Intrinsic signaling function of APP as a novel target of three V642 mutations linked to familial Alzheimer's disease.

APP695 is a transmembrane precursor of Abeta amyloid. In familial Alzheimer's disease (FAD), three mutations V642I/F/G were discovered in APP695, which has been suggested by multiple studies to be a cell surface signaling receptor. We previously reported that normal APP695 encodes a potential GO-linked receptor with ligand-regulated function and that expression of the three FAD mutants (FAD-APPs), not normal APP, induces cellular outputs by GO-dependent mechanisms. This suggests that FAD-APPs are constitutively active GO-linked receptors. Here, we provide direct evidence for this notion. Reconstitution of either recombinant FAD-APP with GO vesicles induced activation of GO, which was inhibitable by pertussis toxin, sensitive to Mg2+ and proportional in quantity to the reconstituted amounts of FAD-APP. Consistent with the dominant inheritance of this type of FAD, this function was dominant over normal APP, because little activation was observed in APP695-GO vesicles. Experiments with antibody competition and sequence deletion indicated that His657-Lys676 of FAD-APP, which has been specified as the ligand-dependent GO-coupling domain of normal APP, was responsible for this constitutive activation, confirming that the three FAD-APPs are mutationally activated APP695. This study identifies the intrinsic signaling function of APP to be a novel target of hereditary Alzheimer's disease mutations, providing an in vitro system for the screening of potential FAD inhibitors.     

Photo-oxidation of membrane-bound and soluble cytochromec in the green sulfur bacteriumChlorobium tepidum

We studied the photosynthetic electron transfer system of membrane-bound and soluble cytochromec inChlorobium tepidum, a thermophilic green sulfur bacterium, using whole cells and membrane preparations. Sulfide and thiosulfate, physiological electron donors, enhanced flash-induced photo-oxidation ofc-type cytochromes in whole cells. In membranes,c-553 cytochromes with two (or three) heme groups served as immediate electron donors for photo-oxidized bacteriochlorophyll (P840) in the reaction center, and appeared to be closely associated with the reaction center complex. The membrane-bound cytochromec-553 had anE _m-value of 180 mV. When isolated soluble cytochromec-553, which has an apparent molecular weight of 10 kDa and seems to correspond to the cytochromec-555 inChlorobium limicola andChlorobium vibrioforme, was added to a membrane suspension, rapid photo-oxidation of both soluble and membrane-bound cytochromesc-553 was observed. The oxidation of soluble cytochromec-553 was inhibited by high salt concentrations. In whole cells, photo-oxidation was observed in the absence of exogenous electron donors and re-reduction was inhibited by stigmatellin, an inhibitor of the cytochromebc complex. These results suggest that the role of membrane-bound and soluble cytochromec inC. tepidum is similar to the role of cytochromec in the photosynthetic electron transfer system of purple bacteria.     

High-resolution crystal structure of beta2-microglobulin formed at pH 7.0

beta(2)-Microglobulin (beta2-m), a light chain of the major histocompatibility complex class I, forms amyloid fibrils in patients undergoing long-term haemodialysis, causing dialysis-related amyloidosis. Based on a comparison of the X-ray structure obtained at pH 5.7 and that of beta2-m in the histocompatibility complex, it has been proposed that the continuous D-strand observed in the crystal structure at pH 5.7 increases the propensity of beta2-m to self-associate via edge-to-edge interactions, thus initiating the formation of fibrils. To obtain further insight into the mechanism by which amyloid fibrils form, we determined the crystal structure of beta2-m at pH 7.0 at a resolution of up to 1.13 A. The crystal structure at pH 7.0 was basically the same as that at pH 5.6, suggesting that the conversion of the beta-bulge in strand D into a contiguous beta-strand is not unique to the crystals formed under slightly acidic conditions. In other words, although the formation of beta2-m fibrils was enhanced under acidic conditions, it remains unknown if it is related to the increased propensity for the disappearance of the beta-bulge in strand D. We consider that the enhanced fibrillation is more directly coupled with the decreased stability leading to the increased propensity of exposing amyloidogenic regions.     

Kinetic analysis of the entire RNA amplification process by Qbeta replicase

The kinetics of the RNA replication reaction by Qbeta replicase were investigated. Qbeta replicase is an RNA-dependent RNA polymerase responsible for replicating the RNA genome of coliphage Qbeta and plays a key role in the life cycle of the Qbeta phage. Although the RNA replication reaction using this enzyme has long been studied, a kinetic model that can describe the entire RNA amplification process has yet to be determined. In this study, we propose a kinetic model that is able to account for the entire RNA amplification process. The key to our proposed kinetic model is the consideration of nonproductive binding (i.e. binding of an enzyme to the RNA where the enzyme cannot initiate the reaction). By considering nonproductive binding and the notable enzyme inactivation we observed, the previous observations that remained unresolved could also be explained. Moreover, based on the kinetic model and the experimental results, we determined rate and equilibrium constants using template RNAs of various lengths. The proposed model and the obtained constants provide important information both for understanding the basis of Qbeta phage amplification and the applications using Qbeta replicase.     

Hepatitis C virus nonstructural protein 5A modulates the toll-like receptor-MyD88-dependent signaling pathway in macrophage cell lines

Hepatitis C virus (HCV) infection induces a wide range of chronic liver injuries; however, the mechanism through which HCV evades the immune surveillance system remains obscure. Blood dendritic cells (DCs) play a pivotal role in the recognition of viral infection and the induction of innate and adaptive immune responses. Several reports suggest that HCV infection induces the dysfunction of DCs in patients with chronic hepatitis C. Toll-like receptor (TLR) has been shown to play various roles in many viral infections; however, the involvement of HCV proteins in the TLR signaling pathway has not yet been precisely elucidated. In this study, we established mouse macrophage cell lines stably expressing HCV proteins and determined the effect of HCV proteins on the TLR signaling pathways. Immune cells expressing NS3, NS3/4A, NS4B, or NS5A were found to inhibit the activation of the TLR2, TLR4, TLR7, and TLR9 signaling pathways. Various genotypes of NS5A bound to MyD88, a major adaptor molecule in TLR, inhibited the recruitment of interleukin-1 receptor-associated kinase 1 to MyD88, and impaired cytokine production in response to TLR ligands. Amino acid residues 240 to 280, previously identified as the interferon sensitivity-determining region (ISDR) in NS5A, interacted with the death domain of MyD88, and the expression of a mutant NS5A lacking the ISDR partially restored cytokine production. These results suggest that the expression of HCV proteins modulates the TLR signaling pathway in immune cells.     

Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing

Virus-induced gene silencing (VIGS) is a powerful tool for functional analysis of genes in plants. A wide-host-range VIGS vector, which was developed based on the Cucumber mosaic virus (CMV), was tested for its ability to silence endogenous genes involved in flavonoid biosynthesis in soybean. Symptomless infection was established using a pseudorecombinant virus, which enabled detection of specific changes in metabolite content by VIGS. It has been demonstrated that the yellow seed coat phenotype of various cultivated soybean lines that lack anthocyanin pigmentation is induced by natural degradation of chalcone synthase ( CHS ) mRNA. When soybean plants with brown seed coats were infected with a virus that contains the CHS gene sequence, the colour of the seed coats changed to yellow, which indicates that the naturally occurring RNA silencing is reproduced by VIGS. In addition, CHS VIGS consequently led to a decrease in isoflavone content in seeds. VIGS was also tested on the putative flavonoid 3'-hydroxylase ( F3'H ) gene in the pathway. This experiment resulted in a decrease in the content of quercetin relative to kaempferol in the upper leaves after viral infection, which suggests that the putative gene actually encodes the F3'H protein. In both experiments, a marked decrease in the target mRNA and accumulation of short interfering RNAs were detected, indicating that sequence-specific mRNA degradation was induced. The present report is a successful demonstration of the application of VIGS for genes involved in flavonoid biosynthesis in plants; the CMV-based VIGS system provides an efficient tool for functional analysis of soybean genes.     

The mechanism of SO2‐induced stomatal closure differs from O3 and CO2 responses and is mediated by nonapoptotic cell death in guard cells

Plants closing stomata in the presence of harmful gases is believed to be a stress avoidance mechanism. SO₂, one of the major airborne pollutants, has long been reported to induce stomatal closure, yet the mechanism remains unknown. Little is known about the stomatal response to airborne pollutants besides O₃. SLOW ANION CHANNEL‐ASSOCIATED 1 (SLAC1) and OPEN STOMATA 1 (OST1) were identified as genes mediating O₃‐induced closure. SLAC1 and OST1 are also known to mediate stomatal closure in response to CO₂, together with RESPIRATORY BURST OXIDASE HOMOLOGs (RBOHs). The overlaying roles of these genes in response to O₃ and CO₂ suggested that plants share their molecular regulators for airborne stimuli. Here, we investigated and compared stomatal closure event induced by a wide concentration range of SO₂ in Arabidopsis through molecular genetic approaches. O₃‐ and CO₂‐insensitive stomata mutants did not show significant differences from the wild type in stomatal sensitivity, guard cell viability, and chlorophyll content revealing that SO₂‐induced closure is not regulated by the same molecular mechanisms as for O₃ and CO₂. Nonapoptotic cell death is shown as the reason for SO₂‐induced closure, which proposed the closure as a physicochemical process resulted from SO₂ distress, instead of a biological protection mechanism.     

Nascent chain, mRNA, and ribosome complexes generated by a pure translation system

Ribosome display is based on the concept that ternary complexes consisting of a nascent chain, ribosome, and mRNA can be generated, thereby establishing the linkage between genotype and phenotype that is essential for evolutionary experiments. With cell extract-based in vitro translation systems, it has been shown that ternary complexes can be generated by omitting the termination codon from the constructs, which can be stabilized at low temperature in the presence of high Mg2+ concentrations. Using an Escherichia coli-based reconstituted in vitro translation system (PURE system), in which all components necessary for the translation reaction were highly purified and reconstituted, ternary complexes could be generated equally well with a variety of sequences at the 3' end of the RNA, even those with a termination codon. Moreover, the generated complexes were stable at temperatures between 4 and 50 degrees C, and are thus highly stable in contrast to previous assumptions.     

Morphology and electrophysiology of water receptors on legs of the cricket Gryllus bimaculatus

To identify the sensory organs that are sensitive to water stimuli in the cricket Gryllus bimaculatus, cuticular structures on the legs and the number of sensory neurons innervating them were studied. Some small hair sensilla on the legs were innervated by 2-5 sensory neurons. All such sensilla had a tiny pore at the tip of their hairs. The diameter of the pore was approximately 0.2 mum. These findings suggest that these are chemosensitive hairs (LCS: leg chemosensillum). Of the three pairs of legs, the anterior legs (forelegs) possessed the largest number of LCSs. Of the five leg segments (i.e., coxa, trochanter, femur, tibia and tarsus), the tarsus possessed the largest number of LCSs on each leg. Electrophysiological investigation by tip recording revealed that some of the LCSs contained water-receptor cells. Because the basitarsus possessed a larger number of LCSs than the other tarsomeres, the distribution of water-receptor-containing LCSs in the basitarsus of a foreleg was investigated morphologically and electrophysiologically. LCSs that contained water-receptor cells were mainly distributed on the ventral surface of the basitarsus. There were two types of water receptor that showed different response patterns to a stimulus, that is, phasic- and tonic-type water receptors. From the distribution of LCSs on the legs, the roles of these different types of water receptors in behavioral selection, that is, the initiation of swimming and the inhibition of flying, will be discussed.