Direct detection of N-H[...]O=C hydrogen bonds in biomolecules by NMR spectroscopy

A nuclear magnetic resonance (NMR) experiment is described for the direct detection of N-H[...]O=C hydrogen bonds (H-bonds) in 15N and 13C isotope-labeled biomolecules. This quantitative 'long-range' HNCO-COSY (correlation spectroscopy) experiment detects and quantifies electron-mediated scalar couplings across the H-bond (H-bond scalar couplings), which connect the magnetically active (15)N and (13)C nuclei on both sides of the H-bond. Detectable H-bonds comprise the canonical backbone H-bonds in proteins as well as other H-bonds in proteins and nucleic acids with N-H donors and O=C (carbonylic or carboxylic) acceptors. Unlike other NMR observables, which provide only indirect evidence of the presence of H-bonds, the H-bond scalar couplings identify all partners of the H-bond, the donor, the donor proton and the acceptor, in a single experiment. The size of the scalar couplings can be related to H-bond geometries. The time required to detect the N-H[...]O=C H-bonds in small proteins (< or = approximately 10 kDa) is typically on the order of 1 d at millimolar concentrations, whereas H-bond detection for larger proteins (< or = approximately 30 kDa) may be possible within several days depending on concentration, isotope composition, magnetic field strength and molecular weight. The proteins ubiquitin (8.6 kDa), dimeric RANTES (2 x 8.5 kDa) and MAP30 (30 kDa) are used as examples to illustrate this procedure.     

Renal hypoplasia, polydactyly, cardiopathy: a new syndrome?

The occurrence of a polymalformation pattern associated with a polydactyly indicates a mendelian inheritance. We report a case with renal hypoplasia, polydactyly, congenital heart defects. A large literature review makes the differential diagnosis and brings this case nearer to an anterior observation of the literature. We discuss an eventual new syndrome with autosomal recessive inheritance.     

Cross-Talk-Free Multi-Color STORM Imaging Using a Single Fluorophore

Multi-color stochastic optical reconstruction microscopy (STORM) is routinely performed; however, the various approaches for achieving multiple colors have important caveats. Color cross-talk, limited availability of spectrally distinct fluorophores with optimal brightness and duty cycle, incompatibility of imaging buffers for different fluorophores, and chromatic aberrations impact the spatial resolution and ultimately the number of colors that can be achieved. We overcome these complexities and develop a simple approach for multi-color STORM imaging using a single fluorophore and sequential labelling. In addition, we present a simple and versatile method to locate the same region of interest on different days and even on different microscopes. In combination, these approaches enable cross-talk-free multi-color imaging of sub-cellular structures.     

High-resolution 3D HNCOCA experiment applied to a 28 kDa paramagnetic protein

Summary A new triple-resonance 3D HNCOCA pulse scheme is presented, designed to identify the backbone nuclei (H^N, N, CO, C) of doubly labelled proteins. The two carbon frequencies are labelled along the same indirect dimension and the corresponding dwell times can be independently scaled in order to account for the relaxation properties and chemical shift ranges of the CO and C. If one takes advantage of the symmetry properties of the spectra in the course of the peak picking, this 3D scheme has the same sensitivity as the 4D experiment, but with an improved resolution. The sequence is illustrated on a 0.5 mM sample of Rhodobacter capsulatus cytochrome c a homodimeric paramagnetic protein of 2×14 kDa. A resonance assignment strategy, based on a low-concentration ¹³C/¹⁵N-labelled sample and a more concentrated ¹⁵N-labelled sample, is proposed for proteins where the expression system shows a limited efficiency.     

In vivo micronucleus test using mouse hepatocytes

The bone-marrow micronucleus (BMM) test is highly specific for clastogenic effects but its sensitivity is determined to a great extent by the substances tested, particularly by their metabolism. Some compounds, such as unstable mutagens or those which generate short-lived metabolites, are not detected in this test because the metabolites produced in the liver do not reach the bone marrow. In an attempt to provide qualitative and quantitative assessments of chromosomal mutations produced in vivo by genotoxic agents not detected in the mouse BMM test, a mouse-liver micronucleus test, adapted from Tates model, was developed. The animals were treated twice, with an interval of 24 h between treatments, and then subjected to partial hepatectomy (PH) 24 h after the second treatment in order to induce mitotic stimulation. The incidence of micronucleated hepatocytes was determined 96 h after PH. The test was evaluated with 5 procarcinogens, each with a complex metabolic pattern: dimethylnitrosamine (DMN), diethylnitrosamine (DEN), 1,1-dimethylhydrazine (1,1-DMH), 4-aminophenol (4-APOL), 4-aminobiphenyl (4-ABPYL) and one direct unstable mutagen, beta-propiolactone (BPL). All these compounds are negative in the mouse BMM test but caused a major increase in the incidence of micronuclei in mouse hepatocytes. This test is simple and can be readily compared with the BMM test. Furthermore, it offers a better assessment of the impact of a compound at the chromosomal level in a metabolically competent cell and can therefore be used for the evaluation of the genotoxic activity of compounds with complex metabolic pathways.     

Connective matrix organization in human pulmonary fibrosis. Collagen polymorphism analysis in fibrotic deposits by immunohistological methods

In the interstitium of the alveolar septa in the peripheral parts of the lung, four molecular types of collagen (I, III, IV and V) each with different morphological appearances, can be identified. The structural integrity of collagens accounts for the physiological efficiency of the lung. Fibrous thickening of alveolar septa is an invariable result of various diseases affecting the interstitium of the lung. The light and electron microscopic findings, and the immunological typing of collagens in six cases of fibrotic alveolar disease, are described. In the alveolar septa, two different compartments (the alveolo-capillary junction and the supportive axis) were affected by fibrosis: the alveolo-capillary junction was widened by the addition of interstitial collagens to basement membranes. In the axis, the increase of interstitial (types I and III) collagen gave rise to different patterns of connective matrix organization, graded as Loose or Dense depending on quantitative alterations of the type I/III ratio. The mode of organization of the fibrotic lung connective matrix, which depends on the quality of deposits in the matrix, may be correlated with the evolution of interstitial pulmonary fibrosis, in terms of its stability, remodelling ability and reversibility.     

Gap junctions in cultured astrocytes: single-channel currents and characterization of channel-forming protein.

Currents from gap junction channels were recorded from pairs of astrocytes in primary culture using the double whole-cell recording technique. In weakly coupled pairs, single-channel events could be resolved without pharmacological uncoupling treatment. Under these conditions, unitary conductance was 56 +/- 7 pS, and except for multiples of this value, no other level of conductance was observed consistently. To characterize the type of junctional protein constituting astrocyte gap junction channels, immunological and biochemical experiments were carried out on the same material. Specific cDNA probes for three connexins identified in mammals (Cx26, Cx32, and Cx43) showed that only Cx43 mRNA was expressed in cultured astrocytes. The presence of Cx43 protein in cultured astrocytes was demonstrated by immunoblotting, immunofluorescence, and immunogold labeling using anti-peptide antibodies specific to Cx43. These results strongly suggest that gap junctions in astrocytes have a 50-60 pS unitary conductance associated with channels composed of Cx43 protein.