Measles virus infects both polarized epithelial and immune cells by using distinctive receptor-binding sites on its hemagglutinin

Measles is one of the most contagious human infectious diseases and remains a major cause of childhood morbidity and mortality worldwide. The signaling lymphocyte activation molecule (SLAM), also called CD150, is a cellular receptor for measles virus (MV), presumably accounting for its tropism for immune cells and its immunosuppressive properties. On the other hand, pathological studies have shown that MV also infects epithelial cells at a later stage of infection, although its mechanism has so far been unknown. In this study, we show that wild-type MV can infect and produce syncytia in human polarized epithelial cell lines independently of SLAM and CD46 (a receptor for the vaccine strains of MV). Progeny viral particles are released exclusively from the apical surface of these polarized epithelial cell lines. We have also identified amino acid residues on the MV attachment protein that are likely to interact with a putative receptor on epithelial cells. All of these residues have aromatic side chains and may form a receptor-binding pocket located in a different position from the putative SLAM- and CD46-binding sites on the MV attachment protein. Thus, our results indicate that MV has an intrinsic ability to infect both polarized epithelial and immune cells by using distinctive receptor-binding sites on the attachment protein corresponding to each of their respective receptors. The ability of MV to infect polarized epithelial cells and its exclusive release from the apical surface may facilitate its efficient transmission via aerosol droplets, resulting in its highly contagious nature.     

Determination of iduronic acid and glucuronic acid in sulfated chondroitin/dermatan hybrid chains by <Superscript>1</Superscript>H-nuclear magnetic resonance spectroscopy

The relative proportion of L-iduronic acid (IdoA) and D-glucuronic acid (GlcA) is of great importance for the structure–function relationship of chondroitin sulfate (CS)/dermatan sulfate (DS). However, determination of the isotypes of uronic acid residues in CS/DS is still a challenge, due to the instability of free uronic acid released by chemical degradation and its conversion to unsaturated uronic acid by digestion with bacterial eliminase. ¹H-Nuclear magnetic resonance (NMR) spectroscopy is a promising tool with which to address this issue, but the traditional method based on the assignment of the ring proton signals of IdoA and GlcA residues still has drawbacks such as the serious overlap of signals in the ¹H-NMR spectrum of CS/DS polysaccharides. We found that the proton signals of the N-acetyl group of N-acetyl-D-galactosamines in CS and DS could be clearly distinguished and accurately integrated in the one-dimensional (1D) ¹H-NMR spectrum. Based on this finding, here we report a novel, sensitive, and nondestructive 1D ¹H-NMR-based method to determine the proportion of IdoA and GlcA residues in CS/DS hybrid chains. The contributions of Fuchuan Li and Shuhei Yamada should be considered equal.     

Reversible monomer-oligomer transition in human prion protein

The structure and the dissociation reaction of oligomers PrP^oligo from reduced human prion huPrP^C(23–231) have been studied by ¹H-NMR and tryptophan fluorescence spectroscopy at varying pressure, along with circular dichroism and atomic force microscopy. The ¹H-NMR and fluorescence spectral feature of the oligomer is consistent with the notion that the N-terminal residues including all seven Trp residues, are free and mobile, while residues 105∼210, comprising the AGAAAAGA motif and S1-Loop-HelixA-Loop-S2-Loop-HelixC, are engaged in intra- and/or inter-molecular interactions. By increasing pressure to 200 MPa, the oligomers tend to dissociate into monomers which may be identified with PrP^C*, a rare metastable form of PrP^C stabilized at high pressure (Kachel et al., BMC Struct Biol 6:16). The results strongly suggest that the oligomeric form PrP^oligo is in dynamic equilibrium with the monomeric forms via PrP^C*, namely huPrP^C ⇆ huPrP^C* ⇆ huPrP^oligo.Key words: human prion, oligomer structure, pressure dissociation, reversible monomer-oligomer transition, circular dichroism, high pressure NMR, atomic force microscopy     

Antinematodal activities of ingenane diterpenes from Euphorbia kansui and their derivatives against the pine wood nematode (Bursaphelenchus xylophilus)

Under the bioassay-guided method, two diterpenes, 3-O-(2",3"-dimethylbutanoyl)-13-O-dodecanoylingenol (1) and 3-O-(2",3"-dimethylbutanoyl)-13-O-decanoylingenol (2) isolated from Euphorbia kansui, showed a pronounced antinematodal activity against the nematode Bursaphelenchus xylophilus at the same minimum effective dose (MED) of 5 microg per cotton ball and still displayed antinematodal activity at a dose of 2.5 microg per cotton ball. Compounds 3-6 were obtained, and the structure of the new compound 6 was elucidated based on 1D- and 2D-NMR analyses and physicochemical data. Preliminary structure-biological activity relationships of ingenane-type compounds were deduced.     

Dynamic function of the spacer region of acetogenins in the inhibition of bovine mitochondrial NADH-ubiquinone oxidoreductase (complex I)

Studies of the action mechanism of acetogenins, the most potent and structurally unique inhibitors of bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase), are valuable in characterizing the inhibitor binding site in this enzyme. Our previous study deepened our understanding of the dynamic function of the spacer region of bis-THF acetogenins [Abe, M., et al. (2005) Biochemistry 44, 14898-14906] but, at the same time, posed new important questions. First, while the two toxophores (i.e., the hydroxylated THF and the gamma-lactone rings) span a distance shorter than that of the extended 13 carbon atoms [-(CH 2) 13-], what is the apparent optimal length of the spacer for the inhibition of 13 carbon atoms? In other words, what is the functional role of the additional methylene groups? Second, why was the inhibitory potency of the mono-THF derivative, but not the bis-THF derivative, drastically reduced by hardening the spacer covering 10 carbon atoms into a rodlike shape [-CH 2-(C identical withC) 4-CH 2-]? This study was designed not only to answer these questions but also to further disclose the dynamic functions of the spacer. We here synthesized systematically designed acetogenins, including mono- and bis-THF derivatives, and evaluated their inhibitory effects on bovine complex I. With regard to the first question, we demonstrated that the additional methylenes enhance the hydrophobicity of the spacer region, which may be thermodynamically advantageous for bringing the polar gamma-lactone ring into the membrane-embedded segment of complex I. With regard to the second question, we observed that a decrease in the flexibility of the spacer region is more adverse to the action of the mono-THF series than that of the bis-THF series. As a cause of this difference, we suggest that for bis-THF derivatives, one of the two THF rings, being adjacent to the spacer, is capable of working as a pseudospacer to overcome the remarkable decrease in the conformational freedom and/or the length of the spacer. Moreover, using photoresponsive acetogenins that undergo drastic and reversible conformational changes with alternating UV-vis irradiation, we provided further evidence that the spacer region is free from steric congestion arising from the putative binding site probably because there is no receptor wall for the spacer region.     

Activation of extracellular signal-regulated kinase by ultraviolet is mediated through Src-dependent epidermal growth factor receptor phosphorylation. Its implication in an anti-apoptotic function.

Ultraviolet (UV) irradiation stimulates stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), which is a member of the mitogen-activated protein kinase (MAPK) superfamily and implicated in stress-induced apoptosis. UV also induces the activation of another MAPK member, extracellular signal-regulated kinase (ERK), which is typically involved in a growth-signaling cascade. However, the UV-induced signaling pathway leading to ERK activation, together with the physiological role, has remained unknown. Here we examined the molecular mechanism and physiological function of UV-induced ERK activation in human epidermoid carcinoma A431 cells that retain a high number of epidermal growth factor (EGF) receptors. UV-induced ERK activation was accompanied with the Tyr phosphorylation of EGF receptors, and both responses were completely abolished in the presence of a selective EGF receptor inhibitor (AG1478) or the Src inhibitor PP2 and by the expression of a kinase-dead Src mutant. On the other hand, SAPK/JNK activation by UV was partially inhibited by these inhibitors. UV stimulated Src activity in a manner similar to the ERK activation, but the Src activation was insensitive to AG1478. UV-induced cell apoptosis measured by DNA fragmentation and caspase 3 activation was enhanced by AG1478 and an ERK kinase inhibitor (U0126) but inhibited by EGF receptor stimulation by the agonist. These results indicate that UV-induced ERK activation, which provides a survival signal against stress-induced apoptosis, is mediated through Src-dependent Tyr phosphorylation of EGF receptors.     

Hus1 acts upstream of chk1 in a mammalian DNA damage response pathway.

The evolutionarily conserved Hus1 proteins function in DNA damage response pathways that serve to maintain genomic stability. Cells lacking mouse Hus1 are hypersensitive to certain genotoxins, and we have explored the molecular basis for this defect by examining how Hus1 inactivation affects genotoxin-induced signaling events. p53 accumulation and activation in response to DNA damage appeared normal in Hus1 null cells. Likewise, Hus1 was dispensable for genotoxin-induced Chk2 phosphorylation. In contrast, Chk1 phosphorylation after genotoxic stress was greatly reduced in the absence of Hus1, but was restored in Hus1 null fibroblasts complemented by infection with a Hus1-expressing retrovirus. These results demonstrate that mouse Hus1 is required for a specific subset of DNA damage signaling events and functions to promote genotoxin-induced Chk1 phosphorylation.     

Coevolution and Hierarchical Interactions of Tomato mosaic virus and the Resistance Gene Tm-1

During antagonistic coevolution between viruses and their hosts, viruses have a major advantage by evolving more rapidly. Nevertheless, viruses and their hosts coexist and have coevolved, although the processes remain largely unknown. We previously identified Tm-1 that confers resistance to Tomato mosaic virus (ToMV), and revealed that it encodes a protein that binds ToMV replication proteins and inhibits RNA replication. Tm-1 was introgressed from a wild tomato species Solanum habrochaites into the cultivated tomato species Solanum lycopersicum. In this study, we analyzed Tm-1 alleles in S. habrochaites. Although most part of this gene was under purifying selection, a cluster of nonsynonymous substitutions in a small region important for inhibitory activity was identified, suggesting that the region is under positive selection. We then examined the resistance of S. habrochaites plants to ToMV. Approximately 60% of 149 individuals from 24 accessions were resistant to ToMV, while the others accumulated detectable levels of coat protein after inoculation. Unexpectedly, many S. habrochaites plants were observed in which even multiplication of the Tm-1-resistance-breaking ToMV mutant LT1 was inhibited. An amino acid change in the positively selected region of the Tm-1 protein was responsible for the inhibition of LT1 multiplication. This amino acid change allowed Tm-1 to bind LT1 replication proteins without losing the ability to bind replication proteins of wild-type ToMV. The antiviral spectra and biochemical properties suggest that Tm-1 has evolved by changing the strengths of its inhibitory activity rather than diversifying the recognition spectra. In the LT1-resistant S. habrochaites plants inoculated with LT1, mutant viruses emerged whose multiplication was not inhibited by the Tm-1 allele that confers resistance to LT1. However, the resistance-breaking mutants were less competitive than the parental strains in the absence of Tm-1. Based on these results, we discuss possible coevolutionary processes of ToMV and Tm-1.