Extent of hypoechogenic area in the thyroid is related with thyroid dysfunction after subacute thyroiditis.

OBJECTIVE: To gain an insight into risk factors for hypothyroidism after subacute thyroiditis (SAT), we examined the correlation between initial laboratory and ultrasonographic findings and sequential thyroid dysfunction among treatment modalities. PATIENTS: We reviewed retrospectively the medical records of 252 patients (26 men and 226 women) with SAT who consecutively visited our thyroid clinic at Kuma Hospital for at least 6 months from 1996 through 2004. RESULTS: Throughout the course, 135 patients (53.6%) developed transient or permanent hypothyroidism. Levels of TSH were most often elevated (greater than 5 IU/ml) 2 months after SAT onset regardless of treatment, and 97.0% of patients who showed transient or permanent hypothyroidism clustered within 6 months from onset. During follow-up, patients treated with prednisone (PSL) were more likely to have normal thyroid function than patients not treated or those receiving anti-inflammatory drug therapy. In patients who developed hypothyroidism with PSL treatment or without treatment, the rates of bilateral hypoechogenic areas (HEA) were 6-fold higher than those of unilateral HEA. Moreover, permanent hypothyroidism occurred in 5.9% of patients, and all patients with permanent hypothyroidism presented initially with bilateral HEA and had consequently small thyroid size with or without abnormal autoimmunity. CONCLUSIONS: The rates of thyroid dysfunction after SAT were significantly lower in patients receiving PSL. Extent of HEA in the thyroid, but not laboratory findings, may be a possible marker for developing thyroid dysfunction after SAT.

Large goiter and multiple rib tumors.

We report an interesting case of a 47-yr-old who had a large goiter and multiple rib tumors. The patient was initially suspected of having thyroid cancer, which had metastasized on the ribs, based on imaging studies. However, laboratory tests revealed a high level of ionized calcium and parathyroid hormone (PTH). The large goiter was diagnosed as having parathyroid tumors owing to the high level of PTH in the tissue fluid. The biopsy specimen from a rib tumor was diagnosed as containing brown tumors associated with primary hyperparathyroidism (PHP). The patient also had prolactinoma and pancreatic gastrinoma. Her daughter had both prolactinoma and PHP, and her brother and her father had PHP. Thus, the patient was diagnosed as having multiple endocrine neoplasia type 1.

Different phenotypes of multiple endocrine neoplasia type 1 (MEN1) in monozygotic twins found in a Japanese MEN1 family with MEN1 gene mutation.

We report monozygotic twins who showed different MEN1 phenotypes. The proband (28 y.o., female) had both primary hyperparathyroidism (PHP) and insulinoma, and genetic analysis revealed a point mutation (569del1, exon 3) of the MEN1 gene. This mutation causes a frameshift and produces a stop codon at codon 184. Restriction digestion (HinfI) analysis confirmed the same mutation of the MEN1 gene in six of the affected members including her two sisters, the monozygotic twins, and no such mutation in two unaffected members. In two generations of this family, eight of eleven family members had PHP and four of them were found to have other MEN1-related lesions. Both of the monozygotic twins had PHP. Interestingly, one had pancreatic tumor but the other had no evidence of it. Pituitary MRI showed no pituitary lesion in either of them. This is the first Japanese case of monozygotic twins with different MEN1 phenotypes.

A novel germline mutation of multiple endocrine neoplasia type 1 (MEN1) gene in a Japanese MEN1 patient and her daughter.

Familial multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant inherited disorder characterized by tumors of the parathyroid, anterior pituitary and gastro-entero-pancreatic endocrine tissues. The MEN1 gene has recently been cloned and its germline mutations have been considered to play an important role in the tumorigenesis of MEN1. We analyzed a Japanese MEN1 patient and her daughter for germline mutations of the MEN1 gene. The proband (60 y.o.) had primary hyperparathyroidism (PHP) and gastrinoma, and her daughter (30 y.o.) had prolactinoma. Clinical examinations revealed no evidence of PHP in the daughter. We identified a novel heterozygous germline mutation (712 A del) at codon 201 in exon 3 of the MEN1 gene in the proband. Restriction digestion analysis revealed the same mutation pattern in her daughter. These findings suggest that this family has familial MEN1 including a rare case of MEN1 with a single lesion of the pituitary. Genetic examinations are useful as diagnostic tools for any rare or variant case of familial MEN1.

No evidence of germline mutation or somatic deletion of the MEN1 gene in a case of familial multiple endocrine neoplasia type 1 (MEN1).

The MEN1 gene has recently been cloned as the gene responsible for multiple endocrine neoplasia type 1 (MEN1) and its germline mutations have been identified in a number of familial MEN1 patients. However, mutation-negative cases have also been reported in some MEN1 families. We report here a Japanese MEN1 family, including a proband with no evidence of MEN1 gene mutation. The proband (51 y.o., female) had three major MEN1 lesions, including primary hyperparathyroidism (HP), prolactinoma, and pancreatic tumor. Her father and brother had HP, and her daughter had both HP and prolactinoma. When we analyzed the proband for a germline mutation of the MEN1 gene, the direct sequencing analysis showed no mutation in the coding region, on the promoter, 5' and 3' untranslated regions of the MEN1 gene. We next examined the loss of heterozygosity (LOH) in the proband's parathyroid tumors using two benign polymorphisms (C2249G in intron 1 and 2248del3 in exon 10) in the MEN1 gene to detect LOH. LOH was not found in any of the four separate regions of the tumor tissues.

Identification of five novel germline mutations of the MEN1 gene in Japanese multiple endocrine neoplasia type 1 (MEN1) families.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by tumours of the parathyroid glands, the anterior pituitary, and endocrine pancreas. The MEN1 gene has recently been cloned and germline mutations have been identified in MEN1 patients in the United States, Canada, and Europe. We examined MEN1 gene mutations in MEN1 and MEN1 related cases in eight unrelated Japanese families. These families include five familial MEN1 (FMEN1), two sporadic MEN1 (SMEN1), and one familial hyperparathyroidism (FHP). Direct sequence analysis of the protein coding regions was carried out in all the probands. We identified six different heterozygous mutations in the coding region, of which five were novel, including one missense mutation (E45G) in both FMEN1 and SMEN1, three deletions (569del, 711del, and 1350del3) in FMEN1 and FHP, and two nonsense mutations (R29X and Y312X) in FMEN1 and SMEN1. Only one of these mutations (Y312X) has previously been reported. One proband with FMEN1 had no mutation in the entire exon sequence including the 5' and 3' untranslated regions. A restriction digestion analysis of 19 relatives from the five families showed a close correlation between the existence of the MEN1 gene mutation and disease onset. Four different polymorphisms, including two novel ones, were identified. These findings imply that a diversity of MEN1 gene mutations exists in Japanese MEN1 and MEN1 related disease, suggesting that analysis of the entire coding region of the MEN1 gene is required for genetic counselling in Japan.

Cellular signaling mechanisms for stimulation of growth hormone secretion and growth hormone primary transcripts by immunosuppressant agents, FK506 and cyclosporin A, in cultured rat pituitary cells.

Although an immunosuppressant, FK506, has been known to stimulate growth hormone (GH) release from rat somatotropes, the cellular signaling mechanism is unknown. In the present study, intracellular signaling pathways were investigated for FK506- and cyclosporin A (CsA)-induced GH release in cultured rat anterior pituitary cells. Northern and Western blot analysis revealed that the FK506-binding protein (FKBP12) and the CsA-binding protein (cyclophilin A) exist at the mRNA and protein level in the rat anterior pituitary tissue. FK506 and CsA increased GH release in a dose-dependent manner and inhibited calcineurin (CaN) activity in the cultured pituitary cells. The third immunosuppressant, rapamycin (RP), inhibited the FK506-induced GH release, although RP alone had no effect. Protein kinase A (PKA) inhibitors, H-89 and HA-1004 and EGTA blocked FK506- and CsA-induced GH release. TGF-beta did not alter basal GH release, but inhibited FK506-induced GH release. GH primary transcripts were increased by FK506, and the effects were blocked by H-89 and HA-1004. These results suggest that the immunosuppressants, FK506 and CsA, stimulate GH release by inhibiting CaN activity which results in the activation of the PKA system in the rat somatotropes. TGF-beta receptors might be involved in FK506-induced GH release as a separate pathway. FK506 also stimulates GH primary transcripts via a PKA-dependent mechanism in a manner similar to its effects on GH release.

Effects of adrenalectomy and glucocorticoid receptor antagonist, RU38486, on pituitary growth hormone-releasing hormone receptor gene expression in rats.

We examined the effects of adrenalectomy and a glucocorticoid receptor antagonist, RU38486, on pituitary GH-releasing hormone (GRH) receptor gene expression in rats. GRH receptor mRNA levels were significantly decreased in adrenalectomized rats and replacement of dexamethasone reversed the decrease to normal. GH secretion was inhibited by adrenalectomy, whereas dexamethasone replacement failed to restore the impaired GH secretion. A high dose of RU38486 had an agonistic effect on GRH receptor mRNA levels. These results suggest that endogenous glucocorticoid is necessary for normal expression of pituitary GRH receptor mRNA in rats.

Molecular cloning and gene expression of growth hormone-releasing peptide receptor in rat tissues.

We cloned a fragment of the rat GH-releasing peptide (GHRP) receptor homologue and examined the tissue distribution of GHRP receptor mRNA in rats. Sequence analysis showed that the open reading frame is well conserved between rat and human with 96% identity in a 364-amino acid overlap. By reverse transcription-polymerase chain reaction we detected GHRP receptor mRNAs in the rat brain including the hypothalamus, anterior pituitary, and renal pelvis in twenty-eight tissues tested. Microdissection revealed that GHRP receptor mRNAs were localized predominantly in the arcuate nucleus and ventromedial hypothalamus.

Germline mutation of the multiple endocrine neoplasia type 1 (MEN1) gene in a family with primary hyperparathyroidism.

Familial primary hyperparathyroidism (FHP) is a rare hereditary disorder characterized by isolated parathyroid tumors without any other lesions related to multiple endocrine neoplasia (MEN). Primary hyperparathyroidism is usually expressed at an early age and is highly penetrated in MEN type 1 (MEN1), suggesting that some FHP may be a variant type or early stage of MEN1. The MEN1 gene has recently been cloned and its germline mutations have been considered to play an important role in the tumorigenesis of MEN1. We studied a Japanese family with primary hyperparathyroidism which included 4 patients. To investigate the possible relationship between primary hyperparathyroidism in this family and the MEN1 gene, we analyzed a proband for a germline mutation of the MEN1 gene in this study. We identified a novel heterozygous mutation (1350del3) at codon 414 in exon 9. Restriction digestion analysis revealed the same mutation pattern in his brother with hyperparathyroidism. These findings suggest that our patients may belong to a variant type of MEN1.

A family of MEN1 with a novel germline missense mutation and benign polymorphisms.

The gene responsible for multiple endocrine neoplasia type 1 (MEN1) has recently been cloned, and its germline mutations were identified in patients with this syndrome. The majority of the mutations, frameshift or nonsense mutations, are expected to result in a loss of function of the gene product menin. Since the consequence of less common missense or in-frame deletion mutations is not clear, careful judgment is necessary regarding the role(s) of such mutations in MEN1 disease. Here we describe a large multigenerational MEN1 family with a novel germline missense mutation and three benign polymorphisms. The proband was a man with hyperparathyroidism and thymic carcinoid. We performed biochemical studies and DNA analyses of the MEN1 gene simultaneously and independently as family screening studies. Seven patients including the proband were identified, and all of them carried a heterozygous germline missense mutation E45G, but 5 members with normal biochemical results did not. This mutation was not observed in 50 normal volunteers. This novel missense mutation is therefore almost conclusively responsible for the disease. Although all of the mutant gene carriers in the present study already had clinical diseases, an MEN1 gene analysis in younger individuals at risk would be very useful in identifying carriers before the onset of the symptoms.

Detection of a novel nonsense mutation of the MEN1 gene in a familial multiple endocrine neoplasia type 1 patient and its screening in the family members.

We identified a novel nonsense mutation(R29X) of the MEN1 gene in a familial multiple endocrine neoplasia type 1 (MEN1) patient. Molecular analysis of the MEN1 gene was performed in the family members by a restriction digestion method. The same mutation pattern was seen in both the proband's younger brother and cousin diagnosed as MEN1, and was also observed in the son of the cousin who showed signs of normal levels of serum PTH associated with mild hypercalcemia and hypophosphatemia. These findings suggest that mutation analysis of the MEN1 gene is very useful in identifying the subclinical state of MEN1 as well as clinical MEN1.

Quantitative analysis of growth hormone (GH) pre-mRNA expression in cultured rat anterior pituitary cells by an intron-specific and competitive PCR method.

We have developed a novel method of quantifying growth hormone(GH) pre-mRNA expression in anterior pituitary cells. DNA-free total RNA extracted from cultured rat anterior pituitary cells was reverse transcribed(RT) to cDNA, and RT products were subsequently quantitated by competitive PCR using intron-specific primers of rat GH gene. After 6-h of incubation in treated cells, dexamethasone(Dex) and triiodo-L-thyronine(T3) significantly increased GH pre-mRNA levels(3.2- and 2.2-fold compared to non-treated cells, respectively). However, Northern blot analysis did not detect significant changes in GH mRNA levels. After 24-h incubation with Dex and T3, significant increases in GH mRNA levels were detected on Northern blots, but GH pre-mRNA levels did not differ between treated and non-treated cells. These findings suggest that both Dex and T3 treatments rapidly increase GH pre-mRNA levels in normal somatotropes. This method has high sensitivity and widespread application to the analysis of pre-mRNAs of target genes.

Biopolymers from marine invertebrates. XIII. Characterization of an antibacterial protein, dolabellanin A, from the albumen gland of the sea hare, Dolabella auricularia.

An antibacterial factor, dolabellanin A, was purified from the albumen gland of a sea hare, Dolabella auricularia. Purified dolabellanin A was a glycoprotein of 250 kilodaltons consisting of 4 subunits, and showed both antibacterial and antineoplastic activities. The two activities were lost in parallel on heating and at low and high pH. This factor was half-maximally active for gram-positive and -negative bacteria at 0.018-0.48 microgram/ml, and its action was not bactericidal but bacteriostatic. Dolabellanin A did not induce morphological elongation of bacteria or the release of adenosine triphosphate, but it completely inhibited the syntheses of deoxyribonucleic acid (DNA) and ribonucleic acid by E. coli within 6 min. These results suggest that dolabellanin A, which is found in a marine invertebrate, the sea hare, is a new antibacterial protein, and that it exerts its action by inhibiting nucleic acid synthesis, as does a DNA-inhibiting chemotherapeutic drug.

Bacteriostatic and cytolytic activity of purple fluid from the sea hare.

A bacteriostatic factor, aplysianin P, was purified from the purple fluid of a sea hare, Aplysia kurodai, as a glycoprotein of 60 K daltons. A cytolytic glycoprotein purified from the purple fluid of Dolabella auricularia did not show antibacterial activity. Aplysianin P was half-maximally active for gram-positive and -negative bacteria at 0.2-5.8 micrograms/mL and its action was not bactericidal but bacteriostatic. Aplysianin P completely inhibited the syntheses of DNA and RNA by E. coli, but it did not induce the release of ATP from bacteria. These results suggest that aplysianin P, found in an invertebrate, the sea hare, is a new bacteriostatic protein and that it exerts its action by inhibiting nucleic acid syntheses, as a DNA-inhibiting, chemotherapeutic drug does.

Biopolymers from marine invertebrates. X. Mode of action of an antibacterial glycoprotein, aplysianin E, from eggs of a sea hare, Aplysia kurodai.

An antibacterial factor, aplysianin E, was purified from the eggs of a sea hare, Aplysia kurodai. Purified aplysianin E was a glycoprotein of 250 kilo daltons consisting of 3 subunits, and showed both antibacterial and antineoplastic activities. The two activities were lost in parallel on heating and at low and high pH. This factor was half-maximally active for gram-positive and -negative bacteria at 0.12-3.3 micrograms/ml and its action was not bactericidal but bacteriostatic. Aplysianin E did not induce morphological elongation of bacteria or their release of adenosine triphosphate (ATP), but it completely inhibited the syntheses of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) by E. coli within 10 min. These results suggest that aplysianin E, found in an invertebrate, the sea hare, is a new antibacterial protein and that it exerts its action by inhibiting nucleic acid synthesis, as a DNA-inhibiting chemotherapeutic drug does.