Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells

Cohesion between sister chromatids is essential for their bi-orientation on mitotic spindles. It is mediated by a multisubunit complex called cohesin. In yeast, proteolytic cleavage of cohesin's alpha kleisin subunit at the onset of anaphase removes cohesin from both centromeres and chromosome arms and thus triggers sister chromatid separation. In animal cells, most cohesin is removed from chromosome arms during prophase via a separase-independent pathway involving phosphorylation of its Scc3-SA1/2 subunits. Cohesin at centromeres is refractory to this process and persists until metaphase, whereupon its alpha kleisin subunit is cleaved by separase, which is thought to trigger anaphase. What protects centromeric cohesin from the prophase pathway? Potential candidates are proteins, known as shugoshins, that are homologous to Drosophila MEI-S332 and yeast Sgo1 proteins, which prevent removal of meiotic cohesin complexes from centromeres at the first meiotic division. A vertebrate shugoshin-like protein associates with centromeres during prophase and disappears at the onset of anaphase. Its depletion by RNA interference causes HeLa cells to arrest in mitosis. Most chromosomes bi-orient on a metaphase plate, but precocious loss of centromeric cohesin from chromosomes is accompanied by loss of all sister chromatid cohesion, the departure of individual chromatids from the metaphase plate, and a permanent cell cycle arrest, presumably due to activation of the spindle checkpoint. Remarkably, expression of a version of Scc3-SA2 whose mitotic phosphorylation sites have been mutated to alanine alleviates the precocious loss of sister chromatid cohesion and the mitotic arrest of cells lacking shugoshin. These data suggest that shugoshin prevents phosphorylation of cohesin's Scc3-SA2 subunit at centromeres during mitosis. This ensures that cohesin persists at centromeres until activation of separase causes cleavage of its alpha kleisin subunit. Centromeric cohesion is one of the hallmarks of mitotic chromosomes. Our results imply that it is not an intrinsically stable property, because it can easily be destroyed by mitotic kinases, which are kept in check by shugoshin.     

Flow cytometric evidence for endopolyploidization in cabbage (Brassica oleracea L.) flowers

Flow cytometric analysis of the nuclear DNA contents of somatic tissues from flowers of cabbage (Brassica oleracea L.) revealed extensive endoreduplication, resulting in tissues that contain cells with multiple ploidy levels (also called ’endopolyploidy’). Multiples of the haploid nuclear genome complement (1C) corresponding to 2C, 4C, 8C, 16C, 32C and 64C were observed in mature flowers. The distribution of cells with the differerent ploidy levels is tissue-specific and is characteristic of the stage of development. Nuclei of young flower buds exclusively gave 2C and 4C peaks, indicating that the tissues maintained diploid level. Endoreduplication was consistently detected during flower development. Endopolyploidy is probably common in differentiation of cabbage plants. Implications of this original feature are discussed. Received: 28 August 2000 / Revision accepted: 20 December 2000     

Incorporation of a high level of vitamin B12 into a vegetable, kaiware daikon (Japanese radish sprout), by the absorption from its seeds

High level of vitamin B(12) was incorporated into kaiware daikon (Japanese radish sprout) by soaking its seeds in B(12) solutions. Vitamin B(12) amount incorporated into kaiware daikon increased up to 1.5 microg/g wet sprout with the soaking time of seeds in 0-200 microg/ml B(12) solution. Vitamin B(12) could be extracted more from the sample heated for a short time than from that of control without heat treatment.     

Release of Arsenic from Soil by a Novel Dissimilatory Arsenate-Reducing Bacterium,Anaeromyxobacter sp. Strain PSR-1

A novel arsenate-reducing bacterium, designated strain PSR-1, was isolated from arsenic-contaminated soil. Strain PSR-1 was phylogenetically closely related to Anaeromyxobacter dehalogenans 2CP-1^T with 16S rRNA gene similarity of 99.7% and coupled the oxidation of acetate with the reduction of arsenate. Arsenate reduction was inhibited almost completely by respiratory inhibitors such as dicumarol and 2-heptyl-4-hydroxyquinoline N-oxide. Strain PSR-1 also utilized soluble Fe(III), ferrihydrite, nitrate, oxygen, and fumarate as electron acceptors. Strain PSR-1 catalyzed the release of arsenic from arsenate-adsorbed ferrihydrite. In addition, inoculation of washed cells of strain PSR-1 into sterilized soil successfully reproduced arsenic release. Arsenic K-edge X-ray absorption near-edge structure (XANES) analysis revealed that the proportion of arsenite in the soil solid phase actually increased from 20% to 50% during incubation with washed cells of strain PSR-1. These results suggest that strain PSR-1 is capable of reducing not only dissolved arsenate but also arsenate adsorbed on the soil mineral phase. Arsenate reduction by strain PSR-1 expands the metabolic versatility of Anaeromyxobacter dehalogenans. Considering its distribution throughout diverse soils and anoxic sediments, Anaeromyxobacter dehalogenans may play a role in arsenic release from these environments.     

Deletion of microsomal prostaglandin E synthase-1 protects neuronal cells from cytotoxic effects of β-amyloid peptide fragment 31-35

Epidemiological studies have suggested that the long-term use of nonsteroidal anti-inflammatory drugs that inhibit cyclooxygenase (COX) activity moderates the onset or progression of Alzheimer's disease (AD). Thus it has been suggested that prostaglandin E(2) (PGE(2)), a major end-product of COX, may play a pathogenic role in AD, but the involvement of PGE synthase (PGES), a terminal enzyme downstream from COX, has not been fully elucidated. To examine the involvement in AD pathology of microsomal PGES-1 (mPGES-1), a PGES enzyme, we here prepared primary cerebral neuronal cells from the cerebri of wild-type and mPGES-1-deficient mice and then treated them with β-amyloid (Aβ) fragment 31-35 (Aβ(31-35)), which represents the shortest sequence of native Aβ peptide required for neurotoxicity. Treatment of wild-type neuronal cells with Aβ(31-35) induced mPGES-1 gene expression and PGE(2) production, followed by significant apoptotic cell death, but apoptosis was not induced in mPGES-1-deficient cells. Furthermore, the combined treatment of Aβ(31-35) and PGE(2) induced apoptosis in mPGES-1-deficient neuronal cells. These results indicated that mPGES-1 is induced during Aβ-mediated neuronal cell death and is involved in Aβ-induced neurotoxicity associated with AD pathology.     

Inhibition of IL-8-induced W3/25+ (CD4+) T lymphocyte recruitment into subcutaneous tissues of rats by selective depletion of in vivo neutrophils with a monoclonal antibody

IL-8 recruits both neutrophils and lymphocytes in vitro and in vivo. To elucidate the mechanisms of lymphocyte recruitment in vivo by IL-8, we examined the role of neutrophil infiltration through selective depletion of circulating neutrophils using a mAb, RP-3. Selective depletion of neutrophils inhibited the IL-8 induced in vivo migration of the W3/25+ (CD4+) T cell subset but did not inhibit that of the MRC-OX8+ (CD8+) subset. These results suggest that CD4+ T cell migration into IL-8-injected s.c. tissues depends on the prior infiltration of neutrophils chemoattracted directly by IL-8.     

Relationship between the earlywood-to-latewood transition and changes in levels of stored starch around the cambium in locally heated stems of the evergreen conifer <Emphasis Type="Italic">Chamaecyparis pisifera</Emphasis>

Key message

We observed the formation of latewood tracheids with narrow diameters and thick walls and the disappearance of stored starch around the cambium on the locally heated region of stems in evergreen conifer Chamaecyparis pisifera during winter cambial dormancy.

Abstract

Wood formation is controlled by cambial cell division, which determines the quantity and quality of wood. We investigated the factors that control cambial activity and the formation of new tracheids in locally heated stems of the evergreen conifer Chamaecyparis pisifera. Electric heating tape was wrapped around one side of the stem, at breast height, of two trees in 2013 and two in 2014. Pairs of stems were locally heated in winter, and small blocks were collected from heated and non-heated regions of stems. Cambial activity and levels of stored starch around the cambium were investigated by microscopy. Cambial reactivation and xylem differentiation occurred earlier in heated than in non-heated regions. New cell plates were formed after 14–18 days of heating. After a few layers of tracheids with large diameters and thin walls had formed, cell division and cell enlargement during differentiation were inhibited. Tracheids with narrow diameters and thick walls, defining those as latewood, were formed near the cambium, and finally, four to six layers of tracheids were induced. After cambial reactivation, amounts of stored starch started to decrease and starch disappeared completely from phloem and xylem cells that were located near the cambium during the differentiation of heated regions. Our results suggest that an increase in temperature induces the conversion of stored starch to soluble sugars for continuous cambial cell division and earlywood formation. By contrast, a shortage of stored starch might be responsible for inhibition of cambial activity and induction of the formation of latewood tracheids.

     

Long-term proliferating early pre B cell lines and clones with the potential to develop to surface Ig-positive, mitogen reactive B cells in vitro and in vivo

Cell lines and clones were established from PB76-positive mouse fetal liver at day 13 and 14 of gestation, which proliferated with division times of a day in serum-substituted cultures under the stimulatory influence of adherent stromal cells and the cytokine IL-7 for periods longer than half a year. These lines expressed varying levels of the B lymphocyte lineage related markers PB76, B220, BP-1, VpreB and lambda 5, but no surface Ig or MHC class II molecules. All clones expressed PB76, VpreB and lambda 5 in a high percentage of cells, while B220 and/or BP-1 expression was low or undetectable in some. A cell line, and several clones established from it, all had kappa and lambda light chain genes in germ-line configuration. Either one or both of their H-chain-gene containing chromosomes carried a DH to JH. These pre B cell lines and clones could be induced to VH to DH and VL to JL rearrangements. This resulted in the development of varying percentages of sIg-positive surface, MHC class II negative, LPS-reactive B cells within 2-3 days, in the absence of contacts with stromal cells and/or IL-7. When injected into SCID mice, the cultured pre B cells populated the spleen of these mice to 5% with surface Ig-, MHC class II-positive LPS-reactive cells for greater than 25 weeks. The long-term in vitro proliferative capacity of these DH-JH rearranged pre B cell clones makes them major candidates for committed stem cells of the B lineage.