Controlled production of proliferating somatic cell hybrids.

The techniques described permit the controlled production of large numbers of proliferating somatic cell hybrids in a relatively short period of time. Sendai virus is used to promote cell hybridization. beta-propriolactone is employed as the inactivating agent of Sendai virus since it produces complete loss of viral infectivity while preserving viral fusion capacity. Cells are fused in monolayer, instead of in suspension, since fixing cells in two dimensions permits one to control cell contacts during the fusion event through the expedient of varying multiplicities of the parental cells and the total cell density. Under the conditions described, a several hundred fold increase in the number of hybrid clones obtained is seen as compared to the controls.

Alpha 5 integrin subunit expression changes during myogenesis.

Fibronectin and its cellular receptor, the alpha 5 beta 1 integrin, are involved in the transmembrane signalling events that control muscle cell differentiation. In this study, the expression of the alpha 5 integrin subunit was followed by reverse transcription-polymerase chain reaction (RT-PCR) to determine alterations during myogenesis. In studies of murine muscle, we found a 90% reduction in the level of the alpha 5 integrin subunit mRNA during early postnatal development. Concurrently, the fibronectin alternative splicing pattern changed markedly in the EIIIB and V exons. In-vitro analyses of these molecules during myoblast differentiation revealed changes that followed trends similar to those observed in vivo, although of lesser magnitude. These observations imply an important role of fibronectin and the alpha 5 integrin subunit in muscle development.

Overview of expression of matrix metalloproteinases (MMP-17, MMP-18, and MMP-20) in cultured human cells.

We recently described the cell type distribution of several matrix metalloproteinases (MMP-1 through MMP-16). In this report we extend this study by analysis of three recently described MMPs. PCR primers for MMP-17, MMP-18, and MMP-20 were optimized for use in RT-PCR. The results demonstrate one or more cell lines or tissue that express mRNA for each of these newly described MMPs.

Neutrophil collagenase (MMP-8) is expressed during early development in neural crest cells as well as in adult melanoma cells.

Matrix metalloproteinase-8 (MMP-8) is a neutral metalloproteinase of the fibrillar collagenase family that also includes MMP-1 and MMP-13. In contrast to the other collagenases, MMP-8 has a very limited tissue distribution, thought to be restricted to neutrophils and chondrocytes. In a previous study, we observed MMP-8 expression in human melanoma cells. This observation led us to assess in more detail the expression of MMP-8 in normal and malignant melanocytic cells. We found that MMP-8 was expressed by 11 out of 12 human melanoma cell lines tested and all 10 primary melanomas we examined, but was not expressed by four primary neonatal melanocyte strains. Since melanocytes arise from highly motile neural crest cells, we examined the hypothesis that MMP-8 might be expressed by neural crest cells. RT-PCR analysis of post-implantation mouse embryos indicated the presence of MMP-8 transcripts at E9.5. In situ hybridization and immunohistochemistry of mouse embryos between E9.5-E14.5 demonstrated MMP-8 expression in the surface ectoderm, neural crest cells and chondrocytes. MMP-8 was also detected in neural crest cell migration located in the circumference of the neural tube, branchial arches and the notochord. In addition, MMP-8 expression was observed between the somites, in circumscriptive areas of the developing brain, heart, and eye, and in the interdigital zones of the limbs. In summary, we found MMP-8 to be the first fibrillar collagenase expressed during development. In contrast to its restricted tissue expression post-partum, MMP-8 was present in multiple embryonic tissues, including neural crest cells. The production of MMP-8 by migrating neural crest cells may contribute to their ability to degrade fibrillar collagen matrices while in transit.

Overview of matrix metalloproteinase expression in cultured human cells.

The matrix metalloproteinases (MMP) have been implicated in tumor invasion and metastasis both by immunohistochemical studies and from the observation that specific metalloproteinase inhibitors block tumor invasion and metastasis. Oligonucleotide primers for thirteen MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16) were optimized for use in RT-PCR. A semi-quantitative RT-PCR assay was used to determine the pattern of MMP mRNA expression in 84 normal and transformed or carcinogen transformed human cell lines and strains derived from different tissues. The results demonstrate one or more cell lines which express thirteen members of the MMP family. In addition, various oncogene transfected human fibroblast cell strains were analyzed for MMP expression. We confirm that over-expression of the H-ras oncoprotein correlates with up-regulation of MMP-9 and demonstrate that over-expression of v-sis also up-regulates MMP-9. A cell line immortalized following myc expression was found to up-regulate MMP-7, MMP-11 and MMP-13. Inappropriate expression of several MMP mRNAs was detected in breast, prostate, bone, colon and oral tumor derived cell lines. Identification of at least one cell line expressing each of thirteen MMPs and the observation of oncogene induced expression of several MMPs should facilitate analysis of the transcriptional mechanisms controlling each MMP.

A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast.

Polyethylene glycol (PEG) can induce genetic transformation in both bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae) without cell wall removal. PEG-mediated transformation of E. coli is technically simple and yields transformants with an efficiency of 10(6)-10(7) transformants/microgram DNA. Detailed analysis of the parameters involved in PEG-mediated transformation of E. coli reveals basic differences between the PEG and standard CaCl2 methods for transformation of E. coli. PEG-mediated transformation of yeast is far simpler than existing protoplast methods and is comparable in efficiency. The new methods described here for PEG-mediated genetic transformation may prove to be of general utility in performing genetic transformation in a wide variety of organisms.

Late passage cells display an increase in contractile behavior.

During aging, alterations in the extracellular matrix and cytoskeleton have been noted. Aging affects both the structure and the biological activity of fibronectin as well as the cellular content and organization of f-actin. The purpose of this study was to examine the functional significance of such alterations. Assays for cell migration, cell spreading, collagen gel contraction, and f-actin organization were conducted using diploid cells strains of high and low population doubling level (PDL). First, an assessment of cell spreading behavior revealed that high PDL cells spread more than low PDL cells. Second, analyses performed with a modified phagokinetic track assay indicate that high PDL cells migrate (phagocytize) faster than low PDL cells. Lastly, it was found that high PDL cells contract collagen gels faster than low PDL cells. Therefore, high PDL cells are capable of exerting more force upon their extracellular matrix than low PDL cells. In agreement with previous observations, we found by phalloidin staining that the f-actin content of high PDL cells was substantially greater than that of low PDL cells. The elevated f-actin content of high PDL cells could promote increased cellular contractility thereby leading to the increase in cell spreading, migration, and collagen gel contraction observed here.

Methods for solid phase peptide synthesis which employ a minimum of instrumentation.

Both automated and manual methods of solid phase peptide synthesis employ three basic steps: (a) Attachment of the first amino acid to a resin, (b) peptide synthesis via successive carbodiimide couplings and (c) cleavage and deblocking of the peptide. Instead of an automated peptide synthesizer, one can manually synthesize peptides with a sintered glass funnel as the only required piece of equipment. Following solid phase synthesis, one can cleave and deblock peptides without the use of anhydrous hydrofluoric acid (HF); hence, the need for specialized equipment required for handling HF can also be eliminated. In the procedure described in this report, cleavage and deblocking is carried out with trifluoromethane sulfonic acid (TFMSA) in glass vessels without the need for high pressure Teflon fittings. Since completion of the coupling reaction can be monitored during each cycle when manual methods are employed, one can avoid repetitive couplings and, thereby economize on reagents. Since TFMSA cleavage and deblocking can be carried out in open glass vessels, one can cleave and deblock large numbers of peptides at the same time. With the methods described, one can satisfactorily prepare large quantities of peptides at minimal cost.

RT-PCR without RNA isolation.

Reverse transcription-PCR (RT-PCR) has traditionally required time-consuming RNA extraction and purification. This report demonstrates that one can completely avoid the RNA extraction step in RT-PCR by basing the comparison of samples on cell number rather than micrograms of total RNA. A new method for lysing cells while preserving RNA is described. RT-PCR is carried out (i) by rapidly freezing cells in the presence of ribonuclease inhibitor (RNase inhibitor) plus dithiothreitol and (ii) by using extracts of 250 or fewer cells directly in the RT-PCR assay. Aldolase mRNA, extracted by freeze-thawing cells in the presence of RNase inhibitor, was found to be stable at 42 degrees C for over three hours. Since the RT step can be completed within 1 h, there is minimal degradation of mRNA. This simple procedure avoids the use of harsh reagents, which may inhibit enzymes involved in RT-PCR, and produces results virtually identical to methods that employ guanidinium thiocyanate and phenol for RNA extraction. Optimized conditions for each parameter of the procedure are described that permit amplification of mRNA from as few as four cells.

Comments on the clinical application of fibronectin in dentistry.

Several studies have demonstrated that citric acid demineralization of the root surface promotes tissue attachment. Since demineralization exposes collagen to which fibronectin binds, the role of fibronectin in the attachment of cells to the tooth surface has been of considerable interest. It is clear that fibronectin and other cell adhesion proteins can promote cell attachment to the tooth surface; therefore, attempts have been made to utilize these findings in a clinical setting. Using a quantitative ELISA procedure to measure the binding of fibronectin to demineralized bone and tooth, we have found that 1 microgram fibronectin can saturate approximately 1 mg of either demineralized bone or demineralized tooth powder. Since serum contains 300 micrograms fibronectin per mL, the bleeding that occurs during oral surgery should saturate exposed tooth surfaces with amounts of fibronectin adequate for cell adhesion. Thus, exogenous fibronectin would appear to be of little clinical benefit.

Amelogenin is a cell adhesion protein.

Amelogenin, the major protein component of tooth enamel, is shown to be a cell adhesion protein. Since it had been shown that an amelogenin-containing preparation, Emdogain, possessed cell-adhesive activity, we tested the hypothesis that amelogenin was responsible for cell-adhesive activity. Recombinant amelogenin was found to promote adhesion at less than 15 micro g/60-mm plate and requires divalent cations for activity. While we found that amelogenin does not bind to collagen or heparin under physiological conditions, it was demonstrated previously that amelogenin does bind to hydroxyapatite. The cell-adhesive activity of amelogenin may play a role in development and may provide a partial explanation for the therapeutic effects of Emdogain in periodontal regeneration.

Monoclonal antibodies to periodontal ligament cells.

Ten mouse monoclonal antibodies were prepared against cultured bovine periodontal ligament cells to be used as reagents for the study of periodontal disease and wound healing. Using standard immunohistochemical methods, these antibodies were found to recognize cell surface antigens in formalin-fixed bovine periodontium. Three of the 10 monoclonal antibodies (i.e., PDL-1, PDL-2, and PDL-10) cross-reacted with cells found in primate periodontium. While the isolated monoclonal antibodies appeared to distinguish subpopulations of cells located in the supporting tissues of teeth, immunohistological examination of other organs (dermis, kidney, skeletal muscle, thyroid, and parotid gland) indicated that a number of cell types of mesenchymal origin share an antigen(s) found on periodontal cells. The monoclonal antibodies described in this report should prove to be useful in studies of periodontal disease and guided tissue regeneration by providing both analytical reagents and immunochemical methods for isolating selected cell populations of the periodontium.

Immunohistological localization of cell adhesion proteins and integrins in the periodontium.

The distribution of the cell adhesion proteins vitronectin, fibronectin, tenascin, and laminin as well as several integrin subunits, alpha 2, alpha 5, and alpha v, was studied in primate periodontal tissues. Full baboon mandibular sections were analyzed by immunohistochemical methods in order to localize the molecules studied in both soft and hard tissues. Vitronectin was associated with the connective tissue of the marginal gingiva, the periodontal ligament, as well as the endosteum and periosteum. A notable finding was the particularly high staining intensity of vitronectin in the periodontal ligament. Fibronectin was widely distributed in the periodontal connective tissue and was also localized to the pericellular matrix of osteocytes and blood vascular elements. Epithelial basement membranes stained positively for both fibronectin and tenascin. These proteins were also expressed in the periosteal and endosteal connective tissues and the periodontal ligament. The staining intensity for tenascin was higher in zones along the cementum and bone surfaces. Laminin was, characteristically, limited to basement membranes of epithelium and endothelium. The distribution of fibronectin, tenascin, and laminin is related to previous findings in other species. The localization of the several integrin alpha-subunits is also described in full baboon mandibular sections. The vitronectin receptor (alpha v) had a uniquely strong expression in osteoclasts of the alveolar bone and was found, at lesser intensity, on periodontal ligament fibroblasts. The fibronectin receptor alpha subunit, alpha 5, was also observed on osteoclasts, and, in addition, was widely distributed on fibroblasts, cementoblasts, and osteoblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibody against human fibronectin which inhibits cell attachment.

Monoclonal antibodies have been prepared against both human and bovine fibronectin. Evidence is provided which indicates that nine different antigenic determinants are recognized by the ten antihuman fibronectin monoclonal antibodies isolated. One monoclonal antibody was identified that blocked fibronectin mediated cell attachment without interfering with fibronectin binding to collagen. Sensitive ELISA assays for fibronectins derived from 32 mammalian species have been developed with the monoclonal reagents characterized in this study.

Cytoscribing: a method for micropositioning cells and the construction of two- and three-dimensional synthetic tissues.

Cells may be positioned in precise, predetermined patterns by a technique, termed cytoscribing, in which cell adhesion proteins are deposited on a substratum under computer control. Using standard office equipment, cells may be positioned within a cell diameter with the techniques described. Cytoscribing involves the use of either a computer-controlled ink jet printer or a graphics plotter to deposit cell adhesion proteins and monoclonal antibodies onto a substrate material. By selecting different cell adhesion proteins and methods that permit the formation of positive or negative patterns of cells (cytoscripts), two-dimensional tissues can be constructed. It is also demonstrated that an optical microlithography technique used in the semiconductor industry can be used to position cells with a precision of less than a micron. By utilizing the properties of cell adhesion proteins, both positive and negative cytoscripts of photoengraved images can be obtained. Techniques for the construction of three-dimensional tissues are described. Thin sheets of collagen were obtained by forming collagen heat gels in a mold. Monolayers of cells growing on thin collagen sheets were then attached to one another by gluing the sheets together with collagen. Sheets of cells do not attach to each other readily in the presence of fibronectin and evidence is provided which indicates that this observation is due to a lack of adhesiveness of the upper surface of cells.