar solvents and electrolyte solutions.     

Aroma von Rotweinblüten: Korrelation sensorischer Daten mit Headspace-Inhaltsstoffen

By headspace-analysis in combination with gas Chromatographic-spectroscopic systems (GC-FID and GC-FTIR-MS) as well as by GC-sniffing-technique it was possible to correlate different vine-varieties by means of the composition of their corresponding vine blossoms fragrance compounds. Red vine blossoms possess a similar odor to white vine blossoms [1, 2] with a beautiful floral and fruity-fresh note as well as a mignonette-like, acriddusty and green side-note. The blossoms differ, depending on the vine-variety, only in the intensity of single odor impressions, but not in the direction of the total odor. The analysis of headspace [3, 4] constituents of different samples of blossoms using of GC-FID, GC-FTIR-MS and the combination with the GC-sniffing-technique allows the identification of odor dominating sesquiterpenes and odor assistant monoterpenes. Various correlations (odor profiles, qualitative and quantitative statistics and multivariate data analyses) give additional information for the characterization of investigated red vine species. A clear separation of the red vine species from the white vine species in respect of the multivariate data analysis [5, 6] of the investigations of headspace constituents of single blossoms is also possible.     

A multidomain boundary element method for two equation turbulence models

The paper deals with the multidomain Boundary Element Method (BEM) for modelling 2D complex turbulent flow using low Reynolds two equation turbulence models. While the BEM is widely accepted for laminar flow this is the first case, where this method is applied for a complex flow problems using k–ε turbulence model. The integral boundary domain equations are discretised using mixed boundary elements and a multidomain method also known as subdomain technique. The resulting system matrix is overdetermined, sparse, block banded and solved using fast iterative linear least squares solver. The simulation of turbulent flow over a backward step is in excellent agreement with the finite volume method using the same turbulent model.     

Comparison of the single-dose pharmacokinetics and tolerability of modafinil and dextroamphetamine administered alone or in combination in healthy male volunteers

An open-label, randomized, crossover study was performed in healthy male volunteers to evaluate the potential pharmacokinetic and pharmacodynamic interactions and tolerability of single oral doses of modafinil (200 mg) and dextroamphetamine (10 mg). Blood samples were collected for determination of plasma levels of modafinil, the acid and sulfone metabolites of modafinil, and dextroamphetamine at intervals through 48 hours after administration for each treatment. Vital signs (blood pressure and pulse rate) were measured through 48 hours, and electrocardiograms were measured through 24 hours after administration. Pharmacokinetic parameters were determined using noncompartmental methods. The data collected in this study of 24 healthy volunteers suggest that concomitant administration of single oral doses of modafinil and dextroamphetamine has no clinically significant effects on the pharmacokinetic profile of either agent. Although there was a slightly greater incidence of adverse events when modafinil and dextroamphetamine were administered together, the concomitant administration of the two drugs was well tolerated.     

Hyaluronan in human cerebrospinal fluid

We studied the concentration of hyaluronan in cerebrospinal fluid (CSF) in various diseases and attempted to define its reference interval. A radioassay utilizing cartilage proteins with affinity for hyaluronan was used in determining the concentration of 200 lumbar and 27 ventricular CSF specimens and 11 brain cyst fluids. Molecular weight distributions were determined by gel chromatography and localization in brain tissue by histochemistry. The hyaluronan level of lumbar CSF showed an increase with age; comparatively healthy children had (mean +/- SD) 50 +/- 41 micrograms/L (n = 40) and adults 166 +/- 77 micrograms/L (n = 9); i.e. significantly different values. The highest level was recorded in a patient with meningitis (> 8000 micrograms/L). More than 4000 micrograms/ L was noted in a patient with tumour metastasis in the cerebellum. Significantly elevated levels were especially found with spinal stenosis, head injury and cerebral infarction, but also in inflammatory medical disorders, hydrocephalus and encephalitis. We found no significant increase in multiple sclerosis and some other neurological diseases. Ventricular CSF of adults contained significantly less hyaluronan (53 +/- 73 micrograms/L; n = 16) than lumbar CSF. Hyaluronan in cyst fluids varied from 31 to 25,000 micrograms/L. Weight average molecular weight of hyaluronan in CSF was 2.9-3.0 x 10(5) and in brain tumour cyst fluid 2.4 x 10(6). In search for the origin of hyaluronan in CSF it was found that its concentration in the choroid plexus and leptomeninges was low, but that hyaluronan was accumulated in the superficial layer of the cerebral cortex. Continued screening for hyaluronan in CSF may be valuable in cases of inflammatory diseases, tumours and obstruction to CSF flow.     

Efficient expression of the gene for spinach phosphoribulokinase in Pichia pastoris and utilization of the recombinant enzyme to explore the role of regulatory cysteinyl residues by site-directed mutagenesis

Phosphoribulokinase (PRK), unique to photosynthetic organisms, is regulated in higher plants by thioredoxin-mediated thiol-disulfide exchange in a light-dependent manner. Prior attempts to overexpress the higher plant PRK gene in Escherichia coli for structure-function studies have been hampered by sensitivity of the recombinant protein to proteolysis as well as toxic effects of the protein on the host. To overcome these impediments, we have spliced the spinach PRK coding sequence immediately downstream from the AOX1 (alcohol oxidase) promoter of Pichia pastoris, displacing the chromosomal AOX1 gene. The PRK gene is now expressed, in response to methanol, at 4-6% of total soluble protein, without significant in vivo degradation of the recombinant enzyme. This recombinant spinach PRK is purified to homogeneity by successive anion-exchange and dye-affinity chromatography and is shown to be electrophoretically and kinetically indistinguishable from the authentic spinach counterpart. Site-specific replacement of all of PRK's cysteinyl residues (both individually and in combination) demonstrates a modest catalytically facilitative role for Cys-55 (one of the regulatory residues) and the lack of any catalytic role for Cys-16 (the other regulatory residue), Cys-244, or Cys-250. Mutants with seryl substitutions at position 55 display non-hyperbolic kinetics relative to the concentration of ribulose 5-phosphate. Sulfate restores hyperbolic kinetics and enhances kinase activity, presumably reflecting conformational differences between the position 55 mutants and wild-type enzyme. Catalytic competence of the C16S-C55S double mutant proves that mere loss of free sulfhydryl groups by oxidative regulation cannot account entirely for the accompanying total inactivation.     

Increased living donor volunteer rates with a formal recipient family education program

As part of our ongoing studies to characterize molecular alterations in a well-defined series of surgically resected esophageal cancers, we examined the expression of 2 ras-regulated genes, whose products (osteopontin and cathepsin L) previously were shown to be associated with tumor invasion and metastasis. RNA was extracted from primary esophageal tumors (adenocarcinomas, 19; squamous-cell carcinomas, 6) and matched histologically normal esophageal mucosa from the distant resection margin. Northern analysis was used to quantitate RNA, relative to an 18S rRNA control, and immunohistochemistry to assess the tissue distribution of osteopontin. In addition, H-, K- and N-ras mutations were studied in the same tissues using PCR and hybridization with allele (mutant)-specific oligonucleotide probes. We demonstrated a K-ras mutation (codon 12, GTT) in one esophageal adenocarcinoma. The ras-regulated gene osteopontin was over-expressed in 100% of squamous-cell carcinomas and in 58% of adenocarcinomas relative to matched normal esophageal mucosa. Patterns of immunoreactivity for osteopontin protein also varied between squamous-cell carcinomas (tumor cell staining) and adenocarcinomas (predominantly tumor-infiltrating macrophages). Expression of cathepsin L also varied with esophageal tumor histology, with over-expression in 58% of primary esophageal adenocarcinomas and 33% of squamous-cell cancers.