Comprehensive behavioural study of GluR4 knockout mice: implication in cognitive function.

Fast excitatory transmission in the mammalian central nervous system is mediated by AMPA-type glutamate receptors. The tetrameric AMPA receptor complexes are composed of four subunits, GluR1-4. The GluR4 subunit is highly expressed in the cerebellum and the early postnatal hippocampus and is thought to be involved in synaptic plasticity and the development of functional neural circuitry through the recruitment of other AMPA receptor subunits. Previously, we reported an association of the human GluR4 gene (GRIA4) with schizophrenia. To examine the role of the GluR4 subunit in the higher brain function, we generated GluR4 knockout mice and conducted electrophysiological and behavioural analyses. The mutant mice showed normal long-term potentiation (LTP) in the CA1 region of the hippocampus. The GluR4 knockout mice showed mildly improved spatial working memory in the T-maze test. Although the retention of spatial reference memory was intact in the mutant mice, the acquisition of spatial reference memory was impaired in the Barnes circular maze test. The GluR4 knockout mice showed impaired prepulse inhibition. These results suggest the involvement of the GluR4 subunit in cognitive function.

Prevention of radiation-induced pneumonitis by recombinant adenovirus-mediated transferring of soluble TGF-beta type II receptor gene.

To investigate whether radiation-induced pneumonitis in the mouse-irradiated lung could be prevented by recombinant adenovirus-mediated soluble transforming growth factor-beta (TGF-beta) type II receptor gene therapy. Radiation fibrosis-prone mice (C57BL/6J) were randomly divided into four groups consisting of a (1) control group (sham-irradiated); (2) radiation (RT)-alone group; (3) RT+AdCMVsTbetaR group and (4) RT+AdCMVluc group. The RT-alone and sham-irradiated mice were killed at several time points after thoracic irradiation with a single dose of 9 Gy, and then the TGF-beta1 concentrations in serum and broncho-alveolar lavage fluid (BALF) were quantified by enzyme-linked immunosorbent assay (ELISA). We used an adenoviral vector expressing a soluble TGF-beta type II receptor (AdCMVsTbetaR), which can bind to TGF-beta and then block the TGF-beta receptor-mediated signal transduction. The C57BL/6J mice were intraperitoneally (i.p.) injected with either 5 x 10(8) plaque-forming units of AdCMVsTbetaR or AdCMVluc, a control adenovirus-expressing luciferase, a week preceding and a week following the X-ray thoracic irradiation. Four weeks after irradiation, the mice were killed and the concentration of TGF-beta1 in the serum and BALF were then measured using ELISA and the lung tissue specimens were examined histopathologically. Following thoracic irradiation with a single dose of 9 Gy, radiation-induced TGF-beta1 release in the serum reached the first peak concentration at 12 h and then declined. It reached a maximal value at 2 weeks after irradiation. In the BALF, the TGF-beta1 concentration was appreciable within the first hour and thereafter declined. It reached a maximal value at 3 days after irradiation. A one-time i.p. injection of AdCMVsTbetaR 1 week before irradiation could not completely suppress the two peaks of the radiation-induced TGF-beta1 increase, whereas an injection a week preceding and a week following thoracic irradiation was able to suppress those two peaks thoroughly. The TGF-beta1 was completely suppressed in the AdCMVsTbetaR-treated mouse serum and BALF; however, no statistical difference was observed in the serum and BALF between the AdCMVluc-infected mice and the control mice at 4 weeks after irradiation (P < 0.05). A histopathological examination showed only mild radiation pneumonitis in the irradiated lungs of AdCMVsTbetaR-treated mice in comparison to the AdCMVluc-infected and RT-alone mice. Our results demonstrated that TGF-beta1 plays an important role in radiation pneumonitis, thus suggesting that the adenovirus-mediated overexpression in soluble TGF-beta type II receptor gene therapy may be a potentially feasible and effective strategy for the prevention of radiation pneumonitis.

The characterisation of hyalocytes: the origin, phenotype, and turnover.

AIM: To determine the characterisation of hyalocytes: the origin, phenotype, and turnover in the rodent. METHODS: To characterise the ultrastructure and distribution of hyalocytes, transmission and scanning electron microscopy was performed in rat eyes. Immunophenotypical analysis was performed by either anti-ED1 or ED2 antibodies. To examine the origin of the hyalocytes, the chimeric mice were created and were used to transplant the bone marrow (BM) cells from enhanced green fluorescent protein (EGFP) transgenic mice. The turnover of hyalocytes was examined at 0, 4, 6, 7, and 12 months after BM transplantation. RESULTS: Hyalocytes were distributed especially in the vitreous cortex and had an irregular shape with a spherical granule. Immunophenotypical studies demonstrated that most of the hyalocytes in rat eyes expressed ED2 but not ED1. In the chimeric mice, the hyalocytes were GFP negative right after BM transplantation. Interestingly, more than 60% of hyalocytes were replaced within 4 months and approximately 90% within 7 months after BM transplantation. CONCLUSIONS: The rodent hyalocytes were shown to express tissue macrophage marker, were derived from BM, and totally replaced within 7 months. These data provide the characterisation of hyalocytes in physiological conditions, especially their origin, distribution, and turnover, and may contribute to the better understanding of the pathogenesis of vitreoretinal disease.

Determination of the orbital polarization in YTiO3 by using soft X-ray linear dichroism.

We report measurements of linear dichroism in x-ray absorption at Ti L(2,3) edges of a Mott-insulating ferromagnet YTiO3, where orbital ordering occurs in the triply degenerate Ti 3d t(2g) states. Dichroic spectra and their integrated intensities are obtained for the incident electric field with polarizations parallel to a, b, and c axes. The comparison of the spectra with atomic multiplet calculations removes the ambiguity about the orbital polarization, i.e., the relative weights of |xy>, |yz>, and |zx> orbits, which are crucial for the origin of ferromagnetism. The result is consistent with the previous analysis of nuclear magnetic resonance in the Mizokawa-Fujimori scheme.

Spontaneous tumorigenesis in mice defective in the MTH1 gene encoding 8-oxo-dGTPase.

Oxygen radicals, which can be produced through normal cellular metabolism, are thought to play an important role in mutagenesis and tumorigenesis. Among various classes of oxidative DNA damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is most important because of its abundance and mutagenicity. The MTH1 gene encodes an enzyme that hydrolyzes 8-oxo-dGTP to monophosphate in the nucleotide pool, thereby preventing occurrence of transversion mutations. By means of gene targeting, we have established MTH1 gene-knockout cell lines and mice. When examined 18 months after birth, a greater number of tumors were formed in the lungs, livers, and stomachs of MTH1-deficient mice, as compared with wild-type mice. The MTH1-deficient mouse will provide a useful model for investigating the role of the MTH1 protein in normal conditions and under oxidative stress.

Analysis of MTH1 gene function in mice with targeted mutagenesis.

Oxidative DNA damage is thought to contribute to carcinogenesis, ageing, and neurological degeneration. Further, the cumulative risk of cancer increases dramatically with age in humans. In general terms, cancer can be regarded as a degenerative disease of ageing. There is evidence for the accumulation of oxidative DNA damage with age based on studies mainly measuring an increase in 8-oxoguanine. 8-Oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is formed in the nucleotide pool of a cell during normal cellular metabolism. When 8-oxoguanine is incorporated into DNA causes mutation. Organisms possess 8-oxo-dGTPase, an enzyme that specifically degrades 8-oxo-dGTP to 8-oxo-dGMP. To analyze the function of MTH1 with 8-oxo-dGTPase activity in vivo, we generated a mouse line carrying a mutant MTH1 allele created by targeted gene disruption. MTH1 homozygous mutant mice were found to have a physically normal appearance, but seemed to have lost 8-oxo-dGTPase activity in liver extracts. When we examined the susceptibility of the mutant mice to spontaneous tumorigenesis, no significant difference was observed in survival rate of MTH1+/+ and MTH1-/- mice. However, pathological examination revealed a statistically significant difference in the incidence of tumors. More tumors were formed in lungs, livers, and stomachs of MTH1-/- mice than in those of the wild type mice. These studies with MTH1-null mutant mice provided an important insight into the role of this nucleotide sanitization enzyme in terms of the spontaneous tumorigenesis as well as mutagenesis caused by the oxygen-induced DNA damage.

Enhancement of hyperthermic killing in L5178Y cells by protease inhibitors.

We have investigated the effect of protease inhibitors on hyperthermic cell killing using cultured mammalian cells (L5178Y) and found that protease inhibitors were potent hyperthermia sensitizers. At 37 degrees C, phenylmethylsulfonyl fluoride (PMSF), a serine protease inhibitor, was not cytotoxic at the concentration of 400 micrograms/ml for up to 6 h. When cells were exposed to PMSF (200-400 micrograms/ml) during heating at 43 degrees C, significant potentiation of hyperthermic cell killing was observed. Other protease inhibitors, such as chymostatin and diisopropylfluorophosphate (both are serine protease inhibitors); (2S,3S)-trans-epoxy-succinyl-L-leucylamido-3-methylbutane ethyl ester (cysteine protease inhibitor) and pepstatin-A (aspartate protease inhibitor) showed similar effects. However, when cells were heated at 43 degrees C in the presence of cycloheximide (a protein synthesis inhibitor) together with PMSF, hyperthermic enhancement by PMSF decreased markedly. A decrease in potentiating the effect of PMSF was also noted with thermotolerant cells. These facts suggest that protease inhibitors may exert their hyperthermic cell killing by inhibiting proteases and ubiquitin, which are necessary to degrade denatured proteins induced by heat.

Enhancement of radiosensitivity of cultured mammalian cells by neocarzinostatin. I. Inhibition of the repair of sublethal damage.

The enhancement of radiosensitivity by neocarzinostatin (NCS), an antitumour drug, was studied using three strains of cultured mammalian cells with different repair capabilities for sublethal damage. NCS enhanced the radiosensitivity of the cells when applied both during and after X-irradiation under aerobic conditions. The enhancement ratios of NCS during X-irradiation were 1.25, 1.27 and 1.38 for mouse lymphoma L5178Y, Chinese hamster V79 and mouse mammary tumour FM 3A cells, respectively. The corresponding ratios after X-irradiation were 1.18, 1.27 and 1.38, respectively. These ratios were proportional to the repair capabilities of the cells for sublethal damage. NCS completely inhibited the repair of sublethal damage regardless of the repair capabilities of the cells for sublethal damage. NCS was equally effective for hypoxic cells. These results suggested that NCS enhanced the radiosensitivity of the cells probably by interacting with the residual damage after X-irradiation, thereby converting the sublethal damage or potentially lethal damage into lethal damage.

Enhancement of radiosensitivity of cultured mammalian cells by neocarzinostatin. II. Fixation of potentially lethal damage.

The effects of neocarzinostatin (NCS), an anti-tumour drug, on the repair of potentially lethal damage (PLD) were studied using cultured Chinese hamster V79, malignant human melanoma and mouse lymphoma L5178Y cells in the stationary phase. The repair of PLD was observed in the melanoma and L5178Y cells but no such repair was observed in the V79 cells, when studied by delayed plating. NCS added to the culture medium immediately after X-irradiation evoked fixation of PLD within 10 min of the addition of NCS. The ratios of D0 values of the survival curves of the cells treated with NCS to those plated immediately after X-irradiation were 0.78, 0.88 and 0.85 for V79, melanoma and L5178Y cells, respectively. The extent of the fixation by NCS was similar to that caused by 0.5 M NaCl solution. The results in the present study and the inhibition of sublethal damage (SLD) by NCS reported previously, suggest that NCS might react with the DNA damage induced by radiation and modify it to lethal damage. The study indicates that SLD and PLD appear to be closely related to one another.

Binucleate cell formation correlates to loss of colony-forming ability in X-irradiated cultured mammalian cells.

The relationship between binucleate cell formation and the loss of colony-forming ability was examined in several cultured mammalian cell lines irradiated with X rays. The maximum fraction of binucleate cells after X irradiation increased dose-dependently within the range in which reproductive cell death might predominate over interphase cell death. When the logarithm of percentage survival was plotted against the percentage binucleate cells, a similar correlation was found for all cell lines tested, with the exception of mouse leukemia L5178Y cells, the most radiosensitive cells used. These observations suggest that the fraction of binucleate cells in the cell population can serve as a measure of cellular radiation damage.