34 research outputs found

    Re-probing of Immunoblots after Storage for More than a Decade

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    In vivo activity and low toxicity of the second-generation antimicrobial peptide DGL13K.

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    Antimicrobial peptides have been evaluated as possible alternatives to traditional antibiotics. The translational potential of the antimicrobial peptide DGL13K was tested with focus on peptide toxicity and in vivo activity in two animal models. DGL13K was effective against Pseudomonas aeruginosa, Staphylococcus aureus and methicillin-resistant S. aureus with minimal bactericidal concentrations similar to the minimal inhibitory concentration. The peptide showed low toxicity to human red blood cells and HEK cells with median lethal dose around 1 mg/ml. The median lethal dose in greater wax moth larvae (Galleria mellonella) was about 125mg/kg while the peptide caused no skin toxicity in a mouse model. A novel high-throughput luminescence assay was used to test peptide activity in infected G. mellonella, thus reducing vertebrate animal use. DGL13K killed P. aeruginosa in both the G. mellonella model and a mouse burn wound infection model, with bacterial viability 3-10-fold lower than in untreated controls. Future experiments will focus on optimizing peptide delivery, dose and frequency to further improve the antibacterial effect

    Text-mining applied to autoimmune disease research: the Sjögren’s syndrome knowledge base

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    Abstract Background Sjögren’s syndrome is a tissue-specific autoimmune disease that affects exocrine tissues, especially salivary glands and lacrimal glands. Despite a large body of evidence gathered over the past 60 years, significant gaps still exist in our understanding of Sjögren’s syndrome. The goal of this study was to develop a database that collects and organizes gene and protein expression data from the existing literature for comparative analysis with future gene expression and proteomic studies of Sjögren’s syndrome. Description To catalog the existing knowledge in the field, we used text mining to generate the Sjögren’s Syndrome Knowledge Base (SSKB) of published gene/protein data, which were extracted from PubMed using text mining of over 7,700 abstracts and listing approximately 500 potential genes/proteins. The raw data were manually evaluated to remove duplicates and false-positives and assign gene names. The data base was manually curated to 477 entries, including 377 potential functional genes, which were used for enrichment and pathway analysis using gene ontology and KEGG pathway analysis. Conclusions The Sjögren’s syndrome knowledge base (http://sskb.umn.edu) can form the foundation for an informed search of existing knowledge in the field as new potential therapeutic targets are identified by conventional or high throughput experimental techniques.</p

    Text-mining applied to autoimmune disease research: the Sjögren¿s syndrome knowledge base

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    Abstract Background Sjögren’s syndrome is a tissue-specific autoimmune disease that affects exocrine tissues, especially salivary glands and lacrimal glands. Despite a large body of evidence gathered over the past 60 years, significant gaps still exist in our understanding of Sjögren’s syndrome. The goal of this study was to develop a database that collects and organizes gene and protein expression data from the existing literature for comparative analysis with future gene expression and proteomic studies of Sjögren’s syndrome. Description To catalog the existing knowledge in the field, we used text mining to generate the Sjögren’s Syndrome Knowledge Base (SSKB) of published gene/protein data, which were extracted from PubMed using text mining of over 7,700 abstracts and listing approximately 500 potential genes/proteins. The raw data were manually evaluated to remove duplicates and false-positives and assign gene names. The data base was manually curated to 477 entries, including 377 potential functional genes, which were used for enrichment and pathway analysis using gene ontology and KEGG pathway analysis. Conclusions The Sjögren’s syndrome knowledge base ( http://sskb.umn.edu) can form the foundation for an informed search of existing knowledge in the field as new potential therapeutic targets are identified by conventional or high throughput experimental techniques

    In vitro aggregation of the regulated secretory protein chromogranin A.

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    Aggregation chaperones, consisting of secretory proteins that contain a hexa-histidine epitope tag, enhance the calcium-induced aggregation of regulated secretory proteins and their sorting to secretory granules. The goal of this study was to gain a better understanding of this unusual aggregation mechanism. Hexa-histidine-epitope-tagged secreted alkaline phosphatase, an aggregation chaperone, enhanced the in vitro aggregation of chromogranin A in the presence of calcium, but not in the presence of magnesium or other divalent cations. As an exception, chromogranin was completely aggregated by zinc, even in the absence of the aggregation chaperone. In addition, fluorescence spectroscopy of the aggregation reaction mixture showed an increase in fluorescence intensity consistent with the formation of protein aggregates. The calcium-induced aggregation of chromogranin A was completely inhibited by 0.2% Triton X-100, suggesting that it involves hydrophobic interactions. In contrast, the detergent did not affect chaperone-enhanced aggregation, suggesting that this aggregation does not depend on hydrophobic interactions. EDTA-treated chaperone did not enhance chromogranin A aggregation, indicating that divalent cations are necessary for chaperone action. Although the structure of the aggregation chaperone was not important, the size of the chaperone was. Thus the free His-hexapeptide could not substitute for the aggregation chaperone. Based on these results, we propose that the hexa-histidine tag, in the context of a polypeptide, acts as a divalent cation-dependent nucleation site for chromogranin A aggregation

    Primers used in the construction of the <i>S</i>. <i>gordoni</i>i DL1 <i>dltA</i> mutant.

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    <p>Primers used in the construction of the <i>S</i>. <i>gordoni</i>i DL1 <i>dltA</i> mutant.</p
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