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31.
Using bioinformatic, proteomic, immunofluorescence, and genetic cross methods, we have functionally characterized a family of putative parasite ligands as potential mediators of cell-cell interactions. We name these proteins the Limulus clotting factor C, Coch-5b2, and Lgl1 (LCCL)-lectin adhesive-like protein (LAP) family. We demonstrate that this family is conserved amongst Plasmodium spp. It possesses a unique arrangement of adhesive protein domains normally associated with extracellular proteins. The proteins are expressed predominantly, though not exclusively, in the mosquito stages of the life cycle. We test the hypothesis that these proteins are surface proteins with 1 member of this gene family, lap1, and provide evidence that it is expressed on the surface of Plasmodium berghei sporozoites. Finally, through genetic crosses of wild-type Pblap1+ and transgenic Pblap1- parasites, we show that the null phenotype previously reported for sporozoite development in a Pblap1- mutant can be rescued within a heterokaryotic oocyst and that infectious Pblap1 sporozoites can be formed. The mutant is not rescued by coparasitization of mosquitoes with a mixture Pblap1+ and Pblap1- homokaryotic oocysts.  相似文献   
32.
Beisel CJ  Rokyta DR  Wichman HA  Joyce P 《Genetics》2007,176(4):2441-2449
In modeling evolutionary genetics, it is often assumed that mutational effects are assigned according to a continuous probability distribution, and multiple distributions have been used with varying degrees of justification. For mutations with beneficial effects, the distribution currently favored is the exponential distribution, in part because it can be justified in terms of extreme value theory, since beneficial mutations should have fitnesses in the extreme right tail of the fitness distribution. While the appeal to extreme value theory seems justified, the exponential distribution is but one of three possible limiting forms for tail distributions, with the other two loosely corresponding to distributions with right-truncated tails and those with heavy tails. We describe a likelihood-ratio framework for analyzing the fitness effects of beneficial mutations, focusing on testing the null hypothesis that the distribution is exponential. We also describe how to account for missing the smallest-effect mutations, which are often difficult to identify experimentally. This technique makes it possible to apply the test to gain-of-function mutations, where the ancestral genotype is unable to grow under the selective conditions. We also describe how to pool data across experiments, since we expect few possible beneficial mutations in any particular experiment.  相似文献   
33.
Xu X  Xie C  Edwards H  Zhou H  Buck SA  Ge Y 《PloS one》2011,6(2):e17138

Background

Pediatric acute myeloid leukemia (AML) remains a challenging disease to treat even with intensified cytarabine-based chemotherapy. Histone deacetylases (HDACs) have been reported to be promising therapeutic targets for treating AML. However, HDAC family members that are involved in chemotherapy sensitivities remain unknown. In this study, we sought to identify members of the HDAC family that are involved in cytarabine sensitivities, and to select the optimal HDACI that is most efficacious when combined with cytarabine for treating children with AML.

Methodology

Expression profiles of classes I, II, and IV HDACs in 4 pediatric AML cell lines were determined by Western blotting. Inhibition of class I HDACs by different HDACIs was measured post immnunoprecipitation. Individual down-regulation of HDACs in pediatric AML cells was performed with lentiviral shRNA. The effects of cytarabine and HDACIs on apoptosis were determined by flow cytometry analysis.

Results

Treatments with structurally diverse HDACIs and HDAC shRNA knockdown experiments revealed that down-regulation of both HDACs 1 and 6 is critical in enhancing cytarabine-induced apoptosis in pediatric AML, at least partly mediated by Bim. However, down-regulation of HDAC2 may negatively impact cytarabine sensitivities in the disease. At clinically achievable concentrations, HDACIs that simultaneously inhibited both HDACs 1 and 6 showed the best anti-leukemic activities and significantly enhanced cytarabine-induced apoptosis.

Conclusion

Our results further confirm that HDACs are bona fide therapeutic targets for treating pediatric AML and suggest that pan-HDACIs may be more beneficial than isoform-specific drugs.  相似文献   
34.
Impaired mitochondrial function and structure and intramyocellular lipid (IMCL) accumulation have been associated with obesity and Type 2 diabetes. We examined whether endurance exercise training and sex influenced IMCL and mitochondrial morphology using electron microscopy, whole-body substrate use, and mitochondrial enzyme activity. Untrained men (n = 5) and women (n = 7) were tested before and after 7 wk of endurance exercise training. Testing included 90 min of cycle ergometry at 60% Vo(2 peak) with preexercise muscle biopsies analyzed for IMCL and mitochondrial size/area using electron microscopy and short-chain beta-hydroxyacyl-CoA dehydrogenase (SCHAD) and citrate synthase (CS) enzyme activity. Training increased the mean lipid area density (P = 0.090), the number of IMCL droplets (P = 0.055), the number of IMCL droplets in contact with mitochondria (P = 0.010), the total mitochondrial area (P < 0.001), and the size of individual mitochondrial fragments (P = 0.006). Women had higher mean lipid area density (P = 0.030) and number of IMCL droplets (P = 0.002) before and after training, but higher individual IMCL area only before training (P = 0.013), compared with men. Women oxidized more fat (P = 0.027) and less carbohydrate (P = 0.032) throughout the study. Training increased Vo(2 peak) (P < 0.001), %fat oxidation (P = 0.018), SCHAD activity (P = 0.003), and CS activity (P = 0.042). In summary, endurance exercise training increased IMCL area density due to an increase in the number of lipid droplets, whereas the increase in total mitochondrial area was due to an increase in the size of individual mitochondrial fragments. In addition, women have higher IMCL content compared with men due mainly to a greater number of individual droplets. Finally, endurance exercise training increased the proportion of IMCL in physical contact with mitochondria.  相似文献   
35.
Blockade of IL-2R with humanized anti-CD25 Abs, such as daclizumab, inhibits Th2 responses in human T cells. Recent murine studies have shown that IL-2 also plays a significant role in regulating Th2 cell differentiation by activated STAT5. To explore the role of activated STAT5 in the Th2 differentiation of primary human T cells, we studied the mechanisms underlying IL-2 regulation of C-MAF expression. Chromatin immunoprecipitation studies revealed that IL-2 induced STAT5 binding to specific sites in the C-MAF promoter. These sites corresponded to regions enriched for markers of chromatin architectural features in both resting CD4 and differentiated Th2 cells. Unlike IL-6, IL-2 induced C-MAF expression in CD4 T cells with or without prior TCR stimulation. TCR-induced C-MAF expression was significantly inhibited by treatment with daclizumab or a JAK3 inhibitor, R333. Furthermore, IL-2 and IL-6 synergistically induced C-MAF expression in TCR-activated T cells, suggesting functional cooperation between these cytokines. Finally, both TCR-induced early IL4 mRNA expression and IL-4 cytokine expression in differentiated Th2 cells were significantly inhibited by IL-2R blockade. Thus, our findings demonstrate the importance of IL-2 in Th2 differentiation in human T cells and support the notion that IL-2R-directed therapies may have utility in the treatment of allergic disorders.  相似文献   
36.
The basidiomycete Chondrostereum purpureum produces several plant cell wall-degrading enzymes, including endopolygalacturonase (endoPG). Degenerate oligonucleotide primers were designed according to conserved regions of endoPG genes from various fungi, plants, and bacteria and used to amplify members of this gene family from C. purpureum. Four different amplification products showed significant similarity to known endoPGs and were used as hybridization probes to screen a library of genomic DNA sequences and to retrieve five full-length endoPG genes (epgA, epgB1, epgB2, epgC, and epgD). The identities between the deduced polypeptides for epgA, epgB1, epgC, and epgD ranged from 61.8 to 80.0%, while the deduced polypeptides for epgB1 and epgB2 shared 97.1% identity. Phylogenetic analysis suggested that the duplication of existing endoPG genes occurred after the divergence of the ascomycetes and basidiomycetes. C. purpureum is the first basidiomycete fungus for which the endoPG gene family has been described.  相似文献   
37.
TMEM41B and VMP1 are integral membrane proteins of the endoplasmic reticulum (ER) and regulate the formation of autophagosomes, lipid droplets (LDs), and lipoproteins. Recently, TMEM41B was identified as a crucial host factor for infection by all coronaviruses and flaviviruses. The molecular function of TMEM41B and VMP1, which belong to a large evolutionarily conserved family, remains elusive. Here, we show that TMEM41B and VMP1 are phospholipid scramblases whose deficiency impairs the normal cellular distribution of cholesterol and phosphatidylserine. Their mechanism of action on LD formation is likely to be different from that of seipin. Their role in maintaining cellular phosphatidylserine and cholesterol homeostasis may partially explain their requirement for viral infection. Our results suggest that the proper sorting and distribution of cellular lipids are essential for organelle biogenesis and viral infection.  相似文献   
38.
High‐throughput (next‐generation) DNA sequencing has removed barriers to data quantity and quality, and it has produced phylogenies with high statistical support. Such data are useful to address phylogenetic congruence among individual genes. Concatenated analyses of unlinked genes often produce well‐resolved phylogenetic trees with bootstrap support on major nodes at or approaching 100%, but they have been criticized for providing incorrect phylogenies for various reasons to include a history of hybridization, introgression, and incomplete lineage sorting. The present study compares next‐generation sequencing results of the same accessions of Daucus with different genomic regions, of which three have been reported before: (i) the entire plastid genome, (ii) 47 mitochondrial genes, and (iii) 94 conserved nuclear orthologs. Here, we report a fourth dataset, (iv) 564 895 nuclear SNPs. There are areas of discordance in all four results using the same accessions analyzed with maximum parsimony, maximum likelihood, and with the nuclear data species trees through a coalescent analysis. The nuclear results show significant areas of discordance that were unexpected, because these studies used the same DNA samples, the nuclear studies were generated from large and high‐quality datasets with the SNPs distributed on all nine linkage groups of Daucus carota, and the results were supported by high bootstrap values. These results raise questions concerning the best data and analytical methods to reconstruct and understand the “truth” of a phylogeny.  相似文献   
39.
Microtubule (MT) dynamic instability is fundamental to many cell functions, but its mechanism remains poorly understood, in part because it is difficult to gain information about the dimer-scale events at the MT tip. To address this issue, we used a dimer-scale computational model of MT assembly that is consistent with tubulin structure and biochemistry, displays dynamic instability, and covers experimentally relevant spans of time. It allows us to correlate macroscopic behaviors (dynamic instability parameters) with microscopic structures (tip conformations) and examine protofilament structure as the tip spontaneously progresses through both catastrophe and rescue. The model's behavior suggests that several commonly held assumptions about MT dynamics should be reconsidered. Moreover, it predicts that short, interprotofilament "cracks" (laterally unbonded regions between protofilaments) exist even at the tips of growing MTs and that rapid fluctuations in the depths of these cracks influence both catastrophe and rescue. We conclude that experimentally observed microtubule behavior can best be explained by a "stochastic cap" model in which tubulin subunits hydrolyze GTP according to a first-order reaction after they are incorporated into the lattice; catastrophe and rescue result from stochastic fluctuations in the size, shape, and extent of lateral bonding of the cap.  相似文献   
40.
Immune-mediated control of tumors may occur, in part, through lysis of malignant cells by CD8(+) T cells that recognize specific Ag-HLA class I complexes. However, tumor cell populations may escape T cell responses by immune editing, by preventing formation of those Ag-HLA complexes. It remains unclear whether the human immune system can respond to immune editing and recognize newly arising escape variants. We report an example of shifting immune responses to escape variants in a patient with sequential metastases of melanoma and long-term survival after surgery alone. Tumor cells in the first metastasis escaped immune recognition via selective loss of an HLA haplotype (HLA-A11, -B44, and -Cw17), but maintained expression of HLA-A2. In the second metastasis, immune escape from an immunodominant MART-1-specific T cell response was mediated by HLA class I down-regulation, resulting in a failure to present this epitope, but persistent presentation of a tyrosinase-derived epitope. Consequent to this modification in tumor Ag presentation, the dominant CTL response shifted principally toward a tyrosinase-targeted response, even though tyrosinase-specific CTL had been undetectable during the initial metastatic event. Thus, in response to immune editing of tumor cells, a patient's spontaneous T cell response adapted, gaining the ability to recognize and to lyse "edited" tumor targets. The observation of both immune editing and immune adaptation in a patient with long-term survival after surgery alone demonstrates an example of immune system reactivity to counteract the escape mechanism(s) developed by tumor cells, which may contribute to the clinical outcome of malignant disease.  相似文献   
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