Temporary membrane distortion of vascular smooth muscle cells is responsible for their apoptosis induced by platelet-activating factor-like oxidized phospholipids and their degradation product, lysophosphatidylcholine

To obtain information about the mechanism of apoptosis induced by oxidized low density lipoproteins (oxLDL) in atherosclerotic plaques, we examined the effects of lysophosphatidylcholine (LPC) and platelet-activating factor (PAF)-like lipids (PAF-LL), which can be derived from oxLDL, on rat vascular smooth muscle cells (VSMC). All the lipids with different structures examined induced apoptosis of VSMC, so we studied the mechanism of induction of apoptosis by LPC. LPC-induced apoptosis was inhibited by alpha-tocopherol (alpha-T) and cholesterol (Chol), but not by other antioxidants such as palmitoyl ascorbic acid and PAF receptor antagonist. The cells temporarily became spherical and highly permeable before induction of apoptosis, and their change in shape was prevented by alpha-T and Chol. From these results, we suggest that the apoptosis induced by oxLDL-derived phospholipids in VSMC is caused by temporary membrane distortion, not through specific receptors.     

Role of microtubules versus myosin heavy chain isoforms in contractile dysfunction of hypertrophied murine cardiocytes

In large mammals there is a correlation between microtubule network densification and contractile dysfunction in severe pressure-overload hypertrophy. In small mammals there is a similar correlation for the shift to beta-myosin heavy chain (MHC), a MHC isoform having a slower ATPase Vmax. In this study, murine left ventricular (LV) pressure overload invoked both mechanisms: microtubule network densification and beta-MHC expression. Cardiac beta-MHC was also augmented without altering tubulin levels by two load-independent means, chemical thyroidectomy and transgenesis. In hypertrophy, contractile function of the LV and its cardiocytes decreased proportionally; microtubule depolymerization restored normal cellular contraction. In hypothyroid mice having a complete shift from alpha-MHC to beta-MHC, contractile function of the LV and its cardiocytes also decreased, but microtubule depolymerization had no effect on cellular contraction. In transgenic mice having a cardiac beta-MHC increase similar to that in hypertrophy, contractile function of the LV and its cardiocytes was normal, and microtubule depolymerization had no effect. Thus, although both mechanisms may cause contractile dysfunction, for the extent of MHC isoform switching seen even in severe murine LV pressure-overload hypertrophy, microtubule network densification appears to have the more important role.     

Identification of surrogate ligands for orphan G protein-coupled receptors

We prepared fusion proteins with an alpha subunit of G protein Gi (Gi1alpha) of 26 orphan G protein-coupled receptors (GPCRs) and with Gsalpha of 10 orphan GPCRs, most of which had been identified from the human genome previously [FEBS Lett 520 (2002) 97]. Ligands for these fusion proteins were screened from a library consisting of approximately 1000 authentic compounds by measuring their effect on [35S]GTPgammaS binding to membrane preparations of insect Sf9 cells expressing these fusion proteins. Eleven compounds were found to act as surrogate agonists for a GPCR-Gsalpha and four GPCR-Gialpha fusion proteins, a compound as an inverse agonist for two GPCR-Gsalpha fusion proteins, and a compound as an endogenous agonist for a GPCR-Gialpha fusion protein.     

Identification of radiation-sensitive mutants in the Medaka, Oryzias latipes

We screened populations of N-ethyl-N-nitrosourea (ENU)-mutagenized Medaka, (Oryzias latipes) for radiation-sensitive mutants to investigate the mechanism of genome stability induced by ionizing radiation in developing embryos. F3 embryos derived from male founders that were homozygous for induced the mutations were irradiated with gamma-rays at the organogenesis stage (48hpf) at a dose that did not cause malformation in wild-type embryos. We screened 2130 F2 pairs and identified three types of mutants with high incidence of radiation-induced curly tailed (ric) malformations using a low dose of irradiation. The homozygous strain from one of these mutants, ric1, which is highly fertile and easy to breed, was established and characterized related to gamma-irradiation response. The ric1 strain also showed higher incidence of malformation and lower hatchability compared to the wild-type CAB strain after gamma-irradiation at the morula and pre-early gastrula stages. We found that the decrease in hatching success after gamma-irradiation, depends on the maternal genotype at the ric1 locus. Terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling assays showed a high frequency of apoptosis in the ric1 embryos immediately after gamma-irradiation at the pre-early gastrula stage but apoptotic cells were not observed before midblastula transition (MBT). The neutral comet assay revealed that the ric1 mutant has a defect in the rapid repair of DNA double-strand breaks induced by gamma-rays. These results suggest that RIC1 is involved in the DNA double strand break repair in embryos from morula to organogenesis stages, and unrepaired DNA double strand breaks in ric1 trigger apoptosis after MBT. These results support the use of the ric1 strain for investigating various biological consequences of DNA double strand breaks in vivo and for sensitive monitoring of genotoxicity related to low dose radiation.     

Protective effect of rhynchophylline and isorhynchophylline on in vitro ischemia-induced neuronal damage in the hippocampus: putative neurotransmitter receptors involved in their action

Rhynchophylline and isorhynchophylline are major tetracyclic oxindole alkaloid components of Uncaira species, which have been long used as medicinal plants. In this study we examined the protective effects of rhynchophylline and isorhynchophylline on in vitro ischemia-induced neuronal damage in the hippocampus and interaction of these alkaloids with neurotransmitter receptors in a receptor expression model of Xenopus oocytes. In vitro ischemia was induced by exposing the hippocampal slices to oxygen- and D-glucose-deprived medium over 8 min. The resultant neuronal damage was elucidated as deterioration of population spike (PS) amplitudes evoked trans-synaptically by electrical stimulation of Schaffer collaterals and recorded in the CA1 area. Rhynchophylline and isorhynchophylline, as well as the N-methyl-D-aspartate (NMDA) antagonist (+/-)-2-amino-5-phosphono-valeric acid (APV), the muscarinic M1 receptor antagonist pirenzepine, and the 5-HT2 receptor antagonist ketanserin, attenuated the in vitro ischemia-induced neuronal damage in a concentration-dependent manner. There was no difference in the extent of protection against the neuronal damage between rhynchophylline and isorhynchophylline treatment. In Xenopus oocytes expressing the rat brain receptors encoded by total RNA, both rhynchophylline and isorhynchophylline reduced muscarinic receptor- and 5-HT2 receptor-mediated current responses in a competitive manner. Together with our previous findings that rhynchophylline and isorhynchophylline have a non-competitive antagonistic effect on the NMDA-type ionotropic glutamate receptors, the present results suggest that these alkaloids exert their protective action against ischemia-induced neuronal damage by preventing NMDA, muscarinic M1, and 5-HT2 receptors-mediated neurotoxicity during ischemia.     

Gene expression of cell surface antigens in the early phase of murine influenza pneumonia determined by a cDNA expression array technique

BACKGROUND: Influenza virus is a worldwide health problem with significant economic consequences. To study the gene expression pattern induced by influenza virus infection, it is useful to reveal the pathogenesis of influenza virus infection; but this has not been well examined, especially in vivo study. AIMS: To assess the influence of influenza virus infection on gene expression in mice, mRNA levels in the lung and tracheal tissue 48 h after infection were investigated by cDNA array analysis. METHODS: Four-week-old outbred, specific pathogen free strain, ICR female mice were infected by intra-nasal inoculation of a virus solution under ether anesthesia. The mice were sacrificed 48 h after infection and the tracheas and lungs were removed. To determine gene expression, the membrane-based microtechnique with an Atlas cDNA expression array (mouse 1.2 array II) was performed in accordance with the manual provided. RESULTS AND CONCLUSIONS: We focused on the expression of 46 mRNAs for cell surface antigens. Of these 46 mRNAs that we examined, four (CD1d2 antigen, CD39 antigen-like 1, CD39 antigen-like 3, CD68 antigen) were up-regulated and one (CD36 antigen) was down-regulated. Although further studies are required, these data suggest that these molecules play an important role in influenza virus infection, especially the phase before specific immunity.     

Asymmetry in the Mitotic Spindle Induced by the Attachment to the Cell Surface during Maturation in the Starfish Oocyte

In order to study the dynamic behavior of the mitotic apparatus leading to unequal cleavage, we investigated the distribution of mitotic microtubules (MTs) during maturation division of starfish oocytes. When the mitotic apparatus attached to the cell surface at metaphase, in both the first and second meiotic division, it is revealed, by immunofluorescence, that the MT distribution in the spindle, as well as in the aster, became asymmetric. MTs in the peripheral half spindle increased in number compared with those in the inner half spindle. Furthermore, these results were confirmed in the living cell by polarization microscopy; shortly after the attachment, the birefringence retardation of the peripheral half spindle became greater than that of the inner one, and the difference increased with time during anaphase. By inhibiting the attachment of the mitotic apparatus by means of centrifugation, the MT distribution maintained a symmetrical pattern through mitosis. These results suggest that the attachment of the mitotic apparatus to the cell surface induces the asymmetrical distribution of MTs not only in the aster but also in the spindle. Such a rich distribution of MTs in the peripheral half spindle appears to ensure chromosome exclusion into the polar body by anchoring them firmly to the cell surface of the animal pole.     

Difference between Maturation Division and Cleavage in Starfish Oocytes: Dependency of Induced Cytokinesis on the Size of the Aster as Revealed by Transplantation of the Centrosome

Using the starfish oocyte and zygote, we investigated the abilities of the centrosome at maturation and cleavage divisions to form the aster and induce cytokinesis, in order to determine differences between these divisions. The transplanted centrosome originated from both maturation and cleavage, induced an additional furrow in cleavage in the recipient cells, but did not induce abnormal polar body formation at maturation. Although it organized an additional aster in the recipient cell in both divisions, a difference in size among asters formed was recognized. Therefore, mitotic asters were stabilized with hexylene glycol in order to measure their radius and clarify this difference. The mean radius (14.4 μm) of the first meiotic aster was significantly smaller than that (20.4 μm) of the aster at the first cleavage. The transplanted cleavage centrosome formed as small an aster as the recipient's own at maturation divisions. When zygotes were briefly treated with colcemid so that the zygotes could not perform cytokinesis but did perform karyokinesis, the size of aster became the same as that in meiosis. These results prove that although any centrosome functions as a microtubule organizing center independent of its origin, the size of the resultant aster decides whether or not cytokinesis would be induced.