Charting a Course for Implementation Research in Oral Health.

Knowledge Transfer Statement: This article provides an overview of implementation science and outlines NIDCR's interest and commitment to research that decreases time from development through implementation of evidence-based oral health interventions.

Insights into steroidogenic acute regulatory protein (StAR)-dependent cholesterol transfer in mitochondria: evidence from molecular modeling and structure-based thermodynamics supporting the existence of partially unfolded states of StAR.

The steroidogenic acute regulatory protein (StAR) is the major entrance for cholesterol in mitochondria under acute stimulation. Under such circumstances, dysfunctional StAR activity can ultimately lead to lipoid congenital adrenal hyperplasia (LCAH). A complete understanding of the StAR's molecular structure and mechanism is essential to comprehend LCAH. Thus far, there is no mechanistic model that can explain experimental results at the molecular level. This is partly due to the lack of the molecular structure of StAR. The closest approximation to the StAR molecular structure is the human MLN64 which has a similar activity to StAR, has a highly homologous primary structure and for which an X-ray structure is known. In this context, we have modeled the structure of StAR through standard homology modeling procedures based on the MLN64 structure. Our StAR model shows the presence of a hydrophobic cavity of 783.9 A(2) in surface area, large enough to fit one molecule of cholesterol. In addition, we have identified a unique charged pair, as in MLN64, lining the surface of the cavity and which could play a key role in the binding of cholesterol through the formation of an H-bond with its OH moiety. This suggests that the cholesterol-binding site of StAR is located inside this cavity. Taking into account that internal cavities are destabilizing to native protein structures and that the lining of the cavity has to become accessible in order to allow cholesterol binding, we have explored the possibility that StAR could exist in equilibrium with partially unfolded states. Using a structure-based thermodynamics approach, we show that partially folded states (with an unfolded C-terminal alpha-helix, and an open cavity) can be significantly populated at equilibrium and therefore allow cholesterol binding. These results are supported by recent experiments that show a loss of StAR helical character upon binding of an analog of cholesterol. Moreover, we show that the replacement of the residues involved in the charged-pair located in the binding site results in the loss of StAR activity, supporting a key role for these residues. Taken together, our results are applicable to StAR functioning both in the mitochondrial intermembrane space as well as outside the mitochondria.

Implementation of the methadone treatment quality assurance system. findings from the feasibility study.

This article describes the development and large-scale testing of the Methadone Treatment Quality Assurance System (MTQAS). MTQAS originated as a NIDA (National Institute on Drug Abuse) funded study to assess the feasibility and utility of implementing a performance-based feedback reporting system for narcotic addiction treatment programs. After several years of research to identify a set of valid, reliable, and clinically useful indicators of program performance using patients' in-treatment outcomes, MTQAS was tested in a feasibility study with more than 70 methadone treatment clinics in seven states. For each of nine quarters, participating clinics received a report summarizing their patient outcomes; reports also included summary data permitting comparisons across states, across clinics within a state, and within a clinic over time. In addition, MTQAS included a case-mix adjustment strategy that permitted fair comparisons of performance among programs with systematically different patient caseloads. The structure and operation of MTQAS, selected analyses of the data, and lessons for future performance measurement systems are discussed.

The in vivo effects of adrenocorticotropin and sodium restriction on the formation of the different species of steroidogenic acute regulatory protein in rat adrenal.

We have studied the in vivo expression of steroidogenic acute regulatory protein (StAR) in adrenals of control, ACTH-treated, and Na+-restricted rats. Indirect immunofluorescence by microscopy revealed the presence of StAR in the zonae glomerulosa (ZG) and fasciculata-reticularis (ZFR). An increased signal was observed in the ZG and zona fasciculata, 5 h after ACTH injection; a few cells of the medulla were also positive. Immunogold electron microscopy showed that StAR was mainly located over mitochondria (MT). By immunoblotting, a major 29-kDa and other minor StAR bands migrating between 30 and 39 kDa were increased 5 h after ACTH treatment but remained unchanged after 1 h. By two-dimensional-PAGE, four StAR species were revealed in homogenates of control ZG, and their intensity was increased 5 h after ACTH treatment but not after 1 h. Also, additional acidic species were seen 5 h after treatment. Other bands with basic isoelectric point were revealed between 29 and 37 kDa. Analyses on whole gland MT and supernatant (SN) revealed four bands in the control SN and five in ACTH SN; the intensity of one band was increased, and that of another one was decreased, in SN of treated rats. ACTH treatment resulted in the localization of many low-isoelectric point StARs in MT. After two-dimensional-PAGE, differences were found in the mobility of some StAR species in the ZG between controls and Na+-restricted rats. In MT, four bands were revealed in the ZG preparations of Na+-restricted and two bands in controls. Four bands were revealed in the ZG SNs of control and Na+-restricted rats; an additional band was observed only in the SN of treated animals, whereas the intensity of another band decreased. Na+ restriction did not affect StAR in the ZFR. In conclusion, StAR was present in the rat adrenal cortex ZG and ZFR and was mainly located in MT. StAR expression was inducible in the ZG and the ZF by ACTH, resulting in the formation of many StAR acidic species; interestingly, such changes were detectable 5 h, but not 1 h, after ACTH administration, suggesting that steroidogenesis stimulation by StAR might occur mainly outside MT. Although less spectacular than for ACTH, Na+ restriction also affected StAR expression in the ZG but not in the ZFR, by increasing two mitochondrial and one SN species, implying that StAR is involved in the mechanism of action of Na+ restriction in promoting aldosterone formation. These results suggest that differential processing and/or changes in phosphorylation may occur in vivo upon ACTH treatment and Na+ restriction. We hypothesize that modification of a relatively small quantity of StAR, mainly located outside MT, is necessary to increase adrenal steroidogenesis challenged either by ACTH or Na+ restriction.

In vivo effects of adrenocorticotrophin on the expression of the hamster steroidogenic acute regulatory protein.

In this study, we report the cDNA cloning of hamster adrenal steroidogenic acute regulatory (StAR) protein and the effect of adrenocorticotrophin (ACTH) on its expression in vivo. A hamster adrenal cDNA library was screened using an 852 bp fragment obtained by polymerase chain reaction; this fragment corresponds to the entire coding sequence (CDS) of the hamster adrenal StAR cDNA. Ten clones of different lengths were isolated and sequenced. The longest clone was 1564 bp and contained 34 bp in the 5'-untranslated region, 852 bp in the CDS, and 678 bp in the 3'-untranslated region (3'-UTR). Two polyadenylation signal sequences were found in the 3'-UTR. The CDS of the ten isolated clones was identical, but six of these lacked the last 132 nucleotides in the 3'-UTR, thus indicating that they had used the first polyadenylation signal. The hamster StAR protein contains 284 amino acid residues, and is 91.9% homologous to mouse, 90.5% to rat, 86.4% to human, 85% to porcine, and 82.5% to bovine StAR protein. Southern blot analysis indicated the presence of only one StAR gene in the hamster genome. Northern blotting analysis revealed the presence of the StAR mRNA in male and female steroidogenic tissues, namely adrenals and gonads, but not in the liver or in the kidneys of either sex. Three mRNA species of 1.7, 3.1 and 5.3 kb were found in whole hamster adrenals. Administration of ACTH to hamsters provoked increases (two- to threefold) in the adrenal content of the StAR mRNA within 1 h in vivo. Western blotting analysis on adrenal mitochondria showed that the level of StAR protein was also significantly elevated (1.5-fold) 1 h after ACTH treatment.

The acute and chronic effects of adrenocorticotropin on the levels of messenger ribonucleic acid and protein of steroidogenic enzymes in rat adrenal in vivo.

The purpose of this study was to evaluate the effects of acute (a single injection) and chronic stimulation (twice daily injection for 9 days) by ACTH on changes occurring in the temporal expression of steroidogenic enzymes in the rat adrenal in vivo. Under acute ACTH stimulation, the level of steroidogenic acute regulatory protein (StAR) messenger RNA (mRNA) was increased within 0.5 h in both zona glomerulosa (ZG) and zona fasciculata-reticularis (ZFR), with maximal increases of 220-370% and 300-350% in the ZG and ZFR, respectively. Increases in the levels of StAR protein in homogenates were also found in the ZG (700%) and the ZFR (300%), but were delayed compared with those of their mRNA. Furthermore, the increase in mitochondrial StAR protein was concomitant with that in the homogenate, indicating that the entry of StAR into mitochondria might not be necessary to increase steroidogenesis during the early stimulatory phase. The levels of c-jun, c-fos, junB, and fosB mRNA in ZG and ZFR were also rapidly maximally elevated within 0.5-1 h after ACTH administration and fell to near control levels 5 h posttreatment. The levels of c-jun protein were already increased in both zones at 1 h, reached 200% at 3 h, and remained elevated 5 h post-ACTH treatment. The levels of c-Fos protein were maximally increased by 240% in both zones after 1 h and decreased thereafter to control values at 5 h. Few changes were observed in the adrenal protein contents of cholesterol side-chain cleavage cytochrome P450 (P450scc), cytochrome P450 11beta-hydroxylase (P450C11), cytochrome P450 21-hydroxylase (P450C21), and 3beta-hydroxysteroid dehydrogenase (3betaHSD). Under chronic stimulation by ACTH, we observed elevations in the levels of plasma corticosteroids and changes in the mRNA and protein levels of many adrenal steroidogenic enzymes in both zones. In the ZG, administration of ACTH for 9 days provoked an increase in the level of StAR mRNA (210-270%) and a decrease in the levels of 3betaHSD, cytochrome P450 aldosterone synthase (P450aldo), and AT1 receptor mRNA (by 40%, 70%, and 90%, respectively), whereas the levels of P450scc and P450C21 mRNA did not differ significantly from the control values. Western blotting analysis showed that the adrenal ZG protein levels of StAR and P450scc were increased (150%), 3betaHSD was not changed, and P450C21 was decreased by 70%. In the ZFR, the levels of P450scc and StAR mRNAs were increased (260% and 570-870%, respectively). The levels of 3betaHSD, P450C21, and P450C11 mRNA did not differ from control values in that zone. Western blotting analysis showed that the ZFR protein level of 3betaHSD was not changed, P450scc and P450C21 were decreased by 40% and 60%, respectively, and StAR was increased by 160%. Although c-fos and fosB mRNAs were undetectable after 9 days of chronic ACTH treatment, c-jun mRNA and its protein were still detectable, suggesting a basic role for this protooncogene in maintaining the integrity and function of the adrenal cortex. When dexamethasone was administered to rats for 5 days to inhibit their ACTH secretion, the mRNA levels of many steroidogenic enzymes were decreased, with the exception of StAR, 3betaHSD, and P450aldo. These results confirm the importance of physiological concentrations of ACTH in maintaining normal levels of adrenocortical enzymes and also indicate that in addition to ACTH, other factors are involved in controlling the expression of StAR, 3betaHSD, and P450aldo. In conclusion, we showed that ACTH acutely increases StAR mRNA followed, after a delay, by an increase in the level of StAR protein; this suggests that posttranslational modifications of the StAR precursor occurred during the early stimulatory phase and before the apparent translation of the newly formed mRNA. The rapid induction of protooncogenes suggests their participation in the action of ACTH to stimulate steroidogenesis. (ABSTRACT TRUNCATED)

Acute in vivo effects of ACTH on the expression of steroidogenic acute regulatory protein in rat adrenal.

We have studied the effects of adrenocorticotropin (ACTH) on the expression of steroidogenic acute regulatory protein (StAR) in rat adrenals in vivo. Following ACTH stimulation, the level of StAR mRNA was increased within 1 h in zona glomerulosa (ZG) and zona fasciculata-reticularis (ZFR), with a maximum increase at 3 h. The increase in StAR protein was delayed when compared to its mRNA. The increase in the mitochondrial StAR protein at 3 h was concomitant with that of the homogenate indicating that the entry of StAR into mitochondria might not be necessary to increase steroidogenesis during the early stimulatory phase. In conclusion, we showed that ACTH increases StAR mRNA followed, after a delay, by an increase in the level of StAR protein; this suggests that post-translational modifications of StAR precursor occur during the early stimulatory phase and this occurs before the apparent translation of the newly formed mRNA.

Influence of dietary sodium restriction on angiotensin II receptors in rat adrenals.

We studied the distribution of angiotensin II (AII) receptors type 1 (AT1) and type 2 (AT2) and the effects of a low sodium intake on these two subtypes of receptors in male rat adrenals. Binding studies on adrenal slices, on cell membranes and on cell suspensions were performed using [125I]AII and specific analogs for AT1 (Losartan) and AT2 (PD 123319) receptors. The distribution of AT1 was also studied by immunofluorescence. Complementary approaches were necessary to reach our goal. Indeed, by autoradiography on adrenal slices, [125I]AII was shown to bind to the zona glomerulosa (ZG) and to the medulla (M). When coincubated with [125I]AII, PD 123319 displaced [125I]AII from the medulla and from the ZG, indicating the presence of AT2 receptors in both zones. Losartan partially displaced [125I]AII from the ZG, indicating the presence of AT1 receptors in that zone. Furthermore, the labeling intensity of the medulla (AT2 receptors) was much stronger in adrenal sections from rats kept on a low sodium regimen than from controls. Immunofluorescence microscopy revealed that AT1 receptors were located mainly in the ZG of control rats. After sodium restriction, AT1 receptors appeared to be uniformly distributed within an enlarged ZG; furthermore AT1 receptor-positive cells were found to a limited degree in the zona fasciculata and possibly in the zona reticularis, and a greater number of these positive cells appeared in these zones under sodium restriction. Cell suspensions from rats fed a low sodium diet showed a 2.7- and 2.1-fold increase in total AII receptors in adrenal ZG and ZFR + M cells when compared with controls. Based on Losartan displacement, we calculated that [125I]AII bound to AT1 and to AT2 receptors was increased in both ZG and ZFR + M cell preparations under sodium restriction. Results of binding studies on cell membranes were also indicative of an increasing effect of sodium restriction on AT1 and AT2 receptors binding capacity. Furthermore, Northern blotting analysis revealed 3.0- and 2.5-fold increases in the level of AT1 receptor mRNA in the ZG and the ZFR + M of rats fed a low sodium diet as compared with those fed a normal diet. The low sodium intake resulted in a weaker increase (1.5-fold) in the level of AT2 receptor messenger RNA in the ZG, with no changes in the ZFR + M preparations. In conclusion, in this study complementary approaches were needed to determine the localization of AT1 and AT2 receptors in the rat adrenal, and to show the increasing effects of a low sodium regimen on the adrenal level of these receptors. Immunofluorescence studies revealed AT1 receptors mainly in the ZG and also in some cells of the inner adrenal cortex zones; in adrenals of rats kept on a low sodium diet the ZG was markedly enlarged, and an increased number of immunoreactive cells with AT1 receptors were observed throughout that zone; also more immunoreactive cells were present in the inner zones of the adrenal cortex. Furthermore in the adrenals of rats kept on a low sodium diet, we observed: 1) an increased number of AT1 and AT2 receptors in cell suspensions from the ZG, and in cell suspensions of the ZFR + M; 2) an increased level of AT1 and AT2 receptor mRNAs in the ZG; 3) an increased level of AT1 receptor mRNA, with no changes in the AT2 mRNA level in the ZFR + M. These results suggest a role for AT1 as well as for AT2 receptors in controlling adrenal function and differentiation under normal as well as under physiological stimulation of AII production following sodium restriction.

Characterization of the adrenal cytochrome P450C17 in the hamster, a small animal model for the study of adrenal dehydroepiandrosterone biosynthesis.

The hamster, like the human produces cortisol as its major glucocorticoid, rather than corticosterone, typical of most enzyme rodents. It is not known, however, if the hamster cytochrome P450C17 (P450C17), a key enzyme for cortisol formation, also exhibits 17,20-lyase activity and if it catalyzes the formation of dehydroepiandrosterone (DHEA) at the adrenal level. To study this, we isolated the cDNA of P450C17 from a hamster adrenal library. This cDNA was sequenced and was found to have an open reading frame for a protein of 511 amino acids, as compared to the human P450C17, which contains 508 amino acids. The hamster P450C17 cDNA, in the coding region, is 76% homologous with the human P450C17 cDNA. The cDNA was then cloned in the expression vector pSV-SPORT 1, which was transiently transfected into COS 1 cells. The transfected cells were used for temporal studies on the transformation of radiolabeled C21-delta5- and C21-delta4-precursors. When transfected cells were incubated with [14C]pregnenolone, rapid formation of [14C]DHEA occurred. The intermediate 17alpha-hydroxypregnenolone accumulated initially with subsequent metabolism to DHEA. Likewise, when incubated with C21-delta4-steroids, [14C]progesterone and [3H]17alpha-hydroxyprogesterone, the 17,20-lyase product androstenedione was produced efficiently. In these studies, with respect to the delta5 pathway, the expressed hamster P450C17 gave similar results to bovine P450C17 cDNA inserted in the same expression vector. However, in contrast to the bovine enzyme, which converted low amounts of progesterone to androstenedione, the expressed hamster P450C17 enzyme showed an active metabolism via the delta4 pathway. Northern blot analysis, using the complete alpha-32P labeled hamster P450C17 cDNA as the probe, demonstrated a strong presence of P450C17 mRNA in hamster adrenals, a weaker presence in testes and ovaries, and no detectable species in brain, mesentery, and kidney. Immunoblotting analysis using an anti-rat P450C17 antibody demonstrated the presence of P450C17 protein in hamster adrenals, testes, and ovaries. Hamster adrenal cell suspensions and microsomal preparations were used to demonstrate the biosynthesis of [14C]17alpha-hydroxypregnenolone and [14C]DHEA from [14C]pregnenolone; both metabolites were formed during incubations. However, the ratio of [14C]DHEA/[14C]17alpha-hydroxypregnenolone was much lower in adrenal cells than in transfected COS 1 cells, indicating the presence of putative factors in hamster adrenal cells, favoring the 17alpha-hydroxylase activity rather than that of the 17,20-lyase. In conclusion, these studies demonstrate that the hamster adrenal is both a DHEA and a cortisol producer, and, therefore, this animal could be a suitable small animal model for the study of the role of DHEA in relation to human biochemistry and physiology.

Acute sporadic non-A, non-B, non-C, non-D, non-D, non-E hepatitis.

Patients presenting with clinical and laboratory features consistent with a diagnosis of acute non-A, non-B hepatitis were evaluated for evidence of hepatitis C or hepatitis E infection and for evidence of severe or prolonged disease. Antibody to hepatitis C virus (anti-HCV) was detected in 75 of 108 (69%) patients, antibody to hepatitis E virus (anti-HEV) in three patients (3%), and neither antibody in 31 (29%) patients. One patient had both anti-HCV and anti-HEV. HCV RNA was not detected in sera from any of 20 patients with seronegative (non-ABCDE) hepatitis, but in all 10 patients with anti-HCV who were tested by polymerase chain reaction (PCR). Compared with patients with acute hepatitis C, those with non-ABCDE hepatitis had a lower incidence of parenteral risk factors (6% vs. 70%; P < .001), higher peak serum bilirubin levels (45% vs. 5% with peak levels > 15 mg/dL; P < .001), more prolonged jaundice (25% vs. 0% with peak bilirubin >5 weeks after onset; P < .01), more severe prothrombin time abnormalities (26% vs. 0% with >3 second prolongation; P < .001), more severe hypoalbuminemia (39% vs. 9% with albumin <3 g/dL; P < .01), and more frequent major clinical complications (13% vs. 0% with encephalopathy; P < .01; 10% vs. 0% with death or transplant; P = .024). Patients with acute non-ABCDE hepatitis were less likely to develop chronic hepatitis than those with acute hepatitis C (23% vs. 68%; P < .05). Thus, patients with acute non-ABCDE hepatitis are epidemiologically distinct from those with acute hepatitis C and have a significantly more severe acute illness.

Adrenocorticotropin regulates the level of the steroidogenic acute regulatory (StAR) protein mRNA in hamster adrenals.

In this study, we report the cloning of a StAR cDNA from a hamster adrenal cDNA library. The library was screened using a PCR fragment specific for the hamster adrenal StAR cDNA. Several clones of different lengths were obtained and one of these was sequenced. Northern blotting analysis revealed the presence of the StAR mRNA in male and female adrenals, in tests and ovaries, but not in the liver or kidneys of either sex. Whole hamster adrenals revealed the presence of four mRNAs of 0.65, 1.7, 3.1 and 5.25 kb, respectively. In addition, ACTH regulates the expression of StAR mRNA in hamster adrenals. Indeed, when groups of hamsters were injected with ACTH and sacrificed at different times after treatment, only the 0.65 kb form of the StAR mRNA did not increase, whereas the other forms increased at varying levels. These results might suggest that the expression of the StAR protein in hamster adrenals depends upon different genes, different promoters, or different polyadenylation signal sites. In conclusion, these results indicate that in vivo, StAR is regulated by ACTH, suggesting the participation of this protein in controlling the transformation of cholesterol to pregnenolone, a key regulatory step in corticosteroidogenesis.

Some effects of a low sodium intake on the expression of P450 aldosterone synthase in the hamster adrenal cortex: immunoblotting, immunofluorescent and immuno-gold electron microscopic studies.

In the current work we studied the effects of a low sodium intake on P450 aldosterone synthase (P450aldo) in the adrenal cortex of male hamsters by Western blotting analysis. We also investigated the zonal distribution of P450aldo with a specific antibody using immunofluorescence and immuno-gold electron microscopy. Western blotting analysis revealed a progressive induction of P450aldo in the adrenals of hamsters kept on a low sodium diet, with two-, four- and eightfold increases after 2, 4 and 21 days on the diet. Immunofluorescence microscopy showed that P450aldo was confined to the zona glomerulosa (ZG) cells. Electron microscopy showed P450aldo to be located in the mitochondria of ZG cells. When hamsters were maintained on a low sodium intake for 2, 11 and 21 days, P450aldo was still found only in the ZG; the ZG appeared either unchanged or sometimes slightly enlarged. Moreover, at days 11 and 21, the intensity of the immunofluorescent signal was much stronger in the ZG of hamsters on the low sodium intake than in controls. Hence, immunocytochemistry using the colloidal-gold technique showed P450aldo to be more abundant in the mitochondria of the experimental animals than in controls. To conclude, P450aldo is present only in the ZG of hamster adrenals and sodium restriction appears to induce its expression by stimulating production within individual ZG cells rather than by stimulating a proliferation of the ZG cells.

Steroid therapy of chronic hepatitis: characteristics associated with response in anti-hepatitis C virus-positive and -negative patients.

OBJECTIVES: The goals of this study were to examine responses to corticosteroid-containing therapy in non-B chronic hepatitis patients with different anti-hepatitis C virus (HCV), autoantibody, and biochemical test results and to determine what factors correlate with response. METHODS: Patients with a prior or current history of steroid therapy for putative autoimmune or chronic non-A, non-B hepatitis were assessed. Responses during the first 6 months of therapy were categorized as "complete" (normal aminotransferases for > or = 1 month), "partial" ( > 50% reduction), or "no response." RESULTS: Sufficient data available to permit evaluation in 32 patients. Complete responses were noted in 17, partial responses in 12, and no response in three subjects. By multivariate analysis, only absence of anti-HCV and presence of cirrhosis were independent predictors of response. Nonresponders were found to have lower scores in a proposed autoimmune hepatitis scoring system, but scores of complete and partial responders were not significantly different. Despite a lower likelihood of a complete response, 80% (12/15) of patients with multiantigen positive anti-HCV tests had either partial or complete initial responses to corticosteroid-containing therapy, and, in nine patients, aminotransferases fell to < 2 times the upper limit of normal. All 15 anti-HCV-negative patients, but only three of 15 anti-HCV-positive patients, entered complete responses that were sustained (aminotransferases < twofold abnormal) on regimens containing < 20 mg/day or prednisolone or prednisone. CONCLUSIONS: Although anti-HCV-positive patients frequently exhibit partial initial responses to immunosuppressive therapy, the absence of specific anti-HCV antibodies was better as a predictor of completeness of response than assessment of autoantibodies or degree of biochemical abnormalities.

The hamster adrenal cytochrome P450C11 has equipotent 11beta-hydroxylase and 19-hydroxylase activities, but no aldosterone synthase activity.

We have isolated a hamster adrenal P45OC11 cDNA which shared 90 and 84% homology, respectively, with the nucleotide sequence and the amino acid sequence of the hamster adrenal P450aldo. Both P450C11 and P450aldo cDNA coding sequences were inserted in the plasmid pBluescript SK, transcribed and then translated using a rabbit reticulocyte system in the presence of [35S]methionine. The reaction products were immunoprecipitated with an anti-bovine P450C11 antibody for P450C11 and with an anti-hamster P450aldo for P450aldo. Immunoprecipitated proteins were analyzed by polyacrylamide gel electrophoresis. A single 35S-labeled protein band was detected for P450C11 and for P450aldo, respectively. P450C11 and P450aldo cDNAs were then both inserted into the expression vector pCMV5 containing a viral sequence specific for the attachment of ribosomes to mRNA. These constructions were transfected in COS-1 cells. 24 h after transfection, the presence of P450C11 and P450aldo mRNAs was determined by Northern blot analysis. In a time study experiment we found that P450C11 transformed the labeled-steroid into [14C]corticosterone, [14C]19-OH-deoxycorticosterone and [14C]18-OH-deoxycorticosterone in ratios of 1:1.11:0.07, after 2 h of incubation; no [14C]aldosterone could be detected. Cells transfected with plasmids harboring the P450aldo cDNA transformed [14C]deoxycorticosterone to [14C]corticosterone, [14C]aldosterone, [14C]18-OH-corticosterone, [14C]18-OH-deoxycorticosterone, [14C]19-OH-deoxycorticosterone and [14C]11-dehydrocorticosterone in ratios of 1:0.25:0.45:0.04:0.04:0.04 after 12 h of incubation. These results indicate that one P450 catalyzes the ultimate step of glucocorticoid formation and a separate P450 is involved in the final steps of aldosterone formation in hamster adrenals. The capacity of the hamster adrenal P450C11 to hydroxylate at positions 11beta and 19 in nearly equal ratio makes this animal an excellent model to study the mechanism of synthesis and inhibition of 19-OH-deoxycorticosterone, the precursor of 19-nor-deoxycorticosterone, a very potent mineralocorticoid involved in the development of essential hypertension.

In vivo effects of adrenocorticotropin on c-jun, jun-B, c-fos and fos-B in rat adrenal.

We have studied the in vivo effects of adrenocorticotropin (ACTH) on mRNA levels of c-jun, jun-B, c-fos and fos-B, in rat adrenals. In control rats, c-jun mRNA was abundant in both zona glomerulosa (ZG) and zona fasciculatareticularis (ZF-R). Although less abundant than c-jun, the mRNA of jun-B could be detected in both zones, whereas that of c-fos could barely be detected and that of fos-B could not. After an injection with short acting ACTH, mRNA levels of c-jun, c-fos, jun-B and fos-B were maximally increased in both zones within 30 min. Within 5h, the mRNA levels decreased towards control levels for c-jun, to below control levels for jun-B, and to undetectable levels for c-fos and fos-B. After a sustained stimulation by two daily administrations of long acting ACTH, the mRNA of c-jun was still abundant in both zones, although its level decreased by 50% and 80% after 36h and 9 days, respectively, after the first injection. Under such conditions, the mRNA level of jun-B was increased, that of fos-B could barely be detected, and that of c-fos could not be detected. To conclude, these results suggest that jun-B, fos-B, and also c-fos play a role in triggering early events leading to an increased steroidogenesis, as well as a basic role in maintaining the integrity of the adrenal cortex in the case of c-jun and jun-B.

P450aldo in hamster adrenal cortex: immunofluorescent and immuno-gold electron microscopic studies.

The zonal distribution of aldosterone synthase cytochrome P450 (P450aldo) in the adrenal cortex of male hamsters was investigated by immunofluorescence and electron microscopy, using an anti-P450aldo peptide antibody. On cryostat sections the immunolocalization of P450aldo was confined to the zona glomerulosa cells. On semi-thin plastic sections, P450aldo was shown to be located in mitochondria. Studies in electron microscopy, using the colloidal gold technique, confirmed that P450aldo was located in mitochondria.

Murine M290 integrin expression modulated by mast cell activation.

Intraepithelial T lymphocytes possess a (TGF beta)-inducible integrin complex that consists of the beta 7 integrin chain in association with a novel alpha chain, murine alpha M290. Using antibodies directed against the murine alpha M290 chain, IL-3-dependent murine bone marrow-derived mast cells demonstrated positive staining after induction with TGF beta. Sequence information from the human intraepithelial T lymphocyte-alpha E gene sequence was used to clone the murine homolog from a TGF beta-induced T cell library. The murine alpha E homolog gene encodes a protein recognized by antibodies to alpha M290. Expression of this gene by bone marrow-derived mast cells was found to be induced by TGF beta or IgE-mediated cross-linking of Fc epsilon R1. Furthermore, exposure of murine TK-1 cells to supernatants from activated mast cells induces the expression of the alpha M290 gene, indicating that a mast cell mediator(s) can act in a paracrine manner to induce expression of this integrin complex by other cells.

The presence of two cytochrome P450 aldosterone synthase mRNAs in the hamster adrenal.

We isolated a cDNA from a hamster adrenal cDNA library which was similar in sequence to those of the mouse and rat P450c18 cDNAs. The hamster P450c18 cDNA, however, was shorter than the rat and mouse P450c18 cDNAs at its 5'-end and the peptide leader sequence was absent. From a hamster genomic library we isolated and sequenced the first seven exons and a 5'-flanking region of the first P450c18 gene exon. With this information we were able to generate a P450c18 cDNA containing the peptide leader sequence using the polymerase chain reaction. Northern analyses were performed on adrenals from hamsters maintained on a low sodium diet for 0, 4, 7 and 10 days using a 32P-labeled sequence specific to P450c18; two mRNA bands were found at 2 and 3.4 kb. The intensity of both bands was increased about 3- to 5-fold under sodium restriction compared to controls. A distinct mRNA band of 2.3 kb hybridized with an oligonucleotide specific to P450(11) beta and its intensity did not change following low sodium intake. Immunoblotting analyses were performed using an antibovine adrenal P450(11) beta antibody that does not discriminate between P450(11) beta and P450c18 proteins. Three bands were detected at 52, 48 and 45 kDa in homogenate preparations of entire glands. Furthermore, the 45 kDa protein band was present in homogenates of the zona glomerulosa and absent in homogenates of the zone fasciculata-reticularis. In conclusion, these results show that the hamster adrenals express P450c18 as do mouse, rat and human adrenal glands. Furthermore, two P450c18 mRNAs, which are inducible by a low sodium intake, are present in the hamster adrenal vs one for the rat. The physiological role of these two hamster adrenal mRNA species remains to be elucidated.

Preferential expression of interleukin-12 or interleukin-4 by murine bone marrow mast cells derived in mast cell growth factor or interleukin-3.

In this report we demonstrate that murine bone marrow cells cultured in either interleukin (IL)-3 or mast cell growth factor (MGF, also known as c-kit ligand and stem cell factor) differentially express cytokine genes. Bone marrow cells cultured in IL-3 differentiate and proliferate, taking on a mucosal mast cell-like phenotype. These cells express the IL-4 gene. Bone marrow cells cultured in MGF take on a connective tissue mast cell-like phenotype and possess transcripts for both of the subunits of the IL-12 cytokine. Bone marrow cells cultured in both IL-3 and MGF express the IL-4 gene at lower levels than that seen for the IL-3 culture alone, but do not possess IL-12 gene transcripts. The level of IL-12 subunit transcripts derived from the MGF-derived bone marrow cells was compared to that found in splenocytes and activated macrophages, the only cells in which IL-12 production has been previously documented. Both of the IL-12 subunit transcripts were found, compared to a beta-actin control, to be present within MGF-derived cells in the same if not higher quantities than the splenocyte or macrophage cultures. Mucosal mast cells have been previously implicated in the development of the T helper type 2 (TH2) T cell phenotype via their expression of IL-4. The finding that the MGF-derived connective tissue-like mast cells possess IL-12 transcripts suggests that the development of the TH1 T cell pathway may be positively influenced by this type of mast cell.