ABSTRACT
Two novel Gram-stain-negative, aerobic, rod shaped bacterial strains BT290T and BT689T were isolated from soil collected in South Korea. Colony morphologies of both strains were circular and convex while the colors of BT290T and BT689T were light-pink and white, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that BT290T and BT689T belong to a distinct lineage within the genus Microvirga (family Methylobacteriaceae, order Rhizobiales, class Alphaproteobacteria, phylum Proteobacteria, kingdom Bacteria). The 16S rR NA gene sequence similarity between two strains was 97.9%. Both strains had the similar quinone system, with ubiquinone 10 (Q-10) as the major respiratory quinone. The major polar lipids of strains BT290T and BT689T were phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylcholine (PC), and phosphatidylglycerol (PG). The major cellular fatty acids of strain BT290T were C18:1 ω7c (58.2%) and C16:0 (17.7%), while those of strain BT689T were C18:1 ω7c (61.8%) and C16:0 (10.8%). On the bases of polyphasic analysis (phylogenetic, chemotaxonomic, and biochemical), strains BT290T and BT689T can be suggested as novel bacterial species within the genus Microvirga and the proposed names are Microvirga terrestris and Microvirga arvi, respectively. The type strain of Microvirga terrestris is BT290T (= KCTC 72367T = NBRC 114844T) and the type strain of Microvirga arvi is BT689T (= KACC 22016T = NBRC 114858T), respectively.
Subject(s)
Alphaproteobacteria , Bradyrhizobiaceae , Methylobacteriaceae , Alphaproteobacteria/genetics , Bacterial Typing Techniques , Base Composition , Bradyrhizobiaceae/genetics , DNA, Bacterial/genetics , Fatty Acids/analysis , Phylogeny , Quinones , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Soil , Soil MicrobiologyABSTRACT
Two novel bacterial strains, designated as BT186T and BT505, were isolated from a soil sample collected in South Korea and characterized. Both strains were Gram-stain-negative, rod-shaped, aerobic, circular, convex, and had red-colored colonies. The level of 16S rRNA gene sequence similarity between the strains BT186T and BT505 was 100%, indicating that they represent an identical species. 16S rRNA sequence analysis indicated that strains BT186T and BT505 belong to a distinct lineage within the genus Hymenobacter (family Hymenobacteraceae, order Cytophagales, class Cytophagia, phylum Bacteroidetes, Kingdom Bacteria). Both strains were closely related to Hymenobacter norwichensis DSM 15439T (98.3% 16S rRNA gene similarity), Hymenobacter aquaticus JCM 31653T (96.8%), and Hymenobacter perfusus LMG26000T (96.5%). Strain BT186T was found to have the MK-7 as the major respiratory quinone. The major polar lipid of strain BT186T was identified to be phosphatidylethanolamine (PE). The major cellular fatty acid profiles of strain BT186T were C16:1 ω5c (24.3%), iso-C15:0 (20.3%) and summed feature 3 (C16:1 ω6c/C16:1 ω7c) (19.9%). Characterization based on polyphasic analysis indicated that strains BT186T and BT505 represent novel species of the genus Hymenobacter and the name Hymenobacter telluris sp. nov. is proposed. The type strain of Hymenobacter telluris is BT186T (= KCTC 72338T = NBRC 114968T).
Subject(s)
Soil Microbiology , Soil , Bacterial Typing Techniques , Base Composition , DNA, Bacterial/genetics , Fatty Acids/analysis , Phylogeny , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNAABSTRACT
Adiponectin, the most abundant adipokine, exhibits various physiological functions. In addition to its critical role in lipid metabolism, recent studies have demonstrated its potent anti-inflammatory and cytoprotective properties. Accumulating evidence suggests that autophagy plays a critical role in various biological responses by adiponectin. However, the underlying mechanisms remain elusive. Herein, we investigated the role of ER stress in adiponectin-induced autophagy and its functional roles in biological responses by adiponectin in macrophages. In this study, globular adiponectin (gAcrp) significantly increased the expression of various ER stress markers in both RAW 264.7 and primary peritoneal macrophages. In addition, inhibition of ER stress by treatment with tauroursodeoxycholic acid (TUDCA) or gene silencing of CHOP prominently suppressed gAcrp-induced autophagy. Treatment with gAcrp also induced significant increase in sestrin2 expression. Interestingly, knockdown of sestrin2 prevented autophagy induction and inhibition of ER stress abrogated sestrin2 induction by gAcrp, collectively implying that ER stress critically contributes to gAcrp-induced autophagy activation via sestrin2 induction. Moreover, pretreatment with TUDCA restored suppression of TNF-α and IL-1ß expression and attenuated the enhanced viability of macrophages induced by gAcrp. Taken together, these findings indicate the potential role of ER stress in autophagy activation, modulation of inflammatory responses, and cell survival by gAcrp in macrophages.
Subject(s)
Adiponectin/metabolism , Autophagy/physiology , Cell Survival/physiology , Endoplasmic Reticulum Stress/physiology , Inflammation/metabolism , Inflammation/pathology , Macrophages/metabolism , Animals , Cell Line , Humans , Interleukin-1beta/metabolism , Macrophages/pathology , Male , Mice , Mice, Inbred C57BL , RAW 264.7 Cells , Sestrins/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Based on the importance of the previous fluorinated and/or hydroxylated chalcones studies, thirty-six compounds were designed as phenyl or hydroxyphenyl bearing fluoro, trifluoromethyl or trifluoromethoxy phenyl propenones and synthesized by applying modified Claisen-Schmidt condensation reaction as a single step. Inhibitory effects of the synthesized compounds on ROS production stimulated by LPS in RAW 264.7 macrophage were evaluated. Structure-activity relationship (SAR) study revealed that the compounds possessing para-hydroxyphenyl group combined with meta-fluoro or meta-trifluoromethyl phenyl group, and meta/para-hydroxyphenyl group combined with ortho-trifluoromethoxyphenyl group have an essential role in inhibiting the LPS-stimulated ROS production in RAW 264.7 macrophages. The most significant inhibitory effect on LPS-stimulated ROS production in RAW 264.7 macrophages was observed in compound 30 that possessed para-hydroxyphenyl group along with ortho-trifluoromethoxyphenyl group.
Subject(s)
Chalcones/pharmacology , Lipopolysaccharides/antagonists & inhibitors , Reactive Oxygen Species/metabolism , Animals , Cell Line , Chalcones/chemistry , Fluorine/chemistry , Hydroxylation , Lipopolysaccharides/pharmacology , Mice , Structure-Activity RelationshipABSTRACT
OBJECTIVES: Clinical features of primary Sjögren's syndrome (pSS) overlap with those of fibromyalgia (FM). This cross-sectional study was conducted to investigate the prevalence of FM in pSS patients and to compare the clinical features of pSS patients with FM to those without FM. METHODS: One hundred pSS patients were consecutively assessed to identify the presence of FM according to the American College of Rheumatology (ACR) 2010 criteria. Clinical and laboratory data were collected from all patients. Additional assessments included EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI) and EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI). The severity of depression was measured by Hamilton depression rating scale 17-items (HAM-D scale). RESULTS: The prevalence of FM was 31.0% (31/100) in pSS. Widespread pain index and symptom severity scale were significantly correlated with ESSPRI (r=0.6542 and r=0.7173, both p<0.0001) and HAM-D scale (r=0.6734 and r=0.6471, both p<0.0001) in pSS. In multivariate analysis, ESSPRI and HAM-D scale were independently associated with increase of tender point count and symptom severity scale. ESSPRI was significantly higher in pSS patients with FM compared to those without FM (p<0.0001). The prevalence of FM in pSS patients with moderate-to-severe depression was significantly higher than those with mild depression or without depression (odds ratio= 10.62, p=0.0009). Serum 25-hydroxy vitamin D3 levels in pSS patients with FM were significantly (p=0.0072) decreased compared to those without FM. CONCLUSIONS: Our study showed that FM was prevalent in pSS. FM was associated with higher ESSPRI and more severe depression.
Subject(s)
Depression , Fibromyalgia , Sjogren's Syndrome , Aged , Cross-Sectional Studies , Depression/diagnosis , Depression/epidemiology , Depression/physiopathology , Female , Fibromyalgia/diagnosis , Fibromyalgia/epidemiology , Fibromyalgia/etiology , Fibromyalgia/psychology , Humans , Male , Middle Aged , Pain Measurement/methods , Pain Measurement/statistics & numerical data , Prevalence , Psychological Tests , Republic of Korea/epidemiology , Severity of Illness Index , Sjogren's Syndrome/complications , Sjogren's Syndrome/epidemiology , Statistics as TopicABSTRACT
The aim of this study is the first time demonstration of cis-12 regio-selective linoleate double-bond hydratase. Hydroxylation of fatty acids, abundant feedstock in nature, is an emerging alternative route for many petroleum replaceable products thorough hydroxy fatty acids, carboxylic acids, and lactones. However, chemical route for selective hydroxylation is still quite challenging owing to low selectivity and many environmental concerns. Hydroxylation of fatty acids by hydroxy fatty acid forming enzymes is an important route for selective biocatalytic oxyfunctionalization of fatty acids. Therefore, novel fatty acid hydroxylation enzymes should be discovered. The two hydratase genes of Lactobacillus acidophilus were identified by genomic analysis, and the expressed two recombinant hydratases were identified as cis-9 and cis-12 double-bond selective linoleate hydratases by in vitro functional validation, including the identification of products and the determination of regio-selectivity, substrate specificity, and kinetic parameters. The two different linoleate hydratases were the involved enzymes in the 10,13-dihydroxyoctadecanoic acid biosynthesis. Linoleate 13-hydratase (LHT-13) selectively converted 10 mM linoleic acid to 13S-hydroxy-9(Z)-octadecenoic acid with high titer (8.1 mM) and yield (81%). Our study will expand knowledge for microbial fatty acid-hydroxylation enzymes and facilitate the designed production of the regio-selective hydroxy fatty acids for useful chemicals from polyunsaturated fatty acid feedstocks.
Subject(s)
Fatty Acids/metabolism , Hydro-Lyases/metabolism , Lactobacillus acidophilus/enzymology , Hydro-Lyases/genetics , Kinetics , Lactobacillus acidophilus/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
The specific activity of a recombinant ß-glucosidase from Pyrococcus furiosus for protopanaxatriol (PPT)-type ginsenosides followed the order Rf > R1 > Re > R2 > Rg2, which were converted to Rh1, Rg1, Rg1, Rh1, and Rh1, respectively. No activity was observed with Rg1 and Rh1. Thus, P. furiosus ß-glucosidase hydrolyzed the outer glycoside at the C-6 position in PPT-type ginsenosides whereas the enzyme did not hydrolyze the inner glucoside at the C-6 position and the glucoside at the C-20 position. The activity for Rf was optimal at 95 °C, pH 5.5, 5 mM ginsenoside, and 32 U enzyme l−1. Under these conditions, P. furiosus ß-glucosidase completely converted from R1 to Rg1 after 10 h, with a productivity of 0.4 g l−1 h−1 and completely converted Rf to Rh1 after 1.2 h, with a productivity of 2.74 g l−1 h−1.
Subject(s)
Archaeal Proteins/metabolism , Ginsenosides/metabolism , Pyrococcus furiosus/enzymology , Sapogenins/metabolism , beta-Glucosidase/metabolism , Biotransformation , Ginsenosides/analysis , Ginsenosides/chemistry , Hot Temperature , Pyrococcus furiosus/genetics , Sapogenins/chemistryABSTRACT
ß-Glucosidase from Thermus thermophilus has specific hydrolytic activity for the outer glucose at the C-20 position in protopanaxadiol-type ginsenosides without hydrolysis of the inner glucose. The hydrolytic activity of the enzyme for gypenoside XVII was optimal at pH 6.5 and 90 °C, with a half-life of 1 h with 3 g enzyme l(-1) and 4 g gypenoside XVII l(-1). Under the optimized conditions, the enzyme converted the substrate gypenoside XVII to ginsenoside F2 with a molar yield of 100 % and a productivity of 4 g l(-1) h(-1). The conversion yield and productivity of ginsenoside F2 are the highest reported thus far among enzymatic transformations.
Subject(s)
Ginsenosides/metabolism , Glucose/metabolism , Thermus thermophilus/enzymology , beta-Glucosidase/metabolism , Enzyme Stability , Gynostemma/metabolism , Hydrogen-Ion Concentration , Hydrolysis , Plant Extracts/metabolism , Temperature , beta-Glucosidase/chemistryABSTRACT
Recombinant Escherichia coli, expressing the oleate hydratase gene of Stenotrophomonas maltophilia, was permeabilized by sequential treatments with 0.125 M NaCl and 2 mM EDTA. The optimal conditions for the production of 10-hydroxy-12,15(Z,Z)-octadecadienoic acid from α-linolenic acid by permeabilized cells were 35 °C and pH 7.0 with 0.1 % (v/v) Tween 40, 50 g permeabilized cells l(-1), and 17.5 g α-linolenic acid l(-1). Under these conditions, permeabilized cells produced 14.3 g 10-hydroxy-12,15(Z,Z)-octadecadienoic acid l(-1) after 18 h, with a conversion yield of 82 % (g/g) and a volumetric productivity of 0.79 g l(-1) h(-1). These values were 17 and 168 % higher than those obtained by nonpermeabilized cells, respectively. The concentration, yield, and productivity of 10-hydroxy-12,15(Z,Z)-octadecadienoic acid obtained by permeabilized cells are the highest reported thus far.
Subject(s)
Escherichia coli/metabolism , Fatty Acids, Unsaturated/metabolism , Hydro-Lyases/biosynthesis , Stenotrophomonas maltophilia/enzymology , alpha-Linolenic Acid/metabolism , Cell Membrane Permeability/drug effects , Escherichia coli/drug effects , Escherichia coli/enzymology , Escherichia coli/genetics , Gene Expression , Hydro-Lyases/genetics , Hydrogen-Ion Concentration , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Stenotrophomonas maltophilia/genetics , TemperatureABSTRACT
As the scale-down and power-saving of silicon-based channel materials approach the limit, oxide semiconductors are being actively researched for applications in 3D back-end-of-line integration. For these applications, it is necessary to develop stable oxide semiconductors with electrical properties similar to those of Si. Herein, a single-crystal-like indium-gallium-zinc-oxide (IGZO) layer (referred to as a pseudo-single-crystal) is synthesized using plasma-enhanced atomic layer deposition and fabricated stable IGZO transistors with an ultra-high mobility of over 100 cm2 Vs-1 . To acquire high-quality atomic layer deposition-processed IGZO layers, the plasma power of the reactant is controlled as an effective processing parameter by evaluating and understanding the effect of the chemical reaction of the precursors on the behavior of the residual hydrogen, carbon, and oxygen in the as-deposited films. Based on these insights, this study found that there is a critical relationship between the optimal plasma reaction energy, superior electrical performance, and device stability.
ABSTRACT
This study investigated the potential of a newly synthesized histone deacetylase (HDAC) inhibitor, MHY446, in inducing cell death in HCT116 colorectal cancer cells and compared its activity with that of suberoylanilide hydroxamic acid (SAHA), a well-known HDAC inhibitor. The results showed that MHY446 increased the acetylation of histones H3 and H4 and decreased the expression and activity of HDAC proteins in HCT116 cells. Additionally, MHY446 was confirmed to bind more strongly to HDAC1 than HDAC2 and inhibit its activity. In vivo experiments using nude mice revealed that MHY446 was as effective as SAHA in inhibiting HCT116 cell-grafted tumor growth. This study also evaluated the biological effects of MHY446 on cell survival and death pathways. The reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) confirmed that ROS play a role in MHY446-induced cell death by reducing poly(ADP-ribose) polymerase cleavage. MHY446 also induced cell death via endoplasmic reticulum (ER) stress by increasing the expression of ER stress-related proteins. NAC treatment decreased the expression of ER stress-related proteins, indicating that ROS mediate ER stress as an upstream signaling pathway and induce cell death. While MHY446 did not exhibit superior HDAC inhibition efficacy compared to SAHA, it is anticipated to provide innovative insights into the future development of therapeutic agents for human CRC by offering novel chemical structure-activity relationship-related information.
ABSTRACT
Achieving high mobility and reliability in atomic layer deposition (ALD)-based IGZO thin-film transistors (TFTs) with an amorphous phase is vital for practical applications in relevant fields. Here, we suggest a method to effectively increase stability while maintaining high mobility by employing the selective application of nitrous oxide plasma reactant during plasma-enhanced ALD (PEALD) at 200 °C process temperature. The nitrogen-doping mechanism is highly dependent on the intrinsic carbon impurities or nature of each cation, as demonstrated by a combination of theoretical and experimental research. The Ga2O3 subgap states are especially dependent on plasma reactants. Based on these insights, we can obtain high-performance indium-rich PEALD-IGZO TFTs (threshold voltage: -0.47 V; field-effect mobility: 106.5 cm2/(V s); subthreshold swing: 113.5 mV/decade; hysteresis: 0.05 V). In addition, the device shows minimal threshold voltage shifts of +0.45 and -0.10 V under harsh positive/negative bias temperature stress environments (field stress: ±2 MV/cm; temperature stress: 95 °C) after 10000 s.
ABSTRACT
The optimized ALD infilling process for depositing Al2O3 in the vertical direction of PbS QDs enhances the photoresponsivity, relaxation rate and the air stability of PbS QDs hybrid IGZO NIR phototransistors. Infilled Al2O3, which is gradually deposited from the top of PbS QDs to the PbS/IGZO interface (1) passivates the trap sites up to the interface of PbS/IGZO without disturbing charge transfer and (2) prevents QDs deterioration caused by outside air. Therefore, an Al2O3 infilled PbS QD/IGZO hybrid phototransistor (AI-PTs) exhibited enhanced photoresponsivity from 96.4 A/W to 1.65 × 102 A/W and a relaxation time decrease from 0.52 to 0.03 s under NIR light (880 nm) compared to hybrid phototransistors without Al2O3 (RF-PTs). In addition, AI-PTs also showed improved shelf stability over 4 months compared to RF-PTs. Finally, all devices we manufactured have the potential to be manufactured in an array, and this ALD technique is a means of fabricating robust QDs/metal oxide hybrids for optoelectronic devices.
ABSTRACT
The tyrosine kinase Fyn is a member of the Src family kinases which are important in many integrin-mediated cellular processes including cell adhesion and migration. Fyn has multiple phosphorylation sites which can affect its kinase activity. Among these phosphorylation sites, the serine 21 (S21) residue of Fyn is a protein kinase A (PKA) recognition site within an RxxS motif of the amino terminal SH4 domain of Fyn. In addition, S21 is critical for Fyn kinase-linked cellular signaling. Mutation of S21A blocks PKA phosphorylation of Fyn and alters its tyrosine kinase activity. Expression of Fyn S21A in cells lacking Src family kinases (SYF cell) led to decreased tyrosine phosphorylation of focal adhesion kinase resulting in reduced focal adhesion targeting, which slowed lamellipodia dynamics and thus cell migration. These changes in cell motility were reflected by the fact that cells expressing Fyn S21A were severely deficient in their ability to assemble and disassemble focal adhesions. Taken together, our findings indicate that phosphorylation of S21 within the pPKA recognition site (RxxS motif) of Fyn regulates its tyrosine kinase activity and controls focal adhesion targeting, and that this residue of Fyn is critical for transduction of signals arising from cell-extracellular matrix interactions.
Subject(s)
Focal Adhesions/physiology , Proto-Oncogene Proteins c-fyn/metabolism , Serine/metabolism , Amino Acid Sequence , Animals , Cell Adhesion/physiology , Cell Line , Cell Movement , Gene Expression Regulation, Enzymologic , Humans , Mice , Molecular Sequence Data , Mutation , Phosphorylation , Proto-Oncogene Proteins c-fyn/genetics , Signal Transduction/physiologyABSTRACT
As one of the energy storage systems, supercapacitors have quite long charge-discharge cycle life. Among many kinds of electrode materials, metal organic frameworks (MOFs) have unique properties such as high specific surface areas and large pore volume as supercapacitor electrode materials. Nickel-MOFs consist of binary ligand such as 1,3,5-Trimesic acid (H3BTC) and terephthalic acid (TPA) were used as working electrode materials in three electrode cell for capacitor system. When synthesizing MOFs, it is possible to prepare uniform crystals using hydrothermal synthesis. The morphology of composites was analyzed by field emission scanning electron microscopy (FE-SEM). Electrochemical properties were measured by cyclic voltammetry (CV), galvanostatic charge-discharge (GCD) in 6M KOH electrolyte.
ABSTRACT
SPIN90 is a key regulator of actin cytoskeletal organization. Using the BioGRID(beta) database (General Repository for Interaction Datasets), we identified IRSp53 as a binding partner of SPIN90, and confirmed the in vivo formation of a SPIN90-IRSp53 complex mediated through direct association of the proline-rich domain (PRD) of SPIN90 with the SH3 domain of IRSp53. SPIN90 and IRSp53 positively cooperated to mediate Rac activation, and co-expression of SPIN90 and IRSp53 in COS-7 cells led to the complex formation of SPIN90-IRSp53 in the leading edge of cells. PDGF treatment induced strong colocalization of SPIN90 and IRSp53 at membrane protrusions. Within such PDGF-induced protrusions, knockdown of SPIN90 protein using siRNA significantly reduced lamellipodia-like protrusions as well as localization of IRSp53 at those sites. Finally, competitive inhibition of SPIN90-IRSp53 binding by SPIN90 PRD dramatically reduced ruffle formation, further suggesting that SPIN90 plays a key role in the formation of the membrane protrusions associated with cell motility.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Membrane/metabolism , Muscle Proteins/metabolism , Nerve Tissue Proteins/metabolism , rac GTP-Binding Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , COS Cells , Cell Membrane/ultrastructure , Cell Movement/physiology , Cell Surface Extensions , Chlorocebus aethiops , Gene Knockdown Techniques , Genetic Vectors/metabolism , Humans , Muscle Proteins/genetics , Platelet-Derived Growth Factor/pharmacology , RNA, Small Interfering/metabolism , TransfectionABSTRACT
We examined the anticancer effects of a novel sirtuin inhibitor, MHY2256, on HCT116 human colorectal cancer cells to investigate its underlying molecular mechanisms. MHY2256 significantly suppressed the activity of sirtuin 1 and expression levels of sirtuin 1/2 and stimulated acetylation of forkhead box O1, which is a target protein of sirtuin 1. Treatment with MHY2256 inhibited the growth of the HCT116 (TP53 wild-type), HT-29 (TP53 mutant), and DLD-1 (TP53 mutant) human colorectal cancer cell lines. In addition, MHY2256 induced G0/G1 phase arrest of the cell cycle progression, which was accompanied by the reduction of cyclin D1 and cyclin E and the decrease of cyclin-dependent kinase 2, cyclin-dependent kinase 4, cyclin-dependent kinase 6, phosphorylated retinoblastoma protein, and E2F transcription factor 1. Apoptosis induction was shown by DNA fragmentation and increase in late apoptosis, which were detected using flow cytometric analysis. MHY2256 downregulated expression levels of procaspase-8, -9, and -3 and led to subsequent poly(ADP-ribose) polymerase cleavage. MHY2256-induced apoptosis was involved in the activation of caspase-8, -9, and -3 and was prevented by pretreatment with Z-VAD-FMK, a pan-caspase inhibitor. Furthermore, the autophagic effects of MHY2256 were observed as cytoplasmic vacuolation, green fluorescent protein-light-chain 3 punctate dots, accumulation of acidic vesicular organelles, and upregulated expression level of light-chain 3-II. Taken together, these results suggest that MHY2256 could be a potential novel sirtuin inhibitor for the chemoprevention or treatment of colorectal cancer or both.
ABSTRACT
We examined the consequences of v-Crk expression in mouse embryo fibroblasts deficient Src family kinases or p130CAS. We found that Src kinases are essential for p130CAS/v-Crk signaling leading to FAK phosphorylation and cell migration in which Src is likely to mediate the focal adhesion targeting of v-Crk. SYF cells showed only low levels of FAK phosphorylation and cell migration, even in the presence of v-Crk. Expression of v-Crk restored migration of p130CAS-deficient cells to the level of wild-type cells, most likely through the targeting of v-Crk to focal adhesions by cSrc. In addition, we identified a new v-Crk-interacting protein that mediates v-Crk signaling in p130CAS-deficient cells. Using RT-PCR and caspase cleavage assays, we confirmed that this protein is not p130CAS and is responsible for maintaining v-Crk/Src signaling and migration in these. These findings suggest that focal adhesion targeting of v-Crk is essential in v-Crk-mediated cellular signaling and that v-Crk must form a complex with p130CAS or a p130CAS substitute to transduce signaling from the extracellular matrix.
Subject(s)
Cell Movement , Crk-Associated Substrate Protein/metabolism , Embryo, Mammalian/cytology , Fibroblasts/enzymology , Focal Adhesion Kinase 1/metabolism , Focal Adhesions/enzymology , Oncogene Protein v-crk/metabolism , Amino Acid Sequence , Animals , Antibodies/pharmacology , Cell Movement/drug effects , Embryo, Mammalian/drug effects , Embryo, Mammalian/enzymology , Fibroblasts/cytology , Fibroblasts/drug effects , Focal Adhesions/drug effects , Mice , Microinjections , Molecular Sequence Data , Myosins/chemistry , Myosins/metabolism , Phosphorylation/drug effects , Protein Binding/drug effects , Wound Healing/drug effects , src-Family Kinases/metabolismABSTRACT
Crk-associated substrate (CAS) is a focal adhesion protein that is involved in integrin signaling and cell migration. CAS deficiency reduces the migration and spreading of cells, both of which are processes mediated by Rac activation. We examined the functions of v-Crk, the oncogene product of the CT10 virus p47gag-crk, which affects cell migration and spreading, membrane ruffling, and Rac activation in CAS-deficient mouse embryonic fibroblasts (CAS-/- MEFs). CAS-/- MEFs showed less spreading than did CAS+/+ MEFs, but spreading was recovered in mutant cells that expressed v-Crk (CAS-/-v-Crk MEF). We observed that the reduction in spreading was linked to the formation of membrane ruffles, which were accompanied by Rac activation. In CAS-/- MEFs, Rac activity was significantly reduced, and Rac was not localized to the membrane. In contrast, Rac was active and localized to the membrane in CAS-/-v-Crk MEFs. Lamellipodia protrusion and ruffle retraction velocities were both reduced in CAS-/- MEFs, but not in CAS-/-v-Crk MEFs. We also found that microinjection of anti-gag antibodies inhibited the migration of CAS-/-v-Crk MEFs. These findings indicate that v-Crk controls cell migration and membrane dynamics by activating Rac in CAS-deficient MEFs.
Subject(s)
Cell Movement/physiology , Cell Surface Extensions/metabolism , Crk-Associated Substrate Protein/metabolism , Embryo, Mammalian/cytology , Fibroblasts/physiology , Oncogene Protein v-crk/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Animals , Crk-Associated Substrate Protein/genetics , Enzyme Activation , Mice , Mice, Inbred BALB C , Mice, Knockout , Oncogene Protein v-crk/genetics , Proto-Oncogene Proteins c-akt/geneticsABSTRACT
BACKGROUND: We examined the relationship between 10-year predicted atherosclerosis cardiovascular disease (ASCVD) risk score and 25-hydroxyvitamin D in Koreans aged 40-79 years. METHODS: A population-based, cross-sectional design was used from data based on the Korea National Health and Nutrition Examination Survey 2014. RESULTS: A total of 1,134 healthy Koreans aged 40-79 years were included. A positive relationship between serum 25-hydroxyvitamin D level and ASCVD score was shown in women (ß=0.015) after adjusting for central obesity, physical activity, and supplement intake. The chances of being in the moderate to high risk (risk group, ASCVD score ≥5%) with vitamin D sufficiency (serum 25-hydroxyvitamin D ≥20 ng/mL) was 1.267-fold (95% confidence interval, 1.039-1.595) greater than the chance of being included in the group with vitamin D deficiency (serum 25-hydroxyvitamin D <20 ng/mL) after adjustments in women. CONCLUSION: Our research indicated a significantly positive association between 25-hydroxyvitamin D and ASCVD score. Further detailed studies to evaluate this correlation are needed.