The biochemical genetics of permethrin resistance in the Learn-PyR strain of house fly

Permethrin resistance in the Learn-PyR strain of house fly was examined in four genetically derived substrains, each being homozygous for a different resistant autosome of the Learn-PyR strain. The resistance of these derivative strains was characterized toxicologically and biochemically. The relative levels of resistance to permethrin conferred by each autosome were 5>3>1>2. Three factors were associated with resistance: (1) increased mixed-function oxidase (MFO) activity associated with elevated levels of cytochrome P-450, cytochrome b₅, and NADPH-cytochrome c reductase (P-450 reductase) activity; (2) target-site insensitivity (kdr); and (3) decreased cuticular penetration. Permethrin resistance factors on chromosome 1 consisted of a piperonyl butoxide (PB)-suppressible mechanism correlated with increased levels of cytochromes P-450 and b₅; on chromosome 2, a PB-suppressible mechanism associated with elevated amounts of cytochrome P-450; on chromosome 3, decreased cuticular penetration, kdr, and increased amounts of P-450 reductase activity; and on chromosome 5, a largely PB-suppressible mechanism correlated with elevated levels of cytochrome P-450 and P-450 reductase activity.     

Differential down-regulation and induction responses of testicular steroidogenic cytochromes P-450(cscc) and P-450(C17α) to human choriogonadotropin

Evidence is presented that the regulation of the cytochrome P-450(C17) of the steroid-17-monooxygenase and of the cytochrome P-450(cscc) of the cholesterolmonooxygenase by human choriogonadotropin (hCG)in vivo is mediated by differential mechanisms in the adult rat testis. An initial down-regulation of the cytochrome P-450(C17) but not of the P-450(cscc) can be demonstrated. Furthermore, induction of the cytochrome P-450(cscc) requires exposure to higher hCG doses (3270 of the maximal induction rate of 43.7 pmol/(testis x d) are achieved with 4 IU hCG/single dose) than induction of the P-450(C17) (59% of the maximal induction rate of 48.4 pmol/(testis x d) with 4 IU hCG/single dose), Finally, induction ofcytochrome P-450(cscc) starts faster after initiation of hCG treatment than induction of P-450(C 17).     

Partial purification of pisatin demethylase,a cytochrome P-450 from the pathogenic fungus Nectria haematococca

The plant pathogen Nectria haematococca can demethylate pisatin, a phytoalexin from pea. Demethylation is apparently necessary for virulence on pea and is catalyzed by a microsomal cytochrome P-450 monooxygenase system. The cytochrome P-450 and NADPH-cytochrome P-450 reductase of this system were solubilized with sodium cholate and partially purified by chromatography on blue A-agarose and -aminohexyl-agarose. The reductase was further purified by chromatography on 2,5-ADP-agarose to a specific activity of about 16 moles cytochrome c reduced per min per mg protein. Upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the reductase fraction contained one major band of molecular weight 84,000. The partially purified cytochrome P-450 fraction contained a number of minor bands and three major bands of molecular weights 52,000, 56,000 and 58,000. This fraction lost all demethylase activity during concentration after -aminohexyl-agarose chromatography, so it could not be purified further. The purified reductase could reconstitute demethylase activity of cytochrome P-450 fractions and appeared to be rate-limiting for demethylase activity in microsomal extracts.     

Interaction of cholesterol hydroxylating cytochrome P-450 with cytochrome b5

Some new relations between cytochrome P-450-dependent monooxygenases were discovered. Cytochrome b5, a representative of "microsomal" monooxygenases, was shown to form a highly specific complex with cytochrome P-450scc, a member of the "ferredoxin" monooxygenase family. This interaction is characterized by a dissociation constant, Kd, of 0.28 microM. The cytochrome P-450scc-cytochrome b5 complex may be cross-linked with water-soluble carbodiimide. Using proteolytic modification of cytochrome b5, it was shown that both hydrophilic and hydrophobic fragments of cytochrome b5 are involved in the interaction with cytochrome P-450scc. Cytochrome b5 immobilized via amino groups is an effective affinity matrix for cytochrome P-450scc purification. The role of some amino acid residues in cytochrome P-450scc interaction with cytochrome b5 was studied. The role and the nature of complexes in cytochrome P-450-dependent monooxygenases as well as interrelationships between "microsomal" and "ferredoxin" monooxygenases are discussed.     

Cytochrome P-448 induction in liver microsomes of mice of inbred strains after administration of xenobiotics of MC-type

Using immunochemical methods, the identity of cytochrome P-448 from liver microsomes of mice of "inducible" and "non-inducible" lines during induction by xenobiotics of MX-type (3-methylcholanthrene, 3,4-benzpyrene, 2,3,7,8-tetrachlorodibenzodioxin) was established. This hemoprotein form was shown to play a role in 3,4-benzpyrene metabolism. Monospecific antibodies to purified cytochromes P-448 and P-450 were obtained; the cytochrome P-448 content in microsomes was measured by rocket immunoelectrophoresis. The content of cytochrome P-448 in control and phenobarbital-induced microsomes makes up to 10-15% of the total hemoprotein content determinable from the CO-spectra. 3-Methylcholanthrene and 3,4-benzpyrene injected into "non-inducible" mice cause no increase in the content of this hemprotein form, whereas in mice induced with 2,3,7,8-tetrachlorodibenzodioxin it rises to 50%. Under these conditions, an almost 100% inhibition of 3,4-benzpyrene metabolism by antibodies to cytochrome P-448 is observed. Antibodies against cytochrome P-448 obtained from liver microsomes of 3-methylcholanthrene-induced mice cause a 90% inhibition of 3,4-benzpyrene in microsomes induced with 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzodioxin.     

Determination of “active” cytochrome P-450 from relaxation kinetics of product formation

We estimate the active part of cytochrome P-450, which is involved in a special substrate transformation, by measuring the initial change of the production rate as a function of the relaxation transitions between two different steady states of the reaction cycle of cytochrome P-450 using the light-reversibility of the carbon monoxide inhibition. The kinetic data of such relaxations are interpreted within a model cycle, which reduces the reaction cycle to three steps. The estimation of the rate constant of the first reduction step, derived from model simulation of the production rate, is confirmed by independent experimental study of the reduction kinetics.An application of our model to the O-deethylation of 7-ethoxycoumarin reveals that — in a time average — 10%–15% of the spectroscopically detectable cytochrome P-450 is involved in that transformation.Abbreviations Cyt. P-450 microsomal cytochrome P-450 - 7-EC 7-ethoxycoumarin     

Purification and characterization of liver cytochrome P-446 isolated from protein energy malnourished rats

A liver cytochrome P-450 isozyme has been purified to homogeneity from protein-energy malnourished rats induced with -naphthoflavone (-NF). The purification steps included chromatography on DEAE-Sephadex-A-25, DEAE-cellulose (DE-53), hydroxylapatite (HA) and carboxymethyl-sephadex (CM) columns. The reduced carbon monoxide difference and absolute spectra showed a Soret peak at 446.5 nm. The wavelength maxima for the oxidized and reduced spectra were at 416 and 408 nm, respectively. Cytochrome P-446 appears to have a predominantly low spin ferric iron, migrates as a single band of molecular weight 56000 in sodium dodecyl sulfate polyacrylamide gels and has a specific content of 14 nmol/mg of protein. P-446 oxidized various substrates at different rates in a reconstituted system with NADPH-cytochrome P-450 reductase and dilauroylphosphatidylcholine. In this system turnover rates for benzo[]pyrene, testosterone and benzphetamine oxidation were: 81.10; 1.85 and 1.42 nmoles product/min/nmol P-446 respectively. While NH₂ terminal amino acid sequence analysis of 18 of the first 20 residues suggests that the cytochrome P-446 isolated from malnourished rats is identical with form c, the catalytic activities suggest that this isozyme may be a more effective or efficient catalyst for some substrates.Abbreviations -NF -napthoflavone - SDS-PAGE Sodium Dodecyl Sulfate polyacrylamide gel electrophoresis - 3-MC 3-Methyl Cholanthrene - PEG Poly Ethylene Glycol - DTT Dithiothreitol - PMSF Phenyl Methyl Sulfonylfluoride - EDTA disodium ethylenediaminetetraacetate - NADPH reduced nicotinamide adenine dinucleotide phosphate - P-450 cytochrome P450, PB-1, PB-4, PB-5 and P-450 isozymes purified from phenobarbital induced rat liver - HPLC High Pressure Liquid Chromatography - B[]P benzo[]pyrene - CM Carboxymethyl Sephadex - PTH-amino acid phenylthiohydantoin amino acid, Cytochrome P-450 EC 1.14.14.1, NADPH Cytochrome P-450 (c) reductase ED 1.6.2.4     

Liver microsomal cytochromes P-450 and azoreductase activity.

Hepatic microsomal azoreductase activity with amaranth (3-hydroxy-4[(4-sulfo-1-naphthalenyl)azo]-2,7-naphthalenedisulfonic acid trisodium salt) as a substrate is proportional to the levels of microsomal cytochrome P-450 from control or phenobarbital-pretreated rats and mice or cytochrome P-448 from 3-methylchol-anthrene-pretreated animals. In the "inducible" C57B/6J strain of mice, 3-methylcholanthrene and phenobarbital pretreatment cause an increase in cytochrome P-448 and P-450 levels, respectively, which is directly proportional to the increase of azoreductase activity. However, in the "noninducible" DBA/2J strain of mice, only phenobarbital treatment causes the increase both in cytochrome P-450 levels and azoreductase activity, while 3-methylcholanthrene has no effect. These experiments suggest that the P-450 type cytochromes are responsible for azoreductase activity in liver microsomes.     

Human Monoclonal Antibodies against Glucagon Receptor Improve Glucose Homeostasis by Suppression of Hepatic Glucose Output in Diet-Induced Obese Mice

Aim

Glucagon is an essential regulator of hepatic glucose production (HGP), which provides an alternative therapeutic target for managing type 2 diabetes with glucagon antagonists. We studied the effect of a novel human monoclonal antibody against glucagon receptor (GCGR), NPB112, on glucose homeostasis in diet-induced obese (DIO) mice.

Methods

The glucose-lowering efficacy and safety of NPB112 were investigated in DIO mice with human GCGR for 11 weeks, and a hyperinsulinemic-euglycemic clamp study was conducted to measure HGP.

Results

Single intraperitoneal injection of NPB112 with 5 mg/kg effectively decreased blood glucose levels in DIO mice for 5 days. A significant reduction in blood glucose was observed in DIO mice treated with NPB112 at a dose ≥5 mg/kg for 6 weeks, and its glucose-lowering effect was dose-dependent. Long-term administration of NPB112 also caused a mild 29% elevation in glucagon level, which was returned to the normal range after discontinuation of treatment. The clamp study showed that DIO mice injected with NPB112 at 5 mg/kg were more insulin sensitive than control mice, indicating amelioration of insulin resistance by treatment with NPB112. DIO mice treated with NPB112 showed a significant improvement in the ability of insulin to suppress HGP, showing a 33% suppression (from 8.3 mg/kg/min to 5.6 mg/kg/min) compared to the 2% suppression (from 9.8 mg/kg/min to 9.6 mg/kg/min) in control mice. In addition, no hypoglycemia or adverse effect was observed during the treatment.

Conclusions

A novel human monoclonal GCGR antibody, NPB112, effectively lowered the glucose level in diabetic animal models with mild and reversible hyperglucagonemia. Suppression of excess HGP with NPB112 may be a promising therapeutic modality for the treatment of type 2 diabetes.     

Use of monoclonal antibody probes against rat hepatic cytochromes P-450c and P-450d to detect immunochemically related isozymes in liver microsomes from different species

Nine distinct monoclonal antibodies raised against purified rat liver cytochrome P-450c react with six different epitopes on the antigen, and one of these epitopes is shared by cytochrome P-450d. None of these monoclonal antibodies recognize seven other purified rat liver isozymes (cytochromes P-450a, b, and e-i) or other proteins in the cytochrome P-450 region of "Western blots" of liver microsomes. Each of the monoclonal antibodies was used to probe "Western blots" of liver microsomes from untreated, or 3-methylcholanthrene-, or isosafrole-treated animals to determine if laboratory animals other than rats possess isozymes immunochemically related to cytochromes P-450c and P-450d. Two protein-staining bands immunorelated to cytochromes P-450c and P-450d were observed in all animals treated with 3-methylcholanthrene (rabbit, hamster, guinea pig, and C57BL/6J mouse) except the DBA/2J mouse, where no polypeptide immunorelated to cytochrome P-450c was detected. The conservation of the number of rat cytochrome P-450c epitopes among these species varied from as few as two (guinea pig) to as many as five epitopes (C57BL/6J mouse and rabbit). The relative mobility in sodium dodecyl sulfate-gels of polypeptides immunorelated to cytochromes P-450c and P-450d was similar in all species examined except the guinea pig, where the polypeptide related to cytochrome P-450c had a smaller Mr than cytochrome P-450d. With the use of both monoclonal and polyclonal antibodies, we were able to establish that purified rabbit cytochromes P-450 LM4 and P-450 LM6 are immunorelated to rat cytochromes P-450d and P-450c, respectively.     

Changes in mixed-function oxidase system in the perfused liver of the cold-acclimated rat

Changes in the hepatic cytochrome P-450-dependent drug-metabolizing system were studied in perfused livers obtained from cold-acclimated male Wistar rats after 30 days of cold exposure (4C) when using hexobarbital as a substrate. In fasted animals the cold-acclimated rats showed higher levels of hexobarbital metabolic rates compared to control rats, but there was no significant difference in fed animals. The maximum rates of hexobarbital metabolism produced by xylitol perfusion were also significantly higher in the perfused liver of cold-acclimated rats. It was concluded that the function of the cytochrome P-450 system for hexobarbital in cold-acclimated rats changed due to both an increase in the activity of the cytochrome P-450 system and to changes in regulation of the cytochrome P-450 system by the supply of reducing equivalents.     

Recombinant Adiponectin Does Not Lower Plasma Glucose in Animal Models of Type 2 Diabetes

Aims/Hypothesis

Several studies have shown that adiponectin can lower blood glucose in diabetic mice. The aim of this study was to establish an effective adiponectin production process and to evaluate the anti-diabetic potential of the different adiponectin forms in diabetic mice and sand rats.

Methods

Human high molecular weight, mouse low molecular weight and mouse plus human globular adiponectin forms were expressed and purified from mammalian cells or yeast. The purified protein was administered at 10–30 mg/kg i.p. b.i.d. to diabetic db/db mice for 2 weeks. Furthermore, high molecular weight human and globular mouse adiponectin batches were administered at 5–15 mg/kg i.p. b.i.d. to diabetic sand rats for 12 days.

Results

Surprisingly, none of our batches had any effect on blood glucose, HbA1c, plasma lipids or body weight in diabetic db/db mice or sand rats. In vitro biological, biochemical and biophysical data suggest that the protein was correctly folded and biologically active.

Conclusions/Interpretation

Recombinant adiponectin is ineffective at lowering blood glucose in diabetic db/db mice or sand rats.     

Modulation of ethylene synthesis in acotyledonous soybean and wheat seedlings

The characteristics of ethylene production and ACC conversion in 8-day-old soybean seedlings were examined and a relationship between cytochrome P-450 activity and ethylene-forming enzyme (EFE) activity was found. An atmosphere containing 10% carbon monoxide (CO) significantly inhibited ethylene production and ACC conversion in control soybean seedlings, but had only a slight effect on soybean seedlings treated with uniconazole. Foliar application of triclopyr, a pyridine analogue of the phenoxy herbicides, significantly increased ethylene production and ACC conversion in control, but not in uniconazoletreated seedlings. Triclopyr treatment also resulted in a three-fold increase in extractable cytochrome P-450 of 5-day-old etiolated soybeans. At equimolar concentrations tetcyclacis was more effective than uniconazole in reducing shoot elongation and endogenous ethylene production. Although uniconazole and tetcyclacis did not inhibit ACC conversion in nonherbicide-treated soybean seedlings, they did prevent the observed increase in ACC-dependent EFE activity following triclopyr application. However, the rate of ACC conversion in etiolated soybean segments was sensitive to uniconazole, and tetcyclacis inhibited the rate of ACC conversion by 2.6-fold in etiolated soybean segments within 4 h after treatment. Microsomal membranes were isolated from 5-day-old naphthalic anhydride-treated etiolated wheat shoots as this tissue contains much higher cytochrome P-450 levels than soybean shoots. Optical difference spectroscopy demonstrated that ACC generated binding spectrum characteristic of a reverse-type-I cytochrome P-450 substrate when combined with reduced microsomes. In vitro conversion of ACC to ethylene by microsomal membranes was NADPH-dependent, inhibited by CO, and had an apparent K_m and V_max of 45 M and 0.345 nl/mg protein/h, respectively. These results suggest that cytochrome P-450-mediated monooxygenase reactions may be intimately involved in the conversion of ACC to ethylene in young soybean and wheat seedlings.     

Aryl hydrocarbons as inducers of cytochrome P-450 in Acinetobacter calcoaceticus

Summary The inducibility of cytochrome P-450 in Acinetobacter calcoaceticus by some compounds known as typical inducers of hepatic cytochromes P-450 was investigated. Besides biphenyl also indene and phenanthrene are inducers, whereas compounds of the so-called phenobarbital type are not. Biphenyl appears to be the most effective inducer with regard to the yield of cytochrome P-450/mg of cell protein. By addition of the compounds in the vapour phase an induction of the protein by naphthalene could be demonstrated. The results are indicative of the existence of bacterial cytochromes P-450 that resemble hepatic cytochromes.     

Effect of carbon source on the biotransformation enzymesin Serratia marcescens

A microorganism capable of degrading camphor as the sole source of carbon was isolated from soil. The strain was identified as Serratia marcescens (NCIM 5115). The strain when grown in the peptone–glucose medium showed a doubling time of 2.7 h. This microorganism showed the presence of cytochrome P-450, cytochrome b₅ and the activities of cytochrome c reductase, dichlorophenol indophenol reductase, aminopyrine-N-demethylase and steroid 11--hydroxylase. A significant increase in all activities was observed when cells were incubated for 3h in a medium containing either 0.2% camphor, 1.0% n-hexadecane or 0.1% naphthalene when compared to the peptone–glucose medium.     

Phosphorylation of hepatic phenobarbital-inducible cytochrome P-450.

The major phenobarbital-inducible cytochrome P-450 purified from rat liver, a member of family II of the cytochrome P-450 gene superfamily, is rapidly phosphorylated by cAMP-dependent protein kinase. The phosphorylation reaches greater than 0.5 mol phosphate/mol P-450 after 5 min and is accompanied by a decrease in enzyme activity. The serine residue in position 128 was shown to be the sole phosphorylation site and a conformational change of the protein was indicated by a shift of the carbon monoxide difference spectrum of the reduced cytochrome from 450 to 420 nm. Comparison of amino acid sequences of various cytochrome P-450 families revealed a highly conserved arginine residue in the immediate vicinity of the phosphorylated serine residue which constitutes the kinase recognition sequence. It also revealed that only the members of the cytochrome P-450 family II carry this kinase recognition sequence. To find out whether this phosphorylation also occurs in vivo, the exchangeable phosphate pool of intact hepatocytes derived from phenobarbital-pretreated rats was labeled with 32Pi followed by an incubation of the cells with the membrane-permeating dibutyryl-cAMP or with the adenylate cyclase stimulator glucagon to activate endogenous kinase. As a result, a microsomal polypeptide with the same electrophoretic mobility as cytochrome P-450 became strongly labeled. Peptide mapping and immunoprecipitation with monospecific antibodies identified this protein as the major phenobarbital-inducible cytochrome P-450. It becomes phosphorylated at the same serine residues as in the cell-free phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aldrin epoxidase activity in liver microsomes from normal or streptozotocin-diabetic rats: Comparison with activity in isolated hepatocytes from normal rats incubated with glucagon

Aldrin epoxidase activity in liver microsomes from streptozotocin-diabetic rats is only 40% of that from normal rats. Epoxidation of aldrin has also been assayed in freshly isolated hepatocytes from normal rats. Addition of 10^–7 M glucagon to the incubation medium leads to a decreased aldrin epoxidase activity. Owing to the previously reported phosphorylation of a purified cytochrome P-450 isozyme, it is postulated that the cytochrome P-450 dependent aldrin epoxidase may be regulated by a glucagon induced phosphorylation process.     

Catalytic activity of isolated cytochromes P-450d and P-450c

Cytochrome P-450d was isolated from isosafrol-induced rat liver microsomes by affinity chromatography on 1.8-diaminooctyl-Sepharose 4B and chromatography on hydroxylapatite using a linear potassium phosphate gradient (45-250 mM). The enzyme has a molecular mass of 54 kDa, CO-maximum 448 nm is characterized by a high spin state; the rate of 4-aminobiphenyl hydroxylation is 54 nmol/min/nmol of cytochrome P-450d (37 degrees C), those, of 7-ethoxyresorufin O-deethylation and benz (a) pyrene oxidation are 1 nmol/min/nmol of cytochrome P-450d (22 degrees C) and 2 nmol/min/nmol of cytochrome P-450d (37 degrees C), respectively. The properties of cytochrome P-450d were compared to those of cytochrome P-450c isolated from 3-methylcholanthrene-induced rats. The yield of these cytochromes under the conditions used (10% P-450d from isosafrol-induced microsomes and 15% P-450c from 3-methylcholanthrene-induced microsomes) was relatively high. Antibodies to cytochromes P-450d and P-450c were obtained. Using rocket immunoelectrophoresis the percentage of these hemoprotein forms in 3-methylcholanthrene-induced (P-450d-20%, P-450c-70%) and isosafrol-induced rat liver microsomes (P-450d-50%, P-450c-15%) was determined.     

Study of benzphetamine-N-demethylase in streptozotocin-diabetic mice and rats: evidence for the induction of catalytically and immunologically specific forms of cytochrome P450

Phenobarbital treatment and streptozotocin-diabetes both increase, in mouse and rat microsomes, a benzphetamine-N-demethylase activity which can be inhibited by a specific antibody raised against purified rat phenobarbital-induced cytochrome P-450. However, similar studies performed on cytochrome P-450 A and B fractions separated by DEAE-cellulose chromatography, clearly proved that streptozotocin-diabetes promotes in mice the synthesis of two new species of cytochrome P-450 and that the streptozotocin diabetes-induced forms are different in mouse and rat. No such modifications were observed in the mixed-function oxidase system of congenitally diabetic mice.     

Character of changes in serotonin-modulating anticonsolidation protein and cytochrome P-450 in tissues of Alburnoides bipunctatus eichwaldi caught in the rivers of Azerbaijan Republic

The work was designed to investigate the levels of a novel serotonin-modulating anticonsolidation protein (SMAP), which activity directly depends on serotonin level, and biomarker cytochrome P-450 in the liver, gills and brain of Alburnoides bipunctatus eichwaldi caught in Khudat, Agstafachay, Kura and Araz rivers running over the territory of Azerbaijan. Noticeable downregulation of cytochrome P-450 and SMAP was observed in the liver and gills of fish caught in Agstafachay river relatively to the values obtained for the fish from unpolluted Khudat river. The levels of cytochrome P-450 and SMAP in liver of fish from Kura and Araz rivers exhibited significant opposite changes: downregulation of cytochrome P-450 level vs. upregulation of SMAP level. In the brain of fish from Agstafachay river the cytochrome P-450 was found to be somewhat downregulated, whereas in the brain of fish from Kura and Araz rivers SMAP was significantly upregulated. The results obtained are analyzed from the standpoint of adaptation and disadaptation processes of water organisms to the effects of pollutants.