Suppression of hepatitis A virus genome translation and replication by siRNAs targeting the internal ribosomal entry site

Small interfering RNAs (siRNAs) targeting the coding region of hepatitis A virus (HAV) were shown to specifically inhibit viral genome replication. Compared to the coding region, the HAV internal ribosomal entry site (IRES) in the 5' non-coding region is highly sequence-conserved and folds into stable secondary structures. Here, we report efficient and sustained RNA interference mediated by both RNase III-prepared siRNA (esiRNA) and vector-derived short hairpin RNAs (shRNAs) that are targeted to various domains of the HAV IRES. Using reporter constructs, and the DNA-based HAV replicon system, we found that shRNAs targeting the HAV IRES domains IIIc and V sustainably suppressed genome translation and replication whereas the IRES domains IIIa and IV were resistant to RNA interference. Our study suggests that some HAV IRES domains might be used as a universal and effective target for specific inhibition of HAV infection.     

Biochemical and medical aspects of the indoleamine 2,3-dioxygenase-initiated L-tryptophan metabolism

Indoleamine 2,3-dioxygenase (EC 1.13.11.42) is a heme-containing dioxygenase which catalyzes the first and rate-limiting step in the major pathway of L-tryptophan catabolism in mammals. Much attention has recently been focused on the dioxygenase because this metabolic pathway is involved not only in a variety of physiological functions but also in many diseases. In this review, the discovery and unique catalytic properties of dioxygenase are described first, and then the recent findings regarding the dioxygenase-initiated tryptophan metabolism are summarized, with special emphasis on the detrimental role of dioxygenase in side effects of interferon-gamma and interleukin-12 (by systemic tryptophan depletion), the escape of malignant tumors from immune surveillance (by immunosuppression caused by tryptophan depletion), several neurodegenerative disorders including Alzheimer's disease (by an aberrant production of neurotoxin, quinolinic acid), and age-related cataract (due to "Kynurenilation," a novel post-translational modification of lens proteins with tryptophan-derived UV filters).     

BCR/ABL activates Rap1 and B-Raf to stimulate the MEK/Erk signaling pathway in hematopoietic cells

The BCR/ABL fusion tyrosine kinase activates various intracellular signaling pathways, thus causing chronic myeloid leukemia (CML). Here we demonstrate that the inducible expression of BCR/ABL in a murine hematopoietic cell line, TonB210, leads to the activation of the Ras family small GTPase Rap1, which is inhibited by the ABL kinase inhibitor imatinib. The Rap1 activity in a CML cell line, K562, was also inhibited by imatinib. Inhibition of Rap1 activation by a dominant negative mutant of Rap1, Rap1-N17, or SPA-1 inhibited the BCR/ABL-induced activation of Elk-1. BCR/ABL also activated in a kinase activity-dependent manner the B-Raf kinase, which is an effector molecule of Rap1 and a potent activator of the MEK/Erk/Elk-1 signaling pathway. Together, these data suggest that, in addition to the well-established Ras/Raf-1 pathway, BCR/ABL activates the alternative signaling pathway involving Rap1 and B-Raf to activate Erk, which may play important roles in leukemogenesis.     

Docosahexaenoic acid-induced amelioration on impairment of memory learning in amyloid beta-infused rats relates to the decreases of amyloid beta and cholesterol levels in detergent-insoluble membrane fractions

We investigated the effects of dietary administration of docosahexaenoic acid (DHA; C22:6n-3) on the levels of amyloid beta (A beta) peptide (1-40) and cholesterol in the nonionic detergent Triton 100 x-insoluble membrane fractions (DIFs) of the cerebral cortex and, also, on learning-related memory in an animal model of Alzheimer's disease (AD) rats infused with A beta peptide (1-40) into the cerebral ventricle. The infusion increased the levels of A beta peptide and cholesterol in the DIFs concurrently with a significant increase in reference memory errors (measured by eight-arm radial-maze tasks) compared with those of vehicle rats. Conversely, the dietary administration of DHA to AD-model rats decreased the levels of A beta peptide and cholesterol in the DIFs, with the decrease being more prominent in the DHA-administered rats. Regression analysis revealed a significant positive correlation between A beta peptide and each of cholesterol, palmitic acid and stearic acid, and between the number of reference memory errors and each of cholesterol, palmitic, stearic and oleic acid; moreover, a significant negative correlation was observed between the number of reference memory errors and the molar ratio of DHA to palmitic plus stearic acid. These results suggest that DHA-induced protection of memory deficits in AD-model rats is related to the interactions of cholesterol, palmitic acid or stearic acid with A beta peptides in DIFs where DHA ameliorates these interactions.     

A neural mechanism of hierarchical discrimination of odors in the olfactory cortex based on spatiotemporal encoding of odor information

We propose a neural mechanism for discrimination of different complex odors in the olfactory cortex based on the dynamical encoding scheme. Both constituent molecules of the odor and their mixing ratios are encoded simultaneously into a spatiotemporal activity pattern (limit cycle attractor) in the olfactory bulb [Hoshino O, Kashimori Y, Kambara T (1998) Biol Cybern 79:109–120]. We present a functional model of the olfactory cortex consisting of some dynamical mapping modules. Each dynamical map is represented by itinerancy among the limit cycle attractors. When a temporal sequence of spatial activity patterns corresponding to a complex odor is injected from the bulb to the network of the olfactory cortex, the neural activity state of each mapping module is fixed to a relevant spatial pattern injected. Recognition of an odor is accomplished by a combination of firing patterns fixed in all the mapping modules. The stronger the response strength of the component, the earlier the component is recognized. The hierarchical discrimination of an odor is made by recognizing the components in order of decreasing response strengths. Received: 28 November 1998 / Accepted in revised form: 17 December 1999     

Dual enhancement of double immunofluorescent signals by CARD: participation of ubiquitin during formation of neurofibrillary tangles

Amplification with catalyzed reporter deposition (CARD) greatly enhances peroxidase signals, which has been utilized to amplify immunohistochemical labelings including fluorochromes. Here we describe a strategy to amplify each of two immunofluorescent signals without crosstalk on double-stained histological sections from human autopsied brains with Alzheimer's disease (AD). One of the two primary antibodies (anti-Abeta or anti-PHF-tau) was probed by a species-specific secondary antibody conjugated with horseradish peroxidase (HRP), which was visualized by FITC-labeled tyramide. After inactivation of HRP, the other primary antibody was probed by another species-specific secondary antibody conjugated with HRP. Amplification with biotinylated tyramide was followed by streptavidin-conjugated Cy-5, which specifically labeled the latter epitope. It was found that Abeta and PHF-tau were localized to senile plaques and neurofibrillary tangles (NFTs), respectively, which verified lack of crosstalk on the double-stained section. Localization of ubiquitin and PHF-tau was looked for at higher magnification in NFT-bearing neurons. Although these two epitopes were colocalized in some neurons, ubiquitin was not always present in PHF-tau positive NFTs. Discrepancy between PFH-tau and ubiquitin, verified inter- and intracellularly, may represent different stages of NFT formation. This is the first report of successful CARD amplification of two different fluorescent signals on double-labeling immunohistochemistry, which is now proved to be powerful in detecting epitopes in relation to AD-related lesions. Improved intensity over tenfold of the two fluorescent signals without crosstalk will expand the application of the multilabeling method with fluorochromes.     

Evolution of the X-linked Zinc Finger Gene and the Y-linked Zinc Finger Gene in Primates

We have sequenced the partial exon of the zinc finger genes (ZFX and ZFY) in 5 hominoids, 2 Old World monkeys, 1 New World monkey, and 1 prosimian. Among these primate species, the percentage similarities of the nucleotide sequence of the ZFX gene were 96-100% and 91.2-99.7% for the ZFY gene. Of 397 sites in the ZFX and ZFY gene sequences, 20 for ZFX gene and 42 for ZFY gene were found to be variable. Substitution causes 1 amino acid change in ZFX, and 5 in ZFY, among 132 amino acids. The numbers of synonymous substitutions per site (Ks) between human and the chimpanzee, gorilla and orangutan for ZFY gene were 0.026, 0.033, and 0.085, respectively. In contrast, the Ks value between human and hominoid primates for the ZFX gene was 0.008 for each comparison. Comparison of the ZFX and ZFY genes revealed that the synonymous substitution levels were higher in hominoids than in other primates. The rates of synonymous substitution per site per year were higher in the ZFY exon than in the SRY exon, and higher in the ZFY exon than in the ZFY intron, in hominoid primates.     

Effects of aryl hydrocarbon receptor signaling on the modulation of TH1/TH2 balance

An orally active antiallergic agent, M50367, skews the Th1/Th2 balance toward Th1 dominance by suppressing naive Th cell differentiation into Th2 cells in vitro. Administration results in the suppression of IgE synthesis and peritoneal eosinophilia in vivo. In this report, we determined that M50354 (an active metabolite of M50367) was a ligand for the aryl hydrocarbon receptor (AhR); the immunological effects of this compound on in vitro Th1/Th2 differentiation from naive Th cells and Th1/Th2 balance in vivo were manifested through binding to AhR. These effects were completely abolished in AhR-deficient mice. AhR expression in the naive Th cell was significantly up-regulated by costimulation of TCR and CD28. Suppression of naive Th cell differentiation into Th2 cells via binding of M50354 to AhR was associated with inhibition of GATA-3 expression in Th cells. In addition, forced expression of a constitutively active form of AhR or activation of AhR by the addition of representative ligands suppressed naive Th cell differentiation into Th2 cells. Based on these results, we conclude that AhR functions as a modulator of the in vivo Th1/Th2 balance through activation in naive Th cells.