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991.
Background
Retroviruses have been observed to bud intracellularly into multivesicular bodies (MVB), in addition to the plasma membrane. Release from MVB is thought to occur by Ca2+-regulated fusion with the plasma membrane.Principal Findings
To address the role of the MVB pathway in replication of the murine leukemia virus (MLV) we took advantage of mouse models for the Hermansky-Pudlak syndrome (HPS) and Griscelli syndrome. In humans, these disorders are characterized by hypopigmentation and immunological alterations that are caused by defects in the biogenesis and trafficking of MVBs and other lysosome related organelles. Neonatal mice for these disease models lacking functional AP-3, Rab27A and BLOC factors were infected with Moloney MLV and the spread of virus into bone marrow, spleen and thymus was monitored. We found a moderate reduction in MLV infection levels in most mutant mice, which differed by less than two-fold compared to wild-type mice. In vitro, MLV release form bone-marrow derived macrophages was slightly enhanced. Finally, we found no evidence for a Ca2+-regulated release pathway in vitro. Furthermore, MLV replication was only moderately affected in mice lacking Synaptotagmin VII, a Ca2+-sensor regulating lysosome fusion with the plasma membrane.Conclusions
Given that MLV spreading in mice depends on multiple rounds of replication even moderate reduction of virus release at the cellular level would accumulate and lead to a significant effect over time. Thus our in vivo and in vitro data collectively argue against an essential role for a MVB- and secretory lysosome-mediated pathway in the egress of MLV. 相似文献992.
993.
Background
Saltational evolution in which a particular lineage undergoes relatively rapid, significant, and unparalleled change as compared with its closest relatives is rarely invoked as an alternative model to the dominant paradigm of gradualistic evolution. Identifying saltational events is an important first-step in assessing the importance of this discontinuous model in generating evolutionary novelty. We offer evidence for three independent instances of saltational evolution in a charismatic moth genus with only eight species.Methodology/Principal Findings
Maximum parsimony, maximum likelihood and Bayesian search criteria offered congruent, well supported phylogenies based on 1,965 base pairs of DNA sequence using the mitochondrial gene cytochrome oxidase subunit I, and the nuclear genes elongation factor-1 alpha and wingless. Using a comparative methods approach, we examined three taxa exhibiting novelty in the form of Batesian mimicry, host plant shift, and dramatic physiological differences in light of the phylogenetic data. All three traits appear to have evolved relatively rapidly and independently in three different species of Proserpinus. Each saltational species exhibits a markedly different and discrete example of discontinuous trait evolution while remaining canalized for other typical traits shared by the rest of the genus. All three saltational taxa show insignificantly different levels of overall genetic change as compared with their congeners, implying that their divergence is targeted to particular traits and not genome-wide.Conclusions/Significance
Such rapid evolution of novel traits in individual species suggests that the pace of evolution can be quick, dramatic, and isolated—even on the species level. These results may be applicable to other groups in which specific taxa have generated pronounced evolutionary novelty. Genetic mechanisms and methods for assessing such relatively rapid changes are postulated. 相似文献994.
Background
Cauliflower mosaic virus (CaMV) and Rice tungro bacilliform virus (RTBV) belong to distinct genera of pararetroviruses infecting dicot and monocot plants, respectively. In both viruses, polycistronic translation of pregenomic (pg) RNA is initiated by shunting ribosomes that bypass a large region of the pgRNA leader with several short (s)ORFs and a stable stem-loop structure. The shunt requires translation of a 5′-proximal sORF terminating near the stem. In CaMV, mutations knocking out this sORF nearly abolish shunting and virus viability.Methodology/Principal Findings
Here we show that two distant regions of the CaMV leader that form a minimal shunt configuration comprising the sORF, a bottom part of the stem, and a shunt landing sequence can be replaced by heterologous sequences that form a structurally similar configuration in RTBV without any dramatic effect on shunt-mediated translation and CaMV infectivity. The CaMV-RTBV chimeric leader sequence was largely stable over five viral passages in turnip plants: a few alterations that did eventually occur in the virus progenies are indicative of fine tuning of the chimeric sequence during adaptation to a new host.Conclusions/Significance
Our findings demonstrate cross-species functionality of pararetroviral cis-elements driving ribosome shunting and evolutionary conservation of the shunt mechanism.We are grateful to Matthias Müller and Sandra Pauli for technical assistance. This work was initiated at Friedrich Miescher Institute (Basel, Switzerland). We thank Prof. Thomas Boller for hosting the group at the Institute of Botany. 相似文献995.
Huber S Schrader J Fritz G Presser K Schmitt S Waisman A Lüth S Blessing M Herkel J Schramm C 《PloS one》2008,3(10):e3302
CD4+CD25+ regulatory T cells (Treg) are important mediators of immune tolerance. A subset of Treg can be generated in the periphery by TGF-beta dependent conversion of conventional CD4+CD25- T cells into induced Treg (iTreg). In chronic viral infection or malignancy, such induced iTreg, which limit the depletion of aberrant or infected cells, may be of pathogenic relevance. To identify potential targets for therapeutic intervention, we investigated the TGF-beta signaling in Treg. In contrast to conventional CD4+ T cells, Treg exhibited marked activation of the p38 MAP kinase pathway. Inhibition of p38 MAP kinase activity prevented the TGF-beta-dependent conversion of CD4+CD25- T cells into Foxp3+ iTreg in vitro. Of note, the suppressive capacity of nTreg was not affected by inhibiting p38 MAP kinase. Our findings indicate that signaling via p38 MAP kinase seems to be important for the peripheral generation of iTreg; p38 MAP kinase could thus be a therapeutic target to enhance immunity to chronic viral infection or cancer. 相似文献
996.
Lemke T Welling A Christel CJ Blaich A Bernhard D Lenhardt P Hofmann F Moosmang S 《The Journal of biological chemistry》2008,283(50):34738-34744
Phosphorylation of serine 1928 (Ser(1928)) of the cardiac Ca(v)1.2 subunit of L-type Ca(2+) channels has been proposed as the mechanism for regulation of L-type Ca(2+) channels by protein kinase A (PKA). To test this directly in vivo, we generated a knock-in mouse with targeted mutation of Ser(1928) to alanine. This mutation did not affect basal L-type current characteristics or regulation of the L-type current by PKA and the beta-adrenergic receptor, whereas the mutation abolished phosphorylation of Ca(v)1.2 by PKA. Therefore, our data show that PKA phosphorylation of Ser(1928) of Ca(v)1.2 is not functionally involved in beta-adrenergic stimulation of Ca(v)1.2-mediated Ca(2+) influx into the cardiomyocyte. 相似文献
997.
Dagda RK Merrill RA Cribbs JT Chen Y Hell JW Usachev YM Strack S 《The Journal of biological chemistry》2008,283(52):36241-36248
The neurodegenerative disorder spinocerebellar ataxia 12 (SCA12) is caused by CAG repeat expansion in the non-coding region of the PPP2R2B gene. PPP2R2B encodes Bbeta1 and Bbeta2, alternatively spliced and neuron-specific regulatory subunits of the protein phosphatase 2A (PP2A) holoenzyme. We show here that in PC12 cells and hippocampal neurons, cell stressors induced a rapid translocation of PP2A/Bbeta2 to mitochondria to promote apoptosis. Conversely, silencing of PP2A/Bbeta2 protected hippocampal neurons against free radical-mediated, excitotoxic, and ischemic insults. Evidence is accumulating that the mitochondrial fission/fusion equilibrium is an important determinant of cell survival. Accordingly, we found that Bbeta2 expression induces mitochondrial fragmentation, whereas Bbeta2 silencing or inhibition resulted in mitochondrial elongation. Based on epistasis experiments involving Bcl2 and core components of the mitochondrial fission machinery (Fis1 and dynamin-related protein 1), mitochondrial fragmentation occurs upstream of apoptosis and is both necessary and sufficient for hippocampal neuron death. Our data provide the first example of a proapoptotic phosphatase that predisposes to neuronal death by promoting mitochondrial division and point to a possible imbalance of the mitochondrial morphogenetic equilibrium in the pathogenesis of SCA12. 相似文献
998.
Clausen JD McIntosh DB Woolley DG Andersen JP 《The Journal of biological chemistry》2008,283(51):35703-35714
ATP plays dual roles in the reaction cycle of the sarcoplasmic reticulum Ca2+-ATPase by acting as the phosphorylating substrate as well as in nonphosphorylating (modulatory) modes accelerating conformational transitions of the enzyme cycle. Here we have examined the involvement of actuator domain residues Arg174, Ile188, Lys204, and Lys205 by mutagenesis. Alanine mutations to these residues had little effect on the interaction of the Ca2E1 state with nucleotide or on the HnE 2 to Ca2E1 transition of the dephosphoenzyme. The phosphoenzyme processing steps, Ca2E1P to E2P and E2P dephosphorylation, and their stimulation by MgATP/ATP were markedly affected by mutations to Arg174, Ile188, and Lys205. Replacement of Ile188 with alanine abolished nucleotide modulation of dephosphorylation but not the modulation of the Ca2E1P to E2P transition. Mutation to Arg174 interfered with nucleotide modulation of either of the phosphoenzyme processing steps, indicating a significant overlap between the modulatory nucleotide-binding sites involved. Mutation to Lys205 enhanced the rates of the phosphoenzyme processing steps in the absence of nucleotide and disrupted the nucleotide modulation of the Ca2E1P to E2P transition. Remarkably, the mutants with alterations to Lys205 showed an anomalous inhibition by ATP of the dephosphorylation, and in the alanine mutant the affinity for the inhibition by ATP was indistinguishable from that for stimulation by ATP of the wild type. Hence, the actuator domain is an important player in the function of ATP as modulator of phosphoenzyme processing, with Arg174, Ile188, and Lys205 all being critically involved, although in different ways. The data support a variable site model for the modulatory effects with the nucleotide binding somewhat differently in each of the conformational states occurring during the transport cycle. 相似文献
999.
Novak I Wang H Revenkova E Jessberger R Scherthan H Höög C 《The Journal of cell biology》2008,180(1):83-90
Meiotic chromosomes consist of proteinaceous axial structures from which chromatin loops emerge. Although we know that loop density along the meiotic chromosome axis is conserved in organisms with different genome sizes, the basis for the regular spacing of chromatin loops and their organization is largely unknown. We use two mouse model systems in which the postreplicative meiotic chromosome axes in the mutant oocytes are either longer or shorter than in wild-type oocytes. We observe a strict correlation between chromosome axis extension and a general and reciprocal shortening of chromatin loop size. However, in oocytes with a shorter chromosome axis, only a subset of the chromatin loops is extended. We find that the changes in chromatin loop size observed in oocytes with shorter or longer chromosome axes depend on the structural maintenance of chromosomes 1β (Smc1β), a mammalian chromosome–associated meiosis-specific cohesin. Our results suggest that in addition to its role in sister chromatid cohesion, Smc1β determines meiotic chromatin loop organization. 相似文献
1000.
Melchers J Diechtierow M Fehér K Sinning I Tews I Krauth-Siegel RL Muhle-Goll C 《The Journal of biological chemistry》2008,283(44):30401-30411
Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three cysteine homologues (Px I-III) of classical selenocysteine-containing glutathione peroxidases. The enzymes obtain their reducing equivalents from the unique trypanothione (bis(glutathionyl)spermidine)/tryparedoxin system. During catalysis, these tryparedoxin peroxidases cycle between an oxidized form with an intramolecular disulfide bond between Cys(47) and Cys(95) and the reduced peroxidase with both residues in the thiol state. Here we report on the three-dimensional structures of oxidized T. brucei Px III at 1.4A resolution obtained by x-ray crystallography and of both the oxidized and the reduced protein determined by NMR spectroscopy. Px III is a monomeric protein unlike the homologous poplar thioredoxin peroxidase (TxP). The structures of oxidized and reduced Px III are essentially identical in contrast to what was recently found for TxP. In Px III, Cys(47), Gln(82), and Trp(137) do not form the catalytic triad observed in the selenoenzymes, and related proteins and the latter two residues are unaffected by the redox state of the protein. The mutational analysis of three conserved lysine residues in the vicinity of the catalytic cysteines revealed that exchange of Lys(107) against glutamate abrogates the reduction of hydrogen peroxide, whereas Lys(97) and Lys(99) play a crucial role in the interaction with tryparedoxin. 相似文献