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A d-galacturonic acid reductase and the corresponding gene were identified from the mold Hypocrea jecorina (Trichoderma reesei). We hypothesize that the enzyme is part of a fungal d-galacturonic acid catabolic pathway which has not been described previously and which is distinctly different from the bacterial pathway. H. jecorina grown on d-galacturonic acid exhibits an NADPH-dependent d-galacturonic acid reductase activity. This activity is absent when the mold is grown on other carbon sources. The d-galacturonic acid reductase was purified, and tryptic digests of the purified protein were sequenced. The open reading frame of the corresponding gene was then cloned from a cDNA library. The open reading frame was functionally expressed in the yeast Saccharomyces cerevisiae. A histidine-tagged protein was purified, and the enzyme kinetics were characterized. The enzyme converts in a reversible reaction from d-galacturonic acid and NADPH to l-galactonic acid and NADP. The enzyme also exhibits activity with d-glucuronic acid and dl-glyceraldehyde.

Experimental conditions for efficient protein radiolabelling and two-dimensional gel electrophoresis were developed for Bifidobacterium longum. Using these tools, protein synthesis in cells before and after heat-shock and bile salts treatment was investigated. Following heat-stress, 13 proteins were upregulated, of which HtrA, DnaK and GroEL were also moderately induced by bile salts, indicating close relationship between the heat and bile salts responses in bifidobacteria. Our work indicated that, as a consequence of prolonged heat-stress, HtrA undergoes sequential modification and proteolysis, and that this mechanism could be employed by bifidobacteria to respond to heat-stress.

Background: Individuals with natural rubber latex allergy often have immediate reactions to plant-derived foods and fresh fruits, such as avocado and banana. IgE of these patients has been shown to bind endochitinases containing an N-terminal hevein-like domain (HLD). However, evidence on 31-kd endochitinase-induced reactions in vivo is lacking., Objective: We sought to assess the clinical significance of 31-kd endochitinases and isolated HLDs in latex-fruit syndrome., Methods: The 31-kd endochitinases and corresponding HLDs were purified or produced from avocado, banana, latex, and wheat germ. Skin prick test reactivities against purified proteins were examined in 15 patients with natural rubber latex allergy. The binding efficiency of IgE to purified proteins was studied by using an inhibition ELISA. Experimentally resolved or modeled structures of the proteins were compared to clarify the molecular basis of clinical reactions., Results: Eleven (73%) patients had skin prick test reactions to isolated HLDs of avocado and banana, but only 1 (7%) patient reacted to their corresponding 31-kd endochitinases. HLDs from avocado and banana inhibited binding of IgE to prohevein (Hev b 6.01) in 59% and 38% of patients, respectively, whereas corresponding percentages for 31-kd endochitinases were 17% and 20%, respectively. Isolated HLDs of wheat germ agglutinin and 18-kd wheat germ agglutinin did not significantly inhibit IgE binding to hevein., Conclusion: The isolated HLD molecules alone, but not when linked to endochitinases, seem to be responsible for IgE-mediated clinical reactions in latex-fruit syndrome. Careful selection of relevant allergens in their proper molecular form is therefore crucial in forming a reliable diagnosis of latex-fruit syndrome.

A D-erythorbic acid-forming soluble flavoprotein, gluconolactone oxidase (GLO), was purified from Penicillium cyaneo-fulvum strain ATCC 10431 and partially sequenced. Peptide sequences were used to isolate a cDNA clone encoding the enzyme. The cloned gene exhibits high levels of similarity with the genes encoding other known eukaryotic lactone oxidases and also with the genes encoding some putative prokaryotic lactone oxidases. Analysis of the coding sequence of the GLO gene indicated the presence of a typical secretion signal sequence at the N terminus of GLO. No other targeting or anchoring signals were found, suggesting that GLO is the first known lactone oxidase that is secreted rather than targeted to the membranes of the endoplasmic reticulum or mitochondria. Experimental evidence, including the N-terminal sequence of mature GLO and data on glycosylation and localization of the enzyme in native and recombinant hosts, supports this analysis. The GLO gene was expressed in Pichia pastoris, and recombinant GLO was produced by using the strong methanol-induced AOX1 promoter. In order to evaluate the suitability of purified GLO for production of D-erythorbic acid, we immobilized it on N-hydroxysuccinimide-activated Sepharose and found that the immobilized GLO retained full activity during immobilization but was rather unstable under reaction conditions. Our results show that both soluble and immobilized forms of GLO can, in principle, be used for production of D-erythorbic acid from D-glucono-delta-lactone or (in combination with glucose oxidase and catalase) from glucose. We also demonstrated the feasibility of glucose-D-erythorbic acid fermentation with recombinant strains coexpressing GLO and glucose oxidase genes, and we analyzed problems associated with construction of efficient D-erythorbic acid-producing hosts.

Background: Patients with wheat-dependent, exercise-induced anaphylaxis (WDEIA) experience recurrent anaphylactic reactions when exercising after ingestion of wheat products. We have identified omega-5 gliadin (Tri a 19) as a major allergen in WDEIA, but the role of exercise in eliciting the symptoms remains obscure., Objective: The aim was to examine whether tissue transglutaminase (tTG)-mediated cross-linking could be involved in modulating the IgE-binding ability and in vivo reactivity of digested omega-5 gliadin peptides in WDEIA., Methods: Purified omega-5 gliadin was digested with pepsin or with pepsin and trypsin and treated with tTG. The binding of IgE antibodies in pooled sera from 10 patients with WDEIA was studied by means of immunoblotting before and after tTG treatment of the digested peptides. The peptides derived from pepsin digestion were separated by means of gel-filtration chromatography, and IgE reactivity of 4 different peptide fractions was studied by immunoblotting before and after tTG treatment. The fraction showing the greatest degree of cross-linking by tTG was further studied by means of IgE ELISA, ELISA inhibition, and skin prick testing., Results: The IgE-binding ability of omega-5 gliadin was retained after pepsin and pepsin-trypsin digestion. tTG treatment of the whole peptic digest formed large peptide complexes, with molecular weights ranging from 40 to greater than 200 kd. These cross-linked aggregates bound IgE antibodies in immunoblotting more intensely than untreated, pepsin-digested, or pepsin-trypsin-digested omega-5 gliadin. A gel-filtration fraction of the whole peptic digest corresponding to the highest peak of the chromatogram and showing the greatest degree of tTG-mediated cross-linking showed an increase in serum IgE reactivity in ELISA after tTG treatment, as well as a shift of reactivity to cross-linked complexes. In the 20 patients with WDEIA, the mean skin prick test wheal elicited by this tTG-treated peptic fraction was 77% larger (P <.001) than that elicited by the untreated peptic fraction and 56% larger (P <.01) than that elicited by intact omega-5 gliadin., Conclusions: Omega-5 gliadin-derived peptides are cross-linked by tTG, which causes a marked increase in IgE binding both in vitro and in vivo. Activation of tTG during exercise in the intestinal mucosa of patients with WDEIA could lead to the formation of large allergen complexes capable of eliciting anaphylactic reactions.

Tissue localization of cysteine proteinases (cathepsins) and their inhibitors (salarin, salmon kininogen) was performed in tissues of the Atlantic salmon. In skin, both epidermis and dermis were strongly stained by antisera against salarin and salmon kininogen. In epidermis the intercellular space seemed to be heavily stained (salarin). In kidney, the inhibitors were mainly localized to the interstitial capillaries. Also, some epithelial cells of the tubules (salarin) and some cells of the interstitium were stained. Mostly, the staining had a diffuse cytoplasmic localization. In the liver some hepatocytes were strongly positive for salarin and salmon kininogen. Purified fish cysteine proteinase inhibitors were not found to inhibit the growth of fish pathogenic bacteria and viruses. In the trunk kidney cathepsins B and L were localized in epithelial cells of the tubules (proximal part) and in cells of the interstitium. Mostly, the staining showed a prominent lysosomal localization. In head kidney large macrophage-like cells were positively stained for cathepsin B. The staining was localized to granula/vacuoles in the cytoplasm. In the liver, some hepatocytes were strongly stained and some were less strongly positive for cathepsin B and L. Mostly, the hepatocytes showed lysosomal staining. Cathepsin L was found in some big macrophage-like cells in the spleen. Mucosal epithelial cells of the esophagus and intestine seemed to be strongly stained for cathepsin B and L. The results show that cathepsins and their inhibitors are specifically and widely distributed in the Atlantic salmon skin indicating that they perform some biologically important and specific but so far unknown functions.

STAT6 is a central mediator of IL-4-induced gene responses. STAT6-mediated transcription is depend ent on the C-terminal transcription activation domain (TAD), but the mechanisms by which STAT6 activates transcription are poorly understood. Here, we have identified the staphylococcal nuclease (SN)-like domain and tudor domain containing protein p100 as a STAT6 TAD interacting protein. p100 was originally characterized as a transcriptional coactivator for Epstein-Barr virus nuclear antigen 2. STAT6 interacted with p100 in vitro and in vivo. The interaction was mediated by the TAD domain of STAT6 and the SN-like domain of p100. p100 did not affect the immediate activation events of STAT6, but enhanced STAT6-mediated transcriptional activation and the IL-4-induced Igepsilon gene transcription in human B-cell line. Finally, p100 associated with the large subunit of RNA polymerase II and was mediating interaction between STAT6 and RNA polymerase II. These findings identify p100 as a novel coactivator for STAT6 and suggest that p100 functions as a bridging factor between STAT6 and the basal transcription machinery.