Intraoperative computed tomography for orbital reconstruction: a systematic review.

Orbital reconstruction is a common procedure with inherent challenges and important consequences. Intraoperative use of computed tomography (CT) is an emerging application that facilitates accurate intraoperative evaluation to improve clinical outcomes. This review aims to investigate the intraoperative and postoperative outcomes of intraoperative CT use in orbital reconstruction. PubMed and Scopus databases were systematically searched. Inclusion criteria were: clinical studies investigating intraoperative CT use in orbital reconstruction. Exclusion criteria were: duplicates; non-English publications; non-full-text publications; studies with insufficient data. Of the 1022 articles identified, seven eligible articles representing 256 cases were included. The mean age was 39 years. Most cases were male (69.9%). With regards to intraoperative outcomes, the mean revision rate was 34.1%, with plate repositioning being the most common type (51.1%). Intraoperative time was variably reported. With regards to postoperative outcomes, there were no revisions, and only one case that had a complication (transient exophthalmos). Mean volumetric difference between the repaired and contralateral orbits was reported in two studies. The findings of this review present an updated evidence-based summary of the intraoperative and postoperative outcomes of intraoperative CT use in orbital reconstruction. Robust longitudinal comparisons of clinical outcomes between intraoperative and non-intraoperative CT cases are required.

E-scooters and maxillofacial fractures: a seven-year multi-centre retrospective review.

BACKGROUND: Standing electric scooters (e-scooters) are a cost-effective and environmentally-friendly transport alternative, but also elicit substantial concern regarding associated craniofacial injuries. This study aims to describe the patient factors, procedural factors and post-operative outcomes of maxillofacial fractures caused by e-scooter accidents. METHODS: Retrospective chart review of patients aged 18 years or older who were surgically treated for these injuries in 2014-2020 at two Australian tertiary hospitals. RESULTS: There were 18 cases included. Most cases were male (66%). The mean age was 35 years. Common risk factors were alcohol use (86%) and lack of helmet use (62%). The most common fracture pattern was zygomatico-maxillary complex (ZMC) fractures (50%). There were no associated systemic injuries. Mean operation timing was 12 days post-injury for ZMC fractures and 3 days post-injury for condyle fractures. For ZMC fractures, the most common method of fixation was 2-point fixation (66%). For condyle fractures, the most common surgical approach was arch bars only (83%). Post-operative complications were reported in six cases, with malocclusion being the most common (n = 3). Revision surgeries were performed in two cases. CONCLUSIONS: Maxillofacial fractures associated with e-scooter accidents appear to be increasing in incidence. Robust longitudinal evaluations with larger sample sizes are required to better understand associated presentations, surgical approaches and post-operative complications.

Traumatic maxillofacial and brain injuries: a systematic review.

Concomitant traumatic brain injury (TBI) and maxillofacial fractures carry the risk of significant morbidity and mortality. The aim of this review was to explore the demographics, types of injury, and complications of traumatic maxillofacial and brain injuries, in order to contribute to comprehensive health strategies. The PubMed and Scopus databases were systematically searched. Inclusion criteria were clinical studies investigating combined traumatic maxillofacial and brain injuries. Exclusion criteria were duplicates, non-English publications, non-full-text publications, publication date before 1990, and studies with insufficient data. Of the 754 articles identified, 15 eligible articles representing 1421 cases were included. The mean age was 38.3 years. Most cases were male (79%). The most common mechanism of injury was traffic accidents (53.4%). The most common fracture pattern was middle third fractures (52.4%). Seven studies had an explicit definition for TBI, using the Glasgow Coma Score (GCS), radiological evidence, and/or specific symptoms. There were 147 complications reported in 62 of 253 cases (24.5%), with the most common being infection (n=54, 36.7%). Significant risk factors for complications included delayed surgical repair, low GCS, and upper third fractures. Robust longitudinal evaluations with clear definitions of TBI are required. Gaps in knowledge include risk factors for complications and fracture pattern-GCS correlations.

Frontal sinus fracture management: a systematic review and meta-analysis.

A systematic review of the frontal sinus fracture management literature was undertaken to document measurable outcomes, with emphasis on complications associated with the various treatment strategies. A comprehensive electronic search was undertaken in October 2018. Randomized controlled trials (RCT), controlled clinical trials, retrospective and prospective studies describing the management of frontal sinus fractures and complications were included. Twenty-four publications were included: one reporting a prospective RCT and 23 reporting consecutive case series studies (four prospective, 19 retrospective). These included 2388 patients (84.1% male, average age 23-43 years); 50.7% of cases were due to motor vehicle accidents and 61.8% had a concomitant intracranial injury. There were six categories for anterior table reconstruction, three endoscopic surgery categories, 11 for obliteration, and six for cranialization. Forest plots demonstrating complications based on the Clavien-Dindo classification of 1 ('low') and >3 ('high') were determined for total, early, and late complications, with heterogeneous effect sizes. Fractures with a nasofrontal outflow tract (NFOT) injury without obstruction can be treated with reconstruction if displaced, or managed conservatively if undisplaced. Obliteration and cranialization should be considered when there is obstruction of the NFOT. A computed tomography scan should be performed at 6 months to evaluate re-ventilation of the sinus. Endoscopic sinus surgery is a reasonable salvage re-ventilation procedure.

Total temporomandibular joint replacement prostheses: a systematic review and bias-adjusted meta-analysis.

The aim of the present study was to determine which prosthesis has resulted in the best outcomes after total temporomandibular joint replacement (TMJR). A comprehensive electronic search was undertaken in September 2015. Inclusion criteria encompassed studies that described one of the three current TMJR systems and that had pre- and postoperative data on at least two of the following TMJR indications: pain, diet, function, and maximum inter-incisal opening (MIO). Sixteen papers were included in the systematic review, reporting 10 retrospective studies and six prospective studies (no randomized controlled or case-controlled trials). A total 312 patients with 505 TMJ Concepts prostheses, 728 patients with 1048 Biomet prostheses, and 125 patients with 196 Nexus prostheses were included in the analysis. There was no real difference between the various TMJR systems in terms of pain or diet scores. Function scores improved with the TMJ Concepts, but this was the only prosthesis for which data were available. Biomet prostheses appeared to have a greater increase in MIO mean gain compared to TMJ Concepts and Nexus prostheses; however this was heavily biased by one study. Without this study, there was no real difference in MIO. It is concluded that the prostheses are similar, but most data are available for the TMJ Concepts prosthesis, with results being favourable.

Combining organic acid treatment with steam pasteurization to eliminate Listeria monocytogenes on fully cooked frankfurters.

An organic acid solution of 2% acetic, 1% lactic, 0.1% propionic, and 0.1% benzoic acids was combined with steam surface pasteurization to treat frankfurters during vacuum packaging to eliminate potential postcook contamination with Listeria monocytogenes. The thermal lethality of L. monocytogenes from steam was evaluated at an inoculation concentration of 1 to 6 log CFU/cm2. About 3-log reductions of L. monocytogenes were achieved when frankfurters were treated by steam for 1.5 s. Combining organic acid treatment with steam pasteurization further inhibited the growth of surviving L. monocytogenes cells for 19 and 14 weeks when the packaged frankfurters were stored at 4 and 7 degrees C, respectively. The results from this study provide meat processors with useful information for controlling L. monocytogenes on ready-to-eat meats.

Eradicating Listeria monocytogenes from fully cooked franks by using an integrated pasteurization-packaging system.

Surface pasteurization by applying steam or hot water before or after packaging of processed foods may be used to eliminate pathogens such as Listeria monocytogenes from ready-to-eat meat and poultry products. Surface pasteurization treatment with a mixture of pressurized steam and hot water was integrated into a continuous vacuum-packaging system to reduce L. monocytogenes from fully cooked franks. The franks (2.54 cm diameter by 15.24 cm length) were surface inoculated to contain up to 6 log CFU/cm2 L. monocytogenes. The inoculated franks were treated at 121 degrees C for 1.5 s in an arrangement of six franks per packaging chamber followed by immediate vacuum sealing of the top films of food packages in the same unit. A 3-log CFU/cm2 reduction of L. monocytogenes on fully cooked franks was obtained using the integrated pasteurization-packaging system. The pasteurization depth was 1.27 mm below the surfaces of the franks. This process provides a commercially applicable means of ensuring food safety by effectively eradicating L. monocytogenes from ready-to-eat meat and poultry products at the very last possible step of food packaging before reaching retail consumers.

Chaperone use by obstetrician/gynecologists.

OBJECTIVE: To describe current practices of chaperone use during pelvic examinations among obstetrician/gynecologists affiliated with a large tertiary care teaching hospital. STUDY DESIGN: Questionnaires were distributed at department of obstetrics and gynecology grand rounds to all practicing attending physicians to obtain physician demographic data, including age, years in practice, practice type (solo or group) and whether chaperones were currently used when performing pelvic examinations at the first obstetric or gynecologic office visit. Physicians were also asked whether they were taught to use chaperones for pelvic examinations during medical school or residency. RESULTS: Of the 59 attending physicians, 100% responded to the questionnaire. As compared to female physicians, male physicians used chaperones more at the first obstetric examination (76.9% vs. 27.8%, P < .002), at gynecologic office visits (70.0% vs. 22.2%, P < .002) and for breast examinations (51.2% vs. 11.1%, P < .01). Physicians greater than 40 years old, in practice longer than 10 years and taught as medical students or residents to use chaperones were statistically more likely to use chaperones. No attendings, male or female, reported losing a patient to another provider or being sued or threatened with legal action because of not using a chaperone. CONCLUSION: Chaperones were used more frequently during pelvic examinations by male physicians, age greater than 40, solo practice, and physicians in practice longer than 10 years. Education affected current practices as specific medical student or residency training influenced the use of chaperones in private practice.

gamma-L-glutamyl-5-hydroxy-L-tryptophan, but not gamma-L-glutamyl-L-tryptophan, causes sodium retention in normal man.

1. This randomized, placebo-controlled, cross-over study compared the relative effectiveness of gamma-L-glutamyl-5-hydroxy-L-tryptophan (glu-5-HTP) and gamma-L-glutamyl-L-tryptophan (glu-TRP) in terms of their ability to act as substrates for renal 5-hydroxytryptamine (5-HT) synthesis and their actions on urinary sodium excretion. 2. Urinary excretion of 5-HT and sodium were determined before, during and after 1 h intravenous infusion of an equimolar amount (45 nmol kg-1 min-1) of glu-5-HTP or glu-TRP or placebo in nine healthy male subjects. 3. Cumulative urinary 5-HT excretion over the 4 h after the start of glu-5-HTP infusion was 350-fold greater than that after placebo, and this was associated with a reduction in the urinary excretion of sodium. 4. In contrast, the urinary excretion values of 5-HT and sodium after administration of glu-TRP were not significantly different from those observed on the placebo day. 5. The marked increase in urinary 5-HT excretion and the retention of sodium after administration of glu-5-HTP have been demonstrated in previous studies and result from increased intrarenal generation of 5-HT. The absence of a rise in urinary excretion of 5-HT after glu-TRP infusion suggests that there was no significant conversion of this glutamyl compound to 5-HT within the kidney. As a result, there was no effect on urinary sodium excretion.

Comparison of protein phosphorylation patterns produced in adrenal cells by activation of cAMP-dependent protein kinase and Ca-dependent protein kinase.

Bovine adrenal fasciculata cells, exposed to either ACTH or AII, synthesize glucocorticoids at an enhanced rate. It is generally accepted that the signaling pathways triggered by these two peptides are not identical. ACTH presumably acts via a cAMP-dependent protein kinase (PKA) and AII, via a calcium-dependent protein kinase. We have found that either peptide hormone stimulates synthesis of a mitochondrial phosphoprotein pp37, leading to accumulation of its proteolytically processed products pp30 and pp29. On the basis of a number of criteria, this 37 kDa protein is the bovine homolog of the 37 kDa protein that we have characterized in rodent steroidogenic tissue (Epstein L. F. and Orme-Johnson N. R.: J. Biol. Chem 266 (1991) 19,739-19,745). Further, bovine pp37 is phosphorylated when PKA or protein kinase C (PKC) is activated directly by (Bu)2 cAMP or PMA, respectively. These studies indicate that either pp37 is a common substrate for PKA and PKC in these cells or there is a common downstream kinase, which is activated by exposure to either ACTH or AII. Rat adrenal glomerulosa cells, exposed to either ACTH or AII, show an enhanced rate of mineralocorticoid synthesis. As for bovine fasciculata cells, it is thought that the signaling pathway triggered by ACTH differs from that triggered by AII. As we found for bovine fasciculata, pp37 is phosphorylated when the rat cells are exposed to either peptide hormone. However, in contrast to the finding for bovine fasciculata, while exposure of the rat glomerulosa cells to (Bu)2cAMP does cause the synthesis of pp37, exposure of the cells to PMA does not. Taken together, these findings provide further evidence that the subcellular signaling events, triggered by the action of AII on bovine adrenal fasciculata and rat adrenal glomerulosa cells, differ. Further, the fact, that pp37 is phosphorylated only when the rate of steroidogenesis is enhanced, reaffirms its potential involvement in the signaling pathway that causes stimulation of steroid hormone biosynthesis.

Influence of acceptor substrate primary amino acid sequence on the activity of human UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase. Studies with the MUC1 tandem repeat.

Synthetic peptides (30 and 20 residues long) corresponding to the native MUC1 tandem repeat sequence (20 residues long) were glycosylated in vitro using UDP-[3H]GalNAc and lysates from the human breast tumor cell line MCF7. Purified glycopeptides were sequenced on a gas-phase sequenator, and glycosylated positions were determined by measuring the incorporated radioactivity in fractions collected following each round of Edman degradation. The results showed that 2 of 3 threonines on the MUC1 tandem repeat peptides were glycosylated at the following positions: GVTSAPDTRPAPGSTAPPAH (underlined Thr residues indicate positions of GalNAc attachment); no glycosylation of serine residues was detected. Determination of the mass of the glycopeptides by mass spectrometry showed that a maximum of two molecules of GalNAc were covalently linked to each 20-residue repeat unit in the peptides. The influence of substrate primary amino acid sequence in determining the substrate specificity of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyl-transferase activity was evaluated using as acceptor substrates a series of overlapping 9-residue peptides that represent a moving set through the tandem repeat of the MUC1 mucin. In addition, the influence of primary amino acid sequence on acceptor substrate activity was evaluated using several peptides that contained single or double amino acid substitutions (relative to the native human MUC1 sequence). These included substitutions in the residues that were glycosylated and substitutions in the surrounding primary amino acid sequence. This study demonstrates that primary amino acid sequence, length, and relative position of the residue to be glycosylated dramatically affect the ability of peptides to serve as acceptor substrates for UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase.

A comparison of the renal and neuroendocrine effects of two 5-hydroxytryptamine renal prodrugs in normal man.

1. The effects of 1 h intravenous infusions of equimolar amounts (45 nmol min-1 kg-1) of two putative 5-hydroxytryptamine renal prodrugs, 5-hydroxy-L-tryptophan and gamma-L-glutamyl-5-hydroxy-L-tryptophan, were investigated in a randomized, placebo-controlled, cross-over study in nine healthy male subjects. 2. Cumulative urinary 5-hydroxytryptamine excretion over the 3 h observation period rose by about 370-fold after 5-hydroxy-L-tryptophan and 390-fold after gamma-L-glutamyl-5-hydroxy-L-tryptophan when compared with placebo infusion. Urinary 5-hydroxy-L-tryptophan excretion was three times higher after administration of gamma-L-glutamyl-5-hydroxy-L-tryptophan than after 5-hydroxy-L-tryptophan infusion. Urinary 5-hydroxyindole-3-acetic acid excretion after 5-hydroxy-L-tryptophan infusion was significantly greater than that after gamma-L-glutamyl-5-hydroxy-L-tryptophan administration. Urinary dopamine excretion was not affected by either compound when compared with placebo. 3. 5-Hydroxy-L-tryptophan significantly reduced urine flow rate and urinary sodium excretion. gamma-L-Glutamyl-5-hydroxy-L-tryptophan was antinatriuretic but did not affect urine output. These changes occurred without significant alterations in effective renal plasma flow and glomerular filtration rate. 4. Both 5-hydroxy-L-tryptophan and gamma-L-glutamyl-5-hydroxy-L-tryptophan significantly increased plasma aldosterone concentration without a concomitant rise in plasma renin activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute action of luteinizing hormone on mouse Leydig cells: accumulation of mitochondrial phosphoproteins and stimulation of testosterone synthesis.

Upon stimulation of Leydig cells with luteinizing hormone (LH) or dibutyryl-3',5'-cyclic AMP (Bt2cAMP) at 37 degrees C, two mitochondrial phosphoproteins accumulate with the same stimulant dose response as the increased rate of testosterone synthesis. The proteins pp32 and pp30 have apparent isoelectric points of 6.6 and 6.5 and molecular weights of approximately 32 30 kDa respectively, as determined by two-dimensional polyacrylamide gel electrophoresis. These two phosphoproteins are not detected in mouse adipose or liver cells nor in the total testicular cell population, of which Leydig cells constitute a small percentage. However, both proteins are also observed in mouse adrenal cells stimulated by ACTH or Bt2cAMP. The appearance of pp32 and pp30 is prevented by inhibitors of cytosolic protein translation, indicating that only newly synthesized protein is available as a substrate for phosphorylation. Proteolytic peptide mapping indicates that both of these mouse Leydig and adrenal proteins have structural similarity to pp30 (formerly denoted as ib), the 30 kDa mitochondrial phosphoprotein that we have observed previously in peptide hormone or Bt2cAMP-stimulated rat adrenal cortex (Pon, L.A., Hartigan, J.A. and Orme-Johnson, N.R. (1986) J. Biol. Chem. 261, 13309-13316; Alberta, J.A., Epstein, L.F., Pon, L.A. and Orme-Johnson, N.R. (1989) J. Biol. Chem. 264, 2368-2372) and rat corpus luteum cells (Pon, L.A. and Orme-Johnson, N.R. (1986) J. Biol. Chem. 261, 6694-6599). Since pp32 is a larger mitochondrial protein of similar primary structure to pp30, it is a potential precursor of this protein. Finally, the detection of the mitochondrial phosphoprotein pp30 in a third steroidogenic tissue type and a third species provides further correlative evidence that the production of pp30 may be an integral part of the subcellular mechanism by which peptide hormones stimulate steroid hormone biosynthesis.

Regulation of steroid hormone biosynthesis. Identification of precursors of a phosphoprotein targeted to the mitochondrion in stimulated rat adrenal cortex cells.

Two short-lived precursor proteins, pp37 and pp32, of the mitochondrial phosphoprotein pp30 (formerly denoted as ib) have been detected in Bt2cAMP-stimulated rat adrenal cortex cells, incubated at 25 degrees C or with 1,10-ortho-phenanthroline at 37 degrees C. Subsequently, these two precursor proteins were also identified in cells incubated at 37 degrees C, where they are present only at low levels due to their short half-life. pp30 is produced in several steroidogenic tissues in response to trophic hormone or second messenger analogue. pp37 and pp32 are also phosphoproteins located in the mitochondrion that are produced in response to cAMP analogue and give rise to proteolytic peptide maps similar to that of pp30. As for pp30, inhibition of cytosolic translation prevents the production of pp37 and pp32. The larger precursor protein pp37 has an apparent molecular mass of 37 kDa, an isoelectric point of approximately 7.1, and a half-life at 37 degrees C of 3-4 min. Pulse-chase studies indicate that this protein is processed into the smaller protein, pp32, which has an apparent molecular mass of 32 kDa, an isoelectric point of approximately 6.4, and a half-life at 37 degrees C of 3-4 min. This latter protein is the immediate precursor of pp30. Since ortho-phenanthroline inhibits the mitochondrial processing protease, while the lower incubation temperature slows both protein import and protease processing, the experimental conditions necessary to detect these proteins are consistent with pp37 being a precursor protein that contains two cleavable presequences and is imported into the mitochondrion. The sequential removal of these sequences produces the mature protein pp30.

Characterization of the adrenal 11 beta-hydroxylase in inbred salt-sensitive and resistant rats.

Rats have been bred for susceptibility and resistance to the hypertensive effect of dietary salt (S/JR & R/JR). S/JR have an abnormal adrenal steroid 11 beta,18-hydroxylase activity resulting in increased production of 18-OH-DOC. S/JR also produce increased quantities of 19-nor-DOC, which may be related, since the 11 beta,18-hydroxylase also catalyzes the 19-hydroxylation of DOC, a pivotal step in 19-nor-DOC biosynthesis. The purpose of the present studies was to further characterize the mutant S/JR adrenal steroid 11 beta,18-hydroxylase. Preliminary studies are also presented on assessing the renal 19-desmolase, the last step in 19-nor-DOC biosynthesis. Adrenal glands were harvested from R/JR and S/JR and prepared for incubation studies, protein immunoblotting, and RNA analysis. Kidneys from Sprague-Dawley rats were also used for isolated renal perfusion studies. Both S/JR and R/JR strains had a single immunostaining band for 11 beta,18-hydroxylase at 51,000 molecular weight which were equal in intensity. Both strains had a single RNA transcript at 4.3 kilobases which hybridized with equal intensity to the bovine cDNA (pB11-9). The Km for 11 beta- and 18-hydroxylation was identical within strains but was different between strains. The Km for 19-hydroxylation was different between S/JR and R/JR, and was much greater than 11 beta- and 18-hydroxylation in both strains. This suggests that the catalytic site for 19-hydroxylation is different than that for 11 beta- and 18-hydroxylation and that the S/JR enzyme binds the substrate with higher affinity than the R/JR enzyme. In the isolated perfusion studies the rat kidneys converted 80% of 19-oxo-DOC to either 19-oic-DOC or 19-nor-DOC. These data demonstrate that the difference in S/JR enzyme activity is probably due to a point mutation in the enzyme or to a change in a regulatory protein.

Inhibition of steroidogenesis in rat adrenal cortex cells by a threonine analogue.

We have investigated the ability of amino acid analogues of serine and threonine to inhibit the increase in steroidogenesis elicited by addition of ACTH or cAMP in cells isolated from the rat adrenal cortex. We have found that the serine analogues, D, L-isoserine, alpha-methyl-D, L-serine and L-homoserine, are almost totally ineffective in inhibiting this process but that the threonine analogue, D, L-beta-hydroxynorvaline, at a concentration of 300 microM inhibits stimulated steroid hormone biosynthesis by ca 95%, while inhibiting overall protein synthesis by only ca 40%. This inhibition was found to occur in a dose-dependent manner and to be reversible by a stoichiometric concentration of threonine. These studies suggest that beta-hydroxynorvaline is functioning as a threonine analogue in our experimental system. Both the onset of inhibition by analogue and reversal of this inhibition by the natural amino acid occurred rapidly, without detectable lag. Since results obtained using cAMP as stimulant parallel those obtained using ACTH, the inhibitory effect of the analogue seems to occur subsequent to the synthesis of cAMP. Additionally, the analogue does not inhibit the conversion of pregnenolone to corticosterone, suggesting the site of action of analogue occurs prior to the synthesis of pregnenolone from cholesterol. Thus, the analogue may be exerting its effect on a protein that is synthesized subsequent to ACTH addition and is important in the acute phase of stimulated steroid hormone biosynthesis. Further, since ACTH action on adrenal cortex cells causes the activation of protein kinase A, which phosphorylates serine and threonine residues, it is possible that the effect of the analogue is to prevent the phosphorylation of a newly-synthesized protein.

Distinctive properties of adrenal cortex mitochondria.

The mitochondria in cells that synthesize steroid hormones not only have enzymes not present in mitochondria of non-steroidogenic cells but also have unique mechanisms for regulating the steroid substrate availability for certain of these enzymes. We have considered in detail the cytochrome P-450scc system that is located in the inner mitochondrial membrane and that catalyzes the initial and rate-determining step in the steroid hormone biosynthetic pathway. The flux through this pathway is regulated both by the levels of these catalysts themselves and by the availability of the substrate cholesterol for conversion to pregnenolone. These two levels of regulation occur in different time frames but are both controlled externally by the action of tissue-specific peptide hormone. We have used the adrenal cortex fasciculata cells as our paradigmatic cell type. The overall picture seems closely similar for mitochondria in other such steroidogenic cells when analogous data are available. Thus, in adrenal cortex fasciculata cells ACTH triggers several long-term (trophic) and short-term (acute) effects upon and within mitochondria that influence the initial and rate-determining step in the steroid hormone biosynthetic pathway. The only second messenger for both effects characterized thus far is cAMP. An increase in membrane-associated cAMP rapidly activates cAMP-dependent protein kinase, which in turn phosphorylates several cellular proteins, e.g., cholesterol ester hydrolase (vide supra). The trophic action, i.e., that produced by exposure of the cells to increased levels of ACTH or cAMP for a prolonged period (minutes to hours), increases the amounts of the steroid hormone synthesizing proteins in the mitochondria by increasing the transcription of the relevant nuclear genes. This latter process is not needed for the acute increase in the rate of steroid hormone biosynthesis. Whether induction of steroidogenic enzymes requires activation of a kinase has not been determined. However, the postulated SHIP proteins provide a mechanism by which cAMP levels and protein synthesis itself may regulate this induction. Mitochondria in steroidogenic tissues exert control over this process by their ability to recognize, import and process correctly the nuclear encoded precursors of the steroidogenic enzymes. Whether control at this level is ultimately dictated by nuclear or mitochondrial gene products or by an interplay between them is still unknown.(ABSTRACT TRUNCATED AT 400 WORDS)

19-Hydroxylase inhibition of adrenal mitochondrial P450 11 beta/18/19-hydroxylase by a suicide inhibitor.

19-Nor-deoxycorticosterone (19-nor-DOC) is a mineralocorticoid that is increased in some forms of experimental and human hypertension. The pivotal step in 19-nor-DOC biosynthesis is adrenal P450 19-hydroxylase, but this enzyme has not been clearly distinguished from P450 11 beta/18-hydroxylase. This study attempted to specifically inhibit adrenal 19-hydroxylation of deoxycorticosterone (DOC) using a suicide aromatase inhibitor, 19-acetylenic androstenedione (19-AA). Purified bovine P450 11 beta/18/19-hydroxylase was incubated with excess substrate DOC, adrenodoxin, and adrenodoxin reductase in the presence of increasing doses of the inhibitor, 19AA. 11 beta-, 18-, and 19-hydroxylation were measured by quantification of corticosterone, 18-OH-DOC, and 19-OH-DOC respectively. Measurements of these products demonstrated that 11 beta- and 18-hydroxylation was not inhibited whereas 19-hydroxylation was inhibited as manifested by decreased 19-OH-DOC formation (p less than .05). The IC50 of 19-AA was approximately 10(-12) M. The specific inhibition of 19-hydroxylation suggests that the 19-hydroxylase may be an enzyme distinct from the P450 11 beta/18-hydroxylase. This further suggests that 19-nor-DOC biosynthesis may be under independent regulation and may be amendable to specific in vivo inhibition.

Mitochondrial localization of a phosphoprotein that rapidly accumulates in adrenal cortex cells exposed to adrenocorticotropic hormone or to cAMP.

We have reported previously that a phosphoprotein, ib, is present in adrenal cortex, corpus luteum, and Leydig cells stimulated with either tissue-specific peptide hormone or with cAMP. The accumulation of protein ib in each of these cell types has been found to parallel the stimulation of steroid synthesis with respect to both time course and stimulant dose response. Thus, protein ib is a potential mediator in the acute stimulation of steroidogenesis by peptide hormone or cyclic AMP. A second protein, pb, the unphosphorylated form of ib, is synthesized constitutively in unstimulated but not stimulated cells and is not converted post-translationally to ib upon stimulation. Using two-dimensional gel electrophoresis of subcellular fractions isolated from rat adrenal cortex cells labeled with [35S] methionine, we have determined the intracellular localization of proteins p and i. We demonstrate that proteins ib and pb are localized predominantly in the mitochondria and are tightly associated with that organelle. We also find that inhibition of mitochondrial protein synthesis by chloramphenicol affects neither the accumulation of these proteins nor the stimulation of steroidogenesis. Thus, protein pb and its phosphorylated counterpart, ib, are synthesized in the cytosol and transported to the mitochondria, the site of the rate-limiting step in steroid hormone biosynthesis.

Subcellular localization of a protein produced in adrenal cortex cells in response to ACTH.

We have reported previously that a protein, ib, is produced in adrenal cortex and other steroidogenic cells with the same tissue-specific peptide hormone or cAMP dose-response and the same kinetics as the increase in steroid hormone biosynthesis. In this study, we have fractionated adrenal cortex cells into subcellular components and used two-dimensional electrophoresis to characterize the proteins in these fractions. We have demonstrated previously that inhibition of cytosolic translation, e.g. by cycloheximide, prevents the production of protein ib. We also report that the production of this protein is not affected by inhibition of mitochondrial translation by chloramphenicol.