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.     

A mutant with constitutive sexual organ development in<Emphasis Type="Italic"> Marchantia polymorpha</Emphasis> L.

In lower land plants, genes controlling the transition from vegetative growth to sexual reproduction have not yet been identified. In the dioecious liverwort Marchantia polymorpha, the transition to sexual reproduction accompanied by the formation of sexual organs on the gametophytic thallus is initiated under long-day conditions. By particle bombardment-mediated mutagenesis, we generated a mutant of M. polymorpha that constitutively forms sexual organs. This mutant is fully fertile, showing that the mutation does not affect formation of male or female sexual organs per se. Genetic analysis reveals that this phenotype is caused by mutation of a single autosomal locus, suggesting that this mutation defines or controls a gene regulating the transition to sexual reproduction in M. polymorpha.     

The Caenorhabditis elegans eukaryotic initiation factor 5A homologue, IFF-1, is required for germ cell proliferation, gametogenesis and localization of the P-granule component PGL-1

Eukaryotic initiation factor 5A (eIF-5A) was originally isolated as a translation initiation factor. However, this function has since been reconsidered, with recent studies pointing to roles for eIF-5A in mRNA metabolism and trafficking [Microbiol. Mol. Biol. Rev. 66 (2002) 460; Eur. Mol. Biol. Org. J. 17 (1998) 2914]. The Caenorhabditis elegans genome contains two eIF-5A homologues, iff-1 and iff-2, whose functions in vivo were examined in this study. The iff-2 mutation causes somatic defects that include slow larval growth and disorganized somatic gonadal structures in hermaphrodites. iff-2 males show disorganized tail sensory rays and spicules. On the other hand, iff-1 mRNA is expressed in the gonad, and the lack of iff-1 activity causes sterility with an underproliferated germline resulting from impaired mitotic proliferation in both hermaphrodites and males. In spite of underproliferation, meiotic nuclei are observed, as revealed by presence of immunoreactivity to the anti-HIM-3 antibody; however, no gametogenesis occurs in the iff-1 gonads. These phenotypes are in part similar to the mutants affected in the components of P granules, which are the C. elegans counterparts of germ granules [Curr. Top Dev. Biol. 50 (2000) 155]. We found that localization of the P-granule component PGL-1 to P granules is disrupted in the iff-1 mutant. In summary, the two C. elegans homologues of eIF-5A act in different tissues: IFF-2 is required in the soma, and IFF-1 is required in the germline for germ cell proliferation, for gametogenesis after entry into meiosis, and for proper PGL-1 localization on P granules.     

Histochemical,ultrastructural, and three-dimensional observation of smooth muscle cells in human gastric mucosa

The present study was undertaken to clarify the histochemical and ultrastructural properties and the three-dimensional distribution of the smooth muscle cells (SMCs) located in the lamina propria (LP) of the human gastric mucosa. Standard paraffin sections obtained from stomachs surgically resected for gastric cancer were immunostained for alpha-smooth muscle actin (alpha-SMA), vimentin, desmin, laminin, and type IV collagen. In addition, 100-m-thick sections were fluorostained for alpha-SMA and CD34, while three-dimensional images were prepared by confocal laser scanning microscope. Ultrastructural studies were carried out using normal gastric biopsy specimens. The results indicated that SMCs in the LP differed between the upper and lower regions, SMCs in the lower LP being fairly typical SMCs, whereas those in the upper LP had apparently lost reactivity for desmin but gained that for vimentin. The basal lamina became sparser, but a fibronexus was occasionally seen in SMCs in the upper LP. Three-dimensional images revealed bundles of SMCs in the upper LP encircling several foveolae to form acinus-like structures and, in the upper LP, SMCs branching into fine fibrils with a brush-like (corpus) or besom-like (i.e., a twiggy witchs broom) appearance (antrum).     

Serum concentrations of androstenediol and androstenediol sulfate, and their relation to cytokine production during and after normal pregnancy

Since it is known that androstenediol (ADIOL) has potent immunoregulatory effects, changes in ADIOL levels during and after pregnancy might affect the maternal immune system. We examined serum concentrations of ADIOL and androstenediol 3-sulfate (ADIOLS) together with IFN-gamma and IL-4 production levels during pregnancy and after delivery up to 10-11 months postpartum. The subjects were 73 normal pregnant, 76 normal postpartum, and 28 normal non-pregnant women. ADIOL and ADIOLS were measured using EIA and GC/MS, respectively. The cytokine levels in the supernatant of whole-blood cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin were measured using ELISA. ADIOL levels significantly decreased compared to non-pregnant levels in the first trimester (P < 0.05) and were reversed in the third trimester (P < 0.05). After pregnancy, ADIOL levels gradually declined, and a significant decrease was observed at 10-11 months postpartum (P < 0.05). ADIOLS levels were significantly lower in the third trimester (P < 0.05) and significantly higher at the first month postpartum (P < 0.001) compared to non-pregnant women. IFN-gamma and IL-4 levels decreased during pregnancy and subsequently increased postpartum. On the other hand, we found significant negative correlations between ADIOL concentrations and production levels of IFN-gamma (P < 0.05) or IL-4 (P < 0.05). These findings suggest that ADIOL may be involved in modifying the maternal immune response during and after pregnancy.     

Engineered sorbitol accumulation induces dwarfism in Japanese persimmon

A cDNA encoding sorbitol-6-phosphate dehydrogenase (S6PDH), which is a key enzyme in sorbitol biosynthesis in Rosaceae, was introduced into the Japanese persimmon (Diospyros kaki) to increase the environmental stress tolerance. Resultant transformants exhibited salt-tolerance with dwarfing phenotypes. Therefore, we studied two transgenic lines to understand the physiological mechanism of this dwarfism: lines PS1 and PS6 accumulated high and moderate levels of sorbitol, respectively. The average length of shoots was significantly shorter as compared with the wild-type in line PS1, while no such decrease was observed in line PS6. The myo-inositol and glucose 6-phosphate (G6P) contents were measured in the transgenic lines because previous work with tobacco transformed with S6PDH had suggested that growth inhibition was due to depletion of these metabolites. Although the myo-inositol content was decreased in PS1 plants, the decrease was much smaller than that observed in transgenic tobacco that accumulates sorbitol. The G6P contents were the same in PS1 plants and phenotypically normal PS6 plants. The level of indole-3-acetic acid (IAA), which affects stem elongation, in line PS1 was similar to the levels in the other lines. A decrease in gibberellin (GA) content generally induces dwarfism in plants. However, GA was not decreased in PS1 plants compared with wild-type or control plants. Therefore, we focused on sorbitol accumulation as the most remarkable feature of PS1 plants. As one possibility, the observed growth inhibition was likely caused by an osmotic imbalance between the cytosol and vacuole.     

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.     

Quaternary structure of the neuronal protein NAP-22 in aqueous solution

NAP-22, a myristoylated, anionic protein, is a major protein component of the detergent-insoluble fraction of neurons. After extraction from the membrane, it is readily soluble in water. NAP-22 will partition only into membranes with specific lipid compositions. The lipid specificity is not expected for a monomeric myristoylated protein. We have studied the self-association of NAP-22 in solution. Sedimentation velocity experiments indicated that the protein is largely associated. The low concentration limiting s value is approximately 1.3 S, indicating a highly asymmetric monomer. In contrast, a nonmyristoylated form of the protein shows no evidence of oligomerization by velocity sedimentation and has an s value corresponding to the smallest component of NAP-22, but without the presence of higher oligomers. Sedimentation equilibrium runs indicate that there is a rapidly reversible equilibrium between monomeric and oligomeric forms of the protein followed by a slower, more irreversible association into larger aggregates. In situ atomic force microscopy of the protein deposited on mica from freshly prepared dilute solution revealed dimers on the mica surface. The values of the association constants obtained from the sedimentation equilibrium data suggest that the weight concentration of the monomer exceeds that of the dimer below a total protein concentration of 0.04 mg/ml. Since the concentration of NAP-22 in the neurons of the developing brain is approximately 0.6 mg/ml, if the protein were in solution, it would be in oligomeric form and bind specifically to cholesterol-rich domains. We demonstrate, using fluorescence resonance energy transfer, that at low concentrations, NAP-22 labeled with Texas Red binds equally well to liposomes of phosphatidylcholine either with or without the addition of 40 mol% cholesterol. Thus, oligomerization of NAP-22 contributes to its lipid selectivity during membrane binding.