Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions

Summary A serum-free primary culture system is described which allows normal rat mammary epithelial cells (RMECs) embedded within a reconstituted basement membrane to undergo extensive growth and functional differentiation as detected by synthesis and secretion of the milk products casein and lipid. RMECs isolated from mammary glands of immature virgin rats were seeded within an extracellular matrix preparation derived from the Engelbreth-Holm-Swarm sarcoma and cultured in a serum-free medium consisting of Dulbecco's modified Eagle's medium-F12 containing insulin, prolactin, progesterone, hydrocortisone, epidermal growth factor, bovine serum albumin, transferrin, and ascorbic acid. Casein synthesis and secretion were documented at the electron microscopic level as well as by an enzyme-linked immunosorbent assay (ELISA) assay using a polyclonal antibody against total rat caseins. Numerous secretory vesicles with casein micelles were noted near the apical surface of the RMECs, and secreted casein was observed in the lumen. These ultrastructural data were confirmed by the ELISA assay which showed that microgram amounts of casein per well were synthesized by the RMECs and that the amount of casein increased with time in culture. Using immunoblot analysis it was demonstrated that the full complement of casein proteins was synthesized. In addition to casein protein, β-casein mRNA levels were shown to increase with time. Synthesized lipid was detected at both the light and electron microscopic levels. Phase contrast photomicrographs demonstrated extensive intracellular lipid accumulation within the ductal and lobuloalveolarlike colonies, and at the electron micrograph level, lipid droplets were predominantly localized near the apical surface of the RMECs. The lipid nature of these droplets was verified by oil red O staining. Results from this study demonstrate that RMECs from immature virgin rats proliferate extensively and rapidly develop the capacity to synthesize and secrete casein and lipid when grown within a reconstituted basement membrane under defined serum-free conditions. This unique system should thus serve as an excellent model in which the regulation of mammary development and gene expression can be investigated. This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health, Bethesda, MD.     

Mouse submandibular salivary epithelial cell growth and differentiation in long-term culture: Influence of the extracellular matrix

Summary The adult mouse submandibular salivary gland provides a good model system to study gene regulation during normal and abnormal cell behavior because it synthesizes functionally distinct products ranging from growth factors and digestive enzymes to factors of relevance to homeostatic mechanisms. The present study describes the long-term growth and differentiation of submandibular salivary epithelial cells from adult male mice as a function of the culture substratum. Using a two-step partial dissociation procedure, it was possible to enrich for ductal cells of the granular convoluted tubules, the site of epidermal growth factor synthesis. Long-term cell growth over a period of 2 to 3 mo. with at least 3 serial passages was obtained only within three-dimensional collagen gels. Cells grew as ductal-type structures, many of which generated lumens with time in culture. Electron microscopic analysis in reference to the submandibular gland in vivo revealed enrichment for and maintenance of morphologic features of granular convoluted tubule cells. Reactivity with a keratin-specific monoclonal antibody established the epithelial nature of the cells that grew within collagen. Maintenance of cell differentiation, using immunoreactivity for epidermal growth factor as criterion, was determined by both cytochemical and biochemical approaches and was found to be dependent on the collagen matrix and hormones. Greater than 50% of the cells in primary collagen cultures contained epidermal growth factor only in the presence of testosterone and triiodothyronine. In contrast, cells initially seeded on plastic or cycled to plastic from collagen gels were virtually negative for epidermal growth factor. Biochemical analysis confirmed the presence of a protein with an apparent molecular weight of 6000 which comigrated with purified mouse epidermal growth factor. Epidermal growth factor was also present in detectable levels in Passage 1 cells. This culture system should permit assessment of whether modulation of submandibular gland ductal cell growth can be exerted via a mechanism that in itself includes epidermal growth factor and its receptor and signal transduction pathway. This work was supported by Public Health Service grant DE07766 from the National Institute of Dental Research, National Institutes of Health, Bethesda, MD.     

Modulation of microvascular growth and morphogenesis by reconstituted basement membrane gel in three-dimensional cultures of rat aorta: A comparative study of angiogenesis in Matrigel,collagen, fibrin,and plasma clot

Summary Rings of rat aorta cultured in Matrigel, a reconstituted gel composed of basement membrane molecules, gave rise to three-dimensional networks composed of solid cellular cords and occasional microvessels with slitlike lumina. Immunohistochemical and ultrastructural studies showed that the solid cords were composed of endothelial sprouts surrounded by nonendothelial mesenchymal cells. The angiogenic response of the aortic rings in Matrigel was compared to that obtained in interstitial collagen, fibrin, or plasma clot. Morphometric analysis demonstrated that the mean luminal area of the microvascular sprouts and channels was significantly smaller in Matrigel than in collagen, fibrin, or plasma clot. The percentage of patent microvessels in Matrigel was also markedly reduced. Autoradiographic studies of³H-thymidine-labeled cultures showed reduced DNA synthesis by developing microvessels in Matrigel. The overall number of solid endothelial cords and microvessels was lower in Matrigel than in fibrin or plasma clot. A mixed cell population isolated from Matrigel cultures formed a monolayer in collagen or fibrin-coated dishes but rapidly reorganized into a polygonal network when plated on Matrigel. The observation that gels composed of basement membrane molecules modulate the canalization, proliferation, and organization into networks of vasoformative endothelial cells in three-dimensional cultures supports the hypothesis that the basement membrane is a potent regulator of microvascular growth and morphogenesis. This work was supported by grants from the W. W. Smith Charitable Trust and grants CA14137 and HL43392 from the National Institutes of Health, Bethesda, MD.     

A dimorphic Alu Sb-like insertion in COL3A1 is ethnic-specific

Alu elements are a class of repetitive DNA sequences found throughout the human genome that are thought to be duplicated via an RNA intermediate in a process termed retroposition. Recently inserted Alu elements are closely related, suggesting that they are derived from a single source gene or closely related source genes. Analysis of the type III collagen gene (COL3A1) revealed a polymorphic Alu insertion in intron 8 of the gene. The Alu insertion in the COL3A1 gene had a high degree of nucleotide identity to the Sb family of Alu elements, a family of older Alu elements. The Alu sequence was less similar to the consensus sequence for the PV or Sb2 subfamilies, subfamilies of recently inserted Alu elements. These data support the observations that at least three source genes are active in the human genome, one of which is distinct from the PV and Sb2 subfamilies and predates either of these two subfamilies. Appearance of the Alu insertion in different ethnic populations suggests that the insertion may have occurred in the last 100,000 years. This Alu insert should be a useful marker for population studies and for marking COL3A1 alleles.     

Role of integrins in differentiation of chick retinal pigmented epithelial cells in vitro

When retinal pigmented epithelial cells (PEC) of chick embryos are cultured under appropriate conditions, the phenotype changes to that of lens cells through a process known as transdifferentiation. The first half of the process, characterized by dedifferentiation of PEC, is accompanied by a marked decrease in adhesiveness of PEC to collagen type I- or type IV-coated dishes. To understand the underlying mechanisms of this change, we analyzed the expression of integrins, which are major receptors for extracellular matrix components. Northern blot analysis with cDNA probes for chicken α3, α6, α8, αv, β1 and β5 integrin mRNA showed that the genes for all these integrins are transcribed at similar levels in PEC and dedifferentiated PEC (dePEC). Further analysis of β1 integrin, which is a major component of integrin heterodimers, showed that although the protein amount of β1 integrin was not changed, its localization at focal contacts seen in PEC was lost in dePEC. When anti-β1 integrin antibody was added to the PEC culture medium, a decrease of cell-substrate adhesiveness occurred, followed by a gradual change in both morphology and gene expression patterns to ones similar to those of dePEC. These findings suggest that an appropriate distribution of β1 integrin plays an essential role in maintaining the differentiated state of PEC through cell-substrate adhesion.     

Use of diethyl squarate for the coupling of oligosaccharide amines to carrier proteins and characterization of the resulting neoglycoproteins by MALDI-TOF mass spectrometry

The 8-methoxycarbonyloctyl glycosides of GlcNAc, Gal1-4Glc, Fuc1-2Fuc1-3GalNac and Fuc1-2Gal1-3[Fuc1-4]GlcNac were converted to primary amines by reaction with neat ethylenediamine and then coupled to bovine serum albumin (BSA) using diethyl squarate as the connector. The average degree of incorporation of the sugar onto the protein, as well as the molecular weight distribution, could be conveniently determined using matrix assisted laser desorption ionization/time of flight (MALDI-TOF) mass spectrometry thus avoiding cumbersome structure-dependent colour-tests or analysis of cleaved ligand. The present coupling method has the advantages of proceeding under very mild conditions, yielding controlled incorporation values and can reliably be used for the coupling of very small amounts (mg) of oligosaccharide.This paper is dedicated to Sen-itiroh Hakomori on the occasion of his 65th birthday     

Mechanical effects of ET-1 in cardiomyocytes isolated from normal and heart-failed rabbits

Endothelin (ET-1) is found at elevated concentrations in the plasma of patients with heart failure and in animal models of cardiomyopathy. The peptide is a potent positive inotropic agent, the effects of which are mediated by increases in cytosolic Ca²⁺ in cardiomyocytes. The object of this study was to investigate at the cellular level, the actions of ET-1 on contractile function and on Ca²⁺ currents in heart-failed ventricular myocardium. Male New Zealand White rabbits (8 wks) were treated with twice weekly injections of epirubicin (4 mg/kg/wk, n=7) or with saline (n=7) for 6 wks, followed by a washout period of 2 wks. Ventricular cardiomyocytes were isolated from rabbit hearts using Langendorff perfusion with collagenase; contractile function was examined using a video microscopy method, and L-type Ca²⁺ currents were recorded using a whole-cell patch-clamp technique. ET-1 produced a concentration-dependent increase in contractile response (% increase from basal value) to a maximum at 1 nM ET-1 of 69 ± 11% (mean ± S.D.) in control cardiomyocytes and 33 ± 6% in heart-failed cells. However, there was no significant change in the EC₅₀ obtained with ET-1 for healthy (0.31 ± 0.1 nM) and for failed cardiomyocytes (0.24 ± 0.1 nM). The effects of ET-1 on L-type Ca²⁺ channels were similar with a peak amplitude at 1 nM ET-1 of –3.26 ± 0.8 in control cardiomyocytes and –3.32 ± 0.9 nA in heart-failed cells. The attenuation of the contractile response to ET-1 in heart-failed cells may reflect a desensitization of ET receptors as a consequence of elevated circulating levels of ET and was not reflected by alteration of transmembrane Ca²⁺ conductance. It is probable, therefore, that multiple signalling pathways are involved in the actions of ET on ventricular myocardium.Recipient of Servier Investigator Award     

β2-Adrenoceptor activation by zinterol causes protein phosphorylation,contractile effects and relaxant effects through a cAMP pathway in human atrium

Evidence from ventricular preparations of cat, sheep, rat and dog suggests that both ₁-adrenoceptors (₁AR) and ₂-adrenoceptors (₂AR) mediate positive inotropic effects but that only ₁AR do it through activation of a cAMP pathway. On the other hand, our evidence has shown that both ₁ AR and ₂ AR hasten relaxation of isolated human myocardium consistent with a common cAMP pathway. We have now investigated in the isolated human right atrial appendage, a tissue whose -AR comprise around 2/3 of ₁AR and 1/3 of ₂AR, whether or not ₂AR-mediated effects occur via activation of a cAMP pathway. We carried out experiments on atria obtained from patients without advanced heart failure undergoing open heart surgery. To activate ₂AR, we used the ₂AR-selective ligand zinterol. Experiments were carried out on paced atrial strips (1 Hz) and tissue homogenates and membrane particles. Zinterol caused positive inotropic and lusitropic (i.e. reduction of t_1:2 of relaxation) effects with EC₅₀ values of 3 and 2 nM, respectively. The zinterol-evoked effects were unaffected by the AR-selective antagonist CGP 20712A (300 nM) but blocked surmountably by the ₂AR-selective antagonist ICI 118551 (50 nM) which reduced both EC₅₀ values to 1 M. Zinterol stimulated adenylyl cyclase activity with an EC₅₀ of 30 nM and intrinsic activity of 0.75 with respect to (–)-isoprenaline (600 M); the effects were resistant to blockade by CGP 20712A (300 nM) but antagonised surmountably by ICI 118551 (50 nM). Zinterol bound to membrane PAR labelled with (–)-[¹²⁵I] cyanopindolol with higher affinity for ₂AR than for - 1 AR; the binding to ₂AR but not to - BAR was reduced by GTPyS (10 M). In the presence of CGP 20712A (300 nM) (–)-isoprenaline (400 M); (to activate both ₁AR and ₂AR maximally) and zinterol (10 M); increased contractile force 3.4-fold and 2.5-fold respectively and reduced relaxation tut by 32% and 18% respectively. These effects of (–)-isoprenaline and zinterol were associated (5 min incubation) with phosphorylation (pmol P/mg supernatant protein) of troponin I and C-protein to values of 8.4 ± 2.0 vs 12.4 ± 2.3 and 10.1 ± 2.5 vs 8.6 ± 1.6 respectively. (–)-Isoprenaline and zinterol also caused phosphorylation of phospholamban (1.8 ± 0.3 vs 0.4 ± 0.1 pmol P/mg respectively) specifically at serine residues. We conclude that in human atrial myocardium activation of both ₁AR and ₂AR leads to cAMP-dependent phosphorylation of proteins involved in augmenting both contractility and relaxation.     

Occurrence of heavy metals,sodium, calcium,and potassium in human hair,teeth, and nails

Heavy metals in biological samples: nails, teeth, and hair were examined during 1991–1993. Investigations of biological samples (hairn=249 samples, teethn=145, nailsn=80 samples) were provided for inhabitants of selected towns in Beskid Śląski. The towns are small mountain towns in southern Poland: Wista, Szczyrk, Istebna, Koniaków, and Jaworzynka. The analysis of ANOVA and MANOVA variances were used for biological samples in the context of age, sex, and type of samples for 12 elements (Pb, Cd, Cr, Zn, Fe, Cu, Mn, Ni, Co, Ca, Na, and K). The matrix correlation and cluster analysis were applied to explain the behavior of metals in human hair, teeth, and nails.     

The heat shock cognate 80 gene of tomato is flanked by matrix attachment regions

Matrix attachment regions (MARs) are thought to participate in the organization and segregation of independent chromosomal loop domains. Although there are several reports on the action of MARs in the context of heterologous genes, information is more limited on the role of MARs associated with plant genes. Transgenic studies suggest that the upstream, intron and downstream regions of the developmentally regulated heat shock cognate 80 gene (HSC80) of tomato participate in chromatin organization. In this study, we tested the in vitro affinity of the HSC80 gene to chromosomal scaffolds prepared from shoot apices of tomato. We found that a 1.5 kb upstream region and a 1.4 kb downstream region, but not the intron region, are MARs. These MARs interact with tomato and pea scaffolds and bind regardless of the expression status of HSC80 in the tissue from which the nuclei were isolated. Comparison to two known yeast MARs ARS1 and CENIII, showed that the HSC80 5MAR binds more avidly to tomato scaffolds than ARS1, while no binding of CENIII was observed. Competition binding between the two HSC80 MARs indicated that the 5 MAR can outcompete the 3 MAR and not vice versa. Last, we observed that the interaction of the 3 MAR with the scaffold could result in an electrophoretic mobility shift resistant to SDS, protease, and phenol treatment. In conclusion, MARs whose binding properties can be clearly differentiated are closely flanking the HSC80 gene. The discovery of MARs in regions which have a distinct function in HSC80 transgenes but not in transient expression assays, is consistent with a chromosomal scaffold role in HSC80 gene regulation.