The development of an innervated epithelial barrier model using a human corneal cell line and ND7/23 sensory neurons

The corneal epithelium is a highly innervated tissue and hence in vitro models that mimic the effects of chemicals or radiation (e.g. ultra violet) on this important barrier should include consideration of the potential role of innervation. A sensory neural cell line, ND7/23, was incorporated into a 2D and 3D model of a corneal epithelium, using a human corneal cell line, and effects on barrier integrity were neither adverse nor stimulatory. In the 3D model the nerve cell bodies were separated from the corneal epithelium, via a porous polycarbonate insert membrane. The ND7/23 cells were induced to form neurites and cease division when cultured in the keratinocyte medium employed for the corneal cells. In the absence of calcium, the epithelial barrier function was lost, shown by enhanced fluorescein leakage and relocation of ZO-1 and E-cadherin from the cell membrane. At 60 microM calcium, and above, the corneal cells formed tight junctions, with peripheral membrane location of ZO-1 and E-cadherin. The presence of the ND7/23 cells did not compromise or enhance the time taken to form these junctions, when monitored at 24-h intervals over 72 h. Both male- and female-derived human corneal cell lines showed a similar tight junction functional response to different medium calcium concentrations in the presence or absence of the ND7/23 cells. Once differentiated in keratinocyte medium, patch-clamped ND7/23 cells were capable of producing a whole-cell current when exposed to low pH (5.4), indicative of the presence of active pH-gated ion channels.     

Further investigations of red cell deformability with nickel mesh

Although the filtration method has been widely employed in red cell deformability studies, the structural irregularity of the pores of a Nuclepore polycarbonate membrane has always been a major problem. Anegawa, T. et al. (Clin. Hemorheol., 7, 1987) obtained a higher reproducibility with the filtration method using a newly designed thin metal film with pores engraved by the photofabrication technique. We further studied the pressure - flow rate relationship of red cell suspension employing this nickel mesh. The filtration of red cell suspensions through the nickel mesh was not influenced by leukocytes contamination or added leukocytes up to a leukocyte count of 250 cells/mm3 within an experimental limitation. On the other hand, the flow was greatly influenced by leukocytes contamination when the polycarbonate membrane was used. The nickel mesh was found to be useful in detecting major determinants of red cell deformability, such as cell geometry and internal cellular viscosity, and in detecting abnormalities of red cell deformability in a patient with microangiopathic hemolytic anemia. In conclusion, the present study clearly shows that the nickel mesh is preferable for investigating red cell deformability to the polycarbonate membrane from a quantitative point of view. This material should contribute to the physiologic and clinical investigation of red cell deformability.     

Skeletal muscle satellite cell diversity: satellite cells form fibers of different types in cell culture

Following skeletal muscle injury, new fibers form from resident satellite cells which reestablish the fiber composition of the original muscle. We have used a cell culture system to analyze satellite cells isolated from adult chicken and quail pectoralis major (PM; a fast muscle) and anterior latissimus dorsi (ALD; a slow muscle) to determine if satellite cells isolated from fast or slow muscles produce one or several types of fibers when they form new fibers in vitro in the absence of innervation or a specific extracellular milieu. The types of fibers formed in satellite cell cultures were determined using immunoblotting and immunocytochemistry with monoclonal antibodies specific for avian fast and slow myosin heavy chain (MHC) isoforms. We found that satellite cells were of different types and that fast and slow muscles differed in the percentage of each type they contained. Primary satellite cells isolated from the PM formed only fast fibers, while up to 25% of those isolated from ALD formed fibers that were both fast and slow (fast/slow fibers), the remainder being fast only. Fast/slow fibers formed from chicken satellite cells expressed slow MHC1, while slow MHC2 predominated in fast/slow fibers formed from quail satellite cells. Prolonged primary culture did not alter the relative proportions of fast to fast/slow fibers in high density cultures of either chicken or quail satellite cells. No change in commitment was observed in fibers formed from chicken satellite cell progeny repeatedly subcultured at high density, while fibers formed from subcultured quail satellite cell progeny demonstrated increasing commitment to fast/slow fiber type formation. Quail satellite cells cloned from high density cultures formed colonies that demonstrated a similar change in commitment from fast to fast/slow, as did serially subcloned individual satellite cell progeny, indicating that the observed change from fast to fast/slow differentiation resulted from intrinsic changes within a satellite cell. Thus satellite cells freshly isolated from adult chicken and quail are committed to form fibers of at least two types, satellite cells of these two types are found in different proportions in fast and slow muscles, and repeated cell proliferation of quail satellite cell progeny may alter satellite cell progeny to increasingly form fibers of a single type.     

Energy of adhesion of human T cells to adsorption layers of monoclonal antibodies measured by a film trapping technique.

A novel method for studying the interaction of biological cells with interfaces (e.g., adsorption monolayers of antibodies) is developed. The method is called the film trapping technique because the cell is trapped within an aqueous film of equilibrium thickness smaller than the cell diameter. A liquid film of uneven thickness is formed around the trapped cell. When observed in reflected monochromatic light, this film exhibits an interference pattern of concentric bright and dark fringes. From the radii of the fringes one can restore the shape of interfaces and the cell. Furthermore, one can calculate the adhesive energy between the cell membrane and the aqueous film surface (which is covered by a layer of adsorbed proteins and/or specific ligands), as well as the disjoining pressure, representing the force of interaction per unit area of the latter film. The method is applied to two human T cell lines: Jurkat and its T cell receptor negative (TCR-) derivative. The interaction of these cells with monolayers of three different monoclonal antibodies adsorbed at a water-air interface is studied. The results show that the adhesive energy is considerable (above 0.5 mJ/m2) when the adsorption monolayer contains antibodies acting as specific ligands for the receptors expressed on the cell surface. In contrast, the adhesive energy is close to zero in the absence of such a specific ligand-receptor interaction. In principle, the method can be applied to the study of the interaction of a variety of biological cells (B cells, natural killer cells, red blood cells, etc.) with adsorption monolayers of various biologically active molecules. In particular, film trapping provides a tool for the gentle micromanipulation of cells and for monitoring of processes (say the activation of a T lymphocyte) occurring at the single-cell level.     

Micropattern immobilization of polysaccharide

Two types of polysaccharides, sulfated hyaluronic acid and heparin, were pattern-immobilized on a poly(ethylene terephthalate) and polystyrene film, respectively, in a specific pattern by photolithography. Sulfated hyaluronic acid was prepared from hylaronic acid by the treatment of sulfur trioxide/pyridine complex. Heparin was purchased and used without further treatment. The polysaccharides were coupled with azidoaniline. The derivatized polysaccharides were cast on a film from aqueous solution. After drying, the film was photo-irradiated in the presence or absence of a photomask. The micropatterning was confirmed by staining with a cationic dye. Platelet adhesion was reduced on the sulfated hyaluronic acid-immobilized areas. The immobilized sulfated hyaluronic acid significantly reduced thrombus formation. On the other hand, cells were cultured on the heparin-immobilized film. In the presence of fibroblast growth factor (FGF), the growth of mouse fibroblast STO cells was enhanced only on the heparin-immobilized regions. This result indicated that micropattern-immobilized heparin activated FGF for cell growth activity.     

Effects of biofilms on zebra mussel postveliger attachment to artificial surfaces

Abstract. The zebra mussel is an introduced fouling organism in North American inland waters. This field study tested whether natural biofilms, formed by covering substrata with a 100-μm mesh that allows microorganisms to attach and films to develop in the absence of postveligers, influenced the attachment of zebra mussel postveligers to artificial surfaces. Low-wettable polycarbonate and wettable glass surfaces were used in the experiments over four field seasons to study biofilm formation (1997–1998) and mussel attachment (1998–2000). The presence of the mesh did not quantitatively affect biofilm development on either substratum as determined by microscopic direct counts and colony-forming units on R2A agar. Natural biofilms on polycarbonate surfaces positively influenced postveliger attachment compared to substrata that initially had no film (ANOVA, p-values ranged from ≤.05 to ≤.001). Biofilms did not influence postveliger attachment to glass surfaces (ANOVA, p>.05). Attachment to both substrata was similar on surfaces with and without previously settled postveligers. Based on these results, we conclude that biofilms can enhance postveliger attachment to some but not all artificial surfaces.     

Relationship between glycocalyx and povidone-iodine resistance in Pseudomonas aeruginosa (ATCC 27853) biofilms.

Biofilm-embedded bacteria are generally more resistant to antimicrobial agents than are planktonic bacteria. Two possible mechanisms for biofilm resistance are that the glycocalyx matrix secreted by cells in a biofilm reacts with and neutralizes the antimicrobial agent and that the matrix creates a diffusion barrier to the antimicrobial agent. This study was therefore conducted to examine the relationship between glycocalyx and enhanced povidone-iodine resistance in biofilms of Pseudomonas aeruginosa (ATCC 27853). Biofilms were generated by inoculation of polycarbonate membranes with broth-grown cells and incubation of them on the surfaces of nutrient agar plates. The quantities of glycocalyx material per cell were found not to be significantly different between biofilm and planktonic samples. Transmission electron microscopy showed that the distributions of glycocalyx material around cells differed in biofilm and in planktonic samples. Addition of alginic acid to planktonic cell suspensions resulted in a slight increase in resistance to povidone-iodine, suggesting some neutralizing interaction. However, the iodine demands created by biofilm and planktonic samples of equivalent biomass were not significantly different and, therefore, do not explain the contrast in resistance observed between biofilm and planktonic samples. Examination of the relationship between cell death and biomass detachment from the glycocalyx matrix revealed that most cell death occurred in the fraction of biomass that detached from a biofilm during treatment. The overall rate of iodine diffusion through biofilms was not different from that of planktonic cells collected on a polycarbonate membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of cell shape change on the function and differentiation of rabbit mammary cells in culture

We examined the role of cell shape, cytodifferentiation, and tissue topography on the induction and maintenance of functional differentiation in rabbit mammary cells grown as primary cultures on two-dimensional collagen surfaces or in three-dimensional collagen matrices. Mammary glands from mid-pregnant rabbits were dissociated into single cells, and epithelial cells were enriched by isopycnic centrifugation. Small spheroids of epithelial cells (approximately 50 cells) that formed on a rotary shaker were plated on or embedded in collagen gels. The cells were cultured for 1 d in serum-containing medium and then for up to 25 d in chemically defined medium. In some experiments, epithelial monolayers on gels were mechanically freed from the dishes on day 2 or 5. These gels retracted and formed floating collagen gels. On attached collagen gels, flat monolayers of a single cell type developed within a few days. The cells synthesized DNA until the achievement of confluence but did not accumulate milk proteins. No morphological changes were induced by prolactin (PRL). On floating gels, two cell types appeared in the absence of cell proliferation. The cells in direct contact with the medium became cuboidal and developed intracellular organelles typical of secretory cells. PRL-induced lipogenesis, resulting in large fat droplets filling the apical cytoplasm and accumulation of casein and α-lactalbumin in vesicles surrounding the fat droplets. We detected tranferrin in the presence or absence of PRL intracellularly in small vesicles but also in the collagen matrix in contact with the cell layer. The second cell type, rich in microfilaments and reminiscent of the myoepithelial cells, was situated between the secretory cell layer and the collagen matrix. In embedding gels, the cells formed hollow ductlike structures, which grew continuously in size. Secretory cells formed typical lumina distended by secretory products. We found few microfilament-rich cells in contact with the collagen gels. Storage and secretion of fat, caseins and alpha-lactalbumin required the presence of PRL, whereas the accumulation and vectorial discharge of transferrin was prolactin independent. There was no differentiation gradient between the tip and the cent of the outgrowth, since DNA synthesis and milk protein storage were random along the tubular structures. These results indicate that establishment of functional polarity and induction of cytodifferentiation are influenced by the nature of the interaction of the cells with the collagen structure. The morphological differentiation in turn plays an important role in the synthesis, storage, and secretion of fat and milk proteins.     

THE ROLE OF THREE CYTOPLASMIC FIBERS IN BHK-21 CELL MOTILITY : I. Microtubules and the Effects of Colchicine

Microtubule breakdown in the presence of 5 or 40 µg/ml of colchicine is observed in BHK-21/C13 fibroblast-like cells. Several morphological and physiological effects are noted in the absence of microtubules: (a) the cells transform from fibroblast-like to epithelial-like cells; (b) the normal pattern of intracellular birefringence changes and a juxtanuclear cap of birefringent filaments is formed; (c) time-lapse cinematography demonstrates that cell locomotion is inhibited in colchicine-treated cells, even though membrane ruffling persists. The results are discussed in terms of the specific roles of microtubules in cultured cell motility and possible functional relationships of the three types of cytoplasmic fibers seen in BHK-21 cells.     

A filtration method for measuring cellular uptake of [14C]methylamine and other highly permeant solutes

A filtration technique has been developed for study of the uptake of [14C]methylamine by Azotobacter vinelandii. This dual filter arrangement requires a precision microporous polycarbonate film which overlays a paper filter. Cellular uptake of radioactivity is terminated by vacuum filtration of the reaction mixture onto the polycarbonate filter without dilution or washing. Filtration was complete in 0.7 s with retention of less than 0.2% of the extracellular radioactivity. The dual filter method gave 20-fold higher levels for intracellular methylamine than filtration followed by washing. Without washing, nitrocellulose filters retained 18 times more extracellular [3H]sorbitol and 80 times more extracellular [14C]methylamine than polycarbonate filters. Use of an underlying paper filter did not significantly improve the performance of nitrocellulose filters. However, addition of a paper filter reduced extracellular methylamine and sorbitol retention on polycarbonate filters by 77 and 86%, respectively. This method is generally applicable to measurement of the uptake of highly permeant molecules by cells and subcellular organelles.     

Increased autocrine production of insulin-like growth factor II (IGF-II) alters serum sensitivity of MCF-7 human breast cancer cell proliferation

Abstract. Regulation of the growth of breast cancer cells is the result of a complex interaction between steroid hormones and growth factors, and in particular of oestrogen and insulin-like growth factors (IGF). Alteration of any one mitogenic component can affect the cell response to other pathways. Previous work has shown that increased autocrine production of IGF-II from a transfected inducible expression vector can result in reduced oestrogen sensitivity of growth of MCF-7 human breast cancer cells. This report describes alterations to non-oestrogen regulated pathways of cell growth following enhanced IGF-II expression in these transfected MI7 cells. Serum sensitivity of cell growth in the absence of oestrogen was found to differ between MI7 and untransfected MCF-7 cells, in that growth of MI7 but not MCF-7 cells was strongly inhibited by high serum levels. Increased serum had no effect on levels of IGF-II mRNA, IGFIR, IGFBP4 mRNA, or IGFBP secreted in MI7 cells. However, growth inhibition by serum in MI7 cells could be overcome by increasing levels of IGF-II in the serum or by removal of IGFBP onto polycarbonate membranes. Thus, the growth inhibition by serum in MI7 cells is concluded to result from the increased levels of IGFBP added with higher serum. This would support an inhibitory role for IGFBP on growth of breast cancer cells when cell growth is being driven by IGF pathways in the absence of oestrogen, and would suggest that cellular sensitivity to such factors can depend on levels of endogenous IGF production.     

Retinal ganglion cell type, size, and spacing can be specified independent of homotypic dendritic contacts

In Brn3b(-/-) mice, where 80% of retinal ganglion cells degenerate early in development, the remaining 20% include most or all ganglion cell types. Cells of the same type cover the retinal surface evenly but tile it incompletely, indicating that a regular mosaic and normal dendritic field size can be maintained in the absence of contact among homotypic cells. In Math5(-/-) mice, where only approximately 5% of ganglion cells are formed, the dendritic arbors of at least two types among the residual ganglion cells are indistinguishable from normal in shape and size, even though throughout development they are separated by millimeters from the nearest neighboring ganglion cell of the same type. It appears that the primary phenotype of retinal ganglion cells can develop without homotypic contact; dendritic repulsion may be an end-stage mechanism that fine-tunes the dendritic arbors for more efficient coverage of the retinal surface.     

Leukemia inhibitory factor enhances formation of germ cell colonies in neonatal mouse testis culture

Spermatogonial stem cells continuously divide in the testis to support spermatogenesis throughout the life of adult male animals. Although very few spermatogonial stem cells are present in vivo, we recently succeeded in expanding these cells in vitro. Germ cells from postnatal testes were able to proliferate in the presence of several types of cytokines, and they formed uniquely shaped colonies of spermatogonia (germline stem or GS cells). These cells reinitiated normal spermatogenesis when transplanted into seminiferous tubules. However, much remains unknown about the contributions of cytokines to successful stem cell culture. In the present study, we examined the role of leukemia inhibitory factor (LIF) in GS cell culture. We found that the addition of LIF to newborn testis cell culture enhances the formation of germ cell colonies. Ciliary neurotrophic factor, but not oncostatin M, had the same effect, although they both bind to the IL-6ST (gp130) receptor. On the other hand, GS cells could be established from pup or adult testes in the absence of LIF. No phenotypic or functional difference was found between GS cells established from different stages, and normal offspring were born from pup-derived GS cells that had been maintained in the absence of LIF, indicating that LIF per se is not involved in the self-renewal of GS cells. These results demonstrate that LIF is useful in the initiation of GS cell culture and suggest that LIF or a related cytokine is involved in the maturation of gonocytes into spermatogonia.     

Sydney rock oyster (Saccostrea glomerata) hemocytes: morphology and function

In this study, three major hemocyte types were identified in the Sydney rock oyster. They were characterized primarily by light and electron microscopy based on the presence or absence of granules and nucleus to cytoplasm ratios. Hemoblast-like cells were the smallest cell type 4.0+/-0.4microm and comprised 15+/-3% of the hemocyte population. They had large nuclei and scanty basic cytoplasm. This cell type also had some endoplasmic reticuli and mitochondria. The second major type were hyalinocytes. Hyalinocytes represented 46+/-6% of all hemocytes. They were large cells (7.1+/-1.0microm) that had low nucleus:cytoplasm ratios and agranular basic or acidic cytoplasm. Hyalinocytes had the ability to phagocytose yeast cells and formed the core of hemocyte aggregates associated with agglutination. Four discrete sub-populations of hyalinocytes were identified. The third major cell type were the granulocytes, comprising 38+/-1% of the hemocyte population. These cells were large (9.3+/-0.3microm) and were characterized by cytoplasm containing many acidic or basic granules. Granulocytes were more phagocytic than hyalinocytes and they formed the inner layer of hemocytes during the encapsulation of fungal hyphae. Five discrete sub-populations of granulocytes were identified based on the types of granules in their cytoplasm. Flow cytometry showed that the hemocytes of rock oysters could be divided into between two and four major cell types based on their light scattering properties. The most common of the cell types identified by flow cytometry corresponded to hyalinocytes and granulocytes. Cytochemical assays showed that most enzymes associated with immunological activity were localized in granulocytes. Their granules contained acid phosphatase, peroxidase, phenoloxidase, superoxide and melanin. Hyalinocytes were positive only for acid phosphatase. All of these observations suggest that Sydney rock oysters have a broad variety of functionally specialized hemocytes, many of which are involved in host defense.     

A Role for Topographic Cues in the Organization of Collagenous Matrix by Corneal Fibroblasts and Stem Cells

Human corneal fibroblasts (HCF) and corneal stromal stem cells (CSSC) each secrete and organize a thick stroma-like extracellular matrix in response to different substrata, but neither cell type organizes matrix on tissue-culture polystyrene. This study compared cell differentiation and extracellular matrix secreted by these two cell types when they were cultured on identical substrata, polycarbonate Transwell filters. After 4 weeks in culture, both cell types upregulated expression of genes marking differentiated keratocytes (KERA, CHST6, AQP1, B3GNT7). Absolute expression levels of these genes and secretion of keratan sulfate proteoglycans were significantly greater in CSSC than HCF. Both cultures produced extensive extracellular matrix of aligned collagen fibrils types I and V, exhibiting cornea-like lamellar structure. Unlike HCF, CSSC produced little matrix in the presence of serum. Construct thickness and collagen organization was enhanced by TGF-ß3. Scanning electron microscopic examination of the polycarbonate membrane revealed shallow parallel grooves with spacing of 200–300 nm, similar to the topography of aligned nanofiber substratum which we previously showed to induce matrix organization by CSSC. These results demonstrate that both corneal fibroblasts and stromal stem cells respond to a specific pattern of topographical cues by secreting highly organized extracellular matrix typical of corneal stroma. The data also suggest that the potential for matrix secretion and organization may not be directly related to the expression of molecular markers used to identify differentiated keratocytes.     

The effect of specific antiserum on the metabolism of three membrane-associated antigens of ASL-1 x LM(TK)- hybrid cells.

Fusion of ASL-1 cells, a murine leukemia forming thymus leukemia (TL) antigens, with LM(TK)- cells, a TL(--) murine cell line, resulted in a stable hybrid forming TL antigens. The hybrids failed to undergo modulation, the reversible dissappearance of TL antigens from the surfaces of the cells, stimulated by TL antiserum. Unlike ASL-1 cells, the rate of disappearance of the antigens from modulation negative hydrid cells was unaffected by TL antiserum. The t 1/2 of TL antigens of the hybrid was approximately 30 h. The t 1/2 of TL antigens of ASL-1 cells was 10 h in the presence of TL antiserum, 18 h in the absence of TL antiserum. The rate of metabolism of a putative tumor-associated antigen of ASL-1 cells formed by the hybrid was unaffected by exposure to specific antiserum, as was the metabolism of H-2 antigens formed by the cell types.     

Population dynamics of human activated natural killer cells in culture

The growth kinetics and population dynamics of recombinent interleukin-2 (rlL-2) stimulated human natural killer (NK) cell-enriched populations were studied in vitro. The NK-enriched populations was obtained from normal peripheral blood mononuclear cells (PBMNC) by immunomagnetic bead depletion of CD3(+) and CD5(+) T cells. The growth kinetics of NK cells, T cells, monocytes, and total cells are shown. In the absence of PBMNC accessory cells, the NK-enriched population showed limited expansion. In the presence of PBMNC accessory cells, the NK-enriched population expanded threefold more than in the absence of accessory cells due to increased NK cell growth rate and increased duration of exponential growth. Using a Transwell system, which separates two cell population by a polycarbonate membrane, the accessory cells were shown to act on the NK-enriched population via a diffusible factor. Accessory cell conditioned media was able to replace the accessory cell population to stimulate NK cell expansion. A monocyte-enriched population prepared by sheep red blood cell rosetting of T cells was extensively phenotyped and compared with the NK-enriched populations. Although the final cultured cells were phenotypically homogeneous for CD56(+)/CD3(-) NK cells, the initial NK precusor populations appear to be different. Namely, the NK cell precursors in the monocyte-enriched population were predominantly CD56(+)/CD2(-). Kinetic equations were formulated for this culture system and the effects of major culture variables are investigated.     

Can retinal adhesion mechanisms determine cell-sorting patterns: a test of the differential adhesion hypothesis

Embryonic chick neural retina cells possess two classes of adhesion mechanism, one Ca2+-independent, one Ca2+-dependent, responsible for short-term cell aggregation. This study investigates the role of these mechanisms in the long-term cell sorting potentially relevant to in vivo histogenesis. Retina cells are prepared either with both (E cells) or with only one mechanism (TC cells, CD; LTE cells, CI), respectively. The two types of cell preparations are differentially labelled using fluorescein or rhodamine isothiocyanate, mixed and allowed to aggregate in the presence or absence of cycloheximide at 0.5 microgram ml-1 to retard metabolic recovery of the removed adhesive mechanism. When observed by fluorescence and phase-contrast microscopy, the aggregates formed in cycloheximide show cell sorting, the cells with both mechanisms assuming a more interior position relative to those with a single adhesion mechanism. In parallel hanging-drop experiments, preformed aggregates of cells with a single adhesion mechanism are seen to spread upon aggregates of cells with both mechanisms. No sorting occurs amongst cells from a given stage prepared using any single dissociation protocol. The observed cell sorting would thus seem to derive exclusively from differential cell adhesiveness dependent upon the different dissociation conditions and maintained in the presence of cycloheximide. The experiments support the hypothesis that the dual CI and CD adhesion mechanisms in question can play a central role in governing cell-sorting behaviour during normal histogenesis.     

Polarized secretion of urokinase-type plasminogen activator by epithelial cells.

Numerous epithelial cell types produce and secrete plasminogen activators (PAs) and/or PA inhibitors (PAIs). When epithelial cells were grown on polycarbonate filters and their apical and basolateral secretion products analyzed, PA activity accumulated in a highly polarized fashion; depending upon the cell line, the compartment of PA accumulation was either apical (MDCK I cells and HBL-100 cells) or basolateral (LLC-PK1, CaCo-2, and HeLa cells). By contrast, PAI-1 was recovered in roughly equal amounts in both compartments. Basolateral accumulation of urokinase-type plasminogen activator (uPA), but not its apical targeting, required an acidic compartment and the integrity of the cytoskeleton. Polarity of uPA accumulation did not result from removal of the free enzyme from the opposite compartment through its binding to the cell surface. Transfection with wild-type or mutated murine uPA demonstrated that neither the "growth factor" domain nor the kringle domain is required for the appropriate sorting of the protein. We propose that polarized secretion of PAs is one mechanism whereby cells spatially control extracellular proteolysis.     

Isolation and culture of glandular epithelial and stromal cells from the endometrium of mares.

Glandular epithelial and stromal cells were isolated from the endometrium of mares by collagenase digestion and were incubated on plastic for 7-9 days until the cells formed confluent monolayers. The cells differed in morphology: epithelial cells appeared polyhedral and stromal cells were spindle like. The monolayers were incubated in the presence and absence of oxytocin. Medium was removed from wells after 2, 8 and 24 h of incubation. Concentrations of prostaglandin F (PGF) in the medium increased significantly during this time. Glandular epithelial cells produced significantly more PGF than did stromal cells. Both types of cell responded significantly to oxytocin stimulation by increased secretion of PGF; the response of glandular epithelial cells tended to be greater than that of stromal cells. Secretion of PGF by cultured cells was not affected by cycle stage or pregnancy.