Invasive dermatophyte infection with Trichophyton interdigitale is associated with prurigo-induced pseudoperforation and a signal transducer and activator of transcription 3 mutation.

Invasive dermatophyte infection, with extension beyond the dermis, in immunocompetent hosts is exceptionally rare. Dermatophytes are keratinophilic and are usually confined to the stratum corneum, hair and nails. Susceptibility to dermatophyte infections is incompletely understood, but inherited mutations in key signalling pathways of the innate immune system have been identified. We report the first case of an invasive dermatophyte infection associated with abrupt onset of a prurigo-induced pseudoperforation and a loss-of-function mutation in signal transducer and activator of transcription 3 (STAT3).

Activating PI3Kδ mutations in a cohort of 669 patients with primary immunodeficiency.

The gene PIK3CD codes for the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ), and is expressed solely in leucocytes. Activating mutations of PIK3CD have been described to cause an autosomal dominant immunodeficiency that shares clinical features with common variable immunodeficiency (CVID). We screened a cohort of 669 molecularly undefined primary immunodeficiency patients for five reported mutations (four gain-of-function mutations in PIK3CD and a loss of function mutation in PIK3R1) using pyrosequencing. PIK3CD mutations were identified in three siblings diagnosed with CVID and two sporadic cases with a combined immunodeficiency (CID). The PIK3R1 mutation was not identified in the cohort. Our patients with activated PI3Kδ syndrome (APDS) showed a range of clinical and immunological findings, even within a single family, but shared a reduction in naive T cells. PIK3CD gain of function mutations are more likely to occur in patients with defective B and T cell responses and should be screened for in CVID and CID, but are less likely in patients with a pure B cell/hypogammaglobulinaemia phenotype.

Anti-oxidative hormetic effects of cellular autophagy induced by nutrient deprivation in a molluscan animal model.

This investigation using a molluscan animal model tested the hypothesis that experimentally induced lysosomal autophagy protects against oxidative cell injury. Induction of augmented lysosomal autophagy has previously been implicated in this protective process. Four treatment groups of blue mussels (Mytilus galloprovincialis) were used: Group 1 (fed - control), Group 2 (fasted), Group 3 (copper + fed) and Group 4 (copper + fasted). Groups 2 and 4 were fasted in order to trigger autophagy; and samples of hepatopancreas (liver analogue or digestive gland) from all 4 groups were taken at 3, 6 and 15 days. Treatment with copper provided a positive reference for oxidative stress: Groups 3 and 4 were treated with copper (10 µg Cu2+/animal/day) for three days only. Oxidative damage and cellular injury in hepatopancreatic digestive cells was found to decrease in Group 2 (fasted) compared to Group 1 (fed - control). Group 3 (fed + copper) showed clear evidence of oxidative stress and cell injury, as well as induction of antioxidant activities. Group 4 (copper + fasted) had a reduced uptake of copper and toxicity of copper was also reduced, compared with Group 3. It was concluded that augmented autophagy had a hormetic cytoprotective anti-oxidant effect.

Oxidative stress, lysosomal damage and dysfunctional autophagy in molluscan hepatopancreas (digestive gland) induced by chemical contaminants.

Autophagy is a highly conserved evolutionary survival or defence process that enables cells and organisms to survive periods of environmental stress by breaking down cellular organelles and macromolecules in autolysosomes to provide a supply of nutrients for cell maintenance. However, autophagy is also a part of normal cellular physiology that facilitates the turnover of cellular constituents under normal conditions: it can be readily augmented by mild environmental stress; but becomes dysfunctional with severe oxidative stress leading to cellular pathology. The molluscan hepatopancreas or digestive gland provides a versatile and environmentally relevant model to investigate lysosomal autophagy and stress-induced dysfunctional autophagy. This latter process has been implicated in many animal and human disease conditions, including degenerative and neurodegenerative diseases, as well as obesity related conditions. Many environmental pollutants have also been found to induce dysfunctional autophagy in molluscan hepatopancreatic digestive cells, and in this study, the marine blue mussel Mytilus galloprovincialis was exposed for 7 days to: 0.1 µM, 1 µM and 10 µM concentrations of fluoranthene and phenanthrene (PAHs); chlorpyrifos and malathion (organophosphorus compounds); atrazine (triazine herbicide); copper (transition metal) and dodecylbenzene sulphonic acid (LAS, surfactant). The marine snail or periwinkle, Littorina littorea, was also exposed to phenanthrene, chlorpyrifos and copper. Indices of oxidative stress, cell injury and dysfunctional autophagy were measured (i.e., lysosomal membrane stability, protein carbonyls, lipofuscin, and lysosomal accumulation of lipid or lipidosis). Evidence of oxidative stress, based on the elevation of lipofuscin and protein carbonyls, was found for all compounds tested; with chlorpyrifos being the most toxic to both species. Dysfunctional autophagy was induced by all of the compounds tested in both species, except for atrazine in mussels. This failure of normal autophagy was consistently associated with oxidative stress. Autophagic dysfunction is an important emerging feature in the aetiology of many disease conditions in animals and humans; and an explanatory conceptual mechanistic model has been developed for dysregulation of autophagy in response to oxidative stress.

Emergent synergistic lysosomal toxicity of chemical mixtures in molluscan blood cells (hemocytes).

The problem of effective assessment of risk posed by complex mixtures of toxic chemicals in the environment is a major challenge for government regulators and industry. The biological effect of the individual contaminants, where these are known, can be measured; but the problem lies in relating toxicity to the multiple constituents of contaminant cocktails. The objective of this study was to test the hypothesis that diverse contaminant mixtures may cause a greater toxicity than the sum of their individual parts, due to synergistic interactions between contaminants with different intracellular targets. Lysosomal membrane stability in hemocytes from marine mussels was used for in vitro toxicity tests; and was coupled with analysis using the isobole method and a linear additive statistical model. The findings from both methods have shown significant emergent synergistic interactions between environmentally relevant chemicals (i.e., polycyclic aromatic hydrocarbons, pesticides, biocides and a surfactant) when exposed to isolated hemocytes as a mixture of 3 & 7 constituents. The results support the complexity-based hypothesis that emergent toxicity occurs with increasing contaminant diversity, and raises questions about the validity of estimating toxicity of contaminant mixtures based on the additive toxicity of single components. Further experimentation is required to investigate the potential for interactive effects in mixtures with more constituents (e.g., 50-100) at more environmentally realistic concentrations in order to test other regions of the model, namely, very low concentrations and high diversity. Estimated toxicant diversity coupled with tests for lysosomal damage may provide a potential tool for determining the toxicity of estuarine sediments, dredge spoil or contaminated soil.

Pathological reactions and recovery of hepatopancreatic digestive cells from the marine snail Littorina littorea following exposure to a polycyclic aromatic hydrocarbon.

The aim of this study was to investigate the cellular pathological responses of hepatopancreatic digestive cells from the periwinkle Littorina littorea exposed to the polycyclic aromatic hydrocarbon (PAH) fluoranthene and to ascertain whether any injurious effects were reversible within the experimental time scale. A secondary objective was to establish the relationship of the various reactions to animal health status, using lysosomal stability as an index of well-being. Exposure of snails to a concentration of 335 microgl(-1) (1.7 microM) fluoranthene (seawater renewed and spiked daily with fluoranthene) for 5 days resulted in a reduction in lysosomal stability (neutral red retention) and endocytosis; and an increase in smooth endoplasmic reticulum (ER) and 7-ethoxycoumarin-o-deethylase (ECOD; measured as cyano-ECOD) activity measured in isolated live digestive cells. Exposed snails treated with clean seawater for a further 8 days resulted in a return to control levels of lysosomal stability, ECOD and ER; endocytosis showed only a partial recovery. Multi-variate and uni-variate analysis showed that there were strong correlations between the various cellular biomarker responses. These findings are interpretable within the current framework of molluscan biomarker responses to PAHs. Principal component analysis was used to derive the first principal component for endocytosis, ER and ECOD reactions and these were plotted against lysosomal stability as a measure of cellular well-being. The resulting significant regression represents the mapping of the individual biomarkers within health status space for a gradient of fluoranthene toxicity. From this analysis, we concluded that endocytosis is an indicator of healthy snails while proliferation of ER and to a lesser extent induced ECOD are indicative of dysfunction and reduced health. Finally, the results indicate that stress induced by chronic exposure to a PAH is reversible.

Cytology and quantitative cytochemistry of a poliferative atypical hemocytic condition in Mytilus edulis (Bivalvia, mollusca).

Proliferative lesions were found in 16 of 994 Mytilus edulis mussels obtained from the mouth of the River Lynher at Plymouth, England. These lesions were characterized by infiltration and replacement of the connective tissue by enlarged, atypical, mitotically active, basophilic, hemocyte-like cells. Cytologic examination indicated the involvement of two cell types in this disorder, both of which were similar in appearance to the much smaller, normal basophallic agranular hemocyte. The abnormal cells were rich in cytoplasmic RNA and had significantly higher DNA levels than normal hemocytes as determined by scanning microdensitometry. A few of the atypical cells contained small cytoplasmic granules that were positive for lysosomal hydrolases. This condition was observed sequentially from what is believed to be the earliest stage of the disease, in which only a few atypical cells were present, to the terminal stage, in which the connective tissue was almost completely replaced and the digestive gland cells were necrotized. Some potentially carcinogenic aromatic hydrocarbons have been identified in the substrate of the mussel bed by gas-liquid chromatography-mass spectrometry analysis.

Mutational analysis of two conserved sequence motifs in HIV-1 reverse transcriptase.

Two conserved sequence motifs, occurring in HIV-1 reverse transcriptase at residues 110-116 and 183-190, have been studied using site-directed mutagenesis of the cloned gene. In particular, aspartates at positions 185 and 186 have each been mutated to either asparagine or glutamate. The resulting mutant proteins were catalytically inactive but still able to bind the template-primer complex, poly rA-oligo dT. Other mutations in these regions resulted in reduced reverse trascriptase activity but the mutation of tyrosine-183 to serine caused a significant increase in the Km for dTTP and the Ki for inhibition by 3'-azidothymidine-triphosphate, 2',3'-dideoxythymidine-triphosphate and phosphonoformic acid.

Engineering of tyrosyl tRNA synthetase.

The gene encoding the enzyme tyrosyl tRNA synthetase from Bacillus stearothermophilus has been systematically altered using synthetic oligonucleotides as mutagens. The construction of mutations has been facilitated by using strains of bacteria defective in mismatch repair and also by utilising a genetic marker in the M13 strain (such as an amber mutation, or an EcoK or EcoB site) which allows selection for the progeny of M13 replication derived from the minus (mutagenized) strand. Several mutations have been constructed in the ATP binding site to elucidate the roles of individual residues in catalysis and substrate binding and it has even been possible to construct mutants which have improved affinity for ATP. Mutations in various surface lysine and arginine residues have allowed us to identify potential contacts with the tRNA, and indicate that a cluster of basic residues close to the C-terminus of the enzyme probably makes important interactions with the tRNA.

Impact of the Sea Empress oil spill on lysosomal stability in mussel blood cells.

Coastal zones are among the most productive and vulnerable areas on the planet. An example of impact on these fragile environments was shown in the case of the "Sea Empress" oil tanker, which ran aground in the Bristol Channel in 1996, spilling 72,000 tons of "Forties" crude oil. The objective was to investigate the sub-lethal cellular pathology and tissue hydrocarbon contamination in marine mussel populations, 4 months after the initial spill, using the neutral red retention (NRR) assay for lysosomal stability in blood cells. NRR was reduced in mussels, and indicative of cell injury, from the two sites closest to the spill in comparison with more distant and reference sites. Lysosomal stability was inversely correlated with polycyclic aromatic hydrocarbon concentrations in mussel tissues. Reduced lysosomal stability has previously been shown to contribute to impaired immunocompetence and to autophagic loss of body tissues. The use of this type of technique is discussed in the context of cost-effective, ecotoxicological tools for Integrated Coastal Zone Management.

Pethidine pharmacokinetics after intramuscular dose: a comparison in Caucasian, Chinese and Nepalese patients.

The pharmacokinetics of pethidine after a single intramuscular injection were studied in 30 male patients of Caucasian, Chinese and Nepalese extraction. There were no significant differences between the three ethnic groups in the mean time for maximum absorption (tmax) and peak plasma concentration (Cmax) of pethidine. The mean (+/- S.D.) elimination half life (t1/2) of pethidine was shorter in Caucasians (4.5 +/- 1.3 h) compared with Nepalese (6.3 +/- 1.6 h) and Chinese (8.1 +/- 3.1 h) (p < 0.01). The plasma clearance of pethidine was greater in Caucasians (14.2 +/- 4.8 ml.min-1.kg-1) than in Nepalese (12.6 +/- 2.9 ml.min-1.kg-1) and Chinese (10.0 +/- 2.9 ml.min-1.kg-1) (p < 0.05); yet the apparent renal clearance was similar (64.1 +/- 22.9, 86.7 +/- 44.5 and 61.4 +/- 30.1 ml.min-1.kg-1, respectively, for the Chinese (n = 6), Caucasian (n = 6) and Nepalese (n = 9) patients). No apparent ethnic differences were found in the tmax and Cmax of norpethidine which emerged as the major metabolite in the plasma in the three races. An apparently higher area under plasma concentration-time curve (AUC infinity [symbol: see text]) and longer elimination t1/2 of the metabolite were observed in the two Asian patient groups. It appears that both the Chinese and Indian groups did not eliminate pethidine as effectively as the Caucasians after a single intramuscular injection, which may be the result of interethnic variability in the metabolism of pethidine. Caution may be required on multiple dosing of pethidine in Asian patients due to the possible accumulation of the parent drug and its toxic metabolite, norpethidine.

Studies on the structure and activity of rabbit Clq (a subcomponent of the first component of complement).

1. The subunit structure of rabbit subcomponent C1q was examined in a previous publication (Reid et al., 1972). The present paper describes some aspects of the structure of the polypeptide chains derived from the molecule. 2. The three polypeptide chains, produced by performic oxidation, of rabbit subcomponent C1q were isolated by ion-exchange chromatography in 8m-urea on DEAE-cellulose. 3. Each chain was found to contain 15-18% glycine and significant amounts of the amino acids hydroxyproline and hydroxylysine. 4. By means of collagenase digestion it was shown that all three chains of rabbit subcomponent C1q contain collagen-like sequences of amino acids which constitute about 40% of each chain. 5. By use of carboxypeptidase A it was established, indirectly, that the collagen-like sequences, in one of the chains, are probably located near, or at, the N-terminal end of the chain. 6. Collagenase digestion and heating at 52 degrees C (but not at 49 degrees C) caused rapid loss of native rabbit subcomponent C1q haemolytic activity.

3-Hydroxy-3-methylglutaryl-coenzyme A synthase from ox liver. Purification, molecular and catalytic properties.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) was purified to homogeneity from ox liver and obtained essentially free from acetoacetyl-CoA thiolase activity. The purification procedure included substrate elution from cellulose phosphate and chromatofocusing. The relative molecular mas was about 100 000 and S20,w0 was 6.36S. The enzyme appears to be a dimer of identical subunits (Mr 47 900). The Km for acetoacetyl-CoA is extremely low (less than 0.5 microM), and acetoacetyl-CoA (Acac-CoA) gives marked substrate inhibition (KiAcac-CoA = 3.5 microM) that is competitive with respect to acetyl-CoA. Both CoA and DL-3-hydroxy-3-methylglutaryl-CoA give mixed product inhibition with respect to acetyl-CoA, which is compatible with a Ping Pong mechanism in which both products can form kinetically significant complexes with two forms of the enzyme. The two forms are most likely to be free enzyme and an acetyl-enzyme intermediate.

3-Hydroxy-3-methylglutaryl-coenzyme A synthase from ox liver. Properties of its acetyl derivative.

Ox liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) reacts with acetyl-CoA to form a complex in which the acetyl group is covalently bound to the enzyme. This acetyl group can be removed by addition of acetoacetyl-CoA or CoA. The extent of acetylation and release of CoA were found to be highly temperature-dependent. At temperatures above 20 degrees C, a maximum value of 0.85 mol of acetyl group bound/mol of enzyme dimer was observed. Below this temperature the extent of rapid acetylation was significantly lowered. Binding stoichiometries close to 1 mol/mol of enzyme dimer were also observed when the 3-hydroxy-3-methylglutaryl-CoA synthase activity was titrated with methyl methanethiosulphonate or bromoacetyl-CoA. This is taken as evidence for a 'half-of-the-sites' reaction mechanism for the formation of 3-hydroxy-3-methylglutaryl-CoA by 3-hydroxy-3-methylglutaryl-CoA synthase. The Keq. for the acetylation was about 10. Isolated acetyl-enzyme is stable for many hours at 0 degrees C and pH 7, but is hydrolysed at 30 degrees C with a half-life of 7 min. This hydrolysis is stimulated by acetyl-CoA and slightly by succinyl-CoA, but not by desulpho-CoA. The site of acetylation has been identified as the thiol group of a reactive cysteine residue by affinity-labelling with the substrate analogue bromo[1-14C]acetyl-CoA.

Succinylation and inactivation of 3-hydroxy-3-methylglutaryl-CoA synthase by succinyl-CoA and its possible relevance to the control of ketogenesis.

Succinyl-CoA (3-carboxypropionyl-CoA) inactivates ox liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) in a time-dependent manner, which is partially prevented by the presence of substrates of the enzyme. The inactivation is due to the enzyme catalysing its own succinylation. Complete inactivation corresponds to about 0.5 mol of succinyl group bound/mol of enzyme dimer. The succinyl-enzyme linkage appears to be a thioester bond and is probably formed with the active-site cysteine residue that is normally acetylated by acetyl-CoA. Succinyl-CoA binds to 3-hydroxy-3-methylglutaryl-CoA synthase with a binding constant of 340 microM and succinylation occurs with a rate constant of 0.57 min-1. Succinyl-enzyme breaks down with a half-life of about 40 min (k = 0.017 min-1) at 30 degrees C and pH 7 and is destabilized by the presence of acetyl-CoA and succinyl-CoA. A control mechanism is postulated in which flux through the 3-hydroxy-3-methylglutaryl-CoA cycle of ketogenesis is regulated according to the extent of succinylation of 3-hydroxy-3-methylglutaryl-CoA synthase.

Preparation of bromo[1-14C]acetyl-coenzyme A as an affinity label for acetyl-coenzyme A binding sites.

Bromo[1-14C]acetyl-CoA has been prepared from CoASH and the N-hydroxysuccinimide ester of bromo[1-14C]acetic acid, and unlabeled bromoacetyl-CoA by reaction of CoASH with bromoacetyl bromide. The products were purified by high-pressure liquid chromatography. Purified bromoacetyl-CoA was characterized, and found to be a potent alkylating agent with a substantial stability in aqueous solution: it decomposed at 30 degrees C and pH 6.6 and 8.0 with halftimes of 3.3 and 2.5 h, respectively. The major breakdown products were CoASH and CoAS X CO X CH2 X SCoA. Bromo[1-14C]acetyl-CoA has been used to affinity label the acetyl-CoA binding site of 3-hydroxy-3-methylglutaryl-CoA synthase from ox liver. It was found to irreversibly inhibit the enzyme activity and bind covalently with a stoichiometry for complete inhibition of about 0.8 mol/mol enzyme dimer.

7th nerve palsy after extradural blood patch.

We describe a patient who developed a 7th cranial nerve palsy following an extradural blood patch; full recovery followed. The likely aetiology is discussed.