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.     

Activation of distinct signal transduction pathways in Trypanosoma cruzi isolates with differential capacity to invade host cells

Mammalian cell invasion by Trypanosoma cruzi requires the activation of signal transduction pathways that result in a Ca(2+) response both in the parasite and the host cell. By using drugs that interfere with the signalling processes, we investigated if the difference in the ability of T. cruzi isolates to invade host cells was associated with the activation of distinct signalling routes in the parasites. Experiments were performed with metacyclic trypomastigotes, the developmental forms that initiate infection in the mammalian host, using the highly invasive isolate CL and the poorly infective isolate G, which belong to distinct phylogenetic lineages. Treatment of parasites with adenylyl cyclase activator forskolin increased the infectivity of the G but not of the CL isolate towards HeLa cells. On the other hand, a specific protein tyrosine kinase inhibitor genistein reduced by approximately 75% the penetration of CL but not of G isolate into HeLa cells. In the CL but not in the G isolate, protein tyrosine kinase mediated the phosphorylation of a 175kDa protein in a manner inducible by a HeLa cell extract. Upon treatment with the phospholipase C inhibitor U73122, or with drugs such as caffeine, which affects Ca(2+) release from inositol-1,4,5-triphosphate-sensitive stores, or thapsigargin, an inhibitor of intracellular Ca(2+) transport ATPases, the infectivity of the CL but not of the G isolate diminished significantly (P<0.005). In both isolates, a combination of ionomycin plus NH(4)Cl or nigericin released Ca(2+) from acidic vacuoles containing a Ca(2+)/H(+) exchange system. This treatment reduced the infectivity of metacyclic forms of the G but not of the CL isolate. Taken together, these data suggest that, for host cell invasion, distinct signalling pathways are activated in metacyclic trypomastigotes of the two isolates, leading to Ca(2+) release from different intracellular compartments.     

Relationship between conidial enzymes and germination of the apple blue mold,Penicillium expansum

The relationship between conidial enzymes of Penicillium expansum and spore germination was examined. The activities of xylanase and pectinase, but not of cellulase and amylase, were detected in the conidia. The levels of xylanase and pectinase were greatly enhanced by xylan and pectin as respective carbon sources in the basal medium. No conidia germinated in the basal medium without a carbon source. The type of carbon source and the enzyme levels of the conidia did not affect the rate of germination. However, a relationship was found between the enzyme levels and the elongation of the germ tubes.     

Plasma protein binding study of oxybutynin by high-performance frontal analysis

Plasma protein binding of oxybutynin (OXY) was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of OXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human alpha1-acid glycoprotein (AGP) solutions. OXY is bound in human plasma strongly and enantioselectively. The bound drug fraction in human plasma containing 2-10 microM (R)- or (S)-OXY was higher than 99%, and the unbound fraction of (R)-OXY was 1.56 times higher than that of (S)-isomer. AGP plays the dominant role in this strong and enantioselective plasma protein binding. The total binding affinities (nK) of (R)- and (S)-OXY to AGP were 6.86 x 10(6) and 1.53 x 10(7) M(-1), respectively, while the nK values of (R)- and (S)-OXY to HSA were 2.64 x 10(4) and 2.19 x 10(-4) M(-1), respectively. The binding affinity of OXY to AGP is much higher than that to HSA, and shows high enantioselectivity (SIR ratio of nK values is 2.2). It was found that both enantiomers are bound competitively at the same binding site on an AGP molecule. The binding property between OXY and low density lipoprotein (LDL) was investigated by using the frontal analysis method incorporated in high-performance capillary electrophoresis (HPCE/FA). It was found the binding is non-saturable and non-enantioselective.     

Conversion of gastric mucosa to intestinal metaplasia in Cdx2-expressing transgenic mice

Gastric intestinal metaplasia occurs as a pathological condition in the gastric mucosa. To clarify how an intestine-specific homeobox gene, Cdx2, affects the morphogenesis of gastric mucosa, we generated transgenic mice expressing Cdx2 in parietal cells. Until Day 18 after birth, the number of parietal cells inthegastric mucosa of transgenic mice was the same as for their normal littermates. However, at Day 19, we detected several glands in which parietal cells disappeared and the proliferating zone moved from the isthmus to the base of the glands. Thereafter, parietal cells decreased gradually and disappeared at Day 37. All of the gastric mucosal cells, except for enterochromaffin-like (ECL) cells, were completely replaced by intestinal metaplasia, consisting of goblet cells, enteroendocrine cells, and absorptive cells expressing alkaline phosphatase. Pseudopyloric gland metaplasia was also formed. The transgenic mouse is a very useful model for clarifying physiological differentiation of gastric and intestinal cell lineages and analyzing the molecular events from intestinal metaplasia to adenocarcinoma.     

Overproduction of highly active trypanosome alternative oxidase in Escherichia coli heme-deficient mutant

Cyanide-insensitive trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms of the African trypanosome, which causes sleeping sickness in humans and nagana in cattle. TAO has been targeted for the development of anti-trypanosomal drugs, because it does not exist in the host. In this study, we established a system for overproduction of highly active TAO in Eschericia coli heme-deficient mutant. Kinetic analysis of recombinant enzyme and TAO in Trypanosoma brucei brucei mitochondria revealed that recombinant TAO retains the properties of native enzyme, indicating that recombinant TAO is quite valuable for further biochemical study of TAO.     

Characterization of Epstein-Barr virus (EBV)-positive NK cells isolated from hydroa vacciniforme-like eruptions

Recently, the involvement of Epstein-Barr virus (EBV) in hydroa vacciniforme (HV)-like eruptions has been suggested. To elucidate the role of EBV in this disease, we isolated EBV-infected cell clones from peripheral blood mononuclear cells (PBMC) and the skin lesions of a patient with HV-like eruptions; cells isolated from PBMC were designated SNK-12, and those from the eruption SNK-11. Both cells expressed CD16, CD56, and HLA-DR and had germline configurations of the T-cell receptor and the immunoglobulin genes, indicating that the cell clones were of NK cell lineage. The analysis of EBV terminal repeats indicated that the cells were monoclonal, had identical clonality, and originated from EBV-positive cells in the PBMC and eruption. Both clones expressed EBNA-1, but not EBNA-2. Although LMP-1 was weakly detected in SNK-11, no LMP-1 was detected in SNK-12. Interestingly, EBV-infected cells required less IL-2 for in vitro growth in the later phase of this disease and this appeared to correlate with the expression of LMP-1, suggesting that the proliferative capacity of the EBV-positive NK cells increased during the time course of the disease, and LMP-1 expression might be responsible for that. This is the first report of the isolation of EBV-infected cells from the skin lesions of HV-like eruptions and strongly suggests that the HV-like eruption in the patient was caused by clonal NK cells with latent EBV infection.     

Nucleoside diphosphate kinases in mammalian signal transduction systems: recent development and perspective

The role of nucleoside diphosphate (NDP) kinase with special reference to mammalian signal transduction systems was described. The interaction between NDP kinases and G proteins was reevaluated in view of their protein structural information and its significance was extended further on the basis of recent findings obtained with small molecular weight G proteins such as Rad, menin, and Rac. Meanwhile, observations suggesting involvement of NDP kinases in the regulation of cell growth and differentiation led to the realization that NDP kinases may play a crucial role in receptor tyrosine kinase signal transduction systems. In fact, a number of experimental results, particularly obtained with PC12 cells, implicate that NDP kinases appear to regulate differentiation marker proteins and cell-cycle-associated proteins cooperatively. Consequently, we propose a hypothesis that NDP kinases might act like a molecular switch to determine the cell fate toward proliferation or differentiation in response to environmental signals.     

Hic-5 communicates between focal adhesions and the nucleus through oxidant-sensitive nuclear export signal

hic-5 was originally isolated as an H(2)O(2)-inducible cDNA clone whose product was normally found at focal adhesions. In this study, we found that Hic-5 accumulated in the nucleus in response to oxidants such as H(2)O(2). Other focal adhesion proteins including paxillin, the most homologous to Hic-5, remained in the cytoplasm. Mutation analyses revealed that the C- and N-terminal halves of Hic-5 contributed to its nuclear localization in a positive and negative manner, respectively. After the finding that leptomycin B (LMB), an inhibitor of nuclear export signal (NES), caused Hic-5 to be retained in the nucleus, Hic-5 was demonstrated to harbor NES in the N-terminal, which was sensitive to oxidants, thereby regulating the nuclear accumulation of Hic-5. NES consisted of a leucine-rich stretch and two cysteines with a limited similarity to Yap/Pap-type NES. In the nucleus, Hic-5 was suggested to participate in the gene expression of c-fos. Using dominant negative mutants, we found that Hic-5 was actually involved in endogenous c-fos gene expression upon H(2)O(2) treatment. Hic-5 was thus proposed as a focal adhesion protein with the novel aspect of shuttling between focal adhesions and the nucleus through an oxidant-sensitive NES, mediating the redox signaling directly to the nucleus.