Antigen-specific suppression of inflammatory arthritis by dendritic cells

Purpose Antigen-specific suppression of a previously primed immune response is a major challenge for immunotherapy of autoimmune disease. We have shown that NF-κB inactivation in dendritic cells (modified DC) converts them into cells that tolerize rather than immunize to specific antigen [1]. Antigen-exposed modified DC prevent priming of immunity, and they suppress previously primed immune responses. Regulatory CD4+ T cells, which can transfer antigen-specific tolerance in an IL-10-dependent fashion, mediate the tolerance. We hypothesized that modified DC exposed to arthritogenic antigen would suppress clinical arthritis after disease onset. Methods Antigen-induced arthritis was induced in C57/Bl6 mice by priming to methylated bovine serum albumin (mBSA) antigen followed by challenge injection of mBSA to one knee. Knee swelling was apparent within 2 days, with peak clinical signs apparent at 5 days. Mice were treated with antigen-exposed modified DC between 2 and 6 days after mBSA challenge to the knee joint. Results Clinical arthritis was suppressed in each group receiving mBSA-exposed modified DC within 4 days compared with mice that received either no DC or keyhole limpet hemocyanin-exposed modified DC. Clinical improvement was associated with mBSA-specific tolerance in mice receiving mBSA-exposed modified DC. Tolerance induction was not impaired by concomitant administration of anti-tumor necrosis factor alpha monoclonal antibody. Subsequent rechallenge with intra-articular IL-1 induced flare of arthritis in all groups, which could be effectively suppressed by a second administration of mBSA-exposed modified DC. Conclusions The data indicate that modified DC induce antigen-specific immune suppression in this model of inflammatory arthritis, even after full clinical expression of the disease. These observations have important implications for antigen-specific therapy of autoimmunity.

Auditory lexical decisions in children with specific language impairment

This study examined spoken-word recognition in children with specific language impairment (SLI) and normally developing children matched separately for age and receptive language ability. Accuracy and reaction times on an auditory lexical decision task were compared. Children with SLI were less accurate than both control groups. Two subgroups of children with SLI, distinguished by performance accuracy only, were identified. One group performed within normal limits, while a second group was significantly less accurate. Children with SLI were not slower than the age-matched controls or language-matched controls. Further, the time taken to detect an auditory signal, make a decision, or initiate a verbal response did not account for the differences between the groups. The findings are interpreted as evidence for language-appropriate processing skills acting upon imprecise or underspecified stored representations.

Biological features of a puzzling symbiotic association between the hermit crab Dardanus insignis and the porcellanid crab Porcellana Sayana (Crustacea)

The symbiotic lifestyle is widespread among porcellanid crabs, which maintain ecological and co-evolutionary associations with annelid polychaetes, poriferans, cnidarians, echinoderms, gastropod. mollusks, and other crustaceans such as shrimps and hermit crabs, among others. We investigated the ecological association between the hermit crab Dardanus insignis and the porcellanid Porcellana sayana, in southeastern Brazil. Porcellanid crabs, hermit crabs, and available shells were collected monthly from July 2001 to June 2003, with a shrimp boat equipped with two double-rig trawl nets. The majority of P. sayana specimens were collected in shells occupied by D. insignis (96.6%); a few were found in empty shells (3.4%). The catch of both symbionts and hosts increased with increasing depth, with the highest occurrence at 35 m. The F. sayana crabs of various sizes could be found solitary or forming aggregations of up to 14 individuals per host, showing no sex or size segregation. In spite of the high diversity of shell species occupied by the hermit crabs and also available in the field, only a few of them were also utilized by P. sayana. The majority (93%) of shells utilized by P. sayana also hosted other symbiont species, constituting the basis of extensive symbiotic complexes. Thus, the ecological relationship between D. insignis and P. sayana may be classified as a non-obligate and non-specific symbiosis that may also involve other facultative organisms such as sea anemones. (C) 2008 Elsevier B.V. All rights reserved.

Expression analysis of putative vitellogenin and lipophorin receptors in honey bee (Apis mellifera L.) queens and workers

Two members of the low density lipoprotein receptor (LDLR) family were identified as putative orthologs for a vitellogenin receptor (Amvgr) and a lipophorin receptor (Amlpr) in the Apis mellifera genome. Both receptor sequences have the structural motifs characteristic of LDLR family members and show a high degree of similarity with sequences of other insects. RT-PCR analysis of Amvgr and Amlpr expression detected the presence of both transcripts in different tissues of adult female (ovary, fat body, midgut, head and specifically hypopharyngeal gland), as well as in embryos. In the head RNA samples we found two variant forms of AmLpR: a full length one and a shorter one lacking 29 amino acids in the O-linked sugar domain. In ovaries the expression levels of the two honey bee LDLR members showed opposing trends: whereas Amvgr expression was upregulated as the ovaries became activated, Amlpr transcript levels gradually declined. In situ hybridization analysis performed on ovaries detected Amvgr mRNA exclusively in germ line cells and corroborated the qPCR results showing an increase in Amvgr gene expression concomitant with follicle growth. (C) 2008 Elsevier Ltd. All rights reserved.

Folate-sensitive fragile site FRA10A is due to an expansion of a CGG repeat in a novel gene, FRA10AC1, encoding a nuclear protein

Fragile sites appear visually as nonstaining gaps on chromosomes that are inducible by specific cell culture conditions. Expansion of CGG/ CCG repeats has been shown to be the molecular basis of all five folate-sensitive fragile sites characterized molecularly so far, i.e., FRAXA, FRAXE, FRAXF, FRA11B, and FRA16A. In the present study we have refined the localization of the FRA10A folate-sensitive fragile site by fluorescence in situ hybridization. Sequence analysis of a BAC clone spanning FRA10A identified a single, imperfect, but polymorphic CGG repeat that is part of a CpG island in the 5'UTR of a novel gene named FRA10ACl. The number of CGG repeats varied in the population from 8 to 13. Expansions exceeding 200 repeat units were methylated in all FRA10A fragile site carriers tested. The FRA10ACl gene consists of 19 exons and is transcribed in the centromeric direction from the FRA10A repeat. The major transcript of similar to 1450 nt is ubiquitously expressed and codes for a highly conserved protein, FRA10ACl, of unknown function. Several splice variants leading to alternative 3' ends were identified (particularly in testis). These give rise to FRA10ACl proteins with altered COOH-termini. Immunofluorescence analysis of full-length, recombinant EGFP-tagged FRA10ACl protein showed that it was present exclusively in the nucleoplasm. We show that the expression of FRA10A, in parallel to the other cloned folate-sensitive fragile sites, is caused by an expansion and subsequent methylation of an unstable CGG trinucleotide repeat. Taking advantage of three cSNPs within the FRA10ACl gene we demonstrate that one allele of the gene is not transcribed in a FRA10A carrier. Our data also suggest that in the heterozygous state FRA10A is likely a benign folate-sensitive fragile site. (C) 2004 Elsevier Inc. All rights reserved.

Tissue-specific expression of head-to-tail cyclized miniproteins in Violaceae and structure determination of the root cyclotide Viola hederacea root cyclotide1

The plant cyclotides are a family of 28 to 37 amino acid miniproteins characterized by their head-to-tail cyclized peptide backbone and six absolutely conserved Cys residues arranged in a cystine knot motif: two disulfide bonds and the connecting backbone segments form a loop that is penetrated by the third disulfide bond. This knotted disulfide arrangement, together with the cyclic peptide backbone, renders the cyclotides extremely stable against enzymatic digest as well as thermal degradation, making them interesting targets for both pharmaceutical and agrochemical applications. We have examined the expression patterns of these fascinating peptides in various Viola species (Violaceae). All tissue types examined contained complex mixtures of cyclotides, with individual profiles differing significantly. We provide evidence for at least 57 novel cyclotides present in a single Viola species (Viola hederacea). Furthermore, we have isolated one cyclotide expressed only in underground parts of V, hederacea and characterized its primary and three-dimensional structure. We propose that cyclotides constitute a new family of plant defense peptides, which might constitute an even larger and, in their biological function, more diverse family than the well-known plant defensins.

Anticonvulsant profile of the alkaloids (+)-erythravine and (+)-11-alpha-hydroxy-erythravine isolated from the flowers of Erythrina mulungu Mart ex Benth (Leguminosae-Papilionaceae)

Neural mechanisms underlying the onset and maintenance of epileptic seizures involve alterations in inhibitory and/or excitatory neurotransmitter pathways. Thus, the prospecting of novel molecules from natural products that target both inhibition and excitation systems has deserved interest in the rational design of new anticonvulsants. We isolated the alkaloids (+)-erythravine and ( +)-11-alpha-hydroxyerythravine from the flowers of Erythrina mulungu and evaluated the action of these compounds against chemically induced seizures in rats. Our results showed that the administration of different doses of (+)-erythravine inhibited seizures evoked by bicuculline, pentylenetetrazole, and kainic acid at maximum of 80, 100, and 100%, respectively, whereas different doses of (+)-11-alpha-hydroxy-erythravine inhibited seizures at a maximum of 100% when induced by bicuculline, NMDA, and kainic acid, and, to a lesser extent, PTZ (60%). The analysis of mean latency to seizure onset of nonprotected animals, for specific doses of alkaloids, showed that (+)-erythravine increased latencies to seizures induced by bicuculline. Although (+)-erythravine exhibited very weak anticonvulsant action against seizures induced by NMDA, this alkaloid increased the latency in this assay. The increase in latency to onset of seizures promoted by (+)-11-alpha-hydroxy-erythravine reached a maximum of threefold in the bicuculline test. All animals were protected against death when treated with different doses of (+)-11-alpha-hydroxy-erythravine in the tests using the four chemical convulsants. Identical results were obtained when using (+)-erythravine in the tests of bicuculline, NMDA, and VIZ, and, to a lesser extent, kainic acid. Therefore, these data validate the anticonvulsant properties of the tested alkaloids, which is of relevance in consideration of the ethnopharmacological/biotechnological potential of E. mulungu. (C) 2010 Elsevier Inc. All rights reserved.

Nestmate recognition in the stingless bee Frieseomelitta varia (Hymenoptera, Apidae, Meliponini): sources of chemical signals

Social insects use cuticular lipids for nestmate recognition. These lipids are chiefly hydrocarbons that can be endogenously produced or acquired from the environment. Although these compounds are already described as coming from different sources for different groups of social insects, nothing is known about the source of cuticular hydrocarbons in stingless bees. We used behavioural recognition tests and cuticle chemical investigation to elucidate the role of endogenous and environmentally based cues for nestmate recognition in the stingless bee Frieseomelitta varia. We found that although newly emerged workers present specific cuticle patterns according to their nest origin, these compounds are not used for nestmate recognition, since newly emerged workers are broadly accepted in different colonies. The cerumen used in nest construction played an important role in recognition behaviour. Twenty minutes of contact with foreign cerumen was sufficient to increase the rejection rates of nestmates and separate the groups of workers according to their chemical profile. On the other hand, tests of feeding on a common diet showed no effect on chemical cuticle pattern or recognition behaviour. (C) 2010 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Non-ribosomal peptides produced by Brazilian cyanobacterial isolates with antimicrobial activity

Cyanobacterial strains isolated from terrestrial and freshwater habitats in Brazil were evaluated for their antimicrobial and siderophore activities. Metabolites of fifty isolates were extracted from the supernatant culture media and cells using ethyl acetate and methanol, respectively. The extracts of 24 isolates showed antimicrobial activity against several pathogenic bacteria and one yeast. These active extracts were characterized by Q-TOF/MS. The cyanobacterial strains Cylindrospermopsis raciborskii 339-T3, Synechococcus elongatus PCC7942, Microcystis aeruginosa NPCD-1, M. panniformis SCP702 and Fischerella sp. CENA19 provided the most active extracts. The 50 cyanobacterial strains were also screened for the presence of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes and microcystin production. Putative fragment genes coding for NRPS adenylation domains and PKS keto-synthase domains were successfully PCR amplified from 92% and 80% of cyanobacterial strains, respectively. The potential therapeutical compounds siderophores were detected in five cyanobacterial isolates. Microcystin production was detected by ELISA test in 26% of the isolates. Further a protease inhibitor substance was detected by LC-MS/MS in the M. aeruginosa NPLJ-4 extract and the presence of aeruginosin and cyanopeptolin was confirmed by PCR amplification using specific primers, and sequenced. This screening study showed that Brazilian cyanobacterial isolates are a rich source of natural products with potential for pharmacological and biotechnological applications. (C) 2010 Elsevier GmbH. All rights reserved.

Short- and long-term, salinity-induced modulation of V-ATPase activity in the posterior gills of the true freshwater crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)

To better understand the biochemical mechanisms underlying anisosmotic extracellular regulation in the freshwater Brachyura, we kinetically characterized the V-ATPase from the posterior gills of Dilocarcinus pagei, acclimated for 10 days to salinities up to 21%.. Specific activity was highest in fresh water (26.5 +/- 2.1 U mg(-1)), decreasing in 5 parts per thousand to 21 parts per thousand, attaining 3-fold less at 15 parts per thousand. Apparent affinities for ATP and Mg(2+) respectively increased 3.2- and 2-fold at 10 parts per thousand, suggesting expression of different isoenzymes. In a 240-h time-course study of exposure to 21%., maximum specific activity decreased 2.5- to 4-fold within 1 to 24 h while apparent affinities for ATP and Mg(2+) respectively increased by 12-fold within 24 h and 2.4-fold after 1 h, unchanged thereafter. K(I) for bafilomycin A(1) decreased 150-fold after 1 h, remaining constant up to 120 h. This is the first kinetic analysis of V-ATPase specific activity in crustacean gills during salinity acclimation. Our findings indicate active gill Cl(-) uptake by D. pagei in fresh water, and short- and long-term down-regulation of V-ATPase-driven ion uptake processes during salinity exposure, aiding in comprehension of the biochemical adaptations underpinning the establishment of the Brachyura in fresh water. (C) 2011 Elsevier Inc. All rights reserved.

Modelling the Distribution of Ischaemic Stroke-Specific Survival Time Using an EM-based Mixture Approach with Random Effects Adjustment

A two-component survival mixture model is proposed to analyse a set of ischaemic stroke-specific mortality data. The survival experience of stroke patients after index stroke may be described by a subpopulation of patients in the acute condition and another subpopulation of patients in the chronic phase. To adjust for the inherent correlation of observations due to random hospital effects, a mixture model of two survival functions with random effects is formulated. Assuming a Weibull hazard in both components, an EM algorithm is developed for the estimation of fixed effect parameters and variance components. A simulation study is conducted to assess the performance of the two-component survival mixture model estimators. Simulation results confirm the applicability of the proposed model in a small sample setting. Copyright (C) 2004 John Wiley Sons, Ltd.

The juvenile hormone (JH) epoxide hydrolase gene in the honey bee (Apis mellifera) genome encodes a protein which has negligible participation in JH degradation

Epoxide hydrolases are multifunctional enzymes that are best known in insects for their role in juvenile hormone (JH) degradation. Enzymes involved in JH catabolism can play major roles during metamorphosis and reproduction, such as the JH epoxide hydrolase (JHEH), which degrades JH through hydration of the epoxide moiety to form JH diol, and JH esterase (JHE), which hydrolyzes the methyl ester to produce JH acid. In the honey bee, JH has been co-opted for additional functions, mainly in caste differentiation and in age-related behavioral development of workers, where the activity of both enzymes could be important for JH titer regulation. Similarity searches for jheh candidate genes in the honey bee genome revealed a single Amjheh gene. Sequence analysis, quantification of Amjheh transcript levels and Western blot assays using an AmJHEH-specific antibody generated during this study revealed that the AmJHEH found in the fat body shares features with the microsomal JHEHs from several insect species. Using a partition assay we demonstrated that AmJHEH has a negligible role in JH degradation, which, in the honey bee, is thus performed primarily by JHE. High AmJHEH levels in larvae and adults were related to the ingestion of high loads of lipids, suggesting that AmJHEH has a role in dietary lipid catabolism. (C) 2010 Elsevier Ltd. All rights reserved.

Maternal investment in egg production: environmental and population-specific effects on offspring performance in the snapping shrimp Alpheus nuttingi (Schmitt, 1924) (Decapoda, Alpheidae)

Among marine invertebrates, the overall biomass invested in egg production varies widely within populations, which can result from the interaction of endogenous and exogenous factors. Species that have constant reproduction throughout the year can be good models to study the influence of environmental factors on reproductive processes. We conducted a seasonal comparison of egg production in the intertidal snapping shrimp Alpheus nuttingi, which shows a continuous reproductive pattern, to examine the hypothesis that differences in egg production are driven by environmental conditions and population features. This population showed an uncommon strategy, characterized by females that produce eggs of varying sizes within their clutches, with reduced egg volume when the number of eggs is higher (Spring-Summer). In these seasons, higher temperatures and greater food availability may allow the production of more eggs compared to the Autumn-Winter seasons. Compared to other alpheid shrimps, this population produces small eggs, but in larger numbers. Despite the higher fecundity, the reproductive output is relatively low, this production being supported by the large size of females from the southern Atlantic region. Our findings showed that the egg production of A. nuttingi was greatly influenced by environmental factors. Therefore, this shrimp, and probably other decapods that possess continuous reproduction, adopt different reproductive strategies during the year. (C) Koninklijke Brill NV, Leiden, 2010.

Morphological and morphometric appraisal of the spermatophore of the southern hermit crab Isocheles sawayai Forest and Saint Laurent, 1968 (Anomura: Diogenidae), with comments on gonopores in both sexes

The spermatophore morphology of the hermit crab Isocheles sawayai from Southwestern Atlantic (Brazil) is described. The spermatophores show similarities with those described for other members of the family Diogenidae, especially with the recently described Loxopagurus loxochelis. The spermatophore is composed of three major regions: a sperm filled head or ampulla, a columnar stalk and a foot or pedestal. The spermatophores show specific morphology in having a circular ampulla, and a constriction or neck between the ampulla (100 mu m) and the thin (27 mu m), long stalk (500 mu m). The stalk penetrates less than half way into the spermatophore head. Most spermatophores show one or the small posterior projections on the underside of the ampulla as being bigger than the other, making it asymmetrical. The size of the spermatophore is related to hermit crab size with direct relationships found between spermatophore ampulla width, total length, and peduncle length with shield length of the hermit crab. The morphological characteristics of the spermatophore of L sawayai. are species-specific distinguishing it from other members of the family, and are useful to infer further phylogenetic relationships. (C) 2008 Elsevier GmbH. All rights reserved.

Mitochondria, endoplasmic reticulum and actin filament behavior after PDT with chloroaluminum phthalocyanine liposomal in HeLa cells

Photodynamic therapy (PDT) for cancer is a therapeutic modality in the treatment of tumors in which visible light is used to activate a photosensitizer. Cell membranes have been identified as an important intracellular target for singlet oxygen produced during the photochemical pathway. This study analyzed the cytotoxicity in specific cellular targets of a photosensitizer used in PDT in vitro. The photosensitizing effects of chloroaluminum phthalocyanine liposomal were studied on the mitochondria, cytoskeleton and endoplasmic reticulum of HeLa cells. Cells were irradiated with a diode laser working at 670 nm, energy density of 4.5 J/cm(2) and power density of 45 mW/cm(2). Fluorescence microscopic analysis of the mitochondria showed changes in membrane potential. After PDT treatment, the cytoskeleton and endoplasmic reticulum presented basic alterations in distribution. The combined effect of AlPHCl liposomal and red light in the HeLa cell line induced photodamage to the mitochondria, endoplasmic reticulum and actin filaments in the cytoskeleton. (c) 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.