Anaphylaxis to pork kidney is related to IgE antibodies specific for galactose-alpha-1,3-galactose.

BACKGROUND: Carbohydrate-specific IgE antibodies present on nonprimate mammalian proteins were incriminated recently in delayed meat anaphylaxis. The aim of this study was to explore whether anaphylaxis to mammalian kidney is also associated with galactose-α-1,3-galactose (αGal)-specific IgE. METHODS: Fourteen patients with anaphylaxis to pork or beef kidney underwent prick tests to meat and kidney. Some patients also underwent skin tests to Erbitux(®) (cetuximab). IgE antibodies to αGal, swine urine proteins, beef and pork meat, serum albumin proteins, cat, and rFel d 1 were measured by ImmunoCAP(®). The αGal levels were estimated in meats and kidney by ELISA inhibition assay. Cross-reactivity between αGal and pork kidney was studied with the ImmunoCAP(®) inhibition assay. RESULTS: Among the 14 patients, 12 presented with anaphylactic shock. Reactions occurred within 2 h from exposure in 67% of patients. Associated risk factors were observed in 10 cases, and alcohol was the main cofactor. Three patients underwent an oral challenge to pork kidney, and anaphylaxis occurred after ingestion of small quantities (1-2 g). Prick tests to kidney were positive in 54% of patients. All tested patients showed positive skin tests to Erbitux(®). All patients tested positive for IgE to αGal, with levels ranging from 0.4 to 294 kU/l. IgE binding to αGal was inhibited by raw pork kidney extract (mean, 77%; range, 55-87%), which showed a high amount of αGal determinants. CONCLUSIONS: Pork or beef kidney anaphylaxis is related to αGal IgE. Its peculiar severity could be due to an elevated content of αGal epitopes in kidney.

An apple a day ... chronic glossitis in a 4-year-old boy.

We report a case of chronic glossitis in a 4-year-old boy due to scurvy. The boy showed up in our department with a patchy depapillated tongue. A detailed dietary history revealed an unbalanced diet without any fruit or vegetable. The biological investigations showed a low serum ascorbic acid. The boy was treated by oral ascorbic acid during 15 days. The glossitis improved within one week and serum levels of vitamin C returned to the normal range. In industrial countries, scurvy became a rare disease in healthy children. However, since a few years, cases are reported in children and teenagers with unbalanced diet coming from economically favoured families. These extreme cases are one of the signs of a more general deterioration of dietary habits in paediatric populations in our societies. This emphasizes the importance of effective nutritional education programs aimed towards both parents and children.

A novel immunoassay using recombinant allergens simplifies peanut allergy diagnosis.

BACKGROUND: Double-blind placebo-controlled food challenge (DBPCFC) is currently considered the gold standard for peanut allergy diagnosis. However, this procedure that requires the hospitalization of patients, mostly children, in specialized centers for oral exposure to allergens may cause severe reactions requiring emergency measures. Thus, a simpler and safer diagnosis procedure is needed. The aim of this study was to evaluate the diagnostic performance of a new set of in vitro blood tests for peanut allergy. METHODS: The levels of IgE directed towards peanut extract and recombinant peanut allergens Ara h 1, Ara h 2, Ara h 3, Ara h 6, Ara h 7, and Ara h 8 were measured in 3 groups of patients enrolled at 2 independent centers: patients with proven peanut allergy (n=166); pollen-sensitized subjects without peanut allergy (n=61), and control subjects without allergic disease (n=10). RESULTS: Seventy-nine percent of the pollen-sensitized patients showed IgE binding to peanut, despite their tolerance to peanut. In contrast, combining the results of specific IgE to peanut extract and to recombinant Ara h 2 and Ara h 6 yielded a peanut allergy diagnosis with a 98% sensitivity and an 85% specificity at a positivity threshold of 0.10 kU/l. Use of a threshold of 0.23 kU/l for recombinant Ara h 2 increased specificity (96%) at the cost of sensitivity (93%). CONCLUSION: A simple blood test can be used to diagnose peanut allergy with a high level of precision. However, DBPCFC will remain useful for the few cases where immunological and clinical observations yield conflicting results.

The Rac1- and RhoG-specific GEF domain of Trio targets filamin to remodel cytoskeletal actin.

Rho GTPases control actin reorganization and many other cellular functions. Guanine nucleotide-exchange factors (GEFs) activate Rho GTPases by promoting their exchange of GDP for GTP. Trio is a unique Rho GEF, because it has separate GEF domains, GEFD1 and GEFD2, that control the GTPases RhoG/Rac1 and RhoA, respectively. Dbl-homology (DH) domains that are common to GEFs catalyse nucleotide exchange, and pleckstrin-homology (PH) domains localize Rho GEFs near their downstream targets. Here we show that Trio GEFD1 interacts through its PH domain with the actin-filament-crosslinking protein filamin, and localizes with endogenous filamin in HeLa cells. Trio GEFD1 induces actin-based ruffling in filamin-expressing, but not filamin-deficient, cells and in cells transfected with a filamin construct that lacks the Trio-binding domain. In addition, Trio GEFD1 exchange activity is not affected by filamin binding. Our results indicate that filamin, as a molecular target of Trio, may be a scaffold for the spatial organization of Rho-GTPase-mediated signalling pathways.

A murine model of cow's milk protein-induced allergic reaction: use for safety assessment of hidden milk allergens.

BACKGROUND: Masked allergens in processed food products can lead to severe allergic reactions following unintentional ingestion. We sought to develop a murine model for the detection of hidden cow's milk proteins (CMP). This study aimed to induce cow's milk allergy in mice, to characterize the anaphylaxis induced by CMP in this model, and to validate its reliability using three margarines manufactured with (A) or without (B, C) milk, sharing the same production line. MATERIALS AND METHODS: Three-week-old BALB/c mice were sensitized intragastrically with CMP plus cholera toxin and boosted 6 times at weekly intervals. CMP-sensitization status was monitored by skin tests, and measurement of CMP-specific IgE and IgG1 levels. On day 44, the minimal threshold of clinical reactivity to CMP in terms of anaphylaxis was determined by performing a dose response of intraperitoneal CMP challenge. Under the same conditions, anaphylaxis was evaluated in CMP-sensitized mice after challenge with protein extracts of margarines A, B or C. RESULTS: Sensitization to CMP was demonstrated by positive skin tests and increased CMP-specific IgE and IgG1. The minimal clinical reactivity threshold corresponding to 0.1 mg CMP elicited detectable anaphylaxis evidenced by clinical symptoms, a decrease in breathing frequency, and increased plasma histamine upon challenge. Similarly, challenges with margarine A containing CMP demonstrated anaphylaxis, whereas those with B or C did not elicit any detectable allergic reaction. CONCLUSION: This study shows that our murine model of CMP-induced anaphylaxis is useful for investigating the allergenic activity and the assessment of margarines with respect to milk.

Sesame allergy threshold dose distribution.

BACKGROUND: Sesame is a relevant food allergen in France. Compared to other allergens there is a lack of food challenge data and more data could help sesame allergy risk management. The aim of this study is to collect more sesame challenge data and investigate the most efficient food challenge method for future studies. METHOD: Records of patients at University Hospital in Nancy (France) with objective symptoms to sesame challenges were collected and combined with previously published data. An estimation of the sesame allergy population threshold was calculated based on individual NOAELs and LOAELs. Clinical dosing schemes at Nancy were investigated to see if the optimal protocol for sesame is currently used. RESULTS: Fourteen patients (10 M/4 F, 22 ± 14.85 years old) with objective symptoms were added to previously published data making a total of 35 sesame allergic patients. The most sensitive patient reacted to the first dose at challenge of 1.02 mg sesame protein. The ED05 ranges between 1.2 and 4.0 mg of sesame protein (Log-Normal, Log-Logistic, and Weibull models) and the ED10 between 4.2 and 6.2 mg. The optimal food challenge dosing scheme for sesame follows semi-log dose increases from 0.3 to 3000 mg protein. CONCLUSION: This article provides a valuable update to the existing clinical literature regarding sesame NOAELs and LOAELs. Establishment of a population threshold for sesame could help in increasing the credibility of precautionary labelling and decrease the costs associated with unexpected allergic reactions. Also, the use of an optimal dosing scheme would decrease time spent on diagnostic and thereafter on the economic burden of sesame allergy diagnosis.

A new mutation in the pufL gene responsible for the terbutryn resistance phenotype in Rubrivivax gelatinosus.

Rubrivivax gelatinosus is a facultative phototrophic non-sulfur bacterium belonging to the beta subclass of the purple bacteria. A terbutryn-resistant mutant of R. gelatinosus has been isolated and characterized. Increased resistance levels to terbutryn (300-fold), atrazine (6-fold) and o-phenanthroline (3-fold) were observed for the mutant compared with wild type. Sequence analysis of the mutant revealed a new mutation in the pufL gene coding for the L subunit of the reaction centre (RC) at codon 192 leading to an amino-acid substitution from Gly in the wild type to Asp in the mutant. This substitution is located in the D helix of the L subunit, suggesting an interaction between terbutryn and this part of the polypeptide in the RC of R. gelatinosus. This is the first report of a mutation leading to herbicide resistance and affecting the D helix in purple bacteria. Furthermore R. gelatinosus wild type is highly sensitive to o-phenanthroline compared with other purple bacteria (Rhodobacter capsulatus and Rhodobacter sphaeroides). Sequence comparison of the L subunit from six purple bacteria in which o-phenanthroline sensitivity was measured suggests that SerL226 might be responsible for this phenotype.

Mutation in phenol-type herbicide resistance maps within the psbA gene in Synechocystis 6714.

A Synechocytis 6714 mutant resistant to the phenol-type herbicide ioxynil was isolated and characterized. Sensitivity to DCMU and atrazine was tf measured in whole cells and isolated thylakoids. The mutant presents the same sensitivity to atrazine as the wild type and a slightly increased sensitivity to DCMU. A point mutation has been found at codon 266 in the psbAI coding locus (AAC to ACC) resulting in an amino acid change from asparagine to threonine in the D1 protein.

Resistance to nitrophenolic herbicides and metronidazole in the cyanobacterium Synechocystis sp. PCC 6803 as a result of the inactivation of a nitroreductase-like protein encoded by drgA gene.

Dinoseb is a herbicide known to inhibit photosystem II electron transfer like DCMU, triazine and phenolic-type herbicides. The mutant Din7 of the cyanobacterium Synechocystis sp. PCC 6803, selected for resistance to dinoseb, and the mutant Ins2, constructed by the insertion of the kanamycin resistance cassette into the drgA gene, were cross-resistant to other nitrophenolic herbicides (DNOC, 2,4-dinitrophenol) and to the cell inhibitor metronidazole but not to the photosystem II inhibitors DCMU or ioxynil. The Din7 mutant had the same characteristics of photosystem II inhibition by dinoseb as the wild type. This result suggested the existence of another site for dinoseb inhibition. The wild type cells modified dinoseb to a non-toxic product that gave an absorption spectrum similar to that of dithionite treated dinoseb containing reduced nitro groups. In contrast, the Din7 mutant did not modify dinoseb. These phenomena were controlled by the drgA gene encoding a protein which showed similarity to several enzymes having nitroreductase activity. The addition of superoxide dismutase to the medium relieved the toxic effect of dinoseb in wild type cells but not in Din7. It is proposed that in wild type cells of Synechocystis sp. PCC 6803 the DrgA protein is involved in detoxification of dinoseb via the reduction of the nitro group(s) and this process is accompanied by the formation of toxic superoxide anions. Mutations blocking the activity of the DrgA protein lead to the development of resistance to nitrophenolic herbicides and metronidazole.

Binding of a native titin fragment to actin is regulated by PIP2.

Titin is a giant protein which extends from Z-line to M-line in striated muscles. We report here the purification of a 150-kDa titin fragment, obtained after V8 protease treatment of myofibrils. This polypeptide was located at the N1-line level, in a titin part known to exhibit stiff properties correlated to an association with actin. By solid or liquid phase binding assays and cosedimentation, we have clearly demonstrated a direct, saturable and relative high affinity binding of the native titin fragment to F-actin. The 150-kDa titin fragment was also shown to accelerate actin polymerization. Furthermore, the actin-titin interaction was found to be inhibited by phosphoinositides.

Differential sensitivity of bicarbonate-reversible formate effects on herbicide-resistant mutants of Synechocystis 6714.

Herbicide-resistant mutants of the cyanobacterium Synechocystis 6714, that are altered in specific amino acids in their D-1 protein, shows differential sensitivity to formate treatment. Measurements on oxygen yield in a sequence of flashes, chlorophyll (Chl) a fluorescence transients and Chl a fluorescence yield decay after a flash reveal that the resistance of cells to formate treatment is in the following (highest to lowest) order: [double mutant] A251V/F211S (Az V) greater than [single mutant] F211S (Az I) congruent to wild type greater than [single mutant] S264A (DCMU II-A). Significance of these results in terms of overlapping between the herbicide and bicarbonate binding niches on the D-1 protein is discussed.

Functional alterations in sarcoplasmic reticulum membranes of magnesium-deficient rat skeletal muscle as consequences of free radical-mediated process.

Free radical-induced physiopathologies are generally thought to be mediated by membrane injuries. Using a pro-oxidant model induced by dietary magnesium deficiency, we have recently shown that skeletal muscle lesions occurred with a rise in the calcium level and enhanced free radical production. In this study, we investigated the physicochemical and biochemical properties of sarcoplasmic reticulum membranes isolated from hind limb muscles of weanling male rats pair fed magnesium-deficient or control diets for 12 d. The calcium-induced calcium efflux from preloaded vesicles was increased in membranes isolated from Mg-deficient rat muscle. In agreement with this latter observation, we demonstrated increased ryanodine binding affinity of the calcium channel. The Ca2(+)-ATPase activity of the pump was shown to be reduced. The viscosity state of the membranes, assessed by 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy, was significantly increased in Mg-deficient membranes. Moreover, these membranes demonstrated an increased content of protein carbonyls as compared with controls. These functional as well as structural changes are closed to those described in sarcoplasmic reticulum membranes oxidatively modified in vitro. Together, these data fitted well with the concept that free radical-induced membrane damages resulting in calcium overload may be at the origin of skeletal muscle lesion during Mg-deficiency.

Towards the understanding of the function of Rb sphaeroides Y wild type reaction center: gene cloning, protein and detergent structures in the three-dimensional crystals.

We report various experiments aimed at the resolution of the 3-dimensional structure of the photosynthetic reaction center from wild type Y Rhodobacter sphaeroides. The genes encoding the L and M polypeptides have been cloned and sequenced. They bear 2 mutations each when compared to those already sequenced in another Rb sphaeroides strain (2.4.1). In the L gene, these codon changes are silent. In the M gene, one is silent and the other one leads to a Leu-Met substitution at position 140. At the present stage of the refinement of the X-ray data (0.3 nm resolution) the structure of the Y reaction center is shown to be highly similar to that of the Rhodopseudomonas viridis reaction center. The binding of spheroidene on the M side of the Y reaction center is shown to be determined by hydrophobic interactions with neighboring amino acids and by steric factors. Preliminary results concerning the localization of the detergent (beta-octylglucoside) in the unit cell are presented. This method combines low angle neutron scattering at different contrasts in H2O/D2O with X-ray crystallographic data.

Analysis of long-range structural effects induced by DNase-I interaction with actin monomeric form or complexed to CapZ.

Two fundamental properties of monomeric actin were examined in this study, ie its interaction with DNase-I, and the inhibition of endonuclease activity consecutive to the association of the two molecules. In particular, the topological independence between catalytic site of DNase-I and interface with actin, structural changes in actin monomer and the absence of conformational changes in DNase-I were described. We demonstrated a loss of flexibility of antigenic structures in actin subdomain I (ie epitopes 18-28 and 95-105) as well as modification in the exposure of Cys10 and Cys374 after DNase-I binding. Furthermore, the conformational changes induced by DNase-I into the actin molecule weakened the interaction of CapZ to its binding site located in the C-terminal region of actin monomer. These structural changes were time-dependent. When actin was cleaved in the DNase-I binding loop (sequence 38-52) at position 42 by E coli A2 strain protease, a tight DNase-I binding to split actin and the conformational changes were still observed, whereas the DNase-I inhibition activity was completely abolished. Finally, when we substitute Ca2+ by Mg2+ (ATP-Mg2+ monomeric actin) which induces a tighter conformation of actin and partially restores the inhibitory ability of split actin, long-range conformational effects of DNase-I are prevented and the ternary complex DNase-I-actin-CapZ is obtained.

Exacerbated immune stress response during experimental magnesium deficiency results from abnormal cell calcium homeostasis.

The aim of this study was to assess the potential mechanism underlying the enhanced inflammatory processes during magnesium deficit. In this study, exacerbated response to live bacteria and platelet activating factors was shown in rats fed a magnesium-deficient diet. Peritoneal cells from these animals also showed enhanced superoxide anion production and calcium mobilising potency following in vitro stimulation. The latter effect occurred very early in the course of magnesium deficiency. These studies first showed that an abnormal calcium handling induced by extracellular magnesium depression in vivo may be at the origin of exacerbated inflammatory response.

Postmortem degradation of white fish skeletal muscle (sea bass, Dicentrarchus labrax): fat diet effects on in situ dystrophin proteolysis during the prerigor stage.

All during fish postmortem evolution, structural muscle proteins are targets for various proteases. During the prerigor period (24 hours at 4 degrees C for sea bass), cytoskeletal proteins are affected by the first proteolytic events. These cleavages disrupt connections between myofibrils and the extracellular matrix, induce segmentation of myofibril cores, and modify the rheological properties of tissue. Dystrophin, a cytoskeletal actin-binding protein, is a relevant in situ marker for muscular proteolysis in the prerigor period. The immunodetection of dystrophin allowed the monitoring of early proteolysis during fish storage. Using antidystrophin antibodies directed toward the carboxy-terminal region, a highly sensitive domain exposed to calpain activity, we showed that proteolysis kinetics are strongly influenced by the muscular lipid content. In particular, comparison between low-fat diets (11.3% lipid) and high-fat diets (30% lipid), used during sea bass farming (90 days), revealed a faster proteolysis rate during the first 8 hours of storage at 0 degrees C with the high-fat diet. The origin of this faster proteolysis is discussed on the basis of a possible activation or translocation of calpains related to lipid accumulation in muscle fibers and cytoskeleton alterations.

Coevolution of actin and associated proteins: an alpha-actinin-like protein in a cyanobacterium (Spirulina platensis).

Actin, together with associated proteins, such as myosin, cross-linking or capping proteins, has been observed in all eukaryotic cells. Presence of actin or actin-like proteins has also been reported in prokaryotic organisms belonging to the cyanobacteria. Our aim was first to extend the characterization of an actin-like protein to another prokaryotic cell, i.e. Spirulina, then to compare the antigenic reactivity of this new protein with that of Synechocystis and skeletal actins. We observed that some of the conserved antigenic epitopes corresponded to actin regions known to interact with cross-linking proteins. We also report for the first time that alpha-actinin and filamin purified from chicken gizzard both interact with a prokaryotic actin-like protein. Finally, we searched for the occurrence of a cross-linking protein in these cyanobacteria and identified a 105-kDa protein as an alpha-actinin-like protein using specific antibodies.