Inhibitory monoclonal antibody to human cytochrome P450 2B6

The human cytochrome P450 2B6 metabolizes, among numerous other substrates, diazepam, 7-ethoxycoumarin, testosterone, and phenanthrene. A recombinant baculovirus containing the human 2B6 cDNA was constructed and used to express 2B6 in Sf9 insect cells. The 2B6 was present at 1.8 +/- 0.4% of the total cellular protein and was purified to a specific content of 13.3 nmol/mg protein. Mice were immunized with the purified 2B6, and a total of 811 hybridomas were obtained from the fusion of NS-1 myeloma cells and spleen cells of the immunized mice. Monoclonal antibodies (MAbs) from 24 of the hybrids exhibited immunobinding to 2B6 as determined by ELISA. One of the MAbs, 49-10-20, showed a strong immunoblotting activity and was highly inhibitory to 2B6 enzyme activity. MAb 49-10-20 inhibited cDNA-expressed 2B6-catalyzed metabolism of diazepam, phenanthrene, 7-ethoxycoumarin, and testosterone by 90-91%. MAb 49-10-20 showed extremely high specificity for 2B6 and did not bind to 17 other human and rodent P450s or inhibit the metabolism of phenanthrene catalyzed by human 1A2, 2A6, 2C8, 2C9, 2D6, 2E1, 3A4, and 3A5. MAb 49-10-20 was used to determine the contribution of 2B6 to the metabolism of phenanthrene and diazepam in human liver. In ten liver samples, MAb 49-10-20 inhibited phenanthrene metabolism variably by a wide range of 8-42% and diazepam demethylation by 1-23%. The degree of inhibition by the 2B6 specific MAb 49-10-20 defines the contribution of 2B6 to phenanthrene and diazepam metabolism in each human liver. This technique using inhibitory MAb 49-10-20 determines the contribution of 2B6 to the metabolism of its substrates in a human tissue containing multiple P450s. This study is a prototype for the use of specific and highly inhibitory MAbs to determine individual P450 function.     

Technical note: a simplified procedure for vitamin E determination in beef muscle

A simplified method for the determination of alpha-tocopherol concentration in beef muscle was developed and evaluated. The method consists of a saponification step applied to 1-g samples of intact, fresh muscle, followed by a single isooctane extraction of the saponified samples. alpha-Tocopherol in the extract was separated by normal phase chromatography and quantified by fluorescence detection. A single extraction with the simplified method accounted for 95% of the total muscle alpha-tocopherol concentration obtained by two extractions with the Arnold et al. (J. Food Sci. 58:28, 1993) method. Recovery of added alpha-tocopherol standard after two extractions of a saponified muscle sample was 91% for the simplified method, which was not different (alpha = .78) from recovery using the Arnold method, and the efficiency of the single extraction in the simplified method was 89%. The coefficients of variation for the simplified and Arnold methods were both 3.1%. This method should permit the duplicate analysis of 100 fresh muscle samples within 24 h.     

Machining of Green Si3N4 Polymer Bonded Ceramic Materials

This research examined the machinability of Si₃N₄, based polymer bonded ceramic green bodies. They were clearly machinable. Surface roughness was comparable to milled steel. The machining forces were lower than those reported for other ceramics and composites. Higher percentages of polymer produced stronger, more machinable green bodies. The areas of greatest concern were: tool wear, distribution of polymer throughout the body, and choice of polymer, for good wettability. Feasible parameters for blank fabrication were determined. Optimal parameters for both blank fabrication and machining are still to be determined. Polymer bonded ceramic bodies may also have their own utility; however applications with these materials will require further investigations.     

Quantification of information transfer via cellular signal transduction pathways

OBJECTIVE: Symptomatic malignant pleural effusions are common sequelae in patients with certain malignancies. Pleurodesis via bedside thoracostomy is the current treatment option most commonly used. To our knowledge, this is the first prospective randomized trial to examine which agent, bleomycin or talc slurry, is superior in terms of effectiveness, safety, and cost. PATIENTS AND METHODS: Between July 1992 and March 1995, 35 patients presenting to our medical center with symptomatic malignant pleural effusions were prospectively randomized to undergo chemical pleurodesis with either bleomycin or talc slurry via bedside thoracostomy. The conditions of patients were assessed and graded before and after treatment concerning pain, dyspnea, and chest radiographs. RESULTS: Twenty-nine patients who underwent 33 treatments (14 with bleomycin and 19 with talc) were available for follow-up. Follow-up ranged from 2 weeks to 8 months (mean, 1.7 months). Both groups demonstrated notable improvement in both pain and dyspnea following treatment, but there were no statistically significant differences between groups in the amount of improvement (two-tailed Student's t test). Permanent control of effusions, defined objectively on chest radiograph, was achieved with 11 bleomycin treatments (79%) and 17 talc treatments (90%) (p=0.388). The procedures were well tolerated and no significant adverse effects were observed. Talc is a much less costly agent than bleomycin ($12.36 cost to our medical center per treatment for talc vs $955.83 for bleomycin). CONCLUSION: Given the similar efficacy and significant cost advantage, we conclude that talc is the agent of choice when utilizing pleurodesis for control of symptomatic malignant pleural effusions.