Expression and characterization of recombinant human UDP-glucuronosyltransferases (UGTs). UGT1A9 is more resistant to detergent inhibition than other UGTs and was purified as an active dimeric enzyme

Eight human liver UDP-glucuronosyltransferases (UGTs) were expressed in baculovirus-infected insect cells as fusion proteins carrying a short C-terminal extension that ends with 6 histidine residues (His tag). The activity of recombinant UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B7, and UGT2B15 was almost fully inhibited by 0.2% Triton X-100. In the case of UGT1A9, however, glucuronidation of alpha-naphthol and scopoletin was resistant to such inhibition, whereas glucuronidation of entacapone and several other aglycones was sensitive. His-tagged UGT1A9 was purified by immobilized metal-chelating chromatography (IMAC). Purified UGT1A9 glucuronidated scopoletin at a high rate, whereas its glucuronidation activity toward entacapone was low and largely dependent on phospholipid addition. Recombinant UGT1A9 in which the His tag was replaced by hemagglutinin antigenic peptide (HA tag) was also prepared. Insect cells were co-infected with baculoviruses encoding both HA-tagged and His-tagged UGT1A9. Membranes from the co-infected cells, or a mixture of membranes from separately infected cells, were subjected to detergent extraction and IMAC, and the resulting fractions were analyzed for the presence of each type of UGT1A9 using tag-specific antibodies. In the case of separate infection, the HA-tagged UGT1A9 did not bind to the column. When co-infected with His-tagged UGT1A9, however, part of the HA-tagged enzyme was bound to the column and was eluted by imidazole concentration gradient together with the His-tagged UGT1A9, suggesting the formation of stable dimers that contain one His-tagged and one HA-tagged UGT1A9 monomers.     

Hyperforin inhibits vesicular uptake of monoamines by dissipating pH gradient across synaptic vesicle membrane

Extracts of Hypericum perforatum (St. John's wort) have antidepressant properties in depressed patients and exert antidepressant-like action in laboratory animals. The phloroglucinol derivative hyperforin has become a topic of interest, as this Hypericum component is a potent inhibitor of monoamines reuptake. The molecular mechanism by which hyperforin inhibits monoamines uptake is yet unclear. In the present study we try to clarify the mechanism by which hyperforin inhibits the synaptic vesicle transport of monoamines. The pH gradient across the synaptic vesicle membrane, induced by vacuolar type H(+)-ATPase, is the major driving force for vesicular monoamines uptake and storage. We suggest that hyperforin, like the protonophore FCCP, dissipates an existing Delta pH generated by an efflux of inwardly pumped protons. Proton transport was measured by acridine orange fluorescence quenching. Adding Mg-ATP to a medium containing 130 mM KCl and synaptic vesicles caused an immediate decrease in fluorescence of acridine orange and the addition of 1 microM FCCP abolished this effect. H(+)-ATPase dependent proton pumping was inhibited by hyperforin in a dose dependent manner (IC(50) = 1.9 x 10(-7) M). Hyperforin acted similarly to the protonophore FCCP, abolishing the ATP induced fluorescence quenching (IC(50) = 4.3 x 10(-7) M). Hyperforin and FCCP had similar potencies for inhibiting rat brain synaptosomal uptake of [3H]monoamines as well as vesicular monoamine uptake. The efflux of [3H]5HT from synaptic vesicles was sensitive to both drugs, thus 50% of preloaded [3H]5HT was released in the presence of 2.1 x 10(-7) M FCCP and 4 x 10(-7) M hyperforin. The effect of hyperforin on the pH gradient in synaptic vesicle membrane may explain its inhibitory effect on monoamines uptake, but could only partially explain its antidepressant properties.     

Effect of chlorhexidine on molecular weight distribution of fructans produced by fructosyltransferase in solution and immobilized on surface

The effect of chlorhexidine (CHX), a potent antibacterial agent, was tested on the molecular weight distribution (MWD) of fructans synthesized by cell-free fructosyltransferase (FTF) in solution in comparison to FTF immobilized onto hydroxyapatite (HA). Size-exclusion chromatography (SEC) analysis has shown that cell-free FTF, both in solution and immobilized on HA, produces both low MW (1.9-2.2 kDa) and high MW (913-1047 kDa) fructans. CHX at a concentration of 0.02% altered the MWD of the fructans by reducing the polydispersity ratio and changing the MWD of the fructans synthesized both by immobilized FTF and by FTF in solution. These changes of the fructans in the presence of CHX adds a new prospective to the anticaries effect of CHX in addition to its antibacterial properties.     

Coordinate induction of glutathione biosynthesis and glutathione-metabolizing enzymes is correlated with salt tolerance in tomato

The acclimation of reduced glutathione (GSH) biosynthesis and GSH-utilizing enzymes to salt stress was studied in two tomato species that differ in stress tolerance. Salt increased GSH content and GSH:GSSG (oxidized glutathione) ratio in oxidative stress-tolerant Lycopersicon pennellii (Lpa) but not in Lycopersicon esculentum (Lem). These changes were associated with salt-induced upregulation of gamma-glutamylcysteine synthetase protein, an effect which was prevented by preincubation with buthionine sulfoximine. Salt treatment induced glutathione peroxidase and glutathione-S-transferase but not glutathione reductase activities in Lpa. These results suggest a mechanism of coordinate upregulation of synthesis and metabolism of GSH in Lpa, that is absent from Lem.     

FolM, a new chromosomally encoded dihydrofolate reductase in Escherichia coli

Escherichia coli (thyA DeltafolA) mutants are viable and can grow in minimal medium when supplemented with thymidine alone. Here we present evidence from in vivo and in vitro studies that the ydgB gene determines an alternative dihydrofolate reductase that is related to the trypanosomatid pteridine reductases. We propose to rename this gene folM.     

A new β-lactoglobulin-based vector targets luciferase cDNA expression to the mammary gland of transgenic mice

A -lactoglobulin (BLG)/luciferase gene vector (p907), composed of a luciferase intronless gene inserted between the second and sixth BLG exons was constructed. Stable transfections of CID-9 cells with this vector, as well as with a series of additional vectors, were performed to define regulatory regions within the BLG sequence, and the contribution of the SV40 polyadenylation (PA) site to luciferase expression. A relatively low level of luciferase activity was supported by vector p907. It was partially rescued by vector p906, in which the BLG 3 region, downstream of the luciferase cDNA, was replaced with the SV40 PA site. Flanking the SV40 region of vector p906, at its 3 end, with BLG sequences of exon 6/intron 6/exon 7 and the 3 region of the gene resulted in vector p904. This vector supported the highest luciferase activity, 10 times or 2.5 times higher than that measured in cells transfected with vectors p907 and p906, respectively. The induced activity supported by vector p904 is attributed to interaction between the SV40 PA site and elements of the distal part of the BLG 3 flanking sequences. The BLG 5 regulatory region of vector p904 encompasses a 3-kb promoter sequences. Deletion of 935 bp of its proximal end resulted in a 60% decrease in luciferase activity. Reduced activity was also seen with vector p915 lacking sequences of exon 1/intron 1/exon 2. This decrease could not be rescued with heterologous sequences of insulin intron 1, inserted upstream of the luciferase cDNA. Two sets of transgenic mice carrying vectors p907 and p904 were generated. Vector p907 supported only marginal luciferase activity in the mammary gland of all transgenic mice tested and luciferase RNA could not be detected by northern analysis. In contrast, 50% of the transgenic mice carrying vector p904 expressed luciferase RNA in the mammary gland and tissue-specific, hormonal-dependent activity was determined. However, the new p904 vector was not able to insulate the transgene from surrounding host DNA sequences, as reflected by its copy number-independent manner of expression. Nevertheless, vector p904 may represent a valuable tool for the expression of cDNAs in the mammary gland of transgenic animals.     

Sildenafil-nitric oxide donor combination promotes ventricular tachyarrhythmias in the swine right ventricle

We tested the hypothesis that sildenafil, singly or in combination with nitric oxide (NO) donors, promotes ventricular tachycardia (VT) and ventricular fibrillation (VF). Vulnerability to VT/VF was tested by rapid pacing in eight isolated normal swine right ventricles (RV). The endocardial activation was optically mapped, and the dynamic action potential duration (APD) restitution curves were constructed with metal microelectrodes. At baseline, no VT/VF could be induced. Sildenafil (0.2 microg/ml) or NO donor singly or in combination did not alter VT/VF vulnerability. However, when 2 microg/ml sildenafil was combined with NO donors, the incidence of VT and VF rose significantly (P < 0.01). VT with a single periodic wavefront was induced in five of eight RVs, and VF with multiple wavefronts was induced in all eight RVs. The sildenafil-NO donor pro-VT/VF combination significantly increased the maximum slope of the APD restitution curve and the amplitude of the APD alternans. The pro-VT/VF effects of sildenafil were reversible after drug-free Tyrode solution perfusion. We conclude that a sildenafil (2 microg/ml) and NO donor combination increases VT/VF vulnerability in the normal RV by a mechanism compatible with the restitution hypothesis.     

Multitrophic interactions of the silverleaf whitefly,host plants,competing herbivores,and phytopathogens

Our laboratory found that silverleaf whitefly (SLW; Bemisia argentifolii Bellows & Perring) feeding alters host plant physiology and chemistry. The SLW induces a number of host plant defenses, including pathogenesis-related (PR) protein accumulation (e.g., chitinases, beta-1,3-glucanases, peroxidases, chitosanases, etc.). Induction of the PR proteins by SLW feeding occurs in various plant species and varieties. The extent and type of induction is dependent on a number of factors that include host plant growing conditions, the length of time the host plant is exposed to SLW feeding, the plant variety, and SLW population densities. The appearance of PR proteins correlates well with reduced infestations of conspecific insect herbivore competitors. Greenhouse and field experiments in which herbivore competitors (cabbage looper, Trichoplusia ni; leaf miner, Liromyza trifolii) were placed on plants previously exposed to SLW feeding demonstrated behavioral differences (oviposition, feeding preferences) and reduced survival rates and development times of these insects. The interaction was asymmetrical, i.e., SLW infestations of plants previously exposed to leaf miners had little or no effect on SLW behavior (oviposition). Induction of plant-defensive proteins by SLW feeding was both local (at the feeding site) and systemic (uninfested leaves distant to the feeding site). There are interactions between diseases such as tomato mottle virus (ToMoV; a geminivirus) and the host plant and SLW. PR proteins were induced in tomato plants infected with ToMoV much as they were via non-viruliferous SLW feeding. The presence of ToMoV in tomato plants significantly increased the number of eggs produced by SLW females. Experiments using tomato plants, powdery mildew (PM), and tobacco mosaic virus (TMV) show that whitefly infestations can affect plant pathogen relationships but the effects vary among pathogen types. Enzyme analyses prior to pathogen inoculation showed that whitefly treatment significantly increased the activities of foliar chitinase and peroxidase. Evaluation of pathogen growth 3 weeks after inoculation showed that whitefly feeding significantly reduced the incidence of PM. However, TMV levels evaluated by ELISA were not significantly affected by whitefly feeding. Six weeks after inoculation with pathogens, the chitinase and peroxidase activities were still elevated in plants initially fed on by whiteflies but continuing pathogen infection had no effect on these enzymes. The possibility that geminivirus infection and/or SLW infestations isolate the host plant for the selected reproduction of the virus and the insect is discussed. Multitrophic cascade effects may contribute to the successful eruptive appearance of SLW on various crops, ranking them as a major pest. They may explain the general observation that when SLW infest a host plant there are few if any competing insect herbivores and pathogens found in the host. However, the results indicate that certain SLW-virus relationships could be mutualistic.     

Salt stress induces up-regulation of an efficient chloroplast antioxidant system in the salt-tolerant wild tomato species Lycopersicon pennellii but not in the cultivated species

The response of the chloroplastic antioxidant system of the cultivated tomato Lycopersicon esculentum (Lem) and its wild salt-tolerant related species L. pennellii (Lpa) to NaCl stress was studied. An increase in H₂O₂ level and membrane lipid peroxidation was observed in chloroplasts of salt-stressed Lem. In contrast, a decrease in these indicators of oxidative stress characterized chloroplasts of salt-stressed Lpa plants. This differential response of Lem and Lpa to salinity, correlates with the activities of the antioxidative enzymes in their chloroplasts. Increased activities of total superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), glutathione-S-transferase (GST), phospholipid hydroperoxide glutathione peroxidase (PHGPX) and several isoforms of non-specific peroxidases (POD) were found in chloroplasts of salt-treated Lpa plants. In these chloroplasts, in contrast, activity of lipoxygenase (LOX) decreased while in those of salt-stressed Lem it increased. Although total SOD activity slightly increased in chloroplasts of salt-treated Lem plants, differentiation between SOD types revealed that only stromal Cu/ZnSOD activity increased. In contrast, in chloroplasts of salt-treated Lpa plants FeSOD activity increased while Cu/ZnSOD activity remained unchanged. These data indicate that salt-dependent oxidative stress and damage, suffered by Lem chloroplasts, was effectively alleviated in Lpa chloroplasts by the selective up-regulation of a set of antioxidative enzymes. Further support for the above idea was supplied by leaf discs experiments in which pre-exposure of Lpa plants to salt-treatment conferred cross-tolerance to paraquat-induced oxidative stress while increased oxidative damage by paraquat-treatment was found in salt-stressed Lem plants.