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K Fujiwara

Publications and source records attributed to K Fujiwara.

At least 415 records · Page 23Linked to original sources
PubMed1994

New hapten-protein conjugation method using N-(m-aminobenzoyloxy) succinimide as a two-level heterobifunctional agent: thyrotropin-releasing hormone as a model peptide without free amino or carboxyl groups.

The use of a two-level heterobifunctional agent N-(m-aminobenzoyloxy)succinimide (m-ABS) allowed us to develop a new method for preparing hapten-protein conjugates. This was demonstrated by a conjugation between thyrotropin-releasing hormone (TRH) and bovine or human serum albumin (BSA or HSA). The conjugation is based on the principle that the succinimidyl ester group of m-ABS immediately acts on an epsilon-amino group of lysine residues of carrier protein BSA (or HSA) and a m-aminobenzoyl group incorporated into the protein is then activated by diazotization to a functional m-diazobenzoyl group (m-DB) acting on a histidyl group of TRH. The TRH-BSA containing about 3.5 mol of TRH per BSA molecule, elicited the production of TRH antibody in rabbits. A new type of enzyme-linked immunosorbent assay (ELISA) for TRH was developed using the antiserum, the solid-phase antigen TRH-HSA and the commercially available horseradish peroxidase-labeled goat anti-rabbit IgG/Fab' as a marker, revealing that the ELISA was monospecific to the hormone and measured as low as 50 pg of the hormone reproducibly. Also, using the antiserum by the indirect immunoperoxidase method the distribution of immunoreactive TRH in the rat brain was demonstrated in neurons of the paraventricular nucleus and neuronal processes of the median eminence. These results strongly suggested that the use of m-ABS provided a simple and efficient new method for preparing immunogens not only for the previously reported haptens with a primary amino group(s) (J. Immunol. Methods 134 (1990) 227), but also for haptens with an imidazole, phenolic, or indole group(s) in the molecule.

Amino Acid Sequence↗
PubMed1994

Purification and characterization of lipoyl-AMP:N epsilon-lysine lipoyltransferase from bovine liver mitochondria.

Lipoyl-AMP:N epsilon-lysine lipolytransferase (lipolytransferase) catalyzes the transfer of the lipoyl group from lipoyl-AMP to a lysine residue of the specific enzyme proteins. We have shown previously that the lipoyltransferase activities locate in mitochondria using apoH-protein of the glycine cleavage system as an acceptor of the lipoyl group (Fujiwara, K., Okamura-Ikeda, K., and Motokawa, Y. (1990) J. Biol. Chem. 265, 17463-17467). Here we describe the purification and the characterization of two isoforms of lipolytransferase termed lipoyltransferase I and lipoyltransferase II from bovine liver mitochondria. Lipoyltransferase II was purified to apparent homogeneity, whereas the final product of lipoyltransferase I still contained a minor contaminant. Although the two forms could be resolved on a hydroxylapatite column chromatography, they were indistinguishable, as judged by: (a) behavior during purification on ion exchange, hydrophobic, or affinity columns; (b) molecular mass determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel exclusion chromatography (40 kDa); and (c) catalytic properties (substrate specificity, kinetic constants, and optimal pH). Both lipoyltransferase I and II could not use lipoic acid plus MgATP as a substrate in place of lipoyl-AMP. Surprisingly, the lipoyltransferases transferred not only the lipoyl group but also the acyl groups from hexanoyl-, octanoyl-, and decanoyl-AMP to apoH-protein to a similar extent.

Acyltransferases↗
PubMed1994

Evidence that impaired intracellular collagen synthesis reduces proliferation in cultured rat hepatocytes.

Contribution of collagen to cell proliferation was studied in cultured hepatocytes. When alpha,alpha'-dipyridyl, an iron chelator which blocks hydroxylation of prolyl and lysyl residues of procollagen and expression of procollagen mRNA, was added to the medium of rat hepatocytes in primary culture, DNA synthesis of the cells was reduced in a dose-related manner without changes in protein synthesis. The reduction of collagen synthesis was parallel with the changes of DNA synthesis. The addition of P-1894B or minoxidil, which inhibits specifically prolyl hydroxylase or lysyl hydroxylase, respectively, also produced similar results. However, the DNA synthesis was not affected by beta-aminopropionitrile fumarate, which inhibits cross linking in extracellular collagen maturation, nor by purified bacterial collagenase. Intracellular processing of collagen synthesis may be required for proliferation in cultured rat hepatocytes.

2,2'-Dipyridyl↗
PubMed1994

Metaphase and anaphase in the artificially induced monopolar spindle.

By using monopolar spindles artificially induced in sea urchin embryos, we examined whether or not the presence of two opposing poles was an indispensable condition for keeping chromosomes at a fixed distance from the pole at metaphase and for the anaphase chromosome movement. Chromosomes were stained with Hoechst dye 33342 and their behavior was followed in the monopolar and the control bipolar spindles. In the monopolar spindle, chromosomes were first arranged on a curved metaphase plate and then spread on a part of the imaginary surface of a sphere whose center was the monopole. The estimated chromosome-to-pole distance was similar to that of bipolar spindles at metaphase and remained fixed until chromosomes started to move toward the pole. The average duration of metaphase in the monopolar spindle was 6 times longer than that in the bipolar spindle. The poleward movement of chromosomes in the monopolar spindle was similar to the anaphase A (chromosome-to-pole movement) in the bipolar spindle with respect to the velocity, duration, distance, and synchronization of migration. These results show that even half of the normal spindle has capacities for the arrangement of chromosomes at metaphase and for the anaphase A chromosome movement. Based on these results, we were able to exclude some existing theories of metaphase, such as the one based on the balance of forces between the two poles.

Anaphase↗
PubMed1994

Immunoglobulin class switch of anti-ganglioside monoclonal antibody from IgM to IgG.

Ganglioside GM2, which is one of the major gangliosides expressed on cell surface of neuroectodermal-origin human tumors, has been focused on as a target molecule for passive immunotherapy. One of the problems in this area was that monoclonal antibodies (mAbs) raised against GM2 were of IgM class even if donors of B cells were varied in mouse, rat or human. We stimulated two kinds of mice hybridomas having membrane-bound anti-GM2 IgM on their surface with GM2 incorporated in synthetic liposomes in the presence of the mouse thymocytes to accelerate the class switch of immunoglobulins (Igs). After the stimulation, protein A-reactive clones were sorted out using a cell sorter. We finally isolated two class switch variants generating mouse IgG3, designated KM796 and KM750, from original hybridomas producing mouse IgM anti-GM2 mAbs, KM696 and KM697, respectively after over 20-time repetitions of the sorting. ELISA with 11 common gangliosides revealed that one of the variant, KM750, retained the same binding specificity to N-acetyl GM2 as that of the parental IgM, KM697. By ELISA using panel of anti-idiotype (Id) mAbs to anti-GM2 mAbs, KM750 was shown to retain the parental KM697 Id. Another variant KM796 almost lost its activity in purification process in acidic condition and changes in Id were suggested. In immunofluorescence assay, KM750 was confirmed to bind to GM2-expressing tumor cell lines. The class switch hybridoma has been stably cultured with the production of the IgG3-class mAb for more than 22 months.

Animals↗
PubMed1994

Specificity of trypsin digestion and conformational flexibility at different sites of unfolded lysozyme.

Fourteen tryptic peptides and nine intermediates were identified as products of trypsin digestion of reduced and S-3-(trimethylated amino) propylated lysozyme. Kinetics of the appearance and disappearance of these products were observed by monitoring the peak areas on the chromatogram. In spite of the complicated reaction pathways, kinetics of the digestion of proteins and several intermediate products show simple decay curves with a single rate constant. In this paper, the trypsin susceptibility of the individual cleavage site is defined as a hydrolytic rate constant of the susceptible peptide bond in the presence of 10 nM trypsin. The cleavage sites of unfolded lysozyme are classified into two groups in terms of the trypsin susceptibility: one has a high susceptibility (10-20 h-1) and the other a low susceptibility (1.0-2.0 h-1). In the unfolded state of lysozyme, in conclusion, the region from residues 15 to 61 has a strong resistance to trypsin digestion; on the other hand, the C-terminal half of the polypeptide chain is flexible enough to fit into the active site of trypsin. In addition, six kinds of pentapeptides were synthesized as analogues of lysozyme fragments including Arg 14, Arg 21, Lys 33, Arg 45, Arg 61, and Arg 73. Kinetics of tryptic digestion of them were observed. Both kcat and KM were determined for these synthetic pentapeptides. The susceptibility of each cleavage site in pentapeptides is determined and compared with that corresponding in proteins. The susceptibility is usually higher when the susceptible peptide bond is included in proteins than in pentapeptides, so long as the conformation of peptide chain is flexible. However, susceptibilities of a few sites in proteins are lower than those in pentapeptides. This means that the peptide chains tend to fold locally to prevent trypsin from binding to the sites. It was found that the sites of Arg 21 and Arg 45 are indeed resistant to trypsin, but the site of Lys 33 is not so much, although the hydrolytic rate at Lys 33 itself is extremely slow.

Amino Acid Sequence↗
PubMed1994

Production and characterization of monoclonal antibodies against the polyamine, spermine: immunocytochemical localization in rat tissues.

Two monoclonal antibodies of types IgG2b and IgG2a, anti-spermine-(Spm)-1 (ASPM-1) and anti-Spm-2 (ASPM-2) respectively were found among five clones of murine monoclonal antibodies, which were raised against Spm conjugated with bovine serum albumin via the cross-linker N-(gamma-maleimidobutyryloxy) succinimide (GMBS). Antibody specificity was evaluated by a recently developed ELISA binding test, and led to the study of tissue sections by immunocytochemistry (ICC). ASPM-1 showed exclusive immunoreactivity with Spm, with the exception of a negligible cross-reactivity (2.0%) with spermidine (Spd). ASPM-2, on the other hand, reacted almost equally with acetylspermine (Ac-Spm) and N1-acetylspermidine (N1-Ac-Spd) but with none of the other polyamine-related compounds tested. Complete agreement was obtained with the results of immunoblot analysis. Furthermore, results for antibody specificity obtained with the ELISA inhibition test and ICC model experiments using Sepharose gel beads strongly suggested that ASPM-1 recognizes the Spm molecule possessing at least a free terminal primary amino group, while ASPM-2 recognizes the Spm molecule acylated at both the terminal primary amino groups. An ICC method using ASPM-2 produced strong staining for polyamines (PAs) in the cytoplasm (but very few in the nuclei) of two different tumor cell lines and protein- or peptide-secreting cell systems, including exocrine and endocrine cell types; ASPM-1 showed immunoreactivity only with the tumor cell lines. These results strongly suggest that ASPM-2 may be useful for studies on actively proliferating and neoplastic cells, supporting our previously proposed idea that in immunocytochemistry PAs were converted to a variety of PA derivatives during the fixation process.

Animals↗
PubMed1994

An evaluation of polyamine immunocytochemistry using immunocytochemical model systems.

Polyamine (PA) immunocytochemistry (ICC) was evaluated by a recently developed enzyme-linked immunosorbent assay (ELISA) binding test (ELISABT) using an anti-spermine (Spm) serum raised against Spm conjugated via the cross-linker N-(gamma-maleimidobutyryloxy)succinimide (GMBS) with bovine serum albumin. In the test the antiserum showed strong immunoreactivity with N1-acetylspermine (Ac-Spm) and acetylspermidine (N1-Ac-Spd and N8-Ac-Spd), and low immunoreactivity with Spm and Spd, which was, however, markedly enhanced after reaction with GMBS, acetic anhydride or glutaraldehyde. Complete agreement with results of immunoblot analysis was observed. PA-like immunoreactivity observed in the present PA ICC in cells in the foveolae and isthmus of rat gastric glands was completely abolished by absorption of the serum with N1,N12-diacetyl-Spm, Ac-Spm or N1-Ac-Spd, but not by Spm or Spd. This absorption test was then simulated by an ELISA inhibition test (ELISAIT) with a solid phase conjugated with Ac-Spm or Spm, and by a PA ICC model system using Sepharose gel beads conjugated with each of several PAs. The results strongly suggest that the immunostaining in the gastric mucosa was mainly due to antibody species in the serum specific to 'acylated' Spm and Spd, but not to Spm or Spd. Acetyl PAs exist at such low concentrations in animal tissues that they are virtually undetectable by current ICC methods. Therefore Spm and Spd are likely candidates for those detected, after having been converted by fixation into such PA derivatives as become reactive with the antiserum.

Animals↗
PubMed1994

Relationship of cell survival, drug dose, and drug uptake after 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine treatment.

The mechanisms that govern the activity and the factors that control the anticancer activity of synthetic ether lipids have not been fully elucidated. In this study, three factors were studied in relationship to cell survival after treatment with 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3): (1) exposure dose, (2) drug uptake, and (3) cell density and cell-cycle distribution. In BG-1 human ovarian carcinoma cells, cell survival was an exponential function of exposure dose and was dependent on drug concentration. Drug uptake was dependent on the concentration of ET-18-OCH3, whereas the reduction in cell survival was directly related to the uptake of drug only in the first decade of cell kill. When the quantity of cells per flask was tripled from 4 to 12 x 10(6) cells, ET-18-OCH3 failed to induce a G2 block. Furthermore, the cell kill induced by a 72-h exposure to 2 microM ET-18-OCH3 was decreased by a factor of 2 when the cell density increased. Therefore, exposure dose and cell density are important parameters in determining the cell kill induced by ET-18-OCH3.

Antineoplastic Agents↗
PubMed1994

Regulating factors of liver regeneration after hepatectomy.

The factors regulating liver regeneration were studied by measuring changes in the liver volume and serum hepatocyte growth factor (HGF) levels after hepatectomy. Changes in the liver volumes were studied in 68 hepatectomized patients, including (A) hepatoma patients who had chronic hepatitis or liver cirrhosis (n = 44) and (B) metastatic liver cancer patients who had normal liver parenchyma (n = 24). The hepatic volume increased by 13.8% of the remnant hepatic volume in group A and by 49.1% in group B. The examined factors included the percentage of resected liver volume (%RLV) and the results of laboratory tests. Regression analysis showed that in group A, both %RLV (beta = 0.46) and the serum total bilirubin (T-Bil) level (beta = -0.33) correlated significantly with the extent of liver regeneration and that in group B, only %RLV (beta = 0.78) correlated significantly with the regeneration. Serum HGF levels after hepatectomy were studied in 21 hepatectomized patients, including 11 hepatoma patients and 10 patients with some types of metastatic liver cancer. Serum HGF levels increased significantly after surgery in all 21 patients. Regression analysis, however, showed that the change in HGF was related to liver cirrhosis (beta = 0.46) and to the maximal postoperative T-Bil level (beta = 0.51) but not to the extent of liver regeneration after hepatectomy. These results suggest that liver regeneration is regulated primarily by factors relating to the percentage of the resected liver parenchyma and that serum HGF levels do not directly relate to liver regeneration after surgery.

Aged↗
PubMed1994

Promotional effects of azathioprine on peripheral B cell lymphomas in BALB/c mice induced by administration of 7,12-dimethylbenz[a]anthracene.

BALB/c mice either were injected subcutaneously with 25 micrograms/g or 50 micrograms/g body weight 7,12-dimethyl-benz[a]anthracene (DMBA) at the age of 13 weeks, or received repeated s.c. injections of azathioprine (0.1 mg/g) starting from the age of 6 or 18 weeks, or they received treatments with both agents. Of the 317 mice treated, 69 developed lymphomas by the age of 50 weeks. None of the 31 non-treated mice developed lymphomas during this period. The lymphomas, classified according to Pattengale-Taylor's classification for murine lymphomas/leukemias, were examined immunohistochemically and immunogenetically. The lymphomas induced with DMBA showed a male preponderance and a higher incidence of the B cell type, especially in the lymph node and spleen. Azathioprine induced predominantly T-lymphoblastic lymphomas in the thymus and these were found more frequently in females. Treatment with both DMBA and azathioprine remarkably increased the incidence of B lymphomas, especially follicular center cell lymphomas in the spleen, which are the predominant type of spontaneous lymphomas occurring in older BALB/c mice. Some of the treated mice also developed prelymphomatous lesions.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1994

Fat-storing cell abnormalities associated with endothelial cell damage after cold ischemic storage of rat liver in UW solution.

Rat liver was stored at 1 degree C in University of Wisconsin solution, and morphological changes were observed after 12, 18, 24, and 36 hr by transmission electron microscopy. There were two types of endothelial cell damage in the hepatic sinusoids. One was disruption of the endothelial linings, and the other detachment of endothelial cells into the sinusoidal space accompanied by fat-storing cell abnormalities. The former damage was seen after storage longer than 12 hr, while the latter developed after 18 hr even in the hepatic sinusoids with no disruption of the linings. Considering that fat-storing cell damage can produce endothelial cell destruction, this damage should be given attention as one of factors of endothelial cell destruction in the hepatic sinusoids after cold storage of the liver.

Adenosine↗
PubMed1994

Do astral microtubules play a role in metaphase chromosome positioning?

From several recent studies on monopolar spindles, it is now clear that a phase analogous to metaphase in bipolar spindles exists in the monopolar spindle, denying the validity of the favored model for metaphase which is based on the balance between two oppositely directed poleward forces acting on the unsplit kinetochores. Faced with this new fact, several investigators have proposed new models for metaphase plate formation which work for both the monopolar and the bipolar spindles. Since astral microtubules are thought to play important roles in certain models, we have investigated the role of astral microtubules in maintaining chromosomes at the metaphase position by using monopolar spindles induced in sea urchin embryo cells. When monopolar spindles were exposed to 1 microM nocodazole, a microtubule depolymerizing agent, most of the astral microtubules were rapidly depolymerized while the kinetochore fibers appeared to be little affected. Chromosomes were locked into the metaphase position for as long as 2 min, indicating that the metaphase chromosome position in sea urchin monopolar spindles can be maintained with little or no astral microtubules. By the effect of the antimitotic drug, kinetochore fibers slowly shortened and chromosomes which were attached to the far end of kinetochore fibers moved toward the pole. On the other hand, the chromosome position rapidly shifted farther away from the pole when monopolar spindles were treated with taxol or D2O. These results indicate that the metaphase chromosome position can be altered by affecting microtubule dynamics, particularly that of the kinetochore fiber microtubules as suggested by the results of the nocodazole experiments.

Animals↗
PubMed1994

Therapy of Graves' ophthalmopathy with intravenous high-dose steroid followed by orbital irradiation.

We investigated the effects of intravenous high-dose steroid therapy followed by orbital irradiation for Graves' ophthalmopathy in eight patients. All patients presented with diplopia or fixed globes. Extraocular muscle dysfunction showed excellent improvement after the combined therapy; diplopia disappeared completely in five of them, and one patient with fixed globes showed normal eye movement. Two other patients also exhibited great improvement, although their diplopia persisted. Their ophthalmopathy index was decreased from (mean +/- SD) 4.25 +/- 0.82 to 0.75 +/- 1.48. Extraocular muscle enlargement, assessed by magnetic resonance imaging study after the radiotherapy, was also reduced after the combined therapy, although two patients did not show remarkable enlarged extraocular muscles. There was no change in extraocular muscle thickness 6-9 months after the therapy, and the ophthalmopathy index did not show change in a long follow-up (maximum 37 months after the therapy). These results suggest that high-dose methylprednisolone followed by orbital radiotherapy is a good therapeutic design for Graves' ophthalmopathy and justify a prospective randomized trial.

Adult↗
PubMed1994

Immunohistochemical distribution of CD44 and desmoplakin I & II in Hailey-Hailey's disease and Darier's disease.

The cell-surface glycoprotein CD44 is found on a wide variety of cells including epidermal cells. It is involved in cell to cell adhesion. Desmoplakin I & II are important components of the attachment plaque of desmosomes. In this study, we compared the distribution patterns of anti-CD44 and anti-desmoplakin I & II in Hailey-Hailey's disease and Darier's disease. In the normal skin, anti-CD44 stained the entire periphery of epidermal keratinocytes while anti-desmoplakin I & II produced dotted staining patterns along the periphery of epidermal keratinocytes. In Hailey-Hailey's disease and Darier's disease, the staining pattern of anti-CD44 on acantholized keratinocytes did not change, but anti-desmoplakin I & II lost their peripheral, dotted patterns and stained diffusely in the cytoplasm in most acantholytic cells. These results suggest that, in Hailey-Hailey's disease and Darier's disease, CD44 may be intact even in acantholytic cells but abnormalities of desmoplakin exist in such cells.

Carrier Proteins↗
PubMed1994

Aberrant expression of the monocyte/macrophage phenotype in a human T cell line immortalized by HTLV-I and an adult T cell leukemia/lymphoma cell line.

An HTLV-I-immortalized human T cell line (JP-2), a N-methyl-N'-nitro-N-nitrosoguanidine-treated JP-2 line (JP-2T), and an adult T cell leukemia cell line (ATL-1T) were examined morphologically and phenotypically. All of these cell lines expressed some T cell markers, including CD4, and showed rearrangement of T cell receptor (TCR) genes, but they lacked CD3 and TCR antigens and expressed some myelomonocytic markers (CD68, HL-21, CD15, CD16). JP-2 cells grew in suspension, but JP-2T and ATL-1T cells, which mostly adhered to the surface of culture vessels, showed macrophage-like morphological features and expressed more monocyte/macrophage markers (lysozyme, alpha 1-antitrypsin) and fibronectin. ATL-1T cells transplanted into SCID mice lost the macrophage features. These results suggest that HTLV-I infected T cells can express some macrophage features and that these cells may provide a model that will be useful in elucidating the phenotypic variability of T cell lymphomas.

Animals↗