Hiroyuki Iwahana

ras Mutations in Endocrine Tumors: Mutation Detection by Polymerase Chain Reaction-Single Strand Conformation Polymorphism

To elucidate the molecular basis for endocrine tumorigenesis, ras mutations in human endocrine tumors were analyzed using polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) analysis. Mutations of the H‐, K‐, N‐ras genes were examined in genomic DNAs from 169 successfully amplified primary endocrine tumors out of 189 samples. Four out of 24 thyroid follicular adenomas analyzed contained mutated N‐ras codon 61, and one contained the mutated H‐ras codon 61. One of the 19 pheochromocytomas revealed mutation of the H‐ras codon 13. No mutations of the ras gene were detected in pituitary adenomas, parathyroid tumors, thyroid cancers, endocrine pancreatic tumors, and adrenocortical tumors. Based on these findings we conclude that activation of the ras gene may play a role in the tumorigenesis of a limited number of thyroid follicular adenomas and pheochromocytomas, and that mutation of the ras gene is not frequent in other human endocrine tumors.

Construction of ELISA system to quantify human ST2 protein in sera of patients.

The human ST2 gene can be specifically induced by growth stimulation in fibroblastic cells, and can also be induced by antigen stimulation in Th2 cells. The gene encodes a soluble secreted protein, ST2, and a transmembrane protein, ST2L, which are closely related to the interleukin-1 receptor. To gain insight into the biological roles of the ST2 gene, three monoclonal antibodies (MAbs) against human ST2 gene products were obtained. To obtain these antibodies, immunization was carried out using two different immunogens: purified soluble human ST2 protein (hST2), and COS7 cells, which express the extracellular portion of human ST2L. 2A5 and FB9 MAbs were derived from the immunization with soluble hST2, and HB12 was derived from the COS7 cell immunization. All three antibodies were shown to detect native forms of the human ST2 gene products by immunoprecipitation, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). In the competitive ELISA using biotinylated and nonlabelled MAbs, neither FB9 nor HB12 affected the binding of 2A5 to ST2 gene products. Based on this result, we constructed a sandwich ELISA system using 2A5 and FB9 to measure the concentration of soluble hST2 in sera. The ELISA, combined with the flow cytometry using these antibodies, will be a useful tool for elucidating the functions of human ST2 gene products in individuals.

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.

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

Factors controlling relative flux rates of thede novo and salvage pathways of purine nucleotide biosynthesis during animal cell growth are not fully understood. To examine the relative role of each pathway for cell growth, three cell lines including CHO K1 (a wild-type Chinese hamster ovary fibroblast cell line), CHO ade −A (an auxotrophic cell line deficient of amidophosphoribosyltransferase (ATase), a presumed rate-limiting enzyme of the de novo pathway), and CHO ade −A transfected with human ATase cDNA (−A+hATase) resulting in 30–350% of the ATase activity of CHO K1, were cultured in purine-rich or purine-free media. Based on the enzyme activities of ATase and hypoxanthine phosphoribosyltransferase, the metabolic rate of the de novo and salvage pathways, the rate of cell growth (growth rate) in three cell lines under various culture conditions, and the effect of hypoxanthine infusion on the metabolic rate of thede novo pathway in rat liver, we concluded the following. 1) In −A+hATase transfectants, ATase activity limits the rate of the de novo pathway, which is closely linked with the growth rate. 2) Purine nucleotides are synthesized preferentially by the salvage pathway as long as hypoxanthine, the most essential source of purine salvage, can be utilized, which was confirmed in rat liver in vivo by hypoxanthine infusion. The preferential usage of the salvage pathway results in sparing the energy expenditure required for de novo synthesis. 3) The regulatory capacity of the de novo pathway (about 200%) was larger than that of the salvage pathway (about 20%) with constant hypoxanthine phosphoribosyltransferase activity.

A novel splice variant of mouse interleukin-1-receptor-associated kinase-1 (IRAK-1) activates nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK)

Interleukin-1 (IL-1)-receptor-associated kinase (IRAK) is an indispensable signalling molecule for host-defence responses initiated by a variety of ligands that bind to members of the Toll/IL-1 receptor family. Here we report a novel splice variant of mouse IRAK-1, IRAK-1-S, which is generated by utilizing a new splicing acceptor site within exon 12. IRAK-1-S cDNA is shorter than the originally reported IRAK-1 (IRAK-1-W) cDNA by 271 nucleotides, and the subsequent frameshift causes a premature termination of translation after 23 amino acids, which are unique to the IRAK-1-S protein. To elucidate the physiological function of IRAK-1-S, we overexpressed it in 293T cells and studied the effects on the IL-1 signalling cascade. As it lacks the C-terminal region of IRAK-1-W that has been reported to contain the TRAF6 (tumour necrosis factor receptor-associated factor 6) binding domain, IRAK-1-S was unable to bind TRAF6 protein, which is a proposed downstream signalling molecule. However, IRAK-1-S overexpressed in 293T cells induced constitutive activation of nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK) independent of stimulation by IL-1, as did IRAK-1-W. To clarify the mechanism of NF-kappaB activation by IRAK-1-S in the absence of binding to TRAF6, we demonstrated that IRAK-1-S binds to IRAK-1-W through its death domain; the findings suggested that overexpressed IRAK-1-S may bind endogenous IRAK-1-W and activate TRAF6 through IRAK-1-W. These results also indicate that this novel variant may play roles in the activation of NF-kappaB and JNK by IL-1 and other ligands whose signal transduction is dependent on IRAK-1 under physiological conditions.

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.

Elevation of ST2 protein levels in cerebrospinal fluid following subarachnoid hemorrhage

Objective –  To study the mode of appearance of ST2 in cerebrospinal fluid (CSF) after subarachnoid hemorrhage (SAH).

Radioimmunoassay of an analog of luteinizing hormone-releasing hormone, [D-Ser(tBu)]6des-Gly-NH210 ethylamide (Buserelin)

A sensitive and specific radioimmunoassay is described for plasma and urinary levels of [D-Ser(tBu)]6des-Gly-NH2(10) ethylamide (buserelin). No appreciable cross-reaction (less than 0.05%) was observed with LH-RH and its analogs other than buserelin fragments (1.6-45%). The sensitivity was 3 pg per tube. At buserelin concentrations of 125, 250 and 500 pg/ml, the intra- and inter-assay coefficients of variation were 7.9, 10.0 and 10.0%, and 19.0, 7.8 and 6.8% respectively. Recovery of buserelin added to plasma was quantitative (62.5 pg/ml, 101.6%; 125 pg/ml, 76.8% and 250 pg/ml, 63.4%). A dose of 5 micrograms buserelin injected subcutaneously into 5 normal male adults, reached a peak plasma level in 45 min (mean value 119.3 +/- 47.3 pg/ml) and remained detectable for at least 4 h. The half disappearance time was 118.8 +/- 26.0 min. Between 9 and 16% of the administered dose was excreted in the urine within 24 h. Buserelin could also be detected in the plasma after intranasal administration of doses of 150, 300 and 450 micrograms. There was a significant difference in the area under the curve (AUC) for plasma levels after subcutaneous injection of 5 micrograms and intranasal administration of 150 micrograms, but not between the AUC values after the three intranasal doses. These results indicate that this method for radioimmunoassay of buserelin is suitable for analyzing the pharmacokinetics and bioavailability of buserelin in man.

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.

Molecular cloning of a human cDNA for the 41-kDa phosphoribosylpyrophosphate synthetase-associated protein

A human cDNA encoding 41-kDa phosphoribosylpyrophosphate (PRPP) synthetase (PRS)-associated protein (PAP41) was cloned from two expressed sequence tag (EST) clones having the nucleotide similarity of 61.5 and 70.0% to human PAP39 cDNA. The predicted open reading frame of 1107 base pairs (bp) has the nucleotide identity of 91.8% to rat PAP41 and encodes a protein of 369 amino acids with a calculated molecular weight (MW) of 40,925. The deduced amino acid sequence exhibits the 98.9% identity to rat PAP41 and 72.2, 50.6, and 50.0% identity with human PAP39, PRS I, and PRS II, respectively, but lacks the PRPP binding site. Southern blot analysis suggested that the PAP41 gene exists as a single copy in the human genome. The single PAP41 mRNA of about 2.1 kb was shown to be present in five human cell lines by Northern blot analysis.