Ketomethylthiobutyric acid formation from methylthioadenosine: A diffusion assay

Typical enzyme kinetics were observed when 5′-methylthioadenosine was used as substrate with extracts of malignant murine cells in a diffusion assay. The volatile product was measured after diffusion into a solution of the sulfhydryl reagent, 5,5′-dithiobis(2-nitrobenzoic acid), which it reduced to a yellow chromophore. Cysteine was required in the system. The volatile product was identified as H₂S derived from the cysteine. The yield of H₂S was similar to the amount of 2-keto-4-methylthiobutyric acid (KMTB) formed from methylthioadenosine when the KMTB was measured simultaneously in an ether extraction assay. KMTB could replace methylthioadenosine as a substrate capable of causing the formation of the diffusible product from cysteine. It is concluded that the following sequence of reactions takes place in the diffusion assay system: (1) 5′-methylthioadenosine + P_i → adenine + 5-methylthioribose-1-P, (2) 5-methylthioribose-1-P → KMTB, (3) KMTB + cysteine → methionine + 3-mercaptopyruvate, (4) 3-mercaptopyruvate + excess R-SH → pyruvate + H₂S, (5) H₂S + 5,5′-dithiobis(2-nitrobenzoic acid) → 5-mercapto-2-nitrobenzoic acid. Thus, the diffusion assay measures the amount of KMTB formed. The key enzyme, cysteine aminotransferase, EC 2.6.1.3, was partially purified from malignant cells and from liver and several of its characteristics are described. The diffusion assay using this enzyme is useful in measuring de novo synthesis of α-keto acids and it is applicable to crude enzyme preparations. The sensitivity is about 5 nmol of keto acid and the accurate range is 5 to 100 nmol.     

Novel Physiological Features of Carboxydothermus hydrogenoformans and Thermoterrabacterium ferrireducens

Carboxydothermus hydrogenoformans is able to grow by conversion of CO to H₂ and CO₂. Besides CO, only pyruvate was described as serving as an energy source. Based on 16S rRNA gene sequence similarity, C. hydrogenoformans is closely related to Thermoterrabacterium ferrireducens. T. ferrireducens is like C. hydrogenoformans a gram-positive, thermophilic, strict anaerobic bacterium. However, it is capable of using various electron donors and acceptors for growth. Growth of C. hydrogenoformans with multiple electron donors and acceptors was tested. C. hydrogenoformans oxidized formate, lactate, glycerol, CO, and H₂ with 9,10-anthraquinone-2,6-disulfonate as an electron acceptor. Sulfite, thiosulfate, sulfur, nitrate, and fumarate were reduced with lactate as an electron donor. T. ferrireducens oxidized CO with 9,10-anthraquinone-2,6-disulfonate as an electron acceptor but did not produce H₂ from CO. In contrast to what was published before, T. ferrireducens was able to grow on lactate with sulfite, sulfur, and nitrate as electron acceptors.     

Effect of Hydrogen Sulfide on Sympathetic Neurotransmission and Catecholamine Levels in Isolated Porcine Iris-Ciliary Body

In the present study, we investigated the pharmacological action of hydrogen sulfide (H₂S, using sodium hydrosulfide, NaHS, and/or sodium sulfide, Na₂S as donors) on sympathetic neurotransmission from isolated, superfused porcine iris-ciliary bodies. We also examined the effect of H₂S on norepinephrine (NE), dopamine and epinephrine concentrations in isolated porcine anterior uvea. Release of [³H]NE was triggered by electrical field stimulation and basal catecholamine concentrations was measured by high performance liquid chromatography (HPLC). Both NaHS and Na₂S caused a concentration-dependent inhibition of electrically evoked [³H]NE release from porcine iris-ciliary body without affecting basal [³H]NE efflux. The inhibitory action of H₂S donors on NE release was attenuated by aminooxyacetic acid (AOA) and propargyglycine (PAG), inhibitors of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), respectively. With the exception of dopamine, NaHS caused a concentration-dependent reduction in endogenous NE and epinephrine concentrations in isolated iris-ciliary bodies. We conclude that H₂S can inhibit sympathetic neurotransmission from isolated porcine anterior uvea, an effect that is dependent, at least in part, on intramural biosynthesis of this gas. Furthermore, the observed action of H₂S donors on sympathetic transmission may be due to a direct action of this gas on neurotransmitter pools.     

Hydrogen sulfide is involved in maintaining ion homeostasis via regulating plasma membrane Na+/H+ antiporter system in the hydrogen peroxide-dependent manner in salt-stress Arabidopsis thaliana root

Hydrogen sulfide (H₂S) and hydrogen peroxide (H₂O₂) function as the signaling molecules in plants responding to salt stresses. The present study presents a signaling network involving H₂S and H₂O₂ in salt resistance pathway of the Arabidopsis root. Arabidopsis roots were sensitive to 100 mM NaCl treatment, which displayed a great increase in electrolyte leakage (EL) and Na⁺/K⁺ ratio under salt stress. The treatment of H₂S donors sodium hydrosulfide (NaHS) enhanced the salt tolerance by maintaining a lower Na⁺/K⁺ ratio. In addition, the inhibition of root growth under salt stress was removed by H₂S. Further studies indicated that H₂O₂ was involved in H₂S-induced salt tolerance pathway. H₂S induced the production of the endogenous H₂O₂ via regulating the activities of glucose-6-phosphate dehydrogenase (G6PDH) and plasma membrane (PM) NADPH oxidase, with the treatment with dimethylthiourea (DMTU, an ROS scavenger), diphenylene iodonium (DPI, a PM NADPH oxidase inhibitor), or glycerol (G6PDH inhibitor) removing the effect of H₂S. Treatment with amiloride (an inhibitor of PM Na⁺/H⁺ antiporter) and vanadate (an inhibitor of PM H⁺-ATPase) also inhibited the activity of H₂S on Na⁺/K⁺ ratio. Through an analysis of quantitative real-time polymerase chain reaction and Western blot, we found that H₂S promoted the genes expression and the phosphorylation level of PM H⁺-ATPase and Na⁺/H⁺ antiporter protein level. However, when the endogenous H₂O₂ level was inhibited by DPI or DMTU, the effect of H₂S on the PM Na⁺/H⁺ antiporter system was removed. Taken together, H₂S maintains ion homeostasis in the H₂O₂-dependent manner in salt-stress Arabidopsis root.     

Pharmacological Actions of Hydrogen Sulfide Donors on Sympathetic Neurotransmission in the Bovine Anterior Uvea,In Vitro

In the present study, we investigated the effect of three different sources of hydrogen sulfide (H₂S) on sympathetic neurotransmission from isolated superfused bovine iris-ciliary bodies. The three agents under consideration were: ACS67, a hybrid of latanoprost and a H₂S-donating moiety; l-cysteine, a substrate for endogenous production of H₂S and GYY 4137, a slow donor of H₂S. We also examined the contribution of prostaglandins to the pharmacological actions of the H₂S donors on release of [³H]-norepinephrine ([³H]NE) triggered by electrical field stimulation. ACS67, l-cysteine and GYY 4137 caused a concentration-dependent inhibition of electrically-evoked [³H]NE release from isolated bovine iris-ciliary bodies without affecting basal [³H]NE efflux. The cyclooxygenase inhibitor, flurbiprofen enhanced the inhibitory action of ACS67 and l-cysteine on stimulated [³H]NE release. Both aminooxyacetic acid, an inhibitor of cystathionine-β-synthase and glibenclamide, a K_ATP channel blocker reversed the inhibition of evoked NE release induced by the H₂S donors. We conclude that H₂S donors can inhibit sympathetic neurotransmission from isolated bovine iris-ciliary bodies, an effect partially dependent on the in situ production of H₂S and prostanoids, and is mediated by an action on K_ATP channels.     

Relative Importance of H2 and H2S as Energy Sources for Primary Production in Geothermal Springs

Geothermal waters contain numerous potential electron donors capable of supporting chemolithotrophy-based primary production. Thermodynamic predictions of energy yields for specific electron donor and acceptor pairs in such systems are available, although direct assessments of these predictions are rare. This study assessed the relative importance of dissolved H₂ and H₂S as energy sources for the support of chemolithotrophic metabolism in an acidic geothermal spring in Yellowstone National Park. H₂S and H₂ concentration gradients were observed in the outflow channel, and vertical H₂S and O₂ gradients were evident within the microbial mat. H₂S levels and microbial consumption rates were approximately three orders of magnitude greater than those of H₂. Hydrogenobaculum-like organisms dominated the bacterial component of the microbial community, and isolates representing three distinct 16S rRNA gene phylotypes (phylotype = 100% identity) were isolated and characterized. Within a phylotype, O₂ requirements varied, as did energy source utilization: some isolates could grow only with H₂S, some only with H₂, while others could utilize either as an energy source. These metabolic phenotypes were consistent with in situ geochemical conditions measured using aqueous chemical analysis and in-field measurements made by using gas chromatography and microelectrodes. Pure-culture experiments with an isolate that could utilize H₂S and H₂ and that represented the dominant phylotype (70% of the PCR clones) showed that H₂S and H₂ were used simultaneously, without evidence of induction or catabolite repression, and at relative rate differences comparable to those measured in ex situ field assays. Under in situ-relevant concentrations, growth of this isolate with H₂S was better than that with H₂. The major conclusions drawn from this study are that phylogeny may not necessarily be reliable for predicting physiology and that H₂S can dominate over H₂ as an energy source in terms of availability, apparent in situ consumption rates, and growth-supporting energy.     

Role of hydrogen sulfide donors in cancer development and progression

In recent years, a vast number of potential cancer therapeutic targets have emerged. However, developing efficient and effective drugs for the targets is of major concern. Hydrogen sulfide (H₂S), one of the three known gasotransmitters, is involved in the regulation of various cellular activities such as autophagy, apoptosis, migration, and proliferation. Low production of H₂S has been identified in numerous cancer types. Treating cancer cells with H₂S donors is the common experimental technique used to improve H₂S levels; however, the outcome depends on the concentration/dose, time, cell type, and sometimes the drug used. Both natural and synthesized donors are available for this purpose, although their effects vary independently ranging from strong cancer suppressors to promoters. Nonetheless, numerous signaling pathways have been reported to be altered following the treatments with H₂S donors which suggest their potential in cancer treatment. This review will analyze the potential of H₂S donors in cancer therapy by summarizing key cellular processes and mechanisms involved.     

Selective inhibition of a plasma fucosyltransferase by N-ethylmaleimide.

Two fucosyltransferases have been found in plasma: the blood group H-dependent GDP-L-fucose:galactoside 2′ fucosyltransferase and a GDP-L-fucose:N-acetylglucosaminide fucosyltransferase. The presence of endogenous acceptors for both enzymes in plasma from normal donors and leukemia patients has complicated measurement of levels of the individual enzymes. We have found that the sulfhydryl reagent N-ethylmaleimide, at 3 mM, inhibits the H-gene specified enzyme without affecting the other. Both enzymes have been partly characterized here with regard to K_m, Mg requirement and sensitivity to inhibitors.     

Regulation of Vascular Tone,Angiogenesis and Cellular Bioenergetics by the 3-Mercaptopyruvate Sulfurtransferase/H2S Pathway: Functional Impairment by Hyperglycemia and Restoration by dl-α-Lipoic Acid

Hydrogen sulfide (H₂S), as a reducing agent and an antioxidant molecule, exerts protective effects against hyperglycemic stress in the vascular endothelium. The mitochondrial enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) is an important biological source of H₂S. We have recently demonstrated that 3-MST activity is inhibited by oxidative stress in vitro and speculated that this may have an adverse effect on cellular homeostasis. In the current study, given the importance of H₂S as a vasorelaxant, angiogenesis stimulator and cellular bioenergetic mediator, we first determined whether the 3-MST/H₂S system plays a physiological regulatory role in endothelial cells. Next, we tested whether a dysfunction of this pathway develops during the development of hyperglycemia and μmol/L to diabetes-associated vascular complications. Intraperitoneal (IP) 3-MP (1 mg/kg) raised plasma H₂S levels in rats. 3-MP (10 1 mmol/L) promoted angiogenesis in vitro in bEnd3 microvascular endothelial cells and in vivo in a Matrigel assay in mice (0.3–1 mg/kg). In vitro studies with bEnd3 cell homogenates demonstrated that the 3-MP-induced increases in H₂S production depended on enzymatic activity, although at higher concentrations (1–3 mmol/L) there was also evidence for an additional nonenzymatic H₂S production by 3-MP. In vivo, 3-MP facilitated wound healing in rats, induced the relaxation of dermal microvessels and increased mitochondrial bioenergetic function. In vitro hyperglycemia or in vivo streptozotocin diabetes impaired angiogenesis, attenuated mitochondrial function and delayed wound healing; all of these responses were associated with an impairment of the proangiogenic and bioenergetic effects of 3-MP. The antioxidants dl-α-lipoic acid (LA) in vivo, or dihydrolipoic acid (DHLA) in vitro restored the ability of 3-MP to stimulate angiogenesis, cellular bioenergetics and wound healing in hyperglycemia and diabetes. We conclude that diabetes leads to an impairment of the 3-MST/H₂S pathway, and speculate that this may contribute to the pathogenesis of hyperglycemic endothelial cell dysfunction. We also suggest that therapy with H₂S donors, or treatment with the combination of 3-MP and lipoic acid may be beneficial in improving angiogenesis and bioenergetics in hyperglycemia.     

Effects of pH and Lactate on Hydrogen Sulfide Production by Oral Veillonella spp.

Indigenous oral bacteria in the tongue coating such as Veillonella have been identified as the main producers of hydrogen sulfide (H₂S), one of the major components of oral malodor. However, there is little information on the physiological properties of H₂S production by oral Veillonella such as metabolic activity and oral environmental factors which may affect H₂S production. Thus, in the present study, the H₂S-producing activity of growing cells, resting cells, and cell extracts of oral Veillonella species and the effects of oral environmental factors, including pH and lactate, were investigated. Type strains of Veillonella atypica, Veillonella dispar, and Veillonella parvula were used. These Veillonella species produced H₂S during growth in the presence of l-cysteine. Resting cells of these bacteria produced H₂S from l-cysteine, and the cell extracts showed enzymatic activity to convert l-cysteine to H₂S. H₂S production by resting cells was higher at pH 6 to 7 and lower at pH 5. The presence of lactate markedly increased H₂S production by resting cells (4.5- to 23.7-fold), while lactate had no effect on enzymatic activity in cell extracts. In addition to H₂S, ammonia was produced in cell extracts of all the strains, indicating that H₂S was produced by the catalysis of cystathionine γ-lyase (EC 4.4.1.1). Serine was also produced in cell extracts of V. atypica and V. parvula, suggesting the involvement of cystathionine β-synthase lyase (EC 4.2.1.22) in these strains. This study indicates that Veillonella produce H₂S from l-cysteine and that their H₂S production can be regulated by oral environmental factors, namely, pH and lactate.     

Hydrogen Sulfide Protects HUVECs against Hydrogen Peroxide Induced Mitochondrial Dysfunction and Oxidative Stress

Background

Hydrogen sulfide (H₂S) has been shown to have cytoprotective effects in models of hypertension, ischemia/reperfusion and Alzheimer''s disease. However, little is known about its effects or mechanisms of action in atherosclerosis. Therefore, in the current study we evaluated the pharmacological effects of H₂S on antioxidant defenses and mitochondria protection against hydrogen peroxide (H₂O₂) induced endothelial cells damage.

Methodology and Principal Findings

H₂S, at non-cytotoxic levels, exerts a concentration dependent protective effect in human umbilical vein endothelial cells (HUVECs) exposed to H₂O₂. Analysis of ATP synthesis, mitochondrial membrane potential (ΔΨm) and cytochrome c release from mitochondria indicated that mitochondrial function was preserved by pretreatment with H₂S. In contrast, in H₂O₂ exposed endothelial cells mitochondria appeared swollen or ruptured. In additional experiments, H₂S was also found to preserve the activities and protein expressions levels of the antioxidants enzymes, superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase in H₂O₂ exposed cells. ROS and lipid peroxidation, as assessed by measuring H₂DCFDA, dihydroethidium (DHE), diphenyl-l-pyrenylphosphine (DPPP) and malonaldehyde (MDA) levels, were also inhibited by H₂S treatment. Interestingly, in the current model, D, L-propargylglycine (PAG), a selective inhibitor of cystathionine γ-lyase (CSE), abolished the protective effects of H₂S donors.

Innovation

This study is the first to show that H₂S can inhibit H₂O₂ mediated mitochondrial dysfunction in human endothelial cells by preserving antioxidant defences.

Significance

H₂S may protect against atherosclerosis by preventing H₂O₂ induced injury to endothelial cells. These effects appear to be mediated via the preservation of mitochondrial function and by reducing the deleterious effects of oxidative stress.     

Emission of volatile sulfur compounds from spruce trees

Spruce (Picea Abies L.) trees from the same clone were supplied with different, but low, amounts of plant available sulfate in the soil (9.7-18.1 milligrams per 100 grams of soil). Branches attached to the trees were enclosed in a dynamic gas exchange cuvette and analyzed for the emission of volatile sulfur compounds. Independent of the sulfate supply in the soil, H₂S was the predominant reduced sulfur compound continuously emitted from the branches with high rates during the day and low rates in the night. In the light, as well as in the dark, the rates of H₂S emission increased exponentially with increasing water vapor flux from the needles. Approximately 1 nanomole of H₂S was found to be emitted per mole of water. When stomata were closed completely, only minute emission of H₂S was observed. Apparently, H₂S emission from the needles is highly dependent on stromatal aperture, and permeation through the cuticle is negligible. In several experiments, small amounts of dimethylsulfide and carbonylsulfide were also detected in a portion of the samples. However, SO₂ was the only sulfur compound consistently emitted from branches of spruce trees in addition to H₂S. Emission of SO₂ mainly proceeded via an outburst starting before the beginning of the light period. The total amount of SO₂ emitted from the needles during this outburst was correlated with the plant available sulfate in the soil. The diurnal changes in sulfur metabolism that may result in an outburst of SO₂ are discussed.     

Removal characteristics of hydrogen sulphide and methanethiol by Thiobacillus sp. isolated from peat in biological deodorization

Thiobacillus sp. HA43 as a dominant strain was isolated from a H₂S-acclimated peat biofilter seeded with aerobically-digested sludge of night soil. Strain IIA43 degraded both H₂S and methanethiol (MT) without lag-time, but degraded neither dimethy sulphide (DMS) nor dimethyl disulphide (DMDS). The removal characteristics for sulphur compounds (H₂S, MT, DMS and DMDS) by strain HA43 well reflected the removal behaviour of the H₂S-acclimated peat biofilter where this strain was isolated. The specific H₂S and MT uptake rates of strain HA43 in batch culture were determined as 1.22 × 10⁻¹² and 8.53 × 10⁻¹⁴ g-S·cell⁻¹·h⁻¹, respectively. The maximum removal rates (V_m = g-S·kg-dry peat⁻¹·d⁻¹) for H₂S and MT by peat biofilter inoculated by strain HA43 were obtained as follows: V_m(H₂S)− 11.3, V_m(MT) = 0.21 in sterilized peat; V_m(H₂S) = 12.4, V_m(MT)− 0.27 in non-sterilized peat; V_m(H₂S) = 33.0, V_m(MT) = 0.27 in peat with aerobically-digested sludge of night soil. The peat biofilter inoculated with strain HA43 enhanced the maximum removal rate for H₂S 6-fold compared with the biofilter without strain HA43.     

Photochemical disproportionation of sulfur into sulfide and sulfate byChlorobium limicola formathiosulfatophilum

The anaerobic bacteriumChlorobium assimilates carbon dioxide in the light with various sulfur compounds as electron donors. The well-known metabolic pathway proceeds from the oxidation of sulfide via sulfur to sulfate. In the dark the reaction is partially reversed when sulfur is reduced to hydrogen sulfide. The fermenting cells thereby release an excess of reductant. We have now found a hydrogen sulfide production from sulfur, which is light-dependent. It is more than ten times faster than the dark reaction. This appears in experiments where the cell suspension is illuminated in absence of CO₂ and flushed continuously with H₂ or Ar. The H₂S is trapped with ZnCl₂ and the S^2- titrated with iodine. The total amount of H₂S evolved in the light increases proportionally with the amount of sulfur added, and about one-half of the added sulfur is converted to H₂S. Another part of the metabolized sulfur appears at the same time as sulfate, but all the sulfur oxidized to sulfate does not account for the larger amount of sulfur reduced to hydrogen sulfide. Very likely other unanalyzed oxidized sulfur compounds must also have been produced. Use of H₂ instead of Ar as the anaerobic gas phase does not increase the amount of H₂S produced, nor does the addition of thiosulfate; sulfur itself is the preferred electron donor for the sulfur reduction. Up to a light intensity of 10000 ergs cm^-2sec^-1 CO₂ does not affect H₂S production. Without CO₂, saturation of the light-dependent evolution of H₂S is reached at about 40000 ergs cm^-2sec^-1. In contrast, presence of CO₂ at this light intensity makes the sulfide production disappear completely. On application of mass spectrometry to the gas exchange upon illumination, at high light intensity a H₂S gush is found during the first 3 min. This is followed by CO₂ fixation, while simultaneously the reductant H₂S is now taken up. WithRhodospirillum rubrum, the addition of sulfur leads to a moderate evolution of H₂S. In contrast toChlorobium this reaction inR. rubrum is not light-sensitive, nor does it produce detectable amounts of sulfate. After addition of malate the rate of H₂S evolution does increase in the light, since the cells use malate as an electron donor during their photochemical metabolism.     

Hydrogen Sulfide Represses Androgen Receptor Transactivation by Targeting at the Second Zinc Finger Module

Androgen receptor (AR) signaling is indispensable for the development of prostate cancer from the initial androgen-dependent state to a later aggressive androgen-resistant state. This study examined the role of hydrogen sulfide (H₂S), a novel gasotransmitter, in the regulation of AR signaling as well as its mediation in androgen-independent cell growth in prostate cancer cells. Here we found that H₂S inhibits cell proliferation of both androgen-dependent (LNCaP) and antiandrogen-resistant prostate cancer cells (LNCaP-B), with more significance on the latter, which was established by long term treatment of parental LNCaP cells with bicalutamide. The expression of cystathionine γ-lyase (CSE), a major H₂S-producing enzyme in prostate tissue, was reduced in both human prostate cancer tissues and LNCaP-B cells. LNCaP-B cells were resistant to bicalutamide-induced cell growth inhibition, and CSE overexpression could rebuild the sensitivity of LNCaP-B cells to bicalutamide. H₂S significantly repressed the expression of prostate-specific antigen (PSA) and TMPRSS2, two AR-targeted genes. In addition, H₂S inhibited AR binding with PSA promoter and androgen-responsive element (ARE) luciferase activity. We further found that AR is post-translationally modified by H₂S through S-sulfhydration. Mutation of cysteine 611 and cysteine 614 in the second zinc finger module of AR-DNA binding domain diminished the effects of H₂S on AR S-sulfhydration and AR dimerization. These data suggest that reduced CSE/H₂S signaling contributes to antiandrogen-resistant status, and sufficient level of H₂S is able to inhibit AR transactivation and treat castration-resistant prostate cancer.     

Hydrogen sulfide effects on stomatal apertures

Hydrogen sulfide (H₂S) has recently been reported to be a signaling molecule in plants. It has been well established that is has such roles in animals and it has been suggested that it is included into the group of gasotransmitters. We have recently shown that hydrogen sulfide causes stomatal opening in the model plant Arabidopsis thaliana. H₂S can be supplied to the plant tissues from donors such as sodium hydrosulfide (NaSH) or more recently from slow release H₂S donor molecules such as GYY4137. Both give similar effects, that is, they cause stomatal opening. Furthermore both H₂S donors reduced the accumulation of nitric oxide (NO) induced by abscisic acid (ABA) treatment of leaf tissues. Here similar work has been repeated in a crop plant, Capsicum anuum, and similar data has been obtained, suggesting that such effects of hydrogen sulfide on plants is not confined to model species.Key words: abscisic acid, GYY4137, hydrogen sulfide, nitric oxide, stomatal apertureThe effects of hydrogen sulfide on plants have been studied for many years, but it is only recently that it has been suggested that this gas is acting as a signaling molecule. In animals this has been well established^1,2 and it has been suggested that H₂S be grouped together with other gasotransmitters.^2,3 This group will also contain nitric oxide (NO) which as well as having established roles in animals is also known to cause stomatal closure in plants.^4,5 With this in mind, we previously investigated whether H₂S may also have an effect on stomatal closure, using a model organism Arabidopsis thaliana.⁶ The study used two different H₂S donors, sodium hydrosulfide (NaSH) and morpholin-4-ium 4 methoxyphenyl(morpholino) phosphinodithionate (GYY4137). The former will release H₂S in an instant burst which soon dissipates, which questions the wisdom of its use. GYY4137 on the other hand will release H₂S much more slowly and in a manner which is more likely to reflect physiological generation of H₂S.^7,8 Both donors caused stomatal that had previously been exposed to light to open even further. If leaf tissues were not light treated H₂S compounds once again caused stomata to open. Furthermore, H₂S treatment prevented stomatal closure caused by dark treatment. To investigate the possible mechanism of this effect, tissues were treated with the plant hormone abscisic acid (ABA) to initiate NO generation and then NO accumulation was measured in the absence and presence of H₂S donors using fluorescent probes and confocal microscopy.⁹ Both NaSH and GYY4137 caused a reduction in the accumulation of NO. This suggests that H₂S may be acting by a disruption of NO signaling, which results in the alteration of guard cell physiology.Others have reported different effects of H₂S on stomatal movements. Garcia-Mata and Lamattina¹⁰ found that both H₂S donors NaSH and GY4137 caused stomatal closure in different plant species including Vicia faba, Arabidopsis thaliana and Impatiens walleriana. Use of glibenclamide, which is an ABC transport inhibitor, reduced the effect. Cystathione γ lyase and L-Cys desulfhydrase are enzymes which may be responsible for H₂S synthesis and stomatal movements were also reduced by propargylglycine, an inhibitor of these enzymes. It was suggested therefore that H₂S helps to mediate ABA signaling pathway in guard cells. This paper was further discussed following its publication by Desikan.¹¹ However, this seems to be in conflict with the work we reported. This would not be the first time that there has been contradictory data when it comes to reporting stomatal movements, as ethylene has been shown to mediate auxin-induced opening¹² and to cause stomatal closure.¹³More recently it has been reported that stomatal conductance was increased by carbonyl sulfide (COS).¹⁴ The authors went on to suggest that this effect was mediated by H₂S which was produced from COS hydrolysis. This seems to support our original data. Therefore, here we report on the effects of both NaSH and GYY4137 on a different plant species and one which has relevance as an important crop, that is Capsicum anuum. GYY4137 was supplied as in our previous paper in reference ⁶ and ⁷. As can be seen in Figure 1A NaSH caused stomata to open further, even though the leaf tissue had been exposed to the light. Stomata were able to close, as ABA treatment demonstrated, therefore showing that the stomata were not defective. When the experiments were repeated with GYY4137 (Fig. 1B) and smaller but similar effect of the addition of the H₂S donor was seen. This would be expected as the release of H₂S from GYY4137 would be slower and more prolonged than from NaSH.^7,8 To investigate if NO accumulation is also effected in Capsicum when treated with H₂S donors, leaf tissue was treated with ABA to initiate NO generation and NO measured by the use of DAF2-DA as previously reported in references ⁶ and ⁹. Once again the presence of H₂S donors dramatically reduced the amount of NO that was measured following ABA treatment (Fig. 2). This once again suggests that H₂S is having an effect on NO metabolism which may account for the stomata aperture measurements. It has been suggested in animal systems that H₂S and NO react, resulting in the formation of nitrosothiols/nitrothiol-like species¹⁵ which could have signaling effects in their own right. NO in plants has been reported to lead to increased cGMP and/or increased nitrosylation of proteins,⁵ but if H₂S was removing the bioavailability of NO both mechanisms are likely to be reduced.Open in a separate windowFigure 1H₂S donors cause stomatal opening in Capsicum anuum. The leaves of analyzed from Capsicum anuum plants which were between 6 and 7 weeks old. Stomatal bioassays were performed as described previously by Desikan et al.⁹ Epidermal peels were incubated in MES-KCl buffer [10 mM 2-morpholino ethane sulfonic acid (MES), 5 mM KCl, 50 µM CaCl₂, pH 6.15] for 2.5 h exposed to the direct lightning (in 60–100 IE m⁻² s⁻¹) before the addition of various compounds. (A) Samples were sheltered from direct lighting and treated with ABA or NaHS for 2.5 h and left under the day light conditions before stomata apertures were analyzed. (B) Samples were sheltered from direct lighting and treated with ABA or GYY 4137 for next 2 h and left under the day light conditions before stomata apertures were analyzed. Apertures were measured using a light microscope and imaging camera with LEICA QWIN image processing and analysis software (Leica Microsystems and Imaging Solutions, Cambridge, UK). n = 40 stomatal apertures, ±SE. GYY4137 was synthesis as previously described in reference ⁷.Open in a separate windowFigure 2H₂S donors reduce NO accumulation in Capsicum anuum. Nitric oxide accumulation was estimated using the specific NO dye DAF2-DA (Calbiochem, Nottingham, UK), using the method described previously by Desikan et al.⁹ Epidermal fragments in MES-KCl buffer (10 mM MES, 5 mM KCl, 50 µM CaCl₂, pH 6.15) were exposed to the direct lightning for 2 h. After 2 h samples were loaded with 30 µM DAF2-DA for 15 min before washing with MES-KCl buffer; three times for 10 min. Fragments were subsequently incubated for a further 30 min in the presence of various compounds (as indicated below) before images were visualized using CLSM (excitation 488 nm, emission 515 nm; Nikon PCM2000, Kingston-upon-Thames, UK). Images acquired were analyzed using SCION IMAGE software (Scion, Frederick, MD, USA). (A) Control with no treatment; (B) ABA (50) treatment; (C) NaHS (100 µm) treatment alone; (D) ABA treatment in the presence of NaHS; (E) GYY4137 (100 µm) treatment alone; (F) ABA treatment in the presence of NaHS.NO metabolism is involved in a wide range of plant functions, including seed germination,¹⁶ floral development,¹⁷ root gravitropism¹⁸ and gene expression¹⁹ as well as controlling stomatal function.⁴ H₂S on the other hand may be present in or around plants for a variety to reasons. H₂S can be produced endogenously by for example by plastid located cysteine desulfhydrases,²⁰ or H₂S may come from the environment,²¹ including the soil and waters.²² This is further discussed in a recent review in reference ²³. Therefore future work should be focused on the interplay between H₂S from a variety of sources on the NO metabolism of a range of plant tissues. Not all affects of H₂S will be mediated by NO, with alterations of glutathione on H₂S treatment being reported for example.²⁴ But the full extent of the modulation of NO accumulation and signal by both exogenous and endogenous H₂S needs to be explored so the role of these gasotransmitters^2,3 in mediating hormone and stress responses in plants can be fully understood.     

Partial characterization of peroxidase isoenzymes from rust-affected wheat leaves

Four anodic peroxidase isoenzymes from wheat leaves were purified by column chromatography and their kinetic behavior with common substrates were examined. One isoenzyme is more active in wheat resistant to stem rust fungi and differed from the others in carbohydrate content and also by a specific activity 2–4-fold higher with non-physiological electron donors. As a substrate, eugenol exhibited kinetic behavior different from p-phenylenediamine, guaiacol or o-dianisidine with all isoenzymes. All four isoenzymes showed similar pH and temperature optima and kinetic behavior and apparent K_m values for both H₂O₂ and non-physiological electron donors.     

Studies on sulphur in vertisols

Summary Some soil and plant test methods were evaluated for predicting response of soybean crop (Glycine max (L.) Merr.) to S application in vertisols. Morgan's reagent, 500 ppm P containing Ca(H₂PO₄)₂.H₂O and KH₂PO₄ solutions, 0.5N NH₄OAc+0.25N HOAc and 0.15% CaCl₂ were found to be suitable extractants for measuring available soil S. The critical limits of extractable S were 9.0 ppm by Morgan's reagent, 10.0 ppm by phosphate solutions, 8.0 ppm by 0.5N NH₄OAc +0.25N HOAc and 14.0 ppm by 0.15% CaCl₂. Morgan's reagent was regarded as superior to other soil test methods in view of its high relationship with S uptake by plants, A values and relative yield. Critical S concentration in soybean plants varied with age. It was 0.15% and 0.185% for 36 and 60 days old plants, respectively. The critical N/S ratio on the other hand appeared to be constant at about 16.5 during vegetative growth period. Constancy of critical N/S ratio in plants was attributed to the near constancy of N/S ratio in plant proteins. There was highly significant relationship between response of soybean to S and to N, supporting the conclusion of some earlier workers that any soil showing large responses to N may not be supplying adequate S from the mineralization of soil organic matter.     

Effect of Hydrogen Sulfide on Cyclic AMP Production in Isolated Bovine and Porcine Neural Retinae

Hydrogen sulfide (H₂S) has been reported to exert pharmacological effects on neural and non-neural tissues from several mammalian species. In the present study, we examined the role of the intracellular messenger, cyclic AMP in retinal response to H₂S donors, sodium hydrosulfide (NaHS) and sodium sulfide (Na₂S) in cows and pigs. Isolated bovine and porcine neural retinae were incubated in oxygenated Krebs buffer solution prior to exposure to varying concentrations of NaHS, Na₂S or the diterpene activator of adenylate cyclase, forskolin. After incubation at different time intervals, tissue homogenates were prepared for cyclic AMP assay using a well established methodology. In isolated bovine and porcine retinae, the combination of both phosphodiesterase inhibitor, IBMX (2 mM) and forskolin (10 μM) produced a synergistic increase (P < 0.001) in cyclic AMP concentrations over basal levels. NaHS (10 nM–100 μM) produced a time-dependent increase in cyclic AMP concentrations over basal levels which reached a maximum at 20 min in both bovine and porcine retinae. At this time point, both NaHS and Na₂S (10 nM–100 μM) caused a significant (P < 0.05) dose-dependent increase in cyclic AMP levels in bovine and porcine retinae. For instance, NaHS (100 nM) elicited a four-fold and three-fold increase in cyclic AMP concentrations in bovine and porcine retinae respectively whilst higher concentrations of Na₂S (100 μM) produced a much lesser effect in both species. In bovine and porcine retinae, the effects caused by forskolin (10 μM) on cyclic AMP production were not potentiated by addition of low or high concentrations of both NaHS and Na₂S. We conclude that H₂S donors can increase cyclic AMP production in isolated neural retinae from cows and pigs. Bovine retina appears to be more sensitive to the stimulatory effect of H₂S donors on cyclic nucleotide production than its porcine counterpart indicating that species differences exist in the magnitude of this response. Furthermore, effects produced by forskolin on cyclic AMP formation were not additive with those elicited by H₂S donors suggesting that these agents may share a common mechanism in their action on the adenylyl cyclase pathway.