SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation

To study the cis- and trans-acting factors that mediate programmed death 1 (PD-1) signaling in primary human CD4 T cells, we constructed a chimeric molecule consisting of the murine CD28 extracellular domain and human PD-1 cytoplasmic tail. When introduced into CD4 T cells, this construct mimics the activity of endogenous PD-1 in terms of its ability to suppress T cell expansion and cytokine production. The cytoplasmic tail of PD-1 contains two structural motifs, an ITIM and an immunoreceptor tyrosine-based switch motif (ITSM). Mutation of the ITIM had little effect on PD-1 signaling or functional activity. In contrast, mutation of the ITSM abrogated the ability of PD-1 to block cytokine synthesis and to limit T cell expansion. Further biochemical analyses revealed that the ability of PD-1 to block T cell activation correlated with recruitment of Src homology region 2 domain-containing phosphatase-1 (SHP-1) and SHP-2, and not the adaptor Src homology 2 domain-containing molecule 1A, to the ITSM domain. In TCR-stimulated T cells, SHP-2 associated with PD-1, even in the absence of PD-1 engagement. Despite this interaction, the ability of PD-1 to block T cell activation required receptor ligation, suggesting that colocalization of PD-1 with CD3 and/or CD28 may be necessary for inhibition of T cell activation.     

Hendecad repeat in segment 2A and linker L2 of intermediate filament chains implies the possibility of a right-handed coiled-coil structure

The conformation adopted by intermediate filament chains (IF) has been described in terms of a central rod domain with four, alpha-helical, left-handed coiled-coil segments (1A, 1B, 2A, and 2B) joined by linkers (L1, L12, and L2, respectively). The rod domain is terminated at its N- and C-terminal ends by "globular" head and tail domains, respectively. This analysis, initially undertaken about 20-25 years ago, was based on the recognition of an underlying heptad substructure in the sequence of the rod domain, the presence of which can be directly associated with an alpha-helical coiled-coil structure. In this work, a hendecad sequence motif that is closely related to the heptad repeat but which is nonetheless significantly different from it has been recognized in the primary structure of segments 2A and linker L2. This motif, which is 11 residues long and structurally equivalent to a true heptad plus another heptad with an inclusive stutter, is consistent with the chains adopting a continuous right-handed coiled-coil structure with a long-period pitch length. It is therefore predicted that segment 2 as a whole may have a coiled-coil conformation with both right-handed (2A+L2) and left-handed (2B) regions. The changeover in handedness would be expected to occur at the C-terminal end of linker L2 and N-terminal end of segment 2B.     

Achieving yield gains in wheat

Wheat provides 20% of calories and protein consumed by humans. Recent genetic gains are <1% per annum (p.a.), insufficient to meet future demand. The Wheat Yield Consortium brings expertise in photosynthesis, crop adaptation and genetics to a common breeding platform. Theory suggest radiation use efficiency (RUE) of wheat could be increased ～50%; strategies include modifying specificity, catalytic rate and regulation of Rubisco, up-regulating Calvin cycle enzymes, introducing chloroplast CO(2) concentrating mechanisms, optimizing light and N distribution of canopies while minimizing photoinhibition, and increasing spike photosynthesis. Maximum yield expression will also require dynamic optimization of source: sink so that dry matter partitioning to reproductive structures is not at the cost of the roots, stems and leaves needed to maintain physiological and structural integrity. Crop development should favour spike fertility to maximize harvest index so phenology must be tailored to different photoperiods, and sensitivity to unpredictable weather must be modulated to reduce conservative responses that reduce harvest index. Strategic crossing of complementary physiological traits will be augmented with wide crossing, while genome-wide selection and high throughput phenotyping and genotyping will increase efficiency of progeny screening. To ensure investment in breeding achieves agronomic impact, sustainable crop management must also be promoted through crop improvement networks.     

Extracellular leucine-rich repeat proteins are required to organize the apical extracellular matrix and maintain epithelial junction integrity in C. elegans

Epithelial cells are linked by apicolateral junctions that are essential for tissue integrity. Epithelial cells also secrete a specialized apical extracellular matrix (ECM) that serves as a protective barrier. Some components of the apical ECM, such as mucins, can influence epithelial junction remodeling and disassembly during epithelial-to-mesenchymal transition (EMT). However, the molecular composition and biological roles of the apical ECM are not well understood. We identified a set of extracellular leucine-rich repeat only (eLRRon) proteins in C. elegans (LET-4 and EGG-6) that are expressed on the apical surfaces of epidermal cells and some tubular epithelia, including the excretory duct and pore. A previously characterized paralog, SYM-1, is also expressed in epidermal cells and secreted into the apical ECM. Related mammalian eLRRon proteins, such as decorin or LRRTM1-3, influence stromal ECM or synaptic junction organization, respectively. Mutants lacking one or more of the C. elegans epithelial eLRRon proteins show multiple defects in apical ECM organization, consistent with these proteins contributing to the embryonic sheath and cuticular ECM. Furthermore, epithelial junctions initially form in the correct locations, but then rupture at the time of cuticle secretion and remodeling of cell-matrix interactions. This work identifies epithelial eLRRon proteins as important components and organizers of the pre-cuticular and cuticular apical ECM, and adds to the small but growing body of evidence linking the apical ECM to epithelial junction stability. We propose that eLRRon-dependent apical ECM organization contributes to cell-cell adhesion and may modulate epithelial junction dynamics in both normal and disease situations.     

Development and Characterization of a New TILLING Population of Common Bread Wheat (Triticum aestivum L.)

Mutagenesis is an important tool in crop improvement. However, the hexaploid genome of wheat (Triticum aestivum L.) presents problems in identifying desirable genetic changes based on phenotypic screening due to gene redundancy. TILLING (Targeting Induced Local Lesions IN Genomes), a powerful reverse genetic strategy that allows the detection of induced point mutations in individuals of the mutagenized populations, can address the major challenge of linking sequence information to the biological function of genes and can also identify novel variation for crop breeding. Wheat is especially well-suited for TILLING due to the high mutation densities tolerated by polyploids. However, only a few wheat TILLING populations are currently available in the world, which is far from satisfying the requirement of researchers and breeders in different growing environments. In addition, current TILLING screening protocols require costly fluorescence detection systems, limiting their use, especially in developing countries. We developed a new TILLING resource comprising 2610 M(2) mutants in a common wheat cultivar 'Jinmai 47'. Numerous phenotypes with altered morphological and agronomic traits were observed from the M(2) and M(3) lines in the field. To simplify the procedure and decrease costs, we use unlabeled primers and either non-denaturing polyacrylamide gels or agarose gels for mutation detection. The value of this new resource was tested using PCR with RAPD and Intron-spliced junction (ISJ) primers, and also TILLING in three selected candidate genes, in 300 and 512 mutant lines, revealing high mutation densities of 1/34 kb by RAPD/ISJ analysis and 1/47 kb by TILLING. In total, 31 novel alleles were identified in the 3 targeted genes and confirmed by sequencing. The results indicate that this mutant population represents a useful resource for the wheat research community. We hope that the use of this reverse genetics resource will provide novel allelic diversity for wheat improvement and functional genomics.     

Rubisco regulation: a role for inhibitors

In photosynthesis Rubisco catalyses the assimilation of CO(2) by the carboxylation of ribulose-1,5-bisphosphate. However, the catalytic properties of Rubisco are not optimal for current or projected environments and limit the efficiency of photosynthesis. Rubisco activity is highly regulated in response to short-term fluctuations in the environment, although such regulation may not be optimally poised for crop productivity. The regulation of Rubisco activity in higher plants is reviewed here, including the role of Rubisco activase, tight binding inhibitors, and the impact of abiotic stress upon them.     

The detection and assessment of the aneugenic potential of selected oestrogens, progestins and androgens using the in vitro cytokinesis blocked micronucleus assay

The use of 17-beta-oestradiol, testosterone, progesterone, zearanol, trenbolone acetate and melengesterol acetate in animal feed as growth promoters has been banned in the European Union since 1989. However, the data available on their genotoxicity is limited. To bridge this gap the present study was carried out with the aim of evaluating these hormones for their ability to induce aneuploidy. Aneuploidy has been recently considered sufficiently important to be included in the routine testing of chemicals and radiation. These types of numerical chromosomal aberrations may arise by at least two mechanisms, chromosome loss and non-disjunction. Over the past few years, the cytokinesis blocked micronucleus (CBMN) technique has evolved into a robust assay for the detection of aneuploidy induction. At the present time, it is the only assay which can reliably detect both chromosome loss and non-disjunction when the basic methodology is coupled with appropriate molecular probing techniques such as immunoflourescent labelling of kinetochores and Fluorescence in situ Hybridisation. In this present study, aneuploidy induction by three groups of hormones was studied using CBMN assay coupled with Fluorescence in situ Hybridisation. The results from the present study demonstrate that 17-beta-oestradiol, diethylstilboestrol, progesterone and testosterone are genotoxic and induce aneuploidy by non-disjunctional mechanism, whereas trenbolone is also genotoxic by a clastogenic mechanism. However, melengesterol acetate and zearanol proved to be non-genotoxic in vitro.     

Exposure of mouse oocytes to bisphenol A causes meiotic arrest but not aneuploidy

Mouse oocytes isolated from large antral follicles were exposed to a wide range of concentrations of bisphenol A (BPA) during maturation in vitro (50 ng/ml to 10 microg/ml BPA in medium). Exposure to high concentrations of BPA (10 microg/ml) affected spindle formation, distribution of pericentriolar material and chromosome alignment on the spindle (termed congression failure), and caused a significant meiotic arrest. However, BPA did not increase hyperploidy at meiosis II at any tested concentration. Some but not all meiosis I arrested oocytes had MAD2-positive foci at centromeres of chromosomes in bivalents, suggesting that they had failed to pass the spindle checkpoint control. In a second set of experiments prepubertal mice were exposed sub-chronically for 7 days to low BPA by daily oral administration, followed by in vitro maturation of the denuded oocytes to metaphase II in the absence of BPA, as this treatment protocol was previously reported to induce chromosome congression failure and therefore suspected to cause aneuploidy in oocytes. The sub-chronic exposure subtly affected spindle morphology and oocyte maturation. However, as with the exposure in vitro, there was no evidence that low BPA doses increased hyperploidy at meiosis II. In conclusion, the data suggest that mouse oocytes from mice respond to BPA-induced disturbances in spindle formation by induction of meiotic arrest. This response might result from an effective checkpoint mechanism preventing the occurrence of chromosome malsegregation and aneuploidy. Low chronic BPA exposure in vivo as such does not appear to pose a risk for induction of errors in chromosome segregation at first meiosis in mouse oocytes. Additional factors besides BPA may have caused the high rate of congression failure and the temporary increase in hyperploidy in mouse metaphase II oocytes reported previously.     

Mechanistic investigations of low dose exposures to the genotoxic compounds bisphenol-A and rotenone

A complete hazard and risk assessment of any known genotoxin requires the evaluation of the mutagenic, clastogenic and aneugenic potential of the compound. In the case of aneugenic chemicals, mechanism of action (MOA) and quantitative responses may be investigated by studying their effects upon the fidelity of functioning of components of the cell cycle. These present studies have demonstrated that the plastics component bisphenol-A (BPA) and the natural pesticide rotenone induce micronuclei and modify the functioning of the microtubule organising centres (MTOCs) of the mitotic spindles of cultured mammalian cells in a dose-dependent manner. BPA and rotenone were used as model compounds in an investigation of dose response relationships for the hazard/risk assessment of aneugens. Thresholds of action for the induction of aneuploidy have been predicted for spindle poisons on the basis of the multiple targets, which may need disabling before a quantitative response can be detected. The cytokinesis blocked micronucleus assay (CBMA) methodology was utilised in the human lymphoblastoid cell lines AHH-1, MCL-5 and Chinese hamster V79 cell lines. A no observable effect level (NOEL) at 10.8 microg/ml BPA was observed for MN induction. Rotenone showed a small increase in MN induction with the first significant effect at 0.25 ng/ml in V79 cells but there was no significant effect in the metabolically competent cell line, MCL-5. For a mechanistic evaluation of the aneugenic effects of BPA and rotenone, fluorescently labelled antibodies were used to visualise microtubules (alpha-tubulin) and MTOCs (gamma-tubulin). The NOELs for tripolar mitotic spindle induction in V79 cells were 7 microg/ml for BPA and 80 pg/ml for rotenone (concentrations which produced similar changes to mitotic index (M.I.)). Interestingly there was close proximity to the NOEL of 10.8 microg/ml BPA for micronucleus (MN) induction in the human lymphoblastoid AHH-1 cell. Multiple MTOCs can therefore be predicted as a possible mechanism for MN induction. The similarity in concentration inducing tripolar mitosis, M.I. and MN changes suggests immunofluorescence analysis to be a useful dose setting assay with emphasis on the mechanism.