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C D Little

Publications and source records attributed to C D Little.

At least 37 records · Page 2Linked to original sources
PubMed1995

Organized type I collagen influences endothelial patterns during "spontaneous angiogenesis in vitro": planar cultures as models of vascular development.

Selected strains of vascular endothelial cells, grown as confluent monolayers on tissue culture plastic, generate flat networks of cellular cords that resemble beds of capillaries--a phenomenon referred to as "spontaneous angiogenesis in vitro". We have studied spontaneous angiogenic activity by a clonal population (clone A) of bovine aortic endothelial cells to identify processes that mediate the development of cellular networks. Confluent cultures of clone A endothelial cells synthesized type I collagen, a portion of which was incorporated into narrow, extracellular cables that formed a planar network beneath the cellular monolayer. The collagenous cables acted as a template for the development of cellular networks: flattened, polygonal cells of the monolayer that were in direct contact with the cables acquired spindle shapes, associated to form cellular cords, and became elevated above the monolayer. Networks of cables and cellular cords did not form in a strain of bovine aortic endothelial cells that did not synthesize type I collagen, or when traction forces generated by clone A endothelial cells were inhibited with cytochalasin D. In a model of cable development, tension applied by a confluent monolayer of endothelial cells reorganized a sheetlike substrate of malleable type I collagen into a network of cables via the formation and radial enlargement of perforations through the collagen sheet. Our results point to a general involvement of extracellular matrix templates in two-dimensional (planar) models of vascular development in vitro. For several reasons, planar models simulate invasive angiogenesis poorly. In contrast, planar models might offer insights into the growth and development of planar vascular systems in vivo.

Animals↗
PubMed1995

Use of starvation promoters to limit growth and selectively enrich expression of trichloroethylene- and phenol-transforming activity in recombinant Escherichia coli [corrected].

The expression of much useful bacterial activity is facilitated by rapid growth. This coupling can create problems in bacterial fermentations and in situ bioremediation. In the latter process, for example, it necessitates addition of large amounts of nutrients to contaminated environments, such as aquifers. This approach, termed biostimulation, can be technically difficult. Moreover, the resulting in situ bacterial biomass production can have undesirable consequences. In an attempt to minimize coupling between expression of biodegradative activity and growth, we used Escherichia coli starvation promoters to control toluene monooxygenase synthesis. This enzyme complex can degrade the environmental contaminants trichloroethylene (TCE) and phenol. Totally starving cell suspensions of such strains degraded phenol and TCE. Furthermore, rapid conversions occurred in the postexponential batch or very slow growth (dilution) rate chemostat cultures, and the nutrient demand and biomass formation for transforming a given amount of TCE or phenol were reduced by 60 to 90%. Strong starvation promoters have recently been clones and characterized in environmentally relevant bacteria like Pseudomonas species; thus, starvation promoter-driven degradative systems can now be constructed in such bacteria and tested for in situ efficacy.

Biodegradation, Environmental↗
PubMed1995

The association of human fibulin-1 with elastic fibers: an immunohistological, ultrastructural, and RNA study.

We examined the pattern of fibulin-1 mRNA and protein expression in human tissues and cell lines. Fibulin-1 transcripts were found in RNA isolated from most tissues and a variety of cultured cells, including fibroblasts, smooth muscle cells, and several epithelial cell lines, but not endothelial cells, lymphomyloid cells, or a number of carcinoma and melanoma lines. Immunohistochemical analysis showed that fibulin-1 is an intercellular component of connective tissues, predominantly associated with matrix fibers in tissues such as the cervix, dermis, intimal and medial layers of blood vessels, heart valves, meningeal tissue of the brain, Wharton's jelly of the umbilical cord, testis, and lung. Most of the fibers that were immunoreactive with fibulin-1 antibodies also stained with antibodies to the elastic fiber proteins elastin and fibrillin, as well as with Verhoeff's elastin stain. Immunoelectron microscopic analysis of elastin fibers of skin and saphenous vein revealed that fibulin-1 was located within the amorphous core of the fibers, similar to elastin, but it was not in the fibrillin-containing, elastin-associated microfibrils. Our finding that fibulin-1 is an elastic fiber component suggests several possible new functions for fibulin-1, e.g., that it is a structural protein that contributes to the elastic properties of connective tissue fibers or that is involved with the process of fibrogenesis.

Calcium-Binding Proteins↗
PubMed1995

An antagonist of integrin alpha v beta 3 prevents maturation of blood vessels during embryonic neovascularization.

Experimental data in this study demonstrate that integrin alpha v beta 3 is fundamentally involved in the maturation of blood vessels during embryonic neovascularization (vasculogenesis). Integrin alpha v beta 3 was specifically expressed on the surface of angioblasts during vessel development in quail embryos and vitronectin, a ligand for alpha v beta 3, localized to the basal surface of these cells. More importantly, microinjection of the anti-alpha v beta 3 monoclonal antibody, LM609, disrupted the normal pattern of vascular development. After exposure to LM609 the angioblasts in experimental embryos appeared as clusters of rounded cells lacking normal cellular protrusions. This led to disruption of lumen formation and abnormal vessel patterning. These findings demonstrate that during vasculogenesis ligation of integrin alpha v beta 3 on the surface of primordial endothelial cells is critical for the differentiation and maturation of blood vessels. Similar studies on chicken chorioallantoic membrane showed that LM609 blocks angiogenesis. Together the two studies suggest that integrin alpha v beta 3 plays a role in neovascularization of tissues.

Animals↗
PubMed1994

Cardiac endothelial heterogeneity defines valvular development as demonstrated by the diverse expression of JB3, an antigen of the endocardial cushion tissue.

The endothelium of the embryonic vertebrate heart evokes a regional specificity that remains an unexplained phenomenon in cardiac morphogenesis. A restricted population of endothelial cells lining the atrioventricular (AV) canal and proximal outflow tract (OT) transforms into mesenchyme, the reputed progenitor of the valves and membranous septa. The remainder of the cells lining these and other regions of the heart, in particular the ventricle, stay epithelial. At the present time there is no information regarding the determinants for endothelial cell diversity. To investigate the molecular basis for functionally distinct endothelial cell populations, we undertook a search for cell surface proteins within the endocardial cushions of Day 4 chicken embryos that might be sensitive to subtle differences in endothelial cell composition. We theorized that monoclonal antibodies raised against proteins expressed during early valve morphogenesis could provide markers for endothelial subpopulations, thereby assisting our efforts in defining, and determining the origin of, endothelial heterogeneity. In the present study, an in vitro collagen gel culture assay was employed to identify an antibody, JB3, that distinguishes between AV/OT endothelium and ventricular endothelium. Based on this assay, JB3-positive material was associated only with AV/OT endothelia or the mesenchyme derived from these epithelia. Also, a network of JB3-positive fibrillar material was observed within the collagen gel surrounding the explanted cells. The JB3 antigen showed a conspicuous distribution in pregastrulation-stage chicken embryos with immunolabeling observed in the initial primitive streak at 5 hr incubation (stage 2). Subsequent detection in the definitive primitive streak, Hensen's mode, and notochord indicate a consistent relationship to midline structures. JB3 antigen also localized to the regions of presumptive precardiac mesoderm and, at later stages, neural crest, somites, and ventral mesocardium. These data suggest that the JB3 antigen may play a role in establishing cardiac endothelial diversity by defining a subpopulation of cells destined to participate in valve formation. Moreover, JB3 may also influence formation of the primary axis and mesoderm structures that form at the midline. Immunochemical analyses showed that JB3 recognizes a polypeptide that migrates near the molecular weight position of fibrillin (350-390 kDa), the extracellular matrix protein linked to the Marfan syndrome. Based on the molecular mass and similar immunostaining patterns in early embryos, we propose that the JB3 antigen is a fibrillin isotype or a fibrillin-associated protein.

Animals↗
PubMed1993

Fibrillin delineates the primary axis of the early avian embryo.

Chicken embryos were immunolabeled with antibodies to fibrillin, an extracellular matrix molecule implicated in the Marfan syndrome. The pattern of labeling, established using both whole-mounted and sectioned embryos, showed a striking localization at the primary axis of the early embryo, including Hensen's node. After stage 8, the labeling of Hensen's node diminished while other structures in the embryo exhibited increased immunolabeling. These structures include the region of the presumptive dorsal aorta, the ventral surface of the notochord, and the mesocardium. Injections of the antibody between the vitelline membrane and the blastoderm in vivo failed to perturb the development of embryos, thus precluding any conclusions regarding the function of fibrillin.

Animals↗
PubMed1992

Antibodies to beta 1-integrins cause alterations of aortic vasculogenesis, in vivo.

Vasculogenesis is the de novo formation of blood vessels from mesoderm. This process occurs very early in development and provides a convenient system for studying morphogenesis in higher vertebrates. The cell-extracellular matrix (ECM) interactions that occur during dorsal aortic vasculogenesis were examined using the monoclonal antibody, CSAT, a reagent known to neutralize the ligand-binding activity of avian beta 1-integrins. We injected CSAT into quail embryos during a period of active vasculogenesis (4-10 somites). The CSAT antibodies, but not controls, had a marked and reproducible effect on aortic vessel formation. Vasculogenesis appeared to be arrested at the stage when slender cord-like assemblies of angioblasts rearrange to form tubules. Indeed, aortic primordia near the site of CSAT injection did not form patent vessels.

Animals↗
PubMed1992

Beta 1 integrins isolated from embryonic chicken fibroblasts bind to monomers and polymers of type I collagen.

The avian integrin beta 1 subfamily consists of multiple alpha-beta subunit heterodimers. We employed two different physical states of type I collagen, monomers and fibrils, in the isolation and characterization of avian collagen integrins. Affinity chromatography showed that three integrins, tentatively designated alpha 155 beta 1 (band 1), alpha 5a beta 1, and alpha 3 beta 1 (band 2), bind fibrillar and monomeric collagen under physiological ionic conditions and require divalent cations for binding activity. Sodium chloride gradients (0-0.5 M) were used to assess the functional ability of the integrins to remain bound to the two forms of type I collagen. The results show that integrins elute from the two forms of collagen with distinct fractionation profiles. One integrin, alpha 155 beta 1, binds fibrillar collagen with relatively higher affinity than the other beta 1 receptors. This same avian integrin, alpha 155 beta 1, is immunoreactive with an antiserum (Hynes et al., 1989) raised against a peptide that corresponds to the entire alpha 5 cytoplasmic domain, and coincidently, part of the alpha 6 cytoplasmic domain (de Curtis et al., 1991). Cell biological studies employing double immunofluorescence show that integrins recognized by this antiserum co-localize with extracellular deposits of type I collagen.

Animals↗
PubMed1992

Fibulin is localized at sites of epithelial-mesenchymal transitions in the early avian embryo.

Fibulin is a 100-kDa calcium-binding, extracellular matrix (ECM), and plasma glycoprotein (Argraves et al., Cell 58, pp. 623-629, 1989; Argraves et al., J. Cell Biol. 111, 3155-3164). Immunoprecipitation analysis showed that antibodies against human fibulin react with an avian isoform (M(r) 100,000). The spatial and temporal distribution of fibulin was examined in the early avian embryo using immunofluorescence microscopy. In stage 15-22 quail embryos fibulin is a constituent of most basement membranes. Areas undergoing epithelial-mesenchymal transitions such as the endocardial cushions, developing myotomes, and neural crest display especially prominent immunostaining. In the early heart fibulin expression was most pronounced in the cardiac jelly at sites where endocardial cushion cells begin the migrations that lead to the formation of valvular and septal primordia. Laser scanning confocal microscopy showed extensive extracellular accumulations of fibulin on the surface of endocardial mesenchyme cells that were motile at the time of fixation (stage 19). These data suggest that enhanced deposition of fibulin at sites of epithelial-mesenchymal transitions may influence cell behavior.

Animals↗
PubMed1992

Perturbation of beta 1 integrin-mediated adhesions results in altered somite cell shape and behavior.

Cell contact and adhesion between somites and the axial extracellular matrix (ECM) is likely to play a fundamental role in vertebrate development. In a preliminary report we showed that injection of the monoclonal antibody CSAT, which recognizes the avian beta 1 integrins, causes a lateral separation of both somites and segmental plate tissue from the embryonic axis (Drake and Little, 1991). In this study we addressed the cell biological response to CSAT injection, particularly the cell-ECM interactions involved in maintaining normal somite-axial relationships. A total of 150 stage 7-10 quail embryos have been injected with CSAT and then cultured for varying periods (1-30 hr). CSAT caused somitic cells to behave abnormally. Changes include, rounding-up, extensive blebbing, and formation of retraction fibers. A majority of separated somites were able to assume normal axial position with further time culture. Whether a somite subsequently aligned at the axis was dependent on the amount of CSAT injected and the postinjection culture period. Embryos in which somites remained separated from the axis after relatively long culture intervals (18-24 hr) displayed abnormal sclerotomal cell migrations. In no case did control injected embryos exhibit cellular alterations. Similarly, the injection of RGD-containing peptides had no detectable effect on somitogenesis or somite/segmental plate adhesion to the axis. On the basis of these data, we conclude that beta 1 integrins are necessary for normal somitic cell adhesions to the axis, but not somite segmentation and differentiation.

Amino Acid Sequence↗
PubMed1991

Integrins play an essential role in somite adhesion to the embryonic axis.

Integrins are proteins that mediate cell adhesion, mainly to the extracellular matrix. One of the first integrins discovered belongs to the beta 1 class of avian integrins and is defined by a monoclonal antibody, CSAT. Using a whole-embryo culture system we injected nanoliter quantities of CSAT caudolateral to the last somite of early quail embryos. The CSAT antibodies, but not control antibodies, resulted in a striking lateral translocation of somites. Relatively higher doses or longer incubation times increased the severity of the effect. We conclude that somite segmentation per se is not influenced by CSAT, but that somite adhesion to axial structures requires integrin-mediated ECM adhesions.

Animals↗
PubMed1990

Avian vasculogenesis and the distribution of collagens I, IV, laminin, and fibronectin in the heart primordia.

The heart-forming regions of the early embryo are composed of splanchnic mesoderm, endoderm, and the associated ECM. The ECM of the heart-forming regions in stage 7-9 chicken embryos was examined using immunofluorescence. Affinity purified antibodies to chicken collagens type I and IV, chicken fibronectin, and mouse laminin were used as probes. We report that (1) the basement membrane of the endoderm contains immunoreactive laminin and collagen IV; (2) the nascent basement membrane of the heart splanchnic mesoderm contains immunoreactive laminin, but not type IV collagen, and (3) the prominent ECM between the splanchnic mesoderm and the endoderm (the primitive-heart ECM) contains collagen IV, collagen I, fibronectin, but not laminin. In addition, we describe microscopic observations on the spatial relationship of cardiogenic cells to the primitive-heart ECM and the endodermal basement membrane.

Animals↗
PubMed1990

Laminin potentiates differentiation of PCC4uva embryonal carcinoma into neurons.

The embryonal carcinoma PCC4uva differentiates into neurons in response to treatment with retinoic acid and dbcAMP. We used this in vitro model system to study the effects of laminin on early neural differentiation. Laminin substrata markedly potentiate neural differentiation of retinoic acid and dbcAMP-treated cultures. Only laminin induced more rapid neural cell body clustering, neurite growth and neurite fasciculation as compared to type IV collagen, type I collagen, and fibronectin substrata. Exogenous laminin substrata promoted greater cell attachment, cellular spreading and growth to confluence than type IV collagen, type I collagen, fibronectin and glass substrata. Laminin-induced effects were inhibited by addition of laminin antibodies or the synthetic laminin-derived peptide Ile-Gly-Ser-Arg-NH2 (YIGSR-NH2). Treatment with YIGSR-NH2 also inhibited neural differentiation in the absence of exogenous laminin substrata, whereas synthetic peptides containing the RGD sequence and a control peptide YIGSK-NH2 showed no inhibitory effects. These results are consistent with the hypothesis that specific interactions between an early differentiating cell population(s) and extracellular laminin are required during neural differentiation.

Amino Acid Sequence↗
PubMed1989

Extracellular matrix components of the peripheral pathway of chick trigeminal axons.

The directed growth of axons to their peripheral targets during development may be influenced by a variety of intrinsic and environmental factors, the latter including the pattern of extracellular matrix components in the region through which they grow. We investigated the localization within the chick trigeminal mesenchyme of a variety of extracellular matrix molecules (laminin, heparan sulfate proteoglycan, entactin, collagen type IV) by using indirect immunofluorescence techniques. The trigeminal mesenchyme contained several of these molecules, but only laminin was specifically localized to trigeminal axon pathways. Double immunofluorescence localization of affinity-purified laminin antibodies and monoclonal antibodies directed against a neuron-specific beta-tubulin (to visualize growing axons and postmitotic V ganglion neurons) demonstrated that the V motor and sensory peripheral nerves confine their growth to two patches of laminin-rich mesenchyme--a ganglionic laminin patch colocalized with V ganglion neurons and their axons, and a mandibular laminin patch colocalized with the V motor axons. Thus, laminin pathways in the mesenchyme may help guide V axons along their appropriate routes in the periphery. Double immunofluorescence localization of these laminin antibodies and monoclonal antibodies that recognize neural crest cells (to visualize precursors of V ganglion neurons and glia) demonstrated that the majority of cells within the ganglionic laminin patch were neural crest and differentiating neurons derived from the trigeminal epidermal placode. Since these cells often were laminin-immunopositive, they might be a source of the ganglionic laminin matrix. The mandibular laminin patch contains nearly no neural crest cells, and probably contains the somitomeric precursors to the mandibular muscle mass. These results suggest that laminin, a matrix molecule implicated in the guidance of trigeminal peripheral axons, might be produced in localized patches by peripheral nervous system components and by muscle targets.

Antibodies, Monoclonal↗
PubMed1989

Distribution of laminin, collagen type IV, collagen type I, and fibronectin in chicken cardiac jelly/basement membrane.

Light microscopic immunolabeling studies were designed to identify and locate structural components within the cell-free extracellular matrix which lies between the embryonic endocardial and myocardial tubes. Affinity-purified antibodies were used to examine stage 15-22 embryonic chicken hearts. Specimens were immunolabeled by using three different methodologies: 1) postembedding labeling of 10 microns cryostat sections, 2) preembedding labeling (en bloc) of whole hearts, and 3) postembedding labeling of ethanol/acetic acid-fixed paraffin sections. Our results establish the spatial distribution of collagen type I and demonstrate for the first time the presence of collagen type IV and laminin in the myocardial-basement-membrane/cardiac jelly.

Animals↗
PubMed1989

Collagen binding proteins derived from the embryonic fibroblast cell surface recognize arginine-glycine-aspartic acid.

Several cell surface proteins (Mr = 120,000, 90,000, 63,000 and 47,000) apparently integral to embryonic fibroblast plasma membranes were extracted with detergent and isolated by collagen affinity chromatography. Certain of these proteins (Mr = 120,000, 90,000, and 47,000) were specifically eluted from collagen affinity columns by synthetic peptides containing the amino acid sequence arginyl-glycyl-aspartic acid (RGD). These data show that a number of collagen binding proteins exist on the embryonic fibroblast cell surface. Some of the proteins may be collagen receptors binding to RGD sequences in the collagen molecule while at least one of the proteins (Mr = 63,000) recognizes features other than RGD.

Animals↗