beta1-4Galactosyltransferase activity of mouse brain as revealed by analysis of brain-specific complex-type N-linked sugar chains.

We previously reported two brain-specific agalactobiantennary N-linked sugar chains with bisecting GlcNAc and alpha1-6Fuc residues, (GlcNAcbeta1-2)(0)(or)(1)Manalpha1-3(GlcNAcbeta1-2M analpha1-6)(GlcNA cbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)Glc NAc [Shimizu, H., Ochiai, K., Ikenaka, K.

Cancer gene therapy with HSV-tk/GCV system depends on T-cell-mediated immune responses and causes apoptotic death of tumor cells in vivo.

To examine the immunological mechanisms involved in cancer gene therapy using the herpes simplex virus thymidine kinase (HSV-tk) gene and ganciclovir (GCV), murine hepatocellular carcinoma (HCC) cells, BNL1ME A.7R.1, were transduced retrovirally with the HSV-tk gene.

Molecular and functional characterization of a novel mouse transient receptor potential protein homologue TRP7. Ca(2+)-permeable cation channel that is constitutively activated and enhanced by stimulation of G protein-coupled receptor.

Characterization of mammalian homologues of Drosophila transient receptor potential protein (TRP) is an important clue to understand molecular mechanisms underlying Ca(2+) influx activated in response to stimulation of G(q) protein-coupled receptors in vertebrate cells. Here we have isolated cDNA encoding a novel seventh mammalian TRP homologue, TRP7, from mouse brain. TRP7 showed abundant RNA expression in the heart, lung, and eye and moderate expression in the brain, spleen, and testis.

Complete cure of established murine hepatocellular carcinoma is achievable by repeated injections of retroviruses carrying the herpes simplex virus thymidine kinase gene.

Although xenotransplantation of retrovirus-producing cells into a tumor has been shown to be effective for the treatment of cancer, injections of recombinant retroviruses are much more feasible for clinical applications. We established a clone producing retroviruses carrying the herpes simplex virus thymidine kinase (HSVtk) gene with titers of up to 4 x 10(7) colony-forming units/ml, and examined the effectiveness of in vivo gene therapy against cancer. Syngeneic mice were inoculated subcutaneously with murine hepatocellular carcinoma (HCC) cells, BNL1ME A.

Expression of Kv3.1 and Kv4.2 genes in developing cerebellar granule cells.

The expression of voltage-gated potassium channels plays an important role in the acquisition of membrane excitability in neurons. We examined the expression pattern of genes in developing cerebellar granule neurons in vivo and in vitro. In situ hybridization of Kv3.

Retroviruses prepared from human DAF expressing murine packaging cells acquire resistance against human serum.

The complement system of the human body inactivates the infectious ability of retroviruses injected as an artificial gene transfer vector. We established new murine leukemia virus (MuLV) packaging cell lines; D2SS and D7S which express decay-accelerating factor (DAF) on their surface. Both D2SS and D7S were resistant against incubation with fresh human serum.

Cytosine deaminase/5-fluorocytosine gene therapy can induce efficient anti-tumor effects and protective immunity in immunocompetent mice but not in athymic nude mice.

Murine hepatocellular carcinoma cells were retrovirally transduced with the bacterial cytosine deaminase (CD) gene. CD-transduced cells exhibited more than 120-fold higher sensitivity to 5-fluorocytosine (5-FC) compared with parental cells. When syngeneic immunocompetent mice were inoculated s.

Programmed cell death without DNA fragmentation in the jimpy mouse: secreted factors can enhance survival.

Jimpy is one of many related mutations affecting the myelin proteolipid protein gene that causes severe hypomyelination in the central nervous system (CNS). Underlying the hypomyelination is a failure of oligodendrocytes (OLs) to differentiate, and the premature death of large numbers of OLs during the developmental period. Previous light and electron microscopic evidence suggested that jimpy OLs die in a manner consistent with programmed cell death.

Proteolipid protein gene product can be secreted and exhibit biological activity during early development.

A gene encoding myelin proteolipid protein (PLP) and its smaller isoform DM20 is expressed at least 1 week before myelination. Mutations within the gene cause abnormalities in the development of premyelinating oligodendrocytes, resulting in hypomyelinating disorders. These findings suggest a premyelinating function of the PLP gene products.

Conversion of brain-specific complex type sugar chains by N-acetyl-beta-D-hexosaminidase B.

The N-linked sugar chains, GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-4)(Manalpha1++ +-3)Manbeta1-4GlcNAcb eta1-4(Fucalpha1-6)GlcNAc (BA-1) and GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-4)(GlcNAcbeta1 -2Manalpha1-3)Manb eta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc (BA-2), were recently found to be linked to membrane proteins of mouse brain in a development-dependent manner [S. Nakakita, S. Natsuka, K.

Systematic analysis of N-linked sugar chains from whole tissue employing partial automation.

A partially automated technique for the isolation and characterization of N-linked sugar chains from glycoproteins of crude tissue samples is established. The N-linked sugar chains from the acetone-extracted tissues are made free by a process of hydrazinolysis and subsequently N-acetylated by GlycoPrep 1000 (Oxford Glycosystems). These free sugar chains are further converted to pyridylamino derivatives by GlycoTag (Takara).

Transduction of glioma cells using a high-titer retroviral vector system and their subsequent migration in brain tumors.

The intracranial migration of transduced glioma cells was investigated in order to improve the treatment of malignant glioma by gene therapy using retroviral vectors. In this study, about half the volume of the tumor mass could be transduced in 14 days after only a single implantation of 3 x 10(5) retrovirus-producing cells into a tumor mass with a diameter of 5 mm. Moreover, we were able to follow the migration of glioma cells transduced by the lacZ-harboring retroviruses originating from the high-titer retrovirus-producing cells.

Expression of a retrovirally transduced gene under control of an internal housekeeping gene promoter does not persist due to methylation and is restored partially by 5-azacytidine treatment.

Although expression of transgenes under the control of a retroviral long terminal repeat (LTR) promoter has been shown not to persist due to methylation, it has been observed that internal promoter may be active even if expression from the LTR promoter is silent. We constructed a retroviral vector carrying the herpes simplex virus thymidine kinase (HSVtk) gene under the control of the albumin gene promoter and transduced the HSVtk gene into hepatocellular carcinoma cells. Three of 14 mice, however, could not eradicate HSVtk-transduced grafts completely despite ganciclovir (GCV) treatment.

Intrathecal 5-fluoro-2'-deoxyuridine (FdUrd) for the treatment of solid tumor neoplastic meningitis: an in vivo study.

To evaluate the possible intrathecal use of 5-fluoro-2'-deoxyuridine (FdUrd) for neoplastic meningitis, its antitumor activity and neurotoxicity in vivo were assessed. FdUrd at doses in the range 5-100 microg/animal was effective against meningeal carcinomatosis using Walker 256 carcinoma cells in rats and MM46 mammary cancer cells in mice and against meningeal gliomatosis using 203 glioma cells in mice. After four intrathecal injections, FdUrd at these doses also showed minimal neurotoxicity in the C57BL/6 mouse brain.

Bystander effect caused by cytosine deaminase gene and 5-fluorocytosine in vitro is substantially mediated by generated 5-fluorouracil.

Because it appears impossible to transfer toxic genes to all the cells of a cancer, the bystander effect is critical to induce effective antitumor effects. In the present study, possible in vitro mechanisms of the bystander effect by the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) were investigated. CD-transduced cancer cells exhibited much higher sensitivity to 5-FC compared to parental cells.

[Clinical application of FdUrd to meningeal dissemination of malignant tumors].

A clinical trial of intrathecal chemotherapy with FdUrd was performed in sixteen patients with meningeal dissemination of malignant tumors. Twelve of the sixteen patients responded to intrathecal FdUrd chemotherapy (1-5 micrograms/dose) through an Ommaya reservoir placed in the lateral ventricle: complete response, 2; partial response, 10; progressive disease, 4. Only slight nausea was observed in two patients and dull headache in one patient.

[In vivo study on intrathecal use of 5-fluoro-2'-deoxyuridine (FdUrd) in meningeal dissemination of malignant tumor].

FdUrd was evaluated in vivo as a potential agent for intrathecal chemotherapy of meningeal carcinomatosis. Neurotoxicity was examined pathologically in normal mice after 4 consecutive intrathecal injections of FdUrd. Using mice models of meningeal carcinomatosis, antitumor activities were studied by evaluating survival time.

Multiple restricted origin of oligodendrocytes.

The plp gene encodes the proteolipid protein and its alternatively spliced product DM-20, major proteins of CNS myelin. In the mouse, plp/dm-20 transcripts are expressed beginning at embryonic day 9.5 (E9.

[In vitro study on intrathecal application of 5-fluoro-2'-deoxyuridine (FdUrd) for meningeal dissemination of malignant tumor].

To evaluate the possible clinical intrathecal use of 5-fluoro-2'-deoxyuridine (FdUrd) for malignant brain tumors, its anti-tumor activity and neurotoxicity were compared with that of 5-fluorouracil (5-FU) and 5-fluorouridine (FUrd) in vitro. FdUrd showed good tumoricidal activity against cultured mouse 203 glioma cells and rat Walker 256 carcinoma cells as well as A172 human glioblastoma cells. Daoy human medulloblastoma cells and CADO-LC4 human lung cancer cells.

Proximal promoter region is sufficient to regulate tissue-specific expression of UDP-galactose: ceramide galactosyltransferase gene.

UDP-galactose:ceramide galactosyltransferase (CGT) is the enzyme which catalyzes the final step of the biosynthesis of galactocerebroside (GalC), the most abundant glycolipid in myelin. We identified regulatory elements which are related to the tissue-specific expression of the mouse CGT gene by promoter assay using chimeric CGT-luciferase constructs. By comparing promoter activity in oligodendroglial CG4 cells and NIH3T3 fibroblasts, only a few hundred base pairs spanning from -309 to -98 were shown to be necessary for the tissue-specific activity of CGT promoter.

Development-dependent expression of complex-type sugar chains specific to mouse brain.

We previously detected a fucosylagalactobiantenna with a bisecting GlcNAc residue (BA-2) and one lacking the GlcNAc residue linked to the Manalpha1-3 residue of BA-2 (BA-1), which were enriched specifically in mouse brain [Shimizu, H., Ochiai, K., Ikenaka, K.

Metabolic labeling of a subset of glial cells by UDP-galactose: implication for astrocyte lineage diversity.

Astrocytes are implicated in many aspects of brain function; however, it remains unclear whether astrocytes arise from a single cell lineage. It is therefore important to obtain new markers for the astrocyte cell lineage. We show that exogenously added UDP-galactose (UDP-Gal) can be used to metabolically label a subset of glial fibrillary acidic protein-positive (GFAP+) cells.

Appearance of a fast inactivating voltage-dependent K+ currents in developing cerebellar granule cells in vitro.

To elucidate the molecular mechanisms that regulate the maturation of action potential, we began by examining voltage-dependent K+ currents, known to contribute to the maturation of action potential, of developing granule cells in mouse cerebellar microexplant cultures. The migration of developing granule cells in this culture is reported to mimic the in vivo process, but their specific identification is still incomplete. In this study, we identified and characterized granule cells in this culture.

In vitro study on intrathecal use of 5-fluoro-2'-deoxyuridine (FdUrd) for meningeal dissemination of malignant brain tumors.

We investigated 5-fluoro-2'-deoxyuridine (FdUrd) as a potential agent for intrathecal treatment of malignant brain tumors with meningeal dissemination. We examined the neurotoxicity of FdUrd in vitro using primary cultures of neurons from C57BL/6 mice (ED14). Tumoricidal activity was also studied in four glioma cell lines and one medulloblastoma cell line.