Isolating cytochrome P450 cDNA and genomic clones: library screening with synthetic DNA oligomers.
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Animals↗
Biomedical subjects
C Hassett
Publications and source records attributed to C Hassett.
Sequence and gene expression of rabbit cytochrome P450 IIC16: comparison to highly related family members.
Cytochrome P450s are heme containing proteins which evolved from an ancestral gene(s) to form a large superfamily of enzymes. We have isolated a unique cDNA from the rabbit P450 IIC subfamily, IIC16, which is 2028 bp in length. Nucleotide sequence determination indicated an ATG start codon 66 bp from the 5' end of the molecule, and an open reading frame coding for a protein of 487 amino acids. P450 IIC16 protein is greater than or equal to 90% identical in sequence to rabbit P450 IIC4, IIC5, and to the partial sequence available for IIC15. Northern and slot blot experiments demonstrated that the P450 IIC16 gene is expressed constitutively in liver, lung, testes, and kidney, and is inducible by phenobarbital in each tissue with the exception of the kidney, where mRNA levels are repressed. Alignment analysis of eight rabbit P450 IIC proteins revealed conserved and variable regions common to all IIC enzymes, and specific areas are suggested which may be important with respect to structure and function.
Amino Acid Sequence↗
Developmental expression and in situ localization of the phenobarbital-inducible rat hepatic mRNAs for cytochromes CYP2B1, CYP2B2, CYP2C6, and CYP3A1.
In this study we examined the differential hepatic expression of four phenobarbital (PB)-inducible rat cytochrome P450 genes, CYP2B1, CYP2B2, CYP2C6, and CYP3A1. The mRNAs encoding these cytochromes were analyzed in the liver with respect to PB responsiveness, developmental expression, and in situ localization. Utilization of the polymerase chain reaction enabled assessment of specific hepatic mRNA expression patterns during early development that were not detectable with standard Northern blot or slot-blot procedures. The polymerase chain reaction data demonstrated that fetal liver from day 15 of gestation was responsive to the inductive effects of transplacental PB administration. Both constitutive and PB-induced levels of each mRNA increased with increasing developmental age, reaching maximal levels approximately 3 weeks postpartum. An exception to this trend was observed for rats of gestational day 22, which exhibited transiently increased constitutive levels of CYP2B1, CYP2B2, and CYP3A1 mRNAs, such that PB-induced levels were not elevated over those observed in untreated animals. In situ hybridization data, obtained with livers form 6-week postpartum animals, revealed striking differences in regional distributions among the cytochrome P450 transcripts. Whereas patterns of PB-induced expression of CYP2C6 mRNAs were relatively homogeneous across the hepatic lobule, CYP3A1 mRNAs in PB-treated livers demonstrated marked centrilobular localization. These results were in contrast to data obtained previously for PB-inducible CYP2B1 and CYP2B2 mRNAs, which were distributed homogeneously across the hepatic lobule except for cells in the immediate vicinity of the periportal tract.
Animals↗
Rabbit microsomal epoxide hydrolase: isolation and characterization of the xenobiotic metabolizing enzyme cDNA.
Many endogenous and xenobiotic chemicals are metabolized to epoxides which may be enzymatically hydrated, via microsomal epoxide hydrolase (mEH), to less reactive dihydrodiol derivatives. On the basis of the reported rabbit mEH amino acid sequence [F. S. Heinemann and J. Ozols (1984) J. Biol. Chem. 259, 797-804], we constructed a 35 base oligonucleotide which was used to screen rabbit liver cDNA libraries. Overlapping rabbit mEH clones were isolated and the full-length cDNA sequence of 1653 bp was determined. The rabbit nucleotide sequence has a high degree of similarity (greater than 75%) with cDNA sequences reported for rat and human mEH. Northern blot analyses with fragments of the rabbit cDNA demonstrate that mEH messenger RNA (mRNA) is expressed constitutively in the liver and induced following exposure to phenobarbital or polychlorinated biphenyls. Constitutive expression of mEH mRNA is also observed in rabbit kidney, testes, and lung. Using benzo[alpha]pyrene-4,5-oxide as substrate, mEH enzymatic activity is shown to correlate closely with tissue levels of mEH mRNA. Southern blot analyses of rabbit DNA suggest that the mEH gene exists as a single copy per haploid genome. The mEH amino acid sequences of the human and rat were compared to that of the deduced rabbit protein in order to analyze the degree of conservation and hydropathy profiles in these species. This comparison permitted the formulation of a computer-assisted model of mammalian mEH as it may relate to the microsomal membrane.
Amino Acid Sequence↗
Quantitative analysis of the thickness of human skin and subcutaneous tissue following controlled expansion with a silicone implant.
Histologic quantitation of the thickness of human tissues that were expanded using silicone expanders showed that the epidermis underwent significant thickening after 5 weeks to 5 months of expansion. The dermis and subcutaneous tissue, on the other hand, were significantly thinner after expansion. Capsules were formed in all 19 patients. The capsule was significantly thickest after 2 to 2.5 months of expansion. Expanded tissues 2 years after cessation of expansion had the same thickness as control tissues and had no remnant fibrous capsule.
Adolescent↗
Use of a conserved consensus oligomer in the identification of cytochrome P-450 mRNAs.
A region of high nucleic acid conservation present in many members of the phenobarbital (PB) family of cytochromes P-450 (P-450s) was targeted for the construction of an 18 base, eight-fold degenerate, consensus oligomer (the PB-mer). When utilized to screen a rabbit liver cDNA library, the PB-mer enabled the isolation of P-450 cDNAs. One isolate, designated pP-450 1a, was characterized by nucleotide sequence, as well as Northern and Southern blot analyses and was demonstrated to be a novel form. Primary nucleotide sequence indicates that this cDNA is highly homologous to several P-450 cDNAs, but only in the protein coding region. The 3'-noncoding segment of P-450 1a is longer and diverges markedly from known P-450 cDNAs in the rabbit PB family.
Amino Acid Sequence↗
Comparison of continuous and interrupted suture techniques in microvascular end-to-side anastomosis.
To compare the speed and patency rates of the continuous and interrupted suture techniques in microvascular end-to-side anastomosis, 10 anastomoses utilizing each technique were performed using a femoral artery-superficial epigastric vein bypass model in Sprague-Dawley rats. The continuous suture technique was found to be almost twice as fast as the interrupted technique with no functional compromise. Patency for both techniques was 100 percent. These results are similar to those of several recent reports comparing the two techniques in end-to-end anastomosis and suggest that the continuous suture technique may be the most efficient technique for end-to-side anastomosis as well.
Animals↗
Identifying alcoholism in medical patients.
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Alcoholism↗
Splenomegaly in mice following exposure to ambient levels of ozone.
Female strain A/J mice were exposed to 0.31 ppm (608 micrograms/m3) ozone continuously for 103 h every other week for 6 mth. Following an additional period of 5 mth in a filtered air environment, animals were killed and examined for evidence of altered spleen weight. It was observed that animals exposed to ozone had a greater spleen weight, and spleen to body weight ratio than air-breathing controls. In some of the ozone-exposed mice, pronounced splenomegaly was noted. Increased spleen weight appears to be another example of an extrapulmonary effect of ozone inhalation.
Animals↗
Reliability of the Behavioral Assessment Scale of Oral Functions in Feeding.
Interrater and test-retest reliability of the Behavioral Assessment Scale of Oral Functions in Feeding were computed for two separate samples of severely and profoundly handicapped students having oral-motor and feeding disorders. Reliability was assessed using the intraclass correlation-generalizability theory approach and resulted in coefficients ranging from .68 to .84. These values were considered only marginally acceptable by current standards of instrument development advocated in the behavioral sciences. This paper discusses the difficulty in developing psychometrically satisfactory assessment instruments for use with the severely and profoundly developmentally disabled and gives implications for practice and future research.
Adolescent↗
Murine lung carcinogenesis following exposure to ambient ozone concentrations.
Inbred strain A/J mice, responsive to the chemical induction of pulmonary adenomas, were used to assess any of several roles that the photochemical air pollutant ozone might play in lung carcinogenesis. In separate experiments, animals were exposed to two concentrations of ozone (0.31 +/- 0.01 and 0.50 +/- 0.02 ppm) intermittently for a 6-month period, to evaluate the potential of ozone to act as either a pulmonary carcinogen, a tumor promoter, or an inhalant capable of increasing lung tumor yield when exposure was in conjunction with a pulmonary carcinogen, urethane. Statistical analyses of results indicated that ozone exposure at both concentrations caused an increase in lung tumor number relative to clean air controls, but that ozone was not an effective tumor promoter under the conditions of our protocol. When ozone exposure immediately preceded treatment with urethane (CAS: 51-79-6), animals were at increased risk for the development of lung adenomas.
Adenoma↗
Characterization of mouse fetal lung cells cultured on a pigskin substrate.
Lung organ bits taken from full-term mice were explanted on the dermal surface of sterile, dead pigskin. The cells migrated onto the pigskin dermis and proliferated to form an organoid culture consisting of ductular structures separated by a matrix of epithelial cells. Cells within the ductular structures were ciliated, produced mucin, and exhibited the activities of nonspecific esterase and gamma-glutamyl transferase; therefore they were considered to be derived from bronchial epithelium. Cells forming the matrix possessed the activities of nonspecific esterase and alkaline phosphatase and contained lamellar structures typical of surfactant-producing pneumocyte Type II cells; therefore they were considered to be derived from alveolar precursor cells.
Alkaline Phosphatase↗
Motility and adhesiveness in human neutrophils. Effects of chemotactic factors.
Human peripheral blood neutrophils (PMN) obtained from healthy adults were examined in vitro with techniques adapted to assess the effects of chemotactic factors (CF) on cellular configuration and adhesiveness. The results were compared with those that use certain conventional techniques for assessing chemotaxis and chemokinesis. Exposure of PMN to N-formyl-l-methionyl-l-phenylalanine (f-Met-Phe), zymosan-activated serum, bacterial chemotactic factor, or a low molecular weight chemotactic factor from activated serum (C5a) in the absence of a gradient resulted in a change in cellular shape from a spherical to a polarized configuration in a high percentage of cells. This occurred rapidly in suspension, under conditions designed to exclude a role for cell adhesiveness, and was reversible upon removal of the CF. Restimulation of cells with the CF resulted in reappearance of the polarized configuration to the same extent as on initial stimulation with one exception: f-Met-Phe pretreated cells failed to respond to f-Met-Phe, though they responded fully to the other CF. Each CF caused a significant increase in PMN attachment to protein-coated glass. This enhanced adhesiveness was not reversible upon removal of the CF when the cells were treated under conditions shown to produce chemotactic deactivation. Cells treated under these conditions also exhibited significantly reduced motility on glass and in micropore filters in the absence of a gradient of CF. Bacterial chemotactic factor, even at high concentrations, failed to produce deactivation and did not cause a sustained enhancement of adhesiveness.
Cell Adhesion↗
Hepatic expression of rat P450 mRNA assessed by in situ hybridization to oligomer probes.
Cell-specific chemical toxicities may be influenced by P450-catalyzed biotransformation reactions. We have undertaken an analysis of P450 expression in isolated rat liver sections to assess better the cellular distribution of P450 gene products. Discriminatory 18-mer oligodeoxynucleotides directed to the phenobarbital (PB) inducible P450s, P450IIB1 (P450b) and P450IIB2 (P450e), were employed as probes for in situ hybridization experiments. With these techniques we demonstrate that P450b and P450e mRNAs are each expressed in the hepatic lobule with similar spatial distribution. In animals pretreated with PB, only cells within the immediate periportal region were refractory to induction. Based on autoradiographic grain densities, responsive hepatocytes accumulated P450b mRNA at levels exceeding that for P450e. We employed in situ hybridization methodology in combination with Northern blot analyses to compare the expression of these mRNAs in two rat strains, Sprague-Dawley and Marshall 520/N (the latter being deficient in the synthesis of P450e isozyme; Rampersaud and Walz, 1987). These strains provided valuable comparative models, demonstrating the specificity and sensitivity of the oligomer probes. The continued development of in situ hybridization methodologies, especially when used in conjunction with synthetic oligomer probes, will permit a detailed analysis of P450 expression in different tissues, under a variety of chemical exposures, and may be a valuable adjuvant to the prediction of target organ toxicities.
Animals↗