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Staphylococcus aureus is a major human pathogen that can cause infections that range from superficial skin and mucosal infections to life threatening disseminated infections. S. aureus can attach to medical devices and host tissues and form biofilms that allow the bacteria to evade the host immune system and provide protection from antimicrobial agents. To counter host-generated oxidative and nitrosative stress mechanisms that are part of the normal host responses to invading pathogens, S. aureus utilizes low molecular weight (LMW) thiols, such as bacillithiol (BSH). Additionally, S. aureus synthesizes its own nitric oxide (NO), which combined with its downstream metabolites may also protect the bacteria against specific host responses. We have previously shown that LMW thiols are required for biofilm formation in Mycobacterium smegmatis and Pseudomonas aeruginosa. Here, we show that the S. aureus bshC mutant strain, which is defective in the last step of the BSH pathway and lacks BSH, is impaired in biofilm formation. We also identify a possible S-nitrosobacillithiol reductase (BSNOR), similar in sequence to an S-nitrosomycothiol reductase found in M. smegmatis and show that the putative S. aureus bsnoR mutant strain has reduced levels of BSH and decreased biofilm formation. Our studies also show that NO plays an important role in biofilm formation and that acidified sodium nitrite severely reduces biofilm thickness. These studies provide insight into the roles of oxidative and nitrosative stress mechanisms on biofilm formation and indicate that BSH and NO are key players in normal biofilm formation in S. aureus., Competing Interests: Declaration of competing interest Clarissa J. Nobile is an associate editor for Microbial Pathogenesis. Clarissa J. Nobile is a cofounder of BioSynesis, Inc., a company developing diagnostics and therapeutics for biofilm infections. Megha Gulati was a consultant for BioSynesis, Inc. The confocal scanning laser microscopy images in this work were taken using a confocal microscope acquired through the National Science Foundation (NSF) MRI award DMR-1625733. This work was supported by the National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS) award R35GM124594 to CJN, by the NIH NIGMS award SC3GM100855 and National Science Foundation (NSF) award MCB1244611 to MR, and by the NIH National Institutes of Allergy and Infectious Diseases (NIAID) award R15AI137975 to ADH. CLE was supported by fellowship F31DE028488 from the NIH National Institute of Dental and Craniofacial Research (NIDCR). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Heat stress-induced oxidative stress in bovine mammary epithelial cells (BMECs) threatens the normal growth and development of bovine mammary tissue, resulting in lower milk production of dairy cows. The aim of the present study is to investigate the protective effects of S-allyl cysteine (SAC), an organosulfur component extracted from aged garlic, on heat stress-induced oxidative stress and apoptosis in BMECs and to explore its underlying mechanisms. Our results showed that heat stress treatment considerably decreased cell viability, whereas SAC treatment dose-dependently restored cell viability of BMECs under heat-stress conditions. In addition, SAC protected BMECs from heat stress-induced oxidative damage by inhibiting the excessive accumulation of reactive oxygen species (ROS) and increasing the activity of antioxidant enzymes. It also inhibited heat stress-induced apoptosis by reducing the ratio of Bax/Bcl-2 and blocking proteolytic the cleavage of caspase-3 in BMECs. Interestingly, we found that the protective effect of SAC on heat stress-induced oxidative stress and apoptosis was dependent on the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. SAC promoted the Nrf2 nuclear translocation in heat stress-induced BMECs. The results were also validated by Nrf2 and Keap1 knockdown experiments further demonstrating that Nrf-2 was indeed involved in the protective effect of SAC on heat stress-induced oxidative damage and apoptosis. In summary, our results showed that SAC could protect BMECs from heat stress-induced injury by mediating the Nrf2/HO-1 signaling pathway, suggesting that SAC could be considered as a therapeutic drug for attenuating heat stress-induced mammary gland diseases., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Pyridoxal 5́-phosphate (PLP) is an important cofactor for amino acid decarboxylases with many biological functions, including the synthesis of signalling molecules, such as serotonin, dopamine, histamine, γ-aminobutyric acid, and taurine. Taurine is an abundant amino acid with multiple physiological functions, including osmoregulation, pH regulation, antioxidative protection, and neuromodulation. In mammalian tissues, taurine is mainly produced by decarboxylation of cysteine sulphinic acid to hypotaurine, catalysed by the PLP-dependent cysteine sulphinic acid decarboxylase (CSAD), followed by oxidation of the product to taurine. We determined the crystal structure of mouse CSAD and compared it to other PLP-dependent decarboxylases in order to identify determinants of substrate specificity and catalytic activity. Recognition of the substrate involves distinct side chains forming the substrate-binding cavity. In addition, the backbone conformation of a buried active-site loop appears to be a critical determinant for substrate side chain binding in PLP-dependent decarboxylases. Phe94 was predicted to affect substrate specificity, and its mutation to serine altered both the catalytic properties of CSAD and its stability. Using small-angle X-ray scattering, we further showed that CSAD presents open/close motions in solution. The structure of apo-CSAD indicates that the active site gets more ordered upon internal aldimine formation. Taken together, the results highlight details of substrate recognition in PLP-dependent decarboxylases and provide starting points for structure-based inhibitor design with the aim of affecting the biosynthesis of taurine and other abundant amino acid metabolites., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Hydrogen sulfide and reactive sulfur species are regulators of physiological functions, have antioxidant effects against oxidative stresses, and are endogenously generated from l-cysteine. Recently, a novel pathway that generates hydrogen sulfide and reactive sulfur species from d-cysteine has been identified. d-Amino acid oxidase (DAO) is involved in this pathway and, among the various brain regions, is especially abundant in the cerebellum. d-Cysteine has been found to be a better substrate in the generation of hydrogen sulfide in the cerebellum than l-cysteine. Therefore, d-cysteine might be a novel neuroprotectant against cerebellar diseases such as spinocerebellar ataxia (SCA). However, it remains unknown if d-cysteine affects cerebellar Purkinje cells (PCs), which are important for cerebellar functions and are frequently degenerated in SCA patients. In the present study, we investigated whether the production of hydrogen sulfide from d-cysteine affects the dendritic development of cultured PCs. d-Cysteine was found to enhance the dendritic development of PCs significantly, while l-cysteine impaired it. The effect of d-cysteine was inhibited by simultaneous treatment with DAO inhibitors and was reproduced by treatment with 3-mercaptopyruvate, a metabolite of d-cysteine produced by the action of DAO, and disodium sulfide, a donor of hydrogen sulfide. In addition, hydrogen sulfide was immediately produced in cerebellar primary cultures after treatment with d-cysteine and 3-mercaptopyruvate. These findings suggest that d-cysteine enhances the dendritic development of primary cultured PCs via the generation of hydrogen sulfide., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A serious consequence of ischemia-reperfusion injury (I/R) is oxidative damage leading to mitochondrial dysfunction. Such I/R-induced mitochondrial dysfunction is observed as impaired state 3 respiration and overproduction of O 2 - . The cascading ROS can propagate cysteine oxidation on mitochondrial complex I and add insult to injury. Herein we employed LC-MS/MS to identify protein sulfonation of complex I in mitochondria from the infarct region of rat hearts subjected to 30-min of coronary ligation and 24-h of reperfusion in vivo as well as the mitochondria of sham controls. Mitochondrial preparations from the I/R regions had enhanced sulfonation levels on the cysteine ligands of iron‑sulfur clusters, including N3 (C 425 ), N1b (C 92 ), N4 (C 226 ), N2 (C 158 /C 188 ), and N1a (C 134 /C 139 ). The 4Fe-4S centers of N3, N1b, N4, and N2 are key redox-active components of complex I, thus sulfonation of metal-binding sites impaired the main electron transfer pathway. The binuclear N1a has a very low redox potential and an antioxidative function. Increased C 134 /C 139 sulfonation by I/R impaired the N1a cluster, potentially contributing to overall O 2 - generation by the FMN moiety of complex I. MS analysis also revealed I/R-mediated increased sulfonation at the core subunits of 51 kDa (C 125 , C 187 , C 206 , C 238 , C 255 , C 286 ), 75 kDa (C 367 , C 554 , C 564 , C 727 ), 49 kDa (C 146 , C 326 , C 347 ), and PSST (C 188 ). These results were consistent with the consensus indicating that 51 kDa and 75 kDa are two of major subunits hosting regulatory thiols, and their enhanced sulfonation by I/R predisposed the myocardium to further oxidant stress with impaired ubiquinone reduction. MS analysis further showed I/R-mediated enhanced sulfonation at the supernumerary subunits of 42 kDa (C 67 , C 112 , C 183 , C 253 ), 15 kDa (C 43 ), and 13 kDa (C 79 ). The 42 kDa protein is metazoan-specific, which was reported to stabilize mammalian complex I. C 43 of the 15 kDa subunit forms an intramolecular disulfide bond with C 56 , which was reported to stabilize complex I structure. C 79 of the 13 kDa subunit is involved in Zn 2+ -binding, which was reported functionally important for complex I assembly. C 79 sulfonation by I/R was found to impair Zn 2+ -binding. No significant enhancement of protein sulfonation was observed in mitochondrial complex I from the rat heart subjected to 30-min ischemia alone in vivo despite a decreased state 3 respiration, suggesting that the physiologic conditions of hyperoxygenation during reperfusion mediated an increase in complex I sulfonation and oxidative injury. In conclusion, sulfonation of specific cysteines of complex I mediates I/R-induced mitochondrial dysfunction via impaired ETC activity, increasing • O 2 - production, and mediating redox dysfunction of complex I., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Trichloroethylene (TCE), a prevalent environmental contaminant, is a potent renal and hepatic toxicant through metabolites such as S-(1, 2-dichlorovinyl)-l-cysteine (DCVC). However, effects of TCE on other target organs such as the placenta have been minimally explored. Because elevated apoptosis and lipid peroxidation in placenta have been observed in pregnancy morbidities involving poor placentation, we evaluated the effects of DCVC exposure on apoptosis and lipid peroxidation in a human extravillous trophoblast cell line, HTR-8/SVneo. We exposed the cells in vitro to 10-100μM DCVC for various time points up to 24h. Following exposure, we measured apoptosis using flow cytometry, caspase activity using luminescence assays, gene expression using qRT-PCR, and lipid peroxidation using a malondialdehyde quantification assay. DCVC significantly increased apoptosis in time- and concentration-dependent manners (p<0.05). DCVC also significantly stimulated caspase 3, 7, 8 and 9 activities after 12h (p<0.05), suggesting that DCVC stimulates the activation of both the intrinsic and extrinsic apoptotic signaling pathways simultaneously. Pre-treatment with the tBID inhibitor Bl-6C9 partially reduced DCVC-stimulated caspase 3 and 7 activity, signifying crosstalk between the two pathways. Additionally, DCVC treatment increased lipid peroxidation in a concentration-dependent manner. Co-treatment with the antioxidant peroxyl radical scavenger (±)-α-tocopherol attenuated caspase 3 and 7 activity, suggesting that lipid peroxidation mediates DCVC-induced apoptosis in extravillous trophoblasts. Our findings suggest that DCVC-induced apoptosis and lipid peroxidation in extravillous trophoblasts could contribute to poor placentation if similar effects occur in vivo in response to TCE exposure, indicating that further studies into this mechanism are warranted., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cysteine hydropersulphide (CysSSH) is a cysteine derivative having one additional sulphur atom bound to a cysteinyl thiol group. Recent advances in the development of analytical methods for detection and quantification of persulphides and polysulphides have revealed the biological presence, in both prokaryotes and eukaryotes, of hydropersulphides in diverse forms such as CysSSH, homocysteine hydropersulphide, glutathione hydropersulphide, bacillithiol hydropersulphide, coenzyme A hydropersulphide, and protein hydropersulphides. Owing to the chemical reactivity of the persulphide moiety, biological systems utilize persulphides as important intermediates in the synthesis of various sulphur-containing biomolecules. Accumulating evidence has revealed another important feature of persulphides: their potent reducing activity, which implies that they are implicated in the regulation of redox signalling and antioxidant functions. In this chapter, we discuss the biological occurrence and possible biosynthetic mechanisms of CysSSH and related persulphides, and we include descriptions of recent advances in the analytical methods that have been used to detect and quantitate persulphide species. We also discuss the antioxidant activity of persulphide species that contributes to protecting cells from reactive oxygen species-associated damage, and we examine the signalling roles of CysSSH in bacteria., (© 2018 Elsevier Ltd. All rights reserved.)

Alliin is a garlic organosulfur compound that possesses various pharmacological properties. In the present study, the protective effects and molecular mechanism of alliin on Lipopolysaccharides (LPS)-induced acute lung injury (ALI) were analyzed. LPS-induced ALI was induced in BALB/c mice by intranasal instillation of LPS. Alliin was administered intraperitoneally to mice 1 h after LPS treatment. The results showed that alliin markedly inhibited lung myeloperoxidase (MPO) activity and wet/dry (W/D) ratio induced by LPS. Alliin also inhibited TNF-α and IL-1β in the bronchoalveolar lavage fluid (BALF) induced by LPS. Furthermore, LPS-induced lung pathological injury was attenuated by treatment of alliin. LPS-induced NF-κB activation was significantly inhibited by alliin. In addition, the expression of peroxisome proliferator-activated receptor γ (PPARγ) was up-regulated by treatment of alliin. Taken together, these results suggested that alliin protected against LPS-induced ALI by activating PPARγ, which subsequently inhibited LPS-induced NF-κB activation and inflammatory response. Alliin might be used as an anti-inflammatory agent in the treatment of ALI., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The thiol moiety of cysteine residues can undergo a number of biologic modifications including oxidation, sulfenylation, nitrosylation, persulfidation, metalation, and other modifications. These modifications can control biological function, including gain as well as loss of function. Herein, we focus attention on the proteomic analysis of S-nitrosylation in health and disease. We describe a novel quantitative approach that combines accurate, sensitive fluorescence modification of cysteinyl-S-nitrosylation that leaves electrophoretic mobility unaffected (SNOFlo), and introduce unique concepts for measuring changes in S-nitrosylation status relative to protein abundance. We present several studies where suitability of this approach for investigating endogenous S-nitrosylation is addressed., (© 2017 Elsevier Inc. All rights reserved.)

Metal ion regulation is essential for living organisms. In prokaryotes metal ion dependent transcriptional factors, the so-called metalloregulatory proteins play a fundamental role in controlling the concentration of metal ions. These proteins recognize metal ions with an outstanding selectivity. A detailed understanding of their function may be exploited in potential health, environmental and analytical applications. Members of the MerR protein family sense a broad range of mostly late transition and heavy metal ions through their cysteine thiolates. The air sensitivity of latter groups makes the expression and purification of such proteins challenging. Here we describe a method for the purification of the copper-regulatory CueR protein under optimized conditions. In order to avoid protein precipitation and/or eventual aggregation and to get rid of the co-purifying Escherichia coli elongation factor, our procedure consisted of four steps supplemented by DNA digestion. Subsequent anion exchange on Sepharose FF Q 16/10, affinity chromatography on Heparin FF 16/10, second anion exchange on Source 30 Q 16/13 and gel filtration on Superdex 75 26/60 resulted in large amounts of pure CueR protein without any affinity tag. Structure and functionality tests performed with mass spectrometry, circular dichroism spectroscopy and electrophoretic gel mobility shift assays approved the success of the purification procedure., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We here present a new method for the expression and purification of recombinant human stem cell factor (rhSCF(164)) in endotoxin-free ClearColi(®) BL21(DE3) cells harboring codon-optimized Profinity eXact™-tagged hSCF cDNA. Previously, we demonstrated that co-expression with thioredoxin increased the solubility of rhSCF in Escherichia coli BL21(DE3), and addition of l-arginine enhanced chromatography performance by removing the endotoxin-masked surface of rhSCF. Initially, we tried to express rhSCF in an endotoxin-free strain using a thioredoxin co-expression system, which resulted in significantly lower expression, possibly due to the stress imposed by overexpressed thioredoxin or antibiotics susceptibility. Therefore, we developed a new expression system without thioredoxin. External redox coupling was tested using persulfides such as glutathione persulfide or cysteine persulfide for the in vivo-folding of hSCF in the cytoplasm. Persulfides improved the protein solubility by accelerating disulfide-exchange reactions for incorrectdisulfides during folding in E. coli. Furthermore, the persulfides enhanced the expression level, likely due to upregulation of the enzymatic activity of T7 RNA polymerase. The recombinant protein was purified via affinity chromatography followed by cleavage with sodium fluoride, resulting in complete proteolytic removal of the N-terminal tag. The endotoxin-free fusion protein from ClearColi(®) BL21(DE3) could bind to the resin in the standard protocol using sodium phosphate (pH 7.2). Furthermore, purified rhSCF enhanced the proliferation and maturation of the human mast cell line LAD2. Thus, we conclude that use of the protein expression system employing E. coli by disulfide shuffling with persulfide addition could be a very useful method for efficient protein production., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The density and diversity of posttranslational modifications (PTMs) observed in histone proteins typically limit their purification to homogeneity from biological sources. Access to quantities of uniformly modified histones is, however, critical for investigating the downstream effects of histone PTMs on chromatin-templated processes. Therefore, a number of semisynthetic methodologies have been developed to generate histones bearing precisely defined PTMs or close analogs thereof. In this chapter, we present two optimized and rapid strategies for generating functional analogs of site-specifically acetylated and sumoylated histones. First, we describe a convergent strategy to site-specifically attach the small ubiquitin-like modifier-3 (SUMO-3) protein to the site of Lys12 in histone H4 by means of a disulfide linkage. We then describe the generation of thialysine analogs of histone H3 acetylated at Lys14 or Lys56, using thiol-ene coupling chemistry. Both strategies afford multimilligram quantities of uniformly modified histones that are easily incorporated into mononucleosomes and nucleosome arrays for biophysical and biochemical investigations. These methods are readily extendable to any desired sites in the four core nucleosomal histones and their variant forms., (© 2016 Elsevier Inc. All rights reserved.)

Molybdenum cofactor deficiency (MoCD) is a severe autosomal recessive inborn error of metabolism first described in 1978. It is characterized by a neonatal presentation of intractable seizures, feeding difficulties, severe developmental delay, microcephaly with brain atrophy and coarse facial features. MoCD results in deficiency of the molybdenum cofactor dependent enzymes sulfite oxidase, xanthine dehydrogenase, aldehyde oxidase and mitochondrial amidoxime reducing component. The resultant accumulation of sulfite, taurine, S-sulfocysteine and thiosulfate contributes to the severe neurological impairment. Recently, initial evidence has demonstrated early treatment with cyclic PMP can turn MoCD type A from a previously neonatal lethal condition with only palliative options, to near normal neurological outcomes in affected patients. We review MoCD and focus on describing the currently published evidence of this exciting new therapeutic option for MoCD type A caused by pathogenic variants in MOCD1., (Copyright © 2015 Elsevier Inc. All rights reserved.)

3-Hydroxyisobutyryl-CoA hydrolase deficiency (HIBCHD) is a rare inborn error of the valine catabolic pathway associated with Leigh-like disease. We report a female patient who presented at the age of 5months with hypotonia, developmental delay and cerebral atrophy on MRI. Pyruvate dehydrogenase deficiency was initially suspected and decreased activity was shown in fibroblasts. Urine tandem mass spectrometry screening showed large increases in the cysteine conjugate of methacrylate previously described in HIBCHD. 3-hydroxyisobutyryl-CoA hydrolase activity in fibroblasts was below the limit of detection of the enzymatic assay and two novel HIBCH mutations were identified (c.[129dupA];[1033G>A]). Urine metabolite investigations also showed increases in 3-hydroxyisobutyryl carnitine, 2,3-dihydroxy-2-methylbutyrate and several metabolites indicating accumulation and subsequent metabolism of methacrylyl-CoA and acryloyl-CoA. The metabolites derived from acryloyl-CoA were also increased in patients with inborn errors of propionyl-CoA metabolism, indicating the involvement of a secondary propionyl-CoA pathway utilising 3-hydroxyisobutyryl-CoA hydrolase. With the exception of 3-hydroxyisobutyryl carnitine, the metabolite abnormalities were essentially the same as those observed in patients with ECHS1 mutations, a recently described disorder that also affects valine metabolism. Our findings demonstrate the benefits of urine tandem mass spectrometry screening for diagnosing HIBCH and ECHS1 defects and that propionate metabolism may play a role in their pathogenesis. These disorders should be considered during the differential diagnosis of Leigh like-diseases and hypotonia., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Acetaminophen (APAP) overdose causes severe and occasionally fatal liver injury. Numerous drugs that attenuate APAP toxicity have been described. However these compounds frequently protect by cytochrome P450 inhibition, thereby preventing the initiating step of toxicity. We have previously shown that pretreatment with allopurinol can effectively protect against APAP toxicity, but the mechanism remains unclear. In the current study, C3HeB/FeJ mice were administered allopurinol 18h or 1h prior to an APAP overdose. Administration of allopurinol 18h prior to APAP overdose resulted in an 88% reduction in liver injury (serum ALT) 6h after APAP; however, 1h pretreatment offered no protection. APAP-cysteine adducts and glutathione depletion kinetics were similar with or without allopurinol pretreatment. The phosphorylation and mitochondrial translocation of c-jun-N-terminal-kinase (JNK) have been implicated in the progression of APAP toxicity. In our study we showed equivalent early JNK activation (2h) however late JNK activation (6h) was attenuated in allopurinol treated mice, which suggests that later JNK activation is more critical for the toxicity. Additional mice were administered oxypurinol (primary metabolite of allopurinol) 18h or 1h pre-APAP, but neither treatment protected. This finding implicated an aldehyde oxidase (AO)-mediated metabolism of allopurinol, so mice were treated with hydralazine to inhibit AO prior to allopurinol/APAP administration, which eliminated the protective effects of allopurinol. We evaluated potential targets of AO-mediated preconditioning and found increased hepatic metallothionein 18h post-allopurinol. These data show metabolism of allopurinol occurring independent of P450 isoenzymes preconditions the liver and renders the animal less susceptible to an APAP overdose., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Protein S-nitrosylation is considered as one of the molecular mechanisms by which nitric oxide regulates signaling events and protein function. The present review presents an updated method which allows for the site-specific detection of S-nitrosylated proteins in vivo. The method is based on enrichment of S-nitrosylated proteins or peptides using organomercury compounds followed by LC-MS/MS detection. Technical aspects for determining the reaction and binding efficiency of the mercury resin that assists enrichment of S-nitrosylated proteins are presented and discussed. In addition, emphasis is given to the specificity of the method by providing technical details for the generation of four chemically distinct negative controls. Finally it is provided an overview of the key steps for generation and evaluation of mass spectrometry derived data., (Copyright © 2012 Elsevier Inc. All rights reserved.)

G protein-coupled receptors (GPCRs) are the most abundant and diverse type of cell surface receptors. GPCR signal duration and amplitude are both controlled by posttranslational modifications, principally phosphorylation. Emerging evidence demonstrates that the GCPRs and their effectors are also subject to S-nitrosylation modification. Protein S-nitrosylation involves the covalent attachment of a nitric oxide (NO) group to the thiol side chain of select cysteine residues (S-NO) that impacts the protein function. Progress in this area of research has been hampered by technical limitations to measure biologic S-nitrosylation, but obstacles have been substantially alleviated over the past few years. The two most commonly used methods to detect S-nitrosylation require decomposition of the S-NO covalent bond and consequent detection of reduced thiol or released NO groups. In this review, we summarize current methods for detection of protein S-nitrosylation with a focus on the biotin switch technique and related methods., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Regional cerebral blood flow (rCBF) is a useful surrogate marker of neuronal activity and a parameter of primary interest in the diagnosis of many diseases. The increasing use of mouse models spawns the demand for in vivo measurement of rCBF in the mouse. Small animal SPECT provides excellent spatial resolution at adequate sensitivity and is therefore a promising tool for imaging the mouse brain. This study evaluates the feasibility of mouse brain perfusion SPECT and assesses the regional pattern of normal Tc-99m-HMPAO uptake and the impact of age and gender. Whole-brain kinetics was compared between Tc-99m-HMPAO and Tc-99m-ECD using rapid dynamic planar scans in 10 mice. Assessment of the regional uptake pattern was restricted to the more suitable tracer, HMPAO. Two HMPAO SPECTs were performed in 18 juvenile mice aged 7.5 ± 1.5weeks, and in the same animals at young adulthood, 19.1 ± 4.0 weeks (nanoSPECT/CTplus, general purpose mouse apertures: 1.2kcps/MBq, 0.7mm FWHM). The 3-D MRI Digital Atlas Database of an adult C57BL/6J mouse brain was used for region-of-interest (ROI) analysis. SPECT images were stereotactically normalized using SPM8 and a custom made, left-right symmetric HMPAO template in atlas space. For testing lateral asymmetry, each SPECT was left-right flipped prior to stereotactical normalization. Flipped and unflipped SPECTs were compared by paired testing. Peak brain uptake was similar for ECD and HMPAO: 1.8 ± 0.2 and 2.1 ± 0.6 %ID (p=0.357). Washout after the peak was much faster for ECD than for HMPAO: 24 ± 7min vs. 4.6 ± 1.7h (p=0.001). The general linear model for repeated measures with gender as an intersubject factor revealed an increase in relative HMPAO uptake with age in the neocortex (p=0.018) and the hippocampus (p=0.012). A decrease was detected in the midbrain (p=0.025). Lateral asymmetry, with HMPAO uptake larger in the left hemisphere, was detected primarily in the neocortex, both at juvenile age (asymmetry index AI=2.7 ± 1.7%, p=0.000) and at young adult age (AI=2.4 ± 1.7%, p=0.000). Gender had no effect on asymmetry. Voxel-wise testing confirmed the ROI-based findings. In conclusion, high-resolution HMPAO SPECT is a promising technique for measuring rCBF in preclinical research. It indicates lateral asymmetry of rCBF in the mouse brain as well as age-related changes during late maturation. ECD is not suitable as tracer for brain SPECT in the mouse because of its fast clearance from tissue indicating an interspecies difference in esterase activity between mice and humans., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Déjà vu is a common experience among the normal population. However, in individuals with temporal lobe epilepsy, it often occurs as a seizure manifestation. The specific cause of such déjà vu is not yet known. Here, we report a case of epilepsy with persistent déjà vu. The patient described the state as if he were living the same life he had lived before. Blood perfusion single-photon-emission computed tomography (SPECT) performed during the persistent déjà vu showed hyperperfusion in the left medial temporal area; discontinuation of déjà vu was accompanied by disappearance of the hyperperfused area on SPECT. Analysis with three-dimensional co-registration of SPECT and MRI revealed that the hyperperfused area during the persistent déjà vu was in the entorhinal cortex of the left temporal lobe. According to recent theories of recognition memory, malfunction of the parahippocampal area may cause déjà vu. It is also suggested that epileptic activity in the parahippocampal area, especially the entorhinal cortex, may elicit déjà vu., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Specific Se-metabolites have been recognized to be the main elements responsible for beneficial effects of Se-enriched diet, and Se-methylselenocysteine (SeMCys) is thought to be among the most effective ones. Here we show that an engineered Saccharomyces cerevisiae strain, expressing a codon optimized heterologous selenocysteine methyltransferase and endowed with high intracellular levels of S-adenosyl-methionine, was able to accumulate SeMCys at levels higher than commercial selenized yeasts. A fine tuned carbon- and sulfate-limited fed-batch bioprocess was crucial to achieve good yields of biomass and SeMCys. Through the coupling of metabolic and bioprocess engineering we achieved a ∼24-fold increase in SeMCys, compared to certified reference material of selenized yeast. In addition, we investigated the interplay between sulfur and selenium metabolism and the possibility that redox imbalance occurred along with intracellular accumulation of Se. Collectively, our data show how the combination of metabolic and bioprocess engineering can be used for the production of selenized yeast enriched with beneficial Se-metabolites., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objectives: This study aimed to determine whether regional cerebral blood flow (rCBF) is abnormal in patients who have Transient Global Amnesia (TGA)., Methods: We obtained noninvasive rCBF measurements using Tc-99m-ethyl cysteinate diamer Single Photon Emission Computed Tomography (SPECT) in 7 patients diagnosed with TGA within 4 days of onset of the amnestic episode while the patients were still symptomatic and in 17 age-matched healthy control subjects. We assessed memory functioning using the Hopkins's Verbal Learning Test (HVLT) and Statistical Parametric Mapping to compare rCBF across diagnostic groups., Results: The patients with TGA were significantly impaired in their performance on the 20-minute delayed recall of the HVLT. They also exhibited significantly decreased rCBF on their SPECT scans in the inferior and middle frontal gyrus bilaterally, with more prominent left-sided reductions in the superior temporal, precentral, and postcentral gyri, as well as increased rCBF primarily in the right hemisphere within the middle temporal, superior temporal, and inferior frontal gyri, cerebellum, and thalamus, compared with the normal control group., Conclusion: These findings suggest that lateralized abnormalities in brain functioning are an important component of the pathophysiology of TGA. Lateralized abnormalities may disrupt functions that are relatively specific to the left hemisphere, including receptive language, symbolic representation, and the processing of local features in the environment, while preserving anterograde memory processes. Increased flow to the right hemisphere centered on regions that subserve the functions of expressive language and visuospatial processing, and may represent processes that compensate for flow reductions to the left hemisphere.

Trichloroethylene (TCE) is a well-known carcinogen in rodents and concerns exist regarding its potential carcinogenicity in humans. Oxidative metabolites of TCE, such as dichloroacetic acid (DCA) and trichloroacetic acid (TCA), are thought to be hepatotoxic and carcinogenic in mice. The reactive products of glutathione conjugation, such as S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and S-(1,2-dichlorovinyl) glutathione (DCVG), are associated with renal toxicity in rats. Recently, we developed a new analytical method for simultaneous assessment of these TCE metabolites in small-volume biological samples. Since important gaps remain in our understanding of the pharmacokinetics of TCE and its metabolites, we studied a time-course of DCA, TCA, DCVG and DCVG formation and elimination after a single oral dose of 2100 mg/kg TCE in male B6C3F1 mice. Based on systemic concentration-time data, we constructed multi-compartment models to explore the kinetic properties of the formation and disposition of TCE metabolites, as well as the source of DCA formation. We conclude that TCE-oxide is the most likely source of DCA. According to the best-fit model, bioavailability of oral TCE was approximately 74%, and the half-life and clearance of each metabolite in the mouse were as follows: DCA: 0.6 h, 0.081 ml/h; TCA: 12 h, 3.80 ml/h; DCVG: 1.4 h, 16.8 ml/h; DCVC: 1.2 h, 176 ml/h. In B6C3F1 mice, oxidative metabolites are formed in much greater quantities (approximately 3600 fold difference) than glutathione-conjugative metabolites. In addition, DCA is produced to a very limited extent relative to TCA, while most of DCVG is converted into DCVC. These pharmacokinetic studies provide insight into the kinetic properties of four key biomarkers of TCE toxicity in the mouse, representing novel information that can be used in risk assessment.

This study was designed to evaluate the utility of subtraction ictal SPECT coregistered to MRI (SISCOM) in atypical psychogenic nonepileptic seizures (PNES). Video-EEG monitoring (vEEG) is the gold standard for PNES diagnosis but, like any modality, has limitations. In difficult cases in which the diagnosis is suspected but remains in question after vEEG, a complementary study that could help differentiate epilepsy from PNES would be desirable. Thirteen SISCOM studies performed in patients with a final diagnosis of PNES were retrospectively reviewed. Common indications for SISCOM were semiology consistent with partial epilepsy (9/13), abnormal head MRI (5/13), and reported abnormal routine EEG (5/13). SISCOM was negative in 85% (11/13) of patients and was helpful in increasing the diagnostic certainty of PNES in these exceptional cases.

The lantibiotics are a class of ribosomally synthesized and posttranslationally modified peptide antibiotics containing the thioether cross-links lanthionine (Lan) and 3-methyllanthionine (MeLan) and typically also the dehydroamino acids dehydroalanine (Dha) and (Z)-dehydrobutyrine (Dhb). The modifications are formed by dehydration of Ser/Thr residues to produce the Dha and Dhb structures, and subsequent conjugate additions of Cys residues onto the unsaturated amino acids to form thioether rings (Lan and MeLan). Because of their ribosomal origin, investigations of the substrate specificity of lantibiotic synthetases have typically focused on site-directed mutagenesis. With the in vitro reconstitution of lacticin 481 synthetase, its substrate specificity was explored in much greater detail by the incorporation of a series of nonproteinogenic Ser, Thr, and Cys analogues into a truncated prelacticin peptide LctA using a combination of solid-phase peptide synthesis (SPPS) and expressed protein ligation (EPL). The strategy described can be used for the growing number of ribosomally synthesized and posttranslationally modified natural products such as the microcins and patellamides.

It has been proposed that Se-methylselenocysteine (MeSeCys) and methylseleninic acid (MSA(IV)) are efficiently transformed through the beta-lyase and reduction reactions, respectively, into methylselenol, the assumed biologically active selenometabolite responsive for the anti-carcinogenicity and anti-oxidant actions of selenium. The bioavailability and distribution of the two selenium sources in major organs/tissues were compared under exactly identical conditions. Namely, labeled selenium sources (76)Se-MeSeCys and (77)Se-MSA(IV), at a single oral dose of 10 microg Se/kg body weight each, were administered simultaneously to rats that had been depleted of natural abundance selenium with a single isotope (78)Se. The same dose of (82)Se-selenite was also administered as a reference selenium source. The distributions of the three labeled selenium isotopes were determined 3 h after the administration in 13 organs/tissues/blood. MeSeCys was taken up more efficiently by most organs, especially the pancreas and duodenum, than MSA(IV) and selenite, the latter two sources being taken up similarly to each other except for in the kidney, liver, and spleen, where the three labeled isotopes were detected at comparable concentrations. The labeled selenium in the liver supernatant was speciated by HPLC inductively coupled argon plasma-mass spectrometry (ICP-MS), and it was suggested that MeSeCys was delivered in its intact form to organs, and then transformed into methylselenol. In addition to the known properties of that MeSeCys is chemically more stable than MSA(IV) and is a naturally occurring edible product, and that MeSeCys produces methylselenol much more efficiently than a homologous selenoamino acid selenomethionine, the present study revealed that MeSeCys is more efficiently distributed than MSA(IV) in its intact form, and then produces methylselenol, suggesting that MeSeCys is the best methylselenol source in most organs/tissues.

The Golgi apparatus processes intracellular proteins, but undergoes disassembly and fragmentation during apoptosis in several neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer's disease. It is well known that other cytoplasmic organelles play important roles in cell death pathways. Thus, we hypothesized that Golgi fragmentation might participate in transduction of cell death signals. Here, we found that Golgi fragmentation and dispersal precede neuronal cell death triggered by excitotoxins, oxidative/nitrosative insults, or ER stress. Pharmacological intervention or overexpression of the C-terminal fragment of Grasp65, a Golgi-associated protein, inhibits fragmentation and decreases or delays neuronal cell death. Inhibition of mitochondrial or ER cell death pathways also decreases Golgi fragmentation, indicating crosstalk between organelles and suggesting that the Golgi may be a common downstream-effector of cell death. Taken together, these findings implicate the Golgi as a sensor of stress signals in cell death pathways.

Arsenite adsorption onto a protein-rich biomass and, more specifically, the chemical groups involved in the uptake were investigated using Raman spectroscopy and DFT calculations. The study was based on spectroscopic analyses of raw and arsenic-loaded biomass as well as standard samples of amino acids and arsenic salts. The predominant secondary structure of the protein was identified as the beta-sheet type, with some contribution from alpha-helix structures. The participation of sulphydryl groups from cystine/cysteine molecules during the adsorption of arsenite was demonstrated. Only the gauche-gauche-gauche (g-g-g) conformation type of the disulfide bonds was involved in arsenic complexation. The formation of a pyramidal trigonal As(HCys)(3) complex was modeled according to the density functional theory (DFT). The agreement of the DFT harmonic frequencies with the RAMAN spectra of the As(HCys)(3) complex demonstrated the relevant features of the cysteine-rich biomaterial regarding arsenic uptake as well as of the mechanism involved in the As(III)/biomass interaction at a molecular level. The results also illustrate that Raman spectroscopy can be successfully applied to investigate the mechanism of metal adsorption onto amorphous biomaterials.

All members in the protein tyrosine phosphatase (PTP) family of enzymes contain an invariant Cys residue which is absolutely indispensable for catalysis. Due to the unique microenvironment surrounding the active center of PTPs, this Cys residue exhibits an unusually low pKa characteristic, thus being highly susceptible to oxidation or S-nitrosylation. While oxidation-dependent regulation of PTP activity has been extensively examined, the molecular details and biological consequences of PTP S-nitrosylation remain unexplored. We hypothesized that the catalytic Cys residue is targeted by proximal nitric oxide (NO) and its derivatives collectively termed reactive nitrogen species (RNS), leading to nitrosothiol formation concomitant with reversible inactivation of PTPs. To test this hypothesis, we have developed novel strategies to examine the redox status of Cys residues of purified PTP1B that was exposed to NO donor S-Nitroso-N-penicillamine (SNAP). A gel-based method in conjunction with mass spectrometry (MS) analysis revealed that the catalytic Cys215 of PTP1B was reversibly modified when PTP1B was briefly treated with SNAP. In order to further identify the exact mode of NO-induced modification, we employed an online LC-ESI-MS/MS analysis incorporating a mass difference-based, data-dependent acquisition function that effectively mapped the S-nitrosylated Cys residues. Our results demonstrated that treating PTP1B with SNAP led to S-nitrosothiol formation of the catalytic Cys215. Interestingly, SNAP-induced modifications were strictly reversible as highly oxidized Cys derivatives (Cys-SO(2)H or Cys-SO(3)H) were not identified by MS analyses. Thus, the methods introduced in this study provide direct evidence to prove the direct link between S-nitrosylation of the catalytic Cys residue and reversible inactivation of PTPs.

Simultaneous or prior exposure to one chemical may alter the concurrent or subsequent response to another chemical, often in unexpected ways. This is particularly true when the two chemicals share common mechanisms of action. The present study uses the paradigm of prior exposure to study the interactive toxicity between inorganic mercury (Hg(2+)) and trichloroethylene (TRI) or its metabolite S-(1,2-dichlorovinyl)-l-cysteine (DCVC) in rat and human proximal tubule. Pretreatment of rats with a subtoxic dose of Hg(2+) increased expression of glutathione S-transferase-alpha1 (GSTalpha1) but decreased expression of GSTalpha2, increased activities of several GSH-dependent enzymes, and increased GSH conjugation of TRI. Primary cultures of rat proximal tubular (rPT) cells exhibited both necrosis and apoptosis after incubation with Hg(2+). Pretreatment of human proximal tubular (hPT) cells with Hg(2+) caused little or no changes in GST expression or activities of GSH-dependent enzymes, decreased apoptosis induced by TRI or DCVC, but increased necrosis induced by DCVC. In contrast, pretreatment of hPT cells with TRI or DCVC protected from Hg(2+) by decreasing necrosis and increasing apoptosis. Thus, whereas pretreatment of hPT cells with Hg(2+) exacerbated cellular injury due to TRI or DCVC by shifting the response from apoptosis to necrosis, pretreatment of hPT cells with either TRI or DCVC protected from Hg(2+)-induced cytotoxicity by shifting the response from necrosis to apoptosis. These results demonstrate that by altering processes related to GSH status, susceptibilities of rPT and hPT cells to acute injury from Hg(2+), TRI, or DCVC are markedly altered by prior exposures.

Se-Methylated selenoamino acids, Se-methylselenocysteine (MeSeCys) and selenomethionine (SeMet), are chemically inert storage forms of selenium in selenium-accumulators, and a nutritional and supplemental source. The metabolic pathway for MeSeCys was precisely traced by referring to those for SeMet and selenite by applying a new tracer method involving multiple homo-elemental stable isotopes. Male Wistar rats were depleted of endogenous natural abundance selenium with a single (80)Se-enriched isotope, and then (76)Se-MeSeCys, (77)Se-SeMet and (82)Se-selenite were orally administered simultaneously at 25 microg Se/kg body weight each. Organs and body fluids were obtained at 3, 6, 9 and 12 h, and 1 and 2 days later, and subjected to speciation analysis. The main characteristics of the metabolism were as follows; MeSeCys was incorporated into selenoprotein P slightly more than or at a comparable level to that of SeMet but less than that of selenite. MeSeCys and SeMet but not selenite was taken up by organs in their intact forms. MeSeCys and SeMet were delivered specifically to the pancreas and present in a form bound to an identical or similar protein. Trimethylselenonium (TMSe) was only produced from MeSeCys, i.e., not from SeMet or selenite, in the kidneys. Both selenosugars A and B of MeSeCys, SeMet and selenite origin were detected in the liver but only selenosugar B in the kidneys. These results suggest that MeSeCys can be a similar or better selenium source than SeMet, and supplies methylselenol much more efficiently in organs than SeMet and selenite. TMSe was produced much efficiently from MeSeCys than from SeMet and selenite, suggesting a role of methylselenol through the beta-lyase reaction in the metabolism of Se-methylated selenoamino acids.

Epidemiology studies suggest that there may be a weak association between high level exposure to trichloroethylene (TCE) and renal tubule cell carcinoma. Laboratory animal studies have shown an increased incidence of renal tubule carcinoma in male rats but not mice. TCE can undergo metabolism via glutathione (GSH) conjugation to form metabolites that are known to be nephrotoxic. The GSH conjugate, S-(1,2-dichlorovinyl)glutathione (DCVG), is processed further to the cysteine conjugate, S-(1,2-dichlorovinyl)-l-cysteine (DCVC), which is the penultimate nephrotoxic species. We have cultured human renal tubule cells (HRPTC) in serum-free medium under a variety of different culture conditions and observed growth, respiratory control and glucose transport over a 20 day period in medium containing low glucose. Cell death was time- and concentration-dependent, with the EC(50) for DCVG being about 3 microM and for DCVC about 7.5 microM over 10 days. Exposure of HRPTC to sub-cytotoxic doses of DCVC (0.1 microM and 1 microM for 10 days) led to a small number of changes in gene expression, as determined by transcript profiling with Affymetrix human genome chips. Using the criterion of a mean 2-fold change over control for the four samples examined, 3 genes at 0.1 microM DCVC increased, namely, adenosine kinase, zinc finger protein X-linked and an enzyme with lyase activity. At 1 microM DCVC, two genes showed a >2-fold decrease, N-acetyltransferase 8 and complement factor H. At a lower stringency (1.5-fold change), a total of 63 probe sets were altered at 0.1 microM DCVC and 45 at 1 microM DCVC. Genes associated with stress, apoptosis, cell proliferation and repair and DCVC metabolism were altered, as were a small number of genes that did not appear to be associated with the known mode of action of DCVC. Some of these genes may serve as molecular markers of TCE exposure and effects in the human kidney.

Background: The pathophysiological role of the extensive interictal cerebral hypometabolism in complex partial seizures (CPS) in refractory mesial temporal lobe epilepsy with hippocampal sclerosis (mTLE-HS) is poorly understood. Our aim was to study ictal-interictal SPECT perfusion versus interictal fluorodeoxyglucose (FDG)-PET metabolic patterns., Methods: Eleven adults with refractory unilateral mTLE-HS, who were rendered seizure free after epilepsy surgery, were included. All had an interictal FDG-PET and an interictal and ictal perfusion SPECT scan. FDG-PET data were reconstructed using an anatomy-based reconstruction algorithm, which corrected for partial volume effects, and analyzed semi-quantitatively after normalization to white matter activity. Using Statistical Parametric Mapping (SPM), we compared interictal metabolism of the patient group with a control group. We correlated metabolic with ictal perfusion changes in the patient group., Results: Global cerebral grey matter glucose metabolism in patients was decreased 10-25% compared with control subjects. Interictal PET hypometabolism and ictal SPECT hypoperfusion were maximal in the ipsilateral frontal lobe. Ictal frontal lobe hypoperfusion was associated with crossed cerebellar diaschisis. The ipsilateral temporal lobe showed maximal ictal hyperperfusion and interictal hypometabolism, which was relatively mild compared with the degree of hypometabolism affecting the frontal lobes., Conclusion: Interictal hypometabolism in mTLE-HS was greatest in the ipsilateral frontal lobe and represented a seizure-related dynamic process in view of further ictal decreases. Crossed cerebellar diaschisis suggested that there is a strong ipsilateral frontal lobe inhibition during CPS. We speculate that surround inhibition in the frontal lobe is a dynamic defense mechanism against seizure propagation, and may be responsible for functional deficits observed in mTLE.

Streptozotocin (STZ)-induced diabetic (DB) rats are protected from nephrotoxicity of gentamicin, cisplatin and mercuric chloride, although the mechanisms remain unclear. Ninety percent of DB mice receiving a LD90 dose (75 mg/kg, ip) of S-1,2-dichlorovinyl-l-cysteine (DCVC) survived in contrast to only 10% of the nondiabetic (NDB) mice surviving the same dose. We tested the hypothesis that the mechanism of protection is upregulated tissue repair. In the NDB mice, DCVC produced steep temporal increases in blood urea nitrogen (BUN) and plasma creatinine, which were associated with proximal tubular cell (PTC) necrosis, acute renal failure (ARF), and death within 48 h. In contrast, in the DB mice, BUN and creatinine increased less steeply, declining after 36 h to completely resolve by 96 h. HPLC analysis of plasma and urine revealed that DB did not alter the toxicokinetics of DCVC. Furthermore, activity of renal cysteine conjugate beta-lyase, the enzyme that bio-activates DCVC, was unaltered in DB mice, undermining the possibility of lower bioactivation of DCVC leading to lower injury. [3H]-thymidine pulse labeling and PCNA analysis indicated an early onset and sustained nephrogenic tissue repair in DCVC-treated DB mice. BRDU immunohistochemistry revealed a fourfold increase in the number of cells in S-phase in the DB kidneys even without exposure to DCVC. Blocking the entry of cells into S-phase by antimitotic intervention using colchicine abolished stimulated nephrogenic tissue repair and nephro-protection. These findings suggest that pre-placement of S-phase cells in the kidney due to diabetes is critical in mitigating the progression of DCVC-initiated renal injury by upregulation of tissue repair, leading to survival of the DB mice by avoiding acute renal failure.

After isoprenylation, the Ras proteins and other proteins terminating with a so-called CAAX motif undergo two additional modifications: (1) endoproteolytic cleavage of the -AAX by Ras converting enzyme 1 (Rce1) and (2) carboxyl methylation of the isoprenylated cysteine residue by isoprenylcysteine carboxyl methyltransferase (Icmt). Although CAAX protein isoprenylation has been studied in great detail, until recently, very little was known about the biological role and functional importance of Icmt in mammalian cells. Studies over the past few years, however, have begun to fill in the blanks. Genetic experiments showed that Icmt-deficient embryos die at mid-gestation, whereas conditional inactivation of Icmt in the liver, spleen, and bone marrow is not associated with obvious pathology. One potential explanation for the embryonic lethality is that Icmt is the only enzyme in mouse cells capable of methylating isoprenylated CAAX proteins--including the Ras proteins. Furthermore, in addition to the CAAX proteins, Icmt methylates the CXC class of isoprenylated Rab proteins. In the absence of carboxyl methylation, the Ras proteins are mislocalized away from the plasma membrane and exhibit a shift in electrophoretic mobility. Given the important role of oncogenic Ras proteins in human tumorigenesis and the mislocalization of Ras proteins in Icmt-deficient cells, it has been hypothesized that inhibition of Icmt could be a strategy to block Ras-induced oncogenic transformation. Recent data provide strong support to that hypothesis: conditional inactivation of Icmt in mouse embryonic fibroblasts and treatment of cells with a novel selective inhibitor of Icmt, termed cysmethynil, results in a striking inhibition of Ras-induced oncogenic transformation.

To investigate abnormal cerebral perfusion in narcoleptics with cataplexy, 25 narcoleptics with cataplexy and 25 normal controls were enrolled in this study. Cerebral perfusion was measured by brain single photon emission computed tomography (SPECT) using 99mTc-ethylcysteinate dimer. Patients and normal controls had not received any medication prior to the SPECT scan. Differences in cerebral perfusion between narcoleptics and normal controls were subjected to statistical parametric mapping (SPM) analysis. Overnight polysomnography and multiple sleep latency test (MSLT) were performed in all patients. Brain SPECT was carried out on all patients and normal controls during the waking state. Clinical symptoms and MSLT results of all patients are in accord with the International Classification of Sleep Disorders criteria for narcolepsy. MSLT showed a short mean sleep latency (1.69 +/- 1.0 min) and 2-5 sleep onset REM periods in individual patient. SPM analysis of brain SPECT showed hypoperfusion of the bilateral anterior hypothalami, caudate nuclei, and pulvinar nuclei of thalami, parts of the dorsolateral/ventromedial prefrontal cortices, parahippocampal gyri, and cingulate gyri in narcoleptics [P < 0.05 by Student's t test with false discovery rate (FDR) correction]. Significant hypoperfusion in the white matter of frontal and parietal lobes was also noted in narcoleptics. This study shows reduced cerebral perfusion in subcortical structures and cortical areas in narcoleptics. The distribution of abnormal cerebral perfusion is concordant with the pathway of the cerebral hypocretin system and may explain the characteristic features of narcolepsy, i.e., cataplexy, emotional lability, and attention deficit.

Difficulties in evaluation of trichloroethylene (TRI)-induced toxicity in humans and extrapolation of data from laboratory animals to humans are due to the existence of multiple target organs, multiple metabolic pathways, sex-, species-, and strain-dependent differences in both metabolism and susceptibility to toxicity, and the lack or minimal amount of human data for many target organs. The use of human tissue for mechanistic studies is thus distinctly advantageous. The kidneys are one target organ for TRI and metabolism by the glutathione (GSH) conjugation pathway is responsible for nephrotoxicity. The GSH conjugate is processed further to produce the cysteine conjugate, S-(1,2-dichlorovinyl)-l-cysteine (DCVC), which is the penultimate nephrotoxic species. Confluent, primary cultures of human proximal tubular (hPT) cells were used as the model system. Although cells in log-phase growth, which are undergoing more rapid DNA synthesis, would give lower LD(50) values, confluent cells more closely mimic the in vivo proximal tubule. DCVC caused cellular necrosis only at relatively high doses (>100 muM) and long incubation times (>24 h). In contrast, both apoptosis and enhanced cellular proliferation occurred at relatively low doses (10-100 muM) and early incubation times (2-8 h). These responses were associated with prominent changes in expression of several proteins that regulate apoptosis (Bcl-2, Bax, Apaf-1, Caspase-9 cleavage, PARP cleavage) and cellular growth, differentiation and stress response (p53, Hsp27, NF-kappaB). Effects on p53 and Hsp27 implicate function of protein kinase C, the mitogen activated protein kinase pathway, and the cytoskeleton. The precise pattern of expression of these and other proteins can thus serve as molecular markers for TRI exposure and effect in human kidney.

Acetaminophen (APAP) nephrotoxicity has been observed both in humans and research animals. Our recent investigations have focused on the possible involvement of glutathione-derived APAP metabolites in APAP nephrotoxicity and have demonstrated that administration of acetaminophen-cysteine (APAP-CYS) potentiated APAP-induced renal injury with no effects on APAP-induced liver injury. Additionally, APAP-CYS treatment alone resulted in a dose-responsive renal GSH depletion. This APAP-CYS-induced renal GSH depletion could interfere with intrarenal detoxification of APAP or its toxic metabolite N-acetyl-p-benzoquinoneimine (NAPQI) and may be the mechanism responsible for the potentiation of APAP nephrotoxicity. Renal-specific GSH depletion has been demonstrated in mice and rats following administration of amino acid gamma-glutamyl acceptor substrates for gamma-glutamyl transpeptidase (gamma-GT). The present study sought to determine if APAP-CYS-induced renal glutathione depletion is the result of disruption of the gamma-glutamyl cycle through interaction with gamma-GT. The results confirmed that APAP-CYS-induced renal GSH depletion was antagonized by the gamma-glutamyl transpeptidase (gamma-GT) inhibitor acivicin. In vitro analysis demonstrated that APAP-CYS is a gamma-glutamyl acceptor for both murine and bovine renal gamma-GT. Analysis of urine from mice pretreated with acivicin and then treated with APAP, APAP-CYS, or acetaminophen-glutathione identified a gamma-glutamyl-cysteinyl-acetaminophen metabolite. These findings are consistent with the hypothesis that APAP-CYS contributes to APAP nephrotoxicity by depletion of renal GSH stores through interaction with the gamma-glutamyl cycle.

Acetaminophen (APAP) nephrotoxicity has been observed both in humans and research animals. Recent studies suggest a contributory role for glutathione (GSH)-derived conjugates of APAP in the development of nephrotoxicity. Inhibitors of either gamma-glutamyl transpeptidase (gamma-GT) or the probenecid-sensitive organic anion transporter ameliorate APAP-induced nephrotoxicity but not hepatotoxicity in mice and inhibition of gamma-GT similarly protected rats from APAP nephrotoxicity. Protection against APAP nephrotoxicity by disruption of these GSH conjugate transport and metabolism pathways suggests that GSH conjugates are involved. APAP-induced renal injury may involve the acetaminophen-glutathione (APAP-GSH) conjugate or a metabolite derived from APAP-GSH. Acetaminophen-cysteine (APAP-CYS) is a likely candidate for involvement in APAP nephrotoxicity because it is both a product of the gamma-GT pathway and a probable substrate for the organic anion transporter. The present experiments demonstrated that APAP-CYS treatment alone depleted renal but not hepatic glutathione (GSH) in a dose-responsive manner. This depletion of renal GSH may predispose the kidney to APAP nephrotoxicity by diminishing GSH-mediated detoxification mechanisms. Indeed, pretreatment of male CD-1 mice with APAP-CYS before challenge with a threshold toxic dose of APAP resulted in significant enhancement of APAP-induced nephrotoxicity. This was evidenced by histopathology and plasma blood urea nitrogen (BUN) levels at 24 h after APAP challenge. APAP alone was minimally nephrotoxic and APAP-CYS alone produced no detectable injury. By contrast, APAP-CYS pretreatment did not alter the liver injury induced by APAP challenge. These data are consistent with there being a selective, contributory role for APAP-GSH-derived metabolites in APAP-induced renal injury that may involve renal-selective GSH depletion.

N-(2,3,4,5,6-pentahydroxylhexyl)-L-cysteine (4) and N,N'-di(2,3,4,5,6-pentahydroxylhexyl)-L-cystine (5) were synthesized. As antagonists of cadmium or lead intoxication, they were found to be effective by oral administration on repeated exposure. The bioassay in vivo indicated that their cadmium-mobilizing potencies are significantly superior to those of N-acetyl-L-cysteine and their lead-mobilizing properties are equivalent to those of DL-penicillamine (DL-PA). The stability constants of the Cd-4 and Cd-5 complexes or Pb-4 and Pb-5 complexes were determined by use of pH titration. The membrane permeability of compounds 4 and 5 was evaluated by transporting across the Caco-2 cell monolayer. The effect of compounds 4 and 5 on the concentrations of essential metal ions in the renal cortex is negligible in comparison with that of the group treated with lead only. The acute toxicity of compounds 4 was tested by oral gavage and the resulting LD50 value for both mice and rats is larger than 10 g kg(-1) bw. The cytogenetic effects of compound 4 were evaluated in the Ames assay, mouse bone marrow micronucleus test, and sperms shape abnormality assay, and all of them show the negative reactions.

Visual assessment, with significant inter- or intraobserver variability, is still the norm for the evaluation of Single Photon Emission Computerized Tomography (SPECT) cerebral perfusion studies. We present in this paper an automated method for screening SPECT studies to detect diffuse disseminated abnormalities based on a computerized atlas of normal regional cerebral blood flow (rCBF). To generate the atlas, a set of normal brain SPECT studies are registered together. The atlas contains the intensity mean, the nonlinear displacement mean, and the variance of the activity pattern. A patient is then evaluated by registering his or her SPECT volume to the atlas and computing the nonlinear 3-D displacement of each voxel needed for the best shape fit to it. A voxel is counted as "abnormal" if the intensity difference between the atlas and the registered patient (or if the 3-D motion necessary to move the voxel to its registered position) is superior to 3 SD of normal mean. The number of abnormal voxels is used to classify studies. We validated this approach on 24 SPECT perfusion studies selected visually for having clear diffuse anomalies and 21 normal studies. A Markovian segmentation algorithm is also used to identify the white and gray matters for regional analysis. Based on the number of abnormal voxels, two supervised classifiers were tested: (1) minimum distance-to-mean and (2) Bayesian. The analysis of the intensity and displacement "abnormal" voxels allow one to achieve an 80% correct classification rate for the whole brain and a 93% rate if we consider only voxels in the segmented gray matter region.

S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a metabolite of a common environmental contaminant, trichloroethylene, is a selective proximal tubular nephrotoxicant. The objective of our study was to examine the dose-response relationship of renal injury and repair following DCVC administration. Male Swiss-Webster mice were injected with DCVC [15, 30, or 75 mg/kg ip in distilled water (10 ml/kg)] and the extent of nephrotoxicity and tissue repair was assessed over a 14-day period. The renal injury due to the low and medium doses of DCVC peaked at 36 and 72 h after dosing, respectively, and then regressed over time due to a timely and adequate tissue repair response. At the highest dose tissue repair was inhibited, thereby causing progression of renal injury, which led to acute renal failure and death of the mice. The possibility that compromised tissue repair was a result of the extensive nephrotoxic injury attendant to the high dose of DCVC was investigated via an equinephrotoxicity study in which separate groups of mice received 40 (LD40) and 75 (LD90) mg DCVC/kg, respectively. Bioactivation-based renal proximal tubular injury measured in these two groups over a time course was identical but there was a marked difference in mortality due to an early and robust tissue repair in the first group relative to the second group. These results support the concept that quantitative evaluation of renal tissue repair in parallel with injury is useful in the assessment of the likely toxic outcome associated with exposure to nephrotoxic drugs and toxicants., (Copyright 2003 Elsevier Science (USA))

Eight patients (seven women), mean +/- SD T1 age 68.57 +/- 12.43 years, average educational level 5.83 +/- 3.70 years, had two Tc-99m ECD SPECT examinations separated by an average 8.49 +/- 5.59 months. Patients were imaged using standard Harlem Hospital acquisition and processing protocols with approximately 30 mCi of ECD on a Prism 3000 triple head gamma camera. Images were interpreted by an independent reader blinded to the patients' clinical history and imaging date. T1 psychiatric diagnosis was seven Alzheimer's dementia (AD) and one depression. Eight T1 images were interpreted as abnormal, six indicative of AD. Binomial 95% two-tail confidence interval for T1 agreement between diagnosis and interpretation was 0.25 0.63 0.92. T2 diagnosis was seven AD and one none. Seven T2 images were abnormal and indicative of AD, and one was normal. T2 confidence interval was 0.34 0.75 0.97. These findings suggest SPECT's value in assessing AD in uneducated socioeconomically disadvantaged geriatric patients.

C165S AhpC in its sulfenate (Cys-SO-) and presumed thiolate (Cys-S-) forms at pH 7 (pKa for sulfenic acid about pH 6.1) exhibit low extinction absorbance bands around 367 and 324 nm, respectively. Sulfenic acid content of the protein can be assessed by its reactivity with the chromophoric TNB anion. Using this technique, H2O2 titrations of C165S AhpC give a maximum of about 1 SOH per subunit on addition of 1.0 to 1.2 equivalents of H2O2. Cys46-SO- is moderately air stable at neutral pH and room temperature and is oxidized at a steady rate of about 10% per half hour. Cys46-SO- of C165S AhpC is reduced in the presence of catalytic amounts of AhpF by approximately 1 equivalent of NADH to regenerate the Cys46-S- species. NBD chloride is extremely useful as a trapping agent for cysteine sulfenic acid. The Cys46-S(O)-NBD adduct absorbs maximally at 347 nm and is 16 amu larger than the Cys46-S-NBD adduct (lambda max = 420 nm) as shown by ESI-MS. Other electrophilic thiol reagents also react with Cys46-SO-; however, iodoacetamide and N-ethylmaleimide reactivities are much lower with Cys46-SO- than with Cys46-S-. These methods are applicable to other sulfenic acid-containing proteins, although in some cases the proteins must be denatured in order to provide accessibility of this species toward labeling agents.

Apoptosis, necrosis, and cell proliferation induced by S-(1,2-dichlorovinyl)-L-cysteine (DCVC), the cysteine conjugate of the environmental and occupational contaminant trichloroethylene, were studied in primary cultures of human proximal tubular (hPT) cells. Cells from male and female donors were incubated with a range of concentrations of DCVC (10 to 1000 microM) for up to 48 h, and assessments of cellular morphology (phase-contrast microscopy), necrosis (lactate dehydrogenase (LDH) release), apoptosis(cell cycle analysis, annexin V staining, and caspase activation), and proliferation (cell cycle analysis and DNA synthesis) were made. Time- and concentration-dependent changes in cellular morphology, including elongation of cell shape, formation of intracellular vesicles, and formation of apoptotic bodies, were observed. Significant increases in LDH release occurred in hPT cells incubated with < or =100 microM DCVC for at least 24 h. hPT cells from males were modestly more sensitive to DCVC than those from females, with maximal LDH release of 78 and 65% in cells from males and females, respectively. Flow cytometry analysis of propidium iodide-stained and DCVC-treated hPT cells showed that apoptosis occurred at markedly lower concentrations (10 microM) and at much earlier incubation times (2 h) than necrosis. A small increase was also noted in the percentage of cells in S-phase after a 4-h treatment with as little as 10 microM DCVC, suggesting that cell proliferation was stimulated. This was supported further by increased DNA synthesis. These results show that DCVC causes apoptosis and enhances cell proliferation in hPT cells at environmentally relevant doses and at earlier time points and lower concentrations than necrosis., (Copyright 2001 Academic Press.)

To determine whether nitric oxide (NO)/peroxynitrite plays any role in neurodestruction observed in ischemic cochlea of the guinea pig, the effects of NO donors like S-nitrosocysteine (S-NC) and nitroglycerin (NTG), peroxynitrite generators like 3-morpholinosydnonimine (SIN-1), peroxynitrite inhibitors like superoxide dismutase plus catalase (SOD/Cat), as well as NOS inhibitors like N(G)-nitro-l-arginine methyl ether (L-NAME), were tested on normal and ischemic cochleae. Various concentrations of S-NC and SIN-1 were introduced into the perilymphatic space of normal guinea pig cochlea. Quantitative scanning electron microscopy of inner and outer hair cells was carried out 2 days later. To determine the level of NO in the cochlea after 20 to 120 min of ischemia, nitrites/nitrates in the perilymph were measured. The effects of NO on the ischemic cochlea were tested by infusion of SOD/Cat, L-NAME, S-NC, and NTG into the perilymphatic space just before decapitation. Introduction of fixative into the cochlea was delayed for 15 min to investigate the effects of the chemicals on nerve endings at the base of inner hair cells. The results showed that the level of nitrites/nitrates tended to decline with increasing time of ischemia. There was no significant hair cell loss in the cochleae treated with SIN-1 or S-NC. At 15 min after ischemia, most of the nerve endings at the base of the inner hair cells were protected from damage when 1 mM S-NC or NTG was infused into the perilymph. Taken together, the results indicate that NO/peroxynitrite is unlikely to be involved in the neurodestruction in the ischemic cochlea. In fact, exogenous NO may have a neural protective effect., (Copyright 2001 Academic Press.)

The nephrotoxicity of trichloroethylene and dichloroacetylene has previously been linked to mitochondrial dysfunction induced by the metabolite S-(1,2-dichlorovinyl)-l-cysteine (DCVC). In this study, we examined whether key biochemical steps associated with mitochondria occur in DCVC-induced apoptosis in cultured porcine proximal tubular LLC-PK1 cells. DCVC caused a decrease in mitochondrial membrane potential (mt Delta Psi) beginning at 4 h and a release of cytochrome c into the cytoplasm at 6 h. Caspase-3-like activity was detected at 6 h and extensive DNA fragmentation was observed at 8 h. Decreases in cellular ATP were not evident until 8 h and later, even though electron microscopy showed that the mitochondria were extensively swollen. Aminooxyacetic acid (AOAA), an inhibitor of cysteine-conjugate beta-lyase, protected against mitochondrial changes and apoptosis. Overexpression of the antiapoptotic Bcl-2 protein desensitized LLC-PK1 cells to DCVC-induced apoptosis. These results support the interpretation that mitochondrial release of cyt c and cyt c-dependent activation of caspase-3 could have a central role in nephrotoxicity due to haloalkene-derived cysteine S-conjugates., (Copyright 2001 Academic Press.)

A primary objective of this review is to facilitate the application of the chemical and structural approaches that are currently being employed in the identification of Cys-SOH, as both transient intermediates and stable redox forms, in biochemical systems where these derivatives are suspected of playing key roles in redox catalysis or regulation. These range from high-resolution crystallographic analyses benefiting from recent technological advances in rapid data collection at cryogenic temperatures to 13C NMR investigations of [3-(13)C]Cys-labeled proteins and chemical modification protocols that can be integrated with both UV-visible and fluorescence spectroscopic as well as mass spectrometric (especially ESI, MALDI-TOF, and even FT ion-cyclotron-resonance) analyses. In summarizing the diversity of biological functions currently identified with Cys-SH reversible Cys-SOH redox cycles (Fig. 17), it should also be [figure: see text] emphasized that in at least one protein (nitrile hydratase) stable Cys-SOH and Cys-SO2H derivatives play important structural roles while also modulating the electronic properties of the iron center; in neither case is the Cys-SOH residue itself involved in reduction and oxidation. The somewhat incomplete structural descriptions of the oxidized Cys forms involved in redox regulation of some transcription factors (e.g., BPV-1 E2 protein and activator protein-1) indicate that there is ample room for the application of the types of investigations employed, for example, with NADH peroxidase and the AhpC peroxiredoxin, with a view toward defining the potential roles of Cys-SOH in these very important contexts of intracellular redox signaling. These advances will also build on the recent progress in defining sulfenic acid stabilization and properties in small molecule model systems, as evidenced in the work of Okazaki, Goto, and others. When viewed in the perspective of Allison's 1976 review on the subject of sulfenic acids in proteins, the reader will hopefully come to appreciate the conclusion that the concept of protein-sulfenic acids has now become a very well-defined and established principle of biochemistry, with current efforts in this and other laboratories being directed to bring about still more detailed understanding of Cys-SOH function in both redox and nonredox modes of enzyme catalysis and regulation of protein function.

We have shown that renal proximal tubular cells (RPTC) recover cellular functions following sublethal injury induced by the oxidant t-butylhydroperoxide but not by the nephrotoxic cysteine conjugate dichlorovinyl-L-cysteine (DCVC). This study investigated whether L-ascorbic acid phosphate (AscP) promotes recovery of RPTC functions following DCVC-induced injury. DCVC exposure (200 microM; 100 min) resulted in 60% RPTC death and loss from the monolayer at 24 h independent of physiological (50 microM) or pharmacological (500 microM) AscP concentrations. Likewise, the DCVC-induced decrease in mitochondrial function (54%), active Na(+) transport (66%), and Na(+)-K(+)-ATPase activity (77%) was independent of the AscP concentration. Analysis of Na(+)-K(+)-ATPase protein expression and distribution in the plasma membrane using immunocytochemistry and confocal laser scanning microscopy revealed the loss of Na(+)-K(+)-ATPase protein from the basolateral membrane of RPTC treated with DCVC. DCVC-injured RPTC cultured in the presence of 50 microM AscP did not proliferate nor recover their physiological functions over time. In contrast, RPTC cultured in the presence of 500 microM AscP proliferated, recovered all examined physiological functions, and the basolateral membrane expression of Na(+)-K(+)-ATPase by day 4 following DCVC injury. These results demonstrate that pharmacological concentrations of AscP do not prevent toxicant-induced cell injury and death but promote complete recovery of mitochondrial function, active Na(+) transport, and proliferation following toxicant-induced injury., (Copyright 2000 Academic Press.)