[Electrophile counter-attack response].
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Animals↗
Biomedical subjects
K Itoh
Publications and source records attributed to K Itoh.
[Ultrasound-guided fine-needle aspiration cytology for neck lesions].
In 1997, 240 cases of ultrasound-guided fine-needle aspiration cytology (FNAC) were performed for neck lesion at Jichi Medical School Hospital. We compared the preoperative cytology findings with postoperative pathologic results for 63 cases (56 lesions) from patients who underwent surgery or open biopsy. The sensitivity, specificity and accuracy of cytological diagnosis were 93.5%, 100% and 96.2%, respectively. There were 2 false negative cases in thyroid gland, which were follicular and medullary carcinomas. Insufficient material for cytological analysis was obtained in 62 of 240 cases (25.8%). Since FNAC can provide high accuracy, we need to reduce insufficient material cases.
Biopsy, Needle↗
[Acetaldehyde adducts in the cerebral cortex of long-term ethanol-fed mice].
It is assumed that the acetaldehyde binds to the proteins after the consumption of ethanol to form an adduct. Such acetaldehyde adducts are related to organ diseases. We examined 8-week-old female BALB/cAJcl mice which had been on a liquid diet for six months. The alcohol-fed mice's liquid diet comprised vitamins, minerals, amino acid and a ethanol solution. The five groups of mice examined were 1.5% ethanol-fed, 2% ethanol-fed, 2.5% ethanol-fed, 3% ethanol-fed, and 3.5% ethanol-fed mice respectively. All mice fed by 2.5% or more ethanol died within a month. Tissue samples were taken from the 1.5% and 2% ethanol-fed mice to investigate the distribution of acetaldehyde adducts in various organs. We demonstrated the immunohistochemical staining of the cerebral cortex and liver from the ethanol-fed mice with antibodies against acetaldehyde adducts. In the 2% ethanol-fed mice, the protein epitope related to acetaldehyde was found in the cerebral cortex and the liver. There was a significant difference in the amount of cell degeneration between the 1.5% and 2% ethanol-fed mice. The results suggest that the neurotoxicity of ethanol starts to cause serious cell degeneration in concentrations of more than 1.5%. The neurotoxicity of ethanol in the cerebral cortex was found to be more prominent than in the liver, resulting in more cell degeneration.
Acetaldehyde↗
Biomechanical behavior of hydroxyapatite as bone substitute material in a loaded implant model. On the surface strain measurement and the maximum compression strength determination of material crash.
Many investigators have advocated that hydroxyapatite ceramics may be extremely prospective bone substitute material mainly through evidence with its given biocompatibility towards bone and demonstrated continuity between living bone and hydroxyapatite. Its mechanical brittleness and strength, however, have been the most serious considerations. In this paper, a study has been performed using animal experiments, massive hydroxyapatite ceramics are implanted into rectangular bone defect created by operation at the load-bearing area in close contact with tibia plateau. The changes of surface strain at the hydroxyapatite implantation of retrieved tibiae are measured at any week after implantation when the compressive stress were applied in the direction of long axis in order to clarify the mechanical behaviors of hydroxyapatite living bone complex. The compression strength of hydroxyapatite implant crash was also determined at each week after implantation. Results revealed that the hydroxyapatite living bone complex has been proven to have sufficient flexibility such that it shows no hysteresis in stress versus strain relationship up to 200 kg of applied load. The strain pattern on the surface of hydroxyapatite implant develops quite similar to that of natural cortical bone. The material crash of hydroxyapatite implant tolerates up to 500 kg of load at 52 weeks after implantation. These characteristics suggest that it achieves normal skeletal function in the points of biomechanical properties in vivo.
Animals↗
Stabilizing effect of lysosomal beta-galactosidase on the catalytic activity of protective protein/cathepsin A secreted by human platelets.
The 32/20-kDa two-chain form of protective protein/cathepsin A (CathA) secreted by human platelets was thermostable in the aggregation supernatant at acidic pH, but was denatured at neutral pH. Leupeptin partly protected the CathA against denaturation, which was not observed in the supernatant after depletion of the cosecreted lysosomal acid beta-galactosidase (beta-Gal) by affinity separation with p-aminophenylthiogalactose (PATG)-agarose beads even at pH 4.8. The purified recombinant human beta-Gal proteins, the 84-kDa precursor and 64-kDa mature-like enzyme (the tryptic product of the 84-kDa precursor), also protected the CathA against denaturation at neutral pH in part. Biospecific interaction analysis revealed that the CathA secreted by platelets dose dependently bound to the immobilized recombinant beta-Gal proteins. The association rate constant of CathA with the 64-kDa mature-like beta-Gal was 4.0 x 10(6) (M-1 s-1) at acidic pH, which was three times larger than that with the 84-kDa beta-Gal precursor. The calculated affinity constants for the enzyme molecules at acidic pH were approximately 1 x 10(9) (M-1), and those at neutral pH were two orders lower. These results first demonstrated that beta-Gal stabilizes the catalytic activity of CathA through direct binding in vitro. The affinity was shown to increase with removal of the carboxy-terminal domain of the beta-Gal precursor.
Blood Platelets↗
Reorganization of stress fiber-like structures in spreading platelets during surface activation.
Alpha-Actinin and myosin were associated into reorganized actin cable networks and partly formed stress fiber-like structures in platelets during surface activation. Double-label immunofluorescence staining using antibodies against alpha-actinin and platelet myosin heavy chain (MHC) showed that alpha-actinin and myosin were colocalized in the cell center at the early stage of activation and dynamically redistributed with shape change. In the later stage, two proteins were colocalized around the granulomeres. alpha-Actinin was also seen beneath the surface membrane while myosin was not. Occasionally, both proteins were segregated, revealed granular staining in the cell body of flattened platelets and often aligned irregular alternate arrangement in the actin cables. Immunoelectron microscopy (immunogold) employing antibodies against MHC and myosin light chain (MLC) demonstrated that myosin, associated with actin cytoskeleton was precisely filamentous (328 nm in average length, 15 nm in width) and bipolar with a central bare zone, since MLCs were located at both ends. Myosin formed a cluster composed of several filaments with repeating alignment, suggesting each cluster corresponded to the granular staining pattern of immunofluorescence. These observations indicated that the organization of alpha-actinin and myosin in actin cables in activated platelets resembled that in stress fibers in various cultured cells.
Actinin↗
Evidence for a voltage-dependent enhancement of neurotransmitter release mediated via the synaptic protein interaction site of N-type Ca2+ channels.
Secretion of neurotransmitters is initiated by voltage-gated calcium influx through presynaptic, voltage-gated N-type calcium channels. These channels interact with the SNARE proteins, which are core components of the exocytosis process, via the synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their alpha1B subunit. Interruption of this interaction by competing synprint peptides inhibits fast, synchronous transmitter release. Here we identify a voltage-dependent, but calcium-independent, enhancement of transmitter release that is elicited by trains of action potentials in the presence of a hyperosmotic extracellular concentration of sucrose. This enhancement of transmitter release requires interaction of SNARE proteins with the synprint site. Our results provide evidence for a voltage-dependent signal that is transmitted by protein-protein interactions from the N-type calcium channel to the SNARE proteins and enhances neurotransmitter release by altering SNARE protein function.
Action Potentials↗
Monoclonal antibody 14F7, which recognizes a stage-specific immature oligodendrocyte surface molecule, inhibits oligodendrocyte differentiation mediated in co-culture with astrocytes.
Cells at an intermediate stage of oligodendrocyte lineage are not only well characterized by biochemical studies but also are likely to relate to the outcome of physiological events. To elucidate the molecular events leading to the development of oligodendrocyte lineage cells, we have raised monoclonal antibodies against stage-specific immature oligodendrocytes, which have previously been isolated by a novel oligodendrocyte-lineage cell culture technique (Sakurai et al.: J Neurosci Res 52:17-26, 1998). We have isolated a mouse monoclonal antibody termed 14F7 which predominantly labels stage-specific immature oligodendrocytes and have found that the expression of 14F7 immunoreactivity in the developing neonatal rat forebrain is closely associated with cells expressing the oligodendrocyte progenitor marker A2B5 and to immature oligodendrocyte expressing O4 antigen. 14F7+ cells were distributed in the ventricular and subventricular zone and the nearby forming corpus callosum as non-myelinating cells. In contrast to cell culture observations, 14F7+ cells were seen only in oligodendrocyte lineage cells. For instance, dissociated cell culture studies indicated that 14F7 labels a cell surface molecule, and its cellular distribution is coincident with all of O4+ cells and A2B5+ cells, and even A2B5- cells. By contrast, 14F7-positive cells did not label astrocytes and, furthermore, did not label myelin basic protein (MBP)-positive oligodendrocytes. 14F7 recognized a 48-kDa protein on sodium dodecyl sulfate polyacrylamide gel electrophoresis. 14F7 immunoreactivity was detectable in rat brain as early as embryonic day 18. Furthermore, in these cells, the total time for differentiation was extended, and on maturation, these cells subsequently expressed an array of myelin-specific proteins, which normally occurs by direct contact with type-1 astrocytes. However, in the presence of 14F7, stage-specific oligodendrocytes co-cultured with astrocytes completely failed to express MBP. These data suggest that the 14F7 antigen is a novel cell surface molecule that is expressed in the intermediate stage of oligodendrocyte-lineage cells, and it is expected that it regulates the differentiation of oligodendrocyte throughout development.
Animals↗
Expression profile of active genes in granulocytes.
A number of genes active in granulocytes have been intensively studied as to the function of their products and their expression controls. However, the intensities and relative order of these gene activities have not been studied. This report describes an expression profile of 748 different species of active genes in human peripheral granulocytes obtained by analyzing a 3'-directed cDNA library that faithfully represents the mRNA population in the source cells. A significant fraction (20.3% of the total) of the expressed genes in granulocytes consisted of nuclear proteins such as DNA binding proteins, of secretory proteins such as cytokines, and of membrane proteins such as major histocompatibility complex (MHC) proteins and receptors. By comparing this expression profile with 11 profiles similarly obtained with unrelated human cells/tissues, we discovered 10 novel genes that are likely to act specifically in granulocytes. Comparison of this expression profile with that obtained with granulocytoids widely used as a granulocyte model by inducing a cultured promyelocytic leukemia cell line HL60 showed similarities and dissimilarities of gene expressions.
Adult↗
Induction of Apg-1, a member of the heat shock protein 110 family, following transient forebrain ischemia in the rat brain.
Apg-1 (Osp94) and apg-2 belong to the heat shock protein (hsp) 110 family. In mouse somatic cells the apg-1 and hsp105/110 transcripts are inducible by a 32 degrees C to 39 degrees C heat shock, while apg-2 is not heat-inducible. Since ischemia is known to induce expression of hsp70, its effect on expression of apg-1 was assessed by using the 20-min forebrain ischemia model of the rat. In the cerebral cortex, Northern blot analysis and in situ hybridization histochemistry demonstrated an increased expression in neuronal cells of apg-1 transcripts 3 h after the onset of reperfusion, with a peak at 12 h, followed by a decline. In the hippocampus, the level was increased at 3 h, remained constant until 24 h, and then declined. Transcript levels of apg-2 as well as hsp 105 were also increased under the present conditions, indicating that the expression of apg-2 was differentially regulated in response to heat and ischemic stresses. The induction kinetics of hsp 105, but neither apg-2 nor hsp 70, were identical to those of apg-1. These results demonstrated that brain ischemia/reperfusion induced expression of each member of the hsp 110 family, although the regulatory mechanisms may not be the same. They also suggest a significant role of apg-1 in both the ischemic- and heat-stress responses and in the normal functioning of the non-stressed neuronal cells.
Animals↗
Protective protein/cathepsin A loss in cultured cells derived from an early-infantile form of galactosialidosis patients homozygous for the A1184-G transition (Y395C mutation).
Galactosialidosis is a human autosomal recessive lysosomal storage disease caused by a genetic defect of protective protein/cathepsin A (PPCA). The patients in a Japanese family with the severe early-infantile form of galactosialidosis were revealed to be homozygous for the A1184-G transition in the PPCA gene in both alleles, which leads to the Y395C substitution. The acid carboxypeptidase (cathepsin A) and lysosomal neuraminidase activities were markedly decreased in cultured fibroblasts and chorionic villus cells derived from the patients, although the decrease in beta-galactosidase activity was less. Immunoblot and immunocytochemical analyses showed that neither the precursor nor the mature form of the PPCA gene product was present in the cultured cells. The Y395C mutation was revealed to cause the loss of the translated product, that determines the severity of the clinical phenotype.
Amino Acid Substitution↗
Axis determination in Xenopus involves biochemical interactions of axin, glycogen synthase kinase 3 and beta-catenin.
Signaling by the Wnt family of extracellular proteins is critical in a variety of developmental processes in which cell and tissue polarity are established [1-5]. Wnt signal transduction has been studied mostly by the genetic approach in Drosophila and Caenorhabditis elegans [1,2,5], but the biochemical mechanisms involved remain to be elucidated. The Wnt pathway also operates during axis determination in vertebrates [3,5]. Frizzled receptors transduce a signal to Dishevelled, leading to inactivation of glycogen synthase kinase 3 (GSK3) and regulation of gene expression by the complex of beta-catenin with LEF/TCF (lymphocyte enhancer factor/T-cell factor) transcription factors [3,5]. Axin is a negative regulator of Wnt signaling and dorsal axial development in vertebrates [6]. Here, we demonstrate that axin is associated with GSK3 in the Xenopus embryo and we localize the GSK3-binding domain to a short region of axin. Binding of GSK3 correlates with the ability of axin to inhibit axial development and with the axis-inducing activity of its dominant-negative form (delta RGS). We also find that wild-type axin, but not delta RGS, forms a complex with beta-catenin. Thus, axin may act as a docking station mediating negative regulation of beta-catenin by GSK3 during dorsoventral axis determination in vertebrate embryos.
Animals↗
Establishment and characterization of a murine osteosarcoma cell line (LM8) with high metastatic potential to the lung.
We established a murine osteosarcoma cell line (LM8) with high metastatic potential to the lung from murine Dunn osteosarcoma using 8 repeated Fidler's procedures. We performed the biological characterization of the LM8 and the maternal Dunn cell lines in vitro and in vivo. Morphologically, LM8 possesses many fillopodial protrusions, lamellipodial structures surrounding the cell surface and membrane ruffles suggesting enhanced cell motility. The increased matrix metalloproteinase (MMP) 2 activity of this cell line might help cell invasion after penetration of the endothelial (mesothelial) cell layer. This cell line exhibited high in vitro invasive activity when seeded onto the mesothelial cell monolayer. Higher expression of VEGF mRNA in this cell line might facilitate neovascularization at the site of metastasis, resulting in extremely high metastatic potency after i.v. injection. LM8 also showed a high metastatic incidence (7/7) to the lung even after s.c. transplantation into the back space of mice. This cell line can provide an excellent tool for studying inhibitory agents against pulmonary metastasis as well as the various important factors involved in metastasis of osteosarcoma.
Alkaline Phosphatase↗
Immunohistochemical characterization of transgenic mice highly expressing human lysosomal alpha-galactosidase.
Human lysosomal alpha-galactosidase predominantly hydrolyzes ceramide trihexoside. A transgenic mouse line, C57BL/6CrSIc-TgN(GLA) 1951 Rin, highly expressing human alpha-galactosidase, has been established and investigated biochemically and immunohistochemically in order to clarify the distribution of the expressed enzyme proteins and to evaluate it as a donor model of organ transplantation therapy for Fabry disease caused by a genetic defect of alpha-galactosidase. In these transgenic mice, about five copies of the transgene were integrated, and alpha-galactosidase activity was expressed in liver, kidney, heart, spleen, small intestine, submaxillary gland, skeletal muscle, cerebrum, cerebellum, bone marrow cells and serum. The enzyme activity was about 22 to 11,080-fold higher than that in non-transgenic mice. In liver, heart and kidney tissues, which are important organs for transplantation studies, sufficient amounts of alpha-galactosidase mRNAs were transcribed, and the expressed enzymes, with molecular weights of 54-60 kDa, are abundant in the liver (enzyme activity: 53,965 nmol h-1 mg-1 protein) and heart (39,906 nmol h-1 mg-1 protein), followed by in the kidney tissue (9177 nmol h-1 mg-1 protein), respectively. An immunohistochemical microscopic study clearly demonstrated the distribution of the expressed enzyme proteins in kidney and liver tissues. Highly expressed alpha-galactosidase was detected in glomerular cells, tubular cells and hepatocytes. These transgenic mice will be useful as a donor model for experimental organ transplantation, and also it will enable recurrent biopsies and long-term observation. The organ transplantation data on mice will provide us with important information.
Animals↗
Modification of dopamine release by nociceptin in conscious rat striatum.
Nociceptin (NOC), an endogenous ligand for the orphan receptor ORL1, has recently been recognized as a neuropeptide. We used brain microdialysis and on-line high performance liquid chromatography to examine the effect of NOC on the basal outflow of dopamine (DA) and its metabolite in the freely moving rat striatum in vivo. The percent change of DA release induced by NOC at concentrations of 10-6 and 10-5 M were 383% and 398%, respectively. This effect of NOC was attenuated by naloxone, suggesting that NOC activates classic (micro, delta, kappa) receptors in a very little way. These data indicates that NOC may act as a neuropeptide which enhances DA release from the striatum of rat brain via an opioid receptor.
Animals↗
Prevalence of verotoxin-producing Escherichia coli harbored in the intestine of cattle in Japan.
The production of verotoxin was examined in 2152 Escherichia coli isolates from 387 cattle in Japan from 1992 to 1994. The toxin was detected in 263 isolates from 94 cattle (detection rate: 24.3%). Verotoxin-producing E. coli (VTEC) was isolated from the cattle in 15 out of 17 prefectures, and the strains were divided into 33 serotypes. E. coli O157:H7 was isolated from 7 out of 387 cattle (detection rate: 1.8%) in four prefectures. These results suggest that VTEC is widely distributed in Japan and include a wide variety of serotypes.
Animals↗
Both stroma and stem cell factor maintain long-term growth of ELM erythroleukemia cells, but only stroma prevents erythroid differentiation in response to erythropoietin and interleukin-3.
Defining how the stromal requirements of hematopoietic progenitors change during leukemia progression is an important topic that is not well understood at present. The murine ELM erythroleukemia is an interesting model because the erythroid progenitors retain dependence on bone marrow-derived stromal cells for long-term growth in vitro, and they also undergo erythroid differentiation in the presence of erythropoietin (EPO) and interleukin-3 (IL-3). In this report, we have shown using neutralizing antibodies that stem cell factor (SCF), insulin-like growth factor (IGF)-1, and integrin signaling pathways are all involved. We then determined whether ELM cells can be maintained long-term without stroma in various combinations of growth factors produced by stroma cells or growth factors for which ELM cells have receptors. This showed that ELM cells could be maintained with high efficiency in SCF alone; furthermore, the cells remained absolutely SCF-dependent and did not become more tumorigenic than cells maintained on stroma. In contrast, ELM cells underwent clonal extinction when serially cloned in IGF1; any cells that survived long-term growth in IGF-1 were found to be IGF1-independent. One important difference between maintaining ELM cells on stroma and growth in SCF is that stroma reversibly inhibits their differentiation in response to EPO and IL-3, whereas SCF does not.
Animals↗
Small GTP-binding protein Rho stimulates the actomyosin system, leading to invasion of tumor cells.
We have shown previously that Rho plays a pivotal role in 1-oleoyl-lysophosphatidic acid (LPA)-dependent invasion of rat hepatoma cells (MM1). Herein we made stable transfectants of MM1 expressing active and Botulinum exoenzyme C3 (C3)-sensitive (Val14), or active and C3-insensitive (Val14/Ile41) forms of human RhoA. Both transfectants showed greatly promoted invasive ability in vitro in the absence of LPA as well as in vivo, adherence to the dish with scattered shape, and enhanced phosphorylation level of 20-kDa myosin light chain (MLC20). A specific MLC kinase inhibitor (KT5926) could inhibit their invasion and the phosphorylation level of MLC20. Stable active RhoA transfectants of W1 cells (low invasive counterpart of MM1) also demonstrated promoted invasive ability in vitro and in vivo, and enhanced phosphorylation level of MLC20. C3 treatment inhibited the invasiveness of the Val14 RhoA transfectant but not that of the Val14/Ile41 RhoA transfectant. LPA enhanced the invasiveness of both transfectants, and this enhancement was abolished by the C3 treatment. These results suggested that 1) the Rho signaling pathway and actomyosin system were linked in the transmigration of tumor cells, and 2) expressed active RhoA enhanced LPA-induced tumor cell invasion via the activation of endogenous RhoA pathway, indicating a positive feedback mechanism in the activation of RhoA.
ADP Ribose Transferases↗