A 4-base pair deletion mutation of Gs alpha gene in a Japanese patient with pseudohypoparathyroidism.

Mutations in the guanine nucleotide binding protein alpha subunit (Gs alpha) have been found in patients with pseudohypoparathyroidism (PHP). We have screened the Gs alpha gene for mutations in a Japanese patient with this disorder and identified a novel 4-base pair deletion in exon 7 in codons 189-190. This deletion causes a frameshift and if synthesis of a truncated form of Gs alpha occurred, it would likely be biologically inactive. The patient was heterozygous for this deletion. The patient's mother and an unaffected brother were tested for the presence of this mutation. His mother had the same mutation, and although her serum calcium and parathyroid hormone levels were within the normal range, she had subcutaneous calcifications. Thus, this mutation appears to be necessary but not sufficient to cause the complete pseudohypoparathyroidism phenotype and thus, other unknown factors, either genetic or acquired, may be necessary for the full syndrome to occur.

Absence of p53 mutation in Japanese patients with malignant thyroid lymphoma.

Abnormalities of the gene encoding the sequence-specific DNA-binding protein, nuclear phosphorprotein p53, are among the most common genetic alterations observed in human cancers. A mutation of this tumor suppressor gene has been reported with a low prevalence in differentiated thyroid carcinomas, while the prevalence was high in undifferentiated thyroid carcinomas. We used denaturing gradient gel electrophoresis (DGGE) to probe for mutations of p53 gene in order to determine its role in the genesis of malignant thyroid lymphoma. Involvement of 27 samples had been proven by histopathologic examination of specimen obtained by open biopsy of the thyroid gland or from cervical lymph nodes. DNA was extracted from tissues embedded in paraffin blocks and exons 5-8 of p53 gene were examined for the presence of mutations by DGGE following amplification by PCR using GC-clamped primers. To confirm accuracy of the method, samples with known p53 mutations were included in the study. No mutations were detected in any of the amplified exons of malignant thyroid lymphoma samples. These results suggest that p53 mutations are not present or are uncommon in Japanese patients with malignant thyroid lymphomas. The role of p53 in this form of carcinogenesis cannot be fully excluded since we have not examined the occurrence of mutations in regions upstream of exon 5.

Plasma CRH response to water immersion-restraint stress in rats bearing a hypothalamic knife cut.

We reported earlier that the plasma level of corticotropin-releasing hormone (CRH) remained high 120 min after the onset of such strong sustained stress as ether-laparotomy or water immersion-restraint, which reflected the persistent secretion of CRH from the hypothalamic median eminence (ME). We investigated the change in plasma CRH during water immersion-restraint stress in rats bearing an anterolateral cut around the medial basal hypothalamus (MBH) which cuts the CRH neurons from the PVN to the ME. Concentrations of CRH in the hypothalamus, extrahypothalamic tissues and peripheral blood were measured by radioimmunoassay. Plasma ACTH was measured with an immunoradiometric assay kit. Plasma baseline ACTH and CRH concentrations did not differ significantly in the sham vs. cut groups. At 120 min after the onset of stress, plasma ACTH concentrations were definitely higher in both groups. In the cut group, plasma CRH at 120 min after stress did not differ significantly from the baseline level, whereas plasma CRH at 120 min was definitely higher in the sham group. Baseline CRH concentrations in the ME did not differ greatly in the two groups. CRH concentrations in the ME of both groups had decreased appreciably 120 min after the onset of stress as compared with baseline CRH, and the CRH decrease was greater in the cut group than in the sham group. CRH in the neurointermediate lobe (NIL) and adrenal gland of both groups showed no significant change at 120 min, compared with the control. These findings confirm that the continuous CRH increase in plasma during sustained stress is derived mainly from the hypothalamus.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene screening in Japanese families with complete deficiency of thyroxine-binding globulin demonstrates that a nucleotide deletion at codon 352 may be a race specific mutation.

OBJECTIVE: Thyroxine-binding globulin (TBG) is a serum protein that transports 75% of circulating thyroxine. Eleven naturally occurring mutations in the human TBG gene have been identified, ten of which alter the properties of the molecule. Three of these mutations produce complete deficiency of TBG (TBG-CD) and four are associated with a second mutation in codon 283 (TBG-poly) which is polymorphic in some ethnic groups but, when present alone, does not alter the properties of the TBG molecule. In this communication we investigate whether two unrelated Japanese families with TBG-CD harboured the TBG-CDJ mutation in codon 352 associated with TBG-CD in families residing in more distant locations of the Japanese Islands. In addition we examined the possible association with TBG-poly and its incidence in the Japanese population. DESIGN: Mutant alleles were identified by amplification of genomic DNAs by the polymerase chain reaction, using allele-specific oligonucleotide primers. PATIENTS: Eight family members and 25 normal subjects. MEASUREMENTS: Serum free thyroxine and TBG concentration were measured by a conventional radioimmunoassay and a more sensitive enzyme immunoassay. Genomic DNAs were extracted from white blood cells and specific mutations at codons 352 and 283 were identified by allele-specific amplification. RESULTS: Three males and three females, whose serum TBG levels were decreased, had mutations at codon 352 as hemizygous and heterozygous, respectively. This mutation was not present in the DNA of any of the related or unrelated subjects with normal TBG concentration. The presence of TBG-poly was demonstrated in only one heterozygous family member and in six out of 30 alleles (20%) in normal unrelated subjects. The frequency of this TBG polymorphism in the Japanese is similar to that of 16% reported in French Canadians. CONCLUSIONS: We conclude that TBG-CDJ might be a prevalent cause of complete deficiency of thyroxine-binding globulin in the Japanese and that TBG-poly probably appeared before the divergence of human races.

Effects of ether-laparotomy and water immersion-restraint stress on CRH concentration in the hypothalamus, extrahypothalamic tissues and peripheral blood.

The effect of sustained stress on the plasma CRH level was studied in rats subjected to the stress of laparotomy conducted under ether anesthesia or water immersion-restraint. The role of AVP in ACTH secretion during such stress was also investigated. Concentrations of CRH and AVP in the hypothalamus, extrahypothalamic tissues and peripheral blood were measured by radioimmunoassays. Persistent secretion of ACTH was observed from 10 or 30 min to 120 min after the onset of each stress. Plasma CRH levels rose significantly 10 min after the onset of ether-laparotomy stress and remained significantly elevated at 120 min compared with controls. In the animals subjected to water immersion-restraint stress, plasma CRH tended to increase during the time course of the stress, reaching levels that were at least two times higher than the control. CRH concentrations in the median eminence (ME) during both types of stress decreased significantly at 120 min. In the ether-laparotomy stressed rats, CRH in the neurointermediate lobe (NIL) decreased significantly at 120 min, similar to the ME. Although a significant change in the adrenal CRH content was observed in the ether-laparotomy stressed rats, the involvement of adrenal CRH in ACTH secretion is unlikely as the absolute change in CRH was very small. These findings suggest that continuous CRH increase reflects a persistent secretion of CRH from the hypothalamic median eminence to the hypophysial portal vessels. It is possible that CRH secretion from the posterior pituitary gland is at least partly responsible for the persistent plasma ACTH increase in ether-laparotomy stress.(ABSTRACT TRUNCATED AT 250 WORDS)

[Hyperresponsiveness of TSH and prolactin and impaired responsiveness of GH in Japanese patients with isolated ACTH deficiency].

Two hundred and forty-one cases of isolated ACTH deficiency have been reported in Japan since 1969. Pituitary hormone responsiveness to stimulation tests before and after hydrocortisone supplementation was investigated in these cases. Plasma ACTH level showed no or little change in response to lysine vasopressin, metyrapone, CRF or insulin-induced hypoglycemia in 97.3-100% of the cases. Serum GH level changed little or not at all in response to GRF, insulin-induced hypoglycemia, glucagon, 1-dopa and arginine in 26.9, 29.3, 40.0, 50.0 and 56.1%, respectively. Serum TSH and prolactin (PRL) levels showed hyperresponse to TRH in 34.7 and 35.6%, respectively. After hydrocortisone therapy, GH secretion was more responsive than before therapy in 78.9% of the cases. After supplementation, TSH level was less responsive to TRH stimulation than before therapy in 59.3% of the cases. After hydrocortisone supplementation, TSH response to TRH decreased in 75% of ACTH-deficient patients without primary hypothyroidism but did not decrease in more than half of those with primary hypothyroidism. TSH response to TRH decreased after supplementation in 76.5% of the patients with TSH hyperresponsiveness before therapy, and increased after therapy in 66.7% of those with normal TSH responses before therapy. After supplementation, PRL response to TRH was less than that before therapy in 43.5% of ACTH--deficient patients, and greater than that before therapy in 30.4%. PRL response to TRH decreased after therapy in 66.7% of the patients with PRL hyperresponsiveness before therapy, and increased in 63.6% of those with normal PRL response before therapy. Primary hypothyroidism and Hashimoto's thyroiditis were complicated in 21.6 and 11.6%, respectively, of the 241 patients with isolated ACTH deficiency. In patients who had TSH hyperresponsiveness and/or high basal TSH levels and PRL hyperresponsiveness and/or high basal PRL levels, primary hypothyroidism was complicated in 58.4 and 42.3%, respectively. Hashimoto's thyroiditis was complicated in 29.8 and 20.5%, respectively, of these patients. Pituitary cell antibody (PCA) was detected in 36.6% of ACTH-deficient patients who were examined. Pituitary cell surface antibody (PCSA) to AtT-20 cells and GH3 cells was detected in 50.0 and 28.0% of the examined cases, respectively. The prevalence of PCA and PCSA did not differ between TSH-hyperresponsive patients and those with normal TSH basal levels and response, whereas PCA and PCSA were significantly more prevalent in PRL-hyperresponsive patients than in those with normal PRL levels and response. An empty sella was found in 30.2% of the examined case.(ABSTRACT TRUNCATED AT 400 WORDS)

[Chronic myelomonocytic leukemia (CMMoL) with systemic lymph node swelling and paroxysmal nocturnal hemoglobinuria (PNH)-like complication].

A 37-year-old male was diagnosed as having chronic myelomonocytic leukemia (CMMoL) with chief complaint of systemic lymph node swelling. On admission, his peripheral blood revealed mild anemia and mild thrombocytopenia with giant platelets, and monocytosis (1480/microliters). NAP score was low. Serum lysozyme increased. The bone marrow showed normal cellularity consisting of 4% myeloblasts and 14.4% promyelocytes, and a few myeloid cells were positive for double staining by alpha-naphthyl butyrate and naphthol ASD chloroacetate esterase. Biopsied specimens of the cervical lymph node showed infiltration of monocytoid cells, which were positive for lysozyme staining, into interfollicular tissue. As for chromosome variation, 21 large satellite was observed in all dividing cells from his bone marrow and peripheral blood. Furthermore, hemolytic anemia with hemoglobinuria developed during his course. Sugar water test was positive, but Ham test negative. Coombs test and Donath-Landsteiner reaction were negative. Abnormal hemoglobin, spherocyte and fragmentation were not found. Hemolysis disappeared about two months later. However, blastic crisis appeared and he died. We showed a case of CMMoL with 21 large satellite and paroxysmal nocturnal hemoglobinuria (PNH)-like complication. Satellite have usually been reported as asymptomatic, and thus this chromosome variant and CMMoL may have been coincidentally observed.

Electron microscopic observation of the cells floating in the anterior chamber in a case of phacolytic glaucoma.

The floating cells in the anterior chamber and a trabeculectomy specimen obtained from a 63-year-old man with phacolytic glaucoma were observed under a light microscope and a transmission electron microscope. Numerous macrophages which had phagocytized degenerated lens material (phacolytic cells) were observed in the anterior chamber, and free-floating degenerated lens material was also conspicuous. Melanin-laden macrophages accompanied by a small number of erythrocytes, ghost erythrocytes, lymphocytes, and macrophages showing erythrophagocytosis and leukophagocytosis and phagocytizing degenerating macrophages were also observed in the anterior chamber. In the trabeculectomy specimen, the intertrabecular space was obstructed by the phacolytic cells, melanin-laden macrophages, cell debris and free-floating degenerated lens material. It is concluded that these substances may cause obstruction of the aqueous humor outflow pathways, resulting in glaucoma.

Light and electron microscopic studies of malignant melanoma of conjunctiva and palpebra.

The authors experienced one case of malignant melanoma of the superior palpebral conjunctiva and the inferior palpebral skin, originating from benign acquired melanosis of the bulbar conjunctiva which developed in a 48-year-old man. Orbital exenteration including superior and inferior palpebrae was performed, and the removed tissue was examined by light and electron microscopy. In the temporal bulbar conjunctiva, melanocytes containing abundant fine melanin granules proliferated over almost the whole area of the epithelial layer. Cellular atypia and karyomitosis were not observed. The basement membrane remained intact with no proliferation of melanocytes in the substantia propria. However, in the perivascular area, in addition to the moderately prominent infiltration by lymphocytes and plasma cells, infiltration by melanophages was observed relatively frequently. Based on these findings, it was confirmed that the primary lesion of the bulbar conjunctiva was an acquired melanosis of Stage IB as classified by Zimmerman. The black nodular tumors of the superior palpebral conjunctiva and inferior palpebral skin consisted of melanoma cells showing markedly prominent cellular and nuclear atypia. The nuclei showed indentations in the membrane with electron-lucent karyoplasm, each having one nucleolus. The cytoplasm contained many round or elongated melanosomes in various developmental stages, with sizes of about 0.7 micron. Scattered among melanoma cells were a number of melanophages which had phagocytized many melanosomes at various stages of maturity.(ABSTRACT TRUNCATED AT 250 WORDS)