Sequence and developmental expression of Cyp18, a member of a new cytochrome P450 family from Drosophila.

Preliminary studies of a partial cDNA clone of the Eig17-1 gene from Drosophila melanogaster have shown that it encodes a probable cytochrome P450 of unknown function. To further characterize the Eig17-1 gene product, a full-length cDNA clone was isolated from a late-larval cDNA library and sequenced. Eig17-1 encodes a protein of 538 amino acids. The predicted protein is a cytochrome P450 that has been assigned to a new family, CYP18. The CYP18 protein is most closely related to steroid and xenobiotic metabolizing P450s of family CYP2 (30-33% identity), and to vertebrate steroidogenic P450s of families CYP17 and CYP21 (25-28% identity). Developmental Northern blot analysis revealed five distinct periods of Cyp18 expression during postembryonic development. Each period lasted 12-15 h, and was tightly correlated with reported ecdysteroid pulses in the first, second and third larval instars, at the time of pupariation and in pupae. This pattern of expression is consistent with the known induction of Cyp18 transcription by 20-hydroxyecdysone at the time of pupariation and suggests that ecdysteroids are major regulators of Cyp18 expression throughout postembryonic development. Northern blot analysis of RNA isolated from different prepupal tissues indicates that Cyp18 is differentially expressed in various ecdysteroid-responsive tissues. High Cyp18 expression was observed in body wall and gut while negligible expression was observed in salivary glands and fat body.

Focal calcifications in otherwise ultrasonographically normal ovaries.

PURPOSE: To analyze the natural history, clinical importance, and need for follow-up in patients with discrete calcifications in otherwise ultrasonographically (US) normal ovaries. MATERIALS AND METHODS: US scans obtained in 28 women with ovarian calcifications but without masses or other structural abnormalities were retrospectively reviewed. Follow-up findings were available in 15 women (mean age, 38.0 years; range, 26-72 years). RESULTS: Two of the 15 women had trilateral calcifications. Thus, 17 ovaries had focal calcifications. Histopathologic confirmation was obtained in 10 cases; follow-up US findings, five cases; and follow-up laparoscopic findings, two cases. In 13 (76%) of the 17 ovaries, calcifications were not clinically important. In four (24%) of the 17, the calcification was the initial or only manifestation of a neoplasm. Lesions were benign in all four of these ovaries: There was one dermoid, one mucinous cystadenoma, and two adenofibromas. CONCLUSION: Until more data are available, findings of calcifications in ovaries with otherwise normal US findings warrant some form of follow-up.

Human ovarian tumor cells: a potential model for thecal cell steroidogenesis.

Ovarian thecal cell production of C19 steroids (i.e. dehydroepiandrosterone, androstenedione, and testosterone) is necessary to provide substrate for granulosa cell biosynthesis of estrogen; however, excessive production of C19 steroids can lead to disorders associated with androgen excess. Because of difficulties in obtaining adequate numbers of thecal cells, the biomolecular regulation of C19 steroid production and expression of steroidogenic enzymes is not well defined. We have overcome this obstacle by developing a highly dependable and unique human ovarian thecal-like tumor (HOTT) cell culture model system from an ovarian tumor found to produce excessive amounts of C19 steroids. Aliquots of freshly dispersed tumor cells were frozen for future use. Once placed in monolayer culture, HOTT cells proliferated and could be maintained for extended periods. Acutely, cultured HOTT cells increased progesterone and cAMP production in response to 2 h of forskolin treatment. These cells were, however, unresponsive to treatment with LH. Steroid hormone production continued in cells that were maintained in culture for up to 2 months. Analysis of the steroids produced by HOTT cells was accomplished using RIA and high performance liquid chromatography. Under basal conditions, HOTT cells produced mainly 17 alpha-hydroxyprogesterone and progesterone. Treatment with forskolin or dibutyryl cAMP (dbcAMP) increased the production of progesterone and 17 alpha-hydroxyprogesterone as well as C19 steroids. Treatment of monolayer cultures of HOTT cells with forskolin (0.01 to 20 mumol/L) or dbcAMP (0.01 to 1 mmol/L) for 48 h increased the production of androstenedione (8- to 15-fold) and progesterone (2- to 5-fold). In HOTT cells chronically treated with forskolin or dbcAMP (up to 72 h), progesterone production was observed to plateau, although the amount of androstenedione continued to increase. The enzymatic activities of both 3 beta-hydroxysteroid dehydrogenase (6-fold), and 17 alpha-hydroxylase P450 (P450c17; 9-fold) were also increased by activation of the protein kinase A messenger pathway. Treatment of HOTT cells with forskolin caused a time-dependent induction of the messenger RNAs for cholesterol side-chain cleavage P450, 3 beta-hydroxysteroid dehydrogenase, and P450c17. No changes in steroidogenic enzyme expression were observed following treatment with LH. In conclusion, these data demonstrate that certain ovarian tumor cells may serve well as appropriate models to study the molecular mechanisms regulating human ovarian thecal cell C19 steroidogenesis and the expression of steroid-metabolizing enzymes.

Human NCI-H295 adrenocortical carcinoma cells: a model for angiotensin-II-responsive aldosterone secretion.

Excessive secretion of aldosterone from the adrenal results in the most common form of endocrine hypertension. An understanding of the regulatory processes involved in aldosterone synthesis and release is needed to define the biomolecular mechanisms controlling excessive production of aldosterone. However, in vitro studies regarding the regulatory mechanisms of human aldosterone production have been limited because of difficulties in obtaining tissue and the subsequent isolation of aldosterone-secreting glomerulosa cells. Herein we describe an adrenocortical carcinoma cell line, NCI-H295, which provides a suitable angiotensin-II (AII)-responsive model system to investigate the acute and chronic regulation of aldosterone synthesis. The cells were characterized with regard to the effects of AII on second messenger systems, aldosterone release, and levels of aldosterone synthase (P450c18) mRNA. In the presence of lithium, AII caused a rapid, but transient, increase in the production of inositol tris- and bisphosphates, whereas a prolonged gradual accumulation of inositol monophosphate occurred. Treatment with AII resulted in a 4.5-fold increase in total inositol phosphates in a concentration-dependent manner and an increase in intracellular cytoplasmic free Ca2+. Significant increases in aldosterone (3.5-fold) were detected within 1 h of AII addition. Aldosterone release occurred in a concentration-and time-dependent manner. The type 1 AII (AT1) receptor was shown to be responsible for activation of phosphoinositidase-C, increased intracellular free Ca2+, and aldosterone production, as determined by use of the AT1 receptor antagonist DuP753. In addition, AII treatment resulted in a time-dependent increase in levels of P450c18 mRNA, as detected by RNAse protection assay. In summary, NCI-H295 cells provide a valuable model system to define mechanisms regulating human aldosterone production.

Regulation of human adrenal carcinoma cell (NCI-H295) production of C19 steroids.

The regulation of biosynthesis of the adrenal C19 steroids (the so-called adrenal androgens) remains unclear. Understanding adrenal production of C19 steroids is important when the benefits of these steroids are considered on processes and diseases associated with aging. In vitro studies defining the mechanisms that regulate the production of human adrenal C19 steroids have been limited because of the difficulties in obtaining adrenal tissue. A cell line that retains differentiated adrenal functions would greatly facilitate research in this area. Herein, we describe the use of the human adrenocortical tumor H295 cell line as a model to evaluate mechanisms controlling C19 and C21 steroid production. The cells were characterized with regard to ACTH, forskolin, and dibutyryl cAMP (dbcAMP) responsiveness, as measured by increased cAMP production, synthesis of steroids, and induction of 17 alpha-hydroxylase cytochrome P450 (P450c17). Forskolin and dbcAMP, which were more effective than ACTH, enhanced the production of cortisol, dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), and androstenedione over a 48-h treatment period. Comparison of the relative amounts of measured steroid secreted under forskolin treatment indicated that the primary product was cortisol (70%), followed by androstenedione (14%), DHEA (9%), and DHEAS (7%). Cortisol was also demonstrated to be the major steroid product by examination of UV-detectable steroids after high performance liquid chromatographic separation. The increases in steroid production caused by ACTH, forskolin, and dbcAMP occurred in a concentration- and time-dependent manner. A key enzyme in the production of C19 steroids is P450c17. ACTH, forskolin, and dbcAMP increased the activity of 17 alpha-hydroxylase by approximately 2.5-, 10-, and 10-fold, respectively. These effects on enzyme activity occurred in a concentration-dependent manner and coincided with increased levels of P450c17 mRNA. In summary, H295 cells should provide a much-needed model to study mechanisms controlling the secretion of glucocorticoids and C19 steroids, because steroid production in these cells is hormonally controlled and associated with the induction of P450c17.

Prospective evaluation of patients with suspected hip fracture and indeterminate radiographs: use of T1-weighted MR images.

To assess the diagnostic efficacy of T1-weighted magnetic resonance (MR) images when findings on radiographs of the hip are indeterminate, 20 patients with suspected hip fracture and indeterminate radiographs were prospectively evaluated. MR imaging was performed with surface coil technique; one coronal T1-weighted (repetition time msec/echo time msec = 400/19) MR image was obtained. The total charge for the examination was $448 (compared with the following usual charges: radionuclide bone scan, $455; tomography, $299; and computed tomography, $797). When MR imaging and clinical outcome were used as the standard of reference, the prospective accuracy of MR imaging in diagnosis of the presence or absence of hip fracture was 100% (20 correct diagnoses). All MR studies were diagnostic. Of the 13 patients with fracture (eight with trochanteric and five with subcapital fracture), 10 patients (77%) underwent surgery and three patients (23%) received conservative treatment. T1-weighted MR images can enable diagnosis or exclusion of hip fracture whenever radiographs are indeterminate. The imaging time is approximately 7 minutes, and the cost of the examination can be made competitive with costs of other advanced imaging examinations.

The effects of immediate continuous passive motion on pain during the inflammatory phase of soft tissue healing following anterior cruciate ligament reconstruction.

Continuous passive motion (CPM) may have potential application as a physical modality in decreasing acute pain. The purpose of this study was to examine the effects of CPM immediately following an arthroscopically-assisted anterior cruciate ligament (ACL) reconstruction utilizing bone-patella-bone autograft on acute pain during the inflammatory phase of soft tissue healing. Acute pain was measured by assessing the amount of pain medication (amount of narcotic delivered from the patient-controlled analgesia (PCA) pump during the first postoperative 24 hours and the total intake of oral medication during the second and third postoperative days), the need for pain medication (number of times the patient pushed the PCA button during the first postoperative 24 hours), and perceived pain (graphic pain scale measuring antalgic sensation). Thirty patients (15-45 years old) participated in this study. The patients were prospectively randomized into two groups, CPM and non-CPM. Both groups followed an identical postoperative rehabilitation program except for the CPM groups using a CPM device. The design of this study included the collection of data during the inflammatory phase of soft tissue healing. The results indicated that the initiation of CPM immediately following an ACL reconstruction had a significant (p < .05) effect on decreasing the amount of medication consumed by the patient and a significant (p < .05) decrease in the patient's need for medication during the inflammatory phase. There was no statistical significance in the level of perceived pain between the groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The clinical use of continuous passive motion in physical therapy.

Continuous passive motion (CPM) has become a common rehabilitation modality for athletes with orthopaedic problems. The purpose of this review is to examine the current literature on the historical perspective of motion, physiological effects of immobilization on bone and soft tissue, clinical CPM studies, animal CPM studies, descriptions of the CPM device, and clinical uses of CPM in physical therapy. The authors conclude that there is little clinical research evaluating the efficacy of protocols for CPM usage in numerous applications for orthopaedic problems. They recommend that further studies regarding the efficacy of CPM treatment be initiated. J Orthop Sports Phys Ther 1992;15(3):132-140.

Sports medicine in Hawaii: care of the high school athlete in Oahu's public schools.

A recent study by the National Athletic Trainers Association indicated that injuries occur more often during practice than during games in high school athletics. Results of the 3-year study indicated that 60% of basketball injuries occurred in boys and young men during practice and 59% occurred in women. About 2/3 of the estimated 120,000 injuries suffered by prep wrestlers each year happen during practice. The study found that an average of 331,865 high school football players (1/3 of the million who play football each year) were sidelined by an injury at least once. With these statistics in mind, our study looks at the approach to the management of injury in the public schools that make up the Oahu Inter-scholastic Association (OIA) in the state of Hawaii. The estimated number of student athletes that participate in organized athletics in the OIA is 7,960 and the number of coaches is 1,189.

Rat P450(17 alpha) from testis: characterization of a full-length cDNA encoding a unique steroid hydroxylase capable of catalyzing both delta 4- and delta 5-steroid-17,20-lyase reactions.

A cDNA clone encoding the complete rat 17 alpha-hydroxylase (P450(17 alpha] from testis has been identified and sequenced. The deduced amino acid sequence is found to have 69% similarity with human P450(17 alpha), 64% similarity with bovine P450(17 alpha), and 47% similarity with chicken P450(17 alpha). The protein contains 507 amino acids being one amino acid shorter than the human P450(17 alpha) as the result of a codon being absent at the position of amino acid 139 in the human sequence. The cDNA hybridizes to a single mRNA (approximately 2.0 kilobases) in rat testis RNA and Southern analysis indicates the presence of a single CYP17 gene in the rat genome. Expression of this cDNA in COS1 cells leads to production of a steroid hydroxylase which is capable of converting both 17 alpha-hydroxypregnenolone and 17 alpha-hydroxyprogesterone into C19 steroids, dehydroepiandrosterone, and androstenedione, respectively. This activity profile is distinct from that of either the human or bovine forms of P450(17 alpha) which are unable to catalyze 17,20-lyase conversion of delta 4-C21 steroids to delta 4-C19 steroids at significant rates.

Deduced amino acid sequence of mature chicken testis ferredoxin.

The cDNA sequence encoding the complete mature form of the steroidogenic ferredoxin from chicken testis has been determined and the amino acid sequence deduced therefrom has been compared with the sequences of bovine, human and porcine steroidogenic ferredoxins. The chicken sequence is between 84% and 88% identical with those of the other mitochondrial iron-sulfur proteins. Thus, the amino acid structure of steroidogenic ferredoxins which transfer electrons to mitochondrial forms of cytochrome P-450 has been very highly conserved over evolutionary time.

Gerbil adrenal 11 beta- and 19-hydroxylating activities respond similarly to inhibitory or stimulatory agents: two activities of a single enzyme.

A high level of steroid 19-hydroxylation is exhibited by adrenal mitochondria of the gerbil, Meriones, unguiculatus, that accounts for the ability of that species to produce nearly equal amounts of corticosterone and 19-hydroxycorticosterone (Proc. Soc. exp. Biol. Med. 165 (1980) 69-74). Inhibitors of steroidogenesis and a polyclonal antibody against bovine cytochrome P-450(11 beta) were used to determine if the agents would effect differential or parallel suppression of 19- vs 11 beta-hydroxylation by gerbil adrenal mitochondria in vitro. The inhibitors (0.1-60 microM) tested (listed in order of decreasing effectiveness) were imazalil, metyrapone, miconazole and 4-hydroxyandrostenedione. With each inhibitor the degree of suppression of 11 beta-hydroxylation was accompanied by a parallel decline in 19-hydroxylation. The addition of the polyclonal antibody preparation also produced equivalent declines in the rates of the two hydroxylation reactions. The addition of ACTH 1 microM to primary cultures of gerbil adrenal cells brought about nearly equal increases in the secretion of 11 beta- and 19-hydroxylated steroids into the culture media. These results support the hypothesis that the 11 beta-hydroxylase of gerbil adrenal mitochondria has the capacity to carry out 11 beta- and 19-hydroxylations with nearly equal facility.

A role for elevated plasma corticosterone in modulation of host response during infection with Trichinella pseudospiralis.

Suppression of host inflammatory response in mice infected with Trichinella pseudospiralis was associated with host plasma corticosterone levels significantly higher than those seen in uninfected mice or in mice infected with T. spiralis. Increases in the population of mitochondria and depletion of lipid droplets in cells of the zona fasciculata were seen in the adrenals of mice infected with T. pseudospiralis. Elevations in enteritis, myositis and myocarditis accompanied 100% mortality in adrenalectomized mice infected with T. pseudospiralis, while lower levels of inflammation and no mortality were observed in sham operated or intact animals infected with this parasite. The severe myositis normally accompanying infection with T. spiralis was suppressed by concurrent infection with 1000 or 2000 T. pseudospiralis to levels equivalent to those seen in animals receiving 0.15 and 0.41 mg cortisone acetate/25 g mouse/day, respectively.

Co-induction of 17 alpha-hydroxylase and C-17,20-lyase activities in primary cultures of bovine adrenocortical cells in response to ACTH treatment.

Bovine adrenocortical cells in primary culture were used to examine the trophic effect of ACTH on the induction of the 17 alpha-hydroxylase and C-17,20-lyase activities. The addition of exogenous pregnenolone to bovine adrenal microsomes showed the appearance of 17 alpha-hydroxy-pregnenolone before the formation of dehydroepiandrosterone. The same sequence of activities was evident in postmitochondrial supernate from bovine adrenocortical cells cultured 36 h in the presence of 1 microM ACTH but not in postmitochondrial supernate from control cells. In another study, bovine adrenocortical cells were cultured for 36 h after which 30 microM 17 alpha-hydroxypregnenolone was added to the medium and the incubation continued 1 h; there was a 4-fold increase in androgen content in the media from ACTH-treated cells over controls. Measurement of the 17 alpha-hydroxylase and C-17,20-lyase reactions in postmitochondrial supernate from cells cultured 0-72 h in the presence of ACTH or 1 mM dibutyryl cAMP showed concomitant increases in the two activities and both activities were inhibited by the same compounds known to inhibit 17 alpha-hydroxylase activity. These observations support the concept of the co-induction of 17 alpha-hydroxylase and C-17,20-lyase activities in response to ACTH; results in keeping with previous studies indicating that the two activities are catalyzed by a single gene product, the polypeptide chain P-45017a.

Discriminatory inhibition of adrenocortical 17 alpha-hydroxylase activity by inhibitors of cholesterol side chain cleavage cytochrome P-450.

Two inhibitors of the cholesterol side chain cleavage reaction were tested for their ability to inhibit bovine adrenocortical 17 alpha-hydroxylase and 21-hydroxylase activities. One inhibitor, 22-amino-23,24-bisnor-5-cholen-3 beta-ol (22-ABC), was found to be a potent inhibitor of 17 alpha-hydroxylation of either progesterone or pregnenolone but was inactive on 21-hydroxylase activity. 22-ABC was found to be a competitive inhibitor of 17 alpha-hydroxylase (cytochrome P-45017 alpha) activity, having an apparent inhibitor constant of 29 nM when using pregnenolone as the substrate. Spectral binding studies showed that 22-ABC produces a type II difference spectrum when added to a bovine adrenocortical microsomal preparation, due presumably to a coordination of its amine nitrogen atom to the heme-iron of cytochrome P-45017 alpha. The second cholesterol side chain cleavage inhibitor tested, (20R)-20-phenyl-5-pregnene-3 beta,20-diol (20-PPD), was found not to inhibit either the 21- or 17 alpha-hydroxylase activities. It is proposed that the phenyl group projecting from C-20 of 20-PPD prevents this steroid from binding to cytochrome P-45017 alpha. The discriminatory interaction of these two steroids with adrenocortical cytochromes P-450 provides some insight with respect to possible structural features of the active-site regions of these enzymes.

3 Beta-hydroxysteroid dehydrogenase-isomerase activity in bovine adrenocortical cells in culture: lack of response to ACTH treatment.

Primary cultures of bovine adrenocortical cells (BAC) were used to determine whether the adrenal microsomal 3 beta-hydroxysteroid dehydrogenase-isomerase complex (3 beta-HSD), like the 17 alpha-hydroxylase (17-OHase), responded to ACTH treatment with an increase in activity. Both enzymes influence the steroidogenic path leading to 17 alpha-hydroxyprogesterone formation and thus could affect adrenal androgen biosynthesis. 3 beta-HSD Activity in postmitochondrial supernatant fluid, homogenates or cell monolayers remained unchanged after cells had been maintained in 1 microM ACTH up to 48 h. Since ACTH exposure led to a marked increase in 17-OHase activity over the same time period, it is concluded that, under the conditions used, the 3 beta-HSD-isomerase complex in BAC is nonresponsive to tropic hormone treatment.

NADP-isocitric dehydrogenase of gerbil adrenal mitochondria: support of steroid hydroxylation.

In gerbil adrenal cortex the activity of intramitochondrial NADP-linked isocitric dehydrogenase (IDH) is up to 10-fold greater than the NAD-linked IDH. The NADP-IDH, apparent Km 0.58 mM, Vmax 280 nmoles/min/mg mitochondrial protein, appears to be the major source of reducing equivalents to support adrenal mitochondrial steroid 11B- and 19-hydroxylation in this species.

Effect of adrenocorticotropin on steroid 21-hydroxylase synthesis and activity in cultured bovine adrenocortical cells. Increased synthesis in the absence of increased activity.

The action of adrenocorticotropin (ACTH) to stimulate synthesis of steroid 21-hydroxylase was studied in bovine adrenocortical cells maintained in primary culture. Continuous treatment with ACTH (1 microM) caused an increased incorporation of [35S]methionine into cytochrome P-450C21 (21-hydroxylase cytochrome P-450); a maximum value (15-fold increase) was attained 24 h after initiation of ACTH treatment. Also, a 3-fold increase in cytochrome P-450C21 synthesis was observed in a cell-free translation system programmed by RNA isolated from cells that were exposed to ACTH for 24 h. The rate of synthesis of cytochrome P-450C21 declined after longer periods of treatment of the cells with ACTH. The increase in synthesis of cytochrome P-450C21 was associated with an increase (3-6 fold) in both total cytochrome P-450 content and in the type I absorbance change induced by 17 alpha-hydroxyprogesterone in microsomes prepared from ACTH-treated cells, as compared with that in microsomes from control cells. By contrast, ACTH did not act to increase steroid 21-hydroxylase activity in cultured intact cells, as determined by the rate of secretion of cortisol and 11-deoxycortisol, after addition of 17 alpha-hydroxyprogesterone to the medium. Similarly, there was no difference in steroid 21-hydroxylase activity in postmitochondrial supernatant fractions prepared from non-treated or ACTH-treated cells. Cytochrome P-450C21 was found to be synthesized as a form identical in molecular weight to the mature form. These results are indicative that ACTH acts to stimulate the synthesis of steroid 21-hydroxylase, yet is without a demonstrable effect on the activity of this enzyme which is high throughout the time period of the experiment.