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Biomedical subjects

D E Ryan

Publications and source records attributed to D E Ryan.

At least 91 records · Page 5Linked to original sources
PubMed1983

The accumulation of distinct mRNAs for the immunochemically related cytochromes P-450c and P-450d in rat liver following 3-methylcholanthrene treatment.

Treatment of rats with 3-methylcholanthrene leads not only to a marked accumulation in the liver of translatable mRNA coding for a 56-kilodalton polypeptide representing cytochrome P-450c, the major 3-methylcholanthrene-induced cytochrome P-450 of rat liver, but also to the accumulation of comparable amounts of mRNA encoding a 52-kilodalton polypeptide which is immunoprecipitated with antibodies prepared against rat liver cytochrome P-450c. Further electrophoretic and immunochemical characterization of the latter translation product demonstrates that it corresponds to cytochrome P-450d, the major isosafrole-induced form of rat liver cytochrome P-450. The mRNAs for cytochromes P-450c and P-450d can be completely separated by electrophoresis in denaturing agarose gels and have chain lengths of approximately 4000 and 2000 nucleotides, respectively. These two mRNAs do not show detectable sequence homology to the mRNAs coding for the major phenobarbital-induced forms of cytochrome P-450 (P-450b and P-450e) since in Northern blotting experiments they fail to hybridize under conditions of low to moderate stringency to cloned probes for the latter mRNAs.

Animals↗
PubMed1983

On the glycosylation state of five rat hepatic microsomal cytochrome P-450 isozymes.

The glycosylation states of five rat hepatic microsomal cytochrome P-450 isozymes (cytochromes P-450a, P-450b, P-450c, P-450d, and P-450e) were examined by quantitative carbohydrate analysis. Carbohydrate content of the purified enzymes as determined by acid hydrolysis, reduction, and gas chromatography of the alditol acetates revealed only trace amounts of neutral and amino hexoses in each of the five isozymes. Levels of mannose ranged from 0.3 to 1.7 mol/mol of cytochrome P-450 whereas levels of galactose were less than or equal to 0.2 mol/mol of cytochrome P-450 for the five hemoproteins. The amino sugars glucosamine and galactosamine were usually present at levels less than or equal to 0.2 mol/mol of cytochrome P-450, although one preparation of cytochrome P-450b had as much as 0.5 mol of glucosamine/mol of cytochrome P-450. Other carbohydrate residues (xylose and arabinose) were not detected in significant quantities. Since N- and O-glycosylation of proteins occurs primarily through N-acetylglucosaminyl and N-acetylgalactosaminyl residues, respectively, the lack of significant amounts of these amino sugars indicates that these five cytochrome P-450 isozymes are not normally glycosylated in the native state. Purified NADPH-cytochrome c reductase, which functions as an electron donor for microsomal cytochrome P-450, contained no detectable quantities of hexose sugars.

Animals↗
PubMed1983

Differential time course of induction of rat liver microsomal cytochrome P-450 isozymes and epoxide hydrolase by Aroclor 1254.

The time course of induction of rat liver microsomal cytochromes P-450a, P-450b + P-450e, P-450c, and P-450d and epoxide hydrolase has been determined in immature male rats administered a single large dose [1500 mumol (500 mg)/kg body wt] of the polychlorinated biphenyl mixture Aroclor 1254. Differential regulation of these xenobiotic-metabolizing enzymes was indicated by their characteristic patterns of induction. The rate of induction of cytochrome P-450a and epoxide hydrolase was relatively slow, and steady-state levels of these enzymes were maintained from approximately Days 9 to 15 after Aroclor 1254 treatment. In contrast, cytochrome P-450c was maximally induced 2 days after Aroclor 1254 treatment and remained at a constant level through Day 15. Steady-state levels of cytochrome P-450d, beginning 1 week after Aroclor 1254 treatment, were preceded by a fairly rapid rate of induction and possibly by a small decline from maximal levels observed around Days 4 to 5. Like those of the other cytochrome P-450 isozymes and epoxide hydrolase, the levels of cytochromes P-450b + P-450e were constant from Day 9 to 15 after Aroclor 1254 treatment. However, an unexpected but reproducible decline (approximately 25%) in total cytochrome P-450 content observed between Days 4 and 9 after Aroclor 1254 treatment principally reflected a dramatic and totally unanticipated decrease (approximately 45%) in the level of cytochromes P-450b + P-450e. This transient decline in the level of cytochromes P-450b + P-450e was not due to an unusual effect of a mixture of polychlorinated biphenyls, since identical results were obtained with two individual congeners, namely 2,3,4,5,4'-penta- and 2,3,4,5,3',4'-hexachlorobiphenyl, that induced the same isozymes as Aroclor 1254. In contrast, when rats were treated with 2,4,5,2',4',5'-hexachlorobiphenyl, which induces cytochromes P-450a and P-450b + P-450e and epoxide hydrolase but not cytochromes P-450c or P-450d, maximal levels of cytochromes P-450b + P-450e were attained on Day 4 and no decrease was observed over the next 11 days. These results suggest that there may be an interaction in the regulation of induction of certain individual cytochrome P-450 isozymes.

Animals↗
PubMed1983

The in vivo turnover of rat liver microsomal epoxide hydrolase and both the apoprotein and heme moieties of specific cytochrome P-450 isozymes.

The in vivo turnover rates of liver microsomal epoxide hydrolase and both the heme and apoprotein moieties of cytochromes P-450a, P-450b + P-450e, and P-450c have been determined by following the decay in specific radioactivity from 2 to 96 h after simultaneous injections of NaH14CO3 and 3H-labeled delta-aminolevulinic acid to Aroclor 1254-treated rats. Total liver microsomal protein was characterized by an apparent biphasic exponential decay in specific radioactivity, with half-lives of 5-9 and 82 h for the fast- and slow-phase components, respectively. Most (approximately 90%) of the rapidly turning over microsomal protein fraction was immunologically distinct from membrane-associated serum protein, and thus appeared to represent integral membrane proteins. The existence of two distinct populations of cytochrome P-450a was suggested by the apparent biphasic turnover of both the heme and apoprotein moieties of the holoenzyme. The half-lives of the apoprotein were estimated to be 12 and 52 h for the fast- and slow-phase components, respectively, and 7 and 34 h for the heme moiety. The turnover of cytochromes P-450b + P-450e was identical to that of cytochrome P-450c, with half-lives of 37 and 28 h for the apoprotein and heme moieties, respectively. In all cases, the shorter half-lives of the heme component compared to the protein component were statistically significant. In contrast to the cytochrome P-450 isozymes, epoxide hydrolase (t1/2 = 132 h) turned over slower than the "average" microsomal protein (t1/2 = 82 h). The differential rates of degradation of these major integral membrane proteins during both the rapid and slow phases of total microsomal protein turnover argue against the concepts of unit membrane degradation and unidirectional membrane flow of liver endoplasmic reticulum.

Animals↗
PubMed1983

The sorption of copper (II), manganese (II), zinc (II) and arsenic (III) onto human hair, and their desorption.

Human hair has been studied in relation to sorption from aqueous solutions of Cu2+, Mn2+, Zn2+ and AsO3-(3). At an equilibrium concentration of 0.3 micrograms ml-1 the sorptions are relatively low for Mn (1.1 micrograms g-1) and As (0.1 microgram g-1), and higher for Zn (10 micrograms g-1) and Cu (35 micrograms g-1). But only in the case of copper is the sorption significant relative to the indigenous levels of the elements in the hair. The greater sorption of Cu2+ may be correlated with better binding to the hair fibre, probably both electrostatically and to the sulphur in the keratin. There appears to be at least three modes of attachment, or three mechanisms of attachment of Cu2+ to the hair. An inter-element effect was observed, where Cu2+ severely inhibits the sorption of Zn2+ and Mn2+. Also the total sorption of Cu2+ is reduced on zinc or manganese-treated hair. Some comments are made regarding the results and the problems of exogenous contamination of human hair.

Adsorption↗
PubMed1983

Identification and localization of amino acid substitutions between two phenobarbital-inducible rat hepatic microsomal cytochromes P-450 by micro sequence analyses.

Two isozymes of rat liver microsomal cytochrome P-450--P-450b and P-450e--were compared by micro sequence analyses of their NH2 termini and tryptic fragments. These two phenobarbital-inducible hemoproteins, which are immunochemically indistinguishable with antibody against cytochrome P-450b, have extensive sequence homology. Automated Edman degradation of the native proteins revealed identical amino acids for the first 35 residues. Sequence determinations of the tryptic peptides, which constitute approximately 75% of each protein molecule, have thus far shown 10 amino acid differences between the two isozymes. Results of our amino acid sequence analyses established that two of the cDNAs, pcP-450pb1 and pcP-450pb4, reported by Fujii-Kuriyama et al. [Fujii-Kuriyama, Y., Mizukami, Y., Kamajiri, K., Sogawa, K. & Muramatsu, M. (1982) Proc. Natl. Acad. Sci. USA 79, 2793-2797] encode cytochrome P-450b whereas pcP-450pb2, a third cDNA whose nucleotide sequence differed slightly from that of the other two (six amino acid substitutions), encodes cytochrome P-450e. In addition to establishing the identity of these cloned cDNAs we provide direct evidence for seven additional amino acid differences between cytochromes P-450b and P-450e that occur beyond the region (Arg358) encoded by the cloned cDNA for cytochrome P-450e. Together, the amino acid sequences determined by micro sequence analysis and recombinant DNA techniques reveal 13 amino acid differences between these two isozymes. This report highlights the complementary nature of two different molecular approaches to elucidation of the amino acid sequences of isozymes with extensive structural homology.

Amino Acid Sequence↗
PubMed1983

Internal derangement of the temporomandibular joint: detection by single-photon emission computed tomography. Work in progress.

The diagnostic accuracy of both planar and single-photon emission computed tomography (SPECT) bone scintigraphy, radionuclide angiography, arthrography, and conventional radiography was evaluated in 36 patients with temporomandibular joint (TMJ) dysfunction undergoing preoperative testing. The sensitivity of SPECT bone scintigraphy (0.94) was comparable with arthrography (0.96) and significantly better than planar bone scintigraphy (0.76), radionuclide angiography (0.35), and transcranial lateral radiographs (0.04). While data for a larger asymptomatic control population are needed, preliminary results give SPECT a diagnostic specificity of 0.70 for internal derangements of the TMJ requiring surgical correction. It is concluded that SPECT bone scintigraphy is a useful noninvasive imaging test to screen for internal derangement of the TMJ.

Adolescent↗
PubMed1982

Comparisons of highly purified hepatic microsomal cytochromes P-450 from Holtzman and Long-Evans rats.

The present study describes the purification and characterization of strain variant forms of a major phenobarbital-inducible microsomal hemoprotein, cytochrome P-450b, from Holtzman and Long-Evans rats. The strain variant hemoproteins cannot be resolved by sodium dodecyl sulfate gel electrophoresis, but can be partially separated in two-dimensional isoelectric focusing SDS gels. If, however, sodium tetradecyl sulfate is incorporated into the one-dimensional gel system, separation of the cytochromes P-450b is achieved. Minor structural differences are detected in the peptides of the cytochromes P-450b following limited proteolysis by Staphylococcus aureus V8 protease, cleavage by cyanogen bromide, or reverse-phase high-pressure liquid chromatography of tryptic peptides. The strain variant cytochromes P-450b are immunochemically and spectrally indistinguishable. The optical spectra of the ferric and ferrous hemoproteins are identical, as are the CO- and ethylisocyanide-reduced difference spectra. Ferrous cytochromes P-450b from both rat strains effectively bind metyrapone with equivalent affinities. In addition, the cytochromes P-450b do not differ in their catalytic activities toward benzphetamine, hexobarbital, benzo [a]pyrene, zoxazolamine, 7-ethoxycoumarin, estradiol-17 beta and testosterone. Cytochrome P-450c, the predominant isozyme inducible in rat liver by 3-methylcholanthrene, was purified from Holtzman and Long-Evans rats. Cytochromes P-450c from both rat strains are indistinguishable based on electrophoretic, immunological, spectral and catalytic properties. Minor structural differences in the cytochromes P-450c were revealed in the reverse-phase high-pressure liquid chromatographic profiles of the tryptic peptides of these hemoproteins, but not in the peptides generated by limited proteolysis or cleavage with cyanogen bromide.

Animals↗
PubMed1982

Polypeptide patterns of hepatic microsomes from Long-Evans rats treated with different xenobiotics.

Two-dimensional gel electrophoresis was used to analyze hepatic microsomal polypeptides after treatment of immature, male Long-Evans rats with 3-methylcholanthrene, pregnenolone-16 alpha-carbonitrile, isosafrole, SKF-525A, Aroclor-1254, gamma-chlordane, or trans-stilbene oxide. Epoxide hydrolase and cytochromes P-450a, P-450bLE, P-450c, P-450d, and P-450e were all identified as resolved polypeptides in these electrophoretograms. Idiosyncratic polypeptide patterns characterized the microsomal preparations following treatment of rats with each inducing agent. Immunochemically identical cytochromes P-450bLE and P-450e were always present at the same relative levels even though their total amount varied 3-fold after induction by isosafrole, SKF-525A, Aroclor-1254, gamma-chlordane, and trans-stilbene oxide. Cytochromes P-450c and P-450d were coinduced by 3-methylcholanthrene, isosafrole, and Aroclor-1254, but their relative amounts varied. Pregnenolone-16 alpha-carbonitrile treatment resulted in an increase of a single major microsomal polypeptide which was also induced by phenobarbital, isosafrole, SKF-525A, Aroclor-1254, and trans-stilbene oxide. Only one polypeptide was identified as epoxide hydrolase in all of the microsomes analyzed. The results suggest that the levels of cytochromes P-450bLE and P-450e may be subject to coordinate control, whereas the other cytochromes P-450 are independently regulated.

Animals↗
PubMed1982

Multiple, immunoidentical forms of phenobarbital-induced rat liver cytochromes P-450 are encoded by different mRNAs.

It was shown previously that four immunochemically identical forms of phenobarbital-induced hepatic cytochrome P-450 exist in unique combinations which characterize different strains and colonies of rats (Vlasuk, G. P., Ghrayeb, J., Ryan, D. E., Reik, L., Thomas, P. E., Levin, W., and Walz, F. G., Jr. (1982) Biochemistry 21, 789-798). One colony of Long-Evans rats exhibited only cytochromes P-450bLE and P-450e among these immunorelated enzymes; whereas, one colony of Holtzman rats was characterized by cytochromes P-450bH and P-450e. After phenobarbital treatment, hepatic poly(A)+-mRNA was isolated from these groups of rats and translated in vitro. The 35S-labeled products were immunoisolated with antibody to cytochrome P-450bLE and analyzed by two-dimensional gel electrophoresis. The results indicated that the products synthesized in vitro correspond exactly to the two particular forms of the enzyme that characterize liver microsomes from each of these groups of rats. It is concluded that different structural genes encode these immunorelated forms of cytochrome P-450 and that significant post-translational processing of their polypeptide products does not occur in vivo.

Animals↗
PubMed1982

Multiplicity strain differences, and topology of phenobarbital-induced cytochromes P-450 in rat liver microsomes.

The multiplicity of phenobarbital-induced cytochromes P-450 in live microsomes from male rats was investigated by using two-dimensional gel electrophoresis, peptide fingerprinting, and immunoaffinity chromatography. Two colonies each of Holtzman and Long-Evans rats were studied. Four molecular forms of phenobarbital-induced cytochromes P-450 were distinguished as polypeptides (designated PB3, variant PB3, PB4, and PB5) which showed apparent immunochemical identity and greater than or equal to 95% fingerprint homology. Two of these polypeptides corresponded to cytochrome P-450b [Ryan, D., Thomas, P. E., Korzeniowski, D., & Levin, W. (1979) J. Biol. Chem. 254, 1365-1374] and cytochrome P-450e [Ryan, D., & Levin, W. (1981) Fed. Proc., Fed. Am. Soc. Exp. Biol. 40, 1640] which had been purified from Long-Evans rats (variant PB3 and PB5, respectively). Each rat colony was characterized by unique combinations of two or three of these immunochemically related forms of cytochromes P-450. Cytochrome P-450e was present in rats from all four colonies, but cytochrome P-450b was only found in Long-Evans rats. Polypeptide PB3 was only found in the two colonies of Holtzman rats, whereas polypeptide PB4 was present in one colony each of Holtzman and Long-Evans rats. In addition to these forms of cytochrome P-450, rats from each colony also evidenced three other major phenobarbital-induced polypeptides which gave unique fingerprints, and one of these was identified as representing epoxide hydrolase. Proteolytic digestion studies of intact microsomes demonstrated that the four immunochemically identical forms of cytochrome P-450 were partially exposed on the outer (cytoplasmic) surface of microsomes. However, polypeptide PB3 was characterized by the greatest rate of proteolytic degradation. These results clearly demonstrate that phenobarbital-induced cytochromes P-450 include microheterogeneous proteins which show remarkable variations related to rat strains and/or colony.

Animals↗