Regulation of the activity of the proinflammatory cytokine IL-1 is complex, involving transcriptional and translational control, precursor processing, a receptor antagonist (IL-1ra), and a decoy ...receptor. Here we report that the soluble form of the IL-1 receptor accessory protein (AcP) increases the affinity of binding of human IL-1α and IL-1β to the soluble human type II IL-1 receptor by approximately 100-fold, while leaving unaltered the low binding affinity of IL-1ra. Soluble AcP is present in normal human serum at an average concentration greater than 300 ng/ml. These findings suggest that the soluble form of IL-1R AcP contributes to the antagonism of IL-1 action by the type II decoy receptor, adding another layer of complexity to the regulation of IL-1 action.
We report here the cloning and characterization of four new members of the interleukin-1 (IL-1) family (FIL1δ, FIL1ε, FIL1ζ, and FIL1η, with FIL1 standing for “Family of IL-1”). The novel genes ...demonstrate significant sequence similarity to IL-1α, IL-1β, IL-1ra, and IL-18, and in addition maintain a conserved exon-intron arrangement that is shared with the previously known members of the family. Protein structure modeling also suggests that the FIL1 genes are related to IL-1β and IL-1ra. The novel genes form a cluster with the IL-1s on the long arm of human chromosome 2.
DNA was extracted from 71 meat samples obtained from UK retail outlets. All of these DNA preparations gave the expected polymerase chain reaction products when amplified with primers specific for the ...species from which the meat originated. A second polymerase chain reaction analysis, using primers specific for the
Toxoplasma gondii
SAG2 locus, revealed the presence of this parasite in 27 of the meat samples. Restriction analysis and DNA sequencing showed that 21 of the contaminated meats contained parasites genotyped as type I at the
SAG2 locus, whilst six of the samples contained parasites of both types I and II.
Toxoplasma- positive samples were subjected to further polymerase chain reaction analysis to determine whether any carried an allele of the dihydropteroate synthase gene that has recently been shown to be causally associated with sulfonamide resistance in
T. gondii. In all cases, this analysis confirmed that parasites were present in the samples and, additionally, revealed that none of them carried the drug-resistant form of dihydropteroate synthase. These results suggest that a significant proportion of meats commercially available in the UK are contaminated with
T. gondii. Although none of the parasites detected in this study carried the sulfonamide-resistance mutation, a simplified procedure for monitoring this situation merits development.
We have exploited the recently developed ability to trans‐ fect the malaria parasite Plasmodium falciparum to investigate the role of polymorphisms in the enzyme dihydropteroate synthase (DHPS), ...identified in sulfadoxine‐resistant field isolates. By using a truncated form of the dhps gene, specific mutations were introduced into the endogenous gene by allelic replacement such that they were under the control of the endogenous promoter. Using this approach a series of mutant dhps alleles that mirror P.falciparum variants found in field isolates were found to confer different levels of sulfadoxine resistance. This analysis shows that alteration of Ala437 to Gly (A437G) confers on the parasite a 5‐fold increase in sulfadoxine resistance and addition of further mutations increases the level of resistance to 24‐fold above that seen for the transfectant expressing the wild‐type dhps allele. This indicates that resistance to high levels of sulfadoxine in P.falciparum has arisen by an accumulation of mutations and that Gly437 is a key residue, consistent with its occurrence in most dhps alleles from resistant isolates. These studies provide proof that the mechanism of resistance to sulfadoxine in P.falciparum involves mutations in the dhps gene and determines the relative contribution of these mutations to this phenotype.
Abstract
Introduction
Parathyroid carcinoma is very rare, and intraoperative definitive diagnosis can be elusive with currently available diagnostics. Near-infrared (NIR) autofluorescence is an ...emerging tool that identifies parathyroid glands in real time. It is not known whether NIR autofluorescence can detect parathyroid carcinoma intraoperatively.
Methods
Patients with preoperative suspicion for parathyroid carcinoma were identified from ongoing studies examining parathyroid autofluorescence with a NIR camera and probe. Specimens from these patients were examined intraoperatively to determine their autofluorescence patterns.
Results
Three patients with suspected parathyroid carcinoma were identified preoperatively. Intraoperative NIR autofluorescence imaging showed a relative lack of autofluorescence for all cases, in contrast to parathyroid adenomas and normal parathyroid glands, which typically exhibit significant autofluorescence. Final pathology confirmed parathyroid carcinoma in all cases.
Conclusion
Parathyroid carcinoma can be difficult to confirm prior to final pathology review. Our 3 cases suggest that absence of NIR autofluorescence may suggest the likelihood of parathyroid carcinoma, but more studies are needed to investigate this experience.
Plasmodium species are difficult to study using proteomic technology because they contain large amounts of haemoglobin-derived products (HDP), generated by parasite breakdown of host haemoglobin. HDP ...are known to interfere with isoelectric focussing, a cornerstone of fractionation strategies for the identification of proteins by mass spectrometry. In addition to the challenge presented by this material, as in most proteomes, there exists in this parasite a considerable dynamic range between proteins of high and low abundance. The enzymes of the folate pathway, a proven and widely used drug target, are included in the latter class.
This report describes a work-flow utilizing a parasite-specific extraction protocol that minimizes release of HDP into the lysate, followed by in-solution based OFFGEL™ electrophoresis at the protein level, trypsin digestion and mass spectrometric analysis.
It is demonstrated that, by removing HDP from parasite lysates, OFFGEL™-mediated protein separation is able to deliver reduced complexity protein fractions. Importantly, proteins with similar and predictable physical properties are sharply focussed within such fractions.
By following this novel workflow, data have been obtained which allow the unequivocal experimental identification by mass spectrometry of four of the six proteins involved in folate biosynthesis and recycling.
Selected reaction monitoring mass spectrometry has been combined with the use of an isotopically labelled synthetic protein, made up of proteotypic tryptic peptides selected from parasite proteins of ...interest. This allows, for the first time, absolute quantification of proteins from Plasmodium falciparum. This methodology is demonstrated to be of sufficient sensitivity to quantify, even within whole cell extracts, proteins of low abundance from the folate pathway as well as more abundant "housekeeping" proteins.
Type I interleukin-1 receptor is the prototype for a family of proteins, which play a central role in early responses to injury and infection. The similarity of function across the family is ...reflected in similarity in signaling: all members tested couple to activation of NFκB and stress kinases. The coupling to these pathways is mediated by a 200-residue intracellular domain (the Toll/interleukin-1 receptor domain), in which sequence conservation is primarily confined to three short motifs (boxes 1, 2, and 3) located at amino acid residue positions 10 (box 1), 60 (box 2), and 170 (box 3). We have analyzed the contribution of these motifs to function by alanine scanning mutagenesis of the human interleukin-1 receptor type I. Mutant receptors were tested for expression, ligand binding, activation of receptor-associated kinase(s), NFκB, stress kinases, and transcription. Mutations in all three motifs led to low cell surface expression. Mutants in box 3 were, however, wild type for signaling, whereas mutants in boxes 1 and 2 were defective. We conclude that the conserved motifs box 1 and box 2 mediate the coupling of molecules in the family to inflammation signaling pathways.
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