A missense mutant myostatin causes hyperplasia without hypertrophy in the mouse muscle

マウスの筋肉形成を抑制する因子としてマイオスタチンが知られている．この遺伝子が欠損するとマウスの筋肉が増大することが報告されている．家畜の肉牛においてはこの遺伝子の1塩基の突然変異により筋肉が増大することが報告されているので，その原因を解明するために同様な実験を行なった．その結果，突然変異マイオスタチンを過剰に発現させたトランスジェニックマウスの筋肉は増大することがわかった．この作用はドミナントネガティブな効果であることが示唆された．

Biochemical and Biophysical Research Communications


Transgenic expression of FGF8 and FGF10 induces transdifferentiation of pancreatic islet cells into hepatocytes and exocrine cells

FGF signaling is essential for normal development of pancreatic islets. To examine the effects of overexpressed FGF8 and FGF10 on pancreatic development, we generated FGF8- and FGF10-transgenic mice (Tg mice) under the control of the glucagon promoter. In FGF8-Tg mice, hepatocyte-like cells were observed in the periphery of pancreatic islets, but areas of alpha and beta cells did not decrease, whereas in FGF10-Tg mice, pancreatic ductal and acinar cells were found in islets, concomitantly with disturbed beta-cell differentiation. These results suggest that FGF8 and FGF10 play important roles in development of hepatocytes and exocrine cells, respectively, and explain the absence of FGF8 expression in normal islets and pancreatic hypoplasia in FGF10-deficient mice.


Identification of cis-elements regulating expression of Fgf10 during limb development.

Fibroblast growth factor 10 (FGF10) is known to be expressed in limb mesenchymal cells and to function as a mesenchymal signaling factor involved in epithelial-mesenchymal interactions during limb development. To elucidate regulation of Fgf10 expression, we isolated the promoter region of Fgf10 containing its 2.0 kb upstream 5'-fragment from the initiation codon and its 0.9 kb downstream fragment. Transcriptional activity of the fragment was examined with transgenic mice, using a lacZ-reporter system. Although no significant expression of the reporter gene was observed for the 0.2 kb 5'-fragment, expression was detected in the apical ectodermal ridge of the limb bud and developing cartilage of the limb for the 2.0 kb and 0.7 kb 5'-fragments, respectively. From comparison of the mouse sequences of the 2.0 kb fragment with corresponding sequences of human and chicken Fgf10, we identified 17 conserved putative enhancer motifs for AER expression and other unidentified expressions. For limb cartilage expression, we found putative enhancer sequences conserved among the three species in the 0.7 kb 5'-fragment. In the fragment, three DNA binding motifs were identified in the mouse and human sequence, although they are not conserved in the corresponding chicken sequence.

The International Journal of Developmental Biology


Familial Juvenile Gouty Nephropathy: Exclusion of 16p12 from the Candidate Locus

Familial juvenile gouty nephropathy (FJGN, MIM 162000) is an autosomal-dominant renal disease characterized by underexcretion-type hyperuricemia, gouty arthritis, and progressive renal disease at younger ages. We analyzed the localization of the responsible gene for FJGN concerning the chromosomal region of 16p12 using parametric linkage analysis in our FJGN. The affected members of this family were accompanied with polyuria due to nephrogenic diabetes insipidus and without hypertension. Fifteen samples were collected from 9 affected and 6 nonaffected members of the family. By using microsatellite markers mainly focused on the short arm of chromosome 16, two point and multipoint linkage analyses were carried out. All of the 2-point logarithm of odds (LOD) scores were typically negative and all of the multipoint LOD scores were less than -3.0 in our FJGN family. The results suggested that the localization of the responsible gene to 16p12 can be excluded in our FJGN family. This finding means that the responsible gene for FJGN is not common.


Feedback Inhibition of Amidophosphoribosyltransferase Regulates the Rate of Cell Growth via Purine Nucleotide, DNA, and Protein Syntheses

To clarify the contributions of amidophosphoribosyltransferase (ATase) and its feedback regulation to the rates of purine de novo synthesis, DNA synthesis, protein synthesis, and cell growth, mutated human ATase (mhATase) resistant to feedback inhibition by purine ribonucleotides was engineered by site-directed mutagenesis and expressed in CHO ade (-)A cells (an ATase-deficient cell line of Chinese hamster ovary fibroblasts) and in transgenic mice (mhATase-Tg mice). In Chinese hamster ovary transfectants with mhATase, the following parameters were examined: ATase activity and its subunit structure, the metabolic rates of de novo and salvage pathways, DNA and protein synthesis rates, and the rate of cell growth. In mhATase-Tg mice, ATase activity in the liver and spleen, the metabolic rate of the de novo pathway in the liver, serum uric acid concentration, urinary excretion of purine derivatives, and T lymphocyte proliferation by phytohemagglutinin were examined. We concluded the following. 1) ATase and its feedback inhibition regulate not only the rate of purine de novo synthesis but also DNA and protein synthesis rates and the rate of cell growth in cultured fibroblasts. 2) Suppression of the de novo pathway by the salvage pathway is mainly due to the feedback inhibition of ATase by purine ribonucleotides produced via the salvage pathway, whereas the suppression of the salvage pathway by the de novo pathway is due to consumption of 5-phosphoribosyl 1-pyrophosphate by the de novo pathway. 3) The feedback inhibition of ATase is more important for the regulation of the de novo pathway than that of 5-phosphoribosyl 1-pyrophosphate synthetase. 4) ATase superactivity leads to hyperuricemia and an increased bromodeoxyuridine incorporation in T lymphocytes stimulated by phytohemagglutinin.


The Rate of Cell Growth Is Regulated by Purine Biosynthesis via ATP Production and G to Sphase Transition

We recently showed that an increased supply of purine nucleotides increased the growth rate of cultured fibroblasts. To understand the mechanism of the growth rate regulation, CHO K1 (a wild type of Chinese hamster ovary fibroblast cell line) and CHO ade (-)A (a cell line deficient in amidophosphoribosyltransferase, a rate-limiting enzyme of the de novo pathway) were cultured under various conditions. Moreover, a defective de novo pathway in CHO ade (-)A cells was exogenously restored by 5-amino-4-imidazole-carboxamide riboside, a precursor of the de novo pathway. The following parameters were determined: the growth rate of CHO fibroblasts, the metabolic rate of the de novo pathway, the enzyme activities of amidophosphoribosyltransferase and hypoxanthine phosphoribosyltransferase, the content of intracellular nucleotides, and the duration of each cell-cycle phase. We concluded the following: (i) Purine de novo synthesis, rather than purine salvage synthesis or pyrimidine synthesis, limits the growth rate. (ii) Purine nucleotides are synthesized preferentially by the salvage pathway as long as hypoxanthine is available for energy conservation. (iii) The GTP content depends on the intracellular ATP content. (iv) Biosynthesis of purine nucleotides increases the growth rate mainly through ATP production and promotion of the G(1)/S transition.


Localization of a gene for familial juvenile hyperuricemic nephropathy causing underexcretion-type gout to 16p12 by genome-wide linkage analysis of a large family.

Familial juvenile hyperuricemic nephropathy (FJHN, MIM 162000) is an autosomal-dominant disease characterized by underexcretion-type hyperuricemia, gout, and chronic renal failure. No loci responsible for this disease or any underexcretion-type hyperuricemia/gout have ever been identified. The aim of the study was to localize a gene responsible for FJHN by linkage analysis. A single large family with at least 20 affected members was analyzed. DNA was obtained from 13 affected and 18 non-affected members after lymphoblastoid cell lines were established. Initially, polymorphic data were obtained for 343 microsatellite loci covering all chromosomes except the X chromosome. Parametric linkage analysis was performed using the obtained data with LINKAGE package software. Following a genome-wide search using a set of highly polymorphic microsatellite markers, initial evidence for linkage was obtained for a marker on chromosome 16p. We subsequently genotyped the same subjects for 12 additional markers spanning approximately 30 cM on the short arm of chromosome 16. We obtained a maximum 2-point logarithm of odds (LOD) score of 6.04 at theta = 0 with the marker D16S401; multipoint linkage analysis yielded a maximum LOD score of 6.14 with markers D16S401 and D16S3113, and established a minimum candidate interval of approximately 9 cM. A gene for FJHN was localized to a candidate interval of approximately 9 cM at 16p12. These findings will be useful for the presymptomatic diagnosis of FJHN in some families and for testing genetic heterogeneity of FJHN in general.


Molecular Cloning and Tissue-specific Expression of a New Member of the Regenerating Protein Family, Islet Neogenesis-associated Protein-related Protein.

Islet neogenesis-associated protein (INGAP) is a protein expressed during islet neogenesis. We have cloned a novel cDNA having a similar sequence to INGAP cDNA. The cDNA encodes 175 amino acids designated INGAP-related protein (INGAPrP). INGAP is expressed in cellophane-wrapped pancreas, but not in normal pancreas, whereas INGAPrP was abundantly expressed in normal pancreas.

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease


Diabetes and tumor formation in transgenic mice expressing reg I.

To examine the effect of overexpressed regenerating gene (Reg) I on pancreatic beta-cells, we generated transgenic mice expressing Reg I in islets (Reg-Tg mice). Three lines of Reg-Tg mice were established. In line-1 Reg-Tg mice, the expression level of Reg I mRNA in islets was 7 times higher than those in lines 2 and 3 of Reg-Tg mice, and line 1 mice developed diabetes by apoptosis of beta-cells, as well as various malignant tumors. In addition to the decrease in beta-cells, compensatory islet regeneration and proliferation of ductal epithelial cells were observed in line-1 Reg-Tg mice. Because Reg I protein was secreted primarily into pancreatic ducts from acinar cells, it may primarily stimulate the proliferation of ductal epithelial cells, and not beta-cells, and their differentiation into islets. Moreover, the tumor-promoting activity of Reg I protein should be considered for its possible clinical applications.

Biochemical and Biophysical Research Communications


The Rate of Cell Growth is Regulated by Purine Biosynthesis via ATP Production and G1 to S Phase Transition.


Diabetes and pancreatic tumours in transgenic mice expressing Pax6.

Both endocrine and exocrine cells of the pancreas differentiate from epithelial cells of primitive pancreatic ducts, and four types of pancreatic islet cells (alpha, beta, delta, and PP cells) are derived from the common pluripotent precursor cells. Although Pa x 6 is expressed in all islet cells, Pa x 4 is detected only in beta cells. In homozygous Pa x 4-null mice, beta cells are absent, whereas the number of alpha cells is increased. Therefore, we hypothesized that the balance of Pa x 4 and 6 is one of the determinants by which the common progenitor cells differentiate into alpha or beta cells. To change this balance, we generated transgenic mice overexpressing Pa x 6 driven by the insulin promoter or the PDX1 promoter. In both types of transgenic mice, normal development of beta cells was disturbed, resulting in apoptosis of beta cells and diabetes. In Insulin/Pa x 6-Tg mice, beta cells were specifically affected, whereas in PDX/Pa x 6-Tg mice, developmental abnormalities involved the whole pancreas including hypoplasia of the exocrine pancreas. Furthermore, PDX/Pa x 6-Tg mice experienced proliferation of both ductal epithelia and islet cells and subsequent cystic adenoma of the pancreas. These findings suggest that Pa x 6 promotes the growth of ductal epithelia and endocrine progenitor cells and that the suppression of Pa x 6 is necessary for the normal development of beta cells and the exocrine pancreas.


Molecular Cloning and Expression Analysis of a Putative Nuclear Protein, SR-25.

We cloned a full-length mouse cDNA and its human homologue encoding a novel protein designated as "SR-25." In Northern blot analysis, SR-25 mRNA was expressed in all organs tested, and relatively abundant in testis and thymus. Deduced amino acid sequences of mouse SR-25 and human SR-25 showed 77.7% identity. SR-25 has a serine-arginine repeat (SR repeat) and two types of amino acid clusters: a serine cluster and a highly basic cluster. Based on the presence of many nuclear localizing signals and a similarity to RNA splicing proteins, SR-25 is strongly suggested to be a nuclear protein and may contribute to RNA splicing.


A Germline Mutation of the Multiple Endocrine Neoplasia Type 1 (MEN1) Gene in Japanese Prolactinoma Variant.

The Journal of Clinical Endocrinology and Metabolism


Expression of somatostatin receptor subtypes and growth inhibition in human exocrine pancreatic cancers.

The antiproliferative effects of somatostatin and its analogs on human pancreatic cancers were studied: (1) by evaluating the gene expression of somatostatin receptor (sstr) subtypes in human pancreatic cancer cell lines and cancer tissue specimens, (2) by evaluating the antiproliferative effects of somatostatin analogs, and (3) by evaluating the effect of sstr-2 cDNA transduction. Using a reverse transcriptase polymerase chain reaction (RT-PCR), the gene expression of five sstr subtypes (sstr-1 to -5) was examined in eight cell lines, and in ten pancreatic cancer tissues and in the normal surrounding pancreatic tissues. The antiproliferative effects of somatostatin (SS) -14 and its two analogs (SMS 201-995, RC-160) were examined by means of an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (thiazolyl blue)) assay on three cell lines and Panc-1 transfectants with human sstr (hsstr)-2A cDNA. Sstr-2 was expressed in all samples tested. All examined cell lines simultaneously expressed sstr-2 and -5, while most of the examined pancreatic cancer tissues did not express both of these subtypes simultaneously. Somatostatin analogs inhibited epidermal growth factor (EGF)-stimulated pancreatic cancer cell proliferation. The cell proliferation was further and significantly inhibited by 14% in stable transfectants of Panc-1 cells with hsstr-2A. Based on these findings, it is concluded that somatostatin analogs with their antiproliferative effects mediated by sstr-2 could be potentially useful in the treatment of pancreatic cancers.

Journal of Hepato-Biliary-Pancreatic Surgery


Differential Regulation of the Human Insulin Gene Transcription by GG1 and GG2 Elements with GG- and C1-binding Factors.

Using a human growth hormone reporter system, the introduced mutations in GG1 alone or both GG elements of GG1 and GG2 in the human insulin promoter abolished 94 or 96% of the beta-cell-specific transcriptional activity in a pancreatic islet beta-cell line of MIN6, while the mutations in GG2 or its total deletion abolished 85 or 86% of the transcriptional activity. When linked to the thymidine kinase promoter, mutations in GG1 or both GG elements abolished 74% of the transcriptional activity in MIN6 cells, while the mutations in GG2 or its total deletion abolished 55 or 54%. In the electrophoretic mobility shift assay (EMSA), one nuclear factor was shown to interact with two GG elements, and another C1-binding factor with GG1 and C1. The differential effects of deletions or selective mutations in the GG2 or GG1 sequence in the oligonucleotide probes on the binding activity of GG- or C1-binding factors in EMSA proved the requirement of both GG1 and GG2 or both GG1 and C1, respectively, for the transaction of these two factors. The molecular size of the GG-binding factor was estimated about 30 kDa. Based on these, we conclude that two GG elements contribute, with GG1 more critically than GG2, to the beta-cell-specific transcription of the human insulin gene through transaction with the GG- and C1-binding factors.

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression


Apotosis and Remodelling of Beta Cells by Paracrine Interferon-g without Insulitis in Transgenic Mice.


Infrequent Detectable Somatic Mutations of the RET and Glial Cell Line-Derived Neurotrophic Factor (GDNF) Genes in Human Pituitary Adenomas.

RET is a receptor tyrosine kinase expressed in neuroendocrine cells and tumors. RET is activated by a ligand complex comprising glial cell line-derived neurotrophic factor (GDNF) and GDNF receptor-alpha (GDNFR-alpha). Activating mutations of the RET proto-oncogene were found in multiple endocrine neoplasia (MEN) 2 and in sporadic medullary thyroid carcinoma and pheochromocytoma of neuroendocrine origin. Mutations of the RET proto-oncogene and the glial cell line-derived neurotrophic factor (GDNF) gene were examined in human pituitary tumors. No mutations of the RET proto-oncogene including the cysteine-rich region or codon 768 and 918 in the tyrosine kinase domain were detected in 172 human pituitary adenomas either by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) or by PCR-restriction fragment length polymorphism (RFLP). Further, somatic mutations of the GDNF gene in 33 human pituitary adenomas were not detected by PCR-SSCP. One polymorphism of the GDNF gene at codon 145 of TGC or TGT was observed in a prolactinoma. The RET proto-oncogene message was detected in a normal human pituitary gland or 4 of 4 human pituitary adenomas with reverse transcription (RT)-PCR, and in rodent pituitary tumor cell lines with Western blotting. The expression of GDNF gene was detected in 1 of 4 human somatotroph adenomas, 1 of 2 corticotroph adenomas, and 2 of 6 rodent pituitary tumor cell lines with RT-PCR. Based on these, it is concluded that somatic mutations of the RET proto-oncogene or the GDNF gene do not appear to play a major role in the pituitary tumorigenesis in examined tumors.


Polymorphisms of Thymidine Kinase Gene in Herpes Simplex Virus Type 1 --- Analysis of Clinical Isolates From Herpetic Keratitis Patients and Laboratory Strains

Drug-resistance of herpes simplex virus (HSV) is caused most frequently by mutation of the viral thymidine kinase (TK) gene. To elucidate the significance of detecting nucleotide changes of the TK gene for screening drug-resistant viruses, the frequency and variation of the genetic polymorphisms in the whole coding region of the TK gene were studied in 14 acyclovir-susceptible HSV type 1 (HSV-1) clinical isolates from 14 patients with epithelial herpetic keratitis. Two reference HSV-1 laboratory strains, McKrae and PH, and two acyclovir-resistant variants of the PH strain were also studied as controls. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct sequencing detected nucleotide differences at 24 positions, and amino acid substitutions at 12 codons in the TK gene of the examined viruses. Nucleotide diversity of 0.0029 per base (the average number of nucleotide substitutions of 3.3 per 1,131 base pairs) in the TK gene in the clinical isolates was comparable to 0.0037 per base of the whole HSV-1 genome in Japanese isolates reported previously. PCR-SSCP analysis of the acyclovir-resistant strains easily detected aberrantly shifted bands by comparing them with those of the parental strain, followed by the quick determination of mutated sequences. These results suggest that detection of nucleotide changes of the TK gene is useful for serial observation of persistent or recurrent HSV infection as observed in immunocompromised hosts, but that it is not useful for screening drug-resistant viruses from nonepidemic clinical isolates because of the comparable genetic polymorphisms in the TK gene as in the whole HSV-1 genome.


Analysis of loss of heterozygosity on chromosome 11 and infrequent inactivation of the MEN1 gene in sporadic pituitary adenomas

To investigate the role of tumor suppressor genes in sporadic pituitary adenomas, we first analyzed loss of heterozygosity on 11q13 with microsatellite analysis in 31 tumors. Loss of heterozygosity on 11q13 was detected in 1 mixed GH/PRL adenoma, and the somatic 22-bp deletion of the multiple endocrine neoplasia type 1 (MEN1) gene encoding menin was detected in this tumor. Trisomy 11 suggested by the decreased mean allelic ratios of 66% or 65% for 16 or 13 microsatellite markers, respectively, in 2 of 31 pituitary adenomas was confirmed by interphase fluorescence in situ hybridization. Screening for mutations of the MEN1 gene did not find mutations with PCR-single strand conformation polymorphism analysis in other pituitary adenomas retaining heterozygosity on 11q13. Based on these, it is concluded that inactivation of the MEN1 gene comprises a rare etiology for tumorigenesis of the pituitary gland, and that trisomy 11 or another gene(s) may contribute to the pathogenesis of sporadic pituitary adenomas.


Hypoplasia of pancreatic islets in transgenic mice expressing activin receptor mutants

Activin, a member of the TGF-beta superfamily, regulates the growth and differentiation of a variety of cell types. Based on the expression of activin in pancreatic rudiments of rat embryos and stimulation of insulin secretion from adult rat pancreatic islets by activin, activin is implicated in the development and function of islets. To examine the significance of activin signaling in the fetal and postnatal development of islets, transgenic mice expressing a dominant negative form of activin receptor (dn-ActR) or a constitutively active form of activin receptor (ActR-T206D) in islets were generated together with the transgenic mice expressing intact activin receptor (intact ActR) as a negative control. Transgenic mice with both dn-ActR and ActR-T206D showed lower survival rates, smaller islet area, and lower insulin content in the whole pancreas with impaired glucose tolerance when compared with transgenic mice with intact ActR or littermates, but they showed the same alpha cell/beta cell ratios as their littermates. In addition to islet hypoplasia, the insulin response to glucose was severely impaired in dn-ActR transgenic mice. It is suggested that a precisely regulated intensity of activin signaling is necessary for the normal development of islets at the stage before differentiation into alpha and beta cells, and that activin plays a role in the postnatal functional maturation of islet beta cells.


Abrogation of Autoimmune Diabetes in Nonobese Diabetic Mice and Preotection against Effector Lymphocytes by Transgenic Paracrine TGFb-1.


Germ-Line Mutations in MEN1 Patients-Authors' Response.

The Journal of Clinical Endocrinology and Metabolism


Analysis of Loss of Heterozygosity on Chromosome 11 and Infrequent Inactivation of MEN1 Gene in Sporadic Pituitary Adenomas.


Absence of Germline Mutations of the MEN1 Gene in Familial Pituitary Adenoma in Contrast to Multiple Endocrine Neoplasia Type 1 in Japanese.


Familial Isolated Primary Hyperparathyroidism with Parathyroid Carcinomas: Clinical and Molecular Features.

Familial isolated primary hyperparathyroidism (FIHP) is a rare hereditary disorder. We present four patients from a single family with FIHP, and genetic analysis of their parathyroid adenomas and parathyroid carcinoma. DNA was extracted from tumours resected at surgery. Tumours were examined for loss of heterozygosity (LOH) with microsatellite polymorphic markers. The 27-year-old proband (Patient 1) died of parathyroid carcinoma with metastases to the lungs and chest wall. Sixteen years later, his 34-year-old sister (Patient 2) presented with a neck tumour and primary hyperparathyroidism. Family screening revealed parathyroid tumours in his 36-year-old sister (Patient 3) and 29-year-old cousin (Patient 4). Histological examination of resected tumours showed parathyroid carcinoma and adenoma in Patient 2, a parathyroid adenoma in Patient 3, and an atypical parathyroid adenoma in Patient 4. Autopsy of the proband ruled out multiple endocrine neoplasia (MEN) type 1, and the three patients who underwent parathyroidectomy did not exhibit any abnormalities in the pancreas or the pituitary gland. Analysis of tumour DNA from one parathyroid carcinoma (Patient 2), the atypical parathyroid adenoma (Patient 4), and two parathyroid adenomas (Patients 2 and 3) showed limited LOH on chromosomes 13q12.3-q32 in an adenoma of Patient 2 and 9p21-p22 and 13q12.3-q32 in an adenoma of Patient 3. These results suggest the possible contribution of tumour suppressor genes including the retinoblastoma gene and the hereditary breast cancer susceptibility gene (BRCA2) on 13q to parathyroid tumours in this family.


Current micro-and Macroangiopathic Complication of Diabetic Patients by Primary Care Physicians on Multicenter-study

プライマリ·ケア医による糖尿病合併症の調査

日本プライマリ·ケア学会誌

日本プライマリ·ケア学会誌


Infrequent Mutations of p27Kip1 Gene and Trisomy 12 in a Subset of Human Pituitary Adenomas.


Amidophosphoribosyltransferase Limits the Rate of Cell Growth-linked de Novo Purine Biosynthesis in the Presence of Constant Capacity of Salvage Purine Biosynthesis.


p53 Gene Mutation in N-(4-hydroxybutyl) nitrosamine-induced Urinary Bladder Tumors and N-methyl-N-nitrosourea-induced Colon Tumors of Rats.


Cystic Glucagonoma with Loss of Heterozygosity on chromosome 11 in Multiple Endocrine Neoplasia Type 1.

A 52-year-old man with a past history of a pituitary adenoma and hyperparathyroidism due to a parathyroid adenoma was admitted because of a solitary tumour of the pancreas revealed by ultrasonography. His family history was unremarkable. Plasma glucagon levels were slightly elevated (280 ng/l, normal range, 40-180 ng/l) with decreased plasma amino acid levels. Plasma glucagon levels disclosed an exaggerated response during an arginine infusion test and paradoxical elevation during a 75 g oral glucose tolerance test. Endoscopic ultrasonography revealed a monolocular cystic mass of about 3 cm in diameter in the body of the pancreas. A pancreatic tumour was diagnosed before surgery as a cystic glucagonoma. Intra-operative ultrasonography showed one cystic mass in the body of pancreas and two other solid tumours, about 1 cm and 0.5 cm in diameter, in the tail of the pancreas. Histologically, all three tumours showed neoplastic epithelial cells with round nuclei forming cords and/or a ribbon-like arrangement. They showed positive staining for Grimelius' silver stain and immunopositive cells for glucagon. Genetic analysis of the main cystic tumour revealed loss of heterozygosity (LOH) on chromosome 11. After the operation, the responses of plasma glucagon to arginine infusion and oral glucose became normal. Here we describe the usefulness of these provocation tests for early diagnosis and post-operative follow-up in a rare cystic glucagonoma associated with multiple endocrine neoplasia type 1 (MEN 1) which had LOH on chromosome 11.


p53 Gene Mutation Analysis in Porokeratosis and Porokeratosis-associated Squamous Cell Carcinoma.

In this and previous studies, we have shown p53 overexpression immunohistochemically in 14 of 17 porokeratotic specimens obtained from 14 lesions of nine cases, and in all six specimens of squamous cell carcinoma (SCC) arising on porokeratotic lesions of two cases. We screened mutations in exons 5 to 10 of the p53 gene in all these specimens by polymerase chain reaction-single strand conformation polymorphism analysis. Mutations of the p53 gene were detected in two of the six SCCs but not in any of the 17 porokeratotic specimens. These two mutations were C to T transitions at codons 146 and 175 in exon 5, which were a nonsense mutation at a dipyrimidine site and a missense mutation at a CG site, respectively. To our knowledge, neither of these mutations has been identified in skin cancers before. Our observations indicate that mutations of the p53 gene are not the major molecular etiology for porokeratosis, but are related to its skin carcinogenesis, and that p53 overexpression in porokeratosis is not due to p53 gene mutations.


Infrequent Mutations of p16INK4A and p15INK4B Genes in Human Pituitary Adenomas.


Inactivation of the Tumor Suppressor Gene on 11q13 in Brothers with Familial Acrogigantism without Multiple Endocrine Neoplasia Type 1.


Prevention of Adoptively Transferred Diabetes in Non-Obese Diabetic Mice with IL-10-Transduced Islet-Specific Th1 Lymphocytes: A Gene Therapy Model for Autoimmune Diabetes.


Molecular Cloning of a cDNA Encoding Human Ribosomal Protein L39.

A cDNA clone encoding a human ribosomal protein L39 (hRPL39) was isolated through a random cDNA sequencing approach to a cDNA library constructed from a human colon carcinoma cell line of COLO 205. Although levels of hRPL39 mRNA were different in several cell lines including carcinoma cell lines from different tissues, they were shown not to be cell cycle-dependent in a human fibroblast cell line of TIG-1.

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression


Molecular Cloning of a Human cDNA Encoding a Trifunctional Enzyme of Carbamoyl- Phosphate Synthetase-Aspartate Transcarbamoylase-Dihydroorotase in de Novo Pyrimidine Synthesis.

Biochemical and Biophysical Research Communications


Detection of Different Quasispecies of Hepatitis C Virus Core Region in Cancerous and Non-cancerous Regions.


GGAAT Motifs Play a Major Role in Transcriptional Activity of the Human Insulin Gene in a Pancreatic Islet Beta Cell Line of MIN6.


Multiple Fluorescence-Based PCR-SSCP Analysis Using Internal Fluorescent Labeling of PCR Products.


Absence of Mutations at Codon 768 of the RET Proto-Oncogene in Sporadic and Hereditary Pheochromocytomas.

Sixteen sporadic pheochromocytomas, 3 pheochromocytomas in neurofibromatosis 1, and 4 pheochromocytomas in multiple endocrine neoplasia (MEN) 2A or 2B were screened for mutations at codon 768 of the RET proto-oncogene by AluI digestion of polymerase chain reaction PCR products and mutations in exon 13 by PCR-single strand conformation polymorphism (SSCP) analysis. Although mutations at codon 768 (GAG --> GAC; Glu --> Asp) of the RET proto-oncogene were recently reported to be found in 40% of sporadic medullary thyroid carcinomas (MTCs), the absence of missense mutations at codon 768 was confirmed both with PCR-restriction fragment length polymorphism (RFLP) and PCR-SSCP analysis in all examined cases of pheochromocytomas. These results suggest that mutations at codon 768 of the RET proto-oncogene do not represent a frequent mechanism of tumorigenesis for both sporadic and hereditary pheochromocytomas.


Obvious mRNA and Protein Expression but Absence of Mutations of the RET Proto-Oncogene in Parathyroid Tumors.


Frequent p53 Accumulation in the Chronically Sun-Exposed Epidermis and Clonal Expansion of p53 Mutant Cells in the Epidermis Adjacent to Basal Cell Carcinoma.

The Journal of Investigative Dermatology


Rat Genomic Structure of Amidophosphoribosyltransferase, cDNA Sequence of Aminoimidazole Ribonucleotide Carboxylase, and Cell Cycle-Dependent Expression of These Two Physically Linked Genes.

Genomic structure of rat amidophosphoribosyltransferase (ATase; EC 2.4.2.14), which catalyzes the first committed step in de novo purine nucleotide synthesis, was determined by polymerase chain reaction (PCR)-based methods. There are 11 exons and all exon-intron boundaries were conserved among rat, human, and chicken ATase genes. A rat aminoimidazole ribonucleotide carboxylase (AIRC) cDNA encoding a bifunctional enzyme of AIRC (EC 4.1.1.21) at step 6 and SAICAR synthetase (EC 6.3.2.6) at step 7 in de novo purine nucleotide synthesis was cloned and sequenced. The size of the cloned rat AIRC cDNA was 1329 bp, and amino acid identity with human and chicken AIRC was 96 and 85%, respectively. The intergenic sequence using a phage clone and the PCR product disclosed that ATase and AIRC genes are physically linked with the 736 bp sequence between the translation start sites, and the determination of the transcriptional start sites by the primer extension assay for these genes disclosed that distance between the two major transcriptional start sites is 585 bp. The amount of mRNAs of both genes showed approx. 5-6-fold increase in G1/S phase of the cell cycle over those in G0 phase in synchronized rat 3Y1 fibroblasts.


Lack of CD54 Expression and Mutation of p53 Gene Relate to the Prognosis of Childhood Burkitt's Lymphoma.


Two Different Pituitary Adenomas in a Patient with Multiple Endocrine Neoplasia Type 1Associated with Growth Hormone-Releasing Hormone-Producing Pancreatic Tumor: Clinical and Genetic Features.

The clinical and genetic features of a 43-year-old male patient with multiple endocrine neoplasia type 1 were reported. He developed hyperparathyroidism, a GHRH-producing pancreatic tumor, and acromegaly between 1980 and 1983. Because his pituitary gland increased in size even after resecting the GHRH-producing pancreatic tumor, transsphenoidal hypophysectomy was performed six years later. The pituitary contained two histologically-different adenomas composed of somatotroph cells and null cells. Genetic analyses revealed loss of heterozygosity on chromosome 11 in common in the pituitary adenomas, the pancreatic endocrine tumors, and a parathyroid hyperplasia. On the other hand, mutations of ras, p53, Gs alpha, and Gi2 alpha genes were not found in these tumors. The loss of the tumor suppressor gene on chromosome 11q12-13 was involved in the formation of two pituitary adenomas, two pancreatic endocrine functioning tumors, and a parathyroid hyperplasia in this patient, but the tumorigenic factors in the specific endocrine organs remain to be studied.


Galactose-Induced Albuminuria in Transgenic Mice Expressing Human Aldose Reductase.


Multiple Fluorescence-Based PCR-SSCP Analysis with Postlabeling.


Tumor-Specific Mutations in the Tyrosine Kinase Domain of the RET Proto-Oncogene in Pheochromocytomas of Sporadic Type.

Sporadic pheochromocytomas, sporadic medullary thyroid carcinomas (MTCs), pheochromocytomas and/or MTCs in multiple endocrine neoplasia (MEN) 2A or 2B were screened for mutations in the tyrosine kinase domain of the RET proto-oncogene by direct sequencing of PCR-amplified products or sequencing subcloned DNAs from PCR-products. All tumors of 4 MEN 2B patients were confirmed to contain a heterozygous missense mutation at codon 918 (ATG-->ACG; Met-->Thr) of the RET proto-oncogene as well as their leukocytes. The same tumor-specific mutations at codon 918 were also found in 5/16 (31%) sporadic pheochromocytomas. These results suggest that mutations of the RET proto-oncogene in its tyrosine kinase domain play a role not only as the predisposing gene for MEN 2B, but also as a tumorigenic factor for pheochromocytomas of sporadic type.


Human Calgizzarin; One Colorectal Cancer-Related Gene Selected by a Large Scale Random cDNA Sequencing and Northern Blot Analysis.


Molecular Cloning of a Group of Mouse Pancreatic Islet B Cell and Glucose Concentration-Related Genes by Random cDNA Sequencing.


p53 Gene Mutations in Human Skin Cancers and Precancerous Lesions: Comparison with Immunohistochemical Analysis.


Transgenic Expression of IL-10 in Pancreatic Islet A cells Accelerates Autoimmune Insulitis and Diabetes in Non-Obese Diabetic Mice.


PCR with End Trimming and Cassette Ligation: A Rapid Method to Clone Exon-Intron Boundaries and a 5'-Upstream Sequence of Genomic DNA Based on a cDNA Sequence.


T-Cassette Ligation: A Method for Direct Sequencing and Cloning of PCR-Amplified DNA Fragments.


Multiple Fluorescence-Based PCR-SSCP Analysis.


An End-Trimming Method to Amplify Adjacent cDNA Fragments by PCR.


Relatively Good Prognosis of Multiple Endocrine Neoplasia Type 2B in Japanese: Review of Cases in Japan and Analysis of Genetic Changes in Tumors.

Since 1968, a total of 23 patients with multiple endocrine neoplasia type 2B (MEN 2B) have been identified in Japan. The mean age at diagnosis was 20.3 years (range, 6 to 39 years). All patients had neuromas and bumpy lips. All patients underwent thyroidectomy for medullary thyroid carcinoma (MTC). Ten of 23 patients had pheochromocytomas. One patient died of cerebral bleeding at the age of 43, 2 patients died of MTC at the age of 35 and 12. Five-, 10-, and 15-year survival rates were 100%, 92%, and 88%, respectively. The clinical course of MTC in MEN 2B in Japanese is not so aggressive when compared with about a 50% 10-year survival reported for Caucasians. Genetic analysis of 4 MTC and 1 pheochromocytoma from 4 patients with MEN 2B revealed no common changes in regard to loss of heterozygosity on 21 chromosomes or point mutations of the ras, Gs alpha, or p53 genes.


PCR-SSCP Screening of b-Amyloid Precursor Protein Mutations in Two Japanese Pedigrees with Familial Early Onset Alzheimer's Disease.


Rare Mutation of Gs Alpha Subunit Gene in Human Endocrine Tumors: Mutation Detection by Polymerase Chain Reaction-Primer-Introduced Restriction Analysis.

The Gs alpha (Gs alpha) gene can be activated to the putative oncogene gsp by specific point mutations at codons 201 or 227. Such mutations have been reported in growth hormone (GH)-secreting pituitary adenomas and thyroid tumors. To clarify the role of Gs alpha gene in human endocrine tumors, 197 tumors were screened for point mutations at codons 201 or 227 of the Gs alpha gene. Mutations were detected by primer-introduced restriction analysis (PIRA) of the polymerase chain reaction (PCR) product of genomic DNA. These Gs alpha mutations were present in 4 of 53 pituitary adenomas (4 of 43 GH-secreting adenomas; 1 of these 4 was a GH- and prolactin-secreting adenoma from a patient with familial multiple endocrine neoplasia Type 1), 4 of 66 thyroid tumors (4 of 30 papillary carcinomas), and 1 of 19 adrenocortical adenomas (1 of 6 aldosterone-secreting adenomas). In contrast, none of these Gs alpha mutations were detected in parathyroid tumors, endocrine pancreatic tumors, or pheochromocytomas. Gs alpha mutations at these two loci may play a role in the pathogenesis of a small population of GH-secreting pituitary adenomas, papillary thyroid carcinomas, and adrenocortical adenomas, but that they are not involved in the pathogenesis of other types of endocrine tumors.


Molecular Cloning of Rat Amidophosphoribosyltransferase.


Molecular Cloning of Human Amidophosphoribosyltransferase.

Biochemical and Biophysical Research Communications


Ras Mutations in Endocrine Tumors: Mutation Detection by PCR-SSCP.


Molecular and Genetic Analysis of a Compound Heterozygote for Dysprothrombinemia of Prothrombin Tokushima and Hypoprothrombinemia.


Two New Nonsense Mutations in Type Ia Antithrombin III Deficiency at Leu 140 and Arg 197.


Detection of a New Polymorphism of the Human Prothrombin (F2) Gene by Combination of PASA and Mutated Primer-Mediated PCR-RFLP.


Role of p53 Gene Mutations for Endocrine Tumorigenesis: Mutation Detection by Polymerase Chain Reaction-Single Strand Conformation Polymorphism.


Highly Polymorphic Region of the Human Prothrombin (F2) Gene.


Detection of a Single Base Substitution of the Gene for Prothrombin Tokushima: The Application of PCR-SSCP for the Genetic and Molecular Analysis of Dysprothrombinemia.

The genetic and molecular basis of a mutant prothrombin of 'prothrombin Tokushima' was studied by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP) analyses. The abnormal gene was detected by altered migration by PCR-SSCP and by the loss of an MspI site by PCR-RFLP. The gene for prothrombin Tokushima was shown to be inherited from the mother of the proband. Sequencing analysis using PCR-amplified genomic DNA clarified a substitution of thymine (T) for cytosine (C) at position 9,490, changing arginine (Arg) to tryptophan (Trp) at position 418 of the polypeptide chain. This point mutation is assumed to be the molecular basis of prothrombin Tokushima, firstly, because of the absence of distinct changes in Southern blot analysis of the proband's DNA (using a full-length human prothrombin cDNA as a probe), secondly, because it has the same molecular weight as the abnormal gene product, and, thirdly, because of the absence of other amino acid abnormalities in the proteolytic peptide-fragments. It is concluded that PCR-SSCP and PCR-RFLP were useful for detecting the abnormal gene and for directly diagnosing the carrier status of dysprothrombinemia. This is the first report of gene analysis of dysprothrombinemia.


Detection of Point Mutations by SSCP of PCR-Amplified DNA after Endonuclease Digestion.


Allele Loss on Chromosome 11 in a Pituitary Tumor from a Patient with Multiple Endocrine Neoplasia Type I.