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Biomedical subjects

A Aoki

Publications and source records attributed to A Aoki.

At least 127 records · Page 7Linked to original sources
PubMed1992

Gene introduction into granulocyte-macrophage progenitor cells by electroporation: the relationship between introduction efficiency and the proportion of cells in S-phase.

Our previous study demonstrated the positive relationship between the gene introduction rate into hematopoietic cell lines by electroporation and the percentage of cells in S-phase. In the present study, granulocyte-macrophage progenitor cells (CFU-C) rich marrow cell fraction were cultured in suspension with IL-3, GM-CSF and G-CSF for 4 days. The number of CFU-C were increased three times after the culture, and 3H-thymidine suicide tests of cultured cells demonstrated that the proportion of CFU-C in S-phase was increased by two to four times. The efficiency of gene transfer into CFU-C with the plasmid pMoZtk (containing the beta-galactosidase gene) by electroporation was nearly doubled by culturing marrow cells with these growth factors. These findings confirm that the introduction rate of the gene into CFU-C by electroporation is more efficient in cell populations with a higher percentage of CFU-C in S-phase.

Cell Count↗
PubMed1992

Glomerulonephritis induced by murine chronic graft-versus-host reaction.

Severe glomerulonephritis was induced successfully in (B10 x DBA/2)F1 (BDF1) mice by injection of parental DBA/2 lymphoid cells. The mice manifested typical nephrotic syndrome dying around 10 weeks post injection. Electron microscopical examination demonstrated electron dense deposits first in the mesangial matrix, then in the subepithelium compatible with immune complex glomerulonephritis. Subendothelial deposits were not observed. Immunofluorescent study revealed IgG deposition in the capillary wall and IgM in the mesangium early in the process. As the lesion progressed, both IgG and IgM were present in the mesangial area and along the capillary wall. Some glomeruli showed segmental mesangiolysis, suggesting that altered mesangial cells have a role in the development of glomerular change, which together with rise in serum anti-DNA antibody titer suggest that autoantibodies promote the glomerular lesions in this model system.

Animals↗
PubMed1992

In vitro monitoring activation by the ligands and specific DNA-binding of the glucocorticoid receptors.

The glucocorticoid receptor is a member of the steroid and thyroid hormone receptor superfamily and acts as a ligand-activated transcription factor. To reconstitute the molecular mechanisms underlying the cellular response to soluble receptor ligands, we have exploited a cell-free system that exhibits glucocorticoid-induced activation of the latent cytosolic glucocorticoid receptor to an active DNA-binding species. We demonstrate here that cytosol from a rat hepatoma cell, M1.19, contains glucocorticoid receptor-specific immunoreactivities and target DNA-binding activities. Moreover, specific DNA-binding activities of M1.19 cytosol were dose-dependently induced by dexamethasone treatment, and linearly correlated with the hormonal induction of chloramphenicol acetyltransferase activity at the corresponding concentrations. These results indicate that the cytosolic glucocorticoid receptor could be converted in a DNA-binding form under cell-free conditions and the ligand appears to play a crucial role in the direct control of the level of functional activity of a given ligand-receptor complex.

Animals↗
PubMed1992

Influence of lactotroph cell density on prolactin secretion in rats.

The relationships between the stimulation of prolactin secretion and proliferation of lactotrophs was studied from a multidisciplinary standpoint in three experimental models. Administration of both oestrogen and sulpiride resulted in a significant increase in prolactin secretion and in the lactotroph population. A single injection of 10 micrograms oestradiol benzoate (OB) induced a twofold increase in the proliferation of lactotrophs (morphometrically as volume density), which increased further (2.5-fold) after three OB injections. Parallel changes were observed in the net counts made on lactotrophs sectioned through the nucleus to avoid possible distortions in volume density caused by hypertrophic cytoplasms. Comparable results were obtained with the mitotic index in the same groups of rats exposed to treatment with colchicine. The effect of sulpiride on proliferation of lactotrophs was also significant (1.7-fold) but less pronounced than in rats treated with oestrogens. The treatments with oestrogen and sulpiride did not stimulate lactotrophic activity in a similar way, as judged by the levels of serum prolactin and the storage patterns of small and big prolactin in pituitary glands. Serum prolactin (mean +/- S.E.M.) in control ovariectomized rats was 4.0 +/- 0.9 micrograms/l and one and three injections of OB raised these levels to 14.4 +/- 5.0 and 28.8 +/- 4.6 micrograms/l respectively. The highest levels of serum prolactin were seen in sulpiride-treated rats (467.2 +/- 28.7 micrograms/l). Striking differences occurred in the pituitary contents of big prolactin, the control values increasing from 5.3 +/- 0.5 to 10.2 +/- 1.3 micrograms/mg after one OB injection and to 14.7 +/- 0.7 micrograms/mg after three OB injections.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Microwell cluster for processing electron microscope sections for immunocytochemistry.

A microwell cluster was manufactured with silicone rubber for incubating thin sections for postembedding electron microscopic immunocytochemistry. The small size of the wells requires only minute amounts (as little as 5 microliters) of antisera and other valuable immunoreagents. The capillary action of the wells holds the incubation media and grids in place, even if the cluster is turned upside down, thus facilitating safe transport and storage of the sections to be stained. This silicone rubber microwell cluster can also be used as a die to imprint wells in Parafilm sheets to be used for the same purpose.

Immune Sera↗
PubMed1992

Differentiation of endocrine myocardiocytes in the developing heart of the toad (Bufo arenarum Hensel).

The differentiation of endocrine myocardiocytes was investigated in the heart of developing toad Bufo arenarum Hensel, combining ultrastructural and immunocytochemical procedures. The distribution of immuno-reactive atrial natriuretic peptide (ANP) in the whole heart was appraised by light microscopy, applying biotin-streptavidin and immunofluorescence techniques. With the latter procedures ANP was first recognized at embryonic stage 22, in both atrium and ventricle. In the ensuing stages the ANP-reactivity became stronger in the atrium, while it became dimmer in the ventricle. At the end of the larval prometamorphic stage, atrial myocardiocytes acquired almost all the features of adult myoendocrine cells. At electron microscope level, small inclusions, about 110-120 nm in diameter, resembling secretory granules were found in myoendocrine cells beginning at embryonic stage 22. However, no immunogold labeling of ANP occurred until stage 25. The number of secretory granules diminished in the ventricles and increased in the atrium of the larval heart and at the end of the prometamorphic stage the atrial myoendocrine cells presented the ultrastructural characteristics of active secretory cells. The synthesis of ANP in larvae is enhanced at a critical period of development when the developing toad switches from an aquatic environment to terrestrial life. The cardiac hormones seem to play a key role in the regulation of the osmolarity of body fluids at this developmental stage.

Animals↗
PubMed1992

In vitro study of erythropoiesis in patients with aplastic anemia and myelodysplastic syndromes: a possible tool for prospective determination of the clinical effectiveness of growth factors.

Effects of recombinant human erythropoietin (rhEpo) and the combination of recombinant human interleukin-3 (rhIL-3) or recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) with rhEpo on erythroid colony formation were examined in vitro in 13 patients with aplastic anemia and 16 with myelodysplastic syndromes (MDS). The methylcellulose cultures of marrow cells from normals and the patients yielded no erythroid colonies in the absence of rhEpo. In normals, CFU-E and BFU-E colony formation was significantly increased by adding either rhIL-3 or rhGM-CSF with rhEpo, compared with rhEpo alone, and rhIL-3 was more potent than rhGM-CSF to form colony-forming units and burst-forming units of erythroid (CFU-E) (BFU-E) colonies. By adding rhIL-3 with rhEpo, CFU-E colony formation was increased in half of patients with RA, compared with rhEpo alone, and by rhGM-CSF, in one third. Approximately one third or one fourth of the patients with MDS showed increased BFU-E colonies when rhIL-3 or rhGM-CSF were added to rhEpo. Cultures containing rhIL-3 or rhGM-CSF with rhEpo yielded larger numbers of BFU-E colonies in half of the patients with nonsevere aplastic anemia than those containing rhEpo alone. These observations suggest that the combination of these growth factors, especially rhIL-3 with rhEpo, is applicable to the treatment of anemia in some patients with aplastic anemia and MDS.

Adolescent↗
PubMed1992

Successful aggressive treatment against multiple intra-abdominal metastases from renal cell carcinoma 18 years after nephrectomy.

The management of late metastases from renal cell carcinoma is often difficult because of multiple organ involvement. We report a case of multiple metastases from renal cell carcinoma in the duodenum, pancreas, intestine, falciform ligament and liver, 18 years after nephrectomy. The patient underwent a total pancreatectomy following a gastroduodenal arterial embolization to control duodenal bleeding, a resection of the ileum and falciform ligament at a second laparotomy and repeated hepatic arterial embolizations to control the growth of liver metastases. Aggressive treatment should be undertaken in cases of late recurrence of renal cell carcinoma after nephrectomy because of the possibly slow-growing biological character of the tumor.

Abdominal Neoplasms↗
PubMed1992

[Natural interferon-alpha induced cytogenetic complete remission in a patient with chronic myelocytic leukemia, diagnosed in early chronic phase with basophilia and normal blood cell counts].

A 57-year-old woman visited to our hospital complaining of paresthesia in the right leg. She had no abnormal physical findings. However, the peripheral blood examination demonstrated 7% basophilia with 8000/microliters WBC count and decreased neutrophil alkaline phosphatase activity (score 37, rate 19%). She was diagnosed as Ph1 chromosome positive CML in early phase by the chromosomal analysis of bone marrow cells. She received subcutaneous injection of natural interferon-alpha at a dosage of 600 x 10(4) IU daily from March 10, 1987. The dosage and administration interval were gradually reduced and prolonged. Since November 1988, weekly injections of 300 x 10(4) IU has been administered as maintenance therapy. Cytogenetic improvement was seen at 4 months after the start of IFN. Disappearance of Ph1 chromosome positive cells was observed on December 11, 1987. It was suggested that the administration of IFN from the early chronic phase played an important role in the control of the disease.

Basophils↗
PubMed1991

Stereological analysis of the hypothalamic ventromedial nucleus. II. Hormone-induced changes in the synaptogenic pattern.

Stereological electron microscopic analysis of the rat hypothalamic ventromedial nucleus (VMN) throughout postnatal development revealed that synaptogenesis takes place up to day 45. Our results disclosed a sexual dimorphism in the synaptic organization of the neuropil of the ventrolateral (VL) subdivision of the VMN. The numerical densities of spine and shaft synapses in the adult male were higher than in the female. A dimorphic pattern in the numerical density of spine synapses occurred as early as day 5, and was present throughout postnatal life, even though in the adult rats both spine and shaft synapses were sexually dimorphic. Neonatal treatment of female rats with testosterone increased the numerical density of axodendritic synapses, inducing a pattern similar to the adult male. On the other hand, administration of tamoxifen to newborn male rats significantly reduced the numerical density of spine synapses to levels comparable to normal female rats.

Aging↗
PubMed1991

Gene transfer into human leukemia cell lines by electroporation: experience with exponentially decaying and square wave pulse.

The efficiency of gene transfer into human leukemia cell lines by electroporation was investigated. For both transient expression (beta-galactosidase gene) and stable transformation (neomycin resistance gene), the transfer efficiency into leukemia cell lines using a square wave pulse was superior to that using an exponentially decaying wave. The transfer rate of pMoZtk (containing beta-galactosidase gene) into K562 by electroporation using a square wave was approximately 5%, compared with 1% by an exponentially decaying pulse. Whereas the transfer rate of pMAM-neo into K562 by electroporation using an exponentially decaying pulse was less than 10(-5), a square wave generated much more efficient introduction rate of nearly 10(-3). In the other leukemia cell lines also, some square wave yields were better than exponential yields and all square wave yields were at least as good as the exponential yields.

Blotting, Southern↗
PubMed1991

Presence of two transcribed malate synthase genes in an n-alkane-utilizing yeast, Candida tropicalis.

The presence of two genomic DNA regions encoding malate synthase (MS) was shown by Southern blot analysis of the genomic DNA from an n-alkane-assimilating yeast, Candida tropicalis, using a partial MS cDNA probe, in accordance with the fact that two types of partial MS cDNAs have previously been isolated. This was also confirmed by the restriction mapping of the two genes screened from the yeast lambda EMBL library. Nucleotide sequence analysis of the respective genomic DNAs, named MS-1 gene and MS-2 gene, revealed that both regions encoding MS had the same length of 1,653 base pairs, corresponding to 551 amino acids (molecular mass of MS-1, 62,448 Da; MS-2, 62,421 Da). Although 29 nucleotide pairs differed in the sequences of the coding regions, the number of amino acid replacements was only one: 159Asn (MS-1)----159Ser (MS-2). In the 5'-flanking regions, there were replacements of four nucleotide pairs, deletion of one pair, and insertion of four pairs. In spite of the fact that two genomic genes were present and transcribed, RNA blot analysis demonstrated that only one band (about 2 kb) was observable even when the carbon sources in the cultivation medium were changed. A comparison of the amino acid sequences was made with MSs of rape (Brassica napus L.), cucumber seed, pumpkin seed, Escherichia coli, and Hansenula polymorpha. A high homology was observed among these enzymes, the results indicating that the protein structure was relatively well conserved through the evolution of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkanes↗
PubMed1991

Preferential proliferation of anti-DNA producing cells of NZB mice in NZB.xid recipients.

B cells from autoimmune NZB mice were transferred into unmanipulated non-autoimmune NZB.xid mice. The number of antibody-producing cells against various antigens in recipient mice was monitored at varying time after cell transfer using ELISPOT assay. NZB B cells producing antibody against all antigens we examined were able to proliferate in NZB.xid mice, which supports the idea of polyclonal B cell activation. However, anti-DNA producing cells proliferated most rapidly, and anti-BrMRBC producing cells proliferated more rapidly than B cells of other antigenic specificities. The percentage of anti-DNA producing cells in total immunoglobulin-producing cells increased over time whereas the percentage of anti-ovalbumin producing cells kept the same level. This indicates directly the preferential proliferation of NZB anti-DNA producing cells in NZB.xid mice. The result shows the responsibility of antigen-specific stimulation or activation on autoimmunity in the context of polyclonal B cell activation.

Actins↗
PubMed1991

Alpha-interferon broadens the difference between surviving fractions of normal and leukemic progenitor cells in vitro by heat: its application to marrow purging.

Alpha-interferon (IFN) may inhibit the proliferation of human leukemic progenitor cells (L-CFU) in vitro and enhance the anti-tumor effects by heat. In this study, the combined effects of IFN and hyperthermia on the growth of L-CFU and human granulocyte-macrophage progenitors (CFU-GM) were examined to determine if this combination resulted in a greater selective killing of L-CFU than that obtained by heat treatment alone. The survival of normal CFU-GM without IFN decreased at elevated temperatures (42-44 degrees C). However, IFN added during heating (42 and 43 degrees C) appeared significantly to protect against the hyperthermic killing of CFU-GM in vitro leaving over 50% of CFU-GM surviving. The optimal dose to protect CFU-GM in vitro dropped to a rather low dose (100 U/ml). On the other hand, the addition of IFN to leukemic cell suspensions enhanced the hyperthermic killing of myeloid leukemic cell lines (HEL and KG-1) as well as a T lymphoblastic cell line (CEM) in a dose-related manner. In addition, similar results were observed in the study of L-CFU from patients with acute myelogenous leukemia. These results suggest that IFN can be used to broaden the difference between surviving fractions of CFU-GM and L-CFU by heat. Thus, this combination could be applied effectively and safely for the elimination of residual clonogenic leukemic cells in autologous remission marrow graft before autologous bone marrow transplantation.

Bone Marrow Purging↗
PubMed1991

Efficient introduction of a gene into hematopoietic cells in S-phase by electroporation.

Cells of the hematopoietic cell line K562 were synchronized by three different methods: single aphidicolin treatment, thymidine treatment followed by hydroxyurea exposure, and double hydroxyurea treatment. The synchronized cells were transfected via electroporation with plasmid pMoZtk, which contains the beta-galactosidase gene, using a square wave pulse immediately after synchronization or at various time points during culture. Simultaneously, synchronized cells were fluorescence-activated cell sorter (FACS) analyzed to determine their stage in the cell cycle using double staining with bromodeoxyuridine (BrdU) and propidium iodide. Highly efficient introduction of pMoZtk was observed for the cell fraction, which predominantly consisted of the cells in S-phase. These results suggest that by increasing the proportion of cells in S-phase, the efficiency of gene transfer into hematopoietic cells such as hematopoietic stem cells can be improved.

Aphidicolin↗
PubMed1991

Effect of hyperthermia on both primary proliferation and self-renewal of human leukemic progenitor cells in vitro: its application to in vitro purging.

Self-renewal, as defined by the capacity to yield new colonies following replating, is an important function of leukemic progenitors (L-CFU) to originate self-maintaining clones. In this work, we studied the effect of hyperthermia (41-44 degrees C) on the growth of human L-CFU derived primary colonies and of secondary colonies formed by replating to evaluate the purging effect of human L-CFU by heat. The survival curves clearly demonstrated much greater hyperthermic sensitivity of L-CFU compared to normal granulocyte-macrophage progenitors (CFU-GM) at all temperatures (41-44 degrees C) studied. At 42 degrees C or higher, L-CFU decreased (by more than 2 log reduction) dramatically and therefore were unable to form colonies in vitro. At 42 degrees C and 43 degrees C, 65 and 30%, respectively, of CFU-GM obtained from remission and normal marrows were left after 1 h exposure. At 44 degrees C, however, CFU-GM derived colonies disappeared after a 2 h exposure. In the four available patients with acute myelogenous leukemia, secondary colonies formed by replating were decreased in proportion to the decreasing primary colonies during heating (42 and 43 degrees C). However, their self-renewal capacity was retained in vitro until the primary colonies disappeared. These observations suggest that heat exposure at 43 degrees C for 1 h could be the most effective conditions for in vitro purging of human L-CFU because of the wide difference between surviving fractions of CFU-GM and L-CFU.

Bone Marrow Transplantation↗