Actomyosin-Mediated Tension Orchestrates Uncoupled Respiration in Adipose Tissues

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Aberrant alternative splicing of RHOA is associated with loss of its expression and activity in diffuse-type gastric carcinoma cells

RhoA is a member of Rho family small GTPases that regulates diverse cellular functions. Recent large-scale sequencing studies have identified recurrent somatic mutations of RHOA in diffuse-type gastric carcinoma (DGC), indicating that RHOA is a driver of DGC. In this study, we investigated the possible abnormalities of RHOA in a panel of gastric carcinoma (GC) cell lines. Pulldown assay and immunoblot analysis showed that the activity and expression of RhoA were detectable in all GC cell lines tested, except for two DGC cell lines, HSC-59 and GSU. RHOA coding region sequencing revealed that aberrant alternative splicing of RHOA occurred in these cell lines. Quantitative real-time PCR analysis showed that the expression of wild-type RHOA was nearly undetectable, whereas splicing variants were almost exclusively expressed in HSC-59 and GSU cell lines. However, the expression levels of RHOA splicing variants were very low and the corresponding proteins were not detected by immunoblotting. Moreover, the splicing isoforms of RhoA protein were neither efficiently expressed nor activated even if ectopically expressed in cells. These results indicate that aberrant alternative splicing of RHOA results in the loss of its activity and expression in DGC cells.     

Species composition and arthropod pest feeding of phytoseiid mites in a Japanese pear greenhouse

A population survey of spider mites and phytoseiid mites was conducted on Japanese pear leaves in a greenhouse. For the survey, the method to estimate phytoseiid mite species composition using quantitative sequencing was modified to be applicable for phytoseiid mite species inhabiting in the greenhouse. Results show the dominant appearance of Neoseiulus californicus (McGregor), Neoseiulus womersleyi (Schicha), and Neoseiulus makuwa (Ehara) from the end of June to late September and their contribution in spider mite control. PCR-based method to detect the ribosomal internal transcribed spacer (ITS) sequences of spider mites from phytoseiid mites was developed. The method shows sensitivity to detect the ITS sequences of Tetranychus urticae Koch from single N. californicus adult at 168 h after ingestion of the spider mite. PCR-based method to detect the cytochrome c oxidase subunit I sequences of several arthropod pests belonging to Hemiptera, Thysanoptera, and Acari from phytoseiid mites was also developed. Results show that phytoseiid mites prey on Eriophyes chibaensis (Kadono) and Aphis gossypii (Glover), in addition to spider mites.     

Involvement of nitric oxide/reactive oxygen species signaling via 8-nitro-cGMP formation in 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in PC12 cells and rat cerebellar granule neurons

To investigate the role of nitric oxide (NO)/reactive oxygen species (ROS) redox signaling in Parkinson's disease-like neurotoxicity, we used 1-methyl-4-phenylpyridinium (MPP⁺) treatment (a model of Parkinson's disease). We show that MPP⁺-induced neurotoxicity was dependent on ROS from neuronal NO synthase (nNOS) in nNOS-expressing PC12?cells (NPC12?cells) and rat cerebellar granule neurons (CGNs). Following MPP⁺ treatment, we found production of 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), a second messenger in the NO/ROS redox signaling pathway, in NPC12?cells and rat CGNs, that subsequently induced S-guanylation and activation of H-Ras. Additionally, following MPP⁺ treatment, extracellular signal-related kinase (ERK) phosphorylation was enhanced. Treatment with a mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor attenuated MPP⁺-induced ERK phosphorylation and neurotoxicity. In conclusion, we demonstrate for the first time that NO/ROS redox signaling via 8-nitro-cGMP is involved in MPP⁺-induced neurotoxicity and that 8-nitro-cGMP activates H-Ras/ERK signaling. Our results indicate a novel mechanism underlying MPP⁺-induced neurotoxicity, and therefore contribute novel insights to the mechanisms underlying Parkinson's disease.     

Stimulation of RNA Synthesis in Isolated Nuclei by Auxin-Binding Proteins-I and -II

The auxin-binding proteins (ABP-I and ABP-II) purified frometiolated mung bean seedlings stimulated RNA synthesis in isolatednuclei both in the presence and absence of (NH₄)₂SO₄. In theabsence of (NH₄)₂SO₄, maximum stimulation of RNA synthesis occurredat 100 µg ABP-I (25%) and 300 µg ABP-II (60%), whereasin the presence of 50 mM (NH₄)₂SO₄ maximum stimulation occurredat 60 µg ABP-I (10%) and 100 µg ABP-II (40%). Thesestimulatory effects on RNA synthesis by ABP-I and ABP-II werecompletely abolished by the addition of -amanitin (4 µg/0.5ml reaction mixture). IAA had no effect on the stimulation ofRNA synthesis by ABP-I and ABP-II. (Received September 30, 1985; Accepted March 5, 1986)     

Purification and Properties of an Extracellular Endo-1,4-r{beta}-Glucanase from Suspension-Cultured Poplar Cells

An endo-1,4-rß-glucanase (EC 3.2.1.4 [EC] ) was purifiedto apparent homogeneity from the culture medium of poplar (Populusalba L.) cells by sequential anion-exchange, hydrophobic, andgel-filtration chromatography. The preparation of extracellularrß-glucanase was homogeneous on SDS-polyacrylamidegel electrophoresis (PAGE) and native PAGE. The molecular weight,as determined by SDS-PAGE was 50,000, whereas that determinedby gel filtration was 40,000. The isoelectric point (pI) was5.5. The purified enzyme catalyzed the endohydrolysis of carboxy-methylcellulosewith a pH optimum of 6.0 and a k_m of 1.0 mg ml^–1. Theenzyme specifically cleaved the 1,4-rß-glucosyl linkagesof carboxymethylcellulose, swollen cellulose, lichenan and xyloglucan,although the last was hydrolyzed more slowly than the othertested substrates. The activity of the endo-1,4-rß-glucanaseincreased up to the early stage of the mid-logarithmic phaseof growth and then decreased rapidly, suggesting that the rß-glucanaseis induced before cell development. (Received April 28, 1993; Accepted July 19, 1993)     

Purification and Properties of an Auxin-Binding Protein from the Shoot Apex of Peach Tree

A soluble auxin-binding protein was purified from the shootapices of peach trees by chromatography on columns of CM-Toyopearl,Sephacryl S-200, 2,4-D-linked-Sepharose 4B and ConA-Sepharose.The molecular mass of the purified protein was estimated tobe about 100 kDa. After electrophoresis on a denaturing gel,the protein gave a single band with a molecular mass of 20 kDa.From Scatchard analyses, the dissociation constant for 2,4-Dwas calculated to be 4.1 10^–5 M and the specific bindingof 2,4-D at saturating concentration was 42 nmol (mg protein)^–1.The binding of [¹⁴C]-2,4-D to the protein was reversible andwas inhibited by IAA, 1-naphthylacetic acid and p-chlorophenoxyisobutyricacid. (Received June 25, 1992; Accepted October 20, 1992)     

Partial Purification of a Soluble Gibberellin-Binding Protein from Mung Bean Hypocotyls

A soluble binding protein specific for GA₄, GA₇ and GA₉ waspartially purified from mung bean hypocotyls, and its characteristicswere examined. Affinity chromatography using immobilized GA₃coupled to Sepharose 4B via the C-7 carboxyl group was veryeffective for purification of the protein. The molecular weightof the protein in its native state was estimated to be 150–200kDa by gel-permeation chromatography. This protein may be aheterooligomer consisting of two subunits (23 kDa and 35 kDa).The optimum pH for binding of GA₄ to the protein was around6.0 and the apparent dissociation constant (K_d) was 310^-7 M. (Received April 24, 1992; Accepted December 16, 1992)     

Detection of hypoxia in the pulmonary tissues of Xenopus laevis over repeated dives

The oxygen environment in African clawed frogs (Xenopus laevis) continuously changes during their development, which involves a rapid increase in the body size, metamorphosis, and transition to adulthood. Nevertheless, there are limited reports on experimental models that are available for studying fluctuations in the oxygen environment in X. laevis. Thus, this study aimed to develop an experimental model on intermittent hypoxia in X. laevis and evaluate hypoxia and oxidative stress in the same. X. laevis were submerged in water with a dissolved oxygen concentration of 2 mg/L for 30 min; they were then removed from the water and allowed to freely absorb oxygen for 5 min. Immunostaining of pimonidazole-containing frozen tissue sections of the lung and liver using anti-pimonidazole antibodies as the hypoxia probes revealed that more than 95% of the submerged X. laevis cells were pimonidazole positive, providing direct evidence of tissue hypoxia. When the amount of oxidative stress in the lungs and liver was evaluated in terms of the amount of lipid peroxides, the diving group showed a 2.08-fold and 3.20-fold increase over the normal group, respectively. Following hypoxia exposure, the dry-to-wet weight ratios of the lung tissues was 1.27 times higher (p < .05), while the liver tissues was 1.06 times higher (although not significant). Thus, the degree of damage depended on the tissues affected. In the future, we believe that this model will be a promising option for analyzing the physiological responses of X. laevis to hypoxia and oxidative stress.     

Isolation of a Drosophila Gene Encoding a Head-Specific Guanylyl Cyclase

Abstract: We have isolated and characterized a new guanylyl cyclase gene ( dgcl) in Drosophila. The deduced amino acid sequence (683 amino acids) most closely resembled the mammalian solubletype guanylyl cyclase α subunit. The cyclase catalytic domain was highly conserved between the mammalian and Drosophila guanylyl cyclases. The dgcl mRNA was detected in wild-type heads but not in bodies, and its level was reduced in the mutant eyes absent (eya) , indicating that dgcl is preferentially expressed in the CNS and in the eye. The enriched distribution in the eye suggests that dgcl may have a role in phototransduction.