Immune-responsive gene 1 (IRG1) and dimethyl itaconate are involved in the mussel immune response.

Immune-responsive gene 1 (irg1) is a gene that is well-conserved among different taxa and is highly expressed in the mussel Mytilus galloprovincialis at the constitutive level. The expression of this gene increases after a bacterial infection, primarily in haemocytes. irg1 catalyses the production of itaconic acid from cis-aconitic acid in the Krebs cycle. Recently, itaconate has been revealed as an immune metabolite involved in macrophage polarization. In this work, we studied the effects of exogenous dimethyl itaconate (DI) on mussels in vitro and in vivo at relevant previously described endogenous concentrations and in mussels infected with Vibrio splendidus. DI did not have adverse effects on the haemocytes viability, apoptotic cells, proliferation and phagocytic activity; however, haemocyte size, velocity and accumulated distance were decreased. The antibacterial activity of DI in vitro and in vivo was observed with high concentrations of DI, that is, 30 and 50 mM, respectively. Furthermore, DI inhibited total ROS, increased mitochondrial ROS and modulated antioxidant genes, such as SOD and CAT, related to Nrf2 activation. In this research, we have demonstrated some important pathways in haemocytes in which itaconate can be involved after its production in a bacterial infection.

IgE-mediated anaphylaxis to Thiomucase, a mucopolyssacharidase: allergens and cross-reactivity.

BACKGROUND: Thiomucase is a mucopolysaccharidase obtained from ovine tissues mainly used to facilitate the diffusion of local anaesthetics and in the treatment of cellulitis. A patient with an anaphylaxis in relation to the intramuscular administration of Thiomucase is reported. OBJECTIVE: To investigate Thiomucase allergens and their possible relationship with dander allergens and animal albumins. MATERIAL AND METHODS: Skin prick tests (SPT) and serum-specific IgE were performed with Thiomucase and danders. Thiomucase SDS-PAGE immunoblotting was performed in order to study allergens. RAST/CAP inhibition and SDS-PAGE immunoblotting inhibition were carried out to study the cross-reactivity. RESULTS: Skin prick tests (SPT) were positive to Thiomucase, animal dander (cat, dog, sheep, other), bovine serum albumin (BSA), and echinococcus. Specific IgE was also positive to Thiomucase, animal dander (cat, dog, sheep, other), BSA and echinococcus. In the RAST-CAP inhibition assays BSA was nearly completely inhibited by Thiomucase, Thiomucase was partially inhibited by BSA and cat and Echinococcus granulosus was partially inhibited by sheep and Thiomucase. In the Thiomucase SDS-PAGE immunoblotting several proteins fixed IgE, ranging from 20 kDa to > 94 kDa, the strongest with 43 kDa. The IgE fixation to BSA, cat and sheep in the SDS-PAGE immunoblotting was completely inhibited by the preincubation of the serum with Thiomucase. CONCLUSIONS: An IgE-mediated anaphylaxis to Thiomucase is documented. Multiple allergens are recognized in Thiomucase by the patient serum, the main with 43 kDa. Partial cross-reactivity with BSA, cat dander and sheep dander is documented.

Magnesium chelatase subunit D from pea: characterization of the cDNA, heterologous expression of an enzymatically active protein and immunoassay of the native protein.

Mg-chelatase catalyzes the insertion of Mg into protoporphyrin and lies at the branchpoint of heme and (bacterio)chlorophyll synthesis. In prokaryotes, three genes--BchI, D and H--encode subunits for Mg-chelatase. In higher plants, homologous cDNAs for the I, D and H subunits have been characterized. Since the N-terminal half of the D subunit is homologous to the I subunit, the C-terminal portion of the pea D was used for antigen production. The antibody recognized the chloroplast D subunit and was used to demonstrate that this subunit associated with the membranes in the presence of MgCl2. The antibody immunoprecipitated the native protein and inhibited Mg-chelatase activity. Expression in Escherichia coli with a construct for the full-length protein (minus the putative transit peptide) resulted in induction of 24.5 kDa (major) and 89 kDa (minor) proteins which could only be solubilized in 6 M urea. However, when host cells were co-transformed with expression vectors for the full-length D subunit and for the 70 kDa HSP chaperonin protein, a substantial portion of the 89 kDa protein was expressed in a soluble form which was active in a Mg-chelatase reconstitution assay.

Purification of chorismate synthase from a cell culture of the higher plant Corydalis sempervirens Pers.

Chorismate synthase (EC 4.6.1.4) was purified from a cell suspension culture of Corydalis sempervirens almost 1000-fold to near homogeneity. The subunit Mr estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was 41,900. The Mr of the native enzyme was estimated to be 80,100 by gel filtration, suggesting a dimeric structure. Antisera directed against the 41.9-kDa protein also reacted with the native enzyme. Further confirmation of the identity of the purified protein was obtained by sequence comparison of a tryptic peptide with known sequences of the Escherichia coli and Neurospora crassa chorismate synthases.

Cysteine conjugate beta-lyase of rat kidney cytosol: characterization, immunocytochemical localization, and correlation with hexachlorobutadiene nephrotoxicity.

Cysteine conjugate beta-lyase (beta-lyase) was purified to electrophoretic homogeneity from the kidney cytosol of male Wistar rats. The highly purified enzyme exhibited a monomeric molecular weight of 50,000 Da and was active in the alpha-beta elimination of cysteine conjugates including S-(1,2-dichlorovinyl)-L-cysteine (DCVC), S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC), and S-(2-benzothiazolyl)-L-cysteine, particularly toward DCVC and TFEC. The purified enzyme also exhibited glutamine transaminase K activity with phenylalanine and alpha-keto-gamma-methiolbutyrate as substrates. An antibody was raised to the purified rat protein in sheep and the crude immune serum affinity purified, yielding a specific antibody that recognized only the beta-lyase protein in whole kidney homogenates. Immunocytochemical studies on rat kidney sections stained with the purified antibody revealed that the cytosolic beta-lyase enzyme was mainly localized in the pars recta of the proximal tubule in untreated rats. This localization is coincident with the site-specific kidney necrosis produced by hexachloro-1,3-butadiene (HCBD). These results indicate that the tissue localization of beta-lyase in the proximal tubule plays an important role in determining the specific nephrotoxicity produced by halogenated alkenes such as HCBD.

Immunohistochemical localization of glutamine transaminase K, a rat kidney cysteine conjugate beta-lyase, and the relationship to the segment specificity of cysteine conjugate nephrotoxicity.

Rat kidney glutamine transaminase K is a major rat kidney cysteine conjugate beta-lyase and is a key enzyme in the nephrotoxicity of some cysteine conjugates. However, it has not been demonstrated that the beta-lyase is present in the target cells. Furthermore, although all segments of the proximal tubule are affected by high doses of nephrotoxic cysteine conjugates, the S3 segment is the most sensitive. Because heterogeneous distribution of the beta-lyase could account for the enhanced sensitivity, antibody raised against rat kidney cysteine conjugate beta-lyase has been prepared and used to investigate the distribution of the enzyme in kidney and other tissues. The data show that the enzyme is highest in rat kidney, consistent with enzyme activity data. By immunohistochemical staining, no enzyme is present in the glomeruli or distal tubular elements of the kidney. The enzyme is present only in the target cells, the renal proximal tubular epithelium. However, the distribution of the beta-lyase within the proximal tubule is not consistent with the hypothesis that a higher concentration of the enzyme in the S3 segment accounts for the greater sensitivity of S3 to nephrotoxic cysteine conjugates compared to S1 and S2. Several alternative hypotheses are discussed.

Chemical modification of tryptophanase from E. coli with polyethylene glycol to reduce its immunoreactivity towards anti-tryptophanase antibodies.

Escherichia coli tryptophanase was modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine (activated PEG2, MW 5,000 x 2). The modified tryptophanase, in which approximately 43% of the total 120 amino groups and 38% of the total 16 sulfhydryl groups in the molecule were coupled, completely lost the immunoreactivity towards anti-tryptophanase serum from rabbit. Approximately 10% of the enzymic activity was retained. The modified enzyme showed the same physicochemical properties as the native enzyme: Km value for L-tryptophan (0.3 mmol/l), optimum pH (8.0) and optimum temperature (50 degrees C). The modified enzyme was more resistant than the native counterpart against proteolytic digestion with trypsin.