Thymic alterations in EphA4-deficient mice

In the present work, we have demonstrated in vivo an altered maturation of the thymic epithelium that results in defective T cell development which increases with age, in the thymus of Eph A4-deficient mice. The deficient thymi are hypocellular and show decreased proportions of double-positive (CD4+CD8+) cells which reach minimal numbers in 4-wk-old thymi. The EphA4 (-/-) phenotype correlates with an early block of T cell precursor differentiation that results in accumulation of CD44-CD25+ triple-negative cells and, sometimes, of CD44+CD25- triple-negative thymocytes as well as with increased numbers of apoptotic cells and an important reduction in the numbers of cycling thymocytes. Various approaches support a key role of the thymic epithelial cells in the observed phenotype. Thymic cytoarchitecture undergoes profound changes earlier than those found in the thymocyte maturation. Thymic cortex is extremely reduced and consists of densely packed thymic epithelial cells. Presumably the lack of forward Eph A4 signaling in the Eph A4 -/- epithelial cells affects their development and finally results in altered T cell development.     

酵母双杂交系统筛选与RapGAP相互作用的蛋白质

目的筛选与Rap GAP相互作用的蛋白质,为进一步研究人源Rap1GAP介导的信号转导通路、揭示其与肿瘤的关系提供实验依据。方法选用与Rap1GAP同源的来自美丽线虫的Rap GAP作为饵蛋白,以来源于美丽线虫的c DNA文库作为靶蛋白,应用p PC97、p PC86组成的酵母双杂交系统筛选c DNA文库中与Rap GAP相互作用的蛋白质。结果通过营养缺陷平板(-LTH)筛选出63个拟似阳性菌落。经过Lac Z鉴定,19个菌落为阳性,其中7个为强阳性。提取来自19个酵母菌落中的重组DNA,经PCR扩增,12个菌落出现阳性结果。将该19个重组DNA分别电转化入DH5α细菌,涂板培养后,每板挑取4~10个克隆,通过Sal I和Not I双酶切鉴定进行阳性克隆筛选。将阳性克隆的重组DNA进行序列测定。测序结果与Gen Bank比较,其中4个克隆的DNA片段为Y39b6a基因片段、2个为Rap GAP、1个为苯丙氨酸-4-羟化酶、1个为细胞色素C氧化酶,还有1个DNA片段编码美丽线虫特有的小分子蛋白的基因片段,其余11个DNA片段不编码已知蛋白质。结论初步筛选出与Rap GAP相互作用的蛋白质,特别是其中有2个克隆为Rap GAP,提示Rap GAP可能以二聚体的方式存在。     

Genomic and experimental evidence for multiple metabolic functions in the RidA/YjgF/YER057c/UK114 (Rid) protein family

Background

It is now recognized that enzymatic or chemical side-reactions can convert normal metabolites to useless or toxic ones and that a suite of enzymes exists to mitigate such metabolite damage. Examples are the reactive imine/enamine intermediates produced by threonine dehydratase, which damage the pyridoxal 5''-phosphate cofactor of various enzymes causing inactivation. This damage is pre-empted by RidA proteins, which hydrolyze the imines before they do harm. RidA proteins belong to the YjgF/YER057c/UK114 family (here renamed the Rid family). Most other members of this diverse and ubiquitous family lack defined functions.

Results

Phylogenetic analysis divided the Rid family into a widely distributed, apparently archetypal RidA subfamily and seven other subfamilies (Rid1 to Rid7) that are largely confined to bacteria and often co-occur in the same organism with RidA and each other. The Rid1 to Rid3 subfamilies, but not the Rid4 to Rid7 subfamilies, have a conserved arginine residue that, in RidA proteins, is essential for imine-hydrolyzing activity. Analysis of the chromosomal context of bacterial RidA genes revealed clustering with genes for threonine dehydratase and other pyridoxal 5''-phosphate-dependent enzymes, which fits with the known RidA imine hydrolase activity. Clustering was also evident between Rid family genes and genes specifying FAD-dependent amine oxidases or enzymes of carbamoyl phosphate metabolism. Biochemical assays showed that Salmonella enterica RidA and Rid2, but not Rid7, can hydrolyze imines generated by amino acid oxidase. Genetic tests indicated that carbamoyl phosphate overproduction is toxic to S. enterica cells lacking RidA, and metabolomic profiling of Rid knockout strains showed ten-fold accumulation of the carbamoyl phosphate-related metabolite dihydroorotate.

Conclusions

Like the archetypal RidA subfamily, the Rid2, and probably the Rid1 and Rid3 subfamilies, have imine-hydrolyzing activity and can pre-empt damage from imines formed by amine oxidases as well as by pyridoxal 5''-phosphate enzymes. The RidA subfamily has an additional damage pre-emption role in carbamoyl phosphate metabolism that has yet to be biochemically defined. Finally, the Rid4 to Rid7 subfamilies appear not to hydrolyze imines and thus remain mysterious.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1584-3) contains supplementary material, which is available to authorized users.     

Optimizing Human Synovial Fluid Preparation for Two-Dimensional Gel Electrophoresis

Background

Prenatal screening for Down Syndrome (DS) would benefit from an increased number of biomarkers to improve sensitivity and specificity. Improving sensitivity and specificity would decrease the need for potentially risky invasive diagnostic procedures.

Results

We have performed an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers. We have used maternal plasma samples obtained from first and second trimesters from mothers carrying DS affected fetuses compared with mothers carrying normal fetuses. Plasma samples were albumin/IgG depleted and expanded pH ranges of pH 4.5 - 5.5, pH 5.3 - 6.5 and pH 6 - 9 were used for two-dimensional gel electrophoresis (2DE). We found no differentially expressed proteins in the first trimester between the two groups. Significant up-regulation of ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4, complement proteins C1s subcomponent, C4-A, C5, and C9 and kininogen 1 were detected in the second trimester in maternal plasma samples where a DS affected fetus was being carried. However, ceruloplasmin could not be confirmed as being consistently up-regulated in DS affected pregnancies by Western blotting.

Conclusions

Despite the in depth 2DE approach used in this study the results underline the deficiencies of gel-based proteomics for detection of plasma biomarkers. Gel-free approaches may be more productive to increase the number of plasma biomarkers for DS for non-invasive prenatal screening and diagnosis.     

Testing for temporal variation in diversification rates when sampling is incomplete and nonrandom

A common pattern found in phylogeny-based empirical studies of diversification is a decrease in the rate of lineage accumulation toward the present. This early-burst pattern of cladogenesis is often interpreted as a signal of adaptive radiation or density-dependent processes of diversification. However, incomplete taxonomic sampling is also known to artifactually produce patterns of rapid initial diversification. The Monte Carlo constant rates (MCCR) test, based upon Pybus and Harvey's gamma (γ)-statistic, is commonly used to accommodate incomplete sampling, but this test assumes that missing taxa have been randomly pruned from the phylogeny. Here we use simulations to show that preferentially sampling disparate lineages within a clade can produce severely inflated type-I error rates of the MCCR test, especially when taxon sampling drops below 75%. We first propose two corrections for the standard MCCR test, the proportionally deeper splits that assumes missing taxa are more likely to be recently diverged, and the deepest splits only MCCR that assumes that all missing taxa are the youngest lineages in the clade, and assess their statistical properties. We then extend these two tests into a generalized form that allows the degree of nonrandom sampling (NRS)to be controlled by a scaling parameter, α. This generalized test is then applied to two recent studies. This new test allows systematists to account for nonrandom taxonomic sampling when assessing temporal patterns of lineage diversification in empirical trees. Given the dramatic affect NRS can have on the behavior of the MCCR test, we argue that evaluating the sensitivity of this test to NRS should become the norm when investigating patterns of cladogenesis in incompletely sampled phylogenies.     

Dendritic cells take up and present antigens from viable and apoptotic polymorphonuclear leukocytes

Dendritic cells (DC) are endowed with the ability to cross-present antigens from other cell types to cognate T cells. DC are poised to meet polymorphonuclear leukocytes (PMNs) as a result of being co-attracted by interleukin-8 (IL-8), for instance as produced by tumor cells or infected tissue. Human monocyte-derived and mouse bone marrow-derived DC can readily internalize viable or UV-irradiated PMNs. Such internalization was abrogated at 4°C and partly inhibited by anti-CD18 mAb. In mice, DC which had internalized PMNs containing electroporated ovalbumin (OVA) protein, were able to cross-present the antigen to CD8 (OT-1) and CD4 (OT-2) TCR-transgenic T cells. Moreover, in humans, tumor cell debris is internalized by PMNs and the tumor-cell material can be subsequently taken up from the immunomagnetically re-isolated PMNs by DC. Importantly, if human neutrophils had endocytosed bacteria, they were able to trigger the maturation program of the DC. Moreover, when mouse PMNs with E. coli in their interior are co-injected in the foot pad with DC, many DC loaded with fluorescent material from the PMNs reach draining lymph nodes. Using CT26 (H-2(d)) mouse tumor cells, it was observed that if tumor cells are intracellularly loaded with OVA protein and UV-irradiated, they become phagocytic prey of H-2(d) PMNs. If such PMNs, that cannot present antigens to OT-1 T cells, are immunomagnetically re-isolated and phagocytosed by H-2(b) DC, such DC productively cross-present OVA antigen determinants to OT-1 T cells. Cross-presentation to adoptively transferred OT-1 lymphocytes at draining lymph nodes also take place when OVA-loaded PMNs (H-2(d)) are coinjected in the footpad of mice with autologous DC (H-2(b)). In summary, our results indicate that antigens phagocytosed by short-lived PMNs can be in turn internalized and productively cross-presented by DC.     

Kinetics, molecular basis, and differentiation of L-lactate transport in spermatogenic cells

Round spermatid energy metabolism is closely dependent on the presence of L-lactate in the external medium. This L-lactate has been proposed to be supplied by Sertoli cells in the seminiferous tubules. L-Lactate, in conjunction with glucose, modulates intracellular Ca²⁺ concentration in round spermatids and pachytene spermatocytes. In spite of this central role of L-lactate in spermatogenic cell physiology, the mechanism of L-lactate transport, as well as possible differentiation during spermatogenesis, has not been studied in these cells. By measuring radioactive L-lactate transport and intracellular pH (pH_i) changes with pH_i fluorescent probes, we show that these cells transport L-lactate using monocarboxylate-H⁺ transport (MCT) systems. RT-PCR, in situ mRNA hybridization, and immunocyto- and immunohistochemistry data show that pachytene spermatocytes express mainly the MCT1 and MCT4 isoforms of the transporter (intermediate- and low-affinity transporters, respectively), while round spermatids, besides MCT1 and MCT4, also show expression of the MCT2 isoform (high-affinity transporter). These molecular data are consistent with the kinetic data of L-lactate transport in these cells demonstrating at least two transport components for L-lactate. These separate transport components reflect the ability of these cells to switch between the generation of glycolytic L-lactate in the presence of external glucose and the use of L-lactate when this substrate is available in the external environment. The supply of these substrates is regulated by the hormonal control of Sertoli cell glycolytic activity. cell differentiation; seminiferous tubules; spermatogenesis; testicle; meiosis     

Phylogenetic relationships and evolutionary history of the reef fish family Labridae

The family Labridae (including scarines and odacines) contains 82 genera and about 600 species of fishes that inhabit coastal and continental shelf waters in tropical and temperate oceans throughout the world. The Labridae (the wrasses) is the fifth largest fish family and second largest marine fish family, and is one of the most morphologically and ecologically diversified families of fishes in size, shape, and color. Labrid phylogeny is a long-standing problem in ichthyology that is part of the larger question of relationships within the suborder Labroidei. A phylogenetic analysis of labrids was conducted to investigate relationships among the six classical tribes of wrasses, the affinities of the wrasses to the parrotfishes (scarines), and the broad phylogenetic structure among labrid genera. Four gene fragments were sequenced from 98 fish species, including 84 labrid fishes and 14 outgroup taxa. Taxa were chosen from all major labrid clades and most major global ocean regions where labrid fishes exist, as well as cichlid, pomacentrid, and embiotocid outgroups. From the mitochondrial genome we sequenced portions of 12S rRNA (1000 bp) and 16S rRNA (585 bp), which were aligned by using a secondary structure model. From the nuclear genome, we sequenced part of the protein-coding genes RAG2 (846 bp) and Tmo4C4 (541 bp). Maximum likelihood, maximum parsimony, and Bayesian analyses on the resulting 2972 bp of DNA sequence produced similar topologies that confirm the monophyly of a family Labridae that includes the parrotfishes and butterfishes and strong support for many previously identified taxonomic subgroups. The tribe Hypsigenyini (hogfishes, tuskfishes) is the sister group to the remaining labrids and includes odacines and the chisel-tooth wrasse Pseudodax moluccanus, a species previously considered close to scarines. Cheilines and scarines are sister-groups, closely related to the temperate Labrini, and pseudocheilines and cheilines are split in all phylogenies. The razorfishes (novaculines) and temperate pseudolabrines form successive sister clades to the large crown group radiation of the Julidini. The cleaner wrasses (Labrichthyini) are nested within this radiation and several julidine genera do not appear to be monophyletic (e.g., Coris and Halichoeres). Invasion of temperate water by this predominantly tropical group has occurred multiple times and the reconstruction of biogeography assuming an Indo-Pacific ancestor results in five different lineages invading the Atlantic/Caribbean region. Functional novelties in the feeding apparatus have allowed labrid fishes to occupy nearly every feeding guild in reef environments, and trophic variation is a central axis of diversification in this family.