Cdc2 Gene Research Articles

The role of reactive oxygen species and hemeoxygenase-1 expression in the cytotoxicity, cell cycle alteration and apoptosis of dental pulp cells induced by BisGMA

Biocompatibility of dentin bonding agents (DBAs) and resin composite is important to preserve the pulp vitality after operative restoration. Bisphenol-glycidyl-methacrylate (BisGMA) is one common monomer adding into DBAs and resin. In this study, we found that exposure of human dental pulp cells to BisGMA (>0.1 m m) led to cytotoxicity, G2/M cell cycle arrest and apoptosis as analyzed by propidium iodide (PI) and PI/annexin V dual fluorescent flow cytometry. These events were associated with a decline of cdc2, cdc25C and cyclinB1 expression at both mRNA and protein levels. BisGMA also induced the expression of hemeoxygenase-1 (HO-1), an oxidative stress responsive gene, in pulp cells. Catalase could prevent the BisGMA-induced alteration of cell cycle-related genes (cdc2, cdc25C, cyclinB1) and HO-1 expression in dental pulp cells. Interestingly, Zn-protoporphyrin (2.5–5 μ m), a HO inhibitor, enhanced the BisGMA-induced reactive oxygen species (ROS) production and cytotoxicity. These results suggest that exposure to higher concentrations of BisGMA may stimulate ROS production, cell cycle arrest, apoptosis and cell death. Inducing the expression of HO-1 in dental pulp cells by BisGMA is mediated by ROS production and important to protect dental pulp against the toxicity by monomers present in composite resin and DBAs.

Y-box binding protein 1 is up-regulated in proliferative breast cancer and its inhibition deregulates the cell cycle

The Y-box-binding protein 1 (YB-1), a member of the cold-shock domain RNA-and DNA-binding protein family, has pleiotropic functions such as regulation of the cell cycle. The aim of this study was to evaluate if YB-1 is a proliferative marker in breast cancer and elucidate potential downstream targets involved in YB-1-mediated cell cycle regulation using RNA interference technology. YB-1 protein expression was evaluated in tissue microarrays of 131 breast invasive ductal carcinomas by immunohistochemistry, while the YB-1 gene expression profile was evaluated in the T-47D, MDA-MB-231, ZR-75-1 and MCF7 breast cancer cell lines. Silencing of the YB-1 gene in T-47D breast cancer cells was performed using siRNA and the effects of down-regulation of YB-1 on cell growth and regulation of the cell cycle were ascertained. A focused panel of 84 genes involved in cell cycle progression was also examined. In tissue microarrays, YB-1 expression was shown to be associated with proliferating cell nuclear antigen (PCNA) immunostaining. siRNA-mediated silencing of the YB-1 gene inhibited cell proliferation and induced G1 phase cell cycle arrest in T-47D breast cancer cells. Knockdown of the YB-1 gene induced up-regulation of two genes which contribute to G1-arrest (RAD9A and CDKN3 genes) and down-regulation of ten genes associated with positive regulation of the cell cycle (SKP2, SUMO1, ANAPC4, CCNB1, CKS2, MNAT1, CDC20, RBBP8, KPNA2 and CCNC genes). The data obtained from the tissue microarrays and cell lines provide evidence that YB-1 is a reliable marker of cell proliferation and possibly a potential molecular target in breast cancer therapy.

A Genome-Wide Association Study of Optic Disc Parameters

The optic nerve head is involved in many ophthalmic disorders, including common diseases such as myopia and open-angle glaucoma. Two of the most important parameters are the size of the optic disc area and the vertical cup-disc ratio (VCDR). Both are highly heritable but genetically largely undetermined. We performed a meta-analysis of genome-wide association (GWA) data to identify genetic variants associated with optic disc area and VCDR. The gene discovery included 7,360 unrelated individuals from the population-based Rotterdam Study I and Rotterdam Study II cohorts. These cohorts revealed two genome-wide significant loci for optic disc area, rs1192415 on chromosome 1p22 (p = 6.72×10−19) within 117 kb of the CDC7 gene and rs1900004 on chromosome 10q21.3-q22.1 (p = 2.67×10−33) within 10 kb of the ATOH7 gene. They revealed two genome-wide significant loci for VCDR, rs1063192 on chromosome 9p21 (p = 6.15×10−11) in the CDKN2B gene and rs10483727 on chromosome 14q22.3-q23 (p = 2.93×10−10) within 40 kbp of the SIX1 gene. Findings were replicated in two independent Dutch cohorts (Rotterdam Study III and Erasmus Rucphen Family study; N = 3,612), and the TwinsUK cohort (N = 843). Meta-analysis with the replication cohorts confirmed the four loci and revealed a third locus at 16q12.1 associated with optic disc area, and four other loci at 11q13, 13q13, 17q23 (borderline significant), and 22q12.1 for VCDR. ATOH7 was also associated with VCDR independent of optic disc area. Three of the loci were marginally associated with open-angle glaucoma. The protein pathways in which the loci of optic disc area are involved overlap with those identified for VCDR, suggesting a common genetic origin.

Thermodynamic and Kinetic Studies of a Stable Imperfect DNA Triplex by Spectroscopic and Calorimetric Methods

AbstractAn imperfect DNA triplex, containing a pyrimidine/purine duplex with two sites of purine/pyrimidine base pair inversions, is formed and recognized. The unusual triplex is examined for the possibility of triplex DNA formation in a living system by way of non‐ideal sequence matching. The duplex part of it consists of a DNA oligomer with 17 nucleotidyl units, 5′‐TTCTTCTGATTCTCTCC‐3′ (C), which has two purine bases flanked by fifteen pyrimidine bases. The sequence of oligomer C is picked from a segment of cell cycle protein cdc25 gene (696 to 712) in Pneumocystis jirovecii, a yeast‐like parasitic fungus, which can cause pneumonia (PCP) in patients with impaired immunity. The complementary oligomer, 5′‐GGAGAGAATCAGAAGAA‐3′ (W), forms a DNA duplex WC with C, accompanied by CG/GC and TA/AT base pair inversions, in contrast to homo pyrimidine/purine duplexes. A third strand, 5′‐CCTCTCTTAGTCTTCTT‐3′ (H), combines with WC, to give an imperfect, but stable, triplex WCH+.UV absorption, circular dichroism, and native gel electrophoresis mobility shift assay are used to monitor the reactions at each stage. The triplex is stable in mild acidic solution, with a 1:1:1 stoichiometric ratio and a UV melting temperature (Tm) of 47.3 °C at pH 4.5. Isothermal titration calorimetry (ITC) and surface Plasmon resonance (SPR) unravel the thermodynamic information (ΔH = −123.1 kcal mol−1, ΔS = −378 cal mol−1K−1 and ΔG = −10.5 kcal mol−1) and kinetic rate constants (ka = 3.71 × 104 M−1 s−1 and kd = 7.49 × 10−4 s−1), respectively, for the WCH+ triplex formation process. WCH+ is termed an imperfect triplex since it contains two non‐canonical, non‐purine/purine/pyrimidine or non‐pyrimidine/purine/pyrimidine, base triads in the middle. This is the first time an imperfect but stable triplex, containing two purine bases in the middle of a pyrimidine‐rich strand (H), is formed and studied. The fact that an imperfect but stable DNA triplex can be formed gives great promise for drug design through more flexible targeting capabilities with much less stringent requirements, e.g., designs against Pneumocystis jirovecii using triplex formation in our example.

Abstract 1065: MicroRNA-1296 and genistein regulate DNA licensing proteins (MCM-genes) in prostate cancer

Abstract Introduction and Objective: The minichromosome maintenance (MCM) gene-family is essential for DNA replication and is found to be frequently up-regulated in a variety of cancers. In this study we examined the role of MCM2 in prostate cancer and the effect of microRNA-1296, genistein and trichostatin A on the MCM-complex. Methods: Prostate cancer cell lines (LNCaP and PC3) were treated with different doses (0-50umol/L) of genistein (72 hours) and 100 ng/ml trichostatin-A (24 hours). Quantitative real-time PCR, immunohistochemical staining and Western analysis were performed to check mRNA and protein expression in cells and tissues. The small interfering RNA (siRNA) approach was employed to understand the functional significance of MCM2 gene. Fluorescence activated cell sorting (FACS) analysis was performed to monitor cell cycle distribution and apoptosis. PC3 cells were transfected with miR-1296 and miR-1296 inhibitor to investigate its effect on the MCM2 gene. Results: Profiling results showed that expression of MCM-genes was higher in prostate tumor sample and cell lines. Genistein and trichostatin A (TSA) significantly down-regulated expression of all MCM-genes. A specific siRNA was used to knock-down MCM2 mRNA and protein expression. FACS results showed that genistein, TSA and siRNA caused a significant decrease in the S-phase of the cell cycle. There was also down-regulation of CDT1, CDC7 and CDK2 genes which govern the loading of MCM-complex on chromatin. We found that microRNA-1296 targets the MCM2 mRNA sequence. MicroRNA-1296 expression is significantly down-regulated in prostate cancer compared to normal tissues. To investigate the relationship between MCM2 and miR-1296, a miR-1296 inhibitor was transfected into PC3 cells. Inhibition of miR-1296 up-regulated both MCM2 mRNA and protein, whereas over-expression caused a significant decrease in MCM2 mRNA, protein and the S phase of cell cycle. Conclusion: MCM-genes are excellent anti-cancer drug targets because they are essential DNA replication factors that are highly expressed in cancer cells. This is the first report showing an anti-MCM effect by miR-1296, genistein and TSA. TSA is undergoing clinical trials as a prostate cancer treatment but has high toxicity. Genistein, a natural non-toxic dietary isoflavone, may be an advantageous therapeutic agent for treating prostate cancer. The use of RNAi is currently being implemented as a gene-specific approach for molecular medicine. By the same principle, the specific down-regulation of oncogenes by micro-RNA may contribute to novel therapeutic approaches in the treatment of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1065.

Abstract 2488: γ-Secretase inhibitor blocks cell proliferation in gastric cancers through mitotic arrest and apoptotic cell death

Abstract Gastric cancer is one of the most common cancers with mortal malignancy. Most patients with advanced gastric cancer have been treated with anti-cancer chemotherapies such as fluorouracil (5-FU), cisplatin and anthracycline which block mitotic cell division and induce consequent apoptotic cell death. However, there are needed novel gastric therapies and/or develop the new agents to overwhelm the resistance for traditional chemotherapeutic agents. Recent studies have reported that the Notch signaling pathway is associated with human gastric cancer and other cancers. Notch receptors are transmembrane proteins which are cleaved by protelolytic emzymes (gamma-secretase) and release their intracellular domain (ICD) upon activation status by ligand binding. In previous reports, notch intracellular domain (NICD) turns on target genes which are related with tumorigenesis such as COX-2, c-Myc and Snail in cancers, including gastric cancer cells. Thus we speculated that the blockage of notch signal would be useful strategy for chemotherapy of gastric cancers. We initially showed that Notch receptors and ligands are expressed in various gastric cancer cells through RT-PCR and western blotting. We also confirmed that Notch signals are activated in gastric cancer cells by Notch reporter gene analysis. Furthermore, GSI treatment reduced cell survival rate in gastric cancer cells, including AGS, SNU1, SNU601, and SNU638 cells. We observed G2/M mitotic arrest and subG1 arrest by GSI treatment in gastric cancer cells, accompanying with the increase of mitotic gene cyclin B1, cdc2, p21, and apototic caspase-3 activation. To demonstrate the effect of p21 increasing by GSI treatment, cell proliferation and G2/M cell cycle arrest was inhibited by GSI treatment in Myr-AKT lentivirus infection cell. Also, p21 and cyclinB1 protein levels were not increased after rapamycin treatment in Myr-AKT lentivirus infection cell. Collectively, these results suggest that GSI, which block Notch signal, would be a potential therapeutic drug in gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2488.

Abstract DD01-01: NMS-1116354: More than an inhibitor of Cdc 7 kinase in S-phase

Abstract DD01-01: NMS-1116354: More than an inhibitor of Cdc 7 kinase in S-phase

Oocyte maturation failure: a syndrome of bad eggs

To show that disruption of meiotic competence results in cell cycle arrest, and the production of immature oocytes that are not capable of fertilization. Through an extensive review of animal studies and clinical case reports, we define the syndrome of oocyte maturation failure as a distinct oocyte disorder, present a classification system based on clinical parameters, and discuss the potential molecular origins for the disease.

Expression and functional characterization of a Rho-family small GTPase CDC42 from Trichinella spiralis

A full-length cDNA encoding a Rho-family small GTPase gene cdc42 of Trichinella spiralis was expressed in E. coli. The recombinant protein of TsCDC42 was purified and used to raise the polyclonal antibodies. The expression of TsCDC42 in different stages of parasite was investigated. The western blot showed that TsCDC42 was expressed in all stages of T. spiralis, including newborn larvae, muscle larvae and adult worms. The immuno-localization revealed that TsCDC42 was ubiquitously distributed in the newborn larvae, muscle larvae and adult worm. Cross-species RNAi was done by knockdown Tscdc42 RNAi in C. elegans. The results revealed that endogenous expression level of CDC42 was decreased by knockdown Tscdc42 RNAi in C. elegans, and this knockdown reduced the progeny of C. elegans. It suggested that Tscdc42 might play the same roles in the early development of T. spiralis.

PH‐Domain‐driven targeting of collybistin but not Cdc42 activation is required for synaptic gephyrin clustering

Collybistin (Cb) is a brain-specific guanine nucleotide exchange factor (GEF) that is essential for the synaptic clustering of gephyrin and GABAA receptors in selected regions of the mammalian central nervous system. It has been previously proposed that Cb regulates gephyrin clustering by activating Cdc42, and thus acts as a signal transducer in a membrane activation process which labels postsynaptic membrane domains for inhibitory synapse formation. Here, we dissected the functional roles of the Dbl-homology (DH) and pleckstrin homology (PH) domains of the constitutively active splice variant Cb II by substituting conserved amino acid residues that are required for GEF activity towards Cdc42 and phosphoinositide binding, respectively. A Cb II mutant lacking any detectable GEF activity towards Cdc42 was still fully active in inducing gephyrin scaffold formation, both in transfected NIH-3T3 cells and in cultured hippocampal neurons. Furthermore, mice with a forebrain-specific inactivation of the Cdc42 gene displayed normal densities of gephyrin and GABA(A) receptor clusters in the hippocampus. In contrast, substitution of Cb II PH-domain residues essential for phosphoinositide binding abolished gephyrin recruitment to synaptic sites. Our results provide evidence that the formation of gephyrin scaffolds at inhibitory synapses requires an intact Cb II PH-domain but is Cdc42-independent.

Regulation of Minichromosome Maintenance Gene Family by MicroRNA-1296 and Genistein in Prostate Cancer

The minichromosome maintenance (MCM) gene family is essential for DNA replication and is frequently upregulated in various cancers. Here, we examined the role of MCM2 in prostate cancer and the effect of microRNA-1296 (miR-1296), genistein, and trichostatin A (TSA) on the MCM complex. Profiling results showed that expression of MCM genes was higher in tumor samples. Genistein and TSA significantly downregulated the expression of all MCM genes. Genistein, TSA, and small interfering RNA duplexes caused a significant decrease in the S phase of the cell cycle. There was also downregulation of CDT1, CDC7, and CDK2 genes, which govern loading of the MCM complex on chromatin. We also found that miR-1296 was significantly downregulated in prostate cancer samples. In PC3 cells, inhibition of miR-1296 upregulated both MCM2 mRNA and protein, whereas overexpression caused a significant decrease in MCM2 mRNA, protein, and the S phase of the cell cycle. MCM genes are excellent anticancer drug targets because they are essential DNA replication factors that are highly expressed in cancer cells. This is the first report showing anti-MCM effect by miR-1296, genistein, and TSA. TSA is undergoing clinical trials as a prostate cancer treatment but has high toxicity. Genistein, a natural, nontoxic dietary isoflavone, may be an advantageous therapeutic agent for treating prostate cancer. The use of RNA interference is currently being implemented as a gene-specific approach for molecular medicine. The specific downregulation of oncogenes by miR may contribute to novel therapeutic approaches in the treatment of prostate cancer.

The Sos-recruitment system as a tool to analyze cellular localization of plant proteins: membrane localization of Arabidopsis thaliana PEPINO/PASTICCINO2

We have explored a modified cytosolic yeast-two-hybrid Sos-recruitment system (SRS) in order to test for membrane localization of a protein. In this system, membrane localization is assessed by rescue of a yeast strain carrying a temperature-sensitive mutation in the CDC25 gene (cdc25-2) at restrictive temperature. The homologous human Sos (hSos) is capable to replace cdc25-2 provided that it is attached to the membrane because only then hSos is functional. This can be achieved when hSos is artificially fused to a protein containing trans-membrane domains (Tms). GFP/YFP fusion construct analyses of the Arabidopsis thaliana PEPINO/PASTICCINO2 (PEP/PAS2) protein have previously shown disparate cellular localizations although this protein possesses clear Tms. Analysis of N-terminal and C-terminal hSos-PEP/PAS2 fusions respectively suggests, that PEP/PAS2 is an integral membrane protein with cytosolic N- and C-termini. This implies that the protein has an even number of Tms and that the first Tm, a signal peptide, is not cleaved off. Our study shows that SRS is suitable to test for protein membrane localization and possibly for more detailed topological analysis of membrane proteins.

Cdc-25.2, aC. elegansortholog ofcdc25, is required to promote oocyte maturation

Cdc25 is an evolutionarily conserved protein phosphatase that promotes progression through the cell cycle. Some metazoans have multiple isoforms of Cdc25, which have distinct functions and different expression patterns during development. C. elegans has four cdc-25 genes. cdc-25.1 is required for germline mitotic proliferation. To determine if the other members of the cdc-25 family also contribute to regulation of cell division in the germ line, we examined phenotypes of loss-of-function mutants of the other cdc-25 family genes. We found that cdc-25.2 is also essential for germline development. cdc-25.2 homozygous mutant hermaphrodites exhibited sterility as a result of defects in oogenesis: mutant oocytes were arrested as endomitotic oocytes that were not fertilized successfully. Spermatogenesis and male germline development were not affected. Through genetic interaction studies, we found that CDC-25.2 functions upstream of maturation-promoting factor containing CDK-1 and CYB-3 to promote oocyte maturation by counteracting function of WEE-1.3. We propose that cdc-25 family members function as distinct but related cell cycle regulators to control diverse cell cycles in C. elegans germline development.

Molecular cloning and phylogenetic analysis of small GTPase protein Tscdc42 from Trichinella spiralis

A full-length of complementary deoxyribonucleic acid (Tscdc42) encoding a putative Rho-family small GTPase gene cdc42 was isolated from Trichinella spiralis, an economically important parasitic nematode of zoonosis. The uninterrupted open reading frame of TsCDC42 encodes a predicted protein of 147 amino acids and containing a highly conserved domain of CDC42. Comparison with selected sequences from the free-living nematode Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, Danio rerio, Mus musculus, and human showed that Tscdc42 is highly conserved. The highest identity of TsCDC42 with CDC42 from Drosophila is 67%, the similarity is up to 73%, the identity of TsCDC42 with the CDC42 homologue of C. elegans is 62%, and the similarity is up to 71%. Phylogenetic analysis of the amino acid sequence data, using the neighbor-joining and maximum parsimony methods, revealed that TsCDC42 is closely related to the molecule inferred from the cdc-42 gene of C. elegans. The transcript of TsCDC42 was analyzed during different stages of the worm.

ARF-induced downregulation of Mip130/LIN-9 protein levels mediates a positive feedback that leads to increased expression of p16Ink4a and p19Arf

The ARF-MDM2-p53 pathway constitutes one of the most important mechanisms of surveillance against oncogenic transformation, and its inactivation occurs in a large proportion of cancers. Here, we show that ARF regulates Mip130/LIN-9 by inducing its translocation to the nucleolus and decreasing the expression of the Mip130/LIN-9 protein through a post-transcriptional mechanism. The knockdown of Mip130/LIN-9 in p53(-/-) and Arf(-/-) mouse embryonic fibroblasts (MEFs) mimics some effects of ARF, such as the downregulation of B-Myb, impaired induction of G2/M genes, and a decrease in cell proliferation. Importantly, although the knockdown of Mip130/LIN-9 reduced the proliferation of p53 or Arf-null MEFs, only p53(-/-) MEFs showed a senescence-like state and an increase in the expression of Arf and p16. Interestingly, the increase in p16 and ARF is indirect because the Mip130/LIN-9 knockdown decreased the transcription of negative regulators of the Ink4a/Arf locus, such as BUBR1 and CDC6. Chromatin immunoprecipitation assays also reveal that Mip130/LIN-9 occupies the promoters of the BubR1 and cdc6 genes, suggesting that Mip130/LIN-9 is necessary for the expression of these genes. Altogether, these results indicate that there is a feedback mechanism between ARF and Mip130/LIN-9 in which either the increase of ARF or the decrease in Mip130/LIN-9 causes a further increase in the expression of Arf and p16.

Y-box binding protein-1 (YB-1) promotes cell cycle progression through CDC6-dependent pathway in human cancer cells

Y-box binding protein-1 (YB-1) plays pivotal roles in acquisition of global drug resistance and cell growth promotion through transcriptional activation of genes for both drug resistance and growth factor receptors. In this study, we investigated whether YB-1 is involved in regulation of the cell cycle and cell proliferation of human cancer cells. Treatment with YB-1 siRNA caused a marked suppression of cell proliferation and expression of a cell cycle related gene, CDC6 by cancer cells. Of cell cycle of cancer cells, S phase content was specifically reduced by knockdown of YB-1. The overexpression of CDC6 abrogated this inhibition of both cell proliferation and S phase entry. ChIP assay demonstrated that YB-1 binds to a Y-box located in the promoter region of the CDC6 gene. Expression of cyclin D1, CDK1 and CDK2 was also reduced with increased expression of p21 Cip1 and p16 INK4A when treated with YB-1 siRNA. Furthermore, the nuclear YB-1 expression was significantly associated with the level of CDC6 nuclear expression in patients with breast cancer. In conclusion, YB-1 plays an important role in cell cycle progression at G1/S of human cancer cells. YB-1 thus could be a potent biomarker for tumour growth and cell cycle in its close association with CDC6.

Identification of cdc25 gene in pinewood nematode, Bursaphelenchus xylophilus, and its function in reproduction.

The cdc25 gene, which is highly conserved in many eukaryotes, encodes a phosphatase that plays essential roles in cell cycle regulation. We identified a cdc25 ortholog in the pinewood nematode, Bursaphelenchus xylophilus. The B. xylophilus ortholog (Bx-cdc25) was found to be highly similar to Caenorhabditis elegans cdc-25.2 in sequence as well as in gene structure, both having long intron 1. The Bx-cdc25 gene was determined to be composed of seven exons and six introns in a 2,580 bp region, and was shown to encode 360 amino acids of a protein containing a highly-conserved phosphatase domain. Bx-cdc25 mRNA was hardly detectable throughout the juvenile stages but was highly expressed in eggs and in both female and male adults. Functional conservation during germline development between C. elegans cdc25 and Bx-cdc25 was revealed by Bx-cdc25 RNA interference in C. elegans.

Cia5a Regulates the Synovial Expression of Pro-inflammatory Cytokines, Proteases, and Nuclear Receptors

Background: The Cia5a locus regulates disease severity and joint damage in pristane-induced arthritis (PIA). Synovial transcriptome differences between DA (severe disease) and DA.F344(Cia5a) congenic rats (mild disease) could facilitate the identification of the Cia5a gene and point to novel pathways involved in the regulation of arthritis severity and joint damage. Methods: RNA from DA (n=6) and DA.F344 (Cia5a) (n=8) synovial tissue collected 21 days post-PIA was used for cRNA transcription and labeling. Samples were hybridized to RatRef-12 BeadChips (21,922 genes), scanned on a BeadArray reader, and normalized using BeadStudio 2.0 (Illumina). Genes with a difference between DA and DA.F344 (Cia5a) of ≥1.5-fold and P value≤0.01 (t-test) were used for pathway detection analyses using IPA 5.5.1 (Ingenuity) and NCBI databases. Results: A total of 8029 genes were expressed in all DA.F344(Cia5a) and DA samples. A total of 1416 genes were significantly up-regulated in DA, including cytokines (Il18 and Ltb), cytokine receptors (Ifngr1, Ifngr2, and Il17ra), innate immunity mediators (Tlr2, Fcer1g, Fcgr3a, and Ncf1), proliferation and survival genes (Apaf1, Casp3, and Cdc2), and proteases (Mmp3, Mmp14, and Mmp19). A total of 1281 genes were up-regulated in synovial samples from DA.F344(Cia5a) congenics, including nuclear receptors (Pparg, Lxra, Lxrg, Rxrg, and Rora) and lipid metabolism genes. A total of 83 differentially expressed genes mapped to the Cia5a interval. Conclusions: F344 alleles at Cia5a are associated with significant reduction of inflammatory and proliferative gene expression signatures observed in DA, and with increased expression of anti-inflammatory nuclear receptors. These observations suggest a potential role for the Cia5a gene in maintaining synovial homeostasis and limiting the synovial inflammatory response that contributes to articular damage.

The germinal centre kinase Don3 triggers the dynamic rearrangement of higher‐order septin structures during cytokinesis in Ustilago maydis

The dimorphic phytopathogenic fungus Ustilago maydis grows in its haploid phase by budding. Cytokinesis and separation of daughter cells are accomplished by the consecutive formation of two distinct septa. Here, we show that both septation events involve the dynamic rearrangement of septin assemblies from hourglass-shaped collars into ring-like structures. Using a chemical genetic approach we demonstrate that the germinal centre kinase Don3 triggers this septin reorganization during secondary septum formation. Although chemical inhibition of an analogue-sensitive version of Don3 prevented septation, a stable septin collar was assembled at the presumptive septation site. Interestingly, the essential light chain of type II myosin, Cdc4, was already associated with this septin collar. Release of Don3 kinase inhibition triggered immediate dispersal of septin filaments and concomitant incorporation of Cdc4 into a contractile actomyosin ring, which also contained the F-BAR domain protein Cdc15. Inhibition of actin polymerization or deletion of the cdc15 gene, did not affect assembly of the initial collar consisting of septin and myosin light chain. However, reassembly of septin filaments into a ring-like structure was prevented in the absence of either F-actin or Cdc15, indicating that septin ring formation in U. maydis depends on a functional contractile actomyosin ring.

Molecular characterization and polyadenylation‐regulated expression of cyclin B1 and Cdc2 in porcine oocytes and early parthenotes

Meiotic maturation of mammalian oocytes is controlled by the maturation/M-phase promotion factor (MPF), a complex of Cdc2 kinase and cyclin B protein. To better understand the molecular mechanism of oocyte maturation, we characterized porcine cyclin B1 and Cdc2 genes, both of which are widely expressed in pig tissues. We further analyzed their expression profiles during in vitro maturation of pig oocyte and early embryonic development at both the mRNA and protein level. Two isoforms of cyclin B1, comprising the same open reading frame but differing in 3'-UTR length, were identified. Cyclin B1 transcripts was up-regulated after 30 hr of maturation, while Cdc2 mRNA levels were unchanged during maturation except for a sharp decline at 44 hr. Cyclin B1 protein synthesis increased with oocyte maturation. Cdc2 protein expression was relatively low during 0-18 hr, followed by a higher level of expression up to 44 hr of maturation. Poly(A)-test PCR clearly revealed that both cyclin B1 isoforms underwent cytoplasmic polyadenylation starting around 18-24 hr during maturation, while a substantial de-adenylation and degradation of Cdc2 isoforms were observed in metaphase II oocytes and during embryo development after parthenogenetic activation. Porcine MII oocytes derived from small follicles (< or = 3 mm) and bad quality 2-cell parthenotes showed lower developmental competence and lower levels of cyclin B1 protein, and Cdc2 mRNA or both gene mRNAs, respectively, compared to their control counterparts. These results suggested that cyclin B1 was regulated posttranscriptionally by cytoplasmic polyadenylation during porcine oocyte maturation. Further, the decreased expression of maternal cyclin B1 and Cdc2 at the mRNA or protein level in developmentally incompetent oocytes and embryos was responsible for, at least in part, a profound defect in further embryonic development.