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M R Waterman

Publications and source records attributed to M R Waterman.

At least 127 records · Page 7Linked to original sources
PubMed1991

The heterologous expression of the cytochromes P450: a new approach for the study of enzyme activities and regulation.

The superfamily of cytochrome P450s encompasses a vast arena of biologically important reactions. The ever-increasing numbers of P450s and the diversity of their enzymatic properties dictate the need to develop new approaches for studying their chemical, physical and catalytic properties. The heterologous expression of P450s in various cell systems (e.g., COS cells, yeast, E. coli, etc.) now provides a means of producing recombinant proteins for such studies. The example is presented of the expression of P450(17)alpha in COS cells and the use of this technique for the comparison of the enzymatic properties of the rat, bovine and human enzymes. Further, studies are described whereby cotransfection results in the simultaneous expression of more than one P450 permitting the construction of 'designer membranes' for assessing protein-protein interactions and the reconstruction of complex pathways of metabolism. Recent advances with genetically engineered systems point to the power of the transfection technique for the study of structure-function relationships with this class of important hemoproteins.

Animals↗
PubMed1991

Deletion within the CYP17 gene together with insertion of foreign DNA is the cause of combined complete 17 alpha-hydroxylase/17,20-lyase deficiency in an Italian patient.

The molecular basis of 17 alpha-hydroxylase/17,20-lyase deficiency syndrome in a 14-yr-old 46,XY Italian patient was investigated by amplification, subcloning, and sequencing of specific exonic sequences from genomic DNA samples. A homozygous mutation, consisting of a 518-basepair (bp) deletion combined with a 469-bp insertion, was identified in the CYP17 gene of the patient. The deletion spans much of exon II, the whole intron 2, and a portion of exon III. A part (156 bp) of the inserted sequence shows 95.5% identity to the nuclear antigen-binding site on Marek disease virus DNA and sequences found in rearranged mitochondrial DNA of rat hepatoma cells. A similar degree of sequence identity (99%) was also found between the above sequences and part of the lac operon of E. coli. The inserted sequence is lacking the BamHI site in intron 2 of CYP17 and contains an in-frame stop codon (TAA). Thus, the mutated gene encodes a truncated nonfunctional steroid hydroxylase, giving rise to symptoms associated with complete combined 17 alpha-hydroxylase/17,20-lyase deficiency. The family history revealed that the patient is the child of a consanguineous marriage and has two genotypically and phenotypically female sisters also suffering from symptoms of the disease. Investigation of genomic DNA from these sisters revealed that in each case both CYP17 alleles contained the same mutation. On the other hand, the parents were found to be heterozygous for this mutation. The insertion could not be found in DNA from normal individuals or in the CYP17 gene of other Italian patients with the 17 alpha-hydroxylase deficiency syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1991

Expression of mRNA encoding basic fibroblast growth factor (bFGF) in bovine corpora lutea and cultured luteal cells.

A radiolabelled cRNA was synthesized using a 1.4 kb cDNA complementary to mRNA encoding bovine basic fibroblast growth factor (bFGF) as a template, and used as a probe to investigate the expression of mRNA encoding bFGF in bovine ovarian tissue, and luteal cells in primary culture. Northern analysis of poly(A +)RNA prepared from follicles and corpora lutea of various stages revealed a major mRNA species of 7 kb in corpora lutea of all stages, the amount of which was higher late in the luteal phase. No hybridizable message was detectable in follicles of any size. When luteal cells were established in primary culture, expression of the 7 kb mRNA species was maintained. This expression was increased markedly when cells were treated with LH/hCG or Bt2cAMP. Prostaglandin F-2 alpha treatment caused a marked decrease in the basal content of this 7 kb mRNA, and also severely impaired the ability of LH to stimulate this expression.

Animals↗
PubMed1991

Adrenaline stimulates cholesterol side-chain cleavage cytochrome P450 mRNA accumulation in bovine adrenocortical cells.

The effect of adrenaline on the accumulation of mRNA encoding cholesterol side-chain cleavage cytochrome P450 (P450scc) and cortisol secretion was studied in bovine adrenocortical cells in primary culture. Treatment of cultured cells with adrenaline resulted in a 2-fold increase in mRNA encoding P-450scc, as revealed by Northern blot analysis. Under these conditions the maximal stimulation with ACTH resulted in a 6-fold accumulation of mRNA encoding P450scc. The effect of adrenaline on the expression of P450scc was abolished by the beta-blocker propranolol, while propranolol had no effect on ACTH-induced P450scc mRNA accumulation. Adrenaline stimulated the secretion of cortisol in a dose-dependent manner with a median effective dose of 0.5 mumol/l. The adrenaline-stimulated cortisol secretion amounted to 42% of the effect of ACTH (0.1 nmol/l). Upon adrenaline treatment, cAMP concentration in the culture medium increased about 50-fold over the basal value. It is concluded that the stimulatory action of adrenaline upon cortisol formation requires beta-adrenergic receptors and is due, at least in part, to a cAMP-mediated increases in the accumulation of mRNA encoding P450scc.

Adrenal Cortex↗
PubMed1991

Expression of genes encoding steroidogenic enzymes in the bovine corpus luteum.

To examine the regulation of P-450SCC expression at the molecular level, a transfection protocol specific for bovine luteal cell cultures was developed. Among several commonly used transfection methods, electroporation yielded highest transfection efficiencies. Transfection of primary cultures of bovine luteal cells with chimaeric DNA constructs containing increasing deletions of the 5'-flanking region of P-450SCC fused to the chloramphenicol acetyl transferase (CAT) reporter gene allowed two conclusions. Firstly, sequences of the P-450SCC 5'-flanking region were capable of conferring basal expression and cAMP responsiveness to the CAT reporter gene and secondly, the region -186 to -100 bp appears to be required for these two types of regulation of gene expression. On the other hand, 5'-flanking regions of P-450(17 alpha) were not capable of conferring any regulation of gene expression to the CAT reporter gene in these cells. Thus, the physiologically observed regulation of the endogenous cytochromes P-450SCC and P-450(17 alpha) is closely reflected by CAT reporter gene expression. These experiments will allow investigation of the molecular mechanisms underlying the regulation of the genes encoding steroidogenic enzymes throughout the ovarian cycle.

Animals↗
PubMed1990

Amino acid substitutions Phe66----Leu and Ser126----Pro abolish cortisol and aldosterone synthesis by bovine cytochrome P450(11)beta.

A cDNA clone encoding the complete protein sequence of the precursor form of bovine cytochrome P450(11)beta has been constructed using a combined technique of first strand cDNA synthesis by reverse transcription followed by polymerase chain reaction. Upon expression of this cDNA in COS 1 cells the P450(11)beta is found to be proteolytically processed and localized in the mitochondrion. This cDNA encodes the major form of P450(11)beta found in bovine adrenal cortex (designated 11 beta-3; Kirita, S., Morohashi, K., Hashimoto, T., Yoshioka, H., Fujii-Kuriyama, Y., and Omura, T. (1988) J. Biochem. 104, 683-686) and is capable of catalyzing 11 beta-hydroxylation of deoxycorticosterone, 11-deoxycortisol, and androstenedione in COS 1 cells as well as aldosterone synthesis from deoxycorticosterone. In addition, a second form of P450(11)beta (herein designated 11 beta-4), having no detectable 11 beta-hydroxylase activity or aldosterone synthase activity, was found in the local bovine population by this cloning procedure. These two forms of P450(11)beta (11 beta-3 and 11 beta-4) contain five amino acid differences between them, all located within the amino-terminal half of the molecules. By changing 2 of the amino acids in the inactive form to the corresponding amino acids in the active form (Leu66----Phe and Pro126----Ser) both 11 beta-hydroxylase and aldosterone synthetase activities were completely restored. Neither of these changes alone led to detectable activity. Thus, upon expression in mitochondria of heterologous cells, bovine P450(11)beta catalyzes both 11 beta-hydroxylation and aldosterone synthesis as reported previously for the purified enzyme in an in vitro reconstituted system, and Phe66 and Ser126 seem to be important residues in maintaining both activities.

Adrenal Cortex↗
PubMed1990

Construction and expression of human/bovine P45017 alpha chimeric proteins: evidence for distinct tertiary structures in the same P450 from two different species.

In the human and bovine adrenal cortex, 17 alpha-hydroxylase (P45017 alpha) catalyzes reactions involved in the production of C21-glucocorticoids (17 alpha-hydroxylation) and C19-androgens (17,20-lyase). The bovine and human forms of P45017 alpha share 71% primary sequence identity. Using naturally occurring restriction sites common to cDNAs encoding both human and bovine P45017 alpha, we have constructed bovine/human (bovine amino terminus and human carboxy terminus) and human/bovine (human amino terminus and bovine carboxy terminus) cDNAs that have been expressed in COS 1 cells, and the enzymatic properties of the resultant chimeric proteins have been examined. The three bovine/human chimeras studied have 17 alpha-hydroxylase activities intermediate between those of the wild-type bovine and wild-type human enzymes, although the 17,20-lyase activity of these chimeras is significantly lower than that of either of the wild-type enzymes. Surprisingly, the opposite chimeras (those containing a human amino-terminal sequene and a bovine carboxy-terminal sequence) are all virtually inactive, even though they appear to be expressed at normal levels. These results indicate that the folding of P45017 alpha initiated by the bovine amino terminus can accommodate human P45017 alpha sequences of various lengths to produce a relatively normal 17 alpha-hydroxylase having decreased 17,20-lyase activity. On the other hand, folding initiated by the human P45017 alpha amino terminus does not easily accommodate bovine carboxy-terminal sequences to produce a functional enzyme. Presumably this difference arises from the fact that the tertiary structures of the bovine and human forms of P45017 alpha are sufficiently different so that interchanging sequences will not lead to functional enzymes in a predictable fashion.

Amino Acid Sequence↗
PubMed1990

Incorporation of steroidogenic pathways which produce cortisol and aldosterone from cholesterol into nonsteroidogenic cells.

Cortisol production from cholesterol requires the activity of four steroid hydroxylases: cholesterol side chain cleavage cytochrome P-450 (P-450scc), 17 alpha-hydroxylase cytochrome P-450 (P-45017 alpha), 21-hydroxylase cytochrome P-450 (P-450C21) and 11 beta-hydroxylase cytochrome P-450 (P-45011 beta). We have previously shown that transformed, nonsteroidogenic COS 1 cells derived from monkey kidney are a useful system for expression of various forms of cytochrome P-450. The present study shows that COS 1 cell cultures multiply transfected with six plasmids containing all four steroid hydroxylases, 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase (3 beta HSD) and adrenodoxin produce cortisol and aldosterone when 22(R)-hydroxycholesterol is supplied to the system. When pregnenolone is used as substrate, various intermediate metabolites are detected at different time points further establishing the incorporation of complete functional steroidogenic pathways into the nonsteroidogenic cell cultures. Since the first and the last reactions in these pathways take place in the mitochondrion, the movement of various intermediate metabolites from mitochondrion to endoplasmic reticulum and back to mitochondrion occurs in and between COS 1 cells.

Adrenodoxin↗
PubMed1990

cAMP-dependent transcription of the human CYP21B (P-450C21) gene requires a cis-regulatory element distinct from the consensus cAMP-regulatory element.

By utilizing chimeric genes constructed from 5'-flanking sequences of the human CYP21B (P-450C21) gene and reporter genes (chloramphenicol acetyltransferase or rabbit beta-globin), a 34-nucleotide sequence has been found to be required for cAMP-dependent transcription. This sequence (-129/-96 base pairs) shows no homology to that of the consensus (CRE) cAMP-regulatory element. Gel retardation analysis shows that a protein-DNA complex is formed between this DNA sequence and nuclear proteins from mouse adrenal Y1 tumor cells or bovine adrenal cortical cells or human fetal adrenal tissue and that formation of this complex cannot be competed by DNA containing the consensus CRE sequence. Even though cAMP-enhanced accumulation of P-450C21 mRNA in primary cultures of bovine adrenocortical cells is inhibited by the protein synthesis inhibitor, cycloheximide, reporter gene transcription enhanced by the cAMP-responsive -129/-96-base pair fragment of the human CYP21B gene is not. We conclude that cAMP-dependent transcription of the human P-450C21 gene (CYP21B), an event required for maintenance of optimal steroidogenic capacity in the adrenal cortex, involves a stable transcription factor(s) distinct from the CRE-binding protein. Furthermore the cAMP-dependent cis-regulatory element of the human P-450C21 gene is distinct from those found associated with the other steroid hydroxylase genes, 17 alpha-hydroxylase cytochrome P-450, cholesterol side chain cleavage cytochrome P-450, and 11 beta-hydroxylase cytochrome P-450, suggesting that each of these genes may require its own set of specific transcription factors for cAMP-dependent regulation.

Adrenal Cortex↗
PubMed1990

Transcriptional regulation of the bovine CYP17 (P-450(17)alpha) gene. Identification of two cAMP regulatory regions lacking the consensus cAMP-responsive element (CRE).

Regions within the 5'-flanking sequence of the bovine CYP17 (P-450(17)alpha) gene which are required for cAMP-dependent regulation of transcription have been localized by transient transfection of chimeric reporter gene constructs into mouse adrenal tumor Y1 cells. Two sequences have been found which individually confer cAMP responsiveness to reporter genes; they are located at -243/-225 and -80/-40 base pairs (bp). Obvious sequence homology between these two regions is not apparent. Gel shift competition analysis indicates that nuclear protein(s) binding to the -243/-225-bp region can be competed for by the addition of a double-stranded oligonucleotide containing a consensus cAMP-responsive element (CRE) from the human chorionic gonadotropin alpha gene, whereas addition of this CRE does not abolish protein-DNA complexes formed with fragments containing the -80/-40-bp sequence. Gel shift and Southwestern analysis indicate that the -243/-225-bp region of the P-450(17)alpha gene and the CRE both bind a 47-kDa protein and that the CRE binds additional proteins (43 and 68 kDa) not apparently recognized by the -243/-225-bp sequence. Thus cAMP-dependent regulation of the bovine P-450(17)alpha gene appears to involve two independent cis-regulatory regions, neither of which contains a consensus CRE. Based on protein binding analysis, one of these regions (that including -80/-40 bp) is distinct from the consensus CRE while the other (that containing -243/-225 bp) may be related to the consensus CRE.

Adrenal Gland Neoplasms↗
PubMed1990

Characterization of the promoter/regulatory region of the bovine CYP11A (P-450scc) gene. Basal and cAMP-dependent expression.

The promoter/regulatory region of the bovine CYP11A (P-450scc) gene was cloned from a bovine genomic library. One major start site of transcription was identified by primer extension analysis with a minor start site four nucleotides further upstream. A putative TATA box is located at position -31, and at position -68 resides a putative binding site for the transcription factor Sp1. Transient transfection of chimeric reporter gene constructs into mouse adrenal tumor Y1 cells was used to locate regions within the P-450scc 5'-flanking sequences that are important for basal and cAMP-dependent transcription of the reporter genes. While cAMP-dependent accumulation of mRNA derived from expression of the endogenous bovine P-450scc gene can be inhibited by protein synthesis inhibitors, transcription of reporter gene constructs containing the promoter/regulatory region of the P-450scc gene is not affected by cycloheximide following transient transfection of Y1 cells or primary bovine adrenocortical cells. Basal expression of these constructs as well as cAMP responsiveness is reduced upon deletion of sequences between -186 and -101, further deletion to -50 leading to loss of virtually all the remaining cAMP responsiveness. The sequence between -183 and -83 alone will direct both basal and cAMP-enhanced transcription when fused to a heterologous promoter and is equally active in either the correct or reverse orientation. No homology to the consensus cAMP-responsive element (CRE) or AP-2 binding site is found in this region whereas an activator protein 1-like sequence is found at position -116. It is concluded that the cAMP responsiveness of P-450scc gene expression is mediated by sequences different from canonical consensus regulatory elements. Whether or not there are sequences conferring cAMP responsiveness which are common both to P-450scc and the other steroidogenic P-450 genes remains to be established.

Adrenal Gland Neoplasms↗
PubMed1990

Angiotensin II inhibits luteinizing hormone-stimulated cholesterol side chain cleavage expression and stimulates basic fibroblast growth factor expression in bovine luteal cells in primary culture.

Angiotensin II has been identified immunohistochemically in the ovaries of both rats and humans. Here we present evidence that angiotensin II (an extremely vasoactive agent in a wide range of tissues) may be involved in the regulation of the major steroidogenic enzyme in the ovary, cholesterol side chain cleavage cytochrome P-450 (P-450scc), as well as of basic fibroblast growth factor (bFGF), which has been implicated as an angiogenic factor in the bovine corpus luteum. We have used primary cultures of bovine luteal cells to examine the effect of angiotensin II and its receptor antagonist, saralasin, on expression of mRNA encoding bFGF as well as on progesterone production and the expression of mRNA encoding cholesterol side chain cleavage cytochrome P-450 (P-450scc). Neither angiotensin II nor saralasin when added alone to the culture medium had any effect on basal progesterone production. Luteinizing hormone (LH) caused a 15-fold increase in progesterone accumulation after 24 h of exposure which was reduced to 5-fold in the presence of angiotensin II. This appeared to be receptor-mediated in that although saralasin alone had no effect on LH-stimulated progesterone accumulation, it significantly reversed the inhibition by angiotensin II. This pattern was mirrored by the levels of mRNA encoding P-450scc, i.c., LH induced the highest levels of expression of this message, these levels were reduced by angiotensin II, and saralasin partially overcame this reduction. Levels of mRNA encoding bFGF were elevated by both LH and angiotensin II. Treatment with saralasin, however, resulted in complete inhibition of bFGF mRNA expression in the presence of both LH and angiotensin II. These results suggest a role for angiotensin II to mediate the action of LH as a regulator of bFGF expression and hence, potentially, angiogenesis. Local production of angiotensin II might also contribute to the refractoriness of luteal progesterone secretion to LH at the time of luteal regression.

Angiotensin II↗
PubMed1990

Combined 17 alpha-hydroxylase/17,20-lyase deficiency due to a 7-basepair duplication in the N-terminal region of the cytochrome P45017 alpha (CYP17) gene.

17 alpha-Hydroxylase deficiency is characterized by defects in either or both of the 17 alpha-hydroxylase/17,20-lyase activities. We have elucidated the molecular basis of the combined deficiency of these activities in a Japanese female who is genotypically male and the child of a consanguineous marriage. The complete exonic sequence of the patient's CYP17 (P45017 alpha) gene revealed a seven-basepair duplication (GCGCACA) in exon 2 which leads to a frame shift and, subsequently, a premature stop codon. Because this stop codon occurs N-terminal to the heme-binding sequence, the presence of this mutation leads to the absence of a functional P45017 alpha-protein in adrenal cortex and testis. This, in turn, leads to an absence of sex steroids and excessive secretion of steroids with mineralocorticoid activity and, consequently, female external genitalia and hypertension in this 46XY patient.

Adolescent↗