Ligand-receptor interactions and membrane structure investigated by AFM and time-resolved fluorescence microscopy

The atomic force microscope (AFM) and the associated dynamic force spectroscopy technique have been exploited to quantitatively assess the interaction between proteins and their binding to specific ligands and membrane surfaces. In particular, we have studied the specific interaction between lung surfactant protein D and various carbohydrates. In addition, we have used scanning AFM and time-resolved fluorescence microscopy to image the lateral structure of different lipid bilayers and their morphological changes as a function of time. The various systems studied illustrate the potential of modern AFM techniques for application to biomedical research, specifically within immunology and liposome-based drug delivery.     

Amyloid adhesins are abundant in natural biofilms

Surface-associated amyloid fibrils have been described by bacteria in the family Enterbacteriaceae, but it is unknown to what extent amyloid adhesins are present in natural biofilms. In this study, amyloid adhesins were specifically stained with Thioflavin T and two conformationally specific antibodies targeting amyloid fibrils. These three independent detection methods were each combined with fluorescence in situ hybridization using fluorescently labelled oligonucleotide probes in order to link phenotype with identity. Escherichia coli mutants with and without amyloid adhesins (curli) served as controls. In biofilms from four different natural habitats, bacteria producing extracellular amyloid adhesins were identified within several phyla: Proteobacteria (Alpha-, Beta-, Gamma- and Deltaproteobacteria), Bacteriodetes, Chloroflexi and Actinobacteria, and most likely also in other phyla. Quantification of the microorganisms producing amyloid adhesins showed that they constituted at least 5-40% of all prokaryotes present in the biofilms, depending on the habitat. Particularly in drinking water biofilms, a high number of amyloid-positive bacteria were identified. Production of amyloids was confirmed by environmental isolates belonging to the Gammaproteobacteria, Bacteriodetes, Firmicutes and Actinobacteria. The new approach is a very useful tool for further culture-independent studies in mixed microbial communities, where the abundance and diversity of bacteria expressing amyloid adhesins seems much greater than hitherto anticipated.     

Dynamic strength of the interaction between lung surfactant protein D (SP-D) and saccharide ligands

In order to investigate the dynamic strength of the interaction between lung surfactant protein D (SP-D) and different sugars, maltose, mannose, glucose, and galactose, we have used an atomic force microscope to monitor the interaction on a single molecule scale. The experiment is performed by measuring the rupture force when the SP-D-sugar bond is subjected to a continuously increasing force. Under these dynamic conditions, SP-D binds strongest to d-mannose and weakest to maltose and d-galactose. These results differ from equilibrium measurements wherein SP-D exhibits preference for maltose. On the basis of this finding, we propose that the binding of the disaccharide maltose to SP-D, which is energetically stronger than the binding of any of the monosacchrides, alters the structure of the binding site in a way that lowers the dynamic strength of the bond. We conclude that determining the strength of a protein-ligand bond under dynamic stress using an atomic force microscope is possibly more relevant for mimicking the actual nonequilibrium physiological situation in the lungs.     

Evaluating the risk of releasing genetically engineered organisms

This article considers the question of a priori assessment of the safety of releasing recombinant DNA engineered organisms. Now and for the foreseeable future, decisions to release such an organism must be based on the results of limited, case-by-case risk assessment studies. The criteria calling for the termination of release programs must be agreed upon in advance of these studies. There is no justification for excluding classes of release organisms from risk assessment. Theory is useful in suggesting a hierarchy of risks, raising the questions that have to be addressed in case-by-case risk assessment and providing protocols for the standardization and execution of these studies. We do not believe that theory can be used to argue categorically for or against the safety of specific releases of recombinant DNA engineered organisms.     

FYVE finger proteins as effectors of phosphatidylinositol 3-phosphate.

Phosphatidylinositol 3-phosphate (PtdIns(3)P), generated via the phosphorylation of phosphatidylinositol by phosphatidylinositol 3-kinase (PI 3-kinase), plays an essential role in intracellular membrane traffic. The underlying mechanism is still not understood in detail, but the recent identification of the FYVE finger as a protein domain that binds specifically to PtdIns(3)P provides a number of potential effectors for PtdIns(3)P. The FYVE finger (named after the first letter of the four proteins containing it; Fab1p, YOTB, Vac1p and EEA1) is a double-zinc binding domain that is conserved in more than 30 proteins from yeast to mammals. It is found in several proteins involved in intracellular traffic, and FYVE finger mutations that affect zinc binding are associated with the loss of function of several of these proteins. The interaction of FYVE fingers with PtdIns(3)P may serve three alternative functions: First, to recruit cytosolic FYVE finger proteins to PtdIns(3)P-containing membranes (in concert with accessory molecules); second, to enrich for membrane bound FYVE finger proteins into PtdIns(3)P containing microdomains within the membrane; and third, to modulate the activity of membrane bound FYVE finger proteins.     

Regional blood flow during exercise in humans measured by near-infrared spectroscopy and indocyanine green.

Using near-infrared spectroscopy (NIRS) and the tracer indocyanine green (ICG), we quantified blood flow in calf muscle and around the Achilles tendon during plantar flexion (1-9 W). For comparison, blood flow in calf muscle was determined by dye dilution in combination with magnetic resonance imaging measures of muscle volume, and, for the peritendon region, blood flow was measured by (133)Xe washout. From rest to a peak load of 9 W, NIRS-ICG blood flow in calf muscle increased from 2.4+/-0.2 to 74+/-5 ml x 100 ml tissue(-1) x min(-1), similar to that measured by reverse dye (77+/-6 ml x 100 ml tissue(-1) x min(-1)). Achilles peritendon blood flow measured by NIRS-ICG rose with exercise from 2.2+/-0.5 to 15.1+/-0.2 ml x 100 ml(-1) x min(-1), which was similar to that determined by (133)Xe washout (2.0+/-0.6 to 14.6+/-0.3 ml x 100 ml tissue(-1) x min(-1)). This is the first study using NIRS and ICG to quantify regional tissue blood flow during exercise in humans. Due to its high spatial and temporal resolution, the technique may be useful for determining regional blood flow distribution and regulation during exercise in humans.