Functional neuronal cells generated by human parthenogenetic stem cells

Parent of origin imprints on the genome have been implicated in the regulation of neural cell type differentiation. The ability of human parthenogenetic (PG) embryonic stem cells (hpESCs) to undergo neural lineage and cell type-specific differentiation is undefined. We determined the potential of hpESCs to differentiate into various neural subtypes. Concurrently, we examined DNA methylation and expression status of imprinted genes. Under culture conditions promoting neural differentiation, hpESC-derived neural stem cells (hpNSCs) gave rise to glia and neuron-like cells that expressed subtype-specific markers and generated action potentials. Analysis of imprinting in hpESCs and in hpNSCs revealed that maternal-specific gene expression patterns and imprinting marks were generally maintained in PG cells upon differentiation. Our results demonstrate that despite the lack of a paternal genome, hpESCs generate proliferating NSCs that are capable of differentiation into physiologically functional neuron-like cells and maintain allele-specific expression of imprinted genes. Thus, hpESCs can serve as a model to study the role of maternal and paternal genomes in neural development and to better understand imprinting-associated brain diseases.     

Endoplasmic reticulum stress response promotes cytotoxic phenotype of CD8αβ+ intraepithelial lymphocytes in a mouse model for Crohn's disease-like ileitis

Endoplasmic reticulum (ER) unfolded protein responses (UPR) are implicated in the pathogenesis of inflammatory bowel disease. Cytotoxic CD8αβ(+) intraepithelial lymphocytes (IEL) contribute to the development of Crohn's disease-like ileitis in TNF(ΔARE/+) mice. In this study, we characterized the role of ER-UPR mechanisms in contributing to the disease-associated phenotype of cytotoxic IEL under conditions of chronic inflammation. Inflamed TNF(ΔARE/+) mice exhibited increased expression of Grp78, ATF6, ATF4, and spliced XBP1 in CD8αβ(+) IEL but not in CD8αα(+) IEL or in lamina propria lymphocytes. Chromatin immunoprecipitation analysis in CD8αβ(+) T cells showed selective recruitment of ER-UPR transducers to the granzyme B gene promoter. Heterozygous Grp78(-/+) mice exhibited an attenuated granzyme B-dependent cytotoxicity of CD8αβ(+) T cells against intestinal epithelial cells, suggesting a critical activity of this ER-associated chaperone in maintaining a cytotoxic T cell phenotype. Granzyme B-deficient CD8αβ(+) T cells showed a defect in IL-2-mediated proliferation in Grp78(-/+) mice. Adoptively transferred Grp78(-/+) CD8αβ(+) T cells had a decreased frequency of accumulation in the intestine of RAG2(-/-) recipient mice. The tissue pathology in TNF(ΔARE/+) × Grp78(-/+) mice was similar to TNF(ΔARE/+) mice, even though the cytotoxic effector functions of CD8αβ(+) T cells were significantly reduced. In conclusion, ER stress-associated UPR mechanisms promote the development and maintenance of the pathogenic cytotoxic CD8αβ(+) IEL phenotype in the mouse model of Crohn's disease-like ileitis.     

Pro-inflammatory properties of stromal cell-derived factor-1 (CXCL12) in collagen-induced arthritis

CXCL12 (stromal cell-derived factor 1) is a unique biological ligand for the chemokine receptor CXCR4. We previously reported that treatment with a specific CXCR4 antagonist, AMD3100, exerts a beneficial effect on the development of collagen-induced arthritis (CIA) in the highly susceptible IFN-γ receptor-deficient (IFN-γR KO) mouse. We concluded that CXCL12 plays a central role in the pathogenesis of CIA in IFN-γR KO mice by promoting delayed type hypersensitivity against the auto-antigen and by interfering with chemotaxis of CXCR4⁺ cells to the inflamed joints. Here, we investigated whether AMD3100 can likewise inhibit CIA in wild-type mice and analysed the underlying mechanism. Parenteral treatment with the drug at the time of onset of arthritis reduced disease incidence and modestly inhibited severity in affected mice. This beneficial effect was associated with reduced serum concentrations of IL-6. AMD3100 did not affect anti-collagen type II antibodies and, in contrast with its action in IFN-γR KO mice, did not inhibit the delayed type hypersensitivity response against collagen type II, suggesting that the beneficial effect cannot be explained by inhibition of humoral or cellular autoimmune responses. AMD3100 inhibited the in vitro chemotactic effect of CXCL12 on splenocytes, as well as in vivo leukocyte infiltration in CXCL12-containing subcutaneous air pouches. We also demonstrate that, in addition to its effect on cell infiltration, CXCL12 potentiates receptor activator of NF-κB ligand-induced osteoclast differentiation from splenocytes and increases the calcium phosphate-resorbing capacity of these osteoclasts, both processes being potently counteracted by AMD3100. Our observations indicate that CXCL12 acts as a pro-inflammatory factor in the pathogenesis of autoimmune arthritis by attracting inflammatory cells to joints and by stimulating the differentiation and activation of osteoclasts.     

A homozygous mutation in the tight-junction protein JAM3 causes hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts

The tight junction, or zonula occludens, is a specialized cell-cell junction that regulates epithelial and endothelial permeability, and it is an essential component of the blood-brain barrier in the cerebrovascular endothelium. In addition to functioning as a diffusion barrier, tight junctions are also involved in signal transduction. In this study, we identified a homozygous mutation in the tight-junction protein gene JAM3 in a large consanguineous family from the United Arab Emirates. Some members of this family had a rare autosomal-recessive syndrome characterized by severe hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts. Their clinical presentation overlaps with some reported cases of pseudo-TORCH syndrome as well as with cases involving mutations in occludin, another component of the tight-junction complex. However, massive intracranial hemorrhage distinguishes these patients from others. Homozygosity mapping identified the disease locus in this family on chromosome 11q25 with a maximum multipoint LOD score of 6.15. Sequence analysis of genes in the candidate interval uncovered a mutation in the canonical splice-donor site of intron 5 of JAM3. RT-PCR analysis of a patient lymphoblast cell line confirmed abnormal splicing, leading to a frameshift mutation with early termination. JAM3 is known to be present in vascular endothelium, although its roles in cerebral vasculature have not been implicated. Our results suggest that JAM3 is essential for maintaining the integrity of the cerebrovascular endothelium as well as for normal lens development in humans.     

Fatty Acid Composition of the Peribacteroid Membrane and the ER in Nodules of Glycine max Varies after Infection by Different Strains of the Microsymbiont Bradyrhizobiumjaponicum

The fatty acid composition of ER, Golgi and peribacteroid membrane (PBM) from root nodules formed on Glycine max after infection with different strains of Bradyrhizobium japonicum has been analysed by gas chromatography. In each plant-microsymbiont combination the fatty acid composition (FAC) of the PBM is distinct from ER and Golgi. The similarity between ER and PBM fatty acid composition is significantly stronger than between Golgi and PBM. In addition the fatty acid composition of all membrane systems in nodules is affected by the microsymbiont strain. A comparison of four strains of Bradyrhizobium japonicum grown in agar surface culture and isolated as the symbiotic bacteroids reveals a decrease in oleic acid during bacteroid differentiation.     

Abortive Infection of F-Plasmid-Containing Escherichia coli Cells by Bacterial Virus T7 Is Determined by the Right End of T7 Gene 1

Phage T7 infects male (F-plasmid-carrying) Escherichia coli cells abortively, whereas the closely related phage T3 grows normally. The inability or ability of phage to replicate in male host cells depends on whether the right end of gene 1 (coding for the phage-specific RNA polymerase) consists of T7 or T3 DNA base sequences.     

The photosynthetic apparatus of C3 and CAM-induced Mesembryanthemum crystallinum L.

Accompanying the CAM induction of Mesembryanthemum crystallinum L. grown in high salinity there are changes in the enzymes of carbon metabolism. However, there are no changes in the electron transport activities, Chla/b ratios or in the distribution of chlorophyll amongst the various pigment-protein complexes of isolated thylakoids. Hence with CAM induction there are no changes in the photochemical apparatus of M. crystallinum thylakoids. Despite comparable amounts of chlorophylla/b-proteins of photosystem II to those found in typical C₃ sun plants, both the C₃ and CAM M. crystallinum chloroplasts have relatively more photosystem II, and, concommitantly, less photosystem I complex. This is consistent with greater fluorescence emission at 685 and 695 nm, and lower emission at 735 nm (measured at 77 K) than typically found for C₃ plants, whether sun or shade species. Photoinhibition of isolated C₃ and CAM thylakoids by white light led to comparable decreases in electron transport capacities and fluorescence emission at 77 K with photosystem II being more affected than PSI. We suggest however, that the presence of more core PSII complexes relative to PSI complexes in this CAM-inducible plant, may provide an additional strategy to mitigate photoinhibition in the short-term.     

New encounters in Arctic waters: a comparison of metabolism and performance of polar cod (<Emphasis Type="Italic">Boreogadus saida</Emphasis>) and Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>) under ocean acidification and warming

Oceans are experiencing increasing acidification in parallel to a distinct warming trend in consequence of ongoing climate change. Rising seawater temperatures are mediating a northward shift in distribution of Atlantic cod (Gadus morhua), into the habitat of polar cod (Boreogadus saida), that is associated with retreating cold water masses. This study investigates the competitive strength of the co-occurring gadoids under ocean acidification and warming (OAW) scenarios. Therefore, we incubated specimens of both species in individual tanks for 4 months, under different control and projected temperatures (polar cod: 0, 3, 6, 8 °C, Atlantic cod: 3, 8, 12, 16 °C) and PCO₂ conditions (390 and 1170 µatm) and monitored growth, feed consumption and standard metabolic rate. Our results revealed distinct temperature effects on both species. While hypercapnia by itself had no effect, combined drivers caused nonsignificant trends. The feed conversion efficiency of normocapnic polar cod was highest at 0 °C, while optimum growth performance was attained at 6 °C; the long-term upper thermal tolerance limit was reached at 8 °C. OAW caused only slight impairments in growth performance. Under normocapnic conditions, Atlantic cod consumed progressively increasing amounts of feed than individuals under hypercapnia despite maintaining similar growth rates during warming. The low feed conversion efficiency at 3 °C may relate to the lower thermal limit of Atlantic cod. In conclusion, Atlantic cod displayed increased performance in the warming Arctic such that the competitive strength of polar cod is expected to decrease under future OAW conditions.     

Role of the programmed death-1 pathway in regulation of alloimmune responses in vivo

Programmed death-1 (PD-1), an inhibitory receptor up-regulated on activated T cells, has been shown to play a critical immunoregulatory role in peripheral tolerance, but its role in alloimmune responses is poorly understood. Using a novel alloreactive TCR-transgenic model system, we examined the functions of this pathway in the regulation of alloreactive CD4+ T cell responses in vivo. PD-L1, but not PD-1 or PD-L2, blockade accelerated MHC class II-mismatched skin graft (bm12 (I-Abm12) into B6 (I-Ab)) rejection in a similar manner to CTLA-4 blockade. In an adoptive transfer model system using the recently described anti-bm12 (ABM) TCR-transgenic mice directly reactive to I-Abm12, PD-1 and PD-L1 blockade enhanced T cell proliferation early in the immune response. In contrast, at a later time point preceding accelerated allograft rejection, only PD-L1 blockade enhanced T cell proliferation. In addition, PD-L1 blockade enhanced alloreactive Th1 cell differentiation. Apoptosis of alloantigen-specific T cells was inhibited significantly by PD-L1 but not PD-1 blockade, indicating that PD-1 may not be the receptor for the apoptotic effect of the PD-L1-signaling pathway. Interestingly, the effect of PD-L1 blockade was dependent on the presence of CD4+ CD25+ regulatory T cells in vivo. These data demonstrate a critical role for the PD-1 pathway, particularly PD-1/PD-L1 interactions, in the regulation of alloimmune responses in vivo.     

Potential for Marker-Assisted Simultaneous Improvement of Grain and Biomass Yield in Triticale

Triticale is a promising crop for agricultural biomass production but breeding has until now mainly focused on grain yield. Here, we evaluated the potential of marker-assisted simultaneous improvement of grain yield and biomass yield. To this end, we employed a large triticale doubled haploid population with 647 individuals derived from four families that were phenotyped for grain yield and biomass yield, as well as thousand-kernel weight, tiller density, and plant height in multi-environment field trials. Employing an association mapping approach, we identified quantitative trait loci (QTL) for all the five traits. The phenotypic correlation between grain yield and biomass yield was low, and we detected only one overlapping QTL suggesting different genetic architectures underlying both traits. Our results indicate that a marker-based selection for either grain yield or biomass yield does not adversely affect the other traits. Furthermore, an improvement of the multiplicative yield traits can to some extent also be achieved by selection for QTL identified for the component traits. Taken together, our results suggest that marker-assisted breeding can assist the establishment of dual-purpose triticale cultivars with high grain and biomass yield.