Development of three parallel cytochrome P450 enzyme affinity detection systems coupled on-line to gradient high-performance liquid chromatography.

A high resolution screening (HRS) technology is described, in which gradient high-performance liquid chromatography (HPLC) is connected on-line to three parallel placed bioaffinity detection systems containing mammalian cytochromes P450 (P450s). The three so-called enzyme affinity detection (EAD) systems contained, respectively, liver microsomes from rats induced by beta-naphthoflavone (CYP1A activity), phenobarbital (CYP2B activity), and dexamethasone (CYP3A activity). Each P450-EAD system was optimized for enzyme, substrate, and organic modifier (isopropyl alcohol, methanol, and acetonitrile) in flow injection analysis mode. Characteristic P450 ligands were used to validate the P450-EAD systems. IC(50) values of the ligands were measured and found to be similar to those obtained with conventional microtiter plate reader assays. Detection limits (n = 3; signal-to-noise ratio = 3) of potent inhibitors ranged from 1 to 3 pmol for CYP1A activity, 4 to 17 pmol for CYP2B activity, and 4 to 15 pmol for CYP3A activity. The three optimized P450-EAD systems were subsequently coupled to gradient HPLC and used to screen compound mixtures for individual ligands. Finally, to increase analysis efficiency, a HRS system was constructed in which all three P450-EAD systems were coupled on-line and in parallel to gradient HPLC. The triple parallelized P450-EAD system was shown to enable rapid profiling of individual components in complex mixtures for inhibitory activity to three different P450s.     

Anxiety and hippocampus volume in the rat.

In depressed patients as well as healthy controls, a positive relationship between hippocampal volume and trait anxiety has been reported. This study sought to explore the possible inter-relation between hippocampal volume and trait anxiety further. Magnetic resonance imaging at 7 T was used to measure hippocampal volumes in a rat model of extremes in trait anxiety (experiment 1) and in a Wistar population with normal anxiety-related behavior (experiment 2). In addition to anxiety-related behavior, potentially confounding factors (depression-like, exploratory, and locomotor behavior) were assessed. Experiment 1 globally supported the hypothesis of a positive relationship between hippocampus volume and trait anxiety but did not allow for ruling out possible confounds arising from cosegregation of other behavioral traits. Experiment 2 yielded strong evidence for a negative relationship which was specific for trait anxiety. Thus, the relationship between hippocampal volume and anxiety may be more complex than expected. Interestingly, anxiety-related behavior in experiment 2 had a stronger influence on hippocampal volume than depression-like behavior. In the light of hippocampal volume loss in anxiety disorder and frequent comorbidity of anxiety and depression, this finding suggests that further research into the relationship between anxiety and hippocampal volume may be critical for understanding hippocampal contributions to normal and pathological behavior.     

Reliability of movement control tests in the lumbar spine

Background  

Movement control dysfunction [MCD] reduces active control of movements. Patients with MCD might form an important subgroup among patients with non specific low back pain. The diagnosis is based on the observation of active movements. Although widely used clinically, only a few studies have been performed to determine the test reliability. The aim of this study was to determine the inter- and intra-observer reliability of movement control dysfunction tests of the lumbar spine.     

Decorin inhibition of PDGF-stimulated vascular smooth muscle cell function: potential mechanism for inhibition of intimal hyperplasia after balloon angioplasty

Decorin is a small proteoglycan that binds to transforming growth factor-beta (TGF-beta) and inhibits its activity. However, its interaction with platelet-derived growth factor (PDGF), involved in arterial repair after injury, is not well characterized. The objectives of this study were to assess decorin-PDGF and decorin-PDGF receptor (PDGFR) interactions, the in vitro effects of decorin on PDGF-stimulated smooth muscle cell (SMC) functions and the in vivo effects of decorin overexpression on arterial repair in a rabbit carotid balloon-injury model. Decorin binding to PDGF was demonstrated by solid-phase binding and affinity cross-linking assays. Decorin potently inhibited PDGF-stimulated PDGFR phosphorylation. Pretreatment of rabbit aortic SMC with decorin significantly inhibited PDGF-stimulated cell migration, proliferation, and collagen synthesis. Decorin overexpression by adenoviral-mediated gene transfection in balloon-injured carotid arteries significantly decreased intimal cross-sectional area and collagen content by approximately 50% at 10 weeks compared to beta-galactosidase-transfected or balloon-injured, non-transfected controls. This study shows that decorin binds to PDGF and inhibits its stimulatory activity on SMCs by preventing PDGFR phosphorylation. Decorin overexpression reduces intimal hyperplasia and collagen content after arterial injury. Decorin may be an effective therapy for the prevention of intimal hyperplasia after balloon angioplasty.     

Assessment of Resolution and Intercenter Reproducibility of Results of Genotyping Staphylococcus aureus by Pulsed-Field Gel Electrophoresis of SmaI Macrorestriction Fragments: a Multicenter Study

Twenty well-characterized isolates of methicillin-resistant Staphylococcus aureus were used to study the optimal resolution and interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. Five identical isolates (one PFGE type), 5 isolates that produced related PFGE subtypes, and 10 isolates with unique PFGE patterns were analyzed blindly in 12 different laboratories by in-house protocols. In several laboratories a standardized PFGE protocol with a commercial kit was applied successfully as well. Eight of the centers correctly identified the genetic homogeneity of the identical isolates by both the in-house and standard protocols. Four of 12 laboratories failed to produce interpretable data by the standardized protocol, due to technical problems (primarily plug preparation). With the five related isolates, five of eight participants identified the same subtype interrelationships with both in-house and standard protocols. However, two participants identified multiple strain types in this group or classified some of the isolates as unrelated isolates rather than as subtypes. The remaining laboratory failed to distinguish differences between some of the related isolates by utilizing both the in-house and standardized protocols. There were large differences in the relative genome lengths of the isolates as calculated on the basis of the gel pictures. By visual inspection, the numbers of restriction fragments and overall banding pattern similarity in the three groups of isolates showed interlaboratory concordance, but centralized computer analysis of data from four laboratories yielded percent similarity values of only 85% for the group of identical isolates. The differences between the data sets obtained with in-house and standardized protocols could be the experimental parameters which differed with respect to the brand of equipment used, imaging software, running time (20 to 48 h), and pulsing conditions. In conclusion, it appears that the standardization of PFGE depends on controlling a variety of experimental intricacies, as is the case with other bacterial typing procedures.The use of electric field pulsing techniques in conjunction with agarose gel electrophoresis for discrimination of large DNA molecules was introduced by Schwarz and Cantor in 1984 (9). During the past decade the methodology has been adapted and improved by various research groups to the point that pulsed-field gel electrophoresis (PFGE) for bacterial strain typing is now utilized with relative ease in a variety of laboratories (1). The combination of contour-clamped homogeneous field electrophoresis and PFGE for the molecular analysis of Staphylococcus aureus has been reported since the late 1980s (7, 19). At present, PFGE is considered to have both the reproducibility and resolving power of a standard technique for the epidemiological typing of bacterial isolates (10, 15).Molecular typing systems can identify different strains within a species, generating data useful for taxonomic or epidemiologic purposes (10, 14). A frequently observed shortcoming of typing systems in general is their lack of reproducibility: most typing systems do not provide a definitive strain identification, which is usually due to the variability of the technique and the lack of large databases containing fragment patterns from a wide variety of organisms to which unknowns can be compared. These problems were recently described in detail for two molecular typing systems. A multicenter study on random amplification of polymorphic DNA for discrimination of S. aureus strains revealed a lack of interlaboratory reproducibility among the banding patterns generated by the participating centers, although the epidemiological interpretation of the data was similar for all the centers involved (16). For PFGE, a similar lack of interlaboratory reproducibility of patterns was observed, although the interpretation of the experimental data also differed per participating center (2). The latter study analyzed 12 different methicillin-resistant S. aureus (MRSA) strains with different techniques optimized in each center and different sources and types of equipment. Since interlaboratory discrepancies with respect to classification of the strains were observed, the study concluded that there is a clear need for standardization of the technique, including the construction of a panel of reference strains to assist the individual researcher in the optimization of the PFGE protocol.The aim of the present study was to compare the fragment patterns of a well-defined collection of MRSA isolates in 12 laboratories using in-house and a standard set of PFGE parameters to determine whether standardization of experimental parameters (DNA preparation and switching protocols) would improve intercenter reproducibility of PFGE analysis.     

Dose-effect relations in time-limited combined psycho-pharmacological treatment for depression

BACKGROUND: A limited number of psychotherapy sessions in combination with medication is preferable to pharmacotherapy only in the treatment of ambulatory patients with major depression. Whether there is a relation between the number of sessions and the efficacy of the treatment is uncertain. METHOD: Randomized clinical trial comparing two treatment conditions in outpatients with major depression. All patients studied had a baseline score of at least 14 points on the 17-item Hamilton Depression Rating Scale. The two conditions consist of 8-session or 16-session Short Psychodynamic Supportive Psychotherapy, both in combination with pharmacotherapy. Efficacy was assessed using the 17-item HDRS, the CGI of Severity and of Improvement, the depression subscale of the SCL-90 and the Quality of Life Depression Scale. RESULTS: The rate of change would seem to indicate that eight sessions are preferable for both moderately and severely depressed patients, although the results converged again at the end. Furthermore, in terms of satisfaction with the number of sessions and drop-out percentages during treatment, no differences were found between the conditions. CONCLUSION: In the light of the outcome analysis (faster remission after fewer sessions), a short version of the psychotherapy treatment in a combined course of treatment seems to be justified.     

Stage-associated risk of transmission of the lyme disease spirochete by EuropeanIxodes ticks

To more closely define the risk of infection by the agent of Lyme disease in Europe, we determined whether spirochetal prevalence increases throughout the development of theIxodes ricinus vector tick. Of all ticks that could be flagged from vegetation,I. ricinus were by far the most abundant. Spirochetal infection rates in the adult stage of this tick (15%) are no higher than those in nymphs (18%) but greatly exceed those in larvae (0.7%). This tick therefore appears to attain infection mainly from the host of its larval stage, generally feeds on hosts that are noncompetent as reservoirs in its nymphal stage, and rarely inherits infection. Risk of human infection mainly derives from contact with the nymphal stage of the vector tick because the larva is rarely infected and the adult is large enough to be noticed and promptly removed.This work was supported in part by grant AI 19693 from the National Institute of Allergy and Infectious Disease and by grants from the city of Berlin