Expression and secretion of active mouse TIMP-1 using a baculovirus expression vector

Mouse TIMP-1, one of the tissue inhibitors of metalloproteinases important in regulating turnover of extracellular matrix in both normal and pathological tissues, was previously expressed inE. coli in an inactive, nonglycosylated state that required refolding to become functional. Due to the difficulty of renaturation, an alternative to the prokaryotic expression system was sought. Since we are interested in studying the pharmacodynamics and pharmacokinetics of TIMP locally administered by controlled delivery to mice with experimentally induced arthritis, we also needed an efficient way of producing active TIMP in large quantities. Using the pBlueBacII transfer vector, we generated a recombinant baculovirus that in Sf9 cells could express glycosylated mouse TIMP-1 to about 3 mg of active protein/liter conditioned medium.     

A genomewide linkage analysis for prostate cancer susceptibility genes in families from Germany

Prostate cancer is a complex disease with a substantial genetic contribution involved in the disease risk. Several genomewide linkage studies conducted so far have demonstrated a strong heterogeneity of susceptibility. In order to assess candidate regions that are particularly relevant for the German population, we performed a genomewide linkage search on 139 prostate cancer families. A nonparametric method (Zlr scores), using GENEHUNTERPLUS, was applied at 500 markers (panel P1400, deCODE), with an average spacing of 7.25 cM. In the entire family collection, linkage was most evident at 8p22 (Zlr=2.47, P=0.0068), close to the previously identified susceptibility gene MSR1. Further local maxima with Zlr>2 (P<0.025) were observed at 1q, 5q and 15q. In a subgroup of 47 families, which matched the Johns Hopkins criteria of hereditary prostate cancer, suggestive linkage was found on 1p31 (Zlr=3.37, P=0.00038), a previously not described candidate region. The remaining 92 pedigrees, with no strong disease history, revealed a maximum Zlr=3.15 (P=0.00082) at 8q13, possibly indicating a gene with reduced penetrance or recessive inheritance. Our results suggest pronounced locus heterogeneity of prostate cancer susceptibility in Germany. In the present study population, the MSR1 gene could play a significant role. Other conspicuous loci, like 1p31 and 8q13, need further investigation in order to verify their relevance and to identify candidate genes.     

Upregulation of TGF-beta, FOXP3, and CD4+CD25+ regulatory T cells correlates with more rapid parasite growth in human malaria infection

Understanding the regulation of immune responses is central for control of autoimmune and infectious disease. In murine models of autoimmunity and chronic inflammatory disease, potent regulatory T lymphocytes have recently been characterized. Despite an explosion of interest in these cells, their relevance to human disease has been uncertain. In a longitudinal study of malaria sporozoite infection via the natural route, we provide evidence that regulatory T cells have modifying effects on blood-stage infection in vivo in humans. Cells with the characteristics of regulatory T cells are rapidly induced following blood-stage infection and are associated with a burst of TGF-beta production, decreased proinflammatory cytokine production, and decreased antigen-specific immune responses. Both the production of TGF-beta and the presence of CD4+CD25+FOXP3+ regulatory T cells are associated with higher rates of parasite growth in vivo. P. falciparum-mediated induction of regulatory T cells may represent a parasite-specific virulence factor.     

Characterization of DEAE-dextran by means of light scattering and combined size-exclusion chromatography/low-angle laser light scattering/viscometry

Ten DEAE (2-(diethylamino)ethyl) dextran samples were investigated by means of static and dynamic light scattering, viscometry and size-exclusion chromatography (SEC) in combination with on-line small-angle laser light scattering (LALLS) and viscometry (VISC). In dilute solution the behavior of DEAE-dextran was compared with that of unsubstituted dextran and the molecular weight M dependences of the radius of gyration R_g, hydrodynamic radius R_h, intrinsic viscosity [η], second virial coefficient A₂ and z-average diffusion coefficient D _z were determined. The relationships for DEAE-dextran dissolved in a 0,8 molar sodium nitrate solution were nearly the same as for dextran dissolved in water with 0,05 wt.-% sodium azide and gave the same exponents. The molecular weight dependence of the intrinsic viscosity cannot be described by a Kuhn-Mark-Houwink relationship with a constant exponent. The slope in the plot of log [η] versus log M decreases with increasing molecular weight which indicates the occurrence of branching. By means of SEC/LALLS/VISC measurements the molecular weight distributions were determined. The distributions were calculated (1) directly from the light scattering signal, (2) from a calibration line obtained by light scattering data of a DEAE-dextran sample with a broad distribution and (3) from the intrinsic viscosity distribution obtained by the on-line viscosity/refractive index detector in combination with the [η]-M relationship. In order to get the correct molecular-weight dependence of the intrinsic viscosity it is necessary to determine the molecular weight distribution directly by LALLS (technique 1) and to combine this with the appropriate intrinsic viscosity data from the viscometer. Only the third technique, which is an extension of technique 1, gave satisfactory results over the whole molecular weight region observed.     

Basecalling with LifeTrace

A pivotal step in electrophoresis sequencing is the conversion of the raw, continuous chromatogram data into the actual sequence of discrete nucleotides, a process referred to as basecalling. We describe a novel algorithm for basecalling implemented in the program LifeTrace. Like Phred, currently the most widely used basecalling software program, LifeTrace takes processed trace data as input. It was designed to be tolerant to variable peak spacing by means of an improved peak-detection algorithm that emphasizes local chromatogram information over global properties. LifeTrace is shown to generate high-quality basecalls and reliable quality scores. It proved particularly effective when applied to MegaBACE capillary sequencing machines. In a benchmark test of 8372 dye-primer MegaBACE chromatograms, LifeTrace generated 17% fewer substitution errors, 16% fewer insertion/deletion errors, and 2.4% more aligned bases to the finished sequence than did Phred. For two sets totaling 6624 dye-terminator chromatograms, the performance improvement was 15% fewer substitution errors, 10% fewer insertion/deletion errors, and 2.1% more aligned bases. The processing time required by LifeTrace is comparable to that of Phred. The predicted quality scores were in line with observed quality scores, permitting direct use for quality clipping and in silico single nucleotide polymorphism (SNP) detection. Furthermore, we introduce a new type of quality score associated with every basecall: the gap-quality. It estimates the probability of a deletion error between the current and the following basecall. This additional quality score improves detection of single basepair deletions when used for locating potential basecalling errors during the alignment. We also describe a new protocol for benchmarking that we believe better discerns basecaller performance differences than methods previously published.     

Über die Behandlung idiopathischer Ödeme mit Bromocriptin

Zusammenfassung Die Therapie idiopathischer Ödeme mit Diuretica ist problematisch, da sie die Symptome nicht beseitigt, sondern einen sekundären Hyperaldosteronismus induziert, der die Ödembildung unterhält. Die Beobachtung einer verminderten Dopaminausscheidung bei Patientinnen mit idiopathischen Ödemen läßt einen Behandlungsversuch mit dem oral wirksamen Dopaminagonisten Bromocriptin als sinnvoll erscheinen.Wir behandelten daher neun Patientinnen mit idiopathischen Ödemen mit typischer Symptomatik und mehrjähriger Anamnese mit 2 × 2,5 mg/die Bromocriptin (Pravidel®). Der Therapieerfolg wurde nach der Normalisierung der circadianen Gewichtsschwankungen und dem subjektiven Befinden beurteilt. Sieben Patientinnen zeigten einen guten, eine nur einen unsicheren Therapieerfolg, und eine Patientin brach die Therapie wegen Nausea ab.Vor und während Bromocriptinbehandlung sind Serumelektrolyte, Blutdruck, Plasmareninaktivität und Aldosteron normal. Bromocriptin normalisiert die circadianen Gewichtsschwankungen, ohne das morgendliche Körpergewicht zu senken. Die Ergebnisse der vorliegenden Pilotstudie legen nahe, daß Bromocriptin bei einigen Patientinnen mit idiopathischen Ödemen eine wirksame Alternative zur herkömmlichen diuretischen Therapie sein kann. Es bleibt offen, ob die Bromocriptinwirkung durch einen Dopaminmangel zu erklären ist, oder ob Bromocriptin eine rein symptomatische Behandlung darstellt.