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Biomedical subjects

J H Hoh

Publications and source records attributed to J H Hoh.

At least 19 recordsLinked to original sources
PubMed1999

Cationic silanes stabilize intermediates in DNA condensation.

In vitro condensation of DNA has been widely studied to gain insight into the mechanisms of DNA compaction in biological systems such as chromosomes and phage heads and has been used to produce nanostructured particles with novel material and functional properties. Here we report on the condensation of DNA in aqueous solutions by cationic silanes, which combine the condensing properties of polyamines with the cross-linking chemistry of silanes. DNA can be reversibly condensed into classical toroidal and rod-shaped structures with these agents. At low silane concentrations DNA forms a variety of looped structures with well-defined characteristics, including flower- and sausage-shaped forms. These structures suggest that at low silane concentrations a DNA-DNA contact in which the strands are at very large angles to each other is stabilized. Changes in these structures observed as a function of silane concentration suggest possible pathways for the formation of toroids and rods.

Cations↗
PubMed1999

Ethanol-induced structural transitions of DNA on mica.

The effect of ethanol on the structure of DNA confined to mica in the presence of Mg2+was examined by varying the ethanol concentration and imaging the DNA by atomic force microscopy. Contour length measurements of the DNA show a transition from all-B-form at 0% ethanol to all-A-form at >25% ethanol. At intermediate ethanol concentrations, contour lengths suggest that individual molecules of air-dried DNA are trapped with mixed compositions of A-form and B-form. The relative composition depends on the ethanol concentration. Fitting the length distributions at intermediate ethanol concentrations to a simple binomial model results in an upper bound estimate for the A-form and B-form domains of approximately 54 bp in the individual molecules. In addition to length changes, the apparent persistence length of DNA decreases with increasing ethanol concentration. At high concentrations of ethanol (>20%), DNA formed several higher order structures, including flower shaped condensates and toroids.

Aluminum Silicates↗
PubMed1999

Relative surface charge density mapping with the atomic force microscope.

An experimental approach for producing relative charge density maps of biological surfaces using the atomic force microscope is presented. This approach, called D minus D (D-D) mapping, uses isoforce surfaces collected at different salt concentrations to remove topography and isolate electrostatic contributions to the tip-sample interaction force. This approach is quantitative for surface potentials below 25 mV, and does not require prior knowledge of the cantilever spring constant, tip radius, or tip charge. In addition, D-D mapping does not require tip-sample contact. The performance of D-D mapping is demonstrated on surfaces of constant charge and varying topography (mechanically roughened mica and stacked bilayers of dipalmitolphosphatidylserine), a surface of varying charge and varying topography (patches of dipalmitolphosphatidylcholine on mica), and bacteriorhopsin membranes adsorbed to mica.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1999

Spatially resolved force spectroscopy of biological surfaces using the atomic force microscope.

The spatial distribution of intermolecular forces governs macromolecular interactions. The atomic force microscope, a relatively new tool for investigating interaction forces between nanometer-scale objects, can be used to produce spatially resolved maps of the surface or material properties of a sample; these include charge density, adhesion and stiffness, as well as the force required to break specific ligand-receptor bonds. Maps such as these will provide fundamental insights into biological structure and will become an important tool for characterizing technologically important biological systems.

Cell Adhesion↗
PubMed1998

Functional protein domains from the thermally driven motion of polypeptide chains: a proposal.

It is proposed that the thermally driven motion of certain polypeptide chains, including those that are part of an otherwise stable folded protein, produces time-averaged three-dimensional domains that confer unique functions to a protein. These domains may be controlled by collapsing the polypeptide into an enthalpically favored structure, or extending it into an entropically dominated form. In the extended form, these domains occupy a relatively large space, which may be used to regulate protein-protein interactions and confer mechanical properties to proteins. This "entropic bristle" model makes several predictions about the structure and properties of these domains, and the predictions are used to reevaluate a range of biophysical studies on proteins. The outcome of the analysis suggests that the entropic bristle can be used to explain a wide range of disparate and apparently unrelated experimental observations.

Allosteric Regulation↗
PubMed1998

Solid-state DNA sizing by atomic force microscopy.

Atomic force microscopy (AFM) allows rapid, accurate, and reproducible visualization of DNA adsorbed onto solid supports. The images reflect the lengths of the DNA molecules in the sample. Here we propose a solid-state DNA sizing (SSDS) method based on AFM as an analytical method for high-throughput applications such as finger-printing, restriction mapping, +/- screening, and genotyping. For this process, the sample is first deposited onto a solid support by adsorption from solution. It is then dried and imaged under ambient conditions by AFM. The resulting images are subjected to automated determination of the lengths of the DNA molecules on the surface. The result is a histogram of sizes that is similar to densitometric scans of DNA samples separated on gels. A direct comparison of SSDS with agarose gel electrophoresis for +/- screening shows that it produces equivalent results. Advantages of SSDS include reduced sample size (i.e., lower reagent costs), rapid analysis of single samples, and potential for full automation using available technology. The high sensitivity of the method also allows the number of polymerase chain reaction cycles to be reduced to 15 or less. Because the high signal-to-noise ratio of the AFM allows for direct visualization of DNA-binding proteins, different DNA conformations, restriction enzymes, and other DNA modifications, there is potential for dramatically improving the information content in this type of analysis.

Adsorption↗
PubMed1998

Surface-directed DNA condensation in the absence of soluble multivalent cations.

Multivalent cations are known to condense DNA into higher ordered structures, including toroids and rods. Here we report that solid supports treated with monovalent or multivalent cationic silanes, followed by removal of soluble molecules, can condense DNA. The mechanism of this surface-directed condensation depends on surface-mobile silanes, which are apparently recruited to the condensation site. The yield and species of DNA aggregates can be controlled by selecting the type of functional groups on surfaces, DNA and salt concentrations. For plasmid DNA, the toroidal form can represent >70% of adsorbed structures.

Adsorption↗
PubMed1998

Automated sizing of DNA fragments in atomic force microscope images.

Current techniques used to measure lengths of DNA fragments in atomic force microscope (AFM) images require a user to operate interactive software and execute tedious error-prone cursor selections. An algorithm is proposed which provides an automated method for determining DNA fragment lengths from AFM images without interaction from the computer operator (e.g. cursor selections or mouse clicks). The approach utilises image processing techniques tailored to characteristics of AFM images of DNA fragments. The automated measurements have a mean absolute deviation of less than 1 pixel when compared to manual image-based measurements. The DNA length determined from the histogram of calculated lengths is accurate to within 3% of the actual DNA length in solution. For fragments that are 250 base-pairs long, the precision is estimated to be within 17 nm, which is about 20% of the total length. This precision was confirmed when the algorithm easily resolved fragments in one image that differed by only 17 nm. Fragment sizes up to 2000 base-pairs have been tested and successfully sized. This algorithm is being developed as part of a new solid-state DNA sizing technique for applications such as genotyping and construction of physical genome maps.

Algorithms↗
PubMed1998

Relative microelastic mapping of living cells by atomic force microscopy.

The spatial and temporal changes of the mechanical properties of living cells reflect complex underlying physiological processes. Following these changes should provide valuable insight into the biological importance of cellular mechanics and their regulation. The tip of an atomic force microscope (AFM) can be used to indent soft samples, and the force versus indentation measurement provides information about the local viscoelasticity. By collecting force-distance curves on a time scale where viscous contributions are small, the forces measured are dominated by the elastic properties of the sample. We have developed an experimental approach, using atomic force microscopy, called force integration to equal limits (FIEL) mapping, to produce robust, internally quantitative maps of relative elasticity. FIEL mapping has the advantage of essentially being independent of the tip-sample contact point and the cantilever spring constant. FIEL maps of living Madine-Darby canine kidney (MDCK) cells show that elasticity is uncoupled from topography and reveal a number of unexpected features. These results present a mode of high-resolution visualization in which the contrast is based on the mechanical properties of the sample.

Animals↗
PubMed1998

Neurofilament-L homopolymers are less mechanically stable than native neurofilaments.

Neurofilaments are cytoskeletal components of neurones that are thought to play an important structural role in the axon. Specific functions of neurofilaments are not yet well defined; however, other intermediate filaments are known to have structural and mechanical functions in different cell types. The atomic force microscope (AFM) can be used to visualize and manipulate biological structures through direct physical contact. This allows the AFM to be used to probe the mechanical properties of these structures. In this paper we present AFM images of native neurofilaments isolated from bovine spinal cord, composed of NF-L, NF-M and NF-H, and filaments polymerized in vitro from purified NF-L. Morphologically these structures, in solution and under ambient conditions, are in agreement with previous data from electron microscopy. However, repeated scanning of NF-L homopolymers (in solution) produced significant disruptions of segments of filaments, both within and at the ends of the filaments. This disruption resulted in complete loss of portions of the filaments and in breaks in the continuity of the filaments. Repeated scanning of isolated native neurofilaments under similar conditions produced no detectable structural changes. Under extremely high applied forces the native neurofilaments were bent and distorted by the action of the AFM tip, but were never broken. These data suggest that purified NF-L is not sufficient to confer complete mechanical stability to neurofilaments.

Animals↗
PubMed1997

Entropic exclusion by neurofilament sidearms: a mechanism for maintaining interfilament spacing.

A long-range repulsive force near isolated neurofilaments was detected by exclusion of large molecules and by direct force measurements with atomic force microscopy. Adsorption of isolated native neurofilaments to a solid substrate in a high-salt solution (170 mM NaCl), in the presence of coisolating contaminants, shows that the contaminants are excluded from a zone that extends from 50-100 nm from the core of the filament. Force-distance measurements by AFM show the presence of a weak repulsive force that extends >50 nm from the core of the filament; this repulsive force is absent in homopolymers of neurofilament L or trypsinized native filaments that lack the long sidearms present in native filaments. These results suggest that neurofilament sidearms form an entropic brush, thereby providing a mechanism for maintaining interfilament spacing.

Animals↗
PubMed1994

Slow cellular dynamics in MDCK and R5 cells monitored by time-lapse atomic force microscopy.

We have examined dynamic events that occur on a time scale of minutes in an epithelial monolayer of Madine-Darby Canine Kidney (MDCK) cells and in ras-transformed MDCK cells by atomic force microscopy (AFM). Cells were imaged under physiological conditions, and time-lapse movies representing approximately 60 s real time per frame were assembled. In normal MDCK cells, two types of protrusions in the apical plasma membrane exhibit dynamic behavior. First, smooth bulges formed transiently over the time scale of minutes to tens of minutes. Second, spike-like protrusions appear initially as bulges, extend well above the apical surface and, finally, seem to detach. R5, an oncogenic transformant derived from MDCK cells, grows very flat on glass. During AFM imaging, these cells sometimes round up and detach from the substrate. In light microscopic observations of parallel preparations, cells rarely detach, suggesting that this is an active response of these cells to irritation by the AFM tip. R5 cells often extend processes that are supported by actin stress fibers. During imaging with the AFM, these processes withdraw at a rate of 1-5 microns/min, similar to that observed by light microscopy. During the withdrawal, movement of the stress fibers can be clearly seen. In the flat periphery of these cells, the transport of intracellular particles along cytoskeletal elements was seen. In addition, we have observed two types of wave-like movements through the cell, which appear to be an organized rearrangement of cytoplasm. One type of wave moves radially out from center of the cell while the other moves circularly along the cell periphery.

Animals↗
PubMed1994

Surface morphology and mechanical properties of MDCK monolayers by atomic force microscopy.

We describe the morphology and mechanical stability of the apical surface of MDCK monolayers by atomic force microscopy (AFM). Living cells could be imaged in physiological solution for several hours without noticeable deterioration. Cell boundaries appear as ridges that clearly demarcate neighboring cells. In some cases the nucleus of individual cells could be seen, though apparently only in very thin areas of the monolayer. Two types of protrusions on the surface could be visualized. Smooth bulges that varied in width from a few hundred nanometers to several micrometers, which appear to represent relatively rigid subapical structures. Another type of protrusion extended well above the membrane and was swept back and forth during the imaging. However, the microvilli that are typically present on the apical surface could not be resolved. For comparison, a transformed MDCK cell line expressing the K-ras oncogene was also examined. When cultured on solid substrata at low density, the R5 cells spread out and are less than 100 nm thick over large areas with both extensive processes and rounded edges. Many intracellular structures such as the nucleus, cytoskeletal elements and vesicles could be visualized. None of the intracellular structures seen in the AFM images could be seen by scanning electron microscopy. Both R5 cells and MDCK monolayers required imaging forces of > 2 nN for good image contrast. Force measurements on the MDCK monolayers show that they are very soft, with an effective spring constant of approximately 0.002 N/m for the apical plasma membrane, over the first micrometer of deformation, resulting in a height deformation of approximately 500 nm per nanoNewton of applied force. The mechanical properties of the cells could be manipulated by addition of glutaraldehyde. These changes were monitored in real time by collecting force curves during the fixation reaction. The curves show a stiffening of the apical plasma membrane that was completed in approximately 1 minute. On the basis of these measurements and the imaging forces required, we conclude that deformation of the plasma membrane is an important component of the contrast mechanism, in effect 'staining' structures based on their relative rigidity.

Animals↗
PubMed1993

Structure of the extracellular surface of the gap junction by atomic force microscopy.

The extracellular surface of the gap junction cell-to-cell channels was imaged in phosphate-buffered saline with an atomic force microscope. The fully hydrated isolated gap junction membranes adsorbed to mica were irregular sheets approximately 1-2 microns across and 13.2 (+/- 1.3) nm thick. The top bilayer of the gap junction was dissected by increasing the force applied to the tip or sometimes by increasing the scan rate at moderate forces. The exposed extracellular surface revealed a hexagonal array with a center-to-center spacing of 9.4 (+/- 0.9) nm between individual channels (connexons). Images of individual connexons with a lateral resolution of < 3.5 nm, and in the best case approximately 2.5 nm, were reliably and reproducibly obtained with high-quality tips. These membrane channels protruded 1.4 (+/- 0.4) nm from the extracellular surface of the lipid membrane, and the atomic force microscope tip reached up to 0.7 nm into the pore, which opened up to a diameter of 3.8 (+/- 0.6) nm on the extracellular side.

Animals↗
PubMed1992

Aspects of gap junction structure and assembly.

The development of ideas about gap junction structure is summarized, including some recent results obtained by use of atomic force microscopy. Particular attention is paid to novel aspects of the biosynthesis and assembly of connexons and to the formation of new junctions.

Animals↗
PubMed1991

Atomic force microscopy and dissection of gap junctions.

An atomic force microscope (AFM) was used to study the structure of isolated hepatic gap junctions in phosphate-buffered saline (PBS). The thickness of these gap junctions appears to be 14.4 nanometers, close to the dimensions reported by electron microscopy (EM). When an increasing force is applied to the microscope tip, the top membrane of the gap junction can be "dissected" away, leaving the extracellular domains of the bottom membrane exposed. When such "force dissection" is performed on samples both trypsinized and fixed with glutaraldehyde, the hexagonal array of gap junction hemichannels is revealed, with a center-to-center spacing of 9.1 nanometers.

Animals↗
PubMed1991

Molecular cloning and characterization of a new member of the gap junction gene family, connexin-31.

A new member of the connexin gene family has been identified and designated rat connexin-31 (Cx31) based on its predicted molecular mass of 30,960 daltons. Cx31 is 270 amino acids long and is coded for by a single copy gene. It is expressed as a 1.7-kilobase mRNA that is detected in placenta, Harderian gland, skin, and eye. Cx31 is highly conserved and can be detected in species as distantly related to rat as Xenopus laevis. It exhibits extensive sequence similarity to the previously identified connexins, 58, 50, and 40% amino acid identity to Cx26, Cx32, and Cx43, respectively. When conservation of predicted phosphorylation sites is used to adjust the alignment of Cx31 to other connexins, a unique alignment of three predicted protein kinase C phosphorylation sites near the carboxyl terminus of Cx31 with three sites at the carboxyl terminus of Cx43 is revealed.

Amino Acid Sequence↗