Adhesion of human dermal reticular fibroblasts on complementary fragments of fibronectin: aging in vivo or in vitro

Attachment, spreading, and microfilament reorganization have been evaluated in human dermal reticular fibroblasts isolated from the inner, upper aspect of the arm of a newborn male (RET5 cells) and a 78-year-old male (RET8 cells). Substrata were tested using a set of complementary fragments from individual polypeptide chains of human plasma fibronectin (pFN) or cellular FNs (cFN). With both cell classes, fragments containing the C-terminal heparin-binding (HepII) domain only elicited linear bundles of microfilaments in spreading cells but no stress fibers; fragments containing the RGDS-dependent cell-binding (CellI) domain elicited only partial spreading with condensations of F-actin at ruffling membranes and at other regions along the plasma membrane. The minimum sequence required to obtain responses identical to those on intact pFN (broad spreading with extensive stress fiber formation) was found in fragment 155 (F155) from the beta chain of pFN; F155 contains both HepII and CellI domains. In contrast, the analogous fragment from the alpha chain of pFN (F145) was notably less effective for generating stress fibers. This evidence along with the better attachment, spreading, and microfilament bundle formation on the HepII fragment from the beta chain than the analogous fragment from the alpha chain indicates that the extra type III homology unit permits more effective interaction of beta chain fragments with cell-surface heparan sulfate proteoglycan and possibly integrin (binding efficiency to the substratum was similar for fragments from both chains). Therefore, alternatively spliced sequences that neighbor binding domains can play significant roles in the interaction of the domain with cell-surface receptors of dermal fibroblasts. Comparison of RET5 responses with those of RET8 cells has identified changes in adhesive mechanisms as cells undergo "aging" processes. Attachment and microfilament bundle formation were far more effective for RET5 cells than for RET8 cells on any of the HepII fragments. Conversely, RET8 cells were far more sensitive to an RGDS-containing peptide in their medium on CellI fragments than RET5 cells. These results together indicate that in vivo aging leads to greater dependence upon cell-surface integrin binding and less dependence upon heparan sulfate proteoglycan binding for responses on FN matrices. When RET5 cells entered senescence (in vitro aging), they also became much more sensitive to peptide A. On several fragments and on intact pFN, RET8 cells generated very thick stress fibers that were observed only on one fragment with RET5 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Low colonisation on artificial substrata in arctic Spitsbergen

High polar communities tend to be young because of the frequent and intense impact of ice (scour), so colonisation patterns are particularly important. Yet, despite a wealth of studies at temperate and tropical latitudes, we know of no hard substratum settlement/colonisation experiments reported north of 60°N, to date. Here we report on fauna encrusting square panels immersed at 12 m depth in Isforden, Spitsbergen (Svalbard) after 2 and 3 days, a week and a year. Arctic colonisation is slow but is not species poor. We found no colonists present after 2 and 3 days but two panels were colonised by a bryozoan and polychaetes after a week. After a year immersion, three panels were 3, 5 and 11% covered with a mean of ∼247 colonists. This is about an order of magnitude lower than has been described from most studies elsewhere, but not as low as has been recorded at an Antarctic locality. Most individual colonists (80–93%) were polychaetes (Spirorbis tridentatus) but most of the species were bryozoans. The Arctic is widely described as taxon poor compared with elsewhere, but at the local scale we investigated, species richness (20) was as high or higher than in many similar colonisation studies along the north Pacific or Atlantic coasts. In striking contrast, no settlement panel study has yielded fewer higher taxa (2 phyla, 3 classes) than this high arctic study.     

Seaweed succession on artificial reefs on different bottom substrata

Artificial rest reefs were set on sandy and rocky bottoms at 5–10 m depth along the coast of southern Japan. Mature thalli ofSargassum, Gelidium and other seaweds were transported from other coastal areas, packed in mesh bags and attached to the reefs to start the beds. After one year, the seaweed flora on the reef on a sandy bottom consisted of more than 20 species, includingSargassum spp. andGelidium amansii, which are important animal food species. Coralline algae were the dominants on the rocky bottom reefs. The lower biomass on reefs on the rocky bottom was due to grazing by urchins. The same number of species was present in the first and second years on reefs on sandy bottoms, but there were moreSargassum thalli the second year.Maximum algal biomass of the artificial reef in May of the second year was 9998 g wet wt m^–2 in sandy areas, 441 g wet wt m^–2 in boulder areas and 228 g wet wt m^–2 in rocky areas. Reefs on rocky bottoms continued to be covered by coralline algae and several species ofCodium andDictyota.     

Studies of extracellular fibronectin matrix formation with fluoresceinated fibronectin and fibronectin fragments

Fluorescein isothiocyanate conjugated human plasma fibronectin, 70-kDa collagen-binding, 60-kDa central, 60-kDa heparin-binding, 180-kDa heparin, collagen-binding fibronectin fragments and gelatin were used to study extracellular fibronectin matrix formation. Exogenous fibronectin, gelatin, 70-kDa collagen-binding and 180-kDa heparin, collagen-binding fragments were shown to be able to bind specifically to preexisting extracellular matrix of living fibroblasts. The results suggest that: (i) Fibronectin matrix formation may occur through a self-assembly process; (ii) the NH2-terminal part of fibronectin is responsible for fibronectin-fibronectin interaction during fibronectin fibril formation; (iii) plasma fibronectin may be the source for tissue fibronectin.     

Adhesion of glycosaminoglycan-deficient chinese hamster ovary cell mutants to fibronectin substrata

We have examined the role of cell surface glycosaminoglycans in fibronectin-mediated cell adhesion by analyzing the adhesive properties of Chinese hamster ovary cell mutants deficient in glycosaminoglycans. The results of our study suggest that the absence of glycosaminoglycans does not affect the initial attachment and subsequent spreading of these cells on substrata composed of intact fibronectin or a fibronectin fragment containing the primary cell-binding domain. However, in contrast to wild-type cells, the glycosaminoglycan- deficient cells did not attach to substrate composed of a heparin- binding fibronectin fragment. Furthermore, the wild-type but not the glycosaminoglycan-deficient cells formed F-actin-containing stress fibers and focal adhesions on substrata composed of intact fibronectin. We propose, therefore, that cell surface proteoglycan(s) participate in the transmembrane linking of intracellular cytoskeletal components to extracellular matrix components which occurs in focal adhesions.     

Near ultraviolet circular dichroism spectroscopy of plasma fibronectin and fibronectin fragments

Comparison of the near uv CD spectrum of human plasma fibronectin with the spectra of the three major leukocyte elastase fragments 25 Kd, 60 Kd and $\text{140}\text{160}$ shows that the aromatic residues of these fragments are in different environments. In particular, the ellipticity bands associated with tryptophan in the 290–300 nm region differ for each fragment. The results also show that intrachain disulfide bridges are important in stabilizing regions of the fibronectin molecule against the structure-disrupting effects of 8M urea.     

A comparison of diatom community structure on natural and artificial substrata

Artificial substrata have been used in diatom studies for almost 100 years. However, concern still exists over whether diatom communities developing on artificial substrata accurately represent communities developing on natural substrata. This study compares the diatom communities colonising glass slides and clay tiles in two coastal dune lakes, and compares these communities to the naturally occurring communities in the epipelon, epilithon, and epiphyton. Both glass microslides and clay tiles, incubated for three separate periods ranging from 29 to 68 days, resulted in replicate substratum samples supporting similar diatom community compositions at each site. The degree of variation between artificial substrata communities at different sites, and between the two artificial substrata types, was generally no more than the degree of variation between communities on different types of natural substrata. Additionally, the composition of the diatom communities on the artificial substrata was representative of the community composition on the natural substrata. The effects of incubation period and siting are discussed.     

Binding of fibronectin to alpha-granule-deficient platelets

Most of the proposed functions for fibronectin involve its interaction with cells, yet the molecular nature of cellular fibronectin binding site(s) has remained obscure. Thrombin induces saturable platelet binding sites for plasma fibronectin and concurrently stimulates surface expression of a number of platelet alpha-granule constituents including thrombospondin and fibrin which are known to interact with fibronectin. To test the hypothesis that these (or other alpha-granule proteins) mediate plasma fibronectin binding, we used platelets of patients with the Gray Platelet Syndrome. These cells were deficient in thrombospondin, beta-thromboglobulin, platelet factor 4, fibronectin, and fibrinogen as measured in radioimmunoassay. They also had reduced von Willebrand factor content as judged by immunofluorescence. At plasma fibronectin inputs from 0.03 to 3 times the apparent kilodalton, these Gray platelets bound virtually identical quantities of fibronectin as normal cells. Thus, platelets containing 1,500 molecules of thrombospondin per platelet could bind more than 100,000 molecules of plasma fibronectin per cell following thrombin stimulation. These data preclude any simple model in which newly surface expressed thrombospondin (or other alpha-granule protein) functions as the major thrombin-stimulated plasma fibronectin receptor in this cell type.     

Phosphorus removal from eutrophic lakes using periphyton on submerged artificial substrata

The potential of periphyton for phosphorus removal from lakes has been investigated using a novel method involving polypropylene (PP) substrate carriers submerged in the pelagial. The study area Lake 'Fühlinger See' in Cologne (Germany) is a complex of mesoeutrophic gravel pit lakes. The whole site is intensively used as a recreation area. Visitors are thought to be the most important single contributors to lake eutrophication. Carriers were exposed at different depths (2, 3.5, 5 m), for different time intervals (1–8 months) and from March to November PP-sheets were readily colonised by periphyton and a biofilm consisting mainly of benthic diatoms developed. Seasonal variability of periphyton on substrates was observed since filamentous green algae colonised the artificial substrates mainly between July and November. Chlorophyll a content of periphyton on the PP-fleece was up to 240-fold higher than chlorophyll a concentrations in the same volume in the epilimnion. Up to around 100 mg of total phosphorus per m² PP-fleece was bound and can be eliminated from the lake by removal of the substrate carriers together with the periphyton after four months of exposure. Though large-scale validations are needed, this method may be applicable as a technique to harvest phosphorus from the water column in larger-scale settings.     

A photographic method for estimating chlorophyll in periphyton on artificial substrata

The collection of time-series of periphyton biomass is a difficult task due to the destructive nature of the standard methods. A non-destructive method based on photography and digitalization, for the estimation of Chla of periphyton colonizing artificial substrata is presented. The standard spectrophotometric method was used to obtain a calibration curve. The relative errors of the proposed method were similar to those of other published methods. The photographic method should be used when a large quantity of samples from the same community is needed and a high precision on the individual measurement is not required.     

Evidence for a dual mechanism of chick embryo fibroblast adhesion on fibronectin and laminin substrata

Eight-day-old chick embryo fibroblasts were shown to adhere specifically to fibronectin and laminin substrata. Moreover, the Scatchard analysis reveals 540,000 binding sites per cell for the fibronectin with a dissociation constant (Kd) of 1.35 microM and 5,500 binding sites per cell for laminin with a Kd of 1.5 nM. Furthermore, cell-fibronectin interactions are mediated by plasma membrane proteins of high molecular weight (HMW) (150K and 125K) insensitive to trypsin treatment and low molecular weight (LMW) proteins (95K, 80K, 65K and 45K) sensitive to trypsin treatment. Adhesion of 8-day-old chick embryo fibroblasts on laminin is mediated by plasma membrane proteins highly sensitive to trypsin treatment. Regarding the paucity of laminin-binding sites, the identification of laminin receptor could not be achieved. Nevertheless, this study provides quantitative and qualitative evidences for different mechanisms of 8-day-old chick embryo fibroblasts on laminin and fibronectin.     

Effect of fibronectin fragments on macrophage phagocytosis of gelatinized particles

Rat plasma fibronectin enhances the binding and ingestion of gelatin-coated, formalin-fixed, or tanned sheep erythrocytes by elicited rat peritoneal macrophages. Fibronectin binding to the gelatinized erythrocytes is required for this enhancement, because macrophages preferentially recognize the surface bound molecule. This enhancement of particle uptake by fibronectin required the presence of a renewable, trypsin-sensitive component(s) on the macrophage surface (fibronectin receptor). When subjected to plasminolysis for 3 hr, fibronectin was degraded into gelatin-binding fragments (170 to 210 kd) and a 25-kd nongelatin binding fragment. The 170 to 210 kd gelatin binding fragments retained uptake-enhancing activity but were less active on a weight and molar basis than intact, dimeric fibronectin. The nongelatin binding 25 kd fragment alone did not enhance uptake. These results indicate that the sites for interaction with both the gelatinized erythrocyte surface and macrophages are retained on 170 to 210 kd fragments. However, the fibronectin dimeric structure is required for maximal expression of opsonic activity.     

The effect of fibronectin fragments on proliferative activity of fibroblasts

The effects of fibronectin and fragments of its limited proteolysis by plasmin on the proliferative activity of human embryo fibroblasts in culture were studied. It was found that native fibronectin and its fragments with Mr greater than or equal to 120 kD do not exert either a stimulating or inhibiting influence, whereas the 15-43 kD fragments significantly stimulate cell proliferation. The stimulating effect increases with a rise in the fragment concentration, reaching a maximum at 12-25 micrograms/ml and decreases at their higher concentrations. The preparation of proliferation-stimulating fragments contains no proteinases admixtures that are active at neutral pH and does not possess any intrinsic proteolytic activity. The proliferation-stimulating activity does not change after removal of collagen-binding fragments.     

Small fibronectin fragments induce endothelium-dependent vascular relaxations

Strips of rabbit thoracic aorta precontracted with phenylephrine relaxed when exposed to selected synthetic peptides derived from the cell attachment domain of fibronectin. The relaxations elicited by both acetylcholine and the hexapeptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) were dependent on the presence of an intact endothelium, were resistant to indomethacin, and were inhibited by hemoglobin. The structure-activity relationship of four oligopeptides derived from fibronectin was in fair agreement with their ability to prevent fibronectin-mediated cell adhesion in other experimental systems. Human plasma fibronectin (up to 2.3 microM) did not relax this preparation and did not prevent the relaxant effect of the synthetic hexapeptide GRGDSP. On the rabbit isolated mesenteric artery, the relaxations induced by GRGDSP were significantly inhibited by indomethacin treatment, suggesting a contribution of locally produced prostaglandins. The displacement of fibronectin by soluble peptides from its binding sites on endothelial cells may result in significant pharmacologic responses, probably resulting from perturbations of the endothelial cell membranes.     

Reversible cross-linking of cellular components of adherent fibroblasts to fibronectin and lectin-coated substrata

Using matrix-immobilized proteins containing photo-labile cross-linking reagents we have been able to probe the underside of cells in the early stages of active spreading. Similar cellular components of hamster fibroblasts appear to be present in closest proximity to substrata containing fibronectin, ricin, concanavalin A (conA) or soybean agglutinin (SBA).     

Heparin inhibits retrovirus binding to fibronectin as well as retrovirus gene transfer on fibronectin fragments

Fibronectin fragments have been shown to improve retrovirus gene transfer efficiency by binding retrovirus and target cells. Using a novel virus adhesion assay, we confirmed binding of type C oncoretrovirus vectors to the heparin II domain of fibronectin and demonstrated inhibition of viral binding and gene transfer by heparin.     

Tests of artificial substrata as nursery habitat for young fish

Recruitment areas for freshwater fish are often negatively affected by eutrophication and physical disturbances. Vegetated areas, which are important nursery habitats, are reduced and water turbidity increased. As a method of compensation, we tested artificial substrata for young-of-the-year fish. The structures were made of spruce bundles, with and without surrounding nets, and placed in a hyper-eutrophic very turbid environment and in an undisturbed area with clear water. Both habitats were devoid of submerged vegetation. Young fish abundance in treated areas was compared with adjacent reference sites. In the clear water area, the abundance of all investigated species – perch, pike, bream, silver-bream and roach – was higher in areas with artificial refuges. A similar response was evident for cyprinids in the turbid environment. High abundance of pikeperch and ruffe appeared in the hyper-eutrophic test area. Neither of these species, nor perch, was attracted to the artificial refuges. The lack of response in perch and pikeperch suggests that the importance of structural refuge decreases in very turbid water for these species. Of the two methods tested, spruce bundles with surrounding nets generally attracted most young fish, implying that the nets further increased the refuge capacity by reducing predation risk. The conclusion is that artificial habitats could improve recruitment habitats and that protective devices can increase refuge capacity.     

The interaction of fibronectin fragments with fibroblastic cells

We have examined the interaction of the purified cell-binding domain of fibronectin with fibroblastic baby hamster kidney cells. When the cell-binding region of fibronectin is part of a large 75,000-dalton fragment, the direct binding of the tritium-labeled fragment to cells in suspension can be observed. There is a single class of 10(5) sites/cell with an apparent dissociation constant of 4 X 10(-7) M. When the cell-binding region is part of a smaller 11,500-dalton fragment, an interaction with cells can only be observed indirectly via inhibition assays. The apparent affinity of this fragment for the cell surface fibronectin receptor is low. This 11,500-dalton fragment competitively inhibits both the direct binding of soluble [3H]fibronectin to cells in suspension and the spreading of cells on fibronectin-coated substrates, suggesting that the fragment binds to the same receptor site as intact fibronectin. Possible models describing the mechanism of the interaction of fibronectin with its receptor are proposed.     

Binding of calcium by parvalbumin fragments

Parvalbumin fragments from carp pI 4.47 parvalbumin corresponding to its residues 1–75 and 76–108 bind Ca²⁺ with affinities corresponding to K_d 0.9 · 10⁻⁴ M and K_d 3 · 10⁻³ M, respectively.     

Binding of calpain fragments to calpastatin

Their cDNA-derived amino acid sequences predict that the 80-kDa subunits of the micromolar and millimolar Ca(2+)-requiring forms of the Ca(2+)-dependent proteinase (mu- and m-calpain, respectively) each consist of four domains and that the 28-kDa subunit common to both mu- and m-calpain consists of two domains. The calpains were allowed to autolyze to completion, and the autolysis products were separated and were characterized by using gel permeation chromatography, calpastatin affinity chromatography, and sequence analysis. Three major fragments were obtained after autolysis of either calpain. The largest fragment (34 kDa for mu-calpain, 35 kDa for m-calpain) contains all of domain II, the catalytic domain, plus part of domain I of the 80-kDa subunit of mu- or m-calpain. This fragment does not bind to calpastatin, a competitive inhibitor of the calpains, and has no proteolytic activity in either the absence or presence of Ca2+. The second major fragment (21 kDa for mu-calpain and 22 kDa for m-calpain) contains domain IV, the calmodulin-like domain, plus approximately 50 amino acids from domain III of the 80-kDa subunit of mu- or m-calpain. The third major fragment (18 kDa) contains domain VI, the calmodulin-like domain of the 28-kDa subunit. The second and third major fragments bind to a calpastatin affinity column in the presence of Ca2+ and are eluted with EDTA. The second and third fragments are noncovalently bound, so the 80- and 28-kDa subunits of the intact calpain molecules are noncovalently bound at domains IV and VI. After separation in 1 M NaSCN, the 28-kDa subunit binds completely to calpastatin, approximately 30-40% of the 80-kDa subunit of mu-calpain binds to calpastatin, and the 80-kDa subunit of m-calpain does not bind to calpastatin in the presence of 1 mM Ca2+.