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Biomedical subjects

Masahiro Kawahara

Publications and source records attributed to Masahiro Kawahara.

At least 19 recordsLinked to original sources
PubMed2006

Antigen-mediated growth control of hybridoma cells via a human artificial chromosome.

Human artificial chromosome (HAC) vectors possess several characteristics sufficient for the requirements of gene therapy vectors, including stable episomal maintenance and mediation of long-term transgene expression. In this study, we adopted an antigen-mediated genetically modified cell amplification (AMEGA) system employing an antibody/cytokine receptor chimera that triggers a growth signal in response to a cognate non-toxic antigen, and applied it to growth control of HAC-transferred cells by adding an antigen that differed from cytokines that may manifest pleiotropic effects. We previously constructed a novel HAC vector, 21 Delta qHAC, derived from human chromosome 21, housed in CHO cells. Here, we constructed an HAC vector harboring an ScFv-gp130 chimera responsive to fluorescein-conjugated BSA (BSA-FL) as well as a model transgene, enhanced green fluorescent protein (EGFP), in CHO cells. The modified HAC was transferred into interleukin (IL)-6-dependent hybridoma 7TD1 cells by microcell-mediated chromosome transfer, and the cells were subsequently found to show BSA-FL-dependent cell growth and sustained expression of EGFP in the absence of IL-6. The AMEGA system in combination with HAC technology will be useful for increasing the efficacy of gene therapy by conferring a growth advantage on the genetically modified cells.

Animals↗
PubMed2006

Identification of HLA class I-restricted tumor-associated antigens in adult T cell leukemia cells by mass spectrometric analysis.

OBJECTIVE: In this study, we attempted a comprehensive analysis of MHC class I-bound peptides in adult T cell leukemia (ATL) cells in order to identify as many tumor-associated antigens (TAAs) as possible that could be used for CTL-based immunotherapy. METHODS AND RESULTS: Using mass spectrometry combined with reversed-phase liquid chromatography, we could sequence 188 HLA class I-restricted candidate peptides from three ATL-derived cell lines. In accordance with the restrained expression of HTLV-I viral RNA in these cell lines, there were no HTLV-I-encoded peptides among these candidates. Based on the differential expression between ATL cells and normal CD4+ T cells, we selected 10 novel peptides as T cell epitopes of overexpressed source proteins. RT-PCR analysis revealed that 5 source proteins including PRAME, a known tumor-testis antigen, were highly expressed in the majority of 16 ATL cases. Furthermore we could induce PRAME-specific CTLs in vitro from an HLA-B62+ healthy donor that showed specific cytotoxicity against HLA-B62+ PRAME+ ATL cells. CONCLUSION: These results demonstrate that comprehensive analysis of HLA class I-bound peptides by mass spectrometry is useful for identification of TAA-derived peptides in ATL. Considering that expression patterns of leukemia/lymphoma-associated antigens vary from case to case, this approach appears to be suitable for the tailor-made immunotherapy of hematological malignancies.

Antigens, Neoplasm↗
PubMed2005

Exogenous gene expression and growth regulation of hematopoietic cells via a novel human artificial chromosome.

A number of gene delivery systems are currently being developed for potential use in gene therapy. Here, we demonstrate the feasibility of 21deltaqHAC, a newly developed human artificial chromosome (HAC), as a gene delivery system. We first introduced a 21deltaqHAC carrying an EGFP reporter gene and a geneticin-resistant gene (EGFP-21deltaqHAC) into hematopoietic cells by microcell-mediated chromosome transfer. These HAC-containing hematopoietic cells showed resistance to geneticin, expressed EGFP and retained the ability to differentiate into various lineages, and the EGFP-21deltaqHAC was successfully transduced into primary hematopoietic cells. Hematopoietic cells harboring the EGFP-21deltaqHAC could still be detected at two weeks post-transplantation in immunodeficient mice. We also showed effective expansion of hematopoietic cells by introducing the 21deltaqHAC containing ScFvg, a gp130-based chimeric receptor that transmits growth signals in response to specific-antigen of this receptor. All of these results demonstrate the usefulness of HAC in gene therapy.

Animals↗
PubMed2005

Phosphorylation of tau at serine 416 by Ca2+/calmodulin-dependent protein kinase II in neuronal soma in brain.

It is well known that tau is a good in vitro substrate for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). However, it is not clear at present whether CaM kinase II phosphorylates tau in vivo or not. Serine 416, numbered according to the longest human tau isoform, has been reported to be one of the major phosphorylation sites by CaM kinase II in vitro. In this study, we produced a specific antibody against tau phosphorylated at serine 416 (PS416-tau). Immunoblot analysis revealed that the antibody reacted with tau in the rat brain extract which was prepared in the presence of protein phosphatase inhibitors. Developmental study indicated that serine 416 was strongly phosphorylated at early developmental stages in rat brain. We examined the localization of PS416-tau in primary cultured hippocampal neurons and the immortalized GnRH neurons (GT1-7 cells), which were stably transfected with CaM kinase IIalpha cDNA. Immunostaining of these cells indicated that tau was phosphorylated mainly in neuronal soma. Interestingly, tau in neuronal soma in Alzheimer's disease (AD) brain was strongly immunostained by the antibody. These results suggest that CaM kinase II is involved in the accumulation of tau in neuronal soma in AD brain.

Aged↗
PubMed2005

Effects of aluminum on the nervous system and its possible link with neurodegenerative diseases.

Aluminum is environmentally abundant, but not an essential element. Aluminum has been associated with several neurodegenerative diseases, such as dialysis encephalopathy, amyotrophic lateral sclerosis and Parkinsonism dementia in the Kii peninsula and Guam, and in particular, Alzheimer's disease. Although this association remains controversial, there is increasing evidence which suggests the implication of metal homeostasis in the pathogenesis of Alzheimer's disease. Aluminum, zinc, copper, and iron cause the conformational changes of Alzheimer's amyloid-beta protein. Al causes the accumulation of tau protein and amyloid-beta protein in experimental animals. Aluminum induces neuronal apoptosis in vivo as well as in vitro. Furthermore, a relationship between aluminum and the iron-homeostasis or calcium-homeostasis has been suggested. Based on these findings, the characteristics of aluminum neurotoxicity are reviewed, and the potential link between aluminum and neurodegenerative diseases is reconsidered.

Aluminum↗
PubMed2004

An antigen-mediated selection system for mammalian cells that produce glycosylated single-chain Fv.

Selection and production of specific antibodies are limiting the development of high-throughput immunoassays such as antibody chips. In this study, we propose an antigen-mediated selection of antibody producers (ASAP) system in mammalian cells. As a model system, transgenes encoding anti-fluorescein ScFv fused to cytokine receptors were introduced to IL-3-dependent cell lines. Addition of fluorescein-conjugated BSA induced growth signal through the ScFv/receptor chimeras, leading to selective expansion of the transduced cells. Cre recombinase was then used to excise the receptor gene flanked by two loxP recognition sites in the introns, resulting in secretion of his-myc-tagged ScFv to the culture medium. When the first loxP site was used in the exon as a linker between ScFv and receptor, enhanced antigen-mediated cell proliferation and production of unexpectedly glycosylated ScFv were achieved. ASAP is the first mammalian selection/production system of recombinant human ScFvs, without need for subcloning and with the advantage of glycosylated product.

Animals↗
PubMed2004

Improved growth response of antibody/receptor chimera attained by the engineering of transmembrane domain.

Structure-based design of antibody/cytokine receptor chimeras has permitted a growth signal transduction in response to non-natural ligands such as fluorescein-conjugated BSA as mimicry of cytokine-cytokine receptor systems. However, while tight on/off regulation is observed in the natural cytokine receptor systems, many chimeras constructed to date showed residual growth-promoting activity in the absence of ligands. Here we tried to reduce the basal growth signal intensity from a chimera by engineering the transmembrane domain (TM) that is thought to be involved in the interchain interaction of natural cytokine receptors. When the retroviral vectors encoding the chimeras with either the wild-type erythropoietin receptor (EpoR) TM or the one bearing two mutations in the leucine zipper motif were transduced to non-strictly interleukin-6-dependent 7TD1 cells, a tight antigen-dependent on/off regulation was attained, also demonstrating the first antigen-mediated genetically modified cell amplification of non-strictly factor-dependent cells. The results clearly indicate that the TM mutation is an effective means to improve the growth response of the antibody/receptor chimera.

Animals↗
PubMed2004

Developmental changes of GABAergic synapses formed between primary cultured cortical neurons.

The characteristics of functional changes of GABAergic synapses between cultured rat cortical neurons were observed by monitoring intracellular calcium level ([Ca2+]in) during development in vitro. After 5 days in vitro (DIV), cultured cortical neurons spontaneously exhibited synchronous oscillatory changes in [Ca2+]in, which were derived from synaptic activity. Exposure to bicuculline, antagonist of gamma-aminobutyric acid (GABA)(A) receptors, caused a marked decrease in the frequency of [Ca2+]in oscillations at 7-20 DIV. Although the frequency of spontaneous oscillations increased during this culture period, the ratio of the decrease in the frequency following bicuculline treatment did not significantly change. Thereafter, to investigate the detailed morphological changes of GABAergic synapses during development in vitro, the cultured neurons were immunostained with antibodies to glutamic acid decarboxylase (GAD), synaptophysin and GABA(A) receptor and were observed under a confocal laser microscope. Most of the GAD-positive puncta colocalized with synaptophysin-positive puncta and were opposed to GABA(A) receptor-positive structures. The images of GAD-positive puncta were reconstructed from the confocal three-dimensional data to analyze their number, volume, and surface area. The number of these puncta increased with culture time at 7-20 DIV. Although the volume of individual GAD-positive puncta did not significantly change, the surface area decreased in a time-dependent manner over the culture period. This system that we developed enabled us to investigate in detail the morphological and functional changes of GABAergic synapses during neuronal development.

Animals↗
PubMed2004

Reversal of antigen-dependent signaling by two mutations in antibody/receptor chimera: implication of inverse agonism in cytokine receptor superfamily.

Understanding the receptor activation mechanism is essential for the rational design of pharmacologically active ligand molecules. However, the activation mechanism of most cytokine receptors remains still unclear, and while agonism and antagonism have been described for ligand-mimetic peptides, there has been no report of inverse agonism that has been characterized for G protein-coupled receptors (GPCRs). To explore the activation mechanism of cytokine receptors, here we tried to investigate how agonism and antagonism could be altered by randomizing antibody variable region of an antibody/cytokine receptor chimera recognizing hen egg lysozyme (HEL) as an agonist. Based on our previous finding that the co-expression of V(H)-gp130 and V(L)-erythropoietin receptor (EpoR) chimeras transduced strict and efficient HEL-dependent cell growth signal, a V(H)-gp130 library encoding four randomized CDR2 residues was retrovirally infected to IL-3-dependent Ba/F3 cells already transfected with V(L)-EpoR. The selection without IL-3 resulted in a clonal expansion of the transduced cells, and interestingly some of which showed HEL dose-dependent growth suppression. Our results clearly indicate that agonism and antagonism of the antibody/cytokine receptor chimera can be readily switched by a subtle modification of the ligand binding domain as well as that of GPCRs, also implying the existence of inverse agonism in cytokine receptor superfamily.

Animals↗
PubMed2004

Selection of genetically modified cell population using hapten-specific antibody/receptor chimera.

Efficient selection of the genetically modified cell population is a critical step to obtain the cells with desired properties. In this study, we propose an antigen-mediated genetically modified cell amplification (AMEGA) system employing an antibody/receptor chimera that triggers a growth signal in response to a non-toxic hapten dimer. An anti-fluorescein single-chain Fv fused to the extracellular D2 domain of erythropoietin receptor and transmembrane/intracellular domains of gp130 was expressed together with a model transgene, enhanced green fluorescent protein (EGFP) downstream of IRES sequence, by retroviral infection to IL-3-dependent Ba/F3 cells. Addition of fluorescein dimers connected by various oligo-DNA linkers induced selective growth of transfectants, thus leading to efficient expansion of EGFP-positive cell population. Also, digestion of the oligonucleotides by specific restriction endonuclease completely suppressed cell growth. Because these hapten dimers are not harmful for normal cells, the approach will be especially useful for reversible in vitro or in vivo expansion of genetically modified cell population employed for cell therapy and tissue engineering.

Animals↗
PubMed2004

AMEGA: antigen-mediated genetically modified cell amplification.

Selection of genetically modified cells is a critical step to engineer the cells with desired properties. While antibiotic selection has been commonly used, administration of cytotoxic drugs often leads to deleterious effects not only to inert cells but also to transfected or transduced ones. To overcome this problem, a positive screening method for genetically modified cells is proposed using a pair of chimeric receptors that trigger a growth signal in response to a specific antigen. Either V(H) or V(L) region of anti-hen egg lysozyme (HEL) antibody HyHEL-10 was fused to extracellular D2 domain of erythropoietin receptor (EpoR) and transmembrane/cytoplasmic domains of either EpoR or gp130. A model transgene, enhanced green fluorescent protein (EGFP) and the chimeric receptor genes that reconstituted functional Fv were retrovirally co-infected to interleukin (IL)-3-dependent Ba/F3 cells, followed by direct HEL selection in the absence of IL-3. Consequently, a single round of selection led to a single population of EGFP-positive cells. The detailed protocol of the method termed antigen-mediated genetically modified cell amplification (AMEGA) is described.

Antigens↗
PubMed2004

[Assay system for synapse formation between primary cultured neurons].

Synapse plasticity, in particular, formation of new synapses, plays crucial roles in learning and memory. We have developed a convenient assay system for measuring the number of newly formed synapses between cultured rat cerebrocortical neurons using the multisite fluorometry system of intracellular calcium. We found that cultured neurons exhibited spontaneous oscillatory changes in intracellular calcium levels and that the frequency of the oscillation was strongly correlated with synaptic density. Combined with immunohistochemical studies, this assay system enables us to study the molecular mechanism of synapse formation, in particular, the involvement of ecto-protein kinase. Other applications of the assay system are discussed here.

Animals↗
PubMed2004

Disruption of calcium homeostasis in the pathogenesis of Alzheimer's disease and other conformational diseases.

Conformational changes of Alzheimer's beta-amyloid protein (AbetaP) enhance its neurotoxicity and play important roles in the pathogenesis of Alzheimer's disease. Recent studies have suggested that a common mechanism is based on diverse "conformational diseases". They share similarity in their formation of beta-sheet containing amyloid fibrils by disease-related proteins and the introduction of apoptotic degeneration. Numerous studies, including our own, have revealed that AbetaP and several disease-related proteins are capable of directly incorporating into membranes, forming calcium-permeable ion channels, and causing abnormal elevation of intracellular calcium levels. This article reviews the current understanding of the pathology of conformational diseases based on the hypothesis that the disruption of calcium homeostasis through amyloid channels may form the molecular basis of neurotoxicity of AbetaP and other disease-related proteins. The roles of membrane lipids and potential development of preventive agents are also discussed.

Alzheimer Disease↗
PubMed2003

Brain-derived neurotrophic factor protects cultured rat hippocampal neurons from aluminum maltolate neurotoxicity.

Aluminum is environmentally abundant but not an essential trace element. Although there is increasing evidence suggesting the implication of aluminum in the pathogenesis of Alzheimer's disease, it is still controversial. We found and report here that aluminum maltolate, a stable and hydrophilic aluminum complex, causes death of primary cultured rat hippocampal neurons in a time- and dose-dependent manner. Degenerated neurons were TUNEL-positive. Immunohistochemical detection of synapsin I and microtubule associated protein 2 revealed the synapse loss between neurons intoxicated by aluminum maltolate. To explore the mechanism underlying its neurotoxicity, we administered various pharmacological compounds prior to the application of aluminum maltolate, and found that brain-derived neurotrophic factor (BDNF) markedly attenuated the neurotoxicity. Furthermore, aluminum maltolate inhibited the elevation of intracellular calcium levels caused by BDNF. Our results suggest the involvement of BDNF in the molecular mechanism underlying neurotoxicity induced by aluminum maltolate.

Animals↗
PubMed2003

Bypassing antibiotic selection: positive screening of genetically modified cells with an antigen-dependent proliferation switch.

While antibiotic selection has been routinely used for the selection of genetically modified cells, administration of cytotoxic drugs often leads to deleterious effects not only to inert cells but also to transfected or transduced ones. In this study, we propose an Antigen-MEdiated Genetically modified cell Amplification (AMEGA) system employing antibody/receptor chimeras without antibiotic selection. Based on a rational design where the extracellular domains of dimeric erythropoietin receptor (EpoR) or gp130 were substituted with heterodimeric V(H)/V(L) regions of anti-hen egg lysozyme (HEL) antibody and EpoR D2 domains, the genes encoding the chimeras as well as a model transgene, enhanced green fluorescent protein (EGFP), were retrovirally infected into IL-3-dependent Ba/F3 cells followed by direct HEL selection in the absence of IL-3. After a single round of selection, EGFP-positive cells were selectively amplified, resulting in a population of almost 100% positive cells. The AMEGA without antibiotic selection will not harm normal cells, which will be especially useful for increasing the efficacy for stem cell-based gene therapy.

Animals↗
PubMed2003

Thyroid hormone enhances the formation of synapses between cultured neurons of rat cerebral cortex.

1. Thyroid hormones play important roles in the development of the brain. Increasing evidence suggests that the deprivation of thyroid hormones in the early developmental stage causes structural and functional deficits in the CNS, but the precise mechanism underlying this remains elusive. In this study, we investigated the effects of thyroid hormones on synapse formation between cultured rat cortical neurons, using a system to estimate functional synapse formation in vitro. 2. Exposure to 10(-9) M thyroid hormones, 3,5,3'-triiodothyronine or thyroxine, caused an increase in the frequency of spontaneous synchronous oscillatory changes in intracellular calcium concentration, which correlated with the number of synapses formed. 3. The detection of synaptic vesicle-associated protein synapsin I by immunocytochemical and immunoblot analysis also confirmed that exposure to thyroxine facilitated synapse formation. 4. The presence of amiodarone, an inhibitor of 5'-deiodinase, or amitrole, a herbicide, inhibited the synapse formation in the presence of thyroxine. 5. In conclusion, we established a useful in vitro assay system for screening of miscellaneous chemicals that might interfere with synapse formation in the developing CNS by disrupting the thyroid system.

Animals↗
PubMed2003

Bicuculline induces synapse formation on primary cultured accessory olfactory bulb neurons.

To investigate the roles of the GABAergic inhibitory system of accessory olfactory bulb (AOB) in pheromonal memory formation, we have developed a primary culture system of AOB neurons, which had numerous excitatory and inhibitory synapses. Using this culture system of AOB neurons, we examined the correlation in rats between neuronal excitation and synaptic morphology by bicuculline-induced disinhibition of cultured AOB neurons. The exposure to bicuculline induced long-lasting oscillatory changes in the intracellular calcium level ([Ca2+]in) of cultured non-GABAergic multipolar neurons, which were identified as mitral/tufted cells (MT cells). These MT cells exhibited the appearance of dendritic filopodia structures after a 10-min treatment with bicuculline. By labelling presynaptic terminals with FM4-64, the appearance of new presynaptic terminals was clearly observed on newly formed filopodia after 120 min treatment with bicuculline. These results suggest that bicuculline-induced [Ca2+]in oscillation of MT cells induces the growth of filopodia and subsequently the formation of new presynaptic terminals. Furthermore, tetrodotoxin or the deprivation of extracellular calcium blocked bicuculline-induced synapse formation. The present results indicate that the long-lasting [Ca2+]in oscillation caused by bicuculline-induced disinhibition of cultured MT cells is significantly implicated in the mechanism underlying synapse formation on cultured AOB neurons. Our established culture system of AOB neurons will aid in clarifying the mechanism of synapse formation between AOB neurons and the molecular mechanism of pheromonal memory formation.

Actins↗