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Biomedical subjects

R O Kelley

Publications and source records attributed to R O Kelley.

At least 19 recordsLinked to original sources
PubMed1999

Scientists, senators, and "software": the keys to unlocking our future.

Scientists, educators, and researchers in the nation's medical schools, teaching hospitals, and research universities have responsibilities for ensuring a bright future for medical research. First, they must define science and communicate its wonder to their students, be their role models and mentors, and nurture and encourage the best and brightest to enter careers in medical research, since they are a precious resource for solving the many challenging and complex research problems that await them and which can bring great benefits to society. Second, they must learn to participate even more effectively and actively in the ongoing partnerships between the federal government, private enterprise, and the medical school and in the processes that lead to appropriations for the funding necessary to support the research enterprise. And finally, they need to recognize the importance of the increasingly interdisciplinary nature of medical research by urging support for the physical and social sciences, mathematics, and engineering.

Academic Medical Centers↗
PubMed1993

Bacterial collagenase disrupts extracellular matrix and opens blood-brain barrier in rat.

Bacterial collagenase causes hemorrhagic necrosis of brain. We studied the enzyme's effect on blood-brain barrier (BBB) permeability and extracellular matrix (ECM) structure by radiolabeled tracers and electron microscopy. Adult rats had intracerebral injection of bacterial collagenase. Brain uptake from blood of [14C]sucrose was measured in 24 rats 0.5 h to 14 days after injection. 12 rats had ultrastructural studies 1 h after collagenase injection. Brain uptake of [14C]sucrose is maximally increased at 0.5 h, remaining significantly increased for 7 days. Ultrastructurally, some vessels had widening of basal lamina while others had severe disruption of basal lamina with stretching of endothelial cells. We conclude that bacterial collagenase disrupts ECM and opens BBB.

Animals↗
PubMed1992

TIMP-2 reduces proteolytic opening of blood-brain barrier by type IV collagenase.

Intracerebral hemorrhage occurs in tumors, stroke and head trauma. Proteolysis of the extracellular matrix around cerebral capillaries by naturally occurring mammalian 72-kDa type IV collagenase may initiate this pathologic event. To investigate this hypothesis adult rats underwent intracerebral injection of type IV collagenase purified from human melanoma cells. Histologically, at 4 h there was perivascular cellular infiltration with hemorrhage, and by 24 h there was infarction with necrosis, edema and hemorrhage. Ultrastructurally, the basal lamina of endothelial cells was disrupted at 2 h. Brain uptake of [14C]dextran and [3H]sucrose increased after intracerebral injection of type IV collagenase compared to controls (P less than 0.0001). Tissue inhibitor of metalloproteinase-2 (TIMP-2) reduced the tracer uptake (P less than 0.02). Metalloproteinase inhibitors reduce extracellular matrix proteolysis and protect the blood-brain barrier.

Analysis of Variance↗
PubMed1991

Ultrastructural and immunocytochemical analysis of the circumferential microfilament bundle in avian retinal pigmented epithelial cells in vitro.

The dedifferentiated phenotype of pigmented epithelial cells in vitro is bipotential and is effected by environmental alterations mediated by the cell surface and associated cytoskeleton. We have begun an investigation into the role that contractile microfilaments play in maintaining cell contact and cell shape in retinal pigmented epithelial cells in vitro. In this paper, we report a structural analysis of the intersection of the circumferential microfilament bundle with the cell membrane of cultured pigmented epithelial cells from chick retina. Techniques of electron microscopy, including freeze-fracturing and deep-etching, reveal that microfilaments of this bundle associate with a junctional complex in the apical cell compartment and with membrane domains which are not components of the junction. Microfilaments link with the cell membrane either at their termini or along the membrane-apposed surface of the circumferential bundle. Furthermore, we report the immunocytochemical localization of filamin (a high molecular weight actin-binding protein, which forms fiber bundles and sheet-like structures when bound with F-actin in solution) in the circumferential/microfilament bundle.

Actin Cytoskeleton↗
PubMed1989

Antibacterial activity of human natural killer cells.

The in vitro effects of human NK cells on viability of Gram-negative and Gram-positive bacteria was investigated. PBLs depleted of glass-adherent cells showed a significant antibacterial activity that was increased as the concentration of NK cells became higher. Leu-11-enriched cells exhibited the most efficient bactericidal activity. Stimulation of NK cells with staphylococcal enterotoxin B for 16 h produced a significant increase in the antibacterial activity of all NK cells tested. The antibacterial activity of monocyte-depleted cells and Leu-11-enriched cells was also enhanced after culturing in vitro for 16-24 h without exogenous cytokines. Dependence of the antibacterial activity on the presence of serum in the culture medium was not found. Ultrastructural studies revealed close contact between NK cell membranes and bacteria, no evidence of phagocytosis, and extracellular bacterial ghosts, after incubation at 37 degrees C. Supernatants from purified NK cells exhibited potent bactericidal activity with kinetics and target specificity similar to that of effector cells. These results document the potent antibacterial activity of purified NK cells and suggest an extracellular mechanism of killing.

Adult↗
PubMed1989

Selective proliferation of natural killer cells among monocyte-depleted peripheral blood mononuclear cells as a result of stimulation with staphylococcal enterotoxin B.

In vitro stimulation of monocyte-depleted peripheral blood mononuclear cells with staphylococcal enterotoxin B (SEB) resulted in selective proliferation of cells which express the phenotypic and functional characteristics of natural killer (NK) cells. This culture system provides an easy method for obtaining highly purified NK cells, by sequential incubation of monocyte-depleted cells with SEB and then with interleukin-2 (IL-2). After culture for 4 to 5 days in the presence of SEB, 98 to 100% of the cells expressed the CD16 (Leu11) and HNK-1 (Leu19) antigens. This purification occurred through the death of lymphocytes lacking NK cell markers and marked proliferation of NK cells themselves, which leads to an enrichment of the NK cell population. Activation of NK cells was detected by the appearance of the gamma interferon receptor and IL-2 receptor antigens. This homogeneous population showed the morphology of large granular lymphocytes, were potent effectors of cell-mediated cytotoxicity against K562 and Daudi tumor cell lines, and were able to kill gram-positive and gram-negative bacteria. IL-2 was necessary to maintain the activation and proliferation after SEB stimulation for 4 days. Moreover, the maximum frequency of binding to K562 cells (60.6%) was similar to that recently found (58 +/- 3%) (P. Garcia-Peñarrubia, F. T. Koster, and A. D. Bankhurst, J. Immunol. Methods 118:199-208, 1989) with fresh and highly purified NK cells. This method can be used as a source of highly purified NK cells to study their functional properties and applications to the treatment of cancer.

Adult↗
PubMed1987

Cell surface oligosaccharide modulation during differentiation. I. Modulation of lectin binding.

IMR-90 human lung fibroblasts exhibit quantitative and qualitative changes in their cell surface oligosaccharide expression as a result of low-density growth, contact inhibition of growth and changes in population doubling level (PDL). Binding of Concanavalin-A (CON-A), wheat germ agglutinin (WGA) and Ricinus communis (RCA-120) was shown to decrease by 20-40% as the cells advanced through their lifespan (but significantly before morphological manifestations commonly associated with senescence, which occur at approximately PDL 45). The binding of Dolichos biflorus agglutinin (DBA) increased by 100% over the same time interval. These changes were detected by quantitation of FITC conjugated lectins on single cells and the use of a sensitive analytical biotin-avidin-enzyme amplification assay on whole populations of cells. These studies were performed on randomly growing cultures. These results suggest an important and dynamic role for the cell surface oligosaccharide in growth control and cellular senescence.

Cell Differentiation↗
PubMed1987

Cell surface oligosaccharide modulation during differentiation. II. Membrane mobility of oligosaccharide lectin conjugates.

The quantitative, population doubling level (PDL) dependent changes in cell surface oligosaccharides on IMR-90 cells, were investigated from the perspective of membrane mobility of the lectin-oligosaccharide conjugates. Concanavalin-A (CON-A), wheat germ agglutinin (WGA), Ricinus communis agglutinin (RCA-120), and Dolichos biflorus agglutinin (DBA) were all observed to cluster, cap, and endocytose in cultured human diploid fibroblasts (IMR-90). Quantitative photometry at 37 degrees C over defined periods of time indicated that as the IMR-90s approached cellular senescence a specific lectin-dependent inability to either endocytose or process the capped complex occurred. The development of a biotin/avidin/enzyme amplification assay permitted the assignment of the accumulating signal to the internal compartment. Kinetic data indicate that there are at least three separate (and separable) mechanisms for the PDL related changes in lectin binding. Data for the CON-A complex indicates that at least two classes of functional complexes are present. Regression analysis of the kinetic data for the RCA-120 complex indicates a similar membrane clearance for the IMR-90s at all population doubling levels (PDL), suggesting that the quantitative differences observed earlier were due to simple quantitative reductions in the RCA complexing molecules. Data for WGA mobility on the membrane indicates that they are both changes in the number and mobility status of the complexes. These results indicate that the quantitative changes in lectin binding observed previously as IMR-90 cells approach senescence are correlated with alterations in membrane mobility patterns of the lectin oligosaccharide conjugates.

Cell Compartmentation↗
PubMed1985

Reduction of filamin in late passage human diploid fibroblasts (IMR-90).

Progressive subcultivation of IMR-90 cells results in non-proliferative, heterogeneous cultures which may reflect aging of the diploid line (Hayflick, Exp. Cell Res., 37 (1965) 614). We have observed that late passage cells exhibit different rates of spreading and morphogenesis when compared to early passage groups, phenomena which we attribute to altered reassembly of the cytoskeleton in senescent cells (Kelley et al. Mech. Ageing Dev., 13 (1980) 127). To determine whether potential differences in cytoskeletal proteins develop with progressive subcultivation, early and late passage cultures were extracted with 0.5% Triton X-100 for 1 min followed by 1.0% sodium dodecyl sulfate (SDS) prior to separation and characterization of extracted proteins by electrophoresis on 7.5-15% gradient SDS gels. Extractions were made of both culture groups 3, 6 and 24 h after reseeding. Cytoskeletal ultrastructure at each stage of spreading was examined either in replicas of extracted cells or directly by scanning electron microscopy. Although considerable variation in cytoskeletal organization was observed, qualitative differences in gel banding patterns of actin, myosin and tubulin were not apparent at selected time points. However, late passage cells at 6 h and 24 h did not exhibit filamin associated with the Triton insoluble fraction as did early passage cells. Since it has been demonstrated that filamin is capable of cross-linking actin microfilaments into bundles or sheets, we suggest that it is a principal element for the variant cell shape and cytoskeletal morphology observed during altered spreading behavior of late passage human diploid fibroblasts.

Cell Survival↗
PubMed1985

Early development of the vertebrate limb: an introduction to morphogenetic tissue interactions using scanning electron microscopy.

The developing limbs of most vertebrates serve as a model system for studies of morphogenesis, pattern formation, cell and tissue interactions and cell differentiation. Mesoderm in the flank of the embryo induces overlying ectoderm to form a thickened, stratified or pseudo-stratified epithelium which becomes the highly specialized apical ectodermal ridge. In turn, the apical ridge specifies individual limb parts (first from structures proximal to the body axis, then to more distal components) and is required for those elements to form. If the ridge is removed, subsequent limb development ceases and no further limb parts appear. The series of ectodermal-mesodermal interactions is poorly understood at the molecular level, but scanning electron microscopy permits the visualization of tissues and cells which participate in this remarkable process of morphogenesis and differentiation. This paper is intended to serve as an introduction for the student beginning an investigation into the multiple, integrated biological processes which culminate in the establishment of a normal vertebrate limb.

Animals↗
PubMed1985

Antibodies to lymphocyte membrane preparations in systemic lupus erythematosus.

IgG and IgM antilymphocyte antibodies were determined in sera from patients with systemic lupus erythematosus (SLE) by an ELISA method using isolated lymphocyte plasma membranes as antigens. Lymphocyte membranes prepared from T or B cell lines or normal peripheral blood showed a high degree of purity by enzyme assay and electron microscopic monitoring. Sera from patients with active SLE showed high IgG and IgM ELISA reactivity against T and B cell plasma membranes compared with normal sera. IgM antibodies to lymphocyte membranes showed a correlation with microcytotoxicity results. Serial studies showed a relationship of antimembrane antibody to disease activity in some patients, but not in others. The ELISA method for antilymphocyte antibody assay provides a sensitive technique for monitoring reactivity against antigens retained in isolated lymphocyte membrane preparations.

Adsorption↗
PubMed1983

Isolation by flow sorting of cytokinetic and morphological heterogeneity in late-passage cultures of human diploid fibroblasts (IMR-90).

Considerable structural, metabolic, and proliferative heterogeneity develops in populations of cultured diploid cells which have reached advanced levels of population doubling. Isolation of noncycling cells from late-passage cultures would permit more definitive investigation of the structure and behavior of individual senescent cells. In this paper, we report the viable sorting of late-passage cultures of human diploid fibroblasts (IMR-90) into two subpopulations of cells with different proliferative potentials. Sorting is based on cellular light-scattering properties and autofluorescence. Structural and behavioral features of the subpopulation exhibiting increased forward-angle light scatter are more characteristic of senescent cells than the subpopulation sorted by decreased forward-angle light scatter.

Autoradiography↗
PubMed1983

A simple technique for the visualization of whole mount cytoskeletons with transmission electron microscopy.

Examination of whole mount cells in the transmission electron microscope has been useful in studies of cellular architecture. The common technique is to grow cells directly on formvar-coated, gold grids for direct observation through a cell. We report a technique for obtaining whole mount preparations which requires neither fragile formvar films nor expensive, gold grids. Cells are grown on palladium-coated coverslips and processed for electron microscopy. The cells and the palladium substrate are separated from the coverslip. The cell-palladium complex is then picked up on copper grids as in thin section processing. We compare images of the cytoskeleton using our technique with images using previously described techniques and present preliminary observations of contracting cell models. Such contractions would tear formvar films if attempted on cells grown in the conventional manner for whole mount examination. Our technique allows cells to contract without tearing the underlying substrate.

Cytological Techniques↗
PubMed1982

Organization of the cytoskeleton in square fibroblasts.

The relationship between the cytoskeleton, stress fiber formation, and cell shape has been difficult to determine in fibroblasts grown in tissue culture. Vagaries in cell shape are complicated, as well, by stochastic cell movements. We dictated the attachment sites and shape of fibroblasts by growing them on square adhesive substrates surrounded by nonadhesive substrates. Cytoskeletal models were made by treating the cells with buffered Triton X-100 and glycerol. The residues were then examined by scanning electron microscopy followed by light microscopy of the same cells. The cytoskeletons of randomly moving cells were examined with whole mount transmission microscopy to confirm images seen with scanning microscopy. The cells thus examined demonstrated definite relationships between ruffling activity and stress fiber terminations, which were limited to the more adhesive, palladium substrate. No stress fibers were seen to end on the lesser adhesive substrate, agarose, and ruffling did not occur across the agarose. Cells too small to fill an entire square tended to extend across one diagonal of the square, and the stress fibers ran parallel to the longest axis of these cells. Larger cells were able to completely fill their squares. The cytoskeletons of these cells were organized in a spatial relation to the square shape of the cells. The cortical meshwork was aligned circularly and diagonally within the cells. Stress fibers appeared to form from the microfilaments of the meshwork and were aligned diagonally across the cells. We conclude that the diagonal arrangement of the stress fibers and cortical meshwork is caused by the same mechanism by which smaller cells spread over the longest axis of a square. Regions of cells where the meshwork was absent or where stress fibers were tightly bundled were occupied by more randomly arranged cytoskeletal components. Regions of tightly bundled stress fibers did not seem to coincide with regions of cortical meshwork as seen by either whole mount transmission or scanning electron microscopy. Stress fibers were revealed in the light microscope to course beneath more randomly oriented cytoskeletal elements. These "lacework-like" elements were found frequently in square cells. Conspicuous structures in this random lacework were focal points of radially arranged filaments. Our observations suggest a continuity between stress fibers and the cortical microfilaments. The orientation of fibers and filaments was, in turn, dependent on cell shape for organization within the cell.

Cell Adhesion↗
PubMed1981

Loss of organized fibronectin matrix from the surface of aging diploid fibroblasts (IMR-90).

Indirect immunofluorescence was used to investigate the production of a fibrillar fibronectin matrix by human diploid fibroblasts (IMR-90) as cells progress through their in vitro lifespan. Early and mid-passage cultures displayed a prominent fibrillar reticulum over the cell layer which formed within 24 hours of seeding. Even sparsely seeded early-passage cells exhibited fibrils of fibronectin on external surfaces. In contrast, fibrillar fibronectin was reduced or absent on surfaces of late-passage cells. However, the larger, non-proliferating, late-passage cells were producing fibronectin, 1h determined by radioimmunoassay of the medium.

Aging↗