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Biomedical subjects

R M Philpot

Publications and source records attributed to R M Philpot.

At least 73 records · Page 4Linked to original sources
PubMed1991

Functional expression of mammalian cytochromes P450IIB in the yeast Saccharomyces cerevisiae.

Three mammalian cytochromes P450 from the IIB subfamily, P450IIB11 from canine and P450IIB4 and P450IIB5 from rabbit, have been expressed in the yeast Saccharomyces cerevisiae by use of an autonomously replicating vector containing the galactose-inducible gal10 promoter. Cytochromes P450IIB4 and P450IIB5 are closely related proteins, with only 11 amino acid substitutions between them. P450IIB11 is a homologous protein, likely orthologous with IIB4 or IIB5, with 102 amino acid substitutions compared with the P450IIB4 protein and 106 compared with the P450IIB5 protein. The expressed proteins are functional in yeast microsomes, exhibiting activity toward androstenedione, 7-ethoxycoumarin, and, in some cases, progesterone. Expressed cytochromes P450IIB4 and P450IIB11 hydroxylate androstenedione with regio- and stereoselectivity characteristic of the purified, reconstituted proteins. A striking difference in the androstenedione metabolite profiles of IIB4 and IIB5 was observed, with IIB4 producing almost exclusively the 16 beta-hydroxy metabolite and IIB5 producing the 16 alpha-hydroxy and 15 alpha-hydroxy products. This is the first time that 15 alpha-hydroxylase activity has been associated with IIB4/IIB5. This activity has also been detected in liver microsomes from some, but not all, individual phenobarbital-induced rabbits tested and is largely inhibited by anti-rabbit P450IIB immunoglobulin G. These studies illustrate the utility of the yeast expression system for defining catalytic activities of individual mammalian cytochromes P450 and identifying new marker activities that can be utilized in liver microsomes.

Androstenedione↗
PubMed1991

The major alpha-class glutathione S-transferases of rabbit lung and liver. Primary sequences, expression, and regulation.

The complete primary structures of two distinct rabbit alpha-class glutathione S-transferase (GST) subunits, rbGST alpha I and rbGST alpha II, have been derived from cDNA sequences. Clones encoding rbGST alpha I were isolated from both hepatic and pulmonary cDNA libraries, whereas clones encoding rbGST alpha II were isolated only from the hepatic library. Immunochemical and peptide sequence data confirmed that rbGST alpha I corresponds to the 27-kDa alpha-class subunit purified from rabbit lung (Serabjit-Singh, C. J., and Bend, J. R. (1988) Arch. Bioch. Biophys. 267, 184-194). Expression of rbGST alpha II in liver but not in lung and expression of rbGST alpha I in both liver and lung was substantiated by Northern and immunochemical analyses. rbGST alpha I and rbGST alpha II are composed of 223 and 221 amino acids, respectively, and are 78% identical in amino acid sequence. Compared to published GST sequences, both proteins are most closely related to the human Ha subunit (greater than 80% identity). On the basis of sequence comparison and Northern and Southern analyses, we conclude that rbGST alpha I and rbGST alpha II are products of different genes that are independently regulated. Further, the regulatory elements of the alpha-class GST genes may be significantly different in the rabbit as compared to the rat, as evidenced by the lack of induction by phenobarbital of rabbit hepatic or pulmonary alpha-class GST subunits, enzymatic activity, or mRNA. This tissue- and species-dependent expression of the predominant class of cytosolic GST implies unique functions for each isozyme and may contribute to the differential susceptibility of tissues and animals to toxicants.

Amino Acid Sequence↗
PubMed1991

Metabolic activation of 4-ipomeanol by complementary DNA-expressed human cytochromes P-450: evidence for species-specific metabolism.

4-Ipomeanol is a pulmonary toxin in cattle and rodents that is metabolically activated by cytochromes P-450 (P-450s). P-450-mediated activation of 4-ipomeanol to DNA binding metabolites was evaluated using a vaccinia virus complementary DNA expression system and an in situ DNA-binding assay. Twelve human P-450s and two rodent P-450s were expressed in human hepatoma Hep G2 cells and examined for their abilities to metabolically activate this toxin. Three forms, designated CYP1A2, CYP3A3, and CYP3A4, were able to catalyze significant production of DNA-bound metabolites of 20-, 8-, and 5-fold, respectively, above binding catalyzed by Hep G2 cells infected with wild-type vaccinia virus. These enzymes, with highest activities, are not known to be expressed in human or rodent lung. CYP2F1 and CYP4B1, two enzymes that are expressed in lung, display only modest 3- and 2-fold respective increased abilities to metabolically activate 4-ipomeanol. Two human forms were inactive and seven other human forms showed activities ranging from 0.5- to 2-fold above control level. Surprisingly, rabbit complementary DNA-expressed CYP4B1 was the most active enzyme (180-fold above control) among all P-450s tested in producing DNA-binding metabolites from this mycotoxin. These studies demonstrate a species difference in 4-ipomeanol metabolism and suggest caution when attempting to extrapolate rodent data to humans.

Biotransformation↗
PubMed1991

Distribution of cytochrome P-450 monoxygenase enzymes in the nasal mucosa of hamster and rat.

Deposition of inhaled particulates onto the respiratory mucosa is relatively great in that portion of the nasal cavity unprotected by ciliated, goblet, or keratinized superficial cells. The cytochrome P-450 system is an important enzyme system involved in the biotransformation of xenobiotics into metabolites that are more readily absorbed. To examine the transitional region caudal to the nasal vestibule, nasal tissues of hamster and rat were prepared for immunocytochemistry. Blocks of tissue representing four levels along the long axis of the nasal cavity were examined. Paraffin sections were processed through the avidin-biotin peroxidase procedure, with diaminobenzidine tetrahydrochloride as the chromagen. Enzyme localization was accomplished through the use of antibodies for three rabbit cytochrome P-450 isozymes; 2, 5, and 6 (subfamilies IIB, IVB, and IA, respectively); and for rabbit NADPH-cytochrome P-450 reductase. Enzyme distribution was similar in both hamster and rat nasal tissues except in cells of striated and intercalated ducts of nasal glands and in cells of the nasolacrimal duct where immunoreactivity was greater in the hamster. Immunoreactivity for reductase and isozyme 2 was intense in nonciliated cells lining the nonolfactory epithelium, in sustentacular cells of the olfactory epithelium, and in acinar cells of olfactory glands. Distribution of reaction products to isozyme 5 and 6 were similar to but not so intense as those of reductase and isozyme 2. Reaction products for reductase and isozyme 2 occurred generally in the same cellular and intracellular regions with the following exceptions: isozyme 2 was more concentrated in cells of striated ducts and of the nasolacrimal duct, and reductase was more abundant in intercalated ducts of nasal glands.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Characterization of the cytochrome P-450 monooxygenase system in nonciliated bronchiolar epithelial (Clara) cells isolated from mouse lung.

The nonciliated bronchiolar epithelial (Clara) cell of the mouse is highly susceptible to toxicants that undergo metabolic activation, presumably because this cell type has high levels of cytochrome P-450 monooxygenases. As a first step in further defining the role of Clara cells in pulmonary xenobiotic activation and detoxication, we have isolated Clara cells (75 to 80% purity) and characterized them morphologically and biochemically. The identity of Clara cells, confirmed by transmission electron microscopy, was based on several features, including abundant agranular endoplasmic reticulum, large mitochondria, and dense secretory granules. Immunocytochemistry of isolated mouse cells showed that the majority were positive with antibodies against three major components of the pulmonary cytochrome P-450 monooxygenase system, cytochrome P-450 isozymes 2 (IIB), 5 (IVB), and NADPH cytochrome P-450 reductase, purified from rabbit lung. The isolated cells also showed a positive reaction with an antibody against the cytochrome P-450 isozyme that is active in the stereoselective metabolism of naphthalene, cytochrome P-450 mN (mN). Immunocytochemistry using the antibody against cytochrome P-450 isozyme 6 (IA1), purified from rabbit lung, showed no reaction in the isolated cells. The presence of intact cytochrome P-450 protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blots of homogenates of isolated cell preparations. The N-demethylation of benzphetamine and epoxidation of naphthalene occurred at easily measurable rates in incubations of isolated Clara cells. In contrast, diols, quinones, and monohydroxylated benzo(a)pyrene metabolites, analyzed by high performance liquid chromatography, were undetectable in extracts of Clara cells incubated with 3H-labeled substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Pyridine effects on P450IIE1, IIB and IVB expression in rabbit liver: characterization of high- and low-affinity pyridine N-oxygenases.

The effects of pyridine exposure on expression of cytochromes P450IIE1, IIB and IVB in rabbit hepatic microsomes and their respective role in pyridine N-oxide production has been examined. Immunoblot analysis revealed that pyridine administration caused a substantial increase in P450IIE1 levels, failed to affect P450IIB content and marginally increased the expression of P450IVB. In an effort to implicate specific forms of P450 in pyridine N-oxide production, the kinetics of pyridine N-oxide formation in uninduced and induced rabbit hepatic microsomal preparations were obtained. Pyridine-induced microsomes exhibited a single low Km value of 81 microM with a approximately 2.5-fold increase in Vmax (2.44 nmol/min/mg protein) relative to uninduced microsomes. Interestingly, pyridine N-oxide production in phenobarbital-induced microsomes were also monophasic, exhibiting a single, high Km value of 949 microM and a Vmax of 3.3 nmol/min/mg protein, a approximately 10-fold increase over the uninduced preparations. In contrast, uninduced and isosafrole-induced rabbit hepatic microsomes both exhibited biphasic kinetics; uninduced microsomes gave Km values of 85 and 973 microM, whereas isosafrole-induced microsomes yielded Km values of 229 and 1733 microM, respectively, with a Vmax somewhat less than uninduced microsomes. When kinetic data were normalized for P450 content, a pronounced substrate specificity was detected for both pyridine- and phenobarbital-induced microsomes. para-Nitrophenol hydroxylase activity was enhanced approximately 6-fold in pyridine-induced microsomes consistent with elevated levels of P450IIE1. para-Nitrophenol competitively inhibited (Ki = 13 microM) the production of pyridine N-oxide in pyridine-induced microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Properties of expressed and native flavin-containing monooxygenases: evidence of multiple forms in rabbit liver and lung.

Our laboratory recently isolated and sequenced cDNAs encoding the microsomal flavin-containing monooxygenases (FMOs) from rabbit liver and rabbit lung. As a first step in understanding the molecular bases for the catalytic and physical differences between these enzymes, we have expressed them in COS-1 cells and compared the properties of the recombinant and native microsomal proteins. Microsomes from transfected cells were examined immunochemically by immunoblotting and catalytically by following methimazole oxidation in the presence and absence of various modulators. The expressed and native FMOs have the same mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the same responses to pH, sodium cholate, magnesium, and temperature, all of which serve to differentiate between the lung and liver enzymes. Analysis of methimazole metabolism in microsomes isolated from rabbit liver or lung showed biphasic kinetics, indicative of two or more enzymes taking part in the reaction. In contrast, the kinetics of methimazole oxidation catalyzed by the expressed FMOs were clearly linear and matched one of the phases observed with the native preparations. Chlorpromazine and imipramine, which are not substrates for the pulmonary FMO, were found to be competitive inhibitors of the high affinity reaction in pulmonary microsomes. These results, and others, indicate that both rabbit lung and liver contain more than one form of FMO.

Animals↗
PubMed1990

The flavin-containing monooxygenase enzymes expressed in rabbit liver and lung are products of related but distinctly different genes.

Preparations of mRNA isolated from rabbit lung and liver were used in the construction of libraries that were screened for cDNAs encoding the pulmonary or hepatic isozyme of the flavin-containing monooxygenase. The hepatic library was screened with cDNA encoding the flavin-containing monooxygenase expressed in pig liver, and a clone containing a 2.0-kilobase insert was detected and isolated. This cDNA insert encoded a protein of 535 amino acids with a primary structure 87% identical to that of the pig flavin-containing monooxygenase. The pulmonary library was screened with polyclonal antibodies to the flavin-containing monooxygenase expressed in rabbit lung, and a clone containing a 2.6-kilobase insert was detected and isolated. Although the protein encoded by this insert also contained 535 amino acids, its primary sequence was only 56% identical to that of the liver enzyme. The sequences of several peptides obtained by digestion of the purified rabbit pulmonary flavin-containing monooxygenase with trypsin matched exactly with sequences derived from the cDNA structure. Tissue-specific distribution of mRNA for the hepatic and pulmonary isozymes of the flavin-containing monooxygenase was consistent with the distribution of protein, an indication that expression of flavin-containing monooxygenase is controlled at the level of transcription. Analysis of genomic DNA indicates that both the hepatic and pulmonary enzymes may be products of single genes.

Amino Acid Sequence↗
PubMed1990

The flavin-containing monooxygenase expressed in pig liver: primary sequence, distribution, and evidence for a single gene.

The primary sequence of the flavin-containing monooxygenase expressed in pig liver has been derived from the nucleotide sequence of cloned cDNA. The derived sequence is composed of 532 amino acids and represents a protein having a molecular weight of 58,952. The complete sequence was obtained from a single clone containing 2070 bases. A second clone, obtained from an independent library, yielded an identical sequence for the 1374 bases present. The amino acid composition compiled from the derived sequence is very similar to that obtained previously from the purified protein. In addition, a 10 amino acid sequence in a peptide formed from the purified protein by digestion with V8 protease exactly matches the derived sequence for residues 309-318. The flavin-containing monooxygenase expressed in pig liver is also expressed in pig lung and kidney as determined by analysis of both microsomal proteins and mRNA. The ratio of mRNA to protein for the enzyme in kidney is about 5 times greater than the same ratio for liver and about twice the ratio for lung. The reasons for these differences are not understood. Southern analysis of genomic DNA indicates that there is a single gene encoding the flavin-containing monooxygenase expressed in pig liver. Therefore, the broad activity of this enzyme in liver appears to be the result of the catalytic diversity of a single protein.

Amino Acid Sequence↗
PubMed1990

Immunohistochemical demonstration of cytochrome P-450 monooxygenase in regenerating tracheal epithelium: a recapitulation of fetal development.

The cytochrome P-450 monooxygenase enzymes, NADPH-reductase and form 2, were demonstrated immunohistochemically in hamster tracheal epithelium that was regenerating after mechanical injury. Bromodeoxyuridine (BrdU), a thymidine analogue, was used to map the location and extent of the wound sites between 8 and 144 h post-injury. In the control and non-wounded areas of the epithelium, the secretory cells were labelled for the monooxygenase enzymes. Label was heaviest in the apical cytoplasm of these columnar cells. At 8 h, secretory cells at the wound margins migrated to cover the wound sites, becoming progressively flattened. Reaction product for monooxygenase enzymes was strong in these flat cells but immunolabelling for BrdU was very low. At 24 h many cells at the wound sites were labelled for BrdU (indicative of a high rate of cell division). Some cells were labelled for monooxygenase but many were not stained at this time. At 48 and 72 h post-injury, none of the cells within the wound sites (regenerating epithelium) were stained. Immunochemical labelling for the monooxygenase enzymes was restored to the nascent secretory cells as they differentiated in the wound sites, beginning at 96 h post-injury. Labelling was stronger at 120 and 144 h post-injury, comparable to that in the control epithelium. The observations suggest that the monooxygenase enzymes were retained by the secretory cells in the wound sites before they divided but were lost from their progeny. Then, the temporal sequence of monooxygenase expression was similar to the pattern of differentiation of nascent secretory cells during fetal development of the tracheal epithelium.

Animals↗
PubMed1990

Use of inactivated foot-and-mouth disease virus antigen in liquid-phase blocking ELISA.

A liquid-phase blocking ELISA is used by the World Reference Laboratory for Foot-and-Mouth Disease for the quantification of antibodies to foot-and-mouth disease virus. The potential for using inactivated FMDV antigens in the assay has been assessed by titrating bovine convalescent sera to all seven serotypes and comparing the titres obtained with live or inactivated antigens. The titres were similar indicating that either live or inactivated antigens can be used in the liquid-phase blocking ELISA. Removing the need to use live antigens in tests for FMD antibody would reduce disease security risk and widen the acceptability of kits for FMD antibody detection and assay.

Animals↗
PubMed1990

Freeze-drying foot-and-mouth disease virus antigens. I. Infectivity studies.

The ability of foot-and-mouth disease virus strains type O1 BFS 1860 and type A22 IRQ 24/64 to retain infectivity after freeze-drying with or without additives being made to virus suspensions was studied. The infectivity titres of freeze-dried antigens was assessed at intervals over a six month storage period at various temperatures and also after reconstitution to the liquid phase and storage with or without glycerination. Certain additive solutions were necessary to prevent degradation of virus during the freeze-drying procedure which reduced any loss of infectivity caused by storage of products at 4 degrees C and 20 degrees C. Additive solutions composed of 10% sucrose and 5% lactalbumin hydrolysate; 10% skimmed milk; 4% peptone and 1% gelatin; and 5% dextran, 1% sodium glutamate and 5% sucrose all prolonged the keeping qualities of virus at the elevated temperature of 37 degrees C. The results indicate that short-term storage and shipment of freeze-dried foot-and-mouth disease virus antigens is possible without the need for refrigeration, thereby reducing transportation and storage costs. Reconstituted antigens survived better after glycerination and storage at -20 degrees C than did non-glycerinated samples stored at 4 degrees C.

Animals↗
PubMed1990

Freeze-drying foot-and-mouth disease virus antigens. II. For use in the ELISA.

Live and inactivated preparations of foot-and-mouth disease virus strains 01 BFS 1860 and A22 IRQ 24/64 were freeze-dried in the presence or absence of additive solutions and assessed for their reactivity by ELISA at intervals over a six month storage period at various temperatures and also after reconstitution and subsequent storage with or without glycerination. The type specificity of all antigen preparations was maintained throughout the study period and the potency of antigens, judged by titration in ELISA, remained constant during the freeze-drying procedure and throughout subsequent storage at -20 degrees C and 4 degrees C with or without additives having been made to virus suspensions prior to freeze-drying. This was also the case with antigens reconstituted and stored at either -20 degrees C with glycerol or at 4 degrees C without glycerol. Certain additive solutions were necessary, however, to preserve the activity of antigens stored at the elevated temperature of 37 degrees C. The reactivity of all freeze-dried antigens was not unduly affected in the liquid-phase blocking ELISA using bovine convalescent antisera of each of the seven serotypes of foot-and-mouth disease virus and known negative, non-immune bovine sera. The results suggest that shipment and long-term storage of freeze-dried foot-and-mouth disease virus antigens is possible for use in the ELISA in the absence of refrigeration. This has attractive advantages for reducing both shipment and storage costs of antigens and for the development of ELISA kits for the diagnosis of foot-and-mouth disease virus.

Animals↗
PubMed1990

The immunocytochemical detection of cytochrome P-450 monooxygenase in the lungs of fetal, neonatal, and adult hamsters.

Antibodies against rabbit cytochrome P-450 reductase (reductase), cytochrome P-450 isozyme 2 (P-450 IIB), and cytochrome P-450 isozyme 5 (P-450 IVB) were used to detect homologous enzymes in the developing lung of the Syrian golden hamster. No immunocytochemical labeling was observed on gestational days 11, 12, and 13. On gestational day 14, light immunoperoxidase labeling for reductase and P-450 IIB was observed over cells lining the trachea and cranial portions of lobar bronchi. On gestational day 15, these enzymes were detected in conducting airways at all anatomic levels, and in the media of the pulmonary vein and its branches. Light labeling for P-450 IVB was first observed over cells lining the trachea and lobar bronchi on gestational day 15, but the smallest bronchioles and the media and endothelium of the pulmonary vein did not label for this enzyme until gestational day 16 (neonatal day 1). Type II pneumocytes and the pleural mesothelium first labeled for each of the three enzymes on neonatal day 1. Although the mesothelium no longer labeled for reductase or P-450 IIB in hamsters 3.5 wk old, the other labeling sites persisted in adult hamsters. Because cytochrome P-450 enzymes are associated with the endoplasmic reticulum, an ultrastructural examination of differentiating secretory cells was made to detect its appearance. At each conducting airway level, smooth endoplasmic reticulum was present in the cells 2 d before cytochrome P-450 enzymes could be detected immunocytochemically. The appearance of these enzymes paralleled the development of the hamster lung; they were first present in the trachea and lobar bronchi, then in the bronchioles, and finally in the alveoli.

Animals↗
PubMed1989

Primary structures of cytochrome P-450 isozyme 5 from rabbit and rat and regulation of species-dependent expression and induction in lung and liver: identification of cytochrome P-450 gene subfamily IVB.

The primary structure of rabbit cytochrome P-450 isozyme 5 has been derived from the nucleotide sequence of cloned cDNA. Identical sequences were obtained for cDNAs constructed with mRNA from four different sources, lung and liver of untreated rabbits and liver from rabbits treated once or four times with phenobarbital. Isozyme 5 shows significant sequence identity only with rabbit P-450p2 (54%) and rat P-450LA omega (53%), which places it in a previously unrecognized cytochrome P-450 gene subfamily (IVB). A cDNA library was also constructed from rat pulmonary mRNA and screened with cDNA encoding rabbit isozyme 5. The amino acid sequence derived from a positive clone was compared with that of rabbit isozyme 5 and found to be 87% identical, significantly greater than observed between other similar forms of cytochrome P-450 from rabbit and rat. Alignment of the primary structures of rabbit isozyme 5 (506 residues), rat isozyme 5 (511 residues), rabbit P-450p2, and rat P-450LA omega shows 43% structural identity and a common 16-residue peptide near position 300 that is unique to these forms of cytochrome P-450. Analysis of mRNA from lung and liver of rabbit, rat, guinea pig, and hamster indicates that species and tissue differences in the expression and induction of isozyme 5 are likely regulated at the level of transcription. These differences fall into one of the following three groups: first, expression in lung and liver and induction in liver by phenobarbital (rabbit); second, expression in lung and liver but no hepatic induction (hamster); and third, expression in lung and little or no expression in liver regardless of treatment (rat and guinea pig).

Amino Acid Sequence↗
PubMed1988

Primary structures of multiple forms of cytochrome P-450 isozyme 2 derived from rabbit pulmonary and hepatic cDNAs.

Rabbit pulmonary and hepatic mRNA was used to construct cDNA libraries that were screened with a cDNA probe (pf 3/46) to murine cytochrome P-450 homologous with rat cytochrome P-450b. Three types of cDNA clones were identified on the basis of restriction analysis with Bst EII. Two types of clones (B0 and B1) were present in a library constructed from pulmonary mRNA. The nucleotide sequence of B0 cDNA encodes a protein of 491 amino acids having a sequence identical to one reported for isozyme 2. The sequence derived from the b1 cDNA also contains 491 amino acids but differs from the B0 sequence at six positions. B1 clones were also obtained from a library constructed from hepatic mRNA isolated 12 hr after a single treatment with phenobarbital. A third type of clone (B2) was also obtained from this library, but no B0 clones were found. The sequence derived from the B2 cDNA contains 491 amino acids and differs from the B0 and B1 sequences at 11 and 15 positions, respectively. A second hepatic cDNA library, constructed from mRNA from the liver of a rabbit treated with phenobarbital daily for 4 days, contained B0 clones. The sequence of one of these was found to be identical to that of the pulmonary B0 clones.

Amino Acid Sequence↗