Biomedical subjects

W M Heckl

Publications and source records attributed to W M Heckl.

At least 19 recordsLinked to original sources

PubMed2004

Structural studies of oligonucleotides containing G-quadruplex motifs using AFM.

G-quadruplex DNAs are cyclic arrays of four guanine bases binding by Hoogsteen hydrogen bonds, found in the telomeric regions of chromosomes and in transcriptional regulatory regions of several important oncogenes. Here, we used high resolution atomic force microscopy (AFM) to observe a specific guanine (G) tetrad mediated complex formation of oligonucleotides containing a G-quadruplex motifs (G-ODN) combined with a palindromic sequence under physiological extracellular conditions. These oligonucleotides have been investigated in correlation to their immunostimulatory effects. We observed structural dependence on ion concentration and G-ODN concentration, where high concentration self-assembled DNA networks were formed.

Adjuvants, Immunologic↗

PubMed2004

LexA-DNA bond strength by single molecule force spectroscopy.

The SOS system of Escherichia coli is coordinated by two proteins: LexA, a repressor protein of several unlinked genes, and the coprotease RecA. As known to date LexA controls 31 genes with slightly different DNA binding motifs allowing for a variable degree of repression from one gene to the other. Besides the SOS system LexA plays an important role in the regulation of transcription. The protein regulates transcription by using particular motifs to bind DNA, the helix-turn-helix motif. Here, we employed AFM-based single molecule force spectroscopy to characterize the interaction of LexA protein with two different DNA motifs: recA and yebG. We measured the dissociation rates to be 0.045 s(-1) for recA and 0.13 s(-1) for yebG, respectively, which is in accordance with the predicted higher affinity between LexA-recA compared to LexA-yebG. The widths of the binding potentials were determined to be 5.4 +/- 1 angstroms and 4.9 +/- 0.5 angstroms, respectively. This short-ranged potential is characteristic for a stiff hydrogen-bonding network between protein and DNA. The unbinding occurs in a breakup rather than a gradual sliding.

Bacterial Proteins↗

PubMed2003

Selection of chloroplasts by laser microbeam microdissection for single-chloroplast PCR.

Laser microbeam microdissection and laser pressure catapulting offer the possibility of separating cell compartments, thus allowing for contamination-free analysis. Using these methods, we were able to select single chloroplasts of Nicotiana tabacum. Starting from homogenized leaf material, chloroplasts were purified by differential centrifugation and applied directly onto a poly-ethylene-naphthalate membrane that was mounted on a microscope slide. Single chloroplasts were dissected under microscopic control and catapulted into a PCR tube. Subsequent PCR of a spacer region between the trnT and trnF genes verified the successful amplification of DNA from a single chloroplast. The advantage of this method compared to the use of capillaries or optical tweezers is that one is able to prepare high numbers of samples in a short time.

Base Sequence↗

PubMed2002

Scanning probe microscopy for Bio & Nanotechnology onboard the ISS.

Since February 2002 Kayser-Threde GmbH, Munich (Germany) leads a study under ESA contract in order to study the technical feasibility and the applications of "Scanning Probe Microscopy for Bio & Nanotechnology onboard the ISS (SONOS)". The objective of this effort is to demonstrate the feasibility of an SPM instrument on the ISS. An appropriate breadboard model will be manufactured and tested within the present study. Its development will be based upon the developed pocket size SPM instrument by Professor W. Hecki of the Center for Crystallography and NanoScience (CeNS) at the Ludwig-Maximilians University (LMU) in Munich. Scanning probe microscopy (SPM) investigates surface structures at very high resolution and can perform nanoengineering. These techniques can be applied to non organic as well as to organic or biological materials.

Journal Article↗

PubMed2001

Differential adsorption of nucleic acid bases: Relevance to the origin of life.

The adsorption of organic molecules onto the surfaces of inorganic solids has long been considered a process relevant to the origin of life. We have determined the equilibrium adsorption isotherms for the nucleic acid purine and pyrimidine bases dissolved in water on the surface of crystalline graphite. The markedly different adsorption behavior of the bases describes an elutropic series: guanine > adenine > hypoxanthine > thymine > cytosine > uracil. We propose that such differential properties were relevant to the prebiotic chemistry of the bases and may have influenced the composition of the primordial genetic architecture.

Adenine↗

PubMed2001

Thermomechanical noise of a free v-shaped cantilever for atomic-force microscopy.

We have calculated the thermal noise of a v-shaped AFM cantilever (Microlever, Type E, Thermomicroscopes) by means of a finite element analysis. The modal shapes of the first 10 eigenmodes are displayed as well as the numerical constants, which are needed for the calibration using the thermal noise method. In the first eigenmode, values for the thermomechanical noise of the z-displacement at 22 degrees C temperature of square root of u2(1) = A/square root of c(cant) and the photodiode signal (normal-force) of S2(1) = A/square root of c(cant) were obtained. The results also indicate a systematic deviation ofthe spectral density of the thermomechanical noise of v-shaped cantilevers as compared to rectangular beam-shaped cantilevers.

Journal Article↗

PubMed2001

Scaling-index method as an image processing tool in scanning-probe microscopy.

The scaling-index method (SIM) is a novel tool for image processing in scanning-probe microscopy. Originating from the theory of complex systems, the SIM can be used in order to extract structural information from arbitrary data sets. This method can readily be applied to the analysis of digital atomic-force microscopy (AFM) images. Especially for biomedical diagnostics, where genetic material is investigated by various microscopic methods, a reliable image segmentation based on the SIM algorithm is helpful. As a first application, AFM-images of GTG-banded human metaphase chromosomes (with G bands obtained by Trypsin using Giemsa) are compared with micrographs from conventional light microscopy by means of a scaling-index analysis. While the grey-level distributions of the optical and the AFM-images are largely different from each other, the scaling-index images are remarkably similar. Using this method, a fingerprint of an image can be produced which helps in the classification and interpretation of the measured data.

Algorithms↗

PubMed2001

GTG banding pattern on human metaphase chromosomes revealed by high resolution atomic-force microscopy.

Surface topography of human metaphase chromosomes following GTG banding was examined using high resolution atomic force microscopy (AFM). Although using a completely different imaging mechanism, which is based on the mechanical interaction of a probe tip with the chromosome, the observed banding pattern is comparable to results from light microscopy and a karyotype of the AFM imaged metaphase spread can be generated. The AFM imaging process was performed on a normal 2n = 46, XX karyotype and on a 2n = 46, XY, t(2;15)(q23;q15) karyotype as an example of a translocation of chromosomal bands.

Azure Stains↗

PubMed2000

Self-programmable, self-assembling two-dimensional genetic matter.

Putative two-dimensional coding systems can be constructed from aqueous solutions of purine and pyrimidine nucleic acid bases evaporated at moderate temperatures on the surfaces of inorganic solids. The resultant structures are monolayers which are formed spontaneously by molecular self-assembly and they have been observed with molecular resolution by scanning tunnelling microscopy (STM). When formed from solutions of a single base, the monolayers of adenine and uracil have crystalline characteristics and the STM images can be interpreted in terms of the geometrical placement of planar arranged molecules that interact laterally by intermolecular hydrogen bonding. When formed from solutions containing a mixture of adenine and uracil, the monolayers have aperiodic structures. Small crystalline domains within these monolayers can be interpreted in terms of the single phase configurations of the molecules and the remaining aperiodic structures can presumably be interpreted, geometrically, in terms of the 21 theoretically possible adenine-adenine, uracil-uracil and adenine-uracil hydrogen bonding interactions. We propose that combinatorial arrangements of planar arranged purine and pyrimidine bases could provide the necessary complexity to act as a primitive genetic mechanism and may have relevance to the origin of life.

Adenine↗

PubMed1998

The role of self-assembled monolayers of the purine and pyrimidine bases in the emergence of life.

The experimental evidence for the spontaneous formation and structure determination of two-dimensional monolayers of the purine and pyrimidine bases is examined. The plausibility of such structures forming spontaneously at the solid-liquid interface following their prebiotic synthesis suggests a functional role for them in the emergence of life. It is proposed that prebiotic interactions of enantiomorphic monolayers of mixed base composition with racemic amino acids might be implicated in a simultaneous origin of a primitive genetic coding mechanism and biomolecular homochirality. The interactions of these monolayers with carbohydrates and other derivatives is also discussed.

Adsorption↗

PubMed1997

The atomic force microscope as a new microdissecting tool for the generation of genetic probes.

The atomic force microscope (AFM) can be used to visualize and to manipulate biological material with relative case and high resolution. This study was carried out to investigate whether probe sets, specific for subregions of the human genome and useful for the painting of chromosome bands, can be established by PCR amplification of AFM-dissected chromosome regions. Compared to standard microdissection techniques, the AFM can be used with much higher precision for the dissection of the region of interest and subsequent nanoextraction of DNA material. After scanning the area of interest in noncontact mode AFM, chromosome bands were cut by the AFM tip at high force. The genetic material of a single cut attached itself to the tip and was extracted and amplified using degenerate oligonucleotide-primed-PCR. Subsequent to hapten labeling, fluorescence in situ hybridization was performed and chromosome band-specific probes were visualized by standard fluorescence microscopy.

Chromosomes, Human, Pair 2↗

PubMed1997

Cut out or poke in--the key to the world of single genes: laser micromanipulation as a valuable tool on the look-out for the origin of disease.

The optical micromanipulation systems UV(ultraviolet)-Laser Microbeam and Optical Tweezers Trap, already proven to be powerful tools for 'non-contact' micro-manipulation of gametes, cells and organelles, have now made their way into the nanocosmos of genes and molecules. Force measurements of DNA transcription have been performed and selective DNA molecule micromanipulation gives insight into single molecule behaviour. Retrievement of selected single cells without contamination is an import prerequisite for further processing with modern methods of molecular biology. Laser micro-dissection allows to precisely eliminate any unwanted material or to isolate pieces of chromosomes or single cells of interest with high accuracy and efficiency. This enables the cell or chromosome specific molecular analysis of genes and genetic defects underlying disease, such as cancer or infection. This review article gives an overview of current topics of laser microbeam application in biological or medical research and advanced molecular diagnosis.

Animals↗

PubMed1996

Chiral symmetry breaking during the self-assembly of monolayers from achiral purine molecules.

Scanning tunneling microscopy was used to investigate the structure of the two-dimensional adsorbate formed by molecular self-assembly of the purine base, adenine, on the surfaces of the naturally occurring mineral molybdenite and the synthetic crystal highly oriented pyrolytic graphite. Although formed from adenine, which is achiral, the observed adsorbate surface structures were enantiomorphic on molybdenite. This phenomenon suggests a mechanism for the introduction of a localized chiral symmetry break by the spontaneous crystallization of these prebiotically available molecules on inorganic surfaces and may have some role in the origin of biomolecular optical asymmetry. The possibility that purine-pyrimidine arrays assembled on naturally occurring mineral surfaces might act as possible templates for biomolecular assembly is discussed.

Adenine↗

PubMed1993

Analysis of banded human chromosomes and in situ hybridization patterns by scanning force microscopy.

Scanning force microscopy was used to analyze banded human chromosomes and in situ hybridization patterns of biotinylated DNA probes. In standard human GTG-banded metaphase chromosome preparations (where GTG is G-banding with trypsin-Giemsa), chromosomal morphology and banding patterns were well preserved during the scanning procedure. The smallest identifiable features were in the range of about 100 nm and are similar to the typical structures seen by electron microscopy. In addition, in situ hybridization of human DNA probes of known chromosomal localization was used to map specific hybridization signals. Imaging of the precipitated crystals at the hybridization site clearly demonstrates the superior resolution of scanning force microscopy compared to conventional microscopy.

Chromosome Banding↗

PubMed1992

Domain walls on graphite mimic DNA.

We show that domain walls on graphite are very likely to mimic features of extended macromolecules like DNA strands, when imaged with an STM. We explain with a simple model how different translational periods along a grain boundary originate from different relative orientations of the graphite lattice at the domain wall. We show how simple geometrical analysis of the images can be used to distinguish true macromolecular features from artifacts.

Artifacts↗

PubMed1992

Scanning force microscopy studies of the S-layers from Bacillus coagulans E38-66, Bacillus sphaericus CCM2177 and of an antibody binding process.

In many prokaryotic cells (eubacteria and archaebacteria) the outermost cell envelope component is composed of a regularly structured protein surface layer (S-layer). The two-dimensional S-layer from Bacillus coagulans E38-66 and Bacillus sphaericus CCM2177 has been investigated by SFM at molecular resolution under physiological conditions (i.e., in buffer solution). We find the E38-66 S-layer lattice to be oblique with lattice parameters of a = 9-10 nm, b = 7-8 nm and gamma = 80 degrees -90 degrees (E38-66). The CCM2177 lattice is square with a = 12-14 nm, in good agreement with TEM data. We have used the unique possibility of the SFM to study the kinematics of biological processes and have performed experiments on the adhesion of polyclonal antibodies to the recrystallized E38-66 protein layer on a time scale of about two to ten seconds per image frame. This represents a first step in directly visualizing molecular recognition reactions.

Antibodies, Bacterial↗

PubMed1991

Two-dimensional ordering of the DNA base guanine observed by scanning tunneling microscopy.

Guanine, one of the four DNA bases, has been observed by tunneling microscopy to form a two-dimensional ordered structure on two crystalline substrates, graphite and MoS2. The two-dimensional lattice formed by guanine is nearly identical on the two surfaces, and heteroepitaxy appears to be the growth mechanism in both cases. Although the resolution of molecular details is superior for the graphite substrate, the simpler results on MoS2 are not only easier to interpret but also facilitate the understanding of the more complex images on graphite. We propose that the interfacial structure is composed of linear chains of hydrogen-bonded molecules aligned into a closely packed two-dimensional array.

DNA↗

PubMed1991

Determination of the physical structure of biological materials at biosensor interfaces by techniques of increasing magnification from microscopic to molecular scale.

Chemical selectivity of biosensors is derived from biological materials interfaced to the surface of transducing devices. Molecular recognition events lead to macroscopic function suitable for analytical measurements. The structure-function relationships of biochemical species at interfaces must be established to characterize and optimize biosensor operation. The techniques of ellipsometry, fluorescence microscopy, electron microscopy, and scanning tunneling microscopy are used to investigate the structure of monolayers and multilayers of proteins and lipids at interfaces that are prepared by Langmuir-Blodgett techniques and by self-assembly from bulk solution. The relative merits and limitations of the measurement techniques in the determination of aspects of interfacial structure are considered.

1,2-Dipalmitoylphosphatidylcholine↗