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Biomedical subjects

K Sugano

Publications and source records attributed to K Sugano.

At least 145 records · Page 8Linked to original sources
PubMed1996

Sensitive detection of loss of heterozygosity in the TP53 gene in pancreatic adenocarcinoma by fluorescence-based single-strand conformation polymorphism analysis using blunt-end DNA fragments.

We have developed a fluorescence-based single-strand conformation polymorphism analysis to detect Haelil-sensitive polymorphic sites in intron 1 of the TP53 gene. It is important to treat the PCR products with Klenow fragment to remove a 3'-protruding nucleotide from the amplified DNA fragments added during the reaction in order to obtain a single peak for each allele. A comparison of the signal profiles of two alleles with those of normal heterozygotes by data processing using computer software has enabled sensitive detection of loss of heterozygosity (LOH) from clinical materials with a fraction of tumor cells below 10%. In analysis of 14 pancreatic carcinomas in which the proportion of the tumor cells is usually low due to the abundance of the stromal component, 7 samples (50%) were informative and 5 of the 7 (71.4 %) were positive for LOH at the TP53 locus. This approach would be useful for allelotyping tumors with low cellularity, as well as other clinical samples such as biopsied specimens and paraffin embedded tissues.

Adenocarcinoma↗
PubMed1996

Analysis of Ki-ras codon 12 mutations in the duodenal juice of patients with pancreatic cancer.

BACKGROUND & AIMS: Point mutations of the Ki-ras gene at codon 12 have been frequently identified in pure pancreatic juice of patients with pancreatic cancer in studies examining pancreatic cancer tissues. The aim of this study was to examine mutations of the Ki-ras codon 12 in the duodenal juice collected from patients with various pancreatic disorders. METHODS: The duodenal juice was collected through a Dreiling tube installed in the duodenum during a secretin test. Analysis of the Ki-ras mutations was performed using the enriched polymerase chain reaction--single-strand conformation polymorphism technique. RESULTS: Point mutations were detected in 12 of 19 patients with pancreatic cancer; of the 12 patients, 10 had ductal tubular adenocarcinoma and 2 intraductal papillary adenocarcinoma. Mutational patterns included GAT (n = 4), GTT (n = 3), CGT (n = 1), and double mutations of GTT and GAT (n = 3) and GAT and CGT (n = 1). In 41 patients with benign pancreatic disorders, a point mutation was detected in only 1 patient with chronic pancreatitis. CONCLUSIONS: Analysis of the Ki-ras codon 12 mutations in the duodenal juice is useful in the diagnosis of pancreatic cancer.

Base Sequence↗
PubMed1996

Detection of K-ras point mutations in mesenteric venous blood from colorectal cancer patients by enriched polymerase chain reaction and single-strand conformation polymorphism analysis.

In order to confirm the presence of cancer cells in mesenteric venous blood and to examine their relationship with the occurrence of liver metastases, we attempted to detect K-ras codon 12 point mutations in perioperative mesenteric blood using enriched polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analysis in 25 patients with primary colorectal tumors carrying K-ras point mutations. Among these patients, three with synchronous liver metastases were included. The same K-ras point mutation (substitution of GAT for GGT) was detected in both the blood and the primary tumor in a Dukes' C patient. We confirmed this result by colony hybridization and estimated the tumor-to-normal cell ratio to be 1:400. This patient has no liver metastases two years after surgery and her carcinoembryonic antigen (CEA) level remains normal. We demonstrated that considerable numbers of cancer cells can be found in mesenteric venous blood during colorectal cancer surgery. However, their potential role in the formation of liver metastases remains unclear.

Colorectal Neoplasms↗
PubMed1996

Role of sulfatides in adhesion of Helicobacter pylori to gastric cancer cells.

We have demonstrated that clinical isolates of Helicobacter pylori preferentially bind to sulfatides (I3SO3-GalCer) and GM3 gangliosides (II3NeuAcLacCer), two predominant acidic glycosphingolipids in the human gastric mucosa, on thin-layer chromatography plates. However, it has not yet been clarified that these glycospingolipids truly serve as adhesion receptors for H. pylori in live cells. In this study, we used a gastric cancer cell line, KATO III, as a cellular model of H. pylori adhesion and examined the role of sulfatides in attachment. The adhesion of H. pylori (i.e., a standard strain of H. pylori, NCTC 11637) to KATO III cells and the effects of various substances on this adhesion were monitored and semiquantitated by flow cytometric analysis. Sulfated glycoconjugates, such as heparin and gastric mucin, significantly inhibited H. pylori adhesion to KATO III cells. Membrane preparations from KATO III cells strongly inhibited this adhesion. In the membrane preparations, sulfatides were present as a major acidic glycosphinoglipid. With the exception of sulfatides, no distinct adhesion of H. pylori to glycospingolipids from KATO III cells were observed. Moreover, H. pylori did not bind to any membrane proteins of KATO III cells. Finally, a monoclonal anti-sulfatide antibody markedly reduced H. pylori adhesion to KATO III cells. These results suggest that sulfatides, and possibly related sulfated compounds, serve as a major receptor for cell adhesion by H. pylori.

Bacterial Adhesion↗
PubMed1996

Detection of microsatellite instability in cancers by arbitrarily primed-PCR fingerprinting using a fluorescently labeled primer (FAP-PCR).

The microsatellite mutator phenotype (MMP), detected as a change in the number of repeating units in hundreds of thousands of microsatellite sequences in the tumor cell genome, underlies the carcinogenesis of a variety of tumors including sporadic and hereditary nonpolyposis colon cancers. This enhanced microsatellite instability was discovered using arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting of DNA from colon cancers. In this study, we found an arbitrary primer that can amplify multiple DNA fragments containing repeated sequences, including the poly A tracts found in the Alu repeats of the human genome. The combined use of primer labeling with fluorescence and an automated DNA sequencing analysis of AP-PCR products (FAP-PCR) detected alterations in fingerprint bands in all DNA samples previously determined to belong to the MMP. Fluorescent AP-PCR fingerprinting using this single arbitrary primer provides a convenient and efficient method for detecting tumor specific fingerprint alterations that are usually undetectable by conventional fingerprinting.

Blotting, Southern↗
PubMed1996

Immunohistochemical evaluation of lymph node micrometastases from breast cancer.

The lymph node micrometastasis was retrospectively immunohistochemically investigated in 185 patients with node-negative breast cancer and compared with the routine conventional findings. The monoclonal antibodies to epithelial membrane antigen (EMA) and mucinous-like carcinoma antigen (MCA) were used. Of 19 EMA-positive cases, 16 were false positive. Therefore only 3 cases were found to show staining with both EMA and MCA. The patients are all living and well and free of recurrence. No micrometastasis was immunhistochemically demonstrated in 11 node-negative breast cancer patients with subsequent metastases. The results of the present study indicate that the detection rate containing occult cells afforded by conventional routine evaluation is sufficient, at least in Japanese.

Adult↗
PubMed1996

[A 32-year-old man who developed a posterior fossa mass 12 years after the radiation therapy for cerebellar arteriovenous malformation].

We report a 32-year-old man who developed cerebellar ataxia and a posterior fossa mass 12 years after the radiation therapy for a cerebellar arteriovenous malformation (AVM). The patient was well until 19 years of the age when he had an acute onset of vertigo and vomiting. A spinal tap was performed and the CSF was bloody. He was admitted to another hospital where an arteriovenous malformation was found in the cerebellum by angiography. Four years after the onset, he developed tingling sensation in the distribution of the second division of the right trigeminal nerve. He was admitted to the neurosurgery service of our hospital where the cerebellar AVM was confirmed. He was transferred to University of California where Bragg peak stereotaxic radiotherapy was successfully performed; this utilizes high energy alpha-ray produced by a cyclotron. Three years after the radiotherapy, marked reduction in the size of the AVM was confirmed by angiography. Twelve years after the onset of his initial symptom, he noted unsteadiness of gait. He was readmitted to our neurosurgery service where obstructive hydrocephalus was found. He was treated by ventriculoperitoneal shunting and placement of a Ommaya reservoir. After these therapy, he noted marked improvement in his gait and ataxia. However, in 1993, his unsteadiness of gait recurred, and he was again admitted to our neurosurgery service on June 20, 1993. On admission, T1-weighted MRI revealed a slightly low signal intensity mass lesion in the right cerebellar hemisphere compressing the brain stem; a spotty high signal intensity lesion and another small low intensity lesion were seen within the mass. Vertebro-basilar angiograms revealed upward displacement of the superior cerebellar arteries. No arteriovenous nidus was visualized. On July, 3rd, the cyst was surgically drained and the Ommaya reservoir was removed. Post-operative course was uneventful, however, he developed head tremor after the surgery. Neurologic examination on July 20, 1993 revealed an alert and well oriented man in no acute distress. General physical examination was unremarkable. Neurologic examination revealed no dementia; higher cerebral functions appeared intact. The optic discs were flat, and visual fields were intact. Ocular movements were full but convergence was restricted. Horizontal gaze nystagmus was noted more in the right lateral gaze. Pupils were intact. Facial sensation and facial muscles were intact. Hearing was normal. His voice was of nasal quality. Pharyngeal reflex was diminished. The tongue showed deviation to the left without atrophy. Head tremor at 5 c/s was noted. He was able to stand with support but was unable to walk. No muscle atrophy or weakness was noted. The finger-to-nose and the heel-to-knee tests showed dysmetria and decomposition more on the right. Rapid alternating movements were ataxic on the right. Muscle tone was diminished on the right. Muscle stretch reflexes were normally elicited and were symmetric. The plantar response was flexor bilaterally. Sensation was intact. On July 21, a posterior fossa exploration was performed. After the surgery, he was treated with 30 mg/day of alotinolol which showed no effect on his head tremor. He was then treated with gradually increasing doses of clonazepam; when he received 8 mg/day of clonazepam, his tremor showed marked improvement. He was discussed in a neurologic CPC on the nature of the posterior fossa lesion and his tremor. Opinions were divided between delayed radiation necrosis and a radiation-induced brain tumor. The chief discussant arrived at the conclusion that the patient had delayed radiation necrosis compressing the brain stem and cerebellar hemispheres. Regarding the nature of his tremor, he thought that his head tremor was of cerebellar type of postural tremor. Histologic examination of the biopsied specimen revealed accumulation of relatively fresh blood constituents in the deep area of the cerebellum forming a mass. Most of the

Adult↗
PubMed1996

[Structural analysis of the histamine H2 receptor].

In gastric parietal cells the histamine H2 receptor is involved in gastric acid secretion. Molecular cloning of the cDNA for the histamine H2 receptor has revealed that the receptor is a member of the G-protein coupled receptor family. We expressed histamine H2 receptors in CHO cells, COS7 cells, and Sf9 cells to analyze N-glycosylation, palmitoylation, and desensitization of the H2 receptor, which are difficult to analyze in isolated parietal cells. The findings obtained in these systems will be useful for studying the gastric acid secretion mechanisms in parietal cells.

Amino Acid Sequence↗
PubMed1996

Body mass index is a determinant of blood pressure in young adults with essential hypertensive parents. Health Care Programme of University of Tokyo.

We analysed blood pressure (BP) and various parameters related to glucose metabolism in 10,867 Japanese university students aged 19.22 +/- 0.01 years, and investigated the relationship between the subject's own BP and hypertension in close relatives. After 2 years we also examined a subset of these students enrolled in 1993. BP levels in subjects having hypertensive parents were normotensive, but significantly higher than those in subjects without hypertensive relatives or only with hypertensive grandparents. The close the genetic relationship was, the higher the subject's own BP levels. In subjects with hypertensive parents, the subject's own mean BP levels correlated significantly with the subject's own body mass indices. This correlation was much weaker in subjects without hypertensive relatives and those having only second degree relatives with hypertension. In addition, the BP levels in the offspring of hypertensive parents correlated with glutamyl-oxaloacetic transaminase. In the follow-up study in 1995, the correlation between BP levels and body mass index (BMI) was observed in this subgroup. Moreover, the subjects with hypertensive parents tended to have a higher plasma insulin level than those without hypertensive relatives. It is suggested that BP is more likely to be determined by BMI in the individuals with a genetic predisposition to hypertension.

Adult↗
PubMed1996

Immunohistochemical detection of c-erbB-2 oncoprotein in patients with breast cancer.

Using a polyclonal antibody against the c-erbB-2 gene product, an immunohistochemical study on the expression of c-erbB-2 oncoprotein was performed in routine formalin-fixed, paraffin-embedded tissue sections from 119 patients with primary breast cancer. Overexpression of c-erbB-2 protein was detected in 35 cases (29%). There was a strongly negative association between c-erbB-2 protein overexpression and estrogen receptor status (p < 0.001). Expression of c-erbB-2 protein was found to be related to clinical stage and tumor size (p < 0.05) but not to the number of involved nodes or age of patients at diagnosis. In addition, this study demonstrated that the c-erbB-2 protein over-expression was an effective independent prognostic indicator in patients with breast cancer.

Adult↗
PubMed1995

Structural and functional analysis of the canine histamine H2 receptor by site-directed mutagenesis: N-glycosylation is not vital for its action.

G-protein-coupled receptors generally share a similar structure containing seven membrane-spanning domains and extracellular site(s) for N-glycosylation. The histamine H2 receptor is a member of the family of G-protein-coupled receptors, and has three extracellular potential sites for N-glycosylation (Asn-4, Asn-162 and Asn-168). To date, however, no information has been presented regarding N-glycosylation of the H2 receptor. To investigate the presence, location and functional roles of N-glycosylation of the H2 receptor, site-directed mutagenesis was performed to eliminate the potential site(s) for N-glycosylation singly and collectively. The wild-type and mutated H2 receptors were expressed stably in Chinese hamster ovary (CHO) cells or transiently in COS7 cells. Immunoblotting of the wild-type and mutated H2 receptors with an antiserum directed against the C-terminus of the H2 receptor showed that mutation at Asn-162, but not at Asn-168, resulted in a substantial decrease in the molecular mass. A mutation at Asn-4 led to a further decrease in the molecular mass. Tunicamycin treatment of the transfected cells yielded a sharp band with a molecular mass identical to that of the mutant devoid of all three potential sites for N-glycosylation. These findings indicate that the H2 receptor is N-glycosylated, and that N-glycosylation takes place mainly at two sites, Asn-4 and Asn-162. Neither the affinity for tiotidine nor that for histamine was affected by the mutagenesis. Immunocytochemistry and tiotidine binding showed that the mutated receptors were exclusively distributed on the cell surface in a fashion similar to that of the wild-type. In addition, the glycosylation-defective receptor was capable of activating adenylate cyclase and elevating the intracellular Ca2+ concentration in response to histamine in stable CHO cell lines. Thus N-glycosylation of the H2 receptor is not required for cell surface localization, ligand binding or functional coupling to G-protein(s).

1-Methyl-3-isobutylxanthine↗
PubMed1995

Single-strand conformation polymorphism analysis by perpendicular temperature-gradient gel electrophoresis.

Using a newly developed temperature-gradient gel electrophoresis apparatus, mutations in the K-ras oncogene and p53 tumor suppressor gene were analyzed for single-strand conformation polymorphism (SSCP). The mobilities of single-stranded DNAs, carrying various mutations, change--depending on the gel temperature during electrophoresis. Therefore, temperature-gradient single-strand conformation polymorphism (TG-SSCP) analysis can provide useful information concerning the optimum temperature for SSCP and may also be used to screen for various mutations or polymorphisms in a single electrophoretic run.

Base Sequence↗
PubMed1995

Identification of mycobacteria by nonradioisotopic single-strand conformation polymorphism analysis.

Clinical isolates of mycobacteria were identified to species levels using nonradioisotopic single-strand conformation polymorphism (non-RI SSCP) analysis of 16S rRNA gene fragments amplified by polymerase chain reaction with primers common to all of mycobacterial species. The method is based on a hypervariable region within the 16S rRNA in mycobacteria, which is characterized by species-specific nucleotide sequences. A total of 92 mycobacterial strains (Mycobacterium tuberculosis, M. avium, M. gordonae, M. intracellulare, M. kansasii, M. chelonae, M. nonchromogenicum, M. xenopi, and unidentified strain) were studied. They were classified into nine types of pattern showing single-strand DNA bands having different mobilities. Each strain was shown in the species-specific mobility by non-RI SSCP analysis. The results of non-RI SSCP analysis were identical to those of standard biochemical methods and 16S rRNA sequencing.

DNA Primers↗