Gemcitabine alters sialic acid binding of the glycocalyx and induces inflammatory cytokine production in cultured endothelial cells.

Gemcitabine (GEM) is an anticancer drug inhibiting DNA synthesis. Glomerular thrombotic microangiopathy (TMA) has been reported as an adverse effect. However, the precise mechanism of GEM-induced endothelial injury remains unknown. Cultured human umbilical vein endothelial cells (HUVECs) in the confluent phase were exposed to GEM (5-100 µM) for 48 h and evaluated cell viability and morphology, lectin binding concerning sialic acid of endothelial glycocalyx (GCX), and immunofluorescent staining of platelet-endothelial cell adhesion molecule (PECAM) and vascular endothelial growth factor receptor 2 (VEGFR2). The mRNA expression of α2,6-sialyltransferase (ST6Gal1), sialidase (neuraminidase-1: NEU-1), and interleukin (IL)-1ß and IL-6 was also evaluated. GEM exposure at 5 µM induced cellular shrinkage and intercellular dissociation, accompanied by slight attenuation of PECAM and VEGFR2 immunostaining, although cell viability was still preserved. At this concentration, lectin binding showed a reduction of terminal sialic acids in endothelial GCX, probably associated with reduced ST6Gal1 mRNA expression. IL-1ß and IL-6 mRNA expression was significantly increased after GEM exposure. GEM reduced terminal sialic acids in endothelial GCX through mRNA suppression of ST6Gal1 and induced inflammatory cytokine production in HUVECs. This phenomenon could be associated with the mechanism of GEM-induced TMA.

High concentration of extracellular nucleotides suppresses cell growth via delayed cell cycle progression in cancer and noncancer cell lines.

Tumor necrosis frequently occurs in malignant tumors, showing rapid growth and invasion. This phenomenon is generally regarded as simple ischemic necrosis due to insufficient tumor vessels and blood supply. However, the necrotic tissue contains high amount of nuclear substances, DNA, and nucleoproteins that may affect the surrounding tumor cells by promoting or suppressing the tumor cell growth in vivo. This study focused on the effects of an externally administered water-soluble nuclear crude extract (SNE) containing nuclear protein and oligonucleotides on several human cancer and noncancer cell lines. The results demonstrated that the SNE suppressed cell growth in cancer and noncancer cells in vitro. Through the flow cytometry analysis of the nuclear DNA content, it was observed that the SNE increased and decreased cell proportion in the S and G2/M phases, respectively, thereby suggesting that the cell growth inhibition was due to cell cycle delay, and not due to apoptosis. These studies suggest that the high-concentration of extracellular nucleotides generated as a result of tumor necrosis and/or released from infiltrated neutrophils could suppress the growth of surrounding cancer and intrinsic cells, which provides us some insights into an alternative anticancer strategy for patients with highly malignant necrotic tumor.

Identification of dynamin-2-mediated endocytosis as a new target of osteoporosis drugs, bisphosphonates.

Nitrogen-containing bisphosphonates are pyrophosphate analogs that have long been the preferred prescription for treating osteoporosis. Although these drugs are considered inhibitors of prenylation and are believed to exert their effects on bone resorption by disrupting the signaling pathways downstream of prenylated small GTPases, this explanation seems to be insufficient. Because other classes of prenylation inhibitors have recently emerged as potential antiviral therapeutic agents, we first investigated here the effects of bisphosphonates on simian virus 40 and adenovirus infections and, to our surprise, found that viral infections are suppressed by bisphosphonates through a prenylation-independent pathway. By in-house affinity-capture techniques, dynamin-2 was identified as a new molecular target of bisphosphonates. We present evidence that certain bisphosphonates block endocytosis of adenovirus and a model substrate by inhibiting GTPase activity of dynamin-2. Hence, this study has uncovered a previously unknown mechanism of action of bisphosphonates and offers potential novel use for these drugs.

Establishment of an improved mouse model for infantile neuroaxonal dystrophy that shows early disease onset and bears a point mutation in Pla2g6.

Calcium-independent group VIA phospholipase A(2) (iPLA(2)beta), encoded by PLA2G6, has been shown to be involved in various physiological and pathological processes, including immunity, cell death, and cell membrane homeostasis. Mutations in the PLA2G6 gene have been recently identified in patients with infantile neuroaxonal dystrophy (INAD). Subsequently, it was reported that similar neurological impairment occurs in gene-targeted mice with a null mutation of iPLA(2)beta, whose disease onset became apparent approximately 1 to 2 years after birth. Here, we report the establishment of an improved mouse model for INAD that bears a point mutation in the ankyrin repeat domain of Pla2g6 generated by N-ethyl-N-nitrosourea mutagenesis. These mutant mice developed severe motor dysfunction, including abnormal gait and poor performance in the hanging grip test, as early as 7 to 8 weeks of age, in a manner following Mendelian law. Neuropathological examination revealed widespread formation of spheroids containing tubulovesicular membranes similar to human INAD. Molecular and biochemical analysis revealed that the mutant mice expressed Pla2g6 mRNA and protein, but the mutated Pla2g6 protein had no glycerophospholipid-catalyzing enzyme activity. Because of the significantly early onset of the disease, this mouse mutant (Pla2g6-inad) could be highly useful for further studies of pathogenesis and experimental interventions in INAD and neurodegeneration.

Development of a chemical screening system using aqueorin-fused protein.

We developed a unique screening system that consists of combination of high photo-sensitivity of photoprotein aequorin (AQ) and our developed high-performance affinity purification system. In the present study, we demonstrated to detect the specific interaction between methotrexate (MTX) and its target dihydrofolate reductase (DHFR) fused with AQ. We succeeded to prepare highly purified AQ-fused DHFR, which showed high sensitive light emission. To test the screening system, we prepared the complex of MTX-immobilized magnetic nanobeads and AQ-fused DHFR. Bound AQ-fused DHFR with the beads was specifically released by addition of MTX. Thus, this methodology enables us to search a novel chemical that binds to target proteins without complicated processes. Furthermore, thank to the highly sensitive luminescence intensity of AQ, this methodology would be performed in very small scale with high responsibility, leading to development of high throughput screening systems.

Bromodomain factor 1 (Bdf1) is phosphorylated by protein kinase CK2.

Bromodomain factor 1 (Bdf1) associates with Saccharomyces cerevisiae TFIID and corresponds to the C-terminal half of higher eukaryotic TAF1. It also associates with the SWR-C complex, which is important for Htz1 deposition. Bdf1 binds preferentially to acetylated histone H4. Bdf1 is phosphorylated, but the mechanism and significance of this modification have been unclear. Two distinct regions within Bdf1 are phosphorylated; one is just C terminal to the bromodomains and the other is near the C terminus. Mutational analysis shows that phosphorylation is necessary for Bdf1 function in vivo. Endogenous protein kinase CK2 purifies with Bdf1 and phosphorylates both domains. A similar mechanism may be responsible for phosphorylation of the C-terminal region of mammalian TAF1. These findings suggest that CK2 phosphorylation of Bdf1 may regulate RNA polymerase II transcription and/or chromatin structure.

A nucleoprotein-enriched diet suppresses dopaminergic neuronal cell loss and motor deficit in mice with MPTP-induced Parkinson's disease.

Parkinson's disease (PD) is an obstinate progressive neurodegenerative disease and characterized by locomotor impairment and dopaminergic neuronal degeneration in the substantia nigra pars compacta (SNc). We examined in here the dietary effect of nucleoprotein (NP) extracted from salmon soft roe on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-injected PD-like mice model to prevent the symptom as an alternative medicine. Male C57/BL6 mice were given either an artificially modified NP-free diet (NF) or NF supplied with 1.2% NP for 1 week. Then, mice were injected intraperitoneally four times with 20 mg/kg MPTP. Seven days later, locomotor activity was examined, and the brains were immunostained with tyrosine hydroxylase (TH) and Iba1 antibodies. Moreover, in situ detection of superoxide anion (O2(-)) and gene expression of mitochondrial electron transfer chain gene, Cox8b was evaluated in midbrains. NP-fed animals showed significantly reduced locomotor impairment and an increased number of TH-positive cells in the SNc compared with NF animals. The NP-fed animals also showed reduced lower levels of O2(-) and up-regulation of Cox8b levels and Iba1 immunoreactivity, suggesting that inflammation and oxidative stress were suppressed and mitochondrial impairment was relieved in these animals. Supplementation of the diet with NP may serve as a useful preventive measure to slow the onset of PD.

Seeking gene candidates responsible for developmental origins of health and disease.

Human epidemiological evidence has led scientists to theorize that undernutrition during gestation is an important early origin of adult diseases. Animal models have successfully demonstrated that maternal diet could contribute to some adult diseases. Undernutrition is perceived harmful in pregnant women, whereas calorie restriction is a strategy proven to extend healthy and maximum lifespan in adult. This diagrammatically opposite effect of nutritional condition might provide us with hints to search for genes underlying health conditions. Here, we have initiated a study examining the effect of undernutrition on maternal and fetal livers, utilizing high-throughput DNA microarray analysis for screening genome-wide changes in their transcriptomes. Briefly, pregnant mice were exposed to food deprivation (FD) on gestation day (GD) 17, and cesarean section was performed on GD18. Control mice were supplied with chow ad libitum until sacrifice. Total RNA extracted from mother and fetal livers for each control and treatment (FD) was analyzed with an Agilent mouse whole genome DNA chip. A total of 3058 and 3126 up- (>1.5-fold) and down- (<0.75-fold) regulated genes, and 1475 and 1225 up- (>1.5-fold) and down- (<0.75-fold) regulated genes showed differential expression at the mRNA level, in the maternal and fetal livers, respectively. Interestingly, 103 genes up-regulated in the mother were down-regulated in the fetus, whereas 108 down-regulated maternal genes were up-regulated in the fetus; these 211 genes are potential candidates related to longevity or health. The role of some of these genes, in context of the proposed mechanisms for developmental origins of health and disease is discussed.

Fucoidan therapy decreases the proviral load in patients with human T-lymphotropic virus type-1-associated neurological disease.

BACKGROUND: Human T-lymphotropic virus type-1 (HTLV-1) is a human retrovirus that causes HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukaemia (ATL). A higher viral load in individuals with HTLV-1 infection increases their risk of developing HAM/TSP and ATL. Moreover, the high proviral load is associated with the clinical progression of HAM/TSP. Reduction of the number of HTLV-1-infected cells is therefore crucial for preventing and treating HTLV-1-associated diseases. Recently, fucoidan, a complex sulphated polysaccharide derived from marine seaweed, has been demonstrated to exert inhibitory effects on HTLV-1 infection in vitro. In this study, we examined the in vivo effects of fucoidan on HTLV-1 infection. METHODS: In this single-centre open-label trial, 13 patients with HAM/TSP were treated with 6 g fucoidan daily for 6-13 months. The HTLV-1 proviral DNA load and frequencies of HTLV-1-specific CD8(+) T-cells, natural killer cells, invariant natural killer T-cells and dendritic cells in the peripheral blood were analysed. Furthermore, the in vitro inhibitory effect of fucoidan on cell-to-cell HTLV-1 infection was examined by using luciferase reporter cell assays. RESULTS: Fucoidan inhibited the cell-to-cell transmission of HTLV-1 in vitro. Furthermore, fucoidan therapy resulted in a 42.4% decrease in the HTLV-1 proviral load without affecting the host immune cells. During the treatment, no exacerbation was observed. Four patients with HAM/TSP developed diarrhoea, which improved immediately after stopping fucoidan administration. CONCLUSIONS: Fucoidan is a new potential therapeutic agent for the prevention and treatment of HTLV-1-associated diseases.

The Saccharomyces cerevisiae histone H2A variant Htz1 is acetylated by NuA4.

The histone H2A variant H2A.Z (Saccharomyces cerevisiae Htz1) plays roles in transcription, DNA repair, chromosome stability, and limiting telomeric silencing. The Swr1-Complex (SWR-C) inserts Htz1 into chromatin and shares several subunits with the NuA4 histone acetyltransferase. Furthermore, mutants of these two complexes share several phenotypes, suggesting they may work together. Here we show that NuA4 acetylates Htz1 Lys 14 (K14) after the histone is assembled into chromatin by the SWR-C. K14 mutants exhibit specific defects in chromosome transmission without affecting transcription, telomeric silencing, or DNA repair. Function-specific modifications may help explain how the same component of chromatin can function in diverse pathways.

GABP, HCF-1 and YY1 are involved in Rb gene expression during myogenesis.

Muscle cell differentiation, or myogenesis, is a well-characterized process and involves the expression of specific sets of genes in an orderly manner. A prerequisite for myogenesis is the exit from the cell cycle, which is associated with the up-regulation of the tumor suppressor Rb. In this study, we set to investigate the regulatory mechanism of the Rb promoter that allows adequate up-regulation in differentiating myoblasts. We report that Rb expression is regulated by the transcription factors GABP, HCF-1 and YY1. Before induction of differentiation, Rb is expressed at a low level and GABP and YY1 are both present on the promoter. YY1, which exerts an inhibitory effect on Rb expression, is removed from the promoter as cells advance through myogenesis and translocates from the nucleus to the cytoplasm. On the other hand, upon induction of differentiation, the GABP cofactor HCF-1 is recruited to and coactivates the promoter with GABP. RNAi-mediated knock-down of HCF-1 results in inhibition of Rb up-regulation as well as myotube formation. These results indicate that the Rb promoter is subject to regulation by positive and negative factors and that this intricate activation mechanism is critical to allow the accurate Rb gene up-regulation observed during myogenesis.

The Caenorhabditis elegans mRNA 5'-capping enzyme. In vitro and in vivo characterization.

Eukaryotic mRNA capping enzymes are bifunctional, carrying both RNA triphosphatase (RTPase) and guanylyltransferase (GTase) activities. The Caenorhabditis elegans CEL-1 capping enzyme consists of an N-terminal region with RTPase activity and a C-terminal region that resembles known GTases, However, CEL-1 has not previously been shown to have GTase activity. Cloning of the cel-1 cDNA shows that the full-length protein has 623 amino acids, including an additional 38 residues at the C termini and 12 residues at the N termini not originally predicted from the genomic sequence. Full-length CEL-1 has RTPase and GTase activities, and the cDNA can functionally replace the capping enzyme genes in Saccharomyces cerevisiae. The CEL-1 RTPase domain is related by sequence to protein-tyrosine phosphatases; therefore, mutagenesis of residues predicted to be important for RTPase activity was carried out. CEL-1 uses a mechanism similar to protein-tyrosine phosphatases, except that there was not an absolute requirement for a conserved acidic residue that acts as a proton donor. CEL-1 shows a strong preference for RNA substrates of at least three nucleotides in length. RNA-mediated interference in C. elegans embryos shows that lack of CEL-1 causes development to arrest with a phenotype similar to that seen when RNA polymerase II elongation activity is disrupted. Therefore, capping is essential for gene expression in metazoans.

YEAF1/RYBP and YAF-2 are functionally distinct members of a cofactor family for the YY1 and E4TF1/hGABP transcription factors.

The transcription factor hGABP/E4TF1 is a heterotetrameric complex composed of two DNA-binding subunits (hGABP alpha/E4TF1-60) and two transactivating subunits (hGABP beta/E4TF1-53). In order to understand the molecular mechanism of transcriptional regulation by hGABP, we searched for proteins that interact with the non-DNA-binding subunit, hGABP beta, using yeast two-hybrid screening. We identified a human cDNA encoding a protein related to YAF-2 (YY1-associated factor 2), which was previously isolated as an interacting partner of the Ying-Yang-1 (YY1) transcription factor. Reflecting this similarity, both YAF-2 and this novel protein (named YEAF1 for YY1- and E4TF1/hGABP-associated factor-1) interacted with hGABP beta and YY1 in vitro and in vivo, indicating that YEAF1 and YAF-2 constitute a cofactor family for these two structurally distinct transcription factors. By using yeast three-hybrid assay, we demonstrated that hGABP beta and YY1 formed a complex only in the presence of YEAF1, indicating that YEAF1 is a bridging factor of these two transcription factors. These cofactors are functionally different in that YAF-2 positively regulates the transcriptional activity of hGABP but YEAF1 negatively regulates this activity. Also, YAF-2 mRNA is highly expressed in skeletal muscle, whereas YEAF1 mRNA is highly expressed in placenta. We speculate that the transcriptional activity of hGABP is in part regulated by the expression levels of these tissue-specific cofactors. These results provide a novel mechanism of transcriptional regulation by functionally distinct cofactor family members.

Total analysis and purification of cellular proteins binding to cisplatin-damaged DNA using submicron beads.

A high-performance affinity purification technique has been developed for cisplatin (CDDP)-damaged DNA binding proteins directly from crude nuclear extracts of HeLaS3 cell using novel submicron beads synthesized by copolymerization of styrene and glycidyl methacrylate (GMA). The beads dramatically decreased both nonspecific protein adsorption on solid surfaces and elution volume and simplified the handling procedure. Preparation of the beads for purification was carried out by immobilization of telomeric repeats, (TTAGGG)(n), on the surface after the reaction with CDDP. At least nine proteins clearly showed higher affinity to CDDP-DNA and were identified by amino acid sequence analysis including HMGB (high mobility group), hUBF (human upstream binding factor), and Ku autoantigen, which were previously reported to be components of CDDP-damaged DNA binding proteins.

Regulation of chromosome stability by the histone H2A variant Htz1, the Swr1 chromatin remodeling complex, and the histone acetyltransferase NuA4.

NuA4, the only essential histone acetyltransferase complex in Saccharomyces cerevisiae, acetylates the N-terminal tails of histones H4 and H2A. Affinity purification of NuA4 revealed the presence of three previously undescribed subunits, Vid21/Eaf1/Ydr359c, Swc4/Eaf2/Ygr002c, and Eaf7/Ynl136w. Experimental analyses revealed at least two functionally distinct sets of polypeptides in NuA4: (i) Vid21 and Yng2, and (ii) Eaf5 and Eaf7. Vid21 and Yng2 are required for bulk histone H4 acetylation and are functionally linked to the histone H2A variant Htz1 and the Swr1 ATPase complex (SWR-C) that assembles Htz1 into chromatin, whereas Eaf5 and Eaf7 have a different, as yet undefined, role. Mutations in Htz1, the SWR-C, and NuA4 cause defects in chromosome segregation that are consistent with genetic interactions we have observed between the genes encoding these proteins and genes encoding kinetochore components. Because SWR-C-dependent recruitment of Htz1 occurs in both transcribed and centromeric regions, a NuA4/SWR-C/Htz1 pathway may regulate both transcription and centromere function in S. cerevisiae.

A Snf2 family ATPase complex required for recruitment of the histone H2A variant Htz1.

Deletions of three yeast genes, SET2, CDC73, and DST1, involved in transcriptional elongation and/or chromatin metabolism were used in conjunction with genetic array technology to screen approximately 4700 yeast deletions and identify double deletion mutants that produce synthetic growth defects. Of the five deletions interacting genetically with all three starting mutations, one encoded the histone H2A variant Htz1 and three encoded components of a novel 13 protein complex, SWR-C, containing the Snf2 family ATPase, Swr1. The SWR-C also copurified with Htz1 and Bdf1, a TFIID-interacting protein that recognizes acetylated histone tails. Deletions of the genes encoding Htz1 and seven nonessential SWR-C components caused a similar spectrum of synthetic growth defects when combined with deletions of 384 genes involved in transcription, suggesting that Htz1 and SWR-C belong to the same pathway. We show that recruitment of Htz1 to chromatin requires the SWR-C. Moreover, like Htz1 and Bdf1, the SWR-C promotes gene expression near silent heterochromatin.