Bee bread increases honeybee haemolymph protein and promote better survival despite of causing higher Nosema ceranae abundance in honeybees.

Adequate protein nutrition supports healthy honeybees and reduces the susceptibility to disease. However little is known concerning the effect of the diet on Nosema ceranae development, an obligate intracellular parasite that disturbs the protein metabolism of honeybees (Apis mellifera). Here we tested the effect of natural (bee bread) and non-natural protein diets (substitute) on haemolymph proteins titers of honeybee and N. ceranae spore production. The natural diet induced higher levels of protein and parasite development, but the survival of bees was also higher than with non-natural diets. The data showed that the administration of an artificially high nutritious diet in terms of crude protein content is not sufficient to promote healthy bees; rather the protein ingested should be efficiently assimilated. The overall results support the idea that the physiological condition of the bees is linked to protein levels in the haemolymph, which affects the tolerance to parasite; consequently the negative impact of the parasite on host fitness is not associated only with the level of infection.

Conservation and divergence in the frog immunome: pyrosequencing and de novo assembly of immune tissue transcriptomes.

BACKGROUND: Frogs are a diverse group of vertebrates for which limited genomic resources are available. Natural frog populations face a multitude of threats, including habitat degradation, infectious disease, and environmental change. Characterizing the functional genomics of anuran tissues in general - and the immune system in particular - will enhance our knowledge of genetic and epigenetic responses to environmental threats and inform conservation and recovery efforts. RESULTS: To increase the number of species with genomic datasets and characterize gene expression in immune-related tissues, we sequenced the transcriptomes of three tissues from two frogs (Espadarana prosoblepon and Lithobates yavapaiensis) on the Roche 454 GS FLX platform. Our sequencing produced 8881 E. prosoblepon and 5428 L. yavapaiensis annotated gene products after de novo assembly and Gene Ontology classification. Transcripts of the innate and acquired immune system were expressed in all three tissues. Inflammatory response and acquired immunity transcripts were significantly more diverged between E. prosoblepon and L. yavapaiensis compared to innate immunity and immune system development transcripts. Immune-related transcripts did not show an overall elevated rate of functional evolution, with the exception of glycosyl proteases, which include lysozymes, central bacterial and fungal-killing enzymes of the innate immune system. CONCLUSIONS: The three frog transcriptomes provide more than 600 Mbp of new genomic data, and will serve as a valuable framework for future comparative studies of non-model anurans. Additionally, we show that immune gene divergence varies by functional group and that transcriptome studies can be useful in comparing rates of evolutionary change across gene families.

Structural differences between human proteins and aero- and microbial allergens define allergenicity.

The current paradigm suggests that structural homology of allergenic proteins to microbial (particularly helminths) or human proteins underlie their allergenic nature. To examine systematically the structural relationships among allergens and proteins of pathogens (helminths, protozoans, fungi and bacteria) as they relate to allergenicity, we compared the amino acid sequence of 499 molecularly-defined allergens with the predicted proteomes of fifteen known pathogens, including Th2 inducing helminths and Th1-inducing protozoans, and humans using a variety of bioinformatic tools. Allergenicity was assessed based on IgE prevalences using publicly accessible databases and the literature. We found multiple homologues of common allergens among proteins of helminths, protozoans, fungi and humans, but not of bacteria. In contrast, 187 allergens showed no homology with any of the microbial genera studied. Interestingly, allergens without homologues or those with limited levels of sequence conservation were the most allergenic displaying high IgE prevalences in the allergic population. There was an inverse relationship between allergenicity and amino acid conservation levels with either parasite, including helminth, or human proteins. Our results suggest that allergenicity may be associated with the relative "uniqueness" of an antigen, i.e. immunogenicity, while similarity would lead to immunological tolerance.

Physiological and proteomic approaches to evaluate the role of sterol binding in elicitin-induced resistance.

Cryptogein is a proteinaceous elicitor secreted by Phytophthora cryptogea that can induce resistance to P. parasitica in tobacco plants. On the basis of previous computer modelling experiments, by site-directed mutagenesis a series of cryptogein variants was prepared with altered abilities to bind sterols, phospholipids or both. The sterol binding and phospholipid transfer activities corresponded well with the previously reported structural data. Induction of the synthesis of reactive oxygen species (ROS) in tobacco cells in suspension and proteomic analysis of intercellular fluid changes in tobacco leaves triggered by these mutant proteins were not proportional to their ability to bind or transfer sterols and phospholipids. However, changes in the intercellular proteome corresponded to transcription levels of defence genes and resistance to P. parasitica and structure-prediction of mutants did not reveal any significant changes in protein structure. These results suggest, contrary to previous proposals, that the sterol-binding ability of cryptogein and its mutants, and the associated conformational change in the ω-loop, might not be principal factors in either ROS production or resistance induction. Nevertheless, the results support the importance of the ω-loop for the interaction of the protein with the high affinity binding site on the plasma membrane.

Analysis of protein composition of red wine in comparison with rosé and white wines by electrophoresis and high-pressure liquid chromatography-mass spectrometry (HPLC-MS).

Wine proteins not only influence wine stability but are also being discussed as potential allergens. Proteins from red, rosé, and white wines were enriched by dialysis and lyophilization followed by separation by SDS-PAGE. Significant differences were detected in the protein compositions of the analyzed wine varieties, and the major protein bands were identified by mass spectrometry after in-gel digestion with trypsin. In German Portugieser red wine, a total of 121 tryptic peptides were identified, which were attributed to 12 grape proteins and 6 proteins derived from yeast. Among the identified constituents are several proteins considered to influence wine stability and previously described potential grape allergens. The pathogenesis-related proteins represent the main proteins in all of the wines, but only some red wines show a band with a molecular mass of 12 kDa, identified as a lipid transfer protein (LTP). The occurrence and distribution of LTP depend on the wine variety.

[Functional role of proteins containing ankyrin repeats].

This review describes and discusses new data about the structure and function of proteins which contain ankyrin-like repeats in their structure. These proteins have been found in cells of different organisms but they are not belonging to the cytoskeletal proteins. Many important functions of such proteins are provided by ankyrin repeats which maintain protein-protein interactions involved in the formation of transcription complexes, initiation of immuno-responses, biogenesis and assembly of cation channels in the membranes, regulation of some cell cycle stages, symbiotic interactions and many other processes. Mutations in genes encoding ankyrin-like proteins can cause defects in gene expression leading to diseases onset and progression in animals and humans. Therefore, the structure, dynamics and function of these proteins is an area of extensive research in modern biology.

Peroxynitrite generation and tyrosine nitration in defense responses in tobacco BY-2 cells.

Peroxynitrite (ONOO(-)) is a compound formed by reaction of superoxide (O(2) (-)) with nitric oxide (NO) and is expected to possess characteristics of both O(2) (-) reactivity and NO mobility in order to function as a signal molecule. Although there are several reports that describe the role of ONOO(-) in defense responses in plants, it has been very difficult to detect ONOO(-) in bioimaging due to its short half-life or paucity of methods for ONOO(-)-specific detection among reactive oxygen species or free radicals. Aminophenyl fluorescein (APF), a recently developed novel fluorophore for direct detection of ONOO(-) in bioimaging, was used for intracellular ONOO(-) detection. ONOO(-) generation in tobacco BY-2 cells treated with INF1, the major elicitin secreted by the late blight pathogen Phytophthora infestans, occurred within 1 h and reached a maximum level at 6-12 h after INF1 treatment. Urate, a ONOO(-) scavenger, abolished INF1-induced ONOO(-) generation. It is well known that ONOO(-) reacts with tyrosine residues in proteins to form nitrotyrosine in a nitration reaction as an ONOO(-)-specific reaction. Western blot analysis using anti-nitrotyrosine antibodies recognized nitrotyrosine-containing proteins in 20 and 50 kDa bands in BY-2 protein extract containing SIN-1 [3-(4-morpholinyl) sydnonimine hydrochloride; an ONOO(-) donor]. These bands were also recognized in INF1-treated BY-2 cells and were found to be slightly suppressed by urate. Our study is the first to report ONOO(-) detection and tyrosine nitration in defense responses in plants.

Inhibitor kappaB and nuclear factor kappaB in granulocyte-macrophage colony-stimulating factor antagonism of dexamethasone suppression of the macrophage response to Aspergillus fumigatus conidia.

BACKGROUND: The dexamethasone (DEX) immunosuppressive effect on macrophage killing activity and cytokine production in response to Aspergillus fumigatus conidia is antagonized by granulocyte-macrophage colony-stimulating factor (GM-CSF). The molecular mechanism is unknown. We postulated that this antagonism is mediated by inhibitor kappaB (I kappaB) induction by DEX and is opposed by acceleration of I kappaB degradation by GM-CSF with or without conidia stimulation, with corresponding effects on translocation and activation of nuclear factor kappa B (NF-kappaB). METHODS: We studied 2 types of cells, resident peritoneal macrophages from CD-1 mice and the murine macrophage RAW264.7 cell line. Cells were unstimulated or stimulated with conidia and simultaneously treated with DEX, GM-CSF, or DEX plus GM-CSF, for 2-4 hours. I kappaB degradation and NF-kappaB activation were assessed by Western blot. RESULTS: Macrophages stimulated with conidia alone increased NF-kappaB translocation. DEX increased I kappaB levels in cytoplasm and blocked translocation of NF-kappaB to the nucleus in unstimulated and conidia-stimulated macrophages. Conversely, GM-CSF decreased I kappaB levels. GM-CSF reversed the effect of DEX on I kappaB levels. NF-kappaB levels were minimal in DEX-treated macrophage nuclear extracts, compared with those from GM-CSF-treated and GM-CSF plus DEX-treated macrophages. CONCLUSION: GM-CSF can reverse the DEX immunosuppressive effect by enhancing I kappaB degradation and promoting NF-kappaB translocation. This would allow macrophage production of proinflammatory cytokines, facilitating resistance to aspergillosis.

Antimicrobial peptide defenses of the mountain yellow-legged frog (Rana muscosa).

The mountain yellow-legged frog (Rana muscosa) inhabits high elevation lakes in California that are largely undisturbed by human activities. In spite of this habitation in remote sites, populations continue to decline. Although predation by non-native fish is one cause for declines, some isolated populations in fishless lakes are suffering new declines. One possible cause of the current wave of declines is the introduction of the pathogenic chytrid fungus (Batrachochytrium dendrobatidis) which invades the adult skin to cause chytridiomycosis. In many amphibian species, the skin is protected by antimicrobial peptides secreted into the mucous. Here we show that R. muscosa produces three previously unknown antimicrobial peptides belonging to the ranatuerin-2 and temporin-1 families of antimicrobial peptides. These three peptides, along with bradykinin, are the most abundant peptides in the skin secretions detected by mass spectrometry. Natural mixtures of peptides and individual purified peptides strongly inhibit chytrid growth. The concentration of total peptides recovered from the skin of frogs following a mild norepinephrine induction is sufficient to inhibit chytrid growth in vitro. A comparison of the species susceptibility to chytridiomycosis and the antichytrid activity of peptides between R. muscosa and R. pipiens suggest that although R. muscosa produces more total skin peptides, it appears to be more vulnerable to B. dendrobatidis in nature. Possible differences in the antimicrobial peptide repertoires and life history traits of the two species that may account for differences in susceptibility are discussed.

Amyloids--a functional coat for microorganisms.

Amyloids are filamentous protein structures approximately 10 nm wide and 0.1-10 mum long that share a structural motif, the cross-beta structure. These fibrils are usually associated with degenerative diseases in mammals. However, recent research has shown that these proteins are also expressed on bacterial and fungal cell surfaces. Microbial amyloids are important in mediating mechanical invasion of abiotic and biotic substrates. In animal hosts, evidence indicates that these protein structures also contribute to colonization by activating host proteases that are involved in haemostasis, inflammation and remodelling of the extracellular matrix. Activation of proteases by amyloids is also implicated in modulating blood coagulation, resulting in potentially life-threatening complications.

Conversion of proepithelin to epithelins: roles of SLPI and elastase in host defense and wound repair.

Increased leukocyte elastase activity in mice lacking secretory leukocyte protease inhibitor (SLPI) leads to impaired wound healing due to enhanced activity of TGFbeta and perhaps additional mechanisms. Proepithelin (PEPI), an epithelial growth factor, can be converted to epithelins (EPIs) in vivo by unknown mechanisms with unknown consequences. We found that PEPI and EPIs exert opposing activities. EPIs inhibit the growth of epithelial cells but induce them to secrete the neutrophil attractant IL-8, while PEPI blocks neutrophil activation by tumor necrosis factor, preventing release of oxidants and proteases. SLPI and PEPI form complexes, preventing elastase from converting PEPI to EPIs. Supplying PEPI corrects the wound-healing defect in SLPI null mice. Thus, SLPI/elastase act via PEPI/EPIs to operate a switch at the interface between innate immunity and wound healing.

Expression of a human polyomavirus oncoprotein and tumour suppressor proteins in medulloblastomas.

AIMS: Although the aetiology of medulloblastoma remains elusive, several lines of evidence suggest an association with the human neurotropic polyomavirus JC and its oncoprotein T antigen. The tumour forming properties of JC virus T antigen are the result, at least in part, of its ability to bind and inactivate tumour suppressor/cell cycle regulatory proteins, such as p53 and the retinoblastoma family of proteins. METHODS: To examine potential relations between these factors, immunohistochemistry was used to determine associations between the T antigen and the expression of p53 and the retinoblastoma proteins pRb, p107, and Rb2/p130 in eight medulloblastomas. RESULTS: Only the three medulloblastomas with T antigen expression also showed nuclear positivity with antibodies to p53. Although immunohistochemistry detected nuclear labelling for pRb in five of the cases, the three that were positive for T antigen showed the highest pRb labelling. The retinoblastoma related proteins p107 and Rb2/p130 were also immunopositive in most T antigen positive medulloblastomas. Double label immunohistochemistry also demonstrated p53 and pRb positivity in the same cells that were T antigen positive. CONCLUSIONS: These correlations suggest that associations between T antigen and p53 and/or T antigen and pRb occur in some of these tumours. These data provide indirect evidence that JC virus, acting through T antigen, might be involved in the formation and progression of medulloblastoma.

Immunoprecipitation.

Immunoprecipitation is a technique in which an antigen is isolated by binding to a specific antibody attached to a sedimentable matrix. It is also used to analyze protein fractions separated by other biochemical techniques such as gel filtration or density gradient sedimentation. The source of antigen for immunoprecipitation can be unlabeled cells or tissues, metabolically or intrinsically labeled cells, or in vitro-translated proteins. This unit describes a wide range of immunoprecipitation techniques, using either suspension or adherent cells lysed by various means (e.g., with and without detergent, using glass beads, etc.). Flow charts and figures give the user a clear-cut explanation of the options for employing the technology.

Immunoprecipitation.

Selective immunoprecipitation of proteins is a useful tool for characterizing proteins and protein-protein interactions. Clear step-by-step protocols are provided for preparing lysates of cells and yeast under a variety of conditions, for binding the antibody to a solid matrix, and for performing the actual immunoprecipitation. An additional method is provided for increasing the specificity of the technique by reprecipitating the antigen with the same or a different antibody.

Wiskott-Aldrich syndrome (role of WASP).

Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by thrombocytopenia, eczema and immunodeficiency. WASP, the gene responsible for WAS, has been identified by positional cloning, contains a PH domain, a GBD domain, a proline rich region, and a verprolin/cofilin homology domain. Subsequent studies suggest that WASP is involved in signal transduction and in the regulation of the cytoskeleton.

Mmf1p, a novel yeast mitochondrial protein conserved throughout evolution and involved in maintenance of the mitochondrial genome.

A novel protein family (p14.5, or YERO57c/YJGFc) highly conserved throughout evolution has recently been identified. The biological role of these proteins is not yet well characterized. Two members of the p14.5 family are present in the yeast Saccharomyces cerevisiae. In this study, we have characterized some of the biological functions of the two yeast proteins. Mmf1p is a mitochondrial matrix factor, and homologous Mmf1p factor (Hmf1p) copurifies with the soluble cytoplasmic fraction. Deltammf1 cells lose mitochondrial DNA (mtDNA) and have a decreased growth rate, while Deltahmf1 cells do not display any visible phenotype. Furthermore, we demonstrate by genetic analysis that Mmf1p does not play a direct role in replication and segregation of the mtDNA. rho(+) Deltammf1 haploid cells can be obtained when tetrads are directly dissected on medium containing a nonfermentable carbon source. Our data also indicate that Mmf1p and Hmf1p have similar biological functions in different subcellular compartments. Hmf1p, when fused with the Mmf1p leader peptide, is transported into mitochondria and is able to functionally replace Mmf1p. Moreover, we show that homologous mammalian proteins are functionally related to Mmf1p. Human p14.5 localizes in yeast mitochondria and rescues the Deltammf1-associated phenotypes. In addition, fractionation of rat liver mitochondria showed that rat p14.5, like Mmf1p, is a soluble protein of the matrix. Our study identifies a biological function for Mmf1p and furthermore indicates that this function is conserved between members of the p14.5 family.

Species-specific elements in the large T-antigen J domain are required for cellular transformation and DNA replication by simian virus 40.

The J domain of simian virus 40 (SV40) large T antigen is required for efficient DNA replication and transformation. Despite previous reports demonstrating the promiscuity of J domains in heterologous systems, results presented here show the requirement for specific J-domain sequences in SV40 large-T-antigen-mediated activities. In particular, chimeric-T-antigen constructs in which the SV40 T-antigen J domain was replaced with that from the yeast Ydj1p or Escherichia coli DnaJ proteins failed to replicate in BSC40 cells and did not transform REF52 cells. However, T antigen containing the JC virus J domain was functional in these assays, although it was less efficient than the wild type. The inability of some large-T-antigen chimeras to promote DNA replication and elicit cellular transformation was not due to a failure to interact with hsc70, since a nonfunctional chimera, containing the DnaJ J domain, bound hsc70. However, this nonfunctional chimeric T antigen was reduced in its ability to stimulate hsc70 ATPase activity and unable to liberate E2F from p130, indicating that transcriptional activation of factors required for cell growth and DNA replication may be compromised. Our data suggest that the T-antigen J domain harbors species-specific elements required for viral activities in vivo.

MAD3 encodes a novel component of the spindle checkpoint which interacts with Bub3p, Cdc20p, and Mad2p.

We show that MAD3 encodes a novel 58-kD nuclear protein which is not essential for viability, but is an integral component of the spindle checkpoint in budding yeast. Sequence analysis reveals two regions of Mad3p that are 46 and 47% identical to sequences in the NH(2)-terminal region of the budding yeast Bub1 protein kinase. Bub1p is known to bind Bub3p (Roberts et al. 1994) and we use two-hybrid assays and coimmunoprecipitation experiments to show that Mad3p can also bind to Bub3p. In addition, we find that Mad3p interacts with Mad2p and the cell cycle regulator Cdc20p. We show that the two regions of homology between Mad3p and Bub1p are crucial for these interactions and identify loss of function mutations within each domain of Mad3p. We discuss roles for Mad3p and its interactions with other spindle checkpoint proteins and with Cdc20p, the target of the checkpoint.

Apg7p/Cvt2p: A novel protein-activating enzyme essential for autophagy.

In the yeast Saccharomyces cerevisiae, the Apg12p-Apg5p conjugating system is essential for autophagy. Apg7p is required for the conjugation reaction, because Apg12p is unable to form a conjugate with Apg5p in the apg7/cvt2 mutant. Apg7p shows a significant similarity to a ubiquitin-activating enzyme, Uba1p. In this article, we investigated the function of Apg7p as an Apg12p-activating enzyme. Hemagglutinin-tagged Apg12p was coimmunoprecipitated with c-myc-tagged Apg7p. A two-hybrid experiment confirmed the interaction. The coimmunoprecipitation was sensitive to a thiol-reducing reagent. Furthermore, a thioester conjugate of Apg7p was detected in a lysate of cells overexpressing both Apg7p and Apg12p. These results indicated that Apg12p interacts with Apg7p via a thioester bond. Mutational analyses of Apg7p suggested that Cys507 of Apg7p is an active site cysteine and that both the ATP-binding domain and the cysteine residue are essential for the conjugation of Apg7p with Apg12p to form the Apg12p-Apg5p conjugate. Cells expressing mutant Apg7ps, Apg7pG333A, or Apg7pC507A showed defects in autophagy and cytoplasm-to-vacuole targeting of aminopeptidase I. These results indicated that Apg7p functions as a novel protein-activating enzyme necessary for Apg12p-Apg5p conjugation.

A homolog of the fungal nuclear migration gene nudC is involved in normal and malignant human hematopoiesis.

The filamentous fungus Aspergillus nidulans nudC gene has an essential function in movement of nuclei following mitosis and is required for normal colony growth. Here, the molecular cloning and role in hematopoiesis of a human gene (designated HnudC) homologous to A. nidulans nudC is reported. The amino terminus of the larger human protein (HNUDC = 45 kDa) does not overlap with A. nidulans NUDC (22 kDa). However, NUDC and the C-terminal 94 amino acids of HNUDC are 67% identical. The C-terminal region of the HnudC gene fully complements the A. nidulans temperature-sensitive nudC3 mutation, suggesting that nudC has an essential function in cell growth that is conserved from filamentous fungi to humans. In initial studies, HNUDC levels were much higher in erythroid precursors compared to most other human tissues. Therefore, the potential role of HnudC in hematopoiesis was explored. In normal human bone marrow, HNUDC protein and mRNA are highly expressed in early myeloid and erythroid precursors and decline as these cells terminally differentiate. To determine whether hematopoietic growth factors induce HnudC expression, TF-1 cells were stimulated by granulocyte-macrophage colony-stimulating factor. This induced a significant increase in HNUDC protein and HnudC mRNA, suggesting that enhancement of HnudC expression in response to growth factor stimulation may be mediated at the transcription level. Furthermore, HNUDC was significantly enhanced in lysates of bone marrow aspirates from patients with acute myelogenous and acute lymphoblastic leukemia compared to aspirates from normal controls, suggesting that HnudC is involved in malignant hematopoietic cell growth as well. These data demonstrate that HNUDC is highly expressed in normal and malignant human hematopoietic precursors and suggest it is of functional importance in the proliferation of these cells.