OBJECTIVETo investigate the effect of different dynamic tensional and compressive stress on the mRNA expression of collagen type I and fibronectin in cultured human periodontal ligament fibroblasts (hPDLF), and explore the regularity of functional change in hPDLF.

METHODSA new cyclic strain loading apparatus was used for mechanically loading. Cells cultured in vitro were loaded with three levels (1000 microstrain, 2000 microstrain, 4000 microstrain) of tensional and compressive forces and collected at different time (0 h, 0.5 h, 1 h, 4 h, 8 h,12 h) course after strain loading. The quantity of collagen type I and fibronectin mRNA was analyzed by means of quantitative real-time PCR with special primers of up- and down-regulated genes. Data were analyzed using SPSS version 10.0 software.

RESULTSDifferent magnitude and different kinds of mechanical forces as well as the force application time significantly changed the expression of collagen type I and fibronectin mRNA in hPDLF.

CONCLUSIONSDynamic mechanical forces could regulate the expression of collagen type I and fibronectin mRNA in hPDLF. Collagen type I and fibronectin participated in the mechanical signal transduction in human periodontal ligament fibroblasts.

Cells, Cultured ; Collagen Type I ; metabolism ; Fibroblasts ; metabolism ; Fibronectins ; metabolism ; Humans ; Periodontal Ligament ; cytology ; metabolism ; RNA, Messenger ; genetics ; Stress, Mechanical

BACKGROUNDThe efficacies of current treatments for invasive aspergillus (IA) are unsatisfactory and new therapeutic targets or regimens to treat IA are urgently needed. Previous studies have indicated that the ability of conidia to invade host cells is critical in IA development and fibronectin has a hand in the conidia adherence process. In the clinical setting, many patients who receive glucocorticoid for extended periods are susceptible to Aspergillus fumigatus (A. fumigatus) infection, for this reason we investigated the effect of glucocorticoid on conidia invasiveness by comparing the invasiveness of A. fumigatus conidia in the type II human alveolar cell line (A549) cultured with different concentrations of dexamethasone. We also explored the relationships between dexamethasone and fibronectin expression.

METHODSFollowing culture with anti-fibronectin antibodies and/or dexamethasone, type II human alveolar A549 cells were infected with conidia of A. fumigatus. After 4 hours, the extracellular free conidia were washed away and the remaining immobilized conidia were released using Triton-X 100 and quantified by counting the colony-forming units. The invasiveness of conidia was measured by calculating the invasion rate (%). The transcription of the fibronectin gene in cells cultured with different concentrations of dexamethasone for 24 hours was tested by fluorogenic quantitative RT-PCR while the expression of fibronectinin cells cultured for 48 hours was tested by Western blotting and immunocytochemistry.

RESULTSA significant reduction in the invasiveness of conidia was seen in the cells cultured with anti-fibronectin antibody ((14.42 ± 1.68)% vs. (19.17 ± 2.53)%, P < 0.05), but no significant difference was observed in cells cultured with a combination of anti-fibronectin antibody and dexamethasone (6.37 ± 10(-5) mol/L). There was no correlation between the dexamethasone concentration and the invasiveness of conidia after dexamethasone pretreatment of cells for 4 hours. In contrast, after pretreated for 24 hours, the invasiveness of conidia in the presence of 6.37×10(-5) mol/L dexamethasone ((24.66 ± 2.41)%) was higher than for the control ((19.17 ± 2.53)%) and the 0.25×10(-5) mol/L group ((19.93 ± 3.06)%), and the invasiveness in the 1.27×10(-5) mol/L group ((22.47 ± 2.46)%) was also higher than in the control, P < 0.05. The relative transcripts of the fibronectin gene after exposure to 6.37×10(-5) mol/L dexamethasone (9.19×10(-3)±1.2×10(-3)) was higher than for the control (4.61×10(-3)± 1.54×10(-3)) and the 0.25×10(-5) mol/L group (6.20×10(-3)± 1.93×10(-3)), and expression in the 1.27×10(-5) mol/L group (7.94×10(-3)± 2.24×10(-3)) was also higher than for the control, P < 0.05. High concentrations of dexamethasone promoted fibronectin production after culture for 48 hours.

CONCLUSIONSDexamethasone can increase invasiveness of A. fumigatus conidia by promoting fibronectin expression. This may partially explain why patients who are given large doses of glucocorticoids for extended periods are more susceptible to A. fumigatus infection.

Aspergillus fumigatus ; pathogenicity ; Cell Line, Tumor ; Dexamethasone ; pharmacology ; Fibronectins ; genetics ; metabolism ; Gene Expression ; drug effects ; Humans

OBJECTIVE: In order to supply an effective reference of early injury time estimation and explore the time limit of detection of EDA\EDB mRNA in human skin samples, the expression of alternative splicing segment of fibronectin--EDA\EDB in incised wound of human skin were studied. METHODS: Using in situ hybridization with DIG-labeled anti-sense RNA probe, the expression of FN EDA\EDB domain was detected in human skin incised wound at the early stage of injury (from 30 min to 3 h). RESULTS: The positive expression rates of FN-EDA\EDB immediately after injury and area far away from wound were same as the control group. The expression of FN-EDA\EDB in human skin incised wound showed a gradually increased tendency in early injury time (within 3 h). The positive expression cells were mainly distributed in basement cells of epidermis and the expression of EDA is much higher than EDB. It's difficult to detect EDA\EDB mRNA when the samples were deposited in air for 4 hour. CONCLUSION FN-EDA\EDB may be used as a sensitive mark for the estimation of early injury time. The in-situ hybridization technique is not applicable in the application.
Fibronectins/genetics* ; Forensic Medicine ; Humans ; In Situ Hybridization ; Protein Isoforms/genetics* ; RNA, Messenger/genetics* ; Skin/metabolism* ; Time Factors ; Wound Healing/physiology*

To express and identify fibronectin C-terminal heparin-binding domain (FNCH BD) polypeptides in Pichia pastoris expression system and study its function, the fragment of FNCHBD was amplified by PCR and inserted into pGEM-T vector. After sequenced, the fragment was inserted into pAo815SM vector, and then cloned into the expression vector pPIC9k. The recombinant plasmid was linerarized with restrict enzyme Sal I and transferred into the yeast host cell KM71 and GS115. The positive yeast clone was screened by G418 resistant, and the target protein was induced to express in the medium containing 0.5% methanol. The culture supernatant was collected and then was purified with membrane ultrafiltration and ion exchange chromatography. The purified product was analyzed with mass spectrogram, SDS-PAGE, Western blotting and heparin affinity chromatography. The results showed that the target protein was around 32 kDa and the purity of the product was above 95%. FNCHBD could be specifically recognized by fibronectin polyclonal antibody. These results suggest that FNCHBD could be expressed and purified successfully in Pichia pastoris, which provides a good strategy to further studies.
Fibronectins ; biosynthesis ; chemistry ; genetics ; Genetic Vectors ; Heparin ; metabolism ; Peptides ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Protein Binding ; Protein Structure, Tertiary ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics

This study explored the molecular mechanism underlying the Gegen Qinlian Decoction(GQD) promoting the differentiation of brown adipose tissue(BAT) to improve glucose and lipid metabolism disorders in diabetic rats. After the hypoglycemic effect of GQD on diabetic rats induced by high-fat diet combined with a low dose of streptozotocin was confirmed, the total RNA of rat BAT around scapula was extracted. Nuclear transcription genes Prdm16, Pparγc1α, Pparα, Pparγ and Sirt1, BAT marker genes Ucp1, Cidea and Dio2, energy expenditure gene Ampkα2 as well as BAT secretion factors Adpn, Fndc5, Angptl8, IL-6 and Rbp4 were detected by qPCR, then were analyzed by IPA software. Afterward, the total protein from rat BAT was extracted, and PRDM16, PGC1α, PPARγ, PPARα, SIRT1, ChREBP, AMPKα, UCP1, ADPN, NRG4, GLUT1 and GLUT4 were detected by Western blot. The mRNA expression levels of Pparγc1α, Pparα, Pparγ, Ucp1, Cidea, Ampkα2, Dio2, Fndc5, Rbp4 and Angptl8 were significantly increased(P<0.05) and those of Adpn and IL-6 were significantly decreased(P<0.05) in the GQD group compared with the diabetic group. In addition, Sirt1 showed a downward trend(P=0.104), whereas Prdm16 tended to be up-regulated(P=0.182) in the GQD group. IPA canonical pathway analysis and diseases-and-functions analysis suggested that GQD activated PPARα/RXRα and SIRT1 signaling pathways to promote the differentiation of BAT and reduce the excessive lipid accumulation. Moreover, the protein expression levels of PRDM16, PGC1α, PPARα, PPARγ, SIRT1, ChREBP, AMPKα, UCP1, GLUT1, GLUT4 and NRG4 were significantly decreased in the diabetic group(P<0.01), which were elevated after GQD intervention(P<0.05). Unexpectedly, the expression of ADPN protein in the diabetic group was up-regulated(P<0.01) as compared with the control group, which was down-regulated after the administration with GQD(P<0.01). This study indicated that GQD promoted BAT differentiation and maturity to increase energy consumption, which reduced the glucose and lipid metabolism disorders and thereby improved diabetes symptoms.
Adipose Tissue, Brown ; Animals ; Diabetes Mellitus, Experimental/genetics* ; Drugs, Chinese Herbal ; Fibronectins ; Glucose ; Lipid Metabolism ; Lipid Metabolism Disorders ; Rats

OBJECTIVE: To explore the effects of advanced glycation end products (AGEs) on the activity of NF-kappaB and fibronectin (Fn) synthesis in the endothelial cells in aged rats. METHODS: Endothelial cells were cultured in M199 from the aorta of 24 month old rats and divided into 3 groups: Group A (5 mmol/L glucose) as controls, Group B (25 mg/L AGEs for 48 h), and Group C (50 mg/L AGEs for 48 h). The activity of NF-kappaB was evaluated by immunofluorescence and the expression of Fn mRNA was measured by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Compared with the controls, AGEs induced the activity of NF-kappaB and increased the Fn mRNA expression in a concentration-dependent manner (P<0.05). CONCLUSION The activity of NF-kappaB and up-regulated expression of Fn mRNA induced by AGEs may contribute to chronic complications of diabetes.
Animals ; Cells, Cultured ; Endothelial Cells ; drug effects ; metabolism ; Fibronectins ; genetics ; metabolism ; Glycation End Products, Advanced ; pharmacology ; NF-kappa B ; genetics ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the role of connective tissue growth factor (CTGF) in the development of glomerulosclerosis by experimental alteration of fibronectin (FN) and Type IV collagen (Col IV) expression in cultured rat mesangial cells (MsC).

METHODSCTGF expression vector was transfected into MsC by Lipofectimine method. Protein and mRNA expression levels of CTGF, FN and Col IV were studied by Western blot and reverse transcription-polymerase chain reaction (RT-PCR) respectively.

RESULTSTwo of MsC clones (MCT-1 and MCT-2) with CTGF overexpression were successfully established and found to have significant increases of FN and Col IV at both protein and mRNA levels. Compared with the controls, the expression of FN protein and mRNA in the two clones were 3.2 times (P < 0.05) and 2.9 times (P < 0.05) higher respectively. The expression of Col IV protein and mRNA was 3.8 times (P < 0.01) and 2.4 times (P < 0.01) higher respectively.

CONCLUSIONCTGF up-regulates FN and Col IV expression in MsC and may play an important role in the development of glomerulosclerosis.

Animals ; Blotting, Western ; Cells, Cultured ; Collagen Type IV ; genetics ; metabolism ; Connective Tissue Growth Factor ; genetics ; metabolism ; Fibronectins ; genetics ; metabolism ; Genetic Vectors ; genetics ; Mesangial Cells ; cytology ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

Adhesion through microbial surface components that recognize adhesive matrix molecules is an essential step in infection for most pathogenic bacteria. In this study, we report that LigB interacts with fibronectin (Fn) through its variable region. A possible role for LigB in bacterial attachment to host cells during the course of infection is supported by the following observations: (i) binding of the variable region of LigB to Madin-Darby canine kidney (MDCK) cells in a dose-dependent manner reduces the adhesion of Leptospira, (ii) inhibition of leptospiral attachment to Fn by the variable region of LigB, and (iii) decrease in binding of the variable region of LigB to the MDCK cells in the presence of Fn. Furthermore, we found a significant reduction in binding of the variable region of LigB to Fn using small interfering RNA (siRNA). Finally, the isothermal titration calorimetric results confirmed the interaction between the variable region of LigB and Fn. This is the first report to demonstrate that LigB binds to MDCK cells. In addition, the reduction of Fn expression in the MDCK cells, by siRNA, reduced the binding of LigB. Taken together, the data from the present study showed that LigB is a Fn-binding protein of pathogenic Leptospira spp. and may play a pivotal role in Leptospira-host interaction during the initial stage of infection.
Animals ; Antigens, Bacterial/*genetics/metabolism ; Cell Line ; Dogs ; Enzyme-Linked Immunosorbent Assay ; Fibronectins/*metabolism ; Immunoglobulin Variable Region/genetics/*metabolism ; Leptospira/*genetics/metabolism ; Microscopy, Confocal ; Protein Binding/*genetics ; *Protein Structure, Tertiary ; RNA, Small Interfering/genetics

OBJECTIVE: To observe the effect of norcantharidin (NCTD) on the expression of mRNA and protein of fibronectin (FN), collagen IV(Col IV) and transforming growth factor-β1(TGF-β1) in human kidney proximal tubular epithelial (HK)-2 cells induced by high glucose. METHODS: HK-2 cells were incubated with serum-free DMEM for 24 h to synchronize cell growth, and then the cells were divided into 4 groups: Group C (5.5 mmol/L D-glucose), Group M (5.5 mmol/L D-glucose + 24.5 mmol/L-mannitol), Group HG (30 mmol/L D-glucose), and Group HG + NCTD (30 mmol/L D-glucose + 0.5-40 mg/L NCTD). Cytotoxicity of HK-2 cells induced by high glucose of NCTD was detected by Trypan blue dye exclusive assay. The effect of NCTD on the proliferation of HK-2 cells in high glucose was determined by MTT. The cells were collected to extract total RNA and protein at 6, 24 and 48 h after the incubation. The expression of FN, Col IV and TGF-β1 mRNA was examined by RT-PCR, and FN, Col IV and TGF-β1 protein was analyzed by Western blot. RESULTS: Trypan blue dye exclusive assay showed NCTD concentrations over 5 mg/L were rather toxic in HK-2 cells. The proliferation of HK-2 cells in high glucose was interrupted by interfered with 5 mg/L NCTD as measured by MTT (P<0.05). NCTD at 5 mg/L had a stronger inhibitory effect than NCTD at 2.5 mg/L. Real-time PCR and Western blot showed that the mRNA and protein expression of FN, collagen IV and TGF-β1 increased in HK-2 cells treated with high glucose (P<0.05), while that in cells treated by NCTD was dramatically inhibited (P<0.05). No change in these parameters was detected in the 30 mmol/L D-mannitol control group (P>0.05). CONCLUSION NCTD can downregulate FN, collagen IV and TGF-β1 mRNA and protein expression in HK-2 cells stimulated by 30 mmol/L D-glucose.
Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Cell Line ; Collagen Type IV ; genetics ; metabolism ; Down-Regulation ; drug effects ; Epithelial Cells ; cytology ; Fibronectins ; genetics ; metabolism ; Glucose ; pharmacology ; Humans ; Kidney Tubules, Proximal ; cytology ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism

This study was aimed to prepare the polypeptide of N-terminal heparin-binding domain of fibronectin(rhFNHN-29 polypeptide) with pichia expression system, to detect biological activity of recombinant polypeptide and investigate its effect on disseminated intravascular coagulation (DIC) in rats. The sequence of N-terminal heparin-binding domain of fibronectin was amplified from FNcDNA by PCR. The aim gene was cloned into T vector for selection. Then it was cloned into pAo815SM and pPIC9K vectors.Lined pPIC9K vectors were transformed into GS115 Pichia cells so as to express the aim polypeptide in Pichia expression system. The fermentation liquid were precipitated by 80% ammonium sulfate, and the further dissolved sediment were purified using S-100 column and SP column. Its activity of binding with heparin were detected by Western-blot. The established DIC rats (40 rats) were randomly divided into two groups. One group was treated with rhFNHN-29 polypeptide, and the other was treated with normal saline. The rats in the former group were injected with rhFNHN-29 polypeptide (10 mg/kg) through tail vein at 0.5 hour before, 2 hours and 4 hours after injection of LPS respectively. The rats in latter group were injected with equal volume saline. In addition, 20 normal rats injected with normal saline were as normal controls. 500 microl blood was taken from the rat vein, at 6 hours after the injection of LPS. White blood cell (WBC), hemoglobin (Hb) and platelets were tested from 50 microl blood. The rest 450 microl blood was used to isolate plasma for detecting TNFa level and coagulogram. The rats were killed at 24 hours after injection with LPS. Their livers, lungs, hearts, kidneys, and brain tissues were taken for histopathologic examination. The results showed that the aim polypeptide was successfully expressed in Pichia expression system. The expression level reached approximately 30 mg/L. The polypeptide had activity of binding with heparin antibody. In the experiment study of polypeptide effect on DIC in rats, the plasma TNFa level in polypeptide-treated group was lower than that in saline control group, the hemogram, coagulogram and histopathology were more obviously improved in polypeptide-treated group as compared with saline control group. It is concluded that the rhFNHN-29 polypeptide is successfully prepared, this polypeptide can antagonize DIC induced by endotoxin in rats.
Animals ; Disseminated Intravascular Coagulation ; therapy ; Endotoxins ; Female ; Fibronectins ; genetics ; immunology ; therapeutic use ; Heparin ; metabolism ; Male ; Peptides ; genetics ; therapeutic use ; Pichia ; metabolism ; Rats ; Rats, Sprague-Dawley