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Biomedical subjects

M Fukuda

Publications and source records attributed to M Fukuda.

At least 577 records · Page 32Linked to original sources
PubMed1996

DNA typing by triplex affinity capture (TAC) PCR.

Using a triplex affinity capture approach, we extracted PCR-ready DNA from fresh blood, buccal swabs, and bloodstains made on hard objects. We immobilized a biotinylated purine-rich oligonucleotide onto streptavidin-coated magnetic beads, and captured double-stranded DNA via triple-helix formation. We were able to amplify the MCT118, ACTBP2, FGA, D8S320, D11S488, and THO1 loci from the captured DNA. DNA typing by triplex affinity capture PCR is simple, rapid, and well-suited for paternity testing and population studies of microsatellites.

Base Sequence↗
PubMed1996

Characteristics and mechanism of the ossification of posterior longitudinal ligament in the tip-toe walking Yoshimura (twy) mouse.

To elucidate the mechanism of ossification of the posterior longitudinal ligament (OPLL), we examined the serial changes in the intervertebral disc of tip-toe walking Yoshimura (twy) mouse. At the age of 6 weeks, the volume of the nucleus pulposus increased in all intervertebral discs causing anterior and posterior herniation. Secondary to this herniation, the cartilagineous tissue of the annulus fibrosus was disrupted and showed regenerative proliferation with PCNA-positive cartilagineous cells. These cells were S-100 positive and the matrix was positive for chondroitin-4-sulfate proteoglycan, indicating the development of calcification. At the age of 15 weeks, the regenerative cartilagineous tissue of the annulus fibrosus reached the posterior longitudinal ligament together with neovascularization and appearance of PCNA-positive proliferating primitive mesenchymal cells. These cells were considered to be osteoblasts since they were positive for alkaline phosphatase and the matrix contained type I collagen. Using electron microscopic X-ray analysis, vesicles present in the matrix of regenerative cartilagineous cells of the annulus fibrosus were confirmed to contain calcium phosphate crystals, also indicating the development of calcification. In conclusion, our serial analysis indicates that ossification of posterior longitudinal ligament in twy mouse was triggered by enlargement of the nucleus pulposus followed by herniation, disruption and regenerative proliferation of annulus fibrosus cartilagineous tissues. Enchondral ossification of the new annulus fibrosus cartilagineous cells and membraneous ossification by primitive osteoblasts proceed to the final ossification of the posterior longitudinal ligaments.

Alkaline Phosphatase↗
PubMed1996

[An operative case of suture-granuloma which resulted from an intra-pulmonary treatment 10 years ago and manifested hemoptysis].

We experienced a 27-year-old male patient with recurrent hemoptysis manifested by granuloma which resulted from surgical repair that was performed for right pneumothorax using unabsorbable sutures (braided silk) before 10 years. The patient had been suffering from fever and cough for three months before hemoptysis. Chest X-ray and CT scan films showed a mass shadow in the lateral side of the right lung field. Furthermore, bronchoscopy revealed bleeding in B3 of the right lung. The patient underwent right upper lobectomy, which disclosed that hemoptysis was due to a granuloma (2.3 x 3.2 cm in size) formed around sutures. The granuloma was caused not only by foreign body reaction but also by transbronchial infection.

Adult↗
PubMed1996

Production of monoclonal anti-thymine-dimer antibody and its use for detection of STR alleles.

To simplify the manual detection of STRs, we developed an immunoblotting method using an anti-thymine-dimer antibody. Upon ultraviolet irradiation, adjacent thymine residues in a DNA strand readily form thymine dimers which are detectable by anti-thymine-dimer antibody. We simply irradiated the supernatant of a boiled suspension of P3U1 myeloma cells with ultraviolet rays, and immunized BALB/c mice with it. By fusing their popliteal lymph node cells with the P3U1 myeloma cell line, we produced a monoclonal anti-thymine-dimer antibody. Using this antibody, we were able to detect STR alleles containing AAAG, AAGG, or AATG repeats by immunoblotting. Our method is well suited for use in parentage testing.

Alleles↗
PubMed1996

Determination of sibship by microsatellite typing in a deficiency case of disputed maternity.

In a disputed maternity case where the father and his two wives were dead and only the six children were available for testing, we analyzed 24 conventional hemogenetic markers and 11 microsatellite (STR) loci. Putting all the results together, we were able to distinguish sibship from half-sibship. Of all the systems tested, STRs were most informative. The present study demonstrates the power of STRs as a tool for solving a deficiency case.

Alleles↗
PubMed1996

Chronic ethanol consumption enhances endotoxin induced hepatic sinusoidal leukocyte adhesion.

In alcoholic liver disease, endotoxin has been postulated to play an important role in its pathogenesis. Endotoxin is known to lead to impediment of hepatic microcirculation, including the adhesion of leukocytes to sinusoidal endothelial cells. In this study, the effect of chronic ethanol consumption on the leukocyte adhesion elicited by endotoxin was examined. Male Wistar rats were pair-fed with a liquid diet containing ethanol or an isocaloric control diet for 6 weeks. The liver of anesthetized rats were placed on the nonfluorescent cover-glass for observation by an intravital inverted microscope equipped with a silicon intensified target camera. The red blood cell (RBC) velocity in hepatic sinusoids was measured by an off-line temporal correlation velocimeter (Capiflow, Sweden) after intravenous injection of fluorescein isothiocyanate-labeled rat RBC. RBC velocity in sinusoids was more severely disturbed in ethanol fed rats than in controls. Leukocytes were stained by the intravenous injection of carboxyfluorescein succinimidyl ester for a fluorographic observation of leukocyte adhesion. After lipopolysaccharide injection, the number of adherent leukocytes was significantly greater in ethanol-fed rats than in controls. Plasma tumor necrosis factor-alpha levels were also higher in ethanol-fed rats than in controls. These results suggest that chronic ethanol consumption aggravates endotoxin induced leukocytes adhesion that may result in hepatic microcirculatory disturbances. Leukocyte adhesion to the sinusoidal wall may be associated with increased in tumor necrosis factor-alpha levels.

Animals↗
PubMed1996

STR typing in a deficiency case of disputed paternity.

In a case of disputed paternity where the alleged father was dead and the children and their mothers were available for testing, we analyzed 23 conventional hemogenetic markers and six short tandem repeat loci. We deduced the possible types of the alleged father and calculated the probability of paternity. Conventional phenotypings did not provide conclusive proof of paternity. In contrast, typing of the short tandem repeat loci gave a combined probability of paternity of 0.9986, establishing paternity. The present study demonstrates the power of short tandem repeat typing as a tool for solving such a deficiency case.

Alleles↗
PubMed1996

STR typing of plasma DNA in a deficiency case of disputed maternity against a patient dying in the hospital.

In this deficiency duo case, the plasma that had been left over after clinical diagnostic tests was the only blood sample available from the deceased alleged mother. Since her blood had been ABO grouped during hospitalization, we tested the plaintiff for the ABO system first. Then, to draw as much genetic information as possible from their plasma, we phenotyped 13 plasma protein systems and, three and a half years later, analyzed three short tandem repeat (STR) loci. The conventional phenotyping and STR genotyping gave probabilities of maternity of 0.974 and 0.967, respectively, bringing the total probability to 0.999. The present study highlights the utility of plasma STR typing in case of need.

ABO Blood-Group System↗
PubMed1996

[Differential diagnosis of tuberculous pleurisy by the measurement of cytokine concentration in pleural effusion].

Pleural fluids obtained from 26 patients with tuberculous pleurisy (T-group), 11 with parapneumonic pleurisy (B-group) and 21 with malignant pleurisy (M-group) were tested for their biologic parameters and cytokine concentrations. 1) The average age of T-group was over 10 years lower than that of M-group with a statistically significant difference. 2) The average CRP value of B-group and the positivity on PPD skin test of T-group were higher than those of the other groups, respectively. 3) Yellowish pleural fluids were mainly observed in T- and B-group, while bloody pleural fluids were mostly seen in M-group with a statistically significant difference. The average total protein amount and adenosine deaminase value in pleural fluid significantly increased in T-group. The percentage of polymorphonuclear leukocytes showed a significant increase in B-group, while lymphocytes significantly increased in T-group with a statistically significant difference. 4) Although no significant difference in concentrations of IL-1 beta, IL-2, IFN-gamma and TNF-alpha in serum was noticed among the three groups, the average concentrations of IFN-gamma and TNF-alpha in pleural fluid in T-group were significantly higher than those in the other groups. 5) TNF-alpha-mRNA of mononuclear cells in pleural fluid was strongly expressed in 3 out of 11 patients of T-group, while no expression was observed in 6 patients of M-group. In conclusion, the measurement of concentrations of two kinds of cytokines in pleural fluid, IFN-gamma and TNF-alpha, may be clinically useful for the differential diagnosis of tuberculous pleurisy from parapneumonic pleurisy and malignant pleurisy.

Adult↗
PubMed1996

[A case of CEA and CA19-9 producing recurrent transitional cell carcinoma in an Indiana pouch after total cystectomy].

A 73-year-old female with transitional cell carcinoma (TCC) of the bladder underwent total cystectomy and Indiana pouch replacement in April, 1992. Histological examination revealed grade 3 TCC. In February 1995, she complained of gross hematuria. Intravenous pyelography (IVP) revealed a right non-functional kidney and filling defect in the Indiana pouch. We suspected colon cancer in the Indiana pouch because the levels of serum carcino-embryonic antigen (CEA) and CA19-9 were elevated. Endoscopic biopsy of intrapouch tumor was done. Pathological examination revealed grade 2 TCC. In July 1995, right nephroureterectomy with resection of Indiana pouch was performed and the surgical specimen revealed renal pelvic and ureteral cancer, grade 2 TCC. The levels of serum CEA and CA19-9 returned to the normal range 21 days after the operation. CEA and CA19-9 histochemical stain of renal pelvic and ureteral cancer were positive. Also CEA-, CA19-9-positive cells were detected in the specimens of the bladder tumor from the total cystectomy performed in 1992. This rare case is discussed and the literature is reviewed.

Aged↗
PubMed1996

Serum levels of cytokines in patients with untreated primary lung cancer.

To evaluate the relationships between serum endogenous cytokine levels and their clinical implications in cancer patients, we measured the serum levels of endogenous granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), and interleukin 6 (IL-6) in patients with untreated primary lung cancer. The serum G-CSF level was measured using a chemiluminescent ELISA, and the other cytokine levels were measured using ELISA. Fifty healthy adults and 183 patients with primary lung cancer were studied. The mean M-CSF level in the lung cancer patients (1106.4 units/ml) was significantly higher than that in the healthy adults (836 units/ml, P = 0.0001). In patients with large cell carcinoma, endogenous G-CSF, M-CSF, and IL-6 levels were significantly higher than those in patients with carcinomas of other cell types (P < 0.05). Univariate analysis showed that survival of 159 non-small cell lung cancer patients with high (more than cutoff level) G-CSF, M-CSF, and IL-6 levels was significantly poorer than that of patients with low levels (Wilcoxon's test, P = 0.018, P < 0. 0001, and P < 0.0001, respectively). Survival of patients with high levels of two or more cytokines was poorer than that of those with high levels of one cytokine or normal cytokine levels (P < 0.0001). Multivariate analysis using Cox's proportional hazards model showed that high M-CSF and C-reactive protein levels correlated significantly with poor survival (P = 0.037 and 0.037, respectively). Our preliminary data suggest that high M-CSF levels in non-small cell lung cancer may be of poor prognostic value.

Adult↗
PubMed1995

Role of the C2A domain of synaptotagmin in transmitter release as determined by specific antibody injection into the squid giant synapse preterminal.

Squid synaptotagmin (Syt) cDNA, including its open reading frame, was cloned and polyclonal antibodies were obtained in rabbits immunized with glutathione S-transferase (GST)-Syt-C2A. Binding assays indicated that the antibody, anti-Syt-C2A, recognized squid Syt and inhibited the Ca(2+)-dependent phospholipid binding to the C2A domain. This antibody, when injected into the preterminal at the squid giant synapse, blocked transmitter release in a manner similar to that previously reported for the presynaptic injection of members of the inositol high-polyphosphate series. The block was not accompanied by any change in the presynaptic action potential or the amplitude or voltage dependence of the presynaptic Ca2+ current. The postsynaptic potential was rather insensitive to repetitive presynaptic stimulation, indicating a direct effect of the antibody on the transmitter release system. Following block of transmitter release, confocal microscopical analysis of the preterminal junction injected with rhodamine-conjugated anti-Syt-C2A demonstrated fluorescent spots at the inner surface of the presynaptic plasmalemma next to the active zones. Structural analysis of the same preparations demonstrated an accumulation of synaptic vesicles corresponding in size and distribution to the fluorescent spots demonstrated confocally. Together with the finding that such antibody prevents Ca2+ binding to a specific receptor in the C2A domain, these results indicate that Ca2+ triggers transmitter release by activating the C2A domain of Syt. We conclude that the C2A domain is directly related to the fusion of synaptic vesicles that results in transmitter release.

Amino Acid Sequence↗
PubMed1995

Role of the C2B domain of synaptotagmin in vesicular release and recycling as determined by specific antibody injection into the squid giant synapse preterminal.

Synaptotagmin (Syt) is an inositol high-polyphosphate series [IHPS inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate] binding synaptic vesicle protein. A polyclonal antibody against the C2B domain (anti-Syt-C2B), an IHPS binding site, was produced. The specificity of this antibody to the C2B domain was determined by comparing its ability to inhibit IP4 binding to the C2B domain with that to inhibit the Ca2+/phospholipid binding to the C2A domain. Injection of the anti-Syt-C2B IgG into the squid giant presynapse did not block synaptic release. Coinjection of IP4 and anti-Syt-C2B IgG failed to block transmitter release, while IP4 itself was a powerful synpatic release blocker. Repetitive stimulation to presynaptic fiber injected with anti-Syt-C2B IgG demonstrated a rapid decline of the postsynaptic response amplitude probably due to its block of synaptic vesicle recycling. Electron microscopy of the anti-Syt-C2B-injected presynapse showed a 90% reduction of the numbers of synaptic vesicles. These results, taken together, indicate that the Syt molecule is central, in synaptic vesicle fusion by Ca2+ and its regulation by IHPS, as well as in the recycling of synaptic vesicles.

Animals↗
PubMed1995

Functional diversity of C2 domains of synaptotagmin family. Mutational analysis of inositol high polyphosphate binding domain.

Synaptotagmins I and II are inositol high polyphosphate series (inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate) binding proteins, which are thought to be essential for Ca(2+)-regulated exocytosis of neurosecretory vesicles. In this study, we analyzed the inositol high polyphosphate series binding site in the C2B domain by site-directed mutagenesis and compared the IP4 binding properties of the C2B domains of multiple synaptotagmins (II-IV). The IP4 binding domain of synaptotagmin II is characterized by a cluster of highly conserved, positively charged amino acids (321 GKRLKKKKTTVKKK 324). Among these, three lysine residues, at positions 327, 328, and 332 in the middle of the C2B domain, which is not conserved in the C2A domain, were found to be essential for IP4 binding in synaptotagmin II. When these lysine residues were altered to glutamine, the IP4 binding ability was completely abolished. The primary structures of the IP4 binding sites are highly conserved among synaptotagmins I through IV. However, synaptotagmin III did not show significant binding ability, which may be due to steric hindrance by the C-terminal flanking region. These functional diversities of C2B domains suggest that not all synaptotagmins function as inositol high polyphosphate sensors at the synaptic vesicle.

Amino Acid Sequence↗
PubMed1995

Benefits of Medroxyprogesterone Acetate (MPA) in Advanced or Recurrent Breast Cancer with Higher Serum Concertration.

The efficacy of medroxyprogesterone acetate (MPA) therapy in controlling progressive measurable metastatic breast cancer was assessed in 61 patients. In addition serum MPA concentrations were measured by high performance liquid chromatography (HPLC) and subjective effects of treatment were monitored. Overall 24 patients (39.3%) achieved an objective response(2 complete responses [ CR ] and 22 partial responses [ PR ]). There was no significant relationships between response to therapy and menopausal status, metastatic sites, previous therapy, histological type, or disease-free interval. Patients with estrogen (ER) and progesterone (PgR) receptor-positive tumors responded more frequently. Significant differences in serum MPA concentrations were seen between responders and non-responders, objective tumor shrinkage being seen in patients with serum levels in excess of 55 ng/ml. There were few cases responding to the therapy with serum MPA concentrations lower than 25 ng/ml. The serum MPA levels significantly correlated with an improvement in the performance status and survival. Patients with serum MPA concentrations lower than 25 ng/ml had significantly poorer survival. There was a significant relationship between MPA level and dose per area of boby surface (mg/ m(2)) in cases with CR or PR or no change (NC). However, the serum levels of patients with progressive disease despite therapy were lower than the expected levels based on the body surface area. This study demonstrated that serum MPA concentration is a determining factor for therapeutic benefit in advanced or recurrent breast cancer.

Journal Article↗
PubMed1995

A novel expression vector composed of a regulatory element of the human leukosialin-encoding gene in different types of mammalian cells.

The regulatory element (RE) of the human leukosialin (LS)-encoding gene, that encodes a major sialoglycoprotein of human leukocyte and platelet membranes, was used to develop a novel expression vector, pKX. The vector was constructed by cloning a RE fragment and the SV40 fragment containing polyadenylation and splicing signals between HindIII and BamHI sites of the pCAT-Basic vector. The transcription level controlled by this vector was evaluated in six different cell lines using a transient expression assay of chloramphenicol acetyltransferase (CAT). The CAT activity of the pKX vector was compared to the other common expression vectors, namely pMSG (driven by the mouse mammary tumor virus LTR), pcDL-SR alpha (SV40 promoter/enhancer and HTLV-I LTR), pcDNAI (cytomegalovirus promoter/enhancer) and pCAT-Control (SV40 promoter/enhancer). The level of expression provided by the pKX vector was comparable to that observed with pcDNAI and pcDL-SR alpha vectors. In different mammalian cell lines, the highest efficiency of expression of the pKX vector was observed in the human T-cell lines, Jurkat and CEM, although the expression of pcDL-SR alpha-CAT in those cell lines was in the same range. The expression of the pKX vector driven by a non-viral promoter and/or enhancer can be as efficient as that driven by a viral promoter and/or enhancer. Potential uses of this vector may be found in studies of transient gene expression in hematopoietic cells and for gene therapy, particularly the ones involving T-cells.

Animals↗
PubMed1995

Induction of neurite outgrowth by MAP kinase in PC12 cells.

Treatment of PC12 cells with nerve growth factor (NGF) results in neural differentiation of the cells, inducing neurite outgrowth. Ras protein has been shown to play an essential role in this process. To examine whether or not the MAP kinase (MAPK) cascade mediates the NGF- and Ras-induced neural differentiation process, we injected PC12 cells with constitutive active forms of each components of the MAPK cascade. When a moderately active mutant of Xenopus MAPK kinase (S222E-MAPKK) in which Ser 222 was changed into glutamic acid was injected, the neurite outgrowth of PC12 cells occurred to some extent. Injection of an N-terminal truncated STE11 protein (delta N-STE11), a constitutively active form of STE11 which is a yeast MAPKK kinase, induced neurite outgrowth in PC12 cells. Furthermore, injection of thiophosphorylated MAPK, but not purified active MAPK, into PC12 cells resulted in neurite outgrowth. Thiophosphorylated MAPK was resistant to protein phosphatase 2A treatment, while purified active MAPK was inactivated by this treatment. All these results have suggested that sustained activation of MAPK is sufficient for PC12 cell differentiation. In accord with this, the delta N-STE11- or S222E- MAPKK-induced neurite outgrowth was inhibited by coinjection of CL-100 protein, a dual-specificity phosphatase that is capable of inactivating MAPK.

Animals↗