A new simple preparation method for NaK-ATPase-rich membrane fragments

A method for the isolation of highly active membrane bound NaK-ATPase without detergents in quantity from the electric organ of the electric eel (Electrophorus electricus) is described. This method consists of the homogenation of electric organ with an isotonic solution containing sucrose, histidine, EDTA, and arginine, and of the separation of the higher active membrane fraction from the microsomal fraction by density gradient centrifugation. The enzyme has a specific activity of about 20 μmol Pi/min/mg at 37°C, and 13 μmol Pi/min/mg at 30°C. Although it is not as pure as the detergent-treated enzyme preparation based on the level of phosphorylated protein, ouabain binding, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, its enzyme activity is comparable to that of the purified enzymes. This preparation is very stable and is able to change its medium by Sephadex chromatography without any loss of enzyme activity and protein content. This preparation is also expected to keep the original characteristics as well as the enzyme in the tissue.     

Carbon-cycling changes during regeneration of a decinduous broadleaf forest after clear-cutting II. Aboveground net production

Growth and death rates of aboveground plant parts were measured in a mature forest and four different-aged deciduous broadleaf forests regeneratede after clear-cutting, with special reference to rates for woody parts (stems and branches) of different diameters (ø) in rerms of the pipe model theory (Shinozaki et al., 1964). The total biomass increment of woody parts of trees higher than 1.3 m varied within a range of 2.1-4.6 ton ha^?1 yr^?1, the increase beingdue largely to the growth of canopy trees exposed to direct sunlight. Biomass increments of small (ø<1 cm) and medium (1≤ø<5 cm) woody parts were negligibly small except in the youngest forest, and changes in aboveground woody biomass with forest age after clear-cutting mainly resulted from accumulation of large (5 cm<ø) woody parts of canopy trees. Biomass loss of trees due to death and grazing increased with forest age from 4.0 to 8.3 ton ha^?1 yr^?1. Recovery of leaf and small wood falls was observed at the early stage of regeneration, while large wood falls increased during regeneration. Flower and fruit fall was markedly higher in the mature forest than in the other four forest types. Mortality of woody parts became higher with forest age and was 20, 5.0 and 0.46% yr^?1 for small, medium and large parts, respectively, at the mature stage. Aboveground net production of the forest was in therange 7.6-13.3 ton ha^?1 yr^?1 with the undergrowth vegetation lower than 1.3 m being 0.4-1.4 ton ha^?1 yr^?1. Production recovered rapidly at an early stage of regeneration and was highest in mature forest.     

Mutations affecting early distribution of primordial germ cells in Medaka (Oryzias latipes) embryo

The development of germ cells has been intensively studied in Medaka (Oryzias latipes). We have undertaken a large-scale screen to identify mutations affecting the development of primordial germ cells (PGCs) in Medaka. Embryos derived from mutagenized founder fish were screened for an abnormal distribution or number of PGCs at embryonic stage 27 by RNA in situ hybridization for the Medaka vasa homologue (olvas). At this stage, PGCs coalesce into two bilateral vasa-expressing foci in the ventrolateral regions of the trunk after their migration and group organization. Nineteen mutations were identified from a screen corresponding to 450 mutagenized haploid genomes. Eleven of the mutations caused altered PGC distribution. Most of these alterations were associated with morphological abnormalities and could be grouped into four phenotypic classes: Class 1, PGCs dispersed into bilateral lines; Class 2, PGCs dispersed in a region more medial than that in Class 1; Class 3, PGCs scattered laterally and over the yolk sac area; and Class 4, PGCs clustered in a single median focus. Eight mutations caused a decrease in the number of PGCs. This decrease was observed in the offspring of heterozygous mothers, indicating the contribution of a maternal factor in determining PGC abundance. Taken together, these mutations should prove useful in identifying molecular mechanisms underlying the early PGC development and migration.     

Analysis of Wnt8 for neural posteriorizing factor by identifying Frizzled 8c and Frizzled 9 as functional receptors for Wnt8

The dorsal ectoderm of vertebrate gastrula is first specified into anterior fate by an activation signal and posteriorized by a graded transforming signal, leading to the formation of forebrain, midbrain, hindbrain and spinal cord along the anteroposterior (A-P) axis. Transplanted non-axial mesoderm rather than axial mesoderm has an ability to transform prospective anterior neural tissue into more posterior fates in zebrafish. Wnt8 is a secreted factor that is expressed in non-axial mesoderm. To investigate whether Wnt8 is the neural posteriorizing factor that acts upon neuroectoderm, we first assigned Frizzled 8c and Frizzled 9 to be functional receptors for Wnt8. We then, transplanted non-axial mesoderm into the embryos in which Wnt8 signaling is cell-autonomously blocked by the dominant-negative form of Wnt8 receptors. Non-axial mesodermal transplants in embryos in which Wnt8 signaling is cell-autonomously blocked induced the posterior neural markers as efficiently as in wild-type embryos, suggesting that Wnt8 signaling is not required in neuroectoderm for posteriorization by non-axial mesoderm. Furthermore, Wnt8 signaling, detected by nuclear localization of beta-catenin, was not activated in the posterior neuroectoderm but confined in marginal non-axial mesoderm. Finally, ubiquitous over-expression of Wnt8 does not expand neural ectoderm of posterior character in the absence of mesoderm or Nodal-dependent co-factors. We thus conclude that other factors from non-axial mesoderm may be required for patterning neuroectoderm along the A-P axis.     

DYRK2 is targeted to the nucleus and controls p53 via Ser46 phosphorylation in the apoptotic response to DNA damage

Genotoxic stress exerts biological activity by activating downstream effectors, including the p53 tumor suppressor. p53 regulates cell-cycle checkpoint and induction of apoptosis in response to DNA damage; however, molecular mechanisms responsible for committing to these distinct functions remain to be elucidated. Recent studies demonstrated that phosphorylation of p53 at Ser46 is associated with induction of p53AIP1 expression, resulting in commitment to apoptotic cell death. In this regard, the role for Ser46 kinases in p53-dependent apoptosis has been established; however, the kinases responsible for Ser46 phosphorylation have yet to be identified. Here, we demonstrate that the dual-specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) directly phosphorylates p53 at Ser46. Upon exposure to genotoxic stress, DYRK2 translocates into the nucleus for Ser46 phosphorylation. Consistent with these results, DYRK2 induces p53AIP1 expression and apoptosis in a Ser46 phosphorylation-dependent manner. These findings indicate that DYRK2 regulates p53 to induce apoptosis in response to DNA damage.     

Potent effects of,and mechanisms for,modification of crosstalk between macrophages and adipocytes by lactobacilli

The murine macrophage‐like cell line J774.1 was treated with heat‐killed cells of Lactobacillus GG (LGG) and L. gasseri TMC0356 (TMC 0356). Interleukin (IL)‐6, IL‐12, and tumor necrosis factor‐α were profiled from the J774.1 cells using enzyme‐linked immunosorbent assay methods. The conditioned medium from cultured J774.1 cells was transferred to the preadipocyte cell line 3T3‐L1 (which is a mouse embryonic fibroblast‐adipose‐like cell line). Growth and differentiation of 3T3‐L1 cells were monitored by analyzing lipid accumulation and expression of peroxisome proliferator‐activated receptor (PPAR)‐γ mRNA. The medium conditioned by 3T3‐L1 cells was added to J774.1 cells and the cytokines in the supernatant analyzed. Compared with that of cells exposed to a PBS‐conditioned medium, lipid accumulation in 3T3‐L1 cells was significantly suppressed in a dose‐dependent manner by each medium that had been conditioned with LGG and TMC0356. PPAR‐γ mRNA expression in 3T3‐L1 cells was also significantly downregulated (P < 0.01, P < 0.05, respectively). The conditioned medium of 3T3‐L1 adipose phenotype significantly stimulated production of IL‐6 and IL‐12 in J774.1 cells treated with LGG and TMC0356. These results suggest that lactobacilli may suppress differentiation of preadipocytes through macrophage activation and alter the immune responses of macrophages to adipose cells.     

Protein kinase C delta regulates Ser46 phosphorylation of p53 tumor suppressor in the apoptotic response to DNA damage

The p53 tumor suppressor is activated in the cellular response to genotoxic stress. Transactivation of p53 target genes dictates cell cycle arrest and DNA repair or induction of apoptosis; however, a molecular mechanism responsible for these distinct functions remains unclear. Recent studies revealed that phosphorylation of p53 on Ser(46) was associated with induction of p53AIP1 expression, resulting in the commitment of the cell fate into apoptotic cell death. Moreover, upon exposure to genotoxic stress, p53DINP1 was expressed and recruited a kinase(s) to p53 that specifically phosphorylated Ser(46). Here, we show that the pro-apoptotic kinase, protein kinase C delta (PKCdelta), is involved in phosphorylation of p53 on Ser(46). PKCdelta-mediated phosphorylation is required for the interaction of PKCdelta with p53. The results also demonstrate that p53DINP1 associates with PKCdelta upon exposure to genotoxic agents. Consistent with these results, PKCdelta potentiates p53-dependent apoptosis by Ser(46) phosphorylation in response to genotoxic stress. These findings indicate that PKCdelta regulates p53 to induce apoptotic cell death in the cellular response to DNA damage.     

Secretion and localization of invertase and inulinase in recombinant Saccharomyces cerevisiae

Summary Secretion of invertase and inulinase produced by recombinant Saccharomyces cerevisiae cells were investigated under derepression conditions of GALI promoter. Secreted invertase mainly localized in the periplasmic space, but most of inulinase was found in the extracellular culture medium. This high level of extracellular secretion of inulinase was not dependent on the growth phase in which derepression of GALI promoter occurs. Our results indicate that the inulinase polypeptide itself may have a function for the protein secretion into the culture medium.     

A novel role of group VIB calcium-independent phospholipase A2 (iPLA2gamma) in the inducible expression of group IIA secretory PLA2 in rat fibroblastic cells

Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) is a prototypic sPLA(2) enzyme that may play roles in modification of eicosanoid biosynthesis as well as antibacterial defense. In several cell types, inducible expression of sPLA(2) by pro-inflammatory stimuli is attenuated by group IVA cytosolic PLA(2) (cPLA(2)alpha) inhibitors such as arachidonyl trifluoromethyl ketone, leading to the proposal that prior activation of cPLA(2)alpha is required for de novo induction of sPLA(2). However, because of the broad specificity of several cPLA(2)alpha inhibitors used so far, a more comprehensive approach is needed to evaluate the relevance of this ambiguous pathway. Here, we provide evidence that the induction of sPLA(2)-IIA by pro-inflammatory stimuli requires group VIB calcium-independent PLA(2) (iPLA(2)gamma), rather than cPLA(2)alpha, in rat fibroblastic 3Y1 cells. Results with small interfering RNA unexpectedly showed that the cytokine induction of sPLA(2)-IIA in cPLA(2)alpha knockdown cells, in which cPLA(2)alpha protein was undetectable, was similar to that in replicate control cells. By contrast, knockdown of iPLA(2)gamma, another arachidonyl trifluoromethyl ketone-sensitive intracellular PLA(2), markedly reduced the cytokine-induced expression of sPLA(2)-IIA. Supporting this finding, the R-enantiomer of bromoenol lactone, an iPLA(2)gamma inhibitor, suppressed the cytokine-induced sPLA(2)-IIA expression, whereas (S)-bromoenol lactone, an iPLA(2)beta inhibitor, failed to do so. Moreover, lipopolysaccharide-stimulated sPLA(2)-IIA expression was also abolished by knockdown of iPLA(2)gamma. These findings open new insight into a novel regulatory role of iPLA(2)gamma in stimulus-coupled sPLA(2)-IIA expression.