Protein complexes from mouse and chick brain that interact with phospho-KXGS motif tau/microtubule associated protein antibody.

Mouse monoclonal 12E8 antibody, which recognises conserved serine phosphorylated KXGS motifs in the microtubule binding domains of tau/tau-like microtubule associated proteins (MAPs), shows elevated binding in brain during normal embryonic development (mammals and birds) and at the early stages of human Alzheimer's disease (AD). It also labels ADF/cofilin-actin rods that form in neurites during exposure to stressors. We aimed to identify direct and indirect 12E8 binding proteins in postnatal mouse brain and embryonic chick brain by immunoprecipitation (IP), mass spectrometry and immunofluorescence. Tau and/or MAP2 were major direct 12E8-binding proteins detected in all IPs, and actin and/or tubulin were co-immunoprecipitated in most samples. Additional proteins were different in mouse versus chick brain IP. In mouse brain IPs, FSD1l and intermediate filament proteins - vimentin, α-internexin, neurofilament polypeptides - were prominent. Immunofluorescence and immunoblot using recombinant intermediate filament subunits, suggests an indirect interaction of these proteins with the 12E8 antibody. In chick brain IPs, subunits of eukaryotic translation initiation factor 3 (EIF3) were found, but no direct interaction between 12E8 and recombinant Eif3e protein was detected. Fluorescence microscopy in primary cultured chick neurons showed evidence of co-localisation of Eif3e and tubulin labelling, consistent with previous data demonstrating cytoskeletal organisation of the translation apparatus. Neither total tau or MAP2 immunolabelling accumulated at ADF/cofilin-actin rods generated in primary cultured chick neurons, and we were unable to narrow down the major antigen recognised by 12E8 antibody on ADF/cofilin-actin rods.

Intra- and interindividual variability in systemic exposure in humans to 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and 2-amino-1-methyl- 6-phenylimidazo[4,5-b]pyridine, carcinogens present in cooked beef.

During the cooking of beef, the genotoxic heterocyclic aromatic amines 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are formed. Little is known about the fate of these compounds in humans or the factors affecting it. We have developed assays based on capillary column gas chromatography-negative ion mass spectrometry capable of the simultaneous measurement of MeIQx, DiMeIQx, and PhIP in cooked meat and in human urine using stable isotope labeled analogues. Ten normal, healthy male volunteers were invited to consume a standard cooked meat meal (400-450 g lean beef, cooked as patties on a griddle hotplate) on four separate occasions over a period of 14 months. Following consumption of the test meals, urine was collected from 0 to 8 h, during which time all free amines were excreted and analyzed for MeIQx, DiMeIQx, and PhIP. Subjects ingested 240 +/- 9 (SEM) g cooked meat, which contained 2.2 +/- 0.2 ng MeIQx/g meat, 0.7 +/- 0.1 ng DiMeIQx/g meat, and 16.4 +/- 2.1 ng PhIP/g meat. The variability in relative systemic bioavailability was assessed from the percentage of ingested amine excreted unchanged in the urine. Subjects excreted 2.1 +/- 1.1% of MeIQx and 1.1 +/- 0.5% of PhIP ingested as unchanged amine in the urine. Levels of DiMeIQx in urine, if present, were below the sensitivity of our assay (20 pg/ml) and could not be detected in any of the samples analyzed. Irrespective of dose, urinary excretion of unchanged MeIQx or PhIP (expressed as a percentage of the ingested dose) remained constant for each individual subject. The intraindividual coefficients of variation for MeIQx (28.4%) and PhIP (23.7%) were low and the pooled interday (intrasubject) coefficients of variation for both compounds were only 19 and 3.4%, respectively. In contrast, inter-subject (intraday) variation was greater, with pooled coefficients of variation of 145% for MeIQx and 71% for PhIP. Based on these studies, it should be possible to use the percentage excretion of MeIQx and PhIP to assess the relative bioavailability of these compounds in humans.

Mutagenesis of mouse intestine in vivo using the Dlb-1 specific locus test: studies with 1,2-dimethylhydrazine, dimethylnitrosamine, and the dietary mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline.

The ability of three model carcinogens, 1,2-dimethylhydrazine, dimethylnitrosamine, and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, to induce mutation in a novel in vivo assay in mouse intestine has been examined. The assay is based on mutations at the Dlb-1 locus which determines the tissue specific pattern of expressio of the binding site for the lectin Dolichos biflorus agglutinin. In C57BL/6J x SWR F1 mice Dlb-1 mutants are recognized as clones of epithelial cells not staining with a peroxidase conjugate of D. biflorus agglutinin. Chronic administration of 1,2-dimethylhydrazine (20 mg/kg/week s.c. for 10 weeks) induced Dlb-1 mutants, whereas administration of a single dose did not. Similarly, chronic dimethylnitrosamine treatment p.o. (0.001% in drinking water for 8 weeks) induced Dlb-1 mutants, but acute administration did not. In contrast, neither chronic nor acute treatment of the mice with 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline induced Dlb-1 mutations. The activities of 1,2-dimethylhydrazine, dimethylnitrosamine, and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in the Dlb-1 assay more accurately reflect their carcinogenic potential than do many in vitro bioassays.

Relationship between oxidative metabolism of 2-acetylaminofluorene, debrisoquine, bufuralol, and aldrin in human liver microsomes.

The capacity of human liver microsomes from 28 individuals to metabolize debrisoquine and bufuralol, two drugs oxidized polymorphically in humans, as well as the carcinogen 2-acetylaminofluorene (AAF), was determined. In addition, the cytochrome P-450 content and the capacity of these microsomes to carry out the epoxidation of aldrin were measured. Interindividual differences in debrisoquine 4-hydroxylation, bufuralol 1-hydroxylation, and aldrin epoxidation were 12-, 20-, and 2.4-fold, respectively. The metabolism of debrisoquine was not correlated with cytochrome P-450 content (r = 0.26), whereas both the metabolism of bufuralol (r = 0.45; r2 = 0.20) and the epoxidation of aldrin (r = 0.72; r2 = 0.52) were correlated. Rates of debrisoquine and bufuralol metabolism were significantly correlated (r = 0.73), whereas only weak correlations existed between debrisoquine:aldrin (r = 0.49) and bufuralol:aldrin (r = 0.51). Because biphasic kinetics have been observed in human liver microsomes for the 7- and 5-hydroxylation of AAF, two concentrations of this substrate were used. The disappearance of AAF at either 0.37 or 50 microM was not correlated with debrisoquine, bufuralol, or aldrin metabolism. Similarly, at 0.37 microM AAF, no correlation existed between the formation of N-, 1-, 3-, 5-, 7-, and 9-hydroxylation products of AAF and debrisoquine, bufuralol, or aldrin metabolism. At 50 microM AAF, only the 7-hydroxylation of this substrate correlated with bufuralol metabolism (r = 0.47). This lack of, or weak correlation between pathways leading to metabolic activation (N-hydroxylation) or detoxication (C-hydroxylation) of the carcinogen AAF and debrisoquine, bufuralol, and aldrin metabolism strongly suggests that different forms of cytochrome P-450 are involved in these pathways. In contrast, exceptionally high correlations (r greater than 0.94) existed between N-OH-AAF:1-OH-AAF. N-OH-AAF:7-OH-AAF, and 7-OH-AAF:1-OH-AAF at the low concentration of AAF, and imply that similar forms of cytochrome P-450 produce these metabolites. However, at 50 microM AAF, these correlations are considerably weaker and explain less than 35% of the variance in the data. It is concluded, based on these multiple cross-correlations, that common cytochrome P-450 isoenzymes are involved in the formation of AAF metabolites, while the metabolism of debrisoquine, bufuralol, and aldrin is unrelated to the metabolism of this carcinogen in human liver microsomes.

CYP1A2-catalyzed conversion of dietary heterocyclic amines to their proximate carcinogens is their major route of metabolism in humans.

The contribution of CYP1A2 to the metabolism of the dietary heterocyclic amines, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in vivo in humans, has been determined with furafylline, a highly selective inhibitor of this enzyme. The inhibitory potential of furafylline in vivo was first assessed by determining its effect on clearance of phenacetin to paracetamol by the model CYP1A2-dependent O-deethylation pathway. Furafylline inhibited this reaction by > 99% in all subjects, thus demonstrating its applicability to determining the contribution of CYP1A2 to a given reaction in vivo. A group of 6 healthy male volunteers received either placebo or 125 mg furafylline, in a double-blind balanced crossover design, 2 h prior to consuming a test meal of fried beef containing a known amount of amines. The excretion of PhIP and MeIQx in urine was determined during the subsequent 28 h, using gas chromatography-mass spectrometry. Following furafylline, the excretion of unchanged MeIQx increased 14.3-fold, while that of PhIP increased 4.1-fold (P < 0.01, paired t test). Elimination of both amines was first order and very rapid, with half-lives of < 5 h. The elimination rate constants did not change following furafylline, suggesting that total clearance is limited by hepatic blood flow. Because the elimination of the amines was first order, it was possible to calculate the contribution of CYP1A2 to the clearance of the amines. CYP1A2-catalyzed metabolism accounts for 91% of the elimination of ingested MeIQx and 70% of ingested PhIP, most likely via N-hydroxylation.

Inducibility of cytochromes P-450 by dioxin in liver and extrahepatic tissues of the marmoset monkey (Callithrix jacchus).

Cytochrome P-450 induction was investigated in the marmoset monkey, a non-human primate, using dioxins as inducing agents. Animals received a single subcutaneous dose of 1.6 nmol tetrachlorodibenzo-p-dioxin or tetrabromodibenzo-p-dioxin/kg body weight. Microsomal fractions were prepared from liver, lung and kidney, and homogenates were prepared from gut and adrenal glands. Anti-peptide antibodies which bind to CYP1A1, CYP1A2, CYP2B6 and CYP3A4 in human were used to identify related forms in the marmoset. The results indicate that CYP1A2 is constitutively expressed in liver, but not in lung, kidney, gut or adrenal gland and that CYP1A1 is not expressed in any of these tissues in untreated animals. Treatment with dioxin induced both CYP1A1 and CYP1A2 in liver, but only CYP1A1 in lung. No induction of CYP1A1 or CYP1A2 was found in kidney, small intestine or adrenal glands. Methoxy-, ethoxy-, pentoxy- and benzoyloxyresorufin O-dealkylases and high affinity phenacetin O-deethylase activities were induced in the liver, whereas ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase activities were not affected by dioxin treatment. High-affinity phenacetin O-deethylase and CYP1A2 apoprotein were detected only in liver, consistent with this activity being specifically catalysed by CYP1A2. Furafylline was found to be a competitive inhibitor of methoxyresorufin O-demethylase activity with a Ki of 10 microM. In the lung the induction of CYP1A1 was accompanied by 15- and 23-fold increases in ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase activities, respectively, suggesting that both activities are catalysed by CYP1A1. In contrast, there was no induction of aryl hydrocarbon hydroxylase activity in lung or liver showing that, unlike in many other species, marmoset CYP1A1 does not catalyse this reaction efficiently. The expression, distribution, induction and substrate specificities of marmoset monkey P-450 enzymes differ from the situation found in rodents and other species, demonstrating that caution has to be exercised when making cross-species extrapolations.

Identification of CYP2E1 in marmoset monkey.

CYP2E1 is the main enzyme responsible for chlorzoxazone 6-hydroxylase activity in human liver. Here, it is shown that marmoset monkey liver microsomal fraction catalyses this reaction at a similar rate and with a similar Km to human liver and that the activity is increased 4-fold in marmosets treated with isoniazid, a known inducer of CYP2E1. This indicates that CYP2E1 is present in marmoset liver. However conversely, an anti-peptide antibody targeted against the C-terminus of human and cynomolgus monkey CYP2E1 (Val-Ile-Pro-Arg-Ser) failed to bind to marmoset monkey hepatic microsomal fraction. To investigate if there is a difference in the C-terminus of CYP2E1 in these species, this region of marmoset CYP2E1 was sequenced following amplification of marmoset liver cDNA with primers selected according to conserved regions identified in human and cynomolgus monkey CYP2E1. It was found that the deduced amino acid sequence of marmoset CYP2E1 in this region is very similar to human CYP2E1, but due to two base differences in the marmoset nucleic acid sequence, the C-terminus of marmoset CYP2E1 is extended by 2 amino acids, i.e. Val-Ile-Pro-Arg-Ser-Ser-Val. This difference is sufficient to prevent the binding of an antibody raised against the C-terminus of human CYP2E1. The expression of CYP2E1 in the marmoset was confirmed by raising an antibody against the deduced C-terminus of marmoset CYP2E1 (Pro-Arg-Ser-Ser-Val). In immunoblotting, this antibody bound to a single protein of 54 kDa in marmoset liver microsomal fraction. The intensity of the band was increased in isoniazid-treated marmosets, consistent with induction of CYP2E1. The antibody did not recognise human or cynomolgus monkey CYP2E1. This was expected since the immunising peptide sequence does not occur in these enzymes. The results demonstrate the presence of CYP2E1 in marmoset liver and illustrate the importance of the C-terminus for the production of specific antibodies against P450 enzymes.

An inhibitory monoclonal anti-protein antibody and an anti-peptide antibody share an epitope on rat cytochrome P-450 enzymes CYP1A1 and CYP1A2.

A monoclonal antibody, 12/2/3/2, which was raised against purified rat CYP1A1 recognises specifically rat and mouse CYP1A1 and CYP1A2, but not any cytochrome P-450 present in hepatic microsomal fractions from rabbit, guinea pig, hamster or human. By comparing the primary sequences of cytochromes P-450 to which 12/2/3/2 does and does not bind, 10 possible locations for its epitope were found. Of these, one was extremely hydrophilic and, hence, predicted to be the most antigenic in the native protein. An antibody was produced against the synthetic peptide corresponding to this region (Gly-Arg-Asp-Arg-Gln-Pro-Arg-Leu: residues 356-363 and 350-357 of rat CYP1A1 and CYP1A2, respectively). The antibody bound to rat, mouse and hamster CYP1A1 and to rat and mouse CYP1A2, but did not bind to any protein present in hepatic microsomal fractions from the rabbit, guinea pig or human. The binding of the anti-peptide antibody to CYP1A1 or CYP1A2 was partially antagonised by the monoclonal antibody. However, whereas the monoclonal antibody inhibited both CYP1A1- (aryl hydrocarbon hydroxylase) and CYP1A2-(high-affinity phenacetin O-deethylase) dependent monooxygenase activity, the anti-peptide antibody was without effect on these activities. Antigen denaturation by 8 M urea or 0.05% (w/v) SDS had no effect on binding of the anti-peptide antibody to cytochrome P-450, whilst binding of the monoclonal antibody was reduced by more than 1000-fold. The anti-peptide antibody partially antagonised the binding of 12/2/3/2 to urea-denatured but not native cytochrome P-450. These data suggest that whilst the complete binding site for the monoclonal antibody is discontinuous, sufficient of the epitope is linear, so that when the antigen is denatured the monoclonal antibody is still able to bind and this binding is antagonised by the anti-peptide antibody. However, inhibition of catalytic activity by the monoclonal antibody must require binding to discontinuous residues.

Mechanisms of cell death.

There is increasing evidence that the mechanisms of chemically mediated cell death are common to a wide variety of cell types and to a large number of toxic compounds. The perturbation of Ca2+ homeostasis appears to be particularly important and may be due to modification of SH-groups in key enzymes. Donald Davies and colleagues discuss the mechanisms by which early events induced by exposure to toxic chemicals may lead to these changes, and their possible consequences. It is now clear that reduced glutathione plays a pivotal role, not only in detoxifying reactive compounds but also in reversing the early biochemical changes in the cell.

Reduction of the viral load of HIV-1 after the intraperitoneal administration of dextrin 2-sulphate in patients with AIDS.

OBJECTIVE: To determine the safety and efficacy of the sulphated polysaccharide, dextrin 2-sulphate, when delivered to the lymphatic circulation by the peritoneal route. DESIGN: An open Phase I/II dose-escalation clinical study in which six patients with AIDS were treated with seven courses of dextrin 2-sulphate each lasting 1 month. METHODS: During each course of treatment, the drug was administered daily for 28 days using an intraperitoneal catheter. Viral load was measured at frequent intervals using a plasma tissue culture infectious dose (TCID) assay, a cellular TCID assay, p24 antigenaemia, HIV-1 RNA and HIV-1 DNA. Plasma beta-chemokine levels were also measured. RESULTS: Dose escalation was completed without toxicity. A total of 7 patient-months of treatment were completed. With increasing doses of dextrin 2-sulphate, the infectious plasma viraemia, cellular viraemia and p24 antigenaemia all fell during the period of drug administration, but with no significant change in HIV-1 RNA. This was associated with increased plasma levels of macrophage inflammatory protein (MIP)-1alpha and MIP-1beta. Dextrin 2-sulphate accumulated in peritoneal macrophages and induced the release of MIP-1alpha and MIP-1beta from these cells in vitro. These beta-chemokines could have augmented the cell surface-mediated anti-HIV-1 effect of dextrin 2-sulphate in vivo by binding to and blocking the CC-chemokine receptor-5. A second fall in infectious plasma viraemia, cellular viraemia, p24 antigenaemia and HIV-1 RNA was seen at day 100 which was then sustained for several months. A clinical improvement in Kaposi's sarcoma was also seen. CONCLUSIONS: Our results suggest that the intraperitoneal administration of dextrin 2-sulphate can reduce the replication of HIV-1 in patients with AIDS. With increasing doses of dextrin 2-sulphate, the fall in viral load was seen during the period of drug administration and again 2 months after completing treatment.

Oral absorption and concentration-effect relationship of tyramine with and without cimoxatone, a type-A specific inhibitor of monoamine oxidase.

The mechanism of increased sensitivity to oral tyramine in patients taking monoamine oxidase inhibitors was investigated by measurement of plasma norepinephrine and tyramine with a reversible, monoamine oxidase form A selective inhibitor, cimoxatone. In the first open study, the pressor activity of 80 mg oral tyramine after cimoxatone was of the order of that of 800 mg without the drug. In a second double-blind study, the equivalent tyramine dose was between 400 and 800 mg. Absorption of unmetabolized tyramine increased in the presence of cimoxatone. Peak plasma concentration of tyramine after an 80-mg dose was approximately three times that of placebo when given after cimoxatone. The plasma tyramine concentration required to induce a similar pressor effect averaged 130.5 ng ml-1 without pretreatment and 17.4 ng ml-1 after cimoxatone in the open study. Another plasma concentration, 16.4 ng ml-1, elicited a lower pressor effect in the double-blind study. The increased sensitivity to oral tyramine has two components: decreased presystemic clearance in the gut wall and increased sensitivity to circulating tyramine. The tyramine dose required to induce a pressor effect after cimoxatone is relatively higher than after irreversible inhibitors such as phenelzine. This may reflect the dose of cimoxatone used, the activity of monoamine oxidase form B (MAO-B) in the gut and liver or displacement of the predominantly reversible and predominantly competitive inhibitor, cimoxatone, from the enzyme by a high local tyramine concentration.

Clinical pharmacology and pharmacokinetics of clonidine.

A 300-mug oral dose of clonidine was administered to 5 normal volunteers and measurements of plasma concentration and effects upon blood pressure, heart rate, circulatory reflexes, sedation, and dry mouth were made for the following 8 hr. The plasma concentration rose to a peak of 1.02 +/- 0.52 ng/ml (SD) at 90 min and fell with a mean half-life of 12.7 hr. Blood pressure of the group fell from 111.0/77.0 to 87.2/60.4 after 3 hr and was 95.2/62.2 mm Hg at 8 hr. Heart rate in recumbency was slowed. Marked sedation and a fall in salivary flow followed the same time-course as the plasma concentration. The cold pressor response was reduced but the Valsalva overshoot was little affected.

Pharmacokinetics and concentration-effect relationships of intervenous and oral clonidine.

The kinetics of the disposition of intravenous and oral clonidine in five normotensive subjects have been determined. It is proposed that a two-compartment model adequately describes the disposition of the drug. The drug is rapidly distributed (t1/2alpha = 10.8 +/- 4.7 min) but slowly elimainated (t1/2beta = 8.5 +/- 0.9 hr). The bioavailability of oral clonidine in the tablets tested averaged 75.2% and 40 to 50% of the bioavailable dose is excreted unchanged in urine. Renal clearance of the drug showed considerable intersubject variation (1.82 +/- 0.34 ml/min/kg) and exceed the calculated glomerular filtration rate in some subjects. Oral and intravenous clonidine induced significant falls in blood pressure (greater than 20/15 mm Hg) in our normotensive subjects and consistently caused marked sedation and dryness of the mouth. Sedation and salivary flow correlated with plasma clonidine concentration over the range 0 to 4 ng/ml. Falls in blood pressure were related to plasma concentration to 1.5 to 2 ng/ml but at higher concentrations the hypotensive effect was attenuated.

Interindividual variation in kinetics of infused epinephrine.

1-Norepinephrine was infused continuously for 10 hr into 5 normotensive, male laboratory subjects (mean age, 32.4 +/- 1.9 yr) at a mean rate of 0.06 microgram/kg/min. Mean plasma norepinephrine (NE) rose from the preinfusion level of 0.19 +/- 0.02 microgram/l to a steady state level of 1.22 +/- 0.29 microgram/l. The mean increase in blood pressure was 21.8 +/- 0.9 mm Hg systolic and 14.1 +/- 1.0 mm Hg diastolic. The mean depression in heart rate was 12.7 +/- 1.7 beats/min. The clearance of norepinephrine ranged from 27.9 to 100.0 ml/kg/min (mean. 58.0 +/- 13.8) and was little influenced by acute hemodynamic changes. The volume of distribution ranged widely (0.09 to 0.40 l/kg), the mean value being 13.51 1. The mean norepinephrine half-life was brief, ranging from 1.45 to 2.9 min (mean, 2.09 +/- 0.34 min). There was no evidence of a slowly accumulating high-capacity low-affinity pool of norepinephrine. These results support the use of plasma norepinephrine as an index of sympathetic activity within an individual but not its validity in interindividual comparisons.

Maternal and neonatal elimination of amobarbital after treatment of the mother with barbiturates during late pregnancy.

Plasma half-lives of amobarbital were determined in newborn children of 10 mothers who had been treated with barbiturates for hypertension in pregnancy for 6 to 42 days prior to delivery. Five mothers had received amobarbital, 200 mg daily, and 5, phenobarbital, 60 to 180 mg daily. Half-lives in 7 of the babies ranged from 16.6 to 49.4 hr, comparable to those previously reported in babies of mothers who had received only a single dose of amobarbital. Thus there was no evidence of induction of amobarbital hydroxylation in these children. Two babies who had a greater than normal rise in serum bilirubin had longer half-lives (86.1 and 117.7 hr). In 1 baby whose mother had membranous glomerulonephritis, plasma amobarbital concentration did not significantly change over the period of the study.

The potential of acetaminophen as a prodrug in gene-directed enzyme prodrug therapy.

Acetaminophen is oxidized by human CYP1A2 to the cytotoxic metabolite N-acetylbenzoquinoneimine (NABQI). Incubation of cells transfected with human CYP1A2 (H1A2 MZ cells) with 4-20 mM acetaminophen for 6 hours at 37 degrees C caused extensive cytotoxicity (cell viability <10%). In contrast, nontransfected V79 MZ cells were unaffected (viability >95%). By mixing H1A2 MZ cells with V79 MZ cells in various proportions and incubating with 4 mM acetaminophen, it was shown that the NABQI released from H1A2 MZ cells also caused cytotoxicity of bystander cells. Thus, in a mixture containing 5% H1A2 MZ cells, exposure to 4 mM acetaminophen for 6 hours resulted in complete cell killing by 24 hours. A similar bystander effect was found by incubating the same proportion of CYP1A2-containing cells with ovarian tumor-derived SK-OV-3 cells or colon tumor-derived HCT116 cells. However, breast tumor-derived MDA-MB-361 cells displayed resistance to the cytotoxic effect of NABQI, and it was necessary to increase the proportion of H1A2 MZ cells to 50% to achieve complete cell killing. In conclusion, the use of acetaminophen as prodrug and CYP1A2 as an activating enzyme is a promising combination for gene-directed enzyme prodrug therapy.

Cross-reaction of antibodies to coupling groups used in the production of anti-peptide antibodies.

A polyclonal antibody raised against a peptide conjugated using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride to maleic anhydride-derivatised lysozyme showed substantial cross-reactivity with m-maleimidobenzoyl-N-hydroxysuccinimide ester-derivatised haemocyanin. This was due to antibodies produced against maleic anhydride-derivatised groups on lysozyme that reacted with m-maleimidobenzoyl-N-hydroxysuccinimide ester-derivatised groups on haemocyanin. This observation is important because it is common practice, in the production of anti-peptide antibodies, to use two conjugates. The same peptide is coupled to two different protein carriers by two different coupling methods. One conjugate is used for immunisation and the other for testing the serum. This method assumes that the only antigen common to the two conjugates is the peptide and this was not the case here. A method is described for screening sera which involves affinity purification of the anti-peptide antibody and comparison of binding to the immunogen with that to an appropriate control conjugate. This method avoids the problem of any cross-reaction to coupling groups or proteins.

Ontogenetic development of the distribution of constitutive and 3-methylcholanthrene-induced CYP1A1 and CYP1A2 in rabbit liver.

We investigated the expression, distribution, and inducibility of 3-methylcholanthrene (MC)-inducible P450 enzymes, CYP1A1 and 1A2, in livers of rabbits at different stages of development, ranging from 4 days before birth (-4 days of age) to adulthood. These enzymes were identified by immunoblotting and immunocytochemistry and quantified by dot-blotting, utilizing previously characterized monoclonal antibodies, 107 and 3/4/2, specific for CYP1A2 and both CYP1A1 and 1A2, respectively, and a polyclonal antibody that recognizes both enzymes. Expression of CYP1A2 is always greater than that of CYP1A1 in livers of untreated rabbits, regardless of age. Moreover, immunocytochemistry showed that CYP1A1 is evenly distributed throughout the liver at all ages, whereas CYP1A2 is highly localized to only a few scattered cells at 1 day before birth. More hepatocytes express this enzyme perinatally. By 6 days of age, expression of CYP1A2 is confined to a narrow band of centrilobular cells, but with increasing age the enzyme is expressed in more hepatocytes until weaning, when all hepatocytes are positive. Although CYP1A1 is induced by MC treatment at most ages, there is no change in its distribution. In contrast, induction of CYP1A2 was shown immunocytochemically to occur in only a limited number of hepatocytes in fetal rabbits. There is a progressive increase with age in the number of hepatocytes that are inducible for CYP1A2. The greatest fold-induction of hepatic CYP1A2 by MC in the rabbit is a 9-11 days of age, when, for MC-treated rabbits, CYP1A2 represents > 60% of the total P450 pool. The modulation of enzyme expression caused by MC treatment of fetuses/neonates leads to developmentally advanced livers with respect to P450 and could have a significant impact on the fetal and neonatal toxicity of some foreign compounds. These data demonstrate, for the first time, that the ontogenetic expression and localization of CYP1A1 and 1A2 within the liver are differentially regulated at the level of the individual cell.

Dose and concentration-effect relations for rilmenidine.

The dose-effect and concentration-effect relations of rilmenidine, a new alpha 2 agonist, were evaluated for the hemodynamic and side-effect responses to single oral doses. Two studies were performed in a double-blind manner: study I in 8 healthy subjects and study II in 10 hypertensive patients. In the course of five 24-hour periods, each separated by at least 1 week, the following 5 treatments were administered in a random order: placebo, rilmenidine (0.5, 1, 2 and 3 mg). Blood pressure was measured with a Roche arteriosonde (study I) or a sphygmomanometer (study II). Sedation was measured using a visual analog scale. Dry mouth was assessed by measuring the salivary flow (study I) or by visual analog scale (study II). Plasma concentration of rilmenidine was assayed by gas chromatography linked with mass spectrometry. The antihypertensive and sedative effects (area under curve) were directly related to the dose and to the log of the plasma concentration of rilmenidine. In contrast to the 2- and 3-mg doses, rilmenidine (0.5 and 1 mg) did not induce orthostatic hypotension, a significant decrease in heart rate or a significant dryness of mouth. At doses of 0.5 and 1 mg, the effects of rilmenidine on sedation were not consistent in both studies: sedation was significant in study I but did not differ from placebo in study II. These studies show that rilmenidine, in common with other alpha 2 agonists, decreases blood pressure in a dose-dependent manner, in normotensive as well as in hypertensive subjects.(ABSTRACT TRUNCATED AT 250 WORDS)