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Biomedical subjects

K Ikenaka

Publications and source records attributed to K Ikenaka.

At least 73 records · Page 4Linked to original sources
PubMed1997

Usefulness of a mouse myelin basic protein promoter for gene therapy of malignant glioma: myelin basic protein promoter is strongly active in human malignant glioma cells.

We have searched for suitable promoters to regulate the expression of suicide genes for use in gene therapy. We have shown that the 1.3-kb fragment of the mouse myelin basic protein (MBP) promoter region initiates transcription in mouse glioma cells more efficiently than glial fibrillary acidic protein (GFAP) or myelin proteolipid protein (PLP) promoter. Among three different lengths of the MBP promoter, the shortest (256-bp) core promoter region initiates transcription as efficiently as 650-bp or 1.3-kb MBP promoter lengths in RSV-M glioma cells. To assess the suitability of the MBP promoter for use in clinical trials of malignant glioma gene therapy, we also had to show that it (the 1.3-kb length in this case) is effective in human glioma cells, as well as in murine glioma cells. The activity of the MBP promoter is much higher than that of GFAP or PLP promoter in most human glioma cells, suggesting that the MBP promoter would be best for directing toxic gene expression in gene therapy for patients with malignant glioma. Human glioma cells in which the MBP promoter was strongly active were sensitive to ganciclovir when they were transduced with MBP promoter/herpes simplex virus thymidine kinase gene-bearing retroviruses. In conclusion, retrovirus-targeted gene therapy for malignant glioma using this MBP promoter is a promising candidate for clinical trials.

3T3 Cells↗
PubMed1997

Retroviral introduction of the p16 gene into murine cell lines to elicit marked antiproliferative effects.

The p16 gene is a candidate tumor suppressor, because mutation of the gene has been reported in many transformed cell lines and some primary tumor tissues. We have examined this possibility in murine cell lines (NIH3T3 and RSV-M) which lack p16 gene expression. Full-length human p16 cDNA was obtained from a HeLa cell line using polymerase chain reaction amplification. We constructed two separate retrovirus vectors carrying this p16 cDNA. First, we transduced the p16 cDNA into the murine cell lines using a retrovirus vector harboring the neomycin-resistance gene. The p16 gene-transduced cells formed no colonies after selection with G418, in contrast to the vector-transduced cells. Next, we used another retrovirus vector that expresses both the p16 cDNA and the Lac Z gene, which enabled us to distinguish affected cells from unaffected ones. Proliferation of the p16 gene-transduced cells was markedly inhibited and morphological change in the cells was also observed. Thus, we concluded that the p16 gene has an antiproliferative effect on the cell cycle and that the loss of its function may play a major role in dysregulated proliferation of the cells.

3T3 Cells↗
PubMed1997

GFAP gene expression during development of astrocyte.

Glial fibrillary acidic protein (GFAP) is expressed exclusively in astrocytes in the central nervous system. In order to characterize individual cultured cells in which the GFAP promoter is active and to identify the regulatory mechanisms of GFAP expression in these cells, we have developed a unique assay system for promoter activity using retrovirus vectors. Retrovirus containing the mouse GFAP promoter fused to the lacZ gene were used to infect mixed glial cultures. The infected cells, in which the GFAP promoter was active, were visualized by X-Gal staining. From these experiments, we found that a 256 bp fragment 5' of the transcription initiation site was sufficient to confer astrocyte-specific expression of GFAP. The GFAP promoter became active about 3 days before GFAP protein can be detected immunohistochemically, which indicates that detection of GFAP promoter activity can be used to identify astrocyte progenitors. We have also established immortalized astrocyte cell lines in which we detect GFAP promoter activity. Immorto mouse is a transgenic mouse generated by the introduction of thermolabile SV40 T Ag, tsA58. A mixed glial culture prepared from 2-day-old Immorto mouse brain was incubated at 32 degrees C, at which temperature most of the cells expressed T Ag. The culture was then infected with retrovirus containing GFAP promoter-lacZ, and the infected cells were selected. Using the fluorescence-activated cell sorter with fluorescein di-beta-D-galactopyranoside as a substrate (FDG-FACS), these cells were separated into two groups: FDG(+), in which the GFAP promoter was active, and FDG(-), in which it was inactive. Mature astrocyte cell lines were established from the FDG(+) cells by colony isolation. The FDG(-) cells were cloned by colony isolation and cultured at 32 or 39 degrees C. At the latter temperature the expression of T Ag was suppressed and cell differentiation was induced in most cells. The cells which became positive for X-Gal staining only after switching to 39 degrees C were collected as immature astrocyte cell lines. These immortalized cell lines should be useful to investigate the molecular mechanisms of astrocyte differentiation.

Animals↗
PubMed1997

Astrocytic lineage analysis by detection of GFAP promoter activity in vitro.

To survey the emergence and onset of differentiation of the astrocytic lineage in the developing mouse cerebral wall, the promoter activity of a 2.5 kb 5'-flanking region of glial fibrillary acidic protein (GFAP) was measured in individual developing brain cells using a retrovirus-mediated gene transfer system. We identified precursors for astrocytes in primary culture of embryonic mouse cerebral wall cells by detection of GFAP promoter activity, which was detected approximately 3 days prior to the appearance of GFAP immunoreactivity. Since retroviruses only integrate into the chromosomes of actively proliferating cells, cells detected by this method should have been mitotically active at the time of retroviral infection on day 15 postfertilization (E15). Furthermore, we observed that cells activating GFAP promoter were located near the ventricular surface of cultured cerebral wall slices as a cluster of spherical cells. These results demonstrate that precursor cells for astrocytes exist within the germinative zone of developing cerebral wall, and that these cells are mitotically active on day E15, which is a late stage of neuronal production period in the mouse cerebral wall. The morphology, location and mitotic activity of these cells suggest that they are unlikely to be cells that have been transformed from radial glial cells.

Animals↗
PubMed1996

Myelin proteolipid protein (PLP), but not DM-20, is an inositol hexakisphosphate-binding protein.

Myelin proteolipid protein (PLP) and its alternatively spliced isoform, DM-20, are the major integral membrane proteins of central nervous system myelin. It is known that PLP and DM-20 are delivered to myelin by a finely regulated vesicular transport system in oligodendrocytes. Evolutionarily, it is believed that ancestral DM-20 acquired a PLP-specific exon to create PLP, after which PLP/DM-20 became a major component of central nervous system myelin. We purified PLP as an inositol 1,3,4,5-tetrakisphosphate-binding protein after solubilization in a non-organic solvent. However, under the isotonic condition, PLP binds inositol hexakisphosphate (InsP6) significantly, not inositol 1,3,4,5-tetrakisphosphate. Most of the InsP6-binding proteins are involved in vesicular transport, suggesting the involvement of PLP in vesicular transport. We separated DM-20 from PLP by CM-52 chromatography and showed that DM-20 has no InsP6 binding activity. These findings indicate that the PLP-specific domain confers the InsP6 binding activity and this interaction may be important for directing PLP transport to central nervous system myelin.

Animals↗
PubMed1996

Distribution of a reeler gene-related antigen in the developing cerebellum: an immunohistochemical study with an allogeneic antibody CR-50 on normal and reeler mice.

We have immunohistochemically investigated the expression of a reeler gene-related antigen in the mouse cerebellum by using a monoclonal antibody, CR-50. This antibody probes a distinct allelic antigen present in normal but not in reeler mutant mice, and this antigen is localized in the brain regions in which morphological abnormalities occur in reeler mice (Ogawa et al., Neuron 14: 899-912, 1995). The developing normal cerebellum showed transient immunoreactivity to CR-50 in a limited set of neurons and in the extracellular space near the pial surface. An early population of CR-50-labeled cells emerged on embryonic day (E) 13 along the dorsal cerebellar surface, comprising the nuclear transitory zone (NTZ). Bromodeoxyuridine labeling revealed the time of origin of these cells to be at E11-12. From E14 to E18, some CR-50-labeled cells were stacked in the inner border of the external granular layer (EGL), whereas others were scattered in deep areas, such as the cerebellar nuclei and the surrounding intermediate zone or white matter. In the first postnatal week, these subcortical structures became immunonegative. However, CR-50 antigen was continuously produced until the second postnatal week by another population of cells occupying i) the premigratory zone (PMZ), the inner half of the EGL, and ii) the internal granular layer (IGL). These later CR-50-positive cells were smaller than the earlier type and showed the morphology typical of granule neurons. Both types of CR-50-labeled cells were positive for a DNA-binding protein, zic. By treating living cerebellar slices with CR-50, the extracellular antigen was localized as a puncutate staining pattern in the NTZ, PMZ, and molecular layer (ML), but not in the subcortical regions and IGL. Purkinje cells were negative for CR-50 and aligned as a monolayer adjacent to the PMZ, though their dendritic trees were closely associated with the extracellular CR-50-antigen in the PMZ and ML. Staining of dissociated cells suggested that the extracellular antigen is initially present throughout the surfaces of the CR-50/anti-zic double positive neurons, and is then rearranged to concentrate on their processes contacting with Purkinje cells. The spatiotemporal expressions of the CR-50 antigen in the cerebellum are consistent with the possibility that this antigen is involved in cell-cell interactions related to the histogenetic assembly of Purkinje cells.

Animals↗
PubMed1996

Neuron-specific expression of a chicken gicerin cDNA in transient transgenic zebrafish.

Gicerin, a novel cell adhesion molecule which belongs to the immunoglobulin superfamily, is expressed temporally and spatially in the developing chick brain and retina. The previous in vitro experiments using transfected cells showed that gicerin can function as a cell adhesion molecule which has both homophilic and heterophilic binding activities. For the in vivo analyses of gicerin in neural development, we tried to utilize a zebrafish system, a vertebrate suitable for studying early development. We generated transient transgenic animals by microinjecting DNA constructs into zebrafish embryos. Chicken gicerin, under control of the neurofilament gene promoter, was preferentially expressed in neuronal cells and gicerin-expressing neurons exhibited a fasciculation formation with neighboring gicerin-positive axons, which may be partly due to homophilic cell adhesion activity of gicerin. These experimental results suggest that this fast and efficient transgenic animal system is useful for studying the functional roles of neuron-specific genes during the development.

Animals↗
PubMed1996

Cell death of oligodendrocytes or demyelination induced by overexpression of proteolipid protein depending on expressed gene dosage.

The transgenic mice, which were produced by introducing the wild type proteolipid protein (PLP) gene, revealed different neurological symptoms depending on expressed gene dosage. Homozygotes, which bore four more copies of the extra PLP gene, died before 4 weeks old with severe tremors and convulsions, while histologically hypomyelination and death of the oligodendrocytes were particularly noticeable. Heterozygotes, which bore two more copies and survived nearly 1 year, suffered from severe hindlimb palsy and bladder-rectal disturbances before death. These symptoms probably resulted from striking demyelination which occurred abruptly at a later stage of life. The heterozygous PLP-transgenic mice are thus available for a model of demyelination diseases.

Animals↗
PubMed1996

GTP-binding protein activation underlies LTP induction by mast cell degranulating peptide.

Mast cell degranulating peptide (MCD) induces long-term potentiation (LTP) in the CA1 region of the hippocampus. MCD has been shown to bind to a voltage-dependent A-type potassium channel with high-affinity (less than 1 nM). However, the concentration necessary to induce LTP is more than 500 nM, suggesting that some other functions of MCD are also fundamental to LTP induction. The concentration of MCD required for LTP induction was greatly reduced by preactivating G proteins. This fact suggests that G protein activation by MCD also plays an important role in LTP induction. MCD-binding proteins were purified from rat brain. G proteins were found to exist in a non-denatured state in this affinity-purified fraction. When reconstituted into a planar lipid bilayer membrane, a potassium-selective and voltage-dependent current could be observed. This channel was blocked by MCD at a high concentration equal to the effective concentration for G protein activation. Addition of GTP-gamma-S significantly blocked the reconstituted current. Thus, we identified a pathway for LTP induction by MCD in which high concentrations of MCD activate G protein which in turns leads to blocking of a potassium channel.

Adenosine Diphosphate Ribose↗
PubMed1996

Localization of mRNA for UDP-galactose: ceramide galactosyltransferase in the brain during mouse development.

UDP-galactose:ceramide galactosyltransferase (CGT) is considered to be a key enzyme in the biosynthesis of galactocerebroside (GalC), a frequently used marker for oligodendrocytes. CGT gene expression has been shown to be restricted to the oligodendrocyte lineage in young adult rat brain. We detected expression of the CGT gene in embryonic mouse brain and spinal cord from E17 onward using an in situ hybridization technique. Localization of CGT messages was restricted to two longitudinal columns of cells in the basal half of the ventricular zone around the fourth ventricule at the most caudal level of the medulla oblongata. After birth, they were distributed in caudal-to-rostral and ventral-to-dorsal gradients. This study shows that CGT mRNA is a good marker for localizing oligodendrocytes, and appears just before the GalC-positive stage.

Aging↗
PubMed1995

Differentiation between dysmyelination and demyelination using magnetic resonance diffusional anisotropy.

Using magnetic resonance (MR) diffusion-weighted method, we examined the optic and the trigeminal nerves of jimpy and twitcher mice, considered to be animal models of Pelizaeus-Merzbacher disease, hypomyelination disorder, and Krabbe disease, demyelination disorder, respectively. In jimpy mice, diffusional anisotropy of optic nerve did not show a significant difference compared to age-matched control mice, suggesting that diffusional anisotropy does exist in absence of multiple layers of myelin sheath. In twitcher mice, diffusional anisotropy was attenuated remarkably in the optic and trigeminal nerves. Loss of axonal straightness on longitudinal section confirmed by electron microscopy appeared to be the principal explanation for it. It is further suggested that this MR diffusion-weighted imaging method enables us to differentiate hypomyelination from demyelination in vivo.

Animals↗
PubMed1995

The reeler gene-associated antigen on Cajal-Retzius neurons is a crucial molecule for laminar organization of cortical neurons.

In the neurological mutant mouse reeler, the histological organization of the neocortex develops abnormally and essentially results in an inversion of the relative positions of the cortical layers. The reeler mutation, therefore, provides an insight into the molecular mechanisms underlying the formation of the cortical layers. We have generated a monoclonal antibody (CR-50) that probes a distinct allelic antigen present in wild-type but not in reeler mutant mice. CR-50 reacted specifically with Cajal-Retzius neurons, one of the first cortical neurons to differentiate in the neocortex, but whose functional role is not known. When dissociated cerebral cortical cells were incubated with CR-50 in reaggregation culture, the genotype-dependent histogenetic assembly of wild-type cortical cells resembled that of reeler mutants. These findings revealed that the selective expression of a distinct molecule on Cajal-Retzius neurons is critical for the normal lamination of cortical neurons in the mammalian neocortex.

Animals↗
PubMed1995

Expression of proteolipid protein gene is directly associated with secretion of a factor influencing oligodendrocyte development.

Oligodendroglial cell death in the myelin proteolipid protein (PLP) mutants can be partially rescued by the environment factor(s) supplied by the wild-type cells in vivo and in vitro. It is possible that the presence of PLP or DM-20 results in secretion of a factor or factors in the CNS influencing oligodendrocyte development. We previously showed that DM-20 mRNA is produced in G26 mouse oligodendroglioma, B104 rat neuroblastoma, and B16 mouse melanoma but not in NIH3T3 mouse fibroblast cell lines. Culture supernatants from these cell lines were added to primary glial cell cultures from embryonic day 17 mouse brain. After 4 days, the number of oligodendrocytes present in cultures with supernatants from DM-20-producing cells (G26, B104, and B16) was significantly higher than that of control cultures but not with the NIH3T3 supernatant. To investigate more directly whether the PLP gene expression is involved in this process, NIH3T3 cells (nonneural cells) were forced to produce PLP or DM-20. By addition of the supernatants from the PLP/DM-20 transformants, the number of oligodendrocytes in the mixed glial cell cultures increased. This clearly demonstrates that the expression of the PLP gene is sufficient for and directly associated with secretion of a factor, which influences the oligodendrocyte development.

3T3 Cells↗
PubMed1995

Infectious retrovirus is inactivated by serum but not by cerebrospinal fluid or fluid from tumor bed in patients with malignant glioma.

Intravenous gene transfer using recombinant retroviruses tends to suffer from a low infectious viral titer when conducted in vivo. This is, in part, caused by complement-mediated proteolytic inactivation of the retrovirus in human serum. However, if the retroviruses were directly injected into the brain, they might not be inactivated. Supernatant from amphotropic retrovirus-producing cells harboring the BAG vectors was incubated with sera or cerebrospinal fluid (CSF) of patients with gliomas or unrelated disorders. The retroviruses were severely inactivated in sera. However, no such inactivation was noted in CSF or fluid from the tumor bed of glioma patients. These data suggest that gene transfer using recombinant retroviruses could be done into the cavity after removal of the tumor in glioma patients.

3T3 Cells↗
PubMed1995

Transgenic systems in studying myelin gene expression.

The myelin sheath is produced by oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). It is necessary to introduce DNA into these cells to study gene regulation and the functions of myelin proteins. Since these cells are labile to commonly used techniques to introduce DNA, a transgenic system must be used for these studies. Myelin basic protein (MBP) and myelin proteolipid protein (PLP) are both highly abundant in CNS myelin, but are also produced in Schwann cells. Various lengths (6.5 kb to 256 bp) of the promoter region of the (classic) MBP gene directed oligodendrocyte-specific expression of the reporter gene in transgenic mice, but no expression was seen in Schwann cells. Promoter regions of the PLP gene (4.2-2.4 kb) seem to contain all the information required for correct spatiotemporal expression, but the level of expression was low. The first intron of the PLP gene is a candidate for the location of the enhancer-like element. Studies on MBP mutant mice carrying wild-type MBP gene or cDNA clearly demonstrated that one function of MBP is to make myelin lamellae compact by fusing the cytoplasmic surfaces of oligodendrocytes into the form of major dense lines. However, functional analysis of PLP gene products using a similar strategy produced confusing results. The wild-type PLP gene introduced into jimpy mutant mice (one of the PLP mutants) did not complement the mutant phenotype. Moreover, overexpression of the PLP gene itself (in wild-type background) was shown to produce a phenotype similar to that of the mutants, including arrest in oligodendrocyte maturation and hypomyelination. Thus, PLP gene products play a fundamental role in oligodendrocyte maturation as well as in the stabilization of myelin structure, and its expression must be tightly regulated.

Animals↗
PubMed1995

A simplified general method for determination of recombinant retrovirus titers.

To construct recombinant retroviruses with only a single active promoter, we introduced point mutations into the TATA box region of the 3'-LTR, and successfully obtained high-titer virus with sufficient self-inactivating activity. However, the viral titer could not be determined by the number of G418 resistant colonies since the neomycin resistance gene was under 5'-LTR control, because of inactivation of the selection marker in target glioma cells. To overcome this problem, we constructed PCR primers with homology to a gene under the control of the internal promoter of recombinant retrovirus, and to retrovirus-specific sequences. There was good correlation between the amount of PCR-amplified product and the number of colony forming units when glioma cells were transduced with the retroviruses containing both the neomycin resistance gene and the HTK gene. Amplified PCR products quantitated by densitometry after glioma cells were transduced with SIV retrovirus vectors, and there was good correlation between density and sensitivity to GCV following transduction. Therefore, detection of HTK PCR products from glioma cells transduced with HTK-bearing retroviruses is useful for determining the appropriate packaging cell for efficient production of viral particles. This detection system is especially useful for isolating high titer clones producing SIV-type retroviruses.

Base Sequence↗