High conversion in asymmetric hydrolysis during permeation through enzyme-multilayered porous hollow-fiber membranes

We describe a novel porous hollow-fiber support for immobilizing aminoacylase in multilayers. Epoxy-group-containing polymer chains were grafted onto a porous hollow-fiber membrane by radiation-induced graft polymerization of glycidyl methacrylate, and subsequently a diethylamino group as an anion-exchange group was introduced into the graft chain. Aminoacylase was adsorbed in multilayers by allowing the amioacylase buffer solution to permeate through the pores across the hollow fiber; the graft chains provided three-dimensional space for the enzymes because of their electrostatic repulsion. The adsorbed enzyme at a degree of multilayer binding of 15 was cross-linked with glutaraldehyde to prevent leakage. An acetyl-DL-methionine solution was allowed to permeate through the pores surrounded by the aminoacylase-immobilized graft chain. Production of L-methionine was observed at a 4.1 mol/h per L of the fiber for a space velocity of 200 h(-1), defined as the flow rate of the effluent penetrating the outside surface of the hollow fiber divided by the membrane volume including the lumen.     

Effects of mutation in hepatitis C virus nonstructural protein 5A on interferon resistance mediated by inhibition of PKR kinase activity in mammalian cells

The IFN-induced double-stranded RNA (dsRNA)-activated protein kinase PKR is one of the key molecules in the antiviral effects of IFN. To clarify the effects of hepatitis C virus nonstructural protein 5A (NS5A) on antiviral activity of IFN, in particular on PKR kinase activity, in mammalian cells, we established inducible NS5A-expressing cell lines derived from human osteosarcoma (Saos-2). The cells expressing NS5A derived from an IFN-resistant clone (NS5A-lb) that interacted with endogenous PKR in vitro, showed a suppressive effect on IFN function as determined by interference with vesicular stomatitis virus (VSV) infection, whereas NS5A (NS5A-2a) from an IFN-sensitive clone did not block the antiviral effect of IFN. A mutant with deletion of the IFN sensitivity determining region (ISDR) in NS5A-1b (NS5A-AISDR) also interacted with PKR and suppressed its activity in vitro. However, neither NS5A-2a nor the C-terminal truncated mutant of NS5A-1b (NS5A-deltaC) blocked PKR activity. These observations confirmed the previous report that the inhibitory effect of NS5A on IFN activity is mediated at least in part by the repression of PKR. In addition, we showed that IFN sensitivity was determined not only by the ISDR but that the involvement of the C-terminal region of NS5A-1b is important for the suppression of PKR activity.     

Na(+) action potentials in human photoreceptors

Mammalian photoreceptors are hyperpolarized by a light stimulus and are commonly thought to be nonspiking neurons. We used the whole-cell patch-clamp technique on surgically excised human retina to examine whether human photoreceptors can elicit action potentials. We discovered that human rod photoreceptors express voltage-gated Na(+) channels, and generate Na(+) action potentials, in response to membrane depolarization from membrane potentials of -60 or -70 mV. Na(+) spikes in human rods were elicited at the termination of a light response that hyperpolarized the potential well below -50 mV. This served to amplify the release of a neurotransmitter when a bright light is turned off, and thus selectively amplify the off response to the light signal.     

Properties of an extracellular polysaccharide produced by a strain of enterobacter isolated from pond water

A bacterium which was isolated from pond water and identified as Enterobacter cloacae produced a viscous extracellular polysaccharide when it was grown aerobically in a medium containing sucrose as a sole source of carbon. The maximum molecular weight of the polysaccharide was about 9.0 x 10(5). The polysaccharide was composed of fucose, galactose, glucose, and glucuronic acid in a molar ratio of 2:3:2:1, but the molecular weight and the molar ratio of the sugar component were different from those of the polysaccharide produced by the same species reported elsewhere.     

Comprehensive sequence analysis of translation termination sites in various eukaryotes

Recent investigations into the translation termination sites of various organisms have revealed that not only stop codons but also sequences around stop codons have an effect on translation termination. To investigate the relationship between these sequence patterns and translation as well as its termination efficiency, we analysed the correlation between strength of consensus and translation efficiency, as predicted according to Codon Adaptation Index (CAI) value. We used RIKEN full-length mouse cDNA sequences and ten other eukaryotic UniGene datasets from NCBI for the analyses. First, we conducted sequence profile analyses following translation termination sites. We found base G and A at position +1 as a strong consensus for mouse cDNA. A similar consensus was found for other mammals, such as Homo sapiens, Rattus norvegicus and Bos taurus. However, some plants had different consensus sequences. We then analysed the correlation between the strength of consensus at each position and the codon biases of whole coding regions, using information content and CAI value. The results showed that in mouse cDNA, CAI value had a positive correlation with information content at positions +1. We also found that, for positions with strong consensus, the strength of the consensus is likely to have a positive correlation with CAI value in some other eukaryotes. Along with these observations, biological insights into the relationship between gene expression level, codon biases and consensus sequence around stop codons will be discussed.     

Mutual dependence of MDM2 and MDMX in their functional inactivation of p53

MDMX, an MDM2-related protein, has emerged as yet another essential negative regulator of p53 tumor suppressor, since loss of MDMX expression results in p53-dependent embryonic lethality in mice. However, it remains unknown why neither homologue can compensate for the loss of the other. In addition, results of biochemical studies have suggested that MDMX inhibits MDM2-mediated p53 degradation, thus contradicting its role as defined in gene knockout experiments. Using cells deficient in either MDM2 or MDMX, we demonstrated that these two p53 inhibitors are in fact functionally dependent on each other. In the absence of MDMX, MDM2 is largely ineffective in down-regulating p53 because of its extremely short half-life. MDMX renders MDM2 protein sufficiently stable to function at its full potential for p53 degradation. On the other hand, MDMX, which is a cytoplasmic protein, depends on MDM2 to redistribute into the nucleus and be able to inactivate p53. We also showed that MDMX, when exceedingly overexpressed, inhibits MDM2-mediated p53 degradation by competing with MDM2 for p53 binding. Our findings therefore provide a molecular basis for the nonoverlapping activities of these two p53 inhibitors previously revealed in genetic studies.     

Detection and induction of CTLs specific for SYT-SSX-derived peptides in HLA-A24(+) patients with synovial sarcoma

To investigate the immunogenic property of peptides derived from the synovial sarcoma-specific SYT-SSX fusion gene, we synthesized four peptides according to the binding motif for HLA-A24. The peptides, SS391 (PYGYDQIMPK) and SS393 (GYDQIMPKK), were derived from the breakpoint of SYT-SSX, and SS449a (AWTHRLRER) and SS449b (AWTHRLRERK) were from the SSX region. These peptides were tested for their reactivity with CTL precursors (CTLps) in 16 synovial sarcoma patients using HLA-A24/SYT-SSX peptide tetramers and also for induction of specific CTLs from four HLA-A24(+) synovial sarcoma patients. Tetramer analysis indicated that the increased CTLp frequency to the SYT-SSX was associated with pulmonary metastasis in synovial sarcoma patients (p < 0.03). CTLs were induced from PBLs of two synovial sarcoma patients using the peptide mixture of SS391 and SS393, which lysed HLA-A24(+) synovial sarcoma cells expressing SYT-SSX as well as the peptide-pulsed target cells in an HLA class I-restricted manner. These findings suggest that aberrantly expressed SYT-SSX gene products have primed SYT-SSX-specific CTLps in vivo and increased their frequency in synovial sarcoma patients. The identification of SYT-SSX peptides may offer an opportunity to design peptide-based immunotherapeutic approaches for HLA-A24(+) patients with synovial sarcoma.     

Redox-coupled conformational alternations in cytochrome c(3) from D. vulgaris Miyazaki F on the basis of its reduced solution structure

Heteronuclear NMR spectroscopy was performed to determine the solution structure of (15)N-labeled ferrocytochrome c(3) from Desulfovibrio vulgaris Miyazaki F (DvMF). Although the folding of the reduced cytochrome c(3) in solution was similar to that of the oxidized one in the crystal structure, the region involving hemes 1 and 2 was different. The redox-coupled conformational change is consistent with the reported solution structure of D. vulgaris Hildenborough ferrocytochrome c(3), but is different from those of other cytochromes c(3). The former is homologous with DvMF cytochrome c(3) in amino acid sequence. Small displacements of hemes 1 and 2 relative to hemes 3 and 4 were observed. This observation is consistent with the unusual behavior of the 2(1)CH(3) signal of heme 3 reported previously. As shown by the (15)N relaxation parameters of the backbone, a region between hemes 1 and 2 has more flexibility than the other regions. The results of this work strongly suggest that the cooperative reduction of hemes 1 and 2 is based on the conformational changes of the C-13 propionate of heme 1 and the aromatic ring of Tyr43, and the interaction between His34 and His 35 through covalent and coordination bonds.     

Solution structure of a SRP19 binding domain in human SRP RNA

Assembly of the human signal recognition particle (SRP) requires SRP19 protein to bind to helices 6 and 8 of SRP RNA. In the present study, structure of a 29-mer RNA composing the SRP19 binding site in helix 6 was determined by NMR spectroscopy. The two A:C mismatches were continuously stacked to each other and formed wobble type A:C base pairs. The GGAG tetraloop in helix 6 was found to adopt a similar conformation to that of GNRA tetraloop, suggesting that these tetraloops are included in an extensive new motif GNRR. Compared with the crystal structure of helix 6 in complex with SRP19 determined previously, the GGAG tetraloop in the complex was found to adopt a similar conformation to the free form, although the loop structure becomes more open upon SRP19 binding. Thus, SRP19 is thought to recognize the overall fold of the GGAG loop.