Formation Of GS Research Articles

Comparative anatomical and transcriptomic analyses of the color variation of leaves in Aquilaria sinensis.

Color variation in plant tissues is a common phenomenon accompanied with a series of biological changes. In this study, a special-phenotype Aquilaria sinensis (GS) with color variation of leaf was firstly reported, and DNA barcode sequences showed GS samples could not be discriminated clearly with the normal A. sinensis sample (NS), which suggested that the variety was not the cause of the GS formation. To reveal the characteristics of GS compared to NS, the anatomical and transcriptome sequencing studies were carried out. In microscopic observation, the leaves of golden-vein-leaf sample (LGS) and normal-vein-leaf sample (LNS) showed significant differences including the area of the included phloem in midrib and the thickness parameters of palisade and spongy tissues; the stems of golden-vein-leaf sample (SGS) and normal-vein-leaf sample (SNS) were also different in many aspects such as the area of vessels and included phloem. In addition, the structure of chloroplast was more complete in the midrib of LNS than that of LGS, and some particles suspected as virus were found through transmission electron microscope as well. Genes upregulated in LGS in contrast with LNS were mainly enriched in photosynthesis. As for stems, most of the genes upregulated in SGS compared to SNS were involved in translation and metabolism processes. The pathways about photosynthesis and chlorophyll metabolism as well as some important transcription factors may explain the molecular mechanism of the unique phenotypes of leaves and the genes related to suberin biosynthesis may result in the difference of stems. In addition, the genes about defense response especially biotic stress associated with numerous pathogenesis-related (PR) genes upregulated in LGS compared to LNS indicated that the pathogen may be the internal factor. Taken together, our results reveal the macro- and micro-phenotype variations as well as gene expression profiles between GS and NS, which could provide valuable clues for elucidating the mechanism of the color variation of Aquilaria.

The incidence of gall stones after bariatric surgery and its association with weight loss

IntroductionGallstone (GS) formation is common in obese patients, particularly during rapid weight loss. This study aimed to determine the incidence of GS in patients underwent bariatric surgery (BS) and the influence of weight loss on GS formation. MethodsThis was a prospective study including 148 consecutive patients underwent BS. Patients with preoperative negative abdominal ultrasound (US) for GS were followed-up for at least 12 months. Patients underwent abdominal ultrasound (US) at 3, 6, 9 and 12 months and were clinically screened for symptomatic or complicated cholelithiasis. No GS lowering prophylaxis was used. Clinical and demographic characteristics were compared at baseline and 12 months after surgery. ResultsAt time of surgery GS was found in 40.6% of the studied patients and history of previous laparoscopic cholecystectomy (LC) was in 6.8% of the studied patients. The incidence of post bariatric GS at 12 months was 22.7% with significant difference (P-value<0.05), (Laparoscopic Sleeve Gastrectomy (LSG): 18.8%, Single anastomosis sleeve ileal bypass (SASI): 33.3%, Mini Gastric Bypass (MGB): 40%, LRYGB 50%). BS operations, rapid weight loss and gastro esophageal reflux disease (GERD) patients are at a higher risk for GS. ConclusionsThe incidence of post bariatric GS at 12 months was high (22.7%) with significant difference and we recommend routine US every 3, 6, 9 and 12 months during the first year after BS.

Mutant KRAS Enhances Stress Granules and Resistance to Proteasome Inhibition Via 15-d-PGJ2 in Multiple Myeloma

Introduction: Mutant KRAS leads to activation of the MEK/ERK pathway in approximately 20% of multiple myeloma (MM) patients. Stress granules (SGs) are cytosolic non-membranous structures composed of translational mRNAs, ribosomal proteins, and RNA-binding proteins, which form in response to different stress stimuli and chemotherapeutic treatment. We have previously shown that mutant KRAS upregulates SG formation. The molecular mechanisms underlying this process are unclear. The purpose of this study was to elucidate a) the mechanism by which mutant KRAS upregulates SG formation and b) to provide a molecular rationale for the targeting the cascades for SG formation in MM patients. Methods. We determined whether mutant KRAS is necessary for SG upregulation. To this purpose a panel of MM cell lines including mutant KRAS (KRASM), mutant NRAS (NRASm) and wildtype KRAS (KRASWT) was used. The gain- and loss-of-function of mutant KRAS was generated to suppress (KRASM/KO) in KRASM or switch on by expression of mutant KRAS G12A (KRASG12A) in KRASWT cells by a lentiviral vector system. SG formation was assessed by measuring the cellular distribution of SG resident proteins by immunofluorescence using anti-G3BP and anti-eIF4G antibodies. The quantitative readout for SG formation was analyzed by ImageJ analyze-particle tool as the SG index. Immunoblotting was used to detect inactivation of eIF2a or activity of MAPK. qPCR analysis was employed to detect the mRNA of COX2. MTT assays were used to detect the effect of SGs on the survival of MM cells alone or in combination with diclofenac sodium (DS), or bortezomib (Bzb). Results: We compared the induction of SGs in response to oxidative stress across a panel of MM cells with different Ras mutation status (KRASM, NRASM and KRASWT)to investigate the role mutant KRAS in GS formation. The level of SGs were significantly higher (p&lt;0.01) in six KRASM, compared with four KRASWT cell lines.We assessed SG formation in KRASM, KRASM/KO and KRAS/G12A cells to determine whether mutant KRAS is a causative factor leading to elevated SG levels. SG formation in KRASM/KO cells was significantly attenuated, by approximately 5-fold (p&lt;0.001), compared to KRASM in response to oxidative stress. Conversely, SG levels were 3.5-fold elevated in KRAS/G12A cells (P&lt;0.01), compared to KRASWT cells. Upregulation of SGs by mutant KRAS resulted in a larger average size and number of SGs per cells. Together, these results indicate that mutant KRAS is necessary and sufficient for SG upregulation under oxidative stress condition. We investigated whether 15-d-PGJ2-depedent inactivation of elF4A is involved in SG upregulation to determine the mechanism through which mutant KRAS promotes SG formation. To do this, we assessed the effect of 15-d-PGJ2 on SG formation because that elF4A inactivation is controlled by 15-d-PGJ2, which is one of COX2-derived products. Treatment of KRASM cells with 15-d-PGJ2 significantly induced accumulation of SGs compared with untreated cells (40-fold increase vs control cells, p&lt;0.001). To determine whether COX2, which controls the rate of biosynthesis of 15-d-PGJ2, is regulated by mutant KRAS, we first compared COX2 mRNA of KRASM/KO with KRASM and showed that a decrease in COX2 expression in KRASM/KO (2-fould decrease, P&lt; 0.01). Conversely, increased COX2 mRNA was seen in KRAS/G12A, compared with KRASWT cells. Furthermore, treatment of KRASM cells with DS led to a decrease in SG levels and this effect was rescued by the addition of 15-d-PGJ2. These results indicate that increase in prostaglandin levels by upregulating prostaglandin biosynthesis is essential for mutant KRAS-induced up-regulation of SGs. Finally, we determined if inhibition of COX2 activity by DS was able to overcome Bzb resistance in KRASM or in KRASM co-occurring t(14:16)/MAF. A significant decrease of cell viability (P&lt;0.001) was observed when treatment of the cells with Bzb and DS were combined, compared with DS treatment alone. Taken together, these results indicate that inhibition of COX2 activity enhances mutant KRAS MM cell sensitivity to proteasome inhibition. Conclusion: Our results suggest that mutant KRAS mutant upregulates SG formation by 15-d-PGJ2 and inhibition of COX2 activity enhances KRAS sensitivity to proteasome inhibition and targeting SG formation might be represent an effective strategy to treatment of KRAS mutant myeloma. Disclosures Davies: Janssen, Celgene: Other: Research Grant, Research Funding; Amgen, Celgene, Janssen, Oncopeptides, Roche, Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Consultant/Advisor. Morgan:Amgen, Roche, Abbvie, Takeda, Celgene, Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Other: research grant, Research Funding. Walker:Celgene: Research Funding. van Rhee:Karyopharm Therapeutics: Consultancy; Kite Pharma: Consultancy; Adicet Bio: Consultancy; EUSA: Consultancy; Castleman Disease Collaborative Network: Consultancy; Sanofi Genzyme: Consultancy; Takeda: Consultancy.

GEOCHEMICAL FEATURES OF METABASITE OF PAVLOVKA AND ZELENIY GAY GREENSTONE STRUCTURE OF EASTERN AZOV (UKRAINIAN SHIELD)

Pavlovka and Zeleniy Gay greenstone structure different from other greenstone structures of Azov megablock lack of ultramafic volcanics. Metabasites of Pavlovka GS related to the tholeiitic series and discover the trend of iron enrichment and metabasites of Zeleniy Gai – belong to calc-alkaline series. From bottom to top in the section in the metabasites of metatoleite formation of Pavlovka GS increases iron, titaniferous content and rare earth elements and reduced alumina content. Metabasites of lower parts of metatoleitick formation belongs to the type N-MORB and the top – to the type E-MORB. The latter differ of Nb positive anomaly (Nb/Nb* = 1,35). Metabasites of Zeleniy Gay GS among the most leucocratic varieties mafic potassium and related to the type of calc-alkaline basalts. It has been much more content of Rb, Sr, Ba, REE, Th, Pb and smaller – Ni, Cr and V. Metabasites of Pavlovka and Zeleniy Gay greenstone structures of the Eastern Azov formed from magma plume sources (ΔNb = +0,03ч0,43). The presence in a cross-sectional Zeleniy Gay andesites may indicate their formation in island-arc regime unlike rift greenstone structures of West-Priazov block.

95 - Fenamic Acid Analogues Oxidized by Peroxidase Enzymes Cause the Formation of Fibronectin Aggregation and Fibronectin-Derived Radicals

Fenamic acids form the substructure of many non-steroidal anti-inflammatory drugs (NSAIDs); they are amongst the most commonly prescribed drugs globally. Interactions between fenamic acid NSAIDs and peroxidases have shown that reactive metabolites can be produced, although detailed studies of these metabolites or their relative reactivity have not been studied. Adverse drug reactions may be catalyzed by peroxidase enzymes in off-target tissues. Myeloperoxidase (MPO) is a peroxidase enzyme found abundantly in neutrophils, which are recruited during inflammation and it is well known that MPO can catalyze drug oxidation. Moreover, it has been demonstrated that MPO binds to fibronectin (FN); the latter is an important multi-functional protein. The objective of this study was to examine the differential reactivity of fenamic acid NSAIDs when oxidized by MPO and the consequent effects on FN. Studies using ESR spin trapping with DMPO revealed that N-phenanthranilic acid (N-PA) produced a stable radical when oxidized by MPO/H2O2; diphenylamine (DPA) and 3-methyldiphenylamine (3-MDA) produced ●OH (DMPO/●OH). In addition, the analogs showed the formation of GS● after the addition of GSH to the MPO/H2O2 system, with 3-MDA producing the most intense DMPO/GS● spectrum. To determine the cytotoxic potential of these radical metabolites, we used HL-60 promyelocytic leukemia cells which contain MPO. 3-MDA, DPA and N-PA demonstrated MPO-catalyzed cytotoxicity in these cells, but not mefenamic acid (MFA). LC/MS analysis of the DPA product formation showed the formation of a hydroxydiphenylamine and a quinoneimine product, indicating MPO oxidation resulted in a hydroxylation reaction. The quinoneimine products appeared to react with GSH, forming a GSH conjugate. Immuno-spin trapping analysis demonstrated that MFA was the most potent in producing FN-protein free radical formation in a MPO-system. However, FN aggregation analysis by silver staining was most prominent with DPA. MFA and its analogues showed the formation of free radicals in a peroxidase-system which interferes with normal cell functioning. Future studies are needed to determine the functional impact of fenamic acid NSAID mediated damage to FN.

Prevalence of Gall Stones in Liver Cirrhosis

Increased incidence and prevalence of gallstones in liver cirrhosis has already been reported by many studies. This study aimed to investigate the prevalence of gallstone disease in Nepali patients with LC and to identify risk factors for gallstone formation. Consecutive patients of LC presenting to liver clinic from January, 2010 to December, 2012 were evaluated for GS by ultrasonography at their first visit. Liver cirrhosis was diagnosed on the basis of clinical features, laboratory parameters, ultrasonography, and/or histopathology. Two hundred and twenty four LC patients were studied. Male to female ratio was 2.3:1. Alcohol was the major etiological factor for LC followed by hepatitis B, alone or in conjunction with alcohol. Seventy-four patients (33%) were found to have GS. Incidence of GS was more in advance stage of cirrhosis. There was no significant correlation between formation of GS and etiology of LC, except for the HCV related liver cirrhosis, in which it was present in 39% of the patients. More the advance disease,more was the incidence as 57% of Child-C patients had GS. Portal vein diameter was also associated with GS formation. When portal vein diameter was more than 13 mm, there was more GS formation. One third of the patients of LC showed GS at the presentation. Patients with HCV related cirrhosis are more prone to develop GS than other. Severity of the disease and portal vein diameter was found to be associated with GS formation.

Glutathione sulfinamide serves as a selective, endogenous biomarker for nitroxyl after exposure to therapeutic levels of donors

Nitroxyl (HNO) donors exhibit promising pharmacological characteristics for treatment of cardiovascular disorders, cancer, and alcoholism. However, whether HNO also serves as an endogenous signaling agent is currently unknown, largely because of the inability to selectively and sensitively detect HNO in a cellular environment. Although a number of methods to detect HNO have been developed recently, sensitivity and selectivity against other nitrogen oxides or biological reductants remain problematic. To improve selectivity, the electrophilic nature of HNO has been harnessed to generate modifications of thiols and phosphines that are unique to HNO, especially compared to nitric oxide (NO). Given high bioavailability, glutathione (GSH) is expected to be a major target of HNO. As a result, the putative selective product glutathione sulfinamide (GS(O)NH2) may serve as a high-yield biomarker of HNO production. In this work, the formation of GS(O)NH2 after exposure to HNO donors was investigated. Fluorescent labeling followed by separation and detection using capillary zone electrophoresis with laser-induced fluorescence allowed quantitation of GS(O)NH2 with nanomolar sensitivity, even in the presence of GSH and derivatives. Formation of GS(O)NH2 was found to occur exclusively upon exposure of GSH to HNO donors, thus confirming selectivity. GS(O)NH2 was detected in the lysate of cells treated with low-micromolar concentrations of HNO donors, verifying that this species has sufficient stability to server as a biomarker of HNO. Additionally, the concentration-dependent formation of GS(O)NH2 in cells treated with an HNO donor suggests that the concentration of GS(O)NH2 can be correlated to intracellular levels of HNO.

In vitro evaluation of the potential for drug-induced toxicity based on 35S-labeled glutathione adduct formation and daily dose

Drug-induced toxicity such as idiosyncratic drug toxicity is believed to be reduced when either reactive metabolite formation or exposure to a drug is minimized. The objective of the present study was therefore to clarify the relationship between the daily doses, the formation rates of reactive metabolite adduct with (35)S-glutathione (RM-GS) and the safety profiles of compounds. The RM-GS formation rates for 113 test compounds were determined by incubation with human liver microsomes, and the test compounds were classified into three categories of safe, warning and withdrawn/black box warning. A total of 23 out of 28 withdrawn/black box warning drugs showed both a RM-GS formation rate of over 1 pmol/30 min/mg protein and a dose of over 100 mg. These results suggest that when compounds are plotted in this region, the compounds would have a relatively high idiosyncratic drug toxicity potential.

Determination of S-nitrosoglutathione and other nitrosothiols by p-hydroxymercurybenzoate derivatization and reverse phase chromatography coupled with chemical vapor generation atomic fluorescence detection

S-Nitrosoglutathione (GSNO) reacts with the organic mercurial probe, p-hydroxymercury benzoate (PHMB, HO–Hg–(C 6H 4)–COO −Na +) giving the complex GS–Hg(C 6H 4)COOH (GS–PHMB). This reaction has been studied by UV measurements at 334 nm also in the presence of ascorbic acid and the product of reaction, the GS–PHMB complex, characterized by Electrospray Ionization Mass Spectrometry (ESI-MS) and by Reversed Phase Chromatography (RPLC) coupled on-line and sequentially with a UV–visible diode array detector (DAD) followed by a cold vapor generation atomic fluorescence spectrometer (CVGAFS). The simultaneous presence of PHMB and ascorbate produced a synergistic effect on GSNO decomposition rate that can be observed only above a given concentration threshold of ascorbate (ascorbate/GSNO molar ratio ≥ 180). The results indicated that the formation of GS–PHMB, both in the presence and the absence of ascorbic acid, does not involve the formation of free thiolic species but it takes place through a more complex mechanism. The PHMB derivatives of GSH and GSNO obtained by the present method were found to be identical by ESI-MS. GSSG did not interfere because it was not reduced and derivatized to GS–PHMB. Once complexed by the alkylating agent N-ethylmaleimide (NEM), GSH did not interfere with the derivatization reaction. This ensured a good selectivity of the developed PHMB derivatization system for RSNO determination. Thus, we have optimized the operating conditions for the selective reaction of PHMB with GSNO and other nitrosothiols (RSNOs) in order to determinate RSNOs in human plasma. LODc for RSNOs in plasma ultrafiltrate was 30 nM (injected concentration, 50 μL loop), the DLR ranged between 0.08 and 50 μM and the CV% was 6.5% at 300 nM concentration level. Reduced and oxidized thiols spiked to plasma did not interfere with the measurement of RSNOs. We found that the sampling procedure was critical for the recovery of endogenous and spiked RSNOs. The ultrafiltrate samples of plasma of 8 healthy humans contained 1460 ± 310 CysNO, 1000 ± 330 nM HCysNO and 320 ± 60 nM GSNO if blood was sampled in a mixture NEM/ethylendiaminotetracetic acid (EDTA)/serine–borate complex (SBC), where serine–borate complex is a potent inhibitor of γ-glutamyltransferase, an enzyme involved in the conversion of GSNO into CysGlyNO. In the absence of SBC during the sampling of blood GSNO concentration found in the ultrafiltrate was lower (at level of the determination limit in plasma ultrafiltrate, i.e. 75 nM) and the peak of CysGlyNO appeared, which corresponded to a concentration of 200 ± 60 nM ( N = 4 blood samples).

Generation of nitroxyl by heme protein-mediated peroxidation of hydroxylamine but not N-hydroxy-L-arginine

The chemical reactivity, toxicology, and pharmacological responses to nitroxyl (HNO) are often distinctly different from those of nitric oxide (NO). The discovery that HNO donors may have pharmacological utility for treatment of cardiovascular disorders such as heart failure and ischemia reperfusion has led to increased speculation of potential endogenous pathways for HNO biosynthesis. Here, the ability of heme proteins to utilize H 2O 2 to oxidize hydroxylamine (NH 2OH) or N-hydroxy-L-arginine (NOHA) to HNO was examined. Formation of HNO was evaluated with a recently developed selective assay in which the reaction products in the presence of reduced glutathione (GSH) were quantified by HPLC. Release of HNO from the heme pocket was indicated by formation of sulfinamide (GS(O)NH 2), while the yields of nitrite and nitrate signified the degree of intramolecular recombination of HNO with the heme. Formation of GS(O)NH 2 was observed upon oxidation of NH 2OH, whereas NOHA, the primary intermediate in oxidation of L-arginine by NO synthase, was apparently resistant to oxidation by the heme proteins utilized. In the presence of NH 2OH, the highest yields of GS(O)NH 2 were observed with proteins in which the heme was coordinated to a histidine (horseradish peroxidase, lactoperoxidase, myeloperoxidase, myoglobin, and hemoglobin) in contrast to a tyrosine (catalase) or cysteine (cytochrome P450). That peroxidation of NH 2OH by horseradish peroxidase produced free HNO, which was able to affect intracellular targets, was verified by conversion of 4,5-diaminofluorescein to the corresponding fluorophore within intact cells.

Discriminating formation of HNO from other reactive nitrogen oxide species

Nitroxyl (HNO) exhibits unique pharmacological properties that often oppose those of nitric oxide (NO), in part due to differences in reactivity toward thiols. Prior investigations suggested that the end products arising from the association of HNO with thiols were condition-dependent, but were inconclusive as to product identity. We therefore used HPLC techniques to examine the chemistry of HNO with glutathione (GSH) in detail. Under biological conditions, exposure to HNO donors converted GSH to both the sulfinamide [GS(O)NH 2] and the oxidized thiol (GSSG). Higher thiol concentrations generally favored a higher GSSG ratio, suggesting that the products resulted from competitive consumption of a single intermediate (GSNHOH). Formation of GS(O)NH 2 was not observed with other nitrogen oxides (NO, N 2O 3, NO 2, or ONOO −), indicating that it is a unique product of the reaction of HNO with thiols. The HPLC assay was able to detect submicromolar concentrations of GS(O)NH 2. Detection of GS(O)NH 2 was then used as a marker for HNO production from several proposed biological pathways, including thiol-mediated decomposition of S-nitrosothiols and peroxidase-driven oxidation of hydroxylamine (an end product of the reaction between GSH and HNO) and N G-hydroxy- l-arginine (an NO synthase intermediate). These data indicate that free HNO can be biosynthesized and thus may function as an endogenous signaling agent that is regulated by GSH content.

Glutathione Propagates Oxidative Stress Triggered by Myeloperoxidase in HL-60 Cells: EVIDENCE FOR GLUTATHIONYL RADICAL-INDUCED PEROXIDATION OF PHOSPHOLIPIDS AND CYTOTOXICITY

Glutathione acts as a universal scavenger of free radicals at the expense of the formation of the glutathionyl radicals (GS*). Here we demonstrated that GS* radicals specifically interact with a reporter molecule, paramagnetic and non-fluorescent 4-((9-acridinecarbonyl)-amino)-2,2,6,6-tetramethylpiperidine-1-oxyl (Ac-Tempo), and convert it into a non-paramagnetic fluorescent product, identified as 4-((9-acridinecarbonyl)amino)-2,2,6,6-tetramethylpiperidine (Ac-piperidine). Horseradish peroxidase-, myeloperoxidase-, and cyclooxygenasecatalyzed oxidation of phenol in the presence of H2O2 and GSH caused the generation of phenoxyl radicals and GS* radicals, of which only the latter reacted with Ac-Tempo. Oxidation of several other phenolic compounds (e.g. etoposide and tyrosine) was accompanied by the formation of GS* radicals along with a characteristic fluorescence response from Ac-Tempo. In myeloperoxidase-rich HL-60 cells treated with H2O2 and phenol, fluorescence microscopic imaging of Ac-Tempo revealed the production of GS* radicals. A thiol-blocking reagent, N-ethylmaleimide, as well as myeloperoxidase inhibitors (succinyl acetone and azide), blocked formation of fluorescent acridine-piperidine. H2O2/phenolinduced peroxidation of major classes of phospholipids in HL-60 cells was completely inhibited by Ac-Tempo, indicating that GS* radicals were responsible for phospholipid peroxidation. Thus, GSH, commonly viewed as a universal free radical scavenger and major intracellular antioxidant, acts as a pro-oxidant during myeloperoxidase-catalyzed metabolism of phenol in HL-60 cells.

Regioselective Monosulfation and Disulfation of the Phytoestrogens Daidzein and Genistein by Human Liver Sulfotransferases

Regioselective sulfation of the phytoestrogens daidzein (DZ, 7,4'-dihydroxyisoflavone) and genistein (GS, 5,7,4'-trihydroxyisoflavone) was investigated using human liver cytosol and purified recombinant human sulfotransferase (SULT) isoforms, SULT1A1, SULT1A3, SULT2A1, and SULT1E1. 7-Position-preferential sulfation of DZ and GS was observed in human hepatic cytosols from 3 male and 3 female subjects. Average ratios for 7- to 4'-sulfate formation were 4.5:1 from DZ and 8.4:1 from GS in these human liver cytosols. Apparent K(m) values for the 7- and 4'-sulfation of DZ and GS by these cytosols were similar and in a range from 0.46 to 0.66 microM. All recombinant human SULTs had activity for 7- and 4'-sulfation of these phytoestrogens except for 7-sulfating activity of SULT1A3. SULT1A1 and SULT1E1 exhibited much higher catalytic efficiency, k(cat)/K(m), for 7- and 4'-sulfation of these substrates than did the other two, SULT1A3 and SULT2A1. SULT1A1 showed K(m) values of 0.47 and 0.52 microM for the mono-sulfation of DZ and GS, respectively, which were very similar to those of human cytosol. The observed k(cat)/K(m) indicated that SULT1A1 catalyzed 7-sulfation of DZ and GS at rates 4.4- and 8.8-fold higher, respectively, than such 4'-sulfation. However, with SULT1E1, catalytic efficiency was very similar for the sulfation of both positions. These data strongly suggest that SULT1A1 plays a major role in monosulfation of the phytoestrogens and determines the regioselectivity of sulfation in human hepatic cytosol. A kinetic study for 7,4'-disulfate formation of DZ and GS from their 7- and 4'-monosulfates indicated that SULT1E1 most efficiently catalyzed both reactions among human SULTs.

Formation of S-[2-carboxy-1-(1H-imidazol-4-yl) ethyl]glutathione, a new metabolite of L-histidine, from cis-urocanic acid and glutathione by the action of glutathione S-transferase.

Exposure of the skin to sunlight results in an increase of the content of epidermal trans-urocanic acid, a key metabolite of L-histidine, and also in occurrence of the isomerization of trans-urocanic acid to the cis isomer. S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]glutathione (GS(CIE)), an adduct of urocanic acid and glutathione, is a presumed origin of a urinary compound S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-L-cysteine (Cys(CIE)). The formation of GS(CIE) is stimulated by exposing the skin to sunlight irradiation. In this study we investigated an enzymatic formation of GS(CIE) from glutathione and cis-urocanic acid by incubation with rat liver extract that contained glutathione S-transferase (GST) at high activity. The formation of GS(CIE) was suppressed significantly when a liver extract depleted of GST activity was used. Enzymatic degradation of GS(CIE) with gamma -glutamyl transpeptidase resulted in the formation of N-[S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-L-cysteinyl]glycine, a metabolic intermediate between the glutathione adduct and Cys(CIE). A hydrolyzed product of GS(CIE) by HCl was identical with the urinary Cys(CIE). Compounds were analyzed by high-voltage paper electrophoresis, capillary electrophoresis, and fast atom bombardment mass spectrometry. From these results, we suggest that GS(CIE) formed from cis-urocanic acid and glutathione is an origin of the urinary compound Cys(CIE) and that the formation reaction is catalyzed mostly by the action of GST.

Glutathionylation of proteins by glutathione disulfide S-oxide

Aqueous solution of S-nitrosoglutathione (GSNO) underwent spontaneous chemical transformation that generated several glutathione derivatives including glutathione sulfonic acid (GSO3H), glutathione disulfide S-oxide (GS(O)SG), glutathione disulfide S-dioxide, and glutathione disulfide. Surprisingly, GS(O)SG (also called glutathione thiosulfinate), which was not identified as a metabolite of GSNO previously, was one of the major products derived from GSNO. This compound was very reactive toward any thiol and the reaction product was a mixed disulfide. The rate of reaction of GS(O)SG with 5-mercapto-2-nitro-benzoate was nearly 20-fold faster than that of GSNO. The mechanism for the formation of GS(O)SG was believed to involve the sulfenic acid (GSOH) and thiosulfinamide (GS(O)NH2) intermediates; the former underwent self-condensation and the latter reacted with GSH to form GS(O)SG. Many reactive oxygen and nitrogen species were also capable of oxidizing GSH or GSSG to form GS(O)SG, which likely played a central role in integrating both the oxidative and nitrosative cellular responses through thionylation of thiols. Treatments of rat brain tissue slices with oxidants resulted in an enhanced thionylation of proteins with a concomitant increase in cellular level of GS(O)SG, suggesting that this compound might play a second messenger role for stimuli that produced a variety of oxidative species.

Kinetics of the anaerobic reaction of nitric oxide with cysteine, glutathione and cysteine-containing proteins: implications for in vivo S-nitrosation

A study is made of the kinetics of the anaerobic reaction of nitric oxide (˙NO) with cysteine and glutathione in relation to its potential physiological importance for the S-nitrosation of cysteine-containing peptides and proteins. The kinetics of the reaction with cysteine (the basic reagent unit) is studied most extensively and it is found that the rate constant is directly proportional to the degree of ionization (α), k = 0.37 × 103α M−1 s−1, offering clear proof for a mechanism based on electrophilic attack of ˙NO on thiolate anions. The rate constant for glutathione is considerably lower than for cysteine at identical pH, which can be attributed to its higher pKa; steric effects do not appear to affect the reactivity of glutathione significantly. On the basis of the rate equations obtained and of similar data for bovine serum albumin and metallothionein-1 a number of calculations were performed with the aim of determining the relative importance of the reaction of ˙NO with O2vs. the direct reaction of ˙NO with peptide and protein thiols under in vivo conditions. The results clearly show that in cells that as a rule contain an appreciable concentration of glutathione the autoxidation of ˙NO and thus the reaction of higher oxides of nitrogen (˙NO2, N2O3) with thiol groups in peptides and proteins does not play a role of any significance with respect to the formation of S-nitrosothiols, as the direct reaction of ˙NO with the thiolate group in glutathione leading to the formation of GS–˙NO− is much faster than the reaction of ˙NO with O2. The difference in reactivity is less pronounced in the case of bovine serum albumin, but again the electrophilic attack clearly is more important than the autoxidation at most physiological ˙NO concentrations. Direct electrophilic attack of ˙NO on metallothionein is of no practical significance, as the process is very much slower than the attack on glutathione.

Determination of S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]glutathione, a novel metabolite of L-histidine, in tissue extracts from sunlight-irradiated rat by capillary electrophoresis.

Exposure of the skin to sunlight results in an increase in the content of epidermal urocanic acid, a key metabolite of L-histidine, and some portions of the metabolite penetrate into the body fluid. S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]glutathione (GS(CIE)), an adduct of glutathione and urocanic acid, was proposed to be an origin of a urinary compound, S-[2-carboxy-1-(1 H-imidazol-4-yl)ethyl]-L-cysteine (Cys(CIE)). Various catabolites of Cys(CIE) were also isolated from human urine previously. However, no direct evidence to show the existence of GS(CIE) as a biological material had been found. By using capillary electrophoresis, the glutathione adduct has now been found in the extracts of rat tissues from the kidney, liver, skin and blood when the rat was kept under conditions of sunlight irradiation after the fur on the dorsal skin had been clipped. On the other hand, no or a trace of GS(CIE) was determined in rat tissue extracts when the animal was kept indoor in usual manner. The glutathione adduct was isolated from the kidney extract of the sunlight-irradiated rat using ion-exchangers and high-voltage paper electrophoresis, and determined by fast-atom-bombardment mass spectrometry. These results indicate that GS(CIE) formation actually occurs in the body and that the formation is accelerated by exposing the rat to sunlight irradiation. From these findings, we propose an alternative pathway of histidine metabolism which is initiated by the adduction of urocanic acid to glutathione to form GS(CIE) and terminates with the formation of the urinary compounds via Cys(CIE).

Generation of thiyl radical by nitric oxide: a spin trapping study

The free radical intermediates formed during the nitrosation of thiols including glutathione (GSH), L-cysteine (CystSH), captopril (CapSH) and N-acetylpenicillamine (NAPenSH) by NO˙ at physiological pH have been investigated using EPR spectroscopy combined with the spin trap DMPO. We have found that NO˙ in the presence of oxygen reacted with GSH, CystSH and CapSH to generate the corresponding thiyl radicals, which are spin trapped by DMPO to give the spin trapped adducts DMPO-SG, DMPO-SCyst and DMPO-SCap respectively. In the case of NAPenSH no spin trapped adduct is detected. Desferrioxamine, an inhibitor of peroxynitrite (ONOO–), has no effect on the formation of either DMPO-SG or DMPO-SCyst, suggesting that ONOO– is not involved in the formation of GS˙ and Cyst˙. In contrast, desferrioxamine inhibits the detection of DMPO-SCap, pointing to a role for ONOO– in the formation of CapS˙. Our results demonstrate that ˙NO2 is the most likely reactive species in the nitrosation of GSH and CystSH by oxygenated NO˙, thereby generating the corresponding thiyl radicals. Furthermore, our data indicate that the scavenging of NO˙ by thiols may lead to the production of superoxide (O2˙–) via the formation of thiyl radicals.

Kinetics of myosin subfragment-1-induced condensation of G-actin into oligomers, precursors in the assembly of F-actin-S1. Role of the tightly bound metal ion and ATP hydrolysis.

In a low ionic strength buffer and in the absence of free ATP, the interaction of G-actin (G) with myosin subfragment-1 (S1) leads to the formation of arrowhead-decorated F-actin-S1 filaments, through a series of elementary steps. The initial formation of GS and G2S complexes is followed by their condensation into short oligomers. The kinetics of formation of G-actin-S1 oligomers have been monitored in a stopped-flow apparatus using a combination of light scattering and fluorescence of NBD-labeled actin. Oligomers appear more stable and are formed at a faster rate from MgATP-G-actin than from CaATP-G-actin. The actin-bound ATP is hydrolyzed when oligomers are formed from MgATP-G-actin, not when they are formed from CaATP-G-actin. The formation of oligomers is energetically favored in the presence of cytochalasin D. All data are consistent with the view that the actin-actin interactions which take place upon condensation of GS and G2S into oligomers are very similar to lateral actin-actin interactions along the short pitch helix of actin filaments, which are involved in actin nucleation. These interactions trigger ATP hydrolysis on actin.

Migration of metallic clips used during laparoscopic cholecystectomy and formation of gallstones around them: surgical implications from a prospective study.

Two groups of patients, with laparoscopic cholecystectomy (LC) were prospectively studied. All patients had serial plain abdominal X-ray examinations at various intervals after operation, to record the position of clips placed during LC. Seventy-one patients had less cystic duct (CD) dissection and > or =4 clips placed during the procedure. One hundred and fifteen patients had a larger CD dissection and only 4 clips placed (2 on the cystic artery and 2 on the CD, without additional clips on smaller vessels). In the former group, 7 patients had clip migration within 1 month and 11 within 1 year vs 1 either at 1 month or 1 year in the latter group (p = 0.01 and <0.001, respectively). During the follow-up, a 72-year-old man belonging to the former group had a recurrent common duct brown pigment stone containing a metallic clip 26 months after operation. He was treated successfully by endoscopic sphincterotomy. Factors predisposing to clip migration were short cystic stump, inadvertent clip dislodgment or incorrect placement, cystic duct ischemic necrosis, and local suppurative complications. Data from 29 patients with GS formed around suture material or phytobezoars observed during a prospective study and from the physicochemical and structural analysis of a cumulative series of 64 GS containing foreign bodies are also presented and discussed. It is suggested that metallic clips can migrate from their initial sites at various intervals within the peritoneal cavity or into the common duct and serve as a nidus for GS formation. Metallic clip migration in most cases is due to technical factors and can usually be prevented. However, it is not possible to prevent either clip migration or GS formation in every case, since even well-placed clips can migrate due to suppurative complications or local ischemic damage, and, once that penetration within the bile tract has occurred, GS are usually going to form, irrespective of the nature and the shape of the foreign body.