Stent implantation through a self-expanding stent

Jailing of a side-branch is a known complication of stent implantation, and makes access to the side-branch difficult, especially if the stent is of the self-expanding type. Although plain balloon angioplasty is feasible for the jailed side-branches, the use of newer devices (a stent, Rotablation or atherectomy) has not been described. We describe a novel way of treating a side-branch jailed by a self-expanding stent by using stent implantation through the strut of a self-expanding stent.     

Analysis of non-TIR NBS-LRR resistance gene analogs in <Emphasis Type="Italic">Musa acuminata</Emphasis> Colla: Isolation,RFLP marker development,and physical mapping

Background  

Many commercial banana varieties lack sources of resistance to pests and diseases, as a consequence of sterility and narrow genetic background. Fertile wild relatives, by contrast, possess greater variability and represent potential sources of disease resistance genes (R-genes). The largest known family of plant R-genes encode proteins with nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for isolation of candidate genes in banana which may be involved in plant defence.     

Retroviruses human immunodeficiency virus and murine leukemia virus are enriched in phosphoinositides

Retroviruses acquire a lipid envelope during budding from the membrane of their hosts. Therefore, the composition of this envelope can provide important information about the budding process and its location. Here, we present mass spectrometry analysis of the lipid content of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). The results of this comprehensive survey found that the overall lipid content of these viruses mostly matched that of the plasma membrane, which was considerably different from the total lipid content of the cells. However, several lipids are enriched in comparison to the composition of the plasma membrane: (i) cholesterol, ceramide, and GM3; and (ii) phosphoinositides, phosphorylated derivatives of phosphatidylinositol. Interestingly, microvesicles, which are similar in size to viruses and are also released from the cell periphery, lack phosphoinositides, suggesting a different budding mechanism/location for these particles than for retroviruses. One phosphoinositide, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂], has been implicated in membrane binding by HIV Gag. Consistent with this observation, we found that PI(4,5)P₂ was enriched in HIV-1 and that depleting this molecule in cells reduced HIV-1 budding. Analysis of mutant virions mapped the enrichment of PI(4,5)P₂ to the matrix domain of HIV Gag. Overall, these results suggest that HIV-1 and other retroviruses bud from cholesterol-rich regions of the plasma membrane and exploit matrix/PI(4,5)P₂ interactions for particle release from cells.     

The synaptojanin-like protein Inp53/Sjl3 functions with clathrin in a yeast TGN-to-endosome pathway distinct from the GGA protein-dependent pathway

Yeast TGN resident proteins that frequently cycle between the TGN and endosomes are much more slowly transported to the prevacuolar/late endosomal compartment (PVC) than other proteins. However, TGN protein transport to the PVC is accelerated in mutants lacking function of Inp53p. Inp53p contains a SacI polyphosphoinositide phosphatase domain, a 5-phosphatase domain, and a proline-rich domain. Here we show that all three domains are required to mediate "slow delivery" of TGN proteins into the PVC. Although deletion of the proline-rich domain did not affect general membrane association, it caused localization to become less specific. The proline-rich domain was shown to bind to two proteins, including clathrin heavy chain, Chc1p. Unlike chc1 mutants, inp53 mutants do not mislocalize TGN proteins to the cell surface, consistent with the idea that Chc1p and Inp53p act at a common vesicular trafficking step but that Chc1p is used at other steps also. Like mutations in the AP-1 adaptor complex, mutations in INP53 exhibit synthetic growth and transport defects when combined with mutations in the GGA proteins. Taken together with other recent studies, our results suggest that Inp53p and AP-1/clathrin act together in a TGN-to-early endosome pathway distinct from the direct TGN-to-PVC pathway mediated by GGA/clathrin.     

Phosphoinositide profiling in complex lipid mixtures using electrospray ionization mass spectrometry

Phosphoinositides (phosphorylated derivatives of phosphatidylinositol, PI) are versatile intracellular signaling lipids whose occurrence in low concentrations complicates direct mass measurements. Here we present a sensitive method to detect, identify and quantify phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP(2)) with different fatty acid compositions (phosphoinositide profiles) in total lipid extracts by electrospray ionization mass spectrometry (ESI-MS). Using this method, we detected elevated concentrations of PIP2 in human fibroblasts from patients with Lowe syndrome, a genetic disorder that affects phosphoinositide metabolism. Saccharomyces cerevisiae cells deficient in enzymes involved in PIP metabolism--Sac1p, a phosphoinositide phosphatase, and Vps34p and Pik1p, a PI 3-kinase and PI 4-kinase, respectively--showed not only different PIP concentrations but also differential changes in PIP profiles indicating metabolic and/or subcellular pooling. Mass spectrometric analysis of phosphoinositides offers unique advantages over existing approaches and may represent a powerful diagnostic tool for human diseases that involve defective phosphoinositide metabolism.     

ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Igamma

Clathrin-mediated endocytosis of synaptic vesicle membranes involves the recruitment of clathrin and AP-2 adaptor complexes to the presynaptic plasma membrane. Phosphoinositides have been implicated in nucleating coat assembly by directly binding to several endocytotic proteins including AP-2 and AP180. Here, we show that the stimulatory effect of ATP and GTPgammaS on clathrin coat recruitment is mediated at least in part by increased levels of PIP2. We also provide evidence for a role of ADP-ribosylation factor 6 (ARF6) via direct stimulation of a synaptically enriched phosphatidylinositol 4-phosphate 5-kinase type Igamma (PIPKIgamma), in this effect. These data suggest a model according to which activation of PIPKIgamma by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse.     

Anti-human CD4 induces peripheral tolerance in a human CD4+, murine CD4-, HLA-DR+ advanced transgenic mouse model

Selection in vivo of potent mAbs to human CD4 useful for immunotherapy, e.g., for the induction of immunological tolerance, is restricted for ethical reasons. We therefore used multiple transgenic mice that lack murine CD4, but express human CD4 specifically on Th cells, and HLA-DR3 as its natural counterligand (CD4/DR3 mice). The injection of CD4/DR3 mice with anti-human CD4 (mAb Max.16H5) before immunization with tetanus toxoid (TT, day 0) totally blocked the formation of specific Abs. This state of unresponsiveness persisted a subsequent boost again performed in the presence of anti-human CD4. When these mice were left untreated for at least 40 days, and were then re-exposed with TT, but in the absence of anti-human CD4, they consistently failed to induce specific Abs (long-term unresponsiveness). Exposure to second party Ags (hen egg lysozyme, human acetylcholine receptor) induced specific Abs comparable with control mice, demonstrating that the anti-CD4-induced unresponsiveness was Ag specific (immunological tolerance). Importantly, the concurrent injection of TT and anti-human CD4 at day 0, followed by another two anti-CD4 treatments, also led to tolerant animals, indicating that tolerance was inducible at the same day as the Ag exposure is provided. We finally demonstrate a limited ability of spleen cells to respond to TT in vitro, indicating that T cells are essentially involved in the maintenance of TT-specific tolerance. These data show for the first time that the human CD4 coreceptor mediates tolerance-inducing signals when triggered by an appropriate ligand in vivo.     

Activation of p70 ribosomal protein S6 kinase is an essential step in the DNA damage-dependent signaling pathway responsible for the ultraviolet B-mediated increase in interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3) protein levels in human dermal fibroblasts

Ultraviolet B (UVB) irradiation has been shown to stimulate the expression of matrix-degrading metalloproteinases via generation of DNA damage and/or reactive oxygen species. Matrix-degrading metalloproteinases promote UVB-triggered detrimental long term effects like cancer formation and premature skin aging. Here, we were interested in identifying components of the signal transduction pathway that causally link UVB-mediated DNA damage and induction of matrix-degrading metalloproteinase (MMP)-1/interstitial collagenase and MMP-3/stromelysin-1 in human dermal fibroblasts in vitro. The activity of p70 ribosomal S6 kinase, a downstream target of the FK506-binding protein-12/rapamycin-associated protein kinase (FRAP) kinase (RAFT1, mTOR), was identified to be 4.8 +/- 0.8-fold, and MMP-1 and MMP-3 protein levels 2.4- and 11.5-fold increased upon UVB irradiation compared with mock-irradiated controls. The FRAP kinase inhibitor rapamycin and the DNA repair inhibitor aphidicolin significantly suppressed the UVB-mediated increase in p70 ribosomal S6 kinase activity by 50-65% and MMP-1 and MMP-3 protein levels by 34-68% and 42-88% compared with UVB-irradiated fibroblasts. By contrast, the interleukin-1beta-mediated increase in MMP-1 and MMP-3 protein levels could not be suppressed by rapamycin. Collectively, our data suggest that the FRAP-controlled p70 ribosomal S6 kinase is an essential component of a DNA damage-dependent, but not of the interleukin-1/cell membrane receptor-dependent signaling.     

Phosphatidic acid is a major phospholipid class in reproductive organs of Arabidopsis thaliana

Phospholipids are the crucial components of biological membranes and signal transduction. Among different tissues, flower phospholipids are one of the least characterized features of plant lipidome. Here, we report that floral reproductive organs of Arabidopsis thaliana contain high levels of phosphatidic acid (PA), a known lipid second messenger. By using floral homeotic mutants enriched with specific floral organs, lipidomics study showed increased levels of PA species in ap3-3 mutant with enriched pistils. Accompanied gene expression study for 7 diacylglycerol kinases and 11 PA phosphatases revealed distinct floral organ specificity, suggesting an active phosphorylation/dephosphorylation between PA and diacylglycerol in flowers. Our results suggest that PA is a major phospholipid class in floral reproductive organs of A. thaliana.