Fn Levels Research Articles

Clarithromycin inhibits rhinovirus‐induced bacterial adhesions to nasal epithelial cells

We investigated the inhibitory effects of clarithromycin (CM) on the rhinovirus (RV)-induced expression of fibronectin (Fn) and carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), which act as major receptors for Staphylococcus aureus and Haemophilus influenzae, respectively. We further investigated the effects of CM on secondary S. aureus and H. influenzae adhesions to RV-infected primary human nasal epithelial cells (HNECs). Cells were pretreated with 10 microM CM 24 hours before RV-16 infection and for 48 hours thereafter. The expression levels of Fn and CEACAMs were assayed by reverse transcriptase-polymerase chain reaction and Western blotting. Bacterial adhesion to cells was assessed by confocal microscopy and the fluorescence intensity of adherent bacteria was analyzed using Image-Pro Plus 5.1 (Media Cybernetics, Bethesda, MD). Clarithromycin significantly inhibited the RV-induced gene and protein expression of Fn and CEACAMs in HNECs. Compared with RV-infected cells, CM treatment significantly reduced the adhesion levels of S. aureus and H. influenzae in RV-infected HNECs to the levels seen in noninfected control cells. These findings indicate that CM has the potential to prevent secondary bacterial infections in RV-infected HNECs by inhibiting the expression of Fn and CEACAM, thereby interfering with bacterial adhesion.

Seminal fibronectin-like antigen and transferrin concentrations in infertile and fertile men.

Fibronectin like antigen (Fn) and transferrin (Trs) levels were measured in the seminal plasma of 40 fertile and 102 infertile men. The concentrations of both proteins were significantly (P < 0.001) higher in the fertile controls compared to the infertile groups. The levels of Fn and Trs (mean value +/- SEM) in the fertile men were 857.9 +/- 9.8 micrograms ml-1 and 164.0 +/- 6.5 micrograms ml-1, respectively; in the azoospermic men (n = 17) 552.7 +/- 24.65 micrograms ml-1 and 20.7 +/- 2.19 micrograms ml-1, respectively; in the group of severe oligozoospermia (n = 35) 568.34 +/- 25.7 micrograms ml-1 and 31.1 +/- 4.18 micrograms ml-1, respectively; in the moderate oligozoospermic group (n = 8) 572.50 +/- 47.9 micrograms ml-1 and 43.4 +/- 15.4 micrograms ml-1 respectively, and in the asthenozoospermic group (n = 26) 512.76 +/- 40.4 micrograms ml-1 and 47.0 +/- 7.9 micrograms ml-1, respectively. Of special interest was the finding from a group of 16 normospermic men (partners of couples with unexplained infertility) who showed significantly lower levels of Fn like antigen, 632.5 +/- 26.9 micrograms ml-1 (P < 0.001) and Trs 41.8 +/- 6.94 micrograms ml-1 (P < 0.0001) compared to normals. No correlation was found between Fn levels with either Trs or FSH levels or sperm count. In conclusion, our results indicate that male infertility is associated with changes in seminal plasma Fn like antigen concentrations and that it can be possibly used as an index of sperm fertilizing capacity.

Comparative Proteome Analysis of Serum from Acute Pulmonary Embolism Rat Model for Biomarker Discovery

Pulmonary embolism (PE) is a common, potentially fatal disease and its diagnosis is challenging because clinical signs and symptoms are nonspecific. In this study, to investigate protein alterations of a rat PE model, total serum proteins collected at different time points were separated by two-dimensional electrophoresis (2-DE) and identified using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Bioinformatics analysis of 24 differentially expressed proteins showed that 20 had corresponding protein candidates in the database. According to their properties and obvious alterations after PE, changes of serum concentrations of Hp, Fn, DBP, RBP, and TTR were selected to be reidentified by western blot analysis. Semiquantitative RT-PCR showed DBP, RBP, and TTR to be down-regulated at mRNA levels in livers but not in lung tissues. The low serum concentrations of DBP, RBP, and TTR resulted in the up-regulation of 25(OH)D3, vitamin A, and FT4 (ligands of DBP, RBP, and TTR) after acute PE in rat models. The serum levels of Hp and Fn were detected in patients with DVT/PE and controls to explore their diagnostic prospects in acute PE because the mRNA levels of Hp and Fn were found to be up-regulated both in lung tissues and in livers after acute PE. Our data suggested that the concentration of serum Fn in controls was 79.42 +/- 31.57 microg/L, whereas that of PE/DVT patients was 554.43 +/- 136.18 microg/L (P < 0.001), and that the concentration of serum Hp in controls was 824.37 +/- 235.24 mg/L, whereas that of PE/DVT patients was 2063.48 +/- 425.38 mg/L (P < 0.001). The experimental PE rat model selected in this study was more similar to the clinical process than the other existing PE animal models, and the findings indicated instant changes of serum proteins within 48 h after acute PE. The exploration of these differentially expressed proteins or their combination with existent markers such as D-dimer may greatly improve the accuracy of the diagnosis of acute PE, but diagnostic tests are still needed to evaluate the sensitivity and specificity of these markers and also the number of false positives and false negatives.

Fibronectin Is Not the Only Important Molecule Required for Fibrinogen/VWF-Independent Platelet Aggregation: Study of Thrombosis in a New Strain of Triple Deficient Mice.

Fibrinogen (Fg) has been considered essential for platelet aggregation. We demonstrated, however, that thrombi do form in Fg-deficient mice and in mice doubly deficient for both fibrinogen and von Willebrand factor (Fg/VWF−/−). We further reported that β3 integrin and thrombin are critical for this Fg/VWF-independent platelet aggregation. In Fg−/− or Fg/VWF−/− mice, platelet fibronectin (Fn) content is increased 3–5 fold. Furthermore, thrombus growth and stability are impaired in plasma Fn conditional deficient (M×-Cre, Fnflox/flox) mice. These data are consistent with the most recent studies of Fn assembly and suggest that Fn may support platelet thrombus formation. To examine whether Fn is the alternative key molecule which mediates platelet aggregation and thrombus formation in Fg/VWF−/− mice, we developed a novel strain of triple knockout (TKO) mice by breeding Fg/VWF−/− mice with M×-Cre+/− Fnflox/flox conditional knockout mice. Cre- littermates delivered from the same parents were used as a control. Fn depletion was induced by i.p. injections of polyinonic-polycytidylic acid. We found that TKO mice are viable with dramatically decreased levels of Fn in both the plasma (<2% of control) and platelets (<20% of control) as determined by immunoblot. No significant difference was found in peripheral blood cell counts or platelet surface adhesion proteins (GPIbα, P-selectin, β3 and β1 integrins) as compared with control mice. Unexpectedly, TKO platelet aggregation induced by thrombin and thrombin receptor activation peptide (TRAP) was enhanced in both platelet rich plasma and PIPES buffer (P<0.05). This phenomenon was also observed in a parallel-plate flow chamber. Significantly more TKO aggregates formed when fluorescently labelled whole blood was perfused over a collagen surface at 500s−1 (P<0.001). We also studied thrombus formation in TKO and Cre- control mice using intravital microscopy. Our preliminary data showed no obvious decrease in thrombus formation in TKO mice and injured arterioles occluded in one of two experimental mice. Our data suggest that Fn is not the only important molecule required for platelet aggregation and thrombus formation in Fg/VWF−/− mice. Further investigation of how Fn may inhibit Fg/VWF-independent platelet aggregation and the identification of what ligand(s) support this novel aggregation pathway may unveil an alternative haemostatic pathway in the absence of Fg and VWF.

Control of Platelet Fibronectin Internalization and Cell Surface Retention by Fibrinogen: Lessons from Hypofibrinogemic Patients.

Although fibrinogen (Fg) has been considered essential for platelet aggregation, we previously demonstrated that thrombi still formed in Fg-deficient mice. In these animals, platelet fibronectin (Fn) content is increased >3 fold, suggesting that Fn may play a role in thrombosis and hemostasis. However, it is unclear whether Fg controls human platelet Fn internalization. We recently identified severely hypofibrinogenaemic patient born from a consanguineous marriage (Xu X et al. Thromb and Haemost 2006; 95: 931–5). The patient and his father had respective plasma Fg levels of 1.7mg/dL (approximately 20 times less than the normal level) and 10.5 mg/dL. We examined the platelet Fn content of the patient, his parents, and healthy donors by immuno-electron microscopy (IEM), western blot, and flow cytometry (FACS) using a rabbit anti-human Fn-specific antibody (Sigma, USA). Significant increase of patient platelet Fn content was detected by all three methods, in which mean fluorescence intensity (MFI) of the patient's platelet Fn by FACS was 3-fold higher than healthy donors. No concomitant alteration of platelet thrombospondin-1 and vitronectin was detected. These data are consistent with our observations in animal models. To examine the role of platelet Fn content in platelet aggregation, we isolated human platelets via sepharose 2B gel filtration. Cell surface Fn expression after thrombin stimulation was examined by FACS. Surprisingly, although platelet Fn was increased, cell surface Fn was not increased. Rather, surface Fn was approximately 6-fold less than that of his father and 10-fold less than healthy donors. Using ELISA, we observed that the patient's platelet Fn was mainly released into the extracellular medium after thrombin treatment since the supernantant contained 2–3 fold higher levels of Fn than that of healthy donors (472.8 ± 13 ng/mL versus 213.5 ±4.7 ng/mL). Since fibrin has been reported as a receptor of Fn on platelet surface, we tested the hypothesis that high expression of platelet surface Fn in healthy donors was due to fibrin formation on the platelet surface after thrombin treatment. Gel filtered platelets from healthy donors were treated with either thrombin or thrombin receptor activation peptide (TRAP, at 100μM). The latter is able to induce platelet Fg release but not fibrin formation. We found that fibrin was indeed expressed on the platelet surface after thrombin but not TRAP treatment, which were detected by both FACS and IEM with a monoclonal anti-fibrin specific antibody (clone T2G1, Accurate Bilchemicals, USA). Consistent with these data, Fn was only significantly detected on the surface after thrombin but not TRAP treatment (MFI = 10.9 ± 2.5 versus 1.3 ± 0.3). To the best of our knowledge, this is first report that thrombin converts platelet-released Fg to fibrin on the platelet surface. Our data also suggested that Fg not only controls platelet Fn internalization but also regulates platelet surface Fn retention by the fibrin-Fn interaction.

Short-term and seasonal variations of exhaled and nasal NO in healthy subjects

Nitric oxide (NO) is an important biological mediator with a variety of cellular and tissue functions. Fractional exhaled (FE(NO)) and nasal NO (FN(NO)) are known to be altered in several respiratory tract disease and disorders. However, there are rather few studies into the reproducibility of FE(NO) or FN(NO) measurements in healthy subjects. The aim of this study was to examine both short- and long-term variations of FE(NO) and FN(NO). In addition to intraday, day-to-day and week-to-week variations, the seasonal variation was assessed. The data revealed that FE(NO) and FN(NO) levels were lower in the mornings compared to the afternoon values. There were clinically significant correlations in day-to-day (morning values 121.4+/-37.5 vs. 119.8+/-29.6, r=0.868, P=0.000), week-to-week (morning values 121.4+/-37.5 vs. 128.0+/-32.1, r=0.637, P=0.000) and seasonal (winter 133.5+/-29.7, summer 138.1+/-47.6, autumn 121.4+/-37.5, r=0.624, P=0.000) values of FN(NO). Day-to-day (morning values 14.6+/-6.7 vs. 14.2+/-5.1, r=0.784, P=0.000) week-to-week (morning values 14.6+/-6.7 vs. 15.4+/-7.3, r=0.738, P=0.000) and seasonal (autumn 14.6+/-7.2, winter 16.6+/-6.4, summer 17.4+/-8.0, r=0.709, P=0.000) levels of FE(NO) were also highly reproducible. Serial FE(NO) and FN(NO) measurements can be used in the monitoring of respiratory tract inflammation. Due to the diurnal variation of FE(NO) and FN(NO,) in long-term follow-up the measurements should be performed at the same time of day.

Comparative analysis of neuroectodermal differentiation capacity of human bone marrow stromal cells by various conversion protocols

Marrow stromal cells (MSCs) are capable of differentiating into multiple mesodermal tissues, including bone, cartilage, fat, and muscle. In addition, these cells were reported to differentiate in vitro and in vivo into cells expressing neuronal and glial markers. However, some controversy persists regarding the in vitro differentiation potential of MSCs into neurons and glia. Several in vitro studies, could not reproduce some of the transdifferentiation protocols. To clarify the neuroectodermal potential of human MSCs, we reevaluated some recent protocols used for the neuroectodermal differentiation of human MSCs in the present study. hMSCs expressed high levels of FN, FDZ1, PTCH as well as the multipotency marker OCT-4. Importantly, we found that many neuroectodermal genes, such as NES, ß-tubulin III, GFAP are already expressed by hMSCs. Interestingly, when comparing six common protocols for conversion of hMSCs towards neurons and glia only our multistep conversion and differentiation protocol was able to further induce a functional neuroectodermal phenotype with subsequent loss of their mesenchymal potential. All other protocols failed to further induce a functional neuroectodermal phenotype. At present we perform multiple co-culture analyses to further define the differentiation potential of hMSCs. Therefore we provide impetus that hMSCs can be efficiently differentiated into functional neuroectodermal cells by a multistep protocol. Our cell culture system provides a powerful tool for investigating the molecular mechanisms of neural differentiation in adult human NSCs. hmNSCs may therefore ultimately help to treat acute and chronic neurodegenerative diseases.

Rat bone marrow stromal cell osteogenic differentiation and fibronectin adsorption on chitosan membranes: The effect of the degree of acetylation

Cell adhesion, migration, and proliferation of a few anchorage-dependent cells cultured on chitosan (Ch) matrices are influenced by the degree of N-acetylation (DA) of Ch. In the present work, we examined the influence of the DA on the attachment, spreading, proliferation, and osteogenic differentiation of rat bone marrow stromal cells (rBMSCs). Ch membranes were characterized in terms of surface morphology, roughness, and wettability, and in terms of adsorption of an adhesive protein, fibronectin (Fn). Chs with DAs in the range of 4 to 49% were used. Among the Ch samples, the DA of 4% led to the highest Fn surface concentration, both from single protein solution and from diluted serum. Furthermore, the levels of Fn adsorbed from serum found for this DA were threefold higher than for the tissue culture polystyrene control, indicating that in the presence of competitive proteins Ch is more specific toward Fn adsorption than tissue culture polystyrene. rBMSCs cultured on Ch carrying a DA of 4% were able to spread, proliferate, and differentiate, reaching a higher level of osteogenic differentiation than on the control, despite the lower cell attachment observed for all Ch samples. Because the Ch sample with a DA of 4% showed the highest Fn adsorption from serum, we suggest that cell adhesion, spreading, and osteogenic differentiation of rBMSCs on Ch may be mediated by the adsorbed layer of Fn.

Prostaglandin E2 Stimulates Fibronectin Expression through EP1 Receptor, Phospholipase C, Protein Kinase Cα, and c-Src Pathway in Primary Cultured Rat Osteoblasts

Fibronectin (Fn) is involved in the early stages of bone formation, and prostaglandin E (PGE) is an important factor regulating osteogenesis. Here we found that PGE(2) enhanced extracellular Fn assembly in rat primary osteoblasts, as shown by immunofluorescence staining and enzyme-linked immunosorbent assay. PGE(2) also increased the protein levels of Fn by using Western blotting analysis. By using pharmacological inhibitors or activators or genetic inhibition by the EP receptor, antisense oligonucleotides revealed that the EP(1) receptor but not other PGE receptors is involved in PGE(2)-mediated up-regulation of Fn. At the mechanistic level, Ca(2+) chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester)), phosphatidylinositol-phospholipase C inhibitor (U73122), or Src inhibitor (PP2) attenuated the PGE(2)-induced Fn expression. Protein kinase C (PKC) inhibitor (GF109203X) also inhibited the potentiating action of PGE(2). Furthermore, treatment with antisense oligonucleotides of various PKC isoforms, including alpha, beta, epsilon, and delta, demonstrated that alpha isozyme plays an important role in the enhancement action of PGE(2) on Fn assembly. Flow cytometry and reverse transcription-PCR showed that PGE(2) and 17-phenyl trinor PGE(2) (EP(1)/EP(3) agonist) increased the surface expression and mRNA level of alpha5 or beta1 integrins. Fn promoter activity was enhanced by PGE(2) and 17-phenyl trinor PGE(2) in cells transfected with pGL2F1900-Luc. Cotransfection with dominant negative mutants of PKCalpha or c-Src inhibited the potentiating action of PGE(2) on Fn promoter activity. Local administration of PGE(2) or 17-phenyl trinor PGE(2) into the metaphysis of the tibia via the implantation of a needle cannula significantly increased the Fn and alpha5beta1 integrin immunostaining and bone volume of secondary spongiosa in tibia. Taken together, our results provided evidence that PGE(2) increased Fn and promoted bone formation in rat osteoblasts via the EP(1)/phospholipase C/PKCalpha/c-Src signaling pathway.

Reduced Plasma Fibronectin Leads to Delayed Thrombus Growth in the Injured Arterioles in Vivo.

In addition to von Willebrand factor and fibrinogen, plasma fibronectin (pFN) contributes significantly to thrombus development in arteries. Complete deficiency of plasma fibronectin (in pFN conditional knockout mice) affects thrombus formation and growth and subsequent occlusion of injured arterioles in vivo. We wanted to study a more physiological decrease of plasma fibronectin without having to induce interferon production in the mice to excise the FN gene. Reduced pFN levels are common in patients with liver disease, sepsis and following trauma or surgery. To evaluate the effect of a reduced amount of plasma fibronectin on thrombus formation at arterial shear rate, we used the ferric chloride model of arterial injury in fibronectin heterozygote mice which have 50% of the normal plasma level of FN. Two groups of mice were observed by intravital microscopy - FN heterozygote (FN+/−) and corresponding age matched wild-type controls (FN +/+). The reduced level of pFN did not affect the early platelet interaction with subendothelium but caused a delay of several minutes in appearance of the first thrombus in the injured arteriole (15 min in FN +/− vs 6 min in FN+/+, p<0.001). Although the thrombi were stably anchored to the vessel wall, the growth of the thrombus was slowed down because of the constant platelet shedding in fibronectin heterozygote mice. Consequently, this lack of firm adhesion of the platelets led to occlusion delay (36 min in FN +/− vs 28 min in FN+/+, p<0.01) with the majority of arterioles in the FN+/− mice not occluding by 40 minutes after injury. In conlusion, the phenotype of thrombosis in the mice with 50% of normal pFN level strikingly resembles the phenotype observed in mice with induced complete deficiency of pFN. This further emphasizes the fundamental role of this plasma protein in thrombosis in vivo in the arterial system.

Plasma fibronectin levels in cancer patients undergoing radiation therapy

Validated markers to predict ionizing radiation-induced normal tissue injury is required. Fibronectin (Fn), a multifunctional protein with major adhesive and opsonic properties, functions in the maintenance of tissue integrity, hence is a candidate marker of ionizing radiation effect. The specific aim of the present study is to evaluate the levels of plasma Fn in cancer patients (pts) undergoing radiation therapy (RT) in correlation with outcomes in terms of radiation toxicity.

Plasma fibronectin as a marker of sepsis

Objectives: To evaluate the value of plasma fibronectin (pFN) as a diagnostic marker of sepsis. Subjects and methods: Plasma FN was determined in patients showing sepsis-related symptoms who had blood cultures performed. These patients were assigned to one of two groups according to their clinical situation: (1) Clinical Septic Group: patients with sepsis according to American College of Chest Physicians/Society of Critical Care Medicine (ACCP/SCCM) criteria; (2) Fever Peak Group: patients who did not fulfil sufficient ACCP/SCCM criteria for sepsis. Two additional control groups were also established: (3) Non-infectious Diseases Control Group and (4) Healthy Control Group. Results: Plasma FN levels, microbiological and clinical data were compared among the different patient groups. For each group, the number of patients, median and mean pFN levels and the 95% confidence interval of the mean were: (1) n=43, 102 mg/l, 122 mg/l (100–144); (2) n=70, 185 mg/l, 207 mg/l (184–231); (3) n=22, 175 mg/l, 181 mg/l (151–211); and (4) n=22, 256 mg/l, 261 mg/l (229–292). Bonferroni’s test of multiple comparisons was able to detect a significant difference between pFN concentrations corresponding to the septic group, compared to the remaining groups ( p ANOVA<0.001). Conclusion: Plasma FN appears to act as a marker of sepsis in that patients showed diminished pFN levels. Along with other clinical and laboratory variables, the use of this marker would allow a rapid diagnosis of sepsis and limit the number of blood cultures to be processed and the number of antibiotic prescriptions, particularly when symptoms are insidious and diagnosis is doubtful. We propose further and more complex studies using a higher number of patients.

Efficacy of AT in pre-eclampsia: a case-control prospective trial

Pre-eclampsia is an extremely severe condition. It is associated with vasospasm, activation of the coagulation system and abnormal haemostasis. In pre-eclamptic patients increased plasmatic concentrations of fibronectin, laminin, von Willebrand factor (VWF) and endothelin are observed. Experimental studies on rats have also shown that the doses of antithrombin III (AT) needed to mediate anti-inflammatory processes are much higher than those required to obtain the anti-coagulant effect. The study aimed to evaluate the clinical efficacy of treatment with high AT doses (HD) in comparison with standard doses (SD). The primary endpoint was the prolongation of pregnancy defined as time (in days) from enrollment to delivery and to assess the maternal bleeding at and after delivery. The secondary endpoint was to demonstrate a role for AT in controlling haemostasis at conventional doses, and the inflammatory state at higher doses. The biochemical parameters assessed were: AT activity (%), Fibronectin (Fn), Fibrinogen, D-dimer, Uricemia, Proteinuria 24h, Protein C Reactive (PCR), Granulocyte Elastase and Endothelin. This study included 23 pre-eclamptic women. Patients were randomly subdivided into two groups: 10 patients ("cases") were treated with high doses of AT (6 vials: 3000 units) once daily for 5 days, or until delivery, while 13 women ("controls") were treated with doses of AT sufficient to maintain at least 80% of the activity. High-dose therapy was associated with prolongation of pregnancy by 2.5 days more when compared with controls (p = 0.03; Mann-Whitney test). The incidence of clinical significant bleeding was lower in cases than in controls (mean 550 mL vs. 650 mL, respectively). Preventive- and conservative-type treatment of moderate-severe pre-eclampsia, based on the administration high doses of AT, allows a significant prolongation of pregnancy, and thus a better neonatal outcome, as well as less maternal intra- and post-operative bleeding. Fn, PCR and elastase levels (markers of inflammation) decrease in the HD group in comparison with SD group. In the HD group, the AT plasma levels were obviously higher both at the end of the treatment (p < 0.0001) and after delivery (p = 0.03), in comparison with SD group. The fibrinogen and D-dimer levels were above the reference interval in both groups. TPA and PAI 1 were found to be significantly raised in the course of pre-eclampsia. In conclusion, the bio-chemical findings support a role for AT in controlling the haemostasis at conventional doses, and the inflammatory state at higher doses.

Elevated levels of beta-catenin and fibronectin in three-dimensional collagen cultures of Dupuytren's disease cells are regulated by tension in vitro.

BackgroundDupuytren's contracture or disease (DD) is a fibro-proliferative disease of the hand that results in the development of scar-like, collagen-rich disease cords within specific palmar fascia bands. Although the molecular pathology of DD is unknown, recent evidence suggests that β-catenin may play a role. In this study, collagen matrix cultures of primary disease fibroblasts show enhanced contraction and isometric tension-dependent changes in β-catenin and fibronectin levels.MethodsWestern blots of β-catenin and fibronectin levels were determined for control and disease primary cell cultures grown within stressed- and attached-collagen matrices. Collagen contraction was quantified, and immunocytochemistry analysis of filamentous actin performed.ResultsDisease cells exhibited enhanced collagen contraction activity compared to control cells. Alterations in isometric tension of collagen matrices triggered dramatic changes in β-catenin and fibronectin levels, including a transient increase in β-catenin levels within disease cells, while fibronectin levels steadily decreased to levels below those seen in normal cell cultures. In contrast, both fibronectin and β-catenin levels increased in attached collagen-matrix cultures of disease cells, while control cultures showed only increases in fibronectin levels. Immunocytochemistry analysis also revealed extensive filamentous actin networks in disease cells, and enhanced attachment and spreading of disease cell in collagen matrices.ConclusionThree-dimensional collagen matrix cultures of primary disease cell lines are more contractile and express a more extensive filamentous actin network than patient-matched control cultures. The elevated levels of β-catenin and Fn seen in collagen matrix cultures of disease fibroblasts can be regulated by changes in isometric tension.

Rapid Determination of Nitrogen Status in Pansy

Two experiments were conducted with pansy (Viola ×wittrockiana Gams `Bingo Yello') to determine the relationship between foliar nitrogen (% of dry weight) (FN) and either sap nitrate concentration (SN) in petioles or SPAD readings of foliage. FN was highly correlated to SN throughout both experiments (r = 0.80 to 0.91). FN was poorly correlated to SPAD readings early in both experiments (r = 0.54 to 0.65), but more highly correlated later when visual symptoms of N deficiency were apparent (r = 0.84 to 0.90). SN determined with the Cardy sap nitrate meter was a reliable predictor of FN in pansy, while SPAD readings were only reliable after symptoms of N deficiency were visually evident. FN can be predicted with SN using the following equation: log(SN) = 0.47*FN + 1.6 [r2 = 0.80, n = 132]. Growers and landscape professionals can use SN readings to predict FN levels in pansy, and thus rapidly and accurately diagnose the N status of their crop.

In human IgA nephropathy uteroglobin does not play the role inferred from transgenic mice

Background: Uteroglobin (UG)-knockout and UG-antisense transgenic mice develop clinical and pathological features of immunoglobulin A (IgA) nephropathy with heavy proteinuria. These models suggested that UG, an anti-inflammatory protein with high affinity for fibronectin (Fn), prevents the formation of IgA-Fn complexes and mesangial deposits in mice. We aim to elucidate whether similar mechanisms underlie the development and severity of human IgA nephropathy. Methods: Specific enzyme-linked immunosorbent assays were devised to detect serum levels of UG binding to Fn or incorporated into IgA-Fn complexes and IgA binding to Fn or collagen IV. Sera from 75 patients with IgA nephropathy with normal renal function and various degrees of proteinuria (0.2 to 5 g/d of protein) stable over the previous 3 months without therapy were investigated and compared with healthy controls. Results: Levels of UG binding to Fn were similar in patients with IgA nephropathy and healthy controls. UG incorporated into circulating IgA-Fn complexes, as well as levels of IgA-Fn complexes and IgA binding Fn and collagen IV, were significantly greater in patients than healthy controls. Greater amounts of UG incorporated into IgA-Fn complexes reduced the risk for proteinuria with protein greater than 1 g/d (odds ratio = 0.67; P < 0.001). Logistic regression analysis assigned a predictive value for proteinuria persistently greater than 1 g/d of protein to lower amounts of UG incorporated into IgA-Fn complexes (R = −0.267; P = 0.008) and increased binding of IgA to collagen IV (R = 0.214; P = 0.0003). Conclusion: This first report of human IgA nephropathy after the publication of the mouse model shows that UG is not reduced in circulation and is even increased in IgA-Fn complexes. Because aberrant IgA1 glycosylation is the event initiating IgA nephropathy in humans, we speculate that the enhanced incorporation of UG into IgA-Fn complexes might represent feedback to reduce the formation of macromolecular aggregates. © 2002 by the National Kidney Foundation, Inc.

The Low Density Lipoprotein Receptor-related Protein Mediates Fibronectin Catabolism and Inhibits Fibronectin Accumulation on Cell Surfaces

Low density lipoprotein receptor-related protein (LRP) is a member of the low density lipoprotein receptor family, which functions as an endocytic receptor for diverse ligands. In this study, we demonstrate that murine embryonic fibroblasts (MEF-2 cells) and 13-5-1 Chinese hamster ovary cells, which are LRP-deficient, accumulate greatly increased levels of cell-surface fibronectin (Fn), compared with LRP-expressing MEF-1 and CHO-K1 cells. Increased Fn was also detected in conditioned medium from LRP-deficient MEF-2 cells; however, biosynthesis of Fn by MEF-1 and MEF-2 cells was not significantly different. When LRP-deficient cells were dissociated from monolayer culture, increased levels of Fn remained with the cells, as determined by cell-surface protein biotinylation, suggesting an intimate relationship with cell surface-binding sites. The LRP antagonist, receptor-associated protein (RAP), promoted Fn accumulation in association with MEF-1 cells, whereas expression of full-length LRP in MEF-2 cells substantially decreased Fn accumulation, confirming the role of LRP in this process. Purified LRP bound directly to immobilized Fn, and this interaction was inhibited by RAP. Furthermore, MEF-1 cells degraded (125)I-Fn at an increased rate, compared with MEF-2 cells. (125)I-Fn degradation by MEF-1 cells was inhibited by RAP. These results demonstrate that LRP functions as a catabolic receptor for Fn. The function of LRP in Fn degradation and the ability of LRP to regulate levels of other plasma membrane proteins represent possible mechanisms whereby LRP prevents Fn accumulation on cell surfaces.

Predominant integrin ligands expressed by osteoblasts show preferential regulation in response to both cell adhesion and mechanical perturbation

Previous studies have demonstrated that both mechanical perturbation and cell adhesion induced the expression of osteopontin (opn) by osteoblasts (Carvalho et al. [1998] J. Cell. Biochem. 70:376-390). The present study examined if these same stimuli on osteoblasts would induce the expression of other integrin binding proteins, specifically fibronectin (fn) and bone sialoprotein (bsp). All three genes showed three- to four-fold maximal induction in response to both cell adhesion and a single 2-h period of an applied spatially uniform, dynamic biaxial strain of 1.3% at 0.25 Hz. Each gene, however, responded with a different time course of induction to mechanical strain, with bsp, fn, and opn showing their maximal response at 1, 3, and 9 h, respectively, after the perturbation period. In contrast, peak induction to cell adhesion was observed at 24 h for bsp and opn, while fn levels peaked at 8 h. Interestingly, while both opn and fn mRNA expression returned to base line after cell adhesion, bsp mRNA levels remained elevated. Examination of collagen type I and osteocalcin mRNAs showed unaltered levels of expression in response to either type of perturbation. A common feature of the signal transduction pathways, which mediate the gene expression in response to both cell adhesion and mechanical perturbation, was the activation of specific tyrosine kinases based on the ablation of the induction of these genes by the tyrosine kinase inhibitor genistein. While cycloheximide blocked the induction of all three mRNAs in response cell adhesion, it failed to block the induction of any of these genes in response to mechanical perturbation. Such results suggest that the induction of these genes after mechanical perturbation was mediated by an immediate response to signal transduction, while cell adhesion mediated effects secondary to signal transduction. Depolymerization of microfilaments with cytochalasin D had no effect on the overall expression of any of these genes in response to cell adhesion and only blocked the induction of opn expression in response to mechanical perturbation. These results suggest that cytoskeletal integrity is only selectively important in the signal transduction of certain types of stimuli and for the regulation of certain genes. In summary, both mechanical perturbation and cell adhesion stimulated the expression of integrin binding proteins. Furthermore, while there are common features in the signal transduction processes that mediate the induction of these genes in response to both stimuli, specific genes are separately regulated by precise mechanisms that are unique to both forms of stimuli.

Comparison of Fibronectin Content in Urinary Macromolecules between Normal Subjects and Recurrent Stone Formers

Objectives: Fibronectin (FN: 230 kD) is a multifunctional α₂–glycoprotein distributed throughout the extracellular matrix and body fluids. Recent studies have shown that a variety of molecules, including FN, inhibit the endocytosis of calcium oxalate (CaOx) crystals in vitro. We recently reported that FN was oversecreted from the renal tubular cells as a result of the stimulation of CaOx crystals, and inhibited the aggregation of CaOx crystals and the adhesion of CaOx crystals to the renal tubular cells. In the present study, we investigated the difference of FN content in urinary macromolecules (UMMs) between normal subjects and recurrent stone formers. Materials and Methods: Urinary parameters in relation to urolithiasis of normal subjects and recurrent stone formers were measured. Proteins in extracted UMMs from urine of normal subjects and recurrent stone formers were measured with a BioRad protein assay, GAGs in each UMMs with a modified DMB assay and the FN content with the ELISA method. Results: In urinary parameters, citrate was significantly higher in urine from normal subjects (female) than normal subjects (male) or recurrent stone formers, and the other parameters showed no differences between each group. The protein concentrations in UMMs showed no differences between each group. Normal subjects (male and female) showed a significantly higher concentration of GAGs than recurrent stone formers (with and without silent stone). Compared with normal subjects and recurrent stone formers without silent stones, higher FN levels were found in recurrent stone formers with silent stones. Normal subjects showed a significantly higher concentration of FN than recurrent stone formers without silent stones. No difference in FN level was shown between normal subjects (male) and normal subjects (female). Conclusion: Recurrent stone formers with silent stones showed a significantly higher concentration of FN in UMMs than normal subjects. This finding suggests that FN might be oversecreted from the renal tubular cells as a result of the stimulation of CaOx stones in vivo. Recurrent stone formers without silent stones showed a significantly lower concentration of FN in UMMs than normal subjects. From this finding it is suggested that FN might play a role as a potent inhibitor of CaOx urolithiasis in a clinical setting.

Transcriptional repression of the human fibronectin gene in laryngeal squamous cell carcinoma cells.

The aim of the experiments was to analyze the mRNA expression pattern and verify the repression of FN gene expression in laryngeal squamous cell carcinoma (SCC) cells in comparison with benign mucosal keratinocytes. Messenger RNA from SCC cells and benign keratinocytes was reverse transcribed and subjected to PCR following differential display (DD) analysis of the amplicons. Northern hybridization was carried out to confirm the reduction of the FN-mRNA expression in both laryngeal SCC cells and larynx carcinoma biopsies, in contrast to adjacent normal mucosa. Quantitation of protein synthesis was performed with homogenates of fresh tumor biopsies and their normal phenotypes, as well as of benign keratinocytes and laryngeal SCC cell lines, respectively, using ELISA. In the liposome-mediated transient transfection assay, FN promoter activity was analyzed by linking the FN promoter sequence to the chloramphenicol acetyltransferase (CAT) reporter gene. Transfection efficacy was monitored by co-transfection with pGL3 control vector. A 191 bp mRNA fragment revealing a 99% homology with the human FN-mRNA was detected, the expression of which was repressed 20 times as much in SCC cells as compared to benign phenotypes. Northern hybridization confirmed the distinctly reduced expression of FN-mRNA in both laryngeal SCC cells and larynx carcinoma biopsies, in contrast to adjacent normal mucosa. The quantitation experiments showed a correlation between the range of FN synthesis and the expression of FN-mRNA in cell lines and the biopsies which were used. The 1.28 kb FN gene promoter drove expression of the CAT reporter gene, which was similar to the FN-mRNA expression showed by DD and Northern hybridization. The mechanisms leading to the low level of FN in many tumors have not yet been sufficiently investigated. Our findings suggest that the decrease of FN in laryngeal SCC cells is transcriptionally regulated.