Shiga toxin 1 interaction with enterocytes causes apical protein mistargeting through the depletion of intracellular galectin-3

Shiga toxins (Stx) 1 and 2 are responsible for intestinal and systemic sequelae of infection by enterohemorrhagic Escherichia coli (EHEC). However, the mechanisms through which enterocytes are damaged remain unclear. While secondary damage from ischemia and inflammation are postulated mechanisms for all intestinal effects, little evidence excludes roles for more primary toxin effects on intestinal epithelial cells. We now document direct pathologic effects of Stx on intestinal epithelial cells. We study a well-characterized rabbit model of EHEC infection, intestinal tissue and stool samples from EHEC-infected patients, and T84 intestinal epithelial cells treated with Stx1. Toxin uptake by intestinal epithelial cells in vitro and in vivo causes galectin-3 depletion from enterocytes by increasing the apical galectin-3 secretion. This Shiga toxin-mediated galectin-3 depletion impairs trafficking of several brush border structural proteins and transporters, including villin, dipeptidyl peptidase IV, and the sodium-proton exchanger 2, a major colonic sodium absorptive protein. The mistargeting of proteins responsible for the absorptive function might be a key event in Stx1-induced diarrhea. These observations provide new evidence that human enterocytes are directly damaged by Stx1. Conceivably, depletion of galectin-3 from enterocytes and subsequent apical protein mistargeting might even provide a means whereby other pathogens might alter intestinal epithelial absorption and produce diarrhea.     

Cyto-physiological events during radish germination in the presence of a Ruta graveolens L. infusion

ABSTRACT

An infusion of Ruta graveolens L. was tested for its inhibitory effects upon radish germination at the cyto-physiological level. Radish seeds were placed under optimum conditions for germination either in water (control) or in the presence of rue infusion (treated seeds). Morpho-physiological observations indicate that in treated radish seeds the inhibition of germination is accompanied by reduced water uptake and delayed reactivation of the outermost living layer, i.e. the aleurone cells. Compared to the control, aleurone cells of treated seeds present many large lipid droplets and protein bodies, without differentiated organelles. Moreover, chemical and biochemical analyses show that the treatment impairs the metabolic pathways of germination, such as the mobilization and utilization of seed reserves, and the loosening of cell walls. In fact, in treated seeds we found i) increased contents of glucose and galactose, ii) higher concentration of malic acid and iii) lower activities of some glycosidases compared to the control. Results suggest that aleurone cells may play an active part in controlling the embryo's metabolism.     

Mode of action of natural growth inhibitors in radish hypocotyl elongation – influence of raphanusanin on auxin-mediated microtubule orientation

Raphanusanin is a plant growth-inhibiting substance which plays an important role in light growth inhibition and phototropism of radish hypocotyls. We investigated the effect of raphanusanin on indole-3-acetic acid (IAA)-mediated orientation of microtubules (MT) in the outer epidermal cells of radish hypocotyl segments using immunofluorescence microscopy. IAA-mediated MT reorientation preceded cell elongation induced by IAA. A change of IAA-mediated MT orientation from longitudinal to transverse started within less than 15 min after IAA treatment, while significant growth promotion induced by IAA was found within about 30 min. The IAA-mediated transverse MT orientations were significantly inhibited by simultaneously added raphanusanin. We also investigated the effect of raphanusanin on the MT orientation of the segments pretreated with IAA. The change of MT orientation induced by raphanusanin preceded growth inhibition of the segments. Within about 60 min after its application, raphanusanin initiated inhibition of the steady-state elongation pre-induced by IAA, while IAA-mediated transverse MT orientations started to change into longitudinal orientations within less than 30 min after application of raphanusanin. Based on these results, it is suggested that raphanusanin induces growth inhibition through interference with the auxin-mediated MT orientations.     

转萝卜过氧化物酶RsPrx1基因保护拟南芥氧化损伤

为了研究萝卜过氧化物酶RsPrx1的功能,本实验构建了RsPrx1的cDNA克隆和表达载体pBI121-RsPrx1.通过农杆菌用浸花法转染拟南芥,获得转基因植株.对转基因植株的研究表明, 萝卜过氧化物酶RsPrx1可以抑制植物根和株高的生长,但不影响植物的成熟.当植物受到氧化应激时,RsPrx1可以保护植物免受氧化损伤. 因此,萝卜过氧化物酶RsPrx1参与植物的生长发育但不起主要作用,并具有抗氧化作用.     

Inhibition of cytokinin-induced plant growth by jasmonic acid and its methyl ester

Effects of jasmonic acid, methyl jasmonate and ABA on cytokinin-induced soybean ( Glycine max Merrill cv. Kingen No. 1) callus growth and radish ( Raphanus sativus L. cv. Comet) cotyledon growth were studied. Jasmonic acid and methyl jasmonate were powerful inhibitors of kinetin- or N-phenyl-N'-(2-chloro-4-pyridyl)urea-induced soybean callus growth, the former being more effective than the latter, especially at low concentrations (0.45 to 4.5 μ M ). These compounds could completely eliminate kinetin- or N-phenyl-N'-(2-chloro-4-pyridyl)urea-induced soybean callus growth at 45 μ M . At low concentrations ABA had no effect but at 450 μ M it completely eliminated callus growth induced by kinetin. Of the compounds tested ABA was the most powerful inhibitor of radish cotyledon growth in the presence or absence of kinetin. Jasmonic acid and methyl jasmonate were much less effective than ABA in this assay system.     

Electroelution of lipoprotein(a) [Lp(a)] from native polyacrylamide gels: a new, simple method to purify Lp(a)

Lipoprotein(a), Lp(a), is an atherogenic lipoprotein consisting of an LDL like core particle and a covalently linked glycoprotein of variable size. Lp(a), isolated from serum always contains LDL and HDL(2) as contaminants since Lp(a) floats in the density range 1.05-1.12 g/ml which overlaps that of LDL and HDL(2). Purified Lp(a) is increasingly needed as a standard to overcome various problems in the standardization of Lp(a) measurements and for in vitro biological studies. Problems inherent to the purification of Lp(a) include the aggregation of Lp(a) with LDL, overlapping size distribution and the inability of some fractions to bind to affinity columns. Here, we describe the development of a new method to purify Lp(a) from contaminating LDL and HDL(2) particles. Lp(a) was isolated from serum by sequential ultracentrifugation, resolved by native polyacrylamide gel electrophoresis and the gel segments were electroeluted to obtain pure Lp(a). l-Proline was added to the sample to a final concentration of 0.1 M to prevent the aggregation of Lp(a) with LDL.     

Lipopolysaccaride-binding peptides obtained by phage display method

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. It has strong toxicity and might cause sepsis or septic shock. Thus early detection of LPS and neutralization of LPS toxicity are required. We obtained several new LPS-binding peptides using a phage display method. We synthesized 3 of these peptides and analyzed their binding affinity and capacity to LPS. One of these peptides, named Li5-001, showed high binding affinity to LPS and lipid A; the K_d values were 10 and 1 nM, respectively. Li5-001 showed a high binding capacity to LPS, and was estimated to bind 130 ng LPS/mg, which is higher than that of polymyxin B (80 ng LPS/mg); however, its LPS-neutralizing activity was low. Li5-001 coupled with beads will be useful for eliminating endotoxin contamination from pharmaceuticals. Its low LPS-neutralizing activity allows to be used in the Limulus amebocyte lysate test without eluting LPS from the Li5-001 coupled beads.