Phosphorylation of MCM4 by Cdc7 kinase facilitates its interaction with Cdc45 on the chromatin

Cdc7 kinase, conserved from yeasts to human, plays important roles in DNA replication. However, the mechanisms by which it stimulates initiation of DNA replication remain largely unclear. We have analyzed phosphorylation of MCM subunits during cell cycle by examining mobility shift on SDS-PAGE. MCM4 on the chromatin undergoes specific phosphorylation during S phase. Cdc7 phosphorylates MCM4 in the MCM complexes as well as the MCM4 N-terminal polypeptide. Experiments with phospho-amino acid-specific antibodies indicate that the S phase-specific mobility shift is due to the phosphorylation at specific N-terminal (S/T)(S/T)P residues of the MCM4 protein. These specific phosphorylation events are not observed in mouse ES cells deficient in Cdc7 or are reduced in the cells treated with siRNA specific to Cdc7, suggesting that they are mediated by Cdc7 kinase. The N-terminal phosphorylation of MCM4 stimulates association of Cdc45 with the chromatin, suggesting that it may be an important phosphorylation event by Cdc7 for activation of replication origins. Deletion of the N-terminal non-conserved 150 amino acids of MCM4 results in growth inhibition, and addition of amino acids carrying putative Cdc7 target sequences partially restores the growth. Furthermore, combination of MCM4 N-terminal deletion with alanine substitution and deletion of the N-terminal segments of MCM2 and MCM6, respectively, which contain clusters of serine/threonine and are also likely targets of Cdc7, led to an apparent nonviable phenotype. These results are consistent with the notion that the N-terminal phosphorylation of MCM2, MCM4, and MCM6 may play functionally redundant but essential roles in initiation of DNA replication.     

ATP-generating glycolytic substrates prevent N-nitrosofenfluramine-induced cytotoxicity in isolated rat hepatocytes

The relationship between cytotoxicity induced by N-nitrosofenfluramine and mitochondrial or glycolytic adenosine triphosphate (ATP) synthesis-dependent intracellular bioenergetics was studied in isolated rat hepatocytes. The supplementation of fructose, an ATP-generating glycolytic substrate, to hepatocyte suspensions prevented N-nitrosofenfluramine-induced cell injury accompanied by the formation of cell blebs, abrupt loss of intracellular ATP and reduced glutathione and mitochondrial membrane potential (DeltaPsi), and the accumulation of oxidized glutathione and malondialdehyde, indicating lipid peroxidation, during a 2h incubation period. Fructose (1-20mM) resulted in concentration-dependent protection against the cytotoxicity of N-nitrosofenfluramine at a concentration of 0.6mM, a low toxic dose. Pretreatment with xylitol, another glycolytic substrate, at concentration of 15mM also prevented the cytotoxicity caused by the nitroso compound, but neither glucose nor sucrose exhibited protective effects. In addition, fructose inhibited N-nitrosofenfluramine (0.5 and 0.6mM)-induced DNA damage, as evaluated in the comet assay, indicating that nuclei as well as mitochondria are target sites of the compound. These results indicate that (a) the onset of N-nitrosofenfluramine-induced cytotoxicity in rat hepatocytes is linked to mitochondrial failure, and that (b) the insufficient supply of ATP in turn limits the activities of all energy-requiring reactions and consequently leads to acute cell death.     

Cutting Edge: Pivotal function of Ubc13 in thymocyte TCR signaling

The Ubc13 E2 ubiquitin-conjugating enzyme is essential for BCR-, TLR-, and IL-1 receptor (IL-1R)-mediated immune responses. Although Ubc13-deficient mice show defects in BCR-, TLR/IL-1R-, or CD40-mediated activation of mitogen-activated protein kinases, the function of Ubc13 in TCR-mediated signaling and responses remains uncertain. To address this, we here generated T cell-specific conditional Ubc13-deficient mice. The frequency of T lymphocytes was severely reduced in spleens from Ubc13-deficient mice. Moreover, Ubc13-deficient thymocytes displayed defective proliferation in response to anti-CD3/CD28 or PMA/ionophore stimulation. Regarding the signal transduction, although NF-kappaB activation was modestly affected, PMA/ionophore-induced activation of Jnk and p38 was profoundly impaired in Ubc13-deficient thymocytes. In addition, PMA/ionophore-mediated ubiquitination of NF-kappaB essential modulator (NEMO)/IkappaB kinase gamma (IKKgamma) and phosphorylation of TGF-beta-activated kinase 1 (TAK1) were nearly abolished in Ubc13-deficient thymocytes. Thus, Ubc13 plays an important role in thymocyte TCR-mediated signaling and immune responses.     

Acidianus manzaensis sp. nov., a Novel Thermoacidophilic Archaeon Growing Autotrophically by the Oxidation of H2 with the Reduction of Fe3+

A novel thermoacidophilic iron-reducing Archaeon, strain NA−1, was isolated from a hot fumarole in Manza, Japan. Strain NA-1 could grow autotrophically using H₂ or S⁰ as an electron donor and Fe³⁺ as an electron acceptor, and also could grow heterotrophically using some organic compounds. Fe³⁺ and O₂ served as electron acceptors for growth. However, S⁰, NO₃ ⁻, NO₂ ⁻, SO₄ ²⁻, Mn⁴⁺, fumarate, and Fe₂O₃ did not serve as electron acceptors. The ranges of growth temperature and pH were 60–90°C (optimum: 80°C) and pH 1.0–5.0 (optimum: pH 1.2–1.5), respectively. Cells were nearly regular cocci with an envelope comprised of the cytoplasmic membrane and a single outer S-layer. The crenarchaeal-specific quinone (cardariellaquinone) was detected, and the genomic DNA G + C content was 29.9 mol%. From 16S rDNA analysis, it was determined that strain NA-1 is closely related to Acidianus ambivalens (93.1%) and Acidianus infernus (93.0%). However, differences revealed by phylogenetic and phenotypic analyses clearly show that strain NA-1 represents a new species, Acidianus manzaensis, sp. nov., making it the first identified thermoacidophilic iron-reducing microorganism (strain NA-1^T = NBRC 100595 = ATCC BAA 1057). Strain NA-1 has been deposited in the culture collections of the National Institute of Technology and Evolution (NBRC 100595) and American Type Culture Collection (ATCC BAA 1057). The 16S rDNA sequence has been deposited at GenBank under accession number AB182498.     

Identification of human UDP-glucuronosyltransferase isoform(s) responsible for the C-glucuronidation of phenylbutazone

Glucuronidation is a major metabolic pathway in the biotransformation of many xenobiotics and endogeneous compounds. There have been many studies on the formation of O-, N- or S-glucuronides and identification of the UDP-glucuronosyltransferase (UGT) isoforms responsible for the formation of these glucuronides. However, there is no information available on which UGT isoform(s) catalyzes C-glucuronidation. In the present study, 16 human UGTs (UGTs 1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B11, 2B15, 2B17 and 2B28) were cloned and expressed in baculovirus-infected insect cells and investigated to determine their C-glucuronidating activity toward phenylbutazone (PB). Among the UGT isoforms investigated, only UGT1A9 catalyzed PB C-glucuronidation. Human liver and kidney microsomes, which are well known to express UGT1A9, had C-glucuronidating activity toward PB. However, the jejunum, which did not express UGT1A9, had no C-glucuronidating activity. These results demonstrate for the first time that PB C-glucuronidation is catalyzed by only UGT1A9.     

Hormonal stimulation increases the recruitment of bone marrow-derived myoepithelial cells and periductal fibroblasts into the mammary gland

Recent reports have revealed that bone marrow (BM)-derived cells can be constituents in a number of organs, especially in remodeling tissue. Using bone marrow transplantation (BMT) technique, we found that BM can serve as a source of both myoepithelial cells and periductal fibroblasts in the mammary gland. The numbers of BM-derived myoepithelial cell were 4.8-fold, and those of periductal fibroblast were 2.4-fold higher in the mice when BMT which was performed at the pubertal stage, as compared with BMT was performed at the postpubertal stage. Treatment with estrogen+progesterone pellet increased numbers of BM-derived myoepithelial cells and periductal fibroblasts, to levels 4.5- and 2.6-fold higher than in placebo mice, respectively. In situ hybridization revealed BM-derived periductal fibroblasts expressed insulin-like growth factor I mRNAs that are known to regulate mammary gland. These results suggest that drastic structural change that is induced by hormonal stimulation increased the recruitment of BM-derived myoepithelial cells and periductal fibroblasts to the mammary gland context.     

Vascular endothelial growth factor restores erectile function through modulation of the insulin-like growth factor system and sex hormone receptors in diabetic rat

Previous studies have shown that intracavernous injection of vascular endothelial growth factor (VEGF) restored erectile function in diabetic rats. However, the mechanism of VEGF in diabetes-related erectile dysfunction (ED) has not been fully investigated. We hypothesize that intracavernous injection of VEGF may reverse diabetes-related ED through modulation of the insulin-like growth factor system and sex hormone receptors. To test this hypothesis the erectile function of treated and control rats was analyzed by measurement of intracavernous pressure (ICP) following electrostimulation of the cavernous nerves. Mean ICP was significantly lower in non-treated diabetic rats compared to controls. After VEGF injection, ICP was significantly higher than in non-treated diabetic rats. IGFBP-3 mRNA and protein expression was significantly higher in non-treated diabetic rat crura than controls, while VEGF-treated animals had control levels. ER-beta and PR mRNA and protein expression was significantly lower in non-treated diabetic rat crura. After VEGF injection, ER-beta and PR mRNA and protein expression was similar to control levels. Expression of AR and ER-alpha was the same in all groups. These findings suggest that orthotopic injection of VEGF may improve the functional recovery of diabetes-related ED through modulation of the insulin-like growth factor system and sex hormone receptors. To our knowledge, this is the first study demonstrating that VEGF treatment restores erectile function through restoration of the insulin-like growth factor system and sex hormone receptor genes at the mRNA and protein levels in diabetic rat crura. These results may be important in understanding the pathogenesis of diabetes-related ED and also in providing better strategies for management of this disease.     

Verotoxin-1 stimulation of macrophage-like THP-1 cells up-regulates tissue factor expression through activation of c-Yes tyrosine kinase: Possible signal transduction in tissue factor up-regulation

Verotoxin (VT)-producing Escherichia coli (E. coli) O157:H7 infections are frequently complicated by thrombotic angiopathy, hemolytic uremic syndrome (HUS) and neurological symptoms. The present data demonstrate that VT-1 (Shiga toxin) stimulation of macrophage-like THP-1 cells up-regulates the activity, antigen and mRNA levels of tissue factor (TF), a key cofactor of the coagulation-inflammation-thrombosis circuit. This up-regulation is accompanied by phosphorylation of phosphatidylinositol 3-kinase (PI3-kinase), IkappaB kinase beta (IKKbeta) and extracellular signal-regulated kinase 2 (ERK2). Changes in TF mRNA levels were in parallel with the activation of NF-kappaB/Rel and Egr-1 activation, but not with AP-1. Inhibition of PI3-kinase attenuated VT-1-induced phosphorylation of IKKbeta and ERK2, and the up-regulation of TF mRNA levels. VT-1 stimulation rapidly activated c-Yes tyrosine kinase, a member of the Src family. Treatment of the cells with c-Yes antisense oligos attenuated the VT-1-induced phosphorylation of PI3-kinase, IKKbeta and ERK2, activations of NF-kappaB/Rel and Egr-1, and up-regulation of TF mRNA levels. These results suggest that VT-1-induced macrophage stimulation activates c-Yes, which then up-regulates TF expression through activation of the IKKbeta/proteasome/NF-kappaB/Rel and MEK/ERK2/Egr-1 pathways via activation of PI3-kinase. Induction of macrophage TF expression by VT-1 may play an important role in the acceleration of the coagulation-inflammation-thrombosis circuit during infections by VT-producing E. coli.