Interaction between methyl CpG-binding protein and ran GTPase during cell division in tobacco cultured cells

BACKGROUND AND AIMS: Methyl CpG-binding proteins are considered to play critical roles in epigenetic control of gene expression by recognizing and interacting with 5-methylcytosine (m(5)C) in eukaryotes. However, among 13 corresponding genes in Arabidopsis thaliana, designated as featuring a methyl-binding domain (MBD), only four have so far been shown actually to bind to m(5)C. One example, AtMBD5, was selected here to screen for interacting proteins. METHODS: Yeast two-hybrid assays were used for screening, and physical interaction was confirmed by pull-down and bimolecular fluorescence complementation (BiFC) assays. Cellular localization was analysed by fluorescence-tagged fusion proteins using tobacco (Nicotiana tabacum) cultured bright yellow 2 cells. KEY RESULTS: A gene finally identified was found to encode AtRAN3, a protein that belongs to the Ran GTPase family, which plays a critical role in nucleocytoplasmic transport and spindle bipolarization during cell division. AtMBD5 and AtRAN3 were clearly shown to interact in the nucleus by BiFC. On co-expression of AtMBD5-cyan fluorescence protein and yellow fluorescence protein-AtRAN3 in tobacco cells, both localized to the nucleus in the resting stage, migrating to the cytoplasm, primarily around chromatin, during mitosis, particularly at metaphase. CONCLUSIONS: These results suggest that AtMBD5 becomes localized to the vicinity of chromosomes with the aid of AtRAN3 during cell division, and may play an important role not only in maintenance of chromatin structures by binding to m(5)C, but also in progress through mitosis by detaching from m(5)C. The present findings also shed light on the physiological function of Ran GTPases, direct target proteins of which have not thus far been well defined, suggesting their key role in chromatin movements in plant cells.     

A CPG component LE generates depolarization of buccal neurons by producing constant plateau potentials during feeding responses of Aplysia kurodai

In the buccal ganglia of Aplysia kurodai we have identified neurons (here termed LE neurons, or LE) producing plateau potentials lasting several seconds by application of short depolarizing currents. Results obtained from experiments using various bath solutions suggest that generation of these plateau potentials may be an endogenous property of LE. Application of various intensities or lengths of depolarizing currents induced in LE almost constant plateau potentials with fixed duration and depolarizing size. LE spikes produced monosynaptic EPSPs in the ipsilateral multi-action neuron (MA) and the jaw-closing motor neuron (JC) in the buccal ganglia. Conversely, MA spikes produced monosynaptic IPSPs in LE. There was electrical coupling between LE and both MA and JC. During the feeding-like response elicited by electrical stimulation of the nerve, LE showed rhythmic depolarization almost simultaneously with MA and JC, and firing on the plateau potentials occurred during the period of JC firing, the later phase of radula retraction. Hyperpolarization of LE during the feeding-like response suppressed generation of plateau potentials, though rhythmic small depolarization was still induced. During LE hyperpolarization, the duration of the depolarization of MA and JC was shortened. These results suggest that LE may be an element of the feeding CPG circuit and may contribute to part of the depolarization of MA and JC by generating constant plateau potentials during the feeding response, though LE may not have rhythm-generating ability.     

Crystallographic evidence that the dinuclear copper center of tyrosinase is flexible during catalysis

At high resolution, we determined the crystal structures of copper-bound and metal-free tyrosinase in a complex with ORF378 designated as a "caddie" protein because it assists with transportation of two CuII ions into the tyrosinase catalytic center. These structures suggest that the caddie protein covers the hydrophobic molecular surface of tyrosinase and interferes with the binding of a substrate tyrosine to the catalytic site of tyrosinase. The caddie protein, which consists of one six-strandedbeta-sheet and one alpha-helix, has no similarity with all proteins deposited into the Protein Data Bank. Although tyrosinase and catechol oxidase are classified into the type 3 copper protein family, the latter enzyme lacks monooxygenase activity. The difference in catalytic activity is based on the structural observations that a large vacant space is present just above the active center of tyrosinase and that one of the six His ligands for the two copper ions is highly flexible. These structural characteristics of tyrosinase suggest that, in the reaction that catalyzes the ortho-hydroxylation of monophenol, one of the two Cu(II) ions is coordinated by the peroxide-originated oxygen bound to the substrate. Our crystallographic study shows evidence that the tyrosinase active center formed by dinuclear coppers is flexible during catalysis.     

Superoxide dismutase activity of Helicobacter pylori per se from 158 clinical isolates and the characteristics

We investigated the correlation between the SOD activity of Helicobacter pylori (H. pylori) and gastroduodenal diseases and the characteristics of strains exposed to oxidative stress. Two sequenced strains, 26695 and J99, and clinical isolates from 156 Japanese patients with gastroduodenal diseases such as gastric cancer (n= 59) and non-cancer (n= 97) were used. SOD activities of all 158 isolates were measured and were divided into three groups: high-SOD activity (>0.22, n= 2), moderate-SOD activity (0.15≦≦0.22, n= 16) and low-SOD activity (<0.15, n= 140). Expressions of H. pylori Fe-SOD were examined by western blotting with anti-H. pylori Fe-SOD antibody prepared inhouse, and the profiles of Fe-SOD activity were investigated by zymogram with activity staining in native-PAGE. The characteristics of strains from high-SOD and low-SOD groups were examined under oxidative stress by paraquat. The average of H. pylori SOD activity was significantly higher in the cancer group than in the non-cancer group (P < 0.05). However, irrespective of SOD activity level, the amount of Fe-SOD expressed was variable among individual strains. Zymogram revealed a single band in moderate-SOD and low-SOD strains, but multiple bands in high-SOD strains were observed. These bands were confirmed as H. pylori Fe-SOD. Under oxidative stress with paraquat, low-SOD strains were drastically eliminated without inducible SOD activity; however, high-SOD strains were still viable with increased SOD activity. This study is the first to exhibit the characteristics of high-SOD activity strains representing multiple bands in zymograms and the correlation between H. pylori SOD activity and gastric cancer.     

PDGF enhances store-operated Ca2+ entry by upregulating STIM1/Orai1 via activation of Akt/mTOR in human pulmonary arterial smooth muscle cells

Platelet-derived growth factor (PDGF) and its receptor are known to be substantially elevated in lung tissues and pulmonary arterial smooth muscle cells (PASMC) isolated from patients and animals with pulmonary arterial hypertension. PDGF has been shown to phosphorylate and activate Akt and mammalian target of rapamycin (mTOR) in PASMC. In this study, we investigated the role of PDGF-mediated activation of Akt signaling in the regulation of cytosolic Ca(2+) concentration and cell proliferation. PDGF activated the Akt/mTOR pathway and, subsequently, enhanced store-operated Ca(2+) entry (SOCE) and cell proliferation in human PASMC. Inhibition of Akt attenuated the increase in cytosolic Ca(2+) concentration due to both SOCE and PASMC proliferation. This effect correlated with a significant downregulation of stromal interacting molecule (STIM) and Orai, proposed molecular correlates for SOCE in many cell types. The data from this study present a novel pathway for the regulation of Ca(2+) signaling and PASMC proliferation involving activation of Akt in response to upregulated expression of PDGF. Targeting this pathway may lead to the development of a novel therapeutic option for the treatment of pulmonary arterial hypertension.     

Enantiomeric resolution of p-toluenesulfonate of valine benzyl ester by preferential crystallizaion

Preferential crystallization of amino acid derivatives by seeding a pure enantiomer into racemic amino acid solutions has been studied for many years. However, few examples of valine derivatives have been reported so far. Although there have been some reports using valine hydrogen chloride with preferential crystallization, it is difficult to obtain optical isomers for valine derivatives using preferential crystallization. In this study, repeated preferential crystallization of p-toluenesulfonate valine benzyl ester with a 20% e.e. in 2-propanol gave a 94% e.e. on sonication. Sonication accelerated crystallization rate, but there was not a big difference in e.e. between with and without sonication. However, this research demonstrates the first preferential crystallization of p-toluenesulfonate of valine benzyl esters with an acceleration of crystallization using sonication.     

NMR study of xenotropic murine leukemia virus-related virus protease in a complex with amprenavir

Liver steatosis was once believed to be a benign condition, with rare progression to chronic liver disease. Thus, in both clinical and experimental practice, it is fundamental to have a reliable and objective method for its precise quantification. An image analysis algorithm was developed and validated for automatically and rapidly quantifying hepatic fat microvesicles. The image processing algorithms automatically segmented interstitial steatosis areas and analyzed the threshold region. Automatic quantifications did not significantly differ from manual evaluations of means of the same areas. Comparison of our image analysis quantifications with staging of histologic evaluations of liver steatosis presented significant correlations that are based on the distribution patterns and on the area quantity of steatosis, respectively. The use of algorithms for analysis and image processing is a sensitive, precise, objective and reproducible method of quantifying hepatic fat microvesicles, which complements semi-quantitative histologic evaluation systems.     

Cell cycle regulation in mouse heart during embryonic and postnatal stages

The regulation of cardiomyocyte proliferation is important for heart development and function. Proliferation levels of mouse cardiomyocytes are high during early embryogenesis and start to decrease at midgestation. Many cardiomyocytes undergo mitosis without cytokinesis, resulting in binucleated cardiomyocytes during early postnatal stages, following which the cell cycle arrests irreversibly. It remains unknown how the proliferation pattern is regulated, and how the irreversible cell cycle arrest occurs. To clarify the mechanisms, fundamental information about cell cycle regulators in cardiomyocytes and cell cycle patterns during embryonic and postnatal stages is necessary. Here, we show that the expression, complex formation, and activity of main cyclins and cyclin‐dependent kinases (CDKs) changed in a synchronous manner during embryonic and postnatal stages. These levels decreased from midgestation to birth, and then showed one wave in which the peak was around postnatal day 5. Detailed analysis of the complexes suggested that CDK activities were inhibited before the protein levels decreased. Analysis of DNA content distribution patterns in mono‐ and binucleated cardiomyocytes after birth revealed changes in cell cycle distribution patterns and the transition from mono‐ to binucleated cells. These analyses indicated that the wave of cell cycle regulator expression or activities during postnatal stages mainly produced binucleated cells from mononucleated cells. The data obtained should provide a basis for the analysis of cell cycle regulation in cardiomyocytes during embryonic and postnatal stages.     

Fast microscopical dissection of action scenes played by Escherichia coli RNA polymerase

Using fast-scanning atomic force microscopy, we directly visualized the interaction of Escherichia coli RNA polymerase (RNAP) with DNA at the scan rate of 1-2 frames per second. The analyses showed that the RNAP can locate the promoter region not only by sliding but also by hopping and/or segmental transfer. Upon the addition of 0.05 mM NTPs to the stalled complex, the RNAP molecule pulled the template DNA uni-directionally at the rates of 15 nucleotides/s on average. The present method is potentially applicable to examine a variety of protein-nucleic acid interactions, especially those involved in the process of gene regulation.