A pedicled palatal periosteal flap for the closure of oro-antral fistula.

Various surgical techniques have been developed for oro-antral fistula (OAF) closure, all of which have some drawback. Twenty consecutive patients with an OAF were enrolled in this prospective study. A trapezoid full-thickness flap extending from the palatal area to the buccal gingiva was raised, including the fistula at its centre. The palatal free end aspect was split into two layers and the deep periosteal layer was folded deep to the flap over the bony defect, thereby sealing the fistula. The superficial layer was returned to its primary position and sutured. The patients were followed for 3 months. Nineteen patients showed immediate OAF closure. One patient showed a residual oro-antral communication of 0.5mm in diameter that resolved spontaneously within 4 weeks. The pain level (on a visual analogue scale) was highest at the first follow-up week, with a mean score of 5.5, which decreased to a mean level of 2.5 in the second week and 0 in the fourth week. The mean satisfaction level was 9.85 on a scale of 0-10 (10 representing total satisfaction). The pedicled palatal periosteal flap is a simple and effective surgical technique with high predictability and patient satisfaction levels, offering one more alternative for the treatment of OAF.

Stimulation of angiotensin AT2 receptors by the non-peptide agonist, Compound 21, evokes vasodepressor effects in conscious spontaneously hypertensive rats.

BACKGROUND AND PURPOSE: Angiotensin type 2 receptor (AT(2) receptor) stimulation evokes vasodilator effects in vitro and in vivo that oppose the vasoconstrictor effects of angiotensin type 1 receptors (AT(1) receptors). Recently, a novel non-peptide AT(2) receptor agonist, Compound 21, was described, which exhibited high AT(2) receptor selectivity. EXPERIMENTAL APPROACH: Functional cardiovascular effects of the drug candidate Compound 21 were assessed, using mouse isolated aorta and rat mesenteric arteries in vitro and in conscious spontaneously hypertensive rats (SHR). KEY RESULTS: Compound 21 evoked dose-dependent vasorelaxations in aortic and mesenteric vessels, abolished by the AT(2) receptor antagonist, PD123319. In vivo, Compound 21 administered alone, at doses ranging from 50 to 1000 ng.kg(-1).min(-1) over 4 h did not decrease blood pressure in conscious normotensive Wistar-Kyoto rats or SHR. However, when given in combination with the AT(1) receptor antagonist, candesartan, Compound 21 (300 ng.kg(-1).min(-1)) lowered blood pressure in SHR only. Further analysis in separate groups of conscious SHR revealed that, at a sixfold lower dose, Compound 21 (50 ng.kg(-1).min(-1)) still evoked a significant depressor response in adult SHR ( approximately 30 mmHg) when combined with different doses of candesartan (0.01 or 0.1 mg.kg(-1)). Moreover, the Compound 21-evoked depressor effect was abolished when co-infused (50 microg.kg(-1).min(-1) for 2 h) with the AT(2) receptor antagonist PD123319. CONCLUSION AND IMPLICATIONS: Collectively, our results indicate that acute administration of Compound 21 evoked blood pressure reductions via AT(2) receptor stimulation. Thus Compound 21 can be considered an excellent drug candidate for further study of AT(2) receptor function in cardiovascular disease.

Analysis of protein induction in the CNS of SJL mice with experimental allergic encephalomyelitis by proteomic screening and immunohistochemistry.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease characterized by demyelination and inflammatory infiltrates in the CNS, and it is an animal model of multiple sclerosis. Piperonyl butoxide (PBO) suppresses disease in EAE mice, and it exhibits a dual effect on cytochrome P450s that manifests in a transient inhibitory phase followed by induction. In order to identify the expression of proteins associated with EAE, a proteomic screening was performed on hindbrain microsomes from control + vehicle, control + PBO, EAE + vehicle, and EAE + PBO female mice. Glucose regulated protein 94 (Grp94) and coagulation factor VIII were among the proteins identified in EAE + vehicle and EAE + PBO mice. Immunohistochemical staining of Grp94 was present in some neurons and oligodendrocytes in hindbrain sections from control animals, and in some cells within inflammatory infiltrates in EAE animals. Since Grp94 (also known as Gp96) can partake in antigen presentation and induction of proinflammatory cytokine expression, its presence in these cells suggests that it may play a role in the pathogenesis of EAE. Coagulation factor VIII is carried and protected by von Willebrand factor. Immunohistochemical staining of von Willebrand factor revealed its presence in some vessels within hindbrain sections from control animals. In EAE animals, the number of labeled vessels was significantly increased, and extracellular granular deposits were observed around labeled vessels indicating that the breakdown of the blood-brain barrier that occurs in EAE permitted its extravasation into the CNS. Additional proteins were identified in the different groups of mice by proteomic screening, but confirmation of their expression profile awaits investigations by independent measures.

Resistance profiles of cyclic and linear inhibitors of HIV-1 protease.

Resistance to anti-HIV protease drugs is a major problem in the design of AIDS drugs with long-term efficacy. To identify structural features associated with a certain resistance profile, the inhibitory properties of a series of symmetric and asymmetric cyclic sulfamide, cyclic urea and linear transition-state analogue inhibitors of HIV-1 protease were investigated using wild-type and mutant enzyme. To allow a detailed structure-inhibition analysis, enzyme with single, double, triple and quadruple combinations of G48V, V82A, 184V and L90M substitutions was used. Kinetic analysis of the mutants revealed that catalytic efficiency was 1-30% of that for the wild-type enzyme, a consequence of reduced kcat in all cases and an increased KM for all mutants except for the G48V enzyme. The overall structure-inhibitory profiles of the cyclic compounds were similar, and the inhibition of the V82A, 184V and G48V/L90M mutants were less efficient than of the wild-type enzyme. The greatest increase in Ki was generally observed for the 184V mutant and least for the G48V/L90M mutant, and additional combinations of mutations did not result in improved inhibition profiles for the cyclic compounds. An extended analysis of additional mutants, and including a set of linear compounds, showed that the profile was unique for each compound, and did not reveal any general structural features associated with a certain inhibition profile. The effects of structural modifications in the inhibitors, or of mutations, were not additive and they differed depending on their context. The results demonstrate the difficulties in predicting resistance, even for closely related compounds, and designing compounds with improved resistance profiles.

P1/P1' modified HIV protease inhibitors as tools in two new sensitive surface plasmon resonance biosensor screening assays.

The commonly used HIV-1 protease assays rely on measurements of the effect of inhibitions on the hydrolysis rate of synthetic peptides. Recently an assay based on surface plasmon resonance (SPR) was introduced. We have taken advantage of the fact that the SPR signal is proportional to the mass of the analyte interacting with the immobilised molecule and developed two new improved efficient competition assay methods. Thus, high molecular weight binders were used as amplifiers of the surface plasmon resonance signal. Linkers were attached by a Heck reaction to the para-positions of the P1/P1' benzyloxy groups of a linear C2-symmetric C-terminal duplicated inhibitor to enable (a) biotin labelling or (b) direct immobilisation of the inhibitor to the biosensor surface matrix. The interaction properties of a series of 17 structurally diverse inhibitors was assessed and compared to previously reported data. The most sensitive assay was obtained by immobilising the enzyme and amplifying the signal with an antibody, giving a detection range between 0.1 nM and 10 microM. Immobilisation of the inhibitor resulted in a stable and durable surface but a narrower detection range (1-100 nM). The two competition assays are anticipated to be very suitable for fast screening of potential HIV inhibitors.

Fast microwave-assisted preparation of aryl and vinyl nitriles and the corresponding tetrazoles from organo-halides

Aryl and vinyl nitriles have been prepared in very high yields from the corresponding bromides using palladium-catalyzed reactions with microwave irradiation employed as the energy source. Furthermore, flash heating was used successfully for the conversion of these nitriles into aryl and vinyl tetrazoles by cycloaddition reactions. One-pot transformation of aryl halides directly to the aryl tetrazoles could be accomplished both in solution and on solid support. All reactions were completed in minutes rather than in hours or days as previously reported with the standard thermal heating technique. A very potent HIV-1 protease inhibitor (K(i) = 0. 56 nM), comprising two tetrazole heterocycles as carboxyl group bioisosteres, was prepared in one pot by microwave-promoted cyanation of a bromo precursor and a subsequent cycloaddition reaction. The temperature-time profiles at 13, 20, and 60 W magnetron input power in DMF are presented.

Membrane fusion proteins are required for oskar mRNA localization in the Drosophila egg chamber.

We used a genetic screen in Drosophila to identify mutations which disrupt the localization of oskar mRNA during oogenesis. Based on the hypothesis that some cytoskeletal components which are required during the mitotic divisions will also be required for oskar mRNA localization during oogenesis, we designed the following genetic screen. We screened for P-element insertions in genes which slow down the blastoderm mitotic divisions. A secondary genetic screen was to generate female germ-line clones of these potential cell division cycle genes and to identify those which cause the mislocalization of oskar mRNA. We identified mutations in ter94 which disrupt the localization of oskar mRNA to the posterior pole of the oocyte. Ter94 is a member of the CDC48p/VCP subfamily of AAA proteins which are involved in homotypic fusion of the endoplasmic reticulum during mitosis. Consistent with the function of the yeast ortholog, ter94-mutant egg chambers are defective in the assembly of the endoplasmic reticulum. We tested whether other membrane biosynthesis genes are required for localizing oskar mRNA during oogenesis. We found that ovaries that are mutant for syntaxin-1a, rop, and synaptotagmin are also defective in oskar mRNA localization during oogenesis. We suggest a pathway for the role of membrane assembly proteins on oskar mRNA localization.

Design and fast synthesis of C-terminal duplicated potent C(2)-symmetric P1/P1'-modified HIV-1 protease inhibitors.

An analysis of the X-ray structure of a complex of HIV-1 protease with a linear C(2)-symmetric C-terminal duplicated inhibitor guided the selection of a series of diverse target compounds. These were synthesized with the objective to identify suitable P1/P1' substituents to provide inhibitors with improved antiviral activity. Groups with various physical properties were attached to the para-positions of the P1/P1' benzyloxy groups in the parent inhibitor. A p-bromobenzyloxy compound, prepared in only three steps from commercially available starting materials, was utilized as a common precursor in all reactions. The subsequent coupling reactions were completed within a few minutes and relied on palladium catalysis and flash heating with microwave irradiation. All of the compounds synthesized exhibited good inhibitory potency in the protease assay, with K(i) values ranging from 0.09 to 3.8 nM. A 30-fold improvement of the antiviral effect in cell culture, compared to the parent compound, was achieved with four of the inhibitors. The differences in K(i) values were not correlated to the differences in antiviral effect, efficiency against mutant virus, or reduced potency in the presence of human serum. The poorest enzyme inhibitors in fact belong to the group with the best antiviral effect. The binding features of two structurally related inhibitors, cocrystallized with HIV-1 protease, are discussed with special emphasis on the interaction at the enzyme/water phase.

A novel follicle-cell-dependent dominant female sterile allele, StarKojak, alters receptor tyrosine kinase signaling in Drosophila.

We describe a new dominant allele, StarKojak, that alters receptor tyrosine kinase signaling in the follicle cells and in the eyes in Drosophila. We isolated StarKojak in a screen for follicle-cell-dependent dominant female sterile mutations. We show that StarKojak and revertants of StarKojak do not complement Star loss-of-function mutations. We propose that StarKojak is a novel type of allele of Star that has both dominant gain-of-function phenotypes early in development and dominant loss-of-function phenotypes later in development. Star encodes a putative transmembrane protein that has previously been shown to be a critical component of the epidermal growth factor receptor tyrosine kinase signaling pathway. Early in oogenesis, Star mRNA expression is higher in StarKojak egg chambers than in wild-type egg chambers, consistent with its gain-of-function phenotype. Later in oogenesis, Star mRNA expression is lower in StarKojak follicle cells than in wild-type follicle cells, consistent with its loss-of-function phenotype. By genetically analyzing StarKojak and its revertants, we present evidence that Star is involved in anterior-posterior axis formation both in the female germline cells and in the somatic follicle cells. We also demonstrate that at least part of the dominant female sterile phenotype of StarKojak is restricted to the posterior-pole follicle cells. We propose that Star functions by processing pro-Gurken to mature Gurken, which is thereby released in the region between the oocyte and the follicle cells and binds to the epidermal growth factor receptor in the follicle cells.

Design and synthesis of new potent C2-symmetric HIV-1 protease inhibitors. Use of L-mannaric acid as a peptidomimetic scaffold.

A study on the use of derivatized carbohydrates as C2-symmetric HIV-1 protease inhibitors has been undertaken. L-Mannaric acid (6) was bis-O-benzylated at C-2 and C-5 and subsequently coupled with amino acids and amines to give C2-symmetric products based on C-terminal duplication. Potent HIV protease inhibitors, 28 Ki = 0.4 nM and 43 Ki = 0.2 nM, have been discovered, and two synthetic methodologies have been developed, one whereby these inhibitors can be prepared in just three chemical steps from commercially available materials. A remarkable increase in potency going from IC50 = 5000 nM (23) to IC50 = 15 nM (28) was observed upon exchanging -COOMe for -CONHMe in the inhibitor, resulting in the net addition of one hydrogen bond interaction between each of the two -NH- groups and the HIV protease backbone (Gly 48/148). The X-ray crystal structures of 43 and of 48 have been determined (Figures 5 and 6), revealing the binding mode of these inhibitors which will aid further design.

A Drosophila kinesin-like protein, Klp38B, functions during meiosis, mitosis, and segmentation.

We show that klp38B, isolated as a mutation that dominantly prolongs blastoderm mitotic cycles in Drosophila, encodes a Drosophila kinesin-like protein. Further genetic analyses show that Klp38B not only functions during mitosis, but is also required for meiosis and abdominal segmentation. Sequence comparisons suggest that Klp38B encodes an amino-terminal microtubule motor domain, a central alpha-helical coiled-coil domain, and a C-terminal globular domain. Evidence that Klp38B is required during meiosis is that flies transheterozygous for mutations in both klp38B and nod have a high frequency of 4th chromosome meiotic nondisjunction. Nod is a chromokinesin, a chromosome binding kinesin, that is believed to provide astral-exclusion forces during the metaphase stage of meiosis. Evidence that Klp38B is required during mitosis is that embryos from female germline clones of klp38B mutations have holes in the cuticle similar to a zygotic string (dCDC25) phenotype. Also, anti-Klp38B antibody injection into precellularization blastoderm embryos causes developmental arrest and the formation of circular mitotic figures. We speculate, based on these phenotypes, that Klp38B is a chromokinesin that provides astral-exclusion forces on the chromosomes during meiosis and mitosis. Consistent with this hypothesis, we have identified an HMG-1 homologous region on Klp38B that could potentially bind AT-rich DNA sequences. Finally, we show that klp38B mutations have defects in abdominal segmentation, suggesting that Klp38B, like Xenopus chromokinesin Xklp1, might be involved in polar granule formation.

Heme-coordinating analogs of lauric acid as inhibitors of fatty acid omega-hydroxylation.

A series of omega-substituted fatty acids with potential heme-coordinating groups was synthesized as inhibitors of lauric acid omega-hydroxylation. The compounds were evaluated using liver microsomes from clofibrate (CF)-induced rats and an engineered expressed CYP4A1-derived fusion protein called f4A1. omega-Imidazolyl-decanoic acid (compound 11) and omega-aminolauric acid (compound 7) were potent Type II ligands and potent inhibitors of lauric acid omega-hydroxylation in both CF-microsomes and f4A1. Replacing their terminal amino or imidazolyl groups with other potential iron-binding groups such as omega-methylsulfinyl-, omega-cyano-, omega-azido-, or omega-formamido all greatly reduced their potency as inhibitors of omega-hydroxylation and their affinity for cytochrome P450 as measured by Ks values. In CF-microsomes, inhibition of (omega-1)-hydroxylation of lauric acid by a homologous series of omega-imidazolyl-alkanoic acids varied only 2-fold but in the same incubations inhibition of omega-hydroxylation increased 22-fold upon going from C-8 to C-12. A similar dependence of binding affinity and inhibitory potency on chain length was also seen in the f4A1 system. In contrast, chain length had little effect on activity among n-alkylamines or N-alkylimidazoles lacking a carboxyl or other polar functional group, suggesting that 7, 11, and related bifunctional compounds interact with CYP4A1 in CF-microsomes and with f4A1 in a specific bidentate fashion. Imidazoles containing phenyl, benzyl, or phenylethyl substituents at N-1 interact less strongly than related N-alkyl-imidazoles of similar carbon number and hydrophobicity, suggesting that the steric bulk and/or rigidity of the phenyl ring is not well accommodated in the active site.

Effect of different solubilizing agents on the aggregation state and catalytic activity of two purified rabbit cytochrome P450 isozymes, CYP1A2 (LM4) and CYP2B4 (LM2).

The effects of different ionic and nonionic detergents and lecithin/Na cholate mixtures on the aggregation state and catalytic activity of two major inducible rabbit cytochromes P450, CYP1A2 (LM4) and CYP2B4 (LM2), both of which are highly aggregated in solution, have been compared. Nonionic detergents Emulgen 913 and Lubrol PX as well as the mixture of lecithin-cholate demonstrate the ability to induce the dissociation of P450 isozymes only to dimers, accompanied by an increase in their catalytic activity, especially in lecithin proteoliposomes.

Heteroatom substitution shifts regioselectivity of lauric acid metabolism from omega-hydroxylation to (omega-1)-oxidation.

In contrast to several isoforms of cytochrome P450 (including 1A1, 1A2, 2B1, 2C2, 2C6 and 2E1), cytochrome P450 4A1 and a fusion protein derived from it, show a strong preference for hydroxylation of lauric acid (C12) at the less chemically reactive omega-CH3 group instead of the more reactive (omega-1)-CH2 group. We have explored the interplay of steric effects on substrate binding vs chemical reactivity at various substrate loci in determining the striking difference in regioselectivity of CYP2B1 and a 4A1-derived fusion protein, through studies with heteroatom substituted analogs of lauric acid, i.e., 10-methoxydecanoic acid and 10-methylthiodecanoic acid. With both enzymes the former undergoes simple omega-hydroxylation (giving 10-hydroxydecanoic acid and HCHO), but the latter undergoes facile S-oxidation at the omega-1 position instead of omega-hydroxylation. The dramatic shift in the regioselectivity of the fusion protein toward the thia analog is consistent with the greater length of C-S bonds and the greater atomic radius and polarizability of sulfur lone-pair electrons within an otherwise restrictive active site.

Fatty acid discrimination and omega-hydroxylation by cytochrome P450 4A1 and a cytochrome P4504A1/NADPH-P450 reductase fusion protein.

The omega-hydroxylation of fatty acids by certain cytochrome P450 enzymes shows a degree of chain-length and regionspecificity which is remarkable in view of the conformational flexibility of these substrates, the strong similarity in properties among homologs, and the lack of polar groups (other than the carboxy terminus) with which to guide and strength enzyme-substrate interactions. To investigate the chemical basis for these features of omega-hydroxylation we designed and synthesized a series of lauric acid analogs and evaluated them as substrates and inhibitors of omega-hydroxylation catalyzed by cytochrome P4504A1 and a cytochrome P450 4A1/NADPH-P450 reductase fusion protein. Among n-alkanoic acids, lauric acid was found to have the optimum chain length for the fusion protein, as it does for native cytochrome P450 4A1. With both enzymes, chain shortening caused a precipitous drop in turnover while chain lengthening caused a gradual drop in turnover. The fusion protein omega-hydroxylated methyl laurate and lauryl alcohol about 1/10th as efficiently as lauric acid, but it did not hydroxylate lauramide. 10-Methoxydecanoic acid underwent O-demethylation (via omega-hydroxylation). The branched substrate 11-methyllauric acid was hydroxylated efficiently and selectively at the omega-position. In contrast, the cyclopropyl analog 11,12-methanolauric acid was not detectably hydroxylated, although it induced Type I binding spectrum and inhibited lauric acid omega-hydroxylation by 43% at equimolar concentrations. omega-(Imidazolyl)-decanoic acid induced a Type II heme-binding spectrum and was an especially potent inhibitor of lauric acid hydroxylation. Collectively these data suggest that the active site of cytochrome P450 4A1 has an elongated tubular shape of definite length (ca. 14 A) with a recognition site for polar groups (including but not limited to carboxyl) at its entrance and the (oxo)heme group at its terminus.

Biochemical characterization of lauric acid omega-hydroxylation by a CYP4A1/NADPH-cytochrome P450 reductase fusion protein.

The binding and hydroxylation of lauric acid by a genetically engineered and expressed fusion protein comprised of an N-truncated form of rat CYP4A1 linked to an N-truncated form of rat NADPH cytochrome P450 oxidoreductase (OR) (constructed by Fisher et al., (1992) Proc. Natl. Acad. Sci. USA 89, 10817-10822) has been characterized biochemically and compared to that shown by purified reconstituted rat CYP4A1 and liver microsomes from clofibrate-induced rats. In all systems lauric acid induced a Type I cytochrome P450 difference spectrum with Ks values in agreement with prior literature (range 10-18 microM). When provided with NADPH and oxygen but no other proteins or lipid, the fusion protein (called f4A1) catalyzed omega-hydroxylation of lauric acid with apparent Km and Vm of 3-4 microM and 4-5 nmol product/min/nmol P450 irrespective of buffer concentration or cation (NaPi or KPi, 25-200 mM); comparable values for reconstituted CYP4A1 and microsomes from clofibrate-induced rats are 9 microM and 34 min-1 and 5 microM and 10 min-1, respectively. (omega-1)-Hydroxylation of lauric acid was barely detectable (omega/(omega-1) = 135) with f4A1 or with reconstituted CYP4A1, but it accounted for up to 50% of total products formed by microsomes from clofibrate-induced rats. When added to the f4A1 system, OR stimulated hydroxylation up to fivefold at a OR:f4A1 ratio of 5:1; additionally, (omega-1)-hydroxylation was routinely observed as a minor process (< 4% of total product) in this system. These effects were also independent of buffer concentration. In contrast addition of cytochrome b5 (b5) caused a small (25%) decrease in omega-hydroxylation, while added phospholipid had no effect. However, the combination of OR, b5, and lipid stimulated turnover approximately 10-fold compared to f4A1 alone, and 11-hydroxylauric acid was regularly formed as a minor (3-4% of total) product. These observations indicate that the fusion protein f4A1 is functionally equivalent to reconstituted CYP4A1 with respect to binding and hydroxylation of lauric acid and suggest that it can be used as an alternative to reconstituted systems for structure-function and mechanistic studies of fatty acid omega-hydroxylation.

Dose-dependent induction of the microsomal monooxygenase system by phenobarbital and 3-methylcholanthrene in the ad libitum and calorie-restricted female rat.

1. We characterized the dose-dependent induction of the microsomal monooxygenase system by phenobarbital (PB) and 3-methylcholanthrene (MC) in the female Fischer 344 rat, which was either calorie restricted (CR) or fed ad libitum (AL). 2. Maximal induction of the major inducible isozymes (2B1/2B2 or 1A1) in rat was achieved at the lowest of the inducer doses employed (10 mg/kg body weight) in both feeding groups. 3. The patterns of dose-dependent PB induction and its magnitude differed between total P450 induction and induction of catalytic activities in AL and CR groups, whereas no differences between CR and AL rat were found in either spectrally detected P450 or EROD activity patterns of dose-dependent MC induction. 4. Calorie restriction increased the inducibility of some hepatic drug-metabolizing enzyme activities. 5. Monoclonal antibody-directed inhibition of MC-induced ethoxyresorufin O-deethylation (EROD) was 55-60% at all induction levels in AL rat and 65-70% in CR rat, while MAb inhibition of PB-induced pentoxyresorufin O-depenthylation (PROD) averaged about 55% in AL and 60% in CR rat.

Ethoxyresorufin and pentoxyresorufin O-dealkylation by hepatic microsomes from female Fischer 344 rats: effects of age and diet.

Ethoxyresorufin (EROD) and pentoxyresorufin (PROD) O-dealkylase activities, and contributions of the P450 cytochromes CYP1A1, CYP2B1 and 2, CYP2C6 and CYP2C12 to these metabolic activities, were evaluated in hepatic microsomes from ad libitum and calorie restricted female Fischer 344 rats across an age continuum from 1 to 26 months. The presence of CYP1A in microsome preparations was confirmed by western blot analysis. Uninduced levels of EROD and PROD peak in very young animals, decline to about 3 months of age, and do not exhibit an additional substantive decline after about 3 months of age. Monoclonal antibody (MAb) 1-7-1 (anti-CYP1A) strongly inhibited EROD activity in all microsome preparations, with the highest levels of inhibition in microsomes from 1- and 3-month-old AL animals. PROD activity in 1-month uninduced animals was apparently not attributable solely to CYP2B1 and 2, since it was inhibited by about 30% in both 1- and 26-month-old AL rats by an MAb specific for CYP2C12. However, in CR rats, CYP2C12 did not contribute to PROD activity. Approximately 40% of PROD activity in old AL rats and 20% of PROD activity in old CR rats was inhibited by an MAb specific for CYP2C6. These data indicate that long-term calorie restriction modulates either the expression or post-translational modification patterns of constitutive P450 isozymes in rats as they age, with P450 patterns in calorie restricted rats more closely resembling those found in young animals.

Microheterogeneity of cytochrome P450(LM2) as revealed by isoelectric focusing.

The homogeneity of different preparations of cytochrome P450(LM2), the major form of cytochrome P450 found in the liver microsomes of phenobarbital-treated rabbits, was checked by SDS polyacrylamide gel electrophoresis and N-terminal amino acid sequencing. In SDS-PAGE all the preparations migrated as a single protein band of molecular mass 49 kDa. The electrophoretic mobility of this band corresponded to that of the major phenobarbital-inducible band in the microsomes of phenobarbital-treated rabbits. A single N-terminal sequence (Met-Glu-Phe) corresponding to the N-terminal sequence of the LM2 form of cytochrome P450 was revealed by N-terminal amino acid sequence analysis. Isoelectric focusing of electrophoretically homogeneous cytochrome P450(LM2) was conducted in polyacrylamide gel in an LKB Ampholine pH 5-8 gradient under denaturing conditions (9.1 M urea). Under these conditions cytochrome P450(LM2) was resolved into six subfractions in the pH range 6.5-7.75. The data obtained reflect the microheterogeneity of this form of cytochrome P450. The existence of different isoelectric points cannot have been due to different protein conformations because denaturing conditions were used, but seem to reflect different primary structures of the cytochrome subfractions.