Microflorae of aquatic moss pillars in a freshwater lake, East Antarctica, based on fatty acid and 16S rRNA gene analyses

Aquatic mosses in the genera Bryum and Leptobryum form unique tower-like ??moss pillars?? underwater in some Antarctic lakes, in association with algae and cyanobacteria. These are communities with a two-layer structure comprising an oxidative exterior and reductive interior. Although habitats and photosynthetic properties of moss pillars have been reported, microfloral composition of the two-layer structure has not been described. Here we report fatty acid analysis of one moss pillar and molecular phylogenetic analysis, based on the 16S rRNA gene, of this and one other moss pillar. Cluster analysis of the phospholipid fatty acid composition showed three groups corresponding to the exterior, upper interior, and lower interior of the pillar. This suggested that species composition differed by section, with the exterior dominated by photosynthetic organisms such as mosses, algae, and cyanobacteria, the upper interior primarily containing gram-positive bacteria and anaerobic sulfate-reducing bacteria, and the lower interior dominated by gram-negative bacteria. Molecular phylogenetic analysis revealed that Proteobacteria dominate the moss pillar as a whole; cyanobacteria were found on the exterior and the gram-positive obligate anaerobe Clostridium in the interior, while gram-positive sulfate-reducing bacteria were present in the lowest part of the interior. Nitrogen-fixing bacteria and denitrifying bacteria were found in all sections. Thus, fatty acid analysis and genetic analysis showed similar patterns. These findings suggest that microorganisms of different phylogenetic groups inhabit different sections of a single moss pillar and form a microbial community that performs biogeochemical cycling to establish and maintain a structure in an oxidation?Creduction gradient between exterior and interior.     

A novel neural-specific BMP antagonist, Brorin-like, of the Chordin family

We identified a gene encoding a novel secreted protein in mice, humans, and zebrafish. As the protein of 222 amino acids is similar to Brorin, a secreted BMP antagonist, which is a member of the Chordin family, we named it Brorin-like. Recombinant Brorin-like protein weakly but significantly inhibited the activity of BMP in mouse preosteoblastic cells and promoted neurogenesis in mouse neural precursor cells. Brorin-like was predominantly expressed in the adult brain and embryonic neural tissues. The inhibition of Brorin-like functions in zebrafish resulted in the impairment of neural development. Brorin-like potentially plays roles in neural development and functions.     

α-Chymotrypsin-catalyzed enantioselective amidation of chiral amines using an N-protected amino acid carbamoylmethyl ester as acyl donor

-Chymotrypsin catalyzed the highly enantioselective amidation of chiral amines such as 1-(1-naphthyl)ethylamine using N-benzyloxycarbonyl-(S)-phenylalanine carbamoylmethyl ester as acyl donor (E = 25–660) in acetonitrile with low water content.     

Membrane filter (pore size, 0.22-0.45 micro m; thickness, 150 micro m) passing-through activity of Pseudomonas aeruginosa and other bacterial species with indigenous infiltration ability

Bacteria growing on MF-Millipore filters (thickness, 150 micro m) passed through the underlying membrane by their infiltration activity. Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae, and Escherichia coli passed through a 0.45- micro m pore size filter within 48-96 h. Pseudomonas aeruginosa, Serratia marcescens, and Listeria monocytogenes passed through a 0.3- micro m pore size filter. P. aeruginosa passed through a 0.22- micro m pore size filter. The membranes which allowed passing-through of bacteria showed normal bubble point values in the integrity test. Studies with isogenic S. marcescens mutants indicated that flagellum-dependent motility or surface-active exolipid were important in the passing-through. P. aeruginosa PAO1 C strain defective in twitching motility was unable to pass through the 0.22- micro m filter. Scanning electron microscopy showed bacteria passing-through the 0.22- micro m filter. Millipore membrane filters having well-defined reticulate structures will be useful in the study of infiltration activity of microbes.     

N-linked glycan structures of human lactoferrin produced by transgenic rice

Human lactoferrin was produced in genetically engineered rice. N-linked glycan structures of recombinant human lactoferrin were determined. The oligosaccharides liberated by hydrazinolysis were labeled with 2-aminopyridine (PA). The PA-labeled glycans were purified by reverse-phase and size-fractionation HPLCs. The structures of these glycans were identified by HPLC, exoglycosidase digestion, and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The glycan structures determined were ManFucXylGlcNAc(2) (3.4%), Man(2)FucGlcNAc(2) (2.1%), Man(3)FucGlcNAc(2) (2.5%), Man(3)FucXylGlcNAc(2) (42.5%), two isomers of Man(2)FucXylGlcNAc(2) (39.1%), Man(3)XylGlcNAc(2) (6.5%), and Man(2)XylGlcNAc(2) (3.9%).     

Down-regulation of Bcl-2-interacting protein BAG-1 confers resistance to anti-cancer drugs

BAG-1 was originally identified as a binding partner of anti-apoptotic factor Bcl-2 [Takayama et al., Cell 80 (1995) 279-284]. Exogenous expression of BAG-1 was reported to confer cells resistance to several stresses [Chen et al., Oncogene 21 (2002) 7050]. We have obtained human cervical cancer HeLa cells with down-regulated BAG-1 levels by using a highly specific and efficient RNA interference approach. Surprisingly, cells with down-regulated BAG-1 exhibited significantly lower sensitivity against several anti-cancer drugs than parental cells expressing normal levels of the protein. Furthermore, growth rate of the cells was reduced when BAG-1 was down-regulated. Activity of ERK pathway appeared to be decreased in BAG-1 down-regulated cells, as shown by the reduced phosphorylation of ERK1/2 proteins. Taken together resistance against anti-cancer drugs acquired by BAG-1 down-regulated cells may well be accounted for by the retardation of cell cycle progression, implicating the importance of BAG-1 in cell growth regulation.     

N-linked glycan structures of mouse interferon-beta produced by Bombyx mori larvae

The full-length mouse interferon-beta (mIFN-beta) cDNA, including the secretion signal peptide coding region under control of the polyhedrin promoter, was introduced into Bombyx mori nucleopolyhedrovirus (BmNPV). Recombinant mIFN-beta (rmIFN-beta) was accumulated in the haemolymph of infected silkworm larvae. Western blot analysis showed isoforms of rmIFN-beta, suggesting that rmIFN-beta is glycosylated. The glycan structures of purified rmIFN-beta were determined. The N-glycans were liberated by hydrazinolysis and the resulting oligosaccharides were labeled with 2-aminopyridine. The pyridylaminated (PA) glycans were purified by gel filtration, reversed-phase HPLC, and size-fractionation HPLC. The structures of the PA-sugar chains were identified by a combination of two-dimensional PA-sugar chain mapping, MS analysis, and exoglycosidase digestions.     

Three-dimensional structure of non-activated cGMP phosphodiesterase 6 and comparison of its image with those of activated forms

Cyclic GMP phosphodiesterase (PDE6) in rod photoreceptors, a key enzyme in vertebrate phototransduction, consists of two homologous catalytic subunits (Palpha and Pbeta) and two identical regulatory subunits (Pgammas). Pgamma regulates the PDE activity through its direct interaction with transducin. Here, using electron microscopy and image analysis of single particles, we show the three-dimensional organization of the basic form of bovine PDE, Palphabetagammagamma, and compare its average image with those of Pgamma-released PDE. The structure of Palphabetagammagamma appears to be a flattened bell-shape, with dimensions of 150 x 108 x 60A, and with a handle-like protrusion attached to the top of the structure. Except for the protrusion, the organization consists of two homologous structures arranged side by side, with each structure having three distinct regions, showing pseudo twofold symmetry. These characteristics are consistent with a model in which the overall structure of Palphabetagammagamma is determined by hetero-dimerization of Palpha and Pbeta, with each subunit consisting of one catalytic and two GAF regions. A comparison of the average image of Palphabetagammagamma with those of Pgamma-released PDE suggests that Pgamma release does not affect the overall structure of Palphabeta, and that the Palphabeta C-terminus, but not Pgamma, is a determinant for the Palphabeta orientation on carbon-coated grids. These observations suggest that the basic structure of PDE does not change during its regulation, which implies that Palphabeta is regulated by its regional interaction with Pgamma.     

Inducible costimulator protein controls the protective T cell response against Listeria monocytogenes

The inducible costimulator protein (ICOS) was recently identified as a costimulatory molecule for T cells. Here we analyze the role of ICOS for the acquired immune response of mice against the intracellular bacterium Listeria monocytogenes. During oral L. monocytogenes infection, low levels of ICOS expression were detected by extracellular and intracellular Ab staining of Listeria-specific CD4(+) and CD8(+) T cells. Blocking of ICOS signaling with a soluble ICOS-Ig fusion protein markedly impaired the Listeria-specific T cell responses. Compared with control mice, the ICOS-Ig treated mice generated significantly reduced numbers of Listeria-specific CD8(+) T cells in spleen and liver, as determined by tetramer and intracellular cytokine staining. In contrast, the specific CD8(+) T cell response in the intestinal mucosa did not appear to be impaired by the ICOS-Ig treatment. Analysis of the CD4(+) T cell response revealed that ICOS-Ig treatment also affected the specific CD4(+) T cell response. When restimulated with listerial Ag in vitro, reduced numbers of CD4(+) T cells from infected and ICOS-Ig-treated mice responded with IFN-gamma production. The impaired acquired immune response in ICOS-Ig treated mice was accompanied by their increased susceptibility to L. monocytogenes infection. ICOS-Ig treatment drastically enhanced bacterial titers, and a large fraction of mice succumbed to the otherwise sublethal dose of infection. Thus, ICOS costimulation is crucial for protective immunity against the intracellular bacterium L. monocytogenes.