Lamellipodia nucleation by filopodia depends on integrin occupancy and downstream Rac1 signaling

Time-lapse video-microscopy unambiguously shows that fibroblast filopodia are the scaffold of lamellipodia nucleation that allows anisotropic cell spreading. This process was dissected into elementary stages by monitoring cell adhesion on micropatterned extracellular matrix arrays of various pitches. Adhesion structures are stabilized by contact with the adhesive plots and subsequently converted into lamellipodia-like extensions starting at the filopodia tips. This mechanism progressively leads to full cell spreading. Stable expression of the dominant-negative Rac1 N17 impairs this change in membrane extension mode and stops cell spreading on matrix arrays. Similar expression of the dominant-negative Cdc42 N17 impairs cell spreading on homogenous and structured substrate, suggesting that filopodia extension is a prerequisite for cell spreading in this model. The differential polarity of the nucleation of lamellipodial structures by filopodia on homogenous and structured surfaces starting from the cell body and of filopodia tip, respectively, suggested that this process is triggered by areas that are in contact with extracellular matrix proteins for longer times. Consistent with this view, wild-type cells cannot spread on microarrays made of function blocking or neutral anti-beta 1 integrin antibodies. However, stable expression of a constitutively active Rac1 mutant rescues the cell ability to spread on these integrin microarrays. Thereby, lamellipodia nucleation by filopodia requires integrin occupancy by matrix substrate and downstream Rac1 signaling.     

Radioprotective properties of eckol against ionizing radiation in mice

We have investigated the radioprotective efficacy of eckol, a component of brown seaweed Ecklonia cava, against the gamma ray-induced damage in vivo. Our results showed that eckol significantly decreased the mortality of lethally irradiated mice. The mechanisms of eckol's protection were found to include: an improvement in hematopoietic recovery, the repair of damaged DNA in immune cells and an enhancement of their proliferation, which had been severely suppressed by ionizing radiation. Thus, we propose eckol as a candidate for adjuvant therapy to alleviate radiation-induced injuries to cancer patients.     

The pre-transmembrane region of the HCV E1 envelope glycoprotein: Interaction with model membranes

The previously identified membranotropic regions of the HCV E1 envelope glycoprotein, a class II membrane fusion protein, permitted us to identify different sequences which might be implicated in viral membrane fusion, membrane interaction and/or protein-protein binding. HCV E1 glycoprotein presents a membrano-active region immediately adjacent to the transmembrane segment, which could be involved in membrane destabilization similarly to the pre-transmembrane domains of class I fusion proteins. Consequently, we have carried out a study of the binding and interaction with the lipid bilayer of a peptide corresponding to segment 309-340, peptide E1_PTM, as well as the structural changes which take place in both the peptide and the phospholipid molecules induced by the binding of the peptide to the membrane. Here we demonstrate that peptide E1_PTM strongly partitions into phospholipid membranes, interacts with negatively-charged phospholipids and locates in a shallow position in the membrane. These data support its role in HCV-mediated membrane fusion and suggest that the mechanism of membrane fusion elicited by class I and II fusion proteins might be similar.     

Immunocytochemical analysis for intracellular dynamics of C1GalT associated with molecular chaperone, Cosmc

The core 1 structure Galβ1-3GalNAcα1-Ser/Thr (T antigen), the major constituent of O-glycan core structure, is synthesized by cooperation of core 1 synthase (C1GalT) and its specific molecular chaperone, Cosmc. The chaperone function of Cosmc has been well investigated biochemically. In this study, we established monoclonal antibodies specifically recognizing either C1GalT or Cosmc, respectively, and investigated the sub-cellular localization of each protein to elucidate how they cooperate to synthesize the core 1 structure.A sequential immunocytochemical analysis of the human colon cancer cell line, LSB, demonstrated different localization of two proteins. C1GalT was localized in Golgi apparatus, while Cosmc was localized in endoplasmic reticulum. In contrast, the LSC cells, which do not have core 1 synthase activity due to a missense mutation in the Cosmc gene, did not express the C1GalT protein. Although the treatment with a proteasome inhibitor, lactacystin, of LSC cells resulted in the increased expression of C1GalT protein, the distribution of C1GalT was not in Golgi apparatus as seen in LSB cells. On the contrary, overexpression of Cosmc but not C1GalT lead to precise localization of C1GalT protein, which distributed in Golgi apparatus and recovered the core 1 synthase activity in LSC cells. These results suggest that the intracellular dynamics of C1GalT is controlled by its specific molecular chaperon, Cosmc, in association with core 1 synthase activity.     

Inhibition of phosphatidylinositide 3-kinase impairs the benzyl isothiocyanate-induced accumulation of autophagic molecules and Nrf2 in human colon cancer cells

The regulating role of phosphatidylinositide 3-kinase (PI3K) in benzyl isothiocyanate (BITC)-induced Nrf2 activation, contributing to the inducible expression of cytoprotective genes, was investigated. BITC significantly enhanced the accumulation of Nrf2 as well as autophagic molecules in human colorectal cancer HCT-116 cells. Experiments using a PI3K-specific inhibitor suggested that PI3K plays the key role in the non-canonical Nrf2 activation by BITC.     

Protein profiling and angiogenic effect of hypoxia-cultured human umbilical cord blood-derived mesenchymal stem cells in hindlimb ischemia

The aim of the present study was to investigate protein profiles of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) cultured in normoxic (21% O₂) and hypoxic (1% O₂) conditions, and evaluate oxygenation effects on angiogenesis in an ischemic hindlimb mouse model using a modified ischemic scoring system. Hypoxic conditions did not change the expression of phenotypic markers and increased adipogenesis and chondrogenesis. Epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), TGF-β RII, and vascular endothelial growth factor (VEGF) were upregulated in the conditioned medium of hypoxic hUCB-MSCs, which are commonly related to angiogenesis and proliferation of biological processes by Gene Ontology. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, significant enrichment of the phosphorylation of abelson murine leukemia viral oncogene homolog 1 (ABL1) (Phospho-Tyr204) and B-cell lymphoma-extra large (BCL-XL) (Phospho-Thr47) as anti-apoptotic pathways was observed in hypoxic hUCB-MSCs. Furthermore, hypoxic conditions induced proliferation and migration, and reduced apoptosis of hUCB-MSCs in vitro. Based on the results of protein antibody array, we evaluated the angiogenic effects of injecting normoxic or hypoxic hUCB-MSCs (1 × 10⁶) into the ischemic hindlimb muscles of mice. Ischemic scores and capillary generation were significantly greater in the hypoxic hUCB-MSC injection group than in the normoxic hUCB-MSC group. Our findings demonstrate that culturing hUCB-MSCs in hypoxic conditions not only significantly enriches phosphorylation in the anti-apoptosis pathway and enhances the secretion of several angiogenic proteins from cells, but also alleviates ischemic injury of hindlimb of mice.     

Allyl Chloride-Induced Time Dependent Changes of Lipid Peroxidation in Rat Nerve Tissue

To accurately know the time-dependent changes of the lipid peroxidation and antioxidative status for elucidating the mechanism of neuropathy induced by allyl chloride (AC), the malondialdehyde (MDA), anti-reactive oxygen species (anti-ROS), glutathione (GSH), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) were investigated in cerebrum, spinal cord and sciatic nerve of rats after 0, 3, 6, 9, 12 weeks of␣AC administration. AC was administrated to Wistar rats by gavage at a single dosage of 200 mg/kg/per dose (three times per week). Rats were sacrificed after 0, 3, 6, 9, 12 weeks of treatment, and cerebrum, spinal cord, sciatic nerves were dissected, homogenized and used for the determination of lipid peroxidation and antioxidative status. The results showed that MDA in cerebrum (112.4%) and sciatic nerve (113.1%) significantly increased (P < 0.05) on third week of AC treatment and at gait score of 2, and further changes of MDA were observed after 6, 9, 12 weeks and at gait score of 3, 4. While a decrease (P < 0.05) in the activities of GSH, CAT, GPx and SOD after 6, 9, 12 weeks intoxication and at gait score of 2, 3, 4 were observed in cerebrum, spinal cord and sciatic nerve. Anti-ROS activities also decreased in all three nerve tissues after 3, 6, 9, 12 weeks intoxication and at gait score of 2, 3, 4. Thus, AC intoxication was associated with elevation of lipid peroxidation and reduction of antioxidative status, and the time-dependent changes of these indexes in Wistar rats nerve tissues occurred. Sciatic nerve was the main target tissue and MDA was most sensitive among all indexes. The changes of lipid peroxidation and antioxidative status might be related to the degradation of nerve fiber and served as one of mechanisms of toxic neuropathy induced by AC.     

Identification of 6-methylsulfinylhexyl isothiocyanate as an apoptosis-inducing component in wasabi

The ethanol extract from Japanese horseradish wasabi was found to inhibit cell proliferation in human monoblastic leukemia U937 cells by inducing apoptotic cell death. Separation by methods including silica gel chromatography and preparative HPLC gave an active compound, which was identified as 6-methylsulfinylhexyl isothiocyanate (6-HITC). Several lines of evidence indicated that 6-HITC induced apoptosis in U937 cells and human stomach cancer MKN45 cells. Thus, 6-HITC is potentially useful as a natural anti-cancer agent.     

Determination of phenethyl isothiocyanate in human plasma and urine by ammonia derivatization and liquid chromatography-tandem mass spectrometry

Phenethyl isothiocyanate (PEITC) is a dietary compound present in cruciferous vegetables that has cancer-preventive properties. Our objective was to develop and validate a novel liquid chromatography-tandem mass spectrometry procedure to analyze PEITC concentrations in human plasma and urine. Following hexane extraction, ammonia was added to samples to derivatize PEITC to phenethylthiourea. Chromatographic separation was achieved on a C(18) column with acetonitrile/5 mM formic acid (60:40, v/v) as the mobile phase followed by tandem mass spectrometry detection in multiple reaction monitoring mode. Deuterium-labeled PEITC was used as the internal standard. The detection limit was 2 nM and calibration curves were linear from 7.8 to 2000 nM. The intra- and inter-day coefficients of variation were less than 5 and 10%, respectively. The intra- and inter-day accuracies ranged from 101.0 to 104.2% and from 102.8 to 118.6%, respectively. The recovery from spiked human plasma and urine ranged from 100.3 to 113.5% and from 98.3 to 103.9%, respectively. The assay was used to measure PEITC in plasma and urine samples obtained from subjects after consumption of 100g of watercress. This novel assay represents the first analytical method with the sensitivity and specificity to determine plasma and urine concentrations of PEITC.     

Binding of a fluorescent lipid amphiphile to albumin and its transfer to lipid bilayer membranes

Kinetics and thermodynamics of the binding of a fluorescent lipid amphiphile, Rhodamine Green(TM)-tetradecylamide (RG-C(14:0)), to bovine serum albumin were characterized in an equilibrium titration and by stopped-flow fluorimetry. The binding equilibrium of RG-C(14:0) to albumin was then used to reduce its concentration in the aqueous phase to a value below its critical micelle concentration. Under these conditions, the only two species of RG-C(14:0) in the system were the monomer in aqueous solution in equilibrium with the protein-bound species. After previous determination of the kinetic and thermodynamic parameters for association of RG-C(14:0) with albumin, the kinetics of insertion of the amphiphile into and desorption off lipid bilayer membranes in different phases (solid, liquid-ordered, and liquid-disordered phases, presented as large unilamellar vesicles) were studied by stopped-flow fluorimetry at 30 degrees C. Insertion and desorption rate constants for association of the RG-C(14:0) monomer with the lipid bilayers were used to obtain lipid/water equilibrium partition coefficients for this fluorescent amphiphile. The direct measurement of these partition coefficients is shown to provide a new method for the indirect determination of the equilibrium partition coefficient of similar molecules between two defined lipid phases if they coexist in the same membrane.