Usefulness and accuracy of segmental adrenal venous sampling on localisation and functional diagnosis of various adrenal lesions in primary aldosteronism.

AIM: To clarify the usefulness and accuracy of segmental adrenal venous sampling (sAVS) on localisation and functional diagnosis of various adrenal lesions in primary aldosteronism. MATERIALS AND METHODS: Consecutive patients (n=162) who underwent adrenalectomy and 138 patients indicated for medication following sAVS were analysed retrospectively. Based on immunohistopathological diagnosis, the positive predictive value (PPV) of computed tomography (CT)-detectable aldosterone-producing adenoma (APA) was calculated. Moreover, endocrinological and sAVS characteristics were analysed quantitatively and qualitatively among APA, CT-undetectable aldosterone-producing nodules (APNs), multiple aldosterone-producing micronodules (MAPM), and medication groups. RESULTS: The PPV of APA by sAVS was 137/141 (97.1%; 95% confidence interval, 92.9-99.2%). Compared to the medication cases, the APA group showed stronger disease activity clinically and significant differences in adrenal hormones, such as a higher aldosterone level and aldosterone-to-cortisol ratio, and lower cortisol levels in the adrenal central vein and aldosterone maximum tributaries on the dominant side after cosyntropin stimulation. The APA group shows focal aldosterone hypersecretion, such as mean number of aldosterone elevated segments (1.7 ± 0.7 versus 2 ± 0.9, p=0.003) and presence of aldosterone-not-elevated segments (93% versus 41%, p<0.001). Clinically and in terms of sAVS, APN and MAPM showed similar characteristics to APA and to the medication cases, respectively. CONCLUSION: sAVS can localise functionally active tissues of CT-detectable and CT-undetectable lesions enabling decisions on surgical or medical treatment.

Sputum-to-serum hydrogen sulphide ratio as a novel biomarker of predicting future risks of asthma exacerbation.

BACKGROUND: Increased level of hydrogen sulphide (H2 S) in sputum is reported to be a new biomarker of neutrophilic airway inflammation in chronic airway disorders. However, the relationship between H2 S and disease activity remains unclear. OBJECTIVE: We investigated whether H2 S levels could vary during different conditions in asthma. METHOD: H2 S levels in sputum and serum were measured using a sulphide-sensitive electrode in 47 stable asthmatic subjects (S-BA), 21 uncontrolled asthmatic subjects (UC-BA), 26 asthmatic subjects with acute exacerbation (AE-BA) and 15 healthy subjects. Of these, H2 S levels during stable, as well as exacerbation states, were obtained in 13 asthmatic subjects. RESULTS: Sputum H2 S levels were significantly higher in the AE-BA subjects compared to the UC-BA and healthy subjects (P < .05). However, serum H2 S levels in the AE-BA subjects were lower than in the S-BA subjects (P < .001) and similar to those in healthy subjects. Thus, the sputum-to-serum ratio of H2 S (H2 S ratio) in the AE-BA subjects was significantly higher than in the S-BA, UC-BA and healthy subjects (P < .05). Among all subjects, sputum H2 S levels showed a trend to decrease with FEV1 %predicted and significantly positive correlations with sputum neutrophils (%), sputum IL-8 and serum IL-8. A multiple linear regression analysis showed that sputum H2 S was independently associated with increased sputum neutrophils (%) and decreased FEV1 %predicted (P < .05). The cut-off level of H2 S ratio to indicate an exacerbation was ≥0.34 (area under the curve; 0.88, with a sensitivity of 81.8% and specificity of 72.7%, P < .001). Furthermore, half of the asthmatic subjects with H2 S ratios higher than the cut-off level experienced asthma exacerbations over the following 3 months after enrolment. CONCLUSIONS: The H2 S ratio may provide useful information on predicting future risks of asthma exacerbation, as well as on obstructive neutrophilic airway inflammation as one of the non-Th2 biomarkers, in asthma.

Key factors for school health policy implementation in Thailand.

Thailand formulated a National School Health Policy (NSHP) in 1998, and it has been widely implemented but has not been evaluated. This case study aimed to identify factors that have influenced the implementation of NSHP in Thailand. For this purpose, we conducted a document review and key informant interviews. We selected key interviewees, from NSHP implementers at national, provincial and school levels in four geographical areas. We adopted a content analysis method, using a framework of 12 influential components of successful policy implementation and triangular policy framework. This study showed that NSHP was well-disseminated and implemented at whole country. We identified seven positive factors influencing NSHP implementation, namely matching with ongoing educational strategy, competition and encouragement by an awarding system, sustainable human capacity building at school level, participation of multiple stakeholders, sufficient understanding and acceptance of school health concepts, sharing information and collaboration among schools in the same clusters and functional fund raising activities. In addition, we identified three negative factors, namely lack of institutional sustainability, vague role of provincial officers and diverse health problems among Thai children. The government should clarify the role of provincial level and set up institutionalized capacity-building system as measures to strengthen monitoring and evaluation activities.

The incidence, treatment and prognosis of cervical carcinoma in young women: a retrospective analysis of 4,975 cases in Japan.

OBJECTIVE: To determine the clinical characteristics of patients (young women) with cervical carcinoma aged less than 35 years. METHODS: Data from patients who were treated for cervical carcinomas from 1990 to 2000 in the Kinki District were retrospectively investigated for clinical stage, histologic type, treatment procedure and prognosis. RESULTS: Of a total of 4,975 cases, 441 patients were aged less than 35 years old. The incidence of cervical carcinoma in these women was 7.9% from 1990 to 1995, 9.1% from 1996 to 2000, and 9.5% from 2001 to 2005. FIGO Stage I included 374 cases, followed by, 49 in Stage II, 11 in Stage III, and seven in Stage IV. Squamous cell carcinoma incidence was 80.7% and non-squamous cell carcinoma incidence was 19.3%. Several types of surgery were performed in patients with Stage I and II, while patients with Stage III and IV were treated with radiotherapy and/or chemotherapy without any type of surgery. In patients who underwent lymphadenectomy, 21.1% cases had nodal involvement. The 5-year survival rate was 95% for Stage I disease, 73% for Stage II, 68% for Stage III, and 19% for Stage IV. CONCLUSION: The incidence of cervical carcinoma in young women slightly increased from 1990 to 2005. The prognosis of cervical carcinoma tends to be better in young women than in older patients, especially in Stage III disease.

Antifouling properties of tough gels against barnacles in a long-term marine environment experiment.

In the marine environment, the antifouling (AF) properties of various kinds of hydrogels against sessile marine organisms (algae, sea squirts, barnacles) were tested in a long-term experiment. The results demonstrate that most hydrogels can endure at least 2 months in the marine environment. In particular, mechanically tough PAMPS/PAAm DN and PVA gels exhibited AF activity against marine sessile organisms, especially barnacles, for as long as 330 days. The AF ability of hydrogels toward barnacles is explained in terms of an 'easy-release' mechanism in which the high water content and the elastic modulus of the gel are two important parameters.

Effects of the short chain sugar acid 2-buten-4-olide on the hypothalamo-pituitary-adrenal axis in normal and adjuvant-induced arthritic rats.

The effects of intraperitoneal (i.p.) administration of 2-buten-4-olide (2-B4O), an endogenous sugar acid, on the hypothalamo-adenohypophysial system were examined in Lewis rats that were normal and in adjuvant-induced arthritic (AA) rats. In comparison with vehicle-treated rats, the plasma corticosterone and c-fos mRNA levels in the paraventricular nucleus (PVN) of normal rats increased significantly after i.p. administration of 2-B4O. Dual immunostaining revealed that almost all corticotrophin-releasing factor (CRF)-immunopositive neurones in the parvocellular division of the PVN exhibited Fos-like immunoreactivity (LI) 120 min after i.p. administration of 2-B4O (100 mg/kg). In the AA rats, repeated i.p. administration of 2-B4O (100 mg/kg) after immunisation significantly suppressed the expression of clinical symptoms and significantly increased plasma concentrations of corticosterone. Further, repeated i.p. administration of 2-B4O significantly increased CRF mRNA levels in the PVN and pro-opiomelanocortin mRNA levels in the anterior pituitary; however, they did not change arginine vasopressin mRNA levels in the parvocellular division of the PVN. These results suggest that i.p. administration of 2-B4O activates the hypothalamo-pituitary-adrenal (HPA) axis via the activation of CRF neurones in the PVN, and the activation of the HPA axis by i.p. administration of 2-B4O may be associated with the inhibition of AA in rats.

Prolactin-releasing peptide is a potent mediator of stress responses in the brain through the hypothalamic paraventricular nucleus.

The effects of i.c.v. administration of prolactin-releasing peptide on neurons in the paraventricular nucleus of rats and plasma corticosterone levels were examined by measuring changes in Fos-like immunoreactivity, c-fos mRNA using in situ hybridization histochemistry, and plasma corticosterone using a specific radioimmunoassay. Approximately 80% of corticotropin-releasing hormone immunoreactive cells exhibited Fos-like immunoreactivity in the parvocellular division of the paraventricular nucleus 90 min after i.c.v. administration of prolactin-releasing peptide. The greatest induction of the c-fos mRNA expression in the paraventricular nucleus was observed 30 min after administration of prolactin-releasing peptide, and occurred in a dose-related manner. Plasma corticosterone levels were also significantly increased 30 min after administration of prolactin-releasing peptide. Next, the effects of restraint stress, nociceptive stimulus and acute inflammatory stress on the expression of the prolactin-releasing peptide mRNA in the dorsomedial hypothalamic nucleus, nucleus of the solitary tract and ventrolateral medulla were examined using in situ hybridization histochemistry for prolactin-releasing peptide mRNA. Restraint stress and acute inflammatory stress upregulated the prolactin-releasing peptide mRNA expression in the nucleus of the solitary tract and ventrolateral medulla. Nociceptive stimulus upregulated the prolactin-releasing peptide mRNA expression in the ventrolateral medulla. Finally, we observed that pretreatment (i.c.v. administration) with an anti-prolactin-releasing peptide antibody significantly attenuated nociceptive stimulus-induced c-fos mRNA expression in the paraventricular nucleus. These results suggest that prolactin-releasing peptide is a potent and important mediator of the stress response in the brain through the hypothalamic paraventricular nucleus.

Risk stratification for in-hospital mortality in spontaneous intracerebral haemorrhage: a Classification and Regression Tree analysis.

BACKGROUND: Risk stratification for mortality in intracerebral haemorrhage (ICH) helps guide care, but existing clinical prediction rules are too cumbersome for clinical practice because of their complexity. AIM: To develop a simple decision tree model of in-hospital mortality risk stratification for ICH patients. METHODS: We collected information on spontaneous ICH patients hospitalized in a teaching hospital in Japan from August, 1998 to December, 2001 (n = 374). All variables were abstracted from data available at the time of initial evaluation. A prediction rule for in-hospital mortality was developed by the Classification and Regression Tree (CART) methodology. The accuracy of the model was evaluated using the area under receiver-operator characteristic curve. RESULTS: Overall in-hospital mortality rate was 20.2%. The CART methodology identified four groups for mortality risk, varying from low (2.1%) to high (58.9%). Level of consciousness (coma) was the best single predictor for mortality, followed by high ICH volume (cut-off 10.4 ml), and then age (cut-off 75 years). The accuracy of our CART model (0.86) exceeded that of a multivariate logistic regression model (0.81). DISCUSSION: ICH patients can easily be stratified for mortality risk, based on three predictors available on admission. This simple decision tree model provides clinicians with a reliable and practical tool.

Fine-scale deletion mapping of the distal long arm of chromosome 6 in 70 human ovarian cancers.

To define a small region on chromosome 6q containing a putative tumor suppressor gene for ovarian cancer, we examined loss of heterozygosity in 70 ovarian tumors of three histological types with nine restriction fragment length polymorphism markers located at 6q24-27. Among 33 cancers of serous type that were informative at one or more loci, 17 showed allelic loss at a few or all loci examined, whereas only 1 of 15 mucinous-type tumors and 2 of 12 clear-cell tumors revealed loss of heterozygosity. This result supported our earlier suggestion that alteration of a gene on chromosome 6q may play an important role during development of serous ovarian tumors (Sato et al., Cancer Res., 51: 5118-5122, 1991). Frequent losses were observed between loci defined by CI6-119 (D6S195) at 6q26 and CI6-49 (D6S161) at 6q27. A detailed deletion map indicated a commonly deleted region between loci defined by CI6-111 (D6S193) and CI6-24 (D6S149); these two markers are estimated to be 1.9 cM apart on the basis of linkage analysis. Our results further define a region containing a tumor suppressor gene involved in ovarian carcinoma within an approximately 2-megabase-long segment of chromosome 6q.

Detailed deletion mapping of chromosome 17q in ovarian and breast cancers: 2-cM region on 17q21.3 often and commonly deleted in tumors.

Using 11 restriction fragment length polymorphism markers, we examined loss of heterozygosity on the long arm of chromosome 17, where one or more genes responsible for hereditary breast and ovarian cancers may be present, in sporadic forms of 94 ovarian and 246 breast cancers. Loss of heterozygosity was observed in 33 of 84 (39.3%) ovarian and in 88 of 214 (41.1%) breast cancers that were informative with at least one marker. Detailed deletion mapping of chromosome 17q in these cancers identified two distinct, commonly deleted regions. One was located between 17q12 and 17q21.3 and the other between 17q25.1 and 17q25.3. In breast cancers, the proximal commonly deleted region was between two loci defined by markers CI17-701 and CI17-730 at 17q21.3, which are 2.4 cM apart. This segment overlaps the region that includes the putative gene for hereditary breast and ovarian carcinomas. The results suggest that at least two tumor suppressor genes associated with sporadic ovarian and breast cancers are present on chromosome 17q and that one of them may be the same gene that is responsible for the hereditary form.

Definition of a commonly deleted region in ovarian cancers to a 300-kb segment of chromosome 6q27.

Allelic deletions of chromosome 6q that occur frequently in ovarian cancers imply the presence of a putative tumor suppressor gene in this chromosomal vicinity. We analyzed DNA from 32 patients with ovarian carcinomas for loss of heterozygosity at loci on the distal portion of chromosome 6q and constructed a detailed deletion map. The map indicated a commonly deleted region between loci D6S149 (defined by CI6-24) and A2, which are estimated to be 300 kb apart on the basis of our cosmid contig map. By means of exon trapping, we found that the human AF-6 gene, which is disrupted in acute myeloid leukemia cells that carry a (6;11)(q27;q23) translocation, is located within the commonly deleted region. Subsequent screening of the AF-6 gene in ovarian carcinomas revealed no mutations. However, our mapping results, which narrowed the region containing the putative tumor suppressor gene to a 300-kb segment of 6q27, will facilitate further efforts to identify a gene associated with ovarian cancer.

Overexpression and overactivation of Akt in thyroid carcinoma.

Enhanced activation of Akt occurs in Cowden's disease, an inherited syndrome of follicular thyroid, breast, colon, and skin tumors, via inactivation of its regulatory protein, PTEN. Whereas PTEN inactivation is uncommon in sporadic thyroid cancer, activation of growth factor pathways that signal through Akt is frequently identified. We hypothesized that Akt overactivation could be a common finding in sporadic thyroid cancer and might be important in thyroid cancer biology. We examined thyroid cancer cells lines and benign and malignant thyroid tissue for total Akt activation and isoform-specific Akt expression. In thyroid cancer cells, Akt 1, 2, and 3 proteins were expressed, total Akt was activated by insulin phosphatidylinositol 3'-kinase, and inhibition of phosphatidylinositol 3'-kinase reduced cell viability. In human thyroid tissue, increased levels of phosphorylated total Akt were identified in follicular but not papillary cancers compared with normal tissue. Levels of Akt 1 and 2 proteins and Akt 2 RNA were elevated only in the follicular cancers. In paired samples, Akt 1, 2, 3, and phospho-Akt levels were higher in five of six cancers, including three of three follicular cancers. These data suggest that Akt activation may play a role in the pathogenesis or progression of sporadic thyroid cancer.

Developmental changes of two distinct ribonuclease H activities from rat brain and characterization of these enzymes.

The specific activities of both Mg2+- and Mn2+-dependent ribonucleases H (RNAase H) from rat cerebella increased up to approx. the 6th day after birth and then decreased in adult rats. Those isolated from the non-cerebellar part decreased gradually toward adult levels after birth. The two enzymes could be separated by phosphocellulose chromatography. They can be distinguished from one another by their ionic requirement, molecular weight, sedimentation coefficient, optimal pH, sensitivity to the -SH reagent, and the effects of salt, polyamine and pyrophosphate. Both enzymes liberate a mixture of oligonucleotides from RAN substrates, with 5'-phosphate and 3'-hydroxyl termini.

Endocardial mapping of ventricular tachycardia in the intact human ventricle. III. Evidence of multiuse reentry with spontaneous and induced block in portions of reentrant path complex.

OBJECTIVES: This study was conducted to characterize the functional nature of the reentrant tract responsible for ventricular tachycardia due to ischemic heart disease. BACKGROUND: A zone of slow conduction forming the return path is though to form a critical component of the reentrant mechanism in ventricular tachycardia. Despite its importance, detailed knowledge of the return path is rare in clinical studies. METHODS: Multielectrode arrays were used intraoperatively to obtain unipolar and high gain bipolar recordings of left ventricular endocardium in patients undergoing map-directed surgical ablation of ventricular tachycardia. A total of 224 local electrograms were analyzed for each tachycardia. RESULTS: Of 10 consecutive patients undergoing intraoperative cardiac mapping, detailed recording of the return tracts of eight ventricular tachycardias were obtained in three patients. The recordings demonstrated that return tracts can be complex and extensive, with multiple paths of entry and exit. Potential and actual alternate paths were observed. Spontaneous and induced block occurred within portions of the complex. Intermittent block in one of two paths of entry resulted in intermittent cycle length changes of the tachycardia without a change in configuration. Block in one exit path resulted in a shift to alternative exit paths, with dramatic changes in ventricular activation and tachycardia configuration. Termination of the tachycardia could result from block close to the entrant or exit portion of the return tract. Different tachycardias were seen to share common portions of a return tract. CONCLUSIONS: These observations enlarge and extend our knowledge of the functional repertoire of complex reentrant tracts that occur in infarct-related ventricular tachycardia. The use of common portions of a reentrant tract by several tachycardias is confirmed. Utilization of alternate pathways can account for changes in configuration and cycle length. Spontaneous and induced block can occur at points of entry and exit in a reentrant tract and may identify optimal targets for ablation attempts. Further advances will require greater emphasis on diastolic activation mapping.

Expression of immediate early genes and vasopressin heteronuclear RNA in the paraventricular and supraoptic nuclei of rats after acute osmotic stimulus.

Monitoring the expression of immediate early genes (IEGs) is useful for following stress-induced cellular responses in the neuroendocrine system. We have examined the transcriptional activities of four IEGs (c-fos, junB, NGFI-A and NGFI-B) and of the arginine vasopressin (AVP) gene in the hypothalamic paraventicular (PVN) and supraoptic nuclei (SON) of rats after acute osmotic stimuli, using in situ hybridization histochemistry. After intraperitoneal (i.p.) administration of hypertonic saline (2% body weight, 900 mOsm/kg), the expression levels of all IEG mRNAs were increased significantly both in the PVN and SON at as early as 10 min, peaked at 30 min and remained elevated until 60 min. The expression of AVP heteronuclear (hn)RNA also peaked at 30 min, and remained elevated until 180 min. Thirty min after i.p. administration of hypertonic saline (600 mOsm/kg), the expression levels of all IEG mRNAs in the PVN and SON were significantly increased in comparison with those after i.p. administration of isotonic saline (290 mOsm/kg). Regression analysis revealed that expression levels of the IEG mRNAs and AVP hnRNA were positively correlated with the plasma concentration of sodium, and the rates of increase of the expression levels of all IEG mRNAs were similar. The expression levels of all IEG mRNAs examined are useful markers for following the changes of the AVP gene transcription in the PVN and SON after acute osmotic stimuli in rats.

Ovary-selective genes I: the generation and characterization of an ovary-selective complementary deoxyribonucleic acid library.

The importance of several ovary-selective/specific genes, i.e. genes preferentially or exclusively expressed in the ovary, has been established. Indeed, null mutant female mice for the c-mos, growth and differentiation factor-9, alpha-inhibin, and zona pellucida-3 genes proved sterile. A loss of function mutation of the human FSH receptor gene established its critical role in ovarian function. These data support the hypothesis that genes expressed selectively or specifically in the ovary are probably essential for the normal functioning of this organ system. We have used the differential screening technique suppression subtractive hybridization to systematically isolate and clone genes that are expressed in an ovary-selective/specific manner. The resultant target complementary DNA (cDNA) library has been exhaustively screened to a point at which additional sequencing was increasingly unlikely (< or = 4%) to yield additional previously unencountered cDNAs. In toto, 844 clones were sequenced and analyzed for homology to known genes using the Basic Local Alignment Tool (BLAST). Of those, 342 were determined to be independent (nonredundant). One hundred and fifty-nine independent clones proved identical to previously characterized genes, whereas an additional 100 independent clones proved significantly homologous (but not identical) to previously characterized genes. Yet 83 other independent clones did not display significant homology to previously characterized genes now listed in the publicly accessible nonredundant databases. As such, these latter genes were deemed novel. Of these 83 novel genes, a total of 36 displayed ovary-specific/selective expression, as determined by probing mouse multitissue Northern blots with 32P-labeled/PCR-amplified cDNA inserts. Under these circumstances, the false positive rate was minimal, as only one novel clone was expressed at a higher level in nonovarian tissues relative to ovary. Of the 36 ovary-specific/ selective novel genes, 22 proved subject to hormonal regulation during a simulated estrous cycle. In this communication we focus on 2 such novel ovary-specific/hormonally-dependent genes, the full-length sequences of which were isolated using rapid amplification of 3'-cDNA ends technology. Taken together, the present study accomplished systematic identification of those genes that are restricted in their expression to the ovary. These ovary-selective genes may have significant implications for the understanding of ovarian function in molecular terms and for the development of innovative strategies for the promotion of fertility or its control.

Follicular thyroglobulin suppresses iodide uptake by suppressing expression of the sodium/iodide symporter gene.

A major function of the thyrocyte is to take up and concentrate iodide. This is needed for thyroid hormone synthesis and is accomplished by the sodium iodide symporter (NIS), whose expression and activity are up-regulated by TSH. Recently, we reported that follicular thyroglobulin (TG) is a potent suppressor ofthyroid-specific gene expression and can overcome TSH-increased gene expression. We suggested this might be a negative feedback, autoregulatory mechanism that counterbalanced TSH stimulation of follicular function. In this report, we support this hypothesis by coordinately evaluating TG regulation of NIS gene expression and iodide transport. We show that physiological concentrations of TG similarly and significantly suppress TSH-increased NIS promoter activity, NIS protein, and NIS-dependent iodide uptake as well as RNA levels. We show, in vivo, that TG accumulation at the apical membrane of a thyrocyte facing the follicular lumen is associated with decreased uptake ofradioiodide. It is likely, therefore, that TG suppresses NIS-dependent iodide uptake and NIS gene expression in vivo, as is the case in vitro. RNA levels of NIS and vascular endothelial growth factor/vascular permeability factor, which has been reported to be TSH regulated and possibly associated with TSH-increased iodide uptake, are coordinately decreased by follicular TG as a function of concentration and time. Also, removal of follicular TG from the medium, but not TSH, coordinately returns NIS and vascular endothelial growth factor/vascular permeability factor RNA levels to their TSH-stimulated state. TG accumulated in the follicular lumen appears, therefore, to be a negative feedback regulator of critical TSH-increased follicular functions, iodide uptake, and vascular permeability.

Thyrotropin induces G1 cyclin expression and accelerates G1 phase after insulin-like growth factor I stimulation in FRTL-5 cells.

We have investigated the mechanism by which TSH pretreatment potentiates insulin-like growth factor I (IGF-I)-induced DNA synthesis in FRTL-5 cells. As previously described, pretreatment with TSH increased IGF-I-induced DNA synthesis, suggesting that the effect of TSH is mediated through the cAMP pathway. TSH and A kinase activators required at least 12 h to precondition cells to respond to IGF-I stimulation. The presence of cycloheximide abolished the effect of TSH to increase IGF-I-induced DNA synthesis. When the time course of thymidine uptake after IGF-I addition was studied, TSH pretreatment increased the maximum DNA incorporation and shortened the G1 phase interval. These results indicated that some proteins induced by TSH are required for the effect of TSH on IGF-I activity, and the proteins are important for cell cycle progression. Cyclins are key regulators of the cell cycle; therefore, we investigated the expression of cyclins D1 and E after TSH stimulation. TSH- and A kinase-activating agents increased the expression of cyclins D1 and E after 24 h. The same amounts of cyclins D1 and E induced by IGF-I were increased after TSH pretreatment. TSH pretreatment induced the expression of G1 cyclin in FRTL-5 cells, and IGF-I caused the accumulation of enough G1 cyclins to drive the cell cycle from G1 to S phase in a short time, which accounts for the effect of TSH on IGF-I induced DNA synthesis.

Regulation of major histocompatibility (MHC) class II human leukocyte antigen-DR alpha gene expression in thyrocytes by single strand binding protein-1, a transcription factor that also regulates thyrotropin receptor and MHC class I gene expression.

The single strand binding protein (SSBP-1) is a positive regulator of TSH receptor gene expression and binds to an element with a GXXXXG motif. The S box of the mouse major histocompatibility class II gene has multiple GXXXXG motifs and can also bind SSBP-1. The S box is one of four highly conserved elements on the 5'-flanking region of class II genes that are necessary for interferon-gamma (IFNgamma) to overcome the normally suppressed state of the gene and induce aberrant class II expression. In this report we show that SSBP-1, when overexpressed in FRTL-5 thyroid cells, is a positive regulator of human leukocyte antigen (HLA)-DR alpha class II gene expression, as is IFNgamma or the class II trans-activator (CIITA). This is evidenced by increased exogenous promoter activity, increased endogenous RNA levels, and increased endogenous antigen expression after transfecting full-length SSBP-1 complementary DNA together with a HLA-DR alpha promoter-reporter gene chimera into TSH-treated FRTL-5 thyroid cells whose endogenous SSBP-1 levels are low. IFNgamma reverses the ability of TSH to decrease endogenous SSBP-1 RNA levels. Also, whereas SSBP-1 transfection does not cause any increase in IFNgamma-induced exogenous promoter activity, transfection of SSBP-1 and CIITA additively increases endogenous class II RNA levels to levels measured in cells treated with IFNgamma. Further, competition studies show that SSBP-1 binding is necessary for formation of the double strand protein/DNA complexes that are seen in electrophoretic mobility shift assays when the class II 5'-flanking region is incubated with extracts from IFNgamma-treated FRTL-5 cells and that have been previously associated with IFNgamma-induced aberrant class II expression. These data suggest that SSBP-1 is involved in the action of IFNgamma to overcome the normally suppressed state of the class II gene; it functions together with CIITA, whose expression is independently increased by IFNgamma. The effect of SSBP-1 as a positive regulator of class II promoter activity is lost in cells maintained without TSH, in which endogenous SSBP-1 RNA levels are already high in the absence of aberrant class II gene expression. These data suggest that high levels of endogenous SSBP-1 are insufficient to cause aberrant class II expression, but, rather, TSH or IFNgamma treatment additionally modulates the cell, albeit differently, such that transfected or endogenous SSBP-1, respectively, can express its positive regulatory activity. The effect of TSH is consistent with reports indicating that TSH enhances the ability of IFNgamma to increase class II gene expression despite the fact IFNgamma increases endogenous SSBP-1 to only the same levels as in cells untreated with TSH. Finally, the effect of SSBP-1 as a positive regulator is lost when GXXXXG motifs, which exist on both the coding and noncoding strands of the S box, are mutated. Consistent with this, mutation and oligonucleotide competition studies show that GXXXXG motifs are necessary for either strand of the S box to bind protein/DNA complexes containing SSBP-1 in FRTL-5 cell extracts or to bind to recombinant SSBP-1. They also suggest that the SSBP-1-binding sites on either strand of the HLA-DR alpha S box are functionally distinct. We conclude from these data that the positive regulatory action of SSBP-1 on class II gene expression involves GXXXXG motifs on each strand of the highly conserved S box of the class II 5'-flanking region. As SSBP-1 is modulated by IFNgamma and is involved in class I and TSH receptor as well as class II gene expression in FRTL-5 cells, the sum of the data supports the hypotheses that common transcription factors regulate all three genes, and their altered activities may contribute to the development of autoimmunity.