Synthesis and properties of 5-azido-UDP-glucose. Development of photoaffinity probes for nucleotide diphosphate sugar binding sites

A new active site directed photoaffinity probe, which is a model compound for studying nucleotide diphosphate sugar binding proteins, has been synthesized by coupling 5-azido-UTP and [32P]Glc-1-P using yeast UDP-glucose pyrophosphorylase to produce [beta-32P]5-azidouridine 5'-diphosphoglucose (5N3UDP-Glc). This probe has photochemical properties similar to that of 5-azidoUTP (Evans, R. K., and Haley, B. E. (1987) Biochemistry 26, 269-276). The efficacy of 5N3UDP-Glc as an active site directed probe was demonstrated using yeast UDP-Glc pyrophosphorylase. Saturation effects of photoinsertion were observed with an apparent Kd of 51 microM and the natural substrate, UDP-Glc, prevented photoinsertion of [beta-32P]5N3UDP-Glc with an apparent Kd of 87 microM. Prevention of photoinsertion was also seen with UTP and pyrophosphate with apparent Kd values less than 200 microM. UMP, UDP, ATP, and GTP were much less effective competitors. Selective photoinsertion was observed with several partially purified enzymes including UDP-Glc dehydrogenase, UDP-Gal-4-epimerase, Gal-1-P uridyltransferase, and phosphorylase a. The absence of nonselective photoinsertion into bulk proteins was demonstrated with crude homogenates of rabbit liver as well as with several UDP-Glc binding proteins. Of the six purified enzymes tested, only phosphoglucomutase has been shown to incorporate radiolabel from the photoprobe in the absence of UV irradiation. These results and a discussion of the utility of 5N3UDP-Glc for detecting UDP-Glc binding proteins and isolating active site peptides are presented.     

A quartz crystal biosensor for measurement in liquids.

The detection of anti-human immunodeficiency virus (HIV) antibodies by means of synthetic HIV peptide immobilized on a piezoelectric quartz sensor is demonstrated. The measurement set-up consists of an oscillator circuit, a suitably modified AT-cut thickness-shear-mode quartz crystal with gold electrodes, which is housed in a special reaction vessel, and a computer-controlled frequency counter for the registration of the measured frequency values. The quartz crystal is adapted for a steady operation in liquids at a frequency of 20 MHz. In phosphate-buffered saline solution the oscillator reaches a stability of about 0.5 Hz within a few seconds, of about 2 Hz within 10 min and about 30 Hz within 1 h. The frequency shift due to the adsorption of various proteins to the uncoated sensor surface has been investigated. It can be shown that a stable adsorptive binding of proteins to an oscillating gold surface is feasible and can be used for the immobilization of a receptor layer (e.g. HIV peptide). Specific binding of the anti-HIV monoclonal antibody to the HIV peptide immobilized on the quartz sensor is demonstrated. Control experiments show, however, additional unspecific binding. According to the experiments, the Sauerbrey formula gives a sufficiently accurate value for the decrease of the resonant frequency due to adsorption or binding of macromolecular proteins on the quartz crystal surface.     

Structural Basis of Heterochromatin Formation by Human HP1

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Nerve growth factor receptors are preaggregated and immobile on responsive cells.

It has been hypothesized that signal transduction occurs by ligand-induced receptor clustering and immobilization. For many peptide receptors, cross-linking by anti-receptor antibodies is sufficient for receptor activation. This is not, however, the case for nerve growth factor receptor (NGFR). Using fluorescence microscopy and fluorescence recovery after photobleaching (FRAP), we have analyzed the distribution and diffusibility of NGFR on a series of cell lines. We have found the following: (1) Cells expressing high-affinity responsive NGFR's display clustered NGFR's even in the absence of ligand. In contrast, NGFR's in nonresponsive cell lines are diffusely distributed. (2) Receptors on responsive cell lines are largely nondiffusing while most receptors on nonresponsive cell lines are relatively free to diffuse. (3) NGF does not greatly alter the distribution or diffusion properties of the NGFR on either nonresponsive or responsive cell lines. Thus, NGFR is preclustered and immobile on responsive cells, which suggests that immobilization of NGFR prior to ligand binding is required for signal transduction.     

Structure of internalin,a major invasion protein of Listeria monocytogenes,in complex with its human receptor E-cadherin

Listeria monocytogenes, a food-borne bacterial pathogen, enters mammalian cells by inducing its own phagocytosis. The listerial protein internalin (InlA) mediates bacterial adhesion and invasion of epithelial cells in the human intestine through specific interaction with its host cell receptor E-cadherin. We present the crystal structures of the functional domain of InlA alone and in a complex with the extracellular, N-terminal domain of human E-cadherin (hEC1). The leucine rich repeat (LRR) domain of InlA surrounds and specifically recognizes hEC1. Individual interactions were probed by mutagenesis and analytical ultracentrifugation. These include Pro16 of hEC1, a major determinant for human susceptibility to L. monocytogenes infection that is essential for intermolecular recognition. Our studies reveal the structural basis for host tro-pism of this bacterium and the molecular deception L. monocytogenes employs to exploit the E-cadherin system.     

Feature-based,automated segmentation of cerebral infarct patterns using T2- and diffusion-weighted imaging

Diffusion-weighted imaging enables the diagnosis of cerebral ischemias very early, thus supporting therapies such as thrombolysis. However, morphology and tissue-characterizing parameters (e.g. relaxation times or water diffusion) may vary strongly in ischemic regions, indicating different underlying pathologic processes. As the determination of the parameters by a supervised segmentation is very time consuming, we evaluated whether different infarct patterns may be segmented by an automated, multidimensional feature-based method using a unified segmentation procedure. Ischemias were classified into 5 characteristic patterns. For each class, a 3D histogram based on T(2)- and diffusion-weighted images as well as calculated apparent diffusion coefficients (ADC) was generated from a representative data set. Healthy and pathologic tissue classes were segmented in the histogram as separate, local density maxima with freely shaped borders. Segmentation control parameters were optimized in a 3-step procedure. The method was evaluated using synthetic images as well as results of a supervised segmentation. For the analysis of cerebral ischemias, the optimal control parameter set led to sensitivities and specificities between 1.0 and 0.9.     

Development of the sperm head in the caddis fly Potamophylax rotundipennis (Insecta,Trichoptera)

The restructuring of the sperm head has been examined in a caddis fly, Potamophylax rotundipennis (Limnephilidae), using light and electron microscopy. The roughly spherical nuclei of young spermatids are transformed into needle-shaped elements in advanced spermatids. During this process, the nuclei transiently become sickle-shaped. Prominent structural changes occur within the nucleus during spermiogenesis. The chromatin of spherical and slightly elongated nuclei has an amorphous appearance, then coarse granules become apparent, chromatin threads are visible in fully elongated nuclei and finally lamellar elements appear. During the changes in chromatin texture, a dense layer, the chromatin rim, develops transiently. This feature of the chromatin surface is interpreted as the structural expression of exchanges between nucleus and cytoplasm. A microtubular manchette is formed at the cytoplasmic face of the nuclear envelope. Whereas the manchette covers the full perimeter of the nucleus in early stages of elongation, gaps in the palisade of microtubules appear before the nuclear diameter decreases and needle-shaped nuclei develop. It is possible that the intermittent deployment of manchette microtubules is involved in reducing the nuclear diameter towards the end of nuclear elongation. The delayed detachment of the chromatin from the posterior pole of the nucleus, observed at the onset of nuclear clongation, points to local modifications of the nuclear envelope responsible for the connection of the centriole adjunct and the flagellum with the posterior pole of the nucleus.     

Red blood cells experience electrostatic repulsion but make molecular adhesions with glass.

We have studied the detachment of unfixed red cells from glass coverslips under unit gravity and by centrifugation in buffered isotonic solutions over a range of ionic strengths. Cell-glass contact areas and separation distances were measured by quantitative interference reflection microscopy. Detachment under unit gravity is highly dependent on ionic strength: dilution increases electrostatic repulsion and greatly reduces the proportion of adherent cells. However, even at 1.5 mM some cells stick. Over the range 3-110 mM such adherent cells are progressively removed by increasing centrifugal forces, but in a manner virtually independent of ionic strength. This fact, together with the irreversibility of pre-adherent cells as ionic strength is progressively reduced, as well as the resistance of cells to lateral shearing forces, provide evidence sufficient to reject the notion of secondary minimum adhesion for unfixed cells at any ionic strength down to 1.5 mM. We conclude that all unfixed cells that stick at ionic strengths from 157 to 1.5 mM make molecular contacts with glass. Comparison with long range force calculations suggests that to penetrate the electrostatic repulsion barrier the contact regions are unlikely to have average surface properties. A new method that compares frequency distributions of contact areas with responses to detachment forces shows that detachment forces are not linearly related to contact areas. This lack of relationship is less clearly evident for rigid glutaraldehyde-fixed cells and may therefore depend on the degree of cellular deformability.