Adhesion of human amnion epithelial cells to extracellular matrix : Evidence for multiple mechanisms

Human amnion epithelial cells attach and flatten slowly (approximately 65 min) onto plastic in the presence of serum but much more rapidly (20-30 min) onto subcellular matrix (SCM) deposited by the same cells. This matrix contains both fibronectin and laminin, but neither molecule on its own can reproduce its adhesive properties. Cells attach on surfaces containing fibronectin and laminin and extend filopodial and lamellipodial areas of cytoplasm without extensive flattening in the perinuclear region. Matrix deposited onto plastic by amnion epithelial cells has trypsin-sensitive and trypsin-resistant, papain-sensitive adhesion-promoting components. Cell spreading triggered by the latter but not the former can be inhibited by pretreating the adhering cells with heparin. Other GAGs are without effect. The results are discussed in terms of multiple interactions between epithelial cells and basal laminae.     

The influence of contact guidance on chemotaxis of human neutrophil leukocytes

Chemotaxis of human neutrophil leukocytes moving on or in aligned 3D fibrin gels is more efficient if the cells are moving along the axis of fibre alignment than if they have to cross the fibres. This was shown by using two assays, one in which the cells were responding to a distant (600 micrometers) gradient source diffusing from a filter paper impregnated with formyl-Met-Leu-Phe and incorporated into the gel, the other in which the cells were responding to nearby (20--30 micrometers) Candida albicans spores in serum. In the former assay, impairment of chemotaxis across the axis of fibre alignment was highly significant. In the latter, cells showed efficient chemotaxis to the spores, but took more irregular paths when crossing the aligned fibres than when running along them. Neutrophils show contact guidance in aligned collagen or fibrin gels (Wilkinson et al., Exp cell res 140 (1982) 55) [1], thus the cells were subjected simultaneously to two directional cues in these experiments, one the chemotactic gradient and the other a contact guidance field. These cues may reinforce or interfere with each other depending on their relative orientation. Since many tissues in vivo show alignment or more complex forms of patterning, tissue architecture is likely to be an important determinant of the efficiency of cellular mobilization in inflamed or infected sites.     

The synaptonemal complex as part of the nuclear matrix of the flour moth, Ephestia kuehniella

A nuclear matrix fraction was prepared from ovaries of the achiasmatic flour moth, Ephestia kuehniella, by removal of the chromatin, using detergent treatment of homogenized ovaries or dissected ovary tips followed by DNase digestion and high salt extraction. Removal of DNA and histones from the nuclei was demonstrated by Feulgen staining and polyacrylamide gel electrophoresis (PAGE), respectively. By light microscopy, ribbon-like structures similar in dimension to the synaptonemal complex were observed in the oocyte after digestion of the chromosomes. Electron microscopic examination of matrix preparations of pachytene cells showed a defined synaptonemal complex structure with both lateral and central elements. Such structures were not found in either the fully differentiated nurse cells or in follicle cells which were exposed to the same preparative technique concurrently. However, in early post-pachytene nurse cells the typical polycomplex structures, formed in these cells from the synaptonemal complex, were found in nuclear matrix preparations. The results suggest an association of synaptonemal complexes with the nuclear matrix.     

Collagen metabolism and spicule formation in sea urchin micromeres

The role of collagen or collagen-like protein(s) in the in vitro formation of the sea urchin embryonic skeleton was investigated using isolated micromeres of Strongylocentrotus purpuratus. Micromeres were cultured in sea water containing 4% horse serum on tissue culture plastic or an extracellular matrix of type I collagen. The effect of proline analogs and an inhibitor of collagen hydroxylation on in vitro spicule formation in both culture systems was monitored. When micromeres are cultured in the presence of proline analogs l-azetidine-2-carboxylic acid and l-3,4-dehydroproline which disrupt collagen metabolism, spicule formation is significantly less inhibited on a collagen substratum than on plastic. Culturing micromeres on plastic in the presence of α,α′-dipyridyl, an inhibitor of collagen hydroxylation, resulted in almost complete inhibition of spicule formation. The inhibition by α,α′-dipyridyl can be overcome by culturing micromeres on collagen substratum. These results do not support the idea of collagen being the calcified organic matrix of the spicule. Rather, they suggest that micromeres synthesize a collagen-like extracellular matrix which is necessary for spicule formation. Inhibition of this activity by proline analogs or a collagen processing inhibitor can be overcome by providing the cells with a previously deposited extracellular matrix.     

Adhesive and migratory behavior of normal and sulfate-deficient sea urchin cells in vitro

Dissociated cells from different stage embryos of the sea urchin Lytechinus pictus were compared in their adhesion to various substrates. Micromeres from 16-cell stage embryos bind to tissue culture and Petri dishes but not to Petri dishes coated with human plasma fibronectin. Other cell types did not adhere to any of the substrates tested. By hatched blastula stage, about 28% of the cells adhered to fibronectin as well as to tissue culture dishes. By the mesenchyme blastula stage, there was a further increase in the proportion of cells adhering to these substrates. At no stage did cells adhere to native rat tail collagen. Primary mesenchymal cells were isolated by their selective adhesion to tissue culture dishes in the presence of horse serum. These cells were then examined for their migratory capacity. Cell spreading and migration followed adhesion and occurred on fibronectin but not on the other substrates tested. Based on analysis of video tapes, greater than 60% of these cells moved faster than 1 micron/min. On the other hand, cells from sulfate-deprived embryos, in which primary mesenchyme migration is blocked in situ, failed to spread and migrated little on the same substratum. This defect was reversed by a 6 h pretreatment of the cells in normal sea water. Thus, the in vitro migratory behavior parallels that observed in vivo. These results support the hypothesis that the primary mesenchymal cells produce a sulfate-dependent component that is required for cell spreading and migration.     

Newborn human skin fibroblasts senesce in vitro without acquiring adult growth factor requirements

Nuclear-envelope nucleoside triphosphatase activity (NTPase), an enzymatic activity thought to participate in RNA transport, was localized in rat liver in situ after brief perfusion with 3% paraformaldehyde. Reaction product was distributed along the nucleoplasmic side of the nuclear envelope (NE) in heterochromatin, was only occasionally found at nuclear pores, and nuclear deposition was selectively blocked by inhibitors of NE NTPase activity. Our results suggest that NTPases, which are active in the NE and which participate in RNA transport, are not specifically associated with nuclear-pore complexes.     

Effect of hemolymph and nervous-tissue components on the in vitro development of flight-muscle myoblasts of Manduca sexta

Abstract. Flight-muscle myoblasts from pupae of Manduca sexta were grown in the presence of either hemolymph or nerve extract. The cells exhibited distinctly different growth patterns: the addition of hemolymph yielded a branched network consisting of short, thin myofibers, while the addition of nerve extract resulted in parallel, extended fiber arrays. Spontaneous contractions were frequently observed. Other medium supplements (muscle extract, mouse-liver extract, β-ecdysone) had no detectable effect on myoblast development. Electron micrographs of explants grown in hemolymph revealed the presence of only a few myofibrils with a parallel filament arrangement but without cross striation, whereas explants grown with nerve extract exhibited cross striation within the voluminous filament arrays.     

Photoaffinity labelling of a nuclear matrix nucleoside triphosphatase and its modulation in the acute-phase response

Photoaffinity labelling has been used to identify the major nuclear matrix nucleoside triphosphatase (NTPase) as a 46 kD polypeptide, which appears to represent the same polypeptide photolabelled in nuclear envelope. Nuclear matrix NTPase and its photolabelling were selectively decreased in the acute phase response of rat liver, which also encompasses decreases (30%) in RNA transport in vitro and nuclear envelope NTPase. These results, and quantitative considerations suggest that the NTPase correlatively linked to RNA transport is not solubilized by detergents; it appears to represent a nuclear matrix component.     

Glycosphingolipids in detergent-insoluble substrate attachment matrix (DISAM) prepared from substrate attachment material (SAM) : Their possible role in regulating cell adhesion

The glycosphingolipids isolated from the detergent-insoluble material (DIM) of whole cells as well as from a similar detergent-insoluble substrate attachment matrix (DISAM) have been investigated in comparison with the glycosphingolipids of whole cells. The proportion of glycolipids in the total lipid extract was enriched in the DISAM as well as DIM fractions as compared to whole cells. The ratio of ganglioside (GM₃) to neutral glycolipids was also higher in the DISAM fractions than in whole cells. The radioactivity incorporated into DISAM glycolipids of BHK cells, metabolically labeled with radioactive glucosamine, was greater in confluent cells than in sparsely growing cells; however, label incorporation into glycolipids of the DISAM fraction of BHKpy cells was 2–3-fold higher than that of confluent BHK cells, although the chemical quantity of GM₃ in whole cells was much lower in BHKpy cells than in BHK cells. In order to confirm the enhanced label in DISAM glycolipids of BHKpy cells by other procedures, the labeled cells were detached by EGTA, washed, and reattached on plates. The amount of label in DISAM glycolipids of the reattached matrix of BHKpy cells was much higher than that of BHK cells.Cell spreading and cell attachment on plastic plate were inhibited by inclusion of GM₃ in the medium. These data suggest that: (i) glycolipids, particularly GM₃, at the cell attachment site have different metabolic activity from those of whole cells; the label in glycolipids goes preferentially into cell attachment sites, and may have some functional role in regulating cell attachment of BHK cells; (ii) metabolic activity and turnover of GM₃ in cell attachment sites of confluent cells are higher than actively growing cells, yet those of transformed cells are much higher than any state of non-transformed cells.     

The effect of concanavalin A on egestion of food vacuoles in Tetrahymena

The ability of concanavalin A (conA) to disrupt food vacuole elimination at the cytoproct of Tetrahymena pyriformis, strain GL-C, was investigated using fluorescence microscopy and thin section electron microscopy. ConA was found to induce tails in Tetrahymena. These tails were specifically stained by fluorescent conA. Thin section observations of conA-treated cells revealed that these tails were the result of abnormal egestion of food vacuole contents at the cytoproct. Tail formation appears to result from an inhibition of endocytosis of food vacuole membrane during egestion. Instead, the food vacuole membrane appears to be cast out of the cell, along with the contents of the vacuole. The mechanism of this inhibition may be related to an apparent absence of microtubules or microfilamentous mat in the cytoproct region of conA-treated cells. Although conA is ingested into food vacuoles in large amounts, conA appears to affect endocytosis only from outside the cell; ingested conA does not appear to be effective. ConA may exert its influence by binding to the cytoproct region. The ability of conA to induce tail formation is inhibited by sugars specific to it. Numerous membranous vesicles are found in association with the oral cilia and cytoproct region of conA-treated cells. These vesicles may be the conA-binding material reported to be secreted by Tetrahymena.     

The effect of mannose6-phosphate on the turnover of cell surface glycosaminoglycans

Human fibroblasts (SL66) were cultured in medium containing 35SO4(2-) to label the glycosaminoglycans (GAGs). After washing, the labeled cells were chased in the presence or absence of mannose6-phosphate (M6P) and the GAGs were analyzed in terms of three arbitrary fractions: 1, Extracellular (soluble medium), 35S radioactivity higher in cultures without M6P than in cultures with M6P. 2, Pericellular (cell surface-associated), 35S radioactivity lower in cultures without M6P than in cultures with M6P. 3, Intracellular (residue within the intact cell), no difference in 35S radioactivity between the two sets of cultures. In addition, when the 35S-labeled GAGs from corresponding cellular compartments derived from cultures with and without M6P were digested with pronase and chondroitin ABC lyase, and then compared by chromatography on Sepharose CL-6B, distinct molecular differences in both the extracellular and pericellular fractions were observed. Several lines of evidence indicate that the effect of M6P on the turnover of 35S-labeled GAGs in our assay system reflects disruption of cell surface lysosomal enzyme activity. For example, when the experiment was performed with I cells, which lack enzymes carrying the M6P marker, no difference was seen in cultures with or without M6P. The addition of lysosomal enzymes derived from normal human fibroblasts to 35SO4-labeled I cells, however, resulted in the turnover of pericellular GAGs and this effect was inhibited by M6P. These results suggest that one possible function of cell surface receptors recognizing the M6P moiety of lysosomal enzymes is to anchor certain of these enzymes proximate to their substrates at the cell surface.     

The effect of monensin on cell aggregation of normal and dystrophic human skin fibroblasts

Measurements of aggregation kinetics using couette viscometry show that freshly trypsinized skin fibroblasts from patients with Duchenne muscular dystrophy have values of intercellular adhesiveness approx. 40% those of normal cells. If cells are allowed to recover from the effects of trypsinization (by incubation for 2 h at 37 degrees C in serum-containing medium) the intercellular adhesiveness of both cell types increases, and normal and Duchenne cells aggregate to the same extent. Exposure to the ionophore monensin during the recovery phase leads to suppression of recovery in both cell types, and this effect of the drug is greater in Duchenne fibroblasts. These results are discussed in relation to other data on the reported differential effects of trypsin and monensin on normal and Duchenne fibroblasts.     

The distribution of calmodulin in living mitotic cells

Calmodulin has been labeled with rhodamine isothiocyanate (CaM-RITC) and used as a probe for the location of calmodulin in vivo. CaM-RITC retains its capacity to regulate the activity of brain phosphodiesterase in a Ca²⁺-dependent manner in vitro, indicating that the labeled protein is still active. After injection into living mammalian cells CaM-RITC incorporates rapidly into the mitotic spindle; the details of its localization there mimic closely the distribution of Calmodulin seen by immunofluorescence. In interphase cells the CaM-RITC is excluded from the nucleus, but shows no region of specific concentration within the cytoplasm. Neither a 2-fold increase in cellular CaM nor the injection of anti CaM has any observable effect on the progress of mitosis.     

The effects of several ion channel blockers and calmodulin antagonists on fertilization-induced acid release and 45Ca2+ uptake in sea urchin eggs

The effects of calcium antagonists, diltiazem and verapamil, and calmodulin antagonists, chlorpromazine, N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), were tested on two responses of the sea urchin egg to insemination: (1) H+ release; (2) Ca2+ uptake. It was found that calcium antagonists inhibited both processes, while calmodulin antagonists only inhibited H+ release but not Ca2+ uptake. Verapamil and diltiazem were effective to inhibit H+ release when added to the egg suspension up to 120 sec and W-7 was effective around 150 sec after insemination. Calcium antagonists became ineffective earlier than W-7 in inhibiting H+ release. A calmodulin-dependent step may thus occur linking the Ca2+ uptake and H+ release. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an anion channel blocker, also inhibited both Ca2+ uptake and H+ release. This result suggests that an uptake of anion(s) occurs along with Ca2+ uptake.     

The effects of retinoic acid on [14C]acetate incorporation into lipids of normal and transformed hamster fibroblasts

This study examined the effects of retinoic acid (RA) on [14C]acetate incorporation and fatty acid composition of hamster embryo fibroblasts (HEF) and two cell lines derived from the same inbred strain but transformed by herpes simplex-2 virus (HSV) or polyoma virus (HFT). Cells were exposed to all trans RA, or dimethylsulfoxide (DMSO), the vehicle for RA, and the lipids labeled with [14C]acetate. Lipids were extracted from the cells, separated by paper chromatography, located by autoradiography, and acetate incorporation determined by liquid scintillation spectrometry. The distribution of fatty acids in total cell lipids was examined by gas chromatography. HEF cells incorporated more acetate into cholesterol than either transformed cell type. The HFT line incorporated more acetate into triglycerides and less into total phospholipids than either the HSV line or the HEF line. RA caused a significant decrease in incorporation of acetate into cholesterol and sphingomyelin in all three cell lines. HEF and HSV cells had decreased incorporation into phosphatidyl inositol-phosphatidyl serine and increased incorporation into triglycerides, changes not evident in the HFT cell. The control fatty acid profiles of the HEF and HSV cells were similar, while the HFT cells had a larger proportion of C16:0 and 18:1 fatty acids. Following treatment with RA all three cell types showed an increase in palmitic and a decrease in oleic acids. The three related cell types showed different [14C]acetate labeling patterns which did not respond uniformly to RA. On the other hand, exposure elicited some like responses in all cell types.     

The endosomal compartment of rat hepatocytes. Its characterization in the course of [125I]insulin internalization

When freshly isolated hepatocytes are incubated with [125I]insulin in the presence of the microtubule-disrupting agent colchicine, internalization of the labelled hormone is not significantly altered. However, the drug limits the endocytosis of the labelled material to a peripheral band of cytoplasm extending 1 micron beyond the plasma membrane. Both in the presence and absence of colchicine, internalized [125I]insulin preferentially associates with clear vesicles (endosomes) and lysosome-like structures, but the relative amount of labelled material associated with clear vesicles is higher in the presence of the drug than in its absence. An inverse pattern is observed for the lysosome-like structures. As demonstrated by cytochemical methods, clear vesicles do not contain the lysosomal enzyme aryl sulfatase. Moreover, colchicine induces an increase of the clear vesicle diameter without affecting their frequency, while it perturbs multivesicular bodies and dense bodies in an opposite way by increasing their frequency without affecting their size. By reducing and/or delaying the fusion between internalized endocytotic vesicles and lysosomes, colchicine allows better characterization of the endosomal compartment of isolated rat hepatocytes and allows it to be distinguished from other compartments, such as multivesicular bodies and the Golgi apparatus.     

The in vitro chondrogenic response of limb-bud mesenchyme to a water-soluble fraction prepared from demineralized bone matrix

Demineralized adult bone matrix initiates de novo ectopic endochondral ossification 2-3 weeks following its intramuscular implantation into adult animals. This phenomenon appears to be similar, in some ways, to inductive cell-matrix interactions which regulate cartilage and bone formation during development. In the present study, we used embryonic chick limb-bud mesenchymal-cell cultures to bioassay extracts of demineralized bone matrix for chondrogenic activity. Guanidinium-chloride (4 M) extracts of demineralized bovine bone were dialyzed against buffers of decreasing ionic strength and then cold water. The cold-water-soluble fraction was found to stimulate chondrogenesis in intermediate-density limb-bud cell cultures (2.2 X 10(6) cells per 35-mm dish), as revealed by visual inspection with phase optics, toluidine-blue staining of fixed plates, and [35S] sulfate incorporation in the cell layer. Further fractionation of this material by anion-exchange, carbohydrate-affinity, and molecular-sieve chromotography produced a semipurified preparation possessing chondrogenic-stimulating activity at doses ranging from 3 to 10 micrograms/ml. The in vitro chondrogenic response of limb-bud mesenchymal cells was dose-dependent, required a minimal initial plating density of 2.08 X 10(5) cells/mm2 of culture dish, and developed gradually over 8-10 days. At an optimal dose of extract, a continuous exposure period of at least 2-3 days was necessary to produce detectable chondrogenic stimulation. In addition, the amount of cartilage formed following an 8-day exposure was markedly influenced by the culture 'age' of the mesenchymal cells (i.e., the time between plating and the start of treatment).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mineralization in vitro of matrix formed by osteoblasts isolated by collagenase digestion

Osteoblasts from calvaria of 18-day-old fetal Sprague-Dawley rats were isolated using a dissecting procedure followed by collagenase digestion. Freshly isolated or previously frozen cells were cultured for up to 4 weeks in a Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 50 micrograms/ml ascorbic acid, with or without 10 mM beta-glycerophosphate. Most of the cells were alkaline phosphatase positive throughout the culture period and expressed a type-I collagen as assessed by immunofluorescence. Cells cultured in the presence of beta-glycerophosphate formed a matrix with type-I collagen in 7 days. The matrix underwent mineralization in less than 2 weeks. In the absence of beta-glycerophosphate, only the formation of a nonmineralized matrix was observed. Electron-microscopic examination revealed osteoblasts embedded in a dense network of collagen fibers, with a well-defined mineralization process in association with matrix vesicles. Scanning electron-microscopy showed that the matrix composed of layers of irregularly shaped spread cells with smooth surfaces trapped in a fiber matrix. No mineralization process was observed when rat skin fibroblasts were cultured under similar conditions. These data demonstrate the ability of enzymatically isolated osteoblasts cultured in the presence of beta-glycerophosphate to form bone in vitro, and that this process is similar to bone formation in vivo.     

The isolation of an antimycin A-resistant human cell line

An antimycin A-resistant derivative of the human cell line, D98, has been obtained by selective mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The derivative, designed MA65, is capable of continuous growth in 15 microM antimycin and the resistant phenotype is stable in the absence of selection. MA65 is not cross-resistant to chloramphenicol or triethyl tin. Crude membrane preparations from MA65 after propagation in medium containing antimycin have normal succinate-cytochrome c oxidoreductase activity and the respiratory activity of whole cells continues in the presence of the drug. The mitochondrially synthesized proteins of D98 and MA65 are similar when compared on sodium dodecyl sulphate (SDS), or isoelectric focusing gels, but there is a reproducible difference in the extent of labelling of one band detected by isoelectric focusing. Genetic analysis is consistent with the existence of a cytoplasmically localized determinant conferring resistance.     

Immunofluorescence studies on proteins B23 and C23 in nucleoli of human lymphocytes

Nucleoli of normal and leukemic lymphocytes were studied by cytochemical and immunofluorescence methods to provide more information on the nucleolar presence and distribution of proteins B23 and C23. Annular nucleoli of human lymphocytes represent a very convenient subject for such studies, since they consist of one centrally located large fibrillar center surrounded by RNP components. In such nucleoli, protein C23 was present mainly in the central nucleolar region and protein B23 was found mostly in the periphery. The nucleolar area immunostained for protein B23 was usually larger than that stained for protein C23. The distribution of protein C23 appeared to be similar to that of intensely stained nucleolar argyrophilic components. No substantial differences were found between the distribution of proteins B23 and C23 in nucleoli of normal and leukemic lymphocytes. In lymphocytes of patients treated with chemotherapy, the immunofluorescence was diminished for protein B23 and particularly so for protein C23.