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Biomedical subjects

Y Shimada

Publications and source records attributed to Y Shimada.

At least 19 recordsLinked to original sources
PubMed1995

Construction of a divalent cell adhesive lysozyme by introducing the Arg-Gly-Asp sequence at two sites.

To increase the cell adhesion activity of 74RGD4, an RGDS-inserted mutant between Val74 and Asn75 of human lysozyme, one more site for the RGD introduction was investigated in the lysozyme molecule. We found that 47RGD4 with RGDS in place of AGDR (residues 47 to 50) in a beta-turn region possesses the same level of adhesion activity as that of 74RGD4. The acceptance of the RGD introduction in the beta-turn region of human lysozyme is in good agreement with recent studies on the functional conformation of RGD. We constructed (47,74)RGD4, a mutant containing RGD at two sites, by combining the N-terminal domain of 47RGD4 and the C-terminal domain of 74RGD4. The (47,74)RGD4 lysozyme, with two functional RGD sequences, exhibits even higher cell adhesion activity than that of 74RGD4 or 47RGD4.

Animals↗
PubMed1995

Reduced fidelity of DNA synthesis in cell extracts from chemically induced primary thymic lymphomas of mice.

To examine whether the fidelity of DNA synthesis is reduced in tumor cells, M13 mp2-based fidelity assays were carried out using 15 samples of whole-cell extracts from primary mouse thymic lymphomas induced by alkylating agents. We found that DNA synthesis activities of thymic lymphomas, detected as incorporation of [3H]TTP into acid-insoluble materials, were 2- to 10-fold higher compared to those of normal thymus. Furthermore, mutant frequencies in the forward mutation assay of DNA synthesis were increased 2- to 7-fold in cell extracts from thymic lymphomas compared to those from normal thymus. As the DNA polymerase beta (pol beta) activity was extremely high in the thymic lymphomas, we screened mutations in the pol beta gene to examine the possibility of involvement of mutated pol beta in reduction of the fidelity of DNA synthesis. Of 20 lymphomas, one case of point mutation (T to A) was found by reverse transcription-PCR single-strand conformation polymorphism analysis. These results suggest that the mutagenic DNA synthesis is involved in murine thymic lymphoma genesis, although mutation of the pol beta gene is not a major causal event.

Animals↗
PubMed1995

Relationship of the ipsilateral rotation in night period and striatal dopamine content reduction in unilateral nigrostriatal 6-OHDA lesioned rats.

In order to discriminate well-lesioned rats after unilateral microinjection of 6-OHDA into the nigrostriatal dopamine system, we measured the spontaneous rotation in the night period and calculated the rate of ipsilateral rotation movement. The rate of ipsilateral rotation movement increased along with the total rotation movement. The rats with over 95% of ipsilateral rotation kept the rate relatively constant for 4 weeks after 6-OHDA lesion and showed a significant increase of contralateral rotation (253.2 +/- 37.9) as compared with the rat that had lower than 95% ipsilateral rotation (3.6 +/- 1.4) after the injection of apomorphine (0.25 mg/kg, s.c.). The reduction percentages of striatal DA contents in animals with unilateral rotation over 95% and under 95% to the lesioned side were 97.8 +/- 0.6% and 59.6 +/- 5.8% (P < 0.001), respectively. The rats with over 90% reduction of striatal DA levels corresponded nicely to rats with 95% ipsilateral rotation among rats injected with apomorphine. These results suggested that the evaluation of ipsilateral rotation, taken the level of 95% rotation to the lesioned side as a standard, was able to discriminate well-lesioned rats without apomorphine treatment after unilateral nigrostriatal 6-OHDA application.

Animals↗
PubMed1995

The sequence of a rat cDNA encoding thrombopoietin.

Overlapping cDNA clones encoding rat thrombopoietin (TPO) were isolated from liver-derived cell line cDNA libraries and the nucleotide sequences were determined. The deduced 326-amino-acid rat TPO showed significant homology to the known TPO of other species, especially in the N-terminal sequence.

Amino Acid Sequence↗
PubMed1995

Congenital dumbbell neuroblastoma.

STUDY DESIGN: New babies with neurologic deficits resulting from intraspinal extension of congenital neuroblastoma is very unusual. Two patients with congenital dumbbell neuroblastoma with paraplegia are described, and 16 other cases are reviewed. OBJECTIVE: To study the clinical features and prognosis of congenital dumbbell neuroblastoma. SUMMARY OF BACKGROUND DATA: Only 16 cases were found in the literature. METHODS: Two girls were diagnosed and underwent surgery. RESULTS: Although no recurrence occurred in both patients, incomplete paresis remained. Corrective surgery was needed because of the post-laminectomy spinal deformity in patient 2. CONCLUSIONS: Although a high survival rate in patients with congenital dumbbell neuroblastoma is encouraging, the poor neurologic recovery is of great concern. The authors recommend an early diagnosis by magnetic resonance imaging and early spinal cord decompression followed by chemotherapy for congenital dumbbell neuroblastoma.

Female↗
PubMed1995

Reaction mechanism of T4 endonuclease V determined by analysis using modified oligonucleotide duplexes.

The reaction mechanism of bacteriophage T4 endonuclease V was investigated using modified oligodeoxyribonucleotide duplexes containing a cis-syn thymine dimer. For the pyrimidine dimer glycosylase step, the formation of a covalent intermediate has been proposed. A fluorine atom was attached to the 2'-position of the 5'-component of the thymine dimer site, which could stabilize the covalent complex and prevent the ring opening of the sugar moiety. The strand cleavage of the 12 base pair substrate analog did not occur, although the glycosyl bond was cleaved by this enzyme. A covalent enzyme--substrate complex was separated by gel electrophoresis under denaturing conditions. It was shown that the enzyme molecules were completely converted to a stable complex in the reaction mixture. Two mechanisms have been proposed for the beta-elimination step. A 12-mer containing a phosphorothioate linkage between adjacent thymidines was prepared. The diastereomers were separated, and the absolute configurations were determined. After formation of the thymine dimer and 32P-labeling of the 5'-terminus, these oligonucleotides were annealed to the complementary 12-mer, and the reaction rates of the pyrimidine dimer glycosylase step and the overall reaction for each duplex were measured under the substrate-saturation conditions. The rate constants indicated that the chemical reaction at the beta-elimination step was rate-limiting. Since no difference was observed in the rate constants for the Rp- and Sp-phosphorothioate substrates, it is concluded that the beta-elimination reaction is catalyzed, not by the internucleotide phosphate, but by an amino acid residue of the enzyme.

Base Sequence↗
PubMed1995

Decreased interleukin-6 level in the cerebrospinal fluid of patients with Alzheimer-type dementia.

We examined IL-6 levels in the cerebrospinal fluid (CSF) of patients clinically diagnosed with Alzheimer-type dementia (ATD) and with vascular dementia (VD) and of age-matched normal subjects. The IL-6 levels in the CSF of ATD, but not VD patients, were significantly decreased. In the early onset ATD patients (disease onset < 65 years), IL-6 levels were reduced to 21% of the control level. The IL-6 levels in the CSF were not associated with the severity of the dementia or the duration of the disease since the identification of the first symptoms.

Acute-Phase Reaction↗
PubMed1995

Relation of angiographically defined coronary artery disease and plasma concentrations of insulin, lipid, and apolipoprotein in normolipidemic subjects with varying degrees of glucose tolerance.

We investigated the association between hyperinsulinemia and changes in lipid, lipoprotein, and apolipoprotein that would increase the risk of coronary artery disease (CAD) independent of glucose tolerance. A coronary angiogram was recorded in 127 male subjects, including 41 with normal glucose tolerance, 41 with impaired glucose tolerance, and 45 with non-insulin-dependent diabetes mellitus (NIDDM). Subjects were divided into 2 groups according to results: the group with CAD (n = 94) and the group with normal coronary arteries (n = 33). All subjects were normolipidemic (total cholesterol < 230 mg/dl and triglycerides < 150 mg/dl). The CAD group had a significantly lower plasma level of high-density lipoprotein (HDL) cholesterol and apolipoprotein A-I (apo A-I) and a higher level of apolipoprotein B (apo B) than the normal group with normal glucose tolerance. In considering subjects with impaired glucose tolerance or NIDDM, the CAD group had a significantly lower plasma level of HDL cholesterol and apo A-I and a significantly higher plasma level of total cholesterol, triglycerides, and apo B than the normal group. In each of the subjects with normal and impaired glucose tolerance, and NIDDM, the elevation of plasma insulin concentration during both the complete test period and the early phase of an oral glucose challenge was significantly higher in the CAD than in the normal group. In all subjects, graded reductions in HDL cholesterol and apo A-I and graded increases in plasma total cholesterol, triglycerides, and apo B were observed with increasing tertiles of the postglucose challenge measurements of insulinemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein A-I↗
PubMed1995

Genomic DNA and messenger RNA expression alterations of the CDKN2B and CDKN2 genes in esophageal squamous carcinoma cell lines.

The genes CDKN2B (MTS2) and CDKN2 (MTS1) encoding the proteins p15 and p16 are both located on chromosomal band 9p21, a locus at which frequent homozygous and heterozygous deletions occur in many primary human tumors, including esophageal carcinoma. CDKN2 and CDKN2B belong to a family of cyclin-dependent kinase 4 inhibitors (INK41) and control cell proliferation during the G1 phase of the cell cycle. Their inactivation may contribute to uncontrolled growth in human cancers. To investigate whether CDKN2B and CDKN2 are involved in esophageal tumorigenesis, we studied homozygous deletion, intragenic mutation, and messenger RNA (mRNA) expression of CDKN2 and CDKN2B in nine esophageal squamous cancer cell lines. Polymerase chain reaction (PCR) amplification revealed that five of the nine cell lines (55%) manifested homozygous deletions of CDKN2B, CDKN2, and/or flanking loci on chromosomal band 9p21. Reverse transcriptase-PCR (RT-PCR) was used to examine CDKN2 and CDKN2B mRNA in the nine cell lines. Lack of CDKN2 and CDKN2B mRNA correlated perfectly with homozygous deletion involving these genes. No subtle intragenic mutations of CDKN2B or CDKN2 were detected by DNA sequencing of their entire coding sequences in any cell lines lacking homozygous deletion. Two of the cell lines manifested homozygous deletions excluding CDKN2; one of these two deletions also excluded CDKN2B. These results suggest that inactivation of CDKN2B and CDKN2 may contribute to the malignant phenotype in esophageal cells and that homozygous deletion may be the predominant mechanism for inactivation of CDKN2B and CDKN2. Alternatively, a gene or genes adjacent to CDKN2B/CDKN2 may constitute the target(s) of deletion at this locus.

Alleles↗
PubMed1995

Changes of cytoskeletal architecture and incorporation of 3H-proline in contracted anterior cruciate ligament.

Changes of cytoskeletal architecture and incorporation of 3H-proline were investigated in contracted anterior cruciate ligaments with use of a model of contracture. In control ligaments, fibroblasts were shown by immunofluorescence microscopy to contain actin, vimentin, and myosin in their cytoplasm. Cytoskeletons were visualized by electron microscopy as a mesh network of microfilaments among cell organelles. In contracted anterior cruciate ligaments, fibroblasts were spindle-shaped and their cytoplasm could not be observed clearly in sections stained with hematoxylin and eosin. Actin staining was distributed irregularly and extensively, whereas vimentin and myosin staining was not scattered so extensively. When compared electromyographically, the actin staining appeared in cytoplasmic pseudopods of the fibroblasts. It was thought that these cytoplasmic pseudopods contained mainly actin and little or no other cytoskeletal elements such as vimentin and myosin. In autoradiographs, contracted anterior cruciate ligaments were shown, with use of 3H-proline, to experience a decrease in the number of labeled cells. On the basis of these findings of cytoskeletal rearrangement and of decreased incorporation of 3H-proline, we hypothesized that fibroblasts of the anterior cruciate ligament had the capacity to change their character during knee immobilization and to play a role in ligament contracture.

Actins↗
PubMed1995

Restoration and analysis of standing-up in complete paraplegia utilizing functional electrical stimulation.

OBJECTIVE: Restoration of stand-up motion in patients with complete paraplegia utilizing multichannel functional electrical stimulation, and analysis of the restored motion. DESIGN: Nonrandomized control trial. SETTING: General community, a referral center, institutional practice, and ambulatory care: PATIENTS: Twelve volunteer samples were used for the collection of normal data. Two complete paraplegics received treatment for the restoration of stand-up motion. MAIN OUTCOME MEASURES: The electromyogram, joint angle, and floor reaction force were investigated during standing-up with arms crossed in front of the chest, and hands-assisted standing-up using parallel bars. The maximum knee joint torque during standing-up without hands-assists was calculated using a three-segment link model. Standing-up motion in complete paraplegics was restored, and then analyzed using the three-dimensional floor reaction force and the hip, knee, and ankle angles. RESULTS: Main muscles used to stand up were the quadriceps, tibialis anterior, and paraspinal muscles. Hands-assists reduced the muscle activity and the vertical floor reaction force. Peak muscle activity was less during hands-assisted standing-up, except for the rectus femoris and the iliopsoas muscle. The maximum knee joint torque during standing-up was 1.6Nm/kg for both knees. Two complete paraplegics were able to stand up smoothly from a wheelchair based on stimulation data obtained from normal subjects. The characteristic pattern during standing-up was knee flexion preceding extension. CONCLUSION: Stand-up motion was restored utilizing electromyogram data and knee joint torque data from normal subjects.

Adult↗
PubMed1995

Purification and some properties of a protease from Streptomyces limosus.

Streptomyces limosus was selected because it secreted a novel protease that catalyzed the synthetic reaction forming Pro-Pro-Pro from Pro-Pro. The protease was purified to an electrophoretically homogeneous state and an activity of more than about 20,000-fold that of the culture broth. The molecular mass of the enzyme was estimated to be 50 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme was most active in alkaline pH for the synthetic reaction producing Pro-Pro-Pro from Pro-Pro, although for the hydrolytic reaction forming proline it was most active in neutral pH. The enzyme was inhibited by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and diazoacetyl-DL-norleucine methyl ester (DAN). It can be considered that this enzyme belongs to the class of aspartic proteases. The substrate specificity indicates that this enzyme has a strong affinity for proline as a N-terminal amino acid of peptides.

Amino Acid Sequence↗
PubMed1995

Induction of differentiation of HL-60 cells by the anti-fungal antibiotic, radicicol.

The anti-fungal antibiotic, radicicol, produced in the culture broth of Neocosmospora tenuicristata, was found to induce differentiation of HL-60 cells into macrophages from the following evidence: (1) it caused morphological changes into macrophage-like cells, (2) induced NBT (Nitrobluetetrazolium) reduction activity, (3) induced phagocytosis, and (4) induced alpha-naphthyl acetate esterase activity. The concentration of radicicol required to differentiate HL-60 cells is 50-100 ng/ml, and the incubation time required for commitment of differentiation is 16 hours. Flow cytometry analysis indicated that radicicol blocks the cell cycle of HL-60 cells at the G1 and G2 sites. In addition, radicicol induced reversal of the transformed phenotype of ras-transformed NIH3T3 cells (DT cells) at 25 ng/ml.

Antifungal Agents↗
PubMed1995

Production of thrombopoietin (TPO) by rat hepatocytes and hepatoma cell lines.

Recently, we purified rat thrombopoietin (TPO) from plasma of irradiated rats (XRP) by measuring its activity that stimulated the production of megakaryocytes from megakaryocyte progenitor cells (CFU-MK) in vitro. We then cloned the cDNAs for rat and human TPO. In this study, we found the production of TPO by hepatocytes isolated with the collagenase perfusion method from both normal and thrombocytopenic rats, by a two-step fractionation of hepatocyte culture medium (CM). Subsequently, CM of rat hepatoma cell lines was screened for the presence of TPO; three cell lines, H4-II-E, McA-RH8994, and HTC, were found to produce TPO. According to the purification procedure for TPO from XRP, TPO was partially purified from 2 L CM of each of three cell lines with a six-step procedure. In the final reverse-phase column, TPO from each cell line was eluted with the same retention time as that from XRP, and the TPO fraction exhibited megakaryocyte colony-stimulating activity (Meg-CSA). TPO-active fraction eluted from the final reverse-phase column was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), extracted from the gel, and assayed. TPO activity from each cell line was found in the respective molecular weight region, indicating the heterogeneity of the TPO molecule. Using reverse transcriptase-polymerase chain reaction (RT-PCR), we detected the expression of TPO mRNA in hepatocytes, three hepatoma cell lines, normal rat liver, and X-irradiated rat liver. Northern blot analysis showed that TPO mRNA was expressed mainly in liver among the various organs tested. These data demonstrate that TPO is produced by rat hepatocytes and hepatoma cell lines and suggest that liver may be the primary organ that produces TPO.

Animals↗
PubMed1995

[Experimental study on effects of omental transposition in cats with spinal cord injury].

The effects of autogenous omental transposition on spinal cord injury in cats were examined using a cord crush injury model produced by a modification of Allen's weight-dropping technique. Twenty-three cats were divided into two groups: an omental transposition group, and a control group. The animals were sacrificed at 1, 2, 3, and 4 weeks after injury. The injured cords were examined histologically using India ink perfusion, morphometry of the spinal cord cavities and also by somatosensory evoked potential (SEP) studies. After 1 week, hemorrhagic necrosis, cavitation and demyelination of almost the same extent and severity were noted in both groups. These changes diminished with time in the omental transposition group, but tended to persist in the control group. In the omental transposition group, fibrous coats of Goldsmith were newly formed on the injured cord between the incised dura edges. After 3 weeks, the fibrous coat was thickened with active proliferation of fibroblasts and collagen fibers. India ink particles were noticed within the fine vessels of the omentum, fibrous coat and injured cord at 4 weeks. No India ink particles were found in the control group even after 4 weeks. In the somatosensory evoked potentials, biphasic or triphasic waves noted before injury were absent immediately after trauma. At 3 and 4 weeks, 3 of 5 cats with the omentum showed reappearance of the waves, whereas no wave-reappearance was noted in the controls. These results suggest that autogenous omental transposition helps to accelerate the healing process after crush injury to the spinal cord.

Animals↗
PubMed1995

A simple and quantitative liquid culture system to measure megakaryocyte growth using highly purified CFU-MK.

A new and quantitative liquid culture system has been developed to measure the production of megakaryocytes from megakaryocyte progenitor cells (colony-forming units-megakaryocyte [CFU-MK]). The system uses as a target population a glycoprotein (Gp) IIb/IIIa+ subpopulation of rat bone marrow cells previously demonstrated to be highly enriched for CFU-MK. GpIIb/IIIa+ cells were cultured at 5 x 10(4) cells/mL (10(4) cells/well) with test samples in 96-well tissue culture plates for 4 days at 37 degrees C. During the final 3 hours of incubation, the cells were pulsed with [14C]5-hydroxytryptamine creatinine sulfate (14C-serotonin). After incubation, the plates were washed and the cell pellets were lysed with Triton-X 100. The cell lysate was infiltrated into a commercially available solid scintillator and dried, and radioactivity was measured. In this assay system, rat interleukin-3 (IL-3) was found to be the most potent among known cytokines tested. Murine granulocyte-macrophage colony-stimulating factor (GM-CSF), human erythropoietin (Epo), human IL-6, and murine stem cell factor (SCF) each alone stimulated megakaryocyte growth but were much less active than rat IL-3. Plasma of rats rendered thrombocytopenic by injection of monoclonal antirat platelet GpIIb/IIIa antibody exhibited significant activity, and the active protein fractions partially purified from the plasma showed much higher activity, but normal rat plasma had no effect. This liquid culture system allows the measurement of a large number of test samples--including a wide variety of cytokines and unknown growth factors, alone or in combinations--and provides a simple method for evaluating the early proliferative events involving CFU-MK in the megakaryocyte differentiation pathway.

Animals↗
PubMed1995

[The effects of glucose and insulin upon functional recovery in the rat heart preserved by continuous perfusion with St. Thomas' Hospital cardioplegic solution].

UNLABELLED: The aim of this study was to determine the effect of the continuous perfusion with Krebs-Henseleit bicarbonate buffer (KHBB) solution or St. Thomas' Hospital cardioplegic solution (ST solution) containing glucose and insulin upon the post-preservation recovery in the isolated rat heart. METHODS: Hearts from male Wistar rats (n = 6/group) were subjected to working perfusion with KHBB solution for measurement of cardiac function. They were continuously perfused (1) with KHBB solution or ST solution for 12 hours at 20 degrees C, (2) with ST solution for 12 hours at 37 degrees C, 20 degrees C or 4 degrees C, (3) with ST solution or ST solution containing glucose (9 mmol/L) for 12 hours at 20 degrees C or 4 degrees C, and (4) with ST solution containing glucose (9 mmol/L) or ST solution containing glucose (9 mmol/L) and insulin (10 U/L solution) for 20 hours at 20 degrees C. This was followed by 15 min of Langendorff perfusion and 20 min of working perfusion with KHBB solution. During the second working perfusion after preservation, cardiac function was measured again. RESULTS: (1) Percent recoveries of cardiac output (%CO) preserved with KHBB and ST solution were 44.9 +/- 4.0 and 57.7 +/- 3.8%, respectively (p < 0.05). (2) %CO after preservation with ST solution at 37, 20, 4 degrees C were 0*, 57.7 +/- 3.8 and 74.4 +/- 2.2*%, respectively (*p < 0.05 vs the 20 degrees C group). (3) At 20 degrees C, glucose addition to ST solution increased %CO to 76.5 +/- 2.4% from 57.7 +/- 3.8% in glucose-free ST solution group (p < 0.05). However, at 4 degrees C, glucose addition to ST solution was rather harmful and decreased %CO to 61.4 +/- 2.8% from 74.4 +/- 2.2 in glucose-free ST solution group (p < 0.05). (4) The addition of insulin to the glucose-containing ST solution resulted in a significant increase in %CO from 24.6 +/- 4.0% in the insulin-free solution to 69.2 +/- 2.0%. CONCLUSIONS: Our results indicate that 1) ST solution might be better than KHBB solution for a continuous perfusion medium of myocardial preservation, and 2) at 20 degrees C, the addition of glucose and insulin to the ST solution could be beneficial for the continuous infusion as a method of preservation, however, at 4 degrees C, these addition might be harmful, suggesting the temperature dependence in the utility of energy substrate.

Animals↗