Amaranth Grain Inhibits Antigen-Specific IgE Production Through Augmentation of the IFN-γ Response in vivo and in vitro

Amaranthus hypochondriacus L. (amaranth) is a nutritionally protein rich plant with a good yield, but there has been no research concerning its immunological effects in vivo or in vitro. In the present study, we examined the effects of amaranth grain on cytokine and IgE production using in vitro helper T cell development and IgE production assays and an animal model of an orally-induced, allergen-specific IgE response. First, we examined the effect of orally administered amaranth on serum IgE concentration which reflects the immune response during allergic disease. We observed significantly decreased (p < 0.05) allergen-specific IgE in the blood of mice in our animal model. We found that orally fed amaranth significantly augmented (p < 0.05) IFN-γ production of spleen cells. In vitro studies demonstrated that the water-soluble fraction of amaranth grain promoted helper T cell type-1 (Th1) phenotype development. Moreover, we found that the amaranth grain extract suppressed antigen-specific IgE production in vitro. These data indicate that there is a component in amaranth grain which has a suppressive effect on IgE production and augments Th1 cytokine production. In conclusion, we found that amaranth grain and its extract inhibited antigen-specific IgE production through augmenting Th1 cytokine responses in vivo and in vitro.     

RNA Polymerase Activity and Specific RNA Structure Are Required for Efficient HCV Replication in Cultured Cells

We have previously reported that the NS3 helicase (N3H) and NS5B-to-3′X (N5BX) regions are important for the efficient replication of hepatitis C virus (HCV) strain JFH-1 and viral production in HuH-7 cells. In the current study, we investigated the relationships between HCV genome replication, virus production, and the structure of N5BX. We found that the Q377R, A450S, S455N, R517K, and Y561F mutations in the NS5B region resulted in up-regulation of J6CF NS5B polymerase activity in vitro. However, the activation effects of these mutations on viral RNA replication and virus production with JFH-1 N3H appeared to differ. In the presence of the N3H region and 3′ untranslated region (UTR) of JFH-1, A450S, R517K, and Y561F together were sufficient to confer HCV genome replication activity and virus production ability to J6CF in cultured cells. Y561F was also involved in the kissing-loop interaction between SL3.2 in the NS5B region and SL2 in the 3′X region. We next analyzed the 3′ structure of HCV genome RNA. The shorter polyU/UC tracts of JFH-1 resulted in more efficient RNA replication than J6CF. Furthermore, 9458G in the JFH-1 variable region (VR) was responsible for RNA replication activity because of its RNA structures. In conclusion, N3H, high polymerase activity, enhanced kissing-loop interactions, and optimal viral RNA structure in the 3′UTR were required for J6CF replication in cultured cells.     

Cell culture and infection system for hepatitis C virus

Hepatitis C virus (HCV) infection causes chronic liver disease and is a worldwide health problem. Despite ever-increasing demand for knowledge on viral replication and pathogenesis, detailed analysis has been hampered by a lack of efficient viral culture systems. We isolated HCV genotype 2a strain JFH-1 from a patient with fulminant hepatitis. This strain replicates efficiently in Huh7 cells. Efficient replication and secretion of recombinant viral particles can be obtained in cell culture by transfection of in vitro-transcribed full-length JFH-1 RNA into Huh7 cells. JFH-1 virus generated in cell culture is infectious for both naive Huh7 cells and chimpanzees. The efficiency of viral production and infectivity of generated virus is substantially improved with permissive cell lines. This protocol describes how to use this system, which provides a powerful tool for studying viral life cycle and for the construction of antiviral strategies and the development of effective vaccines. Viral particles can be obtained in 12 days with this protocol.     

Simultaneous estimation of mixing rates and genetic drift under successive sampling of genetic markers with application to the mud crab (Scylla paramamosain) in Japan

In stock enhancement programs, it is important to assess mixing rates of released individuals in stocks. For this purpose, genetic stock identification has been applied. The allele frequencies in a composite population are expressed as a mixture of the allele frequencies in the natural and released populations. The estimation of mixing rates is possible, under successive sampling from the composite population, on the basis of temporal changes in allele frequencies. The allele frequencies in the natural population may be estimated from those of the composite population in the preceding year. However, it should be noted that these frequencies can vary between generations due to genetic drift. In this article, we develop a new method for simultaneous estimation of mixing rates and genetic drift in a stock enhancement program. Numerical simulation shows that our procedure estimates the mixing rate with little bias. Although the genetic drift is underestimated when the amount of information is small, reduction of the bias is possible by analyzing multiple unlinked loci. The method was applied to real data on mud crab stocking, and the result showed a yearly variation in the mixing rate.     

Beraprost sodium enhances neovascularization in ischemic myocardium by mobilizing bone marrow cells in rats

Beraprost sodium, an orally active prostacyclin analogue, has vasoprotective effects such as vasodilation and antiplatelet activities. We investigated the therapeutic potential of beraprost for myocardial ischemia. Immediately after coronary ligation of Sprague-Dawley rats, beraprost (200 microg/kg/day) or saline was subcutaneously administered for 28 days. Four weeks after coronary ligation, administration of beraprost increased capillary density in ischemic myocardium, decreased infarct size, and improved cardiac function in rats with myocardial infarction. Beraprost markedly increased the number of CD34-positive cells and c-kit-positive cells in plasma. Also, four weeks after coronary ligation of chimeric rats with GFP-expressing bone marrow, bone marrow-derived cells were incorporated into the infarcted region and its border zone. Treatment with beraprost increased the number of GFP/von Willebrand factor-double-positive cells in the ischemic myocardium. These results suggest that beraprost has beneficial effects on ischemic myocardium partly by its ability to enhance neovascularization in ischemic myocardium by mobilizing bone marrow cells.     

Differential regulation of Akt kinase isoforms by the members of the TCL1 oncogene family.

The members of the TCL1 proto-oncogene family (TCL1, MTCP1, and TCL1b) bind to Akt1, increasing its phosphorylation status and kinase activity. This is thought to be secondary to the formation of TCL1-Akt oligomers within which Akt is preferentially phosphorylated. Here we show that, in contrast to Akt1 and Akt2, which bind to all members of the TCL1 family, Akt3 specifically interacts with TCL1 but not with MTCP1 or TCL1b. This association is functional, as the presence of TCL1 but not MTCP1 or TCL1b increased Akt3 kinase activity in in vitro kinase assays. Functional specificity is determined by the Akt pleckstrin homology domain as chimeric Akt1, where Akt1 PH domain was replaced by that of Akt3 was no longer able to interact with MTCP1 or TCL1b and its kinase activity was solely enhanced by TCL1. Moreover, we show that, in TCL1-overexpressing SUPT-11 T-cell leukemia and P3HR-1 Burkitt's lymphoma cell lines, TCL1 interacts with endogenous Akt1, Akt2, and Akt3. TCL1 enhanced hetero-oligomerization of Akt1 with Akt3 and as a consequence facilitated transphosphorylation of Akt molecules, which may contribute to Akt activation and TCL1-induced leukemogenesis in vivo.     

Maternal primary imprinting is established at a specific time for each gene throughout oocyte growth.

Primary imprinting during gametogenesis governs the monoallelic expression/repression of imprinted genes in embryogenesis. Previously, we showed that maternal primary imprinting is disrupted in neonate-derived non-growing oocytes. Here, to investigate precisely when and in what order maternal primary imprinting progresses, we produced parthenogenetic embryos containing one genome from a non-growing or growth-stage oocyte from 1- to 20-day-old mice and one from a fully grown oocyte of adult mice. We used these embryos to analyze the expression of eight imprinted genes: Peg1/Mest, Peg3, Snrpn, Znf127, Ndn, Impact, Igf2r, and p57(KIP2). The results showed that the imprinting signals for each gene were not all imposed together at a specific time during oocyte growth but rather occurred throughout the period from primary to antral follicle stage oocytes. The developmental ability of the constructed parthenogenetic embryos was gradually reduced as the nuclear donor oocytes grew. These studies provide the first insight into the process of primary imprinting during oocyte growth.     

Gate mechanism in breathlessness caused by chest wall vibration in humans

The effects of bilateral alternating out-of-phase vibrations were studied in 10 normal healthy subjects and five asthmatic patients. The second or third intercostal spaces were vibrated during expiration, and the seventh to ninth intercostal spaces were vibrated during inspiration. Most subjects sensed breathlessness during such vibrations, and 100 Hz was most effective. The degree of breathlessness correlated positively with increased respiratory rate. Respiratory rate increased from 14.1 +/- 3.78 (mean +/- SD) to 22.3 +/- 7.14 breaths/min (P less than 0.05) during relatively severe breathlessness and to 20.39 +/- 5.66 breaths/min (P less than 0.05) during less uncomfortable sensation. Slight or negligible breathlessness induced no significant increase in rate (15.33 +/- 4.19 breaths/min). All asthma patients described the sensations during vibration as similar to those during asthma attacks, and their respiratory rates increased 20.7 +/- 11.03% during 100 Hz vibration (P less than 0.01). It is suggested that the uncomfortable sensation of breathlessness may be induced by muscle spindles in the intercostal muscles being activated out of phase with the respiratory cycle. The central mechanism that receives the intercostal afferents may have a certain gate that operates in relation to the sensation of breathlessness.     

MEASUREMENT OF γ-AMINOBUTYRIC ACID IN ISOLATED NERVE CELLS OF CAT CENTRAL NERVOUS SYSTEM

Abstract— A sensitive method for measuring γ-aminobutyric acid (GABA) has been developed. This method consists of a combination of the enzymic GABA assay of J akoby and S cott (1959) with the enzymic cycling technique of L owry , P assonneau , S chulz and R ock (1961) and permits the measurement of as little as 2 × 10⁻¹⁴ mol of GABA. Using this method, GABA analyses were made on single isolated nerve cell bodies of different types from the CNS of the cat. Average GABA concentrations in these cell bodies were: spinal ganglion cells, 0.2 m m ; spinal mononeurons, 0.9 m m ; large cells of the ventral part of Deiters' nucleus, 2.7 m m ; large cells of the dorsal part of Deiters' nucleus, 6.3 m m ; cerebellar nuclei cells, 6.0 m m ; cerebellar Purkinje cells, 6.6mM; cerebral Betz cells, 2.5 m m .
The GABA concentrations in the isolated dorsal Deiters' cells were greatly reduced (1.7 m m ) after the removal of the cerebellar vermis while those of the ventral Deiters' cells were unaffected by the denervation. These results suggest that GABA is concentrated within axon terminals, probably of Purkinje neurons, synapsing with the dorsal Deiters' cells. The results of GABA analyses on isolated nerve cells are discussed in relation to the relevant neuronal functions and the possible role of GABA as an inhibitory transmitter.