Induction of allergic contact dermatitis by astigmatid mite-derived monoterpene, alpha-acaridial

α-Acaridial [2(E)-(4-methyl-3-pentenyl)butenedial] is a novel monoterpene secreted from the house dust mites. Because of its molecular nature of a highly reactive, small lipidic compound, we addressed whether α-acaridial might function as a haptenic allergen that induced allergic contact dermatitis. Mice sensitized with α-acaridial were challenged by the same antigen on the ear skin. After 2 days, significant ear swelling with a prominent infiltration of CD4⁺ T lymphocytes was observed. In vitro, α-acaridial exhibited an outstanding ability to quickly interact with and chemically modify a reference protein. Virtually all cysteine residues and a sizable fraction of lysine residues were found to be selectively modified, suggesting that α-acaridial could potentially interact with any proteins. Previously, numerous mite-derived proteinaceous allergens have been associated with contact dermatitis. Our study now emphasizes that small lipidic compounds released from mites comprise a new class of mite allergens, and therefore, is of significant medical implications.     

Catalytically inactive phospholipase A2 homologue binds to vascular endothelial growth factor receptor-2 via a C-terminal loop region

VEGF (vascular endothelial growth factor) regulates neovascularization through binding to its receptor KDR (kinase insert domain-containing receptor; VEGF receptor-2). We recently identified a catalytically inactive PLA(2) (phospholipase A(2)) homologue (KDR-bp) in the venom of eastern cottonmouth (Agkistrodon piscivorus piscivorus) as a third KDR-binding protein, in addition to VEGF(165) and tissue inhibitor of metalloproteinase-3. KDR-bp binds to the extracellular domain of KDR with a K(d) of 10(-8) M, resulting in specific blockade of endothelial cell growth induced by VEGF(165). Inactive PLA(2) homologues are widely distributed in the venoms of Viperidae snakes and are known to act as myotoxins. In the present study, we demonstrated that KDR-binding ability is a common characteristic for inactive PLA(2) homologues in snake venom, but not for active PLA(2)s such as neurotoxic and platelet aggregation-modulating PLA(2)s. To understand better the KDR and KDR-bp interaction, we resolved the binding region of KDR-bp using eight synthetic peptides designed based on the structure of KDR-bp. A synthetic peptide based on the structure of the C-terminal loop region of KDR-bp showed high affinity for KDR, but other peptides did not, suggesting that the C-terminal loop region of KDR-bp is involved in the interaction with KDR. The results of the present study provide insight into the binding of inactive PLA(2) homologues to KDR, and may also assist in the design of novel anti-KDR molecules for anti-angiogenic therapy.     

An O6-methylguanine-DNA methyltransferase-like protein from Thermus thermophilus interacts with a nucleotide excision repair protein

The major damage to DNA caused by alkylating agents involves the formation of O(6)-methylguanine (O(6)-meG). Almost all species possess O(6)-methylguanine-DNA-methyltransferase (Ogt) to repair such damage. Ogt repairs O(6)-meG lesions in DNA by stoichiometric transfer of the methyl group to a cysteine residue in its active site (PCHR). Thermus thermophilus HB8 has an Ogt homologue, TTHA1564, but in this case an alanine residue replaces cysteine in the putative active site. To reveal the possible function of TTHA1564 in processing O(6)-meG-containing DNA, we characterized the biochemical properties of TTHA1564. No methyltransferase activity for synthetic O(6)-meG-containing DNA could be detected, indicating TTHA1564 is an alkyltransferase-like protein. Nevertheless, gel shift assays showed that TTHA1564 can bind to DNA containing O(6)-meG with higher affinity (9-fold) than normal (unmethylated) DNA. Experiments using a fluorescent oligonucleotide suggested that TTHA1564 recognizes O(6)-meG in DNA using the same mechanism as other Ogts. We then investigated whether TTHA1564 functions as a damage sensor. Pull-down assays identified 20 proteins, including a nucleotide excision repair protein UvrA, which interacts with TTHA1564. Interaction of TTHA1564 with UvrA was confirmed using a surface plasmon resonance assay. These results suggest the possible involvement of TTHA1564 in DNA repair pathways.     

Epidermal growth factor receptor phosphorylates protein kinase C {delta} at Tyr332 to form a trimeric complex with p66Shc in the H2O2-stimulated cells

Protein kinase C (PKC) delta is phosphorylated at Tyr311 and Tyr332 and its catalytic activity is enhanced in the H(2)O(2)-stimulated cells, but the enzymes that recognize these tyrosine residues, especially Tyr332, have been remained to be clarified. The analysis of the endogenous proteins in COS-7 cells revealed that PKCdelta binds to p66Shc, an adaptor protein containing two phosphotyrosine-binding domains, in a manner dependent on its tyrosine phosphorylation upon H(2)O(2) stimulation. The studies using the mutated PKCdelta clarified that PKCdelta associates with p66Shc through the phosphorylated Tyr332 residue. Epidermal growth factor (EGF) receptor was detected in the anti-p66Shc immunoprecipitate prepared from the H(2)O(2)-stimulated cells, and this receptor-type tyrosine kinase phosphorylated PKCdelta at Tyr332 in vitro. PKCdelta was, however, not tyrosine phosphorylated in the EGF-stimulated cells, whereas H(2)O(2)-induced tyrosine phosphorylation of PKCdelta and its association with p66Shc were strongly suppressed by EGF receptor kinase inhibitors such as AG1478 and PD153035. These results indicate that EGF receptor phosphorylates PKCdelta at Tyr332 in the H(2)O(2)-stimulated but not in the growth-factor treated cells, and suggest that PKCdelta in the complex with p66Shc and EGF receptor may play a role in the stress-signalling pathway.     

Efficient production of mannosylerythritol lipids with high hydrophilicity by <Emphasis Type="Italic">Pseudozyma hubeiensis</Emphasis> KM-59

Mannosylerythritol lipids (MELs) are one of the most promising biosurfactants known because of their multifunctionality and biocompatibility. A previously isolated yeast strain, Pseudozyma sp. KM-59, mainly produced a hydrophilic MEL, namely MEL-C (4-O-[4′-O-acetyl-2′,3′-di-O-alka(e)noyl-β-d-mannopyranosyl]-d-erythritol). In this study, we taxonomically characterize the strain in detail and investigate the culture conditions. The genetic, morphological, and physiological characteristics of the strain coincided well with those of Pseudozyma hubeiensis. On batch culture for 4 days under optimal conditions, the yield of all MELs was 21.8 g/l; MEL-C comprised approximately 65% of the all MELs. Consequently, on fed-batch culture for 16 days, the yield reached 76.3 g/l; the volumetric productivity was approximately 4.8 g l⁻¹ day⁻¹. We further examined the surface-active and self-assembling properties of the hydrophilic MELs produced by the yeast strain. They showed higher emulsifying activities against soybean oil and a mixture of hydrocarbons (2-methylnaphtarene and hexadecane, 1:1) than the synthetic surfactants tested. On water penetration scans, they efficiently formed lyotropic liquid crystalline phases such as myelines and lamella () in a broad range of their concentrations, indicating higher hydrophilicity than conventional MELs. More interestingly, there was little difference in the liquid crystal formation between the crude product and purified MEL-C. The present glycolipids with high hydrophilicity are thus very likely to have practical potential without further purification and to expand the application of MELs especially their use in washing detergents and oil-in-water-type emulsifiers.     

Roles of three domains of Tetrahymena eEF1A in bundling F-actin

The conventional role of eukaryotic elongation factor 1A (eEF1A) is to transport aminoacyl tRNA to the A site of ribosomes during the peptide elongation phase of protein synthesis. eEF1A also is involved in regulating the dynamics of microtubules and actin filaments in cytoplasm. In Tetrahymena, eEF1A forms homodimers and bundles F-actin. Ca(2+)/calmodulin (CaM) causes reversion of the eEF1A dimer to the monomer, which loosens F-actin bundling, and then Ca(2+)/CaM/eEF1A monomer complexes dissociate from F-actin. eEF1A consists of three domains in all eukaryotic species, but the individual roles of the Tetrahymena eEF1A domains in bundling F-actin are unknown. In this study, we investigated the interaction of each domain with F-actin, recombinant Tetrahymena CaM, and eEF1A itself in vitro, using three glutathione-S-transferase-domain fusion proteins (GST-dm1, -2, and -3). We found that only GST-dm3 bound to F-actin and influences dimer formation, but that all three domains bound to Tetrahymena CaM in a Ca(2+)-dependent manner. The critical Ca(2+) concentration for binding among three domains of eEF1A and CaM were < or =100 nM for domain 1, 100 nM to 1 microM for domain 3, and >1 microM for domain 2, whereas stimulation of and subsequent Ca(2+) influx through Ca(2+) channels raise the cellular Ca(2+) concentration from the basal level of approximately 100 nM to approximately 10 microM, suggesting that domain 3 has a pivotal role in Ca(2+)/CaM regulation of eEF1A.     

Discovery of Pseudozyma rugulosa NBRC 10877 as a novel producer of the glycolipid biosurfactants, mannosylerythritol lipids, based on rDNA sequence

The search for a novel producer of glycolipid biosurfactants, mannosylerythritol lipids (MEL) was undertaken based on the analysis of ribosomal DNA sequences on the yeast strains of the genus Pseudozyma. Pseudozyma rugulosa NBRC 10877 was found to produce a large amount of glycolipids from soybean oil. Fluorescence microscopic observation also demonstrated that the strain significantly accumulates polar lipids in the cells. The structure of the glycolipids produced by the strain was analyzed by ¹H and ¹³C nuclear magnetic resonance and gas chromatography–mass spectrometry methods, and was determined to be the same as MEL produced by Pseudozyma antarctica, a well-known MEL producer. The major fatty acids of the present MEL consisted of C8 and C10 acids. Based on high performance liquid chromatography, the composition of the produced MEL was as follows: MEL-A (68%), MEL-B (12%), and MEL-C (20%). To enhance the production of MEL by the novel strain, factors affecting the production, such as carbon and nitrogen sources, were further examined. Soybean oil and sodium nitrate were the best carbon and nitrogen sources, respectively. The supplementation of a MEL precursor, such as erythritol, drastically enhanced the production yield from soybean oil at a rate of 70 to 90%. Under the optimal conditions in a shake culture, a maximum yield, productivity, and yield coefficient (on a weight basis to soybean oil supplied) of 142 g l⁻¹, 5.0 g l⁻¹ day⁻¹, and 0.5 g g⁻¹ were achieved by intermittent feeding of soybean oil and erythritol using the yeast.     

Effects of Aluminum on the Growth of Tea Plant and Activation of Antioxidant System

The origin and distribution of carbonic anhydrase (CA), which could accelerate karst processes, are explored in this paper. The soil samples used in the study were collected from four different kinds of karst ecosystems of Southwest China. The results indicate that CA activity could be detected in surface layer soils, and that CA activity varied obviously among the soils in different karst ecosystems. Of the four kinds of karst site, the mean CA activity of the surface soil in Misuga, Liupanshui, which had the lowest vegetation cover, was the lowest with 0.02 U g ^–1 dry soil. Nongla and Jinfu mountain, where there is abundant vegetable species diversity and dense vegetation, had higher mean CA activity in the surface soil with 3.83 U g ^–1 dry soil and 3.13 U g ^–1 dry soil, respectively. Moreover, there was certain difference in CA activities of soils among different kinds of karst landscape in the same kind of karst ecosystem. On the other hand, the higher CA activity could be detected in the soils in the vicinity of the plant roots, and CA activity in soil exhibited both obvious seasonal and vertical changes. These facts implied that plant roots and soil microorganisms might serve as important sources of CA. Besides, the results indicate that CA activity is found in bacteria, actinomycetes and fungi. Actinomycetes had higher intracellular CA activity, while fungi had higher extracellular CA activity. This study shows that higher microbial CA activities are found in Jinfu Mountain, while lower microbial CA activities are found in Misuga, Liupanshui.     

Assembly system of direct modified superparamagnetic iron oxide nanoparticles for target-specific MRI contrast agents

We report the direct modification of SPIOs with a biomolecule and the target-specific assembly system of these modified SPIOs for using MRI contrast agents. The transverse relaxation rate of the aqueous solutions containing the modified SPIOs was altered by the dispersion state.