Inverse synaptic tagging of inactive synapses via dynamic interaction of Arc/Arg3.1 with CaMKIIβ

The Arc/Arg3.1 gene product is rapidly upregulated by strong synaptic activity and critically contributes to weakening synapses by promoting AMPA-R endocytosis. However, how activity-induced Arc is redistributed and determines the synapses to be weakened remains unclear. Here, we show targeting of Arc to inactive synapses via a high-affinity interaction with CaMKIIβ that is not bound to calmodulin. Synaptic Arc accumulates in inactive synapses that previously experienced strong activation and correlates with removal of surface GluA1 from individual synapses. A lack of CaMKIIβ either in vitro or in vivo resulted in loss of Arc upregulation in the silenced synapses. The discovery of Arc's role in "inverse" synaptic tagging that is specific for weaker synapses and prevents undesired enhancement of weak synapses in potentiated neurons reconciles essential roles of Arc both for the late phase of long-term plasticity and for reduction of surface AMPA-Rs in stimulated neurons.     

AMPK limits IL-1-stimulated IL-6 synthesis in osteoblasts: involvement of IκB/NF-κB pathway

AMP-activated protein kinase (AMPK) is currently known to act as a key regulator of metabolic homeostasis. Several biosynthetic enzymes for fatty acid or glycogen are recognized as the targets of AMPK. In the present study, we investigated the role of AMPK in the interleukin-1 (IL-1)-stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. IL-1 induced phosphorylation of AMPK-α (Thr-172), which regulates AMPK activities, and acetyl-CoA carboxylase, a direct substrate of AMPK. Compound C, an inhibitor of AMPK, which suppressed the IL-1-induced phosphorylation of acetyl-CoA carboxylase, increased the release and the mRNA level of IL-6 stimulated by IL-1. Transfection of AMPK siRNA-α also amplified the IL-1-stimulated IL-6 release compared to the control cells. On the other hand, IL-1 elicited the phosphorylation of IκB, which caused subsequent decrease of total level of IκB. Wedelolactone, an inhibitor of IκB kinase, which reduced the phosphorylation both of IκB and NF-κB, significantly enhanced the IL-1-stimulated IL-6 synthesis. Compound C remarkably suppressed the IL-1-induced phosphorylation of IκB. These results strongly suggest that AMPK negatively regulates IL-1-stimulated IL-6 synthesis through the IκB/NF-κB pathway in osteoblasts.     

Whole genome sequencing and future breeding of rice

Contemporary crop improvement relies on the genetic analysis of progeny derived from a cross between different lines with contrasting phenotypes. Such analysis allowed positioning of genes for agronomically important traits, enabling development of DNA makers for marker-assisted selection (MAS). So far the identification of loci for desirable traits have been carried out by linkage analysis using DNA markers. This process required the development of DNA markers that are distributed over the genome as well as the genotyping of each progeny. Due to recent development in next generation sequencing (NGS) technology, whole genome sequencing (WGS) is becoming easier and cheaper. Using NGS, we developed a new method called MutMap that allows rapid isolation of useful alleles from rice mutant lines. An important feature of MutMap is that it does not require marker development. We foresee that the era of genetic markers will be eventually eclipsed by that of WGS applied to all the individuals in the breeding processes.     

Insulin/insulin-like growth factor (IGF) stimulation abrogates an association between a deubiquitinating enzyme USP7 and insulin receptor substrates (IRSs) followed by proteasomal degradation of IRSs

Abscisic acid (ABA) is one of the most essential phytohormones, and plays an important role in growth and development regulation, as well as in stress responses. The PYR/PYL/RCAR family (PYL for short)—comprised of 14 proteins in Arabidopsis—was recently identified as soluble ABA receptors that function in the perception and transduction of ABA signaling. In this work, the crystal structures of PYL10 were determined in the apo- and ABA-bound states, with respective resolutions of 3.0 and 2.7 Å. Surprisingly, a closed CL2 conformation was observed in the apo–PYL10 structure, which was different from a previously reported open CL2 conformation. A putative two-conformation dynamical equilibrium model was proposed to explain PYL10’s constitutive binding to PP2Cs in the apo-state and its increased PP2C binding ability in the ABA-bound state.     

p19ARF-induced p53-independent apoptosis largely occurs through BAX

Combined disruption of the ARF gene and the p53 gene causes mouse predisposition to tumors of a wider variety and at a higher frequency than disruption of the p53 gene, indicating that the ARF gene has p53-independent anti-tumor function in addition to p53-dependent function. Coincidentally with this notion, ectopic expression of the p19(ARF) induces apoptosis for wild-type mouse embryo fibroblasts which have been immortalized by introduction of the SV40 virus genome (SV40-MEFs). The protein expression levels of p53, p21(Cip1), and Bax were not upregulated by ectopic expression of p19(ARF) in SV40-MEFs, indicating that expression of p19(ARF) induced apoptosis through p53-independent pathways in this system. Ectopic expression of p19(ARF) induced prominent apoptosis even in SV40-Bak-/-MEFs. In contrast, expression of p19(ARF) induced only a very low grade of apoptosis in Bax-/- or Bax-/-/Bak-/-SV40-MEFs. Remarkable attenuation of p19(ARF)-induced apoptosis by disruption of the Bax gene thus leads to the conclusion that Bax plays a major role in p53-independent apoptosis induced by p19(ARF).     

Rapid analysis of protein interactions: On-chip micropurification of recombinant protein expressed in Esherichia coli

We describe a rapid analysis of interactions between antibodies and a recombinant protein present in total cell lysates. Using a surface plasmon resonance biosensor, a low concentration of glutathione-S-transferase (GST) fused protein expressed in small scale Esherichia coli culture was purified on an anti-GST antibody immobilized sensor chip. The 'on-chip purification' was verified using matrix-assisted laser desorption/ionization-time of flight mass spectrometry by measuring the molecular masses of recombinant proteins purified on the sensor chip. The specific binding of monoclonal antibodies for the on-chip micropurified recombinant proteins can then be monitored, thus enabling kinetic analysis and epitope mapping of the bound antibodies. This approach reduced time, resources and sample consumption by avoiding conventional steps related to concentration and purification.     

Identification and characterization of three novel beta 1,3-N-acetylglucosaminyltransferases structurally related to the beta 1,3-galactosyltransferase family

We have isolated three types of cDNAs encoding novel beta1,3-N-acetylglucosaminyltransferases (designated beta3Gn-T2, -T3, and -T4) from human gastric mucosa and the neuroblastoma cell line SK-N-MC. These enzymes are predicted to be type 2 transmembrane proteins of 397, 372, and 378 amino acids, respectively. They share motifs conserved among members of the beta1,3-galactosyltransferase family and a beta1,3-N-acetylglucosaminyltransferase (designated beta3Gn-T1), but show no structural similarity to another type of beta1,3-N-acetylglucosaminyltransferase (iGnT). Each of the enzymes expressed by insect cells as a secreted protein fused to the FLAG peptide showed beta1,3-N-acetylglucosaminyltransferase activity for type 2 oligosaccharides but not beta1,3-galactosyltransferase activity. These enzymes exhibited different substrate specificity. Transfection of Namalwa KJM-1 cells with beta3Gn-T2, -T3, or -T4 cDNA led to an increase in poly-N-acetyllactosamines recognized by an anti-i-antigen antibody or specific lectins. The expression profiles of these beta3Gn-Ts were different among 35 human tissues. beta3Gn-T2 was ubiquitously expressed, whereas expression of beta3Gn-T3 and -T4 was relatively restricted. beta3Gn-T3 was expressed in colon, jejunum, stomach, esophagus, placenta, and trachea. beta3Gn-T4 was mainly expressed in brain. These results have revealed that several beta1,3-N-acetylglucosaminyltransferases form a family with structural similarity to the beta1,3-galactosyltransferase family. Considering the differences in substrate specificity and distribution, each beta1,3-N-acetylglucosaminyltransferase may play different roles.     

An RNA-interacting protein, SYNCRIP (heterogeneous nuclear ribonuclear protein Q1/NSAP1) is a component of mRNA granule transported with inositol 1,4,5-trisphosphate receptor type 1 mRNA in neuronal dendrites

mRNA transport and local translation in the neuronal dendrite is implicated in the induction of synaptic plasticity. Recently, we cloned an RNA-interacting protein, SYNCRIP (heterogeneous nuclear ribonuclear protein Q1/NSAP1), that is suggested to be important for the stabilization of mRNA. We report here that SYNCRIP is a component of mRNA granules in rat hippocampal neurons. SYNCRIP was mainly found at cell bodies, but punctate expression patterns in the proximal dendrite were also seen. Time-lapse analysis in living neurons revealed that the granules labeled with fluorescent protein-tagged SYNCRIP were transported bi-directionally within the dendrite at approximately 0.05 microm/s. Treatment of neurons with nocodazole significantly inhibited the movement of green fluorescent protein-SYNCRIP-positive granules, indicating that the transport of SYNCRIP-containing granules is dependent on microtubules. The distribution of SYNCRIP-containing granules overlapped with that of dendritic RNAs and elongation factor 1alpha. SYNCRIP was also found to be co-transported with green fluorescent protein-tagged human staufen1 and the 3'-untranslated region of inositol 1,4,5-trisphosphate receptor type 1 mRNA. These results suggest that SYNCRIP is transported within the dendrite as a component of mRNA granules and raise the possibility that mRNA turnover in mRNA granules and the regulation of local protein synthesis in neuronal dendrites may involve SYNCRIP.