NMR Line Shape Analysis of a Multi-state Ligand Binding Mechanism in Chitosanase

Chitosan interaction with chitosanase was examined through analysis of spectral line shapes in the NMR HSQC titration experiments. We established that the substrate, chitosan hexamer, binds to the enzyme through the three-state induced-fit mechanism with fast formation of the encounter complex followed by slow isomerization of the bound-state into the final conformation. Mapping of the chemical shift perturbations in two sequential steps of the mechanism highlighted involvement of the substrate-binding subsites and the hinge region in the binding reaction. Equilibrium parameters of the three-state model agreed with the overall thermodynamic dissociation constant determined by ITC. This study presented the first kinetic evidence of the induced-fit mechanism in the glycoside hydrolases

Genetic Characterization of Hepatitis C Virus in Long-Term RNA Replication Using Li23 Cell Culture Systems

Background　 　 The most distinguishing genetic feature of hepatitis C virus (HCV) is its remarkable diversity and variation. To understand this feature, we previously performed genetic analysis of HCV in the long-term culture of human hepatoma HuH-7-derived HCV RNA-replicating cell lines. On the other hand, we newly established HCV RNA-replicating cell lines using human hepatoma Li23 cells, which were distinct from HuH-7 cells. 　 Methodology/Principal Findings　 　 Li23-derived HCV RNA-replicating cells were cultured for 4 years. We performed genetic analysis of HCVs recovered from these cells at 0, 2, and 4 years in culture. Most analysis was performed in two separate parts: one part covered from the 5′-terminus to NS2, which is mostly nonessential for RNA replication, and the other part covered from NS3 to NS5B, which is essential for RNA replication. Genetic mutations in both regions accumulated in a time-dependent manner, and the mutation rates in the 5′-terminus-NS2 and NS3-NS5B regions were 4.0–9.0×10−3 and 2.7–4.0×10−3 base substitutions/site/year, respectively. These results suggest that the variation in the NS3-NS5B regions is affected by the pressure of RNA replication. Several in-frame deletions (3–105 nucleotides) were detected in the structural regions of HCV RNAs obtained from 2-year or 4-year cultured cells. Phylogenetic tree analyses clearly showed that the genetic diversity of HCV was expanded in a time-dependent manner. The GC content of HCV RNA was significantly increased in a time-dependent manner, as previously observed in HuH-7-derived cell systems. This phenomenon was partially due to the alterations in codon usages for codon optimization in human cells. Furthermore, we demonstrated that these long-term cultured cells were useful as a source for the selection of HCV clones showing resistance to anti-HCV agents. 　 Conclusions/Significance　 　 Long-term cultured HCV RNA-replicating cells are useful for the analysis of evolutionary dynamics and variations of HCV and for drug-resistance analysis

Pneumo- and neurotropism of avian origin Italian highly pathogenic avian influenza H7N1 isolates in experimentally infected mice

AbstractAn experimental infection of mice was performed in order to investigate the potential for interspecies transmission in mammals of Italian HPAI viruses of the H7N1 subtype. Three avian origin isolates were selected, two strains obtained from ostrich (one of which contained a PB2-627 Lysine residue) and one from a chicken. Following intranasal infection of mice, clinical signs and mortality were recorded in the experimental groups challenged with the two ostrich isolates, while only weight loss was observed in those receiving the chicken strain. Viruses were recovered to a varying extent from respiratory and nervous tissues of infected animals. These results suggest that HPAI viruses, other than H5N1 and H7N7, may have zoonotic implications, and support the consensus that AI infections in poultry are to be eradicated rather than contained

The Diagnostic Value of the Interstitial Biomarkers KL-6 and SP-D for the Degree of Fibrosis in Combined Pulmonary Fibrosis and Emphysema

The combined pulmonary fibrosis and emphysema (CPFE) was reported first in 1990, but it has been comparatively underestimated until recently. Although the diagnostic findings of both emphysematous and fibrotic regions are detectable by high-resolution computed tomography (HRCT) of the chest, the degree of progressive fibrosis, which increases with emphysematous lesions, is difficult to evaluate. In this study, we hypothesized that the biomarkers for pulmonary fibrosis, surfactant protein D (SP-D), and KL-6 would serve as good indicators of fibrotic lesions in CPFE. We recruited 46 patients who had been diagnosed in our hospital with both emphysema and fibrosis by their CT scan image from April 2003 to March 2008. The correlation among their pulmonary function tests, composite physiologic index (CPI), and the serum levels of SP-D and KL-6 was evaluated. We found a correlation between KL-6 and %VC, %TLC, or CPI and between SP-D and %VC or CPI. Interestingly, the combined product of KL-6 and SP-D (KL-6xSP-D) was found to highly correlate with %VC and %TLC or CPI. These results show that both KL-6 and SP-D, and especially the product of SP-D and KL-6, are good indicators of the presence of fibrotic lesions in the lungs of CPFE patients

Radical formation in cytochrome c oxidase

AbstractThe formation of radicals in bovine cytochrome c oxidase (bCcO), during the O2 redox chemistry and proton translocation, is an unresolved controversial issue. To determine if radicals are formed in the catalytic reaction of bCcO under single turnover conditions, the reaction of O2 with the enzyme, reduced by either ascorbate or dithionite, was initiated in a custom-built rapid freeze quenching (RFQ) device and the products were trapped at 77K at reaction times ranging from 50μs to 6ms. Additional samples were hand mixed to attain multiple turnover conditions and quenched with a reaction time of minutes. X-band (9GHz) continuous wave electron paramagnetic resonance (CW-EPR) spectra of the reaction products revealed the formation of a narrow radical with both reductants. D-band (130GHz) pulsed EPR spectra allowed for the determination of the g-tensor principal values and revealed that when ascorbate was used as the reductant the dominant radical species was localized on the ascorbyl moiety, and when dithionite was used as the reductant the radical was the SO2− ion. When the contributions from the reductants are subtracted from the spectra, no evidence for a protein-based radical could be found in the reaction of O2 with reduced bCcO. As a surrogate for radicals formed on reaction intermediates, the reaction of hydrogen peroxide (H2O2) with oxidized bCcO was studied at pH 6 and pH 8 by trapping the products at 50μs with the RFQ device to determine the initial reaction events. For comparison, radicals formed after several minutes of incubation were also examined, and X-band and D-band analysis led to the identification of radicals on Tyr-244 and Tyr-129. In the RFQ measurements, a peroxyl (ROO) species was formed, presumably by the reaction between O2 and an amino acid-based radical. It is postulated that Tyr-129 may play a central role as a proton loading site during proton translocation by ejecting a proton upon formation of the radical species and then becoming reprotonated during its reduction via a chain of three water molecules originating from the region of the propionate groups of heme a3. This article is part of a Special Issue entitled: “Allosteric cooperativity in respiratory proteins”

Transcriptional repression and DNA hypermethylation of a small set of ES cell marker genes in male germline stem cells

BACKGROUND: We previously identified a set of genes called ECATs (ES cell-associated transcripts) that are expressed at high levels in mouse ES cells. Here, we examine the expression and DNA methylation of ECATs in somatic cells and germ cells. RESULTS: In all ECATs examined, the promoter region had low methylation levels in ES cells, but higher levels in somatic cells. In contrast, in spite of their lack of pluripotency, male germline stem (GS) cells expressed most ECATs and exhibited hypomethylation of ECAT promoter regions. We observed a similar hypomethylation of ECAT loci in adult testis and isolated sperm. Some ECATs were even less methylated in male germ cells than in ES cells. However, a few ECATs were not expressed in GS cells, and most of them targets of Oct3/4 and Sox2. The Octamer/Sox regulatory elements were hypermethylated in these genes. In addition, we found that GS cells express little Sox2 protein and low Oct3/4 protein despite abundant expression of their transcripts. CONCLUSION: Our results suggest that DNA hypermethylation and transcriptional repression of a small set of ECATs, together with post-transcriptional repression of Oct3/4 and Sox2, contribute to the loss of pluripotency in male germ cells

Growth of H5N1 Influenza A Viruses in the Upper Respiratory Tracts of Mice

Highly pathogenic avian H5N1 influenza A viruses have spread throughout Asia, Europe, and Africa, raising serious worldwide concern about their pandemic potential. Although more than 250 people have been infected with these viruses, with a consequent high rate of mortality, the molecular mechanisms responsible for the efficient transmission of H5N1 viruses among humans remain elusive. We used a mouse model to examine the role of the amino acid at position 627 of the PB2 viral protein in efficient replication of H5N1 viruses in the mammalian respiratory tract. Viruses possessing Lys at position 627 of PB2 replicated efficiently in lungs and nasal turbinates, as well as in cells, even at the lower temperature of 33 °C. Those viruses possessing Glu at this position replicated less well in nasal turbinates than in lungs, and less well in cells at the lower temperature. These results suggest that Lys at PB2–627 confers to avian H5N1 viruses the advantage of efficient growth in the upper and lower respiratory tracts of mammals. Therefore, efficient viral growth in the upper respiratory tract may provide a platform for the adaptation of avian H5N1 influenza viruses to humans and for efficient person-to-person virus transmission, in the context of changes in other viral properties including specificity for human (sialic acid α-2,6-galactose containing) receptors

Genetic and Epigenetic Alterations of Lysophosphatidic Acid Receptor Genes in Rodent Tumors by Experimental Models

Lysophosphatidic acid (LPA) is a bioactive mediator and induces several biological effects, including cell proliferation, migration, morphogenesis and differentiation. LPA interacts with at least six G protein-coupled receptors (GPCRs), including LPA receptor-1 (LPA1), LPA2, LPA3, LPA4, LPA5 and LPA6. These receptors show different biological functions through the binding of LPA, depending on the type of cells. In human malignancies, a high level of LPA production was found in plasma and ascites in ovarian cancer cases. Moreover, aberrant expression levels of LPA receptor genes were detected in some cancer cells. Therefore, it is suggested that LPA receptors may be involved in the pathogenesis of tumor cells as well as LPA per se. Recently, we have reported that alterations of LPA receptor genes also occur in rodent tumors. In this review, we summarize the recent evidence in the investigations of LPA receptor alterations in rodent tumors by experimental models

Influenza A (H5N1) Viruses from Pigs, Indonesia

TOC summary: Pigs may serve as intermediate hosts in which this avian virus can adapt to mammals