Overexpression of VDUP1 mRNA sensitizes HeLa cells to paraquat

5-Bromodeoxyuridine (BrdU) induces or suppresses senescence-associated genes in any types of mammalian cells. From a cDNA library upregulated by BrdU in HeLa cells, we identified the gene encoding VDUP1 as a senescence-associated gene in normal human fibroblasts. To address a role of VDUP1 in senescence, we established HeLa cell clones, V7 and V27, which express its mRNA in a doxycycline-dependent manner. Although their growth in liquid culture was moderately retarded, colony formation on semi-solid medium was strongly inhibited by overexpression of the mRNA. We also examined susceptibility of these clones to various reagents. Consequently, colony formation in liquid culture was strongly inhibited by paraquat in these clones. Their superoxide dismutase activity was normal.     

Extracellular Poly(α-L-guluronate)lyase from Corynebacterium sp.: Purification, Characteristics, and Conformational Properties

Extracellular alginate lyase was purified from the culture supernatant of Corynebacterium sp. isolated from the sewage of a sea tangle processing factory in order to elucidate the structure—function relationship of alginate lyase. The electrophoretically homogeneous enzyme was shown to have a molecular mass of 27 kDa by sodium dodecyl sulfate (SDS)—polyacrylamide gel electrophoresis (PAGE) and by gel filtration, with an isoelectric point of 7.3. The molecular mass from amino acid analysis was 28.644 kDa. The optimal pH and temperature for the enzyme reaction were around 7.0 and 55°C, respectively. Metal compounds such as MnCl₂ and NiCl₂ increased the enzyme activity. The enzyme was identified as the endolytic poly(α-L-guluronate)lyase, which was active on poly(α-L-1,4-guluronate) and caused a rapid decrease in the viscosity of alginate solution. Measurement of the far-UV circular dichroic spectrum of the enzyme molecule gave a spectrum with a deep trough at 215nm accompanied by a shallow one at around 237 nm, and with a high peak at 197 nm and a much lower one at 230 nm. This spectrum was most likely to be that of the β-form of the enzyme molecule and resembled poly(β-D-mannuronate)lyase from Turbo cornutus (wreath shell) and poly(α-L-guluronate)lyase from Vibrio sp. (marine bacterium). The near-UV circular dichroic spectrum was characteristic for aromatic amino acid residues. In the presence of 6 M urea, these spectra changed drastically in the near-UV and a little in the far-UV with the disappearance of the enzyme activity. Removal of the denaturant in the enzyme solution by dialysis restored both the activity and inherent circular dichroic spectra. The β-sheets observed in alginate lyases as the major ordered structure seem to be a common conformation for the lyases.     

Hepatitis C virus core protein activates nuclear factor kappa B-dependent signaling through tumor necrosis factor receptor-associated factor

Hepatitis C virus (HCV) core protein, a viral nucleocapsid, has been shown to affect various intracellular events including the nuclear factor kappaB (NF-kappaB) signaling supposedly associated with inflammatory response, cell proliferation, and apoptosis. In order to elucidate the effect of HCV core protein on the NF-kappaB signaling in HeLa and HepG2 cells, a reporter assay was utilized. HCV core protein significantly activated NF-kappaB signaling in a dose-dependent manner not only in HeLa and HepG2 cells transiently transfected with core protein expression plasmid, but also in HeLa cells induced to express core protein under the control of doxycycline. HCV core protein increased the DNA binding affinity of NF-kappaB in the electrophoretic mobility shift assay. Acetyl salicylic acid, an IKKbeta-specific inhibitor, and dominant negative form of IKKbeta significantly blocked NF-kappaB activation by HCV core protein, suggesting HCV core protein activates the NF-kappaB pathway mainly through IKKbeta. Moreover, the dominant negative forms of TRAF2/6 significantly blocked activation of the pathway by HCV core protein, suggesting HCV core protein mimics proinflammatory cytokine activation of the NF-kappaB pathway through TRAF2/6. In fact, HCV core protein activated interleukin-1beta promoter mainly through NF-kappaB pathway. Therefore, this function of HCV core protein may play an important role in the inflammatory reaction induced by this hepatotropic virus.     

1,1-Diphenyl-2-picrylhydrazyl radical scavenging activity and tyrosinase inhibitory effects of constituents of sugarcane molasses

In the course of our work into the use of cane by-products, we have studied the isolation and structural determination of bioactive compounds in sugarcane molasses. In this study, three stereo isomers of syringyl glycerol 3'-O-beta-D-glucopyranoside, three stereo isomers of guaiacyl glycerol 3'-O-beta-D-glucopyranoside, a syringyl glycerol 2'-O-beta-D-glucopyranoside, tachioside and a 2,3-dihydro-3,5-dihydroxy-6-methyl-4-(H)-pyran-4-one (DDMP) were isolated from the 25% methanol eluate by Amberlite XAD-2 column chromatography of sugarcane molasses. The structures of these compounds were determined on the basis of spectroscopic evidence. These isolated compounds were examined for their scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical species, and for their inhibitory activity against mushroom tyrosinase. All of the isolated compounds showed DPPH radical scavenging activity, while DDMP and tachioside showed mushroom tyrosinase inhibitory activity.     

The nucleotide sequence of the Escherichia coli rts gene

The nucleotide sequence of rts, an essential Escherichia coli gene, has been determined. Transformation of an rts mutant with the plasmid, pJAF1, containing the rts gene resulted in rescue of the defect. The transformation experiments indicate that the rts gene is distinct from the flanking birA, tRNA and tufB genes.     

Role of Hand1/eHAND in the dorso-ventral patterning and interventricular septum formation in the embryonic heart

Molecular mechanisms for the dorso-ventral patterning and interventricular septum formation in the embryonic heart are unknown. To investigate a role of Hand1/eHAND in cardiac chamber formation, we generated Hand1/eHAND knock-in mice where Hand1/eHAND cDNA was placed under the control of the MLC2V promoter. In Hand1/eHAND knock-in mice, the outer curvature of the right and left ventricles expanded more markedly. Moreover, there was no interventricular groove or septum formation, although molecularly, Hand1/eHAND knock-in hearts had two ventricles. However, the morphology of the inner curvature of the ventricles, the atrioventricular canal, and the outflow tract was not affected by Hand1/eHAND expression. Furthermore, expression of Hand1/eHAND in the whole ventricles altered the expression patterns of Chisel, ANF, and Hand2/dHAND but did not affect Tbx5 expression. In contrast, the interventricular septum formed normally in transgenic embryos overexpressing Hand1/eHAND in the right ventricle but not in the boundary region. These results suggested that Hand1/eHAND is involved in expansion of the ventricular walls and that absence of Hand1/eHAND expression in the boundary region between the right and left ventricles may be critical in the proper formation of the interventricular groove and septum. Furthermore, Hand1/eHAND is not a master regulatory gene that specifies the left ventricle myocyte lineage but may control the dorso-ventral patterning in concert with additional genes.     

The Therapeutic Effect on Bone Mineral Formation from Biomimetic Zinc Containing Tricalcium Phosphate (ZnTCP) in Zinc-Deficient Osteoporotic Mice

The aim of this study was to evaluate the therapeutic efficacy of biomimetic zinc-containing tricalcium phosphate (ZnTCP) produced by hydrothermally converting calcium carbonate exoskeletons from foraminifera, in the treatment of osteoporotic mice. X-Ray powder diffraction showed crystallographic structures matching JCPDS profile for tricalcium phosphate. Mass spectroscopy used to calculate total composition amount showed similar amount of calcium (5×10⁴ µg/g) and phosphate (4×10⁴ ppm) after conversion and the presence of zinc (5.18×10³ µg/g). In vitro zinc release showed no release in PBS buffer and <1% zinc release in 7 days. In vivo evaluation was done in ovariectomized mice by implanting the ZnTCP samples in the soft tissues near the right femur bone for four weeks. Thirty ddY mice (5 weeks old, average weight of 21 g) were divided into six experimental groups (normal, sham, OVX, β-TCP, ZnTCP and direct injection of zinc). CT images were taken every two weeks where the bone mineral density (BMD) and bone mineral content (BMC) were calculated by software based on CT images. The ZnTCP group exhibits cortical and cancellous bone growth of 45% and 20% respectively. While sham, OVX and β-TCP suffered from bone loss. A correlation was made between the significant body weight increase in ZnTCP with the significant increase in plasma zinc level compared with OVX. The presented results indicate that biomimetic ZnTCP were effective in preventing and treating bone loss in osteoporotic mice model.     

Cytokeratin 14 is expressed in immature cells in rat taste buds

We analyzed the differentiation of taste bud cells, by precisely describing expression profiles of cytokeratins (CKs) 8 and 14 in relation to those of marker molecules including label of 5-bromo-2′-deoxy uridine (BrdU) injected. In rat circumvallate papillae, cell division was observed at the basal layer of the epithelium expressing CK14 and located outside taste buds. The progenitor cells began to migrate toward the apical surface and maintained CK14 expression at 1 day after BrdU injection (day 1). On the other hand, a minor population of newly divided cells was infrequently incorporated into taste buds and also maintained CK14 expression at day 1. In taste buds, the conversion of CK subtypes occurred from CK14 to cytokeratin 8 (CK8) at day 2–3, showing the differentiation from immature cells expressing CK14 into mature or maturing cells expressing CK8. Functionally matured cells such as taste receptor cells expressing inositol triphospate receptor type 3 (IP₃R3) never expressed CK14, suggesting that CK14 would be expressed only in immature cells. On the other hand, a small but distinct population of BrdU-positive cells still showed CK14 immunoreactivity in taste buds even at day 12, which might correspond to the cells that remain undifferentiated for a long period within taste buds.     

Regulation of Microbe-Associated Molecular Pattern-Induced Hypersensitive Cell Death,Phytoalexin Production,and Defense Gene Expression by Calcineurin B-Like Protein-Interacting Protein Kinases,OsCIPK14/15, in Rice Cultured Cells

Although cytosolic free Ca²⁺ mobilization induced by microbe/pathogen-associated molecular patterns is postulated to play a pivotal role in innate immunity in plants, the molecular links between Ca²⁺ and downstream defense responses still remain largely unknown. Calcineurin B-like proteins (CBLs) act as Ca²⁺ sensors to activate specific protein kinases, CBL-interacting protein kinases (CIPKs). We here identified two CIPKs, OsCIPK14 and OsCIPK15, rapidly induced by microbe-associated molecular patterns, including chitooligosaccharides and xylanase (Trichoderma viride/ethylene-inducing xylanase [TvX/EIX]), in rice (Oryza sativa). Although they are located on different chromosomes, they have over 95% nucleotide sequence identity, including the surrounding genomic region, suggesting that they are duplicated genes. OsCIPK14/15 interacted with several OsCBLs through the FISL/NAF motif in yeast cells and showed the strongest interaction with OsCBL4. The recombinant OsCIPK14/15 proteins showed Mn²⁺-dependent protein kinase activity, which was enhanced both by deletion of their FISL/NAF motifs and by combination with OsCBL4. OsCIPK14/15-RNAi transgenic cell lines showed reduced sensitivity to TvX/EIX for the induction of a wide range of defense responses, including hypersensitive cell death, mitochondrial dysfunction, phytoalexin biosynthesis, and pathogenesis-related gene expression. On the other hand, TvX/EIX-induced cell death was enhanced in OsCIPK15-overexpressing lines. Our results suggest that OsCIPK14/15 play a crucial role in the microbe-associated molecular pattern-induced defense signaling pathway in rice cultured cells.Calcium ions regulate diverse cellular processes in plants as a ubiquitous internal second messenger, conveying signals received at the cell surface to the inside of the cell through spatial and temporal concentration changes that are decoded by an array of Ca²⁺ sensors (Reddy, 2001; Sanders et al., 2002; Yang and Poovaiah, 2003). Several families of Ca²⁺ sensors have been identified in higher plants. The best known are calmodulins (CaMs) and CaM-related proteins, which typically contain four EF-hand domains for Ca²⁺ binding (Zielinski, 1998). Unlike mammals, which possess single molecular species of CaM, plants have at least three distinct molecular species of CaM playing diverse physiological functions and whose expression is differently regulated (Yamakawa et al., 2001; Luan et al., 2002; Karita et al., 2004; Takabatake et al., 2007). The second major class is exemplified by the Ca²⁺-dependent protein kinases, which contain CaM-like Ca²⁺-binding domains and a kinase domain in a single protein (Harmon et al., 2000). In addition, a new family of Ca²⁺ sensors was identified as calcineurin B-like (CBL) proteins, which consists of proteins similar to both the regulatory β-subunit of calcineurin and the neuronal Ca²⁺ sensor in animals (Liu and Zhu, 1998; Kudla et al., 1999).Unlike CaMs, which interact with a large variety of target proteins, CBLs specifically target a family of protein kinases referred to as CBL-interacting protein kinases (CIPKs) or SnRK3s (for sucrose nonfermenting 1-related protein kinases type 3), which are most similar to the SNF family protein kinases in yeast (Luan et al., 2002). A database search of the Arabidopsis (Arabidopsis thaliana) genome sequence revealed 10 CBL and 25 CIPK homologues (Luan et al., 2002). Expression patterns of these Ca²⁺ sensors and protein kinases suggest their diverse functions in different signaling processes, including light, hormone, sugar, and stress responses (Batistic and Kudla, 2004). AtCBL4/Salt Overly Sensitive3 (SOS3) and AtCIPK24/SOS2 have been shown to play a key role in Ca²⁺-mediated salt stress adaptation (Zhu, 2002). The CBL-CIPK system has been shown to be involved in signaling pathways of abscisic acid (Kim et al., 2003a), sugar (Gong et al., 2002a), gibberellins (Hwang et al., 2005), salicylic acid (Mahajan et al., 2006), and K⁺ channel regulation (Li et al., 2006; Lee et al., 2007; for review, see Luan, 2009; Batistic and Kudla, 2009). However, physiological functions of most of the family members still remain largely unknown.Plants respond to pathogen attack by activating a variety of defense responses, including the generation of reactive oxygen species (ROS), synthesis of phytoalexins, expression of pathogenesis-related (PR) genes, cell cycle arrest, and mitochondrial dysfunction followed by a form of hypersensitive cell death known as the hypersensitive response (Nürnberger and Scheel, 2001; Greenberg and Yao, 2004; Kadota et al., 2004b). Transient membrane potential changes and Ca²⁺ influx are involved at the initial stage of defense responses (Kuchitsu et al., 1993; Pugin et al., 1997; Blume et al., 2000; Kadota et al., 2004a). Many kinds of defense responses are prevented when Ca²⁺ influx is compromised by Ca²⁺ chelators (Nürnberger and Scheel, 2001; Lecourieux et al., 2002). Since complex spatiotemporal patterns of cytosolic free Ca²⁺ concentration have been suggested to play pivotal roles in defense signaling (Nürnberger and Scheel, 2001; Sanders et al., 2002), multiple Ca²⁺ sensor proteins and their effectors should function in the defense signaling pathways. Although possible involvement of some CaM isoforms (Heo et al., 1999; Yamakawa et al., 2001), Ca²⁺-dependent protein kinases (Romeis et al., 2000, 2001; Ludwig et al., 2005; Kobayashi et al., 2007; Yoshioka et al., 2009), as well as Ca²⁺ regulation of EF-hand-containing enzymes such as ROS-generating NADPH oxidase (Ogasawara et al., 2008) have been suggested, other Ca²⁺-regulated signaling components still remain to be identified. No CBLs or CIPKs have so far been implicated as signaling components in defense signaling.N-Acetylchitooligosaccharides, chitin fragments, are microbe-associated molecular patterns (MAMPs) that are recognized by plasma membrane receptors (Kaku et al., 2006; Miya et al., 2007) and induce a variety of defense responses, such as membrane depolarization (Kuchitsu et al., 1993; Kikuyama et al., 1997), ion fluxes (Kuchitsu et al., 1997), ROS production (Kuchitsu et al., 1995), phytoalexin biosynthesis (Yamada et al., 1993), and induction of PR genes (Nishizawa et al., 1999), without hypersensitive cell death in rice (Oryza sativa) cells. In contrast, a fungal proteinaceous elicitor, xylanase from Trichoderma viride (TvX)/ethylene-inducing xylanase (EIX), which is recognized by two putative plasma membrane receptors, LeEix1 and LeEix2 (Ron and Avni, 2004), triggers hypersensitive cell death along with different kinetics of ROS production and activation of a mitogen-activated protein kinase, OsMPK6, previously named as OsMPK2 or OsMAPK6, in rice cells (Kurusu et al., 2005). These two fungal MAMPs thus provide excellent model systems to study innate immunity in rice cells.This study identified two CIPKs involved in various MAMP-induced layers of defense responses, including PR gene expression, phytoalexin biosynthesis, mitochondrial dysfunction, and cell death, in rice. Molecular characterization of these CIPKs, including interaction with the putative Ca²⁺ sensors as well as their physiological functions, is discussed.     

Comparative anatomy of root meristem and root cap in some species of Podostemaceae and the evolution of root dorsiventrality

In the unusual aquatic Podostemaceae, the root is the leading organ of the plant body and is variously compressed and submerged as it adheres to rock surfaces in rapid water. In an anatomical comparison of the root apical meristems and root caps of 33 species that represent the major lineages of the family, the dorsiventrality of root meristems varied and was classified into four patterns: (1) The root cap is produced outward from a nearly radially symmetrical meristem. (2) The meristem and root cap are markedly dorsiventral; the outermost cells of the hood-shaped cap are acroscopic derivatives from bifacial initials on the ventral side, while the pattern on the dorsal side is similar to pattern 1. (3) Bifacial initials are on both the dorsal and ventral sides. (4) No root cap is present. An evolutionary polarity may be evident from pattern 1 to 2 and then to 3. Pattern 2 arose in the early evolution of the subfamily Podostemoideae and subsequently, pattern 3 arose in species with crustose roots, while the least specialized pattern 1 is retained in Tristichoideae and Weddellinoideae. Pattern 4 characterized by caplessness may have appeared recurrently in Tristichoideae and Podostemoideae. These evolutionary changes in the meristem preceded the specialization of external root morphologies.