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Biomedical subjects

K Miyazaki

Publications and source records attributed to K Miyazaki.

At least 55 records · Page 3Linked to original sources
PubMed2001

Matrilysin stimulates DNA synthesis of cultured vascular endothelial cells and induces angiogenesis in vivo.

Matrilysin produced by human colon cancer cells may be involved in the progression and metastasis of cancer. In the present study, we investigated the association of matrilysin with angiogenesis. One microgram of recombinant matrilysin is confirmed to have increased [3H]-thymidine uptake in human umbilical vein endothelial cells. Then we used micro encapsulation and a mouse hemoglobin enzyme-linked immunosorbent assay system for in vivo quantitation of angiogenesis with BALB/c nu/nu athymic mice. Hundred micrograms of recombinant matrilysin induced angiogenesis to the same degree as 10 microg of basic fibroblast growth factor (bFGF). Angiogenesis was observed at the site implanted with human colon cancer WiDr cells in agarose micro beads. This was inhibited by subcutaneous injection of matrilysin-specific antisense oligonucleotide significantly by 53%. In conclusion, matrilysin may be associated with angiogenesis of human colon cancer through the direct proliferative action on endothelial cells.

Animals↗
PubMed2001

Exact calculation of the linear term in the density expansion of the dynamic structure factor of a dilute gas.

We evaluate the linear term in the density expansion of the dynamic structure factor for a classical gas using a generalized Enskog theory developed by one of the authors, which enables us to describe the dynamics at small time and length scales where the use of the Boltzmann equation is severely limited. Agreement of the theory with experiment and simulation is very good. We find that the linear term is very sensitive to the shape of the potential so that the dynamic structure factor can serve as a good probe to determine the intermolecular potentials of dilute gases.

Journal Article↗
PubMed2001

Retention of multilineage differentiation potential of mesenchymal cells during proliferation in response to FGF.

Mesenchymal stem cells (MSC) that can differentiate to various connective tissue cells may be useful for autologous cell transplantation to defects of bone, cartilage, and tendon, if MSC can be expanded in vitro. However, a short life span of MSC and a reduction in their differentiation potential in culture have limited their clinical application. The purpose of this study is to identify a growth factor(s) involved in self-renewal of MSC and the maintenance of their multilineage differentiation potential. Fibroblast growth factor-2 (FGF-2) markedly increased the growth rate and the life span of rabbit, canine, and human bone marrow MSC in monolayer cultures. This effect of FGF-2 was more prominent in low-density cultures than in high-density cultures. In addition, all MSC expanded in vitro with FGF-2, but not without FGF-2, differentiated to chondrocytes in pellet cultures. The FGF+ MSC also retained the osteogenic and adipogenic potential throughout many mitotic divisions. These findings suggest that FGFs play a crucial role in self-renewal of MSC.

Animals↗
PubMed2001

Validation of an analytical method for a potent antitumor agent, TZT-1027, in plasma using liquid chromatography-mass spectrometry.

A sensitive and specific analytical method for a potent antitumor agent, TZT-1027, in plasma has been developed using liquid chromatography-mass spectrometry (LC-MS) with [2H4]TZT-1027 as an internal standard (I.S.). A plasma sample was purified by solid-phase extraction on a C18 cartridge, followed by solvent extraction with diethyl ether. The extract was then injected into the LC-MS system. Chromatography was carried out on a C18 reversed-phase column using acetonitrile-0.05% trifluoroacetic acid (TFA) (55:45) as a mobile phase. Mass spectrometric analysis was performed in atmospheric pressure chemical ionization (APCI) mode with positive ion detection, and the protonated molecular ions ([M+H]+) of TZT-1027 and I.S. were monitored to allow quantitation. The method was applied to the determination of TZT-1027 in human, monkey, dog, rat and mouse plasma. As far as the sample preparation was concerned, good recoveries (73.5-99.1%) were obtained. The calibration curves were linear over the range of 0.25-100 ng per 1 ml of human, dog and rat plasma, per 0.5 ml of monkey plasma, and per 0.1 ml of mouse plasma. From the intra- and inter-day accuracy and precision, the present method satisfies the accepted criteria for bioanalytical method validation. TZT-1027 was stable when stored below -15 degrees C for 6 months in human plasma and for 3 weeks in plasma from other species. TZT-1027 was also stable in plasma through at least three freeze-thaw cycles.

Animals↗
PubMed2001

Deoligomerization of siloxanes with dimethyl carbonate over solid-base catalysts.

Hexamethyldisiloxane and hexamethylcyclotrisiloxane were almost completely deoligomerized with dimethyl carbonate over alumina-supported potassium fluoride catalyst to form methoxytrimethylsilane (85% yield) and dimethoxydimethylsilane (94% yield), respectively, and moreover the addition of a small amount of methanol to the hexamethyldisiloxane deoligomerization reaction enhanced the methoxytrimethylsilane yield to 98%.

Journal Article↗
PubMed2001

Different features of angiogenesis between ovarian and breast carcinoma.

Angiogenesis assessed by immunohistochemical staining for endothelial cells has been widely accepted as an independent prognostic factor in human breast carcinoma. However, the clinicopathologic significance of angiogenesis is still being argued in ovarian carcinoma. Therefore, we retrospectively analyzed the clinicopathologic significance of angiogenesis in ovarian carcinoma compared with that in breast carcinoma. After vessels were stained with CD34-monoclonal antibody, the areas with the highest number of intratumoral microvessels were assessed in a 200x field in 42 ovarian carcinoma and 41 breast carcinoma. Intratumoral microvessel density (IMD) in ovarian carcinoma was significantly lower than that in breast carcinoma. Further, the difference of IMD from tumor to tumor in ovarian carcinoma was smaller than that in breast carcinoma. IMD was correlated with tumor grade, but not with other clinicopathologic variables in ovarian carcinoma. Although the patients with high-IMD tumor revealed a poorer prognosis than those with low-IMD tumor in breast carcinoma, IMD had no influential effects on the survival of the patients with ovarian carcinoma. Our comparative analysis of IMD in ovarian carcinoma with that in breast carcinoma indicates that angiogenesis may play an important role in the transient of ovarian neoplasms, but not in the progression of ovarian carcinomas, and that the biological roles of angiogenesis might be different, depending on histologic subtype.

Adult↗
PubMed2001

Patterns of adaptation in a laboratory evolved thermophilic enzyme.

The heat sensitive psychrophilic protease subtilisin S41 was previously subjected to three rounds of mutagenesis/recombination and screening, resulting in variant 3-2G7, whose half-life at 60 degrees C is approx. 500 times that of wild-type. Here we report the results of five additional generations of laboratory evolution starting from 3-2G7. The half-life of 8th generation enzyme 8-4A9 at 60 degrees C is 1200 times that of wild-type, and slightly more than twice that of 3-2G7. This half-life is >20-fold greater than those of homologous mesophilic subtilisins SSII and BPN'. Circular dichroism melting curves indicate that subtilisin 8-4A9 unfolds at temperatures approx. 25 degrees C higher than wild-type. It is also substantially more resistant to proteolysis at 30 degrees C. Nearly half of the 13 amino acid substitutions accumulated in 8-4A9 involve the mutation of serine residues. This mirrors a pattern observed in natural proteins, where serines are statistically less prevalent in thermophilic enzymes compared to mesophilic ones.

Amino Acids↗
PubMed2001

Expression of angiomodulin (tumor-derived adhesion factor/mac25) in invading tumor cells correlates with poor prognosis in human colorectal cancer.

Angiomodulin (tumor-derived adhesion factor/mac25/insulin-like growth factor binding protein-7), a cell-adhesive glycoprotein, is secreted by cancer cells and vascular endothelial cells. It may be involved in angiogenesis and modulation of the vascular functions necessary for tumor development. Although angiomodulin is expressed in colon cancer, there is limited information on it concerning cancer progression. In the present immunohistochemical study, we examined expression of angiomodulin in human colorectal cancer and its relationship with prognosis. A group of 89 surgically resected colorectal cancers was investigated immunohistochemically. In 37 cases (41.6%), angiomodulin was expressed in invading cancer cells. Early recurrence within 12 months after surgery was higher in patients with angiomodulin-expressing cancer than in those without (p < 0.05). The Kaplan-Meier life table revealed that patients with angiomodulin-positive tumor cells had a shorter survival time than those with negative cells (p < 0.01). The prognosis of patients with Dukes' C and angiomodulin-positive cells was apparently worse than that of patients with Dukes' D and angiomodulin-negative cells. Multivariate analysis with logistic regression indicated that only angiomodulin expression in cancer cells, lymph node metastasis and age remained significant prognostic variables for survival (p < 0.05). Angiomodulin showed correlations with poor prognosis, indicating that it may be a useful prognostic marker in patients with colorectal cancer.

Adult↗
PubMed2001

Development of a new system for prediction of drug absorption that takes into account drug dissolution and pH change in the gastro-intestinal tract.

A new system for prediction of drug absorption that takes into account drug dissolution and pH change in the gastro-intestinal tract was developed. In this new system, a drug (solid form) is added into a drug-dissolving vessel (pH 1.0) and the dissolved drug is transferred to a pH adjustment vessel (pH 6.0). Then the drug solution is transferred to the apical surface of Caco-2 cells, and the permeation rate of the drug across a Caco-2 monolayer is determined. This system was able to predict the oral absorption ratios of ten water-soluble drugs in humans. Using this system, it was predicted that drugs that permeated Caco-2 at a rate of more than 0.1% of the dose in 200 min would be almost completely absorbed after oral administration in humans. For a drug whose permeation ratio was less than 0.03%, the absorption ratio was predicted to be less than 30%. This system also enabled prediction of the absorption rate and variability in the absorption of albendazole, a drug with poor water solubility. It also enabled assessment of the improvement in absorption using a solid dispersion of albendazole-polymers that improved the water solubility. The results suggest that this system is useful for oral absorption screening of new drugs and pharmaceutical products.

Administration, Oral↗
PubMed2001

Conservative therapy for adenocarcinoma and atypical endometrial hyperplasia of the endometrium in young women: central pathologic review and treatment outcome.

Thirty-nine patients with endometrioid adenocarcinoma (EA) and atypical hyperplasia (AH) of the endometrium who received conservative treatment to preserve fertility were collected from member institutions of the Japan Gynecologic Oncology Study Group. Twenty-nine and ten were originally diagnosed with EA without myometrial invasion and AH, respectively. We performed a central pathological review to make definite diagnoses, and the diagnosis of EA in 29 cases was changed to AH in ten, complex hyperplasia in three and atypical polypoid adenomyoma in three, and AH in ten was changed to EA in one and simple hyperplasia in one. Nine of 12 women (75%) with EA and 15 of 18 women (83%) with AH had an initial response to medroxyprogesterone acetate (MPA) treatment. Two of nine responders with EA later developed relapse, and one of them had metastasis to the left obturator lymph node. Two became pregnant, and one delivered one full-term infant. One of the responders with AH had a relapse in the endometrium. Five became pregnant, and four delivered four normal infants. The young women with endometrial carcinoma localized in the endometrium who wish to preserve fertility may be treated as successfully with MPA as those with AH.

Adenocarcinoma↗
PubMed2001

Allelotype analysis of gallbladder carcinoma associated with anomalous junction of pancreaticobiliary duct.

Anomalous junction of pancreaticobiliary duct (AJPBD) patients has an increased risk of gallbladder and bile duct carcinomas. However, the relevance of carcinoma with AJPBD is not fully clarified. We performed analysis of loss of heterozygosity (LOH) at p53 locus and immunohistochemistry of p53 and K-ras gene mutation in five cases of gallbladder carcinoma associated with AJPBD. LOH of p53 locus and overexpression of p53 were detected in two out of five (40%) and five out of five (100%), respectively, in the present study. K-ras gene mutation at codon 12 and 13 was not detected (0%, 0/5). These results suggest that aberrations of p53 are involved in carcinogenesis of gallbladder carcinoma associated with AJPBD. Next, in order to find the genetic events besides K-ras mutation and overexpression of mutant p53 in this disease, LOH analysis was performed using 72 microsatellite markers. High frequency of allelic loss (> 50%) was found on 2p (81.8%), 4p (50%), 4q (50%), 8q (60%), 9q (50%), 10p (50%), 14p (60%), 14q (50%), 16p (60%), 19p (50%), 21p (50%) and Xp (66.6%). The highest deletion regions on chromosome 2p24 (3/3, 100%), 14q22 (3/4, 75%) and 21q22 (3/4, 75%) were found. The present study suggests that gallbladder carcinoma associated with AJPBD has high frequent allelic loss and has two new regions which may harbor putative tumor suppressor genes.

Aged↗
PubMed2001

Dynamic structure factor of a dilute Lennard-Jones gas.

We calculate the leading correction term in the density expansion of the dynamic structure factor for a Lennard-Jones fluid using the Boltzmann equation. The qualitative behavior is found to be very similar to the hard-sphere result reported by Kamgar-Parsi et al. [Phys. Rev. A 35, 4781 (1987)]. A comparison was made with the results from the neutron scattering experiment by Verkerk et al. [Phys. Rev. Lett. 67, 1262 (1991)] for dilute argon gases. Several possibilities to explain the discrepancies of the present results from the experiment are proposed.

Journal Article↗
PubMed2001

Identification, tissue expression, and chromosomal position of a novel gene encoding human ubiquitin-conjugating enzyme E2-230k.

Through large-scale human genome mapping and sequencing of a region at chromosome 17q25.1 that is of particular interest because genes associated with breast, ovarian, and esophageal cancer are likely to be located there, we isolated the cDNA of a novel member of the family of ubiquitin-conjugating enzymes. The predicated peptide showed 97% identity in amino-acid sequence to murine ubiquitin-conjugating enzyme E2-230k (Mus UBE2-230k). The human cDNA consisted of 4878 nucleotides with an open reading frame encoding 1138 amino acids; the approximately 5 kb transcript was expressed predominantly in skeletal muscle and heart. The predicted UBE2-230k peptide contained a motif typical of the UBC domain that has been implicated in the ubiquitin-dependent proteolytic system and related pathways.

Amino Acid Sequence↗
PubMed2001

Application of chemical selective cleavage methods to analyze post-translational modification in proteins.

Three chemical specific cleavage reactions, one for the carboxyl side of aspartyl peptide bonds, one for the carboxyl side of asparaginyl peptide bonds and another for the amino side of seryl/threonyl peptide bonds have been recently established. Additionally, these reactions simultaneously react on several post-translationally modified groups in peptides or proteins. The modified groups cover the external modifications N-formyl, N-acetyl, N-pyroglutamyi residues and C-terminal-alpha amide, as well as the internal modifications such as O-acetyl serine, phosphorylated serine/tyrosine, sulfonylated tyrosine, glycosylated serine/threonine and glycosylated asparagine. These three cleavage reactions relate to key amino acids for modifications, deamidation for asparagine, phosphorylation and acetylation for serine, and glycosylation for asparagine, serine and threonine. The chemical reactions on these modifications change the peptide mapping pattern, and information from these reactions may contribute characterization and location of post-translational modified groups in the protein.

Animals↗
PubMed2001

Comprehensive allelotype study of ovarian tumors of low malignant potential: potential differences in pathways between tumors with and without genetic predisposition to invasive carcinoma.

Ovarian tumors of low malignant potential (LMP) are intermediate between adenomas and ovarian carcinomas (OC); however, the relevance of LMP to ovarian carcinogenesis is not clear. We performed a comparative analysis of allelotypes in 50 cases of LMP (42 mucinous and 8 serous) and 23 cases of OC (15 mucinous and 8 serous) to investigate any differences in genetic changes. Analysis of loss of heterozygosity (LOH) using 25 microsatellite markers reportedly associated with OC revealed that the total LOH frequency at each marker was significantly lower in LMP than in OC (p < 0.01). However, 9 (36%) loci showed higher LOH frequency in mucinous LMP than in mucinous OC. A genome-wide scan for LOH using 91 microsatellite markers and fine mapping revealed that LOH at D7S1805 (7q35) is characteristic of mucinous LMP (19.4% in mucinous LMP, 8.3% in mucinous OC). We further studied LOH in 3 cases of mucinous OC that were accompanied by mucinous LMP lesions. In 2 cases, LOH frequency was higher in the carcinoma portion than in the morphologically LMP portion. The other case showed microsatellite instability in the morphologically LMP portion and LOH in the carcinoma portion. Our results suggest the presence of an LMP-to-OC developmental sequence and the existence of a subset of LMP that does not develop into OC in the mucinous subtype of ovarian tumors.

Adenoma↗
PubMed2001

Expression of serine proteinase inhibitor PP5/TFPI-2/MSPI decreases the invasive potential of human choriocarcinoma cells in vitro and in vivo.

OBJECTIVE: PP5/TFPI-2/MSPI is a Kunitz-type serine proteinase inhibitor with broad inhibitory spectra, abundantly produced by placenta and detected in the blood of pregnant women. Expression of PP5/TFPI-2/MSPI is exclusively detected in syncytiotrophoblasts of placenta, but is barely detectable in choriocarcinoma cells, a trophoblast-derived malignant tumor. Chromosome 7, in which the PP5/TFPI-2/MSPI gene is localized, is frequently lost in various types of tumors. We attempted to elucidate the relation between PP5/TFPI-2/MSPI expression and the malignant properties of choriocarcinoma cells. METHODS: Human choriocarcinoma cells, JAR, were transfected with either a human PP5/TFPI-2/MSPI expression vector or an empty vector, and stable clones were obtained. Messenger RNA expression, protein secretion/localization, growth rate, and plating efficiency were evaluated. In vitro migration and invasive activity were determined by transwell chamber experiments. In vivo tumor growth was evaluated by the subcutaneous injection of cells to nude mice and followed by histological examination. RESULTS: Expression of mRNA and protein of PP5/TFPI-2/MSPI were confirmed, and a high producing clone and a low producing clone were chosen for further analysis. The majority of secreted PP5/TFPI-2/MSPI protein was revealed to associate with the extracellular matrix. Expression of PP5/TFPI-2/MSPI did not affect the growth and migration of the tumor cells, but enhanced their plating efficiency. Its expression significantly inhibited invasion through the Matrigel. Invasive growth into the subcutaneous muscle layer was not evident in the nude mouse tumors of the PP5/TFPI-2/MSPI-expressing cells. CONCLUSION: PP5/TFPI-2/MSPI-expressing choriocarcinoma cells showed suppressed potential of invasion in vitro and in vivo. It is suggested that loss or suppression of PP5/TFPI-2/MSPI expression may result in the acquisition of invasiveness in choriocarcinoma cells.

Animals↗
PubMed2001

Enantioselective binding of disopyramide to alpha1-acid glycoprotein and its variants.

OBJECTIVE: alpha1-acid glycoprotein (AAG) has three main genetic variants, F1, S, and A variants. There are few reports on the correlation between AAG variants and binding activity of drug enantiomers. We studied the differences between the binding characteristics of enantiomers of disopyramide (DP), which is a basic drug. The aim of this study was to elucidate the cause of the differences between the binding characteristics of DP enantiomers. METHODS: The variants in human AAG were separated by hydroxyapatite chromatography. Binding of DP enantiomers to AAG variants was studied by the ultrafiltration method. The characteristics of the binding of DP enantiomers to total variants and each variant were examined by Scatchard analysis within a range of concentrations from 0.5 to 50.0 microg/ml. RESULTS: The binding capacity of S-DP was significantly higher than that of R-DP in variant 3, although the binding capacities of DP enantiomers were almost the same in variant 2. On the other hand, the binding capacities for both S-DP and R-DP in variant 3 were significantly higher than those in variant 2. Furthermore, there was an almost 2.4-fold difference in the dissociation constant (Kd) between S-DP and R-DP in variant 3, although no significant difference was observed in the number of binding sites (N). In variant 2 no significant differences between DP enantiomers were observed in either the dissociation constant or number of binding sites per molecule of AAG. On the other hand, significant differences between variants 2 and 3 in the dissociation constant for both S-DP and R-DP were observed. The differences in dissociation constant between variants 2 and 3 were 4.0-fold in S-DP and 1.7-fold in R-DP. CONCLUSION: The difference between the binding capacities of S-DP and R-DP is due to differences in the association of DP to variants 3-6, and the role of the variants 1 and 2 in the binding of drugs to AAG is minor.

Anti-Arrhythmia Agents↗