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Biomedical subjects

M Murray

Publications and source records attributed to M Murray.

At least 397 records · Page 22Linked to original sources
PubMed1987

Protein kinase C activation enhances cAMP accumulation in Swiss 3T3 cells: inhibition by pertussis toxin.

Addition of phorbol 12,13-dibutyrate (PBt2) in the presence of forskolin or cholera toxin caused marked (6- to 8-fold) and rapid accumulation of cAMP in Swiss 3T3 cells. The effect of PBt2 is mediated by protein kinase C because the synthetic diacylglycerol 1-oleoyl-2 acetylglycerol substitutes for PBt2 in enhancing cAMP accumulation and because the enhancing effect of either PBt2 or 1-oleoyl-2-acetylglycerol was prevented by down-regulation of protein kinase C. Vasopressin, which activates protein kinase C but does not directly affect adenylate cyclase in 3T3 cells, also enhanced cAMP accumulation in cells treated with cholera toxin or forskolin. This effect was abolished by down-regulation of protein kinase C. Treatment with pertussis toxin blocked the enhancing effect of PBt2 in a concentration- and time-dependent manner. Pertussis toxin neither prevented protein kinase C activation by PBt2 nor other biologic responses elicited by PBt2. The results presented here suggest an unusual function for a pertussis toxin substrate--namely, coupling protein kinase C activation to cAMP production.

Adenylate Cyclase Toxin↗
PubMed1987

Potential value of localized skin reactions (chancres) induced by Trypanosoma congolense transmitted by Glossina morsitans centralis for the analysis of metacyclic trypanosome populations.

Goats infected with Trypanosoma congolense transmitted by Glossina morsitans centralis and then treated with the trypanocidal drug diminazene aceturate are immune to tsetse-transmitted infection with a homologous, but not heterologous trypanosome clone. Immune animals fail to develop localized skin reactions (chancres) and do not become infected, whereas on heterologous challenge chancres appear followed by parasitaemia. In this study, the feasibility of using chancre reactions to characterize several different metacyclic populations of T. congolense was evaluated. Provided goats were immunized, it was found that the chancre reaction could be used to distinguish different populations of T. congolense. However, problems were encountered when attempts were made to induce immunity against more than one trypanosome population. When goats were infected by tsetse flies with four antigenically unrelated metacyclic populations of T. congolense, either simultaneously or at 4 day intervals, adequate immunological priming was not always achieved. In fact, goats exposed to superinfection 8 to 12 days after a primary infection did not develop chancres or an immune response to the second infection (although they were immune to the organism used for primary infection). Following trypanocidal treatment these animals were subsequently completely susceptible to homologous infection with the metacyclic population used for superinfection, indicating that some form of interference phenomenon effective at the level of the skin appears to prevent or delay the development of the second trypanosome population following superinfection of infected animals.

Animals↗
PubMed1987

Dose and stage dependency for the development of local skin reactions caused by Trypanosoma congolense in goats.

Intradermal inoculation of metacyclic forms of Trypanosoma congolense propagated in vitro caused skin reactions in goats similar to the local skin reaction (chancre) induced by the bite of an infected tsetse fly. The onset, size and duration of these local skin reactions were dose-dependent. Whereas one cultured metacyclic T. congolense was sufficient to cause a local skin reaction in a goat, over 10(7) bloodstream forms of T. congolense were necessary to elicit a detectable skin reaction and while T. congolense parasites present in lymph did not cause local skin reactions, trypanosomes collected from oedematous fluid of the chancre did. - Using non-dividing irradiated bloodstream forms it was estimated that 10(8) T. congolense were required to induce a detectable local skin reaction. - Intradermal needle inoculation of procyclic forms (uncoated trypomastigotes) of T. congolense propagated in vitro induced an intense inflammatory response which was similar to that found in the early phases of the reaction elicited by metacyclic trypanosomes. This suggests that the uncoated trypomastigotes which are known to be present in the saliva of infected tsetse may play a role in the initial development of the chancre. - The data obtained for the local skin reaction suggest the presence of an intracutaneous dividing stage of T. congolense which is intermediate between the metacyclic and bloodstream forms.

Animals↗
PubMed1987

Altered regulation of cytochrome P-450 enzymes in choline-deficient cirrhotic male rat liver: impaired regulation and activity of the male-specific androst-4-ene-3,17-dione 16 alpha-hydroxylase, cytochrome P-450UT-A, in hepatic cirrhosis.

Total microsomal cytochrome P-450 levels were decreased, to about 50% of control, in liver of male rats made cirrhotic by the prolonged intake of a choline-deficient diet. We have suggested previously that this decrease in cytochrome P-450 levels is not a generalized one, but is selective for certain forms of the enzyme. In the present study, levels of six cytochrome P-450 forms including the sex-specific cytochrome P-450 forms, P-450UT-A, P-450PCN-E, and P-450UT-l, were quantitated immunochemically in hepatic microsomes prepared from control and cirrhotic male rats and were related to changes in the regioselectivity of cytochrome P-450-mediated androst-4-ene-3,17-dione hydroxylation in these fractions. The principal finding of this study was that the male-specific androst-4-ene-3,17-dione 16 alpha-hydroxylase was decreased in cirrhotic microsomes to about 20% of control. The content of P-450UT-A decreased concurrently from about 0.40 to less than 0.01 nmol/mg of microsomal protein. Other pathways of androst-4-ene-3,17-dione hydroxylation were also affected, but to different extents than the 16 alpha-hydroxylase. 6 beta-Hydroxylation decreased in cirrhotic microsomes to about 45% of control, despite a marked decrease in P-450PCN-E from 0.27 to less than 0.002 nmol/mg of microsomal protein. The rate of androst-4-ene-3,17-dione 7 alpha-hydroxylation underwent a less pronounced reduction in cirrhosis to about two-thirds of control microsomal activity, and levels of the cytochrome P-450 associated with this activity, P-450UT-F, were decreased in proportion with the decrease in total microsomal cytochrome P-450. 16 beta-Hydroxylase activity was unaffected by the cirrhotogenic process. From spectral binding studies it was apparent that androst-4-ene-3,17-dione elicited a high affinity type I interaction in control microsomal fractions (Ks = 4.5 microM), whereas no interaction was apparent in cirrhotic liver microsomes. Levels of three other forms of cytochrome P-450--P-450PB-C (a constitutive form inducible by phenobarbital), P-450ISF-G (a major isosafrole-inducible form), and P-450UT-I (the major female sexually-differentiated isozyme)--were apparently unaltered in cirrhosis. These findings are consistent with the assertion that specific forms of cytochrome P-450 are subject to altered regulation in hepatic cirrhosis.

Androstenedione↗
PubMed1986

Effects of primaquine on hepatic microsomal haemoproteins and drug oxidation.

Administration of the antimalarial agent primaquine to male rats (50 mg/kg i.p. daily for 4 days) resulted in a 30% decrease in hepatic microsomal cytochrome P-450 (P-450) content; levels of other microsomal haemoproteins were unaltered. Kinetic analysis of 2 mixed-function oxidase activities (aminopyrine N-demethylase and aniline p-hydroxylase) in primaquine-pretreated rat liver microsomes revealed a significant and similar decrease in the maximal reaction velocities of these enzymes (Vmax), but the apparent Michaelis constants (Km) were not changed. The activity of mitochondrial delta-aminolaevulinic acid synthetase (the rate-limiting step in haem biosynthesis) was normal in primaquine-pretreated rat liver but haem oxygenase activity (the rate-limiting step in haem degradation) was elevated approximately 2-fold. Haem availability for haemoprotein assembly (determined as the haem-saturation ratio of the cytosolic haemoprotein tryptophan pyrrolase) was also normal although the absolute activity of tryptophan pyrrolase was decreased after primaquine pretreatment. In order to facilitate an analysis of the P-450 isozyme profile in control and primaquine-treated rat liver, total microsomal P-450 was isolated by hydrophobic affinity chromatography on n-octylamino-Sepharose 4B. Densitometry of stained polyacrylamide gels following electrophoresis of these partially-purified P-450 fractions indicated that primaquine exposure did not selectively decrease any of the 3 protein bands in the P-450 molecular weight region (48-56 kD). These observations, when considered together, suggest that primaquine may affect P-450 and mixed-function oxidase activity by inhibition of protein synthesis. The characteristic rapid turnover rates of P-450 isozymes may predispose these haemoproteins to the toxic effects of primaquine whereas those haemoproteins that turn over less rapidly, such as cytochrome b5, appear to be less susceptible. Microsomal haem oxygenase activity may be elevated after primaquine administration since lowered haemoprotein requirements for haem could result in excess haem levels within the hepatocyte.

Animals↗
PubMed1986

Mechanistic aspects of the inhibition of microsomal drug oxidation by primaquine.

The kinetics of inhibition of microsomal drug oxidation (as aminopyrine N-demethylase activity) by the antimalarial agent primaquine were found to be concentration-dependent. Lower concentrations of primaquine (0-40 microM) elicited slope-hyperbolic, intercept-hyperbolic noncompetitive (mixed) inhibition with an inhibitor equilibrium-dissociation constant (Ki) of 21 microM. On the other hand, primaquine concentrations greater than 40 microM elicited essentially simple competitive inhibition as judged from Lineweaver-Burk and Dixon analysis with appropriate replots (Ki = 23 microM). The coincident Ki values suggest that the same enzyme-inhibitor complex is involved in inhibition over all concentrations of primaquine tested. The apparent change in kinetics was accounted for in terms of a four-step interaction scheme involving a ternary enzyme-substrate-inhibitor complex that catalyses substrate oxidation at a slower rate than the binary enzyme-substrate complex. Competitive inhibition reflects the likelihood that the ternary complex does not form at all, presumably due to reduced accessibility of the active site to substrate. A good correlation was found between the Ki values for the inhibition of aminopyrine N-demethylase activity (21 or 23 microM) and the modulation of aminopyrine binding (26 microM) by primaquine. These findings suggest that the inhibition of aminopyrine N-demethylase activity by primaquine is mediated via an interaction with the oxidised form of cytochrome P-450. In addition, the apparent change in inhibition kinetics suggests a concentration-dependent change in the capacity of primaquine to modulate substrate binding to cytochrome P-450 as well as the formation of a P-450-aminopyrine-primaquine ternary complex.

Aminopyrine N-Demethylase↗
PubMed1986

Isometamidium chloride prophylaxis against Trypanosoma congolense challenge and the development of immune responses in Boran cattle.

Twenty-four Boran cattle were injected with isometamidium chloride (1 mg/kg bodyweight) to investigate the duration of drug-induced prophylaxis against infection by metacyclic forms of Trypanosoma congolense and to determine if specific antibody responses to the organism were mounted by animals under chemoprophylactic cover. Complete protection against either single challenge by five tsetse flies infected with T congolense, or repeated challenge at monthly intervals by five tsetse flies, lasted for five months. Six months after treatment, two-thirds of the cattle were resistant to challenge, irrespective of whether subjected to single or multiple challenge with trypanosome-infected tsetse flies, or titrated doses of in vitro-cultured metacyclic forms of T congolense (5 X 10(2) to 5 X 10(5) organisms), inoculated intradermally. No animal which resisted infection developed detectable skin reactions at the site of deposition of metacyclic trypanosomes or produced trypanosome-specific antibodies. It was concluded that drug residues effectively limited trypanosome multiplication at the site of deposition in the skin, thus preventing subsequent parasitaemia or priming of the host's immune response.

Animals↗
PubMed1986

New heterocyclic modifiers of oxidative drug metabolism--I. 6-Substituted-2-aminobenzothiazoles.

A series of 6-substituted-2-aminobenzothiazoles(2-AB) was synthesized and evaluated as in vitro inhibitors of microsomal mixed-function oxidase activity (as aminopyrine N-demethylase) from phenobarbitone-induced rat liver. Using physiochemical parameters and multiple regression analysis a quantitative structure-activity relationship (QSAR) was derived in which 82% of the data variance was accounted for in terms of the hydrophobic character of the inhibitor and the molar refractivity of the 2-AB 6-substituent. In contrast, literature equations derived from earlier studies with heterocyclic systems possessing non-polar substitutents underestimated by up to an order of magnitude the potency of the present compounds. Kinetic studies revealed that 6-n-propoxy-2-AB, one of the more potent compounds, was a pure competitive inhibitor of aminopyrine N-demethylase activity (Ki = 60 microM from Dixon analysis), suggesting that the binding of substrate and inhibitor is mutually exclusive at the cytochrome P-450 active site. Binding studies indicated that most 2-AB derivatives elicited mixed type I/reverse type I optical difference spectra in phenobarbitone-induced microsomes. The overlap of these components resulted in non-linear double reciprocal plots of the spectral titrations and precluded the determination of binding parameters. In contrast, the more potent inhibitors (the 6-propoxy and 6-butoxy derivatives of 2-AB) were type I ligands with quite high affinity for ferric cytochrome P-450. Although no quantitative relationship was apparent between inhibition and spectral binding affinity a good correlation (r = 0.93) was observed between inhibition potency (I50) and the capacity of ten 2-AB derivatives to prevent substrate(aminopyrine) binding to cytochrome P-450. These findings suggest that 2-AB derivatives may inhibit microsomal oxidation via a direct competitive effect on substrate binding to cytochrome P-450. The present study also demonstrates that substitution of heterocyclic systems with hydrophilic groups does not necessarily produce weak inhibitors of mixed-function oxidase activity, and that extrapolation of existing QSAR equations to new inhibitor series must be interpreted with caution.

Aminopyrine N-Demethylase↗
PubMed1986

Drug metabolism in cirrhosis. Selective changes in cytochrome P-450 isozymes in the choline-deficient rat model.

The effect of a choline-deficient diet on microsomal cytochrome P-450 and mixed-function oxidase (MFO) activity was investigated in relation to the development of nutritional cirrhosis. In rats that received the choline-deficient diet for 28 weeks cirrhosis was evident macroscopically and histologically; control rats that received an identical diet supplemented with choline had normal livers. Microsomal cytochrome P-450 and cytochrome b5 were reduced in cirrhotic liver to 50% of control levels. Three MFO activities (ethylmorphine N-demethylase, aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase) were also reduced to 40-70% of control levels. However, the turnover number for the O-deethylation of 7-ethoxycoumarin was not reduced in cirrhotic liver. This finding suggested that certain drug oxidations may be selectively depressed in nutritional cirrhosis. To examine the possibility that selective changes in MFO activity may reflect the suppression of certain cytochrome P-450 isozymes, partially purified fractions of the cytochrome were prepared after solubilisation and hydrophobic affinity chromatography (on n-octylamino-Sepharose 4B) of cirrhotic and control liver microsomes. Analysis of these fractions by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and laser densitometry indicated that a protein band of apparent minimum molecular weight 50.5 kD was primarily affected in cirrhotic rat liver microsomes. Levels of two other bands (apparent minimum molecular weight 48 and 52.5 kD) appeared essentially unaltered. Additional electrophoretic studies, conducted under non-reduced conditions, indicated the haemoprotein nature of protein bands in the 48-55 kD region. These data strongly suggest that cirrhosis produced in rats by a choline-deficient diet is associated with selective decreases in oxidative drug metabolism and individual cytochrome P-450 isozymes.

Animals↗
PubMed1986

Successful transfer of frozen N'Dama embryos from the Gambia to Kenya.

Frozen embryos from N'Dama cattle were successfully transferred from The Gambia to Kenya. Of the 26 N'Dama cows used 12 were successfully programmed to superovulate and of these seven produced 30 embryos that were collected seven days after oestrus/service. Five N'Dama bulls were used for natural service. In Kenya 29 embryos were implanted into 29 Boran heifers seven days (+/- 1) after the induction of synchronised oestrus. Eleven pregnancies were established and after one abortion of unknown aetiology at seven and a half months five female and five male calves were born and subsequently reared. During programming the N'Dama cows showed prolonged anoestrus leading to the necessity of oestrus induction using intravaginal progesterone releasing coils; pregnant mare serum gonadotrophin gave better superovulation than follicle stimulating hormone. One N'Dama bull proved to be subfertile. The success of the project has demonstrated the potential of this technique to make disease-free N'Dama available for research purposes and for the promotion of livestock development programmes in tsetse-infested areas using trypanotolerant cattle.

Animals↗
PubMed1986

Reactive synaptogenesis in the CNS. A comparison of regenerating and sprouting systems.

Lesion-induced synaptogenesis was compared in the goldfish retinotectal system, which readily regenerates after optic nerve crush, and in the cat spinal cord, in which collateral sprouting has been demonstrated after dorsal rhizotomy. Quantitative electron microscopic methods were used. Reinnervation of the tectum was complete, but was characterized by a prolonged time course. Reinnervation appeared to be achieved by retinal axons and not by sprouting from nonretinal axons. Reinnervation in the cat spinal cord was also virtually complete, but was very rapid and may be mediated by some axons that are similar to those destroyed and by other axons that are different.

Animals↗
PubMed1986

Selective reactivation of steroid hydroxylases following dissociation of the isosafrole metabolite complex with rat hepatic cytochrome P-450.

In order to elucidate the isozyme specificity of complex formation between cytochrome P-450 and the isosafrole metabolite the effect of complex dissociation on different steroid hydroxylation pathways was studied in hepatic microsomal fractions. Isosafrole induction was found to increase the 16 beta- and 7 alpha-hydroxylation of androst-4-ene-3,17-dione approximately 2.8- and 1.7-fold, respectively, whereas the 16 alpha-hydroxylation pathway was decreased to about one-quarter of control activity; 6 beta-hydroxylation was unchanged from control activity. More striking changes were apparent following dissociation of the isosafrole metabolite from its complex with ferricytochrome P-450 by the steroid substrate. Thus an approximate fourfold elevation of 16 beta-hydroxylase activity was observed after displacement and 6 beta-hydroxylation increased about twofold; 7 alpha-hydroxylase activity was decreased to 0.75-fold of undisplaced activity and 16 alpha-hydroxylase activity was unchanged. These data provide convincing evidence that at least two forms of phenobarbital-inducible cytochrome P-450 (cytochromes P-450PB-B and P-450PB/PCN-E) are present to some extent in a catalytically inactive complexed state in isosafrole-induced rat hepatic microsomes. Furthermore, there is now evidence to suggest that the constitutive isozymes cytochrome P-450UT-A and cytochrome P-450UT-F are not complexed to any degree in hepatic microsomes from isosafrole-induced rats.

Androstenedione↗