Hesperidin inhibits expression of hypoxia inducible factor-1 alpha and inflammatory cytokine production from mast cells

The citrus unshiu peel has been used traditionally as a medicine to improve bronchial and asthmatic conditions or cardiac and blood circulation in Korea, China, and Japan. Here, we report the effects of citrus unshiu peel water extract (CPWE) on the phorbol myristate acetate (PMA) + calcium ionophore A23187-induced hypoxia-inducible factor-1α (HIF-1α) activation and inflammatory cytokine production from the human mast cell line, HMC-1 cells. We compared CPWE with hesperidin, a common constituent of citrus unshiu. CPWE and hesperidin inhibited the PMA + A23187-induced HIF-1α expression and the subsequent production of vascular endothelial growth factor (VEGF). In addition, CPWE suppressed PMA + A23187-induced phosphorylation of the extracellular signal-regulated kinase (ERK). We also show that the increased cytokines interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α level was significantly inhibited by treatment of CPWE or hesperidin. In the present study, we report that CPWE and hesperidin are inhibitors of HIF-1α and cytokines on the mast cell-mediated inflammatory responses.     

In vivo magnetic resonance microscopy of differentiation in Xenopus laevis embryos from the first cleavage onwards

Differentiation inside a developing embryo can be observed by a variety of optical methods but hardly so in opaque organisms. Embryos of the frog Xenopus laevis--a popular model system--belong to the latter category and, for this reason, are predominantly being investigated by means of physical sectioning. Magnetic resonance imaging (MRI) is a noninvasive method independent of the optical opaqueness of the object. Starting out from clinical diagnostics, the technique has now developed into a branch of microscopy--MR microscopy--that provides spatial resolutions of tens of microns for small biological objects. Nondestructive three-dimensional images of various embryos have been obtained using this technique. They were, however, usually acquired by long scans of fixed embryos. Previously reported in vivo studies did not cover the very early embryonic stages, mainly for sensitivity reasons. Here, by applying high field MR microscopy to the X. laevis system, we achieved the temporal and spatial resolution required for observing subcellular dynamics during early cell divisions in vivo. We present image series of dividing cells and nuclei and of the whole embryonic development from the zygote onto the hatching of the tadpole. Additionally, biomechanical analyses from successive MR images are introduced. These results demonstrate that MR microscopy can provide unique contributions to investigations of differentiating cells and tissues in vivo.     

Production of phytoestrogen <Emphasis Type="Italic">S</Emphasis>-equol from daidzein in mixed culture of two anaerobic bacteria

An anaerobic incubation mixture of two bacterial strains Eggerthella sp. Julong 732 and Lactobacillus sp. Niu-O16, which have been known to transform dihydrodaidzein to S-equol and daidzein to dihydrodaidzein respectively, produced S-equol from daidzein through dihydrodaidzein. The biotransformation kinetics of daidzein by the mixed cultures showed that the production of S-equol from daidzein was significantly enhanced, as compared to the production of S-equol from dihydrodaidzein by Eggerthella sp. Julong 732 alone. The substrate daidzein in the mixed culture was almost completely converted to S-equol in 24 h of anaerobic incubation. The increased production of S-equol from daidzein by the mixed culture is likely related to the increased bacterial numbers of Eggerthella sp. Julong 732. In the mixture cultures, the growth of Eggerthella sp. Julong 732 was significantly increased while the growth of Lactobacillus sp. Niu-O16 was suppressed as compared to either the single culture of Eggerthella sp. Julong 732 or Lactobacillus sp. Niu-O16. This is the first report in which two metabolic pathways to produce S-equol from daidzein by a mixed culture of bacteria isolated from human and bovine intestinal environments were successfully linked under anaerobic conditions.     

Construction and Characterization of Shuttle Vectors for Succinic Acid-Producing Rumen Bacteria

Shuttle vectors carrying the origins of replication that function in Escherichia coli and two capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. These vectors were found to be present at ca. 10 copies per cell. They were found to be stably maintained in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 216 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0 × 10⁶ and 7.1 × 10⁶ transformants/μg DNA were obtained with M. succiniciproducens and A. succinogenes, respectively. A 1.7-kb minimal replicon was identified that consists of the rep gene, four iterons, A+T-rich regions, and a dnaA box. It was found that the shuttle vector replicates via the theta mode, which was confirmed by sequence analysis and Southern hybridization. These shuttle vectors were found to be suitable as expression vectors as the homologous fumC gene encoding fumarase and the heterologous genes encoding green fluorescence protein and red fluorescence protein could be expressed successfully. Thus, the shuttle vectors developed in this study should be useful for genetic and metabolic engineering of succinic acid-producing rumen bacteria.     

Defective cholesterol traffic and neuronal differentiation in neural stem cells of Niemann-Pick type C disease improved by valproic acid, a histone deacetylase inhibitor

Niemann-Pick type C disease (NPC) is a neurodegenerative and lipid storage disorder for which no effective treatment is known. We previously reported that neural stem cells derived from NPC1 mice showed impaired self-renewal and differentiation. We examined whether valproic acid (VPA), a histone deacetylase inhibitor, could enhance neuronal differentiation and recover defective cholesterol metabolism in neural stem cells (NSCs) from NPC1-deficient mice (NPC1(-/-)). VPA could induce neuronal differentiation and restore impaired astrocytes in NSCs from NPC1(-/-) mice. Importantly, an increasing level of cholesterol within NSCs from NPC1(-/-) mice could be reduced by VPA. Moreover, essential neurotrophic genes (TrkB, BDNF, MnSoD, and NeuroD) were up-regulated through the repression of the REST/NRSF and HDAC complex by the VPA treatment. Up-regulated neurotrophic genes were able to enhance neural differentiation and cholesterol homeostasis in neural stem cells from NPC1(-/-) mice. In this study, we suggested that, along with cholesterol homeostasis, impaired neuronal differentiation and abnormal morphology of astrocytes could be rescued by the inhibition of HDAC and REST/NRSF activity induced by VPA treatment.     

Characterization of DNA methylation change in stem cell marker genes during differentiation of human embryonic stem cells

Pluripotent human embryonic stem cells (hESCs) have the distinguishing feature of innate capacity to allow indefinite self-renewal. This attribute continues until specific constraints or restrictions, such as DNA methylation, are imposed on the genome, usually accompanied by differentiation. With the aim of utilizing DNA methylation as a sign of early differentiation, we probed the genomic regions of hESCs, particularly focusing on stem cell marker (SCM) genes to identify regulatory sequences that display differentiation-sensitive alterations in DNA methylation. We show that the promoter regions of OCT4 and NANOG, but not SOX2, REX1 and FOXD3, undergo significant methylation during hESCs differentiation in which SCM genes are substantially repressed. Thus, following exposure to differentiation stimuli, OCT4 and NANOG gene loci are modified relatively rapidly by DNA methylation. Accordingly, we propose that the DNA methylation states of OCT4 and NANOG sequences may be utilized as barometers to determine the extent of hESC differentiation.     

Hyperpolarization-activated currents control the excitability of principal neurons in the basolateral amygdala

Anxiety is thought to be influenced by neuronal excitability in basolateral nucleus of the amygdala (BLA). However, molecules that are critical for regulating excitability of BLA neurons are yet to be determined. In the present study, we have examined whether hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels, which mediate the depolarizing cation current, can control the neuronal excitability. HCN channel-like activity appeared to be detected in BLA principal neurons. ZD7288, a specific blocker for HCN channels, increased the input resistance of membrane, hyperpolarized resting membrane potential, and enhanced action potential firing in BLA principal neurons. The blockade of HCN channels facilitated temporal summation of repetitively evoked excitatory postsynaptic potentials, suggesting that suppression of HCN channel activity in principal neurons can accelerate the propagation of synaptic responses onto the axon hillock. Thus, our findings have laid foundation for studies to reveal how HCN channel activity in BLA principal neurons regulates anxiety in vivo.     

NM23-H2 involves in negative regulation of Diva and Bcl2L10 in apoptosis signaling

The Bcl-2 family members are evolutionally conserved and crucial regulators of apoptosis. Diva (Boo), an ortholog of Bcl2L10 or Bcl-B, is a member of the Bcl-2 family that has contradictory functions in apoptosis. To understand the signaling mechanisms of Diva, we searched for proteins that interact with Diva using the yeast two-hybrid system. We identified a nucleoside diphosphate kinase isoform, NM23-H2. Here, we show that Diva bound to NM23-H2 in cells in which the transmembrane domain of Diva was required, and both proteins were colocalized in cytoplasm. Of interest, Diva protein level was significantly down-regulated by NM23-H2 as knock down of NM23-H2 restored Diva expression. Overexpression of NM23-H2 induced apoptosis, and the depletion of NM23-H2 led to the increase of Diva's apoptotic activity. Thus, these results indicate the existence of a previously undiscovered mechanism by which NM23-H2 involves in the regulation of Diva-mediated apoptosis.