Purification,cDNA cloning,and characterization of plant chitinase with a novel domain combination from lycophyte Selaginella doederleinii

Chitinase-A from a lycophyte Selaginella doederleinii (SdChiA), having molecular mass of 53 kDa, was purified to homogeneity by column chromatography. The cDNA encoding SdChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1477 nucleotides and its open reading frame encoded a polypeptide of 467 amino acid residues. The deduced amino acid sequence indicated that SdChiA consisted of two N-terminal chitin-binding domains and a C-terminal plant class V chitinase catalytic domain, belonging to the carbohydrate-binding module family 18 (CBM18) and glycoside hydrolase family 18 (GH18), respectively. SdChiA had chitin-binding ability. The time-dependent cleavage pattern of (GlcNAc)₄ by SdChiA showed that SdChiA specifically recognizes the β-anomer in the + 2 subsite of the substrate (GlcNAc)₄ and cleaves the glycoside bond at the center of the substrate. This is the first report of the occurrence of a family 18 chitinase containing CBM18 chitin-binding domains.

Abbreviations: AtChiC: Arabidopsis thaliana class V chitinase; CBB: Coomassie brilliant blue R250; CBM: carbohydrate binding module family; CrChi-A: Cycas revolute chitinase-A; EaChiA: Equisetum arvense chitinase-A; GH: glycoside hydrolase family, GlxChi-B: gazyumaru latex chitinase-B; GlcNAc: N-acetylglucosamine; HPLC: high performance liquid chromatography; LysM; lysin motif; MtNFH1: Medicago truncatula ecotypes R108-1 chitinase; NCBI: national center for biotechnology information; NF: nodulation factor; NtChiV: Nicotiana tabacum class V chitinase; PCR: polymerase chain reaction; PrChi-A: Pteris ryukyuensis chitinase-A; RACE: rapid amplification of cDNA ends; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SdChiA: Selaginella doederleinii chitinase-A.     

Grifolin derivatives from Albatrellus caeruleoporus, new inhibitors of nitric oxide production in RAW 264.7 cells

Two new farnesyl phenols named grifolinones A and B, together with known grifolin and neogrifolin, were isolated from methanolic extract of the inedible mushroom Albatrellus caeruleoporus. Their structures were characterized by a combination of 2D NMR, MS, IR, and UV spectra. Grifolinone B was composed of two grifolin molecules, which were connected by a C-C bond. Grifolinones A and B, grifolin, and neogrifolin exhibited inhibitory activity against nitric oxide (NO) production stimulated by lipopolysaccharide (LPS) in RAW 264.7 cells with IC50values of 23.4, 22.9, 29.0, and 23.3 microM, respectively.     

Orally administered LPS enhances head kidney macrophage activation with down-regulation of IL-6 in common carp (Cyprinus carpio)

Immunostimulants represent a promising aquaculture tool for enhancing disease and stress resistance in cultured fish. Moreover, the term and dose for acting immunostimulants is an important thing for fish farmer. This study investigated the immune parameters of common carp after oral administration of LPS (5, 10, 20 μg/kg/days) for 30 and 60 days, which is considered to be the proper time period for acting in aquaculture. Phagocytic and bactericidal activities of head kidney macrophages and serum lysozyme activities were significantly enhanced in LPS-fed carp. Orally administered LPS augmented the expression of interleukin (IL)-1β and TNF-α mRNAs but reduced the expression of IL-6 mRNA in head kidney. Although LPS was detected in the serum and liver after a high-dose (>15 mg/kg) oral administration, it was not detected by administered LPS-specific ELISA after a low-dose (<20 μg/kg) administration. It is speculated that orally administered LPS enhances the eliminating functions of head kidney macrophages with down-regulation of IL-6.     

New information on host plants and distribution ranges of an invasive gall midge, <Emphasis Type="Italic">Contarinia maculipennis</Emphasis> (Diptera: Cecidomyiidae), and its congeners in Japan

Gall midges of the genus Contarinia (Diptera: Cecidomyiidae) that infest the flower buds of various plant species have been newly found in Japan in recent years. Those infesting the flower buds of Pseuderanthemum laxiflorum (A. Gray) Hubbard ex Baillon (Amaranthaceae) and Jasminum sambac (Linnaeus) Aiton (Oleaceae) in Okinawa Prefecture, and Dendrobium spp. (Orchidaceae) in Mie Prefecture were identified, on the basis of morphological features and molecular information, as an invasive gall midge, C. maculipennis Felt. C. maculipennis was recorded in Mie Prefecture for the first time, and P. laxiflorum is newly regarded as one of the host plants of C. maculipennis. Three other Contarinia gall midges that we found infesting the flower buds of Lycopersicon esculentum Miller, Capsicum annuum Linnaeus (Solanaceae), and Oxalis corniculata Linnaeus (Oxalidaceae) were not identical with C. maculipennis. Among these, the first two, which infested solanaceous plants, were identical. However, the species other than C. maculipennis could not be identified to the species level because morphological differences were obscure and DNA sequencing data of allied congeners have not yet been registered on GenBank.     

The NPC motif of aquaporin-11, unlike the NPA motif of known aquaporins, is essential for full expression of molecular function

The recently identified molecule aquaporin-11 (AQP11) has a unique amino acid sequence pattern that includes an Asn-Pro-Cys (NPC) motif, corresponding to the N-terminal Asn-Pro-Ala (NPA) signature motif of conventional AQPs. In this study, we examined the effect of the mutation of the NPC motif on the subcellular localization, oligomerization, and water permeability of AQP11 in transfected mammalian cells. Furthermore, the effect was also assessed using zebrafish. Site-directed mutation at the NPC motif did not affect the subcellular localization of AQP11 but reduced its oligomerization. A cell swelling assay revealed that cells expressing AQP11 with a mutated NPC motif had significantly lower osmotic water permeability than cells expressing wild-type AQP11. Zebrafish deficient in endogenous AQP11 showed a deformity in the tail region at an early stage of development. This phenotype was dramatically rescued by injection of human wild-type AQP11 mRNA, whereas the effect of mRNA for AQP11 with a mutated NPC motif was less marked. Although the NPA motif is known to be important for formation of water-permeable pores by conventional AQPs, our observations suggest that the corresponding NPC motif of AQP11 is essential for full expression of molecular function.     

Characterization of a Novel Proteinous Toxin from Sea Anemone <Emphasis Type="Italic">Actineria villosa</Emphasis>

The sea anemone Actineria villosa expresses a lethal protein toxin. We isolated a novel 120-kDa protein, Avt120, from partially purified toxin and found it to possess extremely strong lethal activity. The 3,453-bp Avt120 gene translates to a 995-amino acid protein. The 50% lethal dose (LD₅₀) of purified Avt120 in mice was 85.17 ng. Among several tested cell lines, Colo205 cells were most sensitive to Avt120: 50% of them were damaged by 38.4 ng/mL Avt120. Avt120 exerted ATP degradation activity (10 μmol ATP h⁻¹ mg⁻¹), which was strongly inhibited by ganglioside GM1 to decrease the cytotoxicity of Avt120.     

Identification of native Escherichia coli BL21 (DE3) proteins that bind to immobilized metal affinity chromatography under high imidazole conditions and use of 2D-DIGE to evaluate contamination pools with respect to recombinant protein expression level

Immobilized metal affinity chromatography (IMAC) is a widely used purification tool for the production of active, soluble recombinant proteins. Escherichia coli proteins that routinely contaminate IMAC purifications have been characterized to date. The work presented here narrows that focus to the most problematic host proteins, those retaining nickel affinity under elevated imidazole conditions, using a single bind-and-elute step. Two-dimensional difference gel electrophoresis, a favored technique for resolving complex protein mixtures and evaluating their expression, here discerns variation in the soluble extract pools that are loaded in IMAC and the remaining contaminants with respect to varied levels of recombinant protein expression. Peptidyl-prolyl isomerase SlyD and catabolite activator protein (CAP) are here shown to be the most persistent contaminants and have greater prevalence at low target protein expression.