A vascular gene trap screen defines RasGRP3 as an angiogenesis-regulated gene required for the endothelial response to phorbol esters

We identified Ras guanine-releasing protein 3 (RasGRP3) as a guanine exchange factor expressed in blood vessels via an embryonic stem (ES) cell-based gene trap screen to identify novel vascular genes. RasGRP3 is expressed in embryonic blood vessels, down-regulated in mature adult vessels, and reexpressed in newly formed vessels during pregnancy and tumorigenesis. This expression pattern is consistent with an angiogenic function for RasGRP3. Although a loss-of-function mutation in RasGRP3 did not affect viability, RasGRP3 was up-regulated in response to vascular endothelial growth factor (VEGF) stimulation of human umbilical vein endothelial cells, placing RasGRP3 regulation downstream of VEGF signaling. Phorbol esters mimic the second messenger diacylglycerol (DAG) in activating both protein kinase C (PKC) and non-PKC phorbol ester receptors such as RasGRP3. ES cell-derived wild-type blood vessels exposed to phorbol myristate acetate (PMA) underwent extensive aberrant morphogenesis that resulted in the formation of large endothelial sheets rather than properly branched vessels. This response to PMA was completely dependent on the presence of RasGRP3, as mutant vessels were refractory to the treatment. Taken together, these findings show that endothelial RasGRP3 is up-regulated in response to VEGF stimulation and that RasGRP3 functions as an endothelial cell phorbol ester receptor in a pathway whose stimulation perturbs normal angiogenesis. This suggests that RasGRP3 activity may exacerbate vascular complications in diseases characterized by excess DAG, such as diabetes.     

Xantivin suppresses the activity of EGF-CFC genes to regulate nodal signaling

Lefty, antivin and related genes act in a feedback inhibition mechanism for nodal signaling at a number of stages of vertebrate embryogenesis. To analyze the function of the feedback inhibitor of nodal signaling, Xantivin in Xenopus embryos, we designed a morpholino antisense oligonucleotide (XatvMO) for this gene. XatvMO caused the expansion of mesodermal tissue and head defects. XatvMO-injected gastrulae showed up-regulated expression of the mesodermal markers Xbra, Xwnt8, Xnot, and Chordin, suggesting expansion of the trunk-tail organizer. As expected, depletion of Xantivin also up-regulated nodal signaling as confirmed by the enhanced ectopic expression of Xantivin mRNA, a known target gene of nodal signaling. Furthermore, we investigated the relationship between Xantivin and the EGF-CFC gene FRL-1, which is a component of the nodal receptor. In animal cap assays, FRL-1 could not induce expression of nodal-responsive genes, but could up-regulate expression of these genes when FRL-1 was coinjected with a low dose of Xnr1; coinjection of Xantivin suppressed this up-regulation by FRL-1. We also found that Xantivin can rescue the caudalized phenotype induced by overexpression of FRL-1. Co-immunoprecipitation assays showed that Xantivin interacted with the EGF-CFC proteins, FRL-1 and cripto. Taken together, these results suggest that Xantivin opposes the activity of EGF-CFC genes and thereby antagonizes nodal signaling.     

Photoinhibition, bleaching susceptibility and mortality in two scleractinian corals, Platygyra ryukyuensis and Stylophora pistillata, in response to thermal and light stresses

In the present study, we examined the effect of thermal stress on the photoinhibitory light threshold in a bleaching susceptible (Stylophora pistillata) and a bleaching resistant (Platygyra ryukyuensis) coral. Four light (0, 110, 520, 1015 micromol quantam(-2)s(-1)) and three temperature (26, 32 and 34 degrees C) conditions were used over a 3-h period, followed by 24- and 48-h recovery periods at approximately 21 degrees C under dim light. Dynamic photoinhibition could be detected in both P. ryukyuensis and S. pistillata under 520 and 1015 micromol quantam(-2)s(-1) at 26 degrees C and under 110 micromol quantam(-2)s(-1) at 32 degrees C only in S. pistillata. Chronic photoinhibition was recorded under 520 and 1015 micromol quantam(-2)s(-1) at 34 degrees C in P. ryukyuensis, and under 1015 micromol quantam(-2)s(-1) at 32 degrees C and under all light levels at 34 degrees C in S. pistillata. These results show that high temperature reduced the threshold light intensity for photoinhibition differently in two corals with different bleaching susceptibilities under thermal stress. No visual paling and mortality in P. ryukyuensis was observed at any treatment, even in chronically photoinhibited specimens, while paling and high mortality of S. pistillata was noted in all treatments, apart from samples at 26 degrees C. These observations suggest a potential role of the host in differential bleaching and mortality determination.     

Differential susceptibility to oxidative stress of two scleractinian corals: antioxidant functioning of mycosporine-glycine

This study examined the importance of mycosporine-glycine (Myc-Gly) as a functional antioxidant in the thermal-stress susceptibility of two scleractinian corals, Platygyra ryukyuensis and Stylophora pistillata. Photochemical efficiency of PSII (F_v/F_m), activity of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), and composition and abundance of mycosporine-like amino acids (MAAs) in the coral tissue and in symbiotic zooxanthellae were analyzed during 12-h exposure to high temperature (33 °C). After 6- and 12-h exposures at 33 °C, S. pistillata showed a significantly more pronounced decline in F_v/F_m compared to P. ryukyuensis. A 6-h exposure at 33 °C induced a significant increase in the activities of SOD and CAT in both host and zooxanthellae components of S. pistillata while in P. ryukyuensis a significant increase was observed only in the CAT activity of zooxanthellae. After 12-h exposure, the SOD activity of P. ryukyuensis was unaffected in the coral tissue but slightly increased in zooxanthellae, whereas the CAT activity in the coral tissue showed a 2.5-fold increase. The total activity of antioxidant enzymes was significantly higher in S. pistillata than in P. ryukyuensis, suggesting that P. ryukyuensis is less sensitive to oxidative stress than S. pistillata. This differential susceptibility of the corals is consistent with a 20-fold higher initial concentration of Myc-Gly in P. ryukyuensis compared to S. pistillata. In the coral tissue and zooxanthellae of both species investigated, the first 6 h of exposure to thermal stress induced a pronounced reduction in the abundance of Myc-Gly but not in other MAAs. When exposure was prolonged to 12 h, the Myc-Gly pool continued to decrease in P. ryukyuensis and was completely depleted in S. pistillata. The delay in the onset of oxidative stress in P. ryukyuensis and the dramatic increase in the activities of the antioxidant enzymes in S. pistillata, which contains low concentrations of Myc-Gly suggest that Myc-Gly provides rapid protection against oxidative stress before the antioxidant enzymes are induced. These findings strongly suggest that Myc-Gly is functioning as a biological antioxidant in the coral tissue and zooxanthellae and demonstrate its importance in the survival of reef-building corals under thermal stress.     

Proteasome activator PA28gamma-dependent nuclear retention and degradation of hepatitis C virus core protein

Hepatitis C virus (HCV) core protein plays an important role in the formation of the viral nucleocapsid and a regulatory protein involved in hepatocarcinogenesis. In this study, we have identified proteasome activator PA28gamma (11S regulator gamma) as an HCV core binding protein by using yeast two-hybrid system. This interaction was demonstrated not only in cell culture but also in the livers of HCV core transgenic mice. These findings are extended to human HCV infection by the observation of this interaction in liver specimens from a patient with chronic HCV infection. Neither the interaction of HCV core protein with other PA28 subtypes nor that of PA28gamma with other Flavivirus core proteins was detected. Deletion of the PA28gamma-binding region from the HCV core protein or knockout of the PA28gamma gene led to the export of the HCV core protein from the nucleus to the cytoplasm. Overexpression of PA28gamma enhanced the proteolysis of the HCV core protein. Thus, the nuclear retention and stability of the HCV core protein is regulated via a PA28gamma-dependent pathway through which HCV pathogenesis may be exerted.     

Induction of primary root curvature in radish seedlings in a static magnetic field

Primary roots of radish (Raphanus sativus L.) seedlings were exposed to an inhomogeneous static magnetic field generated by a permanent magnet, during continuous rotation on a 0.06 rpm clinostat, thereby reducing the unilateral influence of gravity. The roots responded tropically to the static magnetic field with the tropism appearing to be negative. These roots responded significantly (P < 0.05) to the south pole of the magnet. The significant tropic response was found for a magnetic flux density of 13-68 mT, for a field gradient of 1.8-14.7 T/m, and for the product of magnetic field and field gradient of 0.023-1.0 T(2)/m. A small, but insignificant, response of the roots to the north pole has also been found.     

NEDD8 recruits E2-ubiquitin to SCF E3 ligase

NEDD8/Rub1 is a ubiquitin (Ub)-like post-translational modifier that is covalently linked to cullin (Cul)-family proteins in a manner analogous to ubiquitylation. NEDD8 is known to enhance the ubiquitylating activity of the SCF complex (composed of Skp1, Cul-1, ROC1 and F-box protein), but the mechanistic role is largely unknown. Using an in vitro reconstituted system, we report here that NEDD8 modification of Cul-1 enhances recruitment of Ub-conjugating enzyme Ubc4 (E2) to the SCF complex (E3). This recruitment requires thioester linkage of Ub to Ubc4. Our findings indicate that the NEDD8-modifying system accelerates the formation of the E2-E3 complex, which stimulates protein polyubiquitylation.     

Coexistence of gland mucous cell-type mucin and lysozyme in gastric gland mucous cells

Class III mucin, identified by paradoxical concanavalin A staining, is confined to gastric gland mucous cells and is an essential component of the gastric surface mucous gel layer. The pretreatment required has hampered the application of this method to electron microscopic studies. Antibody HIK1083 reacts selectively with class III mucins. The present study was undertaken to explore, electron microscopically, the immunoreactivity of the human stomach to HIK1083. We examined normal mucosa from resected human stomachs (five cases; formalin-fixed, paraffin-embedded) and gastric biopsy specimens from patients with early gastric cancer [nine cases; glutaraldehyde- and osmium-fixed, epoxy-embedded (seven cases) and half-strength Karnovsky’s solution-fixed, Lowicryl K4M-embedded (two cases)]. Immunostaining with HIK1083 and anti-lysozyme antibody was examined under light and electron microscopes. Gland mucous cells were labeled with HIK1083, and lysozyme was detected in some gland mucous cells and surface mucous cells. Electron microscopically, the secretory granules of gland mucous cells contained a single electron-dense core. HIK1083-positive mucins and lysozyme coexisted in the secretory granules of gastric gland mucous cells. HIK1083-reactive mucins and lysozyme were distributed in the matrix and in the dense core of these secretory granules, respectively. HIK1083 can be used for electron immunohistochemistry. Accepted: 1 December 1999     

Molecular cloning of rat brain mitochondrial carrier protein-1 cDNA and its up-regulation during postnatal development

Brain mitochondrial carrier protein-1 (BMCP1), a new member of the mitochondrial uncoupling carrier, has been shown to be expressed predominantly in the brain of the mice and humans. We cloned rat BMCP1 cDNA and investigated its mRNA level during postnatal development and under various metabolic conditions. The nucleotide sequence of the cDNA revealed that rat BMCP1 protein was composed of 322 amino acid residues, and was 99 and 96% identical to the mouse and human proteins and 29, 33 and 35% identical to rat uncoupling protein (UCP) 1, UCP2 and UCP3, respectively. The molecular weight was predicted to be 36017 Da and the protein of this size was detectable when the cDNA was expressed in vitro. Using Northern blot analysis, the corresponding mRNA, approximately 1.8-kb in size, was found expressed predominantly in the cerebrum, cerebellum and hypothalamus. A unique developmental pattern was identified in the brain, where BMCP1 expression was low in their fetal life, but significantly elevated in the first postnatal week. Thereafter BMCP1 mRNA was maintained to be gradually increased. In 48-h fasted or insulin-induced hypoglycemic rats, BMCP1 mRNA expression in the hypothalamus slightly, but significantly, decreased compared with that in their appropriate controls. The present results indicate that BMCP1 may be involved in pathogenesis of mitochondrial dysfunction in neurons induced by aging or neurodegenerative disorders, and perhaps in energy balance in the brain.