Profiles of Physarum Microplasmodial Phosphatase Activity Crucial to Cytoplasmic Streaming and Spherule Formation.

This study aimed to investigate for the first time, the profile of Physarum microplasmodial phosphatase (PPH) activity toward the phosphorylated light chain of Physarum myosin II (PLCM) at pH 7.6, the velocity of cytoplasmic streaming, and PPH expression in spherule formation during dark starvation (DS). In this study, we cloned the full-length cDNA of PPH using polymerase chain reaction, based on the N-terminal amino acid sequence of the purified enzyme. The cDNA contained an open reading frame (ORF) of 1245 bp, corresponding to 415 amino acids. We confirmed that a rapid increase in PPH activity toward PLCM and a rapid decrease in cytoplasmic streaming velocity precede spherule formation by Physarum microplasmodia. The profiles of increase in PPH activity toward PLCM, PPH expression, and PPH accumulation during DS were correlated with spherule formation in the Physarum microplasmodia. Moreover, application of the wheat germ cell-free expression system resulted in the successful production of recombinant PPH and in the expression of phosphatase activity toward PLCM. These results suggest that PPH is involved in the cessation of cytoplasmic streaming in Physarum microplasmodia during DS.

Accumulation of noncrystalline cellulose in Physarum microplasmodia.

Physarum plasmodium lives as a slimy mass of protoplast in the dark fragments into small multinucleated microplasmodia (mPL) in a liquid medium. When mPL are exposed to several unfavorable environments, they transform into "spherules" with a cell wall. Using a synchronous spherule-induction system for mPL, we examined the effect of 2,6-dichlorobenzonitrile on the synthesis of cellulose in mPL, by observing mPL under a fluorescence microscope, and isolated cellulose from mPL to identify them morphologically under scanning electron microscopy. Moreover, we examined in vivo labeling to determine when cellulose synthesis is activated in step 2. We found that the nourishment medium in step 2 was essential for mPL prior to spherulation and that the conversion starts at 48 h in step 2 of our system. From the experiments using Updegraff reagent for the sedimentation of cellulose in the cell wall fraction from mPL, we propose that cellulose produced in mPL is likely noncrystalline cellulose. We conclude that mPL of multinucleated protoplasts without the cell wall structure synthesize cellulose under constitutive condition and accumulate abundantly noncrystalline cellulose, in preparation for unfavorable environments that may occur in the future in which mPL must initiate the program to form the cell wall of spherules.

Acceleration of cell growth by xyloglucan oligosaccharides in suspension-cultured tobacco cells.

The incorporation of xyloglucan oligosaccharide (XXXG) into the walls of suspension-cultured tobacco cells accelerated cell expansion followed by cell division, changed cell shape from cylindrical to spherical, decreased cell size, and caused cell aggregation. Fluorescent XXXG added to the culture medium was found to be incorporated into the surface of the entire wall, where strong incorporation occurred not only on the surface, but also in the interface walls between cells during cell division. Cell expansion was always greater in the transverse direction than in the longitudinal direction and then, immediately, expansion led to cell division in the presence of XXXG; this process might result in the high level of cell aggregation seen in cultured tobacco cells. We concluded that the integration of this oligosaccharide into the walls could accelerate not only cell expansion, but also cell division in cultured cells.

Purification and characterization of the plasmodial phosphatase that hydrolyses the phosphorylated light chain of Physarum myosin II from Physarum polycephalum.

A phosphatase was purified through a combination of ion-exchange and hydrophobic chromatography followed by native PAGE from Physarum plasmodia. Recently, we demonstrated that this phosphatase isoform has a hydrolytic activity towards the PMLC (phosphorylated light chain of Physarum myosin II) at pH 7.6. The apparent molecular mass of the purified enzyme was estimated at approximately 50 kDa by means of analytical gel filtration. The enzyme was purified 340-fold to a final phosphatase activity of 400 pkat/mg of protein. Among the phosphorylated compounds tested for hydrolytic activity at pH 7.6, the enzyme showed no activity towards nucleotides. At pH 7.6, hydrolytic activity of the enzyme against PMLC was detected; at pH 5.0, however, no hydrolytic activity towards PMLC was observed. The Km of the enzyme for PMLC was 10 microM, and the V(max) was 1.17 nkat/mg of protein. Ca(2+) (10 microM) inhibited the activity of the enzyme, and Mg(2+) (8.5 microM) activated the dephosphorylation of PMLC. Mn(2+) (1.6 microM) highly stimulated the enzyme's activity. Based on these results, we concluded that the enzyme is likely to be a phosphatase with hydrolytic activity towards PMLC.

Potential role for purple acid phosphatase in the dephosphorylation of wall proteins in tobacco cells.

It is not yet known whether dephosphorylation of proteins catalyzed by phosphatases occurs in the apoplastic space. In this study, we found that tobacco (Nicotiana tabacum) purple acid phosphatase could dephosphorylate the phosphoryl residues of three apoplastic proteins, two of which were identified as alpha-xylosidase and beta-glucosidase. The dephosphorylation and phosphorylation of recombinant alpha-xylosidase resulted in a decrease and an increase in its activity, respectively, when xyloglucan heptasaccharide was used as a substrate. Attempted overexpression of the tobacco purple acid phosphatase NtPAP12 in tobacco cells not only decreased the activity levels of the glycosidases but also increased levels of xyloglucan oligosaccharides and cello-oligosaccharides in the apoplast during the exponential phase. We suggest that purple acid phosphatase controls the activity of alpha-xylosidase and beta-glucosidase, which are responsible for the degradation of xyloglucan oligosaccharides and cello-oligosaccharides in the cell walls.

Activation of beta-glucan synthases by wall-bound purple acid phosphatase in tobacco cells.

Wall-bound purple acid phosphatases have been shown to be potentially involved in the regulation of plant cell growth. The aim of this work was to further investigate the function of one of these phosphatases in tobacco (Nicotiana tabacum), NtPAP12, using transgenic cells overexpressing the enzyme. The transgenic cells exhibited a higher level of phosphatase activity in their walls. The corresponding protoplasts regenerating a cell wall exhibited a higher rate of beta-glucan synthesis and cellulose deposition was increased in the walls of the transgenic cells. A higher level of plasma membrane glucan synthase activities was also measured in detergent extracts of membrane fractions from the transgenic line, while no activation of Golgi-bound glycan synthases was detected. Enzymatic hydrolysis and methylation analysis were performed on the products synthesized in vitro by the plasma membrane enzymes from the wild-type and transgenic lines extracted with digitonin and incubated with radioactive UDP-glucose. The data showed that the glucans consisted of callose and cellulose and that the amount of each glucan synthesized by the enzyme preparation from the transgenic cells was significantly higher than in the case of the wild-type cells. The demonstration that callose and cellulose synthases are activated in cells overexpressing the wall-bound phosphatase NtPAP12 suggests a regulation of these carbohydrate synthases by a phosphorylation/dephosphorylation process, as well as a role of wall-bound phosphatases in the regulation of cell wall biosynthesis.

Purple acid phosphatase in the walls of tobacco cells.

Purple acid phosphatase isolated from the walls of tobacco cells appears to be a 220kDa homotetramer composed of 60kDa subunits, which is purple in color and which contains iron as its only metal ion. Although the phosphatase did not require dithiothreitol for activity and was not inhibited by phenylarsine oxide, the enzyme showed a higher catalytic efficiency (k(cat)/K(m)) for phosphotyrosine-containing peptides than for other substrates including p-nitrophenyl-phosphate and ATP. The phosphatase formed as a 120kDa dimer in the cytoplasm and as a 220kDa tetramer in the walls, where Brefeldin A blocked its secretion during wall regeneration. According to our double-immunofluorescence labeling results, the enzyme might be translocated through the Golgi apparatus to the walls at the interphase and to the cell plate during cytokinesis.

Identification of an estrogenic hormone receptor in Caenorhabditis elegans.

Changes in both behavior and gene expression occur in Caenorhabditis elegans following exposure to sex hormones such as estrogen and progesterone, and to bisphenol A (BPA), an estrogenic endocrine-disrupting compound. However, only one steroid hormone receptor has been identified. Of the 284 known nuclear hormone receptors (NHRs) in C. elegans, we selected nhr-14, nhr-69, and nhr-121 for analysis as potential estrogenic hormone receptors, because they share sequence similarity with the human estrogen receptor. First, the genes were cloned and expressed in Escherichia coli, and then the affinity of each protein for estrogen was determined using a surface plasmon resonance (SPR) biosensor. All three NHRs bound estrogen in a dose-dependent fashion. To evaluate the specificity of the binding, we performed a solution competition assay using an SPR biosensor. According to our results, only NHR-14 was able to interact with estrogen. Therefore, we next examined whether nhr-14 regulates estrogen signaling in vivo. To investigate whether these interactions actually control the response of C. elegans to hormones, we investigated the expression of vitellogenin, an estrogen responsive gene, in an nhr-14 mutant. Semi-quantitative RT-PCR showed that vitellogenin expression was significantly reduced in the mutant. This suggests that NHR-14 is a C. elegans estrogenic hormone receptor and that it controls gene expression in response to estrogen.

Isolation and characterization of a novel thraustochytrid-like microorganism that efficiently produces docosahexaenoic acid.

A thraustochytrid-like microorganism (strain 12B) was isolated from the mangrove area of Okinawa, Japan. On the basis of its ectoplasmic net structure and biflagellate zoospores we determined strain 12B to be a novel member of the phylum Labyrinthulomycota in the kingdom Protoctista. When grown on glucose/seawater at 28 degrees C, it had a lipid content of 58% with docosahexaenoic acid (DHA; 22:6 n-3) at 43% of the total fatty acids. It had a growth rate of 0.38 h(-1). The DHA production rate of 2.8 +/- 0.7 g l(-1) day(-1) is the highest value reported for any microorganism.

Rho mediates endocytosis of epidermal growth factor receptor through phosphorylation of endophilin A1 by Rho-kinase.

After binding of epidermal growth factor (EGF), the EGF receptor is activated, internalized by endocytosis, and subsequently degraded in the lysosomal pathway. Endocytotic trafficking of the activated EGF receptor is essential for controlling EGF signaling. Upon ligand-induced activation of EGF receptors, Cbl (ubiquitin ligase) binds to the activated receptor and leads to translocation of the CIN85 (Cbl-interacting protein of 85 kDa)/endophilin complex in the vicinity of the activated EGF receptors. Endophilin is known as a key regulator of clathrin-mediated endocytosis, and the translocation of endophilin in the vicinity of active EGF receptor is thought to promote receptor internalization. The constitutively active mutant of small GTPase Rho inhibits EGF receptor endocytosis. In this study, we found that this inhibitory effect was canceled by the dominant negative form of Rho-associated kinase (Rho-kinase), which is an effector of Rho. To clarify the molecular mechanisms of endocytosis downstream of Rho/Rho-kinase signal, we searched for and identified endophilin A1 as a novel substrate of Rho-kinase. We identified the phosphorylation site of endophilin A1 at Thr-14 and made endophilin T14D (substitution of Thr-14 by Asp), which is expected to mimic the phosphorylation state of endophilin A1. Endophilin T14D inhibited EGF receptor internalization. Furthermore, phosphorylation of endophilin by Rho-kinase inhibited the binding to CIN85. Taken together, these results suggest that Rho-kinase phosphorylates endophilin downstream of Rho and regulates EGF receptor endocytosis through the inhibition of binding between endophilin and CIN85.

Rho-kinase and myosin II activities are required for cell type and environment specific migration.

Cell migration is important in the development of atherosclerotic lesions. Macrophages and smooth muscle cells migrate into the subendothelial space of arteries, leading to plaque formation. Long-term inhibition of the activity of Rho-kinase induces a regression of atherosclerotic coronary lesions, probably by preventing migration of macrophages and smooth muscle cells. Previous reports concerning the effect of Rho-kinase inhibitors on cell migration are contradictory, however. We examined here the cell type specificity of Rho-kinase inhibitors and found that migration of endothelial cells, macrophages, and smooth muscle cells was inhibited by treatment with Rho-kinase inhibitors in a dose-dependent fashion in a three-dimensional migration assay, whereas that of fibroblasts and epithelial cells was not inhibited. Myosin II inhibitor prevented cell migration in a manner similar to Rho-kinase inhibitors. In contrast, in a two-dimensional migration assay, cell migration was not inhibited by Rho-kinase or myosin II inhibitors for any of the cell types examined. Taken together, these results indicate that Rho-kinase inhibitors suppress migration of specific cell types under specific conditions through the regulation of myosin II activity. Our findings suggest that Rho-kinase is the therapeutic target of atherosclerosis accompanied with invasion by leukocytes and smooth muscle cells.

Epstein-Barr virus infection of human natural killer cell lines and peripheral blood natural killer cells.

Although considerable part of natural killer (NK) cell neoplasms possess EBV genome, there has been no direct evidence that EBV infects human NK cells in vitro. In this study, we demonstrated EBV entry into NK cells using a recombinant EBV, which contains enhanced green fluorescent protein (EGFP) gene in its genome (EGFP-EBV). After 48 h of exposure to EGFP-EBV, we detected EGFP signals in approximately 30% of NK-92 and NKL cells and >40% of peripheral blood NK cells from three healthy volunteers. Reverse transcription-PCR analysis of various EBV-associated genes confirmed EBV infection. In situ hybridization for EBERs and BHLFs showed that latent and lytic infections coexisted at the early phase of EBV infection in two NK cell lines. Although BHLF-positive cells in the early lytic phase were round-shaped, EBER-positive cells in latent EBV infection tended to show a bizarre shape. Flow cytometric analysis of EGFP-EBV-exposed NK cell lines showed that most of EBV-infected cells entered early apoptosis after 72 h of EBV exposure, which explains the difficulties to establish EBV-carrying NK clones. Flow cytometry and reverse transcription-PCR analysis indicated that two NK cell lines may fuse with EBV using HLA class II after binding to the virus through a distinct molecule from CD21. We established two EBV-carrying NKL clones showing latency types I and II, both of which are recognized in EBV-associated NK cell neoplasms. Because EBV-infected NKL cells showed only type I latency during the early phase of infection, the temporal profile of latent gene expression is similar to that of T cells. We first report in vitro EBV infection of human NK cells and establishment of EBV-carrying NK clones, which should contribute to elucidate the role of EBV in the development of NK cell neoplasms.

Identification of Tau and MAP2 as novel substrates of Rho-kinase and myosin phosphatase.

Rho-kinase and myosin phosphatase are implicated in the phosphorylation-state of myosin light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fibre formation in non-muscle cells. Here, we found that microtubule-associated proteins, Tau and MAP2, interacted with the myosin-binding subunit (MBS) of myosin phosphatase, and were the possible substrates of both Rho-kinase and myosin phosphatase. We determined the phosphorylation sites of Tau (Thr245, Thr377, Ser409) and MAP2 (Ser1796) by Rho-kinase. We also found that Rho-kinase phosphorylated Tau at Ser262 to some extent. Phosphorylation by Rho-kinase decreased the activity of Tau to promote microtubule assembly in vitro. Substitutions of Ala for Ser/Thr at the phosphorylation sites of Tau (Tau-AAA) did not affect the activity to promote microtubule assembly, while substitutions of Asp for Ser/Thr (Tau-DDD), which are expected to mimic the phosphorylation-state of Tau, slightly reduced the activity. When Tau, or mutated forms of Tau, were expressed in PC12 cells, followed by treatment with cytochalasin D, they promoted extension of the cell process in a cytochalasin-dependent manner. However, Tau-DDD showed the weaker activity in this capacity than wild-type Tau or Tau-AAA. These results suggest that the phosphorylation-state of these residues of Tau affects its activity both in vitro and in vivo. Thus, it is likely that the Rho-kinase/MBS pathway regulates not only the actin-myosin system but also microtubule dynamics.

Isolation and characterization of four cell wall purple acid phosphatase genes from tobacco cells.

Four full-length cDNAs were isolated from a cDNA library prepared from tobacco cultured cells and designated NtPAP4, NtPAP12, NtPAP19 and NtPAP21, which could correspond to purple acid phosphatase (PAP). Levels of both NtPAP12 and NtPAP21 mRNA in the protoplasts immediately increased after the protoplasts were transferred to a medium for cell wall regeneration, and the accumulation of the mRNA was correlated with cell wall regeneration for 3 h. It is likely that the NtPAP12 and NtPAP21 gene products are wall-bound PAPs at the early stage of regenerating walls in tobacco protoplasts.

Behavior of phosphatase isoforms during sclerotium formation in Physarum polycephalum.

The behavior of phosphatase isoforms under dark-starvation from plasmodium of Physarum polycephalum were investigated to determine their possible roles in sclerotium formation. Two and a half days after dark-starvation, approximately 95% of plasmodia plates formed sclerotia. Specific phosphatase activity increased markedly up to ca. two-fold within the first day of starvation, after which the enzymatic activity decreased rapidly to a level less than the initial level within 2 days of the starvation period. Among the two isoforms of enzyme detected just before sclerotization under dark-starvation conditions, the enzymatic activity of the major isoform (Rm value of 0.6) decreased gradually within 1.5 days of starvation, then linearly to less than 20% of that at the beginning of the observation. Those of other major isoform (Rm value of 0.7) increased up to ca. two-fold within the first day of starvation, then decreased linearly to levels less than that of the first 2 days of the starvation period. Behavior of this isoform strongly suggests that it initiates the formation of sclerotium under dark-starvation conditions.