Protonectin (1-6): A novel chemotactic peptide from the venom of the social wasp Agelaia pallipes pallipes

Peptides constitute the largest group of Hymenoptera venom toxins; some of them interact with GPCR, being involved with the activation of different types of leukocytes, smooth muscle contraction and neurotoxicity. Most of these toxins vary from dodecapeptides to tetradecapeptides, amidated at their C-teminal amino acid residue. The venoms of social wasps can also contains some tetra-, penta-, hexa- and hepta-peptides, but just a few of them have been structurally and functionally characterized up to now. Protonectin (ILG-TILGLLKGL-NH(2)) is a polyfunctional peptide, presenting mast cell degranulation, release of lactate dehydrogenase (LDH) from mast cells, antibiosis against Gram-positive and Gram-negative bacteria and chemotaxis for polymorphonucleated leukocytes (PMNL), while Protonectin (1-6) (ILGTIL-NH(2)) only presents chemotaxis for PMNL However, the mixture of Protonectin (1-6) with Protonectin in the molar ratio of 1:1 seems to potentiate the biological activities dependent of the membrane perturbation caused by Protonectin, as observed in the increasing of the activities of mast cell degranulation, LDH releasing from mast cells, and antibiosis. Despite both peptides are able to induce PMNL chemotaxis, the mixture of them presents a reduced activity in comparison to the individual peptides. Apparently, when mixed both peptides seems to form a supra-molecular structure, which interact with the receptors responsible for PMNL chemotaxis, disturbing their individual docking with these receptors. In addition to this, a comparison of the sequences of both peptides suggests that the sequence ILGTIL is conserved, suggesting that it must constitute a linear motif for the structural recognition by the specific receptor which induces leukocytes migration. (C) 2010 Elsevier Ltd. All rights reserved.

Trans-resveratrol inhibits early blood vessel formation (Vasculogenesis) without impairment of embryonic growth

Resveratrol is a stilbene compound found in grapes and other sources. In this study we examined the effects of trans-resveratrol (4.38-438 mu M/implant) in the vasculogenesis of yolk-sac membranes and its capacity to improve chick embryo growth. High concentrations of the stilbene (43.8-438 mu M) significantly inhibited early vessel formation, decreasing the percentage vitelline vessels of 3.5-day embryos by 50% compared to the control. In addition, basic fibroblast growth factor-stimulated vasculogenesis (140% of vessels as compared to control) was partially reversed by t-resveratrol (35% of inhibition) and treatments with cyclooxygenase inhibitors (acetylsalicylic acid and indomethacin) as well a protein-kinase C (PKC) activator (phorbol-12,13-dibutyrate) decreased the vessel number to 60%, 50%, and 44%, respectively. Treatments with t-resveratrol (4.38-43.8 mu M/implant) significantly increased the body length of embryos incubated in vitro uncoupled from any impairment in the body shape or detectable embryotoxic effect. We suggest that the antivasculogenic activity and the enhancement in embryonic growth promoted by non acute treatments with t-resveratrol were, at least in part, due to PKC inhibition. We suggest that t-resveratrol can be usable not only as a reliable functional nutriment, but also is useful for the development of prophylactic and/or therapeutic agents for treatment of angiogenic-degenerative diseases.

Neurophysiological findings of the late-onset, dominant, proximal spinal muscular atrophies with dysautonomia because of the VAPB PR056SER mutation

The vesicle-associated membrane protein/synaptobrevin-associated membrane protein B (VAPB) Pro56Ser Mutation has been identified in Brazilian families showing various motor neuron syndromes. However, the neurophysiological characteristics of these patients have not been detailed, and some questions Still need to be solved, such as the possible presence of myotonia and the origin of the abdominal protrusion seen in most patients. The eventual finding of suggestive electrophysiological characteristics would be helpful not only for clinical diagnosis but also to selection of the appropriate DNA test. To clarify these questions we carried out sensory and motor conduction Studies, including symphatetic skin response, and needle examination in six genetically proven affected members. The electromyographic findings were those of a slowly progressive motor neuron disorder. Topographically, the abdominal muscles were severely affected, but the facial and laryngeal muscles were preserved or very mildly involved. Sensory conduction studies and sympathetic Skin responses were normal. No myotonic discharge was recorded. These findings are indistinguishable from those of other motor neuron disorders, although the predominant involvement of the proximal limbs and of the abdominal muscles may be of some help in the appropriate clinical setting.

Shape alterations in the striatum in chorea-acanthocytosis

Chorea-acanthocytosis (ChAc) is an uncommon autosomal recessive disorder due to mutations of the VPS13A gene, which encodes for the membrane protein chorein. ChAc presents with progressive limb and orobuccal chorea, but there is often a marked dysexecutive syndrome. ChAc may first present with neuropsychiatric disturbance such as obsessive-compulsive disorder (OCD), suggesting a particular role for disruption to striatal structures involved in non-motor frontostriatal loops, such as the head of the caudate nucleus. Two previous studies have suggested a marked reduction in volume in the caudate nucleus and putamen, but did not examine morphometric change. We investigated morphometric change in 13 patients with genetically or biochemically confirmed ChAc and 26 age- and gender-matched controls. Subjects underwent magnetic resonance imaging and manual segmentation of the caudate nucleus and putamen, and shape analysis using a non-parametric spherical harmonic technique. Both structures showed significant and marked reductions in volume compared with controls, with reduction greatest in the caudate nucleus. Both structures showed significant shape differences, particularly in the head of the caudate nucleus. No significant correlation was shown between duration of illness and striatal volume or shape, suggesting that much structural change may have already taken place at the time of symptom onset. Our results suggest that striatal neuron loss may occur early in the disease process, and follows a dorsal-ventral gradient that may correlate with early neuropsychiatric and cognitive presentations of the disease. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

In LipL32, the major leptospiral lipoprotein, the C terminus is the primary immunogenic domain and mediates interaction with collagen wand plasma fibronectin

LipL32 is the major leptospiral outer membrane lipoprotein expressed during infection and is the immunodominant antigen recognized during the humoral immune response to leptospirosis in humans. In this study, we investigated novel aspects of LipL32. In order to define the immunodominant domains(s) of the molecule, subfragments corresponding to the N-terminal, intermediate, and C-terminal portions of the UpL32 gene were cloned and the proteins were expressed and purified by metal affinity chromatography. Our immunoblot results indicate that the C-terminal and intermediate domains of LipL32 are recognized by sera of patients with laboratory-confirmed leptospirosis. An immunoglobulin M response was detected exclusively against the LipL32 C-terminal fragment in both the acute and convalescent phases of illness. We also evaluated the capacity of LipL32 to interact with extracellular matrix (ECM) components. Dose-dependent, specific binding of LipL32 to collagen type IV and plasma fibronectin was observed, and the binding capacity could be attributed to the C-terminal portion of this molecule. Both heparin and gelatin could inhibit LipL32 binding to fibronectin in a concentration-dependent manner, indicating that the 30-kDa heparin-binding and 45-kDa gelatin-binding domains of fibronectin are involved in this interaction. Taken together, our results provide evidence that the LipL32 C terminus is recognized early in the course of infection and is the domain responsible for mediating interaction with ECM proteins.

Bril: A novel bone-specific modulator of mineralization

In the course of attempting to define the bone ""secretome"" using a signal-trap screening approach, we identified a gene encoding a small membrane protein novel to osteoblasts. Although previously identified in silico as ifitm5, no localization or functional studies had been undertaken on this gene. We characterized the expression patterns and localization of this gene in vitro and in vivo and assessed its role in matrix mineralization in vitro. The bone specificity and shown role in mineralization led us to rename the gene bone restricted ifitm-like protein (Bril). Bril encodes a 14.8-kDa 1.34 arnino acid protein with two transmembrane domains. Northern blot analysis showed bone-specific expression with no expression in other embryonic or adult tissues. In situ hybridization and immunohistochemistry in mouse embryos showed expression localized on the developing bone. Screening of cell lines showed Bril expression to be highest in osteoblasts, associated with the onset of matrix maturation/mineralization, suggesting a role in bone formation. Functional evidence of a role in mineralization was shown by adenovirus-mediated Brit overexpression and lentivirus-mediated Bril shRNA knockdown in vitro. Elevated Bril resulted in dose-dependent increases in mineralization in UMR106 and rat primary osteoblasts. Conversely, knockdown of Bril in MC3T3 osteoblasts resulted in reduced mineralization. Thus, we identified Bril as a novel osteoblast protein and showed a role in mineralization, possibly identifying a new regulatory pathway in bone formation.

Role of LMP1 in immune control of EBV infection

The Epstein-Barr virus (EBV) encoded latent membrane protein (LMP1) plays a crucial role in the long-term persistence of this virus within the cells of the immune system. Not only is this protein critical for the transformation of resting B cells by EBV, it also displays pleiotropic effects on various cellular proteins expressed in the host cell. These include up-regulation of expression of B cell activation antigens, adhesion molecules and various components of the antigen processing pathway. Here we discuss how LMP1 acts like an expression 'switch' which, depending on the stage of EBV infection, manoeuvres various pathways that either modulate the immune system towards or against its survival.

Flotillin-1-enriched lipid raft domains accumulate on maturing phagosomes

Flotillin-1 was recently shown to be enriched on detergent-resistant domains of the plasma membrane called lipid rafts. These rafts, enriched in sphingolipids and cholesterol, sequester certain proteins while excluding others. Lipid rafts have been implicated in numerous cellular processes including signal transduction, membrane trafficking and molecular sorting. In this study, we demonstrate both morphologically and biochemically that lipid rafts are present on phagosomes, These structures are enriched in flotillin-1 and devoid of the main phagosomes membrane protein lysosomal-associated membrane protein (LAMP1), The flotillin-1 present on phagosomes does not originate from the plasma membrane during phagocytosis but accumulates gradually on maturing phagosomes, Treatment with bafilomycin A1, a compound that inhibits the proton pump ATPase and prevents the fusion of phagosomes with late endocytic organelles, prevents the acquisition of flotillin-1 by phagosomes, indicating that this protein might be recruited on phagosomes from endosomal organelles. A proteomic characterization of the lipid rafts of phagosomes indicates that actin, the alpha- and beta -subunits of heterotrimeric G proteins, as well as subunits of the proton pump V-ATPase are among the constituents of these domains. Remarkably, the intracellular parasite Leishmania donovani can actively inhibit the acquisition of flotillin-1-enriched lipid rafts by phagosomes and the maturation of these organelles. These results indicate that specialized functions required for phagolysosome biogenesis may occur at focal points on the phagosome membrane, and therefore represent a potential target of intracellular pathogens.

Identification of vaccine candidate antigens from a genomic analysis of Porphyomonas gingivalis

Porphyromonas gingivalis is a key periodontal pathogen which has been implicated in the etiology of chronic adult periodontitis. Our aim was to develop a protein based vaccine for the prevention and or treatment of this disease. We used a whole genome sequencing approach to identify potential vaccine candidates. From a genomic sequence, we selected 120 genes using a series of bioinformatics methods. The selected genes were cloned for expression in Escherichia coli and screened with P. gingivalis antisera before purification and testing in an animal model. Two of these recombinant proteins (PG32 and PG33) demonstrated significant protection in the animal model, while a number were reactive with various antisera. This process allows the rapid identification of vaccine candidates from genomic data. (C) 2001 Elsevier Science Ltd. All rights reserved.

Expression of the hepatitis C virus structural proteins in mammalian cells induces morphology similar to that in natural infection

Like many positive-strand RNA viruses, replication of the hepatitis C virus (HCV) is associated with cytoplasmic membrane rearrangements. However, it is unclear which HCV Proteins induce these ultrastructural features. This work examined the morphological changes induced by expression of the HCV structural proteins, core, E1 and E2, expressed from a Semliki Forest Virus (SFV) recombinant RNA replicon. Electron microscopy of cells expressing these proteins showed cytoplasmic vacuoles containing membranous and electron-dense material that were distinct from the type I cytoplasmic vacuoles induced during SFV replicon replication. Immunogold labelling showed that the core and E2 proteins localized to the external and internal membranes of these vacuoles. At times were also associated with some of the internal amorphous material. Dual immunogold labelling with antibodies raised against the core protein and against an endoplasmic reticulum (ER)-resident protein (protein disulphide isomerase) showed that the HCV-induced vacuoles were associated with ER-labelled membranes. This report has identified an association between the HCV core and E2 proteins with induced cytoplasmic vacuoles which are morphologically similar to those observed in HCV-infected liver tissue, suggesting that the HCV structural proteins may be responsible for the induction of these vacuoles during HCV replication in vivo.

Differential requirements for COPI coats in formation of replication complexes among three genera of Picornaviridae

Picornavirus RNA replication requires the formation of replication complexes (RCs). consisting of virus-induced vesicles associated with viral nonstructural proteins and RNA. Brefeldin A (BFA) has been shown to strongly inhibit RNA replication of poliovirus but not of encephalomyocarditis virus (EMCV). Here, we demonstrate that the replication of parechovirus 1 (ParV1) is partly resistant to BFA, whereas echovirus 11 (EV11) replication is strongly inhibited. Since BFA inhibits COPI-dependent steps in endoplasmic reticulum (ER)-Golgi transport, we tested a hypothesis that different picornaviruses may have differential requirements for COPI in the formation of their RCs. Using immunofluorescence and cryo-immunoelectron microscopy we examined the association of a COPI component, beta-COP, with the RCs of EMCV, ParV1, and EV11 EMCV RCs did not contain beta-COP. In contrast, beta-COP appeared to be specifically distributed to the RCs of EV11 In ParV1-infected cells beta-COP was largely dispersed throughout the cytoplasm, with some being present in the RCs. These results suggest that there are differences in the involvement of COPI in the formation of the RCs of various picornaviruses, corresponding to their differential sensitivity to BFA. EMCV RCs are likely to be formed immediately after vesicle budding from the ER, prior to COPI association with membranes. ParV1 RCs are formed from COPI-containing membranes but COPI is unlikely to be directly involved in their formation, whereas formation of EV11 RCs appears to be dependent on COPI association with membranes.

Phase variation in meningococcal lipooligosaccharide biosynthesis genes

Neisseria meningitidis expresses a range of lipooligosaccharide (LOS) structures, comprising of at least 13 immunotypes (ITs). Meningococcal LOS is subject to phase variation of its terminal structures allowing switching between ITs, which is proposed to have functional significance in disease. The objectives of this study were to investigate the repertoire of structures that can be expressed in clinical isolates, and to examine the role of phase-variable expression of LOS genes during invasive disease. Southern blotting was used to detect the presence of LOS biosynthetic genes in two collections of meningococci, a global set of strains previously assigned to lineages of greater or lesser virulence, and a collection of local clinical isolates which included paired throat and blood isolates from individual patients. Where the phase-variable genes lgtA, lgtC or IgtG were identified, they were amplified by PCR and the homopolymeric tracts, responsible for their phase-variable expression, were sequenced. The results revealed great potential for variation between alternate LOS structures in the isolates studied, with most strains capable of expressing several alternative terminal structures. The structures predicted to be currently expressed by the genotype of the strains agreed well with conventional immunotyping. No correlation was observed between the structural repertoire and virulence of the isolate. Based on the potential for LOS phase variation in the clinical collection and observations with the paired patient isolates, our data suggest that phase variation of LOS structures is not required for translocation between distinct compartments in the host. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

Light and electron microscopic analysis of aquaporin 1-like-immunoreactive amacrine cells in the rat retina

Aquaporin 1 (AQP1; also known as CHIP, a channel-forming integral membrane protein of 28 kDa) is the first protein to be shown to function as a water channel and has been recently shown to be present in the rat retina. We previously showed (Kim et al. [1998] Neurosci Lett 244:52-54) that AQP1-like immunoreactivity is present in a certain population of amacrine cells in the rat retina. This study was conducted to characterize these cells in more detail, With immunocytochemistry using specific antisera against AQP1, whole-mount preparations and 50-mum-thick vibratome sections were examined by light and electron microscopy. These cells were a class of amacrine cells, which had symmetric bistratified dendritic trees ramified in stratum 2 and in the border of strata 3 and 4 of the inner plexiform layer (IPL). Their dendritic field diameters ranged from 90 to 230 mum. Double labeling with antisera against AQP1 and gamma-aminobutyric acid or glycine demonstrated that these AQP1-like-immunoreactive amacrine cells were immunoreactive for glycine. Their most frequent synaptic input was from other amacrine cell processes in both sublaminae a and b of the IPL, followed by a few cone bipolar cells. Their primary targets were other amacrine cells and ganglion cells in both sublaminae a and b of the IPL. In addition, synaptic output Onto bipolar cells was rarely observed in sublamina b of the IPL. Thus, the AQP1 antibody labels a class of glycinergic amacrine cells with small to medium-sized dendritic fields in the rat retina. (C) 2002 Wiley-Liss, Inc.

Proteasomal targeting of a viral oncogene abrogates oncogenic phenotype and enhances immunogenicity

The ability of viral or mutated cellular oncogenes to initiate neoplastic events and their poor immunogenicity have considerably undermined their potential use as immunotherapeutic tools for the treatment of human cancers. Using an EpsteinBarr virus-encoded oncogene, latent membrane protein 1 (LMP1), as a model, we report a novel strategy that both deactivates cellular signaling pathways associated with the oncogenic phenotype and reverses poor immunogenicity. We show that cotranslational ubiquitination combined with Wend rule targeting of LMP1 enhanced the intracellular degradation of LMP1 and total blockade of LMP1-mediated nuclear factor-kappaB (NF-kappaB) and signal transducer and activator of transcription (STAT) activation in human cells. In addition, although murine cells expressing LMP1 were uniformly tumorigenic, this oncogenicity was completely abrogated by covalent linkage of LMP1 with ubiquitin, while an enhanced CD8(+) T cell response to a model epitope fused to the C-terminus of LMP1 was observed following immunization with ubiquitinated LMP1. These observations suggest that proteasomal targeting of tumor-associated oncogenes could be exploited therapeutically by either gene therapy or vaccination.