Biliary, fecal and urinary excretion of [3H]-prostaglandins in the rat

The profiles of biliary, fecal and urinary excretion of tritium labeled prostaglandins (PG's) of differing biological activity were investigated in the rat. The PG's (10 micrograms/kg: 2 to 50 microCi/rat, in 1 ml polyethylene glycol-400) were administered intragastrically. Excretion data were expressed as a percentage of the total administered radioactivity. For the orally administered PG's 11R-methyl-16R-fluoro-15R-hydroxy-9-oxoprosta-ci s-5-trans-13-dienoic acid and its methyl ester, excretion was equally divided between urine and feces. The fecal and urinary profile of excretion of 3H after prostacyclin (PGI2) was similar to that following administration of 11R, 16, 16-trimethyl-15R-hydroxy-9-oxoprosta-cis-5-trans-13-dienoic acid (trimoprostil), a PG with antisecretory-antiulcer potential. However, PGI2 was very poorly absorbed from the intestine, while the absorption of trimoprostil was very efficient. Biliary excretion, with little entero-porto-hepatic biliary circulation, was the main route of elimination of trimoprostil, thereby resulting in rapid elimination of drug-related products and diminishing the potential for systemic liability in the rat.     

Estrogen and the induction of lordosis in female and male prairie voles (Microtus ochrogaster)

Estrogen elicited lordosis in ovariectomized female prairie voles (Microtus ochrogaster). Treatment with estradiol benzoate (EB) was particularly effective if administered as multiple injections. Very high dose levels were not, in general, any more effective than lower doses. Individual animals typically showed lordosis within 24 to 48 hr following the onset of EB treatment and prolonged treatments did not increase the percentage of females responding to EB. Castrated male prairie voles did not respond with lordosis to repeated daily injections of 10 micrograms EB given for a period of 15 consecutive days.     

Purification of phospholipase D from citrus callus tissue

Phospholipase D in extracts of soluble proteins from callus cultures derived from cotyledons of Citrus sinensis (L.) Osbeck is activated by Ca2+ and anionic detergents and has a pH optimum of 6.5. The enzyme was purified 703-fold over the crude protein extract with a yield of 15% by ammonium sulfate precipitation, ion exchange chromatography, gel filtration, hydrophobic interaction chromatography, and preparative acrylamide gel electrophoresis. Preparative electrophoresis was carried out using conventional slab gel equipment and electroelution of the sliced gel. Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified phospholipase revealed two bands of the same staining intensity running at 94.2K and 90.5K.     

Effects of nucleotides and divalent cations on phospholipase activity in Saccharomyces cerevisiae

Divalent cations activate the lysophospholipase and transacylase reactions catalyzed by the same enzymes in the yeast Saccharomyces cerevisiae. The activation was observed at neutral pH, but not at the pH optimum of lysophospholipase/transacylase, near 3.5. Adenine nucleotides, especially AMP and ADP, are strong inhibitors of the same group of enzymes. Half maximal inhibition by AMP was found at a concentration of about 20 M. The inhibition by nucleotides in low concentrations is enhanced by divalent cations.     

Humanization of a mouse monoclonal antibody by CDR-grafting: the importance of framework residues on loop conformation.

A mouse monoclonal antibody (mAb 425) with therapeutic potential was 'humanized' in two ways. Firstly the mouse variable regions from mAb 425 were spliced onto human constant regions to create a chimeric 425 antibody. Secondly, the mouse complementarity-determining regions (CDRs) from mAb 425 were grafted into human variable regions, which were then joined to human constant regions, to create a reshaped human 425 antibody. Using a molecular model of the mouse mAb 425 variable regions, framework residues (FRs) that might be critical for antigen-binding were identified. To test the importance of these residues, nine versions of the reshaped human 425 heavy chain variable (VH) regions and two versions of the reshaped human 425 light chain variable (VL) regions were designed and constructed. The recombinant DNAs coding for the chimeric and reshaped human light and heavy chains were co-expressed transiently in COS cells. In antigen-binding assays and competition-binding assays, the reshaped human antibodies were compared with mouse 425 antibody and to chimeric 425 antibody. The different versions of 425-reshaped human antibody showed a wide range of avidities for antigen, indicating that substitutions at certain positions in the human FRs significantly influenced binding to antigen. Why certain individual FR residues influence antigen-binding is discussed. One version of reshaped human 425 antibody bound to antigen with an avidity approaching that of the mouse 425 antibody.     

Chemical and spectroscopic evidence for the formation of a ferryl Fea3 intermediate during turnover of cytochrome c oxidase

When partially reduced cytochrome c oxidase samples are reoxidized with dioxygen, an EPR-silent dioxygen intermediate, which is at the three-electron level of dioxygen reduction, is trapped at the dioxygen reduction site. The intermediate has novel spectral features at 580 and 537 nm. Combined optical and EPR results reveal that this intermediate reacts rapidly with CO at 277-298 K causing the abolition of the 580/537 mm features and the appearance of a rhombic CuB EPR signal. A ferryl Fea3, or an intermediate at the same formal level of oxidation, is proposed to oxidize CO to CO2 producing an EPR-detectable CuB adjacent to a low-spin ferrous Fea3-dioxygen (or carbon monoxide) adduct.     

Purification of a phospholipase B inhibitor from Saccharomyces cerevisiae

The phospholipase B activity of plasma membrane vesicles from Saccharomyces cerevisiae is inhibited by the 100 000 X g supernatant of mechanically disrupted yeast cells. A 1850-fold purification of the inhibitor activity was achieved by gel filtration, ion exchange chromatography with DEAE-cellulose and hydrophobic interaction chromatography with Octyl-Sepharose. SDS-polyacrylamide gel electrophoresis of the purified inhibitor revealed two main bands with an apparent Mr of 60 000 and 26 500. The phospholipase B activity was strongly reduced but not completely blocked by this preparation, while the lysophospholipase and transacylase reactions, which are catalyzed by the same membrane-bound enzymes (Witt, W. et al. (1984) Biochim. Biophys. Acta 795, 108-116), were not affected.     

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Preparation and characterization of monoclonal antibodies to several frog rod outer segment proteins

Monoclonal antibodies to proteins important in phototransduction in the frog rod outer segment have been obtained. These include 6 different antibodies to rhodopsin, 50 to a guanine nucleotide binding protein (G-protein; 40,000 daltons), and 2 to cytoplasmic proteins. The antigens used were Percoll-purified rod outer segments, a rod outer segment soluble protein fraction, or a soluble plus peripheral membrane protein fraction. Antibodies were assayed by solid phase assay using a fluorogenic detection system. Proteins to which antibodies bound were assayed on Western blots, and the sensitivities of three different detection systems were compared. Most antibodies bound to only one rod outer segment protein band on Western blots. Immunofluorescence microscopy demonstrated binding of both anti-rhodopsin and anti-G-protein to isolated frog rod outer segments. Antibodies were purified from either culture supernatants or ascites fluid on protein A affinity columns. Two purified anti-G-protein antibodies have binding affinities to 125I-labeled G-protein of less than 10(-6) M-1. Of 11 antibodies to frog or bovine G-protein tested in solid phase and Western blot assays, all bind to the alpha rather than the beta or gamma subunits. Procedures developed here are being used in preparing other antibodies that affect reactions in the phototransduction pathway.