Absence Of Cysteine Research Articles

Formation of advanced glycation end products in glucose-amino acid models of Maillard reaction under dry- and wet-heating conditions.

Advanced glycation end products (AGEs) are compounds formed by non-enzymatic processes in the Maillard reaction and can cause various chronic diseases. This study explores the AGE formation process in a glucose-amino acid system under both wet- and dry-heating conditions, and analyzes the effect of cysteine in AGE formation. Under wet-heating conditions, Nε-carboxymethyllysine (CML) and Nε-carboxyethyllysine (CEL) concentrations rose for the initial 90 min and subsequently declined after 120 min; after 90 min of heating, the maximum yields in the absence of cysteine were 1151.04 ± 14.01 and 3386.90 ± 26.55 ng mL-1, respectively. The concentration of pyrraline (Pyr) increased after 30 min and then decreased after 60 min with a maximum yield of 777.68 ± 23.36 ng mL-1. However, in dry-heating models, the AGE concentrations consistently increased with increasing heating time; the maximum yields for CML, CEL and Pyr were 468.66 ± 10.96, 1993.57 ± 14.81 and 1085.74 ± 58.06 ng mL-1, respectively. The addition of cysteine showed an inhibitory effect on AGE formation, especially for Pyr in the dry-heating model, with inhibition rates ranging from 17.14% to 95.60%. Although wet-heating models produced more CML and CEL, they produced less Pyr than dry-heating models. The AGE formation in wet-heating models positively correlated with the reaction rate; however, the dry-heating reaction demonstrated a more complex relationship between reaction rate and reaction protocol. Moreover, cysteine exhibited a significant inhibitory effect on AGE production, and the degree of inhibition was proportional to the cysteine concentration. This study provides important insights into the mechanisms for AGE formation under various heating conditions, such as those representing baking (dry-heating) and steaming conditions (wet-heating). © 2024 Society of Chemical Industry.

Study and optimization of the selectivity of an odorant binding protein-based bioelectronic nose

Over the past two decades, the use of odorant-binding proteins (OBPs) for the development of biosensors and bioelectronic noses (bioeNs) aimed at detecting and analyzing volatile organic compounds (VOCs) has been the subject of considerable research. However, there is a lack of fundamental studies for better understanding the interaction between OBPs and VOCs in gas phase. In this work, we investigated the effect of two key factors, namely relative humidity (RH) level and immobilization technique, on the selectivity of two OBP-based biosensors in gas phase. Concerning the effect of RH, the results showed that our active OBP (wild-type rat OBP3) lost its selectivity at 0% RH but retained good selectivity at 30% and 50% RH. To better understand the effect of this parameter, the hydration mechanism of the OBP was studied both experimentally and through molecular dynamics simulations. The effect of a cysteine residue, genetically added to the N-terminus of OBPs to control their orientation after immobilization on the chip, was evaluated. A significant reduction in selectivity was observed in the absence of cysteine. As expected, the introduction of this amino acid enabled to control the orientation of OBPs, making their binding pocket more accessible to VOCs and favoring specific interactions. Furthermore, we demonstrated that combining OBP-based biosensors with different properties can improve the discrimination capability of our bioeN. Finally, the ability of our system to detect essential oil vapors was tested, providing preliminary evidence that our bioeN is capable of detecting VOCs in complex media.

Electrochemical recognition for D and L-Cysteine using an achiral monodisperse Cu catalyst restricted by nanosized SOD zeolite cage domain

The recognition for D and L-Cysteine (Cys) broke through to 0.008 pM by the electrochemistry catalyst Cu Sodalite (SOD) zeolite without enzymes or noble metals. The SOD structured zeolite supplied nanosized SOD cages which are highly consistent with the spatial configuration of cysteine. And the applied electric field magnified the subtle differences in binding energy between D and L-Cys through the highly active Cu catalyst. Under electrochemical aspects, these results manifest themselves as distinguishable current peaks and potential intervals. Unlike the related literature, the sharp and sensitive current peak created by Cu catalyst is set as baseline with the absence of cysteine. With the continually increasing concentration of D or L-Cys, L-Cys shifts to the positive potential, and D-Cys shifts to the negative potential. The result reveals good reproducibility and high specificity that the catalyst is insensitive to other amino acids. The novel catalyst is featured by low cost and high stability, besides higher activity than enzymes.

Albumin binding Nanofitins, a new scaffold to extend half-life of biologics – a case study with exenatide peptide

A new strategy of peptide half-life extension has been evaluated. We investigated libraries of a small and very stable protein scaffold called Nanofitin, capable of high affinity for protein targets. We have identified Nanofitins targeting Human and mouse Serum Albumin, which could significantly improve the pharmacokinetics of an active associated peptide, mobilizing the patient’s own albumin without external source. To demonstrate the impact of this approach on half-life extension, a genetic fusion of an Exenatide peptide with an Albumin Binding Nanofitin (ABNF) was performed. Specific activity of Exenatide-ABNF was measured and unaffected by the fusion. In vivo mice results provided convincing data (t½ of 8 min for Exenatide peptide compared to 20 h for Exenatide-ABNF) with sustained pharmacological activity over 3 days. This study constitutes a proof-of-concept of in vivo half-life extension of a biologic using an ABNF. Besides, the absence of cysteine in the Nanofitin scaffold, which is therefore devoid of structuring disulfide bonds, allows manufacturing in microbial cost effective systems.

Thermal Stability and Degradation Kinetics of Patulin in Highly Acidic Conditions: Impact of Cysteine.

The thermal stability and degradation kinetics of patulin (PAT, 10 μmol/L) in pH 3.5 of phosphoric-citric acid buffer solutions in the absence and presence of cysteine (CYS, 30 μmol/L) were investigated at temperatures ranging from 90 to 150 °C. The zero-, first-, and second-order models and the Weibull model were used to fit the degradation process of patulin. Both the first-order kinetic model and Weibull model better described the degradation of patulin in the presence of cysteine while it was complexed to simulate them in the absence of cysteine with various models at different temperatures based on the correlation coefficients (R2 > 0.90). At the same reaction time, cysteine and temperature significantly affected the degradation efficiency of patulin in highly acidic conditions (p < 0.01). The rate constants (kT) for patulin degradation with cysteine (0.0036–0.3200 μg/L·min) were far more than those of treatments without cysteine (0.0012–0.1614 μg/L·min), and the activation energy (Ea = 43.89 kJ/mol) was far less than that of treatment without cysteine (61.74 kJ/mol). Increasing temperature could obviously improve the degradation efficiency of patulin, regardless of the presence of cysteine. Thus, both cysteine and high temperature decreased the stability of patulin in highly acidic conditions and improved its degradation efficiency, which could be applied to guide the detoxification of patulin by cysteine in the juice processing industry.

Effects of Linoleic Acid on Gut-Derived Bifidobacterium breve DSM 20213: A Transcriptomic Approach.

Bacterial production of conjugated linoleic acid (CLA) has recently received great attention because of the potential health benefits of this fatty acid. Linoleic acid (LA) can be converted to CLA by several microorganisms, including bifidobacteria, possibly as a detoxification mechanism to avoid the growth inhibition effect of LA. In the present in vitro study, we investigated the gene expression landscape of the intestinal strain Bifidobacterium breve DSM 20213 when exposed to LA. Transcriptomic analysis using RNA-seq revealed that LA induced a multifactorial stress response in the test strain, including upregulation of genes involved in iron uptake and downregulation of genes involved in sugar and oligopeptide transport. We also observed reduced transcription of genes involved in membrane and pili biosynthesis. The upregulation of iron uptake was not related to any putative ability of LA to chelate Fe2+, but was somewhat linked to stress response. Furthermore, we demonstrated that LA increased reactive oxygen species (ROS) production in bacterial cells, activating an oxidative stress response. This response was proved by thioredoxin reductase transcription, and was primarily evident among bacteria cultured in the absence of cysteine. This is the first report of the potential mechanisms involved in bacterial LA transport and stress response in B. breve.

Discovery of Antiamebic Compounds That Inhibit Cysteine Synthase From the Enteric Parasitic Protist Entamoeba histolytica by Screening of Microbial Secondary Metabolites.

Amebiasis is caused by infection with the protozoan parasite Entamoeba histolytica. Although metronidazole has been a drug of choice against amebiasis for decades, it shows side effects and low efficacy against asymptomatic cyst carriers. In addition, metronidazole resistance has been documented for bacteria and protozoa that share its targets, anaerobic energy metabolism. Therefore, drugs with new mode of action or targets are urgently needed. L-cysteine is the major thiol and an essential amino acid for proliferation and anti-oxidative defense of E. histolytica trophozoites. E. histolytica possesses the de novo L-cysteine biosynthetic pathway, consisting of two reactions catalyzed by serine acetyltransferase and cysteine synthase (CS, O-acetylserine sulfhydrylase). As the pathway is missing in humans, it is considered to be a rational drug target against amebiasis. In this study, we established a protocol to screen both a library of structurally known compounds and microbial culture extracts to discover compounds that target de novo cysteine biosynthesis of E. histolytica. The new screening system allowed us to identify the compounds that differentially affect the growth of the trophozoites in the cysteine-deprived medium compared to the cysteine-containing medium. A total of 431 structurally defined compounds of the Kitasato Natural Products Library and 6,900 microbial culture broth extracts were screened on the system described above. Five compounds, aspochalasin B, chaetoglobosin A, prochaetoglobosin III, cerulenin, and deoxyfrenolicin, from the Kitasato Natural Products Library, showed differential antiamebic activities in the cysteine-deprived medium when compared to the growth in the cysteine-containing medium. The selectivity of three cytochalasans apparently depends on their structural instability. Eleven microbial extracts showed selective antiamebic activities, and one fungal secondary metabolite, pencolide, was isolated. Pencolide showed cysteine deprivation-dependent antiamebic activity (7.6 times lower IC50 in the absence of cysteine than that in the presence of cysteine), although the IC50 value in the cysteine-deprived medium was rather high (283 μM). Pencolide also showed inhibitory activity against both CS1 and CS3 isoenzymes with comparable IC50 values (233 and 217 μM, respectively). These results indicated that antiamebic activity of pencolide is attributable to inhibition of CS. Cytotoxicity of pencolide was 6.7 times weaker against mammalian MRC-5 cell line than E. histotytica. Pencolide has the maleimide structure, which is easily attacked by Michael donors including the thiol moiety of cysteine. The cysteine-adducts of pencolide were detected by mass spectrometric analysis as predicted. As CS inhibition by the pencolide adducts was weak and their IC50 values to CS was comparable to that to the parasite in the cysteine-containing medium, the cysteine-adducts of pencolide likely contribute to toxicity of pencolide to the parasite in the cysteine-rich conditions. However, we cannot exclude a possibility that pencolide inactivates a variety of targets other than CSs in the absence of cysteine. Taken together, pencolide is the first compound that inhibits CS and amebic cell growth in a cysteine-dependent manner with relatively low mammalian cytotoxicity.

Transcriptional Regulation of Cysteine and Methionine Metabolism in Lactobacillus paracasei FAM18149.

Lactobacillus paracasei is common in the non-starter lactic acid bacteria (LAB) community of raw milk cheeses. This species can significantly contribute to flavor formation through amino acid metabolism. In this study, the DNA and RNA of L. paracasei FAM18149 were sequenced using next-generation sequencing technologies to reconstruct the metabolism of the sulfur-containing amino acids cysteine and methionine. Twenty-three genes were found to be involved in cysteine biosynthesis, the conversion of cysteine to methionine and vice versa, the S-adenosylmethionine recycling pathway, and the transport of sulfur-containing amino acids. Additionally, six methionine-specific T-boxes and one cysteine-specific T-box were found. Five of these were located upstream of genes encoding transporter functions. RNA-seq analysis and reverse-transcription quantitative polymerase reaction assays showed that expression of genes located downstream of these T-boxes was affected by the absence of either cysteine or methionine. Remarkably, the cysK2-ctl1-cysE2 operon, which is associated with te methionine-to-cysteine conversion and is upregulated in the absence of cysteine, showed high read coverage in the 5′-untranslated region and an antisense-RNA in the 3′-untranslated region. This indicates that this operon is regulated by the combination of cis- and antisense-mediated regulation mechanisms. The results of this study may help in the selection of L. paracasei strains to control sulfuric flavor formation in cheese.

Differential behaviors of silver nanoparticles and silver ions towards cysteine: Bioremediation and toxicity to Phanerochaete chrysosporium

Potential transformations of silver nanoparticles (AgNPs) upon interaction with naturally ubiquitous organic ligands in aquatic environments influence their transport, persistence, bioavailability, and subsequent toxicity to organisms. In this study, differential behaviors of AgNPs and silver ions (Ag+) towards cysteine (Cys), an amino acid representative of thiol ligands that easily coordinate to Ag+ and graft to nanoparticle surfaces, were investigated in the aspects of bioremediation and their toxicity to Phanerochaete chrysosporium. Total Ag removal, 2,4-dichlorophenol (2,4-DCP) degradation, extracellular protein secretion, and cellular viability were enhanced to some extent after supplement of various concentrations of cysteine under stress of AgNPs and Ag+. However, an obvious decrease in total Ag uptake was observed after 5–50 μM cysteine addition in the groups treated with 10 μM AgNPs and 1 μM Ag+, especially at a Cys:Ag molar ratio of 5. More stabilization in uptake pattern at this ratio was detected under Ag+ exposure than that under AgNP exposure. Furthermore, in the absence of cysteine, all Ag+ treatments stimulated the generation of reactive oxygen species (ROS) more significantly than high-dose AgNPs did. However, cysteine supply under AgNP/Ag+ stress aggravated ROS levels, albeit alleviated at 100 μM Ag+, indicating that the toxicity profiles of AgNPs and Ag+ to P. chrysosporium could be exacerbated or marginally mitigated by cysteine. The results obtained were possibly associated with the lability and bioavailability of AgNP/Ag+-cysteine complexes.

The Plastid and Mitochondrial Peptidase Network in Arabidopsis thaliana: A Foundation for Testing Genetic Interactions and Functions in Organellar Proteostasis.

Plant plastids and mitochondria have dynamic proteomes. Protein homeostasis in these organelles is maintained by a proteostasis network containing protein chaperones, peptidases, and their substrate recognition factors. However, many peptidases, as well as their functional connections and substrates, are poorly characterized. This review provides a systematic insight into the organellar peptidase network in Arabidopsis thaliana We present a compendium of known and putative Arabidopsis peptidases and inhibitors, and compare the distribution of plastid and mitochondrial peptidases to the total peptidase complement. This comparison shows striking biases, such as the (near) absence of cysteine and aspartic peptidases and peptidase inhibitors, whereas other peptidase families were exclusively organellar; reasons for such biases are discussed. A genome-wide mRNA-based coexpression data set was generated based on quality controlled and normalized public data, and used to infer additional plastid peptidases and to generate a coexpression network for 97 organellar peptidase baits (1742 genes, making 2544 edges). The graphical network includes 10 modules with specialized/enriched functions, such as mitochondrial protein maturation, thermotolerance, senescence, or enriched subcellular locations such as the thylakoid lumen or chloroplast envelope. The peptidase compendium, including the autophagy and proteosomal systems, and the annotation based on the MEROPS nomenclature of peptidase clans and families, is incorporated into the Plant Proteome Database.

Drug susceptibility testing in microaerophilic parasites: Cysteine strongly affects the effectivities of metronidazole and auranofin, a novel and promising antimicrobial

The microaerophilic parasites Entamoeba histolytica, Trichomonas vaginalis, and Giardia lamblia annually cause hundreds of millions of human infections which are treated with antiparasitic drugs. Metronidazole is the most often prescribed drug but also other drugs are in use, and novel drugs with improved characteristics are constantly being developed. One of these novel drugs is auranofin, originally an antirheumatic which has been relabelled for the treatment of parasitic infections. Drug effectivity is arguably the most important criterion for its applicability and is commonly assessed in susceptibility assays using in vitro cultures of a given pathogen. However, drug susceptibility assays can be strongly affected by certain compounds in the growth media. In the case of microaerophilic parasites, cysteine which is added in large amounts as an antioxidant is an obvious candidate because it is highly reactive and known to modulate the toxicity of metronidazole in several microaerophilic parasites.In this study, it was attempted to reduce cysteine concentrations as far as possible without affecting parasite viability by performing drug susceptibility assays under strictly anaerobic conditions in an anaerobic cabinet. Indeed, T. vaginalis and E. histolytica could be grown without any cysteine added and the cysteine concentration necessary to maintain G. lamblia could be reduced to 20%. Susceptibilities to metronidazole were found to be clearly reduced in the presence of cysteine. With auranofin the protective effect of cysteine was extreme, providing protection to concentrations up to 100-fold higher as observed in the absence of cysteine. With three other drugs tested, albendazole, furazolidone and nitazoxanide, all in use against G. lamblia, the effect of cysteine was less pronounced. Oxygen was found to have a less marked impact on metronidazole and auranofin than cysteine but bovine bile which is standardly used in growth media for G. lamblia, displayed a marked synergistic effect with metronidazole.

Subcritical Water Hydrolysis of Peptides: Amino Acid Side-Chain Modifications

Previously we have shown that subcritical water may be used as an alternative to enzymatic digestion in the proteolysis of proteins for bottom-up proteomics. Subcritical water hydrolysis of proteins was shown to result in protein sequence coverages greater than or equal to that obtained following digestion with trypsin; however, the percentage of peptide spectral matches for the samples treated with trypsin were consistently greater than for those treated with subcritical water. This observation suggests that in addition to cleavage of the peptide bond, subcritical water treatment results in other hydrolysis products, possibly due to modifications of amino acid side chains. Here, a model peptide comprising all common amino acid residues (VQSIKCADFLHYMENPTWGR) and two further model peptides (VCFQYMDRGDR and VQSIKADFLHYENPTWGR) were treated with subcritical water with the aim of probing any induced amino acid side-chain modifications. The hydrolysis products were analyzed by direct infusion electrospray tandem mass spectrometry, either collision-induced dissociation or electron transfer dissociation, and liquid chromatography collision-induced dissociation tandem mass spectrometry. The results show preferential oxidation of cysteine to sulfinic and sulfonic acid, and oxidation of methionine. In the absence of cysteine and methionine, oxidation of tryptophan was observed. In addition, water loss from aspartic acid and C-terminal amidation were observed in harsher subcritical water conditions.Graphical ᅟ

Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Library

The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (Tm of 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability. Yeast-displayed libraries that were based on this reduced charge Sso7d (rcSso7d) scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor. Importantly, starting from a charge-neutralized scaffold facilitated evolutionary adaptation of binders to differentially charged epitopes on mouse serum albumin and human epidermal growth factor receptor, respectively. Interestingly, the distribution of amino acids in the small and rigid binding surface of enriched rcSso7d-based binders is very different from that generally found in more flexible antibody complementarity-determining region loops but resembles the composition of antibody-binding energetic hot spots. Particularly striking was a strong enrichment of the aromatic residues Trp, Tyr, and Phe in rcSso7d-based binders. This suggests that the rigidity and small size of this scaffold determines the unusual amino acid composition of its binding sites, mimicking the energetic core of antibody paratopes. Despite the high frequency of aromatic residues, these rcSso7d-based binders are highly expressed, thermostable, and monomeric, suggesting that the hyperstability of the starting scaffold and the rigidness of the binding surface confer a high tolerance to mutation.

Paracrine Induction of HIF by Glutamate in Breast Cancer: EglN1 Senses Cysteine

The HIF transcription factor promotes adaptation tohypoxia and stimulates the growth of certain cancers, including triple-negative breast cancer (TNBC). The HIFα subunit is usually prolyl-hydroxylated by EglN family members under normoxic conditions, causing its rapid degradation. We confirmed that TNBC cells secrete glutamate, which we found is both necessary and sufficient for the paracrine induction of HIF1α in such cells under normoxic conditions. Glutamate inhibits the xCT glutamate-cystine antiporter, leading to intracellular cysteine depletion. EglN1, the main HIFα prolyl-hydroxylase, undergoes oxidative self-inactivation in the absence of cysteine both in biochemical assays and in cells, resulting in HIF1α accumulation. Therefore, EglN1 senses both oxygen and cysteine.

Abstract A41: Leveraging metabolic dependencies in cancer: Cysteine addiction in pancreatic cancer cells

Abstract Pancreatic cancer is a devastating and lethal malignancy, estimated to kill over 40,000 Americans per year. Little has changed in the treatment of this disease over the past half-century, highlighting the need for an improved understanding of the molecular mechanisms driving tumor development and maintenance. The malignant behavior of tumor cells is underwritten by alterations to their cellular metabolism, which is optimized to meet the catabolic needs of a rapidly dividing cell. One of the consequences of this oncogenic growth is increased generation of reactive oxygen species (ROS), a potentially toxic byproduct of many metabolic reactions. Interestingly, oncogenes such as Kras, the dominant driver of pancreatic cancer, can counteract ROS toxicity through activation of an antioxidant program, creating a highly reducing intracellular environment. Cysteine is the rate-limiting precursor for the synthesis of glutathione (GSH), the preeminent antioxidant of the cell, and as such may represent a key metabolite in the maintenance of redox balance in tumor cells. In this study we explore the importance of cysteine metabolism in the viability of pancreatic cancer cells and test the effects of cysteine deprivation and the pharmacological inhibition of cysteine uptake on a variety of pancreatic cancer cell lines. To explore the role of cysteine in pancreatic cancer growth, we cultured a panel of human and murine pancreatic cancer cell lines in the absence of cysteine. We found that cysteine withdrawal rapidly induced a peculiar form of cell death called ferroptosis, which can result from the catastrophic accumulation of certain lipid peroxides. Cystine, the oxidized form of cysteine, is the predominant extracellular source of cysteine in most cells, and it is normally imported into the cell by a specific amino acid transporter known as system xc-. We show that pharmacological inhibition of sytem xc- causes rapid cell death in most pancreatic cancer cells through ferroptosis. We find, however, that some cells do exhibit baseline resistance to cysteine deprivation or system xc- inhibition. We hypothesized that these cells utilize methionine to synthesize cysteine through transsulfuration. We show that these cells can be sensitized to system xc- inhibition when co-treated with propargyglycine (PPG), an irreversible inhibitor of gamma-cystathionase, an enzyme required for transsulfuration. These findings imply that cysteine is an important substrate for appropriate redox homeostasis in these cells. To further explore if GSH synthesis is the key mediator of cysteine-deprivation induced cell death, we treated pancreatic tumor cells with buthionine sulfoxamine (BSO), a potent and specific inhibitor of GSH synthesis. To our surprise, cells treated with BSO do not exhibit any of the ill-effects we see in cysteine-deprived pancreatic cancer cells. Therefore, it is unlikely that the cell death seen in cells deprived of cysteine is due solely to GSH depletion, but possibly due to the activity of other antioxidant pathways that require cysteine. In conclusion, we find that pancreatic cancer cells are highly sensitive to cysteine-deprivation and to treatment with system xc- inhibitors, and that the cell death caused by this stress is an iron-dependent, oxidative form of cell death called ferroptosis. Furthermore, it appears that GSH, the primary antioxidant molecule of the cell, is not solely responsible for mediating this cell death, arguing that cysteine may play other important roles in maintaining redox homeostasis in these cells. Given that previous studies show that global system xc- knockout mice do not exhibit any changes in cellular GSH content, these findings may represent a targetable metabolic dependency in pancreatic cancer cells. Citation Format: Michael A. Badgley, Carmine F. Palermo, Stephen A. Sastra, Brent R. Stockwell, Kenneth P. Olive. Leveraging metabolic dependencies in cancer: Cysteine addiction in pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A41.

Homology modeling, vasorelaxant and bradykinin-potentiating activities of a novel hypotensin found in the scorpion venom from Tityus stigmurus.

In a recent work by our group involving a transcriptomics approach applied to the venom glands from Tityus stigmurus we identified a new family of peptides called Hypotensins (TSTI0006C) (Almeida etal., 2012). The cluster TSTI0006C was analyzed in the main 25 amino acid residues and named T.stigmurus Hypotensin (TistH), showing a molecular mass of 2.7kDa, an absence of cysteines and the presence of two C-terminal proline residues, which are a bradykinin-potentiating peptide (BPP) signature. Here, we describe the homology modeling of the three-dimensional structure of TistH. In addition, we evaluated the cardiovascular effects elicited by TistH in normotensive rats. Firstly, TistH showed no cytotoxic effect on horse erythrocyte. Furthermore, in normotensive rats TistH was able to potentiate the hypotensive action of bradykinin (BK) and induced a vasorelaxant effect in mesenteric artery rings by endothelium-dependent release of nitric oxide (NO) and demonstrated independent inhibition of angiotensin converting enzyme (ACE). Our data can contribute to a better understanding of the structural and functional characteristics of TistH and suggest its potential use in cardiovascular diseases.

A label-free fluorescence turn-on sensor for rapid detection of cysteine

A Hg2+-mediated fluorescence turn-on sensor for cysteine (Cys) detection was developed using the nucleic acid minor groove binding dye DAPI. In this work, two fully complementary DNA sequences, a T-rich single-stranded molecule (ssDNA) and an A-rich single-stranded molecule, were employed to constitute consecutive “AT/TA” base pairs, which could strongly enhance the fluorescence of DAPI. In the absence of cysteine, Hg2+ reacted with T-rich single-stranded DNA and “T–Hg2+–T” base pairs formed, this seriously disrupted consecutive AT base pairs. As a result, the fluorescence of DAPI was not increased efficiently. However, considering that cysteine binds strongly to Hg2+, the structure of the “T–Hg2+–T” complexes was destroyed in the presence of cysteine, resulting in the re-formation of consecutive AT base pairs and increased DAPI fluorescence. Obviously, the amount of cysteine could be easily measured based on the enhancement of DAPI fluorescence, and it took only 20min to complete the whole cysteine-sensing process. Therefore, a label-free fluorescent “turn-on” sensor for the rapid detection of cysteine was designed, and the detection limit of this sensor was as low as 2.4nM, which was much lower than those of the most of the previously reported cysteine sensors.

Fermentation-induced variation in heat and oxidative stress phenotypes of Lactococcus lactis MG1363 reveals transcriptome signatures for robustness.

BackgroundLactococcus lactis is industrially employed to manufacture various fermented dairy products. The most cost-effective method for the preservation of L. lactis starter cultures is spray drying, but during this process cultures encounter heat and oxidative stress, typically resulting in low survival rates. However, viability of starter cultures is essential for their adequate contribution to milk fermentation, supporting the ambition to better understand and improve their robustness phenotypes.ResultsThis study describes a transcriptome-phenotype matching approach in which the starter L. lactis MG1363 was fermented under a variety of conditions that differed in the levels of oxygen and/or salt, as well as the fermentation pH and temperature. Samples derived from these fermentations in the exponential phase of bacterial growth were analyzed by full-genome transcriptomics and the assessment of heat and oxidative stress phenotypes. Variations in the fermentation conditions resulted in up to 1000-fold differences in survival during heat and oxidative stress. More specifically, aeration during fermentation induced protection against heat stress, whereas a relatively high fermentation temperature resulted in enhanced robustness towards oxidative stress. Concomitantly, oxygen levels and fermentation temperature induced differential expression of markedly more genes when compared with the other fermentation parameters. Correlation analysis of robustness phenotypes and gene expression levels revealed transcriptome signatures for oxidative and/or heat stress survival, including the metC-cysK operon involved in methionine and cysteine metabolism. To validate this transcriptome-phenotype association we grew L. lactis MG1363 in the absence of cysteine which led to enhanced robustness towards oxidative stress.ConclusionsOverall, we demonstrated the importance of careful selection of fermentation parameters prior to industrial processing of starter cultures. Furthermore, established stress genes as well as novel genes were associated with robustness towards heat and/or oxidative stress. Assessment of the expression levels of this group of genes could function as an indicator for enhanced selection of fermentation parameters resulting in improved robustness during spray drying. The increased robustness after growth without cysteine appeared to confirm the role of expression of the metC-cysK operon as an indicator of robustness and suggests that sulfur amino acid metabolism plays a pivotal role in oxidative stress survival.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0148-6) contains supplementary material, which is available to authorized users.

Novel activating mutations lacking cysteine in type I cytokine receptors in acute lymphoblastic leukemia

AbstractGain-of-function somatic mutations introducing cysteines to either the extracellular or to the transmembrane domain (TMD) in interleukin-7 receptor α (IL7R) or cytokine receptor-like factor 2 (CRLF2) have been described in acute lymphoblastic leukemias. Here we report noncysteine in-frame mutations in IL7R and CRLF2 located in a region of the TMD closer to the cytosolic domain. Biochemical and functional assays showed that these are activating mutations conferring cytokine-independent growth of progenitor lymphoid cells in vitro and are transforming in vivo. Protein fragment complementation assays suggest that despite the absence of cysteines, the mechanism of activation is through ligand-independent dimerization. Mutagenesis experiments and ConSurf calculations suggest that the mutations stabilize the homodimeric conformation, positioning the cytosolic kinases in predefined orientation to each other, thereby inducing spontaneous receptor activation independently of external signals. Hence, type I cytokine receptors may be activated in leukemia through 2 types of transmembrane somatic dimerizing mutations.

Differential activation of soluble guanylate cyclase by a series of aryl disulfanyl dinitrate esters

Classic organic nitrates, such as nitroglycerin (GTN), are important vasodilators classed as NO donors, since biological activity is assumed to result entirely from bioactivation to NO and activation of soluble guanylate cyclase (sGC). Para-substituted aryl disulfanyl nitrate esters (SS-nitrates), designed based on a sulfhydryl-dependent mechanism of NO release, were investigated for their sGC activation. To obtain further insights into their interaction with hemoproteins, SS-nitrates were reacted with oxyhemoglobin (O2-Hb). SS-nitrates activated sGC with higher efficacy than GTN in the presence of cysteine. Some SS-nitrates, unlike GTN, activated sGC in the absence of cysteine. Linear correlations between the σp Hammett parameters and sGC activation efficacies or rates of O2-Hb oxidation by SS-nitrates in the presence of cysteine were found. Our results validate the design of SS-nitrates to circumvent the need for cysteine-mediated bioactivation and hint at NO-dependent and NO-independent mechanisms of sGC activation.