Assembly of mitochondrial ribonucleoprotein complexes involves specific guide RNA (gRNA)-binding proteins and gRNA domains but does not require preedited mRNA.

RNA editing in kinetoplastids probably employs a macromolecular complex, the editosome, that is likely to include the guide RNAs (gRNAs) which specify the edited sequence. Specific ribonucleoprotein (RNP) complexes which form in vitro with gRNAs (H. U. Göringer, D. J. Koslowsky, T. H. Morales, and K. D. Stuart, Proc. Natl. Acad. Sci. USA, in press) are potential editosomes or their precursors. We find that several factors are important for in vitro formation of these RNP complexes and identify specific gRNA-binding proteins present in the complexes. Preedited mRNA promotes the in vitro formation of the four major gRNA-containing RNP complexes under some conditions but is required for the formation of only a subcomponent of one complex. The 5' gRNA sequence encompassing the RYAYA and anchor regions and the 3' gRNA oligo(U) tail are both important in complex formation, since their deletion results in a dramatic decrease of some complexes and the absence of others. UV cross-linking experiments identify several proteins which are in contact with gRNA and preedited mRNA in mitochondrial extracts. Proteins of 25 and 90 kDa are highly specific for gRNAs, and the 90-kDa protein binds specifically to gRNA oligo(U) tails. The gRNA-binding proteins exhibit a differential distribution between the four in vitro-formed complexes. These experiments reveal several proteins potentially involved in RNA editing and indicate that multiple recognition elements in gRNAs are used for complex formation.     

Colitogenic Th1 cells are present in the antigen-experienced T cell pool in normal mice: control by CD4+ regulatory T cells and IL-10

CD4(+) regulatory T cells have been shown to prevent intestinal inflammation; however, it is not known whether they act to prevent the priming of colitogenic T cells or actively control these cells as part of the memory T cell pool. In this study, we describe the presence of colitogenic Th1 cells within the CD4(+)CD45RB(low) population. These pathogenic cells enrich within the CD25(-) subset and are not recent thymic emigrants. CD4(+)CD45RB(low) cells from germfree mice were significantly reduced in their ability to transfer colitis to immune deficient recipients, suggesting the presence of commensal bacteria in the donor mice drives colitogenic T cells into the Ag-experienced/memory T cell pool. This potentially pathogenic population of Ag-experienced T cells is subject to T cell-mediated regulation in vivo by both CD4(+)CD25(+) and CD4(+)CD25(-) cells in an IL-10-dependent manner. Furthermore, administration of an anti-IL-10R mAb to unmanipulated adult mice was sufficient to induce the development of colitis. Taken together, these data indicate that colitogenic Th1 cells enter into the Ag-experienced pool in normal mice, but that their function is controlled by regulatory T cells and IL-10. Interestingly, IL-10 was not absolutely required for CD4(+)CD25(+) T cell-mediated inhibition of colitis induced by transfer of naive CD4(+)CD45RB(high) cells, suggesting a differential requirement for IL-10 in the regulation of naive and Ag-experienced T cells.     

Exclusion effects on vegetation characteristics and their correlation to soil factors in the semi-arid rangeland of Mu Us Sandland,China

Exclusion has been applied as a main measure for re-vegetation all over the world.This paper,by comparing the results of year-round exclusion,seasonal exclusion,and non-exclusion,quantified the vegetation variations under three different exclusion measures and their correlation to soil factors.The analysis results for community species component and plant diversity using multi-response permutation procedures (MRPPs)showed that exclusion did change the species component and increase plant diversity remarkably,while the period of exclusion had no significant influence on these two community features.The indicator species analysis and calculation of similarity indices indicated that community for year-round exclusion were becoming xerophytization and unpalatability,and showed highly spatial heterogeneity of plant species distribution,whereas community for seasonal exclusion was under stable non-equilibrium condition.Detrended correspondence analysis (DCA)and detrend canonical correspondence analysis (DCCA)results of relationship between plant species and soil variables demonstrated that soil moisture was a controlling factor for plant species component,microbiotic soil crust cover,soil organic matter,and soil bulk density had significant effects on soil moisture,among which microbiotic soil crust was a leading factor owing to its limitation to rainfall infiltration on the one hand,and its constraints to entrance of herbaceous seeds into soil or to germination of soil seeds on the other hand.As a result of long-term removal of animal grazing,crust kept intact in year-round exclusion community,which was a main reason of community xerophytization.It was also obvious from ordination results that some important environmental factors,such as tempo-spatial change of rainfall and corresponding tempo-spatial change of soil moisture,were neglected during direct gradient analysis.In addition,biodiversity was close related to soil nutrients as well as to soil moisture condition (soil water content and crust cover),and it had positive relation to available N,and negative relation to available P.Higher soil N had advantage to non-leguminous plants growth on nutrition-poor sand land definitely.The impact of P to community component was unclear and should be studied from plant physiology.Further researches on nonequilibrium theory in semi-arid rangeland will provide a scientific and flexible animal development paradigm for being implementing livestock fen-raising and grazing-forbidden policies in China.     

Designer TGFβ superfamily ligands with diversified functionality

Transforming Growth Factor--beta (TGFβ) superfamily ligands, including Activins, Growth and Differentiation Factors (GDFs), and Bone Morphogenetic Proteins (BMPs), are excellent targets for protein-based therapeutics because of their pervasiveness in numerous developmental and cellular processes. We developed a strategy termed RASCH (Random Assembly of Segmental Chimera and Heteromer), to engineer chemically-refoldable TGFβ superfamily ligands with unique signaling properties. One of these engineered ligands, AB208, created from Activin-βA and BMP-2 sequences, exhibits the refolding characteristics of BMP-2 while possessing Activin-like signaling attributes. Further, we find several additional ligands, AB204, AB211, and AB215, which initiate the intracellular Smad1-mediated signaling pathways more strongly than BMP-2 but show no sensitivity to the natural BMP antagonist Noggin unlike natural BMP-2. In another design, incorporation of a short N-terminal segment from BMP-2 was sufficient to enable chemical refolding of BMP-9, without which was never produced nor refolded. Our studies show that the RASCH strategy enables us to expand the functional repertoire of TGFβ superfamily ligands through development of novel chimeric TGFβ ligands with diverse biological and clinical values.     

Calcium measurement in living filamentous fungi expressing codon-optimized aequorin

Calcium signalling is little understood in filamentous fungi largely because easy and routine methods for calcium measurement in living hyphae have previously been unavailable. We have developed the recombinant aequorin method for this purpose. High levels of aequorin expression were obtained in Neurospora crassa, Aspergillus niger and Aspergillus awamori by codon optimization of the aequorin gene. Three external stimuli (mechanical perturbation, hypo-osmotic shock and high external calcium) were found transiently to increase [Ca(2+)](c). Each of the calcium signatures associated with these physico-chemical treatments was unique, suggesting the involvement of three distinct calcium-mediated signal transduction pathways. The fungal calcium channel blocker KP4 inhibited the [Ca(2+)](c) responses to hypo-osmotic shock and high external calcium, but not to mechanical perturbation. The divalent cation chelator BAPTA inhibited [Ca(2+)](c) responses to mechanical perturbation and hypo-osmotic shock. The calcium agonists A23187 and cyclopiazonic acid increased [Ca(2+)](c) levels.     

All‐trans retinoic acid exposure increases connexin 43 expression in cumulus cells and improves embryo development in bovine oocytes

In developing follicles, cellular coupling within cumulus–oocyte complexes (COCs) creates a functional syncytium allowing for the passage of small molecules. In many species, intercellular coupling between granulosa cells results from the expression of connexin 43 (CX43 or Gja1) and the formation of gap junctional plaques. Previously, our lab has shown that oocytes with a higher developmental potential had higher CX43 expression in their cumulus cells compared with developmentally incompetent oocytes. All‐trans retinoic acid (ATRA) has been shown to increase CX43 expression in several different cell types. In this study we investigated the effect of ATRA treatment, during maturation, on CX43 expression and localization in cumulus cells and the developmental competence of bovine oocytes. COCs and granulosa cells exposed to ATRA during maturation had significantly higher CX43 expression and increased gap junctional coupling, respectively. In addition, there was a significant increase in the maturation, cleavage, and blastocyst rates in ATRA treated COCs. Data from these studies suggest that not only can CX43 be used as a biomarker for oocyte health, it can also potentially be manipulated using ATRA to increase the number of oocytes achieving developmental competence.     

Chromosomal translocation and segmental duplication in Cryptococcus neoformans

Large chromosomal events such as translocations and segmental duplications enable rapid adaptation to new environments. Here we marshal genomic, genetic, meiotic mapping, and physical evidence to demonstrate that a chromosomal translocation and segmental duplication occurred during construction of a congenic strain pair in the fungal human pathogen Cryptococcus neoformans. Two chromosomes underwent telomere-telomere fusion, generating a dicentric chromosome that broke to produce a chromosomal translocation, forming two novel chromosomes sharing a large segmental duplication. The duplication spans 62,872 identical nucleotides and generated a second copy of 22 predicted genes, and we hypothesize that this event may have occurred during meiosis. Gene disruption studies of one embedded gene (SMG1) corroborate that this region is duplicated in an otherwise haploid genome. These findings resolve a genome project assembly anomaly and illustrate an example of rapid genome evolution in a fungal genome rich in repetitive elements.     

Monocytes Regulate the Mechanism of T-cell Death by Inducing Fas-Mediated Apoptosis during Bacterial Infection

Monocytes and T-cells are critical to the host response to acute bacterial infection but monocytes are primarily viewed as amplifying the inflammatory signal. The mechanisms of cell death regulating T-cell numbers at sites of infection are incompletely characterized. T-cell death in cultures of peripheral blood mononuclear cells (PBMC) showed ‘classic’ features of apoptosis following exposure to pneumococci. Conversely, purified CD3⁺ T-cells cultured with pneumococci demonstrated necrosis with membrane permeabilization. The death of purified CD3⁺ T-cells was not inhibited by necrostatin, but required the bacterial toxin pneumolysin. Apoptosis of CD3⁺ T-cells in PBMC cultures required ‘classical’ CD14⁺ monocytes, which enhanced T-cell activation. CD3⁺ T-cell death was enhanced in HIV-seropositive individuals. Monocyte-mediated CD3⁺ T-cell apoptotic death was Fas-dependent both in vitro and in vivo. In the early stages of the T-cell dependent host response to pneumococci reduced Fas ligand mediated T-cell apoptosis was associated with decreased bacterial clearance in the lung and increased bacteremia. In summary monocytes converted pathogen-associated necrosis into Fas-dependent apoptosis and regulated levels of activated T-cells at sites of acute bacterial infection. These changes were associated with enhanced bacterial clearance in the lung and reduced levels of invasive pneumococcal disease.     

Stereoscopic Vision in the Absence of the Lateral Occipital Cortex

Both dorsal and ventral cortical visual streams contain neurons sensitive to binocular disparities, but the two streams may underlie different aspects of stereoscopic vision. Here we investigate stereopsis in the neurological patient D.F., whose ventral stream, specifically lateral occipital cortex, has been damaged bilaterally, causing profound visual form agnosia. Despite her severe damage to cortical visual areas, we report that DF''s stereo vision is strikingly unimpaired. She is better than many control observers at using binocular disparity to judge whether an isolated object appears near or far, and to resolve ambiguous structure-from-motion. DF is, however, poor at using relative disparity between features at different locations across the visual field. This may stem from a difficulty in identifying the surface boundaries where relative disparity is available. We suggest that the ventral processing stream may play a critical role in enabling healthy observers to extract fine depth information from relative disparities within one surface or between surfaces located in different parts of the visual field.