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Biomedical subjects

M Miyake

Publications and source records attributed to M Miyake.

At least 181 records · Page 10Linked to original sources
PubMed1996

Development of porcine blastocysts from in vitro- matured and activated haploid and diploid oocytes.

The purpose of this study was to determine the developmental capacity of electro-activated porcine oocytes. Follicular oocytes collected from gilts at local slaughterhouses were matured for 48 h and were then subjected to a single square pulse of direct current for 100 rhojusec at 1,500 V/cm for activation. To obtain activated diploid oocytes, some were treated with 5.0 micro/ml cytochalasin B for 4 h immediately after electro-activation. The frequency of activation ranged from 96 to 100%. While 91% of activated oocytes that had not been treated with cytochalasin B had 2 polar bodies and a nucleus (haploids), 92% of the oocytes treated with cytochalasin B had only the first polar body and 2 nuclei (diploids). Haploids and diploids were further cultured in TCM-199 medium that contained 10% (v/v) heat- treated fetal calf serum (FCS) and 0.1 mg/ml sodium pyruvate (mTCM) or in Whittenk medium plus 0.4% (w/v) bovine serum albumin (BSA). The frequency of abnormal oocytes was significantly higher in mTCM (83%) than in Whitten's medium (65%) 96 h after the electro-activation (P < 0.01), suggesting that Whitten's medium supported the development of activated oocytes beyond the morula stage. In all cases, several oocytes developed to the blastocyst stage 144 h after electro- activation (1 to 12%). The frequency was significantly higher in the case of diploids cultured in Whitten's medium (12%) (P < 0.01) than in the case of haploids cultured in Whitten's medium (4%), or in the case of haploids cultured in mTCM (1%). The mean number of nuclei per blastocyst was significantly lower in mTCM (haploids, 15; diploids, 16.1) than in Whitten's medium (haploids, 35.7; diploids, 40.1; P < 0.01), suggesting that the number of nuclei in blastocysts was affected by the culture medium.

Journal Article↗
PubMed1996

Increased expression of cyclin D1 and the Rb tumor suppressor gene in c-K-ras transformed rat enterocytes.

Activating mutations in the c-K-ras gene occur in about 40% of human colorectal carcinomas, yet the role of this oncogene in tumorigenesis is not known. We have developed a model cell culture system to study this problem, utilizing the immortalized but non-tumorigenic epithelial cell line IEC18, originally derived from normal rat intestine epithelium. These cells were cotransfected with the drug resistance selectable marker tk-neo and the plasmid pMIKcys, which encodes a mini human c-K-ras gene (15 kb) containing a cysteine mutation at codon 12. Drug resistant clones were isolated. Clones which also expressed the activated c-K-ras gene displayed a transformed morphology, decreased doubling time, increased level of diacylglycerol, anchorage independent growth in soft agar and an aneuploid karyotype and they were also tumorigenic when injected into nude mice. These clones also displayed increased expression, at both the mRNA and protein levels, of cyclin D1 and Rb. These findings may be of clinical relevance since human colorectal tumors also frequently display increased expression of both cyclin D1 and Rb. This model system may be useful for understanding the role and interrelationship between activation of the c-K-ras oncogene and increased expression of cyclin D1 and Rb in colorectal tumorigenesis.

Animals↗
PubMed1996

Correlation of KAI1/CD82 gene expression with good prognosis in patients with non-small cell lung cancer.

As part of our evaluation of members of the transmembrane 4 super-family as possible prognostic predictors, we performed a retrospective study on the expression of the recently identified KAI1 gene by tumors of the lung. This gene, which is identical to CD82, suppresses tumor metastasis of prostate cancer, and its decreased expression may be involved in malignant progression. We used reverse transcription-PCR to analyze tumor tissues from 151 lung cancer patients; 74 tumors were stage I, 17 were stage II, and 60 were stage III. Our results indicate that while 35 patients had tumors in which the KAI1/CD82 gene was conserved (positive), 116 patients had tumors with reduced gene expression (negative). The overall survival rate of patients with KAI1/CD82-positive tumors was significantly higher than that of patients with KAI1/CD82-negative tumors (77.4% versus 38.5%; P=0.002). Furthermore, the overall survival rate of patients with KAI1/CD82-positive adenocarcinoma was also much higher than that of individuals whose adenocarcinoma had reduced KAI1/CD82 expression (73.4% versus 27.1%;P=0.009). Multivariate analysis with the Cox regression model indicated that KAII/CD82 positivity correlated best with the overall survival rate, except for lymph node status. Our data suggest that high KAII/CD82 gene expression by tumors of the lung may be associated with a good prognosis. These findings complement our earlier studies on MRP-1/CD9, another member of the transmembrane 4 superfamily, whose reduced expression in non-small cell lung cancer appears to be a factor of poor prognosis. This set of observations suggests that assessment of the expression status of KAI1/CD82 and MRP-1/CD9 by tumors may provide prognostic information on the clinical behavior of lung cancer.

Age Factors↗
PubMed1996

Motility-related protein-1 (MRP-1/CD9) reduction as a factor of poor prognosis in breast cancer.

The application of reliable markers is of major importance for predicting the prognosis of and instituting the appropriate postsurgical treatment of patients with breast cancer. Previously we showed that motility-related protein-1 (MRP-1), which is identical to CD9, regulates cell motility, and that cultured tumor cells transfected with MRP-1/CD9 cDNA have low motility and low metastatic potential. In addition, MRP-1/CD9 immunoblotting and immunohistochemical study with breast cancer revealed that MRP-1/CD9 expression diminished as the clinical stage of a given breast cancer advanced and that the MRP-1/CD9 gene and protein expression in the metastatic lymph nodes was strikingly lower than in the primary breast cancers. In this study, we also investigated the expression of MRP-1/CD9 by immunoblotting and immunohistochemical analysis in 143 freshly resected invasive ductal carcinomas of the breast: 52 tumors were stage I, 61 were stage II, and 30 were stage III. Tumors were classified as MRP-1/CD9 positive when a band intensity of >30% compared with positive control cells, ZR-75-30 were evaluated with the antibody M31-15, and those with intensities <30% as negative. Moreover, these results were ascertained by immunostaining. Tumor specimens classified as MRP-1/CD9 positive using Western blotting had >50% of the cancer cells immunostained with M31-15, and those classified as MRP-1/CD9 reduced had <50% of the cancer cells immunostained with M31-15. There were 97 patients with MRP-1/CD9 positive tumors and 46 patients whose tumors had reduced MRP-1/CD9 levels. The disease-free rate of the former group of patients was strikingly higher than that of the latter (84.7% versus 51.4%, P<0.001). Similarly, the overall survival rate was also significantly different between the two groups (93.6% versus 69.6%, P=0.004). Multivariate analysis with the Cox regression model indicated that MRP-1/CD9 positively correlated better with disease-free survival (P<0.001) than estrogen receptor, tumor, and lymph node status. Our data suggest that low MRP-1/CD9 expression by tumors of the breast may be associated with poor prognosis. It is conceivable that testing for MRP-1/CD9 may identify node-negative breast cancer patients who are at high risk for early disease recurrence.

Antigens, CD↗
PubMed1996

Unfarnesylated transforming Ras mutant inhibits the Ras-signaling pathway by forming a stable Ras.Raf complex in the cytosol.

Farnesyltransferase inhibitors cause the growth arrest of ras-transformed cells, but not that of normal cells. To elucidate the mechanism of this differential effect, we examined the effect of accumulation of unfarnesylated Ras in the cytosol by using RasG12V,C186S and RasC186S, which mimic unfarnesylated form of the oncogenic and the normal Ras, respectively. We found that RasG12,C186S inhibited activation and membrane translocation of Raf by forming a stable complex with Raf in the cytosol. In contrast, RasC186S showed inhibitory effect on neither Raf activation nor Raf translocation. These results indicate that unfarnesylated oncogenic Ras interacts with Raf in the cytosol and inhibits its membrane translocation, a crucial step for the Raf activation, while unfarnesylated normal Ras does not.

Cell Line↗
PubMed1996

Morphological study of the human maxillofacial venous vasculature: examination of venous valves using the corrosion resin cast technique.

BACKGROUND: The venous drainage in the human head and neck, structures located superior to the heart, generally does not counter the force of gravity. Accordingly, venous valves in the human head have been reported to be few or nonexistent, and therefore the direction of venous drainage is variable and obscure. METHODS: We investigated whether venous valves were present in 13 heads of human adult cadavers using scanning electron microscopic observation of corrosive resin casts in the human maxillofacial region. This technique afforded the observation of the venous valves. RESULTS: Venous valves were found throughout the facial region. In particular, there were many valves distributed in the lingual, upper and lower labial, facial and pharyngeal veins. The venous valves were particularly well formed at motile parts of the maxillofacial region. CONCLUSIONS: It is suggested that these valves may aid the venous blood return and permit the blood drainage in one direction.

Aged↗
PubMed1996

Sex and age patterns of quantitative ultrasound densitometry of the calcaneus in normal Japanese subjects.

: The speed of sound (SOS), broadband ultrasound attenuation (BUA), and the stiffness index (stiffness), which was derived from SOS and BUA, were measured on the calcaneus using the Achilles ultrasound densitometer (Lunar) in normal Japanese woman (n = 473) and men (n = 218). Sex differences and age-related changes in these variables were investigated. In addition, the bone mineral density (BMD) of the lumbar vertebrae measured by dual X-ray absorptiometry (QDR-2000, Hologic) was compared with SOS, BUA, and stiffness. There were some decreases of ultrasound variables in both young adult men and women. BMD values were similar in young adults of both sexes. BMD was relatively stable in women from 20 to 49 years, then decreased at about 1.5%/year. Ultrasound variables were higher in males than females at all ages, and they decreased from age 20 onward in both sexes with an annual loss of about 0.6%. Age-related changes in SOS, BUA, and stiffness were different from those in BMD in normal Japanese subjects. The correlations between BMD and SOS and BUA and stiffness were higher in women (r = 0.51-0.64) than in men (r = 0. 43-0.49).

Absorptiometry, Photon↗
PubMed1996

Distribution and morphology of valves in the human external jugular vein: indications for utilization in microvascular anastomosis.

PURPOSE: This study examined the valves in the external jugular vein (EJV) that could affect microvascular anastomosis and increase the risk of thrombogenesis. MATERIALS AND METHODS: The EJVs of 57 sides from 36 cadavers aged 40 to 95 years (20 males and 16 females) were dissected. After opening the veins, the distribution and morphology of the valves were investigated using a stereoscopic microscope. The length of the valves and inner diameter of the vessels were measured and related to the location of the valves. RESULTS: Forty-nine (86%) of 57 EJVs were confluent with the subclavian vein. The EJV possessed bicuspid valves, and in 46 (93.9%) of the 49 specimens there were valves at the junction with the subclavian vein, whereas 44 (89.8%) contained valves in the middle portion of the specimen. The length of the valves was less than twice the inner diameter, and some were occasionally only rudimentary. CONCLUSION: Numerous valves exist in the middle portion of the EJV where microvascular anastomoses are performed. When valves are observed in the site of anastomosis, they can be avoided by resecting a length equal to approximately twice the inner diameter.

Adult↗
PubMed1996

Long-term alcohol intake enhances ADP-ribosylation of the multifunctional enzyme, phosphoglucomutase, in rat liver.

Adenosine diphosphate (ADP)-ribosylation is a posttranslational protein modification that, in turn, alters several regulatory proteins in mammalian cells. We demonstrated that long-term alcohol intake enhanced the ADP-ribosylation of a 58-kd protein in rat liver plasma membranes. To assess the biological significance of this phenomenon, we partially purified the 58-kd acceptor protein from solubilized rat liver homogenates by two sequential preparative high-pressure liquid chromatographies. Microsequencing revealed that it was phosphoglucomutase (PGM) (EC 5,4,2,2). This enzyme underwent negligible auto ADP-ribosylation, but the ADP-ribosylation was remarkably increased by adding rat liver plasma membranes. The extent of the increase was greater in alcohol-fed rats than in pair-fed controls, suggesting enhanced enzyme activities toward ADP-ribosylation of PGM after chronic alcohol consumption. Several important enzymes are ADP-ribosylated, after which their activities are modified. The results of this study showed that PGM is a novel substrate for ADP-ribosylation in the liver and that the ADP-ribosylation is increased after chronic alcohol consumption. In view of the variety of roles of PGM in the liver (carbohydrate metabolism and Ca2+ homeostasis), specific roles of this modification in terms of the effects of alcohol on hepatocytes may deserve further investigation.

Adenosine Diphosphate Ribose↗
PubMed1996

Fructose stimulates shedding of cytoplasmic droplets from epididymal boar spermatozoa.

The aim of the present study was to establish the presence of an inducer(s) for the shedding of cytoplasmic droplets from boar spermatozoa after ejaculation. Cauda epididymal spermatozoa were incubated with seminal plasma, seminal vesicular fluid (SVF) or chemical agents at 39 degrees C for 30 min. After fixation and staining, percentages of spermatozoa without a droplet were determined. In the samples incubated with seminal plasma, SVF and a filtrate of SVF obtained after passage through an ultrafilter (molecular weight cut-off, 10,000), 43%, 60-69% and 43% of the spermatozoa were without a droplet respectively. The percentage of spermatozoa without a droplet after incubation with D-fructose (1.0 mM), which was one of the energy substrates included in SVF, was 76%. Furthermore, percentages increased to 93% and 90% with the addition of caffeine (2.0 mM) and N6, 2'-O-dibutyryl cyclic adenosine 3',5'-monophosphate (1.0 mM), respectively, but decreased to 48% with the addition of imidazole (2.0 mM). Based on these results, it is suggested that the shedding of cytoplasmic droplets from boar spermatozoa is induced by fructose originating from SVF. It also appears that this event is mediated by increasing the concentration of intracellular cyclic adenosine 3',5'-monophosphate.

Animals↗
PubMed1996

Influence of exercise and degenerative vertebral changes on BMD: a cross-sectional study in Japanese men.

The effects of recreational exercise and milk intake on BMD were assessed in Japanese men using analysis of covariance with significant predictor variables, selected from age, height, body weight and body mass index (BMI), as covariates. The influence of degenerative vertebral changes on these effects was also evaluated. Recreational exercise was found to be positively associated with BMD at the lumbar spine and the femoral neck, whereas milk intake, a possible indicator of calcium intake in the Japanese, was positively associated with BMD at the radius and the lumbar spine. Not only vertebral BMD but also radial and femoral neck BMD was higher in the subjects with osteophytosis or sclerosis in the lumbar vertebrae. Neither exercise nor lumbar pain was significantly related to these vertebral changes. These data suggest a protective effect of regular recreational exercise and milk intake on bone health in Japanese men.

Adult↗
PubMed1996

[Development of simple on-line [oxygen-15]water infuser].

A [15O]water production and infusion system was newly developed for blood flow study with positron emission tomography. The system utilizes a electrostatic cooling unit to liquify [15O]vapor in a coiled plastic tube. Labeled [15O]water is collected into an infusion syringe by a wash of the trapping tube with saline. The system is simple and efficient; the recovery rate of [15O]water is over 90% at 10 degrees C of cooling temperature.

Cerebrovascular Circulation↗
PubMed1996

Linkage-disequilibrium mapping narrows the Fukuyama-type congenital muscular dystrophy (FCMD) candidate region to <100 kb.

Fukuyama-type congenital muscular dystrophy (FCMD), the second most common form of muscular dystrophy in Japan, is an autosomal recessive severe muscular dystrophy associated with brain anomalies. After our initial mapping of the FCMD locus to chromosome 9q31-33, we have further defined the locus within a approximately 5-cM region between D9S127 and D9S2111 and have found linkage disequilibrium between FCMD and D9S306 in this candidate region on 9q31. The high prevalence of FCMD among the Japanese, who are a relatively isolated population, provides an opportunity to utilize linkage-disequilibrium mapping. We developed three new microsatellites, near D9S306, from the FCMD YAC contig, determined their positions on YACs, and performed linkage-disequilibrium mapping with these markers and other newly published loci. The maximum value of p(excess), which represents the strength of linkage disequilibrium, was obtained at D9S2107; and this value showed a relatively steady rise and fall across the region that is likely to contain FCMD. Distances between FCMD and each marker were presumed to be approximately 1 Mb, approximately 350 kb, approximately 140 kb, approximately 20 kb, approximately 280 kb, approximately 450 kb, and approximately 740 kb for D9S306, A107XF9, D9S2105, D9S2107, D9S172, D9S299, and D9S2109, respectively. Haplotype analysis using the three closest markers D9S2105, D9S2107, and D9S172 indicated that most FCMD-bearing chromosomes are derived from a single ancestral founder and suggested that these markers can be used for the diagnosis of sporadic FCMD. Thus, the FCMD gene is most likely to lie within a region of <100 kb containing D9S2107.

Alleles↗
PubMed1995

Characterization of the binding of HU and IHF, homologous histone-like proteins of Escherichia coli, to curved and uncurved DNA.

The binding of E. coli histone-like protein HU to curved and uncurved DNA fragments containing adenine tracts was characterized by relative binding affinity assay, and compared with that of other homologous histone-like protein integration host factor (IHF). Both HU and IHF have about 3- to 5-fold higher affinity for overall curved DNA fragments such as (A6N4)11 and (A3T3N4)12 compared to a standard duplex fragment with mixed sequence. The binding manner of HU to the curved fragments was highly cooperative. However, loss of overall curvature for shorter fragments (< approximately 100 bp) reduced the preference of HU binding to curved (A3T3N4)n over uncurved (T3A3N4)n, indicating that the binding specificity of HU to curved DNA is length-dependent. Thus, the curved DNA configuration of the whole molecule facilitates the binding of several HU molecules to form the hierarchy of HU-DNA complex. Furthermore, it was shown that HU and IHF bind less well to (A6N9)n, which has a zig-zag straight structure, whereas they preferentially bind to uncurved (T3A3N4)14. These results suggested that not only intrinsically overall curvature but also the preferred orientations for DNA bending in the protein-DNA complex are important factors for affinities of HU and IHF.

Bacterial Proteins↗