Ghrelin increases anxiety-like behavior and memory retention in rats

Ghrelin is a peptide found in the hypothalamus and stomach that stimulates food intake and whose circulating concentrations are affected by nutritional state. Very little is known about other central behavioral effects of ghrelin, and thus, we investigated the effects of ghrelin on anxiety and memory retention. The peptide was injected intracerebroventricularly in rats and we performed open-field, plus-maze, and step-down tests (inhibitory avoidance). The administration of ghrelin increased freezing in the open field and decreased the number of entries into the open spaces and the time spent on the open arms in the plus-maze, indicating an anxiogenic effect. Moreover, the peptide increased in a dose-dependent manner the latency time in the step-down test. A rapid and prolonged increase in food intake was also observed. Our results indicate that ghrelin induces anxiogenesis in rats. Moreover, we show for the first time that ghrelin increases memory retention, suggesting that the peptide may influence processes in the hippocampus.     

Endogenous Processing and Presentation of T-cell Epitopes from Chlamydia trachomatis with Relevance in HLA-B27-associated Reactive Arthritis

Chlamydia trachomatis triggers reactive arthritis, a spondyloarthropathy linked to the human major histocompatibility complex molecule HLA-B27, through an unknown mechanism that might involve molecular mimicry between chlamydial and self-derived HLA-B27 ligands. Chlamydia-specific CD8⁺ T-cells are found in reactive arthritis patients, but the immunogenic epitopes are unknown. A previous screening of the chlamydial genome for putative HLA-B27 ligands predicted multiple peptides that were recognized in vitro by CD8⁺ T-lymphocytes from patients. Here stable transfectants expressing bacterial fusion proteins in human cells were generated to investigate the endogenous processing and presentation by HLA-B27 of two such epitopes through comparative immunoproteomics of HLA-B27-bound peptide repertoires. A predicted T-cell epitope, from the CT610 gene product, was presented by HLA-B27. This is, to our knowledge, the first endogenously processed epitope involved in HLA-B27-restricted responses against C. trachomatis in reactive arthritis. A second predicted epitope, from the CT634 gene product, was not detected. Instead a non-predicted nonamer from the same protein was identified. Both bacterial peptides showed very high homology with human sequences containing the HLA-B27 binding motif. Thus, expression and intracellular processing of chlamydial proteins into human cells allowed us to identify two bacterial HLA-B27 ligands, including the first endogenous T-cell epitope from C. trachomatis involved in spondyloarthropathy. That human proteins contain sequences mimicking chlamydial T-cell epitopes suggests a basis for an autoimmune component of Chlamydia-induced HLA-B27-associated disease.Chlamydia trachomatis is an obligate intracellular parasite that infects the urogenital epithelium. It is a very common pathogen and one frequently inducing reactive arthritis (ReA)¹ (1). Multiple strategies, including down-regulation of major histocompatibility complex (MHC) class I and class II expression (2–4) and persistence, have been developed by the bacteria to evade the immune system. Yet both CD4⁺ and CD8⁺ T-cell responses are activated upon infection (5). In particular, HLA-B27-restricted CD8⁺ T-lymphocytes are found in patients with Chlamydia-induced ReA (6, 7). The role of these cells in the pathogenesis and evolution of ReA to chronic disease is probably mediated by IFN-γ. Secretion of this cytokine is critical for the protective immunity against Chlamydia (8) because it inhibits the bacterial growth (9). However, this is often insufficient to promote complete clearance of C. trachomatis, and actually IFN-γ-induced depletion of the tryptophan pool induces the differentiation of the metabolically active reticular bodies to persistent forms (10), which sustain chronic infection and ReA. The high prevalence of HLA-B27 among patients with Chlamydia-induced ReA (11), especially in its chronic form, suggests a pathogenetic mechanism based on interactive effects of the bacteria and HLA-B27 that seems unrelated to the capacity of C. trachomatis to infect or replicate into HLA-B27-positive cells (12). One such mechanism could be T-cell-mediated autoimmunity elicited by molecular/antigenic mimicry between chlamydial and self-derived HLA-B27 ligands. Antigenic mimicry between chlamydial and homologous α-myosin-derived peptides is crucial to inducing autoimmune myocarditis in mice (13). Breakdown of cytotoxic T-lymphocyte (CTL) tolerance to HLA-B27 was observed in transgenic rats upon exposure to C. trachomatis (14). Cross-reactivity between HLA-B27-restricted Chlamydia-specific CTL with self-derived HLA-B27 epitopes has not been reported. However, a biochemical basis for it was provided by the finding of an endogenously processed and presented peptide from the DNA primase of C. trachomatis with high homology to a self-derived HLA-B27 ligand (15, 16).Because of the likely involvement of HLA-B27 in the pathogenesis of chronically evolving ReA, the role of CD8⁺ T-cell responses in the protective immunity against C. trachomatis and the presence of HLA-B27-restricted T-cells in patients with Chlamydia-induced ReA, the identification of relevant chlamydial epitopes becomes crucial to establish the pathogenetic mechanism of this disease. Unfortunately a direct analysis of chlamydial HLA-B27 ligands expressed on infected cells is exceedingly difficult because of their extremely low amounts, which challenge even the most sensitive techniques of MS. In the case of Chlamydia, the situation is further complicated by the down-regulation of MHC class I expression shortly after infection (3, 4). To our knowledge, only one MHC class I ligand was recently identified, in the mouse system, from Chlamydia muridarum-infected cells using state-of-the-art MS techniques (17). Due in part to this difficulty, alternative approaches, such as expression cloning and synthetic peptide epitope mapping (18, 19) or MHC class I tetramer arrays (20), have been used to identify MHC class I-restricted chlamydial T-cell epitopes in mice. In a previous study (6) predictive algorithms were used to screen the whole genome of C. trachomatis for nonamer peptide sequences containing the HLA-B*2705 binding motif and a high probability of being generated by proteasomal cleavage. This led to identifying multiple sequences that, when used as synthetic peptides in vitro, stimulated CD8⁺ T-cells from patients with Chlamydia-induced ReA. Such cells could also be detected in the synovial fluid of these patients using HLA-B27 tetramers complexed to some of these peptides (7).Although these strategies identify chlamydial sequences that are recognized by CD8⁺ T-cells they do not prove that these peptides are the endogenously processed epitopes that activated the natural T-cell responses to the bacteria in vivo. Because of the intrinsic cross-reactivity of T-cells (21, 22), it is conceivable that synthetic peptides recognized in vitro may be different from the natural epitopes generated by endogenous processing of the chlamydial proteins that elicit the HLA-B27-restricted T-cell responses in ReA patients. To investigate this issue we focused on two predicted epitopes (6). Stable transfectants expressing the corresponding chlamydial proteins fused to green fluorescent protein (GFP) were generated in a B*2705-positive cell line. The endogenous processing and presentation of the predicted epitopes or other peptides from the same bacterial protein were analyzed by comparative immunoproteomics analysis of the B*2705-bound peptide repertoires from transfected and untransfected cells and sequencing of peptides differentially presented on the bacterial protein transfectant.     

Anxiety-like behavior induced by IL-1beta is modulated by alpha-MSH through central melanocortin-4 receptors

The proinflammatory cytokine interleukin-1beta (IL-1beta) influences neuroendocrine activity and produces other effects, including fever and behavioral changes such as anxiety. The melanocortin neuropeptides, such as alpha-melanocyte-stimulating hormone (alpha-MSH), antagonize many actions of IL-1, including fever, anorexia and hypothalamic-pituitary-adrenal (HPA) axis activation through specific melanocortin receptors (MC-R) in the central nervous system. The objective of the present study was to establish the effect of MSH peptides on IL-1beta-induced anxiety-like behavior and the melanocortin receptors involved. We evaluated the effects of intracerebroventricular (i.c.v.) administration of IL-1beta (30 ng) and melanocortin receptor agonists: alpha-MSH, an MC3/MC4-R agonist (0.2 microg) or gamma-MSH, an MC3-R agonist (2 microg) or HS014, an MC4-R antagonist (2 microg), on an elevated plus-maze (EPM) test. Injection of IL-1beta induced an anxiogenic-like response, as indicated by reduced open arms entries and time spent on open arms. The administration of alpha-MSH reversed IL-1beta-induced anxiety with co-administration of HS014 inhibiting the effect of alpha-MSH. However, the associated treatment with gamma-MSH did not affect the anxiety response to IL-1beta. These data suggest that alpha-MSH, through central MC4-R can modulate the anxiety-like behavior induced by IL-1beta.     

Concerted evolution of members of the multisequence family chAB4 located on various nonhomologous chromosomes

During the last years it became obvious that a lot of families of long-range repetitive DNA elements are located within the genomes of mammals. The principles underlying the evolution of such families, therefore, may have a greater impact than anticipated on the evolution of the mammalian genome as a whole. One of these families, called chAB4, is represented with about 50 copies within the human and the chimpanzee genomes and with only a few copies in the genomes of gorilla, orang-utan, and gibbon. Members of chAB4 are located on 10 different human chromosomes. FISH of chAB4-specific probes to chromosome preparations of the great apes showed that chAB4 is located, with only one exception, at orthologous places in the human and the chimpanzee genome. About half the copies in the human genome belong to two species-specific subfamilies that evolved after the divergence of the human and the chimpanzee lineages. The analysis of chAB4-specific PCR-products derived from DNA of rodent/human cell hybrids showed that members of the two human-specific subfamilies can be found on 9 of the 10 chAB4-carrying chromosomes. Taken together, these results demonstrate that the members of DNA sequence families can evolve as a unit despite their location at multiple sites on different chromosomes. The concerted evolution of the family members is a result of frequent exchanges of DNA sequences between copies located on different chromosomes. Interchromosomal exchanges apparently take place without greater alterations in chromosome structure. Received: 20 March 1997 / Accepted: 13 September 1997     

Proteasome-independent HLA-B27 ligands arise mainly from small basic proteins

Many of the constitutive peptide ligands of HLA-B27, a molecule strongly associated with spondyloarthritis, are proteasome-independent. Stable isotope tagging, mass spectrometry, and epoxomicin-mediated inhibition were used to determine their percentage, structural features, and parental proteins. Of 104 molecular species examined, 29.8% were proteasome-independent, paralleling the level of HLA-B27 re-expression in the presence of epoxomicin after acid stripping. Proteasome-dependent and -independent ligands differed little in peptide motifs, flanking sequences, and cellular localization of the parental proteins. In contrast, whereas the former set arose from proteins whose size and isoelectric point distribution largely reflected those in the human proteome, proteasome-independent ligands, other than a few matching signal sequences, were almost totally derived from small (about 6-16.5 kDa) and basic proteins, which account for only 6.6% of the human proteome. Thus, a non-proteasomal proteolytic pathway with strong preference for small proteins is responsible for a significant fraction of the HLA-B27-bound peptide repertoire.     

Structural and functional divergence of two fish aquaporin-1 water channels following teleost-specific gene duplication

Background  

Teleost radiation in the oceans required specific physiological adaptations in eggs and early embryos to survive in the hyper-osmotic seawater. Investigating the evolution of aquaporins (AQPs) in these vertebrates should help to elucidate how mechanisms for water homeostasis evolved. The marine teleost gilthead sea bream (Sparus aurata) has a mammalian aquaporin-1 (AQP1)-related channel, termed AQP1o, with a specialized physiological role in mediating egg hydration. However, teleosts have an additional AQP isoform structurally more similar to AQP1, though its relationship with AQP1o is unclear.     

Nerve growth factor induces cell cycle arrest of astrocytes

Neurotrophins can influence multiple cellular functions depending on the cellular context and the specific receptors they interact with. These neurotrophic factors have been extensively studied for their ability to support neuronal survival via Trk receptors and to induce apoptosis via the p75(NTR). However, the p75(NTR) is also detected on cell populations that do not undergo apoptosis in response to neurotrophins. In particular, the authors have detected p75(NTR) expression on astrocytes during development and after seizure-induced injury. In this study, the authors investigated the role of Nerve growth factor (NGF) in regulating astrocyte proliferation and in influencing specific aspects of the cell cycle. The authors have demonstrated that NGF prevents the induction of cyclins and their association with specific cyclin-dependent kinases, and thereby prevents progression through the G1 phase of the cell cycle. Since the authors have previously shown that p75(NTR) but not TrkA, is expressed in astrocytes, these data suggest that activation of p75(NTR) promotes withdrawal of astrocytes from the cell cycle, which may have important consequences during development and after injury.     

WEBnm@ v2.0: Web server and services for comparing protein flexibility

Background

Normal mode analysis (NMA) using elastic network models is a reliable and cost-effective computational method to characterise protein flexibility and by extension, their dynamics. Further insight into the dynamics–function relationship can be gained by comparing protein motions between protein homologs and functional classifications. This can be achieved by comparing normal modes obtained from sets of evolutionary related proteins.

Results

We have developed an automated tool for comparative NMA of a set of pre-aligned protein structures. The user can submit a sequence alignment in the FASTA format and the corresponding coordinate files in the Protein Data Bank (PDB) format. The computed normalised squared atomic fluctuations and atomic deformation energies of the submitted structures can be easily compared on graphs provided by the web user interface. The web server provides pairwise comparison of the dynamics of all proteins included in the submitted set using two measures: the Root Mean Squared Inner Product and the Bhattacharyya Coefficient. The Comparative Analysis has been implemented on our web server for NMA, WEBnm@, which also provides recently upgraded functionality for NMA of single protein structures. This includes new visualisations of protein motion, visualisation of inter-residue correlations and the analysis of conformational change using the overlap analysis. In addition, programmatic access to WEBnm@ is now available through a SOAP-based web service. Webnm@ is available at http://apps.cbu.uib.no/webnma.

Conclusion

WEBnm@ v2.0 is an online tool offering unique capability for comparative NMA on multiple protein structures. Along with a convenient web interface, powerful computing resources, and several methods for mode analyses, WEBnm@ facilitates the assessment of protein flexibility within protein families and superfamilies. These analyses can give a good view of how the structures move and how the flexibility is conserved over the different structures.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0427-6) contains supplementary material, which is available to authorized users.