Sulphate repression of ssuD-dependent alkanesulphonate-sulphur assimilation in Escherichia coli.

Escherichia coli cells utilize alkanesulphonates including taurine as the sulphur source. We previously reported that when E. coli cells carrying a double deletion in tauD and cysN were inoculated into a taurine-containing minimal medium, they started to grow only after long-term incubation (Nishikawa et al. 2018, Microbiology 164: 1446-1456). We show here that cells that can induce ssuD-dependent alkanesulphonate-sulphur assimilation (SASSA) are essentially rare, but suppressors that can induce SASSA appear during long-term incubation. Mutant cells carrying ΔtauD and ΔcysN, ΔcysC or ΔcysH generated suppressor cells that can induce SASSA at a frequency of about 10-6 in a population. Whereas ΔtauD ΔcysN cells without prior SASSA did not express ssuD even when necessary, the cells with prior SASSA properly expressed ssuD. Whole-genome DNA sequencing of a clone isolated from ΔtauD ΔcysN cells with prior SASSA revealed that the influx of sulphate or thiosulphate may be related to the regulation of SASSA. To clarify whether sulphate or thiosulphate affects the induction of SASSA, the effect of mutations in sbp and cysP, which are responsible for sulphate and thiosulphate uptake with different preferences for substrates, was examined. Only the ΔtauD ΔcysN Δsbp mutant did not show repression of SASSA when no sulphate was added to the medium. When the concentration of the sulphate added was over 10 µM, the Δsbp mutant showed repression of SASSA. Therefore, it was considered that the influx of extracellular sulphate resulted in repression of SASSA.

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.

Taurine dioxygenase (tauD)-independent taurine assimilation in Escherichia coli.

On the basis of previous studies on taurine assimilation in Escherichia coli, TauD, an iron- and α-ketoglutarate-dependent taurine dioxygenase, has been regarded as an indispensable factor for assimilation. However, we found that tauD-deficient strains did not lose their taurine assimilation ability when there was no deletion of ssuD, which encodes a reduced flavin mononucleotide [FMNH(2)]-dependent alkanesulfonate monooxygenase, which is responsible for the desulfonation of alkanesulfonates. There were no significant differences in lag phase time, growth rate and final growth yield between the tauD-deficient strain and the tauD wild-type strain. Iron increased the growth rate and final growth yield of the ssuD mutant, but not those of the tauD mutant. The double deletion of tauD and ssuD resulted in the loss of the ability to assimilate taurine. When ssuD was artificially expressed in the double-deletion mutant, the mutant recovered its taurine assimilation ability. These findings indicate that there is another taurine assimilation pathway that is dependent on ssuD but independent of tauD.

Loop of Streptomyces Feruloyl Esterase Plays an Important Role in the Enzyme's Catalyzing the Release of Ferulic Acid from Biomass.

Feruloyl esterases (FAEs) are key enzymes required for the production of ferulic acid from agricultural biomass. Previously, we identified and characterized R18, an FAE from Streptomyces cinnamoneus NBRC 12852, which showed no sequence similarity to the known FAEs. To determine the region involved in its catalytic activity, we constructed chimeric enzymes using R18 and its homolog (TH2-18) from S. cinnamoneus strain TH-2. Although R18 and TH2-18 showed 74% identity in their primary sequences, the recombinant proteins of these two FAEs (recombinant R18 [rR18] and rTH2-18) showed very different specific activities toward ethyl ferulate. By comparing the catalytic activities of the chimeras, a domain comprised of residues 140 to 154 was found to be crucial for the catalytic activity of R18. Furthermore, we analyzed the crystal structure of rR18 at a resolution of 1.5 Å to elucidate the relationship between its activity and its structure. rR18 possessed a typical catalytic triad, consisting of Ser-191, Asp-214, and His-268, which was characteristic of the serine esterase family. By structural analysis, the above-described domain was found to be present in a loop-like structure (the R18 loop), which possessed a disulfide bond conserved in the genus Streptomyces Moreover, compared to rTH2-18 of its parental strain, the TH2-18 mutant, in which Pro and Gly residues were inserted into the domain responsible for forming the R18 loop, showed markedly high kcat values using artificial substrates. We also showed that the FAE activity of TH2-18 toward corn bran, a natural substrate, was improved by the insertion of the Gly and Pro residues.IMPORTANCEStreptomyces species are widely distributed bacteria that are predominantly present in soil and function as decomposers in natural environments. They produce various enzymes, such as carbohydrate hydrolases, esterases, and peptidases, which decompose agricultural biomass. In this study, based on the genetic information on two Streptomyces cinnamoneus strains, we identified novel feruloyl esterases (FAEs) capable of producing ferulic acid from biomass. These two FAEs shared high similarity in their amino acid sequences but did not resemblance any known FAEs. By comparing chimeric proteins and performing crystal structure analysis, we confirmed that a flexible loop was important for the catalytic activity of Streptomyces FAEs. Furthermore, we determined that the catalytic activity of one FAE was improved drastically by inserting only 2 amino acids into its loop-forming domain. Thus, differences in the amino acid sequence of the loop resulted in different catalytic activities. In conclusion, our findings provide a foundation for the development of novel enzymes for industrial use.

Relationship between ventral lumbar disc protrusion and contrast medium leakage during sympathetic nerve block.

Ventral disc protrusions have been neglected because they are asymptomatic. Lumbar sympathetic nerve block (LSNB) is one of the clinical choices for refractory low back pain treatment. Leakage of the contrast medium may occur and lead to complications, especially when using a neurolytic agent. In this study, we retrospectively reviewed the magnetic resonance images (MRIs) of 52 consecutive patients with refractory low back pain due to lumbar spinal canal stenosis who underwent LSNB, and graded ventral disc protrusion at the L1/2 to L5/S1 vertebral discs on a three-point scale (grade 0 = no protrusion, grade 1 = protrusion without migration, grade 2 = protrusion with migration). We also determined if there was leakage of contrast medium in LSNB. Ventral disc protrusion was observed in all patients, and 75 % (39/52) had grade 2 protrusion in the L1/2-L3/4 vertebral discs. Moreover, the incidence of contrast medium leakage was significantly higher at the vertebrae that had grade 2 protrusion than at those with less protrusion. We revealed a higher incidence of ventral disc protrusion of the lumbar vertebrae than previously reported, and that the incidence of leakage in LSNB increased when ventral disc protrusion was present. To avoid complications, attention should be paid to ventral disc protrusions before performing LSNB.

Overexpression of a novel Arabidopsis PP2C isoform, AtPP2CF1, enhances plant biomass production by increasing inflorescence stem growth.

In contrast to mammals, higher plants have evolved to express diverse protein phosphatase 2Cs (PP2Cs). Of all Arabidopsis thaliana PP2Cs, members of PP2C subfamily A, including ABI1, have been shown to be key negative regulators of abscisic acid (ABA) signalling pathways, which regulate plant growth and development as well as tolerance to adverse environmental conditions. However, little is known about the enzymatic and signalling roles of other PP2C subfamilies. Here, we report a novel Arabidopsis subfamily E PP2C gene, At3g05640, designated AtPP2CF1. AtPP2CF1 was dramatically expressed in response to exogenous ABA and was expressed in vascular tissues and guard cells, similar to most subfamily A PP2C genes. In vitro enzymatic activity assays showed that AtPP2CF1 possessed functional PP2C activity. However, yeast two-hybrid analysis revealed that AtPP2CF1 did not interact with PYR/PYL/RCAR receptors or three SnRK2 kinases, which are ABI1-interacting proteins. This was supported by homology-based structural modelling demonstrating that the putative active- and substrate-binding site of AtPP2CF1 differed from that of ABI1. Furthermore, while overexpression of ABI1 in plants induced an ABA-insensitive phenotype, Arabidopsis plants overexpressing AtPP2CF1 (AtPP2CF1oe) were weakly hypersensitive to ABA during seed germination and drought stress. Unexpectedly, AtPP2CF1oe plants also exhibited increased biomass yield, mainly due to accelerated growth of inflorescence stems through the activation of cell proliferation and expansion. Our results provide new insights into the physiological significance of AtPP2CF1 as a candidate gene for plant growth production and for potential application in the sustainable supply of plant biomass.

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.

Differences in cerebral blood flow between missed and generalized seizures with electroconvulsive therapy: a positron emission tomographic study.

While examining the acute effects of electroconvulsive therapy (ECT) on regional cerebral blood flow (rCBF), we could compare the changes in rCBF between missed (not generalized) and generalized seizures using H(2)(15)O positron emission tomography in patients with depression under anesthesia. In contrast to missed seizures, rCBF was increased extensively, particularly in the centrencephalic structures in generalized seizures. These results further support the centrencephalic theory of seizure generalization.

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.

A qualitative analysis of the regulation of cyclic electron flow around photosystem I from the post-illumination chlorophyll fluorescence transient in Arabidopsis: a new platform for the in vivo investigation of the chloroplast redox state.

A transient in chlorophyll fluorescence after cessation of actinic light illumination, which has been ascribed to electron donation from stromal reductants to plastoquinone (PQ) by the NAD(P)H-dehydrogenase (NDH) complex, was investigated in Arabidopsis thaliana. The transient was absent in air in a mutant lacking the NDH complex (ndhM). However, in ndhM, the transient was detected in CO(2)-free air containing 2% O(2). To investigate the reason, ndhM was crossed with a pgr5 mutant impaired in ferredoxin (Fd)-dependent electron donation from NADPH to PQ, which is known to be redundant for NDH-dependent PQ reduction in the cyclic electron flow around photosystem I (PSI). In ndhM pgr5, the transient was absent even in CO(2)-free air with 2% O(2), demonstrating that the post-illumination transient can also be induced by the Fd- (or PGR5)-dependent PQ reduction. On the other hand, the transient increase in chlorophyll fluorescence was found to be enhanced in normal air in a mutant impaired in plastid fructose-1,6-bisphosphate aldolase (FBA) activity. The mutant, termed fba3-1, offers unique opportunities to examine the relative contribution of the two paths, i.e., the NDH- and Fd- (or PGR5)-dependent paths, on the PSI cyclic electron flow. Crossing fba3-1 with either ndhM or pgr5 and assessing the transient suggested that the main route for the PSI cyclic electron flow shifts from the NDH-dependent path to the Fd-dependent path in response to sink limitation of linear electron flow.

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.

Inhibition of epsilon-poly-L-lysine biosynthesis in Streptomycetaceae bacteria by short-chain polyols.

Antimicrobial epsilon-poly-L-lysine (ePL) is secreted by Streptomycetaceae bacteria, and the mechanism of ePL biosynthesis remains to be elucidated. We previously reported that an unknown ePL derivative accumulates in the culture medium of ePL-producing bacteria when glycerol is added to the culture medium (Nishikawa and Ogawa, Appl. Environ. Microbiol. 68:3575-3581, 2002). In this study, by using matrix-assisted laser desorption ionization-time of flight mass spectrometry and nuclear magnetic resonance, we identified the unknown derivative as the ester formed between the hydroxyl group of a glycerol molecule and the terminal carboxyl group of an ePL molecule. When a short-chain aliphatic polyol, such as ethylene glycol, propanediol, or butanediol, was added instead of glycerol, a corresponding ePL-polyol monoester accumulated in the culture medium of ePL-producing bacteria. ePL esterification was accompanied by ePL synthesis in intact cells and a cell-free system, but no esterification of exogenous ePL was observed. ePL-polyol esters were formed during lysine polymerization. The number of lysine residues of ePL-polyol esters decreased with increasing polyol concentration. Taken together, these results indicate that ePL synthesis is inhibited by polyols via esterification and that ePL elongation occurs via the incorporation of lysine monomers into the carboxyl terminus of ePL.

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.

History of obesity as a risk factor for both carotid atherosclerosis and microangiopathy.

As a westernized lifestyle becomes widespread in Japan, the number of individuals with obesity, as well as type 2 diabetes, is rapidly increasing. In this investigation, we studied the prevalence of obesity and its association with the development of diabetic macroangiopathy and microangiopathy. The clinical records of 634 patients in our hospital with type 2 diabetes were surveyed. The relationship between obesity and diabetic retinopathy and nephropathy and macroangiopathy (carotid artery intima-media thickness, IMT) was examined using univariate and multivariate analysis. A body mass index (BMI) > or = 25 kg/m2 was used as the diagnostic criterion for obesity. The prevalence of obesity at the time of the survey was 35% and a history of obesity was reported in 70% of the survey population. Multiple regression analysis revealed that the maximum BMI was significantly correlated with IMT thickening. The prevalence of nephropathy in previously obese patients was significantly higher than in non-obese patients. The maximum BMI was significantly associated with the development of retinopathy and nephropathy, as shown by logistic regression analysis. This suggests that a history of obesity may be an important risk factor for the development of micro- and macroangiopathy in Japanese with type 2 diabetes.

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.