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.     

Hemolytic properties and riboflavin synthesis of Helicobacter pylori: cloning and functional characterization of the ribA gene encoding GTP-cyclohydrolase II that confers hemolytic activity to Escherichia coli

Various strains of Helicobacter pylori were able to lyse erythrocytes from sheep, horse, and human when grown on blood agar. The hemolysis did not depend on the production of the vacuolating cytotoxin VacA as demonstrated by the hemolytic behavior of an isogenic vacA-negative mutant strain. The hemolytic activity could be detected in cell-free supernatants and was not regulated by iron. To isolate genes coding for proteins involved in the destruction of erythrocytes, a plasmid-based DNA library was screened for expression of lytic activity on blood agar. This approach revealed that the H. pylori ribA gene confers hemolytic properties to Escherichia coli. The ribA gene encodes the enzyme GTP-cyclohydrolase II [EC 3.5.4.25] that catalyzes the initial step in the synthesis of riboflavin. The predicted amino acid sequence of the H. pylori RibA protein showed a high degree of similarity to equivalent enzymes from microorganisms and from plants. The single gene on a plasmid restored riboflavin synthesis in a ribA mutant of E. coli and induced hemolytic activity. Furthermore, ribA overexpression was associated with the production of a fluorescent yellow molecule that was not identical with riboflavin. Hemolysis was also seen for the ribA gene from E. coli, indicating that this feature was not specific for the H. pylori gene. The presence of ribA in various H. pylori strains was confirmed by Southern blot hybridization and by polymerase chain reaction with specific primers. This analysis revealed that microdiversity exists within the DNA region upstream from ribA, which was further confirmed by nucleotide sequence analysis. Received: 25 July 1997     

The influence of the spring activator on the mobility of the lower jaw in traumatically injured patients

For functional rehabilitation and improvement of mandibular mobility, 14 patients with dislocated collum or collum fracture dislocations were treated with a spring activator after surgical-conservative treatment. With an interincisal distance of<12 mm, therapy was started with a loop spring. The average age of the patients was 26.6 years. Eight patients showed additional traumatic injuries, 2 patients were seriously polytraumatically injured. The clinical investigations referring to the maximum mouth opening and maximal interincisal distance resulted in a good to very good functional rehabilitation in all cases. The average maximal interincisal distance of 31.7 mm at the beginning of the spring activator therapy could be increased to 47.3 mm. The largest increase of the maximal interincisal distance was observed after 4 weeks of therapy. The type of reaction, however, differed interindividually. Three patients showed a spontaneous improvement of the interincisal distance within the first 2 to 3 weeks of removal of intermaxillary fixation, whereas 7 patients whowed no improvement or only slightly improved values until insertion of the spring activator. An enlargement of the interincisal distance could be achieved in almost every patient within a relatively short period of time despite a drastically reduced mouth opening. Consequently, because of its mode of action in the form of a neuromuscular influence, the spring activator is especially suitable for the functional rehabilitation of patients with fractures of the condylar process.     

Pneumatic sequential-compression boots compared with aspirin prophylaxis of deep-vein thrombosis after total knee arthroplasty

This prospective, randomized study was undertaken to compare the effectiveness of pneumatic sequential-compression boots with that of aspirin in preventing deep-vein thrombosis after total knee arthroplasty. Patients were randomly assigned to one of two prophylactic regimens: compression boots or aspirin. One hundred and nineteen patients completed the study. Seventy-two patients had unilateral arthroplasty and forty-seven, one-stage bilateral arthroplasty. In the unilateral group, the incidence of deep-vein thrombosis was 22 per cent for the patients who used compression boots compared with 47 per cent for those who received aspirin (p less than 0.03). In the bilateral group, the incidence of deep-vein thrombosis was 48 per cent for the patients who used compression boots compared with 68 per cent for those who received aspirin (p less than 0.20). The results confirm the effectiveness of compression boots in the treatment of patients who have had unilateral total knee arthroplasty. Despite the use of compression boots, however, patients who had bilateral arthroplasty were at greater risk for the development of deep-vein thrombosis.     

Überbrückende Antikoagulation

Patients on anticoagulants of the vitamin K antagonist type may sometimes be scheduled for invasive procedures or surgical operations. In order to minimize the risk of thromboembolism caused by the interruption of chronic anticoagulation for the procedure, temporary administration of anticoagulants with shorter half-lives is required (so-called bridging anticoagulation). The present review outlines the spectrum of risks during this period regarding both thromboembolism and major bleeding. Low molecular weight heparins may be considered the medication of choice for bridging anticoagulation, mainly for practical reasons. Since they require no coagulation monitoring or dose adjustment, outpatient treatment is feasible. Such heparins are not labelled for the indication of bridging anticoagulation. However, based on recent studies of large patient cohorts, evidence of their efficacy and safety is significantly more solid than for unfractionated heparin. A simple dosing scheme for low molecular weight heparins is given here and all requirements are discussed for safe guidance through episodes of bridging anticoagulation.     

Relation between magnesium and potassium concentrations in myocardium, skeletal muscle, and mononuclear blood cells.

Potassium and magnesium were measured in 26 cardiac surgery patients (right atrial appendage), 23 autopsy subjects (right atrial appendage, left ventricular free wall, and skeletal muscle), and 9 healthy volunteers (mononuclear blood cells) to determine whether there was a relation between these two ions in the tissues measured. In the cardiac surgery patients, the potassium and magnesium concentrations were 46.35 +/- 3.89 and 4.40 +/- 0.58 (mean +/- SD, mumol/g wet weight tissue), respectively, and were significantly correlated (r = 0.54, P = 0.005). In the autopsy group, the respective concentrations were: for right atrial appendage, 30.54 +/- 10.18 and 3.66 +/- 0.70 mumol/g (r = 0.38, P = 0.14); left ventricular free wall, 60.69 +/- 17.93 and 7.74 +/- 1.73 mumol/g (r = 0.92, P = 0.0001); and skeletal muscle, 93.05 +/- 20.49 and 8.64 +/- 2.06 mumol/g (r = 0.91, P = 0.0001). In the healthy volunteer group, the results for potassium and magnesium in mononuclear blood cells were 42 +/- 9.9 and 3.99 +/- 0.70 fmol/cell, respectively (r = 0.94, P = 0.0001). Thus, potassium and magnesium concentrations were significantly correlated in all the tissues measured.