Characterization of attaching and effacing <Emphasis Type="Italic">Escherichia coli</Emphasis> (AEEC) isolated from pigs and sheep

Background  

Attaching and effacing Escherichia coli (AEEC) are characterized by their ability to cause attaching-and-effacing (A/E) lesions in the gut mucosa of human and animal hosts leading to diarrhoea. The genetic determinants for the production of A/E lesions are located on the locus of enterocyte effacement (LEE), a pathogenicity island that also contains the genes encoding intimin (eae). This study reports data on the occurrence of eae positive E. coli carried by healthy pigs and sheep at the point of slaughter, and on serotypes, intimin variants, and further virulence factors of isolated AEEC strains.     

Involvement of c-Src tyrosine kinase in SHP-1 phosphatase activation by Ang II AT2 receptors in rat fetal tissues

Angiotensin II (Ang II) AT(2) receptors are abundantly expressed in rat fetal tissues where they probably contribute to development. In the present study we examine the effects of Ang II type 2 receptor stimulation on SHP-1 activation. Ang II (10(-7) M) elicits a rapid and transient tyrosine phosphorylation of SHP-1, maximal at 1 min, in a dose-dependent form, blocked by the AT(2) antagonist, PD123319. SHP-1 phosphorylation is followed in time by tyrosine dephosphorylation of different proteins, suggesting a sequence of events. Ang II induces association of SHP-1 to AT(2) receptors as shown by co-immunoprecipitation, Western blot and binding assays. SHP-1 activity was determined in immunocomplexes obtained with either anti-AT(2) or anti-SHP-1 antibodies, after Ang II stimulation (1 min), in correlation with the maximal level of SHP-1 phosphorylation. Interestingly, following receptor stimulation (1 min) c-Src was associated to AT(2) or SHP-1 immunocomplexes. Preincubation with the c-Src inhibitor PP2 inhibited SHP-1 activation and c-Src association, thus confirming the participation of c-Src in this pathway. We demonstrated here for the first time the involvement of c-Src in SHP-1 activation via AT(2) receptors present in an ex vivo model expressing both receptor subtypes. In this model, AT(2) receptors are not constitutively associated to SHP-1 and SHP-1 is not constitutively activated. Thus, we clearly establish that SHP-1 activation, mediated by the AT(2) subtype, involves c-Src and precedes protein tyrosine dephosphorylation, in rat fetal membranes.     

Causal role of oxidative stress in liver preconditioning by thyroid hormone in rats

Hepatic ischemia-reperfusion (IR) injury, a major clinical drawback during surgery, is abolished by L-3,3',5-triiodothyronine (T(3)) administration. Considering that the triggering mechanisms are unknown, the aim of this study is to assess the role of oxidative stress in T(3) preconditioning using N-acetylcysteine (NAC) before T(3) administration. Male Sprague-Dawley rats given a single dose of 0.1 mg of T(3)/kg were subjected to 1 h ischemia followed by 20 h reperfusion, in groups of animals pretreated with 0.5 g of NAC/kg 0.5 h before T(3) or with the respective control vehicles. At the end of the reperfusion period, blood and liver samples were taken for analysis of serum aspartate aminotransferase (AST) and hepatic histology, glutathione (GSH) and protein carbonyl contents, and nuclear factor-kappaB (NF-kappaB) and activating protein 1 (AP-1) DNA binding. The IR protocol used led to a 4.5-fold increase in serum AST levels and drastic changes in liver histology, with significant GSH depletion and enhancement of protein carbonyl levels and of the protein carbonyl/GSH content ratio, whereas NF-kappaB and AP-1 DNA binding was decreased and enhanced, respectively. In a time window of 48 h, T(3) exerted protection against hepatic IR injury, with 88% reduction in the protein carbonyl/GSH ratio and normalization of NF-kappaB and AP-1 DNA binding, changes that were suppressed by NAC administration before T(3). Data presented suggest that a transient increase in the oxidative stress status of the liver is an important trigger for T(3) preconditioning, evidenced in a warm IR injury model through antioxidant intervention.     

Galactosyl transferases in mycobacterial cell wall synthesis

Two galactosyl transferases can apparently account for the full biosynthesis of the cell wall galactan of mycobacteria. Evidence is presented based on enzymatic incubations with purified natural and synthetic galactofuranose (Galf) acceptors that the recombinant galactofuranosyl transferase, GlfT1, from Mycobacterium smegmatis, the Mycobacterium tuberculosis Rv3782 ortholog known to be involved in the initial steps of galactan formation, harbors dual β-(1→4) and β-(1→5) Galf transferase activities and that the product of the enzyme, decaprenyl-P-P-GlcNAc-Rha-Galf-Galf, serves as a direct substrate for full polymerization catalyzed by another bifunctional Galf transferase, GlfT2, the Rv3808c enzyme.     

How amyloplasts,water deficit and root tropisms interact?

Hydrotropism, the differential growth of plant roots directed by a moisture gradient, is a long recognized, but not well-understood plant behavior. Hydrotropism has been characterized in the model plant Arabidopsis. Previously, it was postulated that roots subjected to water stress are capable of undergo water-directed tropic growth independent of the gravity vector because of the loss of the starch granules in root cap columella cells and hence the loss of the early steps in gravitropic signaling. We have recently proposed that starch degradation in these cells during hydrostimulation sustain osmotic stress and root growth for carrying out hydrotropism instead of reducing gravity responsiveness. In addition, we also proposed that abscisic acid (ABA) and water deficit are critical regulators of root gravitropism and hydrotropism, and thus mediate the interacting mechanism between these two tropisms. Our conclusions are based upon experiments performed with the no hydrotropic response (nhr1) mutant of Arabidopsis, which lacks a hydrotropic response and shows a stronger gravitropic response than that of wild type (WT) in a medium with an osmotic gradient.Key words: starch, water deficit, auxin, abscisic acid, gravitropism, hydrotropismRoots of land plants sense and respond to different stimuli, some of which are fixed in direction and intensity (i.e., gravity) while other vary in time, space, direction and intensity (i.e., obstacles and moisture gradients). Directed growth of roots in relation to a gradient in moisture is called hydrotropism and begins in the root cap with the sensing of the moisture gradient. However, since gravity is an omnipresent accompaniment of Earthly life and many living process have evolved with it as a background constant, it is not surprising that root hydrotropism interacts with gravitropism.¹ The hydrotropic response in Arabidopsis, compare with other plants such as pea and cucumber^2,3 is readily observed even in the presence of gravity.^4,5 When Arabidopsis roots are subjected to a water gradient, such that the source of water is placed 180° opposed to the gravity vector, the roots will grow upwards, displaying positive hydrotropism. Therefore, it has been feasible to isolate so far two Arabidopsis mutants affected in their hydrotropic response.^5,6 Analysis of these mutants reveals new insights of the mechanism of hydrotropism. For one hand, the no hydrotropic response (nhr1) mutant lacks a hydrotropic response, and shows a stronger gravitropic response than that of wt and a modified wavy growth response in a medium with an osmotic gradient.^5,7 On the other hand, the mizu-kussei1 (miz1) mutant did not exhibit hydrotropism and showed regular gravitropism.⁶ Hence, the root hydrotropic response is both linked and unlinked from the gravitropic one. Nonetheless, miz1 roots also showed a reduced phototropism and a modified wavy growth response. This indicates that both MIZ1 and NHR1 are not exclusive components of the mechanism for hydrotropism and supports the notion that the root cap has assessment mechanisms that integrate many different environmental influences to produce a final integrated response.⁸ Thus, the physiological phenomena distinctively displayed by roots in order to forage resources from the environment are the result of integrated responses that resulted from many environmental influences sensed in the root cap.In the course of studying how gravity and water availability affected the perception and assessment of each other in root cap cells that generated the final root tropic response, we found that ABA is a critical regulator of the signal transduction mechanism that integrated these two-root tropisms.⁷ For this, we analyzed the long-term hydrotropic response of Arabidopsis roots in an osmotic gradient system. ABA, locally applied to seeds or root tips of nhr1, significantly increased root downward growth in a medium with an osmotic gradient (root length of nhr1 seedlings grown in this medium were on average 12.5 mm and plus 10 µM ABA were 25.1 mm). On the other hand, WT roots germinated and treated locally with ABA in this system were strongly gravitropic, albeit they had almost no starch in amyloplasts of root cap columella cells. Hydrotropically stimulated nhr1 roots, with or without ABA, maintained starch in amyloplastas, as opposed to those of WT. Therefore, the near-absence (WT) or abundant presence (nhr1) of starch granules does not affect the extent of downward gravitropism of roots in an osmotic gradient medium. Starch degradation in the wt might participate in osmoregulation by which root cells maintain turgor and consequently carry out hydrotropism, instead of reducing gravity responsiveness. In fact, it was just recently published that salt-induced rapid degradation of starch in amyloplasts is not likely the main reason for a negative gravitropic response seen under salt stress, because sos mutant roots of Arabidopsis showed negative gravitropic growth without any apparent rapid digestion of starch granules.⁹ Additionally, the stems of overwintering tubers of Potamogeton pectinatus are capable of elongating much faster in the absence than in the presence of oxygen for up to 14 days and its stems has an enhanced capacity for gravitropic movements in completely anoxic conditions.¹⁰ These authors hypothesized that ABA and starch degradation in the starchy tuber sustained stem cell elongation and cell division as well as differential growth required for the gravitropic response in these aquatic plants. These data taken together suggest that in conditions of anoxia, or water stress, ABA and degradation of starch play a critical role in the ability to survive relatively prolonged periods of unfavorable growth conditions. These players are critical when water or minerals are scarce since they regulate the enhancement of root downward growth. However, since roots can trail humidity gradients in soil, they can modulate their branching patterns (architecture) and thus respond to hydrotropism once a water-rich patch is found. Then the response of plants to gravity is principally one of nutrition (shoots to light, roots to mineral and water) and consequently must be regulated according to the long- and short-term environmental variables that occur during the development of the plant.Differential growth that occurs during the gravitropic and phototropic response has been explained according to the Cholodny-Went hypothesis, which states that the lateral transport of auxin across stimulated plant tissues is responsible for the curvature response.¹¹ Analysis of hydrotropism in some Arabidopsis agravitropic auxin transport mutants has demonstrated that these mutations do not influence their hydrotropic response.⁴ Furthermore, current pharmacological studies using inhibitors also indicated that both auxin influx and efflux are not required for hydrotropic response whereas auxin response is necessary for it.¹² These authors suggested a novel mechanism for auxin in root hydrotropism. Here, we analyzed whether asymmetric auxin distribution takes place across hydrotropically-stimulated roots using transgenic plants carrying a responsive auxin promoter (DR5) driving the expression of β-glucuronidase (GUS) or green fluorescent protein (GFP)^13,14 in wt and nhr1 backgrounds. Wt and nhr1 roots hydrotropically stimulated in a system with air moisture gradient⁵ showed no asymmetric expression of the DR5:: GUS or DR5::GFP (Fig. 1A and B). Nonetheless, nhr1 roots showed a substantial decrease in the signal driven by the DR5::GUS and GFP reporters in humidity saturated conditions (Fig. 1A, part b and B, part b), which might indicate that auxin-induced gene expression in the root cap was inhibited. It remains to be determined the significance of this inhibition in the no hydrotropic response phenotype displayed by nhr1 roots. Determination of the DR5::GUS expression in wt and nhr1 roots growing in an osmotic gradient medium for testing long-term hydrotropism revealed that the GUS signal was to some extent diminished in both wt or in nhr1 roots (Fig. 2C and D) compared to those roots growing in normal medium (Fig. 2A and B). An inhibitor of auxin response reduced hydrotropism,¹² and also inhibited auxin-dependent DR5::GUS expression.¹⁵ However, a decrease of DR5::GUS in wt root tips was not an impediment for developing an hydrotropic response. On the other hand, nhr1 roots also showed a decrease of DR5::GUS expression (Fig. 2B and D) and a complete absence of DR5::GFP (data not shown), which did not influence the extent of downward root gravitropism in water deficit conditions. Therefore, it is difficult to assign a role of auxin-induce gene expression in hydrotropism and further studies are required in order to unravel this issue. Furthermore, it needs to be resolved whether these expression studies oppose the idea that gradients in auxin precede differential growth in response to humidity gradients.Open in a separate windowFigure 1DR5:: GUS (A) and DR5::GFP (B) activity in the wild type NHR1 and nhr1 backgrounds. (A) Root tips hydrostimulated in a system with air moisture gradient (C and D) or grown in a saturated water conditions (A and B) stained with 1 mM 5-bromo-4-chloro-3-indolyl-β-d-glucuronic (X-Gluc) acid buffer under the same conditions for 80 min. (B) Root tips hydrostimulated as in (A) (C and D) or grown in a saturated water conditions (A and B) whose green fluorescent signal was visualized by confocal microscopy. Shown are images selected from at least 45 representative root tips. Bar = 29 µm.Open in a separate windowFigure 2Expression of DR5::GUS in wild type NHR1 and nhr1 backgrounds. Roots were hydrotropically stimulated for 8 days in a medium with an osmotic gradient (C and D) or grown in normal medium (A and B) and stained with X-Gluc acid buffer under the same conditions for 80 min. Shown are images selected from at least 50 representative root tips. Bar = 25 µm.Our studies⁷ revealed that ABA is a critical regulator of both root gravitropism and hydrotropism in water deficit conditions, and that the role of auxin under these conditions seems to differ from those observed in several studies thus far published on gravitropism made under well-water conditions. The molecular characterization of NHR1 and from other nhr-like mutants already isolated in our lab will clarify the mechanisms involved in this fascinating tropism.¹⁶     

Paradoxical Condensation of Copper with Elevated ��-Amyloid in Lipid Rafts under Cellular Copper Deficiency Conditions: IMPLICATIONS FOR ALZHEIMER DISEASE*

Redox-active copper is implicated in the pathogenesis of Alzheimer disease (AD), β-amyloid peptide (Aβ) aggregation, and amyloid formation. Aβ·copper complexes have been identified in AD and catalytically oxidize cholesterol and lipid to generate H₂O₂ and lipid peroxides. The site and mechanism of this abnormality is not known. Growing evidence suggests that amyloidogenic processing of the β-amyloid precursor protein (APP) occurs in lipid rafts, membrane microdomains enriched in cholesterol. β- and γ-secretases, and Aβ have been identified in lipid rafts in cultured cells, human and rodent brains, but the role of copper in lipid raft amyloidogenic processing is presently unknown. In this study, we found that copper modulates flotillin-2 association with cholesterol-rich lipid raft domains, and consequently Aβ synthesis is attenuated via copper-mediated inhibition of APP endocytosis. We also found that total cellular copper is associated inversely with lipid raft copper levels, so that under intracellular copper deficiency conditions, Aβ·copper complexes are more likely to form. This explains the paradoxical hypermetallation of Aβ with copper under tissue copper deficiency conditions in AD.Imbalance of metal ions has been recognized as one of the key factors in the pathogenesis of Alzheimer disease (AD).² Aberrant interactions between copper or zinc with the β-amyloid peptide (Aβ) released into the glutamatergic synaptic cleft vicinity could result in the formation of toxic Aβ oligomers and aggregation into plaques characteristic of AD brains (reviewed in Ref. 1). Copper, iron, and zinc are highly concentrated in extracellular plaques (2, 3), and yet brain tissues from AD (4–6) and human β-amyloid precursor protein (APP) transgenic mice (7–10) are paradoxically copper deficient compared with age-matched controls. Elevation of intracellular copper levels by genetic, dietary, and pharmacological manipulations in both AD transgenic animal and cell culture models is able to attenuate Aβ production (7, 9, 11–15). However, the underlying mechanism is at present unclear.Abnormal cholesterol metabolism is also a contributing factor in the pathogenesis of AD. Hypercholesterolemia increases the risk of developing AD-like pathology in a transgenic mouse model (16). Epidemiological and animal model studies show that a hypercholesterolemic diet is associated with Aβ accumulation and accelerated cognitive decline, both of which are further aggravated by high dietary copper (17, 18). In contrast, biochemical depletion of cholesterol using statins, inhibitors of 3-hydroxy-3-methyglutaryl coenzyme A reductase, and methyl-β-cyclodextrin, a cholesterol sequestering agent, inhibit Aβ production in animal and cell culture models (19–25).Cholesterol is enriched in lipid rafts, membrane microdomains implicated in Aβ generation from APP cleavage by β- and γ-secretases. Recruitment of BACE1 (β-secretase) into lipid rafts increases the production of sAPP_β and Aβ (23, 26). The β-secretase-cleaved APP C-terminal fragment (β-CTF), and γ-secretase, a multiprotein complex composed of presenilin (PS1 or PS2), nicastrin (Nct), PEN-2 and APH-1, colocalize to lipid rafts (27). The accumulation of Aβ in lipid rafts isolated from AD and APP transgenic mice brains (28) provided further evidence that cholesterol plays a role in APP processing and Aβ generation.Currently, copper and cholesterol have been reported to modulate APP processing independently. However, evidence indicates that, despite tissue copper deficiency, Aβ·Cu²⁺ complexes form in AD that catalytically oxidize cholesterol and lipid to generate H₂O₂ and lipid peroxides (e.g. hydroxynonenal and malondialdehyde), which contribute to oxidative damage observed in AD (29–35). The underlying mechanism leading to the formation of pathological Aβ·Cu²⁺ complexes is unknown. In this study, we show that copper alters the structure of lipid rafts, and attenuates Aβ synthesis in lipid rafts by inhibition of APP endocytosis. We also identify a paradoxical inverse relationship between total cellular copper levels and copper distribution to lipid rafts, which appear to possess a privileged pool of copper where Aβ is more likely to interact with Cu²⁺ under copper-deficiency conditions to form Aβ·Cu²⁺ complexes. These data provide a novel mechanism by which cellular copper deficiency in AD could foster an environment for potentially adverse interactions between Aβ, copper, and cholesterol in lipid rafts.     

The P2X7 receptor and intracellular pathogens: a continuing struggle

The purinergic receptor, P2X₇, has recently emerged as an important component of the innate immune response against microbial infections. Ligation of P2X₇ by ATP can stimulate inflammasome activation and secretion of proinflammatory cytokines, but it can also lead directly to killing of intracellular pathogens in infected macrophages and epithelial cells. Thus, while some intracellular pathogens evade host defense responses by modulating with membrane trafficking or cell signaling in the infected cells, the host cells have also developed mechanisms for inhibiting infection. This review will focus on the effects of P2X₇ on control of infection by intracellular pathogens, microbial virulence factors that interfere with P2X₇ activity, and recent evidence linking polymorphisms in human P2X₇ with susceptibility to infection.     

Temporal dynamics and genetic diversity of chemotactic‐competent microbial populations in the rhizosphere

The contribution of chemotaxis to the competitive colonization of the rhizosphere for the vast majority of the soil community is unknown. We have developed and applied a molecular diagnostic tool, based on a gene encoding the central regulator of bacterial chemotaxis (cheA), to characterize and temporally track specific populations of native microbes with chemotaxis potential that are present in soil exposed to two rhizospheres: wheat and cowpea. The data show that the chemotactic‐competent communities present in the rhizospheres of the two plants are distinct and less diverse than the bulk soil, indicating the development of unique microbial communities. Consistent with the supposition that selection and recruitment of specific soil microbes takes place in the rhizosphere, the dynamics of specific cheA phylotypes provides support for the hypothesis that chemotaxis provides a competitive advantage to some soil microbes. This is the first study to examine and profile the genetic diversity of chemotaxis genes in natural populations. As such, it illustrates our limited understanding of microbial chemotaxis for the majority of soil microbes. It also highlights the value of a culture‐independent approach for examining chemotaxis populations in order to build empirical lines of evidence for its role in structuring of microbial assemblages.