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Biomedical subjects

M Itakura

Publications and source records attributed to M Itakura.

At least 109 records · Page 6Linked to original sources
PubMed1995

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.

Amidophosphoribosyltransferase↗
PubMed1995

Human calgizzarin; one colorectal cancer-related gene selected by a large scale random cDNA sequencing and northern blot analysis.

A cDNA library was constructed from COLO 205 and 1056 clones randomly selected from this library were partially sequenced. Two hundred and two (38.4%) out of 526 independent genes had more than 80% similarity to the genes reported in GenBank. In Northern blot analysis, 96 out of 98 genes were shown to be expressed at the same level in colon and lung carcinoma cell lines and control fibroblasts. Only two clones, including human synovial phospholipase A-2 and a homologue to rabbit calgizzarin, were expressed at different levels among these cell lines. The full sequence of human calgizzarin was determined and its expression was remarkably elevated in colorectal cancers compared with that in normal colorectal mucosa.

Amino Acid Sequence↗
PubMed1995

Acute onset of diabetic pathological changes in transgenic mice with human aldose reductase cDNA.

To investigate the role of human aldose reductase (hAR) in the pathogenesis of diabetic complications, we generated transgenic mice carrying hAR cDNA driven by the murine MHC class I molecule promoter (hAR-Tg). Northern and Western blot analyses and immunoassay of hAR revealed that both hAR mRNA and the protein were expressed in all tissues tested. Thrombosis in renal vessels and fibrinous deposits in Bowman's capsule were observed in 6-week-old hAR-Tg mice fed a normal diet. Ingestion of a 30% glucose diet for 5 days caused sorbitol concentrations in the liver, kidney, and muscle of hAR-Tg mice to be elevated significantly. Seven-week-old hAR-Tg mice fed a 20% galactose diet for 7 days developed cataracts and occlusion of the retinochoroidal vessels, in addition to pathological changes in the kidney. Despite an elevated aldose reductase level in hAR-Tg mice and their intake of an aldose diet, no histopathological changes were found in other tissues, including the brain, lungs, heart, thymus, spleen, intestine, liver, muscle, spinal cord, or sciatic nerve. Results suggest that target organs of diabetic complications, such as the kidney, lens, and retina are sensitive to damage associated with a high level of AR expression, but other organs are not; the susceptibility of each organ to diabetic complications is determined by not only hAR but also other factors.

Aldehyde Reductase↗
PubMed1995

Molecular cloning of a group of mouse pancreatic islet beta-cell-related genes by random cDNA sequencing.

To understand the molecular basis of glucose concentration-responsive insulin synthesis and secretion from pancreatic islet beta cells, a group of pancreatic islet beta-cell-related cDNAs was cloned. A pair of cDNA libraries was constructed from a mouse pancreatic islet beta-cell line of MIN6, which was cultured in either high glucose or low glucose media. By applying a random cDNA sequencing approach, 503 and 395 independent species were obtained from a total of 1,011 and 762 clones in the high glucose and low glucose library, respectively. The unknown genes comprised the majority of about 70% independent clones in both libraries. In Northern blot analysis, 311 (69.4%) of 448 independent clones showed positive signals within 72 h of autoradiographic exposure. Surprisingly, 150 (48.2%) out of 311 positive clones showed positive signals to MIN6 cells, but not to NIH/3T3 fibroblasts. The expression level of three unknown clones were glucose-concentration dependent. Combination of a random cDNA sequencing approach and Northern blot analysis is useful to obtain a large number of novel genes and islet beta-cell-related genes.

Animals↗
PubMed1995

The effect of non-enzymatic glycation on recombinant human aldose reductase.

It has been demonstrated that activation of aldose reductase (AR; EC 1.1.1.21) in diabetic tissues plays an important role in the pathogenesis of diabetic complications. In the present study, the effects of non-enzymatic glycation of recombinant human AR (rhAR) on enzyme activity and affinity for its substrate (glyceraldehyde), co-factor (NADPH) and inhibitors (ARI; Sorbinil, Tolrestat, AL-1576 and Statil) were examined. Although rhAR was successfully non-enzymatically glycated with HPLC-purified [3H]D-glucose, the Michaelis constant (Km) and catalytic efficiency (Kcat/Km) for glyceraldehyde, the Km for NADPH and the inhibitor constant (Ki) for ARI did not change. These results suggest that the mechanism of AR activation and its insensitivity to inhibition observed in diabetic tissues cannot be attributed to its non-enzymatic glycation.

Aldehyde Reductase↗
PubMed1995

Multiple fluorescence-based PCR-SSCP analysis with postlabeling.

Multiple fluorescence-based PCR single-strand conformation polymorphism (MF-PCR-SSCP) with postlabeling was developed. The target sequence was amplified by PCR using unlabeled primers. Free dNTPs were removed from the amplified products by ethanol precipitation. The dNTPs at the 3' ends of the amplified DNA fragments were exchanged with fluorescent dUTPs or ddNTPs using Klenow fragment of DNA polymerase I. The DNA fragments labeled with fluorescent dUTPs or ddNTPs were heat denatured and applied to a nondenaturing polyacrylamide gel set on an automated DNA sequencer with a gel temperature-controlling system. The image data were analyzed by the computer program Genescan 672. By use of MF-PCR-SSCP with postlabeling, seven different single base mutations of the human K-ras oncogene were detected even under one electrophoresis condition.

Automation↗
PubMed1995

Modulation of vascular natriuretic peptide receptor gene expression in hypertensive and obese hyperglycemic rats.

Receptors for natriuretic peptide (NP) consist of three subtypes: NP-A, NP-B, and NP-C. Recent studies in cultured aortic cells have suggested a phenotype-related switching of the vascular NP receptor from NP-A to NP-B. To ascertain the biological significance of the phenomenon in vivo, we developed a sensitive and reproducible ribonuclease protection assay and determined each receptor messenger RNA (mRNA) level in the vascular vessels of stroke-prone spontaneously hypertensive rats, deoxycorticosterone acetate-salt hypertensive rats, and genetically hyperglycemic. Wistar fatty rats and in cultured aortic smooth muscle cells. The aortic NP-A receptor mRNA level was significantly up-regulated in both types of hypertensive rats, whereas the NP-B receptor mRNA level did not show any significant change. Both NP-A and NP-B receptor mRNA levels were significantly up-regulated in Wistar fatty rats compared with the control values. There was no significant up-regulation of NP-A receptor mRNA in the inferior vena cava of the stroke-prone spontaneously hypertensive rats. Although the NP-A receptor was always the predominant subtype in rat aortic tissue, NP-B receptor was the predominant subtype in aortic smooth muscle cells in culture. These findings suggest that up-regulation of the NP-A receptor, but not the subtype switching, is the major modulation of receptor gene expression in both hypertensive and diabetic rats.

Animals↗
PubMed1995

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.

Adolescent↗
PubMed1994

Angiotensin converting enzyme inhibitor but not calcium blocker down-regulates gene expression of vascular natriuretic peptide receptor in hypertensive rats.

We have recently found that vascular natriuretic peptide (NP)-A receptor mRNA is upregulated in genetically hypertensive (SHR-SP/Izm) and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. In the present study, we examined the effects of antihypertensive treatments on aortic NP-A receptor mRNA expression in these hypertensive rats using ribonuclease protection assay. Oral administration of an angiotensin converting enzyme inhibitor, derapril, but not a calcium channel blocker, manidipine, produced a significant decrease of the NP-A receptor mRNA level after 4 weeks, while both antihypertensive agents showed similar hypotensive effects. Plasma renin was high in SHR-SP/Izm and low in DOCA-salt rats. These results suggest that the vascular renin-angiotensin system rather than the blood pressure has an important role in the regulation of the vascular NP-A receptor.

Animals↗
PubMed1994

Autocrine regulation of rat chondrocyte proliferation by natriuretic peptide C and its receptor, natriuretic peptide receptor-B.

Natriuretic peptide receptor-B (NPR-B) was identified in rat chondrocytes, and its physiological functions were investigated. Rat tissues, including the xiphoid cartilage, brain, lung, liver, adrenal gland, and kidney, were screened for NPR-B activity, which we assayed by receptor guanylate cyclase activity specifically stimulated by C-type natriuretic peptide (CNP), a known selective activator of NPR-B. Cartilage showed distinctly higher NPR-B activity. Furthermore, exposure of cultured rat chondrocytes to CNP (10(-6) M) resulted in a large increase in intracellular cGMP production (376 +/- 38 pmol/well), with threshold responses occurring between 10(-10) and 10(-9) M CNP. Atrial natriuretic peptide and brain natriuretic peptide also stimulated cGMP production in rat chondrocytes but with a potency that was at least 10 times less than that of CNP. Polymerase chain reaction analysis also demonstrated NPR-B gene expression in adult rat xiphisternum and cultured chondrocytes. These findings indicate that NPR-B is present in rat chondrocytes. In rat chondrocytes exposed to CNP, [3H]thymidine incorporation was inhibited in a dose-dependent manner (half-maximal response, 10(-11)M). However, much higher concentrations of atrial natriuretic peptide were required to induce the inhibition of thymidine incorporation. Interestingly, CNP-like immunoreactivity was detected in the conditioned medium from chondrocyte cultures. In addition, TGF-beta 1, a multifunctional cytokine, induced a marked increase in CNP secretion and CNP mRNA levels in chondrocytes. These results indicate that autocrine CNP inhibits mitogenesis in chondrocytes via NPR-B under the control of TGF-beta 1.

Animals↗
PubMed1994

Mutational analysis of disulfide bridges in the type C atrial natriuretic peptide receptor.

The type C receptor (ANP-C or NPR-C) for the natriuretic peptides was demonstrated, by site-directed mutagenesis, to have an immunoglobulin-like disulfide bonding pattern that is very similar to that of the cytokine receptor superfamily. The mature form of ANP-C has a disulfide-linked homodimeric structure and contains 5 conserved cysteine residues per subunit, all in the extracellular domain. To identify the cysteine residue involved in the dimerization and further to determine the intramolecular disulfide bridges and their functional roles, cysteine to serine mutations of the 5 cysteine residues were constructed. An analysis of the mutant receptors expressed in COS-1 cells by 125I-ANP binding assay and by measuring difference in their electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels indicated that 1) the first 4 cysteine residues are joined sequentially, forming the Cys104-Cys132 and Cys209-Cys257 loops of 29 and 49 residues, respectively; 2) the two disulfide-linked loops are essential for the ligand binding activity; 3) the 5th cysteine residue Cys469 is used in the formation of covalently linked dimers; and 4) the covalent association of the subunit through the disulfide bond involving Cys469 has no apparent influence on ligand-receptor interactions. The intramolecular disulfide bond Cys104-Cys132 was also confirmed by direct protein sequencing of tryptic fragments of purified ANP-C receptor. The secondary structural features revealed here will be useful in understanding the structure and function relationships of not only the dimeric ANP-C receptor, which has only a short cytoplasmic tail, but also the ANP-A (GC-A) and ANP-B (GC-B) receptor subtypes, which have a guanylate cyclase domain in their long cytoplasmic tail and have recently been shown to possess an oligomeric structure, since they have similarly spaced cysteine residues in their extracellular domains.

Amino Acid Sequence↗
PubMed1994

T-cassette ligation: a method for direct sequencing and cloning of PCR-amplified DNA fragments.

We describe a method to ligate a PCR-amplified DNA fragment with T-protruding cassettes, which have multiple sites for endonuclease, promoter sequences of T3 and T7, and annealing sites for the universal M13 forward and reverse primers. This method, which we named T-cassette ligation, substantially facilitated direct sequencing and subcloning of PCR products. Two T-cassettes with a protruding T at their 3' ends were obtained by annealing adaptor oligomers to XbaI- or XhoI-digested pBluescript. A DNA fragment amplified by the first PCR was ligated separately to one of the two T-cassettes. The second PCR was performed using a primer complementary to one of the two T-cassettes and one of the two primers used for the first PCR to amplify the ligation product in low abundance. The resultant two DNA fragments had the T3 and T7 promoter, and an annealing site for the universal M13 forward and reverse primer, respectively. These DNA fragments were applicable to direct sequencing. For the purpose of subcloning, the third PCR was carried out with two primers each complementary to each of two T-cassettes and two PCR-amplified DNAs by the second PCR, as templates. The PCR product of the third PCR had multiple sites of the pBluescript polycloning site at both ends, facilitating its subcloning into pBluescript.

Base Sequence↗
PubMed1994

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.

We described a method for PCR amplification of unknown flanking genomic DNA fragments. This method is a combination of PCR with "end-trimming method" and "cassettes and cassette-primers method". In this method, genomic DNA was digested with three different groups of restriction enzymes. DNA in group 1 was digested with BamHI, BglII, FbaI, or MboI. DNA in group 2 was digested with BlnI, NheI, SpeI, or XbaI. DNA in group 3 was digested with SalI or XhoI. Digested DNA in each group was end-trimmed with Klenow fragment of DNA polymerase I in the presence of only one dNTP; dGTP, dCTP, and dTTP for group 1, 2, and 3, respectively. The synthesized cassettes, C1, C2, and C3, had 5'protruding sequences of 5'-ATC-3',5'-TAG-3', and 5'-CGA-3', respectively. Each compatible cassette was ligated to the end-trimmed DNAs in group 1-3, respectively. Nested PCR was then performed using an end-trimmed and cassette-ligated DNA as a template. Primers annealing to known sequences and cassettes were used for the nested PCR. The amplified DNA fragments were electrophoresed on a polyacrylamide gel and purified. The sequences of the DNA fragments were determined after cloning into pBluescript.

Amidophosphoribosyltransferase↗
PubMed1994

p53 gene mutations in human skin cancers and precancerous lesions: comparison with immunohistochemical analysis.

Mutations of exons 3 through 9 of the p53 gene in skin lesions were screened in 23 cases of squamous cell carcinoma (SCC), 25 cases of basal cell carcinoma (BCC), two cases of Bowen's disease, 10 cases of solar keratosis, and five cases of keratoacanthoma by polymerase chain reaction--single strand conformation polymorphism analysis. Mutations of the p53 gene were detected in seven of 23 SCCs (30%), three of 25 BCCs (12%), and none in all cases of Bowen's disease, solar keratosis, or keratoacanthoma. Of 23 cases of SCC, mutations were detected in four of 15 SCCs (27%) that originated in the sunlight-exposed skin region, in two of three SCCs (67%) that originated in the scar tissue, and in one of three SCCs (33%) that originated in radiation dermatitis. Mutations of C-->T transition predominated in SCC and BCC that originated in the sunlight-exposed skin region. Mutations of C-->A or CC-->AT observed in tumors that originated in the predisposed conditions, presumably unrelated to UV light, are different from those found in UV light-related SCC or BCC. Twelve cases of SCC were comparatively analyzed with the immunohistochemical staining with anti-p53 antibody. Two of four cases with positive staining had missense mutations, and three of eight cases with negative staining had nonsense mutations. Based on these findings, immunohistochemical results do not necessarily mean the presence or absence of p53 gene mutations in skin tumors, and sequence analysis is essential for determining whether the gene is mutated.

Aged↗