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Biomedical subjects

Gerald Kada

Publications and source records attributed to Gerald Kada.

7 recordsLinked to original sources
PubMed2006

Age determination of blood spots in forensic medicine by force spectroscopy.

We present a new tool for the estimation of the age of bloodstains, which could probably be used during forensic casework. For this, we used atomic force microscopy (AFM) for high-resolution imaging of erythrocytes in a blood sample and the detection of elasticity changes on a nanometer scale. For the analytic procedure we applied a fresh blood spot on a glass slide and started the AFM detection after drying of the blood drop. In a first step, an overview image was generated showing the presence of several red blood cells, which could easily be detected due to their typical "doughnut-like" appearance. The consecutively morphological investigations in a timeframe of 4 weeks could not show any alterations. Secondly, AFM was used to test the elasticity by recording force-distance curves. The measurements were performed immediately after drying, 1.5 h, 30 h and 31 days. The conditions were kept constant at room temperature (20 degrees C) and a humidity of 30%. The obtained elasticity parameters were plotted against a timeline and repeated several times. The elasticity pattern showed a decrease over time, which are most probably influenced by the alteration of the blood spot during the drying and coagulation process. The preliminary data demonstrates the capacity of this method to use it for development of calibration curves, which can be used for estimation of bloodstain ages during forensic investigations.

Blood Coagulation↗
PubMed2006

A combined optical and atomic force microscope for live cell investigations.

We present an easy-to-use combination of an atomic force microscope (AFM) and an epi-fluorescence microscope, which allows live cell imaging under physiological conditions. High-resolution AFM images were acquired while simultaneously monitoring either the fluorescence image of labeled membrane components, or a high-contrast optical image (DIC, differential interference contrast). By applying two complementary techniques at the same time, additional information and correlations between structure and function of living organisms were obtained. The synergy effects between fluorescence imaging and AFM were further demonstrated by probing fluorescence-labeled receptor clusters in the cell membrane via force spectroscopy using antibody-functionalized tips. The binding probability on receptor-containing areas identified with fluorescence microscopy ("receptor-positive sites") was significantly higher than that on sites lacking receptors.

Animals↗
PubMed2005

Single molecule studies of antibody-antigen interaction strength versus intra-molecular antigen stability.

We investigated molecular recognition of antibodies to membrane-antigens and extraction of the antigens out of membranes at the single molecule level. Using dynamic force microscopy imaging and enzyme immunoassay, binding of anti-sendai antibodies to sendai-epitopes genetically fused into bacteriorhodopsin molecules from purple membranes were detected under physiological conditions. The antibody/antigen interaction strength of 70-170 pN at loading rates of 2-50 nN/second yielded a barrier width of x = 0.12 nm and a kinetic off-rate (corresponding to the barrier height) of k(off) = 6s(-1), respectively. Bacteriorhodopsin unfolding revealed a characteristic intra-molecular force pattern, in which wild-type and sendai-bacteriorhodopsin molecules were clearly distinguishable in their length distributions, originating from the additional 13 amino acid residues epitope in sendai purple membranes. The inter-molecular antibody/antigen unbinding force was significantly lower than the force required to mechanically extract the binding epitope-containing helix pair out of the membrane and unfold it (126 pN compared to 204 pN at the same loading rate), meeting the expectation that inter-molecular unbinding forces are weaker than intra-molecular unfolding forces responsible for stabilizing native conformations of proteins.

Antibodies↗
PubMed2002

Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4'-dithiodipyridine.

Since its introduction in 1959, Ellman's reagent (5,5'-dithio-bis(2-nitrobenzoic acid)) has been the favorite reagent for spectrophotometric measurement of protein sulfhydryls. Meanwhile however, evidence has accumulated that many protein sulfhydryls give an incomplete reaction with Ellman's reagent, even during prolonged assay times. In the present study, the kinetic problem was solved by including cystamine as a "mediator" between the protein sulfhydryl and Ellman's reagent, as previously applied in an enzymatic thiol assay [9]. As an alternative, 4,4'-dithiodipyridine (DTDP) was used in place of Ellman's reagent. Due to its small size, amphiphilic nature, and lack of charge, DTDP quickly reacts with poorly accessible protein sulfhydryls, without any catalysis by cystamine. The DTDP method and the Ellman/cystamine method were both optimized for maximal sensitivity, minimal sample consumption (detection limit 0.2 nmol mL(-1), determination limit 0.6 nmol mL(-1)), and minimal assay time (5 min). In validation experiments, both methods gave identical results and the measured sulfhydryls/protein matched the expected values. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00216-002-1347-2.

Animals↗
PubMedDate not supplied

Dynamic force microscopy imaging of native membranes.

We employed magnetic ACmode atomic force microscopy (MACmode AFM) as a novel dynamic force microscopy method to image surfaces of biological membranes in their native environments. The lateral resolution achieved under optimized imaging conditions was in the nanometer range, even when the sample was only weakly attached to the support. Purple membranes (PM) from Halobacterium salinarum were used as a test standard for topographical imaging. The hexagonal arrangement of the bacteriorhodopsin trimers on the cytoplasmic side of PM was resolved with 1.5nm lateral accuracy, a resolution similar to images obtained in contact and tapping-mode AFM. Human rhinovirus 2 (HRV2) particles were attached to mica surfaces via nonspecific interactions. The capsid structure and 2nm sized protein loops of HRV2 were routinely obtained without any displacement of the virus. Globular and filamentous structures on living and fixed endothelial cells were observed with a resolution of 5-20nm. These examples show that MACmode AFM is a favorable method in studying the topography of soft and weakly attached biological samples with high resolution under physiological conditions.

Capsid↗