Chemical synthesis of homogeneous human glycoproteins exhibiting bioactivity in vivo has been a challenging task. In an effort to overcome this long-standing problem, we selected interferon-β and examined its synthesis. The 166 residue polypep-tide chain of interferon-β was prepared by covalent condensation of two synthetic peptide segments, and a glycosylated synthetic peptide bearing a complex-type glycan of biological origin. The peptides were covalently condensed by native chemical ligation. Selective desulfurization followed by deprotection of the two Cys(Acm) residues gave the target full-length polypeptide chain of interferon-β bearing either a complex-type sialyl biantennary oligo-saccharide or its asialo form. Subsequent folding with concomitant formation of the native disulfide bond afforded correctly folded homogeneous glycosyl-interferon-β. The chemically synthesized sialyl interferon-β exhibited potent anti-tumor activity in vivo.

Ec DOS, a heme-regulated phosphodiesterase from Escherichia coli, is an oxygen sensor enzyme composed of a heme-bound O(2) sensor domain at the N-terminus and a catalytic domain at the C-terminus. The catalytic activity of Ec DOS is substantially enhanced with the formation of a Fe(II) heme-O(2) complex. The physiological importance of H(2)S as a fourth signaling gas molecule in addition to O(2), CO and NO is an emerging focus of research, since H(2)S participates in various physiological functions. In the present study, we showed that catalysis by Ec DOS is markedly increased by H(2)S under aerobic conditions. Absorption spectral findings suggest that SH(-)-modified heme iron complexes, such as Fe(III)-SH(-) and Fe(II)-O(2) complexes, represent the active species for H(2)S-induced catalysis. We further examined the role of Cys residues in H(2)S-induced catalysis using Cys→Ala mutant enzymes. Based on the collective data, we speculate that H(2)S-induced catalytic enhancement is facilitated by an admixture of Fe(III)-SH(-) and Fe(II)-O(2) complexes formed during catalysis and modification of specific Cys residue(s) in the catalytic domain.

AIMS To investigate the effects of the combined application of an N-acyl homoserine lactone (HSL) analog and antibiotics on biofilms of Porphyromonas gingivalis, a major pathogen of periodontal disease. METHODS AND RESULTS Antibiotics used were cefuroxime, ofloxacin and minocycline. A flow-cell model was used for biofilm formation. Samples were divided into four groups: control, analog-treated, antibiotic-treated and combined application groups. Biofilm cell survival was determined using adenosine triphosphate (ATP) bioluminescence and confocal laser microscopy (CLSM). In the combined application group, the ATP count in biofilm cells was significantly decreased compared with the antibiotic-treated group (Games-Howell test, P < 0·05). A combination of cefuroxime and the analog was most effective against the P. gingivalis biofilm. CLSM observations revealed that the proportion of dead cells was highest in the combined application group. CONCLUSIONS The combined application of the N-acyl HSL analog and antibiotics was effective at reducing the viability of P. gingivalis cells in biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY The combined application of the N-acyl HSL analog and antibiotics may be successful for eradicating infections involving bacterial biofilms, such as periodontitis.

Differentiation of human neuroblastoma recapitulates neural crest development. In our whole genome DNA methylation screening of tissue-specific differentially methylated regions (T-DMRs) and developmental stage specific differentially methylated regions (DS-DMRs) we reported that the exon 5 CpG island (CpGi) of Zfp206 (human: ZNF206), which was required to maintain embryonic stem cells in a pluripotent state, was one of potent brain and testis-specific T-DMRs in mice. In this study methylation level of the CpG sites at Zfp206-exon 5 CpGi in mouse brain samples at three different developmental stages (15-day-old embryo; E15, new born; NB, 12-week adult; AD) were quantitatively analyzed and it was identified that Zfp206-exon 5 CpGi was the DS-DMRs in mouse brain. In AD brains, Zfp206-exon 5 CpGi was significantly hypomethylated and Zfp206 expression was repressed, compared with E15 and NB brains. Hence, methylation level of human 5'-end of CpGi at ZNF206-exon 5, which is homologous CpGi to mice, was analyzed in neuroblastomas. Although all four adrenal samples showed complete methylation at the homologous region, we found the hypomethylation in 7 out of 26 neuroblastomas and a significant association between the hypomethylation and poor prognosis. In neuroblastoma cell lines and specimens, the hypomethylation was also associated with ZNF206 expression. These data indicated that the changes in DNA methylation levels at the Zfp206-exon 5 might be one of the important factors during neuronal development in mice and that the hypomethylation of the homologous region induced ZNF206 expression in humans and was associated with human neuroblastomagenesis. Even though the function of ZNF206 and its expression regulation in neuroblastoma remain elusive, ZNF206 might be a candidate differentiation suppressor and prognosis marker in neuroblastoma.

YddV is a globin-coupled oxygen sensor enzyme in that O(2) binding to the Fe(II) heme in the sensor domain substantially enhances its diguanylate cyclase activity. The Fe(III) heme-bound enzyme is also the active form. Amino acid sequence comparisons indicate that Leu65 is well conserved in globin-coupled oxygen sensor enzymes. Absorption spectra of the Fe(III) heme complexes of L65G, L65M, L65Q and L65T mutants of the isolated heme domain of YddV (YddV-heme) were substantially different from that of the wild-type protein. Specifically, Soret bands of the 6-coordinated high-spin Fe(III) complexes of mutant proteins (with H(2)O and His98 as axial ligands) were located at around 403-406nm, distinct from that (391nm) of the 5-coordinated high-spin Fe(III) complex of wild-type protein with His98 as the axial ligand. The autooxidation rate constant (>0.10min(-1)) of the Fe(II)-O(2) complex of L65G was substantially higher than that (0.011min(-1)) of the wild-type protein. Affinities of O(2) for the Fe(II) complexes of L65G and L65T were markedly higher than that for the wild-type protein. Thus, we suggest that the well-conserved Leu65 located in the heme distal side is critical for restricting water access to the heme distal side to avoid rapid autooxidation of YddV, which needs a stable Fe(II)-O(2) complex with a low autooxidation rate.

Two-component signal transduction systems regulate numerous important physiological functions in bacteria. In this study we have identified, cloned, overexpressed, and characterized a dimeric full-length heme-bound (heme:protein, 1:1 stoichiometry) globin-coupled histidine kinase (AfGcHK) from Anaeromyxobacter sp. strain Fw109-5 for the first time. The Fe(III), Fe(II)-O(2), and Fe(II)-CO complexes of the protein displayed autophosphorylation activity, whereas the Fe(II) complex had no significant activity. A H99A mutant lost heme binding ability, suggesting that this residue is the heme proximal ligand. Moreover, His-183 was proposed as the autophosphorylation site based on the finding that the H183A mutant protein was not phosphorylated. The phosphate group of autophosphorylated AfGcHK was transferred to Asp-52 and Asp-169 of a response regulator, as confirmed from site-directed mutagenesis experiments. Based on the amino acid sequences and crystal structures of other globin-coupled oxygen sensor enzymes, Tyr-45 was assumed to be the O(2) binding site at the heme distal side. The O(2) dissociation rate constant, 0.10 s(-1), was substantially increased up to 8.0 s(-1) upon Y45L mutation. The resonance Raman frequencies representing ν(Fe-O2)(559 cm(-1)) and ν(O-O)(1149 cm(-1)) of the Fe(II)-O(2) complex of Y45F mutant AfGcHK were distinct from those of the wild-type protein (ν(Fe-O2), 557 cm(-1); ν(O-O), 1141 cm(-1)), supporting the proposal that Tyr-45 is located at the distal side and forms hydrogen bonds with the oxygen molecule bound to the Fe(II) complex. Thus, we have successfully identified and characterized a novel heme-based globin-coupled oxygen sensor histidine kinase, AfGcHK, in this study.

Heme-regulated eukaryotic initiation factor 2α kinase (HRI) functions under conditions of heme shortage caused by blood diseases such as erythropoietic protoporphyria and β-thalassemia, and retains the heme:globin ratio at 1:1 by sensing the heme concentration in reticulocytes. This HRI function is regulated by various factors including autophosphorylation and protein-protein interactions. A heat-shock protein controls HRI function, however, the molecular mechanism of catalytic regulation of HRI by the heat-shock protein is unclear. In the present study, we examined the interactions of HRI with a heat-shock protein, Hsp90, under various conditions, using a pull-down assay and measuring catalytic activity. It was found that [1] an interaction between Hsp90 and phosphorylated HRI was evident, whereas no interaction was observed between Hsp90 and HRI dephosphorylated by treatment with λ protein phosphatase;[2] Hsp90 enhanced the kinase activity of phosphorylated HRI but not dephosphorylated HRI, but this enhancement was not observed in the presence of heme; and,[3] autophosphorylation of HRI was not influenced by Hsp90. Therefore, we propose that autophosphorylation of HRI is critical for catalytic regulation by Hsp90 under heme-shortage conditions.

Real-time simulation of a biologically realistic spiking neural network is necessary for evaluation of its capacity to interact with real environments. However, the real-time simulation of such a neural network is difficult due to its high computational costs that arise from two factors:(1) vast network size and (2) the complicated dynamics of biologically realistic neurons. In order to address these problems, mainly the latter, we chose to use general purpose computing on graphics processing units (GPGPUs) for simulation of such a neural network, taking advantage of the powerful computational capability of a graphics processing unit (GPU). As a target for real-time simulation, we used a model of the basal ganglia that has been developed according to electrophysiological and anatomical knowledge. The model consists of heterogeneous populations of 370 spiking model neurons, including computationally heavy conductance-based models, connected by 11,002 synapses. Simulation of the model has not yet been performed in real-time using a general computing server. By parallelization of the model on the NVIDIA Geforce GTX 280 GPU in data-parallel and task-parallel fashion, faster-than-real-time simulation was robustly realized with only one-third of the GPU's total computational resources. Furthermore, we used the GPU's full computational resources to perform faster-than-real-time simulation of three instances of the basal ganglia model; these instances consisted of 1100 neurons and 33,006 synapses and were synchronized at each calculation step. Finally, we developed software for simultaneous visualization of faster-than-real-time simulation output. These results suggest the potential power of GPGPU techniques in real-time simulation of realistic neural networks.

Taste buds endure extreme changes in temperature, pH, osmolarity, so on. Even though taste bud cells are replaced in a short span, they contribute to consistent taste reception. Each taste bud consists of about 50 cells whose networks are assumed to process taste information, at least preliminarily. In this article, we describe a neural network model inspired by the taste bud cells of mice. It consists of two layers. In the first layer, the chemical stimulus is transduced into an irregular spike train. The synchronization of the output impulses is induced by the irregular spike train at the second layer. These results show that the intensity of the chemical stimulus is encoded as the degree of the synchronization of output impulses. The present algorithms for signal processing result in a robust chemical-sensing system.

Quorum sensing (QS) controls certain behaviors of bacteria in response to population density. In gram-negative bacteria, QS is often mediated by N-acyl-L-homoserine lactones (acyl-HSLs). Because QS influences the virulence of many pathogenic bacteria, synthetic inhibitors of acyl-HSL synthases might be useful therapeutically for controlling pathogens. However, rational design of a potent QS antagonist has been thwarted by the lack of information concerning the binding interactions between acyl-HSL synthases and their ligands. In the gram-negative bacterium Burkholderia glumae, QS controls virulence, motility, and protein secretion and is mediated by the binding of N-octanoyl-L-HSL (C8-HSL) to its cognate receptor, TofR. C8-HSL is synthesized by the acyl-HSL synthase TofI. In this study, we characterized two previously unknown QS inhibitors identified in a focused library of acyl-HSL analogs. Our functional and X-ray crystal structure analyses show that the first inhibitor, J8-C8, binds to TofI, occupying the binding site for the acyl chain of the TofI cognate substrate, acylated acyl-carrier protein. Moreover, the reaction byproduct, 5'-methylthioadenosine, independently binds to the binding site for a second substrate, S-adenosyl-L-methionine. Closer inspection of the mode of J8-C8 binding to TofI provides a likely molecular basis for the various substrate specificities of acyl-HSL synthases. The second inhibitor, E9C-3oxoC6, competitively inhibits C8-HSL binding to TofR. Our analysis of the binding of an inhibitor and a reaction byproduct to an acyl-HSL synthase may facilitate the design of a new class of QS-inhibiting therapeutic agents.