A male-specific renal cytochrome P-450 isozyme(s) responsible for mutagenic activation of 3-methoxy-4-aminoazobenzene in mice

Renal microsomes from male mice (BALB/c, DBA/2 and BALB/c x DBA/2 F1) showed about 10-fold greater activity for mediating mutagenic activation of 3-methoxy-4-aminoazobenzene (3-MeO-AAB) toward Salmonella typhimurium TA98 than did the corresponding hepatic microsomes, as compared on the basis of nmol of microsomal cytochrome P-450. On the other hand, female renal microsomes and other extrahepatic microsomes (lung, small intestine and colon) in both sexes of mice showed little or no activity for converting 3-MeO-AAB to mutagen(s). The mutagenic activation of 3-MeO-AAB with the male renal enzyme(s) was definitely inhibited by cytochrome P-450 inhibitors, 7,8-benzoflavone and SKF 525A. All these findings suggest that in mice, there is a male-specific renal 3-MeO-AAB activation enzyme(s), a cytochrome P-450 isozyme(s), which is different, at least in proportion and/or in nature, from hepatic cytochrome P-450 isozymes.     

Cutaneous metabolism of benzo[a]pyrene: comparative studies in C57BL/6N and DBA/2N mice and neonatal Sprague--Dawley rats

The metabolism of the polycyclic aromatic hydrocarbon (PAH) carcinogen benzo[a]pyrene (BaP) was studied using microsomes prepared from the skin of the mouse and rat. Topical application of the polychlorinated biphenyl (PCB) Aroclor 1254 or the PAH 3-methylcholanthrene (3-MC) to the skin of the C57BL/6N and DBA/2N mouse and the Sprague-Dawley rat caused statistically significant enhancement of cutaneous microsomal aryl hydrocarbon hydroxylase (AHH) activity in each animal. PCB was a more potent inducer of the enzyme than was 3-MC. BaP metabolism by skin microsomes from the same animals was assessed using high performance liquid chromatography (HPLC). The skin of untreated animals metabolized BaP into 9,10-, 7,8- and 4,5-dihydrodiols, phenols and quinones. Skin application of PCB caused greater than 16–18-fold enhancement of BaP metabolism in the C57BL/6N mouse and the rat and 2–5-fold enhancement in the DBA/2N mouse. Skin application of 3-MC enhanced BaP metabolism 2–8-fold in the C57BL/6N mouse and 5–10-fold in the rat and had no effect in the DBA/2N mouse. The formation of procarcinogenic metabolite BaP-7, 8-diol was greatly enhanced (4–12-fold) by treatment with the PCB and 3-MC in the tumor susceptible C57BL/6N mouse and in the tumor-resistant neonatal Sprague-Dawley rat. In contrast, the formation of BaP-7,8-diol was either slightly enhanced (2-fold) or unaffected by treatment with the PCB or 3-MC in the tumor-resistant DBA/2N mouse. Our data indicate that neither the patterns of metabolism nor the amount of BaP-7,8-diol formation in the skin are reliable predictors of tumor susceptibility to the PAH in rodent skin.     

Metabolism of benzo[a]pyrene: Effect of 3-methylcholanthrene pretreatment on metabolism by microsomes from lungs of genetically “responsive” and ”nonresponsive” mice

The metabolism of [¹⁴C]benzo[a]pyrene by microsomes from the lungs of normal and 3-methylcholanthrene-treated DBA/2J, C57BL/6J, and A/HeJ mouse strains was quantitatively analyzed by high-pressure liquid chromatography. The ratio of dihydrodiols of benzo[a]pyrene to total metabolites formed was greater with lung microsomes than with liver microsomes in all three strains. The ratio of epoxide hydrase to monooxygenase activity in mouse lung was shown to be considerably higher than in mouse liver. Benzo[a]pyrene metabolism by control lung microsomes showed some strain differences. C57BL/6J and A/HeJ mice formed twice as much dihydrodiols as a percentage of total metabolism compared to DBA/2J mice. DBA/2J mice produced somewhat less phenol 2 fraction and considerably more quinone 1 and 2 fractions than the other two mouse strains as a percentage of total metabolism. Treatment of C57BL/6J and DBA/2J mice with 3-methylcholanthrene resulted in a 20-fold increase in the metabolism of benzo[a]pyrene, while A/HeJ mice were induced more than 50-fold. The profiles of metabolites from the 3-methylcholanthrene-induced animals were nearly identical in all three mouse strains.     

Purification and characterization of a microsomal cytochrome P-450 with high activity of coumarin 7-hydroxylase from mouse liver

Phenobarbital-induced coumarin 7-hydroxylase is high in DBA/2J and low in C57BL/6N inbred mice; this genetic difference is encoded by the Coh locus on chromosome 7. The aim of this study was to develop an antibody specific for this cytochrome P-450 polymorphism. P-450 fractions, highly specific for phenobarbital-inducible coumarin 7-hydroxylase activity, were purified from DBA/2J and C57BL/6N mouse liver microsomes. Both proteins are 49 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Soret peaks of the reduced cytochrome . CO complexes are 451 nm. Reconstituted DBA/2J coumarin 7-hydroxylase activity exhibits a V twice as high as, and a Km value 10-fold less than, the reconstituted C57BL/6N activity. Antibodies were raised in rabbit. By Ouchterlony immunodiffusion, both antibodies show 100% cross-reactivity with DBA/2J and C57BL/6N microsomes and purified antigens. Yet, DBA/2J but not C57BL/6N 7-hydroxylase activity is inhibited by the antibody to DBA/2J P-450. Both DBA/2J and C57BL/6N activities are blocked by the antibody to C57BL/6N P-450. Neither antibody has any effect on liver microsomal d-benzphetamine N-demethylase, ethylmorphine N-demethylase, aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase, acetanilide 4-hydroxylase, or aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity. The DBA/2J protein most specific for phenobarbital-induced coumarin 7-hydroxylation is designated 'P-450Coh'. Anti-(P-450Coh) precipitates a relatively minor 49-kDa protein from detergent-solubilized microsomes and from in vitro translation of poly(A+)-enriched total RNA of phenobarbital-treated DBA/2J mouse liver, whereas the major phenobarbital-induced P-450 proteins exhibit a molecular mass of about 51 kDa. The immunoprecipitated translation products correspond to a messenger RNA of 2100 +/- 100 nucleotides.     

Anesthesia in the mouse using a combination of ketamine and promazine

An anesthetic combination of ketamine and promazine was tested in 25 male and 25 female mice of each of two strains. The mean induction time was 5 minutes for the C57BL/6We mouse and 8 minutes for the DBA/2We mouse. No sex differences were observed. The mean duration time was 45 minutes for the C57BL/6We male, 53 minutes for the C57BL/TWe female, 29 minutes for the DBA/2We male, and 35 minutes for the DBA/2We female. The mean recovery time was 39 minutes for the C57BL/6We male, 38 minutes for the C57BL/6We female, 21 minutes for the DBA/2We male, and 24 minutes for the DBA/2We female. The drug combination provided effective anesthesia for these strains of the laboratory mouse for a period of 30--50 minutes after a single intramuscular injection.     

Isosafrole-induced cytochrome P2-450 in DBA/2N mouse liver. Characterization and genetic control of induction

Mouse "cytochrome P2-450" is defined as that form of isosafrole-induced P-450 in DBA/2N liver most specifically correlated with isosafrole metabolism. Isosafrole pretreatment does not induce aryl hydrocarbon hydroxylase activity ("cytochrome P1-450") in C57BL/6N or DBA/2N mice, induces acetanilide 4-hydroxylase activity ("cytochrome P3-450") more than 3-fold in C57BL/6N but not in DBA/2N mice, and induces isosafrole metabolite formation more than 3-fold in both C57BL/6N and DBA/2N mice. P2-450 was, therefore, purified from isosafrole-treated DBA/2N liver microsomes having negligible amounts of contaminating P1-450 and P3-450. The apparent molecular weight of P2-450 is 55,000, and the protein appears homogeneous on sodium dodecyl sulfate-polyacrylamide gels. The Soret peak of the reduced purified cytochrome X CO complex is 448 nm. Purified P2-450, reconstituted in vitro, metabolizes acetanilide poorly and benzo[a]pyrene hardly at all. Anti-(P2-450) inhibits (90 to 100%) liver microsomal isosafrole metabolite formation, yet has no effect on aryl hydrocarbon hydroxylase, acetanilide 4-hydroxylase, biphenyl 2- or 4-hydroxylase, or 7-ethoxycoumarin O-de-ethylase activities. 3-Methylcholanthrene induces anti-(P2-450)-precipitable protein about 12-fold in C57BL/6N and 2-fold in DBA/2N liver; 2,3,7,8-tetrachlorodibenzo-p-dioxin (10 micrograms/kg), about 12-fold in both C57BL/6N and DBA/2N liver; isosafrole, more than 3-fold in both C57BL/6N and DBA/2N. Benzo[a]anthracene at maximal doses induces anti-(P2-450)-precipitable protein in C57BL/6N liver no more than 2-fold, yet is known to be a highly potent inducer of P1-450 mRNA in C57BL/6N liver. The sensitivity of the P2-450 induction process to isosafrole is inherited as an autosomal additive trait; studies of offspring from the C57BL/6N(DBA/N)F1 X DBA/2N backcross confirm involvement of the Ah locus or s closely segregating gene. In contrast, among crosses between C57BL/6N and DBA/2N, sensitivity of the P1-450 and P3-450 induction process to 3-methylcholanthrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin is inherited as an autosomal dominant trait. These data suggest that, although P1-450, P2-450, and P3-450 proteins are controlled by the Ah locus, either a P-450 protein polymorphism exists between C57BL/6N and DBA/2N mice or subtle differences may exist in the interaction of various inducers with Ah receptor.     

Role of phosphatidylinositol-3-kinase-gamma (PI3Kgamma)-mediated pathway in 17beta-estradiol-induced killing of L. mexicana in macrophages from C57BL/6 mice

We recently demonstrated that 17beta-estradiol (E2) enhances killing of Leishmania mexicana in macrophages from both male and female DBA/2 mouse by increasing nitric oxide (NO) production. Here, we analyzed the effect of E2 on leishmanicidal activity and cytokine production by bone marrow-derived macrophages (BMDMs) from male and female C57BL/6 mice in vitro, specifically examining the role of phosphatidylinositol-3-kinase-gamma (PI3Kgamma) in E2-induced parasite killing. Unlike its effect on macrophages from both male and female DBA/2 mice, E2 only increased leishmanicidal activity in macrophages from female C57BL/6 mice, which was evident by a significant reduction in both infection rates and infection levels compared to sham controls. E2-treated BMDMs from female C57BL/6 mice expressed higher levels of interferon-gammaRalpha, and also produced more interleukin (IL)-12, IL-6 and NO than both the sham controls and E2-treated male-derived macrophages. Sham-treated BMDMs from female PI3Kgamma-/- C57BL/6 mice displayed lower infection rates and infection levels compared to sham-treated wild-type (WT) macrophages. However E2, unlike its effect on macrophages from female WT C57BL/6 mice, failed to reduce infection rates and infection levels in BMDMs from female PI3Kgamma-/- mice. Interestingly, E2-treated BMDMs from female C57BL/6 mice produced significant amounts of inflammatory cytokines and NO in levels comparable to those observed in sham-treated PI3Kgamma-deficient macrophages as well as E2-treated macrophages from WT mice. These findings show that E2 exerts a distinct effect on leishmanicidal activity of macrophages from male versus female C57BL/6 mice. In addition, they suggest that PI3Kgamma is not required for E2-induced cytokine and NO production in L. mexicana-infected macrophages from female C57BL/6 mice but it may be involved in parasite clearance from these cells.     

Comparative effects of endrin on hepatic lipid peroxidation and DNA damage,and nitric oxide production by peritoneal macrophages from C57BL/6J and DBA/2 mice

1. Endrin is a polyhalogenated cyclic hydrocarbon which produces hepatic and neurologic toxicity. In order to further assess the mechanism of toxicity ofendrin, the dose-dependent effects of endrin on hepatic lipid peroxidation and DNA damage, and nitric oxide (NO) production by peritoneal exudate cells (primarily macrophages) were investigated in C57BL/6J and DBA/2 mice which vary at the Ah receptor genetic locus. C57BL/6J mice are dioxin-responsive, while DBA/2 mice are dioxin-insensitive.2. Mice of both strains were treated with 0, 1, 2 or 4 mg endrin kg⁻¹ as a single oral dose in corn oil, and the animals were killed 24 hr post-treatment. At doses of 1,2 and 4 mg endrin kg⁻¹ in C57BL/6J mice, hepatic mitochondrial lipid peroxidation increased 1.2-, 2.2- and 3.2-fold, respectively, and 1.8-, 2.3- and 3.5-fold with microsomes, respectively. At these same doses in DBA/2 mice, hepatic mitochondrial lipid peroxidation increased 1.3-, 2.0- and 2.6-fold, respectively, and 1.5-, 1.9- and 2.5-fold with microsomes, respectively.3. Increases of 2.3-, 2.4- and 4.9-fold were observed in hepatic DNA damage (elution constants) in C57BL/6J mice at doses of 1, 2 and 4 mg endrin kg⁻¹, respectively, while at these same three doses, increases of 1.9-, 2.1- and 2.3-fold were observed for DBA/2 mice, respectively.4. Nitric oxide production by peritoneal macrophages from C57BL/6J increased by 1.3-, 1.7- and 2.0-fold with doses of 1, 2 and 4 mg endrin kg⁻¹, respectively, while in macrophages from DBA/2 mice at these same doses, increases of 1.7-, 1.7- and 1.8-fold, respectively, were observed.5. The results indicate that the responsiveness of peritoneal macrophages with respect to both DNA damage and nitric oxide production are more dose-dependent in C57BL/6J mice as compared to DBA/2 mice, while similar results are observed with the lipid peroxidation of hepatic mitochondria and microsomes of the two mouse strains. The results suggest that the toxicity of endrin is less reliant on a mechanism which may involve the Ah receptor system as compared to dioxins as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).     

Decline in male mouse pheromone with age

An age-related decline in urinary-borne pheromone was found in male C57BL/6J mice aged from 2 to 30 months. Pheromone activity, estimated by bioassay, declined sharply after about 10 months of age. Two other strains of mice tested (DBA/2J and CBA/HT6J) also appeared to show an age-related decline in pheromone activity. Within each strain, however, pheromone activity was consistently similar to or higher than that of the C57BL/6J male mice. The DBA/2J and BALB/cWt strains appeared to be high pheromone producers, and the C57BL/6J and CBA/HT6J strains, low producers. This report is the first demonstration of a decline with age in male mouse pheromone activity. This decline appears to be synchronized with the well-defined loss of reproductive function in female mice.     

Lipase activity in germinating sunflower seedlings

Studying lipase in germinating sunflower seedlings, we looked for an activator of the lipolytic activity. In the presence of 1.25 mM ATP, the enzyme activity increased 2-fold. Lipid-body lipase solubilization was realized using two detergents: Tween 80 and CHAPS. Lipolytic activity was increased 10-fold in the presence of 2% (w/v) CHAPS, showing the probable 'complexity' of the enzyme. Looking for the possible lipolytic activity of the 10000 g pellet we detected the presence of the enzyme. The pellet extract was mixed, in a range of concentrations, with the oil-body fraction. The resulting lipolytic activity was 4-fold higher. These results give clues as to the subcellular distribution of lipase and its intracellular transport.     

Distinction between octachlorostyrene and hexachlorobenzene in their potentials to induce ethoxyphenoxazone deethylase and cause porphyria in rats and mice

The potentials of octachlorostyrene (OCS) and hexachlorobenzene (HCB) to induce liver microsomal ethoxyphenoxazone deethylation (an indicator of induction of 3-methylcholanthrene and beta-naphthoflavone-like cytochrome P-450 monoxygenase activity) and cause porphyria in male C57BL/6 and C57BL/10 mice and female F344 rats were compared. Ethoxyphenoxazone deethylation was induced much more by HCB than by OCS in both of these strains of mice (although neither OCS nor HCB greatly induced deethylation in the DBA/2 strain). In rats ethoxyphenoxazone deethylase was induced 26-fold by HCB but only four-fold by OCS, whereas dealkylation of pentoxyphenoxazone (an indicator of phenobarbital-like induction) increased 43- and 36-fold, respectively. Both chemicals were poor inducers of dealkylation of pentoxyphenoxazone in mice. When fed HCB continuously but not when given OCS, C57BL/6 and C57BL/10 mice (both after pretreatment with iron) and F344 rats developed porphyria with a depression of hepatic uroporphyrinogen decarboxylase activity. The results illustrate that in these species OCS and HCB cannot be considered as equally efficient agents for inducing ethoxyphenoxazone deethylation or causing porphyria. If these effects are mediated through binding to the aromatic hydrocarbon responsiveness (Ah) receptor, HCB would appear to have a much greater affinity than OCS despite the face that neither chemical possesses a structure currently considered to be necessary for efficient binding.     

Dimethylnitrosamine metabolism: II. In vitro activation of dimethylnitrosamine by hepatic and renal tissues from crosses among BALB/c, DBA and C57BL mice

Crosses among BALB/c, C57BL and DBA mice were performed to investigate the genetic mechanisms involved in metabolism of DMN by renal and hepatic tissues. Liver S-9 fractions from parental strain DBA had the greatest potential to activate DMN and liver fractions from parental strain BALB/c had the lowest. No age or sex-related differences were observed within strain. Crossing of either C57BL or DBA to BALB/c mice resulted in F1 hybrids with liver microsomal enzymes that gave results similar to the BALB/c parental strain. There were no sex or age differences within crossbred strains in the potential of liver to activate DMN. In contrast male DBA and C57BL parental mice renal S-9 fractions did not differ significantly from each other but did differ significantly from male BALB/c renal fractions and from female and immature animals of all strains. Crossing of either DBA or C57BL mice with BALB/c mice resulted in male F1 hybrids whose renal S-9 fractions did not differ significantly from males of the parental BALB/c strain. In all instances, male renal S-9 fractions had a significantly greater potential to activate DMN than female or immature animals. F1 DBA X C57BL hybrids had renal S-9 fractions that did not differ significantly from the parental strains. These data suggest that the gene(s) for low DMN metabolism of BALB/c mice are apparently dominant over the genes from both DBA and C57BL. The exact genetic or physiological mechanism needs further elucidation.     

The genetics of aryl hydrocarbon hydroxylase induction in mice: A single gene difference between C57BL/6J and DBA/2J

Twenty-one inbred strains of mice were surveyed for inducibility of hepatic aryl hydrocarbon hydroxylase (AHH) activity by the carcinogen 3-methylcholanthrene (MC). In 11 strains given MC, AHH activity increased 1.3- to 5-fold (inducible), whereas ten strains responded with a less than 0.5-fold increase (noninducible). Neither the inducible nor the noninducible class was homogeneous, and in each considerable variation was found in both the basal activity of AHH and the response to MC. Strains DBA/2J and C57BL/6J were chosen to represent the noninducible and inducible classes, respectively. In the crosses (C57BL/6 × DBA/2)F₁ × DBA/2 and (C57BL/6 × DBA/2)F₂, inducibility segregated as a single autosomal dominant gene. The gene symbols Ahh ⁱ and Ahh ⁿ are proposed for the alleles present in C57BL/6J and DBA/2J, respectively. No genetic linkage was found between the Ahh locus and the following loci: b, d, Es-1, Es-3, Gpd-1, Hbb, Id-1, Pgm-1, and sex. Some implications of this work in the study of mammalian enzyme induction and chemically induced carcinogenesis are discussed. There is a positive correlation between AHH inducibility and the development of an inflammatory response to the topical application of the carcinogen 7,12-dimethylbenzanthracene.     

Frequency and cross-reactivity od cytolytic T lymphocyte precursors reacting against male alloantigens

It is well established that cytotoxic T lymphocytes (CTL) specific for the male minor histocompatibility antigen (H-Y) are generated by restimulation in vitro of in vivo primed spleen cells from C57BL/6 (H-2b) female mice with syngeneic male spleen cells. When tested on target cells from H-2 different strains, the male-specific C57BL/6 CTL populations exhibited significant lysis of DBA/2 (H-2d), A (H-2a), but not C3H (H-2k), male and female target cells. In an attempt to document this cross-reactivity further at the clonal level, a sensitive technique of limiting dilution analysis was used to determine the specificity of C57BL/6 individual CTL precursors (CTL-P) reactive against the male antigen. The mean frequency of anti-H-Y CTL-P in spleens of primed female mice was about 1/3500. Between one-third to one-tenth of these CTL-P produced a progeny that cross-reacted with H-2d (allogeneic) female target cells. These findings were confirmed by the analysis of the reactivity pattern exhibited by male-specific CTL clones derived by limiting dilution. Of 99 clones tested, 13 were found to cross-react with female DBA/2 target cells. These results thus indicate that a relatively large proportion (greater than 10%) of H-2b CTL-P directed against the H-Y antigen cross-react with target cells expressing H-2d alloantigens in the absence of H-Y antigen.     

Genetic regulation of UDP-glucuronosyltransferase induction by polycyclic aromatic compounds in mice. Co-segregation with aryl hydrocarbon (benzo(alpha)pyrene) hydroxylase induction.

Induction of hepatic 4-methylumbelliferone UDP-glucuronosyltransferase (EC 2.4.1.17) by polycyclic aromatic compounds, such as 3-methylcholanthrene or beta-naphthoflavone, occurs in C57BL/6N, A/J, PL/J, C3HeB/FeJ, and BALB/cJ but not in DBA/2N, AU/SsJ, AKR/J, or RF/J inbred strains of mice. This pattern of five responsive and five nonresponsive mouse strains parallels that of the Ah locus, which controls the induction of aryl hydrocarbon (benzo[alpha]pyrene) hydroxylase (EC 1.14.14.2). Induction of the transferase is maximal in C57BL/6N mice with 200 mg of 3-methylcholanthrene/kg body weight; no induction occurs in nonresponsive DBA/2N mice even at a dose of 400 mg/kg. The rise of inducible transferase activity lags 1 or more days behind the rise of inducible hydroxylase activity and peaks 5 days after a single dose of 3-methylcholanthrene. In offspring from the appropriate backcrosses and intercross between C57BL/6N and DBA/2N parent strains, the genetic expression of 3-methylcholanthrene-inducible transferase activity is inherited as an additive (co-dominant) trait. This expression differs distinctly from that of the inducible hydroxylase activity, which is inherited almost exclusively as a single autosomal dominant trait in these same animals. The more potent inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin induces the transferase more than 3-fold in C57BL/6N mice and less than 2-fold in DBA/2N mice, whereas the hydroxylase is induced equally (about 8-fold) in both strains. A dose of 3-methylcholanthrene given 3 days after 2,3,7,8-tetrachlorodibenzo-p-dioxin, at a time when hydroxylase induction in both strains is very high, does not enhance the rise in inducible transferase activity seen in C57BL/6N or DBA/2N mice which have received 2,3,7,8-tetrachlorodibenzo-p-dioxin alone. These data indicate that (a) the inducibility of two metabolically coordinated membrane-bound enzyme activities may be regulated by a single genetic locus, and (b) although the hydroxylase can be fully induced in the nonresponsive DBA/2N strain by 2,3,7,8-tetrachlorodibenzo-p-dioxin prior to 3-methylcholanthrene treatment, metabolites of the 3-methylcholanthrene treatment, metabolites of the 3-methylcholanthrene treatment, metabolites of the 3-methylcholanthrene, presumably present in the liver, are incapable of inducing further the transferase activity. The difference in sensitivity between 3-methylcholanthrene and the more potent inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin for both the hydroxylase and the transferase activities suggests the possibility of a common receptor in regulating both enzyme induction processes.     

Stimulation of hepatic phosphatidylcholine biosynthesis in rats fed a high cholesterol and cholate diet correlates with translocation of CTP: phosphocholine cytidylyltransferase from cytosol to microsomes

A new model system for the study of phosphatidylcholine biosynthesis is presented. Young rats were fed a diet that contained 5% cholesterol and 2% cholate. After 6 days there was a 2-fold increase in the concentration of plasma phospholipid (243 mg/dl compared to 132 mg/dl for control animals) and a 3-fold increase in the concentration of plasma phosphatidylcholine. The rate of phosphatidylcholine biosynthesis was measured after injection of [Me-3H]choline into the portal veins. The incorporation of tritium into choline, phosphocholine and betaine by liver was similar for experimental and control animals, whereas there was a 3-fold increased incorporation into phosphatidylcholine of the cholesterol/cholate-fed rats. The activities of the enzymes of phosphatidylcholine biosynthesis in cytosol and microsomes were assayed. The only change detected was in the cytosolic and microsomal activities of CTP: phosphocholine cytidylyltransferase which were increased more than 2-fold in specific activity. When total cytidylyltransferase activity per liver was determined, a dramatic translocation of the enzyme to microsomes was observed. The control livers had 24% of the cytidylyltransferase activity associated with microsomes, whereas this value was 61% in the livers from cholesterol/cholate-fed rats. When the cytosolic cytidylyltransferase was assayed in the presence of phospholipid, the enzyme was stimulated several-fold and the difference in specific activity between control and cholesterol/cholate-fed rats was abolished. The increased activity in cytosol appears to be the result of a 2-fold increase in the amount of phospholipid in the cytosol from cholesterol/cholate-fed rats. The data strongly support the hypothesis that the special diet stimulates phosphatidylcholine biosynthesis by causing a translocation of the cytidylyltransferase from cytosol to microsomes where it is activated.     

CHAPS Solubilization and Functional Reconstitution of beta-Glucan Synthase from Red Beet Root (Beta vulgaris L.) Storage Tissue

A method for the solubilization and reconstitution of red beet (1,3)-β-d-glucan synthase with the detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (CHAPS) was developed. Glucan synthase was effectively solubilized from microsomal or plasma membranes by 0.6% CHAPS in the presence of EGTA and EDTA. Chelators were found essential for effective solubilization and divalent cations inhibitory. A preextraction of membranes with 0.3% CHAPS and 5 millimolar Mg²⁺ prior to the solubilization step was found to remove protein contaminants and increase the specific activity of the solubilized enzyme. Conditions for recovering activity from Sepharose 4B gel filtration columns were defined. Addition of phospholipids and low levels of CHAPS in column elution buffers resulted in complete functional reconstitution with 100% recovery of added activity. Specific activities were increased 20- to 22-fold over microsomes. Active vesicles were recovered by centrifugation. These results provide independent and direct confirmation of the enzyme's requirement for a phospholipid environment.     

Genotype modulates testosterone-dependent activity and reactivity in male mice

Adult castration significantly reduced the homecage locomotor activity of both inbred C57BL/6J and DBA/2J and outbred Rockland-Swiss (R-S) male mice. Castrated C57BL animals exhibited greater reductions in this behavior than did the other genotypes. Locomotor activity in a novel environment (reactivity) was also reduced by castration but only for inbred males. In both test situations, postcastration reductions in ambulation were prevented by implants of testosterone (T)-containing Silastic capsules. Thus, testicular hormones promote activity and reactivity in the male mouse in a genotype-dependent fashion.     

Genetic expression of microsomal electron transport in mice. Different patterns of dependence of constitutive cytochrome P-450 reductase activity of pyridine nucleotide concentrations.

Cytochrome P-450 reductase and aryl hydrocarbon hydroxylase activities were investigated in hepatic microsomes from untreated C57BL/6J, DBA/2J, B6D2F1, and (B6D2) D2 mice. The dependence of the rate of P-450 reduction on the concentration of added pyridine nucleotide (NADPH or NADH) was biphasic in DBA/2J microsomes but monophasic in C57BL/6J microsomes. Analogous strain-specific patterns were observed when the dependence of the rate of benzpyrene hydroxylation on NADPH concentration was examined. In crosses between the two inbred strains and between B6D2F1 mice and DBA/2J mice, the biphasic pattern for both the reductase and the hydroxylase activities was found to co-segregate with the recessive allele for aromatic hydrocarbon responsiveness. These results might reflect an architectural difference between the microsomal electron transport systems of responsive and nonresponsive mice.