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C D Little

Publications and source records attributed to C D Little.

53 records · Page 3Linked to original sources
PubMed1989

Embryonic heart mesenchymal cell migration in laminin.

Laminin, a large glycoprotein and major component of basement membranes, influences cell adhesion, migration, morphology, and differentiation. A peptide sequence, YIGSR, from the B1 chain of laminin has been found to correspond to an active site for cell adhesion. We report here that cardiac mesenchymal cells migrate vigorously within three-dimensional gels of laminin and that the YIGSR peptide will completely abolish this migratory activity. In contrast, migration of the mesenchymal cells into three-dimensional gels composed of collagen or collagen + laminin is not effected by YIGSR or other peptides (GRGDS, GRGDTP) reported to mediate cellular adhesion.

Animals↗
PubMed1989

Embryonic chicken fibroblast collagen binding proteins: distribution, role in substratum adhesion, and relationship to integrins.

Collagen binding proteins (CBP) are hydrophobic, cell surface polypeptides, isolated by collagen affinity chromatography. Antibodies to CBPs inhibit the attachment of embryonic chicken heart fibroblasts to native type I collagen fibrils in a dose-dependent manner. The CBP antibodies also induce rounding and detachment of cells adherent to a planar substratum. This process of antibody-mediated substratum detachment resulted in a clustering of CBP and cell-associated extracellular matrix at the cell surface, and the rearrangement of filamentous actin. Other functional studies showed that cells grown within a three-dimensional gel of type I collagen cannot be immunostained at the cell surface with CBP antibodies. However, treatment of cultures with purified collagenase, unmasks immunoreactive sites and permits strong cell surface immunolabeling. This result suggests that collagen sterically blocks antibody access to CBP. Finally, we show that antibodies to CBP recognize purified avian integrin beta subunits; and that antibodies to avian integrins recognize a 100,000 Mr CBP. These data demonstrate that chicken embryonic fibroblasts possess surface polypeptides that mediate adhesion to type I collagen, and suggest that two of these proteins are related to the integrin family.

Animals↗
PubMed1988

Laminin receptors for neurite formation.

Laminin, a basement membrane glycoprotein promotes both cell attachment and neurite outgrowth. Separate domains on laminin elicit these responses, suggesting that distinct receptors occur on the surface of cells. NG108-15 neuroblastoma-glioma cells rapidly extend long processes in the presence of laminin. We report here that 125I-labeled laminin specifically binds to these cells and to three membrane proteins of 67, 110, and 180 kDa. These proteins were isolated by affinity chromatography on laminin-Sepharose. The 67-kDa protein reacted with antibody to the previously characterized receptor for cell attachment to laminin. Antibodies to the 110-kDa and 180-kDa bands demonstrated that the 110-kDa protein was found in a variety of epithelial cell lines and in brain, whereas the 180-kDa protein was neural specific. Antibodies prepared against the 110-kDa and 180-kDa proteins inhibited neurite outgrowth induced by the neurite-promoting domain of laminin, whereas antibodies to the 67-kDa laminin receptor had no effect on neurite outgrowth. We conclude that neuronal cells have multiple cell-surface laminin receptors and that the 110-kDa and 180-kDa proteins are involved in neurite formation.

Antibodies, Neoplasm↗
PubMed1988

Trichloroethylene biodegradation by a methane-oxidizing bacterium.

Trichloroethylene (TCE), a common groundwater contaminant, is a suspected carcinogen that is highly resistant to aerobic biodegradation. An aerobic, methane-oxidizing bacterium was isolated that degrades TCE in pure culture at concentrations commonly observed in contaminated groundwater. Strain 46-1, a type I methanotrophic bacterium, degraded TCE if grown on methane or methanol, producing CO(2) and water-soluble products. Gas chromatography and C radiotracer techniques were used to determine the rate, methane dependence, and mechanism of TCE biodegradation. TCE biodegradation by strain 46-1 appears to be a cometabolic process that occurs when the organism is actively metabolizing a suitable growth substrate such as methane or methanol. It is proposed that TCE biodegradation by methanotrophs occurs by formation of TCE epoxide, which breaks down spontaneously in water to form dichloroacetic and glyoxylic acids and one-carbon products.

Journal Article↗
PubMed1987

Immunolocalization of extracellular matrix proteins and collagen synthesis in first-trimester human decidua.

The extracellular matrix (ECM) of first-trimester human decidua was examined with indirect immunofluorescence using affinity-purified antibodies to human collagen types I, III, IV, V, laminin, and fibronectin. In addition, the validity of the classification "mesenchymal-epithelioid" for differentiated decidual cells was addressed using antibodies to the intermediate filament proteins, vimentin, a mesenchymal marker, and keratin, an epithelial marker. Biosynthesis of extracellular matrix components was examined by radiolabeling of decidual explants in culture with 3H-proline, followed by immunoprecipitations of synthesized proteins with collagen type-specific antibodies. Immunofluorescence showed decidual cells are embedded in an extensive network of collagen types I and III, and intracytoplasmic staining suggested synthesis of these collagens by the decidual cells. Collagen type IV and laminin localized in the external lamina which surrounds the differentiated decidual cell, and some fluorescence was evident in the peripheral cytoplasm. Immunoreactive collagen type V was observed in close association with the external lamina and in the peridecidual matrix. Fibronectin localized throughout the decidual ECM and in fibrillar and punctuate patterns in the decidual cell cytoplasm. Differentiated decidual cells retained a "mesenchymal" intermediate filament cytoskeleton containing an abundance of vimentin filaments, but very few, if any, keratin filaments. Collagen types I, III, V, and to a lesser extent, IV, were immunoprecipitated from the medium of decidual explants after 24 hours of culture, demonstrating in vitro synthesis and secretion of these collagens by first trimester human decidua.

Collagen↗
PubMed1987

Cellular events associated with lung branching morphogenesis including the deposition of collagen type IV.

In this study mouse lung development was examined using an in vitro model system. The culture system permitted examination of a morphogenic process that eventually led to the formation of presumptive alveoli (terminal sacs). The observations included changes in epithelial cell morphology (transition from a columnar to a spindle shape), and evidence for motile activity on the part of primitive airway epithelial cells. The importance of Type IV collagen to the cellular events associated with branching morphogenesis was investigated by immunolocalization. In addition, we assessed the similarity of normal lung development to in vitro development by comparing cultured lungs with equivalent stages of embryonic and fetal mouse lungs. The results show that cultured embryonic lung explants proceed along a morphogenic pathway that parallels normal lung development; that primitive pulmonary epithelial cells engage in motile activity and transiently acquire an extended cell shape both in vitro and in vivo; that, as suggested by others, the pattern of late branching morphogenesis is not dichotomous, but irregular; and that short wisplike fibers of Type IV collagen are present in developing embryonic and fetal lung mesenchyme. Taken together, the results show that early and late lung branching patterns differ significantly, and suggest that later stages of lung branching involve distinct epithelial cell shape transitions. The immunofluorescence data suggest that fibrous Type IV collagen may be the extracellular matrix scaffold within which early epithelial cells accomplish lung branching morphogenesis.

Animals↗
PubMed1985

Cells that emerge from embryonic explants produce fibers of type IV collagen.

Double immunofluorescence staining experiments designed to examine the synthesis and deposition of collagen types I and IV in cultured explants of embryonic mouse lung revealed the presence of connective tissue-like fibers that were immunoreactive with anti-type IV collagen antibodies. This observation is contrary to the widely accepted belief that type IV collagen is found only in sheet-like arrangements beneath epithelia or as a sheath-like layer enveloping bundles of nerve or muscle cells. The extracellular matrix produced by cells that migrate from embryonic mouse lung rudiments in vitro was examined by double indirect immunofluorescence microscopy. Affinity-purified monospecific polyclonal antibodies were used to examine cells after growth on glass or native collagen substrata. The data show that embryonic mesenchymal cells can produce organized fibers of type IV collagen that are not contained within a basement membrane, and that embryonic epithelial cells deposit fibers and strands of type IV collagen beneath their basal surface when grown on glass; however, when grown on a rat tail collagen substratum the epithelial cells produce a fine meshwork. To our knowledge this work represents the first report that type IV collagen can be organized by cells into a fibrous extracellular matrix that is not a basement membrane.

Animals↗
PubMed1983

Distribution of fibronectin and laminin in basal cell epitheliomas.

The distribution of fibronectin (FN) and laminin (LM) in basal cell epithelioma was evaluated by indirect immunofluorescence. FN is a glycoprotein which promotes interaction between cells and the extracellular matrix, and is present at the dermal-epidermal junction (DEJ) and throughout the dermis, but absent in the normal epidermis. LM, a noncollagenous basement membrane protein, plays a role in epithelial adhesion to type IV collagen, and is normally present in the DEJ, but absent in the epidermis. The role of FN and LM in epithelial differentiation has not been established. Therefore, the distribution of FN and LM in a tumor of epithelial origin was studied by indirect immunofluorescence. Using affinity-purified antibodies to FN and LM, and the appropriate fluorescein-conjugated second antibodies, normal skin and 7 basal cell tumors were examined. By immunofluorescence, nests of malignant basal cells were surrounded by linear LM staining. FN immunofluorescence was intense throughout the connective tissue stroma of all tumors. Five tumors also showed FN staining within the nests of neoplastic cells, and 4 of these were also LM-positive in the tumor bulk. These immunofluorescent findings suggest that epidermal neoplasia can be associated with alterations in the distribution of FN and LM.

Basement Membrane↗
PubMed1982

Masking of extracellular collagen and the co-distribution of collagen and fibronectin during matrix formation by cultured embryonic fibroblasts.

Embryonic chicken heart fibroblasts (CHF) were examined by double immunofluorescence in order to study the interactions of collagen and fibronectin. Double-labelling studies of permeabilized whole mounts of CHF, cultured for 15 min to 2 days after plating, detected extracellular collagen at very early times (15 min to 1 h) but not at times later than 3 h. Abundant fibronectin was seen at all times. These observations suggested that the collagen/procollagen antigen was being "masked' by matrix components at the later times. This possibility was tested in two ways: (1) immunolabelling of frozen sections of multilayered 4-day CHF cultures, which showed extensive extracellular collagen superimposable with fibronectin. (2) Limited trypsinization of 4-day CHF cultures, which allowed collagen to be immunolabelled concomitantly with a partial decrease in the degree of fibronectin labelling. The results are discussed in terms of the secretion and assembly of collagen and fibronectin in cultures of embryonic fibroblasts.

Animals↗
PubMed1979

Production and characterization of a monoclonal antibody to chicken type I collagen.

We have shown that lymphocyte-myeloma cell hybridization can be used to produce large amounts of extremely high-titer specific antibodies against type I collagen, a macromolecule normally of low immunogenicity. In a passive hemagglutination assay the antibody had a high titer against chicken type I collagen but showed no activity against chicken type II or rat type I collagen. By using a two-step fluorescence histochemical procedure on sections of embryonic chicken tibia, strong fluorescence was observed in the perichondrium and surrounding connective tissue (known to contain type I collagen) but not over the cartilage (characterized by type II collagen). When used in conjunction with Staphylococcus aureus as a solid phase immunoadsorbant, the antibody was shown to bind to labeled collagen synthesized in vitro by embryonic chicken calvaria.

Animals↗
PubMed1978

Embryonic neural retina collagen: in vitro synthesis of high molecular weight forms of type II plus a new genetic type.

Chicken neural retina cells from 6- to 7-day embryos were labeled with [(3)H]proline for 24 and 72 hr and the collagenous proteins of the medium were analyzed. Ninety percent of the collagenous proteins eluted from DEAE-cellulose columns as a peak near the middle of the gradient. Upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis, this material consisted predominantly of a slowly migrating procollagen, a smaller amount of an intermediate migrating form, and some alpha-chain material. Limited enzymatic digestion of this DEAE peak material plus mapping of its CNBr peptides identified this material as a type II precursor. The remaining 10% of the collagenous proteins eluted from DEAE-cellulose at the end of the gradient. Electrophoresis of this fraction showed four major bands. Two migrated near the type II precursors, a third migrated somewhat more slowly, and the fourth was near beta-chain dimers. In addition, there were two minor bands. Limited pepsin digestion of this DEAE peak material produced two bands: one migrated slightly more slowly than alpha1, and the second, considerably more slowly. The CNBr peptide pattern of this material appears to be different from any previously described. Thus, neural retina cells in culture synthesize at least two genetically distinct classes of collagenous proteins. One represents precursors to type II. The second is composed of multiple, very high molecular weight forms which may represent precursors (procollagens) of a new genetic type(s) of collagen.

Animals↗
PubMed1977

Procollagen and collagen produced by a teratocarcinoma-derived cell line, TSD4: evidence for a new molecular form of collagen.

Procollagen and collagen were isolated from the culture medium and cell layer of line TSD4 (obtained from mouse teratocarcinoma OTT6050). SDS-polyacrylamide gel electrophoresis of the highly purified procollagen fraction demonstrated that the fraction is composed of theta chains (150,000 daltons), pro alpha chains (130,000 daltons), and alpha chains (100,000 daltons). Limited pepsin digestion of this fraction yielded a single species of collagen molecules having a chain composition (alpha1)3, as did collagen isolated from the cell layer. Each alpha1 chain appears to be slightly larger than alpha1 chains from calf or human type I and type III collagen. Amino acid analysis and cyanogen bromide peptide profiles of pepsin-treated TSD4 collagen demonstrated significant differences from those of other collagens (II, III, IV) of the type alpha1(X)3, although similar to that of the alpha1 chain of type I collagen, [alpha1(1)]2alpha2. Taken together, acrylamide gel electrophoresis, amino acid composition, electron microscopy, and cyanogen bromide peptide analysis indicate that this material represents a new molecular species of collagen not previously characterized, probably related to [alpha1(I)]3.

Amino Acids↗
PubMedDate not supplied

Amplification and expression of the c-myc oncogene in human lung cancer cell lines.

Genetic changes involving the c-myc oncogene have been observed in human tumours. In particular, the c-myc gene is translocated in Burkitt's lymphoma and is amplified in the human promyelocytic leukaemia cell line, HL-60, which contains double minute chromosomes (DMs). More recently, an amplified c-myc gene has been positioned on a chromosomal homogeneous staining region (HSR) in a human colon cancer cell line, COLO 320, with neuroendocrine properties. Furthermore, c-myc is expressed in increased amounts in some human tumour lines, and in some cases, human small cell lung cancers (SCLC) contain DMs and HSRs. These findings prompted us to study the c-myc gene and its RNA expression in a series of human lung cancer cell lines. We now report amplification and expression of the c-myc oncogene in a system other than B-cell lymphomas, namely human lung cancer. Of 18 human lung cancer cell lines tested, 8 showed an amplified 12.5-kilobase (kb) EcoRI c-myc DNA band. Of particular interest are five SCLC lines with a high degree of c-myc DNA amplification (20-76-fold) and greatly increased levels of c-myc RNA. All five lines reside in the variant class of SCLC (SCLC-V) characterized by altered morphology, lack of expression of some SCLC-differentiated functions and more malignant behaviour than pure SCLC. Three of the five lines which have been karyotyped also contain DMs or HSRs. The finding of a greatly amplified c-myc gene in all cell lines of the SCLC-V class examined strongly suggests a role for the c-myc gene in the phenotypic conversion and malignant behaviour of human lung cancer.

Cell Line↗
PubMedDate not supplied

Developmental biology of the vascular smooth muscle cell: building a multilayered vessel wall.

The assembly of the vessel wall from its cellular and extracellular matrix components is a critical process in the development and maturation of the cardiovascular system. However, fundamental questions concerning the origin of vessel wall cells and the mechanisms that regulate their development and differentiation remain unanswered. The initial step of vessel wall morphogenesis is formation of a primary vascular network, comprised of nascent endothelial cell tubes, via the processes of vasculogenesis and angiogenesis. Subsequently, primordial vascular smooth muscle cells (VSMCs) are recruited to the endothelium to form a multilayered vessel wall. During the course of development and maturation, the VSMC plays diverse roles: it is a biosynthetic, proliferative, and contractile component of the vessel wall. Although the field of vascular development has blossomed in the past decade, the molecules and mechanisms that regulate this developmental pathway are not well defined. The focus of this review is on those facets of VSMC development important for transforming a nascent endothelial tube into a multilayered structure. We discuss the primordial VSMC with particular attention to its purported origins, the components of the extracellular milieu that contribute to its development, and the contribution of embryonic hemodynamics to vessel wall assembly.

Animals↗