Inactivation of phosphorylase phosphatase by a factor from rabbit liver and its chemical characterization as glutathione disulfide.

A factor inactivating phosphorylase phosphatase was isolated from rabbit liver. The isolation procedure consisted of heat treatment at 85 degrees C, extraction with n-butyl alcohol, and chromatography on Dowex 1 and DEAE-cellulose columns. The purified factor was different from the known protein inhibitors and was shown to be tripeptide composed of equimolar amounts of glutamic acid, cysteine, and glycine. The NH2-terminal and COOH-terminal amino acids were determined as glutamic acid and glycine, respectively. The factor was finally identified as glutathione disulfide by high voltage paper electrophoresis, paper chromatography, and liquid column chromatography using an amino acid analyzer. Addition of the purified factor or glutathione disulfide converted phosphorylase phosphatase to a stable, less active enzyme species, the extent of conversion depending on the amount added. The inactivated phosphatase was completely reactivated by addition of both glutathione (or 2-mercaptoethanol) and Mn2+ and partially reactivated by adding glutathione alone. Injection of glutathione disulfide into the portal vein of rabbits caused a rapid increase in phosphorylase alpha activity in the liver. These results suggest that glutathione disulfide is involved in regulation of phosphorylase activity in vivo, by causing inactivation of phosphorylase phosphatase in the liver.     

Rapidly labelled RNA from conidiating mycelia of Aspergillus niger

A rapidly labelled RNA species was synthesized during the conidiationin Aspergillus niger under the non-growing condition. Hybridizationcompetition experiments between RNA species isolated duringthe conidiophore- and conidia-forming periods showed qualitativedifferences. ¹Present address: Division of Environmental Physiology, Institutefor Agricultural Research, Tohoku University, Sendai, Japan. (Received April 24, 1976; )     

Massively Parallel DNA Sequencing Successfully Identifies New Causative Mutations in Deafness Genes in Patients with Cochlear Implantation and EAS

Genetic factors, the most common etiology in severe to profound hearing loss, are one of the key determinants of Cochlear Implantation (CI) and Electric Acoustic Stimulation (EAS) outcomes. Satisfactory auditory performance after receiving a CI/EAS in patients with certain deafness gene mutations indicates that genetic testing would be helpful in predicting CI/EAS outcomes and deciding treatment choices. However, because of the extreme genetic heterogeneity of deafness, clinical application of genetic information still entails difficulties. Target exon sequencing using massively parallel DNA sequencing is a new powerful strategy to discover rare causative genes in Mendelian disorders such as deafness. We used massive sequencing of the exons of 58 target candidate genes to analyze 8 (4 early-onset, 4 late-onset) Japanese CI/EAS patients, who did not have mutations in commonly found genes including GJB2, SLC26A4, or mitochondrial 1555A>G or 3243A>G mutations. We successfully identified four rare causative mutations in the MYO15A, TECTA, TMPRSS3, and ACTG1 genes in four patients who showed relatively good auditory performance with CI including EAS, suggesting that genetic testing may be able to predict the performance after implantation.     

Proteomic analysis and in vitro developmental toxicity tests for mechanism-based safety evaluation of chemicals

Mechanism-based safety evaluation and reduction of animal use are important issues in recent developmental toxicology. In vitro developmental toxicity tests with proteomic analysis are the most promising solution to these issues. Groebe et al. systematically applied proteomic analysis to the embryonic stem cell test, a validated in vitro developmental toxicity test, and found protein-expression changes induced by model test chemicals selected from various categories of toxicity. Cluster analysis of all the proteins with expression changes classified the test chemicals into two groups: highly embryotoxic chemicals and non- or weakly embryotoxic chemicals. In addition, some protein biomarker candidates that were known to be involved in normal development were identified. Although further mechanistic investigations are needed, the use of in vitro developmental toxicity tests with proteomic analysis will contribute to mechanism-based safety evaluation with minimal use of animals.     

Structural basis for ESCRT-III CHMP3 recruitment of AMSH

Endosomal sorting complexes required for transport (ESCRT) recognize ubiquitinated cargo and catalyze diverse budding processes including multivesicular body biogenesis, enveloped virus egress, and cytokinesis. We present the crystal structure of an N-terminal fragment of the deubiquitinating enzyme AMSH (AMSHΔC) in complex with the C-terminal region of ESCRT-III CHMP3 (CHMP3ΔN). AMSHΔC folds into an elongated 90?? long helical assembly that includes an unusual MIT domain. CHMP3ΔN is unstructured in solution and helical in complex with AMSHΔC, revealing a novel MIT domain interacting motif (MIM) that does not overlap with the CHMP1-AMSH binding site. ITC and SPR measurements demonstrate an unusual high-affinity MIM-MIT interaction. Structural analysis suggests a regulatory role for the N-terminal helical segment of AMSHΔC and its destabilization leads to a loss of function during HIV-1 budding. Our results indicate a tight coupling of ESCRT-III CHMP3 and AMSH functions and provide insight into the regulation of ESCRT-III.     

Comparison of recombinant protein expression in a baculovirus system in insect cells (Sf9) and silkworm

Using a hybrid baculovirus system, we compared the expression of 45 recombinant proteins from six categories using two models: silkworm (larvae and pupae) and an Sf9 cell line. A total of 45 proteins were successfully expressed; preparation of hybrid baculovirus was unsuccessful for one protein, and two proteins were not expressed. A similar pattern of expression was seen in both silkworm and Sf9 cells, with double and multiple bands found in immunoblotting of the precipitate of both hosts. Degraded proteins were seen only in the silkworm system (particularly in the larvae). Production was more efficient in silkworms; a single silkworm produced about 70 times more protein than 10(6) Sf9 cells in 2 ml of culture medium.     

Gravity sensing by Escherichia coli

We investigated the growth and protein profile of Escherichia coli under various gravity strengths to determine the effects of hypergravity on biochemical reactions. E. coli grows at less than 7,500 g without inhibition. Hypergravity induced OmpW and Antigen 43. Changes in gravity strength altered the expression levels of these proteins. This suggests that hypergravity regulates gene expression in bacteria.     

Genomic characterization of Ralstonia solanacearum phage phiRSB1, a T7-like wide-host-range phage

RSΒ1 is a wide-host-range, T7-like bacteriophage that infects and efficiently lyses the phytopathogenic bacterium Ralstonia solanacearum. The RSB1 genome comprises 43,079 bp of double-stranded DNA (61.7% G+C) with 325-bp terminal repeats and contains 47 open reading frames. Strong activity of tandem early promoters and wide specificity of phage promoters of RSB1 were demonstrated.     

Active hippocampal networks undergo spontaneous synaptic modification

The brain is self-writable; as the brain voluntarily adapts itself to a changing environment, the neural circuitry rearranges its functional connectivity by referring to its own activity. How the internal activity modifies synaptic weights is largely unknown, however. Here we report that spontaneous activity causes complex reorganization of synaptic connectivity without any external (or artificial) stimuli. Under physiologically relevant ionic conditions, CA3 pyramidal cells in hippocampal slices displayed spontaneous spikes with bistable slow oscillations of membrane potential, alternating between the so-called UP and DOWN states. The generation of slow oscillations did not require fast synaptic transmission, but their patterns were coordinated by local circuit activity. In the course of generating spontaneous activity, individual neurons acquired bidirectional long-lasting synaptic modification. The spontaneous synaptic plasticity depended on a rise in intracellular calcium concentrations of postsynaptic cells, but not on NMDA receptor activity. The direction and amount of the plasticity varied depending on slow oscillation patterns and synapse locations, and thus, they were diverse in a network. Once this global synaptic refinement occurred, the same neurons now displayed different patterns of spontaneous activity, which in turn exhibited different levels of synaptic plasticity. Thus, active networks continuously update their internal states through ongoing synaptic plasticity. With computational simulations, we suggest that with this slow oscillation-induced plasticity, a recurrent network converges on a more specific state, compared to that with spike timing-dependent plasticity alone.