Multiple histone modifications in euchromatin promote heterochromatin formation by redundant mechanisms in Saccharomyces cerevisiae

Background  

Methylation of lysine 79 on histone H3 by Dot1 is required for maintenance of heterochromatin structure in yeast and humans. However, this histone modification occurs predominantly in euchromatin. Thus, Dot1 affects silencing by indirect mechanisms and does not act by the recruitment model commonly proposed for histone modifications. To better understand the role of H3K79 methylation gene silencing, we investigated the silencing function of Dot1 by genetic suppressor and enhancer analysis and examined the relationship between Dot1 and other global euchromatic histone modifiers.     

Protein-protein interactions of ESCRT complexes in the yeast Saccharomyces cerevisiae

Ten class E Vps proteins in yeast are known components of the ESCRT complexes I, II and III, which are required for the sorting of proteins to the lumenal membranes of multivesicular bodies. We used the yeast 2 hybrid system to analyze the protein–protein interactions of all 17 soluble class E Vps proteins, as well as proteins thought to be required for the ubiquitination and deubiquitination of cargo proteins at multivesicular bodies. We identified novel interactions between yeast ESCRT complex components suggesting that ESCRTI binds to both ESCRTII and ESCRTIII. These interactions were confirmed by GST pull-down experiments. Our data indicate that the link between ESCRTI and ESCRTIII is via Vps28p and Vps37p/Srn2p binding directly to Vps20p, as well as through indirect interactions via ESCRTII. This is in contrast to the situation in mammalian cells where ESCRTI and ESCRTIII interact indirectly via ALIX, the mammalian homologue of yeast proteins Vps31p/Bro1p and Rim20p. Our data also enable us to link all soluble class E Vps proteins to the ESCRT complexes. We propose the formation of a large multimeric complex on the endosome membrane consisting of ESCRTI, ESCRTII, ESCRTIII and other associated proteins.     

Morphological Changes in the Midgut of Aedes aegypti L. (Diptera: Culicidae) Larvae Following Exposure to an Annona coriacea (Magnoliales: Annonaceae) Extract

Bioinsecticides are important in the control of disease vectors, but data regarding their physiological effects on target insects are incomplete. This study describes morphological changes that occur in the midgut of third instar Aedes aegypti L. (Diptera: Culicidae) following treatment with a methanolic extract of Annona coriacea (Magnoliales: Annonaceae). Dissected midguts were subdivided into anterior and posterior regions and analyzed by light and scanning electron microscopy. Insects exposed to the extract displayed intense, destructive cytoplasmic vacuolization in columnar and regenerative midgut cells. The apical surfaces of columnar cells exhibited cytoplasmic protrusions oriented toward the lumen, suggesting that these cells could be involved in apocrine secretory processes and/or apoptosis. We report that A. coriacea extracts induced morphological alterations in the midgut of A. aegypti midgut larvae, supporting the use of plant extracts for control of the dengue vector.     

Population differences in larval hostplant use in the checkerspot butterfly, Euphydryas chalcedona

Larvae from two populations of Euphydryas chalcedona Doubleday & Hewitson (Nymphalidae) were reared on their own hostplant and that of the other population, in both pre-diapause and post-diapause instars. One population, Chico, uses Penstemon breviflorus Lindl. (Scrophulariaceae), and the other, Echo Lake, uses P. newberryi Gray. Growth rate and survival were determined for pre-diapause and post-diapause larvae from both populations on both plant species; and digestive efficiencies were calculated during the prediapause instars. The results showed that larvae from the two populations differed in their responses to the two plant species. Pre-diapause larvae from Chico performed equally well on both plant species—survival and digestive indices were not significantly different for two Penstemon species. In contrast, pre-diapause larvae from Echo Lake performed significantly worse on the non-hostplant—growth and survival were significantly lower on the non-host, P. breviflorus. In addition, comparison of digestive efficiencies for the two plants showed that larvae from Echo Lake digested P. breviflorus better than P. newberryi, but were significantly less able to convert P. breviflorus to body mass. In the post-diapause instars, larvae from Chico grew faster on the host than on the non-host. Larvae from Echo Lake grew quite slowly on both plant species and significantly more of the Echo Lake larvae returned to diapause instead of completing development.

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Résumé Des chenilles de deux populations d'E. chalcedona ont été élevées sur leur propre plante-hôte et sur celle de l'autre population, aux stades avant et après diapause. Les deux populations s'alimentent sur différentes espèces de Penstemon (Scrophulariaceae), et une population—Echo Lake—est monophage sur P. newberry, tandis que l'autre—Chico—utilise d'abord P. breviflorus, mais les chenilles après diapause sont trouvées sur au moins deux autres espèces de plantes. Les taux de croissance et de survie ont été déterminés pour des chenilles avant et après diapause pour les deux populations sur les deux plantes; les efficacités digestives ont été calculées sur les chenilles avant diapause.Les résultats ont montré que les chenilles des deux populations différaient par leur degré de spécialisation digestive sur leur plante hôte normale: les chenilles de Chico ont utilisé aussi bien les deux plantes, tandis que celles d'Echo Lake le faisaient significativement moins bien sur la plante non-hôte, par suite de l'inaptitude à la digérer. Ainsi la population oligophage est alimentairement moins spécialisée et plus capable de se débrouiller avec une plante non-hôte. Après diapause, les chenilles de Chico s'alimentaient significativement mieux sur plante hôte que non-hôte, ce qui était le cas aussi pour la population monophage. Dans l'ensemble, les chenilles de la population monophage semblaient moins capables de se débrouiller dans des conditions défavorables ou moins avantageuses.

     

Cluster of differentiation antigen 4 (CD4) endocytosis and adaptor complex binding require activation of the CD4 endocytosis signal by serine phosphorylation

Cluster of differentiation antigen 4 (CD4), the T lymphocyte antigen receptor component and human immunodeficiency virus coreceptor, is down-modulated when cells are activated by antigen or phorbol esters. During down-modulation CD4 dissociates from p56(lck), undergoes endocytosis through clathrin-coated pits, and is then sorted in early endosomes to late endocytic organelles where it is degraded. Previous studies have suggested that phosphorylation and a dileucine sequence are required for down-modulation. Using transfected HeLa cells, in which CD4 endocytosis can be studied in the absence of p56(lck), we show that the dileucine sequence in the cytoplasmic domain is essential for clathrin-mediated CD4 endocytosis. However, this sequence is only functional as an endocytosis signal when neighboring serine residues are phosphorylated. Phosphoserine is required for rapid endocytosis because CD4 molecules in which the cytoplasmic domain serine residues are substituted with glutamic acid residues are not internalized efficiently. Using surface plasmon resonance, we show that CD4 peptides containing the dileucine sequence bind weakly to clathrin adaptor protein complexes 2 and 1. The affinity of this interaction is increased 350- to 700-fold when the peptides also contain phosphoserine residues.     

Radiation-induced Release of Transforming Growth Factor α Activates the Epidermal Growth Factor Receptor and Mitogen-activated Protein Kinase Pathway in Carcinoma Cells, Leading to Increased Proliferation and Protection from Radiation-induced Cell Death

Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0-10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90-240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor alpha receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor alpha (TGFalpha) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGFalpha cleavage 120-180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGFalpha. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGFalpha. Neutralization of TGFalpha function by an anti-TGFalpha antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGFalpha-EGFR-MAPK signaling module represents a strategy to decrease carcinoma cell growth and survival after irradiation.     

A simple method using alizarin red S for the detection of calcium in epoxy resin embedded tissue

This report presents a simple procedure for staining 1-2 microns epoxy plastic sections of cells and mineralizing matrix present in fetal bovine bone tissue cultures. A 0.3% aqueous toluidine blue O solution was used as a cellular stain and was followed with 2% alizarin red S for the detection of calcium at sites of mineralization. Effects of concentration and pH of alizarin red S on the penetration of epon embedded thick sections were investigated. Optimal staining was achieved with a 2% aqueous alizarin red S solution adjusted to a pH of 5.5-6.5. This staining procedure provides unusually clear contrast between mineral and bone cells in plastic sections for light microscopy.     

On the existence and separation of the follicle stimulating hormone releasing hormone from the luteinizing hormone releasing hormone.

Porcine hypothalamic fragments were extracted by 2M AcOH at 4°C, and the extractives were subsequently processed in the presence of one protease inhibitor and one anti-oxidant. Gel filtration was performed on Bio-Gel P-2, and supplementary [³H]-LHRH and [¹⁴C]- 3H]-LHRH, and was differentiated from [¹⁴C]- 相似文献   

The design of a novel series of muscarinic receptor antagonists leading to AZD8683, a potential inhaled treatment for COPD

A novel series of muscarinic receptor antagonists was developed, with the aim of identifying a compound with high M₃ receptor potency and a reduced risk of dose-limiting side effects with potential for the treatment of COPD.Initial compound modifications led to a novel cycloheptyl series, which was improved by focusing on a quinuclidine sub-series. A wide range of N-substituents was evaluated to determine the optimal substituent providing a high M₃ receptor potency, high intrinsic clearance and high human plasma protein binding. Compounds achieving in vitro study criteria were selected for in vivo evaluation. Pharmacokinetic half-lives, inhibition of bronchoconstriction and duration of action, as well as systemic side effects, induced by the compounds were assessed in guinea-pig models.Compounds with a long duration of action and good therapeutic index were identified and AZD8683 was selected for progression to the clinic.     

The ovulated ovum of the horse: cytology of nonfertilized ova to pronuclear stage ova

Fertilization and early development in the horse were studied by recovering oviductal ova at various times after postovulatory mating. Ova collected between 7 and 22 h post coitum (pc) were examined for evidence of fertilizing sperm, cellular changes accompanying fertilization, and pronuclear development. Five ova collected between 7 and 9 h pc contained a marginal metaphase plate, but had no indication of sperm components; three of these, however, showed reduced numbers of cortical granules. Two activated ova (10 and 14 h pc) were in telophase of the second meiotic division, following incorporation of the fertilizing sperm. The fertilizing sperm was situated in a slight elevation; the nucleus was expanding but lacked a nuclear envelope. The pronuclear stage in the horse began as early as 12 h pc, and lasted at least until 21 h pc. Sperm tail remnants were seen in 5 of 7 pronuclear-stage ova, although the crowding of the cytoplasm with clusters of lipid and vacuoles made discerning sperm tail remnants difficult. The spindles of the metaphase stage of the second meiotic division were oriented radially, that is, at right angles to the cell surface, in all but one ovum, so this orientation is not a response to fertilization.