Efficient infection of cells in culture by type O foot-and-mouth disease virus requires binding to cell surface heparan sulfate.

Foot-and-mouth disease virus (FMDV) enters cells by attaching to cellular receptor molecules of the integrin family, one of which has been identified as the RGD-binding integrin alpha(v)beta3. Here we report that, in addition to an integrin binding site, type O strains of FMDV share with natural ligands of alpha(v)beta3 (i.e., vitronectin and fibronectin) a specific affinity for heparin and that binding to the cellular form of this sulfated glycan, heparan sulfate, is required for efficient infection of cells in culture. Binding of the virus to paraformaldehyde-fixed cells was powerfully inhibited by agents such as heparin, that compete with heparan sulfate or by agents that compete for heparan sulfate (platelet factor 4) or that inactivate it (heparinase). Neither chondroitin sulfate, a structurally related component of the extracellular matrix, nor dextran sulfate appreciably inhibited binding. The functional importance of heparan sulfate binding was demonstrated by the facts that (i) infection of live cells by FMDV could also be blocked specifically by heparin, albeit at a much higher concentration of inhibitor; (ii) pretreatment of cells with heparinase reduced the number of plaques formed compared with that for untreated cells; and (iii) mutant cell lines deficient in heparan sulfate expression were unable to support plaque formation by FMDV, even though they remained equally susceptible to another picornavirus, bovine enterovirus. The results show that entry of type O FMDV into cells is a complex process and suggest that the initial contact with the cell surface is made through heparan sulfate.     

FLT3 Mutations in Early T-Cell Precursor ALL Characterize a Stem Cell Like Leukemia and Imply the Clinical Use of Tyrosine Kinase Inhibitors

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup.     

PKC-beta1 isoform activation is required for EGF-induced NF-kappaB inactivation and IkappaBalpha stabilization and protection of F-actin assembly and barrier function in enterocyte monolayers

Using monolayers of intestinal Caco-2 cells, we reported that activation of NF-kappaB is required for oxidative disruption and that EGF protects against this injury but the mechanism remains unclear. Activation of the PKC-beta1 isoform is key to monolayer barrier integrity. We hypothesized that EGF-induced activation of PKC-beta1 prevents oxidant-induced activation of NF-kappaB and the consequences of NF-kappaB activation, F-actin, and barrier dysfunction. We used wild-type (WT) and transfected cells. The latter were transfected with varying levels of cDNA to overexpress or underexpress PKC-beta1. Cells were pretreated with EGF or PKC modulators +/- oxidant. Pretreatment with EGF protected monolayers by increasing native PKC-beta1 activity, decreasing IkappaBalpha phosphorylation/degradation, suppressing NF-kappaB activation (p50/p65 subunit nuclear translocation/activity), enhancing stable actin (increased F-actin-to-G-actin ratio), increasing stability of actin cytoskeleton, and reducing barrier hyperpermeability. Cells stably overexpressing PKC-beta1 were protected by low, previously nonprotective doses of EGF or modulators. In these clones, we found enhanced IkappaBalpha stabilization, NF-kappaB inactivation, actin stability, and barrier function. Low doses of the modulators led to increases in PKC-beta1 in the particulate fractions, indicating activation. Stably inhibiting endogenous PKC-beta1 substantially prevented all measures of EGF's protection against NF-kappaB activation. We conclude that EGF-mediated protection against oxidant disruption of the intestinal barrier function requires PKC-beta1 activation and NF-kappaB suppression. The molecular event underlying this unique effect of PKC-beta1 involves inhibition of phosphorylation and increases in stabilization of IkappaBalpha. The ability to inhibit the dynamics of NF-kappaB/IkappaBalpha and F-actin disassembly is a novel mechanism not previously attributed to the classic subfamily of PKC isoforms.     

Theta-isoform of PKC is required for alterations in cytoskeletal dynamics and barrier permeability in intestinal epithelium: a novel function for PKC-theta

Using intestinal Caco-2 cells, we previously showed that assembly of cytoskeleton is required for monolayer barrier function, but the underlying mechanisms remain poorly understood. Because the -isoform of PKC is present in wild-type (WT) intestinal cells, we hypothesized that PKC- is crucial for changes in cytoskeletal and barrier dynamics. We have created the first multiple sets of gastrointestinal cell clones transfected with varying levels of cDNA to stably inhibit native PKC- (antisense, AS; dominant negative, DN) or to express its activity (sense). We studied transfected and WT Caco-2 cells. First, relative to WT cells, AS clones underexpressing PKC- showed monolayer injury as indicated by decreased native PKC- activity, reduced tubulin phosphorylation, increased tubulin disassembly (decreased polymerized and increased monomeric pools), reduced architectural integrity of microtubules, reduced stability of occludin, and increased barrier hyperpermeability. In these AS clones, PKC- was substantially reduced in the particulate fractions, indicating its inactivation. In WT cells, 82-kDa PKC- was constitutively active and coassociated with 50-kDa tubulin, forming an endogenous PKC-/tubulin complex. Second, DN transfection to inhibit the endogenous PKC- led to similar destabilizing effects on monolayers, including cytoskeletal hypophosphorylation, depolymerization, and instability as well as barrier disruption. Third, stable overexpression of PKC- led to a mostly cytosolic distribution of -isoform (<10% in particulate fractions), indicating its inactivation. In these sense clones, we also found disruption of occludin and microtubule assembly and increased barrier dysfunction. In conclusion, 1) PKC- isoform is required for changes in the cytoskeletal assembly and barrier permeability in intestinal monolayers, and 2) the molecular event underlying this novel biological effect of PKC- involves changes in phosphorylation and/or assembly of the subunit components of the cytoskeleton. The ability to alter the cytoskeletal and barrier dynamics is a unique function not previously attributed to PKC-. microtubules; tubulin; occludin; epithelial barrier permeability; protein kinase C isoform     

Gas chromatographic method for detection of urinary sucralose: application to the assessment of intestinal permeability

We developed a capillary column gas chromatography (CCGC) method for the measurement of urinary sucralose (S) and three other sugar probes including, sucrose, lactulose (L) and mannitol (M) for use in in vivo studies of intestinal permeability. We compared the capillary method with a packed column gas chromatography (PCGC) method. We also investigated a possible role for sucralose as a probe for the measurement of whole gut permeability. Sample preparation was rapid and simple. The above four sugars were detected precisely, without interference. We measured intestinal permeability using 5- and 24-h urine collections in 14 healthy volunteers. The metabolism of sugars was evaluated by incubating the intestinal bacteria with an iso-osmolar mixture of mannitol, lactulose and sucralose at 37 degrees C for 19 h. Sugar concentrations and the pH of the mixture were monitored. The use of the CCGC method improved the detection of sucralose as compared to PCGC. The average coefficient of variation decreased from 15% to 4%. It also increased the sensitivity of detection by 200-2000-fold. The GC assay was linear between sucralose concentrations of 0.2 and 40 g/l (r=1.000). Intestinal bacteria metabolized lactulose and acidified the media but did not metabolize sucralose or mannitol. The new method for the measurement of urinary sucralose permits the simultaneous quantitation of sucrose, mannitol and lactulose, and is rapid, simple, sensitive, accurate and reproducible. Because neither S nor M is metabolized by intestinal bacteria, and because only a tiny fraction of either sugar is absorbed, this pair of sugar probes appears to be available for absorption throughout the GI tract. Thus, the 24-h urinary concentrations of S and M, or the urinary S/M ratio following an oral dose of a sugar mixture, might be good markers for whole gut permeability.     

Differential membrane binding and diacylglycerol recognition by C1 domains of RasGRPs

RasGRPs (guanine-nucleotide-releasing proteins) are exchange factors for membrane-bound GTPases. All RasGRP family members contain C1 domains which, in other proteins, bind DAG (diacylglycerol) and thus mediate the proximal signal-transduction events induced by this lipid second messenger. The presence of C1 domains suggests that all RasGRPs could be regulated by membrane translocation driven by C1-DAG interactions. This has been demonstrated for RasGRP1 and RasGRP3, but has not been tested directly for RasGRP2, RasGRP4alpha and RasGRP4beta. Sequence alignments indicate that all RasGRP C1 domains have the potential to bind DAG. In cells, the isolated C1 domains of RasGRP1, RasGRP3 and RasGRP4alpha co-localize with membranes and relocalize in response to DAG, whereas the C1 domains of RasGRP2 and RasGRP4beta do not. Only the C1 domains of RasGRP1, RasGRP3 and RasGRP4alpha recognize DAG as a ligand within phospholipid vesicles and do so with differential affinities. Other lipid second messengers were screened as ligands for RasGRP C1 domains, but none was found to serve as an alternative to DAG. All of the RasGRP C1 domains bound to vesicles which contained a high concentration of anionic phospholipids, indicating that this could provide a DAG-independent mechanism for membrane binding by C1 domains. This concept was supported by demonstrating that the C1 domain of RasGRP2 could functionally replace the membrane-binding role of the C1 domain within RasGRP1, despite the inability of the RasGRP2 C1 domain to bind DAG. The RasGRP4beta C1 domain was non-functional when inserted into either RasGRP1 or RasGRP4, implying that the alternative splicing which produces this C1 domain eliminates its contribution to membrane binding.     

The Efficacy of Salicylic Acid Levels on Photosynthetic Activity,Growth, and Essential Oil Content and Composition of Pennyroyal Plants Under Salt Stress

Journal of Plant Growth Regulation - This research was conducted to find out whether the foliar application of salicylic acid (SA) at 0.5, 1, and 1.5&nbsp;mM in comparison with water spray...