Cyanide poisoning: pathophysiology and treatment recommendations.
This paper aims to assess and compare currently available antidotes for cyanide poisoning. Such evaluation, however, is difficult. Thus, extrapolation from the results of animal studies has potential pitfalls, as significant inter-species differences in response may exist, and these experiments often involve administration of toxin and antidote almost simultaneously, rather than incorporating a more realistic time delay before initiation of treatment. Direct inference from human case reports is also problematic; either because of uncertainties over the exposure levels involved (and hence the likely outcome without treatment), or because of difficulties in identifying the specific contribution of a particular antidote within the overall treatment regimen. Certainly an effort to compare the relative efficacy of cyanide antidotes produces equivocal findings, with no single regimen clearly standing out. Indeed, factors such as the risks of antidote toxicity to various individuals and other practical issues, may be more important considerations. There is therefore no single treatment regimen which is best for all situations. Besides individual risk factors for antidote toxicity, the nature of the exposure and hence its likely severity, the evolving clinical features and the number of persons involved and their proximity to hospital facilities, all need to be considered. Clinically mild poisoning may be treated by rest, oxygen and amyl nitrite. Intravenous antidotes are indicated for moderate poisoning. Where the diagnosis is uncertain, sodium thiosulphate may be the first choice. With severe poisoning, an additional agent is required. Given the various risks with methaemoglobin formers or with unselective use of kelocyanor, hydroxocobalamin may be preferred from a purely risk-benefit perspective. However the former alternatives will likely remain important. (+info)
Nucleolar protein B23 has molecular chaperone activities.
Protein B23 is an abundant, multifunctional nucleolar phosphoprotein whose activities are proposed to play a role in ribosome assembly. Szebeni et al. (1997) showed stimulation of nuclear import in vitro by protein B23 and suggested that this effect was due to a molecular chaperone-like activity. Protein B23 was tested for chaperone activities using several protein substrates. The temperature-dependent and -independent aggregation of the HIV-1 Rev protein was measured using a zero angle light scattering (turbidity) assay. Protein B23 inhibited the aggregation of the Rev protein, with the amount of inhibition proportional to the concentration of B23 added. This activity was saturable with nearly complete inhibition when the molar ratio of B23:Rev was slightly above one. Protein B23 also protected liver alcohol dehydrogenase (LADH), carboxypeptidase A, citrate synthase, and rhodanese from aggregation during thermal denaturation and preserved the enzyme activity of LADH under these conditions. In addition, protein B23 was able to promote the restoration of activity of LADH previously denatured with guanidine-HCl. Protein B23 preferentially bound denatured substrates and exposed hydrophobic regions when complexed with denatured proteins. Thus, by several criteria, protein B23 behaves like a molecular chaperone; these activities may be related to its role in ribosome biogenesis. (+info)
NH2-terminal sequence truncation decreases the stability of bovine rhodanese, minimally perturbs its crystal structure, and enhances interaction with GroEL under native conditions.
The NH2-terminal sequence of rhodanese influences many of its properties, ranging from mitochondrial import to folding. Rhodanese truncated by >9 residues is degraded in Escherichia coli. Mutant enzymes with lesser truncations are recoverable and active, but they show altered active site reactivities (Trevino, R. J., Tsalkova, T., Dramer, G., Hardesty, B., Chirgwin, J. M., and Horowitz, P. M. (1998) J. Biol. Chem. 273, 27841-27847), suggesting that the NH2-terminal sequence stabilizes the overall structure. We tested aspects of the conformations of these shortened species. Intrinsic and probe fluorescence showed that truncation decreased stability and increased hydrophobic exposure, while near UV CD suggested altered tertiary structure. Under native conditions, truncated rhodanese bound to GroEL and was released and reactivated by adding ATP and GroES, suggesting equilibrium between native and non-native conformers. Furthermore, GroEL assisted folding of denatured mutants to the same extent as wild type, although at a reduced rate. X-ray crystallography showed that Delta1-7 crystallized isomorphously with wild type in polyethyleneglycol, and the structure was highly conserved. Thus, the missing NH2-terminal residues that contribute to global stability of the native structure in solution do not significantly alter contacts at the atomic level of the crystallized protein. The two-domain structure of rhodanese was not significantly altered by drastically different crystallization conditions or crystal packing suggesting rigidity of the native rhodanese domains and the stabilization of the interdomain interactions by the crystal environment. The results support a model in which loss of interactions near the rhodanese NH2 terminus does not distort the folded native structure but does facilitate the transition in solution to a molten globule state, which among other things, can interact with molecular chaperones. (+info)
Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions.
The chaperone function of the mammalian 70-kDa heat shock proteins Hsc70 and Hsp70 is modulated by physical interactions with four previously identified chaperone cofactors: Hsp40, BAG-1, the Hsc70-interacting protein Hip, and the Hsc70-Hsp90-organizing protein Hop. Hip and Hop interact with Hsc70 via a tetratricopeptide repeat domain. In a search for additional tetratricopeptide repeat-containing proteins, we have identified a novel 35-kDa cytoplasmic protein, carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP is highly expressed in adult striated muscle in vivo and is expressed broadly in vitro in tissue culture. Hsc70 and Hsp70 were identified as potential interaction partners for this protein in a yeast two-hybrid screen. In vitro binding assays demonstrated direct interactions between CHIP and both Hsc70 and Hsp70, and complexes containing CHIP and Hsc70 were identified in immunoprecipitates of human skeletal muscle cells in vivo. Using glutathione S-transferase fusions, we found that CHIP interacted with the carboxy-terminal residues 540 to 650 of Hsc70, whereas Hsc70 interacted with the amino-terminal residues 1 to 197 (containing the tetratricopeptide domain and an adjacent charged domain) of CHIP. Recombinant CHIP inhibited Hsp40-stimulated ATPase activity of Hsc70 and Hsp70, suggesting that CHIP blocks the forward reaction of the Hsc70-Hsp70 substrate-binding cycle. Consistent with this observation, both luciferase refolding and substrate binding in the presence of Hsp40 and Hsp70 were inhibited by CHIP. Taken together, these results indicate that CHIP decreases net ATPase activity and reduces chaperone efficiency, and they implicate CHIP in the negative regulation of the forward reaction of the Hsc70-Hsp70 substrate-binding cycle. (+info)
A human nuclear-localized chaperone that regulates dimerization, DNA binding, and transcriptional activity of bZIP proteins.
We have identified and cloned a human nuclear protein that dramatically increases DNA binding of transcription factors that contain a basic region-leucine zipper (bZIP) DNA binding domain. We show that this bZIP enhancing factor (BEF) functions as a molecular chaperone. BEF stimulates DNA binding by recognizing the unfolded leucine zipper and promoting the folding of bZIP monomers to dimers; the elevated concentration of the bZIP dimer then drives the DNA binding reaction. Antisense experiments indicate that BEF is required for efficient transcriptional activation by bZIP proteins in vivo. Our results reveal protein folding in the nucleus as a step at which sequence-specific DNA binding proteins can be regulated. (+info)
Domain separation precedes global unfolding of rhodanese.
The enzyme rhodanese was investigated for the conformational transition associated with its urea unfolding. When rhodanese was treated with 0 or 3 M urea, the activity was not significantly affected. 4.25 M urea treatment led to a time-dependent loss of activity in 60 min. Rhodanese was completely inactivated within 2 min in 6 M urea. The 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid fluorescence intensity was not significantly increased during 0, 3, and 6 M urea equilibrations, and the fluorescence was dramatically increased with 4.25 M urea, indicating that hydrophobic surfaces are exposed. After 0 and 3 M urea equilibration, rhodanese was not significantly proteolyzed with trypsin. Treatment with 4.25 M urea led to simultaneous formation of major 12-, 15.9-, 17-, and 21.2-kDa fragments, followed by progressive emergence of smaller peptides. The N termini of the 17- and 21.2-kDa bands were those of intact rhodanese. The N terminus of the 15.9-kDa band starts at the end of the interdomain tether. The 12-kDa band begins with either residue 183 or residue 187. The size and sequence information suggest that the 17- and 15.9-kDa bands correspond to the two domains. The 21.2- and 12-kDa bands appear to be generated through one-site tryptic cleavage. It is concluded that urea disrupts interaction between the two domains, increasing the accessibility of the interdomain tether that can be digested by trypsin. The released domains have increased proteolytic susceptibility and produce smaller peptides, which may represent subdomains of rhodanese. (+info)
Characterization of a sulfurtransferase from Arabidopsis thaliana.
A database search for similarities between sequenced parts of the Arabidopsis thaliana genome with known sulfurtransferase sequences from Escherichia coli and mammals was undertaken to obtain information about plant sulfurtransferase-like proteins. One gene and several homologous EST clones were identified. One of the EST clones was used for screening an Arabidopsis cDNA library. The isolated full-length clone consists of 1134 bp and encodes a 42.6 kDa protein that includes a putative transit peptide sequence of about 7.1 kDa. Sequence comparisons with known sulfurtransferases from different organisms confirmed high homology between them and the existence of several highly conserved regions. Results of a Southern blot performed with genomic Arabidopsis DNA showed the occurrence of at least two sulfurtransferase-like isozymes in Arabidopsis. Recombinant proteins with and without the putative transit peptide were expressed in E. coli with an N-terminal His6-tag, purified by affinity chromatography and tested for enzyme activity using different sulfur donors and acceptors. Both recombinant proteins catalyzed the formation of SCN- from thiosulfate and cyanide as a rhodanese per definition; however, both recombinant proteins preferred 3-mercaptopyruvate to thiosulfate. A monospecific antibody produced by using the mature recombinant protein as an antigen recognized a single protein band in total extracts of Arabidopsis plants equating to the full-length protein size. A single band equating to the size of the mature protein was detected from purified Arabidopsis mitochondria, but there was no antigenic reaction with any protein from chloroplasts. The function of the protein is still speculative. Now tools are available to elucidate the roles and substrates of this sulfurtransferase in higher plants. (+info)
Productive and nonproductive intermediates in the folding of denatured rhodanese.
The competition between protein aggregation and folding has been investigated using rhodanese (thiosulfate:cyanide sulfurtransferase, EC 22.214.171.124) as a model. During folding from a urea-denatured state, rhodanese rapidly forms associated species or intermediates, some of which are large and/or sticky. The early removal of such particles by filtration results in a decreased refolding yield. With time, a portion of the smaller aggregates can partition back first to intermediates and then to refolded protein, while a fraction of these irreversibly form unproductive higher aggregates. Dynamic light scattering measurements indicate that the average sizes of the aggregates formed during rhodanese folding increase from 225 to 325 nm over 45 min and they become increasingly heterogeneous. Glycerol addition or the application of high hydrostatic pressure improved the final refolding yields by stabilizing smaller particles. Although addition of glycerol into the refolding mixture blocks the formation of unproductive aggregates, it cannot dissociate them back to productive intermediates. The presence of 3.9 M urea keeps the aggregates small, and they can be dissociated to monomers by high hydrostatic pressure even after 1 h of incubation. These studies suggest that early associated intermediates formed during folding can be reversed to give active species. (+info)