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J V Pastewka

Publications and source records attributed to J V Pastewka.

At least 19 recordsLinked to original sources
PubMed1986

Noncoordinate regulation of the mRNAs encoding cytochromes P-450BNF/MC-B and P-450ISF/BNF-G.

The mRNAs encoding the major polycyclic aromatic hydrocarbon-induced cytochromes P-450 from rat, P-450BNF/MC-B and P-450ISF/BNF-G, were characterized using three classes of recombinant plasmids: those complementary to (a) only P-450BNF/MC-B mRNA, (b) only P-450ISF/BNF-G mRNA, and (c) both mRNAs. These classes were identified by hybridization-selected translation and immunoprecipitation using six monoclonal and polyclonal antibodies and were later sequenced to confirm their identity and specificity. These findings indicated that the mRNAs encoding these two P-450s have regions that are unique, as well as regions that are homologous. Hybridization-selected translation also showed that the primary in vitro translation products of the P-450BNF/MC-B and P-450ISF/BNF-G mRNAs are 55 and 52 kDa, respectively, and have both unique and common structural characteristics that can be distinguished immunologically. By Northern hybridization, the P-450BNF/MC-B mRNA was found to be 2900 bases long, while the P-450ISF/BNF-G mRNA was 2100 bases long. Precursors of 3500 and 5200 bases were detected for P-450BNF/MC-B mRNA, while a 3100-base precursor was detected for P-450ISF/BNF-G mRNA. These two mRNAs were induced by beta-naphthoflavone, isosafrole, and 3-methylcholanthrene, but not by phenobarbital. In untreated rats, the P-450BNF/MC-B mRNA was consistently present at very low levels while the P-450ISF/BNF-G mRNA was present in variable amounts, suggesting that the latter mRNA can be induced by dietary or other environmental factors. The kinetics of induction of the P-450BNF/MC-B and P-450ISF/BNF-G mRNAs were measured by dot blot hybridization. P-450BNF/MC-B mRNA increased rapidly, reaching half-maximum by 4 h after treatment with 3-methylcholanthrene, while the P-450ISF/BNF-G mRNA increased more slowly, reaching half-maximum after 12 h. The levels of both mRNAs peaked at 24 h, but decreased thereafter at different rates; P-450BNF/MC-B mRNA dropped by about 20% during the next 24 h, while P-450ISF/BNF-G mRNA dropped by 50 to 70%. These differences in the kinetics of induction and the apparent stabilities of the P-450BNF/MC-B and P-450ISF/BNF-G mRNAs, in conjunction with the observed differences in their levels in untreated rats, suggested that these two mRNAs were not coordinately regulated even though they were induced by the same compounds.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Monoclonal antibodies to cytochrome P-450 immunopurify a 45-kDa protein from a human lymphoblastoid cell line.

Monoclonal antibodies (MAbs) to rat liver cytochromes P-450 have previously been used for successful immunopurification of cytochromes P-450 from animal tissues. We now report application of this MAb-based immunopurification technique to the human lymphoblastoid AHH-1 cell line. Immunopurification carried out with 3 different MAbs each yielded a 45-kDa polypeptide. The purified protein contains an MAb-specific epitope present on cytochromes P-450, and may therefore be a human cytochrome P-450.

Animals↗
PubMed1984

Induction of cytochrome P-450 mRNAs quantitated by in vitro translation and immunoprecipitation.

Cytochrome P-450 mRNAs were quantitated by in vitro translation of liver RNA followed by immunoprecipitation with antibodies specific for cytochromes P-450. The kinetics of cytochrome P-450 mRNA induction by 3-methylcholanthrene and by phenobarbital were examined, and differences in the types of cytochrome P-450 mRNAs induced by 3-methylcholanthrene (MC), beta-naphthoflavone (BNF), and phenobarbital (PB) in male and female rats were determined. Phenobarbital strongly induced the mRNA encoding a single peptide antigenically related to the phenobarbital-induced cytochrome P-450PB-B. Male rat liver contained 62% more translatable mRNA for this peptide than did female. 3-Methylcholanthrene and beta-naphthoflavone, but not phenobarbital, induced mRNAs encoding three peptides that were immunologically related to cytochrome P-450BNF/MC-B, which is induced by 3-methylcholanthrene. The levels of translatable mRNA coding for these peptides were twice as high in females as in males. Striking sex differences were observed in the levels of translatable mRNAs for peptides related to cytochrome P-450PB/PCN-E, which is induced by phenobarbital and by pregnenolone-16-alpha-carbonitrile. In females, only RNA preparations from the livers of phenobarbital-treated rats had significant levels of mRNAs encoding these peptides. In contrast, significant levels of these RNAs were observed even in untreated males, and the levels of these mRNAs increased markedly following phenobarbital treatment. All cytochrome P-450 inducers examined caused a 50 to 70% decrease in translatable albumin mRNA. This effect was specific for albumin mRNA, since levels of total translatable mRNA were not generally altered by these inducers. The kinetics of induction of cytochrome P-450 mRNA differed from those of induction of aryl hydrocarbon hydroxylase (AHH) activity. Translatable cytochrome P-450 mRNA was increased as early as 4 h after phenobarbital treatment, peaked between 24 and 36 h, and dropped back to control levels by 120 h. The induction of AHH lagged behind the increase in translatable mRNA, remaining at control levels well after levels of translatable mRNA began to increase but then decreasing roughly in parallel with translatable mRNA. These findings suggest that transcription was not rate limiting for regulation of PB-inducible cytochrome P-450 activity. 3-Methylcholanthrene caused parallel increases in AHH activity and translatable cytochrome P-450 mRNA, but when translatable mRNA began to decrease after about 24 h, AHH activity remained high, suggesting that this P-450 mRNA was less stable than the enzyme for which it coded.

Animals↗
PubMed1982

Identification and quantitation of a 2.0-kilobase messenger ribonucleic acid coding for 3-methylcholanthrene-induced cytochrome P-450 using cloned cytochrome P-450 complementary deoxyribonucleic acid.

We have used a plasmid containing DNA complementary to one of the two size classes of mRNA coding for 3-methylcholanthrene-induced cytochrome P-450 from rat liver to characterize and quantitate that mRNA. The plasmid used was constructed and identified as follows: Total poly(A+) RNA from 3-methylcholanthrene-induced liver was used as a template for cDNA synthesis. Double-stranded cDNA was inserted into plasmid pBR322 by the G-C tailing procedure. Recombinants were screened by colony hybridization using as probe [32P]cDNA prepared from partially purified cytochrome P-450 mRNA. A differential screening approach was used in which duplicate filters were hybridized with probe from either 3-methylcholanthrene treated or untreated rats. Plasmid p23 was strongly positive by colony hybridization. It was conclusively shown to contain cytochrome P-450 cDNA sequences by demonstrating that the mRNA which specifically hybridized to nitrocellulose-bound plasmid p23 could be translated in vitro into peptides that were immunoprecipitable with monoclonal antibodies specific for 3-methylcholanthrene-induced cytochrome P-450. The size and quantity of the mRNA complementary to plasmid p23 were determined by hybridization of the 32P-labeled plasmid to rat liver RNA that had been fractionated by electrophoresis under fully denaturing conditions and transferred to diazobenzyl-oxymethyl-paper. Plasmid p23 hybridized strictly to a single size of mRNA that was about 2000 nucleotides in length, the smaller of the two size classes of mRNA coding for 3-methylcholanthrene-induced cytochrome P-450. From this we concluded that, at least within the region of the mRNA contained within the insert of plasmid p23, the two size classes of 3-methylcholanthrene-induced cytochrome P-450 mRNA were very different in sequence. The mRNA complementary to plasmid p23 was increased about 4-fold after in vivo administration of 3-methylcholanthrene under conditions that result in maximal induction of 3-methylcholanthrene-induced cytochrome P-450 enzymatic activity. This increase in cytochrome P-450 mRNA parallels the increase in cytochrome P-450 enzymatic activity observed after 3-methylcholanthrene administration. These data suggest that the regulation of mRNA levels is an important point of control of cytochrome P-450 gene expression.

Animals↗
PubMed1980

Mutagenicity of some lipsticks and their dyes.

Twenty-four lipsticks of various shades and colors were tested for mutagencitiy with the histidine-requiring tester strain Salmonella typhimurium TA98. Nine lipsticks were mutagenic without microsomal (S-9) activation. Dose-response effects were observed. Eight colorants listed as ingredients of the mutagenic lipsticsk were tested with and without S-9. Drug and Cosmetic (D&C) Orange No. 17, a monoazo dye with two nitro groups, was highly mutagenic in the absence of S-9. The mutagenic effect was decreased or lost in the presence of S-9 prepared from livers of male noninbred Sprague-Dawley rats given a single injection of Aroclor 1254. Eight lipsticsk matched for ingredients other than dyes were tested. Two containing D&C Orange No. 17 were directly mutagenic. The mutagenic effect was decreased by the presence of S-9. Only D&C Orange No. 17 was sufficiently mutagenic without microsomal activation to account for the mutagenicity observed in these lipsticks. Lipsticks containing D&C Orange No. 17 and those labeled with the words "may contain" D&C Orange No. 17 should be suspected of being mutagenic for S. typhimurium TA98. This dye and 2,4-dinitrosaniline, which may also be present, are potential health hazards. Assessment of their carcinogenicity awaits evaluation of results obtained by appropriate testing in animals.

Animals↗
PubMed1979

The cationic carbocyanine dyes Stains-all DBTC, and Ethyl-Stains-all, DBTC-3,3',9 triethyl.

A simple method is presented for distinguishing two closely related metachromatic carbocyanine dyes: Ethyl-Stains-all, a triethyl dye, and Stains-all, a diethyl methyl dye. This has become important since one lot of the triethyl dye was distributed erroneously under the diethyl methyl label. The dyes differ in solubility and in differential staining of macromolecules. Studies performed with both dyes are summarized.

Carbocyanines↗
PubMed1978

Lactoferrin is a marker for prolactin response in mouse mammary explants.

We recorded a bioassay for prolactin using densitometric analysis on polyacrylamide gels of caseins produced by mammary explants in vitro. A protein which also increased in the presence of increasing amounts of prolactin in the medium has now been identified. Its migration in several electrophoretic systems is the same as that of a whey protein of mouse milk, lactoferrin. It is an iron-binding glycoprotein with a molecular weight by SDS gel electrophoresis equal to mouse, human and bovine lactoferrin. It gives a precipitin reaction in an immunodiffusion system with monospecific antiserum raised in rabbits against mouse lactoferrin.

Animals↗
PubMed1977

Bioassay for prolactin: densitometric analysis on polyacrylamide gels of milk protein production by mammary explants in vitro.

Mammary explants from mice in midpregnancy cultured in a synthetic medium containing insulin and hydrocortisone respond to the addition of prolactin by the synthesis of milk proteins. The secretory response in alveolar lumina has served as a histologic endpoint in a sensitive bioassay for prolactin developed by Kleinberg and Frantz (J Clin Invest 50: 1557, 1971). This report demonstrates that the quantitative densitometric analysis of stained milk proteins (caseins) made in response to prolactin is a useful modification of that bioassay. Electrophoretic analysis of the proteins extracted from the explants permits a quantitative estimate of casein content. The amount of casein present after 5 days of incubation was found to be a measure of the prolactin concentration in the medium. No radioactive isotopes are used. The use of electrophoretic analysis has practical advantages over the histologic scoring used earlier, and has approximately the same range of sensitivity.

Animals↗
PubMed1976

Characterization of major milk proteins from BALB/c and C3H mice.

Milk proteins from BALB/c and C3H mice were characterized with respect to their electrophoretic migration in polyacrylamide gels under alkaline and acid conditions. The major casein and whey proteins from each strain migrated similarly under the conditions employed. Phosphoproteins were identified by staining with "Stains-all" and by changes in electrophoretic mobility and staining induced by prior treatment with phosphatase. Sialic acid-rich glycoproteins were identified by staining with periodic acid-Schiff and with "Stains-all" by prior treatment with neuraminidase to identify sialic acid as the acidic portion of the molecule. The two major whey proteins were characterized further by their migration in sodium dodecyl sulfate gels. One protein had the same mobility as mouse serum albumin. The other protein migrated with a mobility similar to that of bovine alpha-lactalbumin. The identity of the former protein was confirmed by its reaction with goat anti-mouse serum albumin in an immunodiffusion procedure, and the latter protein by its B protein activity in the lactose synthetase assay.

Animals↗
PubMed1976

Molecular weights of three mouse milk caseins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and kappa-like characteristics of a fourth casein.

Caseins of mouse milk are phosphoproteins which precipitate at pH 4.6, stain blue with "Stains-all," and stain red with "Stains-all" following alkaline phosphatase digestion. Four caseins were separated electrophoretically in sodium dodecyl sulfate-polyacrylamide gels varying from 8.5 to 15% acrylamide. Molecular weights for three of these proteins were 43,200, 27,700, and 25,900. The molecular weights determined for bovine alphas1 and beta caseins by this method were similar to those previously obtained by other methods. A fourth mouse casein contained carbohydrate, phosphorus, and sialic acid. This protein was rennin-sensitive and behaved anomalously on sodium dodecyl sulfate polyacrylamide gels, as did bovine kappa-casein. Because of similarities with bovine kappa-casein, it was designated with "kappa-casein" of mouse milk.

Animals↗
PubMed1975

Hemoglobin binding by isolated polymeric proteins from human haptoglobin types 2-1 and 2-2. Some suggested polymer subunit compositions.

1. Some of the individual members of the polymeric series of proteins from human haptoglobin types 2-1 and 2-2 were isolated by gel electrophoresis. By reacting this purified material with less than an equivalent amount of hemoglobin and analyzing the result by electrophoresis, the number of haptoglobin-hemoglobin complexes could be clearly counted. For the haptoglobin 2-1 series, the number of complexes formed was n+1, where n is the serial order, in decreasing electrophoretic mobility, of the haptoglobin polymeric form used. For the haptoglobin 2-2 series, the number of complexes was n+2. 2. For the first three members of haptoglobin 2-1 series, the haptoglobin-hemoglobin composition of the complexes was estimated from scans of the unstained gels. The data indicated that this series consists of 2,3,4... alpha beta haptoglobin subunits, each of which can combine with an alpha beta subunit of hemoglobin.

Electrophoresis, Polyacrylamide Gel↗