Ultrasonographic contrast-agent imaging of sub-millimeter vessel structures with spatial compounding: in vitro analyses.

In clinical diagnostics, ultrasonographic contrast-agent imaging gives access to medical parameters such as perfusion and vascularization. In addition to the artifacts that are typical for ultrasonic imaging, e.g., speckle noise and depth-dependent sensitivity and resolution, contrast-agent imaging shows more pronounced depth dependence and may suffer from shadowing artifacts that arise from high attenuation of the ultrasound waves by the contrast agent at high concentrations. By imaging an object from different viewing angles in one 2D image plane and summing the images obtained (spatial compounding), image quality can be increased and artifacts can be suppressed. In the present study, we combined both techniques to overcome the limitations of contrast-agent imaging. We used a commercially available ultrasound scanner and a custom-made high-precision mechanical system to rotate the ultrasound transducer fully around the object under investigation. Using this set-up, ultrasound data were acquired in reflection mode to generate a 360 degrees compound scan of a flow-mimicking phantom supplied with contrast agent.     

Identification of an additional protein involved in mannan biosynthesis

Galactomannans comprise a β‐1,4‐mannan backbone substituted with α‐1,6‐galactosyl residues. Genes encoding the enzymes that are primarily responsible for backbone synthesis and side‐chain addition of galactomannans were previously identified and characterized. To identify additional genes involved in galactomannan biosynthesis, we previously performed deep EST profiling of fenugreek (Trigonella foenum‐graecum L.) seed endosperm, which accumulates large quantities of galactomannans as a reserve carbohydrate during seed development. One of the candidate genes encodes a protein that is likely to be a glycosyltransferase. Because this protein is involved in mannan biosynthesis, we named it ‘mannan synthesis‐related’ (MSR). Here, we report the characterization of a fenugreek MSR gene (TfMSR) and its two Arabidopsis homologs, AtMSR1 and AtMSR2. TfMSR was highly and specifically expressed in the endosperm. TfMSR, AtMSR1 and AtMSR2 proteins were all determined to be localized to the Golgi by fluorescence confocal microscopy. The level of mannosyl residues in stem glucomannans decreased by approximately 40% for Arabidopsis msr1 single T‐DNA insertion mutants and by more than 50% for msr1 msr2 double mutants, but remained unchanged for msr2 single mutants. In addition, in vitro mannan synthase activity from the stems of msr1 single and msr1 msr2 double mutants also decreased. Expression of AtMSR1 or AtMSR2 in the msr1 msr2 double mutant completely or partially restored mannosyl levels. From these results, we conclude that the MSR protein is important for mannan biosynthesis, and offer some ideas about its role.     

Reuse potential of agricultural wastes in semi-arid regions: Egypt as a case study

Agricultural wastes represent an important source of bio-energy and valuable products. In Egypt, 18% of the agricultural wastes is used directly as fertiliser. Another 30% is used as animal food. The remainder is burnt directly on the fields or is used for heating in the small villages, using low efficiency burners. These wastes can be used more efficiently as a source of energy and as organic fertiliser. The anaerobic bioconversion of these materials will result in a net energy production. The utilisation of agricultural wastes for the production of energy and compost, combined with using solar energy will save fossil fuel, improve health conditions and the general life quality in the villages. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chloroplast protein import : quantitative analysis of precursor binding

The first step of chloroplast protein import is binding of a precursor protein to the surface of the organelle. Precursor binding for the small subunit of ribulose-1,5-bisphosphate carboxylase to isolated pea chloroplasts was investigated using a receptor-ligand binding assay. Translocation of precursors was blocked by conducting the binding assays at 0°C. Binding of precursor was judged to be receptor mediated by the following criteria: (a) precursor binding was saturable at between 1500 and 3500 molecules per chloroplast; (b) binding is a high affinity interaction with a dissociation constant of 6 to 10 nanomoles; (c) binding is physiologically productive since most of the bound precursors could be imported from the bound state; and (d) precursor binding was sensitive to both protease and the sulfhydryl modifying reagent N-ethylmaleimide. The effects of these two reagents differed in that protease reduced the total number of binding sites from the surface of chloroplasts but had little effect on binding affinity, whereas N-ethylmaleimide reduced the binding affinity but had little or no effect on receptor density.     

Assembly of active zone precursor vesicles: obligatory trafficking of presynaptic cytomatrix proteins Bassoon and Piccolo via a trans-Golgi compartment

Neurotransmitter release from presynaptic nerve terminals is restricted to specialized areas of the plasma membrane, so-called active zones. Active zones are characterized by a network of cytoplasmic scaffolding proteins involved in active zone generation and synaptic transmission. To analyze the modes of biogenesis of this cytomatrix, we asked how Bassoon and Piccolo, two prototypic active zone cytomatrix molecules, are delivered to nascent synapses. Although these proteins may be transported via vesicles, little is known about the importance of a vesicular pathway and about molecular determinants of cytomatrix molecule trafficking. We found that Bassoon and Piccolo co-localize with markers of the trans-Golgi network in cultured neurons. Impairing vesicle exit from the Golgi complex, either using brefeldin A, recombinant proteins, or a low temperature block, prevented transport of Bassoon out of the soma. Deleting a newly identified Golgi-binding region of Bassoon impaired subcellular targeting of recombinant Bassoon. Overexpressing this region to specifically block Golgi binding of the endogenous protein reduced the concentration of Bassoon at synapses. These results suggest that, during the period of bulk synaptogenesis, a primordial cytomatrix assembles in a trans-Golgi compartment. They further indicate that transport via Golgi-derived vesicles is essential for delivery of cytomatrix proteins to the synapse. Paradigmatically this establishes Golgi transit as an obligatory step for subcellular trafficking of distinct cytoplasmic scaffolding proteins.     

Protein import into chloroplasts

Three proteins from the chloroplastic outer envelope membrane and four proteins from the inner envelope membrane have been identified as components of the chloroplastic protein import apparatus. Multiple molecular chaperones and a stromal processing peptidase are also important components of the import machinery. The interactions of these proteins with each other and with the precursors destined for transport into chloroplasts are gradually being described using both biochemical and genetic strategies. Homologs of some transport components have been identified in cyanobacteria suggesting that at least some of import machinery was inherited from the cyanobacterial ancestors that gave rise to chloroplasts.     

A stromal Hsp100 protein is required for normal chloroplast development and function in Arabidopsis

Molecular chaperones are required for the translocation of many proteins across organellar membranes, presumably by providing energy in the form of ATP hydrolysis for protein movement. In the chloroplast protein import system, a heat shock protein 100 (Hsp100), known as Hsp93, is hypothesized to be the chaperone providing energy for precursor translocation, although there is little direct evidence for this hypothesis. To learn more about the possible function of Hsp93 during protein import into chloroplasts, we isolated knockout mutant lines that contain T-DNA disruptions in either atHSP93-V or atHSP93-III, which encode the two Arabidopsis (Arabidopsis thaliana) homologs of Hsp93. atHsp93-V mutant plants are much smaller and paler than wild-type plants. In addition, mutant chloroplasts contain less thylakoid membrane when compared to the wild type. Plastid protein composition, however, seems to be largely unaffected in atHsp93-V knockout plants. Chloroplasts isolated from the atHsp93-V knockout mutant line are still able to import a variety of precursor proteins, but the rate of import of some of these precursors is significantly reduced. These results indicate that atHsp93-V has an important, but not essential, role in the biogenesis of Arabidopsis chloroplasts. In contrast, knockout mutant plants for atHsp93-III, the second Arabidopsis Hsp93 homolog, had a visible phenotype identical to the wild type, suggesting that atHsp93-III may not play as important a role as atHsp93-V in chloroplast development and/or function.     

Structure of membrane-bound annexin A5 trimers: a hybrid cryo-EM - X-ray crystallography study

Annexins constitute a family of phospholipid- and Ca(2+)-binding proteins involved in a variety of membrane-related processes. The property of several annexins, including annexin A5, to self-organize at the surface of lipid membranes into 2D ordered arrays has been proposed to be functionally relevant in cellular contexts. To further address this question, we investigated the high-resolution structure of annexin A5 trimers in membrane-bound 2D crystals by cryo-electron microscopy (Cryo-EM). A new 2D crystal form was discovered, with p32(1) symmetry, which is significantly better ordered than the 2D crystals reported before. A 2D projection map was obtained at 6.5 A resolution, revealing protein densities within each of the four domains characteristic of annexins. A quantitative comparison was performed between this structure and models generated from the structure of the soluble form of annexin A5 in pseudo-R3 3D crystals. This analysis indicated that both structures are essentially identical, except for small local changes attributed to membrane binding. As a consequence, and contrary to the common view, annexin A5 molecules maintain their bent shape and do not flatten upon membrane binding, which implies either that the four putative Ca(2+) and membrane-binding loops present different types of interaction with the membrane surface, or that the membrane surface is locally perturbed. We propose that the trimerization of annexin A5 molecules is the relevant structural change occurring upon membrane binding. The evidence that 2D arrays of annexin A5 trimers are responsible for its in vitro property of blood coagulation inhibition supports this conclusion.