Biomedical subjects

K Yoshimoto

Publications and source records attributed to K Yoshimoto.

At least 127 records · Page 7Linked to original sources

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

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

Human heart-type cytoplasmic fatty acid-binding protein as an indicator of acute myocardial infarction.

Human heart-type cytoplasmic fatty acid-binding protein (HH-FABPc) has been proposed as an early biochemical indicator of acute myocardial infarction (AMI). However, skeletal muscles also contain HH-FABPc identical to that found in the heart. Before HH-FABPc can be clinically employed as an indicator of AMI, its content in various tissues other than the heart must be known. Accordingly, we measured the HH-FABPc content of various human muscles and organs, using a sandwich enzyme-linked immunosorbent assay (ELISA) for HH-FABPc. HH-FABPc was abundant in the ventricles (0.46 mg/g wet weight and 1.5% of the cytoplasmic protein in the left ventricle), while the atria contained slightly less HH-FABPc (0.25 mg/g wet weight and 0.7% of the cytoplasmic protein in the left atrium). Of the skeletal muscles tested, the diaphragm contained about one-quarter of the HH-FABPc content of the heart, but other skeletal muscles contained very low levels of this protein. Other than the muscles, the kidneys contained less than one-tenth of the HH-FABPc in the heart, and negligible amounts were found in the liver and small intestine. The distribution of HH-FABPc in the heart and skeletal muscles was comparable to that of cardiac-specific creatine kinase (CK-MB) activity, and was inverse to the distribution of myoglobin. The plasma myoglobin/HH-FABPc ratio, determined in patients with AMI and those without AMI, closely reflected that in the heart and skeletal muscles. These findings indicate that HH-FABPc may be useful as a specific indicator of AMI, and the plasma myoglobin/HH-FABPc ratio could provide valuable information for the diagnosis of AMI.

Adult↗

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

Frequent p53 accumulation in the chronically sun-exposed epidermis and clonal expansion of p53 mutant cells in the epidermis adjacent to basal cell carcinoma.

p53 expression was studied immunohistochemically to identify a precursor lesion of basal cell carcinoma (BCC) in the epidermis adjacent to BCC. With two different anti-p53 antibodies of CM1 and DO7, p53 expression was frequently detected in the epidermis adjacent to BCCs arising on the face and in the normal epidermis with usual sun exposure. In the epidermis adjacent to BCC, stained cells were occasionally clustered in a small area, but no cluster was found in the normal epidermis with usual sun exposure. The expression was less frequent in the normal epidermis with rare sun exposure. Ten cases of normal skin with usual sun exposure, showing CM1 staining in the epidermis, were screened for p53 gene mutations with polymerase chain reaction-single-strand conformation polymorphism analysis using DNAs obtained from the epidermis. No mutation was detected in exons 2 to 10 of the p53 gene in these 10 cases. The epidermis flanking three BCCs that was stained with CM1, on the other hand, carried a missense mutation of C to G transversion at a dipyrimidine site of codon 249. This alteration replaced arginine with threonine. The mutation of codon 249 was not detected in the three BCCs. Our results first suggest that ultraviolet light irradiating the skin in a daily life induces p53 accumulation in the epidermis and secondly that the frequent clonal expansion of p53 mutant cells occurs in the epidermis adjacent to BCCs. This clonal expansion of mutant p53 may provide a molecular basis for high risk of developing subsequent new skin cancers in patients with BCC.

Adolescent↗

PubMed1995

Identification of human CYP isoforms involved in the metabolism of propranolol enantiomers--N-desisopropylation is mediated mainly by CYP1A2.

1. Studies using human liver microsomes and six recombinant human CYP isoforms (i.e. CYP1A2, 2A6, 2B6, 2D6, 2E1 and 3A4) were performed to identify the cytochrome P450 (CYP) isoform(s) involved in the ring 4-hydroxylation and side-chain N-desisopropylation of propranolol enantiomers in humans. 2. alpha-Naphthoflavone and 7-ethoxyresorufin (selective inhibitors of CYP1A1/2) inhibited the N-desisopropylation of R- and S-propranolol by human liver microsomes by 20 and 40%, respectively, while quinidine (a selective inhibitor of CYP2D6) abolished the 4-hydroxylation of both propranolol enantiomers almost completely. In contrast, sulphaphenazole (CYP2C8/9 inhibitor), S-mephenytoin (CYP2C19 inhibitor), troleandomycin (CYP3A3/4 inhibitor) and diethyldithiocarbamate (CYP2E1 inhibitor) elicited only weak inhibitory effects on propranolol metabolism via the two measured metabolic pathways. 3. Significant (P < 0.01) correlations were observed between the microsomal N-desisopropylation of both propranolol enantiomers and that for the O-deethylation of phenacetin among the 11 different human liver microsome samples (r = 0.98 and 0.77 for R- and S-propranolol, respectively). A marginally significant (r = 0.60, P congruent to 0.05) correlation was also observed between N-desisopropylation of S-, but not of R-propranolol and the 4'-hydroxylation of S-mephenytoin. No significant correlations were observed between the N-desisopropylation of propranolol enantiomers and the 2-hydroxylation of desipramine, the hydroxylation of tolbutamide or the 6 beta-hydroxylation of testosterone. 4. Significant (P < 0.01) correlations were observed between the microsomal 4-hydroxylation of R- and S-propranolol and the 2-hydroxylation of desipramine (r = 0.85 and 0.98, respectively). A weak (r = 0.66), albeit significant (P < 0.05) correlation was observed between the 4-hydroxylation of R-, but not of S-propranolol and the hydroxylation of tolbutamide. No significant correlations were observed between the 4-hydroxylation of propranolol enantiomers and the oxidation of other substrates for CYP1A2, 2C19, and 3A3/4. 5. Recombinant human CYP1A2 and CYP2D6 exhibited comparable catalytic activity with respect to the N-desisopropylation of both propranolol enantiomers; only expressed CYP2D6 exhibited a marked catalytic activity with respect to the 4-hydroxylation of both propranolol enantiomers.(ABSTRACT TRUNCATED AT 400 WORDS)

Aged↗

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↗

PubMed1995

Two different pituitary adenomas in a patient with multiple endocrine neoplasia type 1 associated 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.

Acromegaly↗

PubMed1995

Mutations in the cysteine-rich region of the RET proto-oncogene in patients diagnosed as having sporadic medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) and pheochromocytoma appear in either a sporadic or a hereditary form as components of multiple endocrine neoplasia (MEN). Many germline mutations of the RET proto-oncogene have been reported in patients with MEN 2A and 2B, and familial MTC (FMTC). To elucidate the etiological roles in tumorigenesis of sporadic MTCs and pheochromocytomas, mutations in the cysteine-rich region of the RET proto-oncogene were analyzed by using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Exons 10 and 11 were studied in genomic DNAs from 3 clinically apparent sporadic MTCs, MTCs and pheochromocytomas from 2 patients with MEN 2A, 1 with FMTC, 4 with MEN 2B, 3 with neurofibromatosis type 1 (NF1), 12 sporadic pheochromocytomas and an MTC cell line, TT. All tumors from two patients with MEN 2A and one patient with FMTC had mutations at codon 618 and 634 as well as their leukocytes, reflecting their germline mutations. In this region, no mutations were detected in any tumors from patients with MEN 2B and NF1, and sporadic pheochromocytomas. But mutations were detected and identified in 3 clinically apparent sporadic MTCs and TT cells. A 6 base pair (bp) deletion causing the loss of a cysteine residue at codon 634 and a mutation causing substitution from cysteine to tyrosine at codon 634 were detected in 2 sporadic MTCs as somatic events. In a female patient diagnosed as having sporadic MTC, a mutation at codon 618 was detected not only in tumor tissues, but also in her leukocytes, suggesting a germline mutation of the RET proto-oncogene.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Gland Neoplasms↗

PubMed1995

The effects of neurotoxins 6-hydroxydopamine and 5,7-dihydroxytryptamine into the rat nucleus accumbens on the alcohol drinking behavior.

To elucidate the relationship between drinking behavior and brain monoamines in the brain rewarding system, the effects of injection of 6-hydroxydopamine (6-OHDA) and 5,7-dihydroxytryptamine (5,7-DHT) into the rat nucleus accumbens (ACC), which would affect dopamine- and indoleamine-containing neurons, respectively, on the rat alcohol preference score were examined. The 6-OHDA (4 micrograms/side)-treated rats showed a higher alcohol preference score for 2 weeks following the treatment, and a lowered dopamine (DA) level in the ACC and midbrain after ACC injection of 6-OHDA. On the other hand, although the serotonin (5-HT) and DA levels in the midbrain and 5-HT level in ACC were lowered after the injection of 5,7-DHT, the alcohol preference score was not significantly changed in the 5,7-DHT (4 micrograms/side)-treated rats. Taken together, these findings suggest that the alcohol drinking behavior is more influenced by the ACC DA activity than 5-HT activity. Changes in alcohol drinking behavior might be related to the compensatory mechanism for the rat to restore the original rewarding properties.

5,7-Dihydroxytryptamine↗

PubMed1995

[Parathyroid cells: structure of Ca2+ sensing receptor].

Using expression in Xenopus oocytes, Brown et al. cloned a bovine parathyroid Ca2+ sensing receptor (BoPCaR1). The 1,085 amino acid membrane protein is included in a class of putative seven transmembrane-spanning structures that activate the phosphoinositol pathway through G proteins. Receptor activation presumably elevates intracellular Ca2+ to inhibit secretion of PTH. BoPCaR1 has a big extracellular domain at the amino terminus. BoPCaR1 is most similar to the metabotropic glutamate receptor. BoPCaR1 mRNA is found in cells that have well known Ca2+ sensing function. Inheritance of one inactive Ca2+ sensing receptor gene causes familial hypocalciuric hypercalcemia (FHH) and autosomal dominant hypocalcemia. Mutations in the extracellular domain and in a portion of the third intracellular loop domain decrease the receptor's sensitivity, causing FHH. Extracellular domain mutations increase the receptor's activity at low Ca2+ concentration to cause autosomal familial hypocalcemia.

Animals↗

PubMed1995

[Familial pituitary tumors].

Familial pituitary tumors are relatively rare. Most commonly, they occur as a part of multiple endocrine neoplasia type 1 (MEN 1). However, familial pituitary adenomas unrelated MEN 1 (familial pituitary adenomas) are extremely rare. In review of MEN 1 in Japan, 60% of the patients with MEN 1 had pituitary tumors. Only 45 cases of familial pituitary adenomas have been reported from 20 families. In our review of familial pituitary adenomas, 30 (67%) of 45 reported cases are acromegaly or gigantism. This incidence is much higher than 28% in MEN 1 patients with pituitary tumors. Allelic deletions at 11q13 were identified in MEN 1 associated pituitary adenomas and familial pituitary adenomas in two gigantism brothers.

Acromegaly↗

PubMed1994

Famotidine-associated central nervous system reactions and plasma and cerebrospinal drug concentrations in neurosurgical patients with renal failure.

Central nervous system toxicity of H2-receptor antagonists has rarely been confirmed by the respective elevated cerebrospinal fluid drug concentrations. We observed two hemodialyzed neurosurgical patients in whom mental deterioration and convulsions developed after intravenous famotidine therapy (10 and 40 mg/day). Their cerebrospinal fluid drug concentrations were grossly elevated (i.e., 160 and 249 ng/ml) compared with those obtained from three other hemodialyzed neurosurgical patients who exhibited no central nervous system reactions (i.e., 47 to 85 ng/ml). In addition, the mean cerebrospinal fluid/plasma drug concentration ratio obtained from these five neurosurgical patients with renal failure (i.e., 0.46) and that from 10 other neurosurgical patients with normal renal function (i.e., 0.41) were about four times greater than that previously reported from non-neurosurgical patients with normal renal function (i.e., 0.12). Our observation suggests that patients with not only renal dysfunction but also following neurosurgical operations have an excessive accumulation of famotidine in the central nervous system and are more susceptible to the drug-induced adverse central nervous system reactions.

Aged↗

PubMed1994

Transgenic expression of IL-10 in pancreatic islet A cells accelerates autoimmune insulitis and diabetes in non-obese diabetic mice.

To study the paracrine effect of IL-10 on autoimmune insulitis and diabetes, we produced IL-10 transgenic non-obese diabetic (NOD) mice (NOD-IL-10) in which murine IL-10 was expressed in pancreatic islet A cells under the control of a rat glucagon promoter without directly manipulating pancreatic islet B cells. Among 11 founder mice, four of four males and three of seven females developed diabetes by 10 weeks of age. Histological analysis of six NOD-IL-10 revealed severe insulitis and prominent ductal proliferation. NOD-IL-10 also showed spotty lymphocytic infiltration in the lung and liver in four of six founder mice. The onset of diabetes in NOD-IL-10 was remarkably earlier than that of 14 weeks of age at the earliest in female non-transgenic NOD mice. When the NOD-IL-10 mouse was backcrossed to C57BL/6 mice, none of the resulting F1, B-N2 or B-N3 generation toward C57BL/6 mice showed diabetes even at 39 weeks of age, in spite of the presence of peri-insulitis and prominent ductal proliferation, while two of four mice of the N-N2 generation toward NOD mice showed early-onset diabetes. Thus, transgenic paracrine expression of IL-10 in situ in the NOD genetic background enhances autoimmune insulitis and diabetes in their onset and severity, ignoring gender difference. Because expression of IL-10 was detected by polymerase chain reaction in pancreatic islets of non-transgenic NOD mice after 5 weeks of age, IL-10 secreted in situ is regarded to enhance cell-mediated autoimmune diabetes, in spite of established in vitro anti-Th1 activity of IL-10.

Animals↗

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↗

PubMed1994

Determination of glycated proteins in biological samples based on colorimetry of 2-keto-glucose released with hydrazine.

We developed a novel method for measuring glycated (glc) proteins in biological samples, based on the colorimetry of 2-keto-glucose which is released from the glc protein (ketoamine) on heating with hydrazine. The ketoamine-induced coloration remained constant at room temperature (25-27 degrees C) for 1 h. The method gave reliable precision and accuracy. However, high concentrations of serum pigments caused positive interference, suggesting that hemolytic or hyperbilirubinemic serum would give false-positive results. The concentration of glc protein in clinical serum samples measured by the present method (y) correlated well with those (fructosamine values, x) measured by the nitroblue tetrazolium-reducing method: y = 1.27x-1.69 (r = 0.92, n = 93). The concentrations (microM, mean +/- S.D.) of glc protein in sera from normal and diabetic subjects were 275 +/- 37 (n = 32) and 403 +/- 98 (n = 32), respectively, and the concentrations (nmol/mg hair, mean +/- S.D.) of glc protein in back hairs from non-diabetic and diabetic rats were 3.7 +/- 0.3 (n = 10) and 8.6 +/- 1.5 (n = 10), respectively. Thus, the technique gave reasonable concentrations of glc proteins in humans and rats with diabetes mellitus, indicating it to be reliable and diagnostically useful.

Adult↗