Prevalence of thiamine deficiency in a stable heart failure outpatient cohort on standard loop diuretic therapy.

BACKGROUND & AIMS: The prevalence of thiamine deficiency in heart failure (HF) patients has been reported to be as high as 50% in outpatient settings and has been found to be as high as 96% in the inpatient setting. Results from previous studies, however, have been inconsistent and further investigation is needed to clarify the true prevalence of thiamine deficiency in patients with chronic HF. The aim of this study was to determine the prevalence of thiamine deficiency in a random sample of stable HF outpatients receiving standard of care loop diuretic therapy. METHODS: A cross-sectional study was conducted in 30 HF patients scheduled for regular follow-up visits in the Mayo Heart Failure Clinic. Whole-blood thiamine diphosphate was measured using high-performance liquid chromatography. Additional clinical and demographic features were collected through review of electronic medical records. RESULTS: The estimated prevalence of thiamine deficiency in stable HF patients was calculated to be <11.6%. There was no correlation between diuretic dose and thiamine levels (r = 0.02, P = 0.93) and there was no correlation found between left-ventricular ejection fraction (LVEF) and thiamine levels (r = 0.147, p = 0.44). CONCLUSION: Our findings suggest that the prevalence of thiamine deficiency, based on standard normal values, in a stable outpatient HF cohort on standard loop diuretic therapy is very low. Previous work has demonstrated improvements in myocardial function with high-dose thiamine supplementation regardless of thiamine blood levels, however, suggesting that thiamine may become conditionally essential with HF. Therefore, we suggest that a disease-specific reference range be determined to accurately identify HF patients that would benefit from thiamine supplementation.

Comparison of Macular Thickness in Patients with Keratoconus and Control Subjects Using the Cirrus HD-OCT.

PURPOSE: The aim of the present study was to compare macular thickness in patients with keratoconus (KC) with macular thickness in healthy subjects. SUBJECTS AND METHODS: Twenty-six patients with KC and 52 control subjects were included. The macular structure was evaluated using a Zeiss Cirrus HD-OCT. The scan pattern used was 512 × 128, which covers an area of approximately 6 × 6 mm of the retina. The cube volume was assessed as well as macular thickness in each of the 9 sectors defined by the software. RESULTS: The mean signal strength was significantly lower in the KC group (mean 8.4, range 6-10) compared with the control group (mean 9.7, range 7-10), P < 0.0001 (unpaired t-test). There were no significant differences in cube volume (unpaired t-test), cube average thickness, or macular thickness between the KC group and the control subjects in any of the retinal locations (one-way ANOVA). CONCLUSION: Macular structure as measured by OCT in KC subjects should be expected to lie within the range of age and sex matched controls.

Evaluation of mosquito responses to pyrethroid insecticides topically applied to sheep.

A rise in the incidence of mosquito-transmitted Cache Valley virus (CVV) in lambs in 2011 prompted a study to evaluate on-animal pyrethroid insecticides to reduce mosquito attacks on sheep. Using enclosure traps for 1 night per wk for 6 wk, we compared engorgement rates of mosquitoes given the opportunity to feed on untreated sheep and sheep treated with 1 Python insecticide ear tag (containing 10% zeta-cypermethrin and 20% piperonyl butoxide) per animal or 2 synergized permethrin body spray treatments (containing 2.5% permethrin and 2.5% piperonyl butoxide). During the 6-wk study, 18,920 mosquitoes were collected in the animal-baited enclosure traps. Thirteen species were identified from these collections with the floodwater species Aedes increpitus and Ae. idahoensis making up 68% of the total. Potential CVV vector species, making up 25% of the samples, included Ae. vexans, Ae. dorsalis, Culex tarsalis, and Culiseta inornata. Traps baited with untreated sheep collected 9,701 mosquitoes with 65% of these engorged. Traps baited with sheep treated with Python ear tags or permethrin spray collected 4,034 and 4,555, respectively, with engorgement rates of 23% and 35%. Blood feeding on ear-tagged sheep was significantly reduced by as much as 90% compared to the untreated sheep, and protection lasted 4 wk or longer. Permethrin spray treatments were most effective within 24 h after application and provided better protection against Ae. dorsalis than the Python tag. Effectiveness of the permethrin spray diminished 1 wk after the 2nd application was made. The effect of these treatments appeared to be repellency because negligible mosquito mortality was observed at the time of collection. Further evaluation of these insecticides under conditions of natural exposure to a mosquito-borne pathogen is warranted.

Strong predictive value of TIMI risk score analysis for in-hospital and long-term survival of patients with right ventricular infarction.

BACKGROUND: While right ventricular myocardial infarction is associated with increased in-hospital morbidity and mortality, prognostic risk factors for in-hospital and long-term mortality are poorly defined. OBJECTIVES: To evaluate the prognostic value of TIMI (Thrombolysis in Myocardial Infarction) risk score analysis in patients with right ventricular myocardial infarction (RVI). DESIGN: Retrospective analysis of a community population. SETTING: Mayo Clinic Coronary Care Unit. PATIENTS: One hundred and two patients with RVI from 580 consecutive patients from Rochester, Minnesota admitted to the Coronary Care Unit with acute inferior or lateral wall myocardial infarction from January 1988 through March 1998. MEASUREMENT: Combined TIMI risk score analysis with in-hospital and long-term mortality. RESULTS: In-hospital morbidity (RVI: 54.9% vs non-RVI: 22.2%; P<0.001) and mortality (RVI: 21.6% vs non-RVI: 6.9%;P <0.001) were increased in patients with RVI. The TIMI risk score predicted risk (per one point increase in TIMI score) for in-hospital mortality (OR 1.23, 95% CI 1.02-1.51, P=0.037) and long-term mortality (OR 1.57, 95% CI 1.25-1.96, P<0.001). Patients with RVI whose TIMI risk score was >or=4 had significantly worse long-term survival compared to those patients with RVI and TIMI score <4 (P=0.006). CONCLUSIONS: In-hospital morbidity and mortality, and long-term mortality are increased by right ventricular infarction and can be accurately predicted by the initial TIMI risk score.

Understanding practice from the ground up.

BACKGROUND: Our objective was to understand family practices from the ground up through intensive direct observation of the practice environment and patient care. METHODS: Eighteen practices were purposefully drawn from a random sample of Nebraska family practices that had earlier participated in a study of preventive service delivery. Each practice was studied intensely over a 4- to 12-week period using a comparative case study design that included extended direct observation of the practice environment and clinical encounters, formal and informal interviews of clinicians and staff, and medical record review. DESIGN: This multimethod assessment process (MAP) provided insights into a wide range of practice activities ranging from descriptions of the organization and patient care activities to quantitative documentation of physician- and practice-level delivery of a wide range of evidence-based preventive services. Initial insights guided subsequent data collection and analysis and led to the integration of complexity science concepts into the design. In response to the needs and wishes of the participants, practice meetings were initiated to provide feedback, resulting in a more collaborative model of practice-based research. CONCLUSIONS: Our multimethod assessment process provided rich data for describing multiple aspects of primary care practice, testing a priori hypotheses, discovering new insights grounded in the actual experience of practice participants, and fostering collaborative practice change.

A recessive form of the Ehlers-Danlos syndrome caused by tenascin-X deficiency.

BACKGROUND: The Ehlers-Danlos syndrome is a heritable connective-tissue disorder caused by defects in fibrillar-collagen metabolism. Mutations in the type V collagen genes account for up to 50 percent of cases of classic Ehlers-Danlos syndrome, but many other cases are unexplained. We investigated whether the deficiency of the tenascins, extracellular-matrix proteins that are highly expressed in connective tissues, was associated with the Ehlers-Danlos syndrome. METHODS: We screened serum samples from 151 patients with the classic, hypermobility, or vascular types of the Ehlers-Danlos syndrome; 75 patients with psoriasis; 93 patients with rheumatoid arthritis; and 21 healthy persons for the presence of tenascin-X and tenascin-C by enzyme-linked immunosorbent assay. We examined the expression of tenascins and type V collagen in skin by immunohistochemical methods and sequenced the tenascin-X gene. RESULTS: Tenascin-X was present in serum from all normal subjects, all patients with psoriasis, all patients with rheumatoid arthritis, and 146 of 151 patients with the Ehlers-Danlos syndrome. Tenascin-X was absent from the serum of the 5 remaining patients with Ehlers-Danlos syndrome, who were unrelated. Tenascin-X deficiency was confirmed in these patients by analysis of skin fibroblasts and by immunostaining of skin. The expression of tenascin-C and type V collagen was normal in these patients. All five of these patients had hypermobile joints, hyperelastic skin, and easy bruising, without atrophic scarring. Tenascin-X mutations were identified in all tenascin-X-deficient patients; one patient had a homozygous tenascin-X gene deletion, one was heterozygous for the deletion, and three others had homozygous truncating point mutations, confirming a causative role for tenascin-X and a recessive pattern of inheritance. CONCLUSIONS: Tenascin-X deficiency causes a clinically distinct, recessive form of the Ehlers-Danlos syndrome. This finding indicates that factors other than the collagens or collagen-processing enzymes can cause the syndrome and suggests a central role for tenascin-X in maintaining the integrity of collagenous matrix.

Heterozygous mutation in the cholesterol side chain cleavage enzyme (p450scc) gene in a patient with 46,XY sex reversal and adrenal insufficiency.

Cytochrome P450scc, the mitochondrial cholesterol side chain cleavage enzyme, is the only enzyme that catalyzes the conversion of cholesterol to pregnenolone and, thus, is required for the biosynthesis of all steroid hormones. Congenital lipoid adrenal hyperplasia is a severe disorder of steroidogenesis in which cholesterol accumulates within steroidogenic cells and the synthesis of all adrenal and gonadal steroids is impaired, hormonally suggesting a disorder in P450scc. However, congenital lipoid adrenal hyperplasia is caused by mutations in the steroidogenic acute regulatory protein StAR; it has been thought that P450scc mutations are incompatible with human term gestation, because P450scc is needed for placental biosynthesis of progesterone, which is required to maintain pregnancy. In studying patients with congenital lipoid adrenal hyperplasia, we identified an individual with normal StAR and SF-1 genes and a heterozygous mutation in P450scc. The mutation was found in multiple cell types, but neither parent carried the mutation, suggesting it arose de novo during meiosis, before fertilization. The patient was atypical for congenital lipoid adrenal hyperplasia, having survived for 4 yr without hormonal replacement before experiencing life-threatening adrenal insufficiency. The P450scc mutation, an in-frame insertion of Gly and Asp between Asp271 and Val272, was inserted into a catalytically active fusion protein of the P450scc system (H2N-P450scc-Adrenodoxin Reductase-Adrenodoxin-COOH), completely inactivating enzymatic activity. Cotransfection of wild-type and mutant vectors showed that the mutation did not exert a dominant negative effect. Because P450scc is normally a slow and inefficient enzyme, we propose that P450scc haploinsufficiency results in subnormal responses to ACTH, so that recurrent ACTH stimulation leads to a slow accumulation of adrenal cholesterol, eventually causing cellular damage. Thus, although homozygous absence of P450scc should be incompatible with term gestation, haploinsufficiency of P450scc causes a late-onset form of congenital lipoid adrenal hyperplasia that can be explained by the same two-hit model that has been validated for congenital lipoid adrenal hyperplasia caused by StAR deficiency.

NF-1C, Sp1, and Sp3 are essential for transcription of the human gene for P450c17 (steroid 17alpha-hydroxylase/17,20 lyase) in human adrenal NCI-H295A cells.

Cytochrome P450c17 catalyzes steroid 17alpha-hydroxylase and 17,20 lyase activities, which are required for the biosynthesis of cortisol and sex steroids. Human P450c17 is expressed in a cAMP-responsive, cell-specific, developmentally programmed fashion, but little is known about its transcriptional regulation. Expression of deletion mutants of up to 2,500 bp of human 5'-flanking DNA in human adrenal NCI-H295A cells indicated that most regulatory activity was confined to the first 227 bp. Deoxyribonuclease I footprinting of the proximal promoter identified the TATA box, an steroidogenic factor-1 site, and three previously uncharacterized sites at -107/85, at -178/-152, and at -220/-185. EMSAs and methylation interference assays suggested that the -107/-85 site and the -178/-152 site bind members of the NF-1 (nuclear factor-1) family of transcription factors. An NF-1 consensus sequence generated similar DNA/protein complexes, and antibodies against NF-1C2/CTF2 supershifted the complexes formed by the -107/-85 site, the -178/-152 site, and the NF-1 consensus site. Western blots of nuclear extracts from NCI-H295A cells probed with this NF-1 antiserum identified two NF-1 isoforms between 50 and 55 kDa. The presence of NF-1C2 (CTF2) and CTF5 in NCI-H295A cells was demonstrated by RT-PCR and sequencing. Mutation of both the -107/-85 and the -178/-152 NF-1 sites reduced basal transcription by half. Supershift assays showed that the ubiquitous proteins Sp1 and Sp3 both bind to the -227/-184 region, and that mutation of their binding sites reduced transcription by 75%. Mutation of the Sp1/Sp3 site plus the two NF-1 sites eliminated almost all detectable transcription. Thus, Sp1 and Sp3 binding to the -227/-184 site and NF-1C proteins binding to the -107/-85 and the -178/-152 sites are crucial for adrenal transcription of the human gene for P450c17.

Genetics of vitamin D biosynthesis and its disorders.

The principal hormone regulator of bone mineralization is vitamin D, which must be activated by a metabolic pathway consisting of a 25-hydroxylase and a 1alpha-hydroxylase to yield 1,25 (OH)(2)D. The hormonal regulation of vitamin D activation is at the level of the 1alpha-hydroxylase. We review the biology of vitamin D, the biochemistry of its activation and the molecular biology of the vitamin D-metabolizing enzymes. Recent advances have resulted in the cloning of the human vitamin D 1alpha-hydroxylase and the identification of mutations in its gene that cause Vitamin D Dependent Rickets type I.

Bundle branch block as a predictor of long-term survival after acute myocardial infarction.

Using a community-based population of patients with acute myocardial infarction (AMI), we sought to: (1) determine the prevalence of bundle branch block (BBB) on the presenting electrocardiogram (ECG), (2) compare the clinical characteristics and the treatment administered to patients with and without BBB, and (3) determine the association of BBB with mortality. We analyzed the admission ECGs of 894 consecutive patients with AMI from Olmsted County, Minnesota, seen at our institution from January 1988 to March 1998. Of these, 53 had left BBB (LBBB) (5.9%) and 60 had right BBB (RBBB) (6.7%). Patients with BBB were more likely to be older, have a history of AMI or hypertension, and to be in Killip class >I at presentation. They were less likely to receive primary reperfusion therapy, beta blockers, or heparin, but more likely to receive angiotensin-converting enzyme inhibitors. They had lower mean predischarge ejection fractions (38 +/- 16% vs 50 +/- 15%, p <0.0001). In-hospital mortality was 13.3%, 17.0%, and 9.1% for patients with RBBB, LBBB, and no BBB, respectively (p = 0.11). Respective postdischarge survival at 1, 3, and 5 years was 80%, 60%, and 50% in the RBBB group, 78%, 56%, and 51% in the LBBB group, and 92%, 85%, and 76% in the group without BBB (p <0.0001). Although BBB was not an independent predictor of mortality on multivariate analysis, the presence of transient or persistent BBB with AMI is an easily recognized clinical marker of increased mortality. Our conclusion from this study is that in a community-based population, patients who had LBBB or RBBB at the time of AMI had lower predischarge ejection fractions and higher in-hospital and long-term unadjusted mortality.

Creation and activity of COS-1 cells stably expressing the F2 fusion of the human cholesterol side-chain cleavage enzyme system.

A fusion construct for the human cholesterol side-chain cleavage enzyme system termed F2 (H(2)N-P450scc-adrenodoxin reductase-adrenodoxin-COOH), was stably expressed in nonsteroidogenic COS-1 cells. Multiple clones were obtained and analyzed, identifying the clone COS-F2-130 as the most active in converting 22R-hydroxycholesterol (22R-OH-C) to pregnenolone. The F2 fusion construct was properly transcribed and translated in COS-F2-130 cells, indicating that these cells did not proteolytically cleave the F2 protein. Steroid analyses show that the COS-F2-130 cells do not convert appreciable quantities of pregnenolone to other steroids. Isolated COS-F2-130 mitochondria showed enhanced steroidogenesis when incubated with biosynthetic N-62 StAR protein in vitro. The cells were easily transfectable with StAR expression vectors, showing that COS-F2-130 cells exhibited both StAR-independent and StAR-dependent activity. Transient expression of either full-length or N-62 StAR stimulated steroidogenesis to approximately 45% of the maximal steroidogenic capacity, as indicated by incubation with 22R-OH-C. Single, double, and triple transfections of individual vectors expressing P450scc, adrenodoxin reductase, and adrenodoxin demonstrated that the P450 moiety of the F2 fusion protein could only receive electrons from the covalently linked adrenodoxin moiety, but that free adrenodoxin reductase could foster activity of the fusion enzyme. COS-F2-130 cells provide a useful system for studying steroidogenesis, as these are the only cells described to date that convert cholesterol to pregnenolone but lack downstream enzymes that catalyze other steroidogenic reactions.

Characteristics of presenting electrocardiograms of acute myocardial infarction from a community-based population predict short- and long-term mortality.

To investigate the relevance of presenting electrocardiographic (ECG) patterns to short- and long-term mortality in nonreferral patients with acute myocardial infarction (AMI), 6 ECG patterns were analyzed. A consecutive series of 907 patients from Olmsted County, Minnesota, admitted to the Mayo Clinic Cardiac Care Unit from January 1, 1988 to March 31, 1998 for acute myocardial infarction comprised the study population. ECG patterns and distribution in the population were: (1) ST elevation alone (20.8%), (2) ST elevation with ST depression (35.2%), (3) normal or nondiagnostic electrocardiograms (18.5%), (4) ST depression alone (11.8%), (5) T-wave inversion only (10.7%), and (6) new left bundle branch block (LBBB) (3.0%). Seven- and 28-day mortalities varied significantly (p <0.01) among the 6 ECG groups. Respective mortalities were 3.0% and 6.0% for patients with normal or nondiagnostic electrocardiograms, 3.1% and 5.2% for T-wave inversion only, 7.4% and 10.6% for ST elevation alone, 9.4% and 13.1% for ST depression alone, 10.3% and 13.8% for ST elevation with ST depression, and 18.5% and 22.2% for new LBBB. Length of hospital stay (LOS) also varied among the ECG pattern groups (p <0.001) with the longest average LOS being in the new LBBB group (12.5 days). Long-term survival was similar among 5 ECG pattern groups (45% to 55% at 8 years from discharge) with the exception of LBBB (20% at 8 years). Among non-LBBB groups, ST-segment depression with or without ST elevation was associated with increased short-term mortality. Also, in this community-based population, 18.5% of patients had normal or nondiagnostic electrocardiograms.

The promoter for the ovine follicle-stimulating hormone-beta gene (FSHbeta) confers FSHbeta-like expression on luciferase in transgenic mice: regulatory studies in vivo and in vitro.

Transgenic mice harboring the ovine FSHbeta (oFSHbeta) promoter plus first intron (from -4741 to +759 bp) linked to a luciferase reporter gene (oFSHbetaLuc) were generated to determine whether this promoter can direct tissue-specific expression in vivo and serve as a model for studying hormonal regulation of the FSHbeta gene. Of six lines of transgenic mice analyzed, luciferase was detected uniquely in the pituitaries of five of them. Pituitary luciferase activity was decreased 51-99% by chronic GnRH treatment (Lupron depot). Orchidectomy caused a 2- to 8-fold increase, and ovariectomy caused a 2- to 27-fold increase in pituitary luciferase activity. Furthermore, pituitary luciferase expression was consistently higher on estrus than on diestrus (3- to 20-fold). These data strongly suggested that the transgene was expressed specifically in pituitary gonadotropes and regulated in the same way as the endogenous mouse FSHbeta gene. Using primary pituitary cell cultures prepared from these transgenic mice, basal luciferase expression was maximal on day 3 and then decreased by day 6 of culture, a pattern reflected by endogenous mouse FSH secretion. In these pituitary cultures, basal oFSHbetaLuc expression was decreased 61-82% by follistatin or 59-79% by inhibin. Similarly, mouse FSH secretion was decreased 71% by follistatin or 65% by inhibin. Progesterone inhibited oFSHbetaLuc expression by 44-51%, but it had no effect on endogenous mouse FSH secretion. Estradiol lowered FSH secretion by 21%, but did not decrease oFSHbetaLuc expression significantly. In conclusion, these data demonstrated the ability of the oFSHbeta promoter to direct expression of a reporter gene specifically to pituitary gonadotropes in transgenic mice. Studying oFSHbetaLuc expression in vivo and in cell cultures derived from pituitaries of these transgenic mice should prove useful for understanding many features of FSHbeta regulation.

Transcriptional regulation of the ovine follicle-stimulating hormone-beta gene by activin and gonadotropin-releasing hormone (GnRH): involvement of two proximal activator protein-1 sites for GnRH stimulation.

Previous studies from our laboratory demonstrated that a transgene consisting of the promoter for the ovine FSH beta-subunit gene and a luciferase reporter (wt-oFSHbetaLuc) was expressed and regulated like the FSHbeta gene in vivo and in vitro. In the present study pituitary cultures were prepared from these transgenic mice as well as mice carrying mutated oFSHbetaLuc lacking two functional activator protein-1 (AP-1) sites at -120 and -83 bp (mut-oFSHbetaLuc). These AP-1 sites were reported necessary for induction of oFSHbetaLuc by GnRH in a HeLa cell system. To examine the importance of the two AP-1 sites in mediating GnRH and activin effects in primary gonadotropes, pituitary cultures derived from transgenic mice were pretreated with follistatin to remove activin or activin-like factors present in the cultures. Follistatin lowered luciferase expression in cultures carrying both wt-oFSHbetaLuc and mut-oFSHbetaLuc transgenes by 74-86%, and subsequent addition of activin induced luciferase expression of both wt- and mut-transgenes by 4- to 14-fold within 4 h, suggesting that these AP-1 sites are not involved in activin stimulation of FSHbeta gene transcription. When GnRH was added along with activin, the wt-oFSHbetaLuc transgene was induced 200% compared with activin alone, but this effect was not observed with the mut-oFSHbetaLuc transgene. These data confirmed the HeLa cell studies showing that GnRH signals through two AP-1 sites to increase oFSHbeta transcription in gonadotropes. However, as the mutation of both AP-1 sites had no apparent effect on the expression and regulation of the transgene in vivo (basal, castration, GnRH down-regulation, cycle stage, and GnRH immunoneutralization), it appears that these AP-1 sites have little influence over the major effect of GnRH observed in vivo. These data also showed that activin plays a major role in transcriptional regulation of the FSHbeta gene, and the oFSHbeta promoter contains the activin response element(s) that is as yet undefined.

A novel role for bone morphogenetic proteins in the synthesis of follicle-stimulating hormone.

FSH is produced in pituitary gonadotropes as an alpha/beta heterodimer, and synthesis of the beta-subunit is the rate-limiting step in overall FSH production. Synthesis of FSHbeta can be regulated by activin and inhibin, both of which are members of the transforming growth factor-beta superfamily. Bone morphogenetic proteins (BMPs) also belong to the transforming growth factor-beta family and are multifunctional growth factors involved in many aspects of tissue development and morphogenesis, including regulation of FSH action in the ovary. Here we report a novel function for BMP-7 and BMP-6 in regulating FSH synthesis in the pituitary. Using primary pituitary cell cultures derived from transgenic mice that carry the ovine FSHbeta promoter linked to a luciferase reporter gene (oFSHbetaLuc), BMP-7 or BMP-6 was found to stimulate oFSHbetaLuc expression by 6-fold. Transient expression of the oFSHbetaLuc in a transformed gonadotrope cell line, LbetaT2, was induced 4-fold by BMP-7 or BMP-6 treatment. More importantly, BMP-7 and BMP-6 increased endogenous FSH secretion by 10- and 14-fold, respectively, from LbetaT2 cells, demonstrating for the first time that a functional signaling BMP system is present in gonadotropes. Two bioneutralizing antibodies to BMP-7, which cross-react with BMP-6, but not with activin A, decreased basal oFSHbetaLuc expression and FSH secretion from transgenic mouse pituitary cultures by 83-88% and 47-48%, respectively, suggesting an autocrine or paracrine role for BMP-7 or BMP-6 in FSH synthesis. Neither bioneutralizing antibody to activin A or activin B decreased basal oFSHbetaLuc expression or mouse FSH secretion significantly. Dose-dependent inhibition of FSH synthesis by anti-BMP7 was also observed in rat and sheep pituitary cultures. These results combined with the fact that the messenger RNAs for BMP-7 and BMP-6 were detected in mouse pituitaries and LbetaT2 cells indicate that BMP-7 and/or BMP-6 can function as FSH stimulators and may be significant physiological factors maintaining basal FSH expression in vivo.

Cell-specific transcriptional regulation of follicle-stimulating hormone-beta by activin and gonadotropin-releasing hormone in the LbetaT2 pituitary gonadotrope cell model.

FSH is secreted by gonadotropes of the anterior pituitary and plays a crucial role in mammalian reproduction. However, little is known about FSH gene regulation due to the lack of a gonadotrope cell line that synthesizes FSH. The LbetaT2 mouse pituitary cell line, isolated by targeted tumorigenesis in transgenic mice, has the characteristics of a mature gonadotrope, including expression of GnRH receptor, steroidogenic factor 1, and both the alpha- and beta-subunits of LH, but was thought not to express FSH. Using RT-PCR, we show that these cells synthesize FSH beta- subunit messenger RNA, which is induced by activin and inhibited by follistatin. Furthermore, in transient transfections an ovine FSHbeta 5'-regulatory region (5.5 kb) confers LbetaT2 cell-specific expression to a reporter gene compared with other pituitary and nonpituitary cell lines. This FSHbeta regulatory region responds to activin specifically in LbetaT2 cells, an effect that is blocked by follistatin. The LHbeta, alpha-subunit, and GnRH receptor regulatory regions are induced by activin and blocked by follistatin. Furthermore, LbetaT2 cells express the components of the activin system, and addition of follistatin alone reduces FSHbeta gene expression, demonstrating that an endogenous activin autocrine loop regulates FSH in these cells. In addition, GnRH stimulates both the FSHbeta and LHbeta regulatory regions, specifically in LbetaT2 cells. Surprisingly, GnRH induction is reduced by follistatin, suggesting its dependence on endogenous activin. As the mouse GnRH receptor promoter is inhibited by follistatin, reduction of GnRH receptor levels might be one mechanism by which follistatin interferes with GnRH induction of gonadotropin genes. In summary, LbetaT2 cells exhibit the characteristics of fully differentiated gonadotropes, including the expression of LH, FSH, GnRH receptor, and components of the activin/follistatin system, as well as display the appropriate responses to activin and GNRH:

Molten-globule structure and membrane binding of the N-terminal protease-resistant domain (63-193) of the steroidogenic acute regulatory protein (StAR).

The first step in steroidogenesis is the movement of cholesterol from the outer to inner mitochondrial membrane; this movement is facilitated by the steroidogenic acute regulatory protein (StAR). StAR has molten-globule properties at low pH and a protease-resistant N-terminal domain at pH 4 and pH 8 comprising residues 63-193. To explore the mechanism of action of StAR we investigated the structural properties of the bacterially expressed N-terminal domain (63-193 StAR) using CD, limited proteolysis and NMR. Far- and near-UV CD showed that the amount of secondary structure was greater at acidic than at neutral pH, but there was little tertiary structure at any pH. Unlike 63-193 StAR liberated from N-62 StAR by proteolysis, biosynthetic 63-193 StAR was no longer resistant to trypsin or proteinase K at pH 7, or to pepsin at pH 4. Addition of trifluoroethanol and SDS increased secondary structure at pH 7, and dodecylphosphocholine and CHAPS increased secondary structure at pH 2, pH 4 and pH 7. However, none of these conditions induced tertiary structure, as monitored by near-UV CD or NMR. Liposomes of phosphatidylcholine, phosphatidylserine and their mixture increased secondary structure of 63-193 StAR at pH 7, as monitored by far-UV CD, and stable protein-liposome complexes were identified by gel-permeation chromatography. These results provide further evidence that the N-terminal domain of StAR is a molten globule, and provide evidence that this conformation facilitates the interaction of the N-terminal domain of StAR with membranes. We suggest that this interaction is the key to understanding the mechanism of StAR's action.