Identification and characterization of a novel molecular-recognition and self-assembly domain within the islet amyloid polypeptide

The islet amyloid polypeptide (hIAPP) is a 37 amino acid residue polypeptide that was found to accumulate as amyloid fibrils in the pancreas of individuals with type II diabetes. Previous studies identified various fragments of hIAPP that can form amyloid fibrils in vitro (e.g. hIAPP(8-20), hIAPP(23-27), and hIAPP(30-37)). However, no comparative and systematic information was available on the role of these structural domains (or others) in the process of molecular recognition that mediates fibrillization, in the context of the full-length polypeptide. To systematically map and compare potential recognition domains, we studied the ability of hIAPP to interact with an array of 28 membrane-spotted overlapping peptides that span the entire sequence of hIAPP (i.e. hIAPP(1-10), hIAPP(2-11...), hIAPP(28-37)). Our study clearly identified a major domain of molecular recognition within hIAPP, as the polypeptide was found to bind with high affinity to a defined linear group of peptides ranging from hIAPP(7-16) to hIAPP(12-21). The maximal binding of the full-length polypeptide was to the hIAPP(11-20) peptide fragment (with the sequence RLANFLVHSS). In order to define the minimal fragment, within this apparent recognition motif, that is capable of self-association and thus may serve as the core molecular recognition motif, we examined the ability of truncated analogs of the recognition sequence to self-assemble into amyloid fibrils. The shortest active fragments capable of self-assembly were found to be the pentapeptides FLVHS and NFLVH. The apparent role of this motif in the process of hIAPP self-assembly is consistent with the profile of the hIAAP-binding distribution to the peptide array. The identification of such short recognition motifs is extremely useful in the attempts to develop means to block amyloid fibril formation by hIAPP. It is worth mentioning that this is only the second time in which peptides as short as a pentapeptide were shown to form amyloid fibrils (the other pentapeptide is FGAIL).     

Cutting edge: persistent viral infection prevents tolerance induction and escapes immune control following CD28/CD40 blockade-based regimen

A continuing concern with CD28 and/or CD40 blockade-based strategies to induce tolerance and mixed chimerism is their potential to disrupt protective immunity to preexisting infections. In this report, we find that preexisting persistent infection with lymphocytic choriomeningitis virus (LCMV) clone 13 prevents the induction of tolerance, mixed chimerism, and donor-reactive T cell deletion. Mice continue to be refractory to tolerance induction even after viremia has been resolved and virus is present only at very low levels in peripheral tissues. Conversely, we find that the full tolerance regimen, or costimulation blockade alone, specifically inhibits already ongoing antiviral immune responses, leading to an inability to control viremia. These findings suggest that ongoing T cell responses continue to depend on costimulatory interactions in the setting of a chronic infection and provide insight into potential risks following costimulation blockade posed by chronic or latent viral infections such as hepatitis C, EBV, and CMV.     

Therapeutic use of IL-2 to enhance antiviral T-cell responses in vivo

Interleukin (IL)-2 is currently used to enhance T-cell immunity but can have both positive and negative effects on T cells. To determine whether these opposing results are due to IL-2 acting differently on T cells depending on their stage of differentiation, we examined the effects of IL-2 therapy during the expansion, contraction and memory phases of the T-cell response in lymphocytic choriomeningitis virus (LCMV)-infected mice. IL-2 treatment during the expansion phase was detrimental to the survival of rapidly dividing effector T cells. In contrast, IL-2 therapy was highly beneficial during the death phase, resulting in increased proliferation and survival of virus-specific T cells. IL-2 treatment also increased proliferation of resting memory T cells in mice that controlled the infection. Virus-specific T cells in chronically infected mice also responded to IL-2 resulting in decreased viral burden. Thus, timing of IL-2 administration and differentiation status of the T cell are critical parameters in designing IL-2 therapies.     

Elg1, an alternative subunit of the RFC clamp loader,preferentially interacts with SUMOylated PCNA

Replication‐factor C (RFC) is a protein complex that loads the processivity clamp PCNA onto DNA. Elg1 is a conserved protein with homology to the largest subunit of RFC, but its function remained enigmatic. Here, we show that yeast Elg1 interacts physically and genetically with PCNA, in a manner that depends on PCNA modification, and exhibits preferential affinity for SUMOylated PCNA. This interaction is mediated by three small ubiquitin‐like modifier (SUMO)‐interacting motifs and a PCNA‐interacting protein box close to the N‐terminus of Elg1. These motifs are important for the ability of Elg1 to maintain genomic stability. SUMOylated PCNA is known to recruit the helicase Srs2, and in the absence of Elg1, Srs2 and SUMOylated PCNA accumulate on chromatin. Strains carrying mutations in both ELG1 and SRS2 exhibit a synthetic fitness defect that depends on PCNA modification. Our results underscore the importance of Elg1, Srs2 and SUMOylated PCNA in the maintenance of genomic stability.     

TCR signal transduction in antigen-specific memory CD8 T cells

Memory T cells are more responsive to Ag than naive cells. To determine whether memory T cells also have more efficient TCR signaling, we compared naive, effector, and memory CD8 T cells of the same antigenic specificity. Surprisingly, initial CD3 signaling events are indistinguishable. However, memory T cells have more extensive lipid rafts with higher phosphoprotein content before TCR engagement. Upon activation in vivo, they more efficiently induce phosphorylation of-LAT (linker for activation of T cells), ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), and p38. Thus, memory CD8 T cells do not increase their TCR sensitivity, but are better poised to augment downstream signals. We propose that this regulatory mechanism might increase signal transduction in memory T cells, while limiting TCR cross-reactivity and autoimmunity.     

Spatial and temporal electroselection patterns in electric field stimulation of polarized luminescence from photosynthetic membrane vesicles

Electroselection processes of charge recombination are manifested in the study of electric field induced polarized emission from photosynthetic membrane vesicles. The study explores the coupled spatial-temporal characteristics of electric field induced charge recombination by examining the dependence of the integrated polarized emission and the time dependent polarization on electric field strength. The experimental results were fitted to theoretical models by computer simulations employing empirical parameters. Simulation of the dependence of the integrated polarized components of emission on electric field strength, suggests field-dependent increased ratio between radiative and nonradiative rates of charge recombination. The observation that the initial polarization values are independent of electric field strength supports the assumption that electric field induced emission originates from the pole area and then spreads away from it towards the equator. The propagation rate of this electric field induced charge recombination from the pole area towards the equator is reflected by the decay of polarization which increases upon raising the electric field strength. Simulation of the polarization's decay, based on a calculated angle of 26.3 ± 0.4° between the transition moment of emission and the plane of the membrane, establishes coupled temporal spatial patterns of electroselection in intramembrane electron transfer invoked by exposing preilluminated photosynthetic vesicles to a homogeneous electric field.     

Liver-infiltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD-1 and low levels of CD127 expression

The majority of people infected with hepatitis C virus (HCV) fail to generate or maintain a T-cell response effective for viral clearance. Evidence from murine chronic viral infections shows that expression of the coinhibitory molecule PD-1 predicts CD8+ antiviral T-cell exhaustion and may contribute to inadequate pathogen control. To investigate whether human CD8+ T cells express PD-1 and demonstrate a dysfunctional phenotype during chronic HCV infection, peripheral and intrahepatic HCV-specific CD8+ T cells were examined. We found that in chronic HCV infection, peripheral HCV-specific T cells express high levels of PD-1 and that blockade of the PD-1/PD-L1 interaction led to an enhanced proliferative capacity. Importantly, intrahepatic HCV-specific T cells, in contrast to those in the periphery, express not only high levels of PD-1 but also decreased interleukin-7 receptor alpha (CD127), an exhausted phenotype that was HCV antigen specific and compartmentalized to the liver, the site of viral replication.     

Identification of novel genomic islands associated with small RNAs

Genome evolution in prokaryotes is assisted by integration of gene pools from phages and plasmids. Regions downstream of tRNAs and tmRNAs are considered as hot spots for the integration of these gene pools or genomic islands. Till date, genomic islands have been identified only at tRNA/tmRNA genes in the enterobacterial genomes. Present work reports 10 distinct small RNAs as potent integration sites for genomic islands. A known tool tRNAcc 1.0 has been used to identify genomic islands associated with small RNAs c0362, oxyS, ryaA, rybB, rybD, ryeB, ryeE, rtT, sraE and tmRNA. The coordinates of 25 such small RNA associated genomic islands in three E. coli (strains: CFT073, EDL933 and K12) and Shigella flexneri (strain: 301) genomes are presented. Moreover cross-verification of the genomic sequences encoded within the identified genomic islands in horizontal gene transfer database, GenBank annotation features and atypical sequence compositions support our results. Again, all of the identified 25 genomic integration sites do exhibit genomic block rearrangements with respect to the associated small RNA. Similar to tRNAs/tmRNAs, the downstream regions of the small RNAs are found to be hotspots of integration.     

Junker: An Intergenic Explorer for Bacterial Genomes

In the past few decades, scientists from all over the world have taken a keen interest in novel functional units such as small regulatory RNAs, small open reading frames, pseudogenes, transposons, integrase binding attB/attP sites, repeat elements within the bacterial intergenic regions (IGRs) and in the analysis of those junk regions for ge- nomic complexity. Here we have developed a web server, named Junker, to facilitate the in-depth analysis of IGRs for examining their length distribution, four-quadrant...     

Ghrelin ovarian cell expression in patients with polycystic ovary syndrome: an immunohistochemical evaluation.

INTRODUCTION: The influence of ghrelin on different organs has been studied recently, e.g. in the regulation of pituitary hormone release, regulation of energy homeostasis, glucose metabolism and insulin secretion, cell proliferation, and reproductive function. However, the etiology of polycystic ovary syndrome has not been fully explained. The aim of our study was to estimate the presence of ghrelin in polycystic ovaries cells and evaluation of the relationship between ghrelin occurrence and cells proliferation. METHODS: In the present work we have compared ten polycystic ovaries with ovaries without pathology as the control group. We used immunohistochemical method to detect ghrelin. The cells proliferation was evaluated by Ki 67 proliferation index. RESULTS: Ghrelin immunostaining was demonstrated in cytoplasm of ovarian secondary interstitial cells and in atretic corpus luteum. The cell nuclei were ghrelin positive in granulosa, theca layers of follicular cyst in both groups as well as in luteal cells of young corpus luteum in healthy ovaries. Ki 67 immunostaining was observed in granulosa and theca layers of follicular cyst in polycystic and healthy ovaries. CONCLUSIONS: It is possible that local ghrelin expression plays an important role in the direct control of ovarian development and function and ghrelin may participate in patomechanism of PCOS.