Mind bomb is a ubiquitin ligase that is essential for efficient activation of Notch signaling by Delta

Lateral inhibition, mediated by Notch signaling, leads to the selection of cells that are permitted to become neurons within domains defined by proneural gene expression. Reduced lateral inhibition in zebrafish mib mutant embryos permits too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a RING ubiquitin ligase. Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Cell transplantation studies suggest that mib function is essential in the signaling cell for efficient activation of Notch in neighboring cells. These observations support a model for Notch activation where the Delta-Notch interaction is followed by endocytosis of Delta and transendocytosis of the Notch extracellular domain by the signaling cell. This facilitates intramembranous cleavage of the remaining Notch receptor, release of the Notch intracellular fragment, and activation of target genes in neighboring cells.     

Dependence of functional traits related to growth rates and their CO<Subscript>2</Subscript> response on multiple habitat climate factors across <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> populations

The values of many plant traits are often different even within a species as a result of local adaptation. Here, we studied how multiple climate variables influence trait values in Arabidopsis thaliana grown under common conditions. We examined 9 climate variables and 29 traits related to vegetative growth rate in 44 global A. thaliana accessions grown at ambient or elevated CO₂ concentration ([CO₂]) and applied a multiple regression analysis. We found that genetic variations in the traits related to growth rates were associated with various climate variables. At ambient [CO₂], plant size was positively correlated with precipitation in the original habitat. This may be a result of larger biomass investment in roots at the initial stage in plants adapting to a lower precipitation. Stomatal conductance and photosynthetic nitrogen use efficiency were negatively correlated with vapor pressure deficit, probably as a result of the trade-off between photosynthetic water- and nitrogen-use efficiency. These results suggest that precipitation and air humidity influence belowground and aboveground traits, respectively. Elevated [CO₂] altered climate dependences in some of the studied traits. The CO₂ response of relative growth rate was negatively correlated with altitude, indicating that plants inhabiting a higher altitude have less plasticity to changing [CO₂]. These results are useful not only for understanding evolutionary process but also to predict the plant species that are favored under future global change.     

Effects of peptide molecular mass and PEG chain length on the vasoreactivity of VIP and PACAP(1-38) in pegylated phospholipid micelles

Bioactive properties of certain amphipathic peptides are amplified when self-associated with sterically stabilized micelles (SSM) composed of polyethylene glycol (PEG)-conjugated phospholipids. The purpose of this study was to determine the effects of amphipathic peptide molecular mass and PEG chain length on vasoreactivity evoked by vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide, and pituitary adenylate cyclase-activating peptide(1-38) (PACAP(1-38)), a 38-amino acid neuropeptide, associated with PEGylated phospholipid micelles in vivo. Both peptides were incubated for 2 h with SSM composed of PEG with molecular mass of 2000 or 5000 grafted onto distearoyl-phosphatidylethanolamine (DSPE-PEG2000 or DSPE-PEG5000) before use. We found that regardless of peptide molecular mass, PEG chain length had no significant effects on peptide-SSM interactions. Using intravital microscopy, VIP associated with DSPE-PEG5000 SSM or DSPE-PEG2000 SSM incubated at 25 degrees C evoked similar vasodilation in the intact hamster cheek pouch microcirculation. Likewise, PACAP(1-38)-induced vasodilation was PEG chain length-independent. However, SSM-associated PACAP(1-38) evoked significantly smaller vasodilation than that evoked by SSM-associated VIP (P < 0.05) at 25 degrees C. When the incubation temperature was increased to 37 degrees C, SSM-associated PACAP(1-38)-induced vasodilation was now similar to that of SSM-associated VIP. This response was associated with a corresponding increase in alpha-helix content of both peptides in the presence of phospholipids. Collectively, these data indicate that for a larger amphipathic peptide, such as PACAP(1-38), greater kinetic energy or longer incubation period is required to optimize peptide-SSM interactions and amplify peptide bioactivity in vivo.     

Purification of nitric oxide synthase from bovine brain: immunological characterization and tissue distribution.

Nitric oxide (NO) synthase (EC 1.14.23) was purified to homogeneity from bovine cerebrum. The molecular weight of NO synthase was estimated to be 150 kDa by both SDS/PAGE and gel filtration at high salt concentration. For activity, the enzyme required NADPH, Ca2+, calmodulin and tetrahydrobiopterin as cofactors. Rabbit polyclonal antibody to bovine brain NO synthase reacted with 150 kDa NO synthase in various bovine and rat organs, including the brain, pituitary and adrenal glands, but not with that in stimulated macrophages, indicating that there are at least two immunologically distinct NO synthases.     

Interactions of human secretin with sterically stabilized phospholipid micelles amplify peptide-induced vasodilation in vivo

Secretin, a 27-amino acid neuropeptide, is a member of the glucagon/secretin/vasoactive intestinal polypeptide (VIP) superfamily of amphipathic peptides that elicits transient vasodilation in vivo. The purpose of this study was to determine whether association of human secretin with sterically stabilized phospholipid micelles (SSM) amplifies the vasorelaxant effects of the peptide in the peripheral microcirculation in vivo. We found that secretin in saline evoked significant concentration-dependent vasodilation in the intact hamster cheek pouch microcirculation (P < 0.05). This response was potentiated and prolonged significantly when secretin was associated with SSM (P < 0.05). Vasodilation evoked by secretin in saline and secretin in SSM was abrogated by VIP(10-28), a VIP receptor antagonist, but not by PACAP(6-38), a PACAP receptor antagonist, or Hoe140, a selective bradykinin B(2) receptor antagonist. Collectively, these data indicate that self-association of human secretin with SSM significantly amplifies peptide vasoreactivity in the intact peripheral microcirculation through activation of VIP receptors. We suggest that the vasoactive effects of human secretin in vivo are, in part, phospholipid-dependent.     

Epigenetic regulation of <Emphasis Type="Italic">MdMYB1</Emphasis> is associated with paper bagging-induced red pigmentation of apples

Main conclusion

Paper-bagging treatment can transform non-transcribed MdMYB1 - 2 and MdMYB1 - 3 alleles into transcribed alleles through epigenetic regulations, resulting in the red pigmentation of a normally non-red apple cultivar ‘Mutsu.’ Anthocyanin biosynthesis in apples is regulated by MdMYB1/A/10, an R2R3-Type MYB gene. ‘Mutsu,’ a triploid apple cultivar harboring non-transcribed MdMYB1-2 and MdMYB1-3 alleles, retains green skin color under field conditions. However, it can show red/pink pigmentation under natural or artificial ultraviolet-B (UV-B) light exposure after paper-bagging and bag removal treatment. In the present study, we found that in ‘Mutsu,’ paper bagging-induced red pigmentation was due to the activation of non-transcribed MdMYB1-2/-3 alleles, which triggered the expression of downstream anthocyanin biosynthesis genes in a UV-B-dependent manner. By monitoring the epigenetic changes during UV-B-induced pigmentation, no significant differences in DNA methylation and histone modifications in the 5′ upstream region of MdMYB1-2/-3 were recorded between the UV-B-treated fruit skin (red) and the fruit skin treated only by white light (green). In contrast, bag treatment lowered the DNA methylation in this region of MdMYB1-2/-3 alleles. Similarly, higher levels of histone H3 acetylation and trimethylation of H3 tail at lysine 4, and lower level of trimethylation of H3 tail at lysine 27 were observed in the 5′ upstream region of MdMYB1-2/-3 in the skin of the fruit immediately after bag removal. These results suggest that bagging treatment can induce epigenetic changes, facilitating the binding of trans factor(s) to MdMYB1-2/-3 alleles, resulting in the activation of these MYBs after bag removal.

     

Detection of human T-cell leukemia virus type I (HTLV-I) provirus in an infected cell line and in peripheral mononuclear cells of blood donors by the nested double polymerase chain reaction method: comparison with HTLV-I antibody tests.

Human T-cell leukemia virus type I (HTLV-I) provirus DNA from the cultured cell line HUT 102 and from peripheral mononuclear cells (PBMC) of anti-HTLV-I antibody-positive Japanese blood donors was detected by the nested double polymerase chain reaction (PCR) method. This procedure consists of a first amplification and a second amplification with the products of the first amplification and primers interior to the first primers. Using this method, we demonstrated that it is possible to detect single-template DNA. Polyacrylamide gel electrophoresis of the nested double PCR products, with our primers, revealed three bands with excess amounts of template DNA, two bands with moderate amounts, and a single band with limited amounts. The amount of provirus in PBMC was roughly estimated from the results of the nested double PCR. Particle agglutination (PA) assays and indirect immunofluorescence testing (IF) with mixed MT-2 cells and Molt-4 cells as targets to detect anti-HTLV-I antibody were performed, and the results were compared with those of the nested double PCR of the pX region. None of the 101 PA-negative samples were positive in either the IF or PCR test. Of the 155 samples that were antibody positive by the PA assay, 57 were positive by both PCR and IF. Furthermore, the results of the IF and PCR tests coincided completely. It was therefore concluded that the IF method is most appropriate for confirmation of the PA assay currently used in most diagnostic laboratories and blood centers.