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Biomedical subjects

I Nakamura

Publications and source records attributed to I Nakamura.

At least 145 records · Page 8Linked to original sources
PubMed1996

Three new cytotoxic T cell epitopes identified within the hepatitis C virus nucleoprotein.

Cytotoxic T lymphocytes (CTLs) may play a role in host defence against hepatitis C virus (HCV) infection, and HCV-specific CTL epitopes may be included in vaccines to induce protective CTLs. We identified three new epitopes within the HCV nucleoprotein recognized by CTLs. HCV nucleoprotein residues 28-37 are the minimal epitope recognized by CTLs in association with the class I human leukocyte antigen B60, and epitopes in HCV nucleoprotein residues 111-130 and 161-180 are both recognized by CTLs in association with the class II human leukocyte antigen DRBI*08032.

Amino Acid Sequence↗
PubMed1996

Calcitonin-induced changes in the cytoskeleton are mediated by a signal pathway associated with protein kinase A in osteoclasts.

Calcitonin is known to inhibit osteoclastic bone resorption through its receptor, which is abundantly expressed on the plasma membrane of osteoclasts. Recently, it was reported that calcitonin receptors were coupled to both cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). To examine how the PKA and PKC pathways are involved in the effects of calcitonin, we focused on changes in the cytoskeleton of murine osteoclast-like multinucleated cells (OCLs) formed in vitro. When OCLs were cultured on dentine slices, they formed resorption pits and ringed structures of F-actin dots (actin rings). Elcatonin, a synthetic analogue of eel calcitonin, disrupted actin rings and inhibited pit formation in a dose-dependent manner. Forskolin and dibutyryl cAMP, both of which have the ability to activate PKA, mimicked the effects of elcatonin. Phorbol myristate acetate and phorbol 12,13-dibutyrate, both of which have the ability to activate PKC, also inhibited pit-forming activity, but little affected actin rings of OCLs. The inhibitory effects of elcatonin on the pit formation and actin ring formation were partially restored by the treatment with Rp-cAMPs, a cAMP antagonist. Elcatonin induced a rapid increase in PKA activity within a few minutes, and its activation by elcatonin occurred in a dose-dependent manner. The time- and dose-dependent profiles of elcatonin for the activation of PKA were similar to those for the disruption of actin rings. Moreover, microinjection of activated PKA into OCLs disrupted actin rings within 10 min on culture dishes. Actin rings were little affected by the microinjection of the PKA preincubated with a cAMP-dependent protein kinase inhibitor (IP-20) into OCLs. These results suggest that PKA activation, rather than PKC activation, is involved in mediating the effects of calcitonin, through the disruption of actin organization.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

Osteoclast function is activated by osteoblastic cells through a mechanism involving cell-to-cell contact.

We have established a method for obtaining an enriched preparation of functionally active osteoclast-like multinucleated cells (enriched OCLs) from co-cultures of mouse primary osteoblasts and bone marrow cells. Using these enriched OCLs, the effect of osteoblastic cells on osteoclast function was examined in two assays: a pit formation assay and an assay for actin ring formation. The enriched OCLs cultured for 24 h on dentine slices formed only a few resorption pits. When various numbers of primary osteoblasts were added to the enriched OCLs, the areas of the resorption pits increased proportionally to the number of osteoblasts added. Like primary osteoblasts, the established cell lines of osteoblastic cells (MC3T3-E1 and KS-4) and bone marrow-derived stromal cells (MC3T3-G2/PA6 and ST2) potentiated the pit formation caused by enriched OCLs. In contrast, the fibroblastic cell lines NIH3T3 and C3H10T1/2) and the myoblastic cell line (C2C12) failed to activate OCL function. When cell-to-cell contact between MC3T3-E1 cells and enriched OCLs was prevented, only a few resorption pits were formed. Pit formation by enriched rat osteoclasts placed on dentine slices was also stimulated by adding MC3T3-E1 cells. Actin ring formation and pit forming activity were well correlated in either culture of enriched mouse OCLs or authentic rat osteoclasts on dentine slices. These results indicate that osteoclast function is activated by osteoblastic cells through a mechanism involving cell-to-cell and/or cell-to matrix contact.

3T3 Cells↗
PubMed1996

Osteoclast function is activated by osteoblastic cells through a mechanism involving cell-to-cell contact.

We have established a method for obtaining an enriched preparation of functionally active osteoclast-like multinucleated cells (enriched OCLs) from co-cultures of mouse primary osteoblasts and bone marrow cells. Using these enriched OCLs, the effect of osteoblastic cells on osteoclast function was examined in two assays: a pit formation assay and an assay for actin ring formation. The enriched OCLs cultured for 24 h on dentine slices formed only a few resorption pits. When various numbers of primary osteoblasts were added to the enriched OCLs, the areas of the resorption pits increased proportionally to the number of osteoblasts added. Like primary osteoblasts, the established cell lines of osteoblastic cell (MC3T3-E1 and KS-4) and bone marrow-derived stromal cells (MC3T3-G2/PA6 and ST2) potentiated the pit formation caused by enriched OCLs. In contrast, the fibroblastic cell lines (NIH3T3 and C3H10T1/2) and the myoblastic cell line (C2C12) failed to activate OCL function. When cell-to-cell contact between MC3T3-E1 cells and enriched OCLs was prevented, only a few resorption pits were formed. Pit formation by enriched rat osteoclasts placed on dentine slices was also stimulated by adding MC3T3-E1 cells. Actin ring formation and pit forming activity were well correlated in either culture of enriched mouse OCLs or authentic rat osteoclasts on dentine slices. These results indicate that osteoclast function is activated by osteoblastic cells-through a mechanism involving cell-to-cell and/or cell-to-matrix contact.

Animals↗
PubMed1996

[The changes of complement activities in sera of mice after subcutaneous administration of beryllium chloride].

We studied changes of the complement pathway activities and the content of C3 in sera of mice, administered BeCl2 (containing 5 micrograms of Be per mouse) or CuCl2 (containing 5 micrograms of Cu per mouse) by a single subcutaneous injection. The value of the classical complement pathway activity (CH50) of the Be group 3 days after administration was significantly higher than that of the control group (P < 0.001). It was significantly lower than in the control group after 7 days (P < 0.001). On the other hand, the CH50 value of the Cu group 3 hr after administration tended to increase, however, it was significantly lower than in the control group after 7 days (P < 0.01). The change of the alternative complement pathway activity (ACH50) value of the Be group was similar to the change of the CH50 value of the group. The ACH50 value of the Cu group 3 days after administration tended to increase but it was the same as the ACH50 value of the control group after 7 days. The C3 contents of both the Be and Cu groups 3 days after administration were significantly higher than in the control group (P < 0.001). The aspartate aminotransferase (AST) activity of the Be group 7 days after administration was significantly higher than that of the control group (P < 0.01). By contrast, AST activity of the Cu group 3 hr after administration was significantly higher than in the control group (P < 0.05). The value of the alanine aminotransferase (ALT) activity of the Be group was low (P < 0.01), but that of the Cu group was high (P < 0.05), 3 hr after administration. These values of both groups after 7 days, however, were significantly higher than in the control group (P < 0.05). The AST/ALT ratio in mice was very high at 3 hr, and it remained high by 7 days after Be injection. On the other hand, the ratio of the Cu group was almost constant for 7 days after Cu injection. Thus, these values changed with relative expedition after Be injection. Therefore, we confirmed that measurements of complement activities and the content of C3 were valuable indices for assaying acute effects of Be on mice.

Alanine Transaminase↗
PubMed1996

Influence of calcium concentration on the antimicrobial activity of lasalocid against Selenomonas ruminantium.

The present study was designed to examine the interaction between the effects of lasalocid and Ca2+ on the growth and structure of Selenomonas ruminantium HD-4. Lasalocid, at a dose of 10 microM, inhibited cell growth almost completely after 12 hr incubation in the presence of relatively high extracellular concentrations of Ca2+ (from 5 to 50 mM), but only slightly reduced cell growth in the presence of 0.2 mM Ca2+. With Ca2+ alone, cell growth was also inhibited at 12 hr as a function of the concentration of Ca2+ over the range 5 to 50 mM. In cultures at mid-exponential phase, growth was markedly inhibited by the simultaneous addition of 10 microM lasalocid and 15 mM Ca2+, but only moderately inhibited by lasalocid in the absence of Ca2+. However, there was no significant effect on bacterial growth at the mid-exponential phase when Ca2+ alone was added to the incubation medium. In thin sections of cells treated with lasalocid in the presence of 15 mM Ca2+, abnormal cells were found with cytoplasmic voids and with an outer membrane detached from the inner membrane layer, this change in the outer membrane was also found in cells treated with lasalocid alone. There was no visible abnormality in the outer membrane in thin sections of Ca2(+)-treated cells, while most of these cells showed only a slight contraction of cytoplasmic material or a loss of cytoplasmic contents. These results indicate the presence of a synergistic effect between the actions of lasalocid and Ca2+ on cell growth, presumably due to induced cytoplasmic alterations.

Animals↗
PubMed1996

Use of fura-2/AM to measure intracellular free calcium in Selenomonas ruminantium.

This paper describes a procedure for loading the acetoxymethyl ester of fura-2 (fura-2/AM), and the subsequent measurement of the concentration of intracellular free Ca2+ ([Ca2+]i) in Selenomonas ruminantium (S. ruminantium) using this technique. To ascertain the optimal loading conditions, the effect was examined on the loading of fura-2/AM of ethylenediamine-tetraacetic acid (EDTA), lysozyme, pluronic F127 alone, or the simultaneous application of EDTA and pluronic F127. Individual administration of either EDTA, lysozyme or pluronic F127 did not produce an optimal loading of fura-2/ AM. The co-application of pluronic F127 and bovine serum albumin after treatment with EDTA increased the ratio of the fluorescence due to excitation at 340 nm to that at 380 nm (R340/380) markedly, with a high signal intensity for intracellular fura-2, indicating that adequate loading had been obtained. Using the present loading method, it was found that the resting free [Ca2+]i of S. ruminantium was 48.1 +/- 2.2 nM (n = 6). This is approximately one half that found in Escherichia coli, Propionibacterium acnes, Streptococcus bovis and eucaryote cells. This is the first measurement of free [Ca2+]i using fura-2/AM in the Gram-negative strict anaerobe S. ruminantium.

Animals↗
PubMed1995

Neurons, intracellular and extracellular neurofibrillary tangles in subdivisions of the hippocampal cortex in normal ageing and Alzheimer's disease.

Unaffected neurons, intracellular neurofibrillary tangles (I-NFTs) and extracellular NFTs (E-NFTs) in six normal subjects and six patients with Alzheimer's disease (AD) were morphometrically evaluated in eight subdivisions of the hippocampal cortex, using the Gallyas silver impregnation technique modified by the application of hematoxylin and eosin. The subdivisions examined included CA1-4, prosubiculum (PRO), subiculum and presubiculum (PRE), parasubiculum (PARA) and the entorhinal cortex (ENT). In the AD patients, the number of unaffected neurons in ENT, CA1, PRO and PARA was significantly decreased to one-quarter to two-thirds of that of the normal aged subjects. These four subdivisions in the AD patients had a greater number of both I- and E-NFTs. There were no significant differences in the total number of unaffected neurons, I- and ENTs between the AD patients and normal aged subjects for all the subdivisions. These findings suggest that neuronal loss in the hippocampal cortex in AD is almost entirely due to NFT formation. Furthermore, with regards to neuronal loss and NFT formation, there were two different subdivision groups in the AD patients. One group was composed of severely affected subdivisions (ENT, CA1, PRO and Para) and was distinct from the other group which was composed of mildly affected subdivisions (CA4, CA3, CA2 and PRE). Each subdivision in the normal aged subjects belonged to the mildly affected group as seen in the AD patients. These findings indicate that both neuronal loss and NFT formation associated with normal ageing and with AD are not only quantitatively but also qualitatively different.

Aged↗
PubMed1995

Allospecificities of B6D2F1 hybrid NK cell subsets defined by Ly-49A expression.

More than 90% of IL-2-activated plastic-adherent murine splenocytes (A-LAK4 cells) are NK1.1+ NK cells and both H-2b/b homozygous C57BL/6- and H-2b/d heterozygous (C57BL/6 x DBA/2)F1 (B6D2F1)-derived populations of such cells contain an Ly-49A+ subset. In B6D2F1 A-LAK cells, as well as in freshly isolated spleen cells of the same mice, Ly-49A+ cells represent approximately 10% of NK1.1+ cells. However, the level of Ly-49A expression in B6D2F1 NK cells is lower than in C57BL/6 (B6). The cytolytic activity of B6- and B6D2F1-derived A-LAK cells against normal target cells is specific, and is in agreement with the known patterns of natural resistance in vivo against Hh-1-mismatched bone marrow allografts. H-2b lymphoma cells transfected with the Dd gene, but not the Ld gene, no longer express the Hh-1b phenotype that is recognized by B6D2F1 A-LAK cells, raising the possibility that this selective effect of the Dd gene on Hh-1b phenotype is related to the known inability of Ly-49A+ A-LAK cells to kill Dd-expressing tumor target cells. Depletion of Ly-49A+ A-LAK cells by Ab and complement reduces the lytic capacity of B6D2F1 A-LAK cells against normal B6 target cells of the Hh-1b phenotype to one-third of the original level. Conversely, positively selected Ly-49A+ A-LAK cells are enriched for the same activity. The results, therefore, favor the view that the Hh-1 phenotype of the target cells may be defined largely by the effector cell's recognition of class I Ags on the target cell surface.

Animals↗
PubMed1995

Wortmannin, a specific inhibitor of phosphatidylinositol-3 kinase, blocks osteoclastic bone resorption.

The biological role of phosphatidylinositol (PI)-3 kinase was examined in osteoclast-like multinucleated cells (OCLs) formed in co-cultures of mouse osteoblastic cells and bone marrow cells. The expression of PI-3 kinase in OCLs was confirmed by Western blot analysis. Wortmannin (WT), a specific inhibitor of PI-3 kinase, inhibited PI-3 kinase activity in OCLs both in vitro and in vivo. WT also inhibited pit-forming activity on dentine slices and disrupted a ringed structure of F-actin-containing dots (an actin ring) in OCLs in a dose-dependent manner. The inhibitory profiles of WT for pit and actin ring formation were similar to that for PI-3 kinase activity in OCLs. Electron microscopic analysis revealed that OCLs treated with WT did not form ruffled borders. Instead, numerous electron lucent vacuoles of differing sizes were found throughout the cytoplasm. These results suggest that PI-3 kinase is important in osteoclastic bone resorption.

Actins↗
PubMed1995

Possible involvement of focal adhesion kinase, p125FAK, in osteoclastic bone resorption.

Involvement of tyrosine phosphorylation in osteoclastic bone resorption was examined using osteoclast-like multinucleated cells prepared from co-cultures of mouse osteoblastic cells and bone marrow cells in the presence of 1 alpha,25-dihydroxyvitamin D3. When osteoclast-like cells were plated on culture dishes in the presence of 10% fetal bovine serum, they were sharply stained in their peripheral region by anti-phosphotyrosine antibody. Western blot analysis revealed that 115- to 130-kD proteins were tyrosine-phosphorylated in osteoclast-like cells. Using immunoprecipitation and immunoblotting, one of the proteins with 115-130 kD was identified as focal adhesion kinase (p125FAK), a tyrosine kinase, which is localized in focal adhesions. Immunostaining with anti-p125FAK antibody revealed that p125FAK was mainly localized at the periphery of osteoclast-like cells. Herbimycin A, a tyrosine kinase inhibitor, not only suppressed tyrosine phosphorylation of p125FAK but also changed the intracellular localization of p125FAK and disrupted a ringed structure of F-actin-containing podosomes in osteoclast-like cells. Antisense oligodeoxynucleotides to p125FAK inhibited dentine resorption by osteoclast-like cells, whereas sense oligodeoxynucleotides did not. These results suggest that p125FAK is involved in osteoclastic bone resorption and that tyrosine phosphorylation of p125FAK is critical for regulating osteoclast function.

Animals↗
PubMed1995

A helper T-cell antigen enhances generation of hepatitis C virus-specific cytotoxic T lymphocytes in vitro.

A T-cell helper for generation of hepatitis C virus-specific cytotoxic T lymphocytes was studied in three patients with chronic hepatitis C. In all three, human leukocyte antigen B44-restricted cytotoxic T lymphocytes recognizing an epitope in hepatitis C virus nucleocapsid protein residues 81-100 were generated from the peripheral blood lymphocytes by repeated stimulation with a synthetic hepatitis C virus nucleocapsid peptide. The proliferative response of peripheral blood lymphocytes to hepatitis C virus nucleocapsid protein residues 1-120 was observed in one patient, and was ascribed to CD4+ T cells. The helper T cells recognized a major epitope in residues 21-40 and a minor epitope(s) in residues 81-110. They produced interferon gamma, but interleukin 4 was not detectable in the T-helper cell culture supernatants. The hepatitis C virus nucleocapsid protein residues 1-120 and the major helper T-cell epitope enhanced generation of hepatitis C virus-specific cytotoxic T lymphocytes in vitro, although the protein alone did not generate them. In the other two patients, the protein did not enhance generation of hepatitis C virus-specific cytotoxic T lymphocytes in vitro. The results suggest that a hepatitis C virus-specific helper T-cell epitope is helpful for inducing a strong specific cytotoxic T-lymphocyte response.

Amino Acid Sequence↗
PubMed1995

Modulation of osteoclast differentiation by local factors.

Bone-resorbing osteoclasts are of hemopoietic cell origin, probably of the CFU-M-derived monocyte-macrophage family. Bone marrow-derived osteoblastic stromal cells play an important role in modulating the differentiation of osteoclast progenitors in two different ways: one is the production of soluble factors, and the other is cell-to-cell recognition between osteoclast progenitors and osteoblastic stromal cells. M-CSF is probably the most important soluble factor, which appears to be necessary for not only proliferation of osteoclast progenitors, but also differentiation into mature osteoclasts and their survival. A number of local factors as well as systemic hormones induce osteoclast differentiation. They are classified into three categories in terms of the signal transduction: vitamin D receptor-mediated signals [1 alpha,25(OH)2D3]; protein kinase A-mediated signals (PTH, PTHrP, PGE2, and IL-1); and gp130-mediated signals (IL-6, IL-11, oncostatin M, and leukemia inhibitory factor). All of these osteoclast-inducing factors appear to act on osteoblastic cells to commonly induce osteoclast differentiation factor (ODF), which recognizes osteoclast progenitors and prepares them to differentiate into mature osteoclasts. This line of approach will undoubtedly produce new ways to treat several metabolic bone diseases caused by abnormal osteoclast recruitment such as osteoporosis, osteopetrosis, Paget's disease, rheumatoid arthritis, and periodontal disease.

Animals↗