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Biomedical subjects

K Sugano

Publications and source records attributed to K Sugano.

At least 127 records · Page 7Linked to original sources
PubMed1997

Diagnosis of bladder cancer by analysis of the allelic loss of the p53 gene in urine samples using blunt-end single-strand conformation polymorphism.

The novel approach of blunt-end single-strand conformation polymorphism (SSCP) has been applied in the analysis of urine samples from bladder-cancer patients for detecting loss of heterozygosity (LOH) of 3 polymorphic markers in the p53 gene. Of the 28 urine samples examined by SSCP analysis of blunt-ended DNA fragments using a fluorescence-based automated sequencer, 16 were informative in more than 1 of the 3 polymorphic markers at the p53 locus and 8 (50.0%) showed allelic loss of the p53 gene. In analysis of resected tumor tissues, LOH of the p53 gene was detected in 8 of 8 informative samples (100%) with T1 and higher stages and/or Grade 2 and Grade 3 tumors, while it was detected in 6 (75.0%) urine samples obtained from these 8 patients. This new diagnostic modality enables sensitive detection of tumor cells in urine samples and would be applicable for diagnostic bladder cancer with invasive character.

Alleles↗
PubMed1997

Role of the C terminus in histamine H2 receptor signaling, desensitization, and agonist-induced internalization.

To evaluate the role of the histamine H2 receptor C terminus in signaling, desensitization, and agonist-induced internalization, canine H2 receptors with truncated C termini were generated. Wild-type (WT) and truncated receptors were tagged at their N termini with a hemagglutinin (HA) epitope and expressed in COS7 cells. Most of the C-terminal intracellular tail could be truncated (51 of 70 residues, termed T308 mutant) without loss of functions: cAMP production, tiotidine binding, and plasma membrane targeting. In fact, the T308 mutant produced more cAMP than the WT when cell-surface expression per cell was equivalent. Pretreatment of cells with 10(-5) M histamine desensitized cAMP productions via WT and T308 receptors to similar extents. Incubation of cells expressing WT receptors with 10(-5) M histamine reduced cell-surface anti-HA antibody binding by approximately 30% (by 30 min, t1/2 approximately 15 min), but did not affect the Bmax of tiotidine in membrane fractions, which represents total receptor amounts, suggesting that WT receptors were internalized from the cell surface. In contrast, no internalization was observed with T308 receptors following histamine treatment. A mutant with a deletion of the 30 C-terminal amino acids, termed T329, was functional but was as potent as the WT in terms of cAMP production. Apart from being desensitized by histamine, the internalization of the receptor was indistinguishable from that of the WT. Internalization was observed in the T320 but not in T313 mutant, narrowing the region involved in internalization to that between Glu314 and Asn320 (ETSLRSN). Of these seven residues, either Thr315, Ser316, or both, were replaced with Ala. Thr315 and Ser316 are conserved among species. The mutation at Thr315 (but not that at Ser316) abolished internalization. Taken together, these results demonstrate that Thr315 is involved in agonist-induced internalization. Furthermore, the finding that T308 receptors were desensitized in the absence of internalization suggests that internalization and desensitization are meditated by independent mechanisms.

Amino Acid Sequence↗
PubMed1997

Oligomer formation of histamine H2 receptors expressed in Sf9 and COS7 cells.

A histamine H2 receptor, which had been mutated at its glycosylation site and tagged at its N-terminus with an HA tag (HA-H2 receptor), was expressed in Sf9 cells and COS7 cells. Immunoprecipitation and immunoblotting of HA-H2 receptors with alphaHA antibody revealed four bands of 31.5 +/- 2.5 kDa, 59.0 +/- 6.0 kDa, 80.5 +/- 4.5 kDa and 120 kDa. These bands were also detected by immunoblot using anti-H2 receptor serum (C-terminus). In addition, H2 receptors without the HA-tag coimmunoprecipitated with HA-tagged H2 receptors devoid of the 51 C-terminal amino acids, via immunoprecipitation with alphaHA antibody, when the two receptors were coexpressed. These results suggest that H2 receptors are present as receptor oligomers and that the C-terminal portion is not involved in the formation of these oligomers.

Animals↗
PubMed1997

Detection of K-ras gene mutations at codon 12 in the pancreatic juice of patients with intraductal papillary mucinous tumors of the pancreas.

BACKGROUND: The authors previously found specific mutations of the K-ras gene at codon 12 in the pancreatic juice of 67% of patients (6 of 9) with pancreatic ductal carcinoma, and the detection of these mutations was useful for diagnosis. This study was performed to detect and evaluate K-ras mutations in pancreatic juice from patients with intraductal papillary mucinous tumor of the pancreas, which is considered a low grade malignancy. The results were interpreted from the viewpoint of clinical significance. METHODS: K-ras mutations were examined using seminested polymerase chain reaction analysis combined with restriction enzyme digestion, followed by nonradioisotopic single strand DNA conformation polymorphism. RESULTS: Twelve of thirteen cases (92%) of intraductal papillary mucinous tumor of the pancreas, confirmed histologically (9 adenomas and 4 carcinomas), and 26 of 43 cases (60%) of ductal carcinoma showed specific K-ras gene mutations in the pancreatic juice. Furthermore, 4 of 22 patients (18%) with chronic pancreatitis, followed for more than 1 year without a sign of pancreatic tumor, showed K-ras mutations. In contrast, no mutations of the K-ras gene were detected in the pancreatic juice from 28 normal controls. CONCLUSIONS: K-ras mutations were found in the pancreatic juice of all but one patient with intraductal papillary mucinous tumor of the pancreas, but they were not useful for distinguishing carcinoma from adenoma. The authors concluded that K-ras mutations are not a specific marker for pancreatic neoplasms because similar mutations were detected in the pancreatic juice from patients with chronic pancreatitis. At the present time, the detection of K-ras mutations in pancreatic juice should be used clinically as an adjunct diagnostic modality for pancreatic diseases.

Adenocarcinoma, Mucinous↗
PubMed1997

Expression of c-met proto-oncogene in primary colorectal cancer and liver metastases.

We have examined the expression of c-met mRNA in tissue from 27 colorectal cancers and ten liver metastases using the reverse transcriptase-polymerase chain reaction method. The expression of c-met mRNA in these tissues was quantified and the copy number of c-met mRNA to 10(8.0) copies of beta-actin mRNA was calculated. Mean copy numbers of c-met mRNA in cancer tissue and normal mucosa were 10(5.5) and 10(4.5) respectively. The c-met expression of cancer was significantly higher than that of normal mucosa (P < 0.0001). In 20 of 22 samples in which c-met expression of both tumor and corresponding normal tissue were examined, c-met was overexpressed in the cancer tissue. No correlation was found between c-met expression and the clinicopathologic background. The mean copy numbers of c-met mRNA in the tissue from the ten liver metastases and normal liver were 10(6.1) and 10(6.2) respectively. Although c-met expression in metastatic tissue was higher than that in the primary cancer tissue, the increase was not statistically significant. In three of four patients with synchronous liver metastases, c-met was overexpressed in the metastatic tissue compared with that in the corresponding primary cancer tissue. These results show that c-met is overexpressed in both primary colorectal cancer and liver metastases and suggest that c-met plays a role in the development of colorectal cancer liver metastases.

Actins↗
PubMed1997

Immunohistochemical study of sulfatide expression in gastric carcinoma: alteration of sulfatide expression.

Immunohistochemical staining using a specific monoclonal antibody against sulfatide was performed to examine cellular localization of sulfatides in normal human gastric mucosa and in various types of gastric carcinoma. In normal gastric mucosa without Helicobacter pylori infection, both epithelial and glandular cells were densely stained with anti-sulfatide antibody. Sulfatide staining was more abundant in the apical area of normal epithelium compared with cytosol or basolateral areas. This tendency was stronger in the antrum than the gastric body and was more intensified in glandular cells of the pyloric glands. The levels of sulfatide expression were decreased in papillary and well-differentiated adenocarcinomas compared with normal mucosa, were markedly attenuated in moderately differentiated adenocarcinomas, and were very low in poorly differentiated adenocarcinomas. The polarity of the stains was preserved in gastric cancers that exhibited differentiated tissue organization. The levels of sulfatide expression were highly variable in signet ring carcinoma cells. The cancer cells that expressed sulfatides did not show any polarity of staining.

Adenocarcinoma↗
PubMed1997

Correlation of allelic loss of the P53 gene and tumor grade, stage, and malignant progression in bladder cancer.

BACKGROUND: We examined loss of heterozygosity (LOH) of the P53 gene in bladder cancer, and investigated the role of the P53 gene on malignant progression of papillary tumors. In addition, the clonality of recurrent bladder cancer was examined. METHODS: LOH of the P53 gene was analyzed in 67 bladder cancers from 47 patients. DNA was extracted from formalin-fixed, paraffin-embedded tissues, amplified by the polymerase chain reaction (PCR) at 3 polymorphic loci in the P53 gene, and analyzed with nonradioisotopic single-strand conformation polymorphism (Non-RI SSCP) analysis. RESULTS: Out of 40 informative samples, LOH was detected in 13 samples, containing 4 of 7 in grade 3 (57%), 9 of 23 in grade 2 (39%), and none of 10 in grade 1 (10%). Statistical significance was observed between the LOH in grades 1 and 2, and in grades 1 and 3. An analysis of 5 cases showing malignant progression revealed that 3 (60%) showed an LOH in the primary tumor, and 2 showed LOH in recurrent tumors, in contrast to LOH found in 3 cases of 19 (16%) not showing malignant progression. Four cases with metachronous recurrence exhibited LOH; 2 at recurrent tumors, 1 only at the initial tumor, and 1 at both tumors. CONCLUSIONS: The alterations of the P53 gene were considered to correlate with tumor grade, and contribute to the malignant progression of bladder cancer. LOH in the P53 gene may serve as a clinical indicator for prognosis in superficial bladder cancer.

Alleles↗
PubMed1997

Polymerase chain reaction-based approaches for detection of allelic loss in the p53 tumor suppressor gene in colon neoplasms.

OBJECTIVES: Inactivation of the p53 tumor suppressor gene is considered to be a late event involved in the malignant transformation of colorectal adenoma to cancer. Thus, its detection is thought to provide useful information for the clinical management of colorectal neoplasms. We devised a rapid screening test for allelic loss of the p53 gene by non-radioisotopic single-strand conformation polymorphism analysis. METHODS: Biopsy materials from 119 colorectal tumors obtained at endoscopy were examined. Three intragenic polymorphic sites were amplified by polymerase chain reaction using DNA extracted from these materials, and amplified DNA fragments were subjected to non-radioisotopic single-strand conformation polymorphism. RESULTS: This method can detect a loss of heterozygosity (LOH) of the p53 locus from samples containing over 40% tumor derived DNA, and the combination of the three polymorphic markers encompassed 62.4% of Japanese patients as informative. In adenocarcinoma, an LOH was detected in 51.5% (17 of 33) of the samples and in 12.2% (4 of 33) of tubular and/or tubulovillous adenomas. The p53 gene was mutated only in samples carrying an LOH, that is 64.7% (11 of 17) of carcinomas and 25.0% (1 of 4) of adenomas, but there were no mutation in samples retaining both alleles. The presence of an LOH was statistically correlated both with p53 mutation and malignant histology (chi 2 test, p < 0.05). CONCLUSIONS: This method can detect LOH from biopsy material obtained at endoscopy. LOH in the p53 locus precedes mutation of the p53 gene, and its detection provides useful information of malignancy in colorectal tumors.

Adenocarcinoma↗
PubMed1997

A new T-lymphocyte cloning assay for detection of in vivo mutations in the human hypoxanthine-guanine phosphoribosyltransferase gene.

The X-linked hypoxanthine-guanine phosphoribosyl transferase (hprt) gene is a target of analyses of in vivo mutation frequencies in circulating T-lymphocytes. We established a novel, accessory cell-free cloning method of T-lymphocytes with a hprt mutation by a combined use of recombinant interleukin-2, conditioned medium from activating T-lymphocytes and culture plates coated with anti-CD3 monoclonal antibody. Using the method, we examined mutation frequencies of the hprt gene in T-lymphocytes from six healthy individuals, nine patients with colon cancer including two patients from different families with hereditary nonpolyposis colon cancer and six cancer-free relatives of the patients. In six healthy individuals, the mean cloning efficiency and mutation frequency (MF) of the hprt gene in T-lymphocytes were 0.51 +/- 0.28 and 9.4 +/- 7.5 x 10(-6), respectively. These data were similar to the reported values. The mean MFs in the nine colon cancer patients (10.6 +/- 7.3 x 10(-6)) were not significantly different from those of the 12 cancer-free individuals (11.6 +/- 9.4 x 10(6)). The correlation between mutation frequencies and age of the individuals was significant regardless of the presence or absence of cancers. The single-strand conformation polymorphism analyses of nested RT-PCR products of hprt mRNA were done in 33 mutant clones from five members of a family of which MF values were high. All the analyzed mutant clones show a genetic aberration in the coding region of the hprt gene. At least 28 of 33 mutants were independent. Our method provides a new versatile tool for in vivo analysis for mutations of the hprt gene.

Adult↗
PubMed1997

[Molecular diagnosis of pancreatic cancer].

Genetic alterations such as K-ras mutation, inactivation of the p53, p16 and DPC4 genes and frequent chromosomal loss of the 17p, 9p, 18q and 1p are thought to play a crucial role in the carcinogenesis of pancreatic cancer. Mutations of K-ras oncogene could be detected frequently in pancreatic juice samples from patients with pancreatic carcinoma and intraductal papillary neoplasm (IPN), although they could be detected in some of the samples from patients with chronic pancreatitis and pancreatic cyst. This suggests that K-ras mutation is an early event in the carcinogenesis of the exocine pancreas. In IPN, analysis of other genetic alteration would be available, since pancreatic juice samples from the patient are relatively rich in the proportion of the tumor cells. A new diagnostic modality of sensitive allelotyping would be useful for evaluating malignant potential of these borderline lesions.

Gene Deletion↗
PubMed1997

Role of Y chromosome of hypertensive parent in determination of blood pressure in normotensive offspring.

We investigated whether the sex of hypertensive parents mediates determination of blood pressure in their normotensive offspring. We analyzed medical checkup data from 7279 Japanese university students. Six hundred and forty-one students had one hypertensive parent with or without hypertensive grandparents, and 609 of these were available for the present analysis. Blood pressure levels in the students with one hypertensive parent were within the normotensive range; however, the blood pressure levels were higher than those in students without hypertensive relatives, and lower than those in students with two hypertensive parents. In the students with one hypertensive parent, systolic blood pressure levels were significantly higher in the male than in the female students. However, the sex of the hypertensive parent did not affect the blood pressure values of their offspring. Multivariate analyses revealed that body mass index was an independent predictor of systolic blood pressure solely in male students with a hypertensive father, but not in male students with a hypertensive mother. In female students with one hypertensive parent, body mass index was irrelevant to the determination of blood pressure. These findings suggest that different mechanisms determine blood pressure in the offspring of hypertensive parents, and that blood pressure determination mediated by body mass index in male students is linked to hypertensive Y chromosome.

Adolescent↗
PubMed1997

Pancreatic lymph nodal and plexus micrometastases detected by enriched polymerase chain reaction and nonradioisotopic single-strand conformation polymorphism analysis: a new predictive factor for recurrent pancreatic carcinoma.

K-ras point mutations have been observed in approximately 90% of pancreatic carcinomas. We genetically analyzed cases of pancreatic regional lymph nodal and plexus micrometastases in invasive ductal carcinoma of the pancreas who were node negative or had metastases limited histopathologically to pancreaticoduodenal lymph nodes. These cases underwent curative resection in our institute. The utility of genetic analysis was compared with that of histopathological study, in terms of postoperative clinical outcome, as a predictive factor for recurrent pancreatic carcinoma. Samples for DNA extraction were obtained from formalin-fixed, paraffin-embedded specimens. A 0.5-microg quantity of DNA was subjected to enriched PCR and nonradioisotopic single-strand conformation polymorphism analysis. K-ras codon 12 mutations were detected in 83% (10 of 12) of invasive ductal carcinomas. In four cases, the genetic analysis of regional lymph nodal metastases and pancreatic plexus invasion of the pancreatic carcinoma yielded results concordant with those of histopathological analysis. In six cases, however, the metastases detected by genetic analysis were more advanced than was indicated by the histopathological examination. The survival rate of cases with metastases beyond the pancreaticoduodenal lymph nodes was significantly lower than that of cases with metastases limited to the pancreaticoduodenal lymph nodes or with no nodal involvement based on genetic analysis (P < 0.05). Intraoperative analysis of point mutations at K-ras codon 12 in the regional lymph nodes and the pancreatic plexus by enriched PCR/nonradioisotopic single-strand conformation polymorphism analysis is a highly accurate predictive factor for recurrent pancreatic carcinoma.

Adult↗
PubMed1996

Interaction between the two signal transduction systems of the histamine H2 receptor: desensitizing and sensitizing effects of histamine stimulation on histamine-dependent cAMP production in Chinese hamster ovary cells.

The histamine H2 receptor is a member of the family of G-protein-coupled receptors and is linked to the activation of adenylate cyclase phospholipase C (PLC). In this study we examined the effects of protein kinase C (PKC) activation in Chinese hamster ovary (CHO) cells stably expressing canine histamine H2 receptors. Pretreatment with 100 nM phorbol 12-myristate 13-acetate at 37 degrees C for 15 min led to significant potentiation of histamine-dependent and forskolin-dependent cAMP production, whereas the biologically inactive phorbol ester, 4 alpha-phorbol 12, 13-didecanoate, was without effect. These potentiating effects were abolished by preincubation with 0.5 microM bisindolylmaleimide, a PKC inhibitor. Thus the activation of PKCs seems to be involved in the potentiation of cAMP production by acting on a post-receptor mechanism. Preincubation of a CHO cell line, CHO-H2R, with 10 microM histamine for 30 min had two effects. Maximal histamine-dependent cAMP production and forskolin-dependent cAMP production were potentiated by 36% and 105.2% respectively. The other effect was a desensitization of the histamine-dependent adenylate cyclase response as demonstrated by a three-fold increase in EC50. Administration of 0.5 microM bisindolylmaleimide before preincubation of CHO-H2R with 10 microM histamine did not alter the desensitizing effect on cAMP production, but did abolish the sensitizing effect. Preincubation of CHO-H2R cells with 10 nM histamine resulted in moderate potentiation, which was also abolished by bisindolylmaleimide, but not in desensitization of the histamine-dependent cAMP production. Thus these results suggest that preincubation with histamine had a sensitizing effect on cAMP production mediated by PLC and PKC activation, as well as a desensitizing effect on the H2 receptor. The former effect is dependent on the intensity of PLC and PKC signals delivered by H2 receptors. The latter effect requires a higher concentration of histamine.

Animals↗
PubMed1996

Isolation of DNA sequences amplified at chromosome 19q13.1-q13.2 including the AKT2 locus in human pancreatic cancer.

In the human pancreatic cancer cell line PANC1, we detected several DNA fragments with abnormally intensified signals by restriction landmark genomic scanning. Major five of these fragments were cloned. All of the cloned fragments were mapped at the 19q13.1-13.2 region where the AKT2 oncogene was located. Southern blotting using the cloned DNA fragments and a fragment of AKT2 cDNA as probes revealed that the AKT2 gene was amplified in 3 of 12 pancreatic cancer cell lines analyzed including PANC1 and in 3 of 20 primary pancreatic cancers. The AKT2 gene was overexpressed in the 3 cell lines with the amplified gene. The results suggest that the AKT2 gene is a candidate oncogene activated by amplification in some human pancreatic cancers.

Base Sequence↗
PubMed1996

Relative ratios of mRNA molecules encoded by genes with homologous sequences using fluorescence-based single-strand conformation polymorphism analysis.

The combination of reverse transcribed (RT)-PCR and fluorescence-based single-strand conformation polymorphism (SSCP) analysis is proposed for the quantitative determination of the ratio of mRNA molecules with homologous sequences. We applied this procedure to lactate dehydrogenase (LDH) subunits A and B. We designed fluorescence labeled common PCR primers in the sequences highly homologous between LDH-A and LDH-B cDNAs and performed RT-PCR-SSCP analysis. When PCR efficiency was almost the same between the different target sequences, analysis of mixtures of known amounts of LDH-A and LDH-B revealed linear and precise proportions of LDH-A mRNA. Template concentrations and PCR cycles did not affect the determination of proportions of LDH-A to total LDH. The present procedure could be easily applied to investigation of expression levels of genes encoding mRNAs with homologous sequences.

Amino Acid Sequence↗