Large scale deletions in the <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> genome create strains with altered regulation of carbon metabolism

Saccharomyces cerevisiae, for centuries the yeast that has been the workhorse for the fermentative production of ethanol, is now also a model system for biological research. The recent development of chromosome-splitting techniques has enabled the manipulation of the yeast genome on a large scale, and this has allowed us to explore questions with both biological and industrial relevance, the number of genes required for growth and the genome organization responsible for the ethanol production. To approach these questions, we successively deleted portions of the yeast genome and constructed a mutant that had lost about 5% of the genome and that gave an increased yield of ethanol and glycerol while showing levels of resistance to various stresses nearly equivalent to those of the parental strain. Further systematic deletion could lead to the formation of a eukaryotic cell with a minimum set of genes exhibiting appropriately altered regulation for enhanced metabolite production. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

VAMP2 is expressed in muscle satellite cells and up-regulated during muscle regeneration

Membrane trafficking is one of the most important mechanisms involved in the establishment and maintenance of the forms and functions of the cell. However, it is poorly understood in skeletal muscle cells. In this study, we have focused on vesicle-associated membrane proteins (VAMPs), which are components of the vesicle docking and fusion complex, and have performed immunostaining to investigate the expression of VAMPs in rat skeletal muscle tissue. We have found that VAMP2, but not VAMP1 or VAMP3, is expressed in satellite cells. VAMP2 is also expressed in myofibers in the soleus muscle and nerve endings. This is consistent with previous studies in which VAMP2 has been shown to regulate GLUT4 trafficking in slow-twitch myofibers in soleus muscle and neurotransmitter release in nerve endings. As satellite cells are quiescent myogenic cells, the expression of VAMP2 has further been examined in regenerating muscles after injury by the snake venom, cardiotoxin; we have observed enhanced expression of VAMP2 in immature myotubes with a peak at 3 days after injury. Our findings suggest that VAMP2 plays roles in quiescent satellite cells and is involved in muscle regeneration. The nature of the material transported in the VAMP2-bearing vesicles in satellite cells and myotubes is still under investigation. This work was supported by a research grant (17A-10) for nervous and mental disorders from the Ministry of Health, Labor, and Welfare of Japan, and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.     

Synthesis and seed germination stimulating activity of some imino analogs of strigolactones

Strigolactones are germination stimulants for seeds of the root parasitic weeds, Striga and Orobanche spp. The imino analog of GR24 showed moderate germination stimulating activity against the seeds of S. hermonthica. The seed germination stimulating activity of some phenyliminoacetates and phenyliminoacetonitriles was also examined. The degree of activity of the phenyliminoacetate was less than that of the phenylacrylates. On the other hand, the degree of activity of the phenyliminoacetonitrile was comparable to that of the phenylacrylonitriles. Among the tested compounds, the 3-pyridyliminoacetonitrile showed higher activity against the seeds of O. crenata than GR24. These findings demonstrate that it is not always essential to have the Michael acceptor of the C-D ring junction moiety which has been proposed to react with nucleophilic species presented at the target site to enhance the activity.     

Anti-angiogenic effects of conjugated docosahexaenoic acid in vitro and in vivo

The anti-angiogenic effects of conjugated docosahexaenoic acid (CDHA), which was prepared by an alkaline treatment of docosahexaenoic acid and contained conjugated double bonds, were investigated in vitro and in vivo. CDHA inhibited tube formation by the bovine aortic endothelial cell (BAEC), and also inhibited the proliferation of BAEC at a concentration of CDHA that suppressed tube formation, but did not influence cell migration. The inhibition of BAEC growth caused by CDHA was accompanied by a marked change in cellular morphology. Nuclear condensation and brightness were observed in Hoechst 33342-stained cells treated with CDHA, indicating that CDHA induced apoptosis in BAEC. We also evaluated the angiogenesis inhibition of CDHA in vivo. The vessel formation which was triggered by tumor cells was clearly suppressed in mice orally given CDHA. Our findings suggest that CDHA has potential use as a therapeutic dietary supplement for minimizing tumor angiogenesis.     

Putative mechanism of the itch-scratch circle: repeated scratching decreases the cutaneous level of prostaglandin D2, a mediator that inhibits itching

In atopic dermatitis, scratching of the skin as a reaction to itching causes injury to the skin, which, in turn, further increases the itching resulting in the establishment of the so-called itch-scratch circle. We have shown that prostaglandin (PG) D2 plays an inhibitory role against pruritus in mice with atopic-like dermatitis; therefore, we examined the relationship between scratching and the cutaneous PGD2 level using an artificial scratching model with a wire brush. Mechanical scratching induced a temporary increase of the skin PGs levels (PGE2, PGD2, 6-ketoPGF1alpha, PGF2alpha). The skin PGD2 level and the ability of PGD2 production decreased at 48 h after repeated scratch, compared to that of normal skin, not so after single scratch. Immunohistochemical analysis and Western blotting revealed a decrease in the levels of cyclooxygenase-1 (COX-1) and hematopoietic PGD synthase in mechanically scratched skin. The reduced ability of the skin for PGD2 production following mechanical scratching could be caused by this decrease in the expression levels of COX-1 and PGD2 synthase. The results suggest that repeated scratching in mice decreases the ability of the skin to produce PGD2, which is an endogenous mediator that inhibits pruritus, resulting in the establishment of the itch-scratch circle.     

Bld10p constitutes the cartwheel-spoke tip and stabilizes the 9-fold symmetry of the centriole

Centrioles/basal bodies have a characteristic cylindrical structure consisting of nine triplet microtubules arranged in a rotational symmetry. How this elaborate structure is formed is a major unanswered question in cell biology [1, 2]. We previously identified a 170 kDa coiled-coil protein essential for the centriole formation in Chlamydomonas. This protein, Bld10p, is the first protein shown to localize to the cartwheel, a 9-fold symmetrical structure possibly functioning as the scaffold for the centriole-microtubule assembly [3]. Here, we report results by using a series of truncated Bld10p constructs introduced into a bld10 null mutant. Remarkably, a transformant (DeltaC2) in which 35% of Bld10p at the C terminus was deleted assembled centrioles with eight symmetrically arranged triplets, in addition to others with the normal nine triplets. The cartwheels in these eight-membered centrioles had spokes approximately 24% shorter than those in the wild-type, suggesting that the eight-triplet centrioles were formed because the cartwheel's smaller diameter. From the morphology of the cartwheel spoke in the DeltaC2 centriole and immunoelectron-microscope localization, we conclude that Bld10p is a major spoke-tip component that extends the cartwheel diameter and attaches triplet microtubules. These results provide the first experimental evidence for the crucial function of the cartwheel in centriolar assembly.     

Displacement and re-accumulation of centromeric cohesin during transient pre-anaphase centromere splitting

The ring-shaped cohesin complex links sister chromatids until their timely segregation during mitosis. Cohesin is enriched at centromeres where it provides the cohesive counterforce to bipolar tension produced by the mitotic spindle. As a consequence of spindle tension, centromeric sequences transiently split in pre-anaphase cells, in some organisms up to several micrometers. This ‘centromere breathing’ presents a paradox, how sister sequences separate where cohesin is most enriched. We now show that in the budding yeast Saccharomyces cerevisiae, cohesin binding diminishes over centromeric sequences that split during breathing. We see no evidence for cohesin translocation to surrounding sequences, suggesting that cohesin is removed from centromeres during breathing. Two pools of cohesin can be distinguished. Cohesin loaded before DNA replication, which has established sister chromatid cohesion, disappears during breathing. In contrast, cohesin loaded after DNA replication is partly retained. As sister centromeres re-associate after transient separation, cohesin is reloaded in a manner independent of the canonical cohesin loader Scc2/Scc4. Efficient centromere re-association requires the cohesion establishment factor Eco1, suggesting that re-establishment of sister chromatid cohesion contributes to the dynamic behaviour of centromeres in mitosis. These findings provide new insights into cohesin behaviour at centromeres. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dock6, a Dock-C subfamily guanine nucleotide exchanger, has the dual specificity for Rac1 and Cdc42 and regulates neurite outgrowth

Small GTPases of the Rho family, Rho, Rac, and Cdc42, are critical regulators of the changes in the actin cytoskeleton. Rho GTPases are typically activated by Dbl-homology (DH)-domain-containing guanine nucleotide exchange factors (GEFs). Recent genetic and biochemical studies revealed a new type of GEF for the Rho GTPases. This family is composed of 11 genes, designated as Dock1 to Dock11, and is structurally divided into four classes Dock-A, -B, -C, and -D. Dock-A and -B subfamilies are typically GEFs specific for Rac1, while the Dock-D subfamily is specific for Cdc42. Here we show that Dock6, a member of the Dock-C subfamily, exchanges GDP for GTP for Rac1 and Cdc42 in vitro and in vivo. Furthermore, we find that, in mouse N1E-115 neuroblastoma cells, expression of Dock6 is increased following differentiation. Transfection of the catalytic Dock Homology Region-2 (DHR-2) domain of Dock6 promotes neurite outgrowth mediated by Rac1 and Cdc42. Conversely, knockdown of endogenous Dock6 by small interference RNA reduces activation of Rac1 and Cdc42 and neurite outgrowth. Taken together, these results suggest that Dock6 differs from all of the identified Dock180-related proteins, in that it is the GEF specific for both Rac1 and Cdc42 and may be one of physiological regulators of neurite outgrowth.     

BAF as a caspase-dependent mediator of nuclear apoptosis in Drosophila

BAF is a double-stranded DNA binding protein required for proper nuclear morphology and function in Drosophila development. Imaginal discs of Drosophila baf-null mutants were found to exist only in younger larvae as small degenerative tissues. Immunohistochemical analyses showed diffuse lamin distribution, DNA fragmentation, and activation of caspase drICE in these tissues, suggesting that apoptotic events can be induced by the loss of baf. We therefore investigated the fate of BAF after induction of the pro-apoptotic hid transgene, and found that the loss of DNA binding forms of BAF preceded that of non-DNA binding forms of BAF. Furthermore, the DNA binding forms of BAF disappeared from nuclei before DNA fragmentation and NPC clustering were detected, showing that the loss of BAF occurs at the initial stages of nuclear apoptosis. This BAF loss was not detected before drICE activation and was inhibited by Ac-DEVD-CHO caspase inhibitors. In summary, BAF disappears at an early stage due to caspase activity when apoptosis is induced by hid, and its depletion in mutants is sufficient in itself to induce cell death, suggesting it is an apoptotic mediator.     

TPT1 Supports Proliferation of Neural Stem/Progenitor Cells and Brain Tumor Initiating Cells Regulated by Macrophage Migration Inhibitory Factor (MIF)

Neurochemical Research - One of the key areas in stem cell research is the identification of factors capable of promoting the expansion of Neural Stem Cell/Progenitor Cells (NSPCs) and...