Extended repertoire of permissible peptide ligands for HLA-B*2702.

Recognition of self peptides bound to the class I major histocompatibility complex molecule HLA-B27 is thought to trigger proliferation of autoreactive T cells and result in autoimmune arthritic diseases. Previous work from other laboratories established that a predominant feature of endogenous peptides eluted from purified B27 is an arginine at position 2. We studied the binding of peptides containing both natural and unnatural amino acids by the subtype HLA-B*2702, with the goal of gaining insight into peptide binding by this B27 subtype that is associated with susceptibility to arthritic disease. A soluble from of B*2702 was depleted of endogenous peptides. We tested the binding of peptides substituted with cysteine, homocysteine, or an alpha-amino-epsilon-mercapto hexanoic acid side chain (Amh) instead of the naturally occurring arginine at position 2, to determine whether the peptide sulfhydryl residue could be covalently linked to cysteine 67 in the B*2702 binding cleft. Although none of the altered peptide sequences bound covalently to B*2702, the affinities of the homocysteine- and Amh-substituted peptides were close to that of the native peptide sequence. Substitutions at position 2 with other side chains, such as glutamine and methionine, also resulted in peptides that bound with only slightly reduced affinity. These results demonstrate that peptide side chains other than arginine at position 2 can be accomodated within the B*2702 peptide binding site with only minor reductions in affinity. This extended repertoire of permissible B27-binding peptides should be taken into account for a consideration of disease-associated peptide sequences.     

Kindlin Binds Migfilin Tandem LIM Domains and Regulates Migfilin Focal Adhesion Localization and Recruitment Dynamics

Focal adhesions (FAs), sites of tight adhesion to the extracellular matrix, are composed of clusters of transmembrane integrin adhesion receptors and intracellular proteins that link integrins to the actin cytoskeleton and signaling pathways. Two integrin-binding proteins present in FAs, kindlin-1 and kindlin-2, are important for integrin activation, FA formation, and signaling. Migfilin, originally identified in a yeast two-hybrid screen for kindlin-2-interacting proteins, is a LIM domain-containing adaptor protein found in FAs and implicated in control of cell adhesion, spreading, and migration. By binding filamin, migfilin provides a link between kindlin and the actin cytoskeleton. Here, using a combination of kindlin knockdown, biochemical pulldown assays, fluorescence microscopy, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP), we have established that the C-terminal LIM domains of migfilin dictate its FA localization, shown that these domains mediate an interaction with kindlin in vitro and in cells, and demonstrated that kindlin is important for normal migfilin dynamics in cells. We also show that when the C-terminal LIM domain region is deleted, then the N-terminal filamin-binding region of the protein, which is capable of targeting migfilin to actin-rich stress fibers, is the predominant driver of migfilin localization. Our work details a correlation between migfilin domains that drive kindlin binding and those that drive FA localization as well as a kindlin dependence on migfilin FA recruitment and mobility. We therefore suggest that the kindlin interaction with migfilin LIM domains drives migfilin FA recruitment, localization, and mobility.     

Studies on the expression of 80-kDa human sperm antigen in rat testis and epididymis.

The 80-kDa human sperm antigen (HSA) has demonstrated to be a promising candidate for development of an antifertility vaccine because it is a sperm-specific, conserved, and immunogenic protein. The present study demonstrates the androgen-regulated expression of 80-kDa HSA in testis and epididymis of rat by immunohistochemistry (IHC), using its specific antibodies. Developmental expression of 80-kDa HSA was investigated on days 10, 20, 40, 60, and 90 of age in the testis and epididymis by IHC, and relative staining intensity was estimated by image analysis using BIOVIS software. On days 10 and 20, no significant staining was observed in the testis and epididymis, whereas it gradually increased from day 40 onwards. The highest staining was seen on day 90 in both testis and epididymis. Gradual increase in expression of 80-kDa HSA after day 40 suggests that it is possibly regulated by androgen. To study the androgen-regulated expression of 80-kDa, adult male rats were treated with 75 mg/kg body weight of ethylene dimethane sulfonate (EDS), which selectively destroys Leydig cells and thus induces complete androgen withdrawal. It was observed that the staining intensity decreased following EDS treatment in rat testis as well as epididymis, and it was regained after supplementation with dihydrotestosterone. Increased expression during sexual maturation at the time of testosterone surge and its regulation by antiandrogen/androgen treatment suggest androgen-dependent expression of 80-kDa HSA in rat testis and epididymis.     

Efficient Imputation of Missing Markers in Low-Coverage Genotyping-by-Sequencing Data from Multiparental Crosses

We consider genomic imputation for low-coverage genotyping-by-sequencing data with high levels of missing data. We compensate for this loss of information by utilizing family relationships in multiparental experimental crosses. This nearly quadruples the number of usable markers when applied to a large rice Multiparent Advanced Generation InterCross (MAGIC) study.     

Non-random organization of the Biomphalaria glabrata genome in interphase Bge cells and the spatial repositioning of activated genes in cells co-cultured with Schistosomamansoni

Biomphalaria glabrata is a major intermediate host for the parasitic trematode Schistosoma mansoni, a causative agent of human schistosomiasis. To decipher the molecular basis of this host-parasite interaction, the Bge embryonic cell line provides a unique in vitro model system to assess whether interactions between the snail and parasite affect the cell and genome biology in either organism. The organization of the B. glabrata genome in Bge cells was studied using image analysis through positioning territories of differently sized chromosomes within cell nuclei. The snail chromosome territories are similar in morphology as well as in non-random radial positioning as those found in other derived protostome and deuterostome organisms. Specific monitoring of four gene loci, piwi, BgPrx, actin and ferritin, revealed non-random radial positioning of the genome. This indicates that specific parts of the snail genome reside in reproducible nuclear addresses. To determine whether exposure to parasite is reflected in genome organization, the interphase spatial positioning of genes was assessed after co-culturing Bge cells with either normal or irradiation attenuated miracidia for 30 min to 24 h. The loci of actin and ferritin, genes that are up-regulated in the snail when subjected to infection, were visualized by fluorescence in situ hybridisation (FISH) and their radial nuclear positions i.e. their position in the interphase nucleus with respect to the nuclear edge/envelope, mapped. Interestingly, large scale gene repositioning correlated to temporal kinetics of gene expression levels in Bge cells co-cultured with normal miracidia while irradiated parasites failed to elicit similar gene expression or gene loci repositioning as demonstrated using the ferritin gene. This indicates that normal but not attenuated schistosomes provide stimuli that evoke host responses that are reflected in the host’s nuclear architecture. We believe that this is not only the first time that gene-repositioning studies have been attempted in a mollusc but also demonstrates a parasite influencing the interphase genome organization of its host.     

Mechanism of mutant calreticulin-mediated activation of the thrombopoietin receptor in cancers

Myeloproliferative neoplasms (MPNs) are frequently driven by mutations within the C-terminal domain (C-domain) of calreticulin (CRT). CRT_Del52 and CRT_Ins5 are recurrent mutations. Oncogenic transformation requires both mutated CRT and the thrombopoietin receptor (Mpl), but the molecular mechanism of CRT-mediated constitutive activation of Mpl is unknown. We show that the acquired C-domain of CRT_Del52 mediates both Mpl binding and disulfide-linked CRT_Del52 dimerization. Cysteine mutations within the novel C-domain (C400A and C404A) and the conserved N-terminal domain (N-domain; C163A) of CRT_Del52 are required to reduce disulfide-mediated dimers and multimers of CRT_Del52. Based on these data and published structures of CRT oligomers, we identify an N-domain dimerization interface relevant to both WT CRT and CRT_Del52. Elimination of disulfide bonds and ionic interactions at both N-domain and C-domain dimerization interfaces is required to abrogate the ability of CRT_Del52 to mediate cell proliferation via Mpl. Thus, MPNs exploit a natural dimerization interface of CRT combined with C-domain gain of function to achieve cell transformation.     

TPX: Biomedical literature search made easy

TPX is a web-based PubMed search enhancement tool that enables faster article searching using analysis and exploration features. These features include identification of relevant biomedical concepts from search results with linkouts to source databases, concept based article categorization, concept assisted search and filtering, query refinement. A distinguishing feature here is the ability to add user-defined concept names and/or concept types for named entity recognition. The tool allows contextual exploration of knowledge sources by providing concept association maps derived from the MEDLINE repository. It also has a full-text search mode that can be configured on request to access local text repositories, incorporating entity co-occurrence search at sentence/paragraph levels. Local text files can also be analyzed on-the-fly. Availability: http://tpx.atc.tcs.com     

Role of MicroRNA Processing in Adipose Tissue in Stress Defense and Longevity

The terminal enzyme of the mitochondrial respiratory chain, cytochrome oxidase, transfers electrons to molecular oxygen, generating water. Within the inner?mitochondrial membrane, cytochrome oxidase assembles into supercomplexes, together with other respiratory chain complexes, forming so-called respirasomes. Little is known about how these higher oligomeric structures are attained. Here we report on Rcf1 and Rcf2 as cytochrome oxidase subunits in S.?cerevisiae. While Rcf2 is specific to yeast, Rcf1 is a conserved subunit with two human orthologs, RCF1a and RCF1b. Rcf1 is required for growth in hypoxia and complex assembly of subunits Cox13 and Rcf2, as well as for the oligomerization of?a subclass of cytochrome oxidase complexes into respirasomes. Our analyses reveal that the cytochrome oxidase of mitochondria displays intrinsic heterogeneity with regard to its subunit composition and that distinct forms of respirasomes can be formed by complex variants.     

Endoplasmic Reticulum Stress Pathway Required for Immune Homeostasis Is Neurally Controlled by Arrestin-1

In response to pathogen infection, the host innate immune system activates microbial killing pathways and cellular stress pathways that need to be balanced because insufficient or excessive immune responses have deleterious consequences. Recent studies demonstrate that two G protein-coupled receptors (GPCRs) in the nervous system of Caenorhabditis elegans control immune homeostasis. To investigate further how GPCR signaling controls immune homeostasis at the organismal level, we studied arrestin-1 (ARR-1), which is the only GPCR adaptor protein in C. elegans. The results indicate that ARR-1 is required for GPCR signaling in ASH, ASI, AQR, PQR, and URX neurons, which control the unfolded protein response and a p38 mitogen-activated protein kinase signaling pathway required for innate immunity. ARR-1 activity also controlled immunity through ADF chemosensory and AFD thermosensory neurons that regulate longevity. Furthermore, we found that although ARR-1 played a key role in the control of immunity by AFD thermosensory neurons, it did not control longevity through these cells. However, ARR-1 partially controlled longevity through ADF neurons.     

Exploring the rearrangement of sensory intelligence in proteobacteria: insight of <Emphasis Type=Italic>Pho regulon</Emphasis>

Pho regulon is a highly evolved and conserved mechanism across the microbes to fulfil their phosphate need. In this study, 52 proteobacteria genomes were analyzed for the presence of phosphorus acquisition genes, their pattern of arrangement and copy numbers. The diverse genetic architecture of the Pho regulon genes indicates the evolutionary challenge of nutrient limitation, particularly phosphorus, faced by bacteria in their environment. The incongruence between the Pho regulon proteins phylogeny and species phylogeny along with the presence of additional copies of pstS and pstB genes, having cross similarity with other genera, suggest the possibility of horizontal gene transfer event. The substitution rate analysis and multiple sequence alignment of the Pho regulon proteins were analyzed to gain additional insight into the evolution of the Pho regulon system. This comprehensive study confirms that genes perform the regulatory function (phoBR) were vertically inherited, whereas interestingly, genes whose product involved in direct interaction with the environment (pstS) acquired by horizontal gene transfer. The substantial amino acid substitutions in PstS most likely contribute to the successful adaptation of bacteria in different ecological condition dealing with different phosphorus availability. The findings decipher the intelligence of the bacteria which enable them to carry out the targeted alteration of genes to cope up with the environmental condition.