Microbial communities and their roles in the Cenozoic sulfurous oil reservoirs in the Southwestern Qaidam Basin, Western China.

The latest discovery of sulfurous natural gas marked a breakthrough in the Cenozoic natural gas exploration in the southwestern margin of Qaidam Basin. The 16S rRNA analyses were performed on the crude oil samples from H2S-rich reservoirs in the Yuejin, Shizigou and Huatugou profiles, to understand the sulfurous gas origin, which was also integrated with carbon and hydrogen isotopes of alkane and sulfur isotopes of H2S collected from the Yingxiongling Area. Results show that the microorganisms in samples can survive in the hypersaline reservoirs, and can be classified into multiple phyla, including Proteobacteria, Planctomycetes, Firmicutes, Bacteroidetes, and Haloanaerobiaeota. Methanogens are abundant in all of the three profiles, while sulfate-reducing bacteria are abundant in Yuejin and Huatugou profiles, contributing to the methane and H2S components in the natural gas. The carbon, hydrogen and sulfur isotopes of sulfurous natural gas in the Yingxiongling Area show that the natural gas is a mixture of coal-type gas and oil-type gas, which was primarily derived from thermal degradation, and natural gas from the Yuejin and Huatugou profiles also originated from biodegradation. The isotopic analysis agrees well with the 16S rRNA results, i.e., H2S-rich natural gas from the Cenozoic reservoirs in the southwest margin of the Qaidam Basin was primarily of thermal genesis, with microbial genesis of secondary importance.

Anthropogenic Emission Sources of Sulfate Aerosols in Hangzhou, East China: Insights from Isotope Techniques with Consideration of Fractionation Effects between Gas-to-Particle Transformations.

Sulfate (SO42-) is a major species in atmospheric fine particles (PM2.5), inducing haze formation and influencing Earth's climate. In this study, the δ34S values in PM2.5 sulfate (δ34S-SO42-) were measured in Hangzhou, east China, from 2015 September to 2016 October. The result showed that the δ34S-SO42- values varied from 1.6 to 6.4‰ with the higher values in the winter. The estimated fractionation factor (α34Sg→p) from SO2 to SO42- averaged at 3.9 ± 1.6‰. The higher α34Sg→p values in the winter were mainly attributed to the decrease of ambient temperature. We further compared the quantified source apportionments of sulfate by isotope techniques with and without the consideration of fractionation factors. The result revealed that the partitioned emission sources to sulfate with the consideration of the fractionation effects were more logical, highlighting that fractionation effects should be considered in partitioning emission sources to sulfate using sulfur isotope techniques. With considering the fractionation effects, coal burning was the dominant source to sulfate (85.5%), followed by traffic emissions (12.8%) and oil combustion (1.7%). However, the coal combustion for residential heating contributed only 0.9% to sulfate on an annual basis in this megacity.

Predicting the number of sulfur atoms in peptides and small proteins based on the observed aggregated isotope distribution.

RATIONALE: Identification of peptides and proteins is a challenging task in mass spectrometry-based proteomics. Knowledge of the number of sulfur atoms can improve the identification of peptides and proteins. METHODS: In this article, we propose a method for the prediction of S-atoms based on the aggregated isotope distribution. The Mahalanobis distance is used as dissimilarity measure to compare mass- and intensity-based features from the observed and theoretical isotope distributions. RESULTS: The relative abundance of the second and the third aggregated isotopic variants (as compared to the monoisotopic one) and the mass difference between the second and third aggregated isotopic variants are the most important features to predict the number of S-atoms. CONCLUSIONS: The mass and intensity accuracies of the observed aggregated isotopic variants are insufficient to accurately predict the number of atoms. However, using a limited set of predictions for a peptide, rather than predicting a single number of S-atoms, has a reasonably high prediction accuracy.

Exploring neutron capture therapy with 33S and 10B.

The 33S(n,α)30Si reaction was proposed as cooperative neutron capturer to 10B(n,α)7Li in Neutron Capture Therapy (NCT). At that moment, the available 33S(n,α)30Si cross-section data were scarce and discrepant in key energy ranges for its use in NCT. Since then, three experiments have been carried out at n_TOF facility at CERN and at Institut Laue-Langevin. These new data are used for the calculation of the dose rate on ICRU-4 tissue by using kerma factors, a simplified model of tissue and a 13.45 keV neutron beam, energy of the most important 33S(n,α)30Si resonance. A significant enhancement of the dose rate due to the presence of 33S is shown. In spite of the limitations, the cooperative action of 33S and 10B is an interesting possibility to be studied for accelerator-based neutron sources with non-moderated neutrons.

Plant sulphur metabolism is stimulated by photorespiration.

Intense efforts have been devoted to describe the biochemical pathway of plant sulphur (S) assimilation from sulphate. However, essential information on metabolic regulation of S assimilation is still lacking, such as possible interactions between S assimilation, photosynthesis and photorespiration. In particular, does S assimilation scale with photosynthesis thus ensuring sufficient S provision for amino acids synthesis? This lack of knowledge is problematic because optimization of photosynthesis is a common target of crop breeding and furthermore, photosynthesis is stimulated by the inexorable increase in atmospheric CO2. Here, we used high-resolution 33S and 13C tracing technology with NMR and LC-MS to access direct measurement of metabolic fluxes in S assimilation, when photosynthesis and photorespiration are varied via the gaseous composition of the atmosphere (CO2, O2). We show that S assimilation is stimulated by photorespiratory metabolism and therefore, large photosynthetic fluxes appear to be detrimental to plant cell sulphur nutrition.

Mercury bioaccumulation in tilefish from the northeastern Gulf of Mexico 2 years after the Deepwater Horizon oil spill: Insights from Hg, C, N and S stable isotopes.

Mercury (Hg) concentration in fish of the Gulf of the Mexico (GoM) is a major concern due to the importance of the GoM for U.S. fisheries. The Deepwater Horizon (DWH) oil spill in April 2010 in the northern GoM resulted in large amounts of oil and dispersant released to the water column, which potentially modified Hg bioaccumulation patterns in affected areas. We measured Hg species (methylmercury (MMHg) and inorganic Hg (IHg)) concentrations, and light (C, N and S) and Hg stable isotopes in muscle and liver tissues from tilefish (Lopholatilus chamaleonticeps) sampled in 2012 and 2013 along the shelf break of the northeastern GoM. Fish located close to the mouth of the Mississippi River (MR) and northwest of the DWH well-head (47 km) showed significantly lower Hg levels in muscle and liver than fish located further northeast of the DWH (>109 km), where 98% of tilefish had Hg levels in the muscle above US consumption advisory thresholds (50% for tilefish close to the DWH). Differences in light and Hg stable isotopes signatures were observed between these two areas, showing higher δ15N, and lower δ202Hg, Δ199Hg and δ34S in fish close to the DWH/MR. This suggests that suspended particles from the MR reduces Hg bioavailability at the base of the GoM food chains. This phenomenon can be locally enhanced by the DWH that resulted in increased particles in the water column as evidenced by the marine snow layer in the sediments. On the other hand, freshly deposited Hg associated with organic matter in more oligotrophic marine waters enhanced Hg bioaccumulation in local food webs. Comparing Hg isotopic composition in liver and muscle of fish indicates specific metabolic response in fish having accumulated high levels of MMHg.

Sulfur-34S and 36S Stable Isotope Labeling of Amino Acids for Quantification (SULAQ34/36) of Proteome Analyses.

Quantitative proteome profiling of microorganisms by isotopic labeling of amino acids is still a challenge, because only microorganisms with auxotrophic character are able to embed amino acids into their biomass in a quantitatively correct manner. Here, we describe an isotopic labeling technique (sulfur stable isotope labeling of amino acids for quantification, SULAQ) for the sulfur-containing amino acids cysteine and methionine in a broad range of organisms. The metabolic labeling approach is suitable for gel-based and gel-free protein analysis.

Carbon, nitrogen and sulphur isotopic fractionation in captive juvenile hooded seal (Cystophora cristata): Application for diet analysis.

RATIONALE: Intrinsic biogeochemical markers, such as stable isotope ratios of carbon, nitrogen and sulphur, are increasingly used to trace the trophic ecology of marine top predators. However, insufficient knowledge of fractionation processes in tissues continues to hamper the use of these markers. METHODS: We performed a controlled feeding experiment with eight juvenile hooded seals (Cystophora cristata) that were held on a herring-based diet (Clupea harengus) for two years. Stable isotope ratios were measured via isotope ratio mass spectrometry in three of their tissues and related to values of these markers in their diet. RESULTS: Diet-tissue isotope enrichment (trophic enrichment factor, TEF) values between dietary herring and seal tissues for carbon (Δ13 C) were +0.7 ‰ for red blood cells, +1.9 ‰ for hair and +1.1 ‰ for muscle. The TEFs for nitrogen trophic (Δ15 N) were +3.3 ‰ for red blood cells, +3.6 ‰ for hair and +4.3 ‰ for muscle. For sulphur, the Δ34 S values were +1.1 ‰ for red blood cells, +1.0 ‰ for hair and +0.9 ‰ for muscle. CONCLUSIONS: These enrichment values were greater than those previously measured in adult seals. This increase may be related to the higher rate of protein synthesis and catabolism in growing animals. This study is the first report on sulphur isotope enrichment values for a marine mammal species.

Synthesis of l-cysteine derivatives containing stable sulfur isotopes and application of this synthesis to reactive sulfur metabolome.

Cysteine persulfide is an L-cysteine derivative having one additional sulfur atom bound to a cysteinyl thiol group, and it serves as a reactive sulfur species that regulates redox homeostasis in cells. Here, we describe a rapid and efficient method of synthesis of L-cysteine derivatives containing isotopic sulfur atoms and application of this method to a reactive sulfur metabolome. We used bacterial cysteine syntheses to incorporate isotopic sulfur atoms into the sulfhydryl moiety of L-cysteine. We cloned three cysteine synthases-CysE, CysK, and CysM-from the Gram-negative bacterium Salmonella enterica serovar Typhimurium LT2, and we generated their recombinant enzymes. We synthesized 34S-labeled L-cysteine from O-acetyl-L-serine and 34S-labeled sodium sulfide as substrates for the CysK or CysM reactions. Isotopic labeling of L-cysteine at both sulfur (34S) and nitrogen (15N) atoms was also achieved by performing enzyme reactions with 15N-labeled L-serine, acetyl-CoA, and 34S-labeled sodium sulfide in the presence of CysE and CysK. The present enzyme systems can be applied to syntheses of a series of L-cysteine derivatives including L-cystine, L-cystine persulfide, S-sulfo-L-cysteine, L-cysteine sulfonate, and L-selenocystine. We also prepared 34S-labeled N-acetyl-L-cysteine (NAC) by incubating 34S-labeled L-cysteine with acetyl coenzyme A in test tubes. Tandem mass spectrometric identification of low-molecular-weight thiols after monobromobimane derivatization revealed the endogenous occurrence of NAC in the cultured mammalian cells such as HeLa cells and J774.1 cells. Furthermore, we successfully demonstrated, by using 34S-labeled NAC, metabolic conversion of NAC to glutathione and its persulfide, via intermediate formation of L-cysteine, in the cells. The approach using isotopic sulfur labeling combined with mass spectrometry may thus contribute to greater understanding of reactive sulfur metabolome and redox biology.

Evaluation of sulfur isotopic enrichment of urine metabolites for the differentiation of healthy and prostate cancer mice after the administration of 34S labelled yeast.

Sulfur isotopic enrichment of urine metabolites in healthy and prostate cancer mice using 34S enriched yeast and High Performance Liquid Chromatography coupled to Multicollector Inductively Coupled Plasma Mass Spectrometry (HPLC-MC-ICP-MS) has been evaluated. A 30 weeks experiment (since the eleventh to the fortieth week of life) was carried out collecting the urine of three healthy mice and three transgenic mice with prostate cancer during 24h after a single oral administration of a 34S enriched yeast slurry. The isotopic enrichment of different sulphur metabolites was monitored by coupling a C18 reverse phase HPLC column with a multicollector ICP-MS using a membrane desolvating system. Quantification of sulfur in the chromatographic peaks was carried out by post-column isotope dilution using a 33S enriched spike. Differences between the 34S enrichment in the urine metabolites of healthy and prostate cancer mice were found from the beginning of the disease. Both populations could be differentiated using a principal component analysis (PCA). Finally, 7 unknown mice were correctly classified in each population using a linear discriminant analysis.

Medical applications of Cu, Zn, and S isotope effects.

This review examines recent applications of stable copper, zinc and sulfur isotopes to medical cases and notably cancer. The distribution of the natural stable isotopes of a particular element among coexisting molecular species varies as a function of the bond strength, the ionic charge, and the coordination, and it also changes with kinetics. Ab initio calculations show that compounds in which a metal binds to oxygen- (sulfate, phosphate, lactate) and nitrogen-bearing moieties (histidine) favor heavy isotopes, whereas bonds with sulfur (cysteine, methionine) favor light isotopes. Oxidized cations (e.g., Cu(ii)) and low coordination numbers are expected to favor heavy isotopes relative to their reduced counterparts (Cu(i)) and high coordination numbers. Here we discuss the first observations of Cu, Zn, and S isotopic variations, three elements closely related along multiple biological pathways, with emphasis on serum samples of healthy volunteers and of cancer patients. It was found that heavy isotopes of Zn and to an even greater extent Cu are enriched in erythrocytes relative to serum, while the difference is small for sulfur. Isotopic variations related to age and sex are relatively small. The 65Cu/63Cu ratio in the serum of patients with colon, breast, and liver cancer is conspicuously low relative to healthy subjects. The characteristic time over which Cu isotopes may change with disease progression (a few weeks) is consistent with both the turnover time of the element and albumin half-life. A parallel effect on sulfur isotopes is detected in a few un-medicated patients. Copper in liver tumor tissue is isotopically heavy. In contrast, Zn in breast cancer tumors is isotopically lighter than in healthy breast tissue. 66Zn/64Zn is very similar in the serum of cancer patients and in controls. Possible reasons for Cu isotope variations may be related to the cytosolic storage of Cu lactate (Warburg effect), release of intracellular copper from cysteine clusters (metallothionein), or the hepatocellular and biosynthetic dysfunction of the liver. We suggest that Cu isotope metallomics will help evaluate the homeostasis of this element during patient treatment, notably by chelates and blockers of Cu trafficking, and understand the many biochemical pathways in which this element is essential.

[Investigations on Sulfur and Carbon Isotopic Compositions of Potential Polluted Sources in Atmospheric PM2.5 in Nanjing Region].

Potential pollution sources of atmospheric PM2.5 in Nanjing region were collected, and sulfur and carbon isotopic compositions were determined by EA-IRMS synchronously. The results showed that δ³4S and δ¹³C values ranged from 1.8‰-3.7‰ and -25.50‰- -23.57‰ in coal soot particles; 4.6‰-9.7‰ and -26.32‰- -23.57‰ in vehicle exhaust; 5.2‰-9.9‰ and -19.30‰- -30.42‰ in straw soot particles, respectively. Besides, the δ¹³C value of dust was -13.45‰. It can be observed that sulfur isotopic compositions in coal soot were lower, while the carbon isotopic composition in dust was higher. Comparing with δ³4S and δ¹³C values in domestic and foreign polluted sources, we found that sulfur and carbon isotopes in atmospheric PM2.5 in Nanjing region presented an obvious regional characteristics. Therefore, the source spectrum of sulfur and carbon isotopic compositions in Nanjing region might provide an insight into source apportionment of atmospheric PM2.5.

Role of oxbow lakes in controlling redox geochemistry of shallow groundwater under a heterogeneous fluvial sedimentary environment in an agricultural field: Coexistence of iron and sulfate reduction.

This study aimed to extend the knowledge of the vertical distribution of redox conditions of shallow groundwater in heterogeneous fluvial sediments near oxbow lakes. For this study, we revisited the study area of Kim et al. (2009) to examine the redox zoning in details. Three multi-level samplers were installed along a flow path near two oxbow lakes to obtain vertical profiles of the subsurface geology and hydrochemical and isotopic data (δ(18)O and δD of water, δ(15)N and δ(18)O of nitrate, and δ(34)S of sulfate) of groundwater. Geologic logging showed that characteristics of the heterogeneous subsurface geology are closely related to the pattern of vertical redox zoning. Hydrochemical data in conjunction with nitrogen and sulfur isotope data show that the redox status of groundwater near oxbow lakes is controlled by denitrification, iron reduction, and sulfate reduction. The oxidizing condition of groundwater occurs in the sand-dominant alluvium located in the up-gradient of oxbow lakes, whereas the reducing condition accompanying denitrification, iron reduction, and local sulfate reduction is developed in silt-rich alluvium in and the downgradient of oxbow lakes. The occurrence of sulfate reduction was newly found in this study. However, the vertical profiles of redox-sensitive parameters show that iron reduction and sulfate reduction occur concurrently near oxbow lakes, although the measured redox potentials suggest that thermodynamic conditions are controlled by the stability of Fe(2+)/Fe-oxides. Therefore, this study shows that the redox condition of groundwater in the iron-rich zone should be carefully interpreted. For this purpose, depth-specific sampling and careful examination of sulfur isotope data will be very useful for identifying the redox processes occurring in the zone with overlapping iron reduction and sulfate reduction in heterogeneous fluvial sediments.

Chemical Assignment of Structural Isomers of Sulfur-Containing Metabolites in Garlic by Liquid Chromatography-Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry.

BACKGROUND: The chemical assignment of metabolites is crucial to understanding the relation between food composition and biological activity. OBJECTIVE: This study was designed to detect and chemically assign sulfur-containing metabolites by using LC-Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) in Allium plants. METHODS: Ultrahigh resolution (>250,000 full width at half-maximum) and mass accuracy (<1 mDa) by FTICR-MS allowed us to distinguish ions containing sulfur isotopes ((32)S and (34)S). RESULTS: Putative 69 S-containing monoisotopic ions (S-ions) were extracted from the metabolome data of onion (Allium cepa), green onion (Allium fistulosum), and garlic (Allium sativum) on the basis of theoretical mass differences between (32)S-ions and their (34)S-substituted counterparts and on the natural abundance of (34)S. Eight S-ions were chemically assigned by using the reference data according to the guidelines of the Metabolomics Standards Initiative. Three ions detected in garlic were assigned as derived from the isomers γ-glutamyl-S-1-propenylcysteine and γ-glutamyl-S-2-propenylcysteine and as S-2-propenylmercaptoglutathione on the basis of differences in key product ions identified in reference tandem MS spectra. CONCLUSION: The ability to discriminate between such geometric isomers will be extremely useful for the chemical assignment of unknown metabolites in MS-based metabolomics.

Maturational alterations in constitutive activity of medial prefrontal cortex kappa-opioid receptors in Wistar rats.

Opioid receptors can display spontaneous agonist-independent G-protein signaling (basal signaling/constitutive activity). While constitutive κ-opioid receptor (KOR) activity has been documented in vitro, it remains unknown if KORs are constitutively active in native systems. Using [(35) S] guanosine 5'-O-[gamma-thio] triphosphate coupling assay that measures receptor functional state, we identified the presence of medial prefrontal cortex KOR constitutive activity in young rats that declined with age. Furthermore, basal signaling showed an age-related decline and was insensitive to neutral opioid antagonist challenge. Collectively, the present data are first to demonstrate age-dependent alterations in the medial prefrontal cortex KOR constitutive activity in rats and changes in the constitutive activity of KORs can differentially impact KOR ligand efficacy. These data provide novel insights into the functional properties of the KOR system and warrant further consideration of KOR constitutive activity in normal and pathophysiological behavior. Opioid receptors exhibit agonist-independent constitutive activity; however, kappa-opioid receptor (KOR) constitutive activity has not been demonstrated in native systems. Our results confirm KOR constitutive activity in the medial prefrontal cortex (mPFC) that declines with age. With the ability to presynaptically inhibit multiple neurotransmitter systems in the mPFC, maturational or patho-logical alterations in constitutive activity could disrupt corticofugal glutamatergic pyramidal projection neurons mediating executive function. Regulation of KOR constitutive activity could serve as a therapeutic target to treat compromised executive function.

4'-O-methylhonokiol increases levels of 2-arachidonoyl glycerol in mouse brain via selective inhibition of its COX-2-mediated oxygenation.

BACKGROUND AND PURPOSE: 4'-O-methylhonokiol (MH) is a natural product showing anti-inflammatory, anti-osteoclastogenic, and neuroprotective effects. MH was reported to modulate cannabinoid CB2 receptors as an inverse agonist for cAMP production and an agonist for intracellular [Ca2+]. It was recently shown that MH inhibits cAMP formation via CB2 receptors. In this study, the exact modulation of MH on CB2 receptor activity was elucidated and its endocannabinoid substrate-specific inhibition (SSI) of cyclooxygenase-2 (COX-2) and CNS bioavailability are described for the first time. METHODS: CB2 receptor modulation ([35S]GTPγS, cAMP, and ß-arrestin) by MH was measured in hCB2-transfected CHO-K1 cells and native conditions (HL60 cells and mouse spleen). The COX-2 SSI was investigated in RAW264.7 cells and in Swiss albino mice by targeted metabolomics using LC-MS/MS. RESULTS: MH is a CB2 receptor agonist and a potent COX-2 SSI. It induced partial agonism in both the [35S]GTPγS binding and ß-arrestin recruitment assays while being a full agonist in the cAMP pathway. MH selectively inhibited PGE2 glycerol ester formation (over PGE2) in RAW264.7 cells and significantly increased the levels of 2-AG in mouse brain in a dose-dependent manner (3 to 20 mg kg(-1)) without affecting other metabolites. After 7 h from intraperitoneal (i.p.) injection, MH was quantified in significant amounts in the brain (corresponding to 200 to 300 nM). CONCLUSIONS: LC-MS/MS quantification shows that MH is bioavailable to the brain and under condition of inflammation exerts significant indirect effects on 2-AG levels. The biphenyl scaffold might serve as valuable source of dual CB2 receptor modulators and COX-2 SSIs as demonstrated by additional MH analogs that show similar effects. The combination of CB2 agonism and COX-2 SSI offers a yet unexplored polypharmacology with expected synergistic effects in neuroinflammatory diseases, thus providing a rationale for the diverse neuroprotective effects reported for MH in animal models.

Natural variations of copper and sulfur stable isotopes in blood of hepatocellular carcinoma patients.

The widespread hypoxic conditions of the tumor microenvironment can impair the metabolism of bioessential elements such as copper and sulfur, notably by changing their redox state and, as a consequence, their ability to bind specific molecules. Because competing redox state is known to drive isotopic fractionation, we have used here the stable isotope compositions of copper ((65)Cu/(63)Cu) and sulfur ((34)S/(32)S) in the blood of patients with hepatocellular carcinoma (HCC) as a tool to explore the cancer-driven copper and sulfur imbalances. We report that copper is (63)Cu-enriched by ∼0.4‰ and sulfur is (32)S-enriched by ∼1.5‰ in the blood of patients compared with that of control subjects. As expected, HCC patients have more copper in red blood cells and serum compared with control subjects. However, the isotopic signature of this blood extra copper burden is not in favor of a dietary origin but rather suggests a reallocation in the body of copper bound to cysteine-rich proteins such as metallothioneins. The magnitude of the sulfur isotope effect is similar in red blood cells and serum of HCC patients, implying that sulfur fractionation is systemic. The (32)S-enrichment of sulfur in the blood of HCC patients is compatible with the notion that sulfur partly originates from tumor-derived sulfides. The measurement of natural variations of stable isotope compositions, using techniques developed in the field of Earth sciences, can provide new means to detect and quantify cancer metabolic changes and provide insights into underlying mechanisms.

(33)S as a cooperative capturer for BNCT.

(33)S is a stable isotope of sulfur for which the emission of an α-particle is the dominant exit channel for neutron-induced reactions. In this work the enhancement of both the absorbed and the equivalent biologically weighted dose in a BNCT treatment with 13.5keV neutrons, due to the presence of (33)S, has been tested by means of Monte Carlo simulations. The kerma-fluence factors for the ICRU-4 tissue have been calculated using standard weighting factors. The simulations depend crucially on the scarce (33)S(n,α)(30)Si cross-section data. The presence of a high resonance at 13.5keV was established by previous authors providing discrepant resonance parameters. No experimental data below 10keV are available. All of this has motivated a proposal of experiment at the n_TOF facility at CERN. A setup was designed and tested in 2011. Some results of the successful test will be shown. The experiment is scheduled for the period November to December 2012.

Evidence for several cysteine transport mechanisms in the mitochondrial membranes of Arabidopsis thaliana.

Cysteine is essential for many mitochondrial processes in plants, including translation, iron-sulfur cluster biogenesis and cyanide detoxification. Its biosynthesis is carried out by serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) which can be found in the cytosol, plastids and mitochondria. Mutants lacking one compartment-specific OAS-TL isoform show viable phenotypes, leading to the hypothesis that the organellar membranes are permeable to substrates and products of the cysteine biosynthetic pathway. In this report, we show that exogenouslly supplied [(35)S]cysteine accumulates in the mitochondrial fraction and is taken up into isolated mitochondria for in organello protein synthesis. Analysis of cysteine uptake by isolated mitochondria and mitoplasts indicates that cysteine is transported by multiple facilitated mechanisms that operate in a concentration gradient-dependent manner. In addition, cysteine uptake is dependent mainly on the ΔpH across the inner membrane. The rates of mitochondrial cysteine transport can be mildly altered by specific metabolites in the cyanide detoxification-linked sulfide oxidation, but not by most substrates and products of the cysteine biosynthetic pathway. Based on these results, we propose that the transport of cysteine plays a pivotal role in regulating cellular cysteine biosynthesis as well as modulating the availability of sulfur for mitochondrial metabolism.

Modified method for determination of sulfur metabolites in plant tissues by stable isotope dilution-based liquid chromatography-electrospray ionization-tandem mass spectrometry.

A wide variety of sulfur metabolites play important roles in plant functions. We have developed a precise and sensitive method for the simultaneous measurement of several sulfur metabolites based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and (34)S metabolic labeling of sulfur-containing metabolites in Arabidopsis thaliana seedlings. However, some sulfur metabolites were unstable during the extraction procedure. Our proposed method does not allow for the detection of the important sulfur metabolite homocysteine because of its instability during sample extraction. Stable isotope-labeled sulfur metabolites of A. thaliana shoot were extracted and utilized as internal standards for quantification of sulfur metabolites with LC-MS/MS using S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine (Met), glutathione (GSH), and glutathione disulfide (GSSG) as example metabolites. These metabolites were detected using electrospray ionization in positive mode. Standard curves were linear (r(2)>0.99) over a range of concentrations (SAM 0.01-2.0µM, SAH 0.002-0.10µM, Met 0.05-4.0µM, GSH 0.17-20.0µM, GSSG 0.07-20.0µM), with limits of detection for SAM, SAH, Met, GSH, and GSSG of 0.83, 0.67, 10, 0.56, and 1.1nM, respectively; and the within-run and between-run coefficients of variation based on quality control samples were less than 8%.