Soybean β-Conglycinin Prevents Age-Related Hearing Impairment

Obesity-related complications are associated with the development of age-related hearing impairment. β-Conglycinin (β-CG), one of the main storage proteins in soy, offers multiple health benefits, including anti-obesity and anti-atherosclerotic effects. Here, to elucidate the potential therapeutic application of β-CG, we investigated the effect of β-CG on age-related hearing impairment. Male wild-type mice (age 6 months) were randomly divided into β-CG-fed and control groups. Six months later, the body weight was significantly lower in β-CG-fed mice than in the controls. Consumption of β-CG rescued the hearing impairment observed in control mice. Cochlear blood flow also increased in β-CG-fed mice, as did the expression of eNOS in the stria vascularis (SV), which protects vasculature. β-CG consumption also ameliorated oxidative status as assessed by 4-HNE staining. In the SV, lipofuscin granules of marginal cells and vacuolar degeneration of microvascular pericytes were decreased in β-CG-fed mice, as shown by transmission electron microscopy. β-CG consumption prevented loss of spiral ganglion cells and reduced the frequencies of lipofuscin granules, nuclear invaginations, and myelin vacuolation. Our observations indicate that β-CG ameliorates age-related hearing impairment by preserving cochlear blood flow and suppressing oxidative stress.     

Antitumor studies. Part 1: design, synthesis, antitumor activity, and AutoDock study of 2-deoxo-2-phenyl-5-deazaflavins and 2-deoxo-2-phenylflavin-5-oxides as a new class of antitumor agents

Novel 2-deoxo-2-phenyl-5-deazaflavins and 2-deoxo-2-phenylflavin-5-oxides were prepared as a new class of antitumor agents and showed significant antitumor activities against NCI-H 460, HCT 116, A 431, CCRF-HSB-2, andKB cell lines. In vivo investigation, 2-deoxo-10-methyl-2-phenyl-5-deazaflavin exhibited the effective antitumor activity against A 431 human adenocarcinoma cells transplanted subcutaneously into nude mouse. Furthermore, AutoDock study has been done by binding of the flavin analogs into PTK pp60(c-src), where a good correlation between their IC(50) and AutoDock binding free energy was exhibited. In particular, 2-deoxo-2-phenylflavin-5-oxides exhibited the highest potential binding affinity within the binding pocket of PTK.     

A study on medicinal plants from Malaysia focused on Acalypha siamensis Oliv. ex Gage. isolation and structure of a new tetraterpene, acalyphaser A

As a part of our chemical studies on Malaysian medicinal plants, five Malaysian plant species were evaluated by cytotoxicity assays using P388 murine leukemia cells. Since Acalypha siamensis exhibited the strongest growth inhibition, its constituents were studied as the object of search for bioactive materials. A novel tetraterpene, acalyphaser A (1), was isolated in the course of the purification. Its structure was elucidated on the basis of 1D- and 2D-NMR techniques, and mass spectrometry.     

Expression of microRNA miR-122 facilitates an efficient replication in nonhepatic cells upon infection with hepatitis C virus

Hepatitis C virus (HCV) is one of the most common etiologic agents of chronic liver diseases, including liver cirrhosis and hepatocellular carcinoma. In addition, HCV infection is often associated with extrahepatic manifestations (EHM), including mixed cryoglobulinemia and non-Hodgkin's lymphoma. However, the mechanisms of cell tropism of HCV and HCV-induced EHM remain elusive, because in vitro propagation of HCV has been limited in the combination of cell culture-adapted HCV (HCVcc) and several hepatic cell lines. Recently, a liver-specific microRNA called miR-122 was shown to facilitate the efficient propagation of HCVcc in several hepatic cell lines. In this study, we evaluated the importance of miR-122 on the replication of HCV in nonhepatic cells. Among the nonhepatic cell lines expressing functional HCV entry receptors, Hec1B cells derived from human uterus exhibited a low level of replication of the HCV genome upon infection with HCVcc. Exogenous expression of miR-122 in several cells facilitates efficient viral replication but not production of infectious particles, probably due to the lack of hepatocytic lipid metabolism. Furthermore, expression of mutant miR-122 carrying a substitution in a seed domain was required for efficient replication of mutant HCVcc carrying complementary substitutions in miR-122-binding sites, suggesting that specific interaction between miR-122 and HCV RNA is essential for the enhancement of viral replication. In conclusion, although miR-122 facilitates efficient viral replication in nonhepatic cells, factors other than miR-122, which are most likely specific to hepatocytes, are required for HCV assembly.     

Molecular nature of mutations induced by high-LET irradiation with argon and carbon ions in Arabidopsis thaliana

Linear energy transfer (LET) is an important parameter to be considered in heavy-ion mutagenesis. However, in plants, no quantitative data are available on the molecular nature of the mutations induced with high-LET radiation above 101-124keVμm(-1). In this study, we irradiated dry seeds of Arabidopsis thaliana with Ar and C ions with an LET of 290keVμm(-1). We analyzed the DNA alterations caused by the higher-LET radiation. Mutants were identified from the M(2) pools. In total, 14 and 13 mutated genes, including bin2, egy1, gl1, gl2, hy1, hy3-5, ttg1, and var2, were identified in the plants derived from Ar- and C-ions irradiation, respectively. In the mutants from both irradiations, deletion was the most frequent type of mutation; 13 of the 14 mutated genes from the Ar ion-irradiated plants and 11 of the 13 mutated genes from the C ion-irradiated plants harbored deletions. Analysis of junction regions generated by the 2 types of irradiation suggested that alternative non-homologous end-joining was the predominant pathway of repair of break points. Among the deletions, the proportion of large deletions (>100bp) was about 54% for Ar-ion irradiation and about 64% for C-ion irradiation. Both current results and previously reported data revealed that the proportions of the large deletions induced by 290-keVμm(-1) radiations were higher than those of the large deletions induced by lower-LET radiations (6% for 22.5-30.0keVμm(-1) and 27% for 101-124keVμm(-1)). Therefore, the 290keVμm(-1) heavy-ion beams can effectively induce large deletions and will prove useful as novel mutagens for plant breeding and analysis of gene functions, particularly tandemly arrayed genes.     

Intracellular imaging of targeted proteins labeled with quantum dots

We developed a new method for imaging the movement of targeted proteins in living cancer cells with photostable and bright quantum dots (QDs). QDs were conjugated with various molecules and proteins, such as phalloidin, anti-tubulin antibody and kinesin. These bioconjugated QDs were mixed with a transfection reagent and successfully internalized into living cells. The movements of individual QDs were tracked for long periods of time. Phalloidin conjugated QDs bound to actin filaments and showed almost no movement. In contrast, anti-tubulin antibody conjugated QDs bound to microtubules and revealed dynamic movement of microtubules. Kinesin showed an interesting behavior whereby kinesin came to be almost paused briefly for a few seconds and then moved once again. This is in direct contrast to the smoothly continuous movement of kinesin in an in vitro assay. The maximum velocity of kinesin in cells was faster than that in the in vitro assay. These results suggest that intracellular movement of kinesin is different from that in the in vitro assay. This newly described method will be a powerful tool for investigating the functions of proteins in living cells.     

Development of bioluminescent pyrophosphate assay using pyruvate phosphate dikinase and its application to single-nucleotide polymorphism analysis

DNA analysis is an important technology with respect to diagnosis of infectious disease and tailored medication. In this study, we developed a novel bioluminescent assay for pyrophosphate, and it was applied to single-nucleotide polymorphism (SNP) analysis using one-base extension reaction. The principle of this method is as follows. A specific primer within each aliquot possessing a short 3′ end of the base of interest was hybridized to the single-stranded DNA template. Subsequently, (exo-)Klenow DNA polymerase and one of either α-thio-dATP, dTTP, dGTP, or dCTP were added and incubated for 1 min. Pyrophosphate released by DNA polymerase is converted to ATP by pyruvate phosphate dikinase (PPDK), and the concentration of ATP is determined using the firefly luciferase reaction. This method, which does not require expensive equipment, can be used to rapidly monitor one point mutation in the gene.     

Electroreceptor model of weakly electric fish Gnathonemus petersii: II. Cellular origin of inverse waveform tuning.

In part I (. Biophys. J. 75:1712-1726), we presented a cellular model of the A- and B-electroreceptors of the weakly electric fish Gnathonemus petersii. The model made clear the cellular origin of the differences in the response functions of A- and B-receptors, which sensitively code the intensity of the fish's own electric organ discharge (EOD) and the variations in the EOD waveform, respectively. The main purpose of the present paper is to clarify the cellular origin of the inverse waveform tuning of the B-receptors by using the receptor model. Inverse waveform tuning means that B-receptors respond more sensitively to the 180 degrees inverted EOD than to undistorted or less distorted EODs. We investigated how the A- and B-receptor models respond to EODs with various waveforms, which are the phase-shifted EODs, whose shift angle is varied from -1 degrees to -180 degrees, and single-period sine wave stimuli of various frequencies. We show that the tuning properties of the B-receptors arise mainly from the combination of two attributes: 1) The waveform of the stimuli (Bstim) effectively sensed by the B-receptor cells. This consists of a first smaller and a second larger positive peak, even though in the original phase-shifted EOD stimuli, the amplitudes of the two positive peaks are reversed. 2) The effective time constant of dynamical response of the receptor cells. It is on the order of the duration of a single EOD pulse. We also calculated the response properties of the A- and B-receptor models when stimulated with natural EODs distorted by various capacitive and resistive objects. Furthermore, we investigated the effect of EOD amplitude on the receptor responses to capacitive and resistive objects. The models presented can systematically reproduce the experimentally observed response properties of natural A- and B-receptor cells. The mechanism producing these properties can be reasonably explained by the variation in the stimulus waveforms effectively sensed by the A- and B-receptor cells and by time constants.     

Heterogeneous nuclear ribonucleoprotein A2 participates in the replication of Japanese encephalitis virus through an interaction with viral proteins and RNA

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that is kept in a zoonotic transmission cycle between pigs and mosquitoes. JEV causes infection of the central nervous system with a high mortality rate in dead-end hosts, including humans. Many studies have suggested that the flavivirus core protein is not only a component of nucleocapsids but also an important pathogenic determinant. In this study, we identified heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) as a binding partner of the JEV core protein by pulldown purification and mass spectrometry. Reciprocal coimmunoprecipitation analyses in transfected and infected cells confirmed a specific interaction between the JEV core protein and hnRNP A2. Expression of the JEV core protein induced cytoplasmic retention of hnRNP A2 in JEV subgenomic replicon cells. Small interfering RNA (siRNA)-mediated knockdown of hnRNP A2 resulted in a 90% reduction of viral RNA replication in cells infected with JEV, and the reduction was cancelled by the expression of an siRNA-resistant hnRNP A2 mutant. In addition to the core protein, hnRNP A2 also associated with JEV nonstructural protein 5, which has both methyltransferase and RNA-dependent RNA polymerase activities, and with the 5'-untranslated region of the negative-sense JEV RNA. During one-step growth, synthesis of both positive- and negative-strand JEV RNAs was delayed by the knockdown of hnRNP A2. These results suggest that hnRNP A2 plays an important role in the replication of JEV RNA through the interaction with viral proteins and RNA.     

De novo design of foldable proteins with smooth folding funnel: automated negative design and experimental verification

De novo sequence design of foldable proteins provides a way of investigating principles of protein architecture. We performed fully automated sequence design for a target structure having a three-helix bundle topology and synthesized the designed sequences. Our design principle is different from the conventional approach, in that instead of optimizing interactions within the target structure, we design the global shape of the protein folding funnel. This includes automated implementation of negative design by explicitly requiring higher free energy of the denatured state. The designed sequences do not have significant similarity to those of any natural proteins. The NMR and CD spectroscopic data indicated that one designed sequence has a well-defined three-dimensional structure as well as alpha-helical content consistent with the target.