Subanesthetic Dose of Propofol Activates the Reward System in Rats.

BACKGROUND: Propofol has addictive properties, even with a single administration, and facilitates dopamine secretion in the nucleus accumbens (NAc). Activation of the dopaminergic circuits of the midbrain reward system, including the ventral tegmental area (VTA) and NAc, plays a crucial role in addiction. However, the effects of propofol on synaptic transmission and biochemical changes in the VTA-NAc circuit remain unclear. METHODS: We investigated the effects of subanesthetic doses of propofol on rat VTA neurons and excitatory synaptic transmission in the NAc using slice patch-clamp experiments. Using immunohistochemistry and western blot analyses, we evaluated the effects of intraperitoneal propofol administration on the expression of addiction-associated transcription factor ΔFosB (truncated form of the FBJ murine osteosarcoma viral oncogene homolog B protein) in the NAcs in 5-week-old rats. RESULTS: In the current-clamp mode, a subanesthetic dose (0.5-5 µmol/L) of propofol increased the action potential frequency in about half the VTA neurons (excited neurons: control: 9.4 ± 3.0 Hz, propofol 0.5 µmol/L: 21.5 ± 6.0 Hz, propofol 5 µmol/L: 14.6 ± 5.3 Hz, wash: 2.0 ± 0.7 Hz, n = 14/27 cells; unchanged/suppressed neurons: control: 1.68 ± 0.94 Hz, propofol 0.5 µmol/L: 1.0 ± 0.67 Hz, propofol 5 µmol/L: 0.89 ± 0.87 Hz, wash: 0.16 ± 0.11 Hz, n = 13/27 cells). In the voltage-clamp mode, about half the VTA principal neurons showed inward currents with 5 µmol/L of propofol (inward current neurons: control: -20.5 ± 10.0 pA, propofol 0.5 µmol/L: -62.6 ± 14.4 pA, propofol 5 µmol/L: -85.2 ± 18.3 pA, propofol 50 µmol/L: -17.1 ± 39.2 pA, washout: +30.5 ± 33.9 pA, n = 6/11 cells; outward current neurons: control: -33.9 ± 14.6 pA, propofol 0.5 µmol/L: -29.5 ± 16.0 pA, propofol 5 µmol/L: -0.5 ± 20.9 pA, propofol 50 µmol/L: +38.9 ± 18.5 pA, washout: +40.8 ± 32.1 pA, n = 5/11 cells). Moreover, 0.5 µmol/L propofol increased the amplitudes of evoked excitatory synaptic currents in the NAc, whereas >5 µmol/L propofol decreased them (control: 100.0 ± 2.0%, propofol 0.5 µmol/L: 118.4 ± 4.3%, propofol 5 µmol/L: 98.3 ± 3.3%, wash [within 10 min]: 70.7 ± 3.3%, wash [30 minutes later]: 89.9 ± 2.5%, n = 13 cells, P < .001, Dunnett's test comparing control and propofol 0.5 µmol/L). Intraperitoneally administered subanesthetic dose of propofol increased ΔFosB expression in the NAc, but not in VTA, 2 and 24 hours after administration, compared with the Intralipid control group (propofol 2 hours: 0.94 ± 0.15, 24 hours: 0.68 ± 0.07; Intralipid 2 hours: 0.40 ± 0.03, 24 hours: 0.37 ± 0.06, P = .0002 for drug in the 2-way analysis of variance). CONCLUSIONS: Even a single administration of a subanesthetic dose of propofol may cause rewarding change in the central nervous system. Thus, there is a potential propofol rewarding effect among patients receiving anesthesia or sedation with propofol, as well as among health care providers exposed to propofol.

Overexpression of GSH1 gene mimics transcriptional response to low temperature during seed vernalization treatment of Arabidopsis.

Keeping imbibed seeds at low temperatures for a certain period, so-called seed vernalization (SV) treatment, promotes seed germination and subsequent flowering in various plants. Vernalization-promoting flowering requires GSH. However, we show here that increased GSH biosynthesis partially mimics SV treatment in Arabidopsis thaliana. SV treatment (keeping imbibed seeds at 4°C for 24 h) induced a specific pattern of gene expression and promoted subsequent flowering in WT A. thaliana. A similar pattern was observed at 22°C in transgenic (35S-GSH1) plants overexpressing the γ-glutamylcysteine synthetase gene GSH1, coding for an enzyme limiting GSH biosynthesis, under the control of the cauliflower mosaic virus 35S promoter. This pattern of gene expression was further strengthened at 4°C and indistinguishable from the WT pattern at 4°C. However, flowering in 35S-GSH1 plants was less responsive to SV treatment than in WT plants. There was a difference in the transcript behavior of the flowering repressor FLC between WT and 35S-GSH1 plants. Unlike other genes responsive to SV treatment, the SV-dependent decrease in FLC in WT plants was reversed in 35S-GSH1 plants. SV treatment increased the GSSG level in WT seeds while its level was high in 35S-GSH1 plants, even at a non-vernalizing temperature. Taking into consideration that low temperatures stimulate GSH biosynthesis and cause oxidative stress, GSSG is considered to trigger a low-temperature response, although enhanced GSH synthesis was not enough to completely mimic the SV treatment.

Negative regulation of abscisic acid-induced stomatal closure by glutathione in Arabidopsis.

We found that glutathione (GSH) is involved in abscisic acid (ABA)-induced stomatal closure. Regulation of ABA signaling by GSH in guard cells was investigated using an Arabidopsis mutant, cad2-1, that is deficient in the first GSH biosynthesis enzyme, γ-glutamylcysteine synthetase, and a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene (CDNB). Glutathione contents in guard cells decreased along with ABA-induced stomatal closure. Decreasing GSH by both the cad2-1 mutation and CDNB treatment enhanced ABA-induced stomatal closure. Glutathione monoethyl ester (GSHmee) restored the GSH level in cad2-1 guard cells and complemented the stomatal phenotype of the mutant. Depletion of GSH did not significantly increase ABA-induced production of reactive oxygen species in guard cells and GSH did not affect either activation of plasma membrane Ca(2+)-permeable channel currents by ABA or oscillation of the cytosolic free Ca(2+) concentration induced by ABA. These results indicate that GSH negatively modulates a signal component other than ROS production and Ca(2+) oscillation in ABA signal pathway of Arabidopsis guard cells.

Computed tomography fluoroscopy-guided placement of iliosacral screws in patients with unstable posterior pelvic fractures.

OBJECTIVE: The purpose of this study was to evaluate retrospectively the safety and effectiveness of the computed tomography (CT) fluoroscopy-guided placement of iliosacral screws in patients with unstable posterior pelvic fractures. MATERIALS AND METHODS: Six patients (four women and two men; mean age 55.8 years; range 35-77 years) with unstable posterior pelvic fractures underwent iliosacral screw placement under CT fluoroscopy guidance between November 2007 and August 2008. Unstable pelvic ring injury (AO types B and C) was the indication for this procedure. RESULTS: In all the six patients except one, CT fluoroscopy-guided placement had been technically successful. In one patient, a second screw had been inserted, with a tilt to the caudal site, and slightly advanced into the extrasacral body; afterward, it could be exchanged safely for a shorter screw. Five patients and one patient underwent placement of two screws and one screw, respectively. The mean duration of the procedure was 15.0 min (range 9-30 min) per screw; the duration was 12.3 min and 18.2 min for the first and second screws, respectively. No complications requiring treatment occurred during or after the procedure. The mean clinical and radiologic follow-up period was 14 months (range 6-21 months). All pelvic injuries had healed satisfactorily, without complication, and all patients are now doing well clinically and can walk. CONCLUSION: CT fluoroscopy-guided placement of iliosacral screws is a safe and effective treatment in patients with unstable posterior pelvic fractures.

Deficient glutathione in guard cells facilitates abscisic acid-induced stomatal closure but does not affect light-induced stomatal opening.

We investigated the role of glutathione (GSH) in stomatal movements using a GSH deficient mutant, chlorinal-1 (ch1-1). Guard cells of ch1-1 mutants accumulated less GSH than wild types did. Light induced stomatal opening in ch1-1 and wild-type plants. Abscisic acid (ABA) induced stomatal closure in ch1-1 mutants more than wild types without enhanced reactive oxygen species (ROS) production. Therefore, GSH functioned downstream of ROS production in the ABA signaling cascade.

Change in the redox state of glutathione regulates differentiation of tracheary elements in Zinnia cells and Arabidopsis roots.

Exogenously applied GSH and GSSG can control the in vitro differentiation of mesophyll cells to tracheary elements (TEs) in Zinnia elegans, and de novo GSH synthesis is essential for the early differentiation. The purpose of the present study is to address how GSH and GSSG control TE differentiation. GSSG transiently accumulated during the in vitro TE differentiation and exogenously applied GSSG down-regulated transcript levels of GSSG reductase (GR), an enzyme maintaining glutathione in a reduced redox state, while there were no significant changes in transcript levels of enzymes involved in GSH synthesis. Transgenic Arabidopsis overexpressing the GR gene showed delayed TE formation in the root, which was attributed to the suppression of cell division. Exogenously applied GSH had an effect similar to overexpression of the GR gene. These findings suggest that reduced states of glutathione suppress TE differentiation. In wild-type Arabidopsis, TE formation was promoted by application of GSSG at an appropriate concentration, but was suppressed at higher concentrations. A T-DNA-inserted knockout mutant of cytosolic GR exhibited delayed TE formation; this phenotype was little affected by GSSG application. Taken together, the process of the redox changes in glutathione is considered to be controlled via GR activity for TE differentiation.

Glutathione-associated regulation of plant growth and stress responses.

A reduced form of glutathione (GSH) is considered to protect the cell from oxidative damage, based on its redox buffering action and abundance in the cell. However, in plants, the high redox potential molecule ascorbate exists at comparable or higher concentrations and is used for scavenging hydrogen peroxide as an electron donor. Recently, examples that cannot be explained simply by the antioxidant activity of GSH have been increasing in number. This article summarizes the recent findings on the glutathione-associated events in plants, in particular, growth and development including cell differentiation, cell death and senescence, pathogen resistance, and enzymatic regulation.

Hybrid lethality of cultured cells of an interspecific F1 hybrid of Nicotiana gossei Domin and N. tabacum L.

Cultured cells were established from the hypocotyl of F(1) hybrid seedlings of Nicotiana gossei Domin and N. tabacum L. The cultured cells started to die at 26 degrees C, but not at 37 degrees C, which is similar to what occurred in cells of the original hybrid plants. An increase in the number of cells without cytoplasmic strands and acidification of the cytoplasm followed by decomposition of the mitochondria and chloroplasts indicated that vacuolar collapse plays a central role in the execution of cell death. Oxygen but not light was required for cell death. Cellular levels of the superoxide anion and hydrogen peroxide temporarily increased during the early phase at 26 degrees C, while no such oxidative burst was observed at 37 degrees C. The reactive oxygen intermediates are potentially involved in the death of the hybrid cells.

Induction of PR-1 accumulation accompanied by runaway cell death in the lsd1 mutant of Arabidopsis is dependent on glutathione levels but independent of the redox state of glutathione.

The lesions simulating disease (lsd) mutants of Arabidopsis spontaneously develop hypersensitive-response-like lesions in the absence of pathogens. To address the function of the redox regulator glutathione in disease resistance, we examined the relationship between endogenous glutathione and PR-1 accumulation using one of these mutants, lsd1, as a disease resistance model. Lesion formation on lsd1 was suppressed by weak light and initiated by the subsequent transition to normal light. The application of buthionine sulfoximine, a specific inhibitor of glutathione biosynthesis, suppressed conditionally induced runaway cell death and expression of the PR-1 gene, suggesting that glutathione regulates the conditional cell death and PR-1 gene expression. The application of reduced (GSH) or oxidized (GSSG) glutathione to lsd1 upregulated the level of total glutathione ([GSH]+[GSSG]) accompanied by hastened accumulation of PR-1, and the basal level of total glutathione in lsd1 was higher than that in wild-type plants. The glutathione redox state defined as [GSH]/([GSH]+[GSSG]) decreased following the conditional transition, but the suppression of this decrease by the application of GSH did not inhibit the accumulation of PR-1. Taken together, conditional PR-1 accumulation in lsd1 is regulated not by the redox state but by the endogenous level of glutathione.

Occurrence of D-histidine residues in antimicrobial poly(arginyl-histidine), conferring resistance to enzymatic hydrolysis.

The antimicrobial peptide poly(arginyl-histidine) is secreted by the ergot fungus Verticillium kibiense. We previously showed that poly(arginyl-histidine) from the fungus inhibits the growth of certain microorganisms more effectively than that chemically synthesized from the L-form of arginine and histidine, implying some substantial differences between the fungal and synthetic peptides. To elucidate what causes such differences, we here investigated the structural features of the fungal peptides. The acid hydrolysates of the fungal peptide contained d-histidine. When synthetic poly(L-arginyl-D-histidine) mimicking the fungal peptide was added to the culture of Salmonella typhimurium together with poly(L-arginyl-L-histidine), poly(L-arginyl-D-histidine) was not easily degraded during the incubation compared with poly(L-arginyl-L-histidine). We concluded that the d-form of histidine residues in the fungal peptide prolongs the life of the peptide leading to the enhancement of antimicrobial activity.

Reduced glutathione is a novel regulator of vernalization-induced bolting in the rosette plant Eustoma grandiflorum.

The transition from the vegetative rosette stage to the reproductive growth stage (bolting) in the rosette plant Eustoma grandiflorum has a strict requirement for vernalization, a treatment that causes oxidative stress. Since we have shown that reduced glutathione (GSH) and its biosynthesis are associated with bolting in another rosette plant Arabidopsis thaliana, we here investigated whether a similar mechanism governs the vernalization-induced bolting of E. grandiflorum. Addition of GSH or its precursor cysteine, instead of vernalization, induced bolting but other thiols, dithiothreitol and 2-mercaptoethanol, did not. The inductive effect of vernalization on bolting was nullified by addition of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, without decreasing the plant growth rate. BSO-mediated inhibition of bolting was reversed by addition of GSH but not by cysteine. These indicate that vernalization-induced bolting involves GSH biosynthesis and is specifically regulated by GSH. Plant GSH increased during the early vernalization period along with the activity of gamma-glutamylcysteine synthetase that catalyzes the first step of GSH biosynthesis, although there was little change in amounts of GSH precursor thiols, cysteine and gamma-glutamylcysteine. These findings strongly suggest that vernalization stimulates GSH synthesis and synthesized GSH specifically determines the bolting time of E. grandiflorum.

Level of glutathione is regulated by ATP-dependent ligation of glutamate and cysteine through photosynthesis in Arabidopsis thaliana: mechanism of strong interaction of light intensity with flowering.

Glutathione (GSH) is associated with flowering in Arabidopsis thaliana, but how GSH biosynthesis is regulated to control the transition to flowering remains to be elucidated. Since the key reaction of GSH synthesis is catalyzed by gamma-glutamylcysteine synthetase (gamma-ECS) and all the gamma-ECS cDNAs examined contained extra sequences for plastid targeting, we investigated the relationships among GSH levels, photosynthesis and flowering. The GSH level in Arabidopsis increased with the light intensity. The ch1 mutants defective in a light-harvesting antenna in photosystem II showed reduced GSH levels with accumulation of the GSH precursor cysteine, and introduction of the gamma-ECS gene GSH1 under the control of the cauliflower mosaic virus 35S promoter (35S-GSH1) into the ch1 mutant altered the GSH level in response to the gamma-ECS mRNA level. These indicate that photosynthesis limits the gamma-ECS reaction to regulate GSH biosynthesis. Like the glutathione-biosynthesis-defective cad2-1 mutant, the ch1 mutants flowered late under weak-light conditions, and this late-flowering phenotype was rescued by supplementation of GSH. Introduction of the 35S-GSH1 construct into the ch1 mutant altered flowering in response to the gamma-ECS mRNA and GSH levels. These findings indicate that flowering in A. thaliana is regulated by the gamma-ECS reaction of GSH synthesis that is coupled with photosynthesis.

Antimicrobial activity of a chelatable poly(arginyl-histidine) produced by the ergot fungus Verticillium kibiense.

We have recently developed a convenient method of screening a broad range of microorganisms that produce epsilon-poly-L-lysine (M. Nishikawa and K. Ogawa, Appl. Environ. Microbiol. 68:3575-3581, 2002). Using this method, we found an ergot fungus that secretes a charged polypeptide other than epsilon-poly-L-lysine. It was identified as a new species on the basis of its 28S rRNA sequence and was named Verticillium kibiense (formerly Epichloe kibiensis). Peptide sequencing and mass spectrometry revealed that the polypeptide is a linear peptide composed of repeated units of arginyl-histidine. The numbers of repeated units were in most cases five and in some cases four or six. This peptide showed activity against a broad range of bacteria and fungi but lost its activity under conditions of high ionic strength. Zinc and copper ions specifically changed the circular dichroism spectra of the peptide and restored the antimicrobial activity from abrogation under high ionic conditions, although these ions had no reinforcing effect on antimicrobial activity when they were added to solutions at a low ionic strength. The peptide labeled with fluorescein was able to permeate the cell membranes of target microbes, but its ability to permeate cell membranes decreased under conditions of high ionic strength. This decreased ability was partially recovered specifically by the addition of zinc and copper ions. These results indicate that poly(arginyl-histidine) is a cationic polypeptide characterized by specific metal binding and resistance to salts.

The sugar-metabolic enzymes aldolase and triose-phosphate isomerase are targets of glutathionylation in Arabidopsis thaliana: detection using biotinylated glutathione.

GSH has multiple actions in physiological responses of plants, but the molecular mechanisms are not fully understood. GSH plays an important role in functional alteration of proteins by reversible covalent incorporation (glutathionylation) in vertebrate cells. To investigate the function of glutathionylation in plant cells, we examined glutathionylated proteins in the suspension-cultured cells of Arabidopsis using biotinylated GSH. Biotinylated GSH was incorporated into about 20 proteins. Two of these proteins were identified as the key enzymes for sugar metabolism, triose-phosphate isomerase (TPI) and putative plastidic aldolase. Recombinant TPI was inactivated by GSSG, and it was reactivated by GSH. The physiological roles of glutathionylation of TPI and aldolase in sugar metabolism are discussed.

Cadmium-dependent generation of reactive oxygen species and mitochondrial DNA breaks in photosynthetic and non-photosynthetic strains of Euglena gracilis.

The photosynthetic strain Z of Euglena gracilis is more susceptible to cadmium chloride (Cd) than the non-photosynthetic strain SMZ. We investigated the correlation of intracellular reactive oxygen species (ROS) levels with Cd-induced cellular damage. Flow cytometry with dihydrorhodamine 123 showed that strain Z generated higher levels of ROS, probably H(2)O(2) and/or ONOO(-), than strain SMZ, and that this difference between the two strains became more pronounced with increasing Cd dose. The levels of ROS increased at cytotoxic concentrations of Cd, at over 10 microM Cd for Z and 50 microM Cd for SMZ. These results show an association of Cd cytotoxicity with ROS generation. Considering that strain SMZ is non-photosynthetic, the higher levels of ROS in strain Z might be due to blockage of photosynthetic electron flow by Cd. Using terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labeling analysis in combination with 4',6-diamidino-2-phenylindole, dihydrochloride staining, we observed DNA breaks in the mitochondria of both strains after Cd exposure. The results suggest that the mitochondrion is the primary target organelle of Cd in E. gracilis cells.

Cell death processes during expression of hybrid lethality in interspecific F1 hybrid between Nicotiana gossei Domin and Nicotiana tabacum.

Hybrid lethality, a type of reproductive isolation, is a genetically controlled event appearing at the seedling stage in interspecific hybrids. We characterized the lethality of F(1) hybrid seedlings from Nicotiana gossei Domin and Nicotiana tabacum cv Bright-Yellow 4 using a number of traits including growth rate, microscopic features of tissues and cells, ion leakage, DNA degradation, reactive oxygen intermediates including superoxide radical (O(2)(-)) and hydrogen peroxide (H(2)O(2)), and expression of stress response marker genes. Lethal symptoms appeared at 4 d after germination in the basal hypocotyl and extended toward both the hypocotyl and root of the plants grown at 26 degrees C. Microscopic analysis revealed a prompt lysis of cell components during cell death. Membrane disruption and DNA degradation were found in the advanced stage of the lethality. The death of mesophyll cells in the cotyledon was initiated by the vascular bundle, suggesting that a putative factor inducing cell death diffused into surrounding cells from the vascular tissue. In contrast, these symptoms were not observed in the plants grown at 37 degrees C. Seedlings grown at 26 degrees C generated larger amounts of reactive oxygen intermediate in the hypocotyl than those grown at 37 degrees C. A number of stress response marker genes were expressed at 26 degrees C but not at 37 degrees C. We proposed that a putative death factor moving systemically through the vascular system induced a prompt and successive lysis of the cytoplasm of cells and that massive cell death eventually led to the loss of the hybrid plant.

An intramolecular disulfide bridge as a catalytic switch for serotonin N-acetyltransferase.

Serotonin N-acetyltransferase (EC. 2.3.1.87) (AA-NAT) is a melatonin rhythm-generating enzyme in pineal glands. To establish a melatonin rhythm, AA-NAT activity is precisely regulated through several signaling pathways. Here we show novel regulation of AA-NAT activity, in which an intramolecular disulfide bond may function as a switch for the catalysis. Recombinant AA-NAT activity was irreversibly inhibited by N-ethylmaleimide (NEM) in an acetyl-CoA-protected manner. Oxidized glutathione or dissolved oxygen reversibly inhibited AA-NAT in an acetyl-CoA-protected manner. To identify the cysteine residues responsible for the inhibition, AA-NAT was first oxidized with dissolved oxygen, treated with NEM, reduced with dithiothreitol, and then labeled with [(14)C]NEM. Cys(61) and Cys(177) were specifically labeled in an acetyl-CoA-protected manner. The AA-NAT with the Cys(61) to Ala and Cys(177) to Ala double substitutions (C61A/C177A-AA-NAT) was fully active but did not exhibit sensitivity to either oxidation or NEM, whereas the AA-NATs with only the single substitutions retained about 40% of these sensitivities. An intramolecular disulfide bond between Cys(61) and Cys(177) formed upon oxidation and cleaved upon reduction was identified. Furthermore, C61A/C177A-AA-NAT expressed in COS7 cells was relatively insensitive to H(2)O(2)-evoked oxidative stress, whereas wild-type AA-NAT was strongly inhibited under the same conditions. These results indicate that the formation and cleavage of the disulfide bond between Cys(61) and Cys(177) produce the active and inactive states of AA-NAT. It is possible that intracellular redox conditions regulate AA-NAT activity through switching via an intramolecular disulfide bridge.

Singlet oxygen imaging in Arabidopsis thaliana leaves under photoinhibition by excess photosynthetically active radiation.

Arabidopsis thaliana leaves were infiltrated with DanePy (3-(N-diethylaminoethyl)-N-dansyl)aminomethyl-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrole), a double, fluorescent and spin sensor of singlet oxygen. DanePy fluorescence was imaged by laser scanning microscopy. We found that DanePy penetrated into chloroplasts but did not alter the functioning of the photosynthetic electron transport as assessed by chlorophyll fluorescence induction. In imaging, DanePy fluorescence was well distinct from chlorophyll fluorescence. Photoinhibition by excess photosynthetically active radiation caused quenching of DanePy fluorescence in the chloroplasts but not in other cell compartments. When leaves were infiltrated with dansyl, the fluorescent group in DanePy, there was no fluorescence quenching during photoinhibition. This shows that the fluorescence quenching of DanePy is caused by the conversion of its pyrrol group into nitroxide, i.e. it was caused by the reaction of singlet oxygen with the double sensor and not by artifacts. These data provide direct experimental evidence for the localization of singlet oxygen production to chloroplasts in vivo.