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Full-Text Articles in Cell Biology

Detection Of Hiv-1-Infected Cells From Patients Using Nonisotopic In Situ Hybridization, Robert H. Singer, Kevin S. Byron, Jeanne B. Lawrence, John L. Sullivan Nov 1989

Detection Of Hiv-1-Infected Cells From Patients Using Nonisotopic In Situ Hybridization, Robert H. Singer, Kevin S. Byron, Jeanne B. Lawrence, John L. Sullivan

Open Access Articles

We have demonstrated that a sensitive, nonisotopic in situ hybridization (ISH) assay can be used to detect HIV-infected cells from seropositive, asymptomatic individuals. Our assay is based on the detection of a biotinated HIV DNA probe hybridized to human immunodeficiency virus (HIV)-infected peripheral blood lymphocytes (PBL) using streptavidin and alkaline phosphatase to identify positive cells. This assay is rapid in that it can be performed within a day and is sensitive enough to unambiguously identify a rare, single, positive cell. Patient samples derived from HIV-seropositive hemophiliacs and HIV-seropositive infants were analyzed before and after coculture with normal PBL. The ...


Structural Changes Induced In Ca2+-Regulated Myosin Filaments By Ca2+ And Atp, Ling-Ling Young Frado, Roger W. Craig Aug 1989

Structural Changes Induced In Ca2+-Regulated Myosin Filaments By Ca2+ And Atp, Ling-Ling Young Frado, Roger W. Craig

Open Access Articles

We have used electron microscopy and proteolytic susceptibility to study the structural basis of myosin-linked regulation in synthetic filaments of scallop striated muscle myosin. Using papain as a probe of the structure of the head-rod junction, we find that this region of myosin is approximately five times more susceptible to proteolytic attack under activating (ATP/high Ca2+) or rigor (no ATP) conditions than under relaxing conditions (ATP/low Ca2+). A similar result was obtained with native myosin filaments in a crude homogenate of scallop muscle. Proteolytic susceptibility under conditions in which ADP or adenosine 5'-(beta, gamma-imidotriphosphate) (AMPPNP) replaced ATP ...


Ultrastructural Visualization Of Cytoskeletal Mrnas And Their Associated Proteins Using Double-Label In Situ Hybridization, Robert H. Singer, Gary L. Langevin, Jeanne B. Lawrence Jun 1989

Ultrastructural Visualization Of Cytoskeletal Mrnas And Their Associated Proteins Using Double-Label In Situ Hybridization, Robert H. Singer, Gary L. Langevin, Jeanne B. Lawrence

Open Access Articles

We have been able to visualize cytoskeletal messenger RNA molecules at high resolution using nonisotopic in situ hybridization followed by whole-mount electron microscopy. Biotinated cDNA probes for actin, tubulin, or vimentin mRNAs were hybridized to Triton-extracted chicken embryo fibroblasts and myoblasts. The cells were then exposed to antibodies against biotin followed by colloidal gold-conjugated antibodies and then critical-point dried. Identification of mRNA was possible using a probe fragmented to small sizes such that hybridization of several probe fragments along the mRNA was detected as a string of colloidal gold particles qualitatively and quantitatively distinguishable from nonspecific background. Extensive analysis showed ...


Periodic Organization Of The Contractile Apparatus In Smooth Muscle Revealed By The Motion Of Dense Bodies In Single Cells, Gary J. Kargacin, Peter H. Cooke, Steven B. Abramson, Fredric S. Fay Apr 1989

Periodic Organization Of The Contractile Apparatus In Smooth Muscle Revealed By The Motion Of Dense Bodies In Single Cells, Gary J. Kargacin, Peter H. Cooke, Steven B. Abramson, Fredric S. Fay

Open Access Articles

To study the organization of the contractile apparatus in smooth muscle and its behavior during shortening, the movement of dense bodies in contracting saponin skinned, isolated cells was analyzed from digital images collected at fixed time intervals. These cells were optically lucent so that punctate structures, identified immunocytochemically as dense bodies, were visible in them with the phase contrast microscope. Methods were adapted and developed to track the bodies and to study their relative motion. Analysis of their tracks or trajectories indicated that the bodies did not move passively as cells shortened and that nearby bodies often had similar patterns ...


Access Of Proteinase K To Partially Translocated Nascent Polypeptides In Intact And Detergent-Solubilized Membranes, Timothy Connolly, Pallia G. Collins, Reid Gilmore Feb 1989

Access Of Proteinase K To Partially Translocated Nascent Polypeptides In Intact And Detergent-Solubilized Membranes, Timothy Connolly, Pallia G. Collins, Reid Gilmore

Open Access Articles

We have used proteinase K as a probe to detect cytoplasmically and luminally exposed segments of nascent polypeptides undergoing transport across mammalian microsomal membranes. A series of translocation intermediates consisting of discrete-sized nascent chains was prepared by including microsomal membranes in cell-free translations of mRNAs lacking termination codons. The truncated mRNAs were derived from preprolactin and the G protein of vesicular stomatitis virus and encoded nascent chains ranging between 64 and 200 amino acid residues long. Partially translocated nascent chains of 100 amino acid residues or less were insensitive to protease digestion from the external surface of the membrane while ...