Enteropathogenic Escherichia coli inhibits butyrate uptake in Caco-2 cells by altering the apical membrane MCT1 level

Enteropathogenic Escherichia coli (EPEC), a food-borne human pathogen, is responsible for infantile diarrhea, especially in developing countries. The pathophysiology of EPEC-induced diarrhea, however, is not completely understood. Our recent studies showed modulation of Na+/H+ and Cl-/HCO3- exchange activities in Caco-2 cells in response to EPEC infection. We hypothesized that intestinal short-chain fatty acid absorption mediated by monocarboxylate transporter 1 (MCT1) might also be altered by EPEC infection. The aim of the current studies was to examine the effect of EPEC infection on butyrate uptake. Caco-2 cells were infected with wild-type EPEC, various mutant strains, or nonpathogenic E. coli HS4, and [14C]butyrate uptake was determined. EPEC, but not nonpathogenic E. coli, significantly decreased butyrate uptake. Infection of cells with strains harboring mutations in escN, which encodes a putative ATPase for the EPEC type III secretion system (TTSS), or in the espA, espB, or espD genes encoding structural components of the TTSS, had no effect on butyrate uptake, indicating the TTSS dependence. On the other hand, strains with mutations in the effector protein genes espF, espG, espH, and map inhibited butyrate uptake, similar to the wild-type EPEC. Surface expression of MCT1 decreased considerably after EPEC but not after nonpathogenic E. coli infection. In conclusion, our studies demonstrate inhibition of MCT1-mediated butyrate uptake in Caco-2 cells in response to EPEC infection. This inhibition was dependent on a functional TTSS and the structural proteins EspA, -B, and -D of the translocation apparatus.     

Characterization of a novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis

A novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis has been isolated and characterized. The protein was extracted from the cells with 1 m NaCl, which required 1.5-fold, single-step purification to yield near-homogeneous preparations. In solution, the protein exists as homomeric aggregates, of mean diameter 21.6 nm, consisting of 22-kDa subunits. MS/MS data for peptides obtained by trypsin digestion of the thiosterase did not match any peptide from Escherichia coli thioesterases or any other thioesterases in the database. The thioesterase was associated exclusively with the surface of cells as revealed by ultrastructural studies using electron microscopy and immunogold labeling. It hydrolyzed saturated and unsaturated fatty acyl-CoAs of C12 to C18 chain length with Vmax and Km of 3.58-9.73 micromol x min(-1) x (mg protein)(-1) and 2.66-4.11 microm, respectively. A catalytically important histidine residue is implicated in the active site of the enzyme. The thioesterase was active and stable over a wide range of temperature and pH. Maximum activity was observed at 65 degrees C and pH 10.5, and varied between 60% and 80% at temperatures of 25-70 degrees C and pH 6.5-10. The thioesterase also hydrolyzed p-nitrophenyl esters of C2 to C12 chain length, but substrate competition experiments demonstrated that the long-chain acyl-CoAs are better substrates for thioesterase than p-nitrophenyl esters. When assayed at 37 and 20 degrees C, the affinity and catalytic efficiency of the thioesterase for palmitoleoyl-CoA and cis-vaccenoyl-CoA were reduced approximately twofold at the lower temperature, but remained largely unaltered for palmitoyl-CoA.     

Synthesis of multisubstituted quinolines from Baylis-Hillman adducts obtained from substituted 2-chloronicotinaldehydes and their antimicrobial activity

Baylis-Hillman acetates were synthesized from substituted 2-chloronicotinaldehydes and were conveniently transformed into multisubstituted quinolines and cyclopenta[g]quinolines on reaction with nitroethane or ethyl cyanoacetate via a successive S(N)2'-S(N)Ar elimination strategy. Thus, synthesized quinolines were evaluated for antimicrobial activity and found having substantial antibacterial and antifungal activity.     

Phytotoxicity of the volatile monoterpene citronellal against some weeds

A study was undertaken to assess the phytotoxicity of citronellal, an oxygenated monoterpenoid with an aldehyde group, towards some weedy species [Ageratum conyzoides L., Chenopodium album L., Parthenium hysterophorus L., Malvastrum coromandelianum (L.), Garcke, Cassia occidentalis L. and Phalaris minor Retz.]. A significant effect on weed emergence and early seedling growth was observed in a dose-response based laboratory bioassay in a sand culture. Emergence of all test weeds was completely inhibited at 100 micro/g sand content of citronellal. Seeds of A. conyzoides and P. hysterophorus failed to emerge even at 50 microg/g content. Root length was inhibited more compared to shoot length. The failure of root growth was attributed to the effect of citronellal on the mitotic activity of growing root tips cells as ascertained by the onion root tip bioassay. At 2.5 mM treatment of citronellal, mitosis was completely suppressed and at higher concentrations cells showed various degrees of distortion and were even enucleated. The post-emergent application of citronellal also caused visible injury in the form of chlorosis and necrosis, leading to wilting and even death of test weeds. Among the test weeds, the effect was severe on C. album and P. hysterophorus. There was loss of chlorophyll pigment and reduction in cellular respiration upon citronellal treatment indicating the impairment of photosynthetic and respiratory metabolism. Scanning electron microscopic studies in C. occidentalis leaves upon treatment of citronellal revealed disruption of cuticular wax, clogging of stomata and shrinkage of epidermal cells at many places. There was a rapid electrolyte leakage in the leaf tissue upon exposure to citronellal during the initial few hours. In P. minor electrolyte leakage in response to 2 mM citronellal was closer to the maximum leakage that was obtained upon boiling the tissue. The rapid ion leakage is indicative of the severe effect of citronellal on the membrane structure and loss of membrane integrity. In all, the study concludes that citronellal causes a severe phytotoxicity on the weeds.     

The basis of the immunomodulatory activity of malaria pigment (hemozoin)

The most common and deadly form of the malaria parasite, Plasmodium falciparum, is responsible for 1.5–2.7 million deaths and 300–500 million acute illnesses annually [Bremen in J. Trop. Med. Hyg. 64:1–11 (2001); World Health Organization (2002)]. Hemozoin, the biomineral formed to detoxify the free heme produced during parasitic hemoglobin catabolism, has long been suspected of contributing to the pathological immunodeficiencies that occur during malarial infection. While there is a growing consensus in the literature that native hemozoin maintains immunosuppressive activity, there is considerable controversy over the reactivity of the synthetic form, β-hematin (BH). Given the emerging importance of hemozoin in modulating a host immune response to malarial infection, a careful examination of the effects of the constitutive components of the malaria pigment on macrophage response has been made in order to clarify the understanding of this process. Herein, we present evidence that BH alone is unable to inhibit stimulation of NADPH oxidase and inducible nitric oxide synthase, the key enzymes involved in oxidative burst, and is sensitive to the microbicidal agents of these enzymes both in vitro and in vivo. Further, by systematically examining each of the malaria pigment’s components, we were able to dissect their impact on the immune reactivity of a macrophage model cell line. Reactions between BH and red blood cell (RBC) ghosts effectively reconstituted the observed immunomodulatory reactivity of native hemozoin. Together, these results suggest that the interaction between hemozoin and the RBC lipids results in the generation of toxic products and that these products are responsible for disrupting macrophage function in vivo.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

Neuroprotective effects of NU1025, a PARP inhibitor in cerebral ischemia are mediated through reduction in NAD depletion and DNA fragmentation

Oxidative stress induced cell injury is reported to contribute to the pathogenesis of cerebral ischemia. Reactive oxygen species such as hydrogen peroxide (H2O2) and superoxide radical along with nitric oxide and peroxynitrite generated during ischemia-reperfusion injury, causes the overactivation of poly (ADP-ribose) polymerase (PARP) leading to neuronal cell death. In the present study we have evaluated the effects of PARP inhibitor, 8-hydroxy-2 methyl-quinazolin-4-[3H]one (NU1025) in H2O2 and 3-morphilinosyndonimine (SIN-1) induced cytotoxicity in PC12 cells as well as in middle cerebral artery occlusion (MCAO) induced focal cerebral ischemia in rats. Exposure of PC12 cells to H2O2 (0.4 mM) and SIN-1 (0.8 mM) resulted in a significant decrease in cell viability after 6 h. Pretreatment with NU1025 (0.2 mM) restored cell viability to approximately 73 and 82% in H2O2 and SIN-1 injured cells, respectively. In MCAO studies, NU1025 was administered at different time points (1 h before reperfusion, immediately before reperfusion, 3 h after reperfusion and 6 h after reperfusion). NU1025 at 1 and 3 mg/kg reduced total infarct volume to 25% and 45%, respectively, when administered 1 h before reperfusion. NU1025 also produced significant improvement in neurological deficits. Neuroprotection with NU1025 was associated with reduction in PAR accumulation, reversal of brain NAD depletion and reduction in DNA fragmentation. Results of this study demonstrate the neuroprotective activity of NU1025 and suggest its potential in cerebral ischemia.     

Aldose reductase mediates the lipopolysaccharide-induced release of inflammatory mediators in RAW264.7 murine macrophages

Abnormal production of inflammatory cytokines and chemokines is a key feature of bacterial endotoxin, lipopolysaccharide (LPS)-induced inflammation, and cytotoxicity; however, the mechanisms regulating production of inflammatory markers remain unclear. Herein, we show that inhibition of the aldehyde-metabolizing enzyme aldose reductase (AR; AKR1B3) modulates NF-kappaB-dependent activation of inflammatory cytokines and chemokines in mouse serum, liver, heart, and spleen. Pharmacological inhibition or small interfering RNA ablation of AR prevented the biosynthesis of tumor necrosis factor-alpha, interleukin 1beta, interleukin-6, macrophage-chemoattractant protein-1, and cyclooxygenase-2 and prostaglandin E(2) in LPS-activated RAW264.7 murine macrophages. The AR inhibition or ablation significantly attenuated LPS-induced activation of protein kinase C (PKC) and phospholipase C (PLC), nuclear translocation of NF-kappaB, and phosphorylation and proteolytic degradation of IkappaBalpha in macrophages. Furthermore, treatment of macrophages with 4-hydroxy-trans-2-nonenal (HNE), and cell-permeable esters of glutathionyl-4-hydroxynonanal (GS-HNE) and glutathionyl-1,4-dihydroxynonane (GS-DHN) activated NF-kappaB and PLC/PKC. Pharmacological inhibition or antisense ablation of AR that catalyzes the reduction of GS-HNE to GS-DHN prevented PLC, PKC, IKKalpha/beta, and NF-kappaB activation caused by HNE and GS-HNE, but not by GS-DHN, suggesting that reduced GS-lipid aldehydes catalyzed by AR propagate LPS-induced production of inflammatory markers. Collectively, these data provide evidence that inhibition of AR may be a significant therapeutic approach in preventing bacterial endotoxin-induced sepsis and tissue damage.     

Enhanced alpha1 microglobulin secretion from Hepatitis E virus ORF3-expressing human hepatoma cells is mediated by the tumor susceptibility gene 101

Viruses are known to exploit the host cell machinery for their benefit during different stages of their life cycle within the infected host. One of the major challenges for a virus during the early stages of infection is to escape recognition by the host immune system. Viruses have adopted many novel strategies to evade the host immune response or to create an immune suppressed environment. An earlier study in our laboratory has demonstrated that the ORF3 protein of the hepatitis E virus expedites the secretion of alpha1 microglobulin, an immunosuppressant molecule. Based on this observation, we proposed that enhanced secretion of alpha1 microglobulin may help maintain an immunosuppressed milieu around the infected hepatocyte (Tyagi, S., Surjit, M., Roy, A. K., Jameel, S., and Lal, S. K. (2004) J. Biol. Chem. 279, 29308-29319). In the present study, we discovered that the ability of the ORF3 protein to expedite alpha1 microglobulin secretion is attributed to the PSAP motif present at the C terminus of the former. The ORF3 protein was able to associate with the tumor susceptibility gene 101 (TSG101) through the PSAP motif. Further, a PSAP motif-mutated ORF3 protein was unable to associate with TSG101 and also lost its ability to enhance the secretion of alpha1 microglobulin. In addition, the ORF3 protein was found to associate simultaneously with TSG101 and alpha1 microglobulin because all three of them were co-precipitated as a ternary complex. Finally, a dominant negative mutant of the VPS4 protein was shown to block the enhanced alpha1 microglobulin secretion in ORF3-expressing hepatocytes. These results suggest a mechanism by which the ORF3 protein exploits the endosomal sorting machinery to enhance the secretion of an immunosuppressant molecule (alpha1 microglobulin) from the cultured hepatocytes.     

Nitric oxide (as sodium nitroprusside) supplementation ameliorates Cd toxicity in hydroponically grown wheat roots

Cadmium (Cd) is a non-redox toxic heavy metal present in the environment and induces oxidative stress in plants. We investigated whether exogenous nitric oxide (NO) supplementation as sodium nitroprusside (SNP) has any ameliorating action against Cd-induced oxidative damage in plant roots and thus protective role against Cd toxicity. Cd treatment (50 or 250 μM) alone or in combination with 200 μM SNP was given to hydroponically grown wheat roots for a short time period of 24 h and then these were shifted to distilled water to observe changes in levels of oxidative markers (lipid peroxidation, H₂O₂ content and electrolyte leakage). Supplementation of Cd with SNP significantly reduced the Cd-induced lipid peroxidation, H₂O₂ content and electrolyte leakage in wheat roots. It indicated a reactive oxygen species (ROS) scavenging activity of NO. However, even upon removal of Cd-treatment solution, the levels of oxidative markers increased during 24 h recovery stage and later at 48 h these decreased. Cd treatment resulted in an upregulation of activities of antioxidant enzymes—superoxide dismutase (SOD, 1.15.1.1), guaiacol peroxidase (GPX, 1.11.1.7), catalase (CAT, 1.11.1.6), and glutathione reductase (GR, 1.6.4.2). SNP supply resulted in a reduction in Cd-induced increased activities of scavenging enzymes. The protective role of exogenous NO in decreasing Cd-induced oxidative damage was also evident from the histochemical localization of lipid peroxidation, plasma membrane integrity and superoxides. The study concludes that an exogenous supply of NO protects wheat roots from Cd-induced toxicity.