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At least 19 recordsLinked to original sources
PubMed2001

Dendritic cells pulsed with unfractionated helminthic proteins to generate antiparasitic cytotoxic T lymphocyte.

Dendritic cells (DC) are sentinels of immunity. We determined their role in the induction of immunity against alveolar echinococcosis, caused by the larval stage of the cestode Echinococcus multilocularis. Furthermore, we evaluated if unfractionated protein from E. multilocularis (Em-Ag) can be used as loading agent for DC (comparable to unfractionated tumour proteins) in order to generate antiparasitic cytotoxic T lymphocyte (CTL). Interestingly, immature DC did not mature in the presence of 1 microg/ml Em-Ag as analysed by FACS and mixed leucocyte reactions. Yet, their capacity to take up dextran was markedly reduced. Further maturation of immature Em-Ag pulsed DC could be induced by proinflammatory cytokines. These mature DC were slightly better inducers of T cell proliferation when compared with unpulsed mature DC. Importantly, by repetetive stimulation of autologous CD8+ lymphocytes with the Em-Ag pulsed mature DC, we were able to generate specifically proliferating CTL lines. Thus, immunotherapy with ex vivo generated Em-Ag pulsed DC might be of benefit for patients inheriting this incurable disease.

Animals↗
PubMedDate not supplied

Enantioselective binding of albendazole sulphoxide to cytosolic proteins from helminth parasites.

The pharmacological effect of the active albendazole metabolite, albendazole sulphoxide (ABZSO), depends on its sustained presence at the site of parasite location and its binding to helmith beta-tubulin. ABZSO is found in the plasma and tissues of albendazole-treated animals in two enantiomeric forms: (+)ABZSO and (-)ABZSO. Knowledge of enantioselectivity in drug action is necessary, since any difference in target proteins affinity between enantiomers may have implications on the pharmacological effect of this anthelmintic molecule. The binding of ABZSO to mammalian and helminth parasites cytosolic proteins, as well as the differential binding of both enantiomers, were studied. Cytosolic proteins from Moniezia expansa (cestode), Ascaris suum (nematode), Fasciola hepatica (trematode), rat liver and brain as well as purified porcine brain tubulin were used. Drug analysis was performed by HPLC using both C18 and chiral columns. ABZSO protein binding was quantitatively different between parasite species (4.17, 2.5 and 1.07 ng/mg for cestode, nematode and trematode, respectively); this binding to helminth cytosolic proteins was enantioselective. Enantiomeric ratios of (-)ABZSO/(+)ABZSO as a percentage were: 43/57 (Ascaris), 36/64 (Moniezia) and 91/9 (Fasciola). Conversely, the binding of ABZSO to mammalian cytosolic proteins showed no enantioselectivity. The overall binding affinity of ABZSO for mammalian cytosolic proteins was lower than that observed in helminth proteins. The characterization of the comparative binding pattern of ABZSO enantiomers to cytosolic proteins from helminth parasites and mammalian tissues may contribute to understanding the pharmacological properties of this chiral anthelmintic molecule.

Albendazole↗
PubMed1997

Remarks on the phylogeny and structure of fatty acid binding proteins from parasitic platyhelminths.

Four fatty acid binding proteins (FABPs) have been described in 4 parasitic platyhelminths: Schistosoma mansoni, Schistosoma japonicum, Fasciola hepatica and Echinococcus granulosus. FABPs form a multigenic family of cytosolic proteins widely distributed in metazoan tissues, the function of which is still poorly understood. These helminth proteins have recently received attention, since there are reports to indicate that S. mansoni and F. hepatica FABPs may be protective antigens. In addition, these proteins could play a major role in the parasites' life-cycles because platyhelminths are unable to synthesize de novo most of their lipids. We have undertaken phylogenetic and structural analyses of platyhelminth FABPs in an attempt to characterize features of biological relevance. Phylogenetically, these FABPs appear to be more closely related to those of vertebrate heart, mammary gland, muscle, retina, skin, brain and myelin, although no clear functional relationships were established between them. We describe several conserved motifs characteristic of specific groups of FABPs. Hydrophilicity, flexibility and accessibility analyses revealed several major putative epitopes for the E. granulosus FABP, EgDf1, that appear to be centred in loops of the EgDf1 3-dimensional structure modelled by molecular replacement.

Amino Acid Sequence↗
PubMed2000

The allele-specific suppressor sup-39 alters use of cryptic splice sites in Caenorhabditis elegans.

Mutations in the Caenorhabditis elegans sup-39 gene cause allele-specific suppression of the uncoordination defect of unc-73(e936). e936 is a point mutation that changes the canonical G at the 5' end of intron 16 to a U. This mutation activates three splice donors, two of which define introns beginning with the canonical GU. Use of these two cryptic splice sites causes loss of reading frame; interestingly these messages are not substrates for nonsense-mediated decay. The third splice donor, used in 10% of steady-state e936 messages, is the mutated splice donor at the wild-type position, which defines an intron beginning with UU. In the presence of a sup-39 mutation, these same three splice donors are used, but the ratio of messages produced by splicing at these sites changes. The percentage of unc-73(e936) messages containing the wild-type splice junction is increased to 33% with a corresponding increase in the level of UNC-73 protein. This sup-39-induced change was also observed when the e936 mutant intron region was inserted into a heterologous splicing reporter construct transfected into worms. Experiments with splicing reporter constructs showed that the degree of 5' splice site match to the splicing consensus sequence can strongly influence cryptic splice site choice. We propose that mutant SUP-39 is a new type of informational suppressor that alters the use of weak splice donors.

Alleles↗
PubMed2001

Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans.

RNA interference (RNAi) is a cellular defense mechanism that uses double-stranded RNA (dsRNA) as a sequence-specific trigger to guide the degradation of homologous single-stranded RNAs. RNAi is a multistep process involving several proteins and at least one type of RNA intermediate, a population of small 21-25 nt RNAs (called siRNAs) that are initially derived from cleavage of the dsRNA trigger. Genetic screens in Caenorhabditis elegans have identified numerous mutations that cause partial or complete loss of RNAi. In this work, we analyzed cleavage of injected dsRNA to produce the initial siRNA population in animals mutant for rde-1 and rde-4, two genes that are essential for RNAi but that are not required for organismal viability or fertility. Our results suggest distinct roles for RDE-1 and RDE-4 in the interference process. Although null mutants lacking rde-1 show no phenotypic response to dsRNA, the amount of siRNAs generated from an injected dsRNA trigger was comparable to that of wild-type. By contrast, mutations in rde-4 substantially reduced the population of siRNAs derived from an injected dsRNA trigger. Injection of chemically synthesized 24- or 25-nt siRNAs could circumvent RNAi resistance in rde-4 mutants, whereas no bypass was observed in rde-1 mutants. These results support a model in which RDE-4 is involved before or during production of siRNAs, whereas RDE-1 acts after the siRNAs have been formed.

Animals↗
PubMed2005

Analysing proteomic data.

The rapid growth of proteomics has been made possible by the development of reproducible 2D gels and biological mass spectrometry. However, despite technical improvements 2D gels are still less than perfectly reproducible and gels have to be aligned so spots for identical proteins appear in the same place. Gels can be warped by a variety of techniques to make them concordant. When gels are manipulated to improve registration, information is lost, so direct methods for gel registration which make use of all available data for spot matching are preferable to indirect ones. In order to identify proteins from gel spots a property or combination of properties that are unique to that protein are required. These can then be used to search databases for possible matches. Molecular mass, pI, amino acid composition and short sequence tags can all be used in database searches. Currently the method of choice for protein identification is mass spectrometry. Proteins are eluted from the gels and cleaved with specific endoproteases to produce a series of peptides of different molecular mass. In peptide mass fingerprinting, the peptide profile of the unknown protein is compared with theoretical peptide libraries generated from sequences in the different databases. Tandem mass spectroscopy (MS/MS) generates short amino acid sequence tags for the individual peptides. These partial sequences combined with the original peptide masses are then used for database searching, greatly improving specificity. Increasingly protein identification from MS/MS data is being fully or partially automated. When working with organisms, which do not have sequenced genomes (the case with most helminths), protein identification by database searching becomes problematical. A number of approaches to cross species protein identification have been suggested, but if the organism being studied is only distantly related to any organism with a sequenced genome then the likelihood of protein identification remains small. The dynamic nature of the proteome means that there really is no such thing as a single representative proteome and a complete set of metadata (data about the data) is going to be required if the full potential of database mining is to be realised in the future.

Animals↗
PubMed1997

A conserved RNA-binding protein that regulates sexual fates in the C. elegans hermaphrodite germ line.

The nematode Caenorhabditis elegans has two sexes, males and hermaphrodites. Hermaphrodites Initially produce sperm but switch to producing oocytes. This switch appears to be controlled by the 3' untranslated region of fem-3 messenger RNA. We have now identified a binding factor (FBF) which is a cytoplasmic protein that binds specifically to the regulatory region of fem-3 3'UTR and mediates the sperm/oocyte switch. The RNA-binding domain of FBF consists of a stretch of eight tandem repeats and two short flanking regions. This structural element is conserved in several proteins including Drosophila Pumilio, a regulatory protein that controls pattern formation in the fly by binding to a 3'UTR. We propose that FBF and Pumilio are members of a widespread family of sequence-specific RNA-binding proteins.

Amino Acid Sequence↗
PubMed2004

In vitro genotoxic and cytotoxic effects of protein somatic products from helminths on donor blood lymphocytes.

Protein somatic products of adult helminths Hymenolepis nana and Toxocara canis and secretory-excretory somatic product of Trichinella spiralis larvae in vitro produced genotoxic and cytotoxic effects on donor blood lymphocytes, which manifested in accumulation of single-chain breaks, alkaline-labile sites in nuclear DNA, and apoptotic cells. This effect depended on the concentration of parasitic protein products during coculturing.

Adult↗
PubMed1999

The rde-1 gene, RNA interference, and transposon silencing in C. elegans.

Double-stranded (ds) RNA can induce sequence-specific inhibition of gene function in several organisms. However, both the mechanism and the physiological role of the interference process remain mysterious. In order to study the interference process, we have selected C. elegans mutants resistant to dsRNA-mediated interference (RNAi). Two loci, rde-1 and rde-4, are defined by mutants strongly resistant to RNAi but with no obvious defects in growth or development. We show that rde-1 is a member of the piwi/sting/argonaute/zwille/eIF2C gene family conserved from plants to vertebrates. Interestingly, several, but not all, RNAi-deficient strains exhibit mobilization of the endogenous transposons. We discuss implications for the mechanism of RNAi and the possibility that one natural function of RNAi is transposon silencing.

Amino Acid Sequence↗
PubMed2000

ATP-activated oligomerization as a mechanism for apoptosis regulation: fold and mechanism prediction for CED-4.

Fold recognition algorithm FFAS (Rychlewski et al., Protein Sci, 2000;9:232-241) was used to match the nucleotide-binding adaptor shared by APAF-1, certain R gene products and CED-4 (NB-ARC domain) to the structure of the D2 domain of N-ethylemaleimide-Sensitive Fusion Protein and the delta; subunit of clamp loader of DNA polymerase III. The predicted structure consists of the p-loop ATP-binding domain, followed by two alpha-helical domains that regulate the oligomerization process. This prediction suggests a detailed molecular mechanism for the "induced proximity" hypothesis (Salvesen and Dixit, Proc Natl Acad Sci USA 1999;96:10964-10967) for CED3/caspase-9 activation by CED4/APAF-1 complex. According to this model, the ATP binding acts as a trigger in CED-4 oligomerization and the helical domain immediately following the ATP-binding domain provides additional mechanisms for regulation of the oligomerization process. This model explains most of known experimental data about CED-4-mediated caspase activation and, at the same time, suggest experiments that could test this hypothesis.

Adenosine Triphosphate↗
PubMed2000

Yeast two-hybrid systems and protein interaction mapping projects for yeast and worm.

The availability of complete genome sequences necessitates the development of standardized functional assays to analyse the tens of thousands of predicted gene products in high-throughput experimental settings. Such approaches are collectively referred to as 'functional genomics'. One approach to investigate the properties of a proteome of interest is by systematic analysis of protein-protein interactions. So far, the yeast two-hybrid system is the most commonly used method for large-scale, high-throughput identification of potential protein-protein interactions. Here, we discuss several technical features of variants of the two-hybrid systems in light of data recently obtained from different protein interaction mapping projects for the budding yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans.

Animals↗
PubMed2000

Predicting the function and subcellular location of Caenorhabditis elegans proteins similar to Saccharomyces cerevisiae beta-oxidation enzymes.

The role of peroxisomal processes in the maintenance of neurons has not been thoroughly investigated. We propose using Caenorhabditis elegans as a model organism for studying the molecular basis underlying neurodegeneration in certain human peroxisomal disorders, e.g. Zellweger syndrome, since the nematode neural network is well characterized and relatively simple in function. Here we have identified C. elegans PEX-5 (C34C6.6) representing the receptor for peroxisomal targeting signal type 1 (PTS1), defective in patients with such disorders. PEX-5 interacted strongly in a two-hybrid assay with Gal4p-SKL, and a screen using PEX-5 identified interaction partners that were predominantly terminated with PTS1 or its variants. A list of C. elegans proteins with similarities to well-characterized yeast beta-oxidation enzymes was compiled by homology probing. The possible subcellular localization of these orthologues was predicted using an algorithm based on trafficking signals. Examining the C termini of selected nematode proteins for PTS1 function substantiated predictions made regarding the proteins' peroxisomal location. It is concluded that the eukaryotic PEX5-dependent route for importing PTS1-containing proteins into peroxisomes is conserved in nematodes. C. elegans might emerge as an attractive model system for studying the importance of peroxisomes and affiliated processes in neurodegeneration, and also for studying a beta-oxidation process that is potentially compartmentalized in both mitochondria and peroxisomes.

Acyl-CoA Oxidase↗
PubMed1995

The SMC proteins and the coming of age of the chromosome scaffold hypothesis.

The mechanism of chromosome condensation is one of the classic mysteries of mitosis. A number of years ago, it was suggested that nonhistone proteins of the chromosome scaffold fraction might help chromosomes to condense, possibly by constructing a framework for the condensed structure. Recent results have shown that topoisomerase II and the SMC proteins, two abundant members of the scaffold fraction, are required for chromosome condensation and segregation during mitosis. Topoisomerase II is a well-characterized enzyme. In contrast, nothing is yet known about the function of the SMC proteins. We summarize evidence suggesting that these proteins may be enzymes whose activity is somehow involved in the establishment and maintenance of mitotic chromosome morphology.

Animals↗
PubMed2001

Postembryonic expression of Caenorhabditis elegans mab-21 and its requirement in sensory ray differentiation.

The male tail sensory rays in Caenorhabditis elegans are complex copulatory structures, the normal patterning of which requires a number of regulatory genes. Among them, mab-21 specifies the identity of sensory ray 6. By using green fluorescent protein reporters, we identify multiple cis-acting elements that control the developmental expression of mab-21. Traced with a functional mab-21:gfp gene driven by authentic regulatory sequences, mab-21 expression could be detected in hypodermal, neuronal, muscle, and ray cells. We showed here that the expression of mab-21 in the hypodermis and neuronal cells was dispensable for its function in ray 6. In contrast, its expression in the ray 6 structural cell and neurons as conferred by its 3' enhancer was crucial for determining the correct ray 6 identity.

Amino Acid Sequence↗
PubMed2003

Synaptic Drosophila UNC-13 is regulated by antagonistic G-protein pathways via a proteasome-dependent degradation mechanism.

UNC-13 is a highly conserved plasma membrane-associated synaptic protein implicated in the regulation of neurotransmitter release through the direct modulation of the SNARE exocytosis complex. Previously, we characterized the Drosophila homologue (DUNC-13) and showed it to be essential for neurotransmitter release immediately upstream of vesicular fusion ("priming") at the neuromuscular junction (NMJ). Here, we show that the abundance of DUNC-13 in NMJ synaptic boutons is regulated downstream of GalphaS and Galphaq pathways, which have inhibitory and facilitatory roles, respectively. Both cAMP modulation and PKA function are required for DUNC-13 synaptic up-regulation, suggesting that the cAMP pathway enhances synaptic efficacy via DUNC-13. Similarly, PLC function and DAG modulation also regulate the synaptic levels of DUNC-13, through a mechanism that appears independent of PKC. Our results suggest that proteasome-mediated protein degradation is the primary mechanism regulating DUNC-13 levels at the synapse. Both PLC- and PKA-mediated pathways appear to regulate synaptic levels of DUNC-13 through controlling the rate of proteasome-dependent DUNC-13 degradation. We conclude that the functional abundance of DUNC-13 at the synapse, a key determinant of synaptic vesicle priming and neurotransmitter release probability, is primarily regulated by the rate of protein degradation, rather than translocation or transport, convergently controlled via both cAMP and DAG signal transduction pathways.

Acetylcysteine↗
PubMed2003

Flavors of protein disorder.

Intrinsically disordered proteins are characterized by long regions lacking 3-D structure in their native states, yet they have been so far associated with 28 distinguishable functions. Previous studies showed that protein predictors trained on disorder from one type of protein often achieve poor accuracy on disorder of proteins of a different type, thus indicating significant differences in sequence properties among disordered proteins. Important biological problems are identifying different types, or flavors, of disorder and examining their relationships with protein function. Innovative use of computational methods is needed in addressing these problems due to relative scarcity of experimental data and background knowledge related to protein disorder. We developed an algorithm that partitions protein disorder into flavors based on competition among increasing numbers of predictors, with prediction accuracy determining both the number of distinct predictors and the partitioning of the individual proteins. Using 145 variously characterized proteins with long (>30 amino acids) disordered regions, 3 flavors, called V, C, and S, were identified by this approach, with the V subset containing 52 segments and 7743 residues, C containing 39 segments and 3402 residues, and S containing 54 segments and 5752 residues. The V, C, and S flavors were distinguishable by amino acid compositions, sequence locations, and biological function. For the sequences in SwissProt and 28 genomes, their protein functions exhibit correlations with the commonness and usage of different disorder flavors, suggesting different flavor-function sets across these protein groups. Overall, the results herein support the flavor-function approach as a useful complement to structural genomics as a means for automatically assigning possible functions to sequences.

Algorithms↗
PubMed2004

Tsp36, a tapeworm small heat-shock protein with a duplicated alpha-crystallin domain, forms dimers and tetramers with good chaperone-like activity.

Small heat shock proteins (sHSPs), which range in monomer size between 12 and 42 kDa, are characterized by a conserved C-terminal alpha-crystallin domain of 80-100 residues. They generally form large homo- or heteromeric complexes, and typically have in vitro chaperone-like activity, keeping unfolding proteins in solution. A special type of sHSP, with a duplicated alpha-crystallin domain, is present in parasitic flatworms (Platyhelminthes). Considering that an alpha-crystallin domain is essential for the oligomerization and chaperone-like properties of sHSPs, we characterized Tsp36 from the tapeworm Taenia saginata. Both wild-type Tsp36 and a mutant (Tsp36C-->R) in which the single cysteine has been replaced by arginine were expressed and purified. Far-UV CD measurements of Tsp36 were in agreement with secondary structure predictions, which indicated alpha-helical structure in the N-terminal region and the expected beta-sandwich structure for the two alpha-crystallin domains. Gel permeation chromatography and nano-ESI-MS showed that wild type Tsp36 forms dimers in a reducing environment, and tetramers in a non-reducing environment. The tetramers are stabilized by disulfide bridges involving a large proportion of the Tsp36 monomers. Tsp36C-->R exclusively occurs as dimers according to gel permeation chromatography, while the nondisulfide bonded fraction of wild type Tsp36 dissociates from tetramers into dimers under nonreducing conditions at increased temperature (43 degrees C). The tetrameric form of Tsp36 has a greater chaperone-like activity than the dimeric form.

Amino Acid Sequence↗