Effects of hydroxyurea and aphidicolin on phosphorylation of ataxia telangiectasia mutated on Ser 1981 and histone H2AX on Ser 139 in relation to cell cycle phase and induction of apoptosis.

BACKGROUND: DNA replication stress often induces DNA damage. The antitumor drug hydroxyurea (HU), a potent inhibitor of ribonucleotide reductase that halts DNA replication through its effects on cellular deoxynucleotide pools, was shown to damage DNA inducing double-strand breaks (DSBs). Aphidicolin (APH), an inhibitor of alpha-like DNA polymerases, was also reported to cause DNA damage, but the evidence for induction of DSBs by APH is not straightforward. Histone H2AX is phosphorylated on Ser 139 in response to DSBs and one of the protein kinases that phosphorylate H2AX is ataxia telangiectasia mutated (ATM); activation of ATM is through its phosphorylation of Ser 1981. The present study was undertaken to reveal whether H2AX is phosphorylated in cells exposed to HU or APH and whether its phosphorylation is mediated by ATM. MATERIALS AND METHODS: HL-60 cells were treated in cultures with 0.1-5.0 mM HU or 1-4 muM APH for up to 5 h. Activation of ATM and H2AX phosphorylation was detected immunocytochemically using Ab specific to Ser1981-ATM or Ser 139-H2AX epitopes, respectively, concurrent with measurement of cellular DNA content. RESULTS: While exposure of cells to HU led to H2AX phosphorylation selectively during S phase and the cells progressing through the early portion of S (DI = 1.1-1.4) were more affected than late-S phase (DI = 1.6-1.9) cells, ATM was not activated by HU. In fact, the level of constitutive ("programmed") ATM phosphorylation was distinctly suppressed, in all phases of the cell cycle, at 0.1-5.0 mM HU. Cells' exposure to APH also resulted in H2AX phosphorylation at Ser139 with no evidence of ATM activation, and as in the case of HU, the early-S cells were more affected than the late-S phase cells. The rise in frequency of apoptotic cells became apparent after 2 h of exposure to HU or APH, and all apoptotic cells had markedly elevated levels of both H2AX-Ser139 and ATM-Ser1981 phosphorylation. CONCLUSIONS: The lack of correlation between H2AX phosphorylation and ATM activation indicates that protein kinase(s) other than ATM (ATR and/or DNA-dependent protein kinase) are activated by DSBs induced by replication stress. Interestingly, HU inhibits the constitutive ("programmed") level of ATM phosphorylation in untreated cells. However, DNA fragmentation during apoptosis activates ATM and dramatically increases level of H2AX phosphorylation.     

Construction of a binary transgenic gene expression system for recombinant protein production in the middle silk gland of the silkworm <Emphasis Type="Italic">Bombyx mori</Emphasis>

To construct an efficient system for the production of recombinant proteins in silkworm (Bombyx mori), we investigated the promoter activity of the silkworm sericin 1, 2, and 3 genes (Ser1, Ser2, and Ser3) using a GAL4/UAS binary gene expression system in transgenic silkworm. The promoter activity of the upstream region of Ser1 was strong, yielding high expression of an enhanced green fluorescent protein (EGFP) transgene in the middle and posterior regions of the middle silk gland (MSG) after day 2 of the fifth instar. The Ser3 upstream region exhibited moderate promoter activity in the anterior MSG, but the Ser2 upstream region did not exhibit any promoter activity. Since the strongest promoter activity was observed for Ser1, we devised a system for the production of recombinant proteins using a GAL4–Ser1 promoter construct (Ser1-GAL4). Transgenic silkworms harboring both the Ser1-GAL4 construct and the previously reported upstream activating sequence (UAS)–EGFP construct, which contains the TATA box region of the Drosophila hsp70 gene, yielded approximately 100 μg EGFP per larva. When we then analyzed the TATA box region, signal peptide, and intron sequences for their effects on production from the UAS-EGFP construct, we found that the optimization of these sequences effectively increased production to an average of 500 μg EGFP protein per transgenic larva. We conclude that this binary system is a useful tool for the mass production of recombinant proteins of biomedical and pharmaceutical interest in silkworm.     

Enhanced activation of T lymphocytes by urease-deficient recombinant bacillus Calmette-Guérin producing heat shock protein 70-major membrane protein-II fusion protein

To activate naive T cells convincingly using Mycobacterium bovis bacillus Calmette-Guérin (BCG), recombinant BCG (BCG-D70M) that was deficient in urease, expressed with gene encoding the fusion of BCG-derived heat shock protein (HSP) 70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed. BCG-D70M was more potent in activation of both CD4(+) and CD8(+) subsets of naive T cells than recombinant BCGs including urease-deficient BCG and BCG-70M secreting HSP70-MMP-II fusion protein. BCG-D70M efficiently activated dendritic cells (DCs) to induce cytokine production and phenotypic changes and activated CD4(+) T cells even when macrophages were used as APCs. The activation of both subsets of T cells was MHC and CD86 dependent. Pretreatment of DCs with chloroquine inhibited both surface expression of MMP-II on DCs and the activation of T cells by BCG-D70M-infected APCs. The naive CD8(+) T cell activation was inhibited by treatment of DCs with brefeldin A and lactacystin so that the T cell was activated by TAP- and proteosome-dependent cytosolic cross-priming pathway. From naive CD8(+) T cells, effector T cells producing perforin and memory T cells having migration markers were produced by BCG-D70M stimulation. BCG-D70M primary infection in C57BL/6 mice produced T cells responsive to in vitro secondary stimulation with MMP-II and HSP70 and more efficiently inhibited the multiplication of subsequently challenged M. leprae than vector control BCG. These results indicate that the triple combination of HSP70, MMP-II, and urease depletion may provide a useful tool for inducing better activation of naive T cells.     

A CD36-related Transmembrane Protein Is Coordinated with an Intracellular Lipid-binding Protein in Selective Carotenoid Transport for Cocoon Coloration

The transport pathway of specific dietary carotenoids from the midgut lumen to the silk gland in the silkworm, Bombyx mori, is a model system for selective carotenoid transport because several genetic mutants with defects in parts of this pathway have been identified that manifest altered cocoon pigmentation. In the wild-type silkworm, which has both genes, Yellow blood (Y) and Yellow cocoon (C), lutein is transferred selectively from the hemolymph lipoprotein to the silk gland cells where it is accumulated into the cocoon. The Y gene encodes an intracellular carotenoid-binding protein (CBP) containing a lipid-binding domain known as the steroidogenic acute regulatory protein-related lipid transfer domain. Positional cloning and transgenic rescue experiments revealed that the C gene encodes Cameo2, a transmembrane protein gene belonging to the CD36 family genes, some of which, such as the mammalian SR-BI and the fruit fly ninaD, are reported as lipoprotein receptors or implicated in carotenoid transport for visual system. In C mutant larvae, Cameo2 expression was strongly repressed in the silk gland in a specific manner, resulting in colorless silk glands and white cocoons. The developmental profile of Cameo2 expression, CBP expression, and lutein pigmentation in the silk gland of the yellow cocoon strain were correlated. We hypothesize that selective delivery of lutein to specific tissue requires the combination of two components: 1) CBP as a carotenoid transporter in cytosol and 2) Cameo2 as a transmembrane receptor on the surface of the cells.     

cDNA cloning and phylogenetic and expression analyses of actin in symbiotic dinoflagellates (Symbiodinium spp.)

Three cDNAs encoding actins were identified in two culturable strains (clades A and F) of the symbiotic dinoflagellates Symbiodinium spp. In a molecular phylogenetic analysis these actin sequences formed a monophyletic group with known dinoflagellate actins, remote from Syact-p that had been isolated from a clade A Symbiodinium strain (HG39). One of the newly identified actin sequences (SyAct-F1) was the most closely related to partial actin cDNA sequences (named AGfact-p and AFcact-p) isolated from adult colonies of two reef corals (Galaxea fascicularis and Favites chinensis) that were inhabited by Symbiodinium spp., suggesting the possibility that the latter two were from the symbionts. Partial AFcact-p sequences could be amplified by PCR using genomic DNA prepared from a symbiotic adult colony of F. chinensis as the template, but not from planula larvae in which zooxanthellae could not be detected, also arguing for the origin of AFcact-p in the symbiont. An expression analysis showed that the levels of the SyAct-A1 mRNA were comparable in symbiotic and non-symbiotic states, and also in motile and non-motile phases in a cultured condition, suggesting its usefulness as a constitutively expressed control gene in expression analysis of Symbiodinium mRNAs.     

Recent Fluctuations in Distribution and Biomass of Cold and Warm Temperature Species of Laminarialean Algae at Cape Ohma, Northern Honshu, Japan

The Cape Ohma region of Shimokita Peninsula, the northernmost point of Honshu Island, Japan, is subject to both the warm Tsugaru Current and the cold Kurile Current. As a result, the Laminarialean flora includes both cold temperature species (Laminaria japonica Areschoug, Kjellmaniella crassifolia Miyabe and Costaria costata (C. Agardh) Saunders) and warm temperature species (Undaria peterseniana (Kjellman) Okamura, Ecklonia stolonifera Okamura), as well as Undaria pinnatifida (Yendo) Okamura, which is distributed in both waters. The frequency of occurrence (as a measure of distribution) and the biomass of these species were recorded in June 1976 (at 50 points in depths between 8–30 m), July 1988 (192 points, 2.5–25 m) and July 2001 (78 points, 2.5–25 m). Comparison of these data revealed a decrease in cold temperature species and an increase in warm temperature species from 1976 or 1988 to 2001. Long-term data of seawater temperature measured at 5 m depth near the study site showed that mean temperatures in the middle of winter (late January to February) in 1989–2000 were 0.9–1.1 ^∘C higher than those in 1980–1988. Higher seawater temperatures in the last decade appear to have affected the frequency of occurrence and biomass of the Laminarialean species along the coasts of Cape Ohma. This result supported our previous conclusion that 1 ^∘C higher mean seawater temperature in late January caused a decrease in the biomass of L. japonica (by ca. 64%) along the same coast.     

IL-15 regulates CD8+ T cell contraction during primary infection

During the course of acute infection with an intracellular pathogen, Ag-specific T cells proliferate in the expansion phase, and then most of the T cells die by apoptosis in the following contraction phase, but the few that survive become memory cells and persist for a long period of time. Although IL-15 is known to play an important role in long-term maintenance of memory CD8+ T cells, the potential roles of IL-15 in CD8+ T cell contraction are not known. Using an adoptive transfer system of OT-I cells expressing OVA257-264/Kb-specific TCR into control, IL-15 knockout (KO) and IL-15 transgenic (Tg) mice followed by challenge with recombinant Listeria monocytogenes expressing OVA, we found that the survival of CD44+CD62L-CD127- effector OT-I cells during the contraction phase is critically dependent on IL-15. In correlation with the expression level of Bcl-2 in OT-I cells, the number of OT-I cells was markedly reduced in IL-15 KO mice but remained at a high level in IL-15 Tg mice during the contraction phase, compared with control mice. In vivo administration of rIL-15 during the contraction phase in IL-15 KO mice inhibited the contraction of effector OT-I cells accompanied by up-regulation of Bcl-2 expression. Furthermore, enforced expression of Bcl-2 protected the majority of effector OT-I cells from death in IL-15 KO mice after infection. These results suggest that IL-15 plays a critical role in protecting effector CD8+ T cells from apoptosis during the contraction phase following a microbial infection via inducing antiapoptotic molecules.     

Neverland is an evolutionally conserved Rieske-domain protein that is essential for ecdysone synthesis and insect growth

Steroid hormones mediate a wide variety of developmental and physiological events in multicellular organisms. During larval and pupal stages of insects, the principal steroid hormone is ecdysone, which is synthesized in the prothoracic gland (PG) and plays a central role in the control of development. Although many studies have revealed the biochemical features of ecdysone synthesis in the PG, many aspects of this pathway have remained unclear at the molecular level. We describe the neverland (nvd) gene, which encodes an oxygenase-like protein with a Rieske electron carrier domain, from the silkworm Bombyx mori and the fruitfly Drosophila melanogaster. nvd is expressed specifically in tissues that synthesize ecdysone, such as the PG. We also show that loss of nvd function in the PG causes arrest of both molting and growth during Drosophila development. Furthermore, the phenotype is rescued by application of 20-hydroxyecdysone or the precursor 7-dehydrocholesterol. Given that the nvd family is evolutionally conserved, these results suggest that Nvd is an essential regulator of cholesterol metabolism or trafficking in steroid synthesis across animal phyla.     

Differential regulation of intracellular redox state by extracellular matrix proteins in glomerular mesangial cells: potential role in diabetic nephropathy

Advanced diabetic nephropathy is characterized by abnormal synthesis of extracellular matrix (ECM) proteins, such as collagen I (COL I). The present experiments were designed to test the hypothesis that the presence of abnormal ECM proteins may be responsible for increased generation of reactive oxygen species (ROS) that are thought to have an important role in the pathogenesis of diabetic nephropathy. SV40 MES 13 murine mesangial cells were plated on COL I or collagen IV (COL IV) for 3 h at 5.5 or 25 mM D-glucose concentration. Increased intracellular ROS generation and reduced intracellular nitric oxide (NO) production was measured in cells attached to COL I compared with cells attached to COL IV. Treatment with N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of NO synthase, reduced this difference in ROS generation between cells attached to either COL I or IV. The results using antibodies against integrins also indicated that an alpha(2) integrin-mediated pathway was involved in the different response in ROS generation caused by ECM proteins. These results suggest that contact between altered ECM proteins that are present in advanced diabetic nephropathy and mesangial cells has the potential to increase intracellular oxidative stress, leading to progressive glomerular damage.     

Differential inhibitory effects of mu-opioids on substance P- and capsaicin-induced nociceptive behavior in mice

The antinociceptive mechanisms of the selective mu-opioid receptor agonists [D-Ala2,NMePhe4,Gly(ol)5]enkephalin (DAMGO), H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA) or H-Tyr-D-Arg-Phe-beta-Ala-NH2 (TAPA-NH2) against substance P (SP)- or capsaicin-elicited nociceptive behaviors was investigated in mice. DAMGO, TAPA or TAPA-NH2 given intrathecally inhibited the nociceptive behaviors elicited by intrathecally administered SP or capsaicin, and these antinociceptive effects were completely eliminated by intrathecal co-administration with D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a selective mu-opioid receptor antagonist. Pretreatment subcutaneously with naloxonazine, a selective mu1-opioid receptor antagonist, partially attenuated the antinociceptive effect of TAPA-NH2, but not DAMGO and TAPA, against SP. However, the antinociception induced by TAPA, but not DAMGO and TAPA-NH2, against capsaicin was significantly inhibited by naloxonazine. On the other hand, co-administration intrathecally with Tyr-D-Pro-Trp-Gly-NH2 (D-Pro2-Tyr-W-MIF-1), a selective mu2-opioid receptor antagonist, significantly attenuated the antinociceptive effects of DAMGO, but not TAPA and TAPA-NH2, against capsaicin, while the antinociceptions induced by three opioid peptides against SP were significantly inhibited by D-Pro2-Tyr-W-MIF-1. These results suggest that differential inhibitory mechanisms on pre- and postsynaptic sites in the spinal cord contribute to the antinociceptive effects of the three mu-opioid peptides.