Showing papers in "Genes to Cells in 2011"

Molecular mechanisms of the Keap1–Nrf2 pathway in stress response and cancer evolution.

Abstract: The Keap1–Nrf2 regulatory pathway plays a central role in the protection of cells against oxidative and xenobiotic damage. Under unstressed conditions, Nrf2 is constantly ubiquitinated by the Cul3–Keap1 ubiquitin E3 ligase complex and rapidly degraded in proteasomes. Upon exposure to electrophilic and oxidative stresses, reactive cysteine residues of Keap1 become modified, leading to a decline in the E3 ligase activity, stabilization of Nrf2 and robust induction of a battery of cytoprotective genes. Biochemical and structural analyses have revealed that the intact Keap1 homodimer forms a cherry-bob structure in which one molecule of Nrf2 associates with two molecules of Keap1 by using two binding sites within the Neh2 domain of Nrf2. This two-site binding appears critical for Nrf2 ubiquitination. In many human cancers, missense mutations in KEAP1 and NRF2 genes have been identified. These mutations disrupt the Keap1–Nrf2 complex activity involved in ubiquitination and degradation of Nrf2 and result in constitutive activation of Nrf2. Elevated expression of Nrf2 target genes confers advantages in terms of stress resistance and cell proliferation in normal and cancer cells. Discovery and development of selective Nrf2 inhibitors should make a critical contribution to improved cancer therapy.

Structural basis for improved efficacy of therapeutic antibodies on defucosylation of their Fc glycans.

Abstract: Removal of the fucose residue from the N-glycans of the Fc portion of immunoglobulin G (IgG) results in a dramatic enhancement of antibody-dependent cellular cytotoxicity (ADCC) through improved affinity for Fcγ receptor IIIa (FcγRIIIa). Here, we present the 2.2-A structure of the complex formed between nonfucosylated IgG1-Fc and a soluble form of FcγRIIIa (sFcγRIIIa) with two N-glycosylation sites. The crystal structure shows that one of the two N-glycans of sFcγRIIIa mediates the interaction with nonfucosylated Fc, thereby stabilizing the complex. However, fucosylation of the Fc N-glycans inhibits this interaction, because of steric hindrance, and furthermore, negatively affects the dynamics of the receptor binding site. Our results offer a structural basis for improvement in ADCC of therapeutic antibodies by defucosylation.

SIRT2 down-regulation in HeLa can induce p53 accumulation via p38 MAPK activation-dependent p300 decrease, eventually leading to apoptosis

Abstract: We previously reported that sirtuin 2 (SIRT2), a mammalian member of the NAD+-dependent protein deacetylases, participates in mitotic regulation, specifically, in efficient mitotic cell death caused by the spindle checkpoint. Here, we describe a novel function of SIRT2 that is different from mitotic regulation. SIRT2 down-regulation using siRNA caused apoptosis in cancer cell lines such as HeLa cells, but not in normal cells. The apoptosis was caused by p53 accumulation, which is mediated by p38 MAPK activation-dependent degradation of p300 and the subsequent MDM2 degradation. Sirtuin inhibitors are emerging as antitumor drugs, and this function has been ascribed to the inhibition of SIRT1, the most well-characterized sirtuin that deacetylases p53 to promote cell survival and also binds to other proteins in response to genotoxic stress. This study suggests that SIRT2 can be a novel molecular target for cancer therapy and provides a molecular basis for the efficacy of SIRT2 for future cancer therapy.

Competition between a noncoding exon and introns: Gomafu contains tandem UACUAAC repeats and associates with splicing factor‐1

Abstract: Gomafu (also referred to as RNCR2/MIAT) was originally identified as a noncoding RNA expressed in a particular set of neurons Unlike protein-coding mRNAs, the Gomafu RNA escapes nuclear export and stably accumulates in the nucleus, making a unique nuclear compartment Although recent studies have revealed the functional relevance of Gomafu in a series of physiological processes, the underlying molecular mechanism remains largely uncharacterized In this report, we identified a chicken homologue of Gomafu using a comparative genomic approach to search for functionally important and conserved sequence motifs among evolutionarily distant species Unexpectedly, we found that all Gomafu RNA examined shared a distinctive feature: tandem repeats of UACUAAC, a sequence that has been identified as a conserved intron branch point in the yeast Saccharomyces cerevisiae The tandem UACUAAC Gomafu RNA repeats bind to the SF1 splicing factor with a higher affinity than the divergent branch point sequence in mammals, which affects the kinetics of the splicing reaction in vitro We propose that the Gomafu RNA regulates splicing efficiency by changing the local concentration of splicing factors within the nucleus

Distinct regulation of mitochondrial localization and stability of two human Sirt5 isoforms

Abstract: Seven human Sir2 homologues (sirtuin) have been identified to date In this study, we clarified the mechanism of subcellular localization of two SIRT5 isoforms (ie, SIRT5iso1 and SIRT5iso2) encoded by the human SIRT5 gene and whose C-termini slightly differ from each other Although both isoforms contain cleavable mitochondrial targeting signals at their N-termini, we found that the cleaved SIRT5iso2 was localized mainly in mitochondria, whereas the cleaved SIRT5iso1 was localized in both mitochondria and cytoplasm SIRT5ΔC, which is composed of only the common domain, showed the same mitochondrial localization as that of SIRT5iso2 These results suggest that the cytoplasmic localization of cleaved SIRT5iso1 is dependent on the SIRT5iso1-specific C-terminus Further analysis showed that the C-terminus of SIRT5iso2, which is rich in hydrophobic amino acid residues, functions as a mitochondrial membrane insertion signal In addition, a de novo protein synthesis inhibition experiment using cycloheximide showed that the SIRT5iso1-specific C-terminus is necessary for maintaining the stability of SIRT5iso1 Moreover, genome sequence analysis from each organism examined indicated that SIRT5iso2 is a primate-specific isoform Taken together, these results indicate that human SIRT5 potentially controls various primate-specific functions via two isoforms with different intracellular localizations or stabilities

Critical role of Wnt5a-Ror2 signaling in motility and invasiveness of carcinoma cells following Snail-mediated epithelial-mesenchymal transition.

Abstract: Expression of Snail has been shown to mediate epithelial-mesenchymal transition (EMT) of epithelial cells and carcinomas, characterized by morphological alterations with disappearance and appearance of E-cadherin and vimentin, respectively. Here, we show that ectopic expression of Snail in human epidermoid carcinoma A431 cells (Snail/A431) induces the representative EMT, resulting in remarkable motile and invasive properties of the cells. Expression of Wnt5a, its receptor Ror2 and matrix metalloproteinase (MMP)-2 is induced in Snail/A431, but not in control A431 cells. Interestingly, suppressed expression of either Wnt5a or Ror2 in Snail/A431 cells results in the inhibition of in vitro cell motility and invasiveness, at least partly mediated by MMP-2, without affecting characteristics of EMT, i.e., mesenchymal morphology, and down- and up-regulations of E-cadherin and vimentin, respectively. We further show that endogenous Snail is required for sustained expression of Wnt5a, Ror2 and MMP-13 in human osteosarcoma SaOS-2 cells. The results indicate that expression of both Wnt5a and Ror2 is induced during Snail-mediated EMT or malignant progression of cancer cells and that consequently activated Wnt5a-Ror2 signaling confers highly motile and invasive properties on cancer cells. Thus, Wnt5a-Ror2 signaling can be a target of cancer therapies to prevent cancer cells from undergoing invasion and metastasis.

Formation of a thymus from rat ES cells in xenogeneic nude mouse↔rat ES chimeras

Abstract: Various conditions for differentiating embryonic stem (ES) cells or induced pluripotent stem (iPS) cells into specific kinds of cell lines are under intensive investigation. However, the production of a functional organ with a three-dimensional structure from ES or iPS cells is difficult to achieve in vitro. In the present paper, we describe the establishment of a green fluorescent protein-expressing rat ES cell line and production of mouse↔rat ES chimera by injecting rat ES cells into mouse blastocysts. The rat ES cells contributed to various organs in the chimera, including germ cells. When we injected ES cells into blastocysts of nu/nu mice lacking a thymus, the resultant chimeras produced thymus derived from rat ES cells in their bodies. The chimeric animals may provide a method for the derivation of various organs from ES or iPS cells.

Sodium-driven motor of the polar flagellum in marine bacteria Vibrio

Abstract: The Na+-driven bacterial flagellar motor is a molecular machine powered by an electrochemical potential gradient of sodium ions across the cytoplasmic membrane The marine bacterium Vibrio alginolyticus has a single polar flagellum that enables it to swim in liquid The flagellar motor contains a basal body and a stator complexes, which are composed of several proteins PomA, PomB, MotX, and MotY are thought to be essential components of the stator that are required to generate the torque of the rotation Several mutations have been investigated to understand the characteristics and function of the ion channel in the stator and the mechanism of its assembly around the rotor to complete the motor In this review, we summarize recent results of the Na+-driven motor in the polar flagellum of Vibrio

Central nervous system-specific deletion of transcription factor Nrf1 causes progressive motor neuronal dysfunction

Abstract: Cap'n'Collar (CNC) proteins heterodimerize with small Maf proteins and regulate the transcription of various genes. Small Maf-deficient mice develop severe neurodegeneration, and it remains unclear whether CNC proteins are involved in this process. In this study, we examined the contribution of Nrf1, one of the CNC proteins, to neuronal homeostasis in vivo. As Nrf1 gene knockout mice are embryonic lethal, we developed a central nervous system (CNS)-specific Nrf1 knockout (CKO) mouse line using mice bearing an Nrf1(flox) allele and Nestin-Cre allele. At birth, the CKO mice appeared indistinguishable from control mice, but thereafter they showed progressive motor ataxia and severe weight loss. All Nrf1 CKO mice died within 3 weeks. These phenotypes are similar to those reported in small Maf-deficient mice, suggesting the presence of collaboration between Nrf1 and small Maf proteins. We also found aberrant accumulation of polyubiquitinated proteins in various CNS regions and apparent neuronal loss in the hippocampus of Nrf1 CKO mice. An oxidative stress marker was accumulated in the spinal cords of the mice, but the expression patterns of oxidative stress response genes regulated by Nrf2 did not change substantially. These results show that Nrf1 sustains the CNS homeostasis through regulating target genes distinct from those regulated by Nrf2.

Lectin microarray analysis of pluripotent and multipotent stem cells.

Abstract: Stem cells have a capability to self-renew and differentiate into multiple types of cells; specific markers are available to identify particular stem cells for developmental biology research. In this study, we aimed to define the status of somatic stem cells and the pluripotency of human embryonic stem (hES) and induced pluripotent stem (iPS) cells using a novel molecular methodology, lectin microarray analysis. Our lectin microarray analysis successfully categorized murine somatic stem cells into the appropriate groups of differentiation potency. We then classified hES and iPS cells by the same approach. Undifferentiated hES cells were clearly distinguished from differentiated hES cells after embryoid formation. The pair-wise comparison means based on ‘false discovery rate’ revealed that three lectins -Euonymus europaeus lectin (EEL), Maackia amurensis lectin (MAL) and Phaseolus vulgaris leucoagglutinin [PHA(L)]- generated maximal values to define undifferentiated and differentiated hES cells. Furthermore, to define a pluripotent stem cell state, we generated a discriminant for the undifferentiated state with pluripotency. The discriminant function based on lectin reactivities was highly accurate for judgment of stem cell pluripotency. These results suggest that glycomic analysis of stem cells leads to a novel comprehensive approach for quality control in cell-based therapy and regenerative medicine.

CNOT2 depletion disrupts and inhibits the CCR4-NOT deadenylase complex and induces apoptotic cell death.

Abstract: Eukaryotic mRNA decay is initiated by shortening of the poly (A) tail; however, neither the molecular mechanisms underlying deadenylation nor its regulation is well understood. The human CCR4-NOT complex is a major cytoplasmic deadenylase consisting of a combination of at least nine subunits, four of which have deadenylase activity. The roles of the other subunits remain obscure. Here, we show that CNOT2 depletion by siRNA induces apoptosis. We also show that CNOT2 depletion destabilizes the complex, resulting in the formation of a complex smaller than that formed in control siRNA-treated cells. The deadenylase activity of the CNOT6L subunit-containing complex prepared from CNOT2-depleted cells was less than that from control cells. Intriguingly, the formation of P-bodies, where mRNA degradation supposedly takes place, was largely suppressed in CNOT2-depleted cells. Furthermore, CNOT2 depletion enhanced CHOP mRNA levels, suggesting that endoplasmic reticulum (ER) stress was occurring, which causes apoptosis in a caspase-dependent manner. These results suggest that CNOT2 is important for controlling cell viability through the maintenance of the structural integrity and enzymatic activity of the CCR4-NOT complex.

Nitro-fatty acids and cyclopentenone prostaglandins share strategies to activate the Keap1-Nrf2 system: a study using green fluorescent protein transgenic zebrafish

Abstract: Nitro-fatty acids are electrophilic fatty acids produced in vivo from nitrogen peroxide that have many physiological activities We recently demonstrated that nitro-fatty acids activate the Keap1-Nrf2 system, which protects cells from damage owing to electrophilic or oxidative stresses via transactivating an array of cytoprotective genes, although the molecular mechanism how they activate Nrf2 is unclear A number of chemical compounds with different structures have been reported to activate the Keap1-Nrf2 system, which can be categorized into at least six classes based on their sensing pathways In this study, we showed that nitro-oleic acid (OA-NO₂), one of major nitro-fatty acids, activates Nrf2 in the same manner that of a cyclopentenone prostaglandin 15-deoxy-Δ(12,14) -prostaglandin J₂ (15d-PGJ₂) using transgenic zebrafish that expresses green fluorescent protein (GFP) in response to Nrf2 activators In transgenic embryos, GFP was induced in the whole body by treatment with OA-NO₂, 15d-PGJ₂ or diethylmaleate (DEM), but not with hydrogen peroxide (H₂O₂), when exogenous Nrf2 and Keap1 were co-overexpressed Induction by OA-NO₂ or 15d-PGJ₂ but not DEM was observed, even when a C151S mutation was introduced in Keap1 Our results support the contention that OA-NO₂ and 15d-PGJ₂ share an analogous cysteine code as electrophiles and also have similar anti-inflammatory roles

Evidence for a role of angiopoietin-like 7 (ANGPTL7) in extracellular matrix formation of the human trabecular meshwork: implications for glaucoma.

Abstract: The trabecular meshwork tissue controls the drainage of the aqueous humor of the eye. A dysfunctional trabecular meshwork leads to an altered fluid resistance, which results in increased intraocular pressure (IOP). IOP is the major risk factor of glaucoma, the second-leading cause of blindness in the developed world. In the search for genes altered by glaucomatous insults, we identified angiopoietin-like7 (ANGPTL7), a member of the ANGPTL family. Although structurally related to the angiopoietins, ANGPTL7's function is poorly understood. Because ANGPTL7 is secreted and because extracellular matrix (ECM) deposition and organization is critical for aqueous humor resistance, we investigated the effect of ANGPTL7 on relevant trabecular meshwork ECM genes and proteins. We find that overexpression of ANGPTL7 in primary human trabecular meshwork cells altered the expression of fibronectin, collagens type I, IV & V, myocilin, versican, and MMP1. ANGPTL7 also interfered with the fibrillar assembly of fibronectin. Finally, we find that silencing ANGPTL7 during the glucocorticoid insult significantly affected the expression of other steroid-responsive proteins. These results indicate that ANGPTL7 modulates the trabecular meshwork's ECM as well as the response of this tissue to steroids. Together with previous findings, these properties strengthen ANGPTL7's candidacy for the regulation of IOP and glaucoma.

Inner nuclear membrane protein Ima1 is dispensable for intranuclear positioning of centromeres.

Abstract: Inner nuclear membrane (INM) proteins play a role in spatial organization of chromosomes within the nucleus. In the fission yeast Schizosaccharomyces pombe, Sad1, an INM protein of the conserved SUN-domain family, plays an active role in moving chromosomes along the nuclear membranes during meiotic prophase. Ima1 is another conserved INM protein recently identified. A previous study claimed that Ima1 is essential for mitotic cell growth, linking centromeric heterochromatin to the spindle-pole body. However, we obtained results contradictory to the previously proposed role for Ima1: Ima1 was dispensable for mitotic cell growth or centromere positioning. This discrepancy was attributed to incorrect ima1 deletion mutants used in the previous study. Our results show that Ima1 collaborates with two other conserved INM proteins of the LEM-domain family that are homologous to human Man1 and Lem2. Loss of any one of three INM proteins has no effect on mitotic cell growth; however, loss of all these proteins causes severe defects in mitotic cell growth and nuclear membrane morphology. Considering that all three INM proteins interact with Sad1, these results suggest that Ima1, Lem2 and Man1 play at least partially redundant roles for nuclear membrane organization.

Breakage of the nuclear envelope by an extending mitotic nucleus occurs during anaphase in Schizosaccharomyces japonicus.

Abstract: During open mitosis in higher eukaryotic cells, the nuclear envelope completely breaks down and then mitotic chromosomes are exposed in the cytoplasm. By contrast, mitosis in lower eukaryotes, including fungi, proceeds with the nucleus enclosed in an intact nuclear envelope. The mechanism of mitosis has been studied extensively in yeast, a closed mitosis organism. Here, we describe a form of mitosis in which the nuclear envelope is torn by elongation of the nucleus in the fission yeast Schizosaccharomyces japonicus. The mitotic nucleus of Sz. japonicus adopted a fusiform shape in anaphase, and its following extension caused separation. Finally, a tear in the nuclear envelope occurred in late anaphase. At the same time, a polarized-biased localization of nuclear pores was seen in the fusiform-shaped nuclear envelope, suggesting a compromise in the mechanical integrity of the lipid membrane. It has been known that nuclear membrane remains intact in some metazoan mitosis. We found that a similar tear of the nuclear envelope was also observed in late mitosis of the Caenorhabditis elegans embryo. These findings provide insight into the diversity of mitosis and the biological significance of breakdown of the nuclear envelope.

Activating mutations of TOR (target of rapamycin)

Abstract: Mammalian target of rapamycin (mTOR) is a key regulator of eukaryotic cell growth In particular, mTORC1, one of the two complexes that contain mTOR, is involved in the regulation of protein synthesis, proliferation, cell cycle and autophagy Hyperactivation of the mTOR signaling pathway is observed in human cancer A variety of approaches including deletion analysis, yeast genetic screens and mining of human cancer genome databases were taken that resulted in the identification of activating mutations of TOR These studies suggest that the FAT, FRB and kinase domains are the three regions of TOR where activating mutations can be identified Within the kinase domain, the mutations are clustered in three hot spots that are all located in the kinase active site that was deduced by the alignment with PI3K One of the hot spots corresponds to the region where PI3K oncogenic mutations have been identified These results are beginning to provide important insights into the mechanism of activation of mTOR

Control of body size in C. elegans dependent on food and insulin/IGF-1 signal.

Abstract: The body size of an organism is governed by genetic and environmental factors. As an environmental factor, food appears to be the most important for body size control in animals. C. elegans worms are usually grown on an E. coli strain OP50. We show that the wild-type worms fed on another E. coli strain HB101 grow 1.6 times as large as those fed on OP50. The regression line representing the relationship between the sizes of worms grown on each food for over 30 mutants was drawn, indicating that small mutants tend to be more affected by the change in food. Mutants for the DAF-2 insulin/IGF-1 receptor and downstream SGK-1, a homolog of the serum- and glucocorticoid-inducible kinase, grow less or little larger on HB101, indicating control of body size by these factors. Results on the suppression of mutations in these factors by a mutation in the DAF-16/FOXO transcription factor indicate both DAF-16-dependent and DAF-16-independent control. Furthermore, we show that the food-dependent body size change is because of a change in cell size that is closely related to the protein content per cell.

Positional cloning of silkworm white egg 2 (w‐2) locus shows functional conservation and diversification of ABC transporters for pigmentation in insects

Abstract: The white, scarlet and brown genes of Drosophila melanogaster encode three half-type ATP-binding cassette (ABC) transporters In Drosophila, precursors of ommochromes and pteridines are transported by White/Scarlet and White/Brown heterodimers, respectively The white egg 2 (w-2) mutant of the silkworm, Bombyx mori, has white eggs and eyes because of lack of ommochrome granules in the serosa and eyes Here, we report that the silkworm w-2 locus encodes an ortholog of Drosophila scarlet Our results indicate that Bombyx Scarlet forms a heterodimer with Bombyx White to transport ommochrome precursors, suggesting that formation of a White/Scarlet heterodimer and its involvement in the transport of ommochrome precursors are evolutionarily ancient and widely conserved traits in insects Contrary to dipteran insects, white and scarlet were juxtaposed in a head-to-tail orientation in the silkworm genome, suggesting that the origin of white and scarlet was a tandem duplication of their ancestral transporter gene In Bombyx, White is also essential for the transport of uric acid in larval epidermis However, our results suggest that a Bombyx White/Scarlet heterodimer is not involved in this process Our results emphasize the functional conservation and diversification of half-type ABC transporter families in insects, which may contribute to their extremely diverse color patterns

Distinctive effects of arachidonic acid and docosahexaenoic acid on neural stem /progenitor cells

Abstract: Arachidonic acid (ARA) and docosahexaenoic acid (DHA), which are the dominant polyunsaturated fatty acids in the brain, have crucial roles in brain development and function. Recent studies have shown that ARA and DHA promote postnatal neurogenesis. However, the direct effects of ARA on neural stem/progenitor cells (NSPCs) and the effects of ARA and DHA on NSPCs at the neurogenic and subsequent gliogenic stages are still unknown. Here, we analyzed the effects of ARA and DHA on neurogenesis, specifically maintenance and differentiation, using neurosphere assays. We confirmed that primary neurospheres are neurogenic NSPCs and that tertiary neurospheres are gliogenic NSPCs. Regarding the effects of ARA and DHA on neurogenic NSPCs, ARA and DHA increased the number of neurospheres, whereas neither ARA nor DHA had a detectable effect on NSPCs in the differentiation condition. In gliogenic NSPCs, DHA increased the number of neurospheres, whereas ARA had no such effect. In contrast, ARA increased the number of astrocytes, whereas DHA increased the number of neurons in the differentiation condition. These results suggest that ARA promotes the maintenance of neurogenic NSPCs and might induce the glial differentiation of gliogenic NSPCs and that DHA promotes the maintenance of both neurogenic and gliogenic NSPCs and might lead to the neuronal differentiation of gliogenic NSPCs.

Transcription factors CgUPC2A and CgUPC2B regulate ergosterol biosynthetic genes in Candida glabrata.

Abstract: Zn[2]-Cys[6] binuclear transcription factors Upc2p and Ecm22p regulate the expression of genes involved in ergosterol biosynthesis and exogenous sterol uptake in Saccharomyces cerevisiae. We identified two UPC2/ECM22 homologues in the pathogenic fungus Candida glabrata which we designated CgUPC2A and CgUPC2B. The contribution of these two genes to sterol homeostasis was investigated. Cells that lack CgUPC2A (upc2AΔ) exhibited enhanced susceptibility to the sterol biosynthesis inhibitors, fluconazole and lovastatin, whereas upc2BΔ-mutant cells were as susceptible to the drugs as wild-type cells. The growth of upc2AΔ cells was also severely attenuated under anaerobic conditions. Lovastatin treatment enhanced the expression of ergosterol biosynthetic genes, ERG2 and ERG3 in wild-type and upc2BΔ but not in upc2AΔ cells. Similarly, serum-induced expression of ERG2 and ERG3 was completely impaired in upc2AΔ cells but was unaffected in upc2BΔ cells, whereas serum-induced expression of the sterol transporter gene CgAUS1 was impaired in both upc2AΔ and upc2BΔ cells. These results suggest that in C. glabrata CgUPC2A but not in CgUPC2B is the main transcriptional regulator of the genes responsible for maintaining sterol homeostasis as well as susceptibility to sterol inhibitors.

Roles of Drosophila deltex in Notch receptor endocytic trafficking and activation.

Abstract: Cell signaling mediated by the Notch receptor (N) regulates many cell-fate decisions and is partly controlled by the endocytic trafficking of N. Drosophila deltex (dx) encodes an evolutionarily conserved regulator of N signaling, an E3-ubiquitin ligase, which ubiquitinates N's intracellular domain. Although Dx was shown to function in N endocytosis in studies of dx over-expression, the roles of endogenous Dx have remained hidden. Here, we investigated N endocytosis in a dx-null Drosophila mutant and found that endogenous Dx is required for at least two steps of N trafficking: the incorporation of N into endocytic vesicles from the plasma membrane and the transport of N from early endosomes to lysosomes. In the absence of Dx functions, N was stabilized in unknown endocytic compartments, where it was probably insulated from transport to lysosomes. We also found that canonical N signaling and Dx-mediated N signaling are activated in two different endocytic compartments, before N is incorporated into multivesicular body (MVB) interluminal vesicles and after N is transported from MVBs, respectively. The endocytic compartment in which Dx-mediated N signaling is activated appears to coincide with the activity of endogenous Dx in N trafficking. These findings extend our understanding of how N's trafficking and activation are correlated.

Identification of target genes for the CDK subunits of the Mediator complex.

Abstract: Mediator is a large complex containing up to 30 subunits that consist of four modules each: head, middle, tail and CDK/Cyclin. Recent studies have shown that CDK8, a subunit of the CDK/Cyclin module, is one of the key subunits of Mediator that mediates its pivotal roles in transcriptional regulation. In addition to CDK8, CDK19 was identified in human Mediator with a great deal of similarity to CDK8 but was conserved only in vertebrates. Previously, we reported that human CDK19 could form the Mediator complexes independent of CDK8. To further investigate the in vivo transcriptional activities of the complexes, we used a luciferase assay in combined with siRNA-mediated knockdown to show that CDK8 and CDK19 possess opposing functions in viral activator VP16-dependent transcriptional regulation. CDK8 supported transcriptional activation, whereas CDK19, however, counteracted it. In this study, we further characterized CDK19. We used microarrays to identify target genes for each CDK, and we selected six genes: two target genes of CDK8, two target genes of CDK19 and two genes that were targets for both. Surprisingly, it turned out that both CDKs bound to all six target genes, regardless of their effects in transcription upon binding, suggesting Mediator as a context-specific transcriptional regulator.

miR126 positively regulates mast cell proliferation and cytokine production through suppressing Spred1.

Abstract: The protein known as Spred1 (Sprouty-related Ena/VASP homology-1 domain-containing protein) has been identified as a negative regulator of growth factor-induced ERK/mitogen-activated protein kinase activation. Spred1 has also been implicated as the target of microRNA-126 (miR126), a miRNA located within the Egfl7 gene, and is involved in the regulation of vessel development through its role in regulating VEGF signaling. In this study, we examined the role of miR126 and Spred1 in the hematopoietic system, as miR126 has been shown to be overexpressed in leukemic cells. miR126 levels were down-regulated during mast cell differentiation from bone marrow cells, whereas Spred1 expression was inversely up-regulated. Overexpression of miR126 suppressed Spred1 expression and enhanced ERK activity in primary bone marrow cells and MC9 mast cells, which were associated with elevated FceRI-mediated cytokine production. To confirm the effect of Spred1 reduction in vivo, we generated hematopoietic cell-specific Spred1-conditional knockout mice. These mice showed increased numbers of mast cells, and Spred1-deficient bone marrow-derived mast cells were highly activated by cross-linking of Fce-R stimulation as well as by IL-3 and SCF stimulation. These results suggest that Spred1 negatively regulates mast cell activation, which is modulated by miR126.

How does genome instability affect lifespan?: roles of rDNA and telomeres.

Abstract: The genome is composed not only of genes but also of several noncoding functional elements (NOCs/ncFE, here I use NOCs), such as transcriptional promoters, enhancers, replication origins, centromeres and telomeres. rDNA has both gene and NOC characteristics. Thus, the rDNA encodes ribosomal RNAs, components of the ribosomes, that account for approximately 80% of the total RNA in a cell. However, rDNA may also act as a NOC with respect to cellular senescence by limiting the number of times a cell can divide. Here, I discuss how rDNA might function as a NOC to influence life span in a manner analogous to telomeres.

Multiple factors in the early splicing complex are involved in the nuclear retention of pre-mRNAs in mammalian cells.

Abstract: Intron-containing pre-mRNAs are retained in the nucleus until they are spliced. This mechanism is essential for proper gene expression. Although the formation of splicing complexes on pre-mRNAs is thought to be responsible for this nuclear retention activity, the details are poorly understood. In mammalian cells, in particular, very little information is available regarding the retention factors. Using a model reporter gene, we show here that U1 snRNP and U2AF but not U2 snRNP are essential for the nuclear retention of pre-mRNAs in mammalian cells, showing that E complex is the major entity responsible for the nuclear retention of pre-mRNAs in mammalian cells. By focusing on factors that bind to the 3'-splice site region, we found that the 65-kD subunit of U2AF (U2AF(65) ) is important for nuclear retention and that its multiple domains have nuclear retention activity per se. We also provide evidence that UAP56, a DExD-box RNA helicase involved in both RNA splicing and export, cooperates with U2AF(65) in exerting nuclear retention activity. Our findings provide new information regarding the pre-mRNA nuclear retention factors in mammalian cells.

Mitotic kinase Aurora‐B is regulated by SUMO‐2/3 conjugation/deconjugation during mitosis

Abstract: The small ubiquitin-related modifier (SUMO) system of higher eukaryotes plays important roles in normal cell division, especially in chromosome segregation. However, only a few mitotic SUMO substrates have been identified in mammals. Here, we show that the mitotic kinase Aurora-B can be modified by SUMO. The E3 SUMO-protein ligase PIAS3 [protein inhibitor of activated STAT (signal transducer and activator of transcription)] dramatically enhanced poly-SUMO-2/3 conjugation of Aurora-B, whereas the SUMO-specific isopeptidase SENP2 (Sentrin/SUMO-specific protease) specifically deconjugated SUMO from Aurora-B. The Lys-202 residue on human Aurora-B was preferentially modified by SUMO, and enhancement of SUMOylation in cells facilitated Aurora-B autophosphorylation, which is essential for its activation. Conversely, SENP2-mediated deSUMOylation of Aurora-B down-regulated its autophosphorylation in cells and also impaired its re-activation in Aurora inhibitor VX-680-treated mitotic cells. Poly-SUMO-2 conjugation of Aurora-B occurred during the M phase of the cell cycle, and both SUMO-2 and PIAS3 were localized adjacent to Aurora-B in the kinetochores in early mitosis. Based on these results, we propose that Aurora-B is a novel mitotic SUMO substrate and that its kinase activity is fine-tuned by the SUMO system.

Halenaquinone, a chemical compound that specifically inhibits the secondary DNA binding of RAD51

Abstract: Mutations and single-nucleotide polymorphisms affecting RAD51 gene function have been identified in several tumors, suggesting that the inappropriate expression of RAD51 activity may cause tumorigenesis. RAD51 is an essential enzyme for the homologous recombinational repair (HRR) of DNA double-strand breaks. In the HRR pathway, RAD51 catalyzes the homologous pairing between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), which is the central step of the HRR pathway. To identify a chemical compound that regulates the homologous-pairing activity of RAD51, in the present study, we screened crude extract fractions from marine sponges by the RAD51-mediated homologous-pairing assay. Halenaquinone was identified as an inhibitor of the RAD51 homologous-pairing activity. A surface plasmon resonance analysis indicated that halenaquinone directly bound to RAD51. Intriguingly, halenaquinone specifically inhibited dsDNA binding by RAD51 alone or the RAD51-ssDNA complex, but only weakly affected the RAD51-ssDNA binding. In vivo, halenaquinone significantly inhibited the retention of RAD51 at double-strand break sites. Therefore, halenaquinone is a novel type of RAD51 inhibitor that specifically inhibits the RAD51-dsDNA binding.

Neural crest-derived multipotent cells in the adult mouse iris stroma.

Abstract: The purpose of this study was to characterize neural crest–derived cells within the adult murine iris. The iris was isolated from P0-Cre ⁄ Floxed-EGFP transgenic (TG) mice. The isolated iris cells formed EGFP-positive spheres on non-adhesive culture plates. Immunostaining showed that these EGFP-positive spheres expressed neural crest markers including Sox10 and p75NTR, and these cells showing in vitro sphere-forming ability were originally resided in the iris stroma (IS), in vivo. Real-time RT-PCR showed that the EGFP-positive spheres expressed significantly higher levels of the neural crest markers than EGFP-negative spheres and bone marrow–derived mesenchymal stem cells. Furthermore, the iris stromal sphere had capability to differentiate into various cell lineages including smooth muscle and cartilage. These data indicate that neural crest–derived multipotent cells can be isolated from the murine IS and expanded in sphere culture.

Equine major histocompatibility complex class I molecules act as entry receptors that bind to equine herpesvirus-1 glycoprotein D

Abstract: The endotheliotropism of equine herpesvirus-1 (EHV-1) leads to encephalomyelitis secondary to vasculitis and thrombosis in the infected horse central nervous system (CNS). To identify the host factors involved in EHV-1 infection of CNS endothelial cells, we performed functional cloning using an equine brain microvascular endothelial cell cDNA library. Exogenous expression of equine major histocompatibility complex (MHC) class I heavy chain genes conferred susceptibility to EHV-1 infection in mouse NIH3T3 cells, which are not naturally susceptible to EHV-1 infection. Equine MHC class I molecules bound to EHV-1 glycoprotein D (gD), and both anti-gD antibodies and a soluble form of gD blocked viral entry into NIH3T3 cells stably expressing the equine MHC class I heavy chain gene (3T3-A68 cells). Treatment with an anti-equine MHC class I monoclonal antibody blocked EHV-1 entry into 3T3-A68 cells, equine dermis (E. Derm) cells and equine brain microvascular endothelial cells. In addition, inhibition of cell surface expression of MHC class I molecules in E. Derm cells drastically reduced their susceptibility to EHV-1 infection. These results suggest that equine MHC class I is a functional gD receptor that plays a pivotal role in EHV-1 entry into equine cells.

Zebrafish Dmrta2 regulates neurogenesis in the telencephalon.

Abstract: Although recent findings showed that some Drosophila doublesex and Caenorhabditis elegans mab-3 related genes are expressed in neural tissues during development, their functions have not been fully elucidated. Here, we isolated a zebrafish mutant, ha2, that shows defects in telencephalic neurogenesis and found that ha2 encodes Doublesex and MAB-3 related transcription factor like family A2 (Dmrta2). dmrta2 expression is restricted to the telencephalon, diencephalon and olfactory placode during somitogenesis. We found that the expression of the proneural gene, neurogenin1, in the posterior and dorsal region of telencephalon (posterior-dorsal telencephalon) is markedly reduced in this mutant at the 14-somite stage without any defects in cell proliferation or cell death. In contrast, the telencephalic expression of her6, a Hes-related gene that is known to encode a negative regulator of neurogenin1, expands dramatically in the ha2 mutant. Based on over-expression experiments and epistatic analyses, we propose that zebrafish Dmrta2 controls neurogenin1 expression by repressing her6 in the posterior-dorsal telencephalon. Furthermore, the expression domains of the telencephalic marker genes, foxg1 and emx3, and the neuronal differentiation gene, neurod, are downregulated in the ha2 posterior-dorsal telencephalon during somitogenesis. These results suggest that Dmrta2 plays important roles in the specification of the posterior-dorsal telencephalic cell fate during somitogenesis.