Cys Groups Research Articles

Prognostic Value of Peritoneal Lavage Cytology in Potentially Resectable Pancreatic Cancer Stratified by Cytologic Status.

Patients with pancreatic ductal adenocarcinoma (PDAC) with positive peritoneal lavage cytology (CY) reportedly have poor prognoses. However, the value of diagnosis of suspicious for malignancy on CY is unknown. This study aimed to elucidate the prognostic impact of CY by focusing on CY subgroups. Data were collected from 231 resectable PDAC patients who underwent curative-intent resection. Patients were divided into three CY-based groups: negative (CY0), suspicious for malignancy (CY-S), and positive (CY1). Clinicopathological characteristics and prognostic factors were analyzed. CY1 and CY-S were diagnosed in 7.8% and 3.9% of the patients, respectively. The CY1 group had significantly larger tumors and higher frequencies of distal tumors, anterior pancreatic tissue invasion, retropancreatic tissue invasion, and R1 resection than the CY0 group. Patient characteristics did not differ between the CY0 and CY-S groups. The CY1 group exhibited worse survival than the CY0 and CY-S groups (median survival time: 18.8 vs. 39.6 months, p=0.0021 and vs. 62.2 months, p=0.018). Multivariate analysis for survival indicated that a tumor size >2 cm, preoperative CA19-9 value >100 U/ml, CY1, lymph node metastasis, R1 resection, and lack of adjuvant chemotherapy were associated with poor prognosis. Both the CY1 and CY-S groups had higher frequencies of peritoneal recurrence than the CY0 group (50% vs. 11.8%, p<0.001 and 44.4% vs. 11.8%, p=0.019). The prognosis of the CY1 group was poor. Although CY-S was associated with a higher frequency of peritoneal recurrence than CY0, the long-term outcomes of patients with surgical treatment were acceptable.

Identification of a Phage Display-Derived Peptide Interacting with the N-Terminal Region of Factor VII Activating Protease (FSAP) Enables Characterization of Zymogen Activation.

Factor VII Activating protease (FSAP) has a protective effect in diverse disease conditions as inferred from studies in FSAP–/– mice and humans deficient in FSAP activity due to single-nucleotide polymorphism. The zymogen form of FSAP in plasma is activated by extracellular histones that are released during tissue injury or inflammation or by positively charged surfaces. However, it is not clear whether this activation mechanism is specific and amenable to manipulation. Using a phage display approach, we have identified a Cys-constrained 11 amino acid peptide, NNKC9/41, that activates pro-FSAP in plasma. The synthetic linear peptide has a propensity to cyclize through the terminal Cys groups, of which the antiparallel cyclic dimer, but not the monocyclic peptide, is the active component. Other commonly found zymogens in the plasma, related to the hemostasis system, were not activated. Binding studies with FSAP domain deletion mutants indicate that the N-terminus of FSAP is the key interaction site of this peptide. In a monoclonal antibody screen, we identified MA-FSAP-38C7 that prevented the activation of pro-FSAP by the peptide. This antibody bound to the LESLDP sequence (amino acids 30–35) in an intrinsically disordered stretch in the N-terminus of FSAP. The plasma clotting time was shortened by NNKC9/41, and this was reversed by MA-FSAP-38C7, demonstrating the utility of this peptide. Peptide NNKC9/41 will be useful as a tool to delineate the molecular mechanism of activation of pro-FSAP, elucidate its biological role, and provide a starting point for the pharmacological manipulation of FSAP activity.

A theoretical approach on the ability of functionalized gold nanoparticles for detection of Cd2+

Cadmium (Cd) as a toxic element that is widely present in water, soil, and air has important effects on human health, therefore proposing an accurate and selective method for detection of this element is of importance. In this article, by employing full atomistic molecular dynamics (MD) simulations and density functional theory dispersion corrected (DFT-D3) calculations, the effects of 6-mercaptonicotinic acid (MNA) and l-cysteine (CYS) on the stability of gold nanoparticles (AuNPs) and their sensitivity against Cd2+ were investigated. The obtained results indicate that pure AuNPs are not stable in water, while functionalized AuNPs with CYS and MNA groups have considerable stability without aggregation. In other words, the functional groups on the surface of AuNPs elevate their resistance against aggregation by an increase in the repulsive interactions between the gold nanoparticles. Moreover, functionalized AuNPs have considerable ability for selective detection of Cd2+ in the presence of different metal ions. Based on the MD simulation results, MNA-CYS-AuNPs (functionalized AuNPs with both functional groups) have the maximum sensitivity against Cd2+ in comparison with MNA-AuNPs and CYS-AuNPs due to the strong electrostatic interactions. DFT-D3 calculations reveal that the most probable interactions between the metal ions and functional groups are electrostatic, and Cd2+ can aggregate functionalized AuNPs due to strong electrostatic interactions with MNA and CYS groups. Moreover, charge transfer and donor–acceptor analyses show that molecular orbital interactions between the functional groups and Cd2+ can be considered as the driving force for AuNPs aggregation. A good agreement between the theoretical results and experimental data confirms the importance of the molecular modeling methods as a fast scientific protocol for designing new functionalized nanoparticles for application in different fields.

Maternal cysteine intake influenced oxidative status and lipid-related gut microbiota and plasma metabolomics in male suckling piglets

Cysteine (Cys), a nutritionally semi-essential amino acid, can regulate oxidative stress and lipid metabolism. Since maternal nutrient intake has an impact on offspring, this study aimed to investigate the effect of maternal Cys intake on male suckling piglets. Twenty-four sows in late pregnancy (85–90 days of gestation) were randomly allotted to three groups and fed diets containing 0.3 %, 0.4 % and 0.5 % Cys. The pregnant sows were fed twice daily (06:00 and 14:00) but the feed supply was withheld near delivery. After delivery, the sows were fed the same diet as before, and the amount of feed supply was gradually increased. During lactation, the piglets were not provided with supplemental feed. On day 21 after partitioning, eight piglets per group (one male piglet per little) and a total of 24 piglets were sampled. The birth weight and weaning weight of the piglets in each litter were recorded. Blood samples and intestinal digesta samples of the piglets were collected. The results showed that the birth weight and weaning weight of the piglets were not affected by the treatment. For the plasma metabolomics analysis, 16 differential metabolites were identified among the groups and were involved in the metabolism of proteins (methionyl-histidine, lysyl-leucine), amino acids (L-homocysteine sulfonic acid, selenomethionine) and nucleic acids (5′-deoxyadenosine, 5-methyldeoxycytidine). In addition, compared with 0.3 % Cys, increased maternal Cys intake (0.4 % and 0.5 % Cys) led to lower serum malondialdehyde levels and plasma levels of estradiol and mevalonate and increased plasma methionyl-histidine levels in the male piglets. Intestinal microbial analysis showed an increase in the phylum Bacteroidetes, and a decrease in the Firmicutes/Bacteroidetes ratio in the colon of the male piglets in the 0.4 % and 0.5 % Cys groups compared with the 0.3 % Cys group. A positive correlation between the Firmicutes/Bacteroidetes ratio and plasma metabolites (mevalonate and estradiol) was observed; these changes were associated with the inhibition of fat synthesis. Taken together, increased maternal Cys intake during late gestation and lactation reduces oxidative stress and influences the lipid-related plasma metabolomics and intestinal microbiota in male piglets.

Kinetic assessment of Michael addition reactions of alpha, beta-unsaturated carbonyl compounds to amino acid and protein thiols

Humans have extensive adverse exposure to alpha,beta-unsaturated carbonyl compounds (ABuCs) as these are major toxins in smoke and exhaust fumes, as well as products of lipid peroxidation. In contrast, another ABuC, dimethylfumarate, is used to treat psoriasis and multiple sclerosis. ABuCs undergo Michael adduction with amine, imidazole and thiol groups, with reaction at Cys residues predominating. Here we report rate constants, k2, for ABuCs (acrolein, crotonaldehyde, dimethylfumarate, cyclohex-1-en-2-one, cyclopent-1-en-2-one) with Cys residues present on N-Ac-Cys, GSH, bovine serum albumin, creatine kinase, papain, glyceraldehyde-3-phosphate dehydrogenase, and both wild-type and the C151S mutant of Keap-1. k2 values for N-Ac-Cys and GSH vary by > 250-fold, indicating a marked ABuC structure dependence, with acrolein the most reactive. There is also considerable variation in k2 between protein Cys groups, with these significantly greater than for GSH. A linear inverse correlation for acrolein with the thiol pKa indicates that the thiolate anion is the reactive species. The modest k2 for GSH rationalizes the detection of protein adducts of ABuCs in cells. The k2 values for dimethylfumarate also vary markedly, with the Cys151 residue on Keap-1 being particularly reactive, with the C151S mutant giving a much lower k2 value. The data for crotonaldehyde, dimethylfumarate, and cyclohex-1-en-2-one show little correlation with the Cys pKa values, indicating that steric/electronic interactions, rather than Cys ionization are important. These data indicate that protein Cys residues, and particularly Cys151 on Keap-1, react readily with dimethylfumarate, and this may help rationalize the use of this compound as a therapeutic agent.

Cyst(e)ine fortification in low crude protein diet improves growth performance of broilers by modulating serum metabolite profile

This study was aimed to explore the metabolomical mechanisms for the potentially ameliorative effect of cyst(e)ine (Cys) fortification on growth performance of broilers fed low crude protein (CP) diet. A total of 432 1-d-old broilers were randomly divided into 6 groups, each of which received one of the following diets: normal-CP diet (positive control, PC), low-CP diet (negative control, NC), NC diet fortified with 0.05%, 0.1%, 0.15% or 0.2% of Cys. Samples were collected on d 42. Results showed that increasing Cys fortification quadratically elevated (P < 0.05) the accumulative growth performance and leg muscle yield of broilers fed NC diet, with 0.1% being the optimal dose. Thus, samples from PC, NC and NC plus 0.1% Cys (NCC) groups were selected for further analysis. Both dietary CP reduction and fortification of 0.1% Cys in NC diet caused complex changes (P < 0.05) in serum amino acids and some other metabolites primarily involved in lipid metabolism. Multiple lipogenesis-related pathways were regulated (P < 0.05) following Cys fortification in NC diet, which could at least partially interpret the benefit of Cys fortification in NC diet on broiler performance. In conclusion, fortifying low-CP diet with 0.1% Cys promoted the growth performance of broilers probably through modulating serum metabolite profile.

Serum and Urine Interleukin-6 and Interleukin-8 Levels Do Not Differentiate Acute Pyelonephritis from Lower Urinary Tract Infections in Children.

BackgroundUrinary tract infection (UTI) is common in pediatrics. Infection of the upper urinary tract may cause renal scarring, and subsequently renal failure and hypertension. Permanent renal damage has been suggested to be caused by the host inflammatory response. Therefore, it is crucial to understand the host defense mechanisms against such infection in order to make timely diagnosis. The aim of this study was to evaluate interleukin-6 (IL-6) and IL-8 as potential biomarkers in differentiating acute pyelonephritis (AP) from cystitis (Cys) in children.MethodsForty-three children (21 with AP and 22 with Cys) were included. Serum and urinary IL-6 and IL-8 were measured during the acute phase (within 12 hours of presentation) and the convalescent phase (8 weeks post-infection). Thirty-eight healthy children were included as controls.ResultsDuring the acute phase, the mean urinary IL-6 level in the Cys group was significantly higher than that in the controls (17.8 pg/mL vs 14.8 pg/mL, P=0.03), while the serum levels were significantly higher in both the Cys and AP groups than in the controls (19.5 pg/mL, 19.4 pg/mL, 15 pg/mL, P=0.005 and 0.02, respectively). During the convalescent phase, serum and urinary IL-6 levels were higher in patients than in controls. Urinary IL-8 levels were significantly higher in both the AP and Cys groups compared to controls (206.5 pg/mL, 291.8 pg/mL, 89.4 pg/mL, P=0.05 and 0.02, respectively) during the acute phase. Serum IL-8 was not significantly different between the 3 groups. Nonetheless, no significant differences were found between the AP and Cys groups, in urinary or serum levels of IL-6 or IL-8, during both phases.ConclusionIL-6 and IL-8 levels are elevated in patients with UTI. However, the levels did not differentiate between AP and cystitis. Further studies are warranted to evaluate their roles as indicators of the site of UTIs.

2,2'-Dipyridyl diselenide: A chemoselective tool for cysteine deprotection and disulfide bond formation.

There are many examples of bioactive, disulfide-rich peptides and proteins whose biological activity relies on proper disulfide connectivity. Regioselective disulfide bond formation is a strategy for the synthesis of these bioactive peptides, but many of these methods suffer from a lack of orthogonality between pairs of protected cysteine (Cys) residues, efficiency, and high yields. Here, we show the utilization of 2,2'-dipyridyl diselenide (PySeSePy) as a chemical tool for the removal of Cys-protecting groups and regioselective formation of disulfide bonds in peptides. We found that peptides containing either Cys(Mob) or Cys(Acm) groups treated with PySeSePy in trifluoroacetic acid (TFA) (with or without triisopropylsilane (TIS) were converted to Cys-S-SePy adducts at 37 °C and various incubation times. This novel Cys-S-SePy adduct is able to be chemoselectively reduced by five-fold excess ascorbate at pH 4.5, a condition that should spare already installed peptide disulfide bonds from reduction. This chemoselective reduction by ascorbate will undoubtedly find utility in numerous biotechnological applications. We applied our new chemistry to the iodine-free synthesis of the human intestinal hormone guanylin, which contains two disulfide bonds. While we originally envisioned using ascorbate to chemoselectively reduce one of the formed Cys-S-SePy adducts to catalyze disulfide bond formation, we found that when pairs of Cys(Acm) residues were treated with PySeSePy in TFA, the second disulfide bond formed spontaneously. Spontaneous formation of the second disulfide is most likely driven by the formation of the thermodynamically favored diselenide (PySeSePy) from the two Cys-S-SePy adducts. Thus, we have developed a one-pot method for concomitant deprotection and disulfide bond formation of Cys(Acm) pairs in the presence of an existing disulfide bond.

The impact of different levels of cysteine on the plasma metabolomics and intestinal microflora of sows from late pregnancy to lactation.

Cysteine (Cys) is a semi-essential amino acid that is synthesized from methionine in mammals and affects their physiological state. This study aimed at investigating the effects of different Cys levels on the birth weight and survival rate of piglets as well as the plasma biochemical parameters, intestinal microbial diversity, and plasma metabolome of sows during late pregnancy. The results showed that 0.4% Cys supplementation increased the birth weight of piglets and decreased the calcium, triglyceride, and bilirubin levels in sows, whereas 0.5% Cys supplementation reduced the gamma-glutamyl transpeptidase levels and increased the serum glucose levels in sows at farrowing. Intestinal microbial analysis demonstrated that 0.4% Cys supplementation increased the diversity of fecal and intestinal microbiota compared with 0.5% Cys supplementation. In addition, plasma metabolomics identified 11 differential metabolites among the 0.4% Cys, 0.5% Cys, and control (basal diet) groups. The serum hypotaurine levels in sows increased by 0.4% and 0.5% Cys supplementation, and the serum acetylcysteine levels increased by 0.5% Cys supplementation; however, the differences in hypotaurine and acetylcysteine levels between the 0.4% and 0.5% Cys groups were not significant. Furthermore, Pearson analysis revealed a positive correlation between the hypotaurine levels and the abundance of Lactobacillus or Pseudobutyrivibrio and a negative correlation between the acetylcysteine levels and the abundance of Ruminococcaceae_UCG-014. Overall, the results indicated that 0.4% Cys supplementation increased the birth weight of piglets, increased the differential metabolites beneficial for combating antioxidative stress in embryos enhancing the intestinal microbial abundance in sows, and increased the diversity of fecal microbiota in sows. Thus, these findings suggest that 0.4% Cys supplementation is highly beneficial for maintaining the health of sows during late pregnancy.

REDOX proteomics reveals energy metabolism alterations in the liver of M. spretus mice exposed to p, p′-DDE

The toxicity induced by the pesticide 2,2-bis(p-chlorophenyl)-1,1,1,-trichloroethane (DDT) and its derivative 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p′-DDE) has been associated with mitochondrial dysfunction, uncoupling of oxidative phosphorylation and respiratory chain electron transport, intracellular ion imbalance, generation of reactive oxygen species and impairment of the antioxidant defense system. A disruption in the cellular redox status causes protein Cys-based regulatory shifts that influence the activity of many proteins and trigger signal transduction alterations. Here, we analyzed the ability of p,p′-DDE to alter the activities of hepatic antioxidants and glycolytic enzymes to investigate the oxidative stress generation in the liver of p,p′-DDE-fed M. spretus mice. We also determined the consequences of the treatment on the redox status in the thiol Cys groups. The data indicate that the liver of p,p'-DDE exposed mice lacks certain protective enzymes, and p,p′-DDE caused a metabolic reprogramming that increased the glycolytic rate and disturbed the metabolism of lipids. Our results suggested that the overall metabolism of the liver was altered because important signaling pathways are controlled by p,p′-DDE-deregulated proteins. The histological data support the proposed metabolic consequences of the p,p′-DDE exposure.

Amyloid fibril-supported Pd nanoparticles as electrocatalyst for hydrogen peroxide reduction

Palladium nanoparticles (Pd NPs) were fabricated by using insulin amyloid fibrils (INSAFs) as biotemplates. Atomic force microscopy measurements showed that ultra-small Pd NPs were well adsorbed and dispersed on surfaces of INSAFs. X-ray photoelectron spectroscopy confirmed the partial reduction of Pd ion into metallic Pd(0) probably due to the presence of Cys groups on surface of the insulin fibrils. The electrochemical performance of Pd/INSAFs to reduction of H2O2 was further evaluated by cyclic voltammetry. The remarkably high electrocatalytic activity, low detection limitation and excellent stability make the Pd/INSAFs a promising bio-nanoelectrocatalyst.

Frequency and clinical implications of pathologic complete response (pCR) at cystectomy for muscle-invasive bladder cancer (MIBC) with or without neoadjuvant chemotherapy (NAC).

383 Background: Subsets of patients (pts) with MIBC, with or without NAC, have no pathologic evidence of disease at cystectomy. The frequency and implications of pCR in “real world” pts has not been well characterized. Methods: Using the National Cancer Database, pts with cT2-4aN0M0 bladder cancer who underwent cystectomy alone (CYS) or with NAC from 2003-2011 were identified. pCR was defined as pT0N0M0. In the CYS and NAC cohorts, the association between pCR and overall survival was analyzed using Cox proportional hazard methods. Results: 12,245 pts met eligibility (CYS: 10,940; NAC: 1,305). The pCR rate with CYS was 4.8% (95% CI 4.4-5.3%) and with NAC was 21.6% (95% CI 19.4-24.0%). In both cohorts, logistic regression revealed lower cT stage and higher cystectomy volume were associated with increased pCR. pCR was associated with a significant improvement in survival in both cohorts (Table). Conclusions: The frequency of pCR in real world pts was substantially lower than reported in clinical trials; however, pCR was associated with markedly improved survival in both CYS and NAC groups. Molecular predictors of pCR in both groups warrant evaluation. Whether the survival benefit associated with pCR in the CYS cohort simply reflects the “smallest/best” tumors or whether there is a role, and mechanistic rationale, for surgical approaches to increase pCR (i.e., re-TURBT in MIBC) warrants study. Finally, our findings lend further support for use of pCR as an endpoint in NAC clinical trials. [Table: see text]

Inactivation of the pore-forming toxin Sticholysin I by peroxynitrite: protection by cys groups incorporated in the toxin.

Sea anemones synthesize a variety of toxic peptides and proteins of biological interest. The Caribbean Sea anemone Stichodactyla helianthus, produces two pore-forming toxins, Sticholysin I (St I) and Stichloysin II (St II), with the ability to form oligomeric pores in cell and lipid bilayers characteristically lacking cysteine in their amino acid sequences. Recently, two mutants of a recombinant variant of Sticholysin I (rSt I) have been obtained with a Cys residue in functionally relevant regions for the pore-forming activity of the toxin: r St I F15C (in the amino terminal sequence) and r St I R52C (in the binding site). Aiming at characterizing the effects of oxidants in toxins devoid (r St I) or containing -SH moieties (r St I F15C and r St I R52C), we measured their hemolytic activity and pore forming capacity prior and after their incubation with peroxynitrite (ONOO(-)). At low ONOO(-)/Toxin ratios, nearly 0.8 Trp groups are modified by each added peroxynitrite molecule, and the toxin activity is reduced in ca. 20 %. On the other hand, in -SH bearing mutants only 0.5 Trp groups are modified by each peroxynitrite molecule and the toxin activity is only reduced in 10 %. The results indicated that Cys is the initial target of the oxidative damage and that Trp residues in Cys-containing toxins were less damaged than those in r St I. This relative protection of Trp groups correlates with a smaller loss of hemolytic activity and permeabilization ability in liposomes and emphasizes the relevance of Trp groups in the pore forming capacity of the toxins.

Effect of High-Dose Cysteine Supplementation on Erythrocyte Glutathione: A Double-Blinded, Randomized Placebo-Controlled Pilot Study in Critically Ill Neonates.

This study's objective was to determine if parenteral cysteine when compared with isonitrogenous noncysteine supplementation increases erythrocyte reduced glutathione (GSH) in neonates at high risk for inflammatory injury. Neonates with a score for neonatal acute physiology >10 requiring mechanical ventilation and parenteral nutrition (PN) were randomized in a double-blinded, placebo-controlled study to receive parenteral cysteine-HCl (CYS group) or additional PN amino acids (ISO group) at 121 mg/kg/d for ≥7 days. A 6-hour [(13)C2] glycine IV infusion was administered at study week 1 to determine the fractional synthetic rate of GSH (FSR-GSH). Baseline characteristics were similar between the CYS (n = 17) and ISO groups (n = 21). Erythrocyte GSH and total glutathione concentrations, GSH:oxidized GSH (GSSG), and FSR-GSH after treatment were not different between groups. However, the CYS group had a larger individual positive change in GSH and total glutathione (infusion day - baseline) compared with the ISO group (P = .02 for each). After adjusting for treatment, a lower enrollment weight and rate of red blood cell transfusion were associated with a decreased change in total glutathione and GSH (P < .05 for each). When compared with isonitrogenous noncysteine supplementation, high-dose cysteine supplementation for at least 1 week in critically ill neonates resulted in a larger and more positive individual change in GSH. Smaller infants and those who received transfused blood demonstrated less effective change in GSH with cysteine supplementation. The benefit of cysteine remains promising and deserves further investigation.

Carbamoylating activity associated with the activation of the antitumor agent laromustine inhibits angiogenesis by inducing ASK1-dependent endothelial cell death.

The anticancer agent 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (laromustine), upon decomposition in situ, yields methyl isocyanate and the chloroethylating species 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE). 90CE has been shown to kill tumor cells via a proposed mechanism that involves interstrand DNA cross-linking. However, the role of methyl isocyanate in the antineoplastic function of laromustine has not been delineated. Herein, we show that 1,2-bis(methylsulfonyl)-1-[(methylamino)carbonyl]hydrazine (101MDCE), an analog of laromustine that generates only methyl isocyanate, activates ASK1-JNK/p38 signaling in endothelial cells (EC). We have previously shown that ASK1 forms a complex with reduced thioredoxin (Trx1) in resting EC, and that the Cys residues in ASK1 and Trx1 are critical for their interaction. 101MDCE dissociated ASK1 from Trx1, but not from the phosphoserine-binding inhibitor 14-3-3, in whole cells and in cell lysates, consistent with the known ability of methyl isocyanate to carbamoylate free thiol groups of proteins. 101MDCE had no effect on the kinase activity of purified ASK1, JNK, or the catalytic activity of Trx1. However, 101MDCE, but not 90CE, significantly decreased the activity of Trx reductase-1 (TrxR1). We conclude that methyl isocyanate induces dissociation of ASK1 from Trx1 either directly by carbamoylating the critical Cys groups in the ASK1-Trx1 complex or indirectly by inhibiting TrxR1. Furthermore, 101MDCE (but not 90CE) induced EC death through a non-apoptotic (necroptotic) pathway leading to inhibition of angiogenesis in vitro. Our study has identified methyl isocyanates may contribute to the anticancer activity in part by interfering with tumor angiogenesis.

Acid–Base Parameters and Steroid Concentrations in Pre‐Ovulatory Follicles and Plasma of Lactating Dairy Cows with Spontaneous and Synchronized Oestrus or Follicular Cyst

The study aimed to compare the acid-base balance and steroid concentrations between follicular fluids (FF) of pre-ovulatory follicles derived from a spontaneous oestrus (SO), synchronized or induced oestrus (IO) and follicular cysts (CYS) and between FF and blood in dairy cows. Forty-two dairy cows were included in this study. The animals were allocated to three groups: SO (n=23); IO (n=11) using GnRH at day 0 and day 9 and PGF2 α at day 7; and animals with CYS (n=10). The follicular fluids (FF) were aspirated from the cyst/pre-ovulatory follicles (∅≥15mm) after SO and after second GnRH dose in IO by transvaginal ultrasound-guided ovum pick-up technique. Blood samples (BL) were collected in heparinized vacutainer tubes. The oxygen tension (pO2) in FF of IO was higher (p<0.05) than in SO and CYS groups. There were negative correlations (p<0.001, r=-0.89) between FF and blood pO2. The carbon dioxide tension (pCO2) and lactate level in FF of CYS group were higher (p<0.05) than in SO and IO groups. There were negative correlations (p<0.01, r=-0.73) between blood and FF pO2. Oestradiol-17β concentration in pre-ovulatory follicles and plasma of the SO group was higher (p<0.001) than in IO and CYS groups. Progesterone concentration in pre-ovulatory follicles and plasma of the SO and IO groups was lower (p<0.01) than in CYS group. Plasma androstenedione concentration in SO and IO groups was higher (p<0.05) than in CYS group. In conclusion, acid-base parameters, E2 and P4 levels in the follicular fluid of both IO and CYS groups were deviated greatly from the physiological level (disturbances of intrafollicular/intracystic environment), which may affect the quality of both the oocyte and the granulosa cells.

Protein targets of acrylamide adduct formation in cultured rat dopaminergic cells

Acrylamide (ACR) is an electrophilic unsaturated carbonyl derivative that produces neurotoxicity by forming irreversible Michael-type adducts with nucleophilic sulfhydryl thiolate groups on cysteine residues of neuronal proteins. Identifying specific proteins targeted by ACR can lead to a better mechanistic understanding of the corresponding neurotoxicity. Therefore, in the present study, the ACR-adducted proteome in exposed primary immortalized mesencephalic dopaminergic cells (N27) was determined using tandem mass spectrometry (LTQ-Orbitrap). N27 cells were characterized based on the presumed involvement of CNS dopaminergic damage in ACR neurotoxicity. Shotgun proteomics identified a total of 15,243 peptides in N27 cells of which 103 unique peptides exhibited ACR-adducted Cys groups. These peptides were derived from 100 individual proteins and therefore ~0.7% of the N27 cell proteome was adducted. Proteins that contained ACR adducts on multiple peptides included annexin A1 and pleckstrin homology domain-containing family M member 1. Sub-network enrichment analyses indicated that ACR-adducted proteins were involved in processes associated with neuron toxicity, diabetes, inflammation, nerve degeneration and atherosclerosis. These results provide detailed information regarding the ACR-adducted proteome in a dopaminergic cell line. The catalog of affected proteins indicates the molecular sites of ACR action and the respective roles of these proteins in cellular processes can offer insight into the corresponding neurotoxic mechanism.

Adsorption of Amino Acids (Ala, Cys, His, Met) on Zeolites: Fourier Transform Infrared and Raman Spectroscopy Investigations

Minerals adsorb more amino acids with charged R-groups than amino acids with uncharged R-groups. Thus, the peptides that form from the condensation of amino acids on the surface of minerals should be composed of amino acid residues that are more charged than uncharged. However, most of the amino acids (74%) in today's proteins have an uncharged R-group. One mechanism with which to solve this paradox is the use of organophilic minerals such as zeolites. Over the range of pH (pH 2.66-4.50) used in these experiments, the R-group of histidine (His) is positively charged and neutral for alanine (Ala), cysteine (Cys), and methionine (Met). In acidic hydrothermal environments, the pH could be even lower than those used in this study. For the pH range studied, the zeolites were negatively charged, and the overall charge of all amino acids was positive. The conditions used here approximate those of prebiotic Earth. The most important finding of this work is that the relative concentrations of each amino acid (X=His, Met, Cys) to alanine (X/Ala) are close to 1.00. This is an important result with regard to prebiotic chemistry because it could be a solution for the paradox stated above. Pore size did not affect the adsorption of Cys and Met on zeolites, and the Si/Al ratio did not affect the adsorption of Cys, His, and Met. ZSM-5 could be used for the purification of Cys from other amino acids (Student-Newman-Keuls test, p<0.05), and mordenite could be used for separation of amino acids from each other (Student-Newman-Keuls test, p<0.05). As shown by Fourier transform infrared (FT-IR) spectra, Ala interacts with zeolites through the [Formula: see text] group, and methionine-zeolite interactions involve the COO, [Formula: see text], and CH(3) groups. FT-IR spectra show that the interaction between the zeolites and His is weak. Cys showed higher adsorption on all zeolites; however, the hydrophobic Van der Waals interaction between zeolites and Cys is too weak to produce any structural changes in the Cys groups (amine, carboxylic, sulfhydryl, etc.); thus, the FT-IR and Raman spectra are the same as those of solid Cys.

Abstract 4469: Carbamoylating activity associated with the antitumor prodrug Laromustine inhibits angiogenesis in vitro by inducing ASK1-dependent endothelial cell death

Abstract The prodrug Laromustine (1,2-bis[methylsulfonyl]-1-[2-chloroethyl]-2-[(methylamino) carbonyl] hydrazine; Cloretazine; VNP40101M) yields methyl isocyanate and a chloroethylating species (90CE) upon decomposition in situ. Laromustine's cytotoxicity is principally attributed to 90CE via a proposed mechanism that involves interstrand DNA cross-linking. However, the role of methyl isocyanate in the antineoplastic function has not been fully defined. A derivative of Laromustine that generates only methyl isocyanate and not the chloroethylating species, 1,2-bis[methylsulfonyl]-1-[(methylamino) carbonyl] hydrazine (101MDCE), is reported here to induce endothelial cell (EC) death through a non-apoptotic pathway at both relatively low (50 μM) and relatively high (200 μM) concentrations. 90CE, which lacks carbamoylating activity, did not induce this cell fate. 101MDCE, but not 90CE, also inhibited EC tube formation in vitro, a process involved in angiogenesis, which is critical for tumor growth and metastases. To understand the molecular mechanism for 101MDCE-induced EC death, we examined signaling pathways activated by 101DMCE in ECs. We found that 101DMCE, but not 90CE, activated JNK/p38 pathways and their upstream apoptosis activator signal-regulating kinase 1 (ASK1) using Western blot analyses. In resting ECs, ASK1 forms a complex with the reduced form of thioredoxin (Trx). The single cysteine residue in ASK1 (C250) and C32 or C35 in Trx are critical for this interaction. 101MDCE effected ASK1 dissociation from Trx, but not from the phosphoserine-binding inhibitor 14-3-3, in vitro and in vivo, consistent with the known reactivity of methyl isocyanate with the sulfhydryl groups of cysteine residues. We previously reported that the carbamoylating activity of Laromustine inhibits the enzyme Trx reductase from leukemia cell extracts. We conclude that carbamoylating agents induce ASK1-dependent EC death through dissociation of ASK1 from Trx either directly by targeting the critical Cys groups in the ASK1-Trx complex or indirectly by inhibiting Trx reductase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4469. doi:10.1158/1538-7445.AM2011-4469

The glucose-6-phosphate dehydrogenase from Trypanosoma cruzi: Its role in the defense of the parasite against oxidative stress

The Trypanosoma cruzi glucose-6-phosphate dehydrogenase (G6PDH) is encoded by several genes located in three of the parasite chromosomes. All the sequences present two possible start codons, 111 bp apart, also present in its Trypanosoma brucei counterpart. As the 37 residues comprised between the two candidate initiator methionines of T. brucei and T. cruzi G6PDHs constitute an unusual N-terminal extension only present in trypanosomatids, two forms of the T. cruzi G6PDH were expressed in Escherichia coli: a long one (Tc-G6PDH-L) translated from the first ATG codon, and a short one (Tc-G6PDH-S) translated from the second. Both were purified and their kinetic constants determined. The apparent K m for glucose-6-phosphate was 189.9, 98.4, and 288 μM, for Tc-G6PDH-L, Tc-G6PDH-S and native Tc-G6PDH, respectively. The apparent K m for NADP was similar for both recombinant proteins. The Tc-G6PDH-L as well as the native enzyme, was inactivated by DTT while the Tc-G6PDH-S was unaffected by the reducing agent. This behavior could be related to the presence of two Cys groups in the N-terminal extension of the Tc-G6PDH-L similarly to the redox regulated G6PDHs from chloroplasts and cyanobacteria. This property, together with a remarkable induction (up to 46-fold) of the T. cruzi G6PDH in metacyclic trypomastigotes under oxidative stress conditions, suggests that the enzyme may play a prominent role in the defense mechanisms of the parasite against oxidative stress becoming an important target for chemotherapy. Western blots using antibodies against the N-terminal extension in Tc-G6PDH-L show that this form is expressed in the parasite.