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Biomedical subjects

M Hayakawa

Publications and source records attributed to M Hayakawa.

At least 37 records · Page 2Linked to original sources
PubMed2002

[Extraction of bisphenol-A from a cardiopulmonary bypass circuit].

We measured levels of bisphenol-A (BPA) in a priming solution and blood of a cardiopulmonary bypass (CPB) circuit. Eight circuits were used in the study of a priming solution. Blood samples were obtained from 6 patients who underwent open heart surgery after the commencement of CPB and at the termination of CPB. Another 3 samples were collected directly from the saline in a polyethylene container as a control. Then the concentrations of BPA in them were determined by means of gas chromatography. No detectable BPA was found in controls. However, a small amount was detected in the saline from the circuits (0.9 +/- 1.1 micrograms/l). A very small amount was also detected in the blood after the commencement and at the termination of CPB (0.3 +/- 0.2 microgram/l, and 0.4 +/- 0.3 microgram/l, respectively). BPA was considered to be leached from the circuit to the priming solution and the blood because the parts of the reservoir and the oxygenator were made of polycarbonate containing BPA. We suppose the BPA concentration is probably at a safe level. However, the data on the endocrinologically toxic level of blood BPA are insufficient. Therefore, the use of plastic in a circuit may require closer scrutiny to determine whether BPA contributes to exposure to xenoestrogens.

Benzhydryl Compounds↗
PubMed2002

Detection of peritoneal micrometastases by reverse transcriptase-polymerase chain reaction targeting carcinoembryonic antigen and cytokeratin 20 in colon cancer patients.

Peritoneal recurrence after curative resection of malignant tumor with negative cytology is considered to be caused by microscopic dissemination of the exfoliated cancer cells from primary tumors to serosal surfaces at the time of operation, not detectable with conventional diagnostic tools. We applied the reverse transcriptase-polymerase chain reaction (RT-PCR) for carcinoembryonic antigen (CEA) and cytokeratin 20 (CK 20) to detect micrometastatic foci in the peritoneal cavity of colon cancer patients. Cytological samples taken by peritoneal lavage from a series of 79 colon cancer patients were analyzed microscopically, for CEA levels, and by RT-PCR analysis using nested primers for CEA and CK 20. Cases with both CEA and CK 20 signals were defined as PCR-positive. This RT-PCR method proved both sensitive (1 tumor cell/10(6) non-tumor cells on preparation of serial colorectal cancer cell dilutions) and specific (no false positive results, 0/23 tested in our control experiment). Intraperitoneal micrometastatic cells were detected in peritoneal lavage 7.6% by cytology, 17.7% by CEA levels, and 24.1% by RT-PCR (significantly higher than by cytology: p=0.0046). RT-PCR detection rate increased in parallel with pathological depth of tumor invasion, and also a pathological stage-dependence was suggested according to the tumor-node-metastasis classification of the International Union Against Cancer. Our results suggest that CEA and CK 20 mRNA identification by RT-PCR appeared to be reliable and may be useful for early diagnosis in peritoneal dissemination of colon cancer.

Aged↗
PubMed2001

Three novel mutations of the RPGR gene exon ORF15 in three Japanese families with X-linked retinitis pigmentosa.

We describe three new mutations in a recently identified exon, ORF15, of the retinitis pigmentosa GTPase regulator gene (RPGR) in three unrelated Japanese families (Families 1-3) with X-linked retinitis pigmentosa (XLRP). The affected males had typical retinitis pigmentosa (RP), whereas the obligate carrier females showed a wide clinical spectrum, ranging from minor symptoms to severe visual disability. Some carrier females in Families 1 and 2 showed typical RP, most carriers manifested high myopia and astigmatism, and their corrected visual acuity was insufficient. They showed an impairment of cone function following the rod dysfunction and accompanied by refractive errors. Microsatellite analysis of Family 1 revealed that the RP in the family was linked to the RP3 locus. Although one patient in the family had no mutation in the previously published exons 1-19 including exon 15a, he had a single-nucleotide insertion in exon ORF15 (g.ORF15 + 753-754 insG). Likewise, patients in Families 2 and 3 had two-base insertion/deletion in the exon, i.e., g.ORF15 + 833-834delGG and g.ORF15 + 861-862insGG, respectively. These insertional/deletional mutations observed in the three families are all different and new, and are predicted to lead to a frameshift, resulting in a truncated protein. These findings may support the previous hypothesis that RPGR-ORF15 is a mutational hot spot.

Adult↗
PubMed2001

Tissue distribution and subcellular localization of a variant form of the human ST2 gene product, ST2V.

The human ST2 gene has been known to encode three splice variants; namely, a soluble secreted form of ST2, a transmembrane form of ST2L, and ST2V of undetermined localization. Therefore, analysis of tissue distribution and subcellular localization of ST2V is important to elucidate functional relationships among the three splice variants of the human ST2 gene. RT-PCR procedure revealed that ST2V is predominantly expressed in the stomach, small intestine, and colon. Transfection of ST2V cDNA into COS7 cells in the presence of [(35)S] methionine and cysteine produced radiolabeled 40 kDa protein, which is recognized by specific monoclonal antibody against human ST2. Subcellular fractionation analysis showed that ST2V protein was distributed in the insoluble fraction of the cell lysate. Finally, ST2V protein was detected on the plasma membrane of COS7 cells, which had been transfected with ST2V cDNA, by confocal laser microscopic analysis. These findings taken together, indicate that ST2V protein localizes on the plasma membrane, suggesting its possible role in modification of the ST2L-signaling pathways.

Alternative Splicing↗
PubMed2001

Influence of natural, electrically neutral lipids on the potentiometric responses of cation-selective polymeric membrane electrodes.

Ionophore-free ion exchanger electrodes were found to exhibit quite a high selectivity for the creatininium ion; however, measurements in diluted urine samples revealed large emf drifts. Potentiometric, chromatographic, NMR, and mass spectrometric evidence did not reveal any major cationic interfering agents, and anionic interfering agents cannot trivially explain the consistently positive emf drifts. Ultrafiltration of urine samples showed that the interfering agents have molecular weights below 1000 u. The drifts are apparently caused by electrically neutral lipophilic compounds of low molecular weight that are easily extracted into organic phases. Follow-up experiments showed that p-cresol and cholesterol cause no significant emf responses but that coproporphyrin, phosphatidylserine, taurocholic acid, cholic acid, phosphatidylethanolamine, and octanoic acid cause positive emf drifts of the type that was observed with the urine samples. The extent of the responses and the response time depend not only on the specific compound but also on the cation in the sample solution. These results suggest that the emf drifts are due to extraction of such natural lipids into the organic membrane phase where they interact in an ionophore-like fashion with the analyte and interfering ions. Changes in the potentiometric selectivities after contact with natural lipids support this interpretation. The same effect of natural lipids is also expected for ionophore-based electrodes. Indeed, exposure of a valinomycin-based electrode to a methylene chloride extract of urine resulted in a significant reduction of the Na+ discrimination, increasing log Kpot(K,Na) from -3.9 to -3.1.

Cations↗
PubMed2001

Neutral endopeptidase inhibits neuropeptide-mediated transactivation of the insulin-like growth factor receptor-Akt cell survival pathway.

G-protein coupled receptor (GPCR) agonists such as neuropeptides activate the insulin-like growth factor-1 receptor (IGF-IR) or the serine-threonine protein kinase Akt, suggesting that neuropeptides-GPCR signaling can cross-communicate with IGF-IR-Akt signaling pathways. Neutral endopeptidase 24.11 (NEP) is a cell-surface peptidase that cleaves and inactivates the neuropeptides endothelin-1 (ET-1) and bombesin, which are implicated in progression to androgen-independent prostate cancer (PC). We investigated the mechanisms of NEP regulation of neuropeptide-mediated cell survival in PC cells, including whether neuropeptide substrates of NEP induce phosphorylations of IGF-IR and Akt in PC cells. Western analyses revealed ET-1 and bombesin treatment induced phosphorylation of IGF-IRbeta and Akt independent of IGF-I in TSU-Pr1, DU145, and PC-3 PC cells, which lack NEP expression, but not in NEP-expressing LNCaP cells. Recombinant NEP and induced NEP expression in TSU-Pr1 cells using a tetracycline-repressive expression system inhibited ET-1-mediated phosphorylation of IGF-IRbeta and Akt, and blocked the protective effects of ET-1 against apoptosis induced by serum starvation. Incubation of TSU-Pr1 cells with specific kinase inhibitors together with ET-1 or bombesin showed that IGF-IR activation is required for neuropeptide-induced Akt phosphorylation, and that neuropeptide-induced Akt activation is predominantly mediated by Src and phosphatidylinositol 3-kinase but not by mitogen-activated protein kinase or protein kinase C. These data show that the neuropeptides ET-1 and bombesin stimulate ligand-independent activation of the IGF-IR, which results in Akt activation, and that this cross-communication between GPCR and IGF-IR signaling is inhibited by NEP.

Bombesin↗
PubMed2001

Coexpression of the partial androgen receptor enhances the efficacy of prostate-specific antigen promoter-driven suicide gene therapy for prostate cancer cells at low testosterone concentrations.

The prostate specific antigen (PSA) promoter/enhancer has been clearly demonstrated to be tissue specific, and has been applied to prostate-specific gene therapy. However, the transcription of the PSA gene is strictly androgen dependent, and its promoter activity is very weak at low concentrations of testosterone, which are generally observed in prostatic cancer patients treated with androgen deprivation. In this study, we used a partial androgen receptor (ARf) containing amino acids 232-429 and 481-657 to transactivate the PSA gene without androgens. We made two expression vectors, ARfPPLUC and ARfPPTK. They contained ARf cDNA driven by cytomegalovirus promoter and cDNAs of either firefly luciferase (LUC) or herpes simplex virus thymidine kinase (TK) driven by PSA promoter/enhancer (PP). The expressed ARf enhanced the PP activity by about 110-fold in the PSA-producing prostate cancer cell line, LNCaP, under low testosterone concentrations. Moreover, in a PSA-nonproducing prostate cancer cell line, DU145, ARf also enhanced the PP activity by about 60-fold in an androgen-independent manner. In a growth inhibition assay, ARfPPTK treated with ganciclovir was found to inhibit the cell growth of LNCaP cells much more effectively than PPTK. Furthermore, in contrast to PPTK, ARfPPTK also had an inhibitory effect on DU145 cells. This system is thus considered to provide a useful therapeutic option in patients with prostate cancer who are receiving hormonal therapy.

Cell Division↗
PubMed2001

Evaluation of the electroosmotic medium pump system for preparative disk gel electrophoresis.

This paper describes an improved electroosmotic elution system for preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) based on the epochal idea of H. V. Tan et al. (Nucleic Acids Res. 1988, 16, 1921-1930). In this elution system, a semipermeable membrane, mounted under the gel terminal end, works as the elution pump as well as the partition of the elution chamber. We refer to this system as the "electroosmotic medium pump system." Operation of the constructed apparatus (3.6 cm i.d. disk gel column) and resolution of the protein bands were examined by separation of the model protein mixture (bovine serum albumin (BSA), ovalbumin, bovine carbonic anhydrase, soybean trypsin inhibitor) and purification of the membrane protein, dipeptidyl peptidase IV (DPP IV). The Spectra/Por 7 dialysis membrane provided a better flow profile for the elution buffer. The four model proteins of the protein mixture were able to be completely separated from each other and recovered without dilution. The maximum protein concentration of eluate achieved was 93 mg/ml, when applying a single component, BSA fraction V, as a sample. Furthermore, the multifunctional ectoenzyme, DPP IV, was purified in a single step.

Animals↗
PubMed2001

Spider silk has an ice nucleation activity.

Several ice nucleating substances have been identified, which exist in vivo or can be extracted from biological materials. Spider silk, which has a strong ability for water condensation, has also been found to possess an ice nucleation activity. The freezing temperature of water droplets was higher in the presence than in the absence of spider silk. Moreover, by means of environmental scanning electron microscopy, it was observed that the activity is not due to foreign matter attached to the silk but to the silk fibroin itself.

Animals↗
PubMed2001

Gastrointestinal stromal tumor of the rectum.

Gastrointestinal stromal tumors (GISTs) are characterized by remarkable variability in their differentiation potential, but most of these lesions do not display convincing smooth muscle or neuronal differentiation. The GISTs arising from the rectum or anal canal are extremely uncommon. We present a case of immunohistochemically proven GIST of the rectum, which was characterized by homogenous isointensity mass without necrosis or hemorrhage on T2-weighted image and by enhancement on gadolinium-enhanced study.

Carcinoma↗
PubMed2001

Can 3D-CTA surpass DSA in diagnosis of cerebral aneurysm?

After introducing of Helical scanning CT (HCT) and 3D-CTA (three-dimensional CT angiography), various improvements to these methods have been made every year to achieve better imaging quality. The current resolution permits visualisation of the internal structure of tumors, although as yet insufficiently clearly. We suggest that these improvements can be more efficient than conventional cerebral aneurysms angiography. In this study, we compared HCT and 3D-CTA with conventional cerebral angiography in patients with cerebral aneurysms at our facility. We also examined whether 3D-CTA has the possibility of independent clinical application and can surpass conventional DSA in diagnostic efficacy. In this paper, we found this information insufficient in clipping operations using 3D-CTA only when 1) It was difficult to distinguish a crooked infundibular dilation from an aneurysm, 2) Imaging threshold influenced the measured value of the vascular diameter and 3) It was also difficult to confirm whether the peripheral vessels adhere to the aneurysm. In conclusion, from the result of the comparison between 3D-CTA and DSA in this study, it was concluded that 3D-CTA is a reliable alternative method to conventional angiography in the diagnosis of anterior circulation and most aneurysms of regular size. In such cases it may be possible to obtain the same quality of preoperative information, but it is less invasive.

Adult↗
PubMed2001

Isolation and characterisation of dipeptidyl peptidase IV from Prevotella loescheii ATCC 15930.

A proline-specific dipeptidyl aminopeptidase, dipeptidyl peptidase IV (EC 3.4.14.5), was purified from a cell sonicate soluble fraction of Prevotella loescheii ATCC 15930 by sequential column chromatography. The molecular mass of the native enzyme was estimated as 160 kDa by high-pressure liquid gel filtration column chromatography and unheated sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The subunit molecular mass was 80 kDa when the enzyme was heated to 100 degrees C in the presence of 2-mercaptoethanol before SDS-PAGE, suggesting that the native enzyme consists of two identical subunits and is folded in 2% SDS. The optimum pH, with glycyl-prolyl-4-methyl-coumaryl-7-amide as the substrate, was 8.0; the isoelectric point was 5.2. Purified enzyme showed a strong preference for dipeptide substrates containing proline and, less efficiently, alanine in the P1 position. The enzyme was markedly inhibited by Cd(2+), Zn(2+), Hg(2+), Co(2+), and serine proteinase inhibitor di-isopropylfluorophosphate.

Alanine↗
PubMed2001

Monoclonal antibody against Porphyromonas gingivalis hemagglutinin inhibits hemolytic activity.

Porphyromonas gingivalis has been implicated as an important pathogen in the development of adult periodontitis. This bacterium possesses hemagglutinating and hemolytic activities to attach and lyse erythrocytes. Hemolysis by this oral pathogen functions to provide heme-containing molecules for growth in the periodontal pocket. We previously constructed a monoclonal antibody using P. gingivalis vesicles as the immunogen, designated as MAb-Pg-vc, which inhibited vesicle-associated hemagglutinating activity. Furthermore, we cloned the gene encoding 130-kDa hemagglutinin (130-kDa HAG) and identified its functional motif for attachment to erythrocytes. Generally, bacterial cell attachment to erythrocytes is an important initial step for expressing hemolysis activity. In the present study, we examined the effect of MAb-Pg-vc on the hemolytic activity of P. gingivalis cells. The MAb-Pg-vc significantly inhibited the hemolytic activity and, further, this inhibitory activity was reduced by the synthetic peptide of the 130-kDa HAG functional motif.

Amino Acid Sequence↗
PubMed2001

An integrated method for the enrichment and selective isolation of Actinokineospora spp. in soil and plant litter.

AIMS: To devise and evaluate a method for isolating the rare, zoosporic actinomycetes, Actinokineospora spp. in soil and plant litter. METHODS AND RESULTS: The newly developed method consists of two enrichment stages followed by plating on a selective medium. The source material is initially incubated with calcium carbonate to multiply the population of Actinokineospora spp., and is then air-dried. The second stage consists of rehydration-centrifugation, in which the amended substrate is immersed in phosphate buffer-soil extract to liberate actinomycete zoospores, and nonmotile microbial associates are then eliminated by centrifugation. Portions of the supernatant enriched with zoospores are plated on humic-acid vitamin agar supplemented with fradiomycin, kanamycin, nalidixic acid and trimethoprim. We examined 39 soil and plant-litter samples taken from fields, forests and stream banks. The proposed method consistently enriched and selectively isolated Actinokineospora spp. in 17 samples. Evidence for antimicrobial activity was found in most of the isolates. CONCLUSION: A combination of enrichment and a medium containing selective antibiotics can be used successfully for efficient isolation of certain rare actinomycete taxa. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of new methodologies with which to isolate rare actinomycetes is of great importance to extend our understanding of their ecology, taxonomy and bioactivity.

Actinomycetales↗
PubMed2001

Presence of a genistein-responsive inhibitory mechanism on interleukin-1alpha-induced NF-kappaB activation.

Interleukin 1 (IL-1) is known to activate the signal transduction machinery, including the transcription factor, nuclear factor kappa B (NF-kappaB). The activation mechanism of NF-kappaB has been studied intensively, while the negative regulatory mechanisms of NF-kappaB remain to be clarified. In the present study, we found that genistein, a tyrosine kinase inhibitor, augmented IL-1alpha-dependent NF-kappaB activation, suggesting the presence of a tyrosine kinase mediating a suppression signal on NF-kappaB. As determined by luciferase reporter gene assay using kappaB-responsive element, genistein enhanced IL-1alpha-induced NF-kappaB activation. Although genistein failed to increase luciferase activity at 1 and 3 h after IL-1alpha stimulation, it induced prolonged activation beginning at 6 h after the initial stimulation. We next examined whether genistein augmented the DNA-binding activity of NF-kappaB, using electrophoretic mobility shift assay. In the case of the control experiment, the binding of NF- kappaB to the kappaB-responsive element peaked at 30 min after IL-1alpha stimulation, and decreased thereafter. In contrast, treatment with genistein maintained the maximum binding activity for at least 2 h after stimulation. Moreover, genistein enhanced the IL-1alpha-dependent degradation of IkappaBalpha. Taken together, our results indicate that genistein augments IkappaB degradation, resulting in continuous NF-kappaB activation. This suggests the possibility that tyrosine kinase negatively regulates NF-kappaB.

Base Sequence↗
PubMed2001

Incidentally discovered giant renal arteriovenous malformation.

A case is presented of giant renal arteriovenous malformation (AVM). A 61-year-old woman was admitted to the National Defense Medical College Hospital for further evaluation of a renal cyst. Doppler ultrasonography and magnetic resonance imaging revealed a giant renal AVM, although the patient had no history nor clinical sign suggesting an AVM. Under the diagnosis of a right renal AVM, the patient underwent AVM resection.

Arteriovenous Malformations↗
PubMed2001

Suicide gene therapy on LNCaP human prostate cancer cells.

The efficacy of combination suicide gene therapy was evaluated using a Herpes simplex virus-thymidine kinase/ganciclovir (HSV-TK/GCV) system and an Escherichia coli cytosine deaminase/5-fluorocytosine (CD/5-FC) system on the LNCaP human prostate cancer cell model. Two types of plasmid vectors with the HSV-TK gene were constructed. A constitutive chicken beta-actin promoter drove one and a prostate-specific antigen (PSA) promoter drove the other. Similarly, a pair of plasmids with the CD gene under a cytomegalovirus (CMV) promoter and the PSA promoter was also constructed. LNCaP cells were transfected in vitro with either or both of those plasmids using a cationic lipid reagent. Transfected cells were treated with GCV and/or 5-FC. The percentage of viable LNCaP cells 7 days after treatment with HSV-TK/GCV or CD/5-FC under a constitutive promoter was 40% and 41% of controls, respectively. The cell viability when two suicide genes were combined was 23%. The cell viabilities after four days with PSA promoter-HSV-TK vectors, CD vectors and a combination of both were 79%, 88% and 88%, respectively. Suicide gene therapy using either HSV-TK/GCV, CD/5-FC, or both, was effective in the LNCaP model. An additive effect was observed when the two suicide genes were used together. The PSA promoter did not seem to be effective enough to elicit cytotoxicity under the experimental conditions used here.

Antimetabolites↗