Identification of human scFvs targeting atherosclerotic lesions: selection by single round in vivo phage display

Our aim was to investigate by in vivo biopanning the lesions developed early in atherosclerosis and identify human antibodies that home to diseased regions. We have designed a two-step approach for a rapid isolation of human Monoclonal phage-display single-chain antibodies (MoPhabs) reactive with proteins found in lesions developed in an animal model of atherosclerosis. After a single round of in vivo biopanning, the MoPhabs were eluted from diseased sections of rabbit aorta identified by histology and NMR microscopy. MoPhabs expressed in situ were selected by subtractive colony filter screening for their capacity to recognize atherosclerotic but not normal aorta. MoPhabs selected by our method predominantly bind atherosclerotic lesions. Two of them, B3.3G and B3.GER, produced as scFv fragments, recognized an epitope present on the surface in early atherosclerotic lesions and within the intimal thickness in more complex plaques. These human MoPhabs homed to atherosclerotic lesions in ApoE(-/-) mice after in vivo injection. A protein of approximately 56 kDa recognized by B3.3G was affinity-purified and identified by mass spectrometry analysis as vitronectin. This is the first time that single round in vivo biopanning has been used to select human antibodies as candidates for diagnostic imaging and for obtaining insight into targets displayed in atherosclerotic plaques.     

Biodiversity of Mimosa pudica rhizobial symbionts (Cupriavidus taiwanensis, Rhizobium mesoamericanum) in New Caledonia and their adaptation to heavy metal-rich soils

Rhizobia are soil bacteria able to develop a nitrogen-fixing symbiosis with legumes. They are taxonomically spread among the alpha and beta subclasses of the Proteobacteria. Mimosa pudica, a tropical invasive weed, has been found to have an affinity for beta-rhizobia, including species within the Burkholderia and Cupriavidus genera. In this study, we describe the diversity of M.?pudica symbionts in the island of New Caledonia, which is characterized by soils with high heavy metal content, especially of Ni. By using a plant-trapping approach on four soils, we isolated 96 strains, the great majority of which belonged to the species Cupriavidus taiwanensis (16S rRNA and recA gene phylogenies). A few Rhizobium strains in the newly described species Rhizobium mesoamericanum were also isolated. The housekeeping and nod gene phylogenies supported the hypothesis of the arrival of the C.?taiwanensis and R.?mesoamericanum strains together with their host at the time of the introduction of M.?pudica in New Caledonia (NC) for its use as a fodder. The C.?taiwanensis strains exhibited various tolerances to Ni, Zn and Cr, suggesting their adaptation to the specific environments in NC. Specific metal tolerance marker genes were found in the genomes of these symbionts, and their origin was investigated by phylogenetic analyses.     

Pharmacological inhibition of c-Jun N-terminal kinase signaling prevents cardiomyopathy caused by mutation in LMNA gene

Mutations in LMNA, which encodes A-type nuclear lamins, cause disorders of striated muscle that have as a common feature dilated cardiomyopathy. We have demonstrated an abnormal activation of both the extracellular signal-regulated kinase (ERK) and the c-Jun N-terminal kinase (JNK) branches of the mitogen-activated protein kinase signaling cascade in hearts from Lmna^H222P/H222P mice that develop dilated cardiomyopathy. We previously showed that pharmacological inhibition of cardiac ERK signaling in these mice delayed the development of left ventricle dilatation and deterioration in ejection fraction. In the present study, we treated Lmna^H222P/H222P mice with SP600125, an inhibitor of JNK signalling. Systemic treatment with SP600125 inhibited JNK phosphorylation, with no detectable effect on ERK. It also blocked increased expression of RNAs encoding natriuretic peptide precursors and proteins involved in the architecture of the sarcomere that occurred in placebo-treated mice. Furthermore, treatment with SP600125 significantly delayed the development of left ventricular dilatation and prevented decreases in cardiac ejection fraction and fibrosis. These results demonstrate a role for JNK activation in the development of cardiomyopathy caused by LMNA mutations. They further provide proof-of-principle for JNK inhibition as a novel therapeutic option to prevent or delay the cardiomyopathy in humans with mutations in LMNA.     

The postsynaptic density 95/disc-large/zona occludens protein syntenin directly interacts with frizzled 7 and supports noncanonical Wnt signaling

Wnt signaling pathways are essential for embryonic patterning, and they are disturbed in a wide spectrum of diseases, including cancer. An unresolved question is how the different Wnt pathways are supported and regulated. We previously established that the postsynaptic density 95/disc-large/zona occludens (PDZ) protein syntenin binds to syndecans, Wnt coreceptors, and known stimulators of protein kinase C (PKC)alpha and CDC42 activity. Here, we show that syntenin also interacts with the C-terminal PDZ binding motif of several Frizzled Wnt receptors, without compromising the recruitment of Dishevelled, a key downstream Wnt-signaling component. Syntenin is coexpressed with cognate Frizzled during early development in Xenopus. Overexpression and down-regulation of syntenin disrupt convergent extension movements, supporting a role for syntenin in noncanonical Wnt signaling. Syntenin stimulates c-jun phosphorylation and modulates Frizzled 7 signaling, in particular the PKCalpha/CDC42 noncanonical Wnt signaling cascade. The syntenin-Frizzled 7 binding mode indicates syntenin can accommodate Frizzled 7-syndecan complexes. We propose that syntenin is a novel component of the Wnt signal transduction cascade and that it might function as a direct intracellular link between Frizzled and syndecans.     

Cellular location of (2R, 3R, 7Z)-2-aminotetradec-7-ene-1, 3-diol, a potent antimicrobial metabolite produced by the Caribbean sponge Haliclona vansoesti

The Caribbean sponge Haliclona vansoesti has been found to contain large amounts of a new sphingosine derivative, (2R, 3R, 7Z)-2-aminotetradec-7-ene-1, 3-diol (compound 1). To determine the localization of this compound within the organism, cell distribution and quantitative determination of the aminodiol content of cell fractions obtained by differential centrifugation have been performed. Results show that choanocytes and archaeocytes are the major sponge cell types and that H. vansoesti harbour small photosynthetic symbionts (cyanobacteria) and few heterotrophic bacteria. Reverse-phase HPLC analyses of the cell fractions reveal that the aminodiol 1 is not associated with the prokaryotic endobionts but with the sponge cells, in particular the archaeocytes. This is clearly established by the positive significant correlation existing between the numbers of archaeocytes and the amounts of aminodiol 1. The mean aminodiol concentration is estimated to be 2 microg/10(5) archaeocytes. The aminodiol 1 is also found in substantial amounts in primary cell cultures, so that cell culture can be envisaged as an option for its production. Sponge cell suspensions display potent antibacterial and antiyeast activities, in correlation with their aminodiol content, indicating that this compound is at least in part responsible for these activities in the sponge. The release of the aminodiol I into the external medium suggests that this substance may be involved in the defence mechanisms of the sponge.     

Improved methods for mutacin detection and production

Studies of mutacins have always been hampered by the difficulties in obtaining active liquid preparations of these substances. In order to be commercially produced, good mutacin yields have to be obtained, preferably in inexpensive media. The results presented here indicate that mutacins can be produced in supplemented cheese whey permeate. The influence of carbon and nitrogen supplements on mutacin production varied according to the producer strain. The use of CaCO3 as a buffer in batch cultures resulted in improved yields of mutacin in the supernatants. Antimicrobial activity assays were improve by acidification of the diluent (pH 2) and were less variable in peptone water (0.5%). The culture medium consisting of cheese whey permeate (6% w/v), yeast extract (2% w/v) and CaCO3 (1% w/v) was found to be an inexpensive medium for the efficient production of mutacins.     

Artocarpin is a polyspecific jacalin-related lectin with a monosaccharide preference for mannose

A reinvestigation of the carbohydrate-binding properties revealed that artocarpin, a previously described mannose-specific lectin from jackfruit (Artocarpus integrifolia) seeds, behaves as a polyspecific lectin. Surface plasmon resonance hapten inhibition experiments demonstrated that artocarpin readily interacted with a wide range of monosaccharides covering galactose, N-acetylgalactosamine, mannose, glucose, sialic acid and N-acetylmuramic acid. Molecular docking confirmed this unexpected ability of artocarpin to interact with structurally different sugars. The biological significance of the polyspecificity of the lectin is discussed in terms of the broadening of the range of potential target glycans present on the surface of plant phytopathogens or predators.     

Detecting single DNA copy number variations in complex genomes using one nanogram of starting DNA and BAC-array CGH

Comparative genomic hybridization to bacterial artificial chromosome (BAC)-arrays (array-CGH) is a highly efficient technique, allowing the simultaneous measurement of genomic DNA copy number at hundreds or thousands of loci, and the reliable detection of local one-copy-level variations. We report a genome-wide amplification method allowing the same measurement sensitivity, using 1 ng of starting genomic DNA, instead of the classical 1 microg usually necessary. Using a discrete series of DNA fragments, we defined the parameters adapted to the most faithful ligation-mediated PCR amplification and the limits of the technique. The optimized protocol allows a 3000-fold DNA amplification, retaining the quantitative characteristics of the initial genome. Validation of the amplification procedure, using DNA from 10 tumour cell lines hybridized to BAC-arrays of 1500 spots, showed almost perfectly superimposed ratios for the non-amplified and amplified DNAs. Correlation coefficients of 0.96 and 0.99 were observed for regions of low-copy-level variations and all regions, respectively (including in vivo amplified oncogenes). Finally, labelling DNA using two nucleotides bearing the same fluorophore led to a significant increase in reproducibility and to the correct detection of one-copy gain or loss in >90% of the analysed data, even for pseudotriploid tumour genomes.     

Nuclear envelope alterations in fibroblasts from LGMD1B patients carrying nonsense Y259X heterozygous or homozygous mutation in lamin A/C gene

Mutations in the LMNA gene encoding nuclear lamins A and C are responsible for seven inherited disorders affecting specific tissues. We have analyzed skin fibroblasts from a patient with type 1B limb-girdle muscular dystrophy and from her deceased newborn grandchild carrying, respectively, a heterozygous (+/mut) and a homozygous (mut/mut) nonsense Y259X mutation. In fibroblasts(+/mut), the presence of only 50% lamins A and C promotes no detectable abnormality, whereas in fibroblasts(mut/mut) the complete absence of lamins A and C leads to abnormally shaped nuclei with lobules in which none of the analyzed nuclear proteins were detected, i.e., B-type lamins, emerin, nesprin-1alpha, LAP2beta, and Nup153. These lobules perturb cell division as fibroblast(mut/mut) cultures with large proportions of cells with dysmorphic nuclei grow more slowly than controls and the cell proliferation normalizes when the number of these abnormally shaped nuclei declines. In all fibroblasts(mut/mut), nesprin-1alpha-like emerin exhibited aberrant localization in the endoplasmic reticulum. Transfection of wild-type lamin A or C cDNAs restored the correct localization of both emerin and nesprin-1alpha. These data demonstrate that lamin C, like lamin A, interacts in vivo directly with nesprin-1alpha and with emerin and that lamin A or C is sufficient for the correct anchorage of emerin and nesprin-1alpha at the nuclear envelope in human cells.     

Syndecan recycling [corrected] is controlled by syntenin-PIP2 interaction and Arf6

Syndecans are heparan sulfate proteoglycans that modulate the activity of several growth factors and cell adhesion molecules. PDZ domains in the adaptor protein syntenin interact with syndecans and with the phosphoinositide PIP(2), which is involved in the regulation of the actin cytoskeleton and membrane trafficking. Here, we show that the syntenin PDZ domain-PIP(2) interaction controls Arf6-mediated syndecan recycling through endosomal compartments. FGF receptor accompanies syndecan along the syntenin-mediated recycling pathway, in a heparan sulfate- and FGF-dependent manner. Syndecans that cannot recycle via this pathway become trapped intracellularly and inhibit cell spreading. This syntenin-mediated syndecan recycling pathway may regulate the surface availability of a number of cell adhesion and signaling molecules.