What triggers differential DNA methylation of genes and TEs: contribution of body methylation?

Transposable elements (TEs) are epigenetically silenced with extensive DNA methylation. The silent epigenetic marks should, however, be excluded from active genes. By genetic approaches, we study mechanisms to remove the heterochromatin marks from transcribed genes. Based on our observations on control of TE transcription, we propose a possible trigger for the TE-specific accumulation of DNA methylation. A critical difference between TEs and genes could be their responses to the DNA methylation in the internal part of transcribed regions. When their internal region is methylated, genes are still transcribed, but TEs could be silenced, which may reflect the obligatory position of every critical cis-acting element within the TE itself. This initial difference of TEs and genes will be amplified by positive feedback loops to stabilize active or silent states. Thus, the mechanisms to accumulate heterochromatin marks within transcribed regions could provide a trigger to induce differential DNA methylation between genes and TEs.

Propofol inhibits phorbol 12, 13-dibutyrate-induced, protein kinase C-mediated contraction of rat aortic smooth muscle.

BACKGROUND: Propofol induces dose-dependent vasodilation and hypotension in the clinical situation, and protein kinase C (PKC)-mediated Ca2+ sensitization plays an important role in vascular smooth muscle contraction. This study is designed to examine the effects of propofol on the active phorbol ester (phorbol 12, 13-dibutyrate; PDBu)-induced, PKC-mediated contraction of rat aortic smooth muscle. METHODS: The PDBu-induced contraction of endothelium-denuded rat aortic rings was measured in the presence or absence of PKC inhibitor, bisindolylmaleimide I, or propofol, using isometric force transducers. The PDBu-induced PKC phosphorylation of endothelium-denuded rat aortic strips was detected in the presence or absence of bisindolylmaleimide I or propofol, using Western blotting. RESULTS: PDBu, but not the inactive phorbol ester, 4-alpha-phorbol 12-myristate-13-acetate, dose-dependently induced both a slowly developing sustained contraction and PKC phosphorylation of rat aortic smooth muscle, reaching the peak level at the concentration of 10(-6) M. The PDBu (10(-6) M)-induced contraction was dose-dependently inhibited by bisindolylmaleimide I with reductions of 6.8 +/- 1.8% (P > 0.05), 39.8 +/- 8.7% (P < 0.01) and 96.7 +/- 1.4% (P < 0.01) in response to concentrations of 5 x 10(-7) M, 10(-6)x M and 5 x 10(-6) M, respectively, and by propofol with decreases of 5.2 +/- 1. 6% (P > 0.05), 9.4 +/- 1.7% (P < 0.05), 65.3 +/- 9.2% (P < 0.01) and 96.2 +/- 1.6% (P < 0.01) in response to concentrations of 5 x 10(-7) M, 10(-6) M, 5 x 10(-6) M and 10(-5) M, respectively. Both bisindolylmaleimide I and propofol also inhibited the PDBu-induced increase in the density of the phosphorylated PKC bands in a dose-dependent manner, with decreases of 6.3 +/- 2.8% (P > 0.05), 42.9 +/- 3.2% (P < 0.01) and 96.6 +/- 3.4% (P < 0.01) in response to 5 x 10(-7) M, 10(-6) M or 5 x 10(-6) M bisindolylmaleimide I, respectively, and with decreases of 4.2 +/- 2.5% (P > 0.05), 13.5 +/- 1.7% (P < 0.05), 69.5 +/- 3.5% (P < 0.01) and 95.3 +/- 4.3% (P < 0.01) in response to 5 x 10(-7) M, 10(-6) M, 5 x 10(-6) M and 10(-5) M propofol, respectively. CONCLUSION: Propofol dose-dependently inhibits PDBu-induced, PKC-mediated contraction of rat aortic smooth muscle.

Comparison of the effects of isoflurane and sevoflurane on protein tyrosine phosphorylation-mediated vascular contraction.

BACKGROUND: Isoflurane induces greater effects on vasodilation and decreasing blood pressure than sevoflurane. Tyrosine kinase-catalyzed protein tyrosine phosphorylation plays an important role in regulating vascular smooth muscle contraction. The aim of the present study was to compare the effects of isoflurane and sevoflurane on tyrosine phosphorylation-mediated vascular constriction, by assessing the degree of sodium orthovanadate (Na(3)VO(4), tyrosine phosphatase inhibitor)-induced contraction and protein tyrosine phosphorylation of rat aortic smooth muscle. METHODS: Na(3)VO(4)-induced contraction and protein tyrosine phosphorylation of rat aortic smooth muscle were measured in the presence of genistein, a tyrosine kinase inhibitor, and different concentrations of isoflurane and sevoflurane, using isometric force measurement and Western blot, respectively. RESULTS: Na(3)VO(4) (10(-4) M) induced sustained contraction and tyrosine phosphorylation of substrates that were both markedly attenuated in the presence of genistein (5 x 10(-5) M). Isoflurane and sevoflurane dose-dependently (1, 2, 3 MAC) attenuated the Na(3)VO(4)-induced contraction (P < 0.05-0.005, n = 8), with a greater degree of inhibition by isoflurane than sevoflurane at 2 MAC (P < 0.01) and 3 MAC (P < 0.05). Both anesthetics also attenuated the total band density of the Na(3)VO(4)-induced, tyrosine-phosphorylated substrates in a concentration-dependent manner (P < 0.05-0.005, n = 4), with much greater attenuation by isoflurane than sevoflurane at 1 and 2 MAC (P < 0.05), respectively. CONCLUSION: The results of the present study demonstrate that isoflurane exhibits a greater degree of inhibition on the Na(3)VO(4)-stimulated contraction and protein tyrosine phosphorylation of rat aortic smooth muscle compared with sevoflurane. These findings suggest that isoflurane depresses the protein tyrosine phosphorylation-mediated contraction of vascular smooth muscle to a greater degree than sevoflurane.

Genomic localization of endogenous mobile CACTA family transposons in natural variants of Arabidopsis thaliana.

The differentiation between gene-rich and transposon-rich (gene-poor) regions is a common feature of plant genomes. This may be due to preferential integration of transposons into gene-poor regions or may be due to purifying selection against transposon insertion into gene-rich regions. We examined the distribution of a low-copy-number mobile subfamily of Arabidopsis CACTA transposons in the genomes of 19 natural variants (ecotypes) of A. thaliana, and compared that to the pattern of integrations induced in the laboratory by mutation of the DDM1 (Decrease in DNA Methylation) gene. Sequences similar to mobile CACTA1 copies were distributed among the ecotypes and showed high degrees of polymorphism in genomic localization. Despite the high level of polymorphism, the copy number was low in all the ecotypes examined, and the elements were localized preferentially in pericentromeric and transposon-rich regions. This contrasts with the pattern of transposition induced by the ddm1 mutation, in which the range of integration sites is less biased and the copy number frequently increases. Based on these observations, we discuss the possible contribution of natural selection and chromatin structure to the distribution of transposons.

Linear DNA intermediates of the Tto1 retrotransposon in Gag particles accumulated in stressed tobacco and Arabidopsis thaliana.

The active transcription of some plant retrotransposons under diverse stress conditions suggests active transposition. However, transposition has been demonstrated only during tissue/cell culture. To examine whether transposition is activated under conditions other than tissue/cell culture, DNA intermediates for retrotransposition of the tobacco retrotransposon Tto1 were analysed. Using transgenic Arabidopsis callus expressing high levels of Tto1 RNA in a ddm1 hypomethylation mutant background, the existence of extrachromosomal Tto1 linear DNA molecules in a Gag-particle fraction was demonstrated. By combination with ligation-mediated PCR amplification, we detected Tto1 linear DNA molecules in particle fractions from callus and methyl jasmonate-treated leaves of tobacco, but not from non-stressed leaves. Tto1 DNA intermediates could not be detected in the tobacco corolla where Tto1 is expressed. These results indicate that the transcriptional activation of Tto1 by defence-related stresses leads to the synthesis of DNA intermediates, whereas post-transcriptional suppression of Tto1 activity is suggested in the corolla.

Mobilization of transposons by a mutation abolishing full DNA methylation in Arabidopsis.

A major component of the large genomes of higher plants and vertebrates comprises transposable elements and their derivatives, which potentially reduce the stability of the genome. It has been proposed that methylation of cytosine residues may suppress transposition, but experimental evidence for this has been limited. Reduced methylation of repeat sequences results from mutations in the Arabidopsis gene DDM1 (decrease in DNA methylation), which encodes a protein similar to the chromatin-remodelling factor SWI2/SNF2 (ref. 7). In the ddm1-induced hypomethylation background, silent repeat sequences are often reactivated transcriptionally, but no transposition of endogenous elements has been observed. A striking feature of the ddm1 mutation is that it induces developmental abnormalities by causing heritable changes in other loci. Here we report that one of the ddm1-induced abnormalities is caused by insertion of CAC1, an endogenous CACTA family transposon. This class of Arabidopsis elements transposes and increases in copy number at high frequencies specifically in the ddm1 hypomethylation background. Thus the DDM1 gene not only epigenetically ensures proper gene expression, but also stabilizes transposon behaviour, possibly through chromatin remodelling or DNA methylation.

Antigen-specific T(h)1 cells as direct effectors of Propionibacterium acnes-primed lipopolysaccharide-induced hepatic injury.

T(h)1 cells are cytotoxic effector cells that utilize Fas ligand (FasL) and tumor necrosis factor. The physiological roles of cytotoxic T(h)1 cells are considered to be immunoregulation by eliminating autoreactive lymphocytes or hyper-activated foreign antigen-specific lymphocytes. Their pathological roles, however, remain to be clarified. To investigate whether T(h)1 cells can destroy organs, we generated a Propionibacterium acnes-specific T(h)1 clone from C57BL/6 mice and tested whether the clone could serve as an effector in a P. acnes-primed lipopolysaccharide (LPS)-induced hepatic injury system, one of the septic shock models. B6SMN:C3H-FasL(gld) (B6-gld) mice, which were deficient in functional FasL, were resistant to P. acnes/LPS-induced hepatic shock. The T(h)1 clone rendered B6-gld mice sensitive to the hepatic shock after the i.v. transfer. The hepatic injury in the clone-transferred B6-gld mice, which was evaluated by both biochemical and histological examination, was inhibited by an anti-FasL mAb that we developed. These results suggested that bacterial antigen-specific T(h)1 cells like this clone can participate in organ destruction in vivo as one of the cytotoxic effectors and play a critical role in endotoxin-induced hepatic injury.

Epigenetic developmental mechanisms in plants: molecules and targets of plant epigenetic regulation.

Genetic approaches to understanding the role of epigenetic regulation of gene expression in plants and its mechanisms have revealed several new components. Rapidly accumulating information from other eukaryotes provides complementary knowledge with important implications for plant research. Comparison of epigenetic events across species is proving critical for defining the mechanisms and functions of epigenetic modification, including those specific to plants.

The role of oxygen-derived free radicals in augmented relaxations to levcromakalim in the aorta from hypertensive rats.

Hydrogen peroxide and peroxynitrite induce relaxations via ATP-sensitive K+ channels, indicating that oxygen-derived free radicals may activate these channels. Levels of free radicals are increased throughout the arterial wall in animal models of atherosclerosis, and therefore, vasorelaxation via ATP-sensitive K+ channels may be augmented in chronic hypertension. The present study was designed to determine whether relaxations to an ATP-sensitive K+ channel opener, levcromakalim, are increased in the aorta from spontaneously hypertensive rats (SHR) and whether free radical scavengers reduce these relaxations. Rings of aortas without endothelium taken from age-matched Wistar-Kyoto rats (WKY) and SHR were suspended for isometric force recording. Relaxations to levcromakalim (10(-8) to 10(-5) M), which are abolished by glibenclamide (10(-5) M), were augmented in the aorta from SHR, compared to those in the aorta from WKY. In the aorta from SHR, catalase (1200 U/ml), but neither superoxide dismutase (150 U/ml) nor deferoxamine (10(-4) M), reduced relaxations to levcromakalim, whereas in the aorta from WKY, the free radical scavengers did not affect these relaxations. These results suggest that in chronic hypertension, vasorelaxation to an ATP-sensitive K+ channel opener is augmented and that hydrogen peroxide produced in smooth muscle cells may partly contribute to these relaxations.

Alterations in Ca2+-binding on plasma membrane after adaptation to salt stress of tobacco cells in suspension.

Electrophoresis was used to study effects of salinity on the characteristics of Ca2+ binding to the outer surface of plasma membrane (PM) of protoplasts isolated from two types of tobacco (Nicotiana tabacum L., cv. Bright Yellow) cultured cells that were adapted (tolerant) and unadapted (sensitive) to 50 mM NaCl stress. Electrophoretic analysis of salt-sensitive NaCl-unadapted cells shows that Na+ induced an appreciably higher degree of reduction in the amount of Ca2+ bound to PM compared with K+ with increasing concentration from 0.1 to 30 mM. In salt-tolerant NaCl-adapted cells, however, both Na+ and K+ ions induced almost the same degree of reduction in the amount of Ca2+ bound to PM in the physiological concentration range of Ca2+ in the medium between 2 and 4 mM. These results suggest that, under the physiological conditions, PM of salt-sensitive NaCl-unadapted cells has an appreciable amount of PM-bound Ca2+ that is desorbed much easier by Na+ than K+, whereas PM of salt-tolerant NaCl-adapted cells has the PM-bound Ca2+ that can be equally desorbed by Na+ and K+.

The late flowering phenotype of fwa mutants is caused by gain-of-function epigenetic alleles of a homeodomain gene.

The transition to flowering in Arabidopsis thaliana is delayed in fwa mutant plants. FWA was identified by loss-of-function mutations in normally flowering revertants of the fwa mutant, and it encodes a homeodomain-containing transcription factor. The DNA sequence of wild-type and fwa mutant alleles was identical in the genomic region of FWA. Furthermore, the FWA gene is ectopically expressed in fwa mutants and silenced in mature wild-type plants. This silencing is associated with extensive methylation of two direct repeats in the 5' region of the gene. The late flowering phenotype, ectopic FWA expression, and hypomethylation of the repeats were also induced in the ddm1 hypomethylated background. Mechanisms for establishment and maintenance of the epigenetic mark on FWA are discussed.

Silencing of retrotransposons in arabidopsis and reactivation by the ddm1 mutation.

Gene silencing associated with repeated DNA sequences has been reported for many eukaryotes, including plants. However, its biological significance remains to be determined. One important function that has been proposed is the suppression of transposons. Here, we address transposon suppression by examining the behavior of the tobacco retrotransposon Tto1 and endogenous retrotransposons in Arabidopsis. After an initial increase in copy number because of active transposition in the Arabidopsis genome, Tto1 became silent. The amount of transcript was reduced, and the inactivated Tto1 became methylated. This silencing correlated with an increase in copy number. These phenomena mimic repeat-induced gene silencing. The homozygous ddm1 (for decrease in DNA methylation) mutation of Arabidopsis results in genomic DNA hypomethylation and the release of silencing in repeated genes. To investigate the role of DNA methylation and the gene-silencing machinery in the suppression of Tto1, we introduced the ddm1 mutation into an Arabidopsis line carrying inactivated Tto1 copies. In the homozygous ddm1 background, Tto1 became hypomethylated and transcriptionally and transpositionally active. In addition, one of the newly isolated endogenous Arabidopsis retrotransposon families, named Tar17, also became hypomethylated and transcriptionally active in the ddm1 mutant background. Our results suggest that the inactivation of retrotransposons and the silencing of repeated genes have mechanisms in common.

Lymphotoxin inhibits Chlamydia pneumoniae growth in HEp-2 cells.

Cytokines such as gamma interferon and tumor necrosis factor alpha (TNF-alpha) inhibit the intracellular replication of Chlamydia pneumoniae or Chlamydia trachomatis. In this study, we found that another cytokine, lymphotoxin (TNF-beta), restricts the growth of C. pneumoniae in HEp-2 cells. When lymphotoxin (10 U/ml) was added during incubation from 8 to 16 h postinoculation, inclusion body formation was severely reduced. In addition, we observed activation of nitric oxide production and the nuclear transition of NF-kappaB in HEp-2 cells in response to lymphotoxin. These results suggest that inhibition of chlamydial growth by lymphotoxin is mediated, at least in part, by nuclear transition of NF-kappaB, resulting in induction of nitric oxide synthase to produce nitric oxide, a potent bacteristatic agent. This is the first report on antichlamydial activity of lymphotoxin through induction of nitric oxide.

Meiotically and mitotically stable inheritance of DNA hypomethylation induced by ddm1 mutation of Arabidopsis thaliana.

In contrast to mammalian epigenetic phenomena, where resetting of gene expression generally occurs in each generation, epigenetic states of plant genes are often stably transmitted through generations. The Arabidopsis mutation ddm1 causes a 70% reduction in genomic 5-methylcytosine level. We have previously shown that the ddm1 mutation results in an accumulation of a variety of developmental abnormalities by slowly inducing heritable changes in other loci. Each of the examined ddm1-induced developmental abnormalities is stably transmitted even when segregated from the potentiating ddm1 mutation. Here, the inheritance of DNA hypomethylation induced by ddm1 was examined in outcross progeny by HPLC and Southern analyses. The results indicate that (i) DDM1 gene function is not necessary during the gametophyte stage, (ii) ddm1 mutation is completely recessive, and (iii) remethylation of sequences hypomethylated by the ddm1 mutation is extremely slow or nonexistent even in wild-type DDM1 backgrounds. The stable transmission of DNA methylation status may be related to the meiotic heritability of the ddm1-induced developmental abnormalities.

A novel chemical delivery system for brain targeting.

Two different chemical approaches for brain drug delivery and targeting are proposed in the present review. One is a chemical drug delivery using a ring-closure reaction to the hydrophilic quaternary thiazolium compound in the brain. The other is a chemical drug targeting utilizing the nutrient receptor (transporter) system on the blood-brain barrier. The brain delivery system has been optimized and it was demonstrated that the brain delivery of three drugs, a drug for Parkinson's disease, an excitatory amino acid antagonist and a free radical scavenger, were improved by the conjugation with cis-2-formylaminoethenylthio derivatives in vivo. As for the brain targeting system, it was demonstrated that the conjugation with L-Glu improved the drug's brain distribution via the L-Glu excitatory and/or transport receptors in vitro and in vivo. These findings suggest that the concepts of two chemical approaches will contribute to the development of new central nervous system drugs.

The role of endothelium-derived nitric oxide in relaxations to levcromakalim in the rat aorta.

The present study was designed to examine the role of basally released nitric oxide in relaxations to an ATP-sensitive K+ channel opener. Whether relaxations to levcromakalim are modulated by endothelial removal or the inhibitors of vasodilator effects of endothelium-derived nitric oxide, were investigated in the rat aorta. During contractions to phenylephrine (3 x 10(-7) to 10(-6) M), levcromakalim (10(-8) to 10(-5) M) or a nitric oxide donor, 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC-7, 10(-9) to 10(-5) M), was added in a cumulative fashion. Relaxations to levcromakalim (10(-8) to 10(-5) M) were significantly reduced by the endothelium-removal. In aortas with endothelium, relaxations in response to levcromakalim were decreased by selective inhibitors of nitric oxide synthase (N(G)-nitro-L-arginine methyl ester, 10(-4) M) and soluble guanylate cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one; ODQ, 10(-5) M) and a scavenger of nitric oxide (carboxy-PTIO, 10(-3) M). Relaxations to levcromakalim in aortas treated with these inhibitors are comparable to those seen in aortas without endothelium. KCl (30 mM) and an ATP-sensitive K+ channel inhibitor, glibenclamide (10(-5) M), abolished relaxations to levcromakalim in aortas with or without endothelium, whereas glibenclamide did not alter relaxations to NOC-7 (10(-9) to 10(-5) M) in aortas without endothelium. These results suggest that in rat aortas, inhibition of vasodilator effects of basally released nitric oxide can reduce relaxations via ATP-sensitive K+ channels, although these channels do not mediate relaxations to exogenously applied nitric oxide.

Pharmacokinetic characteristics and therapeutic effects of mitomycin C-dextran conjugates after intratumoural injection.

The pharmacokinetics and therapeutic effects of macromolecular prodrugs of mitomycin C (MMC), MMC-dextran conjugates (MMC-D) were studied after intratumoural injection in rats bearing Walker 256 carcinosarcoma. As the first step, the intratumoural disposition characteristics of these drugs were delineated in perfusion experiments employing a tissue-isolated tumour preparation. While MMC immediately disappeared from the tumour preparation following direct intratumoural injection, cationic and anionic MMC-D were retained in the tumour longer, demonstrating that the intratumoural clearance of MMC can be greatly retarded by dextran conjugation. The effect was more pronounced in the case of the cationic conjugate. Venous outflow data in the perfusion experiments were analyzed based on a compartment model in which the tumour tissue was assumed to consist of two compartments, one well- and the other poorly-perfused. The pharmacokinetic analysis revealed that macromolecular conjugation reduced elimination of MMC from the poorly-perfused region rather than well-perfused region. Simulation of conjugated and free MMC levels in the tissue using the calculated parameters clearly showed that intratumoural injection of MMC-D, especially the cationic form, can maintain a certain level of active free MMC in the tissue for a much longer time period. The long retention of cationic MMC-D in tumour after intratumoural injection was also confirmed by an in vivo pharmacokinetic study and whole body autoradiography in rats bearing subcutaneous Walker 256 carcinosarcoma. In addition, superior antitumour activity of cationic MMC-D was observed against subcutaneous tumours after intratumoural injection. Together with the finding that MMC is selectively toxic to hypoxic tumour cells at low concentrations, these pharmacokinetic studies strongly support the therapeutic efficacy of the macromolecular prodrugs.

Activation of c-jun and c-fos genes in dNTP imbalance cell death induced with 5-fluoro-2'-deoxyuridine in mouse mammary tumor FM3A cell line.

5-Fluoro-2' -deoxyuridine (FdUrd)-induced death of mouse mammary FM3A cells was found to be associated with an increased expression of cellular c-jun and c-fos genes. The increase in these gene expressions was mediated through the protein kinase C-dependent pathway. Blockage of the expressions with the use of antisense oligodeoxynucleotide for c-jun delayed the cell death. These findings suggest that the activation of c-jun and c-fos genes, which encode transcription factors participating in cell proliferation, plays a role in FdUrd-induced cell death.

Different modes of cell death induced by 5-fluoro-2'-deoxyuridine in two clones of the mouse mammary tumor FM3A cell line.

The mode of cell death induced by 1 microM 5-fluoro-2'-deoxyuridine (FdUrd) changed in a wild-type F28-7 clone of mouse mammary tumor FM3A cells after a six-month culture. In the original stocked F28-7 clone, FdUrd-induced cell death was accompanied by necrosis-like cell swelling and DNA fragmentation to 100-200 kbp. In subclone F28-7-A isolated from F28-7 cells, which had been cultured for six months, apoptotic bodies and nucleosomal DNA-ladder fragments were observed with the treatment. Furthermore, we investigated the differences in FdUrd-induced intracellular signals between these clones. In F28-7 cells, FdUrd induced increases in caspase-3-like activity, and the mRNA levels of the c-jun, c-fos and c-myc genes, which were greater and earlier than those in F28-7-A cells. Moreover, intracellular acidification occurred in F28-7-A cells treated with FdUrd, though it was not observed in F28-7 cells. These findings suggest that FdUrd-induced cell death occurred through the death program to cell lysis (necrosis) without apoptosis when the induction of these intracellular signals was very high and when intracellular acidification was deficient. Investigation of the differences in the mode of FdUrd-induced cell death between these clones would be important for elucidating the molecular mechanism of pivotal events guiding cells toward either apoptosis or necrosis.