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Biomedical subjects

H Iwahana

Publications and source records attributed to H Iwahana.

At least 19 recordsLinked to original sources

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.

Amino Acid Sequence

Spherical parasporal inclusions of the lepidoptera-specific and coleoptera-specific Bacillus thuringiensis strains: a comparative electron microscopic study.

Four Lepidoptera-specific Bacillus thuringiensis strains that belong to the four H serogroups (serovars sumiyoshiensis, fukuokaensis, darmstadiensis, and japonensis) and a Coleoptera (Scarabaeidae)-specific strain belonging to serovar japonensis were examined for comparative ultrastructure of spherical parasporal inclusions. The prominent feature of the inclusions of the Lepidoptera-specific strains was the existence of thick, highly electron-dense envelopes surrounding a homogeneous protein matrix. The envelopes were 15.0-66.7 nm thick and consisted of 5-12 layers of membrane. This is also the case with inclusions of a Coleoptera-specific strain. The ultrastructure of inclusions from the five strains was in marked contrast to that of the bipyramidal parasporal inclusions produced by a Lepidoptera-specific serovar sotto strain.

Animals

Bacillus thuringiensis Cry1Aa toxin-binding region of Bombyx mori aminopeptidase N.

The Bacillus thuringiensis Cry1Aa toxin-binding region of Bombyx mori aminopeptidase N (APN) was analyzed, to better understand the molecular mechanism of susceptibility to the toxin and the development of resistance in insects. APN was digested with lysylendopeptidase and the ability of the resulting fragments to bind to Cry1Aa and 1Ac toxins was examined. The binding abilities of the two toxins to these fragments were different. The Cry1Aa toxin bound to the fragment containing 40-Asp to 313-Lys, suggesting that the Cry1Aa toxin-binding site is located in the region between 40-Asp and 313-Lys, while Cry1Ac toxin bound exclusively to mature APN. Next, recombinant APN of various lengths was expressed in Escherichia coli cells and its ability to bind to Cry1Aa toxin was examined. The results localized the Cry1Aa toxin binding to the region between 135-Ile and 198-Pro.

Amino Acid Sequence

Presence and expression of a novel variant form of ST2 gene product in human leukemic cell line UT-7/GM.

A novel variant cDNA from the human ST2 gene other than ST2 or ST2L was identified and tentatively named ST2V. Alternative splicing inserts a new exon which leads to a change in the C-terminal portion of ST2, causing it to gain a hydrophobic tail instead of losing the third immunoglobulin-like domain. ST2V is expressed in human leukemic cell line UT-7 and its sublines UT-7/GM, UT-7/EPO, and UT-7/TPO, in addition to human helper T cell line 5C10. The amount of ST2V mRNA is greatly diminished when UT-7/GM cells are induced to differentiate into either erythroblastic or megakaryoblastic phenotypes. The possible roles of the ST2V in growth and differentiation are intriguing.

Alternative Splicing

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.

Animals

The lipopolysaccharide-binding protein participating in hemocyte nodule formation in the silkworm Bombyx mori is a novel member of the C-type lectin superfamily with two different tandem carbohydrate-recognition domains.

We recently isolated and characterized the lipopolysaccharide (LPS)-binding protein, BmLBP, from the larval hemolymph of the silkworm Bombyx mori. BmLBP is a pattern recognition molecule that recognizes the lipid A portion of LPS and participates in a cellular defense reaction. This paper describes the cDNA cloning of BmLBP. The deduced amino acid sequence of BmLBP revealed that BmLBP is a novel member of the C-type lectin superfamily with a unique structural feature that consists of two different carbohydrate-recognition domains in tandem, a short and a long form.

Acute-Phase Proteins

cDNA cloning and expression of Bacillus thuringiensis Cry1Aa toxin binding 120 kDa aminopeptidase N from Bombyx mori.

Bacillus thuringiensis Cry1Aa toxin binds to a 120 kDa putative receptor protein in the Bombyx mori midgut. Recently, this protein was purified and identified as glycosyl-phosphatidylinositol (GPI) anchored aminopeptidase N (APN). In this study, a full-length cDNA thought to encode this 120 kDa APN was isolated and sequenced. It has a 2958 bp ORF encoding 986 amino acids. In the deduced amino acid sequence, we identified GPI-anchor and zinc-metallopeptidase signals, which are the same as those of APNs of other insects that are reported to be putative Cry1 toxin receptors. The B. mori APN amino acid sequence also has a high similarity with those of the other APNs. Subsequently, the recombinant APN was expressed by Escherichia coli and its Cry1Aa toxin binding ability was analyzed. Ligand blotting showed that Cry1Aa toxin bound to the recombinant APN.

Amino Acid Sequence

Acaloleptins A: inducible antibacterial peptides from larvae of the beetle, Acalolepta luxuriosa.

We purified and characterized three structurally related antibacterial peptides with a molecular mass of 8 kDa (acaloleptins A1, A2, and A3) from the hemolymph of immunized larvae of the Udo longicorn beetle, Acalolepta luxuriosa. These peptides have the same 6 N-terminal amino acid residues and show potent antibacterial activity against some Gram-negative bacteria. The three peptides are thought to be isoforms. Reverse phase HPLC analysis of the hemolymph of immunized and naive larvae showed that acaloleptins A1, A2, and A3 were inducible and suggested that all three peptides were produced in a single insect. We determined the complete amino acid sequence of acaloleptin A1: Acaloleptin A1 consists of 71 amino acid residues and shares significant sequence similarity with coleoptericin and holotricin 2, which were isolated from other coleopteran insects. Furthermore, the 29 C-terminal residues of acaloleptin A1 had 40% identity with the 30 C-terminal residues of hymenoptaecin found in honeybees. Arch. Insect Biochem.

Amino Acid Sequence

Binding of phylogenetically distant Bacillus thuringiensis cry toxins to a Bombyx mori aminopeptidase N suggests importance of Cry toxin's conserved structure in receptor binding.

We investigated the binding proteins for three Cry toxins, Cry1Aa, Cry1Ac, and the phylogenetically distant Cry9Da, in the midgut cell membrane of the silkworm. In a ligand blot experiment, Cry1Ac and Cry9Da bound to the same 120-kDa aminopeptidase N (APN) as Cry1Aa. A competition experiment with the ligand blot indicated that the three toxins share the same binding site on several proteins. The values of the dissociation constants of the three Cry toxins and 120-kDa APN are as low as the case of other Cry toxins and receptors. These results suggest that distantly related Cry toxins bind to the same site on the same proteins, especially with APN. We propose that the conserved structure in these three toxins includes the receptor-binding site.

Amino Acid Sequence

Different promoter usage and multiple transcription initiation sites of the interleukin-1 receptor-related human ST2 gene in UT-7 and TM12 cells.

The ST2 gene encodes receptor-like molecules that are very similar to the type I interleukin-1 receptor. Two distinct types of the ST2 gene products, ST2 (a soluble secreted form) and ST2L (a transmembrane form) are produced by alternative splicing. Here we demonstrate that the human ST2 gene has two alternative promoters followed by distinct noncoding first exons, which are located more than 8 kb apart and are spliced to the common exon 2 containing the translation initiation site. Within 1001 bp upstream of the transcription initiation site of the cloned distal promoter, there are four GATA-1. The main promoter used for the expression of the ST2 gene in UT-7, a human leukaemic cell line, is distinct from that in TM12, a human fibroblastic cell line. Although UT-7 cells use both distal and proximal promoters, the distal promoter is used dominantly for expression of both ST2 and ST2L mRNA. On the other hand, almost all transcription in TM12 cells starts from the proximal promoter. These results contrast with those of former studies on the rat system, in which ST2 and ST2L mRNA were generated by use of the proximal and distal promoters, respectively. Furthermore, UT-7 cells use multiple transcription initiation sites in both the proximal and distal promoters, whereas the transcription of the ST2 gene in TM12 cells starts at a unique site. Intriguingly, these results suggest that ST2 and ST2L proteins have distinct functions in different cells within different biological systems, such as those of growth control, differentiation and immunological responses.

Alternative Splicing

Opposite regulation of the expression of cyclin-dependent kinase inhibitors during contact inhibition.

Contact inhibition is a well-known phenomenon, but the details of its mechanism are poorly understood. Recently, cyclin-dependent kinase inhibitors have been studied intensively with respect to their regulatory role in the cell cycle, and of them, p27(Kip1) is particularly involved in contact inhibition. p27(Kip1) is believed to be regulated primarily through posttranscriptional mechanisms. We now report that cyclin-dependent kinase inhibitors, including p27, are regulated differently at the mRNA level during contact inhibition in murine BALB/c-3T3 fibroblasts. The mRNA expression of p15, p16, and p27 was up-regulated as the cell density increased, but, on the contrary, the mRNA level of p21(Cip1/WAF1/Sdi1) markedly decreased when the cells became confluent. The protein levels of these genes were regulated in the same way as their mRNA levels, and cyclin-dependent kinase-2 activity was markedly inhibited on density-mediated growth arrest of the cells. These results indicate that the regulation of mRNA expression of cyclin-dependent kinase inhibitors appears to contribute to their protein levels and to the arrest of cell growth through contact inhibition.

3T3 Cells

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.

Adenocarcinoma

Familial isolated primary hyperparathyroidism with parathyroid carcinomas: clinical and molecular features.

OBJECTIVE: 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. DESIGN: DNA was extracted from tumours resected at surgery. Tumours were examined for loss of heterozygosity (LOH) with microsatellite polymorphic markers. PATIENTS: 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. RESULTS: 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. CONCLUSION: 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.

Adenoma

Lipopolysaccharide-binding proteins and their involvement in the bacterial clearance from the hemolymph of the silkworm Bombyx mori.

Proteins having the ability to bind to Escherichia coli K12W3110 (rough (R) mutant) were isolated and purified by affinity precipitation from the larval hemolymph of the silkworm Bombyx mori. These proteins were found to consist of two components with molecular masses of 43 kDa and 40 kDa by SDS/PAGE. They bound to all E. coli R mutants (Ra, Rb1, Rc, Rd1 and Re) and Salmonella minnesota R mutants. However, they did not bind to smooth types of the above bacteria. They bound to both lipopolysaccharide(LPS)-coated and lipid-A-coated microtiter plates and have similar dissociation constants for LPS and lipid A. This indicates that the binding proteins recognize the lipid A portion of LPS and thus, we have named these proteins BmLBP (B. mori LPS-binding proteins). We also found that BmLBP participated in the clearance of E. coli cells injected into the body cavity of the silkworm.

Acute-Phase Proteins

p53 gene mutation in N-butyl-N-(4-hydroxybutyl)nitrosamine-induced urinary bladder tumors and N-methyl-N-nitrosourea-induced colon tumors of rats.

We analyzed p53 mutations in 17 N-butyl-N-(4-hydroxybutyl) nitrosamine-induced bladder transitional cell carcinomas (TCCs) with or without areas of squamous cell carcinoma (SCC) of Long-Evans Cinnamon (LEC) and F344 rats, and in 7 N-methyl-N-nitrosourea-induced colon adenocarcinomas of LEC rats by polymerase chain reaction-single strand conformation polymorphism analysis and DNA sequencing. Of these bladder tumors, one TCC with moderately differentiated SCC had a T to G transversion mutation at codon 141, leading to a Val to Gly amino acid change. No p53 mutation was found in colon adenocarcinomas. Thus a p53 gene mutation seems infrequent in these rat bladder and colon carcinogenesis models even in the late stage.

Animals

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 the de 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 the de 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.

Amidophosphoribosyltransferase

Aminopeptidase N from Bombyx mori as a candidate for the receptor of Bacillus thuringiensis Cry1Aa toxin.

Cry1Aa toxin-binding proteins from the midgut brush border membrane vesicles of Bombyx mori, a toxin-susceptible silkworm, were analyzed to find candidates for the toxin receptors. Ligand blotting showed that Cry1Aa toxin bound to a 120-kDa protein. A part of the 120-kDa protein was solubilized from the membrane vesicles with phosphatidylinositol-specific phospholipase C, resulting in a 110-kDa protein which therefore may be linked to a glycosyl-phosphatidylinositol anchor. The 120-kDa and 110-kDa Cry1Aa toxin-binding proteins were solubilized with detergent or pohosphatidylinositol-specific phospholipase C, respectively, and purified using anion-exchange chromatography. Scatchard plot analysis for the specific binding of purified 110-kDa protein to Cry1Aa toxin yielded a Kd value of 7.6 nM, which was similar to that for the binding of intact brush border membrane vesicles to the toxin. N-terminal and internal amino acid sequences of the 120-kDa and 110-kDa proteins showed high degrees of similarity to those of aminopeptidase N, a putative Cry1Ac toxin receptor, reported in Manduca sexta and Heliothis virescens. On this basis, the 120-kDa Cry1Aa toxin-binding protein from B. mori was identified as a member of the aminopeptidase family.

Amino Acid Sequence