Presence of immunoreactive corticotropin-releasing factor in rat testis.

We attempted to demonstrate the presence of immunoreactive (ir) CRF in rat testis by RIA, and by an immunocytochemical technique. The RIA was performed on 27,000 x g supernatants of phosphate buffer extracts of adult rat testes, and revealed a high concentration of irCRF (51 - 74 ng/g testis), with a clear parallelism to the standard curve prepared with synthetic rat CRF. In contrast, the peripheral blood level of irCRF was extremely low (less than 0.05 ng/ml). Immunocytochemical studies of irCRF revealed strong specific staining in the Leydig cells and germ cells in normal adult rat testis. Epididymal spermatozoa also stained positive. Testicular irCRF fluctuated significantly with age. The levels (mean +/- SD) assayed in 10, 20, 60 and 90-day-old rats were 41.6 +/- 4.7, 8.7 +/- 0.2, 55.5 +/- 3.3 and 71.3 +/- 3.4 ng/g testis, respectively (P less than 0.01). The level was drastically reduced after abdominal translocation of a testis (9.6 +/- 1.3 ng/g testis), and after hypophysectomy (16.7 +/- 1.6 ng/g testis) in adult rats. However, neither hemicastration nor unilateral cryptorchidism influenced the irCRF levels in the contralateral testis. These data suggest a local production of irCRF which may play a role in regulation of Leydig cell function and sperm maturation in testis and epididymis.

A syndrome of gonadotropin resistance possibly due to a luteinizing hormone receptor defect.

An 18-yr-old 46,XY man with primary hypogonadism and a microphallus is described whose Leydig cells appear to be partially insensitive to gonadotropin action. The external genitalia were well differentiated though abnormally small. The mean +/- SE baseline plasma testosterone (T) level was 62 +/- 3.9 ng/dl, and androstenedione was 34.5 +/- 7.3 ng/dl. Plasma levels of dehydroepiandrosterone, dehydroepiandrosterone sulfate, 17-hydroxyprogesterone, 17-hydroxypregnenolone, corticosterone, deoxycorticosterone, and 17 beta-estradiol were all normal. After the im administration of hCG, plasma T increased insignificantly from 71 to 78 ng/dl, and androstenedione increased from 22 to 47 ng/dl; there was no significant change in the levels of precursor steroids. The mean +/- SE serum FSH level was 17.4 +/- 3.6 mIU/ml, and LH was 15.4 +/- 1.1 mIU/ml (normal, 5-20); both responded briskly to iv GnRH. Exogenous T therapy resulted in normal virilization, whereas therapy with hCG was ineffectual. Testicular biopsy revealed Leydig cells in normal numbers, some spermatogenesis, and thickened tubular basement membranes. In vitro binding studies using [125I]hCG were performed with testicular homogenates from the patient and three normal subjects. With 7.4 fmol labeled hCG, the specific binding (mean +/- SD), expressed as femtomoles of hCG per mg protein, was 1.16 +/- 0.44 compared to 2.49 +/- 0.41 in normal subjects (P less than 0.05). These data demonstrate partial resistance to hCG and suggest that the defect in Leydig cell function may be at the LH receptor or postreceptor level.

Three-year follow-up of women with the sole diagnosis of depressive personality disorder: subsequent development of dysthymia and major depression.

OBJECTIVE: The authors sought to determine whether subjects with the sole diagnosis of depressive personality disorder are at higher risk for developing dysthymia and major depression than are healthy comparison subjects. METHOD: Eighty-five women with depressive personality disorder who had no comorbid axis I or axis II disorders and 85 age-matched healthy comparison women were initially recruited and reinterviewed 3 years later to evaluate the cumulative incidence rate of dysthymia and major depression. RESULTS: At the 3-year follow-up assessment, the women with depressive personality disorder had a significantly greater odds ratio for developing dysthymia than did the healthy comparison women. The difference in odds ratios for the development of major depression between women with and without depressive personality disorder did not reach statistical significance. CONCLUSIONS: The present study, the first to determine the subsequent development of dysthymia and major depression in subjects with the sole diagnosis of depressive personality disorder, found that subjects with depressive personality disorder had a greater risk of developing dysthymia than did healthy comparison subjects at 3-year follow-up. Findings of the current study also suggest that depressive personality disorder may mediate the effects of a family history of axis I unipolar mood disorders.

Available FSH receptors in adult rat testis in vivo.

It has been suggested that the tight junctions formed between Sertoli cells during the peripubertal period constitute a barrier to circulating FSH in the adult testis, limiting its access to the lumen of the seminiferous tubules. There are also reports of FSH receptor-binding inhibitors. These observations prompted us to study the extent of FSH receptor availability in vivo in the adult rat. Experimental rats were given an intracardiac injection of rat FSH (rFSH), and the occupied receptor was measured by radioimmunoassay of acid-released rFSH from the testis. In the saline-injected control animals, there were 247 fmol occupied FSH receptors/g testis, and as much as 1788 fmol of the unoccupied high-affinity receptors/g testis, as measured by in-vitro binding studies. After intracardiac injection of increasing amounts of rFSH (up to 606 pmol), receptor occupancy increased to a maximum plateau of only 448 fmol/g testis. In contrast, when rFSH was given by intratesticular injection in order to achieve pharmacological doses in the testis, the maximum binding was 662 fmol/g testis. Scatchard analysis of the in-vivo data revealed, however, that the maximum concentration of the high-affinity receptor was 452 fmol/g testis, a value concordant with the highest in-vivo binding observed in animals given intracardiac rFSH (448 fmol/g). A single injection of the hormone did not induce down-regulation of FSH receptors, regardless of the dose, whereas multiple injections of menotrophin were effective, at least to some extent. Despite the receptor loss, the immediate receptor availability was maintained, suggesting the presence of a receptor pool.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of regulatory elements on the promoter region of human ATP-citrate lyase.

ATP-citrate lyase (ACL), an enzyme catalyzing the first step in biosynthesis of fatty acids, is induced during the lipogenesis and cholesterologenesis. We demonstrate that the region -213 to -128 of human ACL promoter is responsible for conferring glucose-mediated transcription. This region in the ACL promoter contains Sp1 binding sites determined by DNase I foot-printing assay. Gel retardation assay using oligonucleotides from -179 to -141 and -140 to -110 showed two specific DNA-protein complexes postulated to be formed by transcription factor Sp1. Competition gel shift and supershift assays have confirmed that these DNA-protein complexes were the result of induced Sp1 as well as another Sp1-related proteins. Western blot analysis also demonstrated that transcription factor Sp1 was slightly increased in the nuclear proteins extracted from Alexander cells following supplementation of glucose. In addition, expression of 110 kDa protein reacting with antibody against Sp3 was dramatically increased by glucose supplementation, while isoforms of Sp3, about 80 kDa in size was decreased in its amounts. Our results suggest that changes in the expression of Sp1 family proteins play an important role in activation of the ACL promoter by glucose.

Differences in p53 gene polymorphisms between Korean schizophrenia and lung cancer patients.

The reduced incidence of cancer observed in schizophrenia patients may be related to differences in genetic background. It has been suggested that genetic predisposition towards schizophrenia is associated with reduced vulnerability to lung cancer, and p53 gene is one of the candidate genes. We tested the genetic association between schizophrenia and lung cancer by analyzing polymorphic sites in the p53 gene. Genotype and allele frequencies at two polymorphic sites in the p53 gene (BstUI and MspI restriction sites in exon 4 and intron 6, respectively) were studied in Korean schizophrenia (n=179) and lung cancer patients (n=104). Comparisons of the genotype and allele frequencies of the MspI polymorphism revealed significant differences between schizophrenia and lung cancer patients. The results suggest that the p53 polymorphism specifically found in schizophrenia patients may be associated with reduced vulnerability to lung cancer.

Cloning and expression of rat liver type glucose transporter and translocation by insulin in Chinese hamster ovary cells.

The 5'- and 3'-side half of liver type glucose transporter (GLUT2) cDNA was amplified from total RNA or mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). The amplified 5'-side fragment of GLUT2 cDNA was inserted into pGEM4Z and named pGLGT1, and the 3'-side fragment of GLUT2 cDNA was inserted into the HindIII site of pGLGT1 to construct pGLGT2 which contains an entire open reading frame of GLUT2 cDNA. The GLUT2 cDNA in pGLGT2 was transferred to an eukaryotic expression vector (pMAM) to construct pMLGT, which was expressed in the insulin-sensitive Chinese hamster ovary (CHO) cells. Western blot analysis showed that the GLUT2 gene in pMLGT was expressed in the transfected CHO cells successfully. The GLUT2 content in the plasma membrane fraction of insulin-treated CHO cells expressing GLUT2 increased 3.8-fold compared to that of the control group. This result suggests that GLUT2, which is not subjected to translocation by insulin in the cells of its major distribution, can be translocated if it is expressed in the suitable cells sensitive to insulin action.

Antisense GLUT1 RNA suppresses the transforming phenotypes of NIH 3T3 cells transformed by N-Ras.

An antisense approach was attempted to investigate the role of antisense GLUT1 RNA in suppressing tumor cell phenotypes using N-ras-transformed NIH 3T3 cells. The established cell line transformed by ras showed typical biological characteristics of cancer cells, such as increased glucose transport, GLUT1 mRNA contents, and the ability to form colonies on the soft agar. In this system, the plasmids (pMAM-GLUT1(rev)) which can transcribe the antisense GLUT1 RNA were transfected and the accompanying changes in the phenotypes of the ras-transformed cells were observed. The expression of antisense GLUT1 RNA by induction with dexamethasone reduced the glucose transport by 30% (1.97 +/- 0.13 nmoles) after 4 min incubation when compared to the non-induction group of transformed cell (2.85 +/- 0.19 nmoles). Also, the number of colonies sized over 50 microns on the soft agar was reduced significantly in the antisense RNA expressing group compared to non-induction group. These results suggest that the expression of antisense GLUT1 RNA reduced the glucose transport and transforming potential in soft agar possibly by hybridization with GLUT1 mRNA in N-ras-transformed NIH 3T3 cells.

Expression and localization of human papillomavirus type 16 E6 and E7 open reading frame proteins in human epidermal keratinocyte.

Over 60 different types of human papillomavirus (HPV) have been identified, and they are classified into high and low risk groups based on the risk for malignant progression of HPV associated lesions. HPVs belonging to a high risk group have been shown to express two major transforming proteins, E6 and E7. With respect to the transforming activity of these proteins, many investigators have reported the location of these proteins in the cell, but their results are still controversial. In the present study, HPV type 16 E6 or E7 open reading frame (ORF) proteins were expressed and localized in human epidermal keratinocytes (RHEK-1) using the vaccinia virus as an expression vector. Immunofluorescence detection using monoclonal antibodies against E6 or E7 ORF proteins revealed that E6 or E7 proteins of HPV type 16 were located in the cytoplasm of RHEK-1 cells. These results suggest that E6 and E7 proteins bind to the tumor suppressor counterparts, thereby preventing transport of these proteins into the nucleus. These antioncogene products that fail to be rapidly transported out of the cytosol may be degraded by certain proteases such as the ubiquitin dependent system. In this way, the precise function of antioncogene products in the regulation of cell growth could be destroyed, and abnormal cell growth could occur.

The effects of wild type p53 tumor suppressor gene expression on the normal human cervical epithelial cells or human epidermal keratinocytes transformed with human papillomavirus type 16 DNA.

The inactivation of p53 and p105RB by viral proteins or by mutations plays a key role in the oncogenesis of cervical carcinoma. The E6 and E7 proteins of HPV type 16 can bind to p53 and p105RB tumor suppressor gene products, respectively. In the present study, we tested a simple in vivo model that could explain the interactions between HPV E6 oncoprotein and p53 tumor suppressor protein. Our results showed that the life span of normal cervical epithelial cells was increased up to 4.5 times when transfected with expression vector containing E6/E7 ORF of HPV type 16. However, these cells did not divide after second crisis. Therefore, we employed an established human epidermal keratinocytes, RHEK-1. When transfected with an expression vector containing E6 ORF of HPV type 16, RHEK-1 cells showed anchorage independent growth character. When RHEK-E6 cells were transfected with wild type p53 expression vector, the growth rate of the RHEK-E6 cells was diminished. After 48 hours of transfection, many cells showed apoptotic signal but no more apoptotic signal was observed thereafter. These results suggested that the overexpression of the wild type p53 could overcome the dysfunction of the p53 on the cell cycle regulation imposed by E6 protein although not being of physiological condition.

Testicular arginine vasopressin in the developing rat: a possible physiological role.

Testicular immunoreactive arginine vasopressin (irAVP) has been shown to inhibit testosterone production by the Leydig cell in vitro. We studied pre- and postpubertal rats and the results are in agreement with the notion that such inhibition might occur in vivo and, therefore, could have physiological significance. Testicular irAVP and serum testosterone levels were measured in 14-, 21-, 28-, 35-, 42-, and 70-day-old rats. The testicular irAVP concentration was lowest in early prepubertal rats, increased significantly to the highest level at 35 days, then decreased at 42 days, and plateaued at 70 days of age. A pubertal increase in serum testosterone was observed at 42 days when AVP was decreasing. Thus, an inverse relationship was observed at the onset of pubertal maturation. These parameters were also measured in 3 groups of rats 1 week after hemicastration which was performed at the ages of 28, 35 and 70 days. In the 28-day-old rats there was a significant decrease in irAVP 1 week later, in contrast to the intact animal, whereas hemicastration performed at 35 and 70 days of age had no impact on irAVP. Our data suggest that the role of AVP may be physiologically relevant during the peripubertal period of testicular maturation in the rat.

Adrenal function in thalassemia major following long-term treatment with multiple transfusions and chelation therapy. Evidence for dissociation of cortisol and adrenal androgen secretion.

Eight patients with beta-thalassemia who were given long-term treatment with combined multiple transfusions and chelation therapy underwent adrenal testing. The six male and two female patients ranged in age from 7 to 19 years. Six of eight patients had delayed bone ages and height greater than 2.5 SDs below the mean. Of the six patients more than 13 years of age, two had clinical evidence of isolated adrenarche and only one had evidence of true puberty. Cortisol levels were similar in patients and controls at zero time (10.6 +/- 1.8 micrograms/dL [292 +/- 50 nmol/L] vs 10.8 +/- 1.4 micrograms/dL [298 +/- 39 nmol/L]) and at 60 minutes (26.6 +/- 2.5 micrograms/dL [734 +/- 69 nmol/L] vs 24.9 +/- 1.9 micrograms/dL [687 +/- 52 nmol/L]) after insulin hypoglycemia (all values are the mean +/- SE). During an eight-hour infusion of ACTH, cortisol responses in the patients with thalassemia were not significantly different from those of controls. Baseline levels of the adrenal androgens dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S) were significantly lower in the subjects with thalassemia compared with controls of similar bone age and pubertal status. The prolonged ACTH infusion caused a significant increase in the DHEA level (79.2 +/- 14.7 ng/dL [2.74 +/- 0.51 nmol/L] vs 538.6 +/- 38.1 ng/dL [18.67 +/- 4.79 nmol/L]) and the DHEA-S level (37.5 +/- 10.8 micrograms/dL [1.02 +/- 0.29 mumol/L] vs 70.5 +/- 18.3 micrograms/dL [1.19 +/- 0.50 mumol/L]) in the patients. The patients' peak stimulated levels of DHEA-S were significantly lower than those of the controls, whereas peak levels of DHEA were similar in the patients and the controls. These results indicate that combined multiple transfusions and chelation therapy preserve the integrity of the ACTH-cortisol axis in patients with thalassemia. The reduced levels of adrenal androgens, short stature, and delayed puberty noted in our patients suggest, however, that alternative approaches to the therapy of thalassemia are needed.

Immunocytochemical localization of hFSH as an index of Sertoli cell function in the human testis.

The FSH receptor in the human testis has not been well characterized in vivo. Using an immunoperoxidase technique we have attempted the immunocytochemical localization of FSH in testicular tissue from patients with a variety of disorders including oligo- or azoospermia (N = 6), cryptorchidism (N = 3), and prostatic carcinoma (N = 3). Specific staining for hFSH was observed inside the seminiferous tubule, generally near the basal membrane in all except the cryptorchid patients. Specific staining was also localized in the luminal area of the seminiferous tubule. In most cases, FSH-positive cells were also found in the interstitium, with a minority of the cells being macrophages. The latter were more prevalent in the undescended testes and in orchiectomy specimens from patients with prostatic cancer. The pattern of FSH localization observed in this study probably represents receptorbound hormone, and may reflect damage to the Sertoli cell and its tight junctions. Further study of the changes in receptor distribution as an indication of Sertoli cell malfunction, may be helpful in our understanding of human testicular disorders.

Adrenal androgens in children with short stature.

Recent data suggest that adolescent individuals with growth hormone (GH) deficiency have subnormal levels of adrenal androgens (AA). In order to determine the developmental pattern of AA in GH deficiency and to assess whether AA levels can help identify children with GH deficiency, we measured plasma concentrations of dehydroepiandrosterone (DHEA), DHEA sulfate (DHEA-S), delta 4-androstenedione (delta 4A), and cortisol in the basal state and during prolonged adrenocorticotropin (ACTH) infusion (8 h) in a group of 34 individuals, 26 males and 8 females, with short stature. Their chronological ages (CA) ranged from 1.75 to 17.5 years (median 10.35 years). The subjects were grouped into two categories according to the results of pituitary testing: group 1 = short, non-GH-deficient (n = 16), and group 2 = GH-deficient, ACTH-sufficient (n = 18). Patients in groups 1 and 2 had similar bone ages (BA: 7.2 +/- 0.7 vs. 7.5 +/- 1.0 years) and Z scores for height (-3.0 +/- 0.2 vs. -3.2 +/- 0.3 units) and height velocity (-2.5 +/- 0.4 vs. -2.6 +/- 0.2 units). For both groups there were significant increases from basal to peak levels for DHEA, DHEA-S, delta 4A and cortisol following prolonged ACTH infusion. Although both basal and peak levels of DHEA-S overlapped in groups 1 and 2 for all CA and BA, levels in group 2 tended to be lower, especially for BA greater than 10 years.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning of human acetyl-CoA carboxylase beta promoter and its regulation by muscle regulatory factors.

The 280-kDa beta-isoform of acetyl-CoA carboxylase (ACCbeta) is predominantly expressed in heart and skeletal muscle, whereas the 265-kDa alpha-isoform (ACCalpha) is the major ACC in lipogenic tissues. The ACCbeta promoter showed myoblast-specific promoter activity and was strongly induced by MyoD in NIH3T3 cells. Serial deletions of the promoter revealed that MyoD acts on the E-boxes located at positions -498 to -403 and on the proximal region including the 5'-untranslated region. Destruction of the E-boxes at positions -498 to -403 by site-directed mutagenesis resulted in a significant decrease of MyoD responsiveness. The "TGAAA" at -32 to -28 and the region around the transcription start site play important roles in basal transcription, probably as a TATA box and an Inr element, respectively. Mutations of another E-box at -14 to -9 and a "GCCTGTCA" sequence at +17 to +24 drastically decreased the MyoD responsiveness. The novel cis-element GCCTGTCA was preferentially bound by MyoD homodimer in EMSA and conferred MyoD responsiveness to a luciferase reporter, which was repressed by the overexpression of E12. This finding is unique since activation via E-boxes is mediated by heterodimers of MyoD and E-proteins. We screened a human skeletal muscle cDNA library to isolate clones expressing proteins that bind to the region around the GCCTGTCA (+8 to +27) sequence, and isolated Myf4 and Myf6 cDNAs. Electrophoretic mobility shift assay showed that recombinant Myf4 and Myf6 bind to this novel cis-element. Moreover, transient expression of Myf6 induced significant activation on the ACCbeta promoter or an artificial promoter harboring this novel cis-element. These findings suggest that muscle regulatory factors, such as MyoD, Myf4, and Myf6, contribute to the muscle-specific expression of ACCbeta via E-boxes and the novel cis-element GCCTGTCA.