Cholera toxin promotes the induction of regulatory T cells specific for bystander antigens by modulating dendritic cell activation

It has previously been reported that cholera toxin (CT) is a potent mucosal adjuvant that enhances Th2 or mixed Th1/Th2 type responses to coadministered foreign Ag. Here we demonstrate that CT also promotes the generation of regulatory T (Tr) cells against bystander Ag. Parenteral immunization of mice with Ag in the presence of CT induced T cells that secreted high levels of IL-4 and IL-10 and lower levels of IL-5 and IFN-gamma. Ag-specific CD4(+) T cell lines and clones generated from these mice had cytokine profiles characteristic of Th2 or type 1 Tr cells, and these T cells suppressed IFN-gamma production by Th1 cells. Furthermore, adoptive transfer of bone marrow-derived dendritic cells (DC) incubated with Ag and CT induced T cells that secreted IL-4 and IL-10 and low concentrations of IL-5. It has previously been shown that IL-10 promotes the differentiation or expansion of type 1 Tr cells. Here we found that CT synergized with low doses of LPS to induce IL-10 production by immature DC. CT also enhanced the expression of CD80, CD86, and OX40 (CD134) on DC and induced the secretion of the chemokine, macrophage inflammatory protein-2 (MIP-2), but inhibited LPS-driven induction of CD40 and ICAM-I expression and production of the inflammatory cytokines/chemokines IL-12, TNF-alpha, MIP-1alpha, MIP-1beta, and monocyte chemoattractant protein-1. Our findings suggest that CT induces maturation of DC, but, by inducing IL-10, inhibiting IL-12, and selectively affecting surface marker expression, suppresses the generation of Th1 cells and promotes the induction of T cells with regulatory activity.     

RNA sequencing reveals two major classes of gene expression levels in metazoan cells

The expression level of a gene is often used as a proxy for determining whether the protein or RNA product is functional in a cell or tissue. Therefore, it is of fundamental importance to understand the global distribution of gene expression levels, and to be able to interpret it mechanistically and functionally. Here we use RNA sequencing (RNA‐seq) of mouse Th2 cells, coupled with a range of other techniques, to show that all genes can be separated, based on their expression abundance, into two distinct groups: one group comprised of lowly expressed and putatively non‐functional mRNAs, and the other of highly expressed mRNAs with active chromatin marks at their promoters. These observations are confirmed in many other microarray and RNA‐seq data sets of metazoan cell types.     

Advances in maize genomics: the emergence of positional cloning

Positional cloning has been and remains a powerful method for gene identification in Arabidopsis. With the completion of the rice genome sequence, positional cloning in rice also took off, including the cloning of several quantitative trait loci. Positional cloning in cereals such as maize whose genomes are much larger than that of rice was considered near impossible because of the vast amounts of repetitive DNA. However, conservation of synteny across the cereal genomes, in combination with new maize resources, has now made positional cloning in maize feasible. In fact, a chromosomal walk is usually much faster than the more traditional method of gene isolation in maize by transposon tagging.     

The fermentation stoichiometry of Thermotoga neapolitana and influence of temperature,oxygen, and pH on hydrogen production

The hyperthermophilic bacterium, Thermotoga neapolitana, has potential for use in biological hydrogen (H₂) production. The objectives of this study were to (1) determine the fermentation stoichiometry of Thermotoga neapolitana and examine H₂ production at various growth temperatures, (2) investigate the effect of oxygen (O₂) on H₂ production, and (3) determine the cause of glucose consumption inhibition. Batch fermentation experiments were conducted at temperatures of 60, 65, 70, 77, and 85°C to determine product yield coefficients and volumetric productivity rates. Yield coefficients did not show significant changes with respect to growth temperature and the rate of H₂ production reached maximum levels in both the 77°C and 85°C experiments. The fermentation stoichiometry for T. neapolitana at 85°C was 3.8 mol H₂, 2 mol CO₂, 1.8 mol acetate, and 0.1 mol lactate produced per mol of glucose consumed. Under microaerobic conditions H₂ production did not increase when compared to anaerobic conditions, which supports other evidence in the literature that T. neapolitana does not produce H₂ through microaerobic metabolism. Glucose consumption was inhibited by a decrease in pH. When pH was adjusted with buffer addition cultures completely consumed available glucose. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Structural and spectroscopic studies of a model for catechol oxidase

A binuclear copper complex, [Cu₂(BPMP)(OAc)₂][ClO₄]·H₂O, has been prepared using the binucleating ligand 2,6-bis[bis(pyridin-2-ylmethylamino)methyl]-4-methylphenol (H-BPMP). The X-ray crystal structure reveals the copper centers to have a five-coordinate square pyramidal geometry, with the acetate ligands bound terminally. The bridging phenolate occupies the apical position of the square-based pyramids and magnetic susceptibility, electron paramagnetic resonance (EPR) and variable-temperature variable-field magnetic circular dichroism (MCD) measurements indicate that the two centers are very weakly antiferromagnetically coupled (J = −0.6 cm⁻¹). Simulation of the dipole–dipole-coupled EPR spectrum showed that in solution the Cu–O–Cu angle was increased from 126° to 160° and that the internuclear distance was larger than that observed crystallographically. The high-resolution spectroscopic information obtained has been correlated with a detailed ligand-field analysis to gain insight into the electronic structure of the complex. Symmetry arguments have been used to demonstrate that the sign of the MCD is characteristic of the tetragonally elongated environment. The complex also displays catecholase activity (k _cat = 15 ± 1.5 min⁻¹, K _M = 6.4 ± 1.8 mM), which is compared with other dicopper catechol oxidase models. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

GRASP55 regulates the unconventional secretion and aggregation of mutant huntingtin

Recent studies demonstrated that the Golgi reassembly stacking proteins (GRASPs), especially GRASP55, regulate Golgi-independent unconventional secretion of certain cytosolic and transmembrane cargoes; however, the underlying mechanism remains unknown. Here, we surveyed several neurodegenerative disease–related proteins, including mutant huntingtin (Htt-Q74), superoxide dismutase 1 (SOD1), tau, and TAR DNA–binding protein 43 (TDP-43), for unconventional secretion; our results show that Htt-Q74 is most robustly secreted in a GRASP55-dependent manner. Using Htt-Q74 as a model system, we demonstrate that unconventional secretion of Htt is GRASP55 and autophagy dependent and is enhanced under stress conditions such as starvation and endoplasmic reticulum stress. Mechanistically, we show that GRASP55 facilitates Htt secretion by tethering autophagosomes to lysosomes to promote autophagosome maturation and subsequent lysosome secretion and by stabilizing p23/TMED10, a channel for translocation of cytoplasmic proteins into the lumen of the endoplasmic reticulum–Golgi intermediate compartment. Moreover, we found that GRASP55 levels are upregulated by various stresses to facilitate unconventional secretion, whereas inhibition of Htt-Q74 secretion by GRASP55 KO enhances Htt aggregation and toxicity. Finally, comprehensive secretomic analysis identified novel cytosolic cargoes secreted by the same unconventional pathway, including transgelin (TAGLN), multifunctional protein ADE2 (PAICS), and peroxiredoxin-1 (PRDX1). In conclusion, this study defines the pathway of GRASP55-mediated unconventional protein secretion and provides important insights into the progression of Huntington’s disease.     

Altered differentiation and clustering of Sertoli cells in transgenic mice showing a Sertoli cell specific knockout of the connexin 43 gene

Histological analysis revealed that Sertoli cell specific knockout of the predominant testicular gap junction protein connexin 43 results in a spermatogenic arrest at the level of spermatogonia or Sertoli cell-only syndrome, intratubular cell clusters and still proliferating adult Sertoli cells, implying an important role for connexin 43 in the Sertoli and germ cell development. This study aimed to determine the (1) Sertoli cell maturation state, (2) time of occurrence and (3) composition, differentiation and fate of clustered cells in knockout mice. Using immunohistochemistry connexin 43 deficient Sertoli cells showed an accurate start of the mature markers androgen receptor and GATA-1 during puberty and a vimentin expression from neonatal to adult. Expression of anti-Muellerian hormone, as a marker of Sertoli cell immaturity, was finally down-regulated during puberty, but its disappearance was delayed. This observed extended anti-Müllerian hormone synthesis during puberty was confirmed by western blot and Real-Time PCR and suggests a partial alteration in the Sertoli cell differentiation program. Additionally, Sertoli cells of adult knockouts showed a permanent and uniform expression of GATA-1 at protein and mRNA level, maybe caused by the lack of maturing germ cells and missing negative feedback signals. At ultrastructural level, basally located adult Sertoli cells obtained their mature appearance, demonstrated by the tripartite nucleolus as a typical feature of differentiated Sertoli cells. Intratubular clustered cells were mainly formed by abnormal Sertoli cells and single attached apoptotic germ cells, verified by immunohistochemistry, TUNEL staining and transmission electron microscopy. Clusters first appeared during puberty and became more numerous in adulthood with increasing cell numbers per cluster suggesting an age-related process. In conclusion, adult connexin 43 deficient Sertoli cells seem to proliferate while maintaining expression of mature markers and their adult morphology, indicating a unique and abnormal intermediate phenotype with characteristics common to both undifferentiated and differentiated Sertoli cells.     

BRCA1 Pathway Function in Basal-Like Breast Cancer Cells

Sporadic basal-like cancers (BLCs) are a common subtype of breast cancer that share multiple biological properties with BRCA1-mutated breast tumors. Despite being BRCA1^+/+, sporadic BLCs are widely viewed as phenocopies of BRCA1-mutated breast cancers, because they are hypothesized to manifest a BRCA1 functional defect or breakdown of a pathway(s) in which BRCA1 plays a major role. The role of BRCA1 in the repair of double-strand DNA breaks by homologous recombination (HR) is its best understood function and the function most often implicated in BRCA1 breast cancer suppression. Therefore, it is suspected that sporadic BLCs exhibit a defect in HR. To test this hypothesis, multiple DNA damage repair assays focused on several types of repair were performed on a group of cell lines classified as sporadic BLCs and on controls. The sporadic BLC cell lines failed to exhibit an overt HR defect. Rather, they exhibited defects in the repair of stalled replication forks, another BRCA1 function. These results provide insight into why clinical trials of poly(ADP-ribose) polymerase (PARP) inhibitors, which require an HR defect for efficacy, have been unsuccessful in sporadic BLCs, unlike cisplatin, which elicits DNA damage that requires stalled fork repair and has shown efficacy in sporadic BLCs.     

Determinants of the interaction of the spinal muscular atrophy disease protein SMN with the dimethylarginine-modified box H/ACA small nucleolar ribonucleoprotein GAR1

Deletion or mutation of the SMN1 (survival of motor neurons) gene causes the common, fatal neuromuscular disease spinal muscular atrophy. The SMN protein is important in small nuclear ribonucleoprotein (snRNP) assembly and interacts with snRNP proteins via arginine/glycine-rich domains. Recently, SMN was also found to interact with core protein components of the two major families of small nucleolar RNPs, fibrillarin and GAR1, suggesting that SMN may also function in the assembly of small nucleolar RNPs. Here we present results that indicate that the interaction of SMN with GAR1 is mediated by the Tudor domain of SMN. Single point mutations within the Tudor domain, including a spinal muscular atrophy patient mutation, impair the interaction of SMN with GAR1. Furthermore, we find that either of the two arginine/glycine-rich domains of GAR1 can provide for interaction with SMN, but removal of both results in loss of the interaction. Finally, we have found that unlike the interaction of SMN with the Sm snRNP proteins, interaction with GAR1 and fibrillarin is not enhanced by arginine dimethylation. Our results argue against post-translational arginine dimethylation as a general requirement for SMN recognition of proteins bearing arginine/glycine-rich domains.