Cholesterol modification of hedgehog is required for trafficking and movement,revealing an asymmetric cellular response to hedgehog

Hedgehog family members are secreted proteins involved in numerous patterning mechanisms. Different posttranslational modifications have been shown to modulate Hedgehog biological activity. We investigated the role of these modifications in regulating subcellular localization of Hedgehog in the Drosophila embryonic epithelium. We demonstrate that cholesterol modification of Hedgehog is responsible for its assembly in large punctate structures and apical sorting through the activity of the sterol-sensing domain-containing Dispatched protein. We further show that movement of these specialized structures through the cellular field is contingent upon the activity of proteoglycans synthesized by the heparan sulfate polymerase Tout-Velu. Finally, we show that the Hedgehog large punctate structures are necessary only for a subset of Hedgehog target genes across the parasegmental boundary, suggesting that presentation of Hedgehog from different membrane compartments is responsible for Hedgehog functional diversity in epithelial cells.     

Oxalic acid: a microbial metabolite of interest for the pulping industry

Oxalate is a common metabolite produced by almost all plant-pathogenic fungi. The degradation of cell wall from poplar chips and poplar sawdust by oxalate is reviewed here. Oxalate treatments decrease slightly the amount of sugars constituting hemicelluloses, but only in fibres and not in sawdust or wood chips. The examination of the cell wall ultrastructure of wood chips by transmission electron microscopy (TEM) after polysaccharide staining showed a characteristic fading of the staining of the S1/S2 and S2/S3 transition areas, supporting the idea that reactivity and organization of polysaccharides had changed after the oxalate treatments. Finally, all these changes enhanced the ability of the wood chips to be defibrated by a thermomechanical (TMP) process, as well as the further refining of the pulps. Looking at the fiber surface, it became apparent that fracture areas during the TMP pulping had moved toward the S2 layer, explaining why defibrating and refining occurred more easily, with less energy input in the process.     

Mouse myodulin, a new potential angiogenic factor, functionally expressed in yeast

Myodulin is a new integral membrane protein down-regulated in skeletal muscle atrophy. A first characterization suggested that myodulin could be a skeletal muscle angiogenic factor operating through direct cell-to-cell interactions. Here, we show that mouse myodulin can be expressed at the plasma membrane of Saccharomyces cerevisiae and purified. Co-culture experiments of myoblasts and cardiac vascular endothelial cells reveal that myodulin, either presented in yeast membranes or in liposomes after purification, increases the invasive potential of endothelial cells with a similar efficiency as when over-expressed in skeletal muscle cells. Functional essays using myodulin expressed in yeast bring new information about the myodulin functional mechanism, suggesting that one or several muscle cell components could be necessary for myodulin to increase the invasive potential of endothelial cells. The yield of purified myodulin should allow structure-function relationships studies for a better understanding of myodulin functional mechanisms.     

Correlations between mean echogenicity and material properties of normal and diseased equine superficial digital flexor tendons: an in vitro segmental approach

The objective of this study was to test the hypothesis that tendon echogenicity is associated with the material properties of the corresponding tendon site, especially in case of lesions, due to local changes in tendon matrix composition. Four normal and nine spontaneously injured equine superficial digital flexor tendons (SDFT) were isolated then ultrasonographically examined under tension, in a special device placed in a water bath. Ultrasonographic transversal images (7.5MHz linear transducer) of five segments along each tendon were digitized, and analyzed in order to measure the mean cross-sectional area (MCSA) and mean echogenicity (ME) of each segment. The tendons were then tested in traction until rupture in a testing machine. For each segment, stress and strain were determined throughout the test, and the elastic modulus (EM) was evaluated. The tendon lesions were also documented by histology. No correlation was found between ME and the material properties of normal tendon segments. At the rupture sites of the nine diseased tendons, ME was positively correlated with maximal stress and EM, whereas no correlation was demonstrated with maximal strain. Besides, a positive correlation was demonstrated between ME and both MCSA and EM, when the three metacarpal segments of the diseased tendons were considered. Although ME gives only rough information about tendon matrix structure, it does show, under these in vitro conditions, significant correlations with material properties of pathological tendon segments, which may improve the functional significance and therefore the prognostic value of the ultrasonographic examination of tendon lesions.     

The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs

Stable cell lines that individually express the eight known human prostanoid receptors (EP(1), EP(2), EP(3), EP(4), DP, FP, IP and TP) have been established using human embryonic kidney (HEK) 293(EBNA) cells. These recombinant cell lines have been employed in radioligand binding assays to determine the equilibrium inhibitor constants of known prostanoid receptor ligands at these eight receptors. This has allowed, for the first time, an assessment of the affinity and selectivity of several novel compounds at the individual human prostanoid receptors. This information should facilitate interpretation of pharmacological studies that employ these ligands as tools to study human tissues and cell lines and should, therefore, result in a greater understanding of prostanoid receptor biology.     

<Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis> induces aponecrosis in human aortic smooth muscle cells

Background  

The intracellular bacterium Chlamydia pneumoniae is suspected to play a role in formation and progression of atherosclerosis. Many studies investigated cell death initiation versus inhibition by Chlamydia pneumoniae in established cell lines but nothing is known in primary human aortic smooth muscle cells, a cell type among others known to be involved in the formation of the atherosclerotic plaque. Type of cell death was analyzed by various methods in primary aortic smooth muscle cells after infection with Chlamydia pneumoniae to investigate a possible pathogenic link in atherosclerosis.     

Physical properties of compounds promoting oral delivery of macromolecular drugs

The spectroscopic and solution properties of a series of amidated acids (delivery agents), which promote the gastrointestinal absorption of USP heparin and other drugs that show poor oral bioavailability, are investigated using Raman and NMR spectroscopy. The results show evidence for self-association at low concentrations of delivery agents that increases as the concentration of the delivery agent is increased. The self-associate is characterized by ring-ring stacking interactions, and the best geometrical arrangement for the stacking is the parallel-shifted arrangement of the rings. In addition, the amide group participates in the formation of intermolecular hydrogen bonds in the self-associate. Unlike the rigid ring, the tails of these delivery agents remain relatively flexible in the self-associate. It is suggested that the limited solubility of the delivery agents at physiological pH arises from a percentage of protonated carboxyls. Their presence promotes the formation of intermolecular hydrophobic and ring stacking interactions, which are otherwise weakened by an ionized carboxyl group.     

Use of an α-d-glucosidase for the specific cytochemical identification of lateral α-d-xylosyl end groups in plant xyloglucans

Summary α-Linked d-xylosyl side chains represent the typical feature common to all xyloglucans not shared by other cell wall polysaccharides. Since no easily available α-d-xyloxidase is known, advantage was taken of the conformational and configurational homologies between α-d-xylopyranose and α-d-glucopyranose to make an α-d-glucosidase-gold complex which was able to recognize α-d-xylosyl terminal residues of xyloglucans. This marker was used together with α-l-fucosidase gold complex for the double labeling on two different structural features of the same macromolecule in plant primary cell wall.     

Study of lignification by noninvasive techniques in growing maize internodes. An investigation by Fourier transform infrared cross-polarization-magic angle spinning 13C-nuclear magnetic resonance spectroscopy and immunocytochemical transmission electron microscopy.

Noninvasive techniques were used for the study in situ of lignification in the maturing cell walls of the maize (Zea mays L.) stem. Within the longitudinal axis of a developing internode all of the stages of lignification can be found. The synthesis of the three types of lignins, p-hydroxyphenylpropane (H), guaiacyl (G), and syringyl (S), was investigated in situ by cross-polarization-magic angle spinning 13C-solid-state nuclear magnetic resonance, Fourier transform infrared spectroscopy, and immunocytochemical electron microscopy. The first lignin appearing in the parenchyma is of the G-type preceeding the incorporation of S nuclei in the later stages. However, in vascular bundles, typical absorption bands of S nuclei are visible in the Fourier transform infrared spectra at the earliest stage of lignification. Immunocytochemical determination of the three types of lignin in transmission electron microscopy was possible thanks to the use of antisera prepared against synthetic H, G, and the mixed GS dehydrogenative polymers (K. Ruel, O. Faix, J.P. Joseleau [1994] J Trace Microprobe Tech 12: 247-265). The specificity of the immunological probes demonstrated that there are differences in the relative temporal synthesis of the H, G, and GS lignins in the different tissues undergoing lignification. Considering the intermonomeric linkages predominating in the antigens used for the preparation of the immunological probes, the relative intensities of the labeling obtained provided, for the first time to our knowledge, information about the macromolecular nature of lignins (condensed versus noncondensed) in relation to their ultrastructural localization and development stage.     

Binding mode and affinity studies of DNA-binding agents using topoisomerase I DNA unwinding assay

A topoisomerase I DNA unwinding assay has been used to determine the relative DNA-binding affinities of a model pair of homologous naphthalene diimides. Binding affinity data were corroborated using calorimetric (ITC) and spectrophotometric (titration and T(m)) studies, with substituent size playing a significant role in binding. The assay was also used to investigate the mode of binding adopted by several known DNA-binding agents, including SYBR Green and PicoGreen. Some of the compounds exhibited unexpected binding modes.