Hypoxia followed by reoxygenation induces secretion of cyclophilin A from cultured rat cardiac myocytes

We previously reported that hypoxia followed by reoxygenation (hypoxia/reoxygenation) rapidly activated intracellular signaling such as mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated protein kinase (ERK) 1/2, p38MAPK, and stress-activated protein kinases (SAPKs). To investigate the humoral factors which mediate cardiac response to hypoxia/reoxygenation, we analyzed the conditioned media from cardiac myocytes subjected to hypoxia/reoxygenation by two-dimensional electrophoresis and mass spectrometry. We identified cyclophilin A (CyPA) as one of the proteins secreted from cardiac myocytes in response to hypoxia/reoxygenation. Hypoxia/reoxygenation induced the expression of CyPA and its cell surface receptor CD147 on cardiac myocytes in vitro. This was also confirmed by ischemia/reperfusion in vivo. Recombinant human (rh) CyPA activated ERK1/2, p38MAPK, SAPKs, and Akt in cultured cardiac myocytes. Furthermore, CyPA significantly increased Bcl-2 in cardiac myocytes. These data strongly suggested that CyPA is released from cardiac myocytes in response to hypoxia/reoxygenation and may protect cardiac myocytes from oxidative stress-induced apoptosis.     

Targeted disruption of the mouse 3-phosphoglycerate dehydrogenase gene causes severe neurodevelopmental defects and results in embryonic lethality

D-3-Phosphoglycerate dehydrogenase (Phgdh; EC 1.1.1.95) is the first committed enzyme of L-serine biosynthesis in the phosphorylated pathway. To determine the physiological importance of Phgdh-dependent L-serine biosynthesis in vivo, we generated Phgdh-deficient mice using targeted gene disruption in embryonic stem cells. The absence of Phgdh led to a drastic reduction of L-serine metabolites such as phosphatidyl-L-serine and sphingolipids. Phgdh null embryos have small bodies with abnormalities in selected tissues and died after days post-coitum 13.5. Striking abnormalities were evident in the central nervous system in which the Phgdh null mutation culminated in hypoplasia of the telencephalon, diencephalon, and mesencephalon; in particular, the olfactory bulbs, ganglionic eminence, and cerebellum appeared as indistinct structures. These observations demonstrate that the Phgdh-dependent phosphorylated pathway is essential for normal embryonic development, especially for brain morphogenesis.     

Fas-mediated apoptosome formation is dependent on reactive oxygen species derived from mitochondrial permeability transition in Jurkat cells

Generation of reactive oxygen species (ROS) and activation of caspase cascade are both indispensable in Fas-mediated apoptotic signaling. Although ROS was presumed to affect the activity of the caspase cascade on the basis of findings that antioxidants inhibited the activation of caspases and that the stimulation of ROS by itself activated caspases, the mechanism by which these cellular events are integrated in Fas signaling is presently unclear. In this study, using human T cell leukemia Jurkat cells as well as an in vitro reconstitution system, we demonstrate that ROS are required for the formation of apoptosome. We first showed that ROS derived from mitochondrial permeability transition positively regulated the apoptotic events downstream of mitochondrial permeability transition. Then, we revealed that apoptosome formation in Fas-stimulated Jurkat cells was clearly inhibited by N-acetyl-L-cysteine and manganese superoxide dismutase by using both the immunoprecipitation and size-exclusion chromatography methods. To confirm these in vivo findings, we next used an in vitro reconstitution system in which in vitro-translated apoptotic protease-activating factor 1 (Apaf-1), procaspase-9, and cytochrome c purified from human placenta were activated by dATP to form apoptosome; the formation of apoptosome was markedly inhibited by reducing reagents such as DTT or reduced glutathione (GSH), whereas hydrogen peroxide prevented this inhibition. We also found that apoptosome formation was substantially impaired by GSH-pretreated Apaf-1, but not GSH-pretreated procaspase-9 or GSH-pretreated cytochrome c. Collectively, these results suggest that ROS plays an essential role in apoptosome formation by oxidizing Apaf-1 and the subsequent activation of caspase-9 and -3.     

Poly(A)- and primer-independent RNA polymerase of Norovirus

Replication of positive-strand caliciviruses is mediated by a virus-encoded RNA-dependent RNA polymerase (RdRp). To study the replication of Norovirus (NV), a member of the family Caliciviridae, we used a recombinant baculovirus system to express an enzymatically active RdRp protein from the 3D region of the NV genome and defined conditions for optimum enzymatic activity. Using an RNA template from the NV 3' genomic region, we observed similar levels of enzymatic activity in assays with and without a poly(A) tail. RdRp activity was not significantly affected by the addition of an RNA primer to the reaction mixture. Thus, the NV RdRp exhibited primer- and poly(A)-independent RNA polymerase activity. While the RdRp inhibitor phosphonoacetic acid inhibited NV RdRp activity, another gliotoxin did not. The active recombinant NV RdRp will be of benefit to studies of NV replication and will facilitate the development of specific inhibitors of NV proliferation.     

An angiotensin II AT1 receptor antagonist, telmisartan augments glucose uptake and GLUT4 protein expression in 3T3-L1 adipocytes

Evidence has accumulated that some of the angiotensin II AT1 receptor antagonists have insulin-sensitizing property. We thus examined the effect of telmisartan on insulin action using 3T3-L1 adipocytes. With standard differentiation inducers, a higher dose of telmisartan effectively facilitated differentiation of 3T3-L1 preadipocytes. Treatment of both differentiating adipocytes and fully differentiated adipocytes with telmisartan caused a dose-dependent increase in mRNA levels for PPARgamma target genes such as aP2 and adiponectin. By contrast, telmisartan attenuated 11beta-hydroxysteroid dehydrogenase type 1 mRNA level in differentiated adipocytes. Of note, we demonstrated for the first time that telmisartan augmented GLUT4 protein expression and 2-deoxy glucose uptake both in basal and insulin-stimulated state of adipocytes, which may contribute, at least partly, to its insulin-sensitizing ability.     

Bipartite 3'-cis-acting signal for replication in yeast 23 S RNA virus and its repair

23 S RNA narnavirus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome is small (2.9 kb) and only encodes its RNA-dependent RNA polymerase. Recently, we have succeeded in generating 23 S RNA virus from an expression vector containing the entire viral cDNA sequence. Using this in vivo launching system, we analyzed the 3'-cis-acting signals for replication. The 3'-non-coding region of 23 S RNA contains two cis-elements. One is a stretch of 4 Cs at the 3' end, and the other is a mismatched pair in a stem-loop structure that partially overlaps the terminal 4 Cs. In the latter element, the loop or stem sequence is not important but the stem structure with the mismatch pair is essential. The mismatched bases should be purines. Any combination of purines at the mismatch pair bestowed capability of replication on the RNA, whereas converting it to a single bulge at either side of the stem abolished the activity. The terminal and penultimate Cs at the 3' end could be eliminated or modified to other nucleotides in the launching plasmid without affecting virus generation. However, the viruses generated regained or restored these Cs at the 3' terminus. Considering the importance of the viral 3' ends in RNA replication, these results suggest that this 3' end repair may contribute to the persistence of 23 S RNA virus in yeast by maintaining the genomic RNA termini intact. We discuss possible mechanisms for this 3' end repair in vivo.     

Enhancement of serum- and platelet-derived growth factor-induced cell proliferation by Necl-5/Tage4/poliovirus receptor/CD155 through the Ras-Raf-MEK-ERK signaling

Necl-5/Tage4/poliovirus receptor/CD155 has been shown to be the poliovirus receptor and to be up-regulated in rodent and human carcinoma. We have found previously that mouse Necl-5 regulates cell motility. We show here that mouse Necl-5 is furthermore involved in the regulation of cell proliferation. Studies using a specific antibody against Necl-5 and a dominant negative mutant of Necl-5 revealed that Necl-5 enhanced the serum-induced proliferation of NIH3T3, Swiss3T3, and mouse embryonic fibroblast cells. Necl-5 enhanced the serum-induced activation of the Ras-Raf-MEK-ERK signaling, up-regulated cyclins D2 and E, and down-regulated p27(Kip1), eventually shortening the period of the G(0)/G(1) phase of the cell cycle in NIH3T3 cells. Necl-5 similarly enhanced the platelet-derived growth factor-induced activation of the Ras-Raf-MEK-ERK signaling and shortened the period of the G(0)/G(1) phase of the cell cycle in NIH3T3 cells. Necl-5 acted downstream of the platelet-derived growth factor receptor and upstream of Ras. Moreover, up-regulated Necl-5 was involved at least partly in the enhanced proliferation of transformed cells including NIH3T3 cells transformed by an oncogenic Ras or v-Src. These results indicate that Necl-5 plays roles not only in cell motility but also in cell proliferation.     

DiaA, a novel DnaA-binding protein, ensures the timely initiation of Escherichia coli chromosome replication

The DnaA protein is the initiator of Escherichia coli chromosomal replication. In this study, we identify a novel DnaA-associating protein, DiaA, that is required for the timely initiation of replication during the cell cycle. DiaA promotes the growth of specific temperature-sensitive dnaA mutants and ensures stable minichromosome maintenance, whereas DiaA does not decrease the cellular DnaA content. A diaA::Tn5 mutation suppresses the cold-sensitive growth of an overinitiation type dnaA mutant independently of SeqA, a negative modulator of initiation. Flow cytometry analyses revealed that the timing of replication initiation is disrupted in the diaA mutant cells as well as wild-type cells with pBR322 expressing the diaA gene. Gel filtration and chemical cross-linking experiments showed that purified DiaA forms a stable homodimer. Immunoblotting analysis indicated that a single cell contains about 280 DiaA dimers. DiaA stimulates minichromosome replication in an in vitro system especially when the level of DnaA included is limited. Moreover, specific and direct binding between DnaA and DiaA was observed, which requires a DnaA N-terminal region. DiaA binds to both ATP- and ADP-bound forms of DnaA with a similar affinity. Thus, we conclude that DiaA is a novel DnaA-associating factor that is crucial to ensure the timely initiation of chromosomal replication.     

Aliphatic acid amides of the fruits of Zanthoxylum piperitum

Six aliphatic acid amides (1-6) were isolated from the pericarp of Zanthoxylum piperitum fruits. MS and NMR spectroscopic investigation revealed that these compounds have a ketone and/or hydroxyl group(s) in the unsaturated aliphatic acid moiety of the structure of the amides. Combinations 3-4 and 5-6 are stereoisomers in terms of the relative configurations of their two asymmetric carbons.