Enhancement of opsin activity by all-trans-retinal

This study examines the hemodynamic performance of small size St. Jude BioImplant aortic prostheses using dobutamine echocardiography. Eleven patients (3 women, mean age 75 years) who had undergone aortic valve replacement with a size 21-mm St. Jude BioImplant aortic prostheses at 10.8 +/- 5.1 months (SD) previously were studied. Dobutamine infusion was started at a rate of 5 microg/kg/min and increased to 10 microg/kg/min, and subsequently to 20 microg/kg/min at 15-minute intervals. Pulsed and continuous-wave Doppler studies were performed at rest and at the end of each stage. Effective orifice area, mean gradient, and the performance index across each prosthesis were calculated and cardiac output was determined by Doppler measurement of flow in the left ventricular outflow tract. Stress dobutamine increased heart rate and cardiac output by 51% and 56%, respectively (both p <0.0001), and the mean transvalvular gradient increased from 30.1 +/- 7.5 mm Hg at rest to 49.3 +/- 11.5 mm Hg at maximum stress (p <0.0005). The performance index increased progressively from 0.29 +/- 0.05 at rest to 0.40 +/- 0.10 at maximum stress (p <0.0005). Regression modeling analyses demonstrated that the maximum stress gradient was independent of all variables except the resting gradient (p = 0.03). Body surface area had no effect on the changes in cardiac output, effective orifice area, or transprosthetic gradient at maximum stress. Thus, these data demonstrate that the size 21-mm St. Jude BioImplant prosthesis exhibits suboptimal hemodynamic performance with transvalvular gradients consistent with mild to moderate aortic stenosis, both at rest and under stress conditions.     

Metabolism of N,N-diethylbenzamide and N,N-diethyl-alpha,alpha'-13C-benzamide by rat liver microsomes

The O2 and NADPH-dependent metabolism of N,N-diethylbenzamide (DEB), a topically applied insect repellent, has been investigated in liver microsomal suspensions from phenobarbital-pretreated male Wistar rats. Incubation conditions for the enzymatic production of N-ethylbenzamide (EB) from DEB (0.2 mM) were studied by use of HPLC with UV detection. With the microsomal enzymes suspended in 10 mM phosphate buffer (pH 7.4) and the substrate delivered in acetone (10 microliter), the yield of EB was 81.9 +/- 2.9% in five replicated experiments with NADPH (2 mM) and MgCl2 (4 mM). Conversion of DEB to EB was strongly inhibited by carbon monoxide, a cytochrome P-450 inhibitor. A sample of N,N-diethyl-alpha,alpha'-13C-benzamide was synthesized and was used, in conjunction with high-resolution 13C NMR spectroscopy, to identify the metabolites arising from oxidation of the ethyl group. Using the distortionless enhancement by polarization transfer pulse sequences and a substrate concentration of 0.4 mM, 13C-labeled acetaldehyde and glycolaldehyde, both detected as the hydrates, were found in the microsomal incubates. N-Ethyl-N-(alpha-hydroxyethyl)benzamide, the presumed metabolic progenitor of acetaldehyde, was not detected in these metabolic experiments. A pathway is proposed to account for the metabolic generation of glycolaldehyde by hydroxylation of both carbons of the N-ethyl group.     

Activation and detoxification of dinitropyrenes by cytosol and microsomes from Aroclor-pretreated rats in the Ames and umu assays

PURPOSE: To evaluate the efficacy of topical mitomycin C in treating conjunctival and corneal epithelial dysplasia and neoplasia. METHODS: Seven eyes of seven patients with conjunctival and corneal epithelial dysplasia and neoplasia were treated with one drop of topical mitomycin C 0.04% four times a day for 7 days in alternate weeks. The patients' charts were reviewed retrospectively. Patients with either multiple recurrences or extensive ocular surface involvement were treated. In all eyes, the diagnosis of epithelial dysplasia or neoplasia was confirmed by histopathology before the onset of therapy. Patients were examined at least every 14 days during treatment and examined at intervals after completion of treatment. RESULTS: With topical mitomycin C, six eyes of seven patients had complete clinical regression of their conjunctival and corneal epithelial dysplasia and neoplasia. One eye of one patient had partial clinical regression of conjunctival and corneal epithelial dysplasia. Follow-up after completion of topical mitomycin C therapy and excision of residual disease ranged from 2 to 16 months (mean, 9 months; SD, 4.3 months) and was without clinical sign of recurrence. Topical mitomycin C therapy was associated with transitory ocular discomfort, conjunctival injection, tearing, photophobia, and punctate epithelial keratopathy. CONCLUSION: In this small series of eyes, topical mitomycin C was effective as a treatment for conjunctival and corneal epithelial dysplasia and neoplasia.     

The formation of threo-11-hydroxy-trans-12: 13-epoxy-9-cis-octadecenoic acid by enzymic isomerisation of 13-L-hydroperoxy-9-cis, 11-transoctadecadienoic acid by soybean lipoxygenase-1

Report on 35 cases of mallet finger treated conservatively: a circular plaster cast was modeled in hyperextension of the distal interphalangeal joint. The immobilisation of the whole finger form the tip to the proximal phalanx was maintained for six weeks. The reported results were good or excellent with the exception of two cases.     

Biotransformation of chlorpromazine and methdilazine by Cunninghamella elegans

When tested as a microbial model for mammalian drug metabolism, the filamentous fungus Cunninghamella elegans metabolized chlorpromazine and methdilazine within 72 h. The metabolites were extracted by chloroform, separated by high-performance liquid chromatography, and characterized by proton nuclear magnetic resonance, mass, and UV spectroscopic analyses. The major metabolites of chlorpromazine were chlorpromazine sulfoxide (36%), N-desmethylchlorpromazine (11%), N-desmethyl-7-hydroxychlorpromazine (6%), 7-hydroxychlorpromazine sulfoxide (36%), N-hydroxychlorpromazine (11%), 7-hydroxychlorpromazine sulfoxide (5%), and chlorpromazine N-oxide (2%), all of which have been found in animal studies. The major metabolites of methdilazine were 3-hydroxymethdilazine (3%). (18)O(2) labeling experiments indicated that the oxygen atoms in methdilazine sulfoxide, methdilazine N-oxide, and 3-hydroxymethdilazine were all derived from molecular oxygen. The production of methdilazine sulfoxide and 3-hydroxymethdilazine was inhibited by the cytochrome P-450 inhibitors metyrapone and proadifen. An enzyme activity for the sulfoxidation of methdilazine was found in microsomal preparations of C. elegans. These experiments suggest that the sulfoxidation and hydroxylation of methdilazine and chlorpromazine by C. elegans are catalyzed by cytochrome P-450.     

DNA mismatch repair catalyzed by extracts of mitotic, postmitotic, and senescent Drosophila tissues and involvement of mei-9 gene function for full activity

Extracts of Drosophila embryos and adults have been found to catalyze highly efficient DNA mismatch repair, as well as repair of 1- and 5-bp loops. For mispairs T.G and G.G, repair is nick dependent and is specific for the nicked strand of heteroduplex DNA. In contrast, repair of A.A, C.A, G.A, C.T, T.T, and C.C is not nick dependent, suggesting the presence of glycosylase activities. For nick-dependent repair, the specific activity of embryo extracts was similar to that of extracts derived from the entirely postmitotic cells of young and senescent adults. Thus, DNA mismatch repair activity is expressed in Drosophila cells during both development and aging, suggesting that there may be a function or requirement for mismatch repair throughout the Drosophila life span. Nick-dependent repair was reduced in extracts of animals mutant for the mei-9 gene. mei-9 has been shown to be required in vivo for certain types of DNA mismatch repair, nucleotide excision repair (NER), and meiotic crossing over and is the Drosophila homolog of the yeast NER gene rad1.     

Ethnic-related differences in coumarin 7-hydroxylation activities catalyzed by cytochrome P4502A6 in liver microsomes of Japanese and Caucasian populations

1. Interethnic differences in cytochrome P4502A6 (CYP2A6) levels and coumarin 7-hydroxylation activities were determined in liver microsomes of 30 Japanese and 30 Caucasians. 2. Although CYP2A6 levels and coumarin 7-hydroxylation activities varied very significantly in the 60 human samples examined, both CYP2A6 levels and coumarin 7hydroxylation activities were found to be higher in Caucasian than Japanese population. 3. Interestingly, eight of the 30 Japanese examined showed very low or undetectable levels of coumarin 7-hydroxylation activities as well as of CYP2A6 in liver microsomes. All of the Caucasians, however, had significant CYP2A6 levels and variable 7-hydroxylation activities. 4. Kinetic analvsis of coumarin 7-hydroxylation activities in liver microsomes of various human samples suggested that although there were 260-fold differences in Vmax's in 10 human samples examined, the Km's were very similar (2.1 + or - 107 mu M); a value consistent with that obtained (1.2 mu M) with purified CYP2A6 in reconstituted system. 5. The results suggest that CYP2A6 is actually involved in the 7-hydroxylation of coumarin in human liver microsomes, and that interethnic differences in coumarin 7-hydroxylation activities in Japanese and Caucasian population may be ascribed to the differences in expression of CYP2A6 protein.     

Glucuronidation of entacapone, nitecapone, tolcapone, and some other nitrocatechols by rat liver microsomes

PURPOSE: Nitrocatechol COMT inhibitors are a new class of bioactive compounds, for which glucuronidation is the most important metabolic pathway. The objective was to characterize the enzyme kinetics of nitrocatechol glucuronidation to improve the understanding and predicting of the pharmacokinetic behavior of this class of compounds. METHODS: The glucuronidation kinetics of seven nitrocatechols and 4-nitrophenol, the reference substrate for phenol UDP-glucuronosyltransferase activity, was measured in liver microsomes from creosote-treated rats and determined by non-linear fitting of the experimental data to the Michaelis-Menten equation. A new method that combined densitometric and radioactivity measurement of the glucuronides separated by HPTLC was developed for the quantification. RESULTS: Apparent K(m) values for the nitrocatechols varied greatly depending on substitution pattern being comparable with 4-nitrophenol (0.11 mM) only in the case of 4-nitrocatechol (0.19 mM). Simple nitrocatechols showed two-fold Vmax values compared with 4-nitrophenol (68.6 nmol min-1 mg-1), while all disubstituted catechols exhibited much lower glucuronidation rate. Vmax/K(m) values were about 10 times higher for monosubstituted catechols compared to disubstituted ones. The kinetic parameters for COMT inhibitors were in the following order: K(m) nitecapone > > entacapone > tolcapone; Vmax nitecapone > entacapone > tolcapone; Vmax/K(m) tolcapone > nitecapone > entacapone. CONCLUSIONS: Nitrocatechols can in principle be good substrates of UGTs. However, substituents may have a remarkable effect on the enzyme kinetic parameters. The different behaviour of nitecapone compared to the other COMT inhibitors may be due to its hydrophilic 5-substituent. The longer elimination half-life of tolcapone in vivo compared to entacapone could not be explained by glucuronidation kinetics in vitro.     

Biotransformation of resveratrol to piceid by Bacillus cereus

Microbial transformation of resveratrol (1), trans-3,4', 5-trihydroxystilbene, was studied. Preparative scale biotransformation of 1 with whole-cell suspensions of Bacillus cereus UI 1477 resulted in the production of metabolite 2 which was identical in all respects to an authentic sample of piceid, resveratrol 3-O-beta-D-glucoside.     

Rhodopsin arginine-135 mutants are phosphorylated by rhodopsin kinase and bind arrestin in the absence of 11-cis-retinal

Arginine-135, located at the border between the third transmembrane domain and the second cytoplasmic loop of rhodopsin, is one of the most highly conserved amino acids in the family of G protein-coupled receptors. The effect of mutation at Arg-135 on the ability of rhodopsin to undergo desensitization was investigated. Four mutants, R135K, R135Q, R135A, and R135L, were examined for their ability to be phosphorylated by rhodopsin kinase, to bind arrestin, and to activate the rod cell G protein, transducin (Gt). All of the mutants were phosphorylated, bound arrestin, and were able to activate Gt when reconstituted with 11-cis-retinal. Surprisingly, several of the mutants could be phosphorylated by rhodopsin kinase and could bind arrestin in the absence of 11-cis-retinal but were not able to activate Gt. These observations represent the first demonstration of a mutant G protein-coupled receptor that assumes a conformation able to interact with its G protein-coupled receptor kinase and arrestin, but not with its G protein, in the absence of ligand.     

Biotransformation of cerivastatin in mice, rats, and dogs in vivo

Biotransformation of cerivastatin was investigated in mice, rats, and dogs in vivo using the 14C-labeled drug. Marked species differences exist, both in pathways and extent of cerivastatin metabolism. Unchanged drug, together with its lactone, predominates in dog plasma and represents 40% of the dose in the excreta, whereas in rat bile they account for approximately 10% of the dose. In mice, the drug is metabolized rapidly and almost completely. Biotransformation of cerivastatin occurs by three distinct phase I routes and by phase II conjugation with sugar-type moieties and taurine. Phase I routes are demethylation of the pyridinyl methyl ether, beta-oxidation of the 3,5-dihydroxy acid side chain, and reductive removal of the side chain 3-hydroxy group. In dogs, demethylation is the dominating phase I biotransformation. Phase II conjugation is equally important. In dog bile, different regioisomeric drug glucuronides and the benzylic glucuronide and glucoside conjugate of the demethylated drug were found. In rats, besides demethylation, beta-oxidation of the dihydroxy acid side chain-followed by reductive removal of the 5-hydroxy group-is the major reaction. The resulting pentenoic acid derivatives are observed in plasma and liver homogenate. These metabolites are subsequently conjugated with taurine and excreted in the bile. This metabolic sequence is also important in mice. Furthermore, only in mice, cerivastatin is subject to reductive removal of the 3-hydroxy group, together with demethylation. The 5-hydroxyheptenoic acids formed predominate in plasma and liver homogenate, whereas the corresponding taurine conjugates are excreted in the bile.     

Separation of the two reactions, oxidation and isomerization, catalyzed by Streptomyces cholesterol oxidase

Site-directed mutagenesis was used to identify key amino acid residues of the cholesterol oxidase from Streptomyces sp., which catalyzes the oxidation of cholesterol and the isomerization of 5-cholesten-3-one. Eight mutant enzymes were constructed and the following amino acid substitutions were identified: N318A, N318H, E356A, E356D, H441A, H441N, N480A and N480Q. Mutants N318A and N318H retained both oxidation and isomerization activities. The mutant E356D retained oxidation but not isomerization activity. On the other hand, mutants N480A and N480Q showed no oxidation activity but retained their isomerization activities. The two catalytic reactions, oxidation and isomerization, in cholesterol oxidase were thus successfully separated. When the H441A or H441N mutation was introduced, both the oxidase and isomerase activities were completely lost. The H441, E356 and N480 residues thus appear to participate in the catalysis of cholesterol oxidase, whereas N318 does not. An analysis of the products of these mutant enzymes suggested that the previously proposed 6-hydroxylation reaction by cholesterol oxidase is actually autooxidation from 5-cholesten-3-one. Kinetic studies of the purified wild-type and mutant enzymes showed that the k(cat)/Km values for oxidation in E356D and for isomerization in N480A increased six- and threefold, respectively, over those in the wild-type. These mutational effects and the reaction mechanisms are discussed in terms of the three-dimensional structure of the enzyme constructed on the basis of homology modeling.     

Biotransformation of monoterpenes, bile acids, and other isoprenoids in anaerobic ecosystems

RGH-2716 is a novel 1-oxa-3,8-diazaspiro[4.5] decan 2-one, which was published to have potent inhibitory effect on neuronal Na and Ca movement and stimulatory action on nerve growth factor (NGF)-production, as well as to show significant antiamnesic activity in experimental amnesia models. The aim of the present experiments was to study the effect of the compound on the learning process and on the different stages of memory using water-labyrinth in normal and memory impaired young animals, as well as to study cognitive effect of RGH-2716 on aged animals. At the doses of 0.5 mg/kg i.p. or 3 mg/kg p.o. given before daily swimming, this compound improved the learning process of young animals impaired by either diazepam (DIA) or scopolamine (SCOP). In retrograde amnesia model RGH-2716 (3 mg/kg p.o.) significantly ameliorated consolidation process and retrieval of information impaired by SCOP or DIA. Nimodipine and vinpocetine (10 mg/kg p.o.) showed moderate effect compared to RGH-2716. Aged rats pretreated with daily i.p. RGH-2716 performed the tasks with significantly fewer errors and shorter swimming time than untreated aged rats. When aged animals had to solve a new labyrinth problem, treated aged rats showed significantly better learning ability than aged controls. One month of oral treatment of aged rats with 3 mg/kg dose of RGH-2716 two times daily resulted in a "tendency-like" improvement in learning of aged Fischer 344 and spontaneously hypertensive (SH) rats. The present results make RGH-2716 an interesting compound for the treatment of cognitive disorders.     

Nutrition and drugs. II. Biotransformation, interactions]

Hypoxitherapy proposed by Russian researchers promotes a 2-3-fold decrease in morbidity of poor-health subjects (high risk groups). As shown by the experience gained in the medical department of the Moskvich car corporation hypoxitherapy provides enhancement of the human resistance to extreme ecological conditions responsible for high mortality of population in the Russian Federation. The information on the production of special devices, hypoxicators, for creation of hypoxic mixtures (mountain air) is given. Large-scale application of hypoxitherapy for prevention, treatment and rehabilitation seems most effective in the conditions of sanatoria.     

Comparative studies on the catalytic roles of cytochrome P450 2C9 and its Cys- and Leu-variants in the oxidation of warfarin, flurbiprofen, and diclofenac by human liver microsomes

A unique immunoliposome has been developed as a drug delivery vehicle for immunotherapy. Human recombinant interleukin-2 (IL-2) has been chemically coupled to the external surface of small unilamellar vesicles (SUVs) containing methotrexate as a candidate immunosuppresive agent in order to specifically direct the drug-bearing liposome to activated T-cells expressing the high affinity IL-2 receptor. This drug delivery system is designed to deliver an immunosuppressive agent to those cells that actively participate in disorders such as graft rejection without delivering an effective but potentially toxic drug to all cells of the immune system as well as other healthy tissues. IL-2 was chemically modified with succinimidyl 4-[p-maleidophenyl butyrate](SMPB) while the receptor binding domain on IL-2 was protected by monoclonal anti-IL-2 bound to Protein A-Silica Gel. The antibody recognizes the receptor binding domain of the IL-2 molecule. The IL-2 was derivatized with S-succinimidyl-S-thioacetate (SATA) in order to add an acetyl thioester group to the lipid and create the complex. The derivatized lipid (SATA-PE) was then part of the liposome formulation containing DSPC:cholesterol: SATA-PE at a mole ratio of 1.5:1.0:0.26. SMPB-IL-2 was covalently coupled to the external surface of the SUV after deacetylation of the thioester moiety at pH 7.4 in PBS. Liposomes prepared by sonication or extrusion had an average diameter of 46-50 nm. SUV-IL-2 bound to the high affinity IL-2 receptor as measured by competitive binding assays and Scatchard analysis using 111InCl2-loaded liposomes The preparation exhibited a binding constant of 30 pM, consistent with values for free IL-2 cited in the literature. SUV IL-2 could be used as the sole source of IL-2 for the murine CTLL-2 T-cell line or for human mitogen-activated PBLs. The presence of IL-2 coupled to the surface was absolutely required for delivery of the drug to the cell. When methotrexate was encapsulated within the internal aqueous space, receptor-mediated endocytosis led to the inhibition of proliferation due to delivery of MTX to the cytoplasm of the cell. More than 90% of the methotrexate was retained within the liposome during storage over a 24-h period at 4 degrees C. This immunoliposome represents a new class of cell specific immunoliposomes whose entry into the cell is controlled by a cell surface receptor.