Comprehensive molecular level characterization of protein- and polyphenol-rich tara (Caesalpinia spinosa) seed germ flour suggests novel hypothesis about possible accidental hazards.

Tara (Caesalpinia spinosa, Leguminosae) seed germ (TSG), a by-product of tara gum (E417) extraction, has been used as a protein- and polyphenol-rich food ingredient for human and animal nutrition. Nevertheless, TSG is the alleged culprit for a recent foodborne outbreak of even severe acute illnesses that have affected hundreds of individuals in the USA, perhaps triggered by nonprotein amino acids such as baikiain. Herein, the composition of TSG has been characterized at molecular level, with a focus on proteins, phenolics, lipids, and mineral composition. TSG contains 43.4 % (w/w) proteins, tentatively identified for the first time by proteomics, and 14 % lipids, consisting of 83.6 % unsaturated fatty acids, especially linoleic acid. Ash is surprising high (6.5 %) because of an elevated concentration of P, K, Ca, and Mg. The detection of a rare earth element such as gadolinium (Gd, 1.6 mg kg-1), likely sourced from anthropogenic pollution, suggests alternative hypotheses for the origin of TSG hazards.

Insights into the Structure-Capacity of Food Antioxidant Compounds Assessed Using Coulometry.

CDAC (coulometrically determined antioxidant capacity) involves the determination of the antioxidant capacity of individual compounds or their mixtures using constant-current coulometry, with electrogenerated Br2 as the titrant, and biamperometric detection of the endpoint via Br2 excess. CDAC is an accurate, sensitive, rapid, and cheap measurement of the mol electrons (mol e-) transferred in a redox process. In this study, the CDAC of 48 individual antioxidants commonly found in foods has been determined. The molar ratio CDAC (CDACχ, mol e- mol-1) of representative antioxidants is ranked as follows: tannic acid > malvidin-3-O-glucoside ≃ curcumin > quercetin > catechin ≃ ellagic acid > gallic acid > tyrosol > BHT ≃ hydroxytyrosol > chlorogenic acid ≃ ascorbic acid ≃ Trolox®. In many cases, the CDACχ ranking of the flavonoids did not comply with the structural motifs that promote electron or hydrogen atom transfers, known as the Bors criteria. As an accurate esteem of the stoichiometric coefficients for reactions of antioxidants with Br2, the CDACχ provides insights into the structure-activity relationships underlying (electro)chemical reactions. The electrochemical ratio (ER), defined as the antioxidant capacity of individual compounds relative to ascorbic acid, represents a dimensionless nutritional index that can be used to estimate the antioxidant power of any foods on an additive basis.

Gliadin-reactive T cells in Italian children from preventCD cohort at high risk of celiac disease.

BACKGROUND: Newborns at high risk of celiac disease (CD) were recruited in Italy in the context of the PreventCD study and closely monitored for CD, from 4 months up to a mean age of 8 years at follow-up. The aim of our study was to investigate intestinal T-cell reactivity to gliadin at the first clinical and/or serological signs of CD. METHODS: Gliadin-reactive T-cell lines were generated from intestinal biopsies of 19 HLA-DQ2-or HLA-DQ8-positive children. At biopsy, 11 children had a diagnosis of acute CD, two of potential CD, and six were non-celiac controls. Immune reactivity was evaluated against gliadin and known immunogenic peptides from α-, γ-, or ω-gliadins. The role of deamidation by transglutaminase (tTG) in determining the immunogenicity of gliadin was also investigated. RESULTS: Most of the children with CD (either acute or potential) had an inflammatory response to gliadin. Notably, signs of T-cell reactivity to gliadin were also found in some non-celiac subjects, in which IFN-γ responses occurred mainly when regulatory IL-10 and TGF-ß cytokines were blocked. Interestingly, PreventCD children reacted to gliadin peptides found active in adult CD patients, and tTG deamidation markedly enhanced gliadin recognition. CONCLUSIONS: T cells reactive to gliadin can be detected in the intestine of children at high risk of developing CD, in some cases also in the presence of a normal mucosa and negative CD-associated antibodies. Furthermore, children at a very early stage of CD recognize the same gliadin epitopes that are active in adult CD patients. Tissue transglutaminase strongly enhances gluten T-cell immunogenicity in early CD.

Protective effects of ID331 Triticum monococcum gliadin on in vitro models of the intestinal epithelium.

A growing interest in developing new strategies for preventing coeliac disease has motivated efforts to identify cereals with null or reduced toxicity. In the current study, we investigate the biological effects of ID331 Triticum monococcum gliadin-derived peptides in human Caco-2 intestinal epithelial cells. Triticum aestivum gliadin derived peptides were employed as a positive control. The effects on epithelial permeability, zonulin release, viability, and cytoskeleton reorganization were investigated. Our findings confirmed that ID331 gliadin did not enhance permeability and did not induce zonulin release, cytotoxicity or cytoskeleton reorganization of Caco-2 cell monolayers. We also demonstrated that ID331 ω-gliadin and its derived peptide ω(105-123) exerted a protective action, mitigating the injury of Triticum aestivum gliadin on cell viability and cytoskeleton reorganization. These results may represent a new opportunity for the future development of innovative strategies to reduce gluten toxicity in the diet of patients with gluten intolerance.

Transport across Caco-2 monolayers of peptides arising from in vitro digestion of bovine milk proteins.

The entire panel of peptides produced from caseins (CN) and whey proteins (WP) that survive in vitro sequential gastro-pancreatic digestion and translocate across monolayers of Caco-2 cells, used as a model of the intestinal epithelium, has been characterised by HPLC and mass spectrometry. Among the milk-derived bioactive peptides, only minor amounts of mono-phosphorylated peptides arising from αs1- and ß-CN were detected. The absorption behaviour of two resistant ß-lactoglobulin (ß-Lg) domains, ß-Lg 125-135 and ß-Lg 40-60, was studied in detail using synthetic peptides. The IgE-binding properties of the digests recovered from the apical and basolateral monolayer compartments were evaluated by dot-blot, using the sera of milk allergic children (N=5). Outcomes indicated ß-Lg 127-135 as a possible "immune sensitising factor"in vivo. The almost complete loss of the IgE-affinity of CN and WP after digestion points out the need to design in vivo experiments to track the metabolic fate of dietary proteins.

Structural analysis and Caco-2 cell permeability of the celiac-toxic A-gliadin peptide 31-55.

Celiac disease is a chronic enteropathy caused by the ingestion of wheat gliadin and other cereal prolamines. The synthetic peptides 31-43 (P31-43) and 31-49 (P31-49) from A-gliadin are considered to be model peptides for studying innate immunity in celiac disease. Our previous study demonstrated that P31-43 and P31-49 are encrypted within peptide 31-55 (P31-55), which is naturally released from gastropancreatic digestion and is not susceptible to hydrolysis by brush border membrane enzymes. Here, we analyzed the permeability of P31-55 through the epithelial cell layer of confluent Caco-2 cells using high-performance liquid chromatography, mass spectrometry, and fluorescence-activated cell sorting. Twenty-three percent of the P31-55 added to the apical chamber was transported to the basolateral chamber after 4 h of incubation without being degraded by hydrolysis. Treatment of Caco-2 cells with whole gliadin digests extracted from a common wheat cultivar increased the epithelial P31-55 translocation by approximately 35%. Moreover, we observed an atypical chromatographic profile consisting of a double peak. Chromatography using different column temperatures and circular dichroism highlighted the presence of more conformational structures around the amide bond of the two adjacent prolines 38 and 39. These findings confirm that P31-55 is gastrointestinally resistant and is permeable across a Caco-2 monolayer. Moreover, we hypothesize that the various conformations of P31-55 may play a role in the activation of innate immunity.

Immunogenic peptides can be detected in whole gluten by transamidating highly susceptible glutamine residues: implication in the search for gluten-free cereals.

Tissue transglutaminase (TG2) plays a central role in celiac disease (CD) pathogenesis by strongly enhancing the immunogenicity of gluten, the CD-triggering antigen. By deamidating specific glutamine (Q) residues, TG2 favors the binding of gluten peptides to DQ2/8 molecules and, subsequently, their recognition by cognate T cells. Six peptides were previously identified within wheat gliadin whole extracts by tagging the TG2-susceptible Q residues with monodansylcadaverine (MDC) and nanospray tandem mass spectrometry (nanoESI-MS/MS). The immunogenicity of these peptides was next tested in gliadin-specific T-cell lines established from CD intestinal mucosa. Four peptides, corresponding to known epitopes of α- and γ-gliadins, induced cell proliferation and interferon (IFN)-γ production. Interestingly, one of the two non-T-cell stimulatory peptides corresponded to the 31-49 α-gliadin peptide implicated in the innate immune activation in CD mucosa. This study describes a strategy for identifying immunogenic gluten peptides potentially relevant for CD pathogenesis in protein extracts from wheat and other edible cereals.

Selection of sourdough lactobacilli with antifungal activity for use as biopreservatives in bakery products.

Two hundred and sixteen LAB cultures from sourdoughs and dough for bread and panettone production were screened for in vitro antifungal properties against three indicator cultures ascribed to Aspergillus japonicus , Eurotium repens , and Penicillium roseopurpureum , isolated from bakery environment and moldy panettone. Nineteen preselected isolates were subjected to minimum inhibitory concentration determination against the indicator cultures. Sourdoughs prepared with the two most promising strains, identified as Lactobacillus rossiae LD108 and Lactobacillus paralimentarius PB127, were characterized. The sourdough extracts were subjected to HPLC analysis coupled with a microtiter plate bioassay against A. japonicus to identify the active fractions. MALDI-TOF MS analysis revealed the occurrence of a series of peptides corresponding to wheat α-gliadin proteolysis fragments in the active fraction from L. rossiae LD108 sourdough. The ability to prevent mold growth on bread was demonstrated for both strains, whereas L. rossiae LD108 also inhibited mold growth on panettone.

Lactosylated casein phosphopeptides as specific indicators of heated milks.

Casein phosphopeptides (CPP) were identified in small amounts in milks heated at various intensities by using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. CPP selectively concentrated on hydroxyapatite (HA) were regenerated using phosphoric acid mixed in the matrix. Unphosphorylated peptides not retained by HA were removed by buffer washing. This procedure enhanced the MALDI signals of CPP that are ordinarily suppressed by the co-occurrence of unphosphorylated peptides. CPP, belonging to the ß-casein (CN) family, i.e., (f1-29) 4P, (f1-28) 4P, and (f1-27) 4P, and the α(s2)-CN family, i.e., (f1-21) 4P and (f1-24) 4P, were observed in liquid and powder milk. The lactosylated counterparts were specific to intensely heated milks, but absent in raw and thermized/pasteurized milk. Most CPP with C-terminal lysines probably arose from the activity of plasmin; an enzyme most active in casein hydrolysis. A CPP analogue was used as the internal standard. The raw milk signature peptide ß-CN (f1-28) 4P constituted ~4.3% of the total ß-CN. Small amounts of lactosylated peptides, which varied with heat treatment intensity, were detected in the milk samples. The limit of detection of ultra-high-temperature milk adjunction in raw or pasteurized milk was ~10%.

Mass spectrometry-based identification of the tumor antigen UN1 as the transmembrane CD43 sialoglycoprotein.

The UN1 monoclonal antibody recognized the UN1 antigen as a heavily sialylated and O-glycosylated protein with the apparent molecular weight of 100-120 kDa; this antigen was peculiarly expressed in fetal tissues and several cancer tissues, including leukemic T cells, breast, and colon carcinomas. However, the lack of primary structure information has limited further investigation on the role of the UN1 antigen in neoplastic transformation. In this study, we have identified the UN1 antigen as CD43, a transmembrane sialoglycoprotein involved in cell adhesion, differentiation, and apoptosis. Indeed, mass spectrometry detected two tryptic peptides of the membrane-purified UN1 antigen that matched the amino acidic sequence of the CD43 intracellular domain. Immunological cross-reactivity, migration pattern in mono- and bi-dimensional electrophoresis, and CD43 gene-dependent expression proved the CD43 identity of the UN1 antigen. Moreover, the monosaccharide GalNAc-O-linked to the CD43 peptide core was identified as an essential component of the UN1 epitope by glycosidase digestion of specific glycan branches. UN1-type CD43 glycoforms were detected in colon, sigmoid colon, and breast carcinomas, whereas undetected in normal tissues from the same patients, confirming the cancer-association of the UN1 epitope. Our results highlight UN1 monoclonal antibody as a suitable tool for cancer immunophenotyping and analysis of CD43 glycosylation in tumorigenesis.

Hydroxyapatite as a concentrating probe for phosphoproteomic analyses.

A novel method for the selective enrichment of casein phosphoproteins/phosphopeptides (CPP) from complex mixtures is reported herein. This method employs ceramic hydroxyapatite (HA) as a solid-phase adsorbent to efficiently capture phosphoproteins and CPP from complex media. Casein was chosen as the model phosphoprotein to test the protocol. CPP immobilized on HA microgranules formed a complex that was included in the matrix-assisted laser desorption/ionization mass spectrometry (MALDI) matrix before desorbing directly from the well plate. Casein fractions with different levels of phosphorylation were desorbed based upon the specific concentration of trifluoroacetic acid (TFA) included in the MALDI matrix. The HA-bound casein enzymolysis was performed in situ with trypsin to remove non-phosphorylated peptides and isolate the immobilized CPP. The latter were recovered by centrifugation, dried, and co-crystallized with a 1% phosphoric acid (PA) solution in the matrix that was appropriate for detecting CPP in MALDI-MS spectra. This approach for the selection of casein/CPP resulted in the identification of 32 CPP by MALDI-time of flight (TOF). The analytical process involved two steps requiring ∼2h, excluding the time required for the enzymatic reaction. The alkaline phosphatase (AP)-assisted de-phosphorylation of tryptic CPP allowed the phosphorylation level of peptides to be calculated concurrently with MALDI-TOF MS and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS). The effectiveness of the extraction procedure assayed on eggshell phosphoproteins resulted in the identification of 5 phosphoproteins and 14 derived phosphopeptides with a phosphoprotein global recovery of ∼70% at least.

Toward milk speciation through the monitoring of casein proteotypic peptides.

The possibility of detecting extraneous milk in singles species cheese-milk has been explored. A mass spectrometry (MS)-based procedure has been developed to detect 'signature peptides', corresponding to the predefined subset of 'proteotypic peptides', as matchless analytical surrogates of the parent caseins. Tryptic digests of skimmed milk samples from four species were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Amongst the candidate signature peptides that are able to differentiate milks from the four species, the alpha(s1)-casein (CN) f8-22 peptide was selected as a convenient marker for bovine, ovine and water buffalo milk while the f4-22 peptide was selected as a marker for the two caprine alpha(s1)-CN A and B variants, which differ by a Pro(16) (B)->Leu(16) (A) substitution. MALDI analysis of the digest allowed the detection of alpha(s1)-CN f8-22 and caprine alpha(s1)-CN f4-22. The accurate evaluation of caprine milk in a quaternary mixture required the development of a liquid chromatography/electrospray ionization (LC/ESI)-MS procedure. Five synthetic signature peptide analogues, which differed from their natural counterparts by a single amino acid substitution, were used as internal standards to quantify the alpha(s1)-CN, which was chosen as a reference milk protein, from the different species. The limits of detection were 0.5% (1% for caprine) for either the MALDI or the LC/ESI-MS method. The isotopic-label-free quantification of isoform- or variant-specific signature peptides has disclosed a convenient approach for targeting proteins in complex mixtures.

In vivo targeting and growth inhibition of the A20 murine B-cell lymphoma by an idiotype-specific peptide binder.

B-cell lymphoma is a clonal expansion of neoplastic cells that may result in fatal outcomes. Here, we report the in vivo targeting and growth inhibition of aggressive A20 murine B-cell lymphoma by idiotype-specific peptide pA20-36. pA20-36 was selected from random peptide libraries and bound specifically to the B-cell receptor (BCR) of A20 cells in mice engrafted with A20 lymphoma, as shown by histology and positron emission tomographic analysis. BCR cross-linking of A20 cells with pA20-36 resulted in massive apoptosis of targeted tumor cells and in an increased survival of the diseased animals without any detectable evidence of toxicity. The pA20-36 treatment reverted the immune suppression of the tumor microenvironment as shown by reduced expression of vascular endothelial growth factor, interleukin-10, and transforming growth factor-beta cytokines together with a lower number of CD11b+Gr-1+ inhibitor myeloid-derived suppressor cells and Foxp3+CD4+ Treg cells. Furthermore, pA20-36 treatment was associated with an increased number of tumor-infiltrating, activated CD8+ T cells that exerted a tumor-specific cytolytic activity. These findings show that a short peptide that binds specifically to the complementarity-determining regions of the A20 BCR allows in vivo detection of neoplastic cells together with significant inhibition of tumor growth in vivo.

Peptides surviving the simulated gastrointestinal digestion of milk proteins: biological and toxicological implications.

Resistance to proteases throughout the gastrointestinal (GI) tract is a prerequisite for milk-derived peptides to exert biological activities. In this work an in vitro multi-step static model to simulate complete digestion of the bovine milk proteins has been developed. The experimental set-up involved the sequential use of: (i) pepsin, (ii) pancreatic proteases, and (iii) extracts of human intestinal brush border membranes, in simulated gastric, duodenal and jejuneal environments, respectively. Enzymatic concentrations and reaction times were selected in order to closely reproduce the in vivo conditions. The aim was to identify the peptide candidates able to exhibit significant bioactive effects. Casein and whey protein peptides which survived the in vitro GI digestion have been identified by the combined application of HPLC and mass spectrometry techniques. While the permanence of the main potentially bioactive peptides from both casein and whey proteins was found of limited physiological relevance, the high resistance to proteolysis of specific regions of beta-lactoglobulin (beta-Lg), and especially that of the peptide beta-Lg f125-135, could have implications for the immunogenic action of beta-Lg in the insurgence of cow's milk allergy.

Intestinal T cell responses to gluten peptides are largely heterogeneous: implications for a peptide-based therapy in celiac disease.

The identification of gluten peptides eliciting intestinal T cell responses is crucial for the design of a peptide-based immunotherapy in celiac disease (CD). To date, several gluten peptides have been identified to be active in CD. In the present study, we investigated the recognition profile of gluten immunogenic peptides in adult HLA-DQ2(+) celiac patients. Polyclonal, gliadin-reactive T cell lines were generated from jejunal mucosa and assayed for both proliferation and IFN-gamma production in response to 21 peptides from wheat glutenins and alpha-, gamma-, and omega-gliadins. A magnitude analysis of the IFN-gamma responses was performed to assess the hierarchy of peptide potency. Remarkably, 12 of the 14 patients recognized a different array of peptides. All alpha-gliadin stimulatory peptides mapped the 57-89 N-terminal region, thus confirming the relevance of the known polyepitope 33-mer, although it was recognized by only 50% of the patients. By contrast, gamma-gliadin peptides were collectively recognized by the great majority (11 of 14, 78%) of CD volunteers. A 17-mer variant of 33-mer, QLQPFPQPQLPYPQPQP, containing only one copy of DQ2-alpha-I and DQ2-alpha-II epitopes, was as potent as 33-mer in stimulating intestinal T cell responses. A peptide from omega-gliadin, QPQQPFPQPQQPFPWQP, although structurally related to the alpha-gliadin 17-mer, is a distinct epitope and was active in 5 out of 14 patients. In conclusion, these results showed that there is a substantial heterogeneity in intestinal T cell responses to gluten and highlighted the relevance of gamma- and omega-gliadin peptides for CD pathogenesis. Our findings indicated that alpha-gliadin (57-73), gamma-gliadin (139-153), and omega-gliadin (102-118) are the most active gluten peptides in DQ2(+) celiac patients.

Fast screening and quantitative evaluation of internally deleted goat alphas1-casein variants by mass spectrometric detection of the signature peptides.

Currently, the internally deleted caprine alphas1-casein (alphas1-CN) variants F and G, associated with low casein expression, are detected by means of ordinary descriptive techniques. No relevant procedure is available to detect internally deleted goat alphas1-CN in bulk milks. The availability of full-length and alphas1-CN F and G variants allowed us to further investigate this issue. Using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and high-performance liquid chromatography (HPLC)/electrospray ionization (ESI)-MS and ESI-MS/MS, tryptic signature peptides alphas1-CN F f59-63/f43-63, alphas1-CN G f4-20/f4-21, alphas1-CN B2 f4-22 Pro16 and alphas1-CN A f4-22 Leu16 were identified. This also helped to solve the interesting question of how the casein variants contribute to the composition of goat's bulk milk. Synthetic peptide analogues with ionization efficiency equivalent to that of tryptic junction peptides were used as internal standards to evaluate alphas1-CN variants, either individually or globally, using bulk milk from a single goat breed as a model system. Here, alphas1-CN F accounted for 0.15+/-0.08% and the alphas1-CN G variant was missing or below the 0.10% detection limit. The analysis of six samples confirmed that alphas1-CN G was missing and that alphas1-CN F occurred at a low frequency in hybrid and local breed bulk milks from Mediterranean areas. In conclusion, a quantitative MS-based application of the signature peptides for full-length and internally deleted variants in goat's casein is provided. The strategy is also suggested for the determination of splice variants in any biological sample.

Physical and functional characterization of the genetic locus of IBtk, an inhibitor of Bruton's tyrosine kinase: evidence for three protein isoforms of IBtk.

Bruton's tyrosine kinase (Btk) is required for B-cell development. Btk deficiency causes X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Btk lacks a negative regulatory domain and may rely on cytoplasmic proteins to regulate its activity. Consistently, we identified an inhibitor of Btk, IBtk, which binds to the PH domain of Btk and down-regulates the Btk kinase activity. IBtk is an evolutionary conserved protein encoded by a single genomic sequence at 6q14.1 cytogenetic location, a region of recurrent chromosomal aberrations in lymphoproliferative disorders; however, the physical and functional organization of IBTK is unknown. Here, we report that the human IBTK locus includes three distinct mRNAs arising from complete intron splicing, an additional polyadenylation signal and a second transcription start site that utilizes a specific ATG for protein translation. By northern blot, 5'RACE and 3'RACE we identified three IBTKalpha, IBTKbeta and IBTKgamma mRNAs, whose transcription is driven by two distinct promoter regions; the corresponding IBtk proteins were detected in human cells and mouse tissues by specific antibodies. These results provide the first characterization of the human IBTK locus and may assist in understanding the in vivo function of IBtk.

Identification of a peptide from alpha-gliadin resistant to digestive enzymes: implications for celiac disease.

Current knowledge indicates that both innate and adaptive immune responses are involved in Celiac disease (CD) driven by different gliadin peptides. By studying a representative recombinant alpha-gliadin form, a further 25-mer peptide resistant to gastric, pancreatic, and human intestinal brush-border membrane enzymes was detected. This peptide latter encompasses the sequence 31-43 known to elicit the innate immune response in CD. The resistance of 25-mer, as well as that of the already described 33-mer related to the CD adaptive immune response, was confirmed on a standard flour wheat sample representative of the most widespread European varieties.

Identification of immunodominant epitopes of alpha-gliadin in HLA-DQ8 transgenic mice following oral immunization.

Celiac disease, triggered by wheat gliadin and related prolamins from barley and rye, is characterized by a strong association with HLA-DQ2 and HLA-DQ8 genes. Gliadin is a mixture of many proteins that makes difficult the identification of major immunodominant epitopes. To address this issue, we expressed in Escherichia coli a recombinant alpha-gliadin (r-alpha-gliadin) showing the most conserved sequence among the fraction of alpha-gliadins. HLA-DQ8 mice, on a gluten-free diet, were intragastrically immunized with a chymotryptic digest of r-alpha-gliadin along with cholera toxin as adjuvant. Spleen and mesenteric lymph node T cell responses were analyzed for in vitro proliferative assay using a panel of synthetic peptides encompassing the entire sequence of r-alpha-gliadin. Two immunodominant epitopes corresponding to peptide p13 (aa 120-139) and p23 (aa 220-239) were identified. The response was restricted to DQ and mediated by CD4+ T cells. In vitro tissue transglutaminase deamidation of both peptides did not increase the response; furthermore, tissue transglutaminase catalyzed extensive deamidation in vitro along the entire r-alpha-gliadin molecule, but failed to elicit new immunogenic determinants. Surprisingly, the analysis of the cytokine profile showed that both deamidated and native peptides induced preferentially IFN-gamma secretion, despite the use of cholera toxin, a mucosal adjuvant that normally induces a Th2 response to bystander Ags. Taken together, these data suggest that, in this model of gluten hypersensitivity, deamidation is not a prerequisite for the initiation of gluten responses.

Studies on structure-activity relationship of sphaeropsidins A-F, phytotoxins produced by Sphaeropsis sapinea f. sp. cupressi.

Six forms of sphaeropsidins (SA-SF), three- and tetra-cyclic unrearranged pimarane diterpenes produced by Sphaeropsis sapinea f. sp. cupressi, as well as eight derivatives obtained by chemical modification of SA-SC, were assayed for their bioactivity. The effect of each compound on plants which are host or non-host of the pathogen was investigated. Activity on some plant pathogenic fungi was also tested. Some structure-activity relationships have been identified for both phytotoxic and antifungal activity. It appears that the integrity of the tricyclic pimarane system, the preservation of the double bond C(8)-C(14), the tertiary hydroxyl group at C-9, the vinyl group at C-13, and the carboxylic group at C-10 as well as the integrity of the A-ring provide these molecules with non selective phytotoxic and antimycotic activity.