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M Sakashita

Publications and source records attributed to M Sakashita.

At least 19 recordsLinked to original sources
PubMed2006

Validation of the quality of histological images obtained of fresh and formalin-fixed specimens of esophageal and gastric mucosa by laser-scanning confocal microscopy.

BACKGROUND AND STUDY AIMS: We have previously reported the success of a method of virtual histology using laser-scanning confocal microscopy (LCM) in vitro on untreated fresh specimens obtained from the gastrointestinal mucosa. In the present study, we aimed to apply LCM to both fresh and formalin-fixed specimens, without additional treatment, in order to validate and compare the quality of the images obtained. METHODS: We obtained 18 specimens from 11 patients, either by endoscopic biopsy or following surgical resection. First, we observed the fresh, saline-immersed specimen with LCM using the Fluroview microscope (Olympus Co. Ltd., Tokyo, Japan). We then fixed the specimen with formalin and obtained further LCM images 1 hour, 3 hours, and 24 hours after fixation. Three independent observers observed the images and were asked to assess the origin of the samples, the treatment of the samples, the time after formalin fixation, and whether they showed benign or malignant lesions. We used kappa statistics to compare the agreement among the three observers in each of these four areas of interest. RESULTS: Between January and March 2003, we obtained 191 LCM images from 18 specimens. Thirty images were randomly selected for observation. The overall accuracy for differentiating between esophagus and stomach specimens was 96.6 %. The accuracy of differentiating normal from cancerous lesions was 92.2 %. The differentiation between saline-immersed and formalin-fixed specimens was 59.7 % accurate and the assessment of the time interval after formalin fixation was only 37.3 % accurate. The kappa statistics showed that there was strong interobserver agreement on the differentiation of specimen origin and of cancerous from benign lesions. However, there was no agreement among the observers on the method of specimen preparation or on the estimated time interval after formalin fixation. CONCLUSIONS: We concluded that images obtained from fresh specimens using LCM were of a quality good enough to make an accurate diagnosis of upper gastrointestinal carcinoma.

Biopsy↗
PubMed2003

Virtual histology of colorectal lesions using laser-scanning confocal microscopy.

BACKGROUND AND STUDY AIMS: Histological examination of gastrointestinal lesions is currently based on light-microscopic examination of thin-slice specimens, with hematoxylin and eosin staining. A study of the use of laser-scanning confocal microscopy (LCM) to obtain immediate microscopic images of untreated specimens for examining colorectal lesions was carried out. A probe-type LCM prototype endomicroscope that can be passed through the working channel of an endoscope has also been developed. MATERIALS AND METHODS: The study materials consisted of colorectal lesions resected either endoscopically or surgically at Showa University Northern Yokohama Hospital. One hundred untreated specimens were examined using LCM. The histopathological findings in the lesions were seven cases of normal colonic mucosa, five hyperplastic polyps, 68 adenomas with low-grade dysplasia, 10 adenomas with high-grade dysplasia, and 10 adenocarcinomas. An argon laser beam with a wavelength of 488 nm was used for the LCM study. Observation of the resected normal colonic mucosa (in vitro) and the rectal mucosa of a healthy volunteer (in vivo) was possible using the endomicroscope. The LCM images for each specimen were compared with the hematoxylin-eosin-stained histopathological cross-sections. RESULTS: The LCM images corresponded well with the conventional hematoxylin-eosin light-microscopic images. The nuclei were not visualized in normal mucosa or hyperplastic polyps. In adenomas with high-grade dysplasia and carcinomas, nuclei were more often visible than in adenomas with low-grade dysplasia. The rate of visualization of nuclei was significantly different ( P < 0.01) between these two groups (60.0 % vs. 10.3 %). In LCM images using endomicroscope, it was possible to recognize the orifices of the colonic glands and goblet cells both in vitro and in vivo. CONCLUSIONS: Laser-scanning confocal microscopy provides immediate images that correspond well with those of hematoxylin-eosin staining. An improved probe-type LCM endomicroscope is being developed which should provide better histological images of colorectal lesions in vivo.

Adenocarcinoma↗
PubMed2003

Development of virtual histology and virtual biopsy using laser-scanning confocal microscopy.

The aim of this project is to acquire a direct image of histology from in vivo gastrointestinal mucosa. In other words, the task of 'endo-microscope' is to observe the cellular architecture of tissue in vivo during routine endoscopic examination. As the first step to completing this study, resected fresh specimens from the oesophagus. stomach and colon were examined by laser-scanning confocal microscopy (LCM) (Fluoview, Olympus, Tokyo). Fresh untreated mucosal specimens obtained by endoscopic pinch biopsy, polypectomy or endoscopic mucosal resection were collected and placed in normal saline and examined by LCM, collecting the reflective light of a 488-nm wavelength argon laser beam. As the second step, a probe-type LCM 'endo-microscope' was designed and applied to observe the human oral-cavity mucosa. The probe has 4.5-mm outer diameter and 20-cm length, which enables easy access to oral cavity mucosa. The estimated special resolution of the probe is 1-5 microm. A real-time microscopic image directly from ex vivo fresh specimens was acquired. The acquired LCM images corresponded well with the conventional H-E light microscopic images. Cell wall, nucleus and cytoplasm were simultaneously visualized by LCM scanning. This novel method enables serial imaginary microscopic sections on fresh specimens. In addition, a probe-type LCM 'endo-microscope' was designed and was applied to observe human oral cavity mucosa. Virtual histological images from the living oral squamous cell were successfully obtained. LCM images from ex vivo fresh specimens demonstrated the features of the H-E staining histological image. In the next step to accomplish our project, we developed a LCM probe with 4.5-mm outer diameter to obtain a virtual image of human oral cavity mucosa.

Biopsy↗
PubMed2002

Protonic diffusion in high-pressure ice VII.

Near ambient pressures, molecular diffusion dominates protonic diffusion in ice. Theoretical studies have predicted that protonic diffusion will dominate at high pressures in ice. We measured the protonic diffusion coefficient for the highest temperature molecular phase of ice VII at 400 kelvin over its entire stable pressure region. The values ranged from 10(-17) to 10(-15) square meters per second at pressures of 10 to 63 gigapascals. The diffusion coefficients extrapolated to high temperatures close to the ice VII melting curve were less by a factor of 10(2) to 10(3) than a superionic criterion of approximately 10(-8) square meters per second, at which protons would diffuse freely.

Journal Article↗
PubMed2002

Variation in MT expression in early-stage depressed-type and polypoid-type colorectal tumours.

Metallothionein (MT) expression is observed in various carcinomas, but its role is not fully understood. To clarify the clinicopathological significance of MT, 87 colorectal adenomas and 128 early-stage carcinomas were immunohistochemically analysed for MT expression. The degree of MT immunostaining of a specimen was graded according to the proportion of MT-positive cells; negative (<5%) and positive (focally 5-50%, diffusely >50%). MT expression significantly decreased with tumour development. For carcinomas, MT-positivity was significantly associated with depth of invasion (T1 60% versus T2 33%; P<0.01), vascular involvement (positive 35% versus negative 61%; P<0.01) and morphology (polypoid 62% versus depressed 26%; P<0.01). Regarding MT-positive distribution, the diffuse-positive rate in MT-positive polypoid lesions was 28%, while MT-positive depressed lesions were all diffusely stained (P<0.01). In conclusion, our results suggested that decreasing MT expression is an early event in colorectal carcinogenesis and may reflect local invasion. Furthermore, MT-positive distribution may reflect genetic differences between the polypoid and depressed-type.

Adult↗
PubMed2001

Synthesis and biological evaluations of condensed pyridine and condensed pyrimidine-based HMG-CoA reductase inhibitors.

A series of 3,5-dihydroxyheptenoic acid derivatives containing pyrazolopyridine, isoxazolopyridine, thienopyridine, and pyrazolopyrimidine as a key scaffold was synthesized from condensed pyridine and condensed pyrimidine carboxylic acid esters by homologation, aldol condensation with ethyl acetoacetate dianion, and stereoselective reduction of the 5-hydroxyketone. Several compounds in the series were found to have potent HMG-CoA reductase inhibitory activities in vitro and marked cholesterol biosynthesis inhibitory activities in vivo. It has been shown that these scaffolds can be used as a suitable replacement for the hexahydronaphthalene ring present in naturally occurring HMG-CoA reductase inhibitors.

Administration, Oral↗
PubMed2001

Glut1 expression in T1 and T2 stage colorectal carcinomas: its relationship to clinicopathological features.

Glucose uptake is mediated by glucose transporter (Glut) proteins, which exhibit altered expression in a variety of malignant neoplasms. Glut1 expression is thought to be a potential marker for malignant transformation. The aim of the present study was to investigate the expression of Glut1 protein in colorectal adenomas, T1 and T2 stage carcinomas. Immunohistochemical detection of Glut1 protein was examined in 141 formalin-fixed and paraffin-embedded colorectal tumour specimens (57 adenomas, 84 carcinomas). The degree of Glut1 immunostaining of a specimen was graded according to the proportion of Glut1-positive cells in it; absent (positive cells are 0%), weakly positive (less than 10%), moderately positive (10-50%), and strongly positive (more than 50%). Glut1 expression was present in 18% of the adenomas with low-grade dysplasia, and in 63% of the adenomas with high-grade dysplasia. The positivity in such lesions was usually weak, but was moderate in 8% of the adenomas with high grade dysplasia. For the carcinomas, there were significant correlations between Glut1-positivity and depth of invasion (T1 45% versus T2 74%, P<0.01), histological differentiation (well 49% versus moderately to poorly 74%, P< 0.05) and morphological type (polypoid 42% versus depressed 73%, P< 0.05), if the cut-off value was set at 10% of cells. In conclusion, we clarified the relationship between Glut1 expression and clinicopathological features in T1 and T2 stage colorectal carcinomas, and our results suggested a high malignant potential of the depressed-type carcinoma.

Adenocarcinoma↗
PubMed2001

Synthesis and biological evaluations of quinoline-based HMG-CoA reductase inhibitors.

A series of quinoline-based 3,5-dihydroxyheptenoic acid derivatives were synthesized from quinolinecarboxylic acid esters by homologation, aldol condensation with ethyl acetoacetate dianion, and reduction of 3-hydroxyketone to evaluate their ability to inhibit the enzyme HMG-CoA reductase in vitro. In agreement with previous literature, a strict structural requirement exists on the external ring, and 4-fluorophenyl is the most active in this system. For the central ring, substitution on positions 6, 7, and 8 of the central quinoline nucleus moderately affected the potency, whereas the alkyl side chain on the 2-position had a more pronounced influence on activity. Among the derivatives, NK-104 (pitavastatin calcium), which has a cyclopropyl group as the alkyl side chain, showed the greatest potency. We found that further modulation and improvement in potency at inhibiting HMG-CoA reductase was obtained by having the optimal substituents flanking the desmethylmevalonic acid portion, that is, 4-fluorophenyl and cyclopropyl, instead of the usual isopropyl group.

Animals↗
PubMed2000

Flat-elevated and depressed, subtypes of flat early colorectal cancers, should be distinguished by their pathological features.

Flat-type colorectal tumors have are being detected with increasing frequency. It has become clear that these flat lesions contain two subtypes; flat-elevated and depressed lesions. However, their clinicopathological features and roles in colorectal carcinogenesis remain obscure. We classified colorectal adenomas and submucosal invasive cancers into three types: polypoid, flat-elevated, and depressed types. A clinicopathological study of 2505 colorectal tumors (2407 adenomas, 98 submucosal invasive cancers) was then performed. Furthermore, 64 tumors (25 adenomas with high-grade dysplasia, 39 submucosal invasive cancers) from which DNA was extracted were examined for K-ras gene mutation. The percentages of each configuration in the resected materials were 62.0%, 36.4%, and 1.6% of the polypoid, flat-elevated, and depressed types, respectively. The rate of submucosal invasive cancer in the depressed type was always high regardless of size. In the polypoid and flat-elevated types, lesions of larger size showed higher rates of invasion. Analysis of submucosal invasive cancers revealed no adenomatous components in any of the depressed-type lesions; in the polypoid and flat-elevated types the frequencies of cancer with adenomatous components were 83.6% and 77.8%, respectively. The flat-elevated type was more frequently located (77.8%) in the proximal colon than the other types (polypoid type 16.4%, depressed type 25.0%). The incidence of K-ras gene mutation was 47.2%, 18.2%, and 0% in the polypoid, flat-elevated, and depressed types, respectively. These findings suggest that the flat-elevated and depressed types are similar in that they are both morphologically flat and have infrequent incidences of K-ras gene mutation, but these two lesions differ in their pathological features. Especially, depressed type lesions have a tendency to invade the submucosal layer even when they are small. Therefore one should always be aware of this type of lesion during colonoscopic examination.

Adenoma↗
PubMed2000

Analysis of Helicobacter pylori vacA gene and serum antibodies to VacA in Japan.

Vacuolating cytotoxin, VacA, is one of the most important pathogenetic factors produced by Helicobacter pylori. However, it is not clear whether the diversity in disease outcome may be ascribed to variations in strain and/or to the host responses to virulence factors. In this study, we analyzed the vacA middle region sequence among 65 Japanese isolates to clarify the variation in strain and assayed antibody titer to VacA by ELISA using purified VacA to evaluate the host response to cytotoxin. The nucleotide sequence identities compared among Japanese isolates were 92.8 +/- 3.56%, and compared to 88.3 +/- 2.89% in tox+ strains reported in GenBank. Positive correlation was found between the antibody titers and the severity of atrophic change of the stomach. In Japan the nucleotide sequences of the vacA middle region were highly homologous and genetically closer to tox+ strains. Antibody titers and host response to cytotoxin may be associated with atrophy of the stomach.

Adult↗
PubMed1998

Molecular cloning of the rat Tpx-1 responsible for the interaction between spermatogenic and Sertoli cells.

We previously showed in a primary culture of rat testicular cells that spermatogenic cells specifically bind to somatic Sertoli cells and that this interaction is needed for spermatogenic cells to differentiate in vitro. Adopting an expression cloning procedure, we here isolated a cDNA coding for a spermatogenic cell protein whose expression gave a cultured cell line the ability to bind to Sertoli cells. The protein, 243 amino acids with a putative N-terminal signal peptide and a C-terminal Cys-rich region, turned out to be the rat homologue of a testicular protein called Tpx-1 whose function had yet to be determined. A polyclonal antibody raised against bacterially expressed Tpx-1 significantly inhibited the binding of spermatogenic cells to Sertoli cells. The above results indicated that Tpx-1 is a testicular cell adhesion molecule responsible for the specific interaction between spermatogenic and Sertoli cells.

Amino Acid Sequence↗
PubMed1997

Determination of a novel anti-psychotic agent AD-5423 and its metabolites in plasma by high-performance liquid chromatography with fluorescence detection.

AD-5423, 2-(4-ethyl-1-piperazinyl)-4-(4-fluorophenyl)-5,6,7,8,9,10-hexahydrocy cloocta [b]pyridine, is a novel anti-psychotic agent. In order to investigate the pharmacokinetics of AD-5423, a simple and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of AD-5423 and its two N-oxidized metabolites (N-desethyl AD-5423 and AD-5423 N-oxide) in plasma. After pretreatment of a plasma sample by solid-phase extraction, AD-5423 and its metabolites were analyzed on a HPLC with fluorescence detection (335/410 nm). Chromatography was performed on two C18 reversed-phase columns connected by a switching system, with a mobile phase of acetonitrile-methanol-25 mM sodium phosphate buffer (pH 2.5) containing 25 mM sodium 1-heptanesulfonate (36:26:38 v/v/v). The method gives satisfactory accuracy and precision for the determination of AD-5423 and its metabolites. In human plasma, accurate determination are possible over the concentration ranges of 0.04-5 ng ml-1 for AD-5423 and 0.1-5 ng ml-1 for N-oxidized metabolites. The intra- and inter-day assay precision (R.S.D.) of AD-5423 (0.5 ng ml-1) were 3.6 and 7.2%, respectively. In plasma of experimental animals, the validated quantitative range are 0.1-100 ng ml-1 for both AD-5423 and its metabolites.

Adult↗
PubMed1997

Pharmacological profile of a novel synthetic inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Pharmacological properties of NK-104 ((+)-monocalcium bis¿(3R,5S,6E)-7-[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolyl++ +]-3,5-dihydroxy-6- heptenoate¿, CAS 147526-32-7), a novel synthetic inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, were investigated. The kinetic study, using rat liver microsomal HMG-CoA reductase, revealed that NK-104 is a competitive inhibitor of HMG-CoA reductase with a Ki of 1.7 nmol/l. To examine the inhibitory effect on sterol synthesis in vivo, de novo synthesis of sterols from [14C]acetate 3 h after oral administration of NK-104 was measured in rats. NK-104 showed marked inhibition in liver (ED50 0.13 mg/kg) and in ileum (ED50 0.20 mg/kg), but much weaker in the other tissues. The inhibitory effect of NK-104 on liver sterol synthesis lasted over 6 h, while that of pravastatin and simvastatin disappeared 6 h after administration of the drugs twice the ED50s. Due to induction of HMG-CoA reductase, initial suppression of hepatic sterol synthesis by pravastatin and simvastatin was compensated, and the cumulative change in hepatic sterol synthesis during 12 h after drug administration was remarkably negative only with long-acting NK-104. Hypolipidemic effects of NK-104 (0.03, 0.1, 0.3 and 1 mg/kg p.o. for 2 weeks) were examined in beagle dogs. NK-104 reduced plasma total cholesterol dose-dependently (13.1, 18.5 and 20.2% at doses of 0.1, 0.3 and 1 mg/kg, respectively), and also plasma triglycerides by 0.1 mg/kg or more. Pravastatin (1 and 3 mg/kg) and simvastatin (3 mg/kg) lowered plasma total cholesterol (14.0, 15.4 and 17.4%, respectively), but did not significantly affect plasma triglyceride levels. These results indicate that NK-104 is a potent, liver-selective, long-acting HMG-CoA reductase inhibitor with a high cholesterol- and triglyceride-lowering potency.

Animals↗
PubMed1995

Effects of efonidipine hydrochloride on cholesterol esterification mediated by beta-very low density lipoprotein in J774 macrophages.

The effects of efonidipine hydrochloride (efonidipine), a dihydropyridine calcium antagonist, on the cholesterol ester metabolism induced by beta-migrating very low density lipoprotein (beta-VLDL) in J774 macrophages were studied. The cholesteryl ester content in the macrophages was increased by incubation with beta-VLDL, and the increase was inhibited by efonidipine. Oleic acid incorporation into cellular cholesteryl ester was increased by beta-VLDL in J774 macrophages. The incorporation at an early phase of beta-VLDL induction (0-3 hr) was inhibited by efonidipine. This inhibitory effect of efonidipine was greater at an early phase of beta-VLDL induction (0-3 hr) than at a late phase of the induction (8-11 hr). Pretreatment of the cells with efonidipine enhanced the inhibitory effect. Efonidipine also inhibited beta-VLDL degradation but not the binding and association in macrophages without pretreatment. beta-VLDL binding and association to macrophages were decreased by pretreatment of the cells with efonidipine. beta-VLDL metabolism was also decreased by dibutyryl cyclic AMP pretreatment. The decrease of beta-VLDL metabolism by efonidipine was prevented by co-treatment with efonidipine and HA1004, a protein kinase A inhibitor. Furthermore, efonidipine increased the intracellular cyclic AMP content in J774 macrophages. These findings suggest that efonidipine suppresses cholesterol ester deposition in atherosclerotic foam cells by inhibiting the modified lipoprotein metabolism and cholesterol esterification mainly through elevation of the cellular cyclic AMP level.

Animals↗