A novel peptidyl inhibitor of voltage-gated K + channels, named parabutoxin 3 (PBTx3), has been purified to homogeneity from the venom of Parabuthus transvaalicus. This scorpion toxin contains 37 residues, has a mass of 4274 Da and displays 41% identity with charybdotoxin (ChTx, also called Ôa-KTx1.1Õ). PBTx3 is the tenth member (called Ôa-KTx1.10Õ) of subfamily 1 of K + channel-blocking peptides known thus far. Electrophysiological experiments using Xenopus laevis oocytes indicate that PBTx3 is an inhibitor of Kv1 channels (Kv1.1, Kv1.2, Kv1.3), but has no detectable effects on Kir-type and ERG-type channels. The dissociation constants (K d ) for Kv1.1, Kv1.2 and Kv1.3 channels are, respectively, 79 lM, 547 nM and 492 nM. A synthetic gene encoding a PBTx3 homologue was designed and expressed as a fusion protein with the maltose-binding protein (MBP) in Escherichia coli. The recombinant protein was purified from the bacterial periplasm compartment using an amylose affinity resin column, followed by a gel filtration purification step and cleavage by factor X a (fX a ) to release the recombinant toxin peptide (rPBTx3). After final purification and refolding, rPBTx3 was shown to be identical to the native PBTx3 with respect to HPLC retention time, mass spectrometric analysis and functional properties. The threedimensional structure of PBTx3 is proposed by homology modelling to contain a double-stranded antiparallel b sheet and a single a-helix, connected by three disulfide bridges. The scaffold of PBTx3 is homologous to most other a-KTx scorpion toxins.
This report describes a DNA amplification procedure for routine identification of heat-labile-toxin-producing Escherichia coli. Two oligonucleotide primers were used in a polymerase chain reaction procedure to amplify a highly conserved region of the A subunit of the heat-labile enterotoxin gene. Amplifications were done directly on E. coli colonies from plates when Salmonella, Shigella, or parasite infections were excluded as agents of the severe diarrhea in the patients. The conditions for the polymerase chain reaction method were empirically determined, and the procedure is inexpensive, sensitive, and specific. Positive results can be obtained over a wide variation in bacterial numbers, with no inhibition of Thermus aquaticus DNA polymerase. Detection of the amplified product can be done by agarose gel electrophoresis, which is specific and sensitive enough for routine diagnosis of this pathogen in clinical isolates. If greater sensitivity and specificity are required, hybridization with 32p_ or alkaline phosphatase-labeled oligonucleotide probes can be used. Our results suggest that heat-labile-toxin-producing E. coli is responsible for about 9% of nondiagnosed diarrhea cases in Tygerberg Hospital, Tygerberg, Republic of South Africa.
Cerebral inflammation plays a role in diseases such as multiple sclerosis (MS), Alzheimer's disease (AD), and depression. Iron is involved in infection and inflammation through free radical production. Theoretically transferrin should prohibit iron from participating in oxidative reactions, but transferrin has also been found to promote free radical damage. We reported previously that isolation of transferrin from plasma by ion exchange column chromatography produced a broad pink protein band that subsequently separated on a gel filtration column into three proteins containing many metals. In this study some properties of the three proteins were studied in 20 volunteers. Protein 3 (identified as transferrin by nephelometry) contained the most iron while Protein 1 (called "toxiferrin") contained significantly less iron (p < 0.00001). Plasma from volunteers obtained under conditions of infection/inflammation with fever (n = 5) had a significantly increased toxiferrin to transferrin ratio compared to healthy volunteers (n = 15; p < 0.001). In vitro, Protein 2 and transferrin inhibited lipid peroxidation, while toxiferrin (possibly a protease degradation product of transferrin), enhanced lipid peroxidation. Also, toxiferrin (1 mg/mL) caused a significant increase in viability of monocytes as measured by the 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) reduction test, as well as the morphological transformation of monocytes to macrophages.
The tryptic digests of blood samples obtained from transferrin C1 and C2 (TfC 1 and TfC2 hereafter) genotypes were analysed by Liquid Chromatography coupled to Electrospray Mass Spectrometry (LC/ESI--MS/MS). The analytical results confirmed the single base change in exon 15 of the Tf gene. The solution behaviour and the iron binding properties of the two Tf variants were studied by UV-visible spectrophotometry and by circular dichroism. It appears that TfC2 globally manifests the same spectral features as the native protein. The local conformation of the two iron binding sites is conserved in the two Tf variants as evidenced by the visible absorption and CD spectra. Also, the iron binding capacities and their pH-dependent profiles are essentially the same. Overall, our investigation points out that the single amino acid substitution in TfC2 (Pro 570 Ser) does not affect the general conformation of the protein nor the local structure of the iron binding sites. The implications of these results for the etiopathogenesis of Alzheimer's disease are discussed.
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