ABSTRACT
BACKGROUND: Identifying dysphagia as a potential complication of sepsis may improve swallowing function and survival while decreasing hospital length of stay. OBJECTIVES: Our goal was to determine the frequency of dysphagia in sepsis survivors on the 7th day after admission, as well as their associated factors and outcomes. METHODS: This single-centre, retrospective, observational study analysed data from sepsis survivors admitted to Okayama Saiseikai General Hospital from 2018 to 2019. Participants with sepsis were assigned to one of two study groups based on the presence or absence of dysphagia using the criterion of Functional Oral Intake Scale score <5 on the 7th day after admission. We used multivariate logistic regression to determine factors independently associated with dysphagia on the 7th day after admission. Multivariate logistic regression was also used to determine associations between groups and outcomes, including dysphagia on hospital discharge, direct discharge home (discharge of patients directly to their home), and total dependency (Barthel Index score ≤20) on hospital discharge. RESULTS: One hundred one patients met the study inclusion criteria, 55 with dysphagia and 46 without dysphagia. Fasting period (adjusted odds ratio [AOR]: 1.31, 95% confidence interval [CI]: 1.07-1.59) and enteral tube feeding (AOR: 8.56, 95% CI: 1.95-37.5) were independently associated with the presence of dysphagia on the 7th day after admission. Dysphagia on the 7th day after admission was associated with dysphagia on hospital discharge (AOR: 46.0, 95%, CI: 7.90-268.3), a lower chance of direct discharge home (AOR: 0.03, 95% CI: 0.01-0.15), and a higher incidence of total dependency (AOR: 9.30, 95% CI: 2.68-32.2). CONCLUSIONS: We found that dysphagia was commonly encountered post sepsis. Fasting period and enteral tube feeding were independently associated with dysphagia on the 7th day after admission. Dysphagia on the 7th day after admission was also associated with dysphagia on hospital discharge, nondirect discharge home, and dependency in activities of daily living at the time of hospital discharge.
Subject(s)
Deglutition Disorders , Sepsis , Humans , Deglutition Disorders/epidemiology , Deglutition Disorders/etiology , Retrospective Studies , Activities of Daily Living , Deglutition , Sepsis/complications , Sepsis/epidemiologyABSTRACT
Identifying the targets of a drug is critical to understand the mechanism of action and predicts possible side effects. The conventional approach is capturing interacting proteins by affinity purification. However, it requires drugs to be immobilized to a solid support or derivatized with chemical moieties used for pulling down interacting proteins. Such covalent modifications to drugs may mask a critical recognition site for or alter the binding affinity to their targets. To overcome the drawback, several methods that do not require covalent modifications to drugs have been developed. These methods identify targets by detecting proteins whose thermodynamic stability is enhanced in the presence of drugs. Although the utility of these methods has been demonstrated, the difficulty in identifying low abundant targets is the common problem of these methods. We have developed a new target identification method that increases the likelihood of identifying low abundant targets. The method uses histidine-hydrogen deuterium exchange (His-HDX) as a readout technique to probe the changes in protein stability induced by drugs. The workflow involves incubating cell lysates in various concentrations of a protein denaturant in the presence and absence of a drug in D2O followed by digestion of the proteins, enrichment of His-containing peptides, and analysis of the enriched His-peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The developed method was successfully applied to identify the interaction between endogenously expressed MAPK14 and its inhibitor in HEK293 cell lysates. The implementation of selective enrichment of histidine-containing peptides in the workflow was a key that enabled identifying the MAPK14-inhibitor interaction.
Subject(s)
Deuterium Exchange Measurement , Histidine , Chromatography, Liquid , Deuterium , Drug Interactions , HEK293 Cells , Humans , Hydrogen , Tandem Mass SpectrometryABSTRACT
Protein-protein interactions (PPIs) lead the formation of protein complexes that perform biochemical reactions that maintain the living state of the living cell. Although therapeutic drugs should influence the formation of protein complexes in addition to PPI network, the methodology analyzing such influences remain to be developed. Here, we demonstrate that a new approach combining HPLC (high performance liquid chromatography) for separating protein complexes, and the SILAC (stable isotope labeling using amino acids in cell culture) method for relative protein quantification, enable us to identify the protein complexes influenced by a drug. We applied this approach to the analysis of thalidomide action on HepG2 cells, assessed the identified proteins by clustering data analyses, and assigned 135 novel protein complexes affected by the drug. We propose that this approach is applicable to elucidating the mechanisms of actions of other therapeutic drugs on the PPI network, and the formation of protein complexes.
Subject(s)
Amino Acids/chemistry , Drug Evaluation, Preclinical/methods , Protein Interaction Maps/drug effects , Proteins/chemistry , Proteins/metabolism , Proteomics , Hep G2 Cells , Humans , Isotope Labeling , Thalidomide/pharmacologyABSTRACT
BACKGROUND: Duffy antigen/chemokine receptor (DARC) is a non-signaling receptor for multiple chemokines. The role of DARC on red blood cells (RBCs) has remained elusive. The purpose of this study was to analyze selective storage of DARC-binding chemokines in RBCs. METHODS: Peripheral blood from healthy volunteers with DARC-positive blood type was collected in EDTA tubes. The concentration of DARC binding chemokines (i.e., MCP-1, RANTES, eotaxin-1, TARC, and IL-8), DARC nonbinding chemokines (i.e., MIP-1α, IP-10), and several cytokines in the supernatant of purified RBCs before and after hemolysis was measured using Bio-Plex and ELISA assays. Storage of chemokines in RBCs and the expression of DARC were evaluated using flow-cytometry. RESULTS: The levels of all DARC-binding chemokines except TARC and IL-8 increased significantly after hemolysis. There was no significant increase in any of the DARC non-binding chemokines or in the other cytokines after hemolysis. RANTES, eotaxin-1, and MCP-1 were detectable intracellularly but not on the RBC surface. RANTES was absorbed by RBCs. DARC was expressed intracellularly in RBCs as well as on the surface. CONCLUSIONS: These data suggested that DARC-positive RBCs store RANTES, MCP-1 and eotaxin-1. DARC on RBC may be internalized from the surface in the process of chemokine absorption.
Subject(s)
Erythrocytes , Carrier Proteins , Chemokine CCL11 , Chemokines , Duffy Blood-Group System , Humans , Interleukin-8 , Receptors, Cell SurfaceABSTRACT
BACKGROUND: A number of outbreaks caused by Bacillus species have been reported to date. Outbreaks reported in the last decade have predominantly arisen in Japanese hospitals. AIM: To elucidate factors contributing to these real or pseudo outbreaks by Bacillus species, and to evaluate the rate of Bacillus species-positive blood culture samples in Japan. METHODS: A systematic review of the literature was performed. Reports including data on outbreaks caused by Bacillus species were searched for in PubMed, Google Scholar and Evidence-based Medicine BMJ from inception through 10 Aug 2014. Japanese nationwide data on bacteriological tests were collected from Japan Nosocomial Infections Surveillance. Regional bacteriological data for Akita prefecture were collected using the Akita Regional Network for Infection Monitoring/Control System. FINDINGS: Contamination of reusable towels was suspected as a cause for the high rate of Bacillus-positive blood cultures in Japan. The rate of Bacillus species in blood cultures was much higher in Japan than in reports from other countries. CONCLUSIONS: The high contamination rate of blood culture samples by Bacillus species in Japan is a matter of concern for infection control and medical treatment. Bacteriological investigation of reusable towels should be considered in hospitals with a high frequency of Bacillus-positive blood cultures.
Subject(s)
Bacillaceae Infections/etiology , Bacillus/isolation & purification , Cross Infection/microbiology , Cross Infection/etiology , Disease Outbreaks , Hospitals , Humans , Infection Control/methods , JapanABSTRACT
Specific detection of protein biomarkers plays an important role in diagnostics and therapeutics. We have fabricated polymeric nanogels, which can specifically interact with the cancer biomarker thrombin to serve as a model. Two types of 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymers bearing a thrombin-binding oligonucleotide aptamer and its complementary chain were independently synthesized by redox-initiated radical polymerization. These MPC polymers associate in a complimentary fashion due to double strand formation of the oligonucleotides in aqueous media, leading to the spontaneous formation of spherical nanogels. Nanogel formation was confirmed by dynamic light scattering (DLS) and transmittance microscopy. The average size of nanogel particles was 124 ± 2 nm and the nanogels were mono-dispersed (polydispersity index 0.21). Functional intercalators could be stably incorporated into nanogels through the physical interaction between the intercalators and the oligonucleotides. The ethidium bromide (EtBr)-incorporating nanogels were used as detectors for thrombin. The fluorescence intensity of solutions containing the EtBr-incorporating nanogels was decreased with an increase in the concentration of thrombin. The transformation of quadruplex-thrombin structure from complementary double-stranded structures resulted in the decrease in fluorescence intensity. In contrast, the intensity did not change when the nanogels were incubated with albumin. Thrombin is only one such model used to demonstrate this technique; oligonucleotide aptamers can be freely designed to interact with versatile bio-substances. Therefore, aptamer-crosslinked nanogels can be appropriate nanomaterials for disease diagnosis and therapy.
ABSTRACT
Implantable sensors for continuous glucose monitoring hold great potential for optimal diabetes management. This is often undermined by a variety of issues associated with: (1) negative tissue response; (2) poor sensor performance; and (3) lack of device miniaturization needed to reduce implantation trauma. Herein, we report our initial results towards constructing an implantable device that simultaneously address all three aforementioned issues. In terms of device miniaturization, a highly miniaturized CMOS (complementary metal-oxide-semiconductor) potentiostat and signal processing unit was employed (with a combined area of 0.665 mm(2)). The signal processing unit converts the current generated by a transcutaneous, Clark-type amperometric sensor to output frequency in a linear fashion. The Clark-type amperometric sensor employs stratification of five functional layers to attain a well-balanced mass transfer which in turn yields a linear sensor response from 0 to 25 mM of glucose concentration, well beyond the physiologically observed (2 to 22 mM) range. In addition, it is coated with a thick polyvinyl alcohol (PVA) hydrogel with embedded poly(lactic-co-glycolic acid) (PLGA) microspheres intended to provide continuous, localized delivery of dexamethasone to suppress inflammation and fibrosis. In vivo evaluation in rat model has shown that the transcutaneous sensor system reproducibly tracks repeated glycemic events. Clarke's error grid analysis on the as-obtained glycemic data has indicated that all of the measured glucose readings fell in the desired Zones A & B and none fell in the erroneous Zones C, D and E. Such reproducible operation of the transcutaneous sensor system, together with low power (140 µW) consumption and capability for current-to-frequency conversion renders this a versatile platform for continuous glucose monitoring and other biomedical sensing devices.
Subject(s)
Blood Glucose Self-Monitoring/instrumentation , Glucose/analysis , Miniaturization/instrumentation , Prostheses and Implants , Skin , Animals , Biosensing Techniques , Electrochemistry , Lactic Acid/chemistry , Male , Metals/chemistry , Oxides/chemistry , Polyglycolic Acid/chemistry , Polylactic Acid-Polyglycolic Acid Copolymer , Polyvinyl Alcohol/chemistry , Rats , Rats, Sprague-Dawley , SemiconductorsABSTRACT
Activities and the understanding of infection control in healthcare facilities have improved in the past decade since a certification system for medical personnel, such as infection control nurse and infection control doctor, were introduced in Japan. These specialists are distributed among tertiary general hospitals, while many small and mid-scale hospitals have no infection control specialists. In 2012, the Japanese Ministry of Health, Labour and Welfare launched a new strategy for further improvement of infection control by supporting a regional network of infection control activities. Through the infection control network, small or mid-scaled hospitals can utilize infection control specialists in tertiary general hospitals, enter educational programs on infection control and consult in cases of nosocomial infection outbreaks. As part of the regional infection control network, we established an information network system, named ReNICS, to share the bacteriological test results of the hospitals in Akita prefecture. ReNICS offers epidemiological data on bacteria identified in the region. We can identify the spread of multi-drug resistant bacteria and can roughly estimate the quality of infection control activities in each facility. As a similar information network is being prepared in Hirosaki University Hospital Infection Control Center in Aomori, a prefecture neighboring Akita, we discussed the roles of university hospitals for a regional infection control network.
Subject(s)
Drug Resistance, Multiple, Bacterial , Hospitals, University , Infection Control/methods , Information Services , Humans , Information Dissemination , RoleABSTRACT
BACKGROUND: Depression is frequently associated with unsuccessful smoking cessation. OBJECTIVE: In this study, we investigated the impact of depression history on smoking cessation success in a clinical setting. METHODS: This retrospective study included 726 patients who visited our smoking cessation clinic between January 1, 2001, and December 31, 2018. Kaplan-Meier analyses and Cox proportional hazards regression models were used to perform univariate and multivariate analyses of smoking cessation success factors. RESULTS: Among the 726 patients, 76 had a history of depression and demonstrated significantly lower 12-week quit rate compared to those without (33.6% vs. 69.6%, p < .001). Multivariate Cox analysis revealed a significant association between abstinence rate and history of depression (hazard ratio 2.251, 95% CI 1.505-3.315, p < .001), history of schizophrenia (hazard ratio 2.716, 95% CI 1.427-4.840, p = .003), and Fagerström Nicotine Dependence Test scores (hazard ratio 1.519, 95% CI 1.053-2.197, p = .025). CONCLUSIONS: Our findings suggested that a history of depression is a significant prognostic factor for smoking cessation, underscoring the need for targeted interventions for patients with a history of depression. The findings of this study are subject to potential selection bias due to recruitment from a single hospital, which may limit the generalizability of our results. This study highlights the necessity for novel, specialized smoking cessation therapies to support patients with a history of depression in their cessation journey.
ABSTRACT
Chondromodulin-I (ChM-I) is a 25-kDa glycoprotein in cartilage matrix that inhibits angiogenesis. It contains two distinctive structural domains: the N-terminal third of the molecule is a hydrophilic domain that contains O-linked and N-linked oligosaccharide chains, and the C-terminal two-thirds is a hydrophobic domain that contains all of the cysteine residues. In the present study, we have attempted to further uncover the structural requirements for ChM-I to exert anti-angiogenic activity by monitoring its inhibition of the vascular endothelial growth factor (VEGF)-A-induced migration of HUVEC in vitro. Site-directed mutagenesis experiments revealed that the cyclic structure formed by the disulfide bridge between Cys(83) and Cys(99) in human ChM-I is indispensable for its anti-angiogenic function. Moreover, the C-terminal hydrophobic tail (from Trp(111) to Val(120) ) was found to play an important role in ensuring the effectiveness of ChM-I activity on HUVEC. A synthetic cyclic peptide corresponding to the ChM-I region between Ile(82) to Arg(100) also inhibited the migration of HUVEC, while replacing the Cys(83) and Cys(99) residues in this peptide with Ser completely negated this inhibitory activity. An additional synthetic cyclic peptide harboring the hydrophobic C-terminal tail of ChM-I clearly mimicked the inhibitory action of this protein on the migration of HUVEC and successfully inhibited tumor angiogenesis and growth in a xenograft mouse model of human chondrosarcoma.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Intercellular Signaling Peptides and Proteins/pharmacology , Membrane Proteins/pharmacology , Peptides, Cyclic/pharmacology , Recombinant Proteins/pharmacology , Amino Acid Sequence , Angiogenesis Inhibitors/chemical synthesis , Angiogenesis Inhibitors/chemistry , Animals , Cattle , Cell Line, Tumor , Cell Movement/drug effects , Cells, Cultured , Chondrosarcoma/blood supply , Chondrosarcoma/drug therapy , Chondrosarcoma/pathology , Cysteine/chemistry , Cysteine/genetics , Disulfides/chemistry , Dose-Response Relationship, Drug , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Intercellular Signaling Peptides and Proteins/chemistry , Intercellular Signaling Peptides and Proteins/genetics , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Mice, Inbred BALB C , Mice, Nude , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Neovascularization, Pathologic/prevention & control , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/chemistry , Recombinant Proteins/chemistry , Vascular Endothelial Growth Factor A/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Protein termini play important roles in biological processes, but there have been few methods for comprehensive terminal proteomics. We have developed a new method that can identify both the amino and the carboxyl termini of proteins. The method independently uses two proteases, (lysyl endopeptidase) Lys-C and peptidyl-Lys metalloendopeptidase (Lys-N), to digest proteins, followed by LC-MS/MS analysis of the two digests. Terminal peptides can be identified by comparing the peptide masses in the two digests as follows: (i) the amino terminal peptide of a protein in Lys-C digest is one lysine residue mass heavier than that in Lys-N digest; (ii) the carboxyl terminal peptide in Lys-N digest is one lysine residue mass heavier than that in Lys-C digest; and (iii) all internal peptides give exactly the same molecular masses in both the Lys-C and the Lys-N digest, although amino acid sequences of Lys-C and Lys-N peptides are different (Lys-C peptides end with lysine, whereas Lys-N peptides begin with lysine). The identification of terminal peptides was further verified by examining their MS/MS spectra to avoid misidentifying pairs as termini. In this study, we investigated the usefulness of this method using several protein and peptide mixtures. Known protein termini were successfully identified. Acetylation on N-terminus and protein isoforms, which have different termini, was also determined. These results demonstrate that our new method can confidently identify terminal peptides in protein mixtures.
Subject(s)
Metalloendopeptidases/metabolism , Peptide Fragments/metabolism , Proteins/chemistry , Proteins/metabolism , Serine Endopeptidases/metabolism , Acetylation , Animals , Chromatography, Liquid , Humans , Lysine/chemistry , Proteomics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
BACKGROUND: Chondromodulin-I (ChM-I) is an anti-angiogenic glycoprotein that is specifically localized at the extracellular matrix of the avascular mesenchyme including cartilage and cardiac valves. In this study, we characterized the expression pattern of ChM-I during early pregnancy in mice in vivo and its effect on invasion of trophoblastic cells into Matrigel in vitro. RESULTS: Northern blot analysis clearly indicated that ChM-I transcripts were expressed in the pregnant mouse uterus at 6.5-9.5 days post coitum. In situ hybridization and immunohistochemistry revealed that ChM-I was localized to the mature decidua surrounding the matrix metalloproteinase-9 (MMP-9)-expressing trophoblasts. Consistent with this observation, the expression of ChM-I mRNA was induced in decidualizing endometrial stromal cells in vitro, in response to estradiol and progesterone. Recombinant human ChM-I (rhChM-I) markedly inhibited the invasion through Matrigel as well as the chemotactic migration of rat Rcho-1 trophoblast cells in a manner independent of MMP activation. CONCLUSIONS: This study demonstrates the inhibitory action of ChM-I on trophoblast migration and invasion, implying the potential role of the ChM-I expression in decidual cells for the regulated tissue remodeling and angiogenesis at feto-maternal interface.
Subject(s)
Intercellular Signaling Peptides and Proteins/analysis , Membrane Proteins/analysis , Trophoblasts/metabolism , Animals , CHO Cells , Cell Line , Cell Movement , Cricetinae , Cricetulus , Decidua/metabolism , Female , Humans , In Situ Hybridization , Intercellular Signaling Peptides and Proteins/metabolism , Intercellular Signaling Peptides and Proteins/pharmacology , Matrix Metalloproteinase 9/metabolism , Membrane Proteins/metabolism , Membrane Proteins/pharmacology , Mice , Mice, Inbred C57BL , Pregnancy , RNA, Messenger/metabolism , Recombinant Proteins/pharmacology , Trophoblasts/cytologyABSTRACT
DEC1 (BHLHB2/Stra13/Sharp2) and DEC2 (BHLHB3/Sharp1) are basic helix-loop-helix (bHLH) transcription factors that are involved in circadian rhythms, differentiation and the responses to hypoxia. We examined whether DEC1 and DEC2 are involved in apoptosis regulation, in human breast cancer MCF-7 cells. We found that siRNA-mediated knockdown of DEC2 resulted in marked enhancement of apoptosis compared with that in control cells transfected with nonspecific siRNA. However, knockdown of DEC1 by siRNA did not affect cell survival. Knockdown of DEC2 affected the expression of mRNA or proteins related to apoptosis, such as Fas, c-Myc, caspase-8, poly (ADP-ribose) polymerase (PARP) and Bax. We also showed that tumor necrosis factor-alpha (TNF-alpha) up-regulates the expression of DEC1 and DEC2. DEC2 over-expression caused by the transfection of an expression vector reduced the amounts of cleaved PARP and caspase-8 induced by TNF-alpha treatment, whereas DEC1 over-expression increased it. Finally, we revealed that treatment with double knockdown against both DEC1 and DEC2 decreased the amounts of cleaved PARP and caspase-8 induced by DEC2 siRNA with or without TNF-alpha. These data indicate that DEC2 has an anti-apoptotic effect, whereas DEC1 has a pro-apoptotic effect, which are involved in the balance of survival of human breast cancer MCF-7 cells.
Subject(s)
Apoptosis/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , Adenosine Diphosphate Ribose/genetics , Adenosine Diphosphate Ribose/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Breast Neoplasms/genetics , Caspase 8/genetics , Caspase 8/metabolism , Cell Line, Tumor , Circadian Rhythm/genetics , Circadian Rhythm/physiology , Female , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Transcription Factors/physiology , Transfection , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , Tumor Suppressor Proteins/geneticsABSTRACT
Chondromodulin-I (ChM-I) is a 25-kDa glycoprotein that specifically localizes in the extracellular matrix of cartilage and negatively regulates angiogenesis. ChM-I comprises two domains: an N-terminal hydrophilic domain (domain 1) containing an N-linked glycosylation site and a C-terminal hydrophobic domain (domain 2) with all four disulfide bonds that are present in this protein. We generated a nonglycosylated recombinant human ChM-I (NG-hChM-I) and compared its bioactivity with that of the glycosylated form of human ChM-I (G-hChM-I) expressed in Chinese hamster ovary cells in vitro. NG-hChM-I exhibited the growth factor/inhibitor activity in the cultures of chondrocytes and vascular endothelial cells but required markedly higher doses. Although domain 1 is predicted to be hydrophilic per se on the basis of its amino acid sequence, NG-hChM-I remains insoluble in aqueous solution as much as ΔN-hChM-I that lacks the N-terminal 37 amino acids containing an N-glycosylation site. Circular dichroism measurements revealed that the content of α-helix was calculated to be 34% in G-hChM-I, whereas the content of the characteristic secondary structures in NG-hChM-I was distinctly lower than those in G-hChM-I. These results indicate that glycosylation in domain 1 is critical for the structural integrity for biological functions of ChM-I in vitro.
Subject(s)
Intercellular Signaling Peptides and Proteins/chemistry , Intercellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Amino Acid Sequence , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/genetics , Angiogenesis Inhibitors/metabolism , Angiogenesis Inhibitors/pharmacology , Animals , CHO Cells , Cell Line , Cells, Cultured , Chondrocytes/drug effects , Chondrocytes/metabolism , Circular Dichroism , Cricetinae , Cricetulus , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Glycosylation , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/pharmacology , Membrane Proteins/genetics , Membrane Proteins/pharmacology , Mice , Molecular Sequence DataABSTRACT
Chondromodulin-I (ChM-I) is a cartilage-derived angiogenesis inhibitor that has been identified as inhibitory to the growth activity of vascular endothelial cells. In our present study, we demonstrate the anti-angiogenic activity of recombinant human ChM-I (rhChM-I) in mouse corneal angiogenesis and examine its action. We focus on the VEGF-A-induced migration of vascular endothelial cells, a critical regulatory step in angiogenesis. In a modified Boyden chamber assay, nanomolar concentrations of rhChM-I inhibited the chemotactic migration of human umbilical vein endothelial cells (HUVECs) induced by VEGF-A as well as by FGF-2 and IGF-I. The ChM-I action was found to be endothelial cell-specific and independent of cell adhesions. Time-lapse analysis further revealed that rhChM-I markedly reduces VEGF-A-stimulated motility of HUVECs and causes frequent alterations of the moving front due to the appearance of multiple transient protrusions. This action involved the inhibition of cell spreading and the disrupted reorganization of the actin cytoskeleton upon VEGF-A stimulation. Consistent with these observations, rhChM-I was found to significantly reduce the activity of Rac1/Cdc42 during cell spreading, and the VEGF-A-induced Rac1 activity but not its basal activity in quiescent cells. Taken together, our present data suggest that ChM-I impairs the VEGF-A-stimulated motility of endothelial cells by destabilizing lamellipodial extensions.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Cell Movement/drug effects , Corneal Neovascularization , Endothelial Cells , Intercellular Signaling Peptides and Proteins/pharmacology , Membrane Proteins/pharmacology , Pseudopodia/drug effects , Vascular Endothelial Growth Factor A/pharmacology , Animals , Cell Movement/physiology , Cells, Cultured , Corneal Neovascularization/metabolism , Culture Media, Serum-Free , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelial Cells/physiology , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred BALB C , Pseudopodia/metabolism , Pseudopodia/ultrastructure , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Signal Transduction/physiologyABSTRACT
BACKGROUND: Claudin-1 is a membrane protein of tight junctions, and is associated with the development of various cancers. However, the significance of claudin-1 expression in cancer cells is not well understood. Here, we showed for the first time the anti-apoptotic effect of claudin-1 in human breast cancer MCF-7 cells. METHODS: Human breast cancer MCF-7 and T47 D cells were treated with or without tamoxifen, siRNA against claudin-1, or tamoxifen and claudin-1 siRNA. The samples were analyzed by RT-PCR, Western blotting or immunofluorescent staining. RESULTS: The expression of claudin-1 was upregulated in tamoxifen-treated MCF-7 cells, whereas the expression of claudin-1 was not altered in tamoxifen-treated T47 D cells. Knockdown of claudin-1 by siRNA increased the amount of poly (ADP-ribose) polymerase (PARP) regardless of tamoxifen treatment in MCF-7 cells, but not T47 D cells. In the cell membranes of the MCF-7 cells, tamoxifen treatment increased the amount of claudin-1, but decreased the amount of ß-catenin. Claudin-1 siRNA increased the amount of E-cadherin in the cytoplasm of the MCF-7 cells as well as the amount of ß-catenin in their cell membranes. CONCLUSION: These results indicate that claudin-1 has anti-apoptotic effects, and is involved in the regulation of the expression and subcellular localization of ß-catenin and E-cadherin in MCF-7, but not T47 D cells.
Subject(s)
Apoptosis , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Membrane Proteins/metabolism , Tamoxifen/pharmacology , Antineoplastic Agents, Hormonal/pharmacology , Cadherins/biosynthesis , Cell Line, Tumor , Cell Nucleus/metabolism , Claudin-1 , Dose-Response Relationship, Drug , Female , Gene Expression Profiling , Humans , RNA, Small Interfering/metabolism , beta Catenin/biosynthesisABSTRACT
DEC1 (BHLHB2/Sharp2/Stra13) and DEC2 (BHLHB3/Sharp1) are basic-helix-loop-helix (bHLH) transcription factors, involved in cellular differentiation, responses to hypoxia and circadian rhythms. We recently showed that the expression of DEC1 and DEC2 was up-regulated by hypoxia; however, the functions of these two factors under hypoxic conditions have not been elucidated in detail. It is well established that the expression of vascular endothelial growth factor (VEGF) is up-regulated by hypoxia, and the expression of VEGF in response to hypoxia depends on transcriptional activation by a heterodimer comprising hypoxia-inducible factor 1alpha (HIF-1alpha) and arylhydrocarbon receptor nuclear translocator 1 (ARNT1). In the present study, we showed that DEC2, but not DEC1, suppressed VEGF gene expression under hypoxic conditions. DEC2 protein was co-immunoprecipitated with HIF-1alpha but not with ARNT1. The binding of HIF-1alpha to the hypoxia response element (HRE) in the VEGF promoter was decreased by DEC2 over-expression, and increased by DEC2 knockdown. We also showed that the circadian expression of VEGF showed a reciprocal pattern to that of DEC2 in cartilage. DEC2 had a circadian oscillation in implanted Sarcoma 180 cells. We conclude that DEC2 negatively regulates VEGF expression and plays an important role in the pathological conditions in which VEGF is involved.
Subject(s)
Cell Hypoxia/genetics , Transcription Factors/metabolism , Vascular Endothelial Growth Factor A/genetics , Animals , Aryl Hydrocarbon Receptor Nuclear Translocator/genetics , Aryl Hydrocarbon Receptor Nuclear Translocator/metabolism , Base Sequence , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Binding Sites/genetics , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , Gene Expression Regulation , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Male , Mice , Mice, Inbred C57BL , NIH 3T3 Cells , Plasmids/genetics , Promoter Regions, Genetic , RNA, Small Interfering/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sarcoma 180/genetics , Sarcoma 180/metabolism , Transcription Factors/genetics , TransfectionABSTRACT
Claudin-1 is a membrane protein with four transmembrane domains, that is exclusively localized at cellular tight junctions. Recent studies have reported that claudin-1 plays an important role in cancer invasion and metastasis. However, the significance of claudin-1 in pancreatic cancer is still unknown. In the present study, we investigated the role of claudin-1 expression in pancreatic cancer growth using the PANC-1 human pancreatic cancer cell line. Treatment with tumor necrosis factor-alpha (TNF-alpha), an inflammatory cytokine, resulted in increased detection of 89 kDa products of poly-(ADP-ribose) polymerase (PARP), a marker of apoptosis, and decreased PANC-1 cell proliferation by 23%. Expression of claudin-1 was up-regulated by TNF-alpha in a concentration-dependent manner in PANC-1 cells. PANC-1 cells treated with TNF-alpha and siRNA against claudin-1 showed a 15% increase in proliferation; i.e. the cells transfected with siRNA against claudin-1 showed resistance to TNF-alpha-induced apoptosis. These results suggest that claudin-1 expression is responsible for TNF-alpha-dependent growth signals and the proliferation of pancreatic cancer cells.
Subject(s)
Cell Proliferation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Membrane Proteins/biosynthesis , Pancreatic Neoplasms/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Cell Line, Tumor , Claudin-1 , Collagen Type XI/metabolism , Dose-Response Relationship, Drug , Humans , Neoplasm Invasiveness , Neoplasm Metastasis , Pancreatic Neoplasms/pathology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Class II release factor 3 (RF3) from the sulfate-reducing bacterium Desulfovibrio vulgaris Miyazaki F, which promotes rapid dissociation of a class I release factor, has been overexpressed, purified and crystallized in complex with GDP at 293 K using the sitting-drop vapour-diffusion method. A data set was collected to 1.8 A resolution from a single crystal at 100 K using synchrotron radiation. The crystal belongs to space group P1, with unit-cell parameters a = 47.39, b = 82.80, c = 148.29 A, alpha = 104.21, beta = 89.78, gamma = 89.63 degrees . The asymmetric unit contains four molecules of the RF3-GDP complex. The Matthews coefficient was calculated to be 2.3 A(3) Da(-1) and the solvent content was estimated to be 46.6%.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/classification , Desulfovibrio vulgaris/chemistry , Peptide Termination Factors/chemistry , Peptide Termination Factors/classification , Sulfates/chemistry , Bacterial Proteins/genetics , Crystallization , Desulfovibrio vulgaris/genetics , Gene Expression Regulation, Bacterial , Peptide Termination Factors/genetics , Sulfates/metabolism , X-Ray DiffractionABSTRACT
The mutagenicities of various carcinogens induced by liver microsomes are increased in the presence of liver cytosol in rodents. It still remains, however, to be clarified which factor or factors in the cytosol enhance(s) the microsome-mediated mutagenicities. In the present study, we sought to identify the enhancing factor in liver cytosol prepared from rats using the microsome-mediated Salmonella mutagenicity induced by 2-amino-6-methyldipyrido [1,2-a:3',2'-d] imidazole (Glu-P-1). By a series of chromatographic steps, we purified a 16-kDa protein on SDS-PAGE from the cytosol of rat livers. Partial amino acid sequences of this protein revealed that the 16-kDa protein was copper, zinc-superoxide dismutase (CuZn-SOD). The purified CuZn-SOD enhanced the microsome-mediated mutagenicities of several heterocyclic amines and aromatic amines. Furthermore, bovine and human CuZn-SOD also enhanced the microsome-mediated mutagenicity of Glu-P-1. The CuZn-SOD caused an increase in the mutagenicity of N-hydroxylated Glu-P-1 formed from Glu-P-1 by the microsomes, although CuZn-SOD did not affect either the formation or the stability of the N-hydroxylated derivative. These findings suggest that the enhancing cytosol factor for the mutagenicity of Glu-P-1 is CuZn-SOD, which stimulates the mutagenicity of N-hydroxylated Glu-P-1 without changing its metabolism.