Sheep pancreatic microsomes as an alternative to the dog source for studying protein translocation.
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ABSTRACT:
SUBMITTER: Kaderbhai MA
PROVIDER: S-EPMC1136481 | biostudies-other | 1995 Feb
REPOSITORIES: biostudies-other
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Project description:Cytoplasmic localization of the prion protein (PrP) has been observed in different species and cell types. We have investigated this poorly understood phenomenon by expressing fusion proteins of sheep prion protein and green fluorescent protein ((GFP)PrP) in N2a cells, with variable sequence context surrounding the start codon Met(1). (GFP)PrP expressed with the wild-type sequence was transported normally through the secretory pathway to the cell surface with acquisition of N-glycan groups, but two N-terminal fragments of (GFP)PrP were detected intracellularly, starting in frame from Met(17). When (GFP)PrP was expressed with a compromised Kozak sequence ((GFP)PrP*), dispersed intracellular fluorescence was observed. A similar switch from pericellular to intracellular PrP localization was seen when analogous constructs of sheep PrP, without inserted GFP, were expressed, showing that this phenomenon is not caused by the GFP tag. Western blotting revealed a reduction in glycosylated forms of (GFP)PrP*, whereas the N-terminal fragments starting from Met(17) were still present. Formation of these N-terminal fragments was completely abolished when Met(17) was replaced by Thr, indicating that leaky ribosomal scanning occurs for normal sheep PrP and that translation from Met(17) is the cause of the aberrant cytoplasmic localization observed for a fraction of the protein. In contrast, the same phenomenon was not detected upon expression of similar constructs for mouse PrP. Analysis of samples from sheep brain allowed immunological detection of N-terminal PrP fragments, indicating that sheep PrP is subject to similar processing mechanisms in vivo.
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Project description:BackgroundAllergy to dog (Canis familiaris) is a worldwide common cause of asthma and allergic rhinitis. However, dander extract in routine diagnostics is not an optimal predictor of IgE-mediated dog allergy. Our objective was to evaluate saliva as an allergen source for improved diagnostics of allergy to dog.MethodsIgE-binding proteins in dog saliva and dander extract were analysed by immunoblot and mass spectrometry (LC-MS/MS) using pooled or individual sera from dog-allergic patients (n = 13). Sera from 59 patients IgE positive to dander and 55 patients IgE negative to dander but with symptoms to dog were analysed for IgE against saliva and dander by ELISA. Basophil stimulation with dog saliva and dander extract was measured by flow cytometry among three dog-allergic patients. Additionally, IgE-binding protein profiles of saliva from different breeds were investigated by immunoblot.ResultsGreater number and diversity of IgE-binding proteins was found in saliva compared to dander extract and varied among dog breeds. In saliva, Can f 1, 2, 3 and 6 were identified but also four new saliva allergen candidates. The majority of the 59 dog dander-positive sera (n = 44) were IgE positive to dog saliva. Among patients IgE negative to dander, but with symptoms to dog, 20% were IgE positive to saliva. The biological activity of saliva was confirmed by basophil degranulation.ConclusionsDog saliva is an allergen source for improved diagnostics of dog allergy. The IgE-binding protein profile of saliva from different dogs varies.
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Project description:Livestock herding dogs are crucial contributors to Australian agriculture. However, there is a dearth of empirical studies of the behavioural interactions between dog and livestock during herding. A statistical approach that may reveal cause and effect in such interactions is lag sequential analysis. Using 48 video recordings of livestock herding dogs and sheep in a yard trial competition, event-based (time between behaviours is irrelevant) and time-based (time between behaviours is defined) lag sequential analyses identified several significant behavioural interactions (adjusted residuals greater than 2.58; the maximum likelihood-ratio chi-squared statistic for all eight contingency tables identified all sequences as highly significant (p < 0.001)). These sequences were: The dog ceasing all movement followed by the sheep also ceasing movement; the dog chasing the sheep and a group of sheep escaping the main flock; a single sheep escaping the flock and the dog chasing; sheep initiating movement followed by the dog following; foot-stamping followed by the dog ceasing all movement; and, foot-stamping by the sheep and the dog lip-licking in response. Log linear regression identified significant relationships among undesirable behaviours in sheep and both observed trial duration (p = 0.001) and trial score (p = 0.009). No differences in the herding styles of dogs were identified between sex of dog and frequency of sheep escape behaviours (p = 0.355) nor the sex of dog and competition level (p = 0.116). The identification of trial score as a predictor of efficient performance confirms the benefits of incorporating extant objective measures to assess livestock herding dogs.
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Project description:Cotranslational protein transport into dog pancreas microsomes involves the Sec61p complex plus a luminal heat shock protein 70. Posttranslational protein transport into the yeast endoplasmic reticulum (ER) involves the so-called Sec complex in the membrane, comprising a similar Sec61p subcomplex, the putative signal peptide receptor subcomplex, and the heat shock protein 40-type subunit, Sec63p, plus a luminal heat shock protein 70. Recently, human homologs of yeast proteins Sec62p and Sec63p were discovered. Here we determined the concentrations of these two membrane proteins in dog pancreas microsomes and observed that the canine homologs of yeast proteins Sec62p and Sec63p are abundant proteins, present in almost equimolar concentrations as compared with Sec61alphap monomers. Furthermore, we detected fractions of these two proteins in association with each other as well as with the Sec61p complex. The J domain of the human Sec63p was shown to interact with immunoglobulin heavy chain binding protein. Thus, the membrane of the mammalian ER contains components, known from the posttranslationally operating protein translocase in yeast. We suggest that these components are required for efficient cotranslational protein transport into the mammalian ER as well as for other transport processes.
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Project description:BACKGROUND: Mycoplasma gallisepticum is a major poultry pathogen and causes severe economic loss to the poultry industry. In mycoplasmas lipoproteins are abundant on the membrane surface and play a critical role in interactions with the host, but tools for exploring their molecular biology are limited. RESULTS: In this study we examined whether the alkaline phosphatase gene (phoA ) from Escherichia coli could be used as a reporter in mycoplasmas. The promoter region from the gene for elongation factor Tu (ltuf) and the signal and acylation sequences from the vlhA 1.1 gene, both from Mycoplasma gallisepticum , together with the coding region of phoA , were assembled in the transposon-containing plasmid pISM2062.2 (pTAP) to enable expression of alkaline phosphatase (AP) as a recombinant lipoprotein. The transposon was used to transform M. gallisepticum strain S6. As a control, a plasmid containing a similar construct, but lacking the signal and acylation sequences, was also produced (pTP) and also introduced into M. gallisepticum . Using a colorimetric substrate for detection of alkaline phosphatase activity, it was possible to detect transformed M. gallisepticum . The level of transcription of phoA in organisms transformed with pTP was lower than in those transformed with pTAP, and alkaline phosphatase was not detected by immunoblotting or enzymatic assays in pTP transformants, eventhough alkaline phosphatase expression could be readily detected by both assays in pTAP transformants. Alkaline phosphatase was shown to be located in the hydrophobic fraction of transformed mycoplasmas following Triton X-114 partitioning and in the membrane fraction after differential fractionation. Trypsin proteolysis confirmed its surface exposure. The inclusion of the VlhA lipoprotein signal sequence in pTAP enabled translocation of PhoA and acylation of the amino terminal cysteine moiety, as confirmed by the effect of treatment with globomycin and radiolabelling studies with [14C]palmitate. PhoA could be identified by mass-spectrometry after separation by two-dimensional electrophoresis. CONCLUSION: This is the first study to express PhoA as a lipoprotein in mycoplasmas. The pTAP plasmid will facilitate investigations of lipoproteins and protein translocation across the cell membrane in mycoplasmas, and the ease of detection of these transformants makes this vector system suitable for the simultaneous screening and detection of cloned genes expressed as membrane proteins in mycoplasmas.
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Project description:Food crises caused by growing global population or environmental changes are predicted in the near future; therefore, sustainable solutions are needed. Edible insects, which are rich in protein and can save feed and environmental resources, have the potential to be a sustainable alternative protein source. However, there is limited evidence on the impact on health. In this study, we investigated the biological effects of ingesting bee larva by examining their effects on amino acid, lipid, and glucose metabolism in animal models. In our animal experiments, the replacement of casein as a protein source, with edible insects, did not seem to cause any deficiency in murine amino acid levels in the plasma and liver. Metabolomic analysis of plasma metabolites showed decreased 3-methylhistidine and increased nicotinamide in the bee larva-derived protein-fed mice. Decreased levels of plasma 3-metylhistidine, an indicator of muscle degradation, implies that replacement to bee-larva protein from casein did not cause muscle degradation in vivo. We further investigated effects of increased plasma nicotinamide on peripheral tissue and found an increase in expression levels of genes involved in glucose uptake in muscle and thermogenesis in adipose tissue. These data imply that bee larva is a potential sustainable, safe and healthy alternative protein source.
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Project description:We demonstrate simultaneous measurements of DNA translocation into glass nanopores using ionic current detection and fluorescent imaging. We verify the correspondence between the passage of a single DNA molecule through the nanopore and the accompanying characteristic ionic current blockage. By tracking the motion of individual DNA molecules in the nanocapillary perpendicular to the optical axis and using a model, we can extract an effective mobility constant for DNA in our geometry under high electric fields.
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Project description:Liver microsomes are widely used to study xenobiotic metabolism in vitro, and covalent binding to microsomal proteins serves as a surrogate marker for toxicity mediated by reactive metabolites. We have applied liquid chromatography-tandem mass spectrometry (LC-MS-MS) to identify protein targets of the biotin-tagged model electrophiles 1-biotinamido-4-(4'-[maleimidoethylcyclohexane]-carboxamido)butane (BMCC) and N-iodoacetyl-N-biotinylhexylenediamine (IAB) in human liver microsomes. The biotin-tagged peptides resulting from in-gel tryptic digestion were enriched by biotin-avidin chromatography and LC-MS-MS was used to identify 376 microsomal cysteine thiol targets of BMCC and IAB in 263 proteins. Protein adduction was selective and reproducible, and only 90 specific cysteine sites in 70 proteins (approximately 25% of the total) were adducted by both electrophiles. Differences in adduction selectivity correlated with different biological effects of the compounds, as IAB- but not BMCC-induced ER stress in HEK293 cells. Targeted LC-MS-MS analysis of microsomal glutathione-S-transferase cysteine 50, a target of both IAB and BMCC, detected time-dependent adduction by the reactive acetaminophen metabolite N-acetyl-p-benzoquinoneimine during microsomal incubations. The results indicate that electrophiles selectively adduct microsomal proteins, but display differing target selectivities that correlate with differences in toxicity. Analysis of selected microsomal protein adduction reactions thus could provide a more specific indication of potential toxicity than bulk covalent binding of radiolabeled compounds.
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