A Common Substrate Recognition Mode Conserved between Katanin p60 and VPS4 Governs Microtubule Severing and Membrane Skeleton Reorganization

Katanin p60 (kp60), a microtubule-severing enzyme, plays a key role in cytoskeletal reorganization during various cellular events in an ATP-dependent manner. We show that a single domain isolated from the N terminus of mouse katanin p60 (kp60-NTD) binds to tubulin. The solution structure of kp60-NTD was determined by NMR. Although their sequence similarities were as low as 20%, the structure of kp60-NTD revealed a striking similarity to those of the microtubule interacting and trafficking (MIT) domains, which adopt anti-parallel three-stranded helix bundle. In particular, the arrangement of helices 2 and 3 is well conserved between kp60-NTD and the MIT domain from Vps4, which is a homologous protein that promotes disassembly of the endosomal sorting complexes required for transport III membrane skeleton complex. Mutation studies revealed that the positively charged surface formed by helices 2 and 3 binds tubulin. This binding mode resembles the interaction between the MIT domain of Vps4 and Vps2/CHMP1a, a component of endosomal sorting complexes required for transport III. Our results show that both the molecular architecture and the binding modes are conserved between two AAA-ATPases, kp60 and Vps4. A common mechanism is evolutionarily conserved between two distinct cellular events, one that drives microtubule severing and the other involving membrane skeletal reorganization.     

Inhibition of orexin-induced wakefulness by pyrilamine,an H1 receptor antagonist

Sleep and Biological Rhythms -     

Early-Stage Induction of SWI/SNF Mutations during Esophageal Squamous Cell Carcinogenesis

The SWI/SNF chromatin remodeling complex is frequently inactivated by somatic mutations of its various components in various types of cancers, and also by aberrant DNA methylation. However, its somatic mutations and aberrant methylation in esophageal squamous cell carcinomas (ESCCs) have not been fully analyzed. In this study, we aimed to clarify in ESCC, what components of the SWI/SNF complex have somatic mutations and aberrant methylation, and when somatic mutations of the SWI/SNF complex occur. Deep sequencing of components of the SWI/SNF complex using a bench-top next generation sequencer revealed that eight of 92 ESCCs (8.7%) had 11 somatic mutations of 7 genes, ARID1A, ARID2, ATRX, PBRM1, SMARCA4, SMARCAL1, and SMARCC1. The SMARCA4 mutations were located in the Forkhead (85Ser>Leu) and SNF2 family N-terminal (882Glu>Lys) domains. The PBRM1 mutations were located in a bromodomain (80Asn>Ser) and an HMG-box domain (1,377Glu>Lys). For most mutations, their mutant allele frequency was 31–77% (mean 61%) of the fraction of cancer cells in the same samples, indicating that most of the cancer cells in individual ESCC samples had the SWI/SNF mutations on one allele, when present. In addition, a BeadChip array analysis revealed that a component of the SWI/SNF complex, ACTL6B, had aberrant methylation at its promoter CpG island in 18 of 52 ESCCs (34.6%). These results showed that genetic and epigenetic alterations of the SWI/SNF complex are present in ESCCs, and suggested that genetic alterations are induced at an early stage of esophageal squamous cell carcinogenesis.     

p150glued-Associated Disorders Are Caused by Activation of Intrinsic Apoptotic Pathway

Mutations in p150^glued cause hereditary motor neuropathy with vocal cord paralysis (HMN7B) and Perry syndrome (PS). Here we show that both overexpression of p150^glued mutants and knockdown of endogenous p150^glued induce apoptosis. Overexpression of a p150^glued plasmid containing either a HMN7B or PS mutation resulted in cytoplasmic p150^glued-positive aggregates and was associated with cell death. Cells containing mutant p150^glued aggregates underwent apoptosis that was characterized by an increase in cleaved caspase-3- or Annexin V-positive cells and was attenuated by both zVAD-fmk (a pan-caspase inhibitor) application and caspase-3 siRNA knockdown. In addition, overexpression of mutant p150^glued decreased mitochondrial membrane potentials and increased levels of translocase of the mitochondrial outer membrane (Tom20) protein, indicating accumulation of damaged mitochondria. Importantly, siRNA knockdown of endogenous p150^glued independently induced apoptosis via caspase-8 activation and was not associated with mitochondrial morphological changes. Simultaneous knockdown of endogenous p150^glued and overexpression of mutant p150^glued had additive apoptosis induction effects. These findings suggest that both p150^glued gain-of-toxic-function and loss-of-physiological-function can cause apoptosis and may underlie the pathogenesis of p150^glued-associated disorders.     

Cathepsin E Is a Marker of Gastric Differentiation and Signet-Ring Cell Carcinoma of Stomach: A Novel Suggestion on Gastric Tumorigenesis

Gastric cancer (GC) presents various histological features, though the mechanism underlying its diversity is seldom elucidated. It is mainly classified into well differentiated tubular adenocarcinoma (tub1), moderately differentiated tubular adenocarcinoma (tub2), poorly differentiated adenocarcinoma (por), signet-ring cell carcinoma (sig), mucinous adenocarcinoma (muc), and papillary adenocarcinoma (pap). By screening, we found cathepsin E (CTSE) expresses universally in sig-type, occasionally in por-type, and rarely in tub1/tub2-type GC cell lines. In surgically-resected specimens, CTSE was immunostained in 50/51 sig-type (98.0%), 3/10 tub1-type (30.0%), 7/18 tub2-type (38.9%), 15/26 por-type (57.7%), 4/10 pap-type (40.0%), and 0/3 muc-type (0.0%) GC. In endoscopically-resected specimens, 6/7 sig-type (85.7%), 7/52 tub1-type (13.7%), 5/12 tub2-type (41.7%), 2/7 pap-type (28.6%) GC and 0/6 adenoma (0.0%) expressed CTSE. For non-malignant tissues, CTSE is universally expressed in normal fundic, pyloric, and cardiac glands of stomach, but hardly in other digestive organs. In the precancerous intestinal metaplasia of stomach, CTSE is mostly observed in mixed gastric-and-intestinal type and deficient in solely-intestinal type. CTSE expression is positively correlated with gastric marker MUC5AC (p<0.0001) and negatively correlated with intestinal marker MUC2 (p = 0.0019). For sig-type GC, in both tumors and background mucosa, expression of MUC5AC and CTSE is high whereas that of MUC2 is low, indicating that sig-type GC reflects the features of background mucosa. For gastric adenoma and tub1/tub2-type GC, more undifferentiated tumors tend to show higher expression of CTSE with MUC5AC and lower expression of MUC2 in tumors, but they tend to present lower expression of CTSE, MUC5AC and MUC2 in background mucosa. These suggest that more malignant gastric adenocarcinoma with stronger gastric and weaker intestinal properties tend to arise from background mucosa with decreased both gastric and intestinal features. In conclusion, CTSE is a marker of both gastric differentiation and signet-ring cell carcinoma, which should shed light on the mechanism of gastric tumorigenesis.     

Enzymatic degradation of amylouronate (α-(1 → 4)-linked glucuronan) by α-glucuronidase from Paenibacillus sp. TH501b

Enzymatic degradation of amylouronate (α-(1 → 4)-linked polyglucuronic acid sodium salt, α-(1 → 4)-linked glucuronan), which was prepared from water-soluble starch by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation, was investigated. A bacterial strain TH501b capable of degrading amylouronate was isolated from soil samples collected in the natural environment. Molecular analysis of the 16S rRNA gene showed that TH501b belongs to the genus Paenibacillus. A hydrolytic enzyme responsible for the degradation of amylouronate, amylouronate hydrolase-I (AUH-I), was detected in the cell-free extract of TH501b. AUH-I was purified by four steps of column chromatography and some properties were characterized. The molecular mass of the native AUH-I was estimated to be approximately 115 kDa by size exclusion chromatography (SEC), whereas sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) showed two major bands at 80 kDa and 46 kDa, respectively. The enzyme was most active at pH 6.0–7.0 and 30 °C. The SEC analysis of reaction products revealed that AUH-I liberated glucuronate as a sole product from amylouronate, indicating that AUH-I hydrolyzed amylouronate exolytically, and thus, was classified as α-glucuronidase.     

Stabilizing factor(s) of nitric oxide (NO) synthetase.

We found that the cytosol of rat peritoneal polymorphonuclear neutrophils contains factor(s) that can stabilize an unstable enzyme, nitric oxide synthetase, in the cytosol. This enzyme has been purified to a single protein from the cytosol. Its half-life was 3 hours at 4 degrees C and was prolonged to greater than 24 hours by the stabilizing factor in the cytosol. The molecular weight of the stabilizing factor was greater than 100,000. Its activity was lost by the treatment with heating or alkali for 1 min or with acid for 5 min. It did not adhere to the carboxymethyl or diethylaminoethyl column at neutral pH. This stabilizing factor(s) may play a role in the regulation of the nitric oxide synthetase.     

Patients with idiopathic cardiomyopathy belong to the same mitochondrial DNA gene family of Parkinson's disease and mitochondrial encephalomyopathy

Comparison of total mitochondrial DNA sequences of patients with idiopathic (deleted or hypertrophic) cardiomyopathy with those of patients with Parkinson's disease and mitochondrial encephalomyopathies revealed distinct clustering of point mutations among patients. Furthermore, an inverse relation was found between the total number of base-substitution and life span of the patients. Among point mutations found in each patient, sequentially diverged six clusters consisting of 14, 10, 7, 1, 2, and 3 mutations, respectively, were detected. Five sub-clusters consisting of 2, 2, 11, 1, and 1 mutations, respectively, were detected. From each cluster, the patient's unique mutations were diverged with three types of the mutations specific for the disease. The divergence allowed construction of a phylogenetic tree which clearly indicated that patients with idiopathic cardiomyopathy belong to the same mitochondrial DNA gene family of Parkinson's disease and mitochondrial encephalomyopathies.     

Synaptic morphology in the neostriatum of the rat: Possible serotonergic synapse

Electron-microscopic examination of the striatum of rats 2–3 days after intraperitoneal injection of 10–15 mg/kgp-chloroamphetamine (PCA) indicated two types of terminal degeneration: a light, filamentous degeneration of symmetrical boutons, and a dark, electron-dense degeneration of asymmetrical boutons. The latter was identical to that seen 2–3 days after 6-hydroxydopamine hydrochloride (6-OHDA), and its frequency after PCA was decreased if the animal had been pretreated 10 days previously with 6-OHDA. The same two morphological types of striatal synapses were preferentially labeled by intraventricularly administered [³H]5-HT. No structural specificity in labeling in the striatum was seen after raphe injections of labeled 5-HTP or 5-HT. The probable morphology of striatal dopaminergic, cholinergic, GABAnergic, and serotonergic synapses is discussed, with a review of the pertinent evidence.