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Biomedical subjects

M R Waterman

Publications and source records attributed to M R Waterman.

At least 163 records · Page 9Linked to original sources
PubMed1988

Levels of adrenodoxin, NADPH-cytochrome P-450 reductase and cytochromes P-45011 beta, P-45021, P-450scc, in adrenal zona fasciculata-reticularis tissue from androgen-treated rats.

Treatment of rats with methylandrostenediol (MAD), an anabolic androgen, caused a profound reduction (65%) in the level of cytochrome P-450 11 beta in rat adrenocortical mitochondria as measured by immunoblots using a specific antibody. The decreases in mitochondrial cytochrome P-450scc (15%) and adrenodoxin (20%) were much less than that observed for cytochrome P-45011 beta. A 35% decrease in adrenal microsomal cytochrome P-450 21 and NADPH-cytochrome P-450 reductase levels was brought about by the treatment with MAD. The data establish that the preferential decrease in adrenal steroid 11 beta-hydroxylase activity associated with androgen treatment results from a decrease in cytochrome P-450 11 beta. This is consistent with the role of 11-deoxycorticosterone in the pathogenesis of androgen-induced hypertension in rats.

Adrenal Cortex↗
PubMed1988

Combined 17 alpha-hydroxylase/17,20-lyase deficiency due to a stop codon in the N-terminal region of 17 alpha-hydroxylase cytochrome P-450.

Steroid 17 alpha-hydroxylase (cytochrome P-450(17)alpha) mediates both 17 alpha-hydroxylase and 17,20-lyase activities. A relatively rare disease, 17 alpha-hydroxylase deficiency is characterized by defects in either or both of these activities. The molecular basis for variability of the defect is not well understood. We have determined the exonic sequence of the mutant P-450(17)alpha gene from one Japanese patient with combined 17 alpha-hydroxylase/17,20-lyase deficiencies. A stop codon (TGA) due to a single point mutation was found at the position of amino acid 17 in exon 1 of the P-450(17)alpha gene. The presence of a stop codon in the N-terminal region of this gene leads to the absence of a functional P-450(17)alpha protein in adrenal cortex and ovary, and consequently hypertension, primary amenorrhea and osteoporosis in this patient.

Adrenal Cortex↗
PubMed1988

Simultaneous transfection of COS-1 cells with mitochondrial and microsomal steroid hydroxylases: incorporation of a steroidogenic pathway into nonsteroidogenic cells.

Transfected, nonsteroidogenic COS-1 cells derived from monkey kidney are found to be capable of supporting the initial and rate-limiting step common to all steroidogenic pathways, the side-chain cleavage of cholesterol to produce pregnenolone. Endogenous COS-1 kidney cell renodoxin reductase and renodoxin are able to sustain low levels of this activity catalyzed by bovine cholesterol side-chain cleavage cytochrome P450 (P450scc) whose synthesis is directed by a transfected plasmid containing P450scc cDNA. Double transfection with both P450scc and adrenodoxin plasmids leads to greater pregnenolone production and indicates that adrenodoxin plays a role as a substrate for this reaction or that bovine adrenodoxin serves as a better electron donor than the endogenous iron-sulfur protein renodoxin. Also it is found that both the bovine adrenodoxin and P450scc precursor proteins are proteolytically processed upon their uptake by COS-1 cell mitochondria to forms having the same electrophoretic mobility as mature bovine adrenodoxin and P450scc. Following triple transfection of COS-1 cells with P450scc, adrenodoxin, and 17 alpha-hydroxylase cytochrome P450 plasmids, pregnenolone produced in mitochondria by the side-chain cleavage reaction can be further metabolized in the endoplasmic reticulum to 17 alpha-hydroxypregnenolone and dehydroepiandrosterone. Although this functional steroidogenic pathway can be incorporated into this nonsteroidogenic cell type, it is found to be nonresponsive to cAMP, a potent activator of steroid hormone biosynthesis in adrenal cortex, testis, and ovary. Thus the cellular mechanisms necessary to support both microsomal and mitochondrial steroid hydroxylase activities appear not to be tissue specific, whereas the acute cAMP-dependent regulation of steroidogenesis is not present in transformed kidney (COS-1) cells.

Adrenodoxin↗
PubMed1988

Structural characterization of normal and mutant human steroid 17 alpha-hydroxylase genes: molecular basis of one example of combined 17 alpha-hydroxylase/17,20 lyase deficiency.

Steroid 17 alpha-hydroxylase (cytochrome P-450 17 alpha) catalyzes both 17 alpha-hydroxylation of pregnenolone and progesterone and 17,20-lysis of 17 alpha-hydroxypregnenolone and 17 alpha-hydroxyprogesterone. In the course of undertaking detailed investigation of the structure-function relationships which exist within this enzyme we have begun to elucidate the molecular basis of human deficiencies in either or both of these activities. Consequently we have determined the exonic structure of the human P-450 17 alpha gene as well as the sequences at the exon/intron boundaries and at the site of initiation of transcription. A single gene in the human genome encodes this protein, being the sole member of a unique gene family (P450XVII) within the P-450 supergene family. A protocol for exonic sequencing of the P-450 17 alpha gene has been established which permits structural analysis of the gene from patients having 17 alpha-hydroxylase and/or 17,20-lyase deficiency. This procedure has been applied to the mutant gene from one individual having combined 17 alpha-hydroxylase/17,20-lyase deficiencies. A four-base duplication is found in exon 8 producing a protein with an altered C-terminal amino acid sequence which results in loss of both enzymatic activities.

Adrenal Hyperplasia, Congenital↗
PubMed1988

Immunoblot analysis of cholesterol side-chain cleavage cytochrome P-450 and adrenodoxin in corpora lutea of cyclic and late-pregnant sheep.

The specific contents of cytochrome P-450scc and adrenodoxin in corpora lutea of late pregnant sheep were, respectively, 1/5 and 1/8 that of corpora lutea of the oestrous cycle, suggesting lower steroidogenic enzyme capacity in the former. The contents of Complex V proteins were also lower in the corpora lutea of late pregnancy. It was observed in the immunoblots of both Complex V and cytochrome P-450scc that immunoreactive bands of molecular weights lower than the native proteins were present in the samples from corpora lutea of late pregnancy, indicative of degradation of the native enzymes. It is concluded that corpora lutea of sheep during late pregnancy have a much lower enzyme capacity for steroidogenesis than do those of the oestrous cycle (mid-luteal phase) due to a reduction in the content of cytochrome P-450scc and adrenodoxin. The reduction in the levels of steroidogenic enzyme proteins appears to be unspecific and probably reflects an overall demise in mitochondrial functions.

Adrenodoxin↗
PubMed1988

Some enzymatic vagaries of a bovine adrenal microsomal cytochrome P-450 introduced and expressed in transformed monkey kidney cells.

The present studies illustrate the ability to carry out kinetic measurements of steroid hydroxylation using a cytochrome P-450 expressed in a tissue culture cell system. For these experiments a single species of cDNA, incorporated into a suitable expression vector, has been introduced via transfection. A number of interesting preliminary observations have been made on the function of the cytochrome P-450 associated with adrenocortical microsomes which catalyses the 17-hydroxylation of progesterone and pregnenolone. In confirmation of earlier reports the adrenal 17-OHase possesses both 17-hydroxylase as well as C17,20-lyase activities. However, the latter is only functional with 17-OH pregnenolone and not with 17-OH progesterone as substrate. This result differs from the numerous reports that a lyase activity for both substrates is associated with this P-450. The reason for this difference between a delta 4 and a delta 5 steroid remains unresolved although initial experiments indicate that the 5-alpha reduced progesterone is a suitable substrate for both the 17-OHase as well as lyase reactions. This result suggests an inhibitory effect of the delta 4 double bond preventing the carbon-carbon cleavage of the C-17,20 bond in 17-OH progesterone. Clearly more experiments will be required to resolve this question. Measurements of substrate affinity for the cytochrome P-450 expressed in COS cells appears to be influenced by a permeability barrier to the steroid effecting the transport of the steroid across the cell membrane into the cells. This conclusion is suggested by the presence of a time lag before the onset of metabolism as well as by the discrepancy in the concentration of substrate required to give half-maximal rates of metabolism, cf. the results obtained where the initial concentration of progesterone present in the reaction medium is altered versus those experiments measuring the kinetics of substrate depletion. The presence of such a barrier to the free movement of steroid across the membrane is interesting to contemplate when considering the build up of 17-OH pregnenolone required for the lyase reaction. Most unexpected where the results obtained when a comparable expression vector containing the cDNA for cytochrome b5 was cotransfected with pCD17 alpha 2. Cytochrome b5 has been postulated to be an electron transfer component participating in the cyclic function of some cytochromes P-450 (Hildebrandt and Estabrook, 1971).(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Cortex↗
PubMed1987

Tetradecanoyl phorbol acetate suppresses follicle-stimulating hormone-induced synthesis of the cholesterol side-chain cleavage enzyme complex in rat ovarian granulosa cells.

The effect of tetradecanoylphorbol acetate (TPA) on follicle-stimulating hormone (FSH)-induced synthesis of the cholesterol side-chain cleavage (SCC) enzyme complex was studied in rat ovarian granulosa cells cultured for 48 h in serum-free medium. Cell proteins were radiolabeled with [35S]methionine, followed by immunoprecipitation of cholesterol side-chain cleavage cytochrome P-450 (P-450SCC) as well as the iron-sulfur protein adrenodoxin. Polyacrylamide gel electrophoresis and fluorography of the immunoprecipitates showed that TPA, when added in combination with FSH (50 ng/ml) or dibutyryl cAMP (Bt2cAMP; 1 mM), suppressed the stimulatory effects of these compounds on the synthesis of the SCC components in a concentration-dependent fashion. The effect of TPA was accompanied by decreased progesterone formation and decreased cAMP accumulation. The structural analog of TPA, phorbol-4 alpha-didecanoate, which does not activate protein kinase C (Ca2+/phospholipid-dependent enzyme), had no effect on the FSH- or Bt2cAMP-stimulated synthesis of SCC and progesterone or on cAMP formation. In addition to inhibiting the synthesis of these proteins, TPA greatly reduced the FSH- and Bt2cAMP-induced increase in levels of mRNA encoding the precursor form of P-450SCC. It is concluded that the effect of the phorbol ester TPA to inhibit FSH-stimulated progesterone formation in cultured ovarian granulosa cells of the rat involves decreased synthesis of the components of the SCC enzyme complex due to reduced levels of mRNA encoding the precursor forms of these proteins. The results are indicative that TPA not only inhibits FSH-mediated stimulation of cAMP formation but also may block cAMP-mediated induction of SCC synthesis. It is postulated that the effects of TPA may reflect the physiological role of protein kinase C in the regulation of ovarian steroidogenesis.

Animals↗
PubMed1987

Multiple species of bovine adrenodoxin mRNA. Occurrence of two different mitochondrial precursor sequences associated with the same mature sequence.

Bovine adrenodoxin mRNA is found to consist of several distinct mRNA species which can be divided into two sets. Each set utilizes at least three of four separate poly(A)+ addition sites providing an explanation of the three sizes of adrenodoxin mRNA (1.75, 1.4, and 0.95 kilobases) previously observed in bovine adrenocortical RNA by this laboratory (Okamura, T., John, M.E., Zuber, M.X., Simpson, E.R., and Waterman, M.R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5705-5709). The two sets are distinguished from one another by a unique 5' sequence leading to two different amino acid sequences (approximately 10% homology) for the precursor portion of this nuclear encoded, mitochondrial protein. A common mature adrenodoxin sequence is encoded by both sets of mRNA. One set of RNAs is 10-fold more abundant than the other, but the levels of both sets can be induced by treatment of primary bovine adrenocortical cell cultures with adrenocorticotropin. The biological significance of these two types of adrenodoxin precursor sequences remains obscure.

Adrenal Cortex↗
PubMed1987

Mature bovine adrenodoxin contains a 14-amino-acid COOH-terminal extension originally detected by cDNA sequencing.

Since the nucleotide sequence of bovine adrenodoxin cDNA is at variance with protein sequencing data in that it encodes an additional 14 amino acids at the COOH terminus, we used a specific antibody raised against this 14-amino-acid segment to examine its presence in: nascent precursor polypeptide chains, the processed mature adrenodoxin and mitochondria of both steroidogenic and nonsteroidogenic tissues. These studies reveal the presence of the extra peptide in the precursor form derived from in vitro translation and in the newly synthesized mature form as shown by [35S]methionine labeling of proteins in adrenocortical cells. Both the purified COOH-terminal synthetic peptide and purified mature adrenodoxin competed with radiolabeled adrenodoxin for immunoprecipitation by the anti-peptide antibody. Immunoblots revealed the presence of the extra peptide in purified adrenodoxin and in bovine adrenocortical, corpus luteal, kidney and liver mitochondria while it was not detectable in heart mitochondria. Thus, we conclude that mature adrenodoxin and its homologs in non-steroidogenic tissues contain the C-terminal extension following uptake into mitochondria. These results indicate structural homology between adrenodoxin and the iron-sulfur proteins of the kidney and liver and also suggest the presence of a second iron-sulfur protein in kidney and liver.

Adrenal Cortex↗
PubMed1987

Studies on the synthesis of sterol carrier protein-2 in rat adrenocortical cells in monolayer culture. Regulation by ACTH and dibutyryl cyclic 3',5'-AMP.

The effects of ACTH or dibutyryl cyclic AMP (Bt2cAMP) on the synthesis of sterol carrier protein-2 (SCP2) have been studied in rat adrenocortical cells in monolayer culture. Radiolabeling of total cellular proteins with [35S]methionine and immunoprecipitation with antibodies directed against rat liver SCP2, followed by polyacrylamide gel electrophoresis and fluorography, showed a 3-4-fold increase in the rate of synthesis of SCP2 in cells treated for 48 h with ACTH (1 microM) or Bt2cAMP (0.1 mM). The induction of SCP2 synthesis depended upon the concentrations of ACTH or Bt2cAMP with an ED50 of 8 and 100 nM, respectively, and increased linearly with time between 12 and 48 h of treatment. Immunoprecipitation of SCP2 synthesized in a rabbit reticulocyte in vitro translation system programmed with RNA isolated from cells treated with ACTH or Bt2cAMP revealed increased synthesis of SCP2 compared to RNA from control cells. The immunoprecipitable rat adrenal SCP2, synthesized in a cell-free translation system, showed mobility corresponding to Mr of 14,400 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was clearly larger than immunodetectable SCP2 synthesized in cultured adrenal cells (Mr = 11,300). The electrophoretic mobilities of rat liver SCP2 synthesized in cultured cells and in a cell-free translation system were the same as the respective forms from rat adrenal. It is concluded that the synthesis of SCP2 in rat adrenocortical cells is induced by ACTH and that the induction is mediated by cAMP and may involve increased levels of translatable mRNA encoding a higher molecular weight precursor form of SCP2, which presumably undergoes post-translational processing yielding the mature form.

Adrenal Cortex↗
PubMed1987

Regulation of the biosynthesis of steroidogenic enzymes.

Recombinant DNA technology can permit study of the regulation of steroid hydroxylase gene expression at three levels. The first of these is cAMP-regulated gene expression. In the adrenal, ACTH, via cAMP, increases the expression of the genes for all of the cytochrome P-450 species involved in the steroid biosynthetic pathway, as well as the iron-sulfur protein, adrenodoxin. This action of cAMP is inhibited by cycloheximide, suggestive of the involvement of a regulatory protein factor in mediating this action of cAMP. The second level is tissue-specific regulation of steroid hydroxylase gene expression. An example of this which we have studied is the expression of cholesterol side-chain cleavage cytochrome P-450 (P-450sec) and 17 alpha-hydroxylase cytochrome P-450 (P-450(17) alpha) in the bovine ovary. P-450sec is expressed at high levels in the corpus luteum but at low levels in follicles, whereas P-450(17)alpha is expressed in follicles, but is undetectable in the corpus luteum. The third level is fetal imprinting. A number of the cytochrome P-450 species involving in the steroidogenic pathway are expressed in the fetal adrenal at a time when exposure of the gland to ACTH is very low, suggestive that factor(s) other than pituitary ACTH mediate this expression in fetal life.

Adrenal Glands↗
PubMed1987

Ontogeny of adrenal steroid hydroxylases: evidence for cAMP-independent gene expression.

Total RNA from normal and anencephalic human fetal adrenals was examined by blot analysis for transcripts encoding P-450scc, P-450(11) beta, P-450(17) alpha, P-450C21 and adrenodoxin using bovine cDNA clones specific for these different enzymes. The specific contents of RNA encoding these components of the adrenocortical steroidogenic pathway were found to be similar in both types of adrenal tissue. Likewise, immunoblot analysis showed comparable concentrations of P-450scc, P450(17) alpha and adrenodoxin protein to be present in adrenal tissues from normal and anencephalic human fetuses. Immunoblot analysis of homogenates of fetal sheep adrenals of increasing gestational age (85-145 days) showed constant levels of P-450scc and P-450(11) beta, but increasing P-450(17) alpha content, especially near term. Both sheep fetuses prior to 136 days gestational age and human anencephalic fetuses are known to have extremely low circulating levels of immunoreactive ACTH as well as very low adrenal adenylate cyclase activity. Thus, it is concluded that factors other than pituitary ACTH which operate independent of adenylate cyclase activation are required for the initial expression (imprinting) of steroid hydroxylase genes.

Adenylyl Cyclases↗
PubMed1987

Effects of epidermal growth factor on the synthesis of the cholesterol side-chain cleavage enzyme complex in rat ovarian granulosa cells in primary culture.

The effect of epidermal growth factor (EGF) on the synthesis of the components of the cholesterol side-chain cleavage enzyme complex (SCC) was studied in rat ovarian granulosa cells. The cells were cultured for 48 h in the presence or absence of EGF (15 ng/ml) and/or FSH (50 ng/ml) after which proteins were radiolabeled by incubation with [35S]methionine followed by immunoprecipitation of newly synthesized P-450scc or adrenodoxin (ISP) with polyclonal antibodies directed against the corresponding proteins from bovine adrenal cortex. In addition the action of EGF on the level of translatable RNA for P-450scc was evaluated using a cell-free translation system programmed with RNA isolated from treated and untreated cells, followed by immunoisolation of newly synthesized proteins. Immunoisolated proteins were separated by polyacrylamide-gel electrophoresis, visualized by fluorography and quantified by densitometry. EGF stimulated progesterone formation by the cells 3-fold and potentiated the FSH-induced stimulation of progesterone formation, but had no effect on cAMP accumulation. EGF also stimulated the synthesis of P-450scc and ISP, and enhanced the FSH-induced synthesis of P-450scc and ISP in a concentration-dependent fashion with a maximal stimulation attained at concentrations ranging from 1.0 to 100 ng/ml. No appreciable changes in the induction pattern were observed when EGF and dibutyryl cyclic AMP (Bt2cAMP) were added together, as compared to when Bt2cAMP was added alone. Neither treatment affected the synthesis of the constitutive mitochondrial enzyme, F1-ATPase. Immunoisolation of P-450scc from the proteins synthesized in a rabbit reticulocyte in vitro translation system programmed with RNA isolated from EGF- and/or FSH-treated cells, revealed that EGF enhanced the FSH-stimulated synthesis of the precursor form of P-450scc.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Regulation of the induction of a cytochrome P-450 prostaglandin omega-hydroxylase by pregnancy in rabbit lung.

The mechanism of induction of an adult rabbit cytochrome P-450, prostaglandin (PG) omega-hydroxylase (P-450PG-omega), during pregnancy has been investigated. This P-450 isozyme regiospecifically hydroxylates PGE1, PGA1, and PGF2 alpha at carbon-20 (the omega position). The specific activity of this enzyme is induced from 0.07 nmol of 20-OH-PGE1 to 3.05 nmol of 20-OH-PGE1 formed per min per mg of microsomal protein in the lungs of 25- to 28-day pregnant rabbits as compared to nonpregnant rabbits. Immunoblotting studies with a polyclonal antibody raised against this P-450 have shown that there is a concomitant gestational age-dependent increase in the amount of P-450PG-omega microsomal protein accompanying the increase of omega-hydroxylase activities. Within 3 days postpartum, both omega-hydroxylase activity and the amount of immunodetectable P-450PG-omega drop precipitously to near control levels. In vitro translation of total cellular RNA, extracted from the lungs of pregnant rabbits at various days throughout gestation, and immunoprecipitation of newly synthesized P-450PG-omega demonstrated a similar gestational age-dependent increase in translatable P-450PG-omega mRNA, as was observed with omega-hydroxylase activity and P-450PG-omega protein levels. These data suggest that the induction of this P-450 may occur at the transcriptional level and, furthermore, that control of cytochrome P-450PG-omega expression in the rabbit lung is tightly regulated at both protein and mRNA levels.

Animals↗
PubMed1987

Regulation of the synthesis of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the bovine ovary in vivo and in vitro.

In order to investigate the pattern of ovarian cholesterol biosynthesis during the bovine estrous cycle, tissue concentrations of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme in the synthesis of cholesterol, were determined by immunoblot techniques. Medium-sized (9-11 mm) and large (14-18 mm) follicles, after removal of follicular fluid by centrifugation, and corpora lutea from the early, early-mid, late-mid, and late stages of the luteal phase were used (n = 5 per group). The specific content (per microgram of tissue homogenate protein) and total content of HMG-CoA reductase in medium-sized and large follicles were substantially lower than those of corpora lutea of the early-mid and late-mid luteal phase. The specific content was elevated in a number of the corpora lutea from the early luteal phase and was low in regressing corpora lutea. Thus during the midluteal phase, when steroid hormone production is elevated, the total and specific contents of HMG-CoA reductase are also elevated. To investigate the mechanisms whereby the levels of HMG-CoA reductase are regulated, primary monolayer cultures of bovine luteal cells (early-mid and late-mid luteal phase) were used. Cells were cultured for 24 h in Dulbecco's modified Eagle's medium containing lipoprotein-poor fetal calf serum (2% vol/vol). At this concentration there was no stimulation of the production of progesterone above that seen with no addition of serum. Under these conditions the total and specific contents, and the synthesis, of HMG-CoA reductase were stimulated by treatment with (Bu)2cAMP (1 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Levels of messenger ribonucleic acid encoding cholesterol side-chain cleavage cytochrome P-450, 17 alpha-hydroxylase cytochrome P-450, adrenodoxin, and low density lipoprotein receptor in bovine follicles and corpora lutea throughout the ovarian cycle.

To investigate the molecular basis for the pattern of ovarian steroid production during the bovine estrous cycle, the relative levels of mRNA specific for cholesterol side-chain cleavage cytochrome P-450, 17 alpha-hydroxylase cytochrome P-450, adrenodoxin, and low density lipoprotein receptor were determined in ovarian antral follicles of differing size (less than 3-18 mm) and corpora lutea from the early, early-mid, late-mid, and regressionary stages. Total and poly(A)+ RNA was size-fractionated on agarose-formaldehyde gels, transferred to nylon filters and hybridized to specific 32P-labeled probes. The levels of mRNAs for the rate-limiting enzymes in the conversion of cholesterol into progesterone, namely cholesterol side-chain cleavage cytochrome P-450 and its electron donor, adrenodoxin, were higher in corpora lutea than in follicles. Conversely the levels of mRNA specific for the key regulatory enzyme in the conversion of pregnenolone or progesterone to androgen, namely 17 alpha-hydroxylase cytochrome P-450, were high in all antral follicles examined but were low in young corpora lutea and undetectable in more mature corpora lutea. Low density lipoprotein receptor mRNA was detectable in antral follicles and corpora lutea but the levels were greater in corpora lutea. These results suggest that the pattern of changes in steroid hormone biosynthesis during the bovine estrous cycle and in the ovarian content of steroidogenic enzymes is related to and probably dependent upon the pattern of change in levels of mRNAs for steroidogenic enzymes and related proteins.

Adrenodoxin↗