Production of D-amino acid oxidase (DAO) of Trigonopsis variabilis in Schizosaccharomyces pombe and the characterization of biocatalysts prepared with recombinant cells

The cDNA of D-amino acid oxidase (DAO) gene isolated from Trigonopsis variabilis was expressed in Schizosaccharomyces pombe. A clone, ASP327-10, transformed with plasmid vector, pTL2M5DAO, expressed catalytically active DAO in the presence of G418, and converted Cephalosprin C to alpha-ketoadipyl-7-cephalosporanic acid (KA-7-ACA) and glutaryl-7-aminocephalosporanic acid (GL-7-ACA). Biocatalysts were prepared using ASP327-10 and T. variabilis, and evaluated to demonstrate the feasibility of recombinant S. pombe for industrial application. The cells were immobilized by crosslinking polyethylene imine after glutardialdehyde (GDA) fixation and permeabilization by alkaline treatment. Although the biocatalyst prepared from ASP327-10 exhibited DAO activity, catalase activity still remained fully even after permeabilization, under which condition, the catalase activity of T. variabilis decreased to 20-30%. Heat treatment was required before cell fixation by GDA to inactivate the catalase in S. pombe. This improved the efficiency of bioconversion to GL-7-ACA, but caused poor mechanical strength in the biocatalyst of S. pombe. To overcome this weakness, a catalase-deficient host strain was obtained by ethylmethansulfate mutagenesis. Moreover, taking economics into consideration, the integrative vector, pTL2M5DAO-8XL, with multi-copies of expression cassette was constructed to express DAO in S. pombe even in the absence of G418. The newly established integrant, ASP417-7, did not exhibit any catalase activity so that heat treatment was not required. The obtained integrant and its biocatalyst were significantly improved in GL-7ACA conversion ability and mechanical strength. This study demonstrates that the established integrant is a potential candidate as an alternative source of DAO enzyme.     

Differential sensitivity between Fks1p and Fks2p against a novel beta -1,3-glucan synthase inhibitor,aerothricin3 [corrected

Fks1p and Fks2p are catalytic subunits of beta-1,3-glucan synthase, which synthesize beta-1,3-glucan, a main component of the cell wall in Saccharomyces cerevisiae. Although Fks1p and Fks2p are highly homologous, sharing 88.1% identity, it has been shown that Fks2p is more sensitive than Fks1p to one of echinocandin derivatives, which inhibits beta-1,3-glucan synthase activity. Here we show a similar differential sensitivity between Fks1p and Fks2p to a novel beta-1,3-glucan synthase inhibitor, aerothricin3 [corrected]. To investigate the molecular mechanism of this differential sensitivity, we constructed a series of chimeric genes of FKSs and examined their sensitivity to aerothricin3 [corrected]. As a result, it was shown that a region around the fourth extracellular domain of Fks2p, containing 10 different amino acid residues from those of Fks1p, provided Fks1p aerothricin3 [corrected] sensitivity when the region was replaced with a corresponding region of Fks1p. In order to identify essential amino acid residues responsible for the sensitivity, each of the 10 non-conserved amino acids of Fks1p was substituted into the corresponding amino acid of Fks2p by site-directed mutagenesis. Surprisingly, only one amino acid substitution of Fks1p (K1336I) conferred Fks1p hypersensitivity to aerothricin3 [corrected]. On the other hand, reverse substitution of the corresponding amino acid of Fks2p (I1355K) resulted in loss of hypersensitivity to aerothricin3 [corrected]. These results suggest that the 1355th isoleucine of Fks2p plays a key role in aerothricin3 [corrected] sensitivity.     

The leukocyte common antigen (CD45) of the Pacific hagfish,Eptatretus stoutii: implications for the primordial function of CD45

CD45, originally known as the leukocyte common antigen, is a prototypical transmembrane protein tyrosine phosphatase that plays a critical role in signal transduction through T-cell and B-cell receptors, as well as in T-cell and B-cell development. In the present study, we show that the Pacific hagfish, widely believed to lack the adaptive immune system, has CD45. The presence of CD45 in jawless fish is consistent with the recent discovery that CD45 also plays a crucial role in innate immunity via the regulation of signaling through type I and type II cytokine receptors. It is likely that CD45 was recruited to activate lymphocytes through antigen receptors encoded by rearranging genes in jawed vertebrates.     

Spatial distribution of total, ammonia-oxidizing, and denitrifying bacteria in biological wastewater treatment reactors for bioregenerative life support

Bioregenerative life support systems may be necessary for long-term space missions due to the high cost of lifting supplies and equipment into orbit. In this study, we investigated two biological wastewater treatment reactors designed to recover potable water for a spacefaring crew being tested at Johnson Space Center. The experiment (Lunar-Mars Life Support Test Project-Phase III) consisted of four crew members confined in a test chamber for 91 days. In order to recycle all water during the experiment, an immobilized cell bioreactor (ICB) was employed for organic carbon removal and a trickling filter bioreactor (TFB) was utilized for ammonia removal, followed by physical-chemical treatment. In this study, the spatial distribution of various microorganisms within each bioreactor was analyzed by using biofilm samples taken from four locations in the ICB and three locations in the TFB. Three target genes were used for characterization of bacteria: the 16S rRNA gene for the total bacterial community, the ammonia monooxygenase (amoA) gene for ammonia-oxidizing bacteria, and the nitrous oxide reductase (nosZ) gene for denitrifying bacteria. A combination of terminal restriction fragment length polymorphism (T-RFLP), sequence, and phylogenetic analyses indicated that the microbial community composition in the ICB and the TFB consisted mainly of Proteobacteria, low-G+C gram-positive bacteria, and a Cytophaga-Flexibacter-Bacteroides group. Fifty-seven novel 16S rRNA genes, 8 novel amoA genes, and 12 new nosZ genes were identified in this study. Temporal shifts in the species composition of total bacteria in both the ICB and the TFB and ammonia-oxidizing and denitrifying bacteria in the TFB were also detected when the biofilms were compared with the inocula after 91 days. This result suggests that specific microbial populations were either brought in by the crew or enriched in the reactors during the course of operation.     

Neurotoxic mechanisms triggered by Alzheimer's disease-linked mutant M146L presenilin 1: involvement of NO synthase via a novel pertussis toxin target

While it has been reported that familial Alzheimer's disease (FAD)-linked mutants of amyloid precursor protein (APP) and presenilin (PS)2 induce neuronal cytotoxicity in a manner sensitive to antioxidant and pertussis toxin (PTX), little of the mechanism for PS1-mediated neuronal cell death has been characterized. We previously found that multiple mechanisms, different in detail, underlie cytotoxicities by two FAD-linked mutants of APP, using neuronal cells with an ecdysone-controlled expression system. Here we report that this system revealed that (i) low expression of FAD-linked M146L-PS1 caused neuronal cell death, whereas that of wild-type (wt)PS1 did not; (ii) mutation-specific cytotoxicity by M146L-PS1 was sensitive to antioxidant glutathione-ethyl-ester and resistant to Ac-DEVD-CHO; (iii) cytotoxicity by higher expression of wtPS1 was resistant to both; and (iv) cytotoxicity by M146L-PS1 was inhibited by PTX. It was also highly likely that the involved superoxide-generating enzyme was nitric oxide synthase (NOS), and that the PTX-sensitive cytotoxic signal by M146L-PS1 was mediated by none of the G(i/o) proteins. We conclude that M146L-PS1 activates a NOS-mediated cytotoxic pathway via a novel PTX target.     

Identification of genes expressed preferentially in the honeybee mushroom bodies by combination of differential display and cDNA microarray

To clarify the molecular basis underlying the neural function of the honeybee mushroom bodies (MBs), we identified three genes preferentially expressed in MB using cDNA microarrays containing 480 differential display-positive candidate cDNAs expressed locally or differentially, dependent on caste/aggressive behavior in the honeybee brain. One of the cDNAs encodes a putative type I inositol 1,4,5-trisphosphate (IP(3)) 5-phosphatase and was expressed preferentially in one of two types of intrinsic MB neurons, the large-type Kenyon cells, suggesting that IP(3)-mediated Ca(2+) signaling is enhanced in these neurons.     

Association of plasma viral RNA load with prognosis in cats naturally infected with feline immunodeficiency virus

We measured the quantity of plasma feline immunodeficiency virus (FIV) RNA using a real-time sequence detecting system. Plasma viral RNA load was shown to correlate with the clinical stage, survival time, and disease progression in naturally FIV-infected cats. The present study indicates that the plasma viral RNA load can be used as a clinical marker representing the impairment of the immune system and predicting the clinical outcome in FIV-infected cats.