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Biomedical subjects

C Bustamante

Publications and source records attributed to C Bustamante.

At least 91 records · Page 5Linked to original sources
PubMed1992

Substrate preparation for reliable imaging of DNA molecules with the scanning force microscope.

A simple method of substrate preparation for imaging circular DNA molecules with the scanning force microscope (SFM) is presented. These biomolecules are adsorbed onto mica that has been soaked in magnesium acetate, sonicated and glow-discharged. The stylus-sample forces that may be endured before sample damage occurs depends on the ambient relative humidity. Images of circular DNA molecules have been obtained routinely using tips specially modified by an electron beam with a radius of curvature, Rc, of about 10 nm [D. Keller and C. Chih-Chung, Surf. Sci. 268 (1992) 333]. The resolution of these adsorbed biomolecules is determined by the Rc. At higher forces individual circular DNA molecules can be manipulated with the SFM stylus. Strategies to develop still sharper probes will be discussed.

Aluminum Silicates↗
PubMed1992

Deposition and imaging of metal-coated biomolecules with the STM.

We have applied a simple and reliable procedure for imaging biomolecules with the scanning tunneling microscope (STM). The biomolecules are adsorbed on glow-discharged mica, then coated with a thin film of platinum-carbon. We have tested this method with linear and circular (plasmid) DNA molecules. The contrast and resolution of the STM images are comparable to electron micrographs of the same molecules when shadowed. Though the present lateral resolution (5-6 nm) is limited by the grain size of the conductive film, some details like supercoiled regions in the DNA are resolved. This method is interesting for two reasons. First, as an alternative technique for imaging biomolecules. Second, for use as a control in STM studies of bare biomolecules.

DNA↗
PubMed1991

Daunomycin inverts the long-range chirality of DNA condensed states.

The effect of daunomycin upon DNA condensed states induced by poly(ethylene glycol) (PEG) was studied by circular dichroism (CD) and circular intensity differential scattering (CIDS). The CD spectra of these aggregates showed psi-type anomalies and intensities 10-100 times greater than those obtained with the dispersed DNA solutions in the absence of PEG. Increasing concentrations of daunomycin, added to the DNA solution prior to its aggregation, led, in the presence of PEG, to CD and CIDS signals which gradually decreased in magnitude and eventually inverted sign. The coincidence of the transition point of both signals and a careful characterization of the CD spectrum at the transition point clearly indicated that the inversion observed corresponds to an inversion of the handedness of the aggregates. The latter result suggests that the structure of the aggregates at the inversion point should resemble that of a nematic liquid-crystalline structure. The characteristic B-DNA spectrum obtained in this case further suggests that the packing process does not affect the secondary structure of the DNA molecules and that small changes in their local structure can induce dramatic changes in their long-range tertiary packing. The results obtained in this study represent a confirmation of a recent theory of psi-type CD in which the anomalous signals are interpreted as a manifestation of the long-range chirality of the aggregates.

Animals↗
PubMed1991

Model and computer simulations of the motion of DNA molecules during pulse field gel electrophoresis.

A model is presented for the motion of individual molecules of DNA undergoing pulse field gel electrophoresis (PFGE). The molecule is represented by a chain of charged beads connected by entropic springs, and the gel is represented by a segmented tube surrounding the beads. This model differs from earlier reptation/tube models in that the tube is allowed to leak in certain places and the chain can double over and flow out of the side of the tube in kinks. It is found that these kinks often lead to the formation of U shapes, which are a major source of retardation in PFGE. The results of computer simulations using this model are compared with real DNA experimental results for the following cases: steady field motion as seen in fluorescence microscopy, mobility in steady fields, mobility in transverse field alternation gel electrophoresis (TFAGE), mobility in field inversion gel electrophoresis (FIGE), and linear dichroism (LD) of DNA in agarose gels during PFGE. Good agreement between the simulations and the experimental results is obtained.

Chemical Phenomena↗
PubMed1991

Differential polarization imaging. IV. Images in higher Born approximations.

The theory of differential polarization imaging developed previously within the framework of the first Born approximation is extended to higher Born approximations, taking into account interactions among the polarizable groups in the object. Several properties of differential polarization images, originally described using first Born approximation are modified when higher Born approximations are used. In particular, (a) when the polarizable groups are spherically symmetric, the off-diagonal Mueller elements Mij (i not equal to j) in bright field do not vanish in higher Born approximations, as they do in the first Born approximation case. (b) In higher Born approximations, the dark field Mi4 and M4i (i = 1, 2, 3) images do not vanish as in the first Born approximation, due to the anisotropy induced by the interactions among the groups. (c) When the polarizability tensor of each group is symmetric and real, the bright field M14 and M41 images always vanish in the first Born approximation. In higher Born approximations, these terms do not vanish if the groups bear a chiral relationship to each other. Quantitative criteria for the validity of the first Born approximation in differential polarization imaging are explicitly derived for three different types of media: (a) linearly anisotropic, (b) circularly anisotropic, and (c) linearly and circularly anisotropic (medium displaying linear birefringence and circular birefringence). These criteria define the limits of thickness and the degree of anisotropy of optically thin media. Finally, the possibility to perform optical sectioning in differential polarization imaging in the presence and absence of group interactions is discussed.

Biophysical Phenomena↗
PubMed1991

Differential polarization imaging. V. Numerical aperture effects and the contribution of preferential scattering and absorption to the circular dichroism images.

Chiral objects which scatter and absorb preferentially left versus right circularly polarized light give rise to bright-field circular dichroism (CD) images containing contributions from both these two phenomena. These contributions are separated and characterized mathematically, and the effect of the dimensions of the chiral object on their relative magnitude is discussed. CD images of the long-range chiral organization of the thylakoid membranes in chloroplasts are obtained at two different wavelengths to illustrate the diverse wavelength dependence of the preferential absorption and scattering contributions to the images. The bright field CD images not only depend on the magnitude and sign of the preferential scattering and preferential absorption contributions, but also on the numerical aperture of the lens used. This dependence is obtained formally and a method to extract the angle dependent preferential scattering contributions to the images is presented. The validity of this method is confirmed experimentally.

Biophysical Phenomena↗
PubMed1991

[A microsurgical technic in infertility due to a tuboperitoneal factor. The results].

The experience with microsurgical technique in 131 infertile women is presented. The 65.7% had distal tubal occlusion and 16.7% had proximal occlusion. In 48.9% of the women the peritoneal factor was present. In 61.8% distal surgery was done: 16.8% fimbrioplasties and 45% neosalphingostomies. The pregnancy rate was 47.4%. The incidence of ectopic pregnancy was 20%. The 85.5% of the pregnancies was performed in the two first years of follow up. With these results the good prognosis factors, with statistical significance, were: a. Age under 30 years. b. Infertility under 10 years of evolution. c. Normal male factor. The results are commented.

Adult↗
PubMed1991

Application of scanning tunneling microscopy to structural biology.

Scanning tunneling microscopy offers the possibility of visualizing biological molecules in conditions similar to those in vivo with molecular resolution. Images of DNA and various proteins have been obtained, but insufficient conductivity through, and inhomogeneous and unstable adsorption of the biomolecules continue to prevent reliable imaging. Applying a metal coating to samples, to separate the conductivity and deposition problems has yielded satisfactory deposition procedures in various laboratories, but extension of this protocol to high resolution imaging of macromolecules has yet to be demonstrated. In this paper we present a review of the main results obtained in our laboratory, which illustrate the main problems encountered by investigators attempting to image metal-coated and uncoated biological specimens.

DNA↗
PubMed1990

Imaging of kinked configurations of DNA molecules undergoing orthogonal field alternating gel electrophoresis by fluorescence microscopy.

The dynamics of individual DNA molecules undergoing orthogonal field alternating gel electrophoresis (OFAGE) have been studied by use of T2 DNA molecules labeled with a dye and visualized with a fluorescence microscope. The mechanism of reorientation used by a molecule to align itself in the direction of the new orthogonal field depends on the degree of extension of the chain immediately before the application of this field. The formation of kinks is promoted when time is allowed between the application of the two orthogonal fields so that the molecule attains a partially relaxed configuration. In this case, the chain appears bunched up in domains moving along the contour of the molecule. These regions are found to be the locations where the kinks are formed upon application of the second field perpendicular to the chain. The formation of kinks provides a significant retardation of the reorientation of the molecules, relative to molecules that do not form kinks, and appears to play an important role in the fractionation attained with OFAGE. A classification of various reorientation mechanisms observed in molecules that form kinks is presented.

DNA↗
PubMed1990

Fluorescence microscopy of the dynamics of supercoiling, folding, and condensation of bacterial chromosomes, induced by acridine orange.

The fluorescent dye, acridine orange, was used to visualize bacterial chromosomes extending from bacteria attached to a glass surface. The acridine-induced condensation of these chromosomes was followed in real-time with a low light level video camera. Acridine orange induced the packing of the bacterial chromosome into thick bundles which underwent various forms of condensation, supercoiling, folding, and rolling into a compact particle. Filaments attached to the surface at both ends were topologically constrained and supercoiled rapidly; whereas all three patterns of condensation were noted among filaments attached at only one end or free from the surface. Kinks often appeared in the filaments prior to supercoiling or folding, and the dynamic events observed often occurred around these kinks. These observations identify several mechanisms of condensation available to higher order structures of DNA, and indicate that kinks are an important intermediate step in many of the transitions.

Acridine Orange↗
PubMed1989

Images of single-stranded nucleic acids by scanning tunnelling microscopy.

The scanning tunnelling microscope has the potential to resolve the structure of biological molecules with atomic detail. Progress has been made in the imaging of dried, unshadowed double helices of DNA4-7 and in recording images of DNA under water. Also, images of unshadowed complexes of DNA with the RecA protein from Escherichia coli indicate that this technique may not be restricted to thin biological samples. Here we present images of polydeoxyadenylate molecules aligned in parallel, with their bases lying flat on a surface of highly oriented pyrolytic graphite and with their charged phosphodiester backbones protruding upwards. Based on these images, a molecular model has been built which suggests the presence of a hydrogen bond that could stabilize the parallel alignment. Our micrographs demonstrate the potential application of scanning tunnelling microscopy in structural studies of nucleic acids and provide evidence that it could be used to sequence DNA.

Adsorption↗
PubMed1989

Imaging of metal-coated biological samples by scanning tunneling microscopy.

A method for imaging biological samples by scanning tunneling microscopy (STM) is presented. There are two main difficulties in imaging biological samples by STM: (1) the low conductivity of biological material and (2) finding a method of reliably depositing the sample on a flat conducting surface. The first of these difficulties was solved by coating the samples with a thin film of platinum-carbon. The deposition problem was solved by a method similar to a procedure used to deposit biological molecules onto field ion microscope (FIM) tips. STM images of bacteriophage T7 and filamentous phage fd are shown. The substrate on which the samples were absorbed was atomically flat gold. The images do not show molecular detail due to the metal coating, but the gross dimensions and morphology are correct for each type of virus. Also, the surface density of virus particles increases and decreases in the way expected when the conditions of deposition are changed. These methods allow reliable and reproducible STM imaging of biological samples.

Bacteriophages↗
PubMed1989

Real-time imaging of single DNA molecules with fluorescence microscopy.

A fluorescence microscopy technique was used to image the dynamics of individual DNA molecules. Lambda, calf thymus, cosmid (circular), and T4 DNA were studied with the fluorescent dye acridine orange. Experiments with DNAase I were conducted, and the results indicate that these observations correspond to DNA molecules. The results of experiments with circular DNA provide strong evidence that these were single DNA molecules. Molecules were observed free in solution or attached to a glass or copper surface at one or several points. The Brownian motion of these molecules was observed, indicating that DNA in solution exists in a partially supercoiled state. Some molecules appeared stretched and were attached to the surface by their termini; the lengths of these molecules were measured. Such molecules also exhibited elastic behavior upon breaking. The power of this technique is demonstrated in images of cosmid DNA molecules, catenanes, and DNA extending from T4 phage particles. These results suggest immediate applications to molecular biology, such as examining the dynamics of protein-DNA interactions. Areas of ongoing research are discussed.

DNA↗
PubMed1989

Imaging of single uncoated DNA molecules by scanning tunneling microscopy.

Scanning tunneling microscope images of DNA molecules absorbed onto highly oriented pyrolytic graphite have been obtained. Three methods of deposition and sample preparation have been utilized. In the first method, a highly concentrated solution of DNA is sonicated, and a drop is deposited on freshly cleaved graphite. Under these conditions, the molecules tend to align in a parallel fashion, forming liquid-crystalline phases. In the second method, a solution of DNA is deposited directly on the graphite surface without sonication. In this case, ammonium acetate, a volatile salt, is used to decrease the amount of the residual salt crystals left after drying. In the third method, a solution containing lysed phage particles and DNA is adsorbed onto a graphite surface. The molecules are seen either isolated or in small bundles. The values of height, periodicity, and thickness observed and the handedness of the molecules are consistent with those expected for DNA. In all cases, the molecules were identified by their characteristic periodic structure and because, at higher magnification, no graphite-like structure was detectable on the surface of the molecules. Often the DNA molecules appear to adsorb in areas of the graphite that have many steps and defects. A mechanism that explains the magnitude of the tunneling currents measured in DNA is proposed. This mechanism, in turn, suggests a general method by which large insulating molecules can be rendered conductive.

DNA↗
PubMed1989

Direct observation of large chiral domains in chloroplast thylakoid membranes by differential polarization microscopy.

Long-range chiral organization of the pigment-protein complexes in mature granal chloroplasts has been established by differential polarization imaging and local circular dichroism spectra. Linear and circular dichroism images of oriented chloroplasts were obtained in a confocal differential polarization microscope. The circular dichroism images display signals of opposite signs emerging from discrete regions with local dichroic values much larger than anticipated, indicating domains in the thylakoid membranes having long-range chiral organization. These domains are associated with positive and negative circular dichroism bands obtained at specific locations on the chloroplasts. Surprisingly, the local circular dichroism spectra do not display the excitonic shape of spectra obtained for macroscopic suspensions, but the latter can be produced by superposition of two local spectra of opposite sign. These data are evidence for the existence of long-range chiral order of the pigment-protein complexes in thylakoid membranes. The possible role of the long-range chiral domains in the efficiency of energy delocalization through the thylakoid membranes is discussed.

Chloroplasts↗
PubMed1988

Helically organized macroaggregates of pigment-protein complexes in chloroplasts: evidence from circular intensity differential scattering.

Angle dependence of circular intensity differential scattering (CIDS) and of nonpolarized scattering was determined in isolated spinach chloroplasts at 514.5 nm. CIDS between 0 degrees and 170 degrees was independent of the nonpolarized scattering and showed intense lobes of alternating signs, exhibiting the negative and positive maxima around 15 degrees and 70 degrees, respectively. These results provide experimental evidence for the existence of large helically organized macroaggregates of pigment-protein complexes in thylakoid membranes. Modeling of the CIDS data by a simple helical array of uniaxial polarizable groups suggests that the chiral structure is left-handed with pitch and radius of the order of 385 nm.

Chloroplasts↗
PubMed1988

Differential polarization imaging. III. Theory confirmation. Patterns of polymerization of hemoglobin S in red blood sickle cells.

In this paper we test the predictions of the differential polarization imaging theory developed in the previous two papers. A characterization of the patterns of polymerization of hemoglobin in red blood cells from patients with sickle cell anemia is presented. This system was chosen because it is relatively easy to handle and because previous studies have been done on it. A differential polarization microscope designed and built in our laboratory was used to carry out this study. This microscope uses an image dissector camera, a photoelastic modulator, and a phase-lock amplifier. This design represents a substantial modification with respect to the instrumentation used in the previous results communicated on this system. Therefore, the results presented here also permit us to confirm the validity of our conclusions. On the basis of the differential polarization images obtained, models of the patterns of polymerization of the hemoglobin S inside the sickle cells are proposed and their M12 and regular images are calculated by the theory. Good agreement between those models and the experimental systems is found, as well as with the results previously reported.

Anemia, Sickle Cell↗