Endothelial cell surfaces play key roles in several important physiological and pathological processes such as blood clotting, angiogenic responses, and inflammation. Here we describe the cloning and characterization of tie, a novel type of human endothelial cell surface receptor tyrosine kinase. The extracellular domain of the predicted tie protein product has an exceptional multidomain structure consisting of a cluster of three epidermal growth factor homology motifs embedded between two immunoglobulinlike loops, which are followed by three fibronectin type III repeats next to the transmembrane region. Additionally, a cDNA form lacking the first of the three epidermal growth factor homology domains was isolated, suggesting that alternative splicing creates different tie-type receptors. Cells transfected with tie cDNA expression vector produce glycosylated polypeptides of 117 kDa which are reactive to antisera raised against the tie carboxy terminus. The fie gene was located in chromosomal region ip33 to ip34. Expression of the tie gene appeared to be restricted in some cell lines; large amounts oftie mRNA were detected in endothelial cell lines and in some myeloid leukemia cell lines with erythroid and megakaryoblastoid characteristics. In addition, mRNA in situ studies further indicated the endothelial expression of the tie gene. The tie receptor tyrosine kinase may have evolved for multiple protein-protein interactions, possibly including cell adhesion to the vascular endothelium.

Abstract. Paraffin sections of human skeletal tissues were studied in order to identify cells responsible for production of types I, II, and HI collagens by in situ hybridization. Northern hybridization and sequence information were used to select restriction fragments of eDNA clones for the corresponding mRNAs to obtain probes with a minimum of cross-hybridization. The specificity of the probes was proven in hybridizations to sections of developing fingers: osteoblasts and chondrocytes, known to produce only one type of fibrillar collagen each (I and II, respectively) were only recognized by the corresponding eDNA probes. Smooth connective tissues exhibited variable hybridization intensities with types I and III collagen eDNA probes.The technique was used to localize the activity of type II collagen production in the different zones of cartilage during the growth of long bones. Visual inspection and grain counting revealed the highest levels of proal(II) collagen mRNAs in chondrocytes of the lower proliferative and upper hypertrophic zones of the growth plate cartilage. This finding was confirmed by Northern blotting of RNAs isolated from epiphyseal (resting) cartilage and from growth zone cartilage.Analysis of the osseochondral junction revealed virtually no overlap between hybridization patterns obtained with probes specific for type I and type 1I collagen mRNAs. Only a fraction of the chondrocytes in the degenerative zone were recognized by the pro~tl(ID collagen eDNA probe, and none by the type I collagen eDNA probe. In the mineralizing zone virtually all cells were recognized by the type I collagen eDNA probe, but only very few scattered cells appeared to contain type II collagen mRNA. These data indicate that in situ hybridization is a valuable tool for identification of connective tissue cells which are actively producing different types of collagens at the various stages of development, differentiation, and growth.T HE majority of total body collagen is found in the specialized connective tissues of the skeletal system in the form of the fibrillar collagens of types I, II, and III. Each of these collagens are synthesized as procollagens containing three proa-chains. Type I collagen is a heterotrimer of two al(I) and one a2(I) chains, while types II and HI collagens are homotrimers of al(ID and al0II) chains, respectively. All these chains share considerable homology both at the level of amino acids, mRNAs, and gene structure (5,17,19). Additionally, at least nine other collagen types with variable functions have been characterized (5).The development and growth of the skeletal tissues is a complicated process involving a number of changes in the expression of collagen genes (29,34). This is exemplified by the growth of long bones which occurs in the growth plate areas by chondrocyte division and deposition of cartilage matrix, containing type II collagen fibers. Having terminally differentiated, the hypertrophic chondrocytes degenerate, the extracellular matrix becomes calcified, and is finally invaded ...

Fluorescent lanthanide chelates with long decay times allow the suppression of the fast decaying autofluorescence in biological specimens. This property makes lanthanide chelates attractive as labels for fluorescence microscopy. As a consequence of the suppression of the background fluorescence the sensitivity can be increased.We modified a standard epifluorescence microscope for time-resolved fluorescence imaging by adding a pulsed light source and a chopper in the narrow aperture plane. A cooled CCD-camera was used for detection and the images were digitally processed.A fluorescent europium chelate was conjugated to antisera and to streptavidin. These conjugates were used for the localization of tumor associated antigen C242 in the malignant mucosa of human colon, for the localization of type I1 collagen mRNA in developing human cartilaginary growth plates, and for the detection of HPV type specific gene sequences in the squamous epithelium of human cer- vix.The specific slowly decaying fluorescence of the europium label could be effectively separated from the fast decaying background fluorescence. It was possible to use the europium label at the cell and tissue level and the autofluorescence was effectively suppressed in in situ hybridization and immunohistochemical reactions in both frozen and formaldehydefixed, wax-embedded specimens. 0 1992 Wiley-Liss, Inc.

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