Transient gene suppression in a red alga, Cyanidioschyzon merolae 10D

Antisense suppression is a powerful tool to analyze gene function. In this study, we show that antisense RNA suppressed the expression of a target gene in the unicellular red alga, Cyanidioschyzon merolae. In this study, the antisense strand of the catalase gene was cloned and inserted into an expression vector upstream of the GFP gene. This plasmid was introduced into C. merolae cells using a polyethylene glycol-mediated transformation protocol. Using the expression of GFP as a marker of transformed cells, the expression of catalase was examined by immunocytochemistry. Decreased expression of catalase was observed in cells that were transformed with the antisense strand of the catalase gene. These results indicate the utility of this antisense suppression system.     

Simultaneous monitoring of excitatory postsynaptic potentials and extracellular L-glutamate in mouse hippocampal slices

Simultaneous monitoring of amperometric currents at a glass capillary sensor based on recombinant GluOx and field excitatory postsynaptic potentials (fEPSPs) were performed in region CA1 of mouse hippocampal slices. A transient increase in the glutamate current relative to the basal one at control stimulation (0.052Hz) was evoked by stimulation at 2 Hz for 2 min. The magnitude of the glutamate current was dependent on the intensity (current) of a 2 Hz stimulus and reflected the slope of the fEPSP. The in situ calibration of the L-glutamate sensor revealed that the extracellular concentration of L-glutamate released by 2 Hz stimulation before tetanus is in the range from 0.8 to 2.2 μM and it is enhanced after tetanic stimulation. The L-glutamate level at a test stimulus (0.052 Hz) was estimated to be 32 nM. The recombinant GluOx-based sensor exhibited weak responses to glutamine above 300 μM and L-aspartic acid above 200 μM. The potential use of a glass capillary sensor in combination with fEPSP measurements for electrophysiological study is discussed.     

Involvement of Rho/Rho kinase pathway in regulation of apoptosis in rat hepatic stellate cells

Hepatic stellate cells (HSCs) play a central role in the development of hepatic fibrosis. Recent evidence has revealed that HSCs also play a role in its resolution, where HSC apoptosis was determined. Moreover, induction of HSC apoptosis caused a reduction of experimental hepatic fibrosis in rats. Thus knowing the mechanism of HSC apoptosis might be important to clarify the pathophysiology and establish the therapeutic strategy for hepatic fibrosis. In HSCs, Rho and Rho kinase are known to regulate contraction, migration, and proliferation with modulation of cell morphology. Controversy exists as to the participation of Rho and Rho kinase on cell survival, and little is known regarding this matter in HSCs. In this study, we directed our focus on the role of the Rho pathway in the regulation of HSC survival. C3, an inhibitor of Rho, increased histone-associated DNA fragmentation and caspase 3 activity with enhanced condensation of nuclear chromatin in rat cultured HSCs. Moreover, Y-27632, an inhibitor of Rho kinase, had the same effects, suggesting that inhibition of the Rho/Rho kinase pathway causes HSC apoptosis. On the other hand, lysophosphatidic acid, which stimulates the Rho/Rho kinase pathway, decreased histone-associated DNA fragmentation in HSCs. Inhibition of the Rho/Rho kinase pathway did not affect p53, Bcl-2, or Bax levels in HSCs. Thus we concluded that the Rho/Rho kinase pathway may play a role in the regulation of HSC survival.     

Controlling cancer through the autotaxin-lysophosphatidic acid receptor axis

LPA (lysophosphatidic acid, 1-acyl-2-hydroxy-sn-glycero-3-phosphate), is a growth factor-like lipid mediator that regulates many cellular functions, many of which are unique to malignantly transformed cells. The simple chemical structure of LPA and its profound effects in cancer cells has attracted the attention of the cancer therapeutics field and drives the development of therapeutics based on the LPA scaffold. In biological fluids, LPA is generated by ATX (autotaxin), a lysophospholipase D that cleaves the choline/serine headgroup from lysophosphatidylcholine and lysophosphatidylserine to generate LPA. In the present article, we review some of the key findings that make the ATX-LPA signalling axis an emerging target for cancer therapy.     

Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells

Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃) has anti-cancer activity in several colon cancers. 1α,25(OH)₂D₃ induces the epithelial differentiation of SW480 colon cancer cells expressing VDR (SW480-ADH) by upregulating E-cadherin expression; however, its precise mechanism remains unknown. We found that phosphatidylinositol-5-phosphate 4-kinase type II beta (PIPKIIβ) but not PIPKIIα is required for VDR-mediated E-cadherin induction in SW480-ADH cells. The syntenin-2 postsynaptic density protein/disc large/zona occludens (PDZ) domain and pleckstrin homology domain of phospholipase C-delta1 (PLCδ1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLCδ1 PHD inhibited 1α,25(OH)₂D₃-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P₂ production mediates E-cadherin expression through PIPKIIβ in a VDR-dependent manner. PIPKIIβ is also involved in the suppression of the cell motility induced by 1α,25(OH)₂D₃. These results indicate that PIPKIIβ-mediated PI(4,5)P₂ signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.     

Alterations in Detergent-Resistant Plasma Membrane Microdomains in Arabidopsis thaliana During Cold Acclimation

Microdomains in the plasma membrane (PM) have been proposedto be involved in many important cellular events in plant cells.To understand the role of PM microdomains in plant cold acclimation,we isolated the microdomains as detergent-resistant plasma membranefractions (DRMs) from Arabidopsis seedlings and compared lipidand protein compositions before and after cold acclimation.The DRM was enriched in sterols and glucocerebrosides, and theproportion of free sterols in the DRM increased after cold acclimation.The protein-to-lipid ratio in the DRM was greater than thatin the total PM fraction. The protein amount recovered in DRMsdecreased gradually during cold acclimation. Cold acclimationfurther resulted in quantitative changes in DRM protein profiles.Subsequent mass spectrometry and Western blot analyses revealedthat P-type H⁺-ATPases, aquaporins and endocytosis-related proteinsincreased and, conversely, tubulins, actins and V-type H⁺-ATPasesubunits decreased in DRMs during cold acclimation. Functionalcategorization of cold-responsive proteins in DRMs suggeststhat plant PM microdomains function as platforms of membranetransport, membrane trafficking and cytoskeleton interaction.These comprehensive changes in microdomains may be associatedwith cold acclimation of Arabidopsis.     

Characterization of bacterial-type phospho<Emphasis Type="Italic">enol</Emphasis>pyruvate carboxylase expressed in male gametophyte of higher plants

Background  

Phosphoenolpyruvate carboxylase (PEPC) is a critical enzyme catalyzing the β-carboxylation of phosphoenolpyruvate (PEP) to oxaloacetate, a tricarboxylic acid (TCA) cycle intermediate. PEPC typically exists as a Class-1 PEPC homotetramer composed of plant-type PEPC (PTPC) polypeptides, and two of the subunits were reported to be monoubiquitinated in germinating castor oil seeds. By the large-scale purification of ubiquitin (Ub)-related proteins from lily anther, two types of PEPCs, bacterial-type PEPC (BTPC) and plant-type PEPC (PTPC), were identified in our study as candidate Ub-related proteins. Until now, there has been no information about the properties of the PEPCs expressed in male reproductive tissues of higher plants.     

Epstein-barr virus (EBV) nuclear protein 2-induced disruption of EBV latency in the Burkitt's lymphoma cell line Akata: analysis by tetracycline-regulated expression

The Burkitt's lymphoma (BL) cell line Akata retains the latency I program of Epstein-Barr virus (EBV) gene expression and cross-linking of its surface immunoglobulin G (IgG) by antibodies results in activation of viral replication. When EBV nuclear antigen 2 (EBNA2) was artificially expressed by a constitutive expression vector, the Cp EBNA promoter remained inactive and accordingly the latency III program was not induced. In contrast, expression of LMP2A and activity of the Fp lytic promoter were activated. Consistent with this Fp activity, the rate of spontaneous activation of the EBV replicative cycle was increased significantly, suggesting the possibility that EBNA2 can induce EBV replication. The efficiency of anti-IgG-induced activation of the viral replication was reduced in Akata cells expressing EBNA2. To obtain more direct evidence for EBNA2-induced activation of the EBV replicative cycle, this protein was next expressed by a tetracycline-regulated expression system. EBNA2 was undetectable with low doses (<0.5 microgram/ml) of tetracycline, while its expression was rapidly induced after removal of the antibiotic. This induced expression of EBNA2 was immediately followed by expression of EBV replicative cycle proteins in up to 50% of the cells, as shown by indirect immunofluorescence and immunoblot analysis. These results suggest an unexpected potential of EBNA2 to disrupt EBV latency and to activate viral replication.