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T Saitoh

Publications and source records attributed to T Saitoh.

At least 307 records · Page 17Linked to original sources
PubMed1991

Expression of active secreted forms of human amyloid beta-protein precursor by recombinant baculovirus-infected insect cells.

Three alternatively spliced forms of the amyloid precursor protein (APP), APP-695, APP-751, and APP-770, were expressed in the baculovirus expression vector system. The recombinant proteins were secreted into the culture medium by infected insect cells, and APP molecules were detected in insect cells and medium 2 days after infection with the recombinant APP-baculoviruses. A partial sequence of the NH2 terminus of the secreted protein revealed identity with the native secreted protein and showed that the signal peptide was recognized and properly cleaved in insect cells. Purified secreted recombinant APP-751 comigrated with protease nexin 2 purified from platelets and fibroblasts. A 15-kDa COOH-terminal fragment of APP was also detected in cells infected with recombinant baculoviruses, suggesting that recombinant APP proteins were cleaved at the COOH-terminal end like native APP protein. Recombinant APP-751 and APP-770 formed complexes with epidermal growth factor-binding protein, whereas APP-695 did not. In addition, recombinant APP-751 and APP-770 inhibited trypsin and chymotrypsin activity, whereas APP-695 did not. Growth of a human fibroblast cell line, A-1, that required APP for complete growth, was restored upon addition of secreted recombinant APP-695 or APP-751. Thus, the appropriately sized, secreted recombinant APP proteins produced in this expression system are biologically active.

Amino Acid Sequence↗
PubMed1991

Concentration of hydrophobic organic compounds and extraction of protein using alkylammoniosulfate zwitterionic surfactant mediated phase separations (cloud point extractions).

The zwitterionic surfactants 3-[nonyl- (or decyl-) dimethyl-ammonio]propyl sulfate, (C9-APSO4 or C10-APSO4) were synthesized using Nilsson's procedure, and their phase separation behavior under different experimental conditions was evaluated. The results indicate that such zwitterionic surfactants can be utilized for the extraction/preconcentration of hydro-phobic species in a manner akin to that previously reported for nonionic surfactants. This was demonstrated for several practical applications including the extraction/preconcentration of some steroidal hormones and vitamin E prior to high-performance liquid chromatography analysis. The zwitterionic surfactant mediated phase separation was also applied to the extraction of the hydrophobic membrane protein, bacterio-rhodopsin, from the hydrophilic cytochrome c protein, both originally present in an aqueous phase. The concentration factors for this aqueous two-phase extraction technique using C10-APSO4 ranged from 26 to 35 with recoveries in the range 88 to greater than 96%. Some comparative studies indicate that the use of zwitterionic surfactants in lieu of nonionic surfactants (e.g. polyoxyethylene(7.5) nonyl phenyl ether PONPE-7.5) in such an extraction method offers some significant advantages such as purer, homogeneous surfactant preparation, minimum background absorbance at UV detection wavelengths, the two-phase region occurring at lower temperatures, and greater extraction efficiencies/concentration factors among others.

Ammonium Sulfate↗
PubMed1991

Visualization of exocytotic secretory processes of mast cells by fluorescence techniques.

Secretory processes via exocytosis in rat peritoneal mast cells were visualized by two complementary fluorescence techniques; one staining pre-exocytotic granules with a basic probe and the other staining post-exocytotic granules with acidic probes. Granules within mast cells were selectively stained with acridine orange and emitted orange yellow fluorescence. Upon stimulation with compound 48/80, release of acridine orange from granules was observed both in population and single cell measurements. This release was seen in some localized area of mast cells. Opening of pores between plasma membranes and granule membranes was monitored using acidic fluorescence probes such as 6-carboxyfluorescein or lucifer yellow CH. Not only granules located at peripheral region, but also granules near the core region participated in exocytosis. The existence of junctions between these granules was suggested. TMA-DPH, a lipophilic membrane probe, which was localized at plasma membrane before stimulation, diffused into granule membranes after stimulation. This shows that after stimulation, some constituents of plasma and granule membranes were mixed. Even after extensive degranulation, mast cells extruded acidic probes, indicating the plasma membranes still play a role of barrier. Activation of lateral motion of granules preceding to exocytosis was not observed. It was concluded that the visualization of secretory processes by fluorescence and image processing techniques will be useful for the study of molecular mechanisms underlying exocytosis.

Acridine Orange↗
PubMed1991

A possible fetal antigen of Mr 70,000 in neurofibrillary tangles.

To test the hypothesis that tau-immunoreactive curly fibers represent a regenerative attempt of cortical neurons in the brain affected with Alzheimer's disease (AD), we established 2 monoclonal antibodies to isolated neurofibrillary tangles. Both antibodies extensively stained curly fibers and specifically labeled a novel protein of Mr 70,000 (p70), which was abundantly expressed in fetal brain, but undetectable in adult brain. Furthermore, the antibodies visualized curly fibers in newborn rat brain, which were very similar to those in the AD brain, and also intensely stained extending neurites of NGF-primed PC 12 cells. These observations suggest that P70 is a fetal antigen associated with growing neurites, and thus support the sprouting hypothesis.

Aging↗
PubMed1991

Structure-function relationships of the mouse inositol 1,4,5-trisphosphate receptor.

The homotetrameric complex of inositol 1,4,5-triphosphate (InsP3) receptors displays a Ca2+ release activity in response to InsP3 molecules. Structure-function relationships of the mouse cerebellar InsP3 receptor have been studied by analyses of a series of internal deletion or C-terminal truncation mutant proteins expressed in NG108-15 cells. Within the large cytoplasmic portion of the InsP3 receptor, approximately 650 N-terminal amino acids are highly conserved between mouse and Drosophila, and this region has the critical sequences for InsP3 binding that probably form the three-dimensionally restricted binding site. The N-terminal region of each InsP3 receptor subunit also binds one InsP3 molecule. Cross-linking experiments have revealed that InsP3 receptors are intermolecularly associated at the transmembrane domains and/or the successive C termini. The interaction between the receptor subunit and InsP3 may cause a conformational change in the tetrameric complex, resulting in the opening of Ca2+ channels.

Amino Acid Sequence↗
PubMed1991

Differential expression of protein kinase C isozymes in rat glial cell cultures.

Protein kinase C (PKC) is a family of closely related enzymes implicated in molecular processes involved in growth and differentiation in a variety of cells. We studied the presence and distribution of 4 PKC isozymes in glial cell cultures of the rat hippocampus employing antisera raised against synthetic peptides predicted from the cDNA sequences corresponding to the C-terminal portion of 4 PKC isoforms, alpha, beta I, beta II, and gamma. PKC(alpha) and -(beta II), but neither PKC(beta I) nor -(gamma) isoforms were detected in glial cultures of the rat hippocampus. Anti-PKC(alpha) immunostained all glial cells, whereas anti-PKC(beta II) faintly stained about 20% of total glial cells resembling the type-2 astrocyte that were GFAP immunopositive, with few processes. Anti-PKC(beta II) did not stain about 80% of the glial fibrillary acidic protein (GFAP)-immunopositive cells with a few thick processes which resembled the type-1 astrocyte. A few cells that stained intensely with anti-PKC(beta II) were GFAP immunopositive and possessed fine, but well-developed, multiple processes. Faint PKC(beta II) immunoreactivity was also detected among anti-MBP-positive cells (possibly oligodendrocytes), RCA-1-positive cells (possibly microglia), and small, oval, anti-GFAP-positive cells. These results suggest the involvement of distinct PKC isoforms in different glial functions.

Animals↗
PubMed1991

Identification of glycolipid receptors for Helicobacter pylori by TLC-immunostaining.

Helicobacter pylori has been identified as a causative agent in active chronic gastritis. The receptor for this bacteria, however, is not known. It is likely that the receptor molecules may be glycosphingolipids as shown in the cases of other bacteria. We explored this possibility by a thin-layer chromatography (TLC)-immunostaining method. Among glycosphingolipids extracted from human gastric mucosa, intact Helicobacter pylori specifically bound to I3SO3-GalCer and II3NeuAc-LacCer, whereas no specific binding to neutral glycosphingolipids, which share the same ceramide moiety with I3SO3-GalCer or II3NeuAc-LacCer, was demonstrated. Sonicated bacteria could still bind to II3NeuAc-LacCer with comparable affinity. In contrast, the binding of bacteria to I3SO3-GalCer was greatly diminished upon sonication. These results suggest that each of the oligosaccharide moieties of II3NeuAc-LacCer and I3SO3-GalCer may be specifically recognized by different ligand molecules of Helicobacter pylori.

Bacterial Adhesion↗
PubMed1991

[Qualitative assessment of centrilobular emphysema using computed tomography].

The ability of computed tomography (CT) in the qualitative assessment of centrilobular emphysema (CLE) was studied, with special reference to the morphological relationship between emphysematous change and relatively large bronchovascular bundle forming the margin of secondary pulmonary lobule. Three autopsied lobes with mild, moderate and severe CLE were fixed by the method of Heitzman. Then high resolution CT (HRCT) was applied to those inflation-fixed lobes, which allowed for direct one-to-one pathologic correlation. Furthermore, HRCT findings of these clinical cases with emphysema were analysed. Emphysematous change in CLE was observed as low-attenuation area (LAA) within the homogeneous background on Ct images. In mild and moderate cases, LAA's were round or oval in shape, however in severe case they were observed to be irregular in shape. The border between LAA and parenchyma was not clearly defined, however in severe case LAA was found to be bordered partially or rather subtotally by the bronchovascular bundle. In mild and moderate cases, LAA's were observed to be distant from relatively large bronchovascular bundle. On the other hand, in severe case, only a small amount of lung parenchyma was left between LAA and bronchovascular bundle. These findings correspond to morphologic characteristics of CLE, that is, the process of emphysematous change tends to take place in the center of the secondary lobule, and even if the lobe is severely destroyed, some portion of the peripheral acinus is still preserved at lobular margin. Thus, CT can help to identify the presence of CLE. Furthermore, CT scan analysis of severe grade emphysema may lead to the differentiation between CLE and panlobular emphysema.

Aged↗
PubMed1991

Advances in Alzheimer's disease.

The problem of the etiology of Alzheimer's disease has not been solved. But in the past several years there have been significant extensions of our knowledge of the disease and advances in determining the molecular changes underlying the disorder. There is now convincing evidence that the dementia per se is caused by loss of neurons and synapses, particularly in neocortex and hippocampus. The molecular aspects of amyloid and its precursor protein have been defined. The nature of intracellular changes leading to accumulation of the paired helical filament is beginning to be understood. For the first time, putative risk factors can be described in terms of pathogenetic mechanisms. Thus, it may become possible in the not-too-distant future to discover interventions that will slow the progress of this devastating disease.

Alzheimer Disease↗
PubMed1991

Spinal cord ischemia reduces calcium/calmodulin-dependent protein kinase activity.

Calcium/calmodulin (Ca2+/CaM)-dependent protein phosphorylation was evaluated in a rabbit spinal cord ischemia model. One hour of ischemia reduced particulate (5% of control) and cytosolic (35% of control) Ca2+/CaM-dependent protein kinase activity significantly (p less than 0.01). In vitro phosphorylation of endogenous proteins by endogenous Ca2+/CaM-dependent protein kinase showed that phosphorylation of 14 particulate and 7 cytosolic proteins was stimulated in the presence of Ca2+/CaM in control tissue. However, after 1 hour of ischemia, Ca2+/CaM-dependent protein phosphorylation was virtually absent in the particulate fraction and significantly reduced in the cytosol. When equal amounts of control and ischemic tissue samples were combined and assayed, Ca2+/CaM-dependent protein kinase activity was 43% of control in particulate and 70% of control in cytosolic fractions. This suggests that reduced Ca2+/CaM-dependent protein phosphorylation is probably not due to the presence of an inhibitory activity in ischemic tissue. These results show that the Ca2+/CaM-dependent protein phosphorylation system is impaired after ischemia durations which cause irreversible damage. These altered phosphorylation reactions may play critical roles in mediating irreversible neurologic injury.

Animals↗
PubMed1991

Expression of protein kinase C isozymes in primary neuronal cultures of the rat cerebellum.

Protein kinase C (PKC), a family of closely related enzymes, has been implicated in molecular processes involved in differentiation in a variety of cells, including neuronal cells. We studied the presence and distribution of four PKC isozymes immunocytochemically in primary neuronal cultures of the rat cerebellum. We employed four anti-PKC antisera raised against synthetic peptides predicted from the cDNA sequence of the C-terminal portion of four PKC isozymes, alpha, beta I, beta II, and gamma. The majority of neurons were PKC(beta II) immunoreactive both in the early and late (14 days) stage of culture, whereas PKC(alpha)-, (beta I)-, and (gamma)-immunoreactive neurons were most abundant in the late stage of culture. Immunoreactivity of each PKC was high in the cytoplasm, processes, and growth cones. Prominent nuclear staining was observed with anti-PKC(gamma) antibody. These results are in contrast with in vivo results where each PKC isozyme is localized in a distinct population of neurons and subcellular compartment, suggesting the presence of regulatory mechanisms for PKC expression and compartmentalization in vivo.

Animals↗
PubMed1991

Immunoreactivity of CD45, a protein phosphotyrosine phosphatase, in Alzheimer's disease.

Both protein kinases and phosphoprotein phosphatases are important components of signal transduction systems in cells. Recent studies in Alzheimer's disease (AD) have shown abnormal protein phosphorylation in the cortex suggesting an alteration in these enzymes. In the present study, an antibody against CD45 was used to analyze the status of this protein phosphotyrosine phosphatase in AD. We studied and quantified the immunohistochemical and immunochemical distribution of this integral membrane protein in control and AD brain. We found that anti-CD45 immunostained the great majority of microglia, both resting and activated. These cells were Ricinus communis agglutinin I positive and glial fibrillary acidic protein and neurofilament negative. The AD frontal cortex showed a 35% (P less than 0.01) increase in the number of anti-CD45 immunoreactive microglia as compared with controls. These results were consistent with the immunoblot quantification of CD45 immunoreactivity following native gel electrophoresis. In AD, 30% of the CD45-immunostained microglia were clustered in the neuritic plaques (about six per plaque) while the remaining 70% were scattered in the neuropil. The AD hippocampus showed an increase in CD45-immunoreactive microglia in the molecular layer of the dentate gyrus. At the ultrastructural level, CD45 immunoreactivity was localized exclusively to the plasma membrane of the microglia. The presence of the anti-CD45 immunoreactivity in microglia suggests the possibility that they may require the presence of CD45 as a cell surface receptor which may regulate cell function through modulation of intracellular signaling.

Aged↗
PubMed1991

Membranous lipodystrophy (Nasu-Hakola disease) with thalamic degeneration: report of an autopsied case.

An autopsied case of membranous lipodystrophy (Nasu-Hakola disease, NHD) with thalamic degeneration was reported. A 34-year-old Japanese man was diagnosed as having NHD by bone biopsy prior to the onset of clinical symptoms. His maternal grandfather and paternal grandmother are cousins, but this family history is negative for NHD. He developed frontal lobe syndrome at the age of 35 with progressive dementia, and died of acute renal failure at the age of 46. Gross inspection of the brain detected atrophy and softening of the cerebral white matter, predominantly in the frontal lobe. Microscopically, numerous spheroids, predominant fibrillary gliosis with less prominent demyelination "dissociation glio-myélinique" and scanty sudanophilic lipid droplets were observed, indicating the sclerosing type of NHD. An unusual patholgoical finding in this case was selective involvement of the thalamic nuclei with preservation of the other gray matter except for focal cortical necrosis. The topography of the affected thalamic nuclei is similar to that of systemic thalamus degeneration. An association with thalamic degeneration in NHD has not been previously reported. The present case suggests that NHD also affects the thalamus.

Adult↗
PubMed1991

Impaired biphasic insulin release in mildly diabetic rats bearing a chronic portal vein catheter.

Using a new technique of chronic portal vein catheterization in freely moving rats, we investigated the insulin release to intravenous (IV) glucose (0.5 g/kg) injection in mildly diabetic (35 mg/kg streptozotocin, IV) rats. In nondiabetic rats, plasma insulin of the portal vein showed a clear biphasic release pattern, which peaked within two min after glucose injection, reached a nadir between three and six min, then began to rise to a second peak between 7 and 10 min, and reached a second nadir at 14 min. In mildly diabetic rats, a biphasic insulin release was evident, but both the first and the second phase insulin releases were impaired. In spite of this impaired insulin release, glucose intolerance was mild. Simultaneous blood sampling from the portal and peripheral veins after IV glucose injection revealed that diabetic rats showed diminished hepatic insulin extraction. These results suggest that the biphasic insulin release to glucose is impaired in mildly diabetic rats, but diminished hepatic insulin extraction contributes at least to keeping glucose tolerance mild.

Animals↗
PubMed1991

L3T4+ T cells induce hepatic lesions resembling primary biliary cirrhosis in mice with graft-versus-host reactions due to major histocompatibility complex class II disparity.

We had shown that the appearance of hepatic lesions such as epithelioid granulomas and chronic nonsuppurative destructive cholangitis (CNSDC)-like bile duct changes characteristic of primary biliary cirrhosis (PBC) in mice undergoing MHC class II-disparate graft-versus-host reactions (GVHR). To further examine the pathogenesis of the disease, we examined in the present study which T cell subset, i.e., L3T4+ or Lyt-2+ T lymphocytes, had an ability to induce such hepatic lesions. (B6 x bm12)F1 recipients were injected with unseparated T cells, L3T4+, or Lyt2+ T cells of B6 mice and on various days postinjection liver specimens were obtained. At Day 14 postinjection, livers of mice injected with whole T cells or L3T4+ T cells showed PBC-like histological changes, but none of the lesions were induced by Lyt-2+ T cells. Immunohistochemical studies revealed that Lyt-2+ as well as L3T4+ T cells were detected around bile ducts and some of them were infiltrating among bile duct epithelial cells. Kinetic studies showed that shortly after injection of L3T4+ T cells, L3T4+ T cells appeared around bile ducts and then Mac-1+ cells emerged. Lyt-2+ T cells and surface IgM+ B cells were detected on Day 5 and increased thereafter. Hepatic granulomas consisted of both L3T4+ and Lyt-2+ T cells with a few B cells. The aberrant expression of MHC class II (Ia) antigen was detected mainly at the lateral surface of bile duct epithelial cells by Day 14 postinjection. Based on these findings, the developmental mechanism of PBC-like hepatic lesions induced in mice with GVHR was discussed.

Animals↗
PubMed1991

Patterns of aberrant sprouting in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by extensive synaptic and neuronal loss and by plaque formation in the cortex, but the mechanisms responsible for synaptic plasticity in the neocortex are still not completely understood. To analyze the sprouting response in AD cortex, we compared the patterns of GAP-43 with synaptophysin immunoreactivity. In AD, GAP-43 immunohistochemistry revealed extensive sprouting in the hippocampal molecular layer, stratum polymorphous, CA1 region, and prosubiculum. These regions presented abundant anti-GAP-43-immunoreactive coiled fibers and dystrophic neurites in association with plaques. Some of these sprouting structures were colocalized with anti-synapto-physin- and anti-neurofilament-positive neurites. The AD neocortex was characterized by an overall decrease in GAP-43 immunoreactivity accompanied by sprouting neurites in the areas of synaptic pathology. We conclude that GAP-43 might be involved in the mechanisms of synaptic plasticity in the AD cortex, as well as in the process of aberrant sprouting in the neuritic plaques.

Aged↗
PubMed1991

Endothelin-1 and endothelin-3 stimulate ovarian steroidogenesis.

The possible direct involvement of endothelin (ET) in preovulatory follicular steroidogenesis was investigated in vitro. Follicular development in immature rats was induced by pregnant mare's serum gonadotropin (PMS). Twenty-four and 48 h after PMS injection, ovarian follicles were incubated in vitro with or without 4 x 10(-11) to 4 x 10(-5) M endothelin-1 (ET-1) or endothelin-3 (ET-3) for 2 h, or perifused in vitro with 4 x 10(-7) M of ET-1 or ET-3 for 3 h. In the incubation study, ET-1 and ET-3 significantly stimulated progesterone, testosterone, and 17 beta-estradiol production by follicles 24 and 48 h after PMS injection. Progesterone, testosterone, and 17 beta-estradiol were increased in a dose-dependent manner by 4 x 10(-11) to 4 x 10(-6) M ET-1 or ET-3; in the case of ET-3, this increase occurred only at 24 h after PMS injection. In addition, ET-1 stimulated ovarian steroid production more effectively than ET-3. In addition to their effects on ovarian steroidogenesis, both ET-1 and ET-3 increased tissue cyclic adenosine-3',5'-monophosphate (cyclic AMP) production. The steroidogenic effect of ET-1 or ET-3 was most pronounced by follicles 48 h after PMS injection. In the perifusion experiment, ET-1 and ET-3 also stimulated progesterone, testosterone, and 17 beta-estradiol secretion. These indicate the involvement of ET-1 or ET-3 in the regulation of ovarian differentiation by stimulating steroidogenesis, at least partially through the mediation of cyclic AMP.

Animals↗