Obesity is a major health problem across the world, but there are few ways to effectively treat or manage it in the long term. Researchers are searching for more convenient, cost-effective and noninvasive therapies for overweight and obese people. Recent studies have illustrated that the microbiome of the body's different organs can be used as a vehicle for in-situ gene therapy. We suggest that the recombinant form of "Pichia pastoris" yeast expressing the hybrid protein of "irisin-furin-transferrin" under the control of the enolase 1 promoter is a new nutraceutical strategy to absorb fewer calories from intestinal nutrients, and induce a higher metabolic rate to expend more calories, similar to that from engaging in physical activity. By comparison, this method can be a long-term, noninvasive treatment and can be used for obese patients who have movement limitations.
Fibronectins ; genetics ; Furin ; genetics ; Genetic Therapy ; Humans ; Obesity ; therapy ; Pichia ; genetics ; Recombinant Fusion Proteins ; genetics ; Transferrin ; genetics ; Weight Loss

Alternative Splicing ; Fibronectins/genetics* ; Forensic Medicine ; Humans ; Protein Conformation ; Protein Isoforms/genetics* ; Wound Healing/physiology*

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*

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

AIMTo observe the effect of FAK-related non-Kinase (FRNK) plasmid on hepatic stellate cell (HSC) proliferation stimulated by fibronectin (FN).

METHODSFRNK plasmid was transfected into HSC with transient liposomal transfection. The proteins of FRNK, FAK and p-FAK(Tyr397) were assayed by Western blotting analysis. The proliferation of HSC was evaluated by improved MTT assay, and cell cycle pattern was determined by flow cytometry (FCM).

RESULTS(1) The expression of FRNK protein increased after FRNK transfected HSC, and it was at 48 h that the expression of FRNK protein was the highest (P < 0.01). The protein level of FAK was no significant difference between before FRNK plasmid transfection and after transfection (P > 0.05). The expression of p-FAK(Tyr397) protein was down-regulated after FRNK had been transfected in HSC, (P < 0.01). (2) The HSC proliferation inhibition rates at 12 h, 24 h and 48 h after FRNK transfection were 20.07%, 26.16%, 29.77%, respectively (P < 0.01). (3) Compared with the non-FRNK plasmid group, the FRNK-transfected HSCs almost arrested in G0/G1 phase (71.4 +/- 2.81 vs 48.9 +/- 1.66, P < 0.01).

CONCLUSIONAfter FRNK were transfected successfully in HSCs using lipofectamine, the phosphorylation of FAK was inhibited. The HSC proliferation was restrained in a time-dependent manner and the HSC was arrested in G0/G1 phase.

Cell Line ; Cell Proliferation ; Fibronectins ; Hepatic Stellate Cells ; cytology ; Humans ; Phosphorylation ; Plasmids ; genetics ; Protein-Tyrosine Kinases ; genetics ; Transfection

Three-dimensional (3D) culture of cells could closely mimic the in vivo situation with regard to cell function and microenvironment compared with plane monolayer cultured cells. In this paper, we established 3D culture of rat WB-F344 cells with rotary cell culture system (RCCS) to simulate microgravity environment, and examined cells proliferation, morphology, microstructure, E-cadherin protein quantity and mRNA expression of adhesion molecules by count the number of cells, optical microscope, transmission electron microscope and reverse transcriptase-polymerase chain reaction (RT-PCR). The results demonstrated that cells were polyhedron with lots of micovilli and mitochondria, which grow well and packed together densely to form irregular aggregates. Adjacent cells were connected with desmosome and tight junction. With the regard, the aggregates behaved 3D growth characteristics. Moreover, compared with control, mRNA level of Fibronectin and E-cadherin protein were increased, the changes maybe is the part mechanism in this microgravity simulated cells culture models which strengthened cells junction. This rotating 3D model might facilitate the study of interactions of cell-cell, cell-matrix and the mechanisms.
Animals ; Cadherins ; genetics ; Cell Adhesion ; Cell Culture Techniques ; methods ; Cell Proliferation ; Fibronectins ; genetics ; Rats ; Spheroids, Cellular ; ultrastructure ; Weightlessness Simulation

OBJECTIVETo investigate the pathogenic mechanism of vocal fold polyps and Reinke's edema.

METHODSA reverse-transcriptase polymerase chain reaction (RT-PCR) technique was adopted, mRNA levels of 9 proteins were measured in 12 vocal fold polyps, 2 Reinke's edema and 5 normal vocal folds (from total laryngectomy).

RESULTSThe results showed that in the vocal fold polyps, mRNA levels of collagenase and fibromodulin descended and levels of fibronectin increased (P < 0.05). mRNA levels of lysyl oxidase and hyaluronic acid synthase 2 had no statistic difference between lesions and normal vocal folds (P > 0.05). mRNA express of tropoelastin exon, elastase and hyaluronidase was positive in part of lesion tissue and positive in all normal vocal folds. mRNA of procollagen I was negative in both groups. In the Reinke's edema, mRNA express of fibronectin was close to vocal fold polyps and mRNA express of fibromodulin was close to normal vocal folds.

CONCLUSIONSIt was speculated phonation trauma and vocal fold restoring to trauma played an important role in pathogenic mechanism. Fibromodulin and fibronectin were two components involved in the disorders.

Adult ; Extracellular Matrix Proteins ; genetics ; Female ; Fibronectins ; genetics ; Humans ; Laryngeal Edema ; genetics ; pathology ; Male ; Middle Aged ; Polyps ; genetics ; pathology ; RNA, Messenger ; genetics ; Vocal Cords

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