Myrothecium verrucaria bilirubin oxidase and its mutants for potential copper ligands

Bilirubin oxidase (EC:1.3.3.5) purified from a culture medium of Myrothecium verrucaria MT-1 (authentic enzyme) catalyzes the oxidation of bilirubin to biliverdin in vitro and recombinant enzyme (wild type) was obtained by using an overexpression system of the bilirubin oxidase gene with Aspergillus oryzae harboring an expression vector. The absorption and ESR spectra showed that both bilirubin oxidases are multicopper oxidases containing type 1, type 2, and type 3 coppers similar to laccase, ascorbate oxidase, and ceruloplasmin. Site-directed mutagenesis has been performed for the possible ligands of each type of copper. In some mutants, Cys457 --> Val, Ala, His94 --> Val, and His134.136 --> Val, type 1 and type 2 copper centers were perturbed completely and the enzyme activity was completely lost. Differing from the holoenzyme, these mutants showed type 3 copper signals. However, the optical and magnetic properties characteristic of type 1 copper were retained even by mutating one of the type 1 copper ligands, i.e., a mutant, Met467 --> Gly, showed a weak but apparent enzyme activity. A double mutant His456.458 --> Val had only type 1 Cu, showing a blue band at 600 nm (epsilon = 1.6 x 10(3)) and an ESR signal with very narrow hyperfine splitting (A parallel = 7.2 x 10(-)3 cm-1). Since the type 2 and type 3 coppers are not present, the mutant did not show enzyme activity. These results strongly imply that the peculiar sequence in bilirubin oxidase, His456-Cys457-His458, forms an intramolecular electron-transfer pathway between the type 1 copper site and the trinuclear center composed of the type 2 and type 3 copper sites.     

Orexins and orexin receptors: implication in feeding behavior

Angiotensin IV, (V-Y-I-H-P-F), binds to AT4 receptors in blood vessels to induce vasodilatation and proliferation of cultured bovine endothelial cells. This latter effect may be important not only in developing tissues but also in injured vessels undergoing remodelling. In the present study, using normal rabbit carotid arteries, we detected AT4 receptors in vascular smooth muscle cells and in the vasa vasorum of the adventitia. Very low receptor levels were observed in the endothelial cells. In keeping with the described binding specificity of AT4 receptors, unlabelled angiotensin IV competed for [125I]angiotensin IV binding in the arteries, with an IC50 of 1.4 nM, whereas angiotensin II and angiotensin III were weaker competitors. Within the first week following endothelial denudation of the carotid artery by balloon catheter, AT4 receptor binding in the media increased to approximately 150% of control tissue. AT4 receptor binding further increased in the media, large neointima and re-endothelialized cell layer to 223% at 20 weeks after injury. In view of the known trophic effects of angiotensin IV, the elevated expression of AT4 receptors, in both the neointima and media of arteries, following balloon injury to the endothelium, suggests a role for the peptide in the adaptive response and remodelling of the vascular wall following damage.     

Viable spermatozoa can be recovered from refrigerated mice up to 7 days after death

To develop a model for utilizing germ cells collected from dead animals, male mice were euthanized and refrigerated for various periods, and the viability of the epididymal spermatozoa was examined by in vitro fertilization, embryo culture, and embryo transfer. Higher proportions of fresh oocytes were fertilized when males had been stored at 4-6 or 8-10 degrees C than at 0 degrees C. By partially dissecting the zona of freshly ovulated oocytes, spermatozoa from ICR male mice could fertilize oocytes (21% fertilization rate) after being stored for 5 days at 4-6 degrees C, and spermatozoa from BDF1 male mice could fertilize oocytes (39%) after being stored for 7 days at 4-6 degrees C. The resulting two-cell embryos had the ability to develop into expanded blastocysts in culture (81-100%) and into live young after transfer (34-47%). With further refinement of this system, it should be applicable not only for rescuing valuable genetic variants in laboratory animals or livestock animals but also for wild species in the future.     

Fas ligand-mediated exocrinopathy resembling Sjögren's syndrome in mice transgenic for IL-10

Although IL-10 has been implicated in the pathogenesis of several autoimmune diseases, the mechanisms by which this cytokine mediates inflammatory lesions remain to be elucidated. Exocrine gland destruction is an important early step in the development of Sj?gren's syndrome. To better understand the role of IL-10 in Sj?gren's syndrome, we made transgenic mice in which the mouse IL-10 gene was regulated by the human salivary amylase promoter. Transgenic expression of IL-10 induced apoptosis of glandular tissue destruction and lymphocyte infiltration consisting primarily of Fas-ligand (FasL)+ CD4+ T cells, as well as in vitro up-regulation of FasL expression on T cells. These data suggest that overexpression of IL-10 in the glands and their subsequent Fas/FasL-mediated bystander tissue destruction is a causal factor in the development of this disease.     

MAGE-A4 interacts with the liver oncoprotein gankyrin and suppresses its tumorigenic activity

Hepatocellular carcinoma ranks among the most common malignancies in Southeast Asia and South Africa. Although there are many modalities of treatment, the recurrence and metastasis rates are high, and the prognosis is unsatisfactory. Gankyrin, a recently found oncoprotein, is a promising target for drug therapy because it is overexpressed in all studied hepatocellular carcinomas. Gankyrin contains six ankyrin repeats and interacts with Rb, Cdk4, and the S6 ATPase of the 26 S proteasome. In this study, a yeast two-hybrid screen with gankyrin has identified MAGE-A4 as another interacting protein. The interaction, mediated by the C-terminal half of MAGE-A4, was reproduced in mammalian cells. The interaction was specific to MAGE-A4, because other MAGE family proteins structurally similar to MAGE-A4, i.e. MAGE-A1, MAGE-A2, and MAGE-A12, did not bind to gankyrin. MAGE-A4 partially suppressed both anchorage-independent growth in vitro and tumor formation in athymic mice of gankyrin-overexpressing cells. The ability of mutant MAGE-A4 to interact with gankyrin correlated with the ability to suppress the anchorage-independent growth. These results demonstrate that MAGE-A4 binds to gankyrin and suppresses its oncogenic activity. So far, the major focus of studies on the MAGE proteins has been on their potential for cancer immunotherapy. Our results may also shed light on novel functions for MAGE-A proteins.     

Calcium disodium edetate enhances type A monoamine oxidase activity in monkey brain

The effects of metal chelators on monoamine oxidase (MAO) isozymes, MAO-A and MAO-B, in monkey brain mitochondria were investigated in vitro. MAO-A activity increased to about 40% with 0.1 μM calcium disodium edetate (CaNa₂EDTA) using serotonin as a substrate, and this activation was proportional to the concentration of CaNa₂EDTA. On the other hand, MAO-A activities were decreased gradually with an increasing concentration of o-phenanthroline and diethyldithiocarbamic acid, but these metal chelators had no effect on MAO-B activity in monkey brain. The activation of MAO-A activity by CaNa₂EDTA was reversible. CaNa₂EDTA did not activate both MAO-A and MAO-B activities in rat brain mitochondria. Zn and Fe ions were found in the mitochondria of monkey brain. Zn ions potently inhibited MAO-A activity, but Fe ions did not inhibit either MAO-A or MAO-B activity in monkey brain mitochondria. These results indicate that the activating action of CaNa₂EDTA on MAO-A was the result of the chelating of Zn ions contained in mitochondria by CaNa₂EDTA. These results also indicate the possibility that Zn ions may regulate physiologically the level of serotonin and norepinephrine content in brain by inhibiting a MAO-A activity.     

Polysaccharide-polynucleotide complexes. Part 7. Hydrogen-ion and salt concentration dependence of complexation between schizophyllan and single-stranded homo RNAs

Schizophyllan belongs to a beta-1,3-D-glucan family, which exists as a random coil in dimethyl sulfoxide (DMSO) and as a triple helix in water, respectively. The schizophyllan single chain forms a complex with single-stranded homo RNAs in water/DMSO mixed solvents. Using circular dichroism, we studied the complexation and its stability as a function of apparent pH (pH(*)) in a mixed solvent system and as a function of the salt concentration. The complex is formed in the pH(*) range 6.5-10, and dissociated in the pH(*) range 4-6. Both poly(A) and poly(C) adopt a double strand in the pH(*) range 4-6 and a single strand in the pH(*) range 6.5-10. Therefore, the conformational change of each polynucleotide is responsible for dissociation/association of the complex, i.e., the single strand of the polynucleotides can form complexes, whereas the double one cannot. This result indicates that hydrogen bonding and similarity of the helix parameters are essential for the complex formation. The melting temperature of the complex reaches the maximum around 0.05 M of NaCl and KCl, and the value of the maximum temperature depends on the cation species.     

Accelerated clearance of Escherichia coli in experimental peritonitis of histamine-deficient mice

IL-5 plays a pivotal role in growth and differentiation of eosinophils. The signal transduction mechanism of IL-5Ralpha is largely unknown. We have demonstrated that IL-5 induces tyrosine phosphorylation of IL-5Ralpha in eosinophils. To identify IL-5Ralpha-associated tyrosine kinases, we have examined the expression of Src family tyrosine kinases in eosinophils. Among the Src family members, Lyn, Hck, Fgr, and Lck are present in eosinophils, and, among these four kinases, only Lyn is associated with the IL-5Ralpha under basal conditions. We also confirm the association of Janus kinase (Jak)2 with IL-5Ralpha. Lyn kinase phosphorylates both IL-5Ralpha and betacR in vitro. The importance of Lyn kinase for eosinophil differentiation was studied using antisense oligodeoxynucleotides. Lyn antisense oligodeoxynucleotide blocks eosinophil differentiation from stem cells in a dose-dependent manner. The Jak2 inhibitor tyrphostin AG490 also inhibits eosinophil differentiation. The importance of Lyn for eosinophil differentiation was further studied using Lyn knockout mice. The IL-5-stimulated eosinophil differentiation from bone marrow cells is significantly inhibited in Lyn(-/-) mice as compared with that in control mice. We conclude that both Lyn and Jak2 play an essential role in IL-5Ralpha signaling, leading to eosinophil differentiation. The effect of Lyn appears to be relatively specific for the eosinophilic lineage.