1H, 13C, and 15N resonance assignment of the first PDZ domain of mouse ZO-1

Zonula occludens-1 (ZO-1) is a scaffolding molecule critical to the formation of intercellular adhesion structures, such as tight junctions (TJs) and adherens junctions (AJs). ZO-1 contains three PDZ domains followed by a GUK domain and a ZU5 domain. The first PDZ of ZO-1 (ZO-1(PDZ1)) serves as a protein–protein interaction module and interacts with the C-termini of almost all claudins to initiate the formation of a belt-like structure on the lateral membranes, thereby promoting TJ formation. It has been recently reported that approximately 15% of all PDZ domains bind phosphoinositides, and ZO-1(PDZ1) is the one of these. Here we report the ¹⁵N, ¹³C, and ¹H chemical shift assignments of the first PDZ domain of mouse ZO-1. The resonance assignments obtained in this work may contribute in clarifying the interplay between the two binary interactions, ZO-1(PDZ1)–claudins and ZO-1(PDZ1)–phospholipids, and suggesting a novel regulation mechanism underlying the formation and maintenance of cell–cell adhesion machinery downstream of the phospholipid signaling pathways.     

Skeletal muscle-specific expression of PGC-1α-b, an exercise-responsive isoform, increases exercise capacity and peak oxygen uptake

Background

Maximal oxygen uptake (VO_2max) predicts mortality and is associated with endurance performance. Trained subjects have a high VO_2max due to a high cardiac output and high metabolic capacity of skeletal muscles. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a nuclear receptor coactivator, promotes mitochondrial biogenesis, a fiber-type switch to oxidative fibers, and angiogenesis in skeletal muscle. Because exercise training increases PGC-1α in skeletal muscle, PGC-1α-mediated changes may contribute to the improvement of exercise capacity and VO_2max. There are three isoforms of PGC-1α mRNA. PGC-1α-b protein, whose amino terminus is different from PGC-1α-a protein, is a predominant PGC-1α isoform in response to exercise. We investigated whether alterations of skeletal muscle metabolism by overexpression of PGC-1α-b in skeletal muscle, but not heart, would increase VO_2max and exercise capacity.

Methodology/Principal Findings

Transgenic mice showed overexpression of PGC-1α-b protein in skeletal muscle but not in heart. Overexpression of PGC-1α-b promoted mitochondrial biogenesis 4-fold, increased the expression of fatty acid transporters, enhanced angiogenesis in skeletal muscle 1.4 to 2.7-fold, and promoted exercise capacity (expressed by maximum speed) by 35% and peak oxygen uptake by 20%. Across a broad range of either the absolute exercise intensity, or the same relative exercise intensities, lipid oxidation was always higher in the transgenic mice than wild-type littermates, suggesting that lipid is the predominant fuel source for exercise in the transgenic mice. However, muscle glycogen usage during exercise was absent in the transgenic mice.

Conclusions/Significance

Increased mitochondrial biogenesis, capillaries, and fatty acid transporters in skeletal muscles may contribute to improved exercise capacity via an increase in fatty acid utilization. Increases in PGC-1α-b protein or function might be a useful strategy for sedentary subjects to perform exercise efficiently, which would lead to prevention of life-style related diseases and increased lifespan.     

Polymorphism of the Tryptophan Hydroxylase 2 (TPH2) Gene Is Associated with Chimpanzee Neuroticism

In the brain, serotonin production is controlled by tryptophan hydroxylase 2 (TPH2), a genotype. Previous studies found that mutations on the TPH2 locus in humans were associated with depression and studies of mice and studies of rhesus macaques have shown that the TPH2 locus was involved with aggressive behavior. We previously reported a functional single nucleotide polymorphism (SNP) in the form of an amino acid substitution, Q468R, in the chimpanzee TPH2 gene coding region. In the present study we tested whether this SNP was associated with neuroticism in captive and wild-born chimpanzees living in Japan and Guinea, respectively. Even after correcting for multiple tests (Bonferroni p = 0.05/6 = 0.008), Q468R was significantly related to higher neuroticism (β = 0.372, p = 0.005). This study is the first to identify a genotype linked to a personality trait in chimpanzees. In light of the prior studies on humans, mice, and rhesus macaques, these findings suggest that the relationship between neuroticism and TPH2 has deep phylogenetic roots.     

Systems biology of symmetry breaking during neuronal polarity formation

Polarization, in which a single axon and multiple dendrites are formed, is crucial for neuronal functions, and symmetry breaking is the initial step of this process. Accumulating studies have revealed a number of molecules that act asymmetrically in neurons, and thereby regulate neuronal polarity. Thus, one of the major goals of current research is to understand how asymmetric signals are generated during the symmetry-breaking step. Current models of neuronal symmetry breaking generally involve "local activation" for induction of axon outgrowth and "global inhibition" to suppress formation of multiple axons and can be categorized into "one-takes-all" and "activator-inhibitor" models. Both types of model incorporate a positive feedback loop to execute local activation, but differ in the manner of global inhibition. Quantitative experimentation combined with computational modeling is a powerful strategy in systems biology, and analyses in this direction have begun to yield a more profound understanding of how neurons break their symmetry during polarity formation.     

Properties of gene knockdown system by vector‐based siRNA in zebrafish

RNA interference (RNAi) has emerged as a powerful tool to silence specific genes. Vector‐based RNAi systems have been developed to downregulate targeted genes in a spatially and temporally regulated fashion both in vitro and in vivo. The zebrafish (Danio rerio) is a model animal that has been examined based on a wide variety of biological techniques, including embryonic manipulations, forward and reverse genetics, and molecular biology. However, a heritable and tissue‐specific knockdown of gene expression has not yet been developed in zebrafish. We examined two types of vector, which produce small interfering RNA (siRNA), the direct effector in RNAi system; microRNA (miRNA) process mimicking vectors with a promoter for RNA polymerase II and short hairpin RNA (shRNA) expressing vector through a promoter for RNA polymerase III. Though gene‐silencing phenotypes were not observed in the miRNA process mimicking vectors, the transgenic embryos of the second vector (Tg(zU6‐shGFP)), shRNA expressing vector for enhanced green fluorescence protein, revealed knockdown of the targeted gene. Interestingly, only the embryos from Tg(zU6‐shGFP) female but not from the male fish showed the downregulation. Comparison of the quantity of siRNA produced by each vector indicates that the vectors tested here induced siRNA, but at low levels barely sufficient to silence the targeted gene.     

Temperature Dependences of Torque Generation and Membrane Voltage in the Bacterial Flagellar Motor

In their natural habitats bacteria are frequently exposed to sudden changes in temperature that have been shown to affect their swimming. With our believed to be new methods of rapid temperature control for single-molecule microscopy, we measured here the thermal response of the Na⁺-driven chimeric motor expressed in Escherichia coli cells. Motor torque at low load (0.35 μm bead) increased linearly with temperature, twofold between 15°C and 40°C, and torque at high load (1.0 μm bead) was independent of temperature, as reported for the H⁺-driven motor. Single cell membrane voltages were measured by fluorescence imaging and these were almost constant (∼120 mV) over the same temperature range. When the motor was heated above 40°C for 1–2 min the torque at high load dropped reversibly, recovering upon cooling below 40°C. This response was repeatable over as many as 10 heating cycles. Both increases and decreases in torque showed stepwise torque changes with unitary size ∼150 pN nm, close to the torque of a single stator at room temperature (∼180 pN nm), indicating that dynamic stator dissociation occurs at high temperature, with rebinding upon cooling. Our results suggest that the temperature-dependent assembly of stators is a general feature of flagellar motors.     

N-myc Downstream-regulated Gene 1 Promotes Tumor Inflammatory Angiogenesis through JNK Activation and Autocrine Loop of Interleukin-1α by Human Gastric Cancer Cells

The expression of N-myc downstream-regulated gene 1 (NDRG1) was significantly correlated with tumor angiogenesis and malignant progression together with poor prognosis in gastric cancer. However, the underlying mechanism for the role of NDRG1 in the malignant progression of gastric cancer remains unknown. Here we examined whether and how NDRG1 could modulate tumor angiogenesis by human gastric cancer cells. We established NU/Cap12 and NU/Cap32 cells overexpressing NDRG1 in NUGC-3 cells, which show lower tumor angiogenesis in vivo. Compared with parental NU/Mock3, NU/Cap12, and NU/Cap32 cells: 1) induced higher tumor angiogenesis than NU/Mock3 cells accompanied by infiltration of tumor-associated macrophages in mouse dorsal air sac assay and Matrigel plug assay; 2) showed much higher expression of CXC chemokines, MMP-1, and the potent angiogenic factor VEGF-A; 3) increased the expression of the representative inflammatory cytokine, IL-1α; 4) augmented JNK phosphorylation and nuclear expression of activator protein 1 (AP-1). Further analysis demonstrated that knockdown of AP-1 (Jun and/or Fos) resulted in down-regulation of the expression of VEGF-A, CXC chemokines, and MMP-1, and also suppressed expression of IL-1α in NDRG1-overexpressing cell lines. Treatment with IL-1 receptor antagonist (IL-1ra) resulted in down-regulation of JNK and c-Jun phosphorylation, and the expression of VEGF-A, CXC chemokines, and MMP-1 in NU/Cap12 and NU/Cap32 cells. Finally, administration of IL-1ra suppressed both tumor angiogenesis and infiltration of macrophages by NU/Cap12 in vivo. Together, activation of JNK/AP-1 thus seems to promote tumor angiogenesis in relationship to NDRG1-induced inflammatory stimuli by gastric cancer cells.     

Benzalkonium Chloride Accelerates the Formation of the Amyloid Fibrils of Corneal Dystrophy-associated Peptides

Corneal dystrophies are genetic disorders resulting in progressive corneal clouding due to the deposition of amyloid fibrils derived from keratoepithelin, also called transforming growth factor β-induced protein (TGFBI). The formation of amyloid fibrils is often accelerated by surfactants such as sodium dodecyl sulfate (SDS). Most eye drops contain benzalkonium chloride (BAC), a cationic surfactant, as a preservative substance. In the present study, we aimed to reveal the role of BAC in the amyloid fibrillation of keratoepithelin-derived peptides in vitro. We used three types of 22-residue synthetic peptides covering Leu110-Glu131 of the keratoepithelin sequence: an R-type peptide with wild-type R124, a C-type peptide with C124 associated with lattice corneal dystrophy type I, and a H-type peptide with H124 associated with granular corneal dystrophy type II. The time courses of spontaneous amyloid fibrillation and seed-dependent fibril elongation were monitored in the presence of various concentrations of BAC or SDS using thioflavin T fluorescence. BAC and SDS accelerated the fibrillation of all synthetic peptides in the absence and presence of seeds. Optimal acceleration occurred near the CMC, which suggests that the unstable and dynamic interactions of keratoepithelin peptides with amphipathic surfactants led to the formation of fibrils. These results suggest that eye drops containing BAC may deteriorate corneal dystrophies and that those without BAC are preferred especially for patients with corneal dystrophies.     

Kinesin-5/Eg5 is important for transport of CARTS from the trans-Golgi network to the cell surface

Here we report that the kinesin-5 motor Klp61F, which is known for its role in bipolar spindle formation in mitosis, is required for protein transport from the Golgi complex to the cell surface in Drosophila S2 cells. Disrupting the function of its mammalian orthologue, Eg5, in HeLa cells inhibited secretion of a protein called pancreatic adenocarcinoma up-regulated factor (PAUF) but, surprisingly, not the trafficking of vesicular stomatitis virus G protein (VSV-G) to the cell surface. We have previously reported that PAUF is transported from the trans-Golgi network (TGN) to the cell surface in specific carriers called CARTS that exclude VSV-G. Inhibition of Eg5 function did not affect the biogenesis of CARTS; however, their migration was delayed and they accumulated near the Golgi complex. Altogether, our findings reveal a surprising new role of Eg5 in nonmitotic cells in the facilitation of the transport of specific carriers, CARTS, from the TGN to the cell surface.