Efficient and directive generation of two distinct endoderm lineages from human ESCs and iPSCs by differentiation stage-specific SOX17 transduction

The establishment of methods for directive differentiation from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is important for regenerative medicine. Although Sry-related HMG box 17 (SOX17) overexpression in ESCs leads to differentiation of either extraembryonic or definitive endoderm cells, respectively, the mechanism of these distinct results remains unknown. Therefore, we utilized a transient adenovirus vector-mediated overexpression system to mimic the SOX17 expression pattern of embryogenesis. The number of alpha-fetoprotein-positive extraembryonic endoderm (ExEn) cells was increased by transient SOX17 transduction in human ESC- and iPSC-derived primitive endoderm cells. In contrast, the number of hematopoietically expressed homeobox (HEX)-positive definitive endoderm (DE) cells, which correspond to the anterior DE in vivo, was increased by transient adenovirus vector-mediated SOX17 expression in human ESC- and iPSC-derived mesendoderm cells. Moreover, hepatocyte-like cells were efficiently generated by sequential transduction of SOX17 and HEX. Our findings show that a stage-specific transduction of SOX17 in the primitive endoderm or mesendoderm promotes directive ExEn or DE differentiation by SOX17 transduction, respectively.     

Cooling Treatment Transiently Increases the Permeability of Brain Capillary Endothelial Cells Through Translocation of Claudin-5

The blood–brain-barrier (BBB) is formed by different cell types, of which brain microvascular endothelial cells are major structural constituents. The goal of this study was to examine the effects of cooling on the permeability of the BBB with reference to tight junction formation of brain microendothelial cells. The sensorimotor cortex above the dura mater in adult male Wistar rats was focally cooled to a temperature of 5 °C for 1 h, then immunostaining for immunoglobulin G (IgG) was performed to evaluate the permeability of the BBB. Permeability produced by cooling was also evaluated in cultured murine brain endothelial cells (bEnd3) based on measurement of trans-epithelial electric resistance (TEER). Immunocytochemistry and Western blotting of proteins associated with tight junctions in bEnd3 were performed to determine protein distribution before and after cooling. After focal cooling of the rat brain cortex, diffuse immunostaining for IgG was observed primarily around the small vasculature and in the extracellular spaces of parenchyma of the cortex. In cultured bEnd3, TEER significantly decreased during cooling (15 °C) and recovered to normal levels after rewarming to 37 °C. Immunocytochemistry and Western blotting showed that claudin-5, a critical regulatory protein for tight junctions, was translocated from the membrane to the cytoplasm after cooling in cultured bEnd3 cells. These results suggest that focal brain cooling may open the BBB transiently through an effect on tight junctions of brain microendothelial cells, and that therapeutically this approach may allow control of BBB function and drug delivery through the BBB.     

Perturbations of a water column of Lake Onogawa by local heavy rainfall

The perturbations of a water column at the deepest part of Lake Onogawa by a local heavy rainfall were analyzed. Mixing throughout the water column (18.8-m deep) was indicated by changes in the distribution of water temperature. This mixing perturbed the hypolimnetic seasonal distributions of dissolved components. All partic-ulate components peaked at 10 m, suggesting a center of inflowing water. Compared with background levels, particulate nitrogen (PN), particulate carbon (PC), particulate phosphorus (PP), and suspended solids (SS) increased from 16-fold for PN to 100-fold for SS. Soluble reactive phosphorus was the only dissolved component that formed a clear maximum at 10 m, as did the particulate components. Assuming that SS consists mainly of mineral particles, SS can be classified into three categories: (1) A rapidly sinking fraction, the main body of the loaded SS, with a sinking rate exceeding 1 m day⁻¹ and radii exceeding 2–3 μm. (2) A slowly sinking fraction with a sinking speed of about 10 cm day⁻¹ and radii of 0.7–0.9 μm; this fraction is calculated to be about 4% of the total loaded SS at most. (3) A fraction that was essentially retained in the water column. The maximum estimate of this fraction was 0.5% of the total. Received: January 1, 2000 / Accepted: August 10, 2000     

Loss of the Xeroderma Pigmentosum Group A Gene (XPA) Enhances Apoptosis of Cultured Cerebellar Neurons Induced by UV but Not by Low-K+ Medium

Abstract: To study the involvement of the xeroderma pigmentosum group A gene ( XPA ) in neuronal apoptosis, we cultured cerebellar neurons from mice lacking XPA gene ( XPA ^−/−) and induced apoptosis by exposure to UV irradiation or medium containing a low concentration of potassium (low-K⁺ medium). When cerebellar neurons from postnatal days 15–16 wild-type mice were treated with UV irradiation, apoptotic neuronal death was observed after 24–48 h. About 60% of neurons survived 48 h after UV irradiation at a dose of 5 J/m². On the other hand, neurons from XPA ^−/− mice showed a significantly increased vulnerability to UV irradiation, and >90% of neurons died 48 h after UV irradiation at a dose of 5 J/m². In contrast, low-K⁺ medium induced apoptosis of neurons from mice of each genotype with the same kinetics. These results suggest that the XPA gene is involved in neuronal DNA repair and that it thereby influences apoptosis induced by DNA damage in cultured cerebellar neurons.     

Brain-derived Neurotrophic Factor Enhances the Basal Rate of Protein Synthesis by Increasing Active Eukaryotic Elongation Factor 2 Levels and Promoting Translation Elongation in Cortical Neurons

The constitutive and activity-dependent components of protein synthesis are both critical for neural function. Although the mechanisms controlling extracellularly induced protein synthesis are becoming clear, less is understood about the molecular networks that regulate the basal translation rate. Here we describe the effects of chronic treatment with various neurotrophic factors and cytokines on the basal rate of protein synthesis in primary cortical neurons. Among the examined factors, brain-derived neurotrophic factor (BDNF) showed the strongest effect. The rate of protein synthesis increased in the cortical tissues of BDNF transgenic mice, whereas it decreased in BDNF knock-out mice. BDNF specifically increased the level of the active, unphosphorylated form of eukaryotic elongation factor 2 (eEF2). The levels of active eEF2 increased and decreased in BDNF transgenic and BDNF knock-out mice, respectively. BDNF decreased kinase activity and increased phosphatase activity against eEF2 in vitro. Additionally, BDNF shortened the ribosomal transit time, an index of translation elongation. In agreement with these results, overexpression of eEF2 enhanced protein synthesis. Taken together, our results demonstrate that the increased level of active eEF2 induced by chronic BDNF stimulation enhances translational elongation processes and increases the total rate of protein synthesis in neurons.The synthesis and post-translational modification of proteins play key roles in neural development, synaptic plasticity, and cognitive brain functions such as learning and memory (1, 2). Recent studies have revealed that activity-dependent regulation of translation affects neural plasticity (3, 4). Previously, we reported that BDNF,² a critical molecule for neural plasticity (5–7), enhances protein synthesis and activates the translational machinery in central nervous system neurons (8). In addition, neurotransmitters such as glutamate (9, 10), dopamine (11), and serotonin (12) are also reported to facilitate translation in neurons. These observations indicate that endogenous molecules can acutely modulate neuronal translation in response to neural activity. Translation of an mRNA molecule comprises three steps: initiation, elongation, and release (or termination) (13). In the first step, mRNA and methionyl-tRNA_i^Met are recruited to a ribosome. During elongation, aminoacyl-tRNAs are sequentially recruited and the nascent peptide chain lengthens incrementally as amino acids are covalently attached via peptide bonds. Finally, the polypeptide chain is released from the ribosome. Each step is regulated by a variety of factors. The activities of these regulatory proteins are predominantly controlled by phosphorylation and GTP binding. BDNF activates both initiation and elongation by modulating these processes (8, 14, 15).In addition to these acute, stimulation-induced changes in the translation rate, the long term regulation of translation plays important roles in developing and mature brains. In fact, recent studies have shown that genetic disruption or overexpression of translation factors or modulator genes alters synaptic plasticity and behavior as well as the basal rate of protein synthesis. Mice lacking the gene encoding GCN2, a kinase that phosphorylates eIF2α, exhibited enhanced translation as well as aberrant long term potentiation and spatial learning (16). Similar phenotypes have been observed in mice carrying a constitutively active mutant variant (Ser⁵² to Ala) of eIF2α (17). Mice lacking eIF4E-binding protein 2 (4EBP2) exhibited increased cap-dependent translation and altered long-term potentiation, long-term depression (LTD), and learning (18, 19). Mice expressing a transgene encoding a dominant-negative version of MEK, which inhibits the phosphorylation of eIF4E and protein synthesis, were found to have learning deficits (20). Thus, modifying the rate of protein synthesis can produce deleterious effects on synaptic plasticity and brain function.Although genetic modifications can affect translation, the mechanisms by which the basal translation rate is controlled in normal neurons are unknown. Here, we demonstrate that chronic treatment of primary cortical neurons with BDNF increases the level of active, unphosphorylated eukaryotic elongation factor 2 (eEF2) and enhances the rates of elongation and protein synthesis. Analysis of BDNF mutant mice supports a role for this neurotrophin in regulating the basal rate of protein synthesis.     

Differing Dynamics of Intrapersonal and Interpersonal Coordination: Two-finger and Four-Finger Tapping Experiments

Finger-tapping experiments were conducted to examine whether the dynamics of intrapersonal and interpersonal coordination systems can be described equally by the Haken—Kelso—Bunz model, which describes inter-limb coordination dynamics. This article reports the results of finger-tapping experiments conducted in both systems. Two within-subject factors were investigated: the phase mode and the number of fingers. In the intrapersonal experiment (Experiment 1), the participants were asked to tap, paced by a gradually hastening auditory metronome, looking at their fingers moving, using the index finger in the two finger condition, or the index and middle finger in the four-finger condition. In the interpersonal experiment (Experiment 2), pairs of participants performed the task while each participant used the outside hand, tapping with the index finger in the two finger condition, or the index and middle finger in the four-finger condition. Some results did not agree with the HKB model predictions. First, from Experiment 1, no significant difference was observed in the movement stability between the in-phase and anti-phase modes in the two finger condition. Second, from Experiment 2, no significant difference was found in the movement stability between the in-phase and anti-phase mode in the four-finger condition. From these findings, different coordination dynamics were inferred between intrapersonal and interpersonal coordination systems against prediction from the previous studies. Results were discussed according to differences between intrapersonal and interpersonal coordination systems in the availability of perceptual information and the complexity in the interaction between limbs derived from a nested structure.     

A novel two-pore domain K+ channel,TRESK, is localized in the spinal cord

To find a novel human ion channel gene we have executed an extensive search by using a human genome draft sequencing data base. Here we report a novel two-pore domain K+ channel, TRESK (TWIK-related spinal cord K+ channel). TRESK is coded by 385 amino acids and shows low homology (19%) with previously characterized two-pore domain K+ channels. However, the most similar channel is TREK-2 (two-pore domain K+ channel), and TRESK also has two pore-forming domains and four transmembrane domains that are evolutionarily conserved in the two-pore domain K+ channel family. Moreover, we confirmed that TRESK is expressed in the spinal cord. Electrophysiological analysis demonstrated that TRESK induced outward rectification and functioned as a background K+ channel. Pharmacological analysis showed TRESK to be inhibited by previously reported K+ channel inhibitors Ba2+, propafenone, glyburide, lidocaine, quinine, quinidine, and triethanolamine. Functional analysis demonstrated TRESK to be inhibited by unsaturated free fatty acids such as arachidonic acid and docosahexaenoic acid. TRESK is also sensitive to extreme changes in extracellular and intracellular pH. These results indicate that TRESK is a novel two-pore domain K+ channel that may set the resting membrane potential of cells in the spinal cord.     

UDP-arabinopyranose mutase gene expressions are required for the biosynthesis of the arabinose side chain of both pectin and arabinoxyloglucan,and normal leaf expansion in Nicotiana tabacum

Journal of Plant Research - Plant cell walls are composed of polysaccharides such as cellulose, hemicelluloses, and pectins, whose location and function differ depending on plant type. Arabinose is...