We examined the fibronectin (FN) secretion of cultured trabecular meshwork (TM) cells in a normal human eye by indirect immunofluorescent technique using mouse anti-human FN monoclonal antibody and FITC-conjugated goat anti-mouse IgG. To localize FN on frozen sections of normal TM, which were obtained from 7 enucleated eyes owing to traumatic eyeball rupture, the same indirect immunofluorescent method was used. Immunoelectron microscopy was applied to demonstrate the distribution pattern of FN in the normal TM of 2 human eyes using an avidin-biotin-peroxidase complex method. In the tissue culture of TM, the TM cell walls and extracellular matrices showed an intense staining with antibody to FN. Indirect immunofluorescent staining of FN on frozen sections of TM showed strong positive reactions in the subendothelial region. There was no reaction in the central core of the trabecular beam. Immunoelectron microscopy revealed the reaction products to FN in the areas lining the trabecular endothelial cells.
Adolescent ; Adult ; Antibodies, Monoclonal ; Cells, Cultured ; Fibronectins/biosynthesis/*metabolism ; Fluorescent Antibody Technique ; Humans ; Male ; Microscopy, Immunoelectron ; Trabecular Meshwork/*metabolism/ultrastructure

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

AIMTo investigate the changes in the extracellular matrix protein expression and the morphology of seminiferous tubules in the testis of 88 azoospermic men.

METHODSThe patients were of the following categories: (1) 22 cases of Sertoli-cell-only syndrome, (2) 20 cases of spermatogenic arrest, and (3) 46 cases with hypospermatogenesis. Testicular sections were immunohistochemically stained for fibronectin, vimentin, laminin and collagen type IV. The seminiferous tubular diameter and the connective matrix zone (CMZ, the acellular zone between the basement membrane [BM] and the peritubular cells) thickness were measured. Seminiferous tubules were typed according to the thickness of the connective matrix in the lamina propria. The predominant tubule type and the Johnsen and Silber scores were determined.

RESULTSThe mean tubular diameter were 119 +/- 27, 117 +/- 20, and 140 +/- 38 microm for Groups 1, 2, and 3, respectively. Both the laminin and the type IV collagen were localized to the epithelial BM and peritubular cells. In most of the tubules, BM and peritubular cells were separated by a homogenous acellular layer, the CMZ, in which laminin, type IV collagen, fibronectin and vimentin were not present. It is perceived that the worse the testicular histology, the higher the thickness of the CMZ.

CONCLUSIONIn testis with no or low sperm production, the diameter of the seminiferous tubules is decreased, the thickness of the seminiferous tubular wall is increased and a CMZ is formed between the peritubular cells and the BM. The thickness of CMZ is increasing with the advancement of testiclar deterioration. The most important morphologic predictive factor for spermiogenesis is the predominant

Adult ; Collagen Type IV ; analysis ; biosynthesis ; Extracellular Matrix Proteins ; analysis ; biosynthesis ; Fibronectins ; analysis ; biosynthesis ; Humans ; Immunohistochemistry ; Laminin ; analysis ; biosynthesis ; Logistic Models ; Male ; Middle Aged ; Oligospermia ; metabolism ; pathology ; Seminiferous Epithelium ; metabolism ; pathology ; Spermatogenesis ; Testis ; chemistry ; metabolism ; pathology ; Vimentin ; analysis ; biosynthesis

OBJECTIVE: To investigate the expression of EBV-encoded latent membrane protein 2A (LMP2A) and epithelial-mesenchymal transformation(EMT) associated markers (E-cadherin and fibronectin) in nasopharyngeal carcinoma (NPC) and its clinical significance. METHOD: The expression of LMP2A, E-cad-herin and fibronectin proteins in 32 cases of chronic nasopharyngeal inflammation, 56 cases of NPC and 18 cases of NPC lymph node metastasis were examined byimmunohistochemical SP method. RESULT: (1)The positive rates of LMP2A in NPC and its lymph node metastasis were significantly higher than those of chronic nasopharyngeal inflammation (89. 3%vs 37. 5%o and 77. 8% vs 37. 5%) respectively (both P<0. 01); The normal expression rates of E-cadherin in NPC and its lymph node metastasis were significantly lower than those of chronic nasopharyngeal inflammation (33. 9% vs 90. 6% and 5. 6% vs 90. 6%) respectively (both P<0. 01); The positive rates of fibronectin in NPC and its lymph node metastasis were significantly higher than those of chronic nasopharyngeal inflammation (83. 9% vs 28. 1% and 72. 2% vs 28. 1%) respectively (both P<0. 01). (2) ZLMP2A expression were negatively correlated with normal expression of E-cadherin (r= -0. 387, P<0. 01), and were positively correlated with fibronectin (r= 0. 421, P<0. 01). (3)LMP2A, E-cadherin and fibronectin expression were significantly correlated with N stage and clinical stage (both P<0. 05), but the three proteins were not significantly correlated with M stage (both P> 0. 05). In addition, LMP2A and E-cadherin expression were significantly correlated with T stage (both P<0. 01). CONCLUSION LMP2A and fibronectin expressions were increased in NPC, but normal expression of E-cadherin were decreased. LMP2A may promote lymph node metastasis and malignant progression of NPC by induce EMT through downregulation of E-cadherin and upregulation of fibronectin.
Cadherins ; biosynthesis ; Carcinoma ; Epithelial-Mesenchymal Transition ; Fibronectins ; biosynthesis ; Humans ; Lymphatic Metastasis ; Membrane Proteins ; biosynthesis ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; Nasopharynx

OBJECTIVE: To determine the mechanism of peritoneal fibrosis of peritoneal mesothelial cells by high glucose. METHODS: The third passage human peritoneal mesothelial cells (HPMCs) from primary culture were divided into a control group (F(12)) and high glucose groups (F(12)+4% glucose) in different times (24, 48 h). The cell proliferation was assayed by the method of MTT (methylthiazoletetrazolium). The cell damage was measured by LDH (lactate dehydrogenase). The protein expression of fibronectin (FN), transforming growth factor-beta1(TGF-beta(1)) and connective tissue growth factor (CTGF) were detected by ELISA. The mRNA expression of FN, TGF-beta(1) and PAI-1 were detected by RT-PCR. RESULTS: High glucose suppressed the cell proliferation. The result of MTT showed that compared with the control group, the value of OD of high glucose groups at 24 or 48 h decreased significantly (P<0.01 or 0.01); The cell damage was enhanced in high glucose groups, at 24 or 48 h compared with the control group at the same time (all P<0.01). The protein expressions of TGF-beta(1), CTGF and FN in supernate fluid of cell culture were significantly enhanced when high glucose stimulated the HPMCs in the high glucose groups at 24 or 48 h compared with the control group at the same time (P<0.05 or 0.001). The expressions of FN, TGF-beta(1) and PAI-1 mRNA were upregulated in 24 h high glucose group compared with that of 24 h control group. CONCLUSION High glucose can suppress the HPMC proliferation and damage HPMCs. Increase of TGF-beta(1), CTGF, FN and PAI-1 of HPMCs stimulated by high glucose can promote the synthesis and decreased degradation of extracellular matrix, which might be related with the mechanism of peritoneal fibrosis of peritoneal mesothelial cells by high glucose.
Cell Proliferation ; drug effects ; Cells, Cultured ; Epithelial Cells ; metabolism ; pathology ; Fibronectins ; biosynthesis ; genetics ; Glucose ; pharmacology ; Humans ; Peritoneal Dialysis ; Peritoneum ; metabolism ; pathology ; Plasminogen Activator Inhibitor 1 ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Transforming Growth Factor beta ; biosynthesis ; genetics

An experimental model of reproducible focal cerebral contusions in rats was made by a free-drop impacting right hemisphere. The expression of fibronectin and its mRNA after cerebral contusion were detected respectively by immunohistochemical staining and in situ hybridization. Results indicated that the expression of fibronectin and its mRNA increased after injury, and there existed a relationship between increased fibronectin and its mRNA and different intervals after brain injury. It is inferred that the expression of fibronectin and its mRNA can be used for timing of brain injuries and distinguishing antemortem and postmortem brain contusions.
Animals ; Brain Injuries/metabolism* ; Fibronectins/biosynthesis* ; Immunohistochemistry ; In Situ Hybridization ; Male ; Postmortem Changes ; RNA, Messenger/biosynthesis* ; Rats ; Rats, Sprague-Dawley ; Time Factors

This study inquired into the formation of the "overlapping growth" of hepatoma cells through quantitative characterization on the growth of hepatoma cells in situ by means of morphological observation, Image Tool computer analytic system, statistical analysis as well as the experimental methods of cell mechanics and biochemistry. The results were as follows: (1) The ability of hepatoma cells to regulate cell morphological deformation was better than that of hepatic cells; (2) While we were using micropipette aspiration technique to suck the "overlapping growth plaque" of hepatoma cells, the "overlapping growth plaque" fell off from the substrate, leaving a blank area; (3) Integrin expression of hepatoma cells was more obvious than that of hepatic cells; (4) Fibronectin (Fn) down-regulated the integrin expression in the hepatoma cells cultured on the Fn coated surface, enhanced the cells' adhesion ability and morphological stability, but reduced the formation and aggregation of the round cells. These results indicated (1) The so-called overlapping growing area was actually formed by many closely arrayed and piled round cells; (2) The production of round cells may be caused by integrin abnormal expression and the effect on the hepatoma cells adhesion stability; (3) The formation of "overlapping growth plaque" in hepatoma cells is related to the round cells' congregation induced by the high frequency morphological transformation.
Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Adhesion ; Cell Proliferation ; Fibronectins ; metabolism ; Humans ; Image Processing, Computer-Assisted ; Integrin beta Chains ; biosynthesis ; genetics ; Liver Neoplasms ; metabolism ; pathology ; Tumor Cells, Cultured

OBJECTIVE: To observe the means of fibronectin(FN) alternative splicing and the expression of EIIIA-FN variant in rat skin after bruise, for the sake of providing some help for forensic estimation of wound interval. METHODS: Total RNA was isolated from wounded skin, and reverse transcription polymerase chain reaction was conducted to amplify target segments. RESULTS: Detectable EIIIA+(526 bp) segments, lacked in normal organize, was amplified at 1 h after experimental wound, and the levels were increased within 24 h. CONCLUSION The alternative splicing EIIIA-fibronectin variant would be a satisfied criterion for research of skin injury.
Alternative Splicing ; Animals ; Epithelium/metabolism* ; Fibronectins/genetics* ; Forensic Medicine ; Integrin alpha4beta1/biosynthesis* ; RNA, Messenger/metabolism* ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Skin/metabolism* ; Time Factors

OBJECTIVETo study the changes of fibronectin (FN) and type IV collagen (ColIV) expression in cultured rat mesangial cells (MsC) transfected with Smad 2 vector and to investigate the molecular mechanism of glomerular extracellular matrix accumulation in glomerulosclerosis via transforming growth factor-beta (TGF-beta)/Smad signal pathway.

METHODSSmad 2 vector was transfected into MsC by calcium phosphate. Western blot analysis was used to detect Smad 2 protein. The expression of FN and ColIV proteins and their mRNAs was determined by Western blot and reverse transcriptase-polymerase chain reaction respectively.

RESULTSFour MsC clones (T-12, T-31, T-35, T-40) with Smad 2 overexpression were established. The expression of FN and ColIV was significantly increased at mRNA and protein levels in two (T-12, T-31). Compared with controls, the expression of FN proteins and mRNAs in these two clones was 2.4 times (P < 0.05) and 2.7 times (P < 0.05) higher respectively. The expression of ColIV proteins and mRNAs was 2.9 times (P < 0.01) and 3.3 times (P < 0.01) higher respectively.

CONCLUSIONSIt is postulated that Smad 2 in TGF-beta/Smad signal pathway is important in promoting the accumulation of FN and ColIV in sclerotic glomeruli of diseased kidneys.

Animals ; Cells, Cultured ; Collagen Type IV ; biosynthesis ; genetics ; Fibronectins ; biosynthesis ; genetics ; Genetic Vectors ; Mesangial Cells ; cytology ; metabolism ; Plasmids ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Signal Transduction ; Smad2 Protein ; genetics ; metabolism ; Transfection ; Transforming Growth Factor beta ; metabolism

OBJECTIVETo investigate the effects of Tongluo Recipe on the expression of collagen IV (Col IV), fibronectin (FN), laminin (LN), transforming growth factor-beta1 (TGF-beta1) in rat renal tissues and explore the mechanism underlying these effects in rats with glomerular sclerosis.

METHODSThe pathological changes in the renal tissues of rats with glomerular sclerosis were observed microscopically, and the expressions of Col IV, FN, LN, and TGF-beta1 were detected using immunohistochemical staining and image analysis system.

RESULTSTongluo Recipe significantly decreased the expressions of Col IV, FN, LN and TGF-beta1 in the renal tissue of rats with glomerular sclerosis (P<0.05 or P<0.01) and obviously alleviated the renal pathologies (P<0.01).

CONCLUSIONThe therapeutic effects of Tongluo Recipe are probably mediated by lowered expressions of Col IV, FN, LN and TGF-beta1.

Animals ; Collagen Type IV ; biosynthesis ; Drugs, Chinese Herbal ; therapeutic use ; Fibronectins ; biosynthesis ; Glomerulosclerosis, Focal Segmental ; drug therapy ; metabolism ; pathology ; Kidney ; drug effects ; metabolism ; pathology ; Male ; Phytotherapy ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; biosynthesis ; Treatment Outcome