We investigated the time course and localization of ovarian tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type-1(PAI-1) expression during the ovulatory period in rat by RNase protection assay and in situ hybridization. Immature female Wistar rats were injected with 25 IU pregnant mare serum gonadotropin (PMSG), followed 50h later by 25 IU human chorionic gonadotropin (hCG). Levels of tPA mRNA were low before hormone treatment and after PMSG treatment. After hCG treatment, tPA mRNA levels increased rapidly, the first peak at 4h after hCG treatment and reached a maximum just prior to ovulation, 12h later, before declining again. PAI-1 mRNA was barely detectable before hormone treatment but was transiently induced by hCG treatment, reaching peak levels after 4h. Subsequently, PAI-1 mRNA levels decreased until early luteinization. The expression of tPA mRNA 4h after hCG treatment occurred mainly in the follicular thecal-interstitial cells, but was barely detectable in the granulosa cells, whereas 12h after hCG treatment it was maximal in the granulosa cells of the large follicles destined to ovulate. PAI-1 mRNA was expressed mainly in ovarian stromal tissue and in the thecal external interstitial cells encapsulating the follicles at 4h after hCG treatment. These results suggest that the temporal regulation of tPA biosynthesis after hCG induction depends on the cell types and size classes in the various ovarian compartments. PAI-1 may be produced by the stormal tissue and the thecal external interstitial cells and is perhaps implicated in structural changes during follicular growth, ovulation and luteinization.

View full abstract

The enzyme-linked immunospot (ELISPOT) assay is an efficient technique for the enumeration of single cells secreting antibodies or cytokines. To assess mice interleukin 6 (IL-6)- secreting cells at a single cell level, a highly sensitive ELISPOT assay with 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt and nitroblue tetrazolium chloride (BLIP/NBT) as a gel-substrate has been developed. The IL-6 specific ELISPOT assay thus developed with an ordinary plastic ELISA plate is as efficient as the previous IL-6 specific ELISPOT assay system which uses a nitrocellulose coated plate, and is much more economic and convenient. We demonstrated with this newly devised ELISPOT assay that the ratio of IL-6 producing cells in the resident peritoneal exuded cells was highest in normal BALB/c mice, and a few IL-6 secreting cells were detected in all organs we tested, namely the spleen, bone marrow, Peyer's patch, axillary lymph node and thymus.

View full abstract

The antidiabetic effects of Lactobacillus casei (LC) on a non-insulin-dependent diabetes mellitus (NIDDM) model, KK-A^y mice, were investigated. The oral administration of LC to male 4-week-old KK- A^y mice, or raising the mice on a 0.05% LC-containing diet significantly decreased the plasma glucose at 8 to 10 weeks of age compared with the control group. The body weights of the LC-treated groups were lower than those of the control group, although the food intake was nearly the same in all groups. Phenotypic analysis of spleen cell surface markers revealed that the increase in CD4⁺ T cells at 12 weeks was significantly inhibited by the oral treatment with LC. Cytokine production, especially that of interferon-γ and interleukin 2, was also inhibited in the oral LC-treated group. The plasma insulin levels of the LC-treated groups were also lower than those of the control group, and the insulin binding potential of red blood cells in the LC-treated mice was augmented more than that in the control group. Taken together, these findings led us to conclude that the oral administration of LC in the NIDDM model mice, KK-A^y, was involved in the decrease in the plasma glucose level and modified the host immune responses.

View full abstract

To determine (1) the relationship between primary hyperparathyroidism with mild hypercalcemia and psychiatric disturbances, bone density, or non-specific symptoms, and (2) the effect of parathyroidectomy on these outcomes, a systematic and critical review of the literature was conducted. Relevant citations were identified using MEDLINE (1966 to August, 1995) and PsycINFO (1967 to August, 1995). Studies were included for the overview if they described patients with mild hypercalcemia (<12mg/dl), and if they dealt with at least one of the following outcomes: psychiatric disturbances, bone density, joint pain, constipation, polyuria/nocturia or weight loss. Either a calculated effect size or Z score was used to estimate the effect of the disease or parathyroidectomy on these outcomes. Seven studies met the inclusion criteria for this overview. Two out of three case-control studies on psychiatric symptoms found a significant association between primary hyperparathyroidism with mild hypercalcemia and psychiatric disturbances (effect sizes; 0.17, 1.2 and 1.6). One of the three studies also examined the effect of parathyroidectomy on psychiatric symptoms, and found an effect size of 1.5. All four cross-sectional studies that measured bone mass showed significantly reduced bone density in the forearm and the lumbar spine. The bone loss ranged from 0.9 to 1.4 standard deviation below the age- and sex-adjusted mean value in the forearm, and was 0.5 in the spine. There was no relevant study regarding non-specific symptoms. Among the seven studies, five did not explicitly indicate whether the patients had classical symptoms of either osteitis fibrosa cystica or renal stones. Primary hyperparathyroidism with mild hypercalcemia is associated with psychiatric disturbances and reduced bone density. Nevertheless, further research is needed to determine the symptoms, particularly for a group of patients without either classical bone disease or renal stones. The effects of parathyroidectomy on these outcomes also remain to be determined.

View full abstract

Retinoic acid induces differentiation of preosteoblastic cells. We have demonstrated that osteoblastic differentiation and down-regulation of transforming growth factor (TGF)-β receptors requires the interaction of type I collagen with α2β1 integrin (J Biol Chem 271: 3938-3944, 1996). The purpose of this study was to clarify the role of collagen in retinoic acid-induced differentiation and down-regulation of TGF-β receptors using preosteoblastic RCT-1 cells. Retinoic acid enhanced the expression of alkaline phosphatase and type I collagen, and reduced TGF-β receptors in these cells. Inhibiting collagen synthesis abolished these changes. Because TGF-β inhibits osteoblastic differentiation, the changes described here may contribute to the osteoblastic differentiation by retinoic acid.

View full abstract

The role of serotonergic neurons in the mesencephalic raphe nuclei in regulating male sexual behavior in female rats was examined. The median or dorsal raphe nucleus lesions (MRL or DRL) were made in ovariectomized rats and behavioral tests were performed after implantation of Silastic tubes containing testosterone and treatment with serotonin synthesis inhibitor p-chlorophenylalanine (pCPA). Half of the animals in each group received 4100mg/kg pCPA injections before the behavioral test. As a result, the incidences and frequencies of mounts and intromissive patterns in the MRL and DRL groups were comparable to those in control females without brain surgery. Mount latency in the MRL females was shorter than that in the control females. When pCPA was given, most females with or without brain surgery showed mounts and intromissive patterns, and frequencies were higher than those in females without pCPA. These results suggest that the median raphe nucleus plays an inhibitory role in the onset mechanism for mounting. On the other hand, a stronger inhibitory influence in regulating male sexual behavior exists in other serotonergic neurons than those in the median and dorsal raphe nuclei in female rats.

View full abstract

To investigate the activation of particulate guanylate cyclase in pregnant women with pregnancy-induced hypertension (PIH), plasma levels of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and guanosine 3', 5'-cyclic phosphate (cGMP) were measured by radioimmunoassays specific to each substance. Ten normal nonpregnant women, and 30 normal pregnant women, 17 pregnant women with mild PIH and 11 pregnant women with severe PIH in the third trimester were included in this retrospective observational study. The diagnosis and classification of hypertension were carried out according to the technical bulletin (No. 91) of the American College of Obstetricians and Gynecologists. In the pregnant women with mild PIH, plasma cGMP levels as well as ANP and BNP levels were significantly (P<0.05) higher than those in gestational age-matched normal pregnant or nonpregnant women. But in the pregnant women with severe PIH, plasma cGMP levels were significantly lower than those in pregnant women with mild PIH (P<0.05), although plasma ANP and BNP levels were higher than those in pregnant women with mild PIH.

View full abstract

A series of 44 patients with acromegaly underwent transsphenoidal surgery between 1987 and 1996. The early postoperative mean basal GH level <5ng/ml or <3ng/ml was achieved in 43 (97.7%) or 38 (86.4%) out of 44 patients, respectively. Preoperative abnormal GH secretory response to TRH, GnRH and oral glucose administration was restored to normal both after surgery and at the time of the final follow-up in all patients whose early postoperative mean basal GH levels were reduced to <3ng/ ml, whereas they remained abnormal in those with mean basal GH levels of ≥3ng/ml. In contrast, insulin-like growth factor 1 (IGF-1) levels, when measured by the extraction method, tended to be reduced gradually to normal between 6 months and 2 years after surgery in some patients with a successful operation. Therefore, 34 (87.1%) out of 39 patients who have been followed up longer than 6 months met the following stringent criteria at the time of the final follow-up: mean basal GH level <3 ng/ml, a normal IGF-1 level, and normal GH response to TRH, GnRH and oral glucose administration. In this series, the most unfavorable preoperative factor influencing operative outcome is tumor invasion of the cavernous sinus. Our results clearly indicate that selective adenomectomy by transsphenoidal surgery is the therapy of first choice in any patient with acromegaly and that the complete biochemical cure of acromegaly can be achieved in 87% of patients by surgery alone with an acceptable low surgical morbidity.

View full abstract

Truncated and full-length epidermal growth factor (EGF) receptors are produced in rat liver cells. The truncated EGF receptor mRNA is almost identical to the full-length EGF receptor mRNA except for the lack of a 3' region of the full-length receptor mRNA. To understand the stability of rat EGF receptor mRNAs, we analyzed the expression of EGF receptor mRNAs in the hepatoma cell line, AH66 and liver cells. Ten, 7 and 5kb full-length and 2.7kb truncated EGF receptor mRNAs were detected in both of them. The half-lives of the 10 and 2.7kb EGF receptor mRNAs were determined in AH66 cells using a transcriptional inhibitor, 5, 6-dichlororibofuranosylbenzimidazole. The half-lives of the 10 and 2.7kb mRNAs were 1.2 and 11h, respectively. These results indicated that the truncated mRNA is 4 times more stable than the full-length mRNA in rat cells. As for the stability, the role of a sequence of 3'-untranslated region of the EGF receptor mRNA was discussed.

View full abstract

Insulin-like growth factors (IGF) and their specific binding proteins (IGFBPs) are believed to be important regulators of fetal growth. IGFBP protease which proteolyzes IGFBPs and changes the biochemical properties of IGFBPs is also presumed to be involved in fetal growth. The aim of this study is to elucidate the physiological significance of IGFBP protease in fetal growth and regulators of protease in placenta and decidua. The intact IGFBP-3 was proteolyzed into fragments when pregnant serum was incubated with ¹²⁵I-IGFBP-3. IGFBP-3 protease activity showed a marked increase at 5 weeks of gestation and reached a plateau in maternal circulation at 15 weeks of gestation. These changes in protease activity correlated with the profiles of IGFBPs in the maternal circulation analyzed by Western ligand blot, where the IGFBP-1 is only the dominant IGFBP. The intact IGFBP-3 was proteolyzed when culture media of decidual cells were incubated with ¹²⁵I-IGFBP-3, but was not proteolyzed when culture media of trophoblast cells were incubated with ¹²⁵I-IGFBP-3. Decidual protease activity was slightly increased by IGF-I and completely inhibited by progesterone. The protease activity was more increased in the mothers with growth retarded infant than in those in the mothers with normal growth infants, suggesting that the protease activity is elevated in compensation for the impaired fetal growth. These results suggest that increased protease activity in maternal blood may be involved in the fetal growth indirectly by reducing the binding activity of IGFBP-3 to IGF-I, and that protease activity in maternal blood may be derived from decidua that is regulated by placental hormones.

View full abstract

In human endometrium insulin-like growth factor binding protein (IGFBP)-1 inhibits the mitogenic action of insulin-like growth factor (IGF)-I by inhibiting the binding of IGF-I to its receptor. Our purpose was to compare circulating levels of IGF-I and IGFBP-1 in women with and without endometrial cancer. We assessed circulating levels of IGF-I and IGFBP-1 and IGFBP-3 in 23 patients with endometrial cancer, 11 patients with uterine cervical cancer and 27 healthy control women. The mean circulating level of IGF-I decreased significantly following menopause but was not correlated with age in the control group. The body mass index was significantly higher in the endometrial cancer group than in the control group. Analysis of covariance showed that even after the data were adjusted to eliminate the influence of the body mass index, the circulating IGF-I concentration was higher in postmenopausal endometrial cancer patients than in postmenopausal control subjects. The mean circulating level of IGFBP-1 was significantly lower in postmenopausal cancer patients than in postmenopausal control subjects. There were no significant differences in the serum levels of IGF-I and IGFBP-1 in the patients with cervical cancer and the control group. In conclusion, an increased circulating concentration of IGF-I and a decreased circulating concentration of IGFBP-1 are associated with endometrial cancer especially in postmenopausal women.

View full abstract

We investigated the association of the apolipoprotein (apo) E isoform phenotype with the basal serum cholesterol level and cholesterol response to diet therapy in outpatients with primary hypercholesterolemia. The basal levels of serum cholesterol, triglyceride and HDL-cholesterol in the 132 subjects were 286±26mg/dl, 154±83mg/dl and 54±14mg/dl, respectively. The frequencies of apo E 3/2, 3/3, 4/2, 4/3 and 4/4 were 1, 104, 2, 24 and 1, respectively. There were no differences in serum lipids between subjects with the two most common apo E phenotypes, i.e., apoE 3/3 (n=104) and apoE 4/3 (n=24). The serum cholesterol response to diet therapy was evaluated by measuring the serum lipids of 52 participants before and 2-3 months after diet therapy. After dietary counselling, serum cholesterol values were reduced significantly from 293±27 to 256±36mg/dl (P<0.01) in the total group of study subjects. There was no significant difference in serum cholesterol reduction in response to diet therapy between subjects with apoE4 (E4/2, E4/3 and E4/4, n=12) and without apoE4 (E3/3 and E3/2, n=40). Following dietary counselling, patients who lost a large amount of body weight (BM I_??_1.0) exhibited a greater reduction in serum cholesterol than those who showed less weight loss. In conclusion, the results of our study demonstrated the primary importance of diet therapy in the treatment of hypercholesterolemia, although other investigators have suggested that apo E phenotypes influence the response of serum cholesterol to dietary changes.

View full abstract

To measure thyroid-stimulating antibody (TSAb) in sera from patients with Graves' disease, we developed a new assay system with using frozen stocks of CHO-K₁ cells. CHO-K₁ cells trasfected with cloned thyrotropin (TSH) receptor on a 96 well plate were frozen in Cell Banker^TM and stored at -70°C. Three days before the assay, they were thawed in the culture medium and allowed to grow in a monolayer until use. The medium was replaced with medium containing IgGs from the patients, then after 2h, it was collected and concentrations of adenosine 3'-5'-cyclicmonophosphate (cAMP) were measured. This method is sensitive enough to detect TSAb and it is simpler and easier than the methods which use FRTL-5 cells.

View full abstract

Dermatitis herpetiformis is an autoimmune skin disorder frequently associated with gastrointestinal diseases. We report a 53-year-old male with a four-year history of refractory dermatitis herpetiformis associated with hypopituitarism. Endocrine testing, ophthalmological examination and magnetic resonance imaging revealed hypopituitarism due to a non-functioning pituitary macroadenoma. Following transsphenoidal removal of the pituitary tumor and appropriate hormone replacement, complete remission of the skin disorder was obtained. We discuss the permissive role of panhypopituitarism in the course of dermatitis herpetiformis.

View full abstract

Congenital lipoid adrenal hyperplasia (CLAN) is an autosomal recessive disorder characterized by impaired synthesis of all adrenal and gonadal steroid hormones. It has recently been reported that mutations in the steroidogenic acute regulatory protein (StAR) gene cause CLAH. We analyzed the nucleotide sequences of exon 7 of the StAR gene in a Japanese CLAH patient with a karyotype of 47, XYY, and her parents. The patient was homozygous for a nonsense mutation Q258X, which changed codon 258 (CAG) encoding Gln to the stop codon TAG, and the her parents were heterozygous for the Q258X mutation. Since the Q258X mutation destroys a MvaI site normally present in the StAR gene sequence, we confirmed the Q258X mutation by means of the restriction endonuclease MvaI digestion of the PCR products. Endocrinological examinations of the parents revealed normal responses of adrenal steroid hormones to exogenous adrenocorticotropin administration, confirming the failure to detect the heterozygous carriers of CLAH by hormonal evaluation.

View full abstract

The thyroid gland is physiologically stimulated in normal early pregnancy. This stimulated thyroid function is occasionally termed “gestational transient thyrotoxicosis”. The cause of this thyrotoxicosis has been clarified to closely it associate with human chorionic gonadotropin (hCG). We encountered a pregnant patient with hyperemesis and thyrotoxicosis, who manifested symptoms of Wernicke encephalopathy. Although her serum hCG concentration transiently increased in accordance with the thyrotoxicosis, it was within normal limits for the gestational week. Both the thyrotoxicosis and a catabolic state due to the hyperemesis were thought to have induced a vitamin B1 deficiency, causing the Wernicke encephalopathy. This case suggests that pregnant patients with hyperemesis should undergo careful endocrinological and neurological evaluations.

View full abstract

We present a 42-year-old woman with concomitant transient hypothyroidism due to Hashimoto's thyroiditis, autoimmune hepatitis and isolated ACTH deficiency. Two months after ceasing prednisolone (5mg/day) for uveitis, she was discovered incidentally to have liver dysfunction with hypergammaglobulinemia, later diagnosed as autoimmune hepatitis by histological examination of the biopsied liver. In addition, primary hypothyroidism due to Hashimoto's thyroiditis and secondary hypocortisolism due to isolated ACTH deficiency were revealed by endocrinological examination. Although not treated, her liver dysfunction and hypothyroid state recovered simultaneously, and the isolated ACTH deficiency was restored six months later. We concluded, after a needle-biopsy of the thyroid, that the transient hypothyroidism was due to Hashimoto's thyroiditis and the reversible ACTH deficiency was probably due to autoimmune hypophysitis. This case shows that cessation of steroid treatment may transiently exacerbate the polyglandular autoimmune syndrome.

View full abstract