Oxidative Degradation of Squalene by Arthrobacter Species

An organism isolated from soil and identified as Arthrobacter sp. was studied for its squalene degradation. The degradation product from squalene, which accumulated in the culture broth, was isolated and identified as trans-geranylacetone by mass spectrometry, gas chromatography, infrared spectrometry, and nuclear magnetic resonance spectrometry. Addition of a high concentration of K₂HPO₄ to the culture medium resulted in accumulation of fairly large amounts of carboxylic acids in addition to geranylacetone. These carboxylic acids were identified as isovaleric, β,β′-dimethylacrylic, geranic, and (+)-(R)-citronellic acids. Among these acids, α,β-saturated carboxylic acids were found to be predominant in quantity.     

Fbxo45 Forms a Novel Ubiquitin Ligase Complex and Is Required for Neuronal Development

Fbxo45 is an F-box protein that is restricted to the nervous system. Unlike other F-box proteins, Fbxo45 was found not to form an SCF complex as a result of an amino acid substitution in the consensus sequence for Cul1 binding. Proteomics analysis revealed that Fbxo45 specifically associates with PAM (protein associated with Myc), a RING finger-type ubiquitin ligase. Mice deficient in Fbxo45 were generated and found to die soon after birth as a result of respiratory distress. Fbxo45^−/− embryos show abnormal innervation of the diaphragm, impaired synapse formation at neuromuscular junctions, and aberrant development of axon fiber tracts in the brain. Similar defects are also observed in mice lacking Phr1 (mouse ortholog of PAM), suggesting that Fbxo45 and Phr1 function in the same pathway. In addition, neuronal migration was impaired in Fbxo45^−/− mice. These results suggest that Fbxo45 forms a novel Fbxo45-PAM ubiquitin ligase complex that plays an important role in neural development.Ubiquitin-dependent proteolysis is indispensable for various biological processes (3, 40). Protein ubiquitylation is mediated by several enzymes that act in concert, with a ubiquitin ligase (E3) playing a key role in substrate recognition (14). E3 enzymes contain specific structural motifs that mediate recruitment of a ubiquitin-conjugating enzyme (E2), with these motifs including HECT, RING finger, U-box, and PHD finger domains (30). The SCF complex consists of Skp1 (adaptor subunit), Cul1 (scaffold subunit), an F-box protein (substrate recognition subunit), and Rbx1 (also known as Roc1 or Hrt1; RING finger-containing subunit). Whereas Skp1, Cul1, and Rbx1 are common to all SCF complexes, the F-box protein is variable (with ∼70 such proteins having been identified in humans) and confers substrate specificity.Fbxo45 is an F-box protein that was originally isolated as an estrogen-induced protein (47). Human and mouse Fbxo45 genes comprise three exons and possess several consensus binding sequences for the estrogen receptor in the promoter region. Fbxo45 mRNA is rapidly induced on exposure of MCF-7 cells to 17β-estradiol (47). FSN-1, the Caenorhabditis elegans ortholog of Fbxo45, binds to RPM-1 (regulator of presynaptic morphology 1) together with CUL-1 and SKR-1, the C. elegans orthologs of mammalian Cul1 and Skp1, respectively (21, 46). RPM-1 belongs to an evolutionarily conserved family of proteins (the PHR family) that include Highwire (HIW) (Drosophila melanogaster), Esrom (Danio rerio), Phr1 (Mus musculus), and protein associated with Myc (PAM) (Homo sapiens), each of which contains a RING-finger domain that is required for its E3 activity (7, 20, 21, 27, 44). Complete loss of function of fsn-1 in C. elegans results in defects that are characterized by the simultaneous presence of overdeveloped and underdeveloped neuromuscular junctions (NMJs) and which are similar to, but not as pronounced as, those observed in rpm-1^−/− mutants. These genetic findings support the notion that the functions of FSN-1 and RPM-1 are partially overlapping (21).Although PHR family members interact with many potential targets (11, 24, 26, 31), genetic data have shown that one key substrate of RPM-1 and HIW is the mitogen-activated protein kinase kinase kinase known as DLK (dual leucine zipper kinase) in C. elegans and known as Wallenda in D. melanogaster, respectively. The abundance of this kinase is increased in rpm-1 or hiw mutants, and synaptic defects in the mutant worms and flies are suppressed by a loss of DLK or Wallenda. Furthermore, an increase in the level of DLK or Wallenda is sufficient to phenocopy the synaptic defects of the rpm-1 or hiw mutants (5, 27). PAM has also been shown to catalyze the ubiquitylation of tuberin (TSC2) and to regulate signaling by mTOR (mammalian target of rapamycin) in human cells (12).To elucidate the physiological functions of Fbxo45 in mammals, we have now generated mice deficient in this protein. Analysis of the mutant mice revealed that Fbxo45 is required for normal neuromuscular synaptogenesis, axon pathfinding, and neuronal migration. Moreover, we found that Fbxo45 does not form an authentic SCF complex as a result of an amino acid substitution in the F-box domain, and we identified PAM as a binding partner of Fbxo45. The phenotype of Fbxo45^−/− mice was found to be similar to that of Phr1^−/− mice, especially with regard to the defects of neuromuscular synapse formation and of axon navigation. Our results indicate that three fundamental processes of neural development— axonal projection, synapse formation, and neuronal migration—may be linked by a common machinery consisting of the Fbxo45-Phr1 complex.     

Differences in liver homogenates from Donryu, Fischer, Sprague-Dawley and Wistar strains of rat in the drug-metabolizing enzyme assay and the salmonella/hepatic S9 activation test

Comparison studies for detecting differences between liver microsome and S9 preparations from 4 strains (Donryu, Fischer, Sprague-Dawley, Wistar) of young male rats were carried out with pretreatment of the animals by inducers such as PCBs and PB plus 5,6-BF. Each microsome fraction was assayed for the enzymic activity of metabolism of model substrates such as aniline, benzophetamine, BP, DMN and 7-ethoxycoumarin. The hepatic S9 sample was also compared, as regards its metabolizing ability to activate 9 pre-mutagens (2AA, AAF, o-AAT, BP, DAB, DMBA, DMN, m-PDA, quinoline) to directly acting mutagens in the Salmonella/hepatic S9 activation test by using TA98, TA100 and TA1537 strains with or without cytochrome P450 inhibitors (SKF-525A, metyrapone, 7,8-benzoflavone).In the enzymic assay with PCBs-induced microsomes, BP hydroxylation revealed a strain-specific difference: the microsomes from Fischer and Wistar rats were more effective for metabolizing BP than those from the other strains of rat. The effect of induction by PB plus 5,6-BF for Fischer rats showed relatively higher enzymic activity in the same induction group. Other microsomes prepared from rats with and without induction by PB plus 5,6-BF did not show a clear-cut strain dependency in the enzymic activities assayed.In the mutation experiments with hepatic S9 samples, the examination of DAB and quinoline revealed a marked strain difference when S9 samples prepared from PCBs-pretreated and PB-plus-5,6-BF-induced rats were used: the S9 sample from Fischer rats was available for activating the two pre-mutagens to directly acting mutagens. No marked difference in the metabolic activation of the remaining 7-pre-mutagens was observed on other S9 preparations.In examinations of mutagenicity activities with the use of three inhibitors, the two S9 preparations made with the two induction methods showed inhibition profiles closely similar to each other. However, there were minor differences in the profiles by these inhibitors.From these findings it was concluded that Fischer rat-liver S9 is useful for detecting mutagens in the metabolic activation test, when induction by PB plus 5,6-BF was used in the Ames Salmonella test.     

IgM-type GM-CSF autoantibody is etiologically a bystander but associated with IgG-type autoantibody production in autoimmune pulmonary alveolar proteinosis

The granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibody (GMAb) is the causative agent underlying autoimmune pulmonary alveolar proteinosis (aPAP). It consists primarily of the IgG isotype. At present, information on other isotypes of the autoantibody is limited. We detected serum the IgM isotype of GMAb (IgM-GMAb) in more than 80% of patients with aPAP and 22% of healthy subjects, suggesting that a continuous antigen pressure may be present in most patients. Levels of the IgM isotype were weakly correlated with IgG-GMAb levels but not IgA-GMAb, suggesting that its production may be associated with that of IgG-GMAb. The mean binding avidity to GM-CSF of the IgM isotype was 100-fold lower than the IgG-GMAb isotype, whereas the IC(50) value for neutralizing capacity was 20,000-fold higher than that of IgG-GMAb, indicating that IgM-GMAb is only a very weak neutralizer of GM-CSF. In bronchoalveolar lavage fluid from nine patients, IgG-GMAb was consistently detected, but IgM-GMAb was under the detection limit in most patients, confirming that IgM-GMAb is functionally a bystander in the pathogenesis of aPAP. It rather may be involved in the mechanism for development of IgG-GMAb in vivo.     

GTDC2 modifies O-mannosylated α-dystroglycan in the endoplasmic reticulum to generate N-acetyl glucosamine epitopes reactive with CTD110.6 antibody

Hypoglycosylation is a common characteristic of dystroglycanopathy, which is a group of congenital muscular dystrophies. More than ten genes have been implicated in α-dystroglycanopathies that are associated with the defect in the O-mannosylation pathway. One such gene is GTDC2, which was recently reported to encode O-mannose β-1,4-N-acetylglucosaminyltransferase. Here we show that GTDC2 generates CTD110.6 antibody-reactive N-acetylglucosamine (GlcNAc) epitopes on the O-mannosylated α-dystroglycan (α-DG). Using the antibody, we show that mutations of GTDC2 identified in Walker–Warburg syndrome and alanine-substitution of conserved residues between GTDC2 and EGF domain O-GlcNAc transferase resulted in decreased glycosylation. Moreover, GTDC2-modified GlcNAc epitopes are localized in the endoplasmic reticulum (ER). These data suggested that GTDC2 is a novel glycosyltransferase catalyzing GlcNAcylation of O-mannosylated α-DG in the ER. CTD110.6 antibody may be useful to detect a specific form of GlcNAcylated O-mannose and to analyze defective O-glycosylation in α-dystroglycanopathies.     

miR‐197 induces epithelial–mesenchymal transition in pancreatic cancer cells by targeting p120 catenin

Invasive ductal adenocarcinoma (IDA) of the pancreas manifests poor prognosis due to the early invasion and distant metastasis. In contrast, intraductal papillary mucinous adenoma or carcinoma (IPMA or IPMC) reveals better clinical outcomes. Various molecular mechanisms contribute to these differences but entire picture is still unclear. Recent researches emphasized the important role of miRNA in biological processes including cancer invasion and metastasis. We previously described that miR‐126 is down‐regulated in IDA compared with IPMA or IPMC, and miR‐126 regulates the expression of invasion related molecule disintegrin and metalloproteinase domain‐containing protein 9 (ADAM9). Assessing the difference of miRNA expression profiles of IDA, IPMA, and IPMC, we newly identified miR‐197 as an up‐regulated miRNA specifically in IDA. Expression of miR‐197 in pancreatic cancer cells resulted in the induction of epithelial–mesenchymal transition (EMT) along with the down‐regulation of p120 catenin which is a putative target of miR‐197. Direct interaction between miR‐197 and p120 catenin mRNA sequence was confirmed by 3′UTR assay, and knockdown of p120 catenin recapitulated EMT induction in pancreatic cancer cells. In situ hybridization of miR‐197 and immunohistochemistry of p120 catenin showed mutually exclusive patterns suggesting pivotal role of miR‐197 in the regulation of p120 catenin. This miR‐197/p120 catenin axis could be a novel therapeutic target. J. Cell. Physiol. 228: 1255–1263, 2013. © 2012 Wiley Periodicals, Inc.     

Heparin/heparan sulfate/CD44-v3 enhances cell migration in term placenta-derived immortalized human trophoblast cells

The function of CD44-v3 and heparin/heparan sulfate (HS) signaling was investigated during trophoblast cell migration to identify their role in the renewal of syncytial layer damage caused by increased hemodynamic turbulence in the intervillous space and maintenance of syncytial integrity in pre-eclampsia. We evaluated the effect of heparin/HS/CD44-v3-mediated processes during scratch wound closure in monolayer immortalized human trophoblast cells derived from term placenta (TCL-1 cells). Western blot analysis showed that these cultured human trophoblast cells express the epidermal growth factor receptor and CD44-v3 but do not express syndecan 4. An in vitro scratch wound healing assay showed enhanced migration of trophoblast cells in a dose-dependent manner in the presence of heparin compared with controls when cultured under serum-free conditions. Conversely, an anti-CD44 function-blocking antibody and CD44 siRNA suppressed the migration of trophoblast cells in the presence of heparin in a similar scratch assay. Furthermore, both heparin treatment and in vitro scratch wounding induced the phosphorylation of p21-activated kinase 1 (PAK1), whereas the anti-CD44-v3 antibody suppressed the heparin-induced phosphorylation of PAK1 in trophoblast cells. These results indicate that heparin/HS/CD44-v3-mediated signaling, in the absence of growth factor networks, enhances the direct repair of the damaged trophoblast layer through the migration of trophoblast cells. This renewed cell coverage may lead to the maintenance of syncytiotrophoblast cell function and an associated reduction in pathogenic soluble factors derived from the damaged trophoblast cells.     

Biochemical systematics of Japanese fireflies of the subfamily Luciolinae and their flash communication systems

Japanese fireflies of the subfamily Luciolinae are biochemically analyzed using 13 allozymes, and the phylogenetic relationships obtained from this analysis are compared with their flash communication systems. As a result, the Japanese Luciolinae can be divided into three groups.Hotaria parvula andH. tsushimana together withLuciola yayeyamana andL. kuroiwae from the first group, and they use the same communication system.L. lateralis, Curtos okinawana, andC. costipennis make up the second group, and their communication systems are also the same.L. cruciata makes up the last one, and its communication system is different from the other fireflies of Luciolinae. Therefore, their taxonomical arrangement and communication systems are not congruent. However, the genetic similarity deduced by allozymic analysis of the members of the Japanese Luciolinae is highly consistent with their flash communication systems.     

2-Cyclohexylcarbonylbenzimidazoles as potent,orally available and brain-penetrable opioid receptor-like 1 (ORL1) antagonists

The synthesis and biological evaluation of new potent opioid receptor-like 1 (ORL1) antagonists are presented. Conversion of the thioether linkage of the prototype [It is reported prior to this communication as a consecutive series.: Kobayashi, K.; Kato, T.; Yamamoto, I.; Shimizu, A.; Mizutani, S.; Asai, M.; Kawamoto, H.; Ito, S.; Yoshizumi, T.; Hirayama, M.; Ozaki, S.; Ohta, H.; Okamoto, O. Bioorg. Med. Chem. Lett., in press] to the carbonyl linker effectively reduces susceptibility to P-glycoprotein (P-gp) efflux. This finding led to the identification of 2-cyclohexylcarbonylbenzimizole analogue 7c, which exhibited potent ORL1 activity, excellent selectivity over other receptors and ion channels, and poor susceptibility to P-gp. Compound 7c also showed satisfactory pharmacokinetic profiles and brain penetrability in laboratory animals. Furthermore, 7c showed good in vivo antagonism. Hence, 7c was selected as a clinical candidate for a brain-penetrable ORL1 antagonist.