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Taurine is an abundant amino acid essential to various physiological functions in many organisms; however, invertebrate taurine biosynthesis pathways are largely unknown. Here, we investigated the activities of taurine biosynthesis enzymes, cysteinesulfinic acid decarboxylase (CSD), cysteic acid decarboxylase (CAD), and cysteamine dioxygenase (CAO) in the livers of spear squid Heterololigo bleekeri and swordtip squid Uroteuthis edulis. When the enzyme reactions were performed at 35 °C for 60 min, CSD and CAD activities of spear squid were 1.49 ± 0.25 and 0.79 ± 0.11 nmol/(min mg protein), respectively; no CAO activity was observed. The CSD and CAD activities of swordtip squid were lower than those of spear squid, but CAO activity was also observed. These results suggest that taurine biosynthesis pathways in squids vary between species, as in fish. Under the conditions of our experiment, kinetic analysis revealed that in spear squid, the Michaelis–Menten constant and maximum reaction rate were 0.20 ± 0.02 mM and 1.78 ± 0.22 nmol/(min mg protein), respectively, for CSD, and for CAD 0.57 ± 0.02 mM and 1.29 ± 0.17 nmol/(min mg protein), respectively. Cysteic acid competitively inhibited spear squid CSD activity, indicating that a single enzyme catalyzes the decarboxylation of cysteinesulfinic and cysteic acids in this squid.

Here, we investigated the effect of dietary cysteic acid on the growth performance, sulfur amino acid content, and gene expression levels of taurine-synthesizing enzymes, growth hormone (GH), and insulin-like growth factor 1 (IGF-1) in Japanese flounder Paralichthys olivaceus. Juvenile flounder (0.9 g) were fed one of four diets for 30 days: with 0.25, 0.5, or 1.0% cysteic acid (C0.25, C0.5, C1.0) supplementation and without supplementation (control). Fish in the C0.25 and C0.5 groups showed significantly better growth than those in the control group (P 0.05), the expression level of IGF-1 in C1.0 fish was significantly higher than that in controls (P

βcysteine 93 residue plays a key role in oxygen (O2)-linked conformational changes in the hemoglobin (Hb) molecule. This solvent accessible residue is also a target for binding of thiol reagents that can remotely alter O2 affinity, cooperativity, and Hb's sensitivity to changes in pH. In recent years, βCys93 was assigned a new physiological role in the transport of nitric oxide (NO) through a process of S-nitrosylation as red blood cells (RBCs) travel from lungs to tissues. βCys93 is readily and irreversibly oxidized in the presence of a mild oxidant to cysteic acid, which causes destabilization of Hb resulting in improper protein folding and the loss of heme. Under these oxidative conditions, ferryl heme (HbFe4+), a higher oxidation state of Hb is formed together with its protein radical (.HbFe4+). This radical migrates to βCys93 and interacts with other "hotspot" amino acids that are highly susceptible to oxidative modifications. Oxidized βCys93 may therefore be used as a biomarker of oxidative stress, reflecting the deterioration of Hb within RBCs intended for transfusion or RBCs from patients with hemoglobinopathies. Site specific mutation of a redox active amino acid(s) to reduce the ferryl heme or direct chemical modifications that can shield βCys93 have been proposed to improve oxidative resistance of Hb and may offer a protective therapeutic strategy.

Among amniotes, reptiles and mammals are differently adapted to terrestrial life. It is generally appreciated that terrestrialization required adaptive changes of vertebrate metabolism, particularly in the mode of nitrogen excretion. However, the current paradigm is that metabolic adaptation to life on land did not involve synthesis of enzymatic pathways de novo, but rather repurposing of existing ones. Here, by comparing the inventory of pyridoxal 5'-phosphate-dependent enzymes in different amniotes, we identify in silico a pathway for sulfur metabolism present in chick embryos but not in mammals. Cysteine lyase contains haem and pyridoxal 5'-phosphate co-factors and converts cysteine and sulfite into cysteic acid and hydrogen sulfide, respectively. A specific cysteic acid decarboxylase produces taurine, while hydrogen sulfide is recycled into cysteine by cystathionine beta-synthase. This reaction sequence enables the formation of sulfonated amino acids during embryo development in the egg at no cost of reduced sulfur. The pathway originated around 300 million years ago in a proto-reptile by cystathionine beta-synthase duplication, cysteine lyase neofunctionalization and cysteic acid decarboxylase co-option. Our findings indicate that adaptation to terrestrial life involved innovations in metabolic pathways, and reveal the molecular mechanisms by which such innovations arose in amniote evolution. Invasion of land required changes of vertebrate metabolism. Here, the authors report a pathway for sulfur metabolism present in chick embryos but not in mammals, which originated around 300 million years ago in a proto-reptile.

Taurine is one of the most abundant amino acids in mammalian tissues. It is obtained from the diet and by de novo synthesis from cysteic acid or hypotaurine. Despite the discovery in 1954 that the oxygenation of hypotaurine produces taurine, the identification of an enzyme catalyzing this reaction has remained elusive. In large part, this is due to the incorrect assignment, in 1962, of the enzyme as an NAD-dependent hypotaurine dehydrogenase. For more than 55 years, the literature has continued to refer to this enzyme as such. Here we show, both in vivo and in vitro, that the enzyme that oxygenates hypotaurine to produce taurine is flavin-containing monooxygenase (FMO) 1. Metabolite analysis of the urine of Fmo1-null mice by 1H NMR spectroscopy revealed a buildup of hypotaurine and a deficit of taurine in comparison with the concentrations of these compounds in the urine of wild-type mice. In vitro assays confirmed that human FMO1 catalyzes the conversion of hypotaurine to taurine, utilizing either NADPH or NADH as cofactor. FMO1 has a wide substrate range and is best known as a xenobiotic- or drug-metabolizing enzyme. The identification that the endogenous molecule hypotaurine is a substrate for the FMO1-catalyzed production of taurine resolves a long-standing mystery. This finding should help establish the role FMO1 plays in a range of biologic processes in which taurine or its deficiency is implicated, including conjugation of bile acids, neurotransmitter, antioxidant and anti-inflammatory functions, and the pathogenesis of obesity and skeletal muscle disorders. SIGNIFICANCE STATEMENT: The identity of the enzyme that catalyzes the biosynthesis of taurine from hypotaurine has remained elusive. Here we show, both in vivo and in vitro, that flavin-containing monooxygenase 1 catalyzes the oxygenation of hypotaurine to produce taurine.

In the last decades, silk fibroin and wool keratin have been considered functional materials for biomedical applications. In this study, fabrics containing silk fibers from Bombyx mori and Tussah silk fibers from Antheraea pernyi, as well as wool keratin fabrics, were grafted with phosmer CL and phosmer M (commercial names, i.e., methacrylate monomers containing phosphate groups in the molecular side chain) with different weight gains. Both phosmers were recently proposed as flame retarding agents, and their chemical composition suggested a possible application in bone tissue engineering. IR and Raman spectroscopy were used to disclose the possible structural changes induced by grafting and identify the most reactive amino acids towards the phosmers. The same techniques were used to investigate the nucleation of a calcium phosphate phase on the surface of the samples (i.e., bioactivity) after ageing in simulated body fluid (SBF). The phosmers were found to polymerize onto the biopolymers efficiently, and tyrosine and serine underwent phosphorylation (monitored through the strengthening of the Raman band at 1600 cm−1 and the weakening of the Raman band at 1400 cm−1, respectively). In grafted wool keratin, cysteic acid and other oxidation products of disulphide bridges were detected together with sulphated residues. Only slight conformational changes were observed upon grafting, generally towards an enrichment in ordered domains, suggesting that the amorphous regions were more prone to react (and, sometimes, degrade). All samples were shown to be bioactive, with a weight gain of up to 8%. The most bioactive samples contained the highest phosmers amounts, i.e., the highest amounts of phosphate nucleating sites. The sulphate/sulphonate groups present in grafted wool samples appeared to increase bioactivity, as shown by the five-fold increase of the IR phosphate band at 1040 cm−1.

Aquatic invertebrates vary in methylmercury (MeHg) levels among systems which has been attributed, in part, to environmental conditions, but may also be linked to differences in their biochemical composition. As MeHg is known to bind to thiol-containing amino acids such as cysteine in proteins of fish, our objective was to determine if these amino acids explain MeHg variability among aquatic invertebrate taxa. Benthic macroinvertebrates from diverse functional feeding groups and bulk zooplankton were collected from six acidic lakes in Kejimkujik National Park, Nova Scotia, Canada, and analyzed for MeHg, cysteine (as cysteic acid), methionine (as methionine sulfone), and nitrogen (relative trophic level, δ 15 N) and carbon (carbon source, δ 13 C) isotopes. MeHg was significantly and positively related to cysteine or methionine in zooplankton, caddisfly and stonefly tissues (R 2 from 0.24 to 0.57). In addition, methionine or cysteine in combination with δ 15 N and/or δ 13 C were better predictors of MeHg levels in stoneflies, mayflies, caddisflies and zooplankton among these lakes (R 2 adj = 0.25–0.91). Overall, these novel findings suggest that the variability in MeHg of aquatic invertebrates can be explained, in part, by their tissue levels of thiol-containing amino acids.

Sulfonolipids (SLs) are bacterial lipids that are structurally related to sphingolipids. Synthesis of this group of lipids seems to be mainly restricted to Flavobacterium, Cytophaga and other members of the phylum Bacteroidetes. These lipids have a wide range of biological activities: they can induce multi-cellularity in choanoflagellates, act as von Willebrand factor receptor antagonists, inhibit DNA polymerase, or function as tumour suppressing agents. In Flavobacterium johnsoniae, their presence seems to be required for efficient gliding motility. Until now, no genes/enzymes involved in SL synthesis have been identified, which has been limiting for the study of some of the biological effects these lipids have. Here, we describe the identification of the cysteate-fatty acyl transferase Fjoh_2419 required for synthesis of the SL precursor capnine in F. johnsoniae. This enzyme belongs to the α-oxoamine synthase family similar to serine palmitoyl transferases, 2-amino-3-oxobutyrate coenzyme A ligase and 8-amino-7-oxononanoate synthases. Expression of the gene fjoh_2419 in Escherichia coli caused the formation of a capnine-derived molecule. Flavobacterium johnsoniae mutants deficient in fjoh_2419 lacked SLs and were more sensitive to many antibiotics. Mutant growth was not affected in liquid medium but the cells exhibited defects in gliding motility.

The aim of this work was to optimize our natural hair dyeing system which we described in our previous work and to compare with other dyeing systems. Therefore, we investigated concentration limits of matcha and mordant and compared this new dyeing method with commercial permanent systems on the market. Completely unpigmented hair tresses were dyed with matcha powder (camelia sinensis) and iron(II)-lactate. To investigate the wash fastness and concentration limits, the differently dyed hair tresses were spectrophotometrically measured. The comparison of the damage potential for which cysteic acid is an indicator was measured by NIR. The concentration of matcha and mordant are responsible for the intensity of the color results. The higher the matcha or the mordant concentration, the darker the color results of the dyed hair tresses. Hair damage of matcha mordant dyeing is comparable with results of commercial permanent hair coloration systems. Moreover, the results of wash fastness of matcha mordant dyed hair tresses is comparable and even better by tendency to permanent colored hair tresses.

The amyloid β peptide, as one of the main components in senile plaque, represents a defining pathological feature for Alzheimer’s disease, and is therefore commonly used as a biomarker for this disease in clinical analysis. However, the selection of suitable standards is limited here, since only a few are commercially available, and these suffer from varying purity. Hence, the accurate characterization of these standards is of great importance. In this study, we developed a method for the traceable quantification of the peptide content using species-specific isotope dilution and ICP-MS/MS detection. It is based on the separation of the sulfur-containing amino acids methionine and cysteine after oxidation and hydrolysis of the peptide. Using a strong anion exchange column, both amino acids could be separated from each other, as well as from their oxidized forms and sulfate. The sulfur content was determined via ICP-MS/MS using oxygen as reaction gas. Species-specific isotope dilution was enabled by using a 34S-labeled yeast hydrolysate, containing methionine sulfone and cysteic acid with different isotopic composition. The peptide contents of Aβ standards (Aβ40,42), as well as myoglobin and lysozyme with different degrees of purity, were determined. For validation purposes, the standard reference material NIST 2389a, which contains the amino acids in a similar concentration, was subjected to the developed sample preparation and analysis method. In addition to accounting for errors during sample preparation, high levels of accuracy and precision could be obtained using this method, making it fit-for-purpose for the characterization of peptide standards. Graphical abstract

4H-chromenes play a significant role in natural and pharmacological products. Despite continuous advances in the synthesis methodology of these compounds, there is still a lack of a green and efficient method. In this study, we have designed cysteic acid chemically attached to magnetic graphene oxide (MNPs·GO-CysA) as an efficient and reusable solid acid catalyst to synthesize 4H-chromene skeletons via a one-pot three components reaction of an enolizable compound, malononitrile, an aldehyde or isatin, and a mixture of water–ethanol as a green solvent. This new heterogeneous catalyst provides desired products with a good to excellent yield, short time, and mild condition. This procedure presents an environmentally friendly approach for the synthesis of a great number of 4H-chromene derivatives.

Extracts from Streptomyces sp. S4.7 isolated from the rhizosphere of edelweiss, an alpine medicinal plant, exhibited activity against Gram-positive bacteria. LC-HRMS analyses of the extracts resulted in the detection of two unknown, structurally related lipopeptides that were assumed to be responsible for the antibiotic activity. LC-MS guided isolation and structure elucidation of viennamycins A and B (1 and 2) by HR-MS/MS, 1D and 2D NMR, and Marfey's analyses revealed them to be novel compounds, with viennamycin A containing cysteic acid, a unique feature for lipopeptides. Tests for antibacterial, antifungal, and cytotoxic activities of purified viennamycins, both with and without divalent cations, did not reveal any bioactivity, suggesting that their biological function, which could not be determined in the tests used, is atypical for lipopeptides. The genome of Streptomyces sp. S4.7 was sequenced and analyzed, revealing the viennamycin biosynthetic gene cluster. Detailed bioinformatics-based analysis of the viennamycin gene cluster allowed elucidation of the biosynthetic pathway for these lipopeptides.

Objective To understand the structural and chemical effects of cosmetic peroxide bleaching on human hair. Methods Human hair was progressively bleached using alkaline peroxide-persulphate treatment. Proteins lost through leaching were examined using amino acid analysis and mass spectrometric sequencing. Fibre damage was assessed using transmission electron microscopy, amino acid analysis and redox proteomics. Results Protein loss through leaching increased with bleaching severity. Leached proteins were not limited to the cuticle, but also included cortical intermediate filaments and matrix keratin-associated proteins. The leached proteins were progressively oxidized as bleaching severity increased. Bleached fibres demonstrated substantial damage to the cuticle layers and to the cortex. Extensive melanin granule degradation was present after the mildest bleach treatment. Protein oxidation in bleached fibres was principally in cortical intermediate filaments - the most abundant hair proteins - and targeted the sulphur-containing amino acids, particularly the conversion of cystine disulphide bonds to cysteic acid. Conclusion Peroxide chemical treatments quickly access the cortex, causing untargeted oxidative damage across the fibre in addition to the desired loss of melanin. Peroxide ingress is likely facilitated by the considerable structural degradation caused to the cuticle layers of hair fibres. The consequences of the peroxide action within the cuticle and cortex are oxidation of the proteins, and subsequent protein loss from the fibre that correlates to bleaching severity.

Previous studies suggest that specific binding to the complex consisting of fibroblast growth factor receptor-1 (FGFR1) and the coreceptor beta-Klotho (KLB) is the premise for human FGF19 and FGF21 activating the downstream signaling cascades, and regulating the metabolic homeostasis. However, it was found that human FGF21 loses its ability to bind to FGFR1-KLB after iodination with Na125 I and chloramine T, whereas human FGF19 retained its affinity for FGFR1-KLB even after iodination. The molecular mechanisms underlying these differences remained elusive. In this study, we first demonstrated that an intramolecular disulfide bond was formed between cysteine-102 and cysteine-121 in FGF21, implying that the oxidation of the cysteine to cysteic acid, which may interfere with the active conformation of FGF21, did not occur during the iodination procedures, and thus ruled out the possibility of the two conserved cysteine residues mediating the loss of FGF21 binding affinity to FGFR1-KLB upon iodination. Site-directed mutagenesis and molecular modeling were further applied to determine the residue(s) responsible for the loss of FGFR1-KLB affinity. The results showed that mutation of a single tyrosine-207, but not the other five tyrosine residues in FGF21, to a phenylalanine retained the FGFR1-KLB affinity of FGF21 even after iodination, whereas replacing the corresponding phenylalanine residue with tyrosine in FGF19 did not alter its binding affinity to FGFR1-KLB, but decreased the receptor binding ability of the iodinated protein, suggesting that tyrosine-207 is the crucial amino acid responsible for the loss of specifying FGFR1-KLB affinity of the iodinated FGF21.

The species Streptomyces lincolnensis is known as a producer of lincomycin A, a clinically important lincosamide antibiotic with activity against Gram-positive bacteria. Here, we report that S. lincolnensis produces a new cysteate-containing lactone product, cysteoamide (1), which arises from nonribosomal peptide synthetase-programmed sequential assembly of the monomers phenylacetic acid, valine, cysteate, threonine, β-hydroxyleucine, and β-alanine and subsequent intramolecular cyclization to form a lactone ring. The structure of 1 was determined by combined analysis of NMR and MS spectra, while the amino acid absolute configurations in 1 were assigned by Marfey's analysis following acid hydrolysis. The biosynthetic gene cluster of 1 was defined in the genome of S. lincolnensis by bioinformatics analysis and in vivo genetic study. In addition, in vitro assay revealed that OrfA, a pyridoxal 5'-phosphate-dependent protein, is responsible for the formation of the unusual cysteate unit. Cysteate-containing nonribosomal peptides appear to be widely present in various Streptomyces strains, and this study generates interest in their intrinsic functions that remain poorly understood.

We examined the performance of an ultra-high performance liquid chromatography method to quantify protein-bound sulphur amino acids in zooplankton. Both cysteic acid and methionine sulfone were linear from 5 to 250 pmol (r2 = 0.99), with a method detection limit of 13 pmol and 9 pmol, respectively. Although there was no matrix effect on linearity, adjacent peaks and co-eluting noise from the invertebrate proteins increased the detection limits when compared to common standards. Overall, performance characteristics were reproducible and accurate, and provide a means for quantifying sulphur amino acids in aquatic invertebrates, an understudied group.

Taurine and hypotaurine are abundantly present in molluskan tissues; however, the pathways for their biosynthesis have not yet been well investigated. In this study, we established a method for simultaneous detection of taurine-related compounds, including taurine, hypotaurine, cysteic acid, cysteine sulfinic acid (CSA), cystathionine, γ-aminobutyric acid, and β-alanine along with 17 free amino acids, using phenylthiocarbamyl derivatization in ammonia-containing solution, followed by injection into reversed-phase high-performance liquid chromatography. Using this method, we analyzed the major tissues (gill, mantle, adductor muscle, foot, and digestive gland) of the hydrothermal-vent-specific mussel Bathymodiolus septemdierum. The gill and digestive gland were suggested to be the major organs for taurine/hypotaurine biosynthesis because taurine and hypotaurine levels tended to be high, and their precursors, i.e., cysteine, cysteic acid, and CSA (only in digestive gland), were detected in these two tissues. Cystathionine was detected at relatively high level in digestive gland and in other tissues at low levels, suggesting its role as storage material for taurine/hypotaurine biosynthesis. Adductor muscle was found to be rich in free amino acids, mainly due to abundance of non-sulfur-containing amino acids.

Novel potential type electrochemical chiral biosensing system with unique capability of distinguishing and quantitating of tyrosine (Tyr) enantiomers by L-cysteic acid and left-handed chiral carbonaceous nanotubes (L-CCNT) modified glassy carbon electrode (L-Cys/L-CCNT/GCE) was first developed. The effect of sweep cycles of L-Cys and the kinds of L-CCNT on electrochemical chiral biosensing performance of L-Cys/L-CCNT/GCE were investigated. The electrochemical identification and quantitative determination of L- and D-tyrosine in their mixed solution were successfully achieved based on the different oxidation potential signals. The chiral structure of L-CCNT, the aromatic ring of Tyr, and also the intermolecular hydrogen bond between cysteic acid (CyA) and Tyr could possibly produce the difference in the free energy, which reflects as potential difference of L- and D-tyrosine. A good linear relationship between the potential, current, and different concentration ratios of L- and D-Tyr was obtained. Our present work realizes the simultaneous detection of Tyr enantiomers in their mixed solution based on the different potential signals, and it is of far-reaching significance in real electrochemical chiral biosensor study.

Taurine is one of the most abundant amino acids in mammalian tissues. It is obtained from the diet and byde novosynthesis, from cysteic acid or hypotaurine. Despite the discovery in 1954 that the oxygenation of hypotaurine produces taurine, the identification of an enzyme catalyzing this reaction has remained elusive. In large part this is due to the incorrect assignment, in 1962, of the enzyme as a NAD-dependent hypotaurine dehydrogenase. For more than 55 years the literature has continued to refer to this enzyme as such. Here we show, bothin vivoandin vitro, that the enzyme that oxygenates hypotaurine to produce taurine is flavin-containing monooxygenase 1 (FMO1). Metabolite analysis of the urine ofFmo1-null mice by1H NMR spectroscopy revealed a build-up of hypotaurine and a deficit of taurine in comparison with the concentrations of these compounds in the urine of wild-type mice.In vitroassays confirmed that FMO1 of human catalyzes the conversion of hypotaurine to taurine utilizing either NADPH or NADH as co-factor. FMO1 has a wide substrate range and is best known as a xenobiotic- or drug-metabolizing enzyme. The identification that the endogenous molecule hypotaurine is a substrate for the FMO1-catalyzed production of taurine resolves a long-standing mystery. This finding should help establish the role FMO1 plays in a range of biological processes in which taurine or its deficiency is implicated, including conjugation of bile acids, neurotransmitter, anti-oxidant and anti-inflammatory functions, the pathogenesis of obesity and skeletal muscle disorders.

We performed computer simulations to investigate the effect of oxidation on the extracellular cystine (CYC) uptake by the xC- antiporter. The latter is important for killing of cancer cells. Specifically, applying molecular dynamics (MD) simulations we studied the transport of CYC across xCT, i.e., the light subunit of the xC- antiporter, in charge of bidirectional transport of CYC and glutamate. We considered the outward facing (OF) configuration of xCT, and to study the effect of oxidation, we modified the Cys327 residue, located in the vicinity of the extracellular milieu, to cysteic acid (CYO327). Our computational results showed that oxidation of Cys327 results in a free energy barrier for CYC translocation, thereby blocking the access of CYC to the substrate binding site of the OF system. The formation of the energy barrier was found to be due to the conformational changes in the channel. Analysis of the MD trajectories revealed that the reorganization of the side chains of the Tyr244 and CYO327 residues play a critical role in the OF channel blocking. Indeed, the calculated distance between Tyr244 and either Cys327 or CYO327 showed a narrowing of the channel after oxidation. The obtained free energy barrier for CYC translocation was found to be 33.9kJmol-1, indicating that oxidation of Cys327, by e.g., cold atmospheric plasma, is more effective in inhibiting the xC- antiporter than in the mutation of this amino acid to Ala (yielding a barrier of 32.4kJmol-1). The inhibition of the xC- antiporter may lead to Cys starvation in some cancer cells, eventually resulting in cancer cell death.

A novel peptide (Cya-Phe-Leu-Ala-Pro, SCP) was formulated through non-protein amino acid-cysteic acid (Cya) modification of collagen peptide (Phe-Leu-Ala-Pro, CP) from Acaudina molpadioides. Introduction of this Cya showed remarkable improvement in the scavenging activities of OH·. SCP exhibited stronger effects than CP in preventing H2O2-induced oxidative damage due to lower levels of ROS and MDA, and higher activities of antioxidant enzymes, such as SOD, GSH-Px, HO-1, and NQO1. It was speculated that SCP could significantly increase the expression level of Nrf2 compared to CP, thereby activating the expression of downstream ARE genes. The expression levels of p38 in the upstream pathway to regulate Nrf2 content were significantly higher in both the CP and SCP-treated groups, while a higher level of JNK was observed only in the SCP-treated groups. The present study provided insights towards the application of cysteic acid modified peptide in protecting cell from oxidative damage through the JNK/Nrf2 pathway.

A generic quantification approach was introduced addressing the characterization of protein standards while fulfilling the principles of metrology. Traceable absolute quantification was achieved combining a proven biochemical method, i.e. protein hydrolysis followed by amino acid quantification with the concept of species specific isotope dilution analysis (IDA). The method relies on the determination of the two sulfur containing amino acids, cysteine and methionine by sulfur speciation analysis and is hence applicable to any protein containing sulfur. In vivo synthesis using 34S as sulfur source in yeast fermentations provided species specific isotopically enriched standards for IDA quantification of cysteine and methionine in the oxidized forms, methionine sulfone and cysteic acid. Reverse isotope dilution mass spectrometry (IDMS) characterization by inductively coupled plasma mass spectrometry (ICP-MS) combined to anion exchange showed that very high concentrated spike material could be produced with μmol amounts of proteinogenic sulfur containing amino acids per g cell dry weight. An enrichment of 34S to 96.3 ± 0.4% (n = 3) and 98.5 ± 0.4% (n = 3) for cysteic acid and methionine sulfone, respectively, was assessed. The established IDA method was validated for the absolute quantification of commercially available lysozyme and ceruloplasmin standards including the calculation of a total combined uncertainty budget.

After reacting with hydrogen peroxide (H2O2), sickle-cell hemoglobin (HbS, βE6V) remains longer in a highly oxidizing ferryl form (HbFe4+=O) and induces irreversible oxidation of "hot-spot" amino acids, including βCys-93. To control the damaging ferryl heme, here we constructed three HbS variants. The first contained a redox-active Tyr in β subunits (F41Y), a substitution present in Hb Mequon; the second contained the Asp (K82D) found in the β cleft of Hb Providence; and the third had both of these β substitutions. Both the single Tyr-41 and Asp-82 constructs lowered the oxygen affinity of HbS but had little or no effects on autoxidation or heme loss kinetics. In the presence of H2O2, both rHbS βF41Y and βF41Y/K82D enhanced ferryl Hb reduction by providing a pathway for electrons to reduce the heme via the Tyr-41 side chain. MS analysis of βCys-93 revealed moderate inhibition of thiol oxidation in the HbS single F41Y variant and dramatic 3- to 8-fold inhibition of cysteic acid formation in rHbS βK82D and βF41Y/K82D, respectively. Under hypoxia, βK82D and βF41Y/K82D HbS substitutions increased the delay time by ∼250 and 600 s before the onset of polymerization compared with the rHbS control and rHbS βF41Y, respectively. Moreover, at 60 °C, rHbS βK82D exhibited superior structural stability. Asp-82 also enhanced the function of Tyr as a redox-active amino acid in the rHbS βF41Y/K82D variant. We conclude that the βK82D and βF41Y substitutions add significant resistance to oxidative stress and anti-sickling properties to HbS and therefore could be potential genome-editing targets.

The UAG termination codon is generally recognized as the least efficient and least frequently used of the three universal stop codons. This is substantiated by numerous studies in an array of organisms. We present here evidence of a translational readthrough of a mutant nonsense UAG codon in the transcript from the cysteine sulfinic acid decarboxylase (csad) gene (ENSDARG00000026348) in zebrafish. The csad gene encodes the terminal enzyme in the taurine biosynthetic pathway. Taurine is a critical amino acid for all animals, playing several essential roles throughout the body, including modulation of the immune system. The sa9430 zebrafish strain (ZDB-ALT-130411-5055) has a point mutation leading to a premature stop codon (UAG) 20 amino acids 5’ of the normal stop codon, UGA. Data from immunoblotting, enzyme activity assays, and mass spectrometry provide evidence that the mutant is making a CSAD protein identical to that of the wild-type (XP_009295318.1) in terms of size, activity, and amino acid sequence. UAG readthrough has been described in several species, but this is the first presentation of a case in fish. Also presented are the first data substantiating the ability of a fish CSAD to utilize cysteic acid, an alternative to the standard substrate cysteine sulfinic acid, to produce taurine.

Thiol-containing compounds such as l-cysteine have been demonstrated to play an important role in metabolism and cellular growth, acting as powerful antioxidants. Consequently, their analytical determination in biological media has received a considerable amount of attention. In this work, an electrochemical sensor for the accurate electroanalytical determination of l-cysteine is proposed, based upon a Co(II)-phthalocyanine bulk modified disposable screen-printed graphite electrode (CoPc-SPE). Cyclic (CV) and Square Wave (SWV) voltammetry experiments have demonstrated an excellent electrocatalytic activity towards the electrochemical oxidation of l-cysteine using CoPc-SPEs within optimum neutral or basic pH. Moreover, the SWV response of l-cysteine is found to exhibit a linear range of 2.6–200 μM, with a low limit of detection of 4 μM (S/N = 3) and a sensitivity of 0.78 μA cm− 2 μM− 1. Coefficient of variation for reproducibility and repeatability of l-cysteine determination using the CoPc-SPE are 3 and 0.4%, respectively. The effect of inherent interferences such as naturally occurring amino acids, cystine and cysteic acid has been also evaluated. Finally, the applicability of the l-cysteine electrochemical sensor based upon CoPc-SPEs has been successfully demonstrated for the first time for the assessment of l-cysteine in a complex embryo cell culture medium. J. Iniesta thanks the programme Salvador de Madariaga from the Ministerio de Economía y Competitividad, grant number PRX14/00363. This work has been also financially supported by the MICINN-FEDER (Spain) through the projects CTQ2013-48280-C3-3-R.

Vialykh, E. A., Ilarionov, S. A., Abdelrahman, H. M. and Vialykh, I. A. 2014. Changes in amino acids content of humic acids sequentially extracted from peat and sod–Podzolic soil. Can. J. Soil Sci. 94: 575–583. Amino acids (AA) and peptides are thought to be part of humic acids (HA), but debate whether they are an integral part of the HA is still going. Humic acids sequentially extracted from peat and sod-podzolic soil were analyzed for their AA content, elemental composition and by Fourier transform infrared spectroscopy. Extracted HA were hydrolyzed in 6 M HCl for 16 h for AA release, which was detected by a capillary electrophoresis system. Alanine, arginine, sum of aspartic acid and asparagine, sum of cysteic acid and cysteine, sum of glutamic acid and glutamine, glycine, histidine, leucine and isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine were identified. The total content of hydrolyzable AA in sod-Podzol HA increased by 6.2–8.2% with increasing the extraction cycles while an inverse tendency was observed for AA released from peat HA. Moreover, individual AA expressed as percentages of total AA were constant values with coefficients of variation lower than 20% for the studied HA.

In order to investigate in detail the influence of chemical treatments (reduction, hydrolyzed eggwhite protein (HEWP) treatment, and oxidation) on damaged hair keratin fibers, the structure of cross-sections at various depths of excessively bleached (damaged) black human hair resulting from a permanent waving process was directly analyzed using Raman spectroscopy. It was found that l -cysteine (CYS) largely reacted with the gauche-gauche-gauche (GGG) conformation of disulfide (–SS–) groups (while CYS did not react with the trans-gauche-trans (TGT) conformation). In particular, not only the GGG content, but also the cysteic acid content existing throughout the cortex region of the excessively bleached human hair remarkably decreased by performing the oxidation process after reduction. On the other hand, the GGG content of the excessively bleached black human hair increased, while the TGT content decreased by performing the oxidation process after reduction and then HEWP treatment processes. From these experiments, the authors concluded that some of the keratin associated protein (KAP), which has a rich –SS– content and cysteic acid content was eluted from the cortex region along with the disconnection of –SS– groups, thereby leading to the remarkable reduction in the reconnection of –SS– groups of the excessively bleached black human hair after the permanent waving process (the reduction and oxidation processes). Also, the authors concluded that the HEWP treatment process in the permanent waving process caused the reconstruction of the KAP, thereby contributing to the acceleration of the reconnection of –SS– groups during the oxidation process.

In order to investigate in detail the internal structure changes in virgin black human hair keratin fibers resulting from bleaching treatments, the structure of cross-sections at various depths of black human hair, which had been impossible due to high melanin grande content, was directly analyzed using Raman spectroscopy. The gauche – gauche – gauche (GGG) content of the SS groups existing from the cuticle region to the center of cortex region of the virgin black human hair remarkably decreased, while the gauche – gauche – trans and trans – gauche – trans contents were not changed by performing the excessive bleaching treatment. In particular, it was found that not only the β-sheet and/or random coil content, but also the α-helix content existing throughout the cortex region of virgin black human hair decreased. In addition, the transmission electron microscope observation shows that the proteins in the cell membrane complex, the cuticle and cortex of the virgin black human hair were remarkably eluted by performing the excessive bleaching treatment. From these experiments, the author concluded that the SS groups, which have a GGG conformation were decomposed and finally converted to cysteic acid, and the α-helix structure of some of the proteins existing in the keratin was changed to the random coil structure, or eluted from the cortex region, thereby leading to the reduction in the protein density of the virgin human hair after the excessive bleaching treatment.

Cordyceps militaris (CM) occurring from a fruiting body by a host insect is a kind of mushroom, which is composed of animal host and plant fruit body. CM contains large amounts of useful ingredients including polysaccharides, vitamins, amino acids, minerals, etc. The essential amino acids from CM including cystine, lysine, and methionine can be expected to improve damaged hair treatment as effective ingredients. In this study, the improvement effect of the CM extracts on chemical and physical properties for damaged-hair treatment was investigated. The cysteic acid and cystine monooxide produced by oxidation of cystine were analyzed their chemical structure by FT-IR spectroscopy. It was confirmed that the vibration absorption peak (1,041 cm-1) of cysteic acid was reduced and increased sulfur content considerably which means meaningful improvement effect on damaged-hair treatment. Apparently, the cuticle morphology of the damaged-hair was improved significantly by treatment with CM extracts. Especially, confocal laser scanning microscope images of the damaged-hair treated with the extract showed highly increased fluorescence intensity which means promising effect in hair treatment. The tensile strength of the damaged hair treated also increased by 168% compared with the damaged hair.

This manuscript concerns the tissue-specific transcription of mouse and cattle glutamate decarboxylase-like protein 1 (GADL1) and the biochemical activities of human GADL1 recombinant protein. Bioinformatic analysis suggested that GADL1 appears late in evolution, only being found in reptiles, birds, and mammals. RT-PCR determined that GADL1 mRNA is transcribed at high levels in mouse and cattle skeletal muscles and also in mouse kidneys. Substrate screening determined that GADL1, unlike its name implies, has no detectable GAD activity, but it is able to efficiently catalyze decarboxylation of aspartate, cysteine sulfinic acid, and cysteic acid to β-alanine, hypotaurine, and taurine, respectively. Western blot analysis verified the presence of GADL1 in mouse muscles, kidneys, C2C12 myoblasts, and C2C12 myotubes. Incubation of the supernatant of fresh muscle or kidney extracts with cysteine sulfinic acid resulted in the detection of hypotaurine or taurine in the reaction mixtures, suggesting the possible involvement of GADL1 in taurine biosynthesis. However, when the tissue samples were incubated with aspartate, no β-alanine production was observed. We proposed several possibilities that might explain the inactivation of ADC activity of GADL1 in tissue protein extracts. Although β-alanine-producing activity was not detected in the supernatant of tissue protein extracts, its potential role in β-alanine synthesis cannot be excluded. There are several inhibitors of the ADC activity of GADL1 identified. The discovery of GADL1 biochemical activities, in conjunction with its expression and activities in muscles and kidneys, provides some tangible insight toward establishing its physiological function(s).

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:27:06Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:42:27Z : No. of bitstreams: 1 2-s2.0-84869206693.pdf: 345052 bytes, checksum: af1f663f5bb60b9d0121f6ed18b7e210 (MD5) Made available in DSpace on 2014-05-27T11:27:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-10-23 Amino acids are well known to be an important class of compounds for the maintenance of body homeostasis and their deficit, even for the polar neuroactive aminoacids, can be controlled by supplementation. However, for the amino acid taurine (2-aminoethanesulfonic acid) this is not true. Due its special physicochemical properties, taurine is unable to cross the blood-brain barrier. In addition of injured taurine transport systems under pathological conditions, CNS supplementation of taurine is almost null. Taurine is a potent antioxidant and anti-inflammatory semi-essential amino acid extensively involved in neurological activities, acting as neurotrophic factor, binding to GABA A/glycine receptors and blocking the excitotoxicity glutamate-induced pathway leading to be a neuroprotective effect and neuromodulation. Taurine deficits have been implicated in several CNS diseases, such as Alzheimer's, Parkinson's, epilepsy and in the damage of retinal neurons. This review describes the CNS physiological functions of taurine and the development of new derivatives based on its structure useful in CNS disease treatment.&; 2012 by the authors; licensee MDPI, Basel, Switzerland. Lapdesf-Laboratory of Drug Design School of Pharmaceutical Sciences University of São Paulo State (UNESP), Rodovia Araraquara-Jaú Km1, CEP 14.801-902, Araraquara, SP Lapdesf-Laboratory of Drug Design School of Pharmaceutical Sciences University of São Paulo State (UNESP), Rodovia Araraquara-Jaú Km1, CEP 14.801-902, Araraquara, SP

A descaling and oxidation pretreatment was employed to maximize the yield of cortical cells in the disintegration process of wool fiber. The results indicated that the productivity of intact cortical cells was greatly increased by moderate oxidation pretreatment in 1.6 % per-acetic acid within 2 h, but the yield would be decreased by further oxidation pretreatment. In order to give a reasonable explanation for this fact, the effect of the increasing time of oxidation pretreatment on the yield of intact cortical cells was investigated by means of spectral analysis using FT-IR and XRD. The intensity of the peak at 1040 and 1173 cm−1 in FT-Infrared spectrum gradually increased with increasing oxidation pretreatment time, suggesting that more and more SS bonds were cleaved to form cysteic acid. X-ray Diffraction investigation showed that the crystallinity of wool fiber obviously decreased when the time of oxidation pretreatment exceeded 2 h. The combined results of FT-IR and XRD revealed that SS bonds in the amorphous region of wool fiber were first cleaved in the fiber components. The selective cleavage of SS bonds in the amorphous region by the appropriate oxidation pretreatment can effectively decrease the bonding force between the components of wool fiber and enhance the yield of intact cortical cells.

Available amino acids are those absorbed from the gastrointestinal tract in a form suitable for body protein synthesis. True ileal digestible amino acids are determined based on the difference between dietary amino acid intake and unabsorbed dietary amino acids at the terminal ileum. The accuracy of ileal digestible amino acid estimates for predicting available amino acid content depends on several factors, including the accuracy of the amino acid analysis procedure. In heat processed foods, lysine can react with compounds to form nutritionally unavailable derivatives that are unstable during the hydrochloric acid hydrolysis step of amino acid analysis and can revert back to lysine causing an overestimate of available lysine. Recently, the true ileal digestible reactive (available) lysine assay based on guanidination has provided a means of accurately determining available lysine in processed foods. Methionine can be oxidised during processing to form methionine sulphoxide and methionine sulphone and cysteine oxidised to cysteic acid. Methionine sulphoxide, but not methionine sulphone or cysteic acid, is partially nutritionally available in some species of animal. Currently, methionine and cysteine are determined as methionine sulphone and cysteic acid respectively after quantitative oxidation prior to acid hydrolysis. Consequently, methionine and cysteine are overestimated if methionine sulphone or cysteic acid are present in the original material. Overall, given the problems associated with the analysis of some amino acids in processed foodstuffs, the available amino acid content may not always be accurately predicted by true ileal amino acid digestibility estimates. For such amino acids specific analytical strategies may be required.

We have previously shown that hydrogen peroxide (H(2)O(2)) triggers irreversible oxidation of amino acids exclusive to the β-chains of purified human hemoglobin (HbAo). However, it is not clear, whether α- or β-subunit Hb variants exhibit different oxidative resistance to H(2)O(2) when compared to their native HbAo. Hb Providence contains two β-subunit variants with single amino acid mutations at βLys82→Asp (βK82D) and at βLys82→Asn (βK82N) positions and binds oxygen at lower affinity than wild type HbA. We have separated Hb Providence into its 3 component fractions, and contrasted oxidative reactions of its β-mutant fractions with HbAo. Relative to HbAo, both βK82N and βK82D fractions showed similar autoxidation kinetics and similar initial oxidation reaction rates with H(2)O(2). However, a more profound pattern of changes was seen in HbAo than in the two Providence fractions. The structural changes in HbAo include a collapse of β-subunits, and α-α dimer formation in the presence of excess H(2)O(2). Mass spectrometric and amino acid analysis revealed that βCys93 and βCys112 were oxidized in the HbAo fraction, consistent with oxidative pathways driven by a ferrylHb and its protein radical. These amino acids were oxidized at a lesser extent in βK82D fraction. While the 3 isolated components of Hb Providence exhibited similar ligand binding and oxidation reaction kinetics, the variant fractions were more effective in consuming H(2)O(2) and safely internalizing radicals through the ferric/ferryl pseudoperoxidase cycle.

Searching for metabolites from Streptomyces sp. RI051-SDHV6 resulted in the discovery of a novel peptide, JBIR-96 (1). The structure of 1 was established as an N-phenylacetylated pentapeptide involving a cysteic acid and a peptide lactone structure by extensive NMR and MS analyses. In addition, the absolute configuration of 1 was established by Marfey's and modified Mosher's methods.

A study on the effect of cysteic acid position on the types of fragment ions formed by collision-induced dissociation (CID) of [M - H](-) ions is presented. Of particular note is the observation of d-type fragment ions for peptides that contain an N-terminal cysteic acid (fixed negative charge) and cleavable amino acid side chains possessing a β-γ carbon-carbon bond. For example, the CID mass spectrum of oxidized cys-kemptide (C(ox)LRRASLG) [M - H + O(3)](-) ions contains abundant series of d-type fragment ions, and similar results are observed for oxidized cysteine-containing ribonuclease A proteolytic peptides. The d(i) fragment ions are assumed to arise by a charge-remote and/or charge-assisted fragmentation mechanism, which both occur at high collision energies and involve consecutive reactions (i.e., the formation of a(i) ions followed by the elimination of the side chain to form d(i) ions).

Human hair fibers are primarily composed of keratin protein, characterized by a very high content of cysteine, a sulfur-containing amino acid, which ordinarily forms cystine via a disulfide bond. It is known that some cystine residues are converted to cysteic acid during permanent waving or hair coloring, although details of their distribution and extent are still unclear. In this study, by using difference in XANES profiles of cystine and cysteic acid at the S-K absorption edge, the formation of cysteic acid was confirmed for homogenized samples of permed or bleached hair. Furthermore chemical mapping of cysteic acid was performed on hair-section samples with X-ray contact microscopy. The peripheral region, cuticle, in bleached hair showed the highest content of cysteic acid compared with the other parts, while permed hair showed relatively uniform distribution. This finding suggests that perming and bleaching damage hair by different mechanisms.

Recent developments in the technique of combinatorial peptide ligand libraries, for enhancing the visibility of the low-abundance proteome, are reviewed here. Novel en bloc elution systems, allowing essentially complete proteome recovery in a single step, are reported here, particularly, en bloc elution with 3–5% boiling sodium dodecyl sulphate (SDS) or in urea–thiourea–CHAPS added with either 40 mM formic acid or 25 mM cysteic acid. Novel capturing systems are also discussed: in particular, although capturing at pH 7.2 in physiological saline has always been recommended, it is shown that capturing also at acidic (pH 3.8) and alkaline (pH 9.5) values substantially increments the total captured protein population. Some examples of detection of novel proteins by the described methodology are also discussed. In particular, in the case of venom proteins, where essentially all components had been detected and fully described by conventional means, the application of the ligand library technology allowed the discovery of two, previously unreported, trace enzymes necessary for the maintenance of the native structure of venom components, namely peroxiredoxin and glutaminyl cyclase. In the case of the red blood cell (RBC) cytoplasmic proteome, where a grand total of 1570 components of the 2% minority proteomes have been identified, these findings allowed to unravel the genetic defect of a rare RBC disease, called congenital dyserythropoietic anemia type II. The mutations are located in the SEC23B gene coding for the SEC23B protein, detected for the first time in the RBC proteome thanks to the peptide capturing technology.

A new process for feltproofing of wool fibers based on the biopolymer sericin is reported. The susceptibility of wool to sericin has been enhanced by creation of new active sites along the wool keratin molecules, such as cysteic acid and S-cystine sulphonate residues, by pretreatment with hydrogen peroxide and sodium sulphite. The sericin-combining capacity of wool has been even enhanced by carrying out the reaction in the presence of different crosslinkersnamely dimethylol dihydroxy ethylene urea, dimethyl dihyroxy ethylene urea, and epichlorohydrin, which are able to link sericin with the available sites of wool. The effect of these treatments on some of the inherent properties of wool as well as on its morphological structure is also reported. The felting resistance of wool tops treated with the system H2O2 /Na2SO3/sericin/ECH (felt ball density 0.045 g /cm3) is close to that of the machine washable wool obtained commercially (felt ball density 0.039 g /cm3).

During the processing of feedstuffs and foods, methionine can be oxidized to methionine sulfoxide and methionine sulfone, and cysteine can be oxidized to cysteic acid. Methionine sulfone and cysteic acid are nutritionally unavailable, but methionine sulfoxide can be utilized, at least to some degree. The degree of utilization depends on the levels of methionine, cysteine, and methionine sulfoxide in the diet, but there is no consensus in the literature on the quantitative impact of these dietary constituents on methionine sulfoxide utilization. Methionine and cysteine are most often determined after quantitative oxidation to methionine sulfone and cysteic acid, respectively, using performic acid oxidation prior to hydrolysis. However, this method may overestimate the methionine content of processed foods, as it will include any methionine sulfoxide and methionine sulfone present. A selection of analytical methods has been developed to allow the separate determination of the 3 oxidized forms of methionine, the merits of which are discussed in this review. An additional consideration for determining methionine and cysteine bioavailability is that not all dietary methionine and cysteine is digested and absorbed from the small intestine. Selected methods designed to determine the extent of digestion and absorption are discussed. Finally, a concept for a new assay for determining methionine bioavailability, which includes determining the digestibility of methionine and methionine sulfoxide as well as the utilization of methionine sulfoxide, is presented.

Accurate determination of cysteic acid, homocysteic acid, and taurine in aqueous solution is essential in many analyses of biological and clinical applications. These sulfonic acids are difficult to be separated and determined using reversed phase chromatography and cation‐exchange chromatography. In this research, an accurate and sensitive determination for the three sulfonic acids has been achieved using anion‐exchange chromatography with post‐column derivatization and spectrometric detection. This technique has outperformed the technique using suppressed conductivity detection. It has enormously enhanced the accuracy, detection limit, sensitivity, and linearity in the determination of these three sulfonic acids. This technique has demonstrated to be excellent for simultaneously determining cysteic acid, homocysteic acid, and taurine.

Five feedstuffs were oxidized using performic acid, and these, along with their unoxidized counterparts, were acid hydrolyzed for multiple times (0-144 h) in degassed and vacuum-sealed glass tubes. The methionine sulfone, cysteic acid, methionine, and cysteine contents were determined for each hydrolysis time. Least-squares nonlinear regression of the sulfur amino acid contents and hydrolysis time was used to predict the actual sulfur amino acid content as well as the hydrolysis and loss rates. Least-squares nonlinear regression estimates for methionine content compared well with those of methionine sulfone for most of the feedstuffs tested. In contrast, the estimates for cysteine agreed poorly with cysteic acid. The loss rates during acid hydrolysis for methionine, methionine sulfone, and cysteic acid were low. Overall, acid hydrolysis in an evacuated sealed tube for 24 h without prior oxidation is suitable for methionine, but not cysteine, quantitation in some complex feedstuffs.

The integrin receptor alphavbeta3 is overexpressed on the endothelial cells of growing tumors and on some tumor cells themselves. A radiolabeled alphavbeta3 antagonists belonging to the quinolin-4-one class of peptidomimetics (TA138) was previously shown to exhibit high affinity for integrin alphavbeta3 and high selectivity versus other integrin receptors. 111In-TA138 exhibited high tumor uptake in the c-neu Oncomouse mammary adenocarcinoma model and produced excellent scintigraphic images. This study describes the synthesis of eight divalent versions of TA138 and their evaluation as potential tumor radiotherapeutic agents. The two main variables in this study were the length of the spacer bridging the biotargeting moieties and the total negative charge of the molecules imparted by the cysteic acid pharmacokinetic modifiers. Receptor affinity was evaluated in a panel of integrin receptor affinity assays, and biodistribution studies using the 111In-labeled derivatives were carried out in the c-neu Oncomouse model. All divalent agents maintained the high receptor affinity and selectivity of TA138, and six of the eight 111In derivatives exhibited blood clearance that was faster than 111In-TA138 at 24 h postinjection (PI). All divalent agents exhibited tumor uptake and retention at 24 h PI that was higher than 111In-TA138. Tumor/organ ratios were improved for most of the divalent agents at 24 h PI in critical nontarget organs marrow, kidney, and liver, with the agents having intermediate-length spacers (29-43 A) showing the largest improvement. As an example, 111In-15 showed tumor uptake of 14.3% ID/g at 24 h PI and tumor/organ ratios as follows: marrow, 3.24; kidney, 7.29; liver, 8.51. A comparison of therapeutic indices for 90Y-TA138 and 177Lu-15 indicate an improved therapeutic index for the divalent agent. The implications for radiotherapeutic applications and the mechanism of this multivalent effect are discussed.

Hydrogen peroxide (H2O2) triggers a redox cycle between ferric and ferryl hemoglobin (Hb) leading to the formation of a transient protein radical and a covalent hemeprotein cross-link. Addition of H2O2 to highly purified human hemoglobin (HbA0) induced structural changes that primarily resided within β subunits followed by the internalization of the heme moiety within α subunits. These modifications were observed when an equal molar concentration of H2O2 was added to HbA0 yet became more abundant with greater concentrations of H2O2. Mass spectrometric and amino acid analysis revealed for the first time that βCys-93 and βCys-112 were oxidized extensively and irreversibly to cysteic acid when HbA0 was treated with H2O2. Oxidation of further amino acids in HbA0 exclusive to the β-globin chain included modification of βTrp-15 to oxyindolyl and kynureninyl products as well as βMet-55 to methionine sulfoxide. These findings may therefore explain the premature collapse of the β subunits as a result of the H2O2 attack. Analysis of a tryptic digest of the main reversed phase-high pressure liquid chromatography fraction revealed two α-peptide fragments (α128 - α139) and a heme moiety with the loss of iron, cross-linked between αSer-138 and the porphyrin ring. The novel oxidative pathway of HbA0 modification detailed here may explain the diverse oxidative, toxic, and potentially immunogenic effects associated with the release of hemoglobin from red blood cells during hemolytic diseases and/or when cell-free Hb is used as a blood substitute.

Variants in the gene encoding the enzyme glutamic acid decarboxylase like 1 (GADL1) have been associated with response to lithium therapy. Both GADL1 and the related enzyme cysteine sulfinic acid decarboxylase (CSAD) have been proposed to be involved in the pyridoxal-5′-phosphate (PLP)-dependent biosynthesis of taurine. In the present study, we compared the catalytic properties, inhibitor sensitivity and expression profiles of GADL1 and CSAD in brain tissue. In mouse and human brain we observed distinct patterns of expression of the PLP-dependent decarboxylases CSAD, GADL1 and glutamic acid decarboxylase 67 (GAD67). CSAD levels were highest during prenatal and early postnatal development; GADL1 peaked early in prenatal development, while GAD67 increased rapidly after birth. Both CSAD and GADL1 are being expressed in neurons, whereas only CSAD mRNA was detected in astrocytes. Cysteine sulfinic acid was the preferred substrate for both mouse CSAD and GADL1, although both enzymes also decarboxylated cysteic acid and aspartate. In silico screening and molecular docking using the crystal structure of CSAD and in vitro assays led to the discovery of eight new enzyme inhibitors with partial selectivity for either CSAD or GADL1. Lithium had minimal effect on their enzyme activities. In conclusion, taurine biosynthesis in vertebrates involves two structurally related PLP-dependent decarboxylases (CSAD and GADL1) that have partially overlapping catalytic properties but different tissue distribution, indicating divergent physiological roles. Development of selective enzyme inhibitors targeting these enzymes is important to further dissect their (patho)physiological roles. publishedVersion

Attempts to make a diamino disulfonic acid derivative of an aza-BODIPY showed it was difficult to add BF2 to a disulfonated azadipyrromethene, and sulfonation of an aza-BODIPY resulted in loss of the BF2 fragment. We conclude the electron-deficient character of aza-BODIPY dyes destabilizes them relative to BODIPY dyes. Consequently, sulfonation of the aza-BODIPY core is not a viable strategy to increase water solubility. This assertion was indirectly supported via stability studies of a BODIPY and an aza-BODIPY in aqueous media. To afford the desired compound type, an aza-BODIPY with two amino and two sulfonic acid groups was prepared via modification of the aryl substituents with cysteic acid.

Isolates of the wheat stem rust fungus Puccinia graminis f. sp. Tritici grow on an agar medium containing Czapek's minerals, glucose, citrate, cysteine and one of the following nitrogen sources: ammonium, aspartate, asparagine, glutamate, glutamine, alanine, serine and glycine, but not nitrate or nitrite. Cysteine can be replaced by homocysteine or methionine, but not by sulphate, reduced inorganic sulphur, thiolacetlc acid, cysteic acid, cysteamine or by glutathione or S-methylcysteine. This fungus incorporates [35S] sulphide but not [35S] sulphate into the amino acids cysteine and methionine, although sulphate uptake into the mycelium occurs. More than 70% of the [35S] sulphide incorporated into organic sulphur leaks into the culture filtrate as S-methylcysteine, cysteine, glutathione and cysteinyl-glycine. Most 35S leaks as cysteinylglycine, with less than 1% of the 35S in methionine or homocysteine. This fungus has very low levels of nitrate reductase compared with Neurospora crassa; but when labelled with [U-14C] glucose in the presence of glutamine incorporates considerable 14C into amino acids. Approximately 70-85% of the 14C labelled amino acids leak into the culture filtrate, including most amino acids, particularly glutamate, glutamine and a peptide believed to be Y-glutamylglutamate. From these results it appears that the fungus is unable to reduce sulphate or nitrate but can synthesise all other nitrogen containing compounds. Rust-infected susceptible wheat leaves synthesise twice as much [35S] cysteine, [35S] methionine and [35S] glutathione from 35S04, as non-infected leaves, although there is no corresponding increase in the incorporation of [35S] cysteine and [35S] methionine into protein a Glutathione breakdown is also accelerated in infected leaves, and is accompanied by a decrease in the metabolically active glutathione pool. From these studies it appears that glutamine is probably the major source of nitrogen and methionine the major source of sulphur for the rust fungus when grown in association with its host. It is suggested that the leakage of amino acids and peptides is due to the operation of the y-glutamyl cycle. The importance of amino acid excretion to the nutrition and metabolism of the rust fungus in this parasitic association is discussed.

The methyl-accepting chemotaxis proteins are a family of receptors in bacteria that mediate chemotaxis to diverse signals. To explore the plasticity of these proteins, we have developed a simple method for selecting cells that swim to target attractants. The procedure is based on establishing a diffusive gradient in semi-soft agar plates and does not require that the attractant be metabolized or degraded. We have applied this method to select for variants of the Escherichia coli aspartate receptor, Tar, that have a new or improved response to different amino acids. We found that Tar can be readily mutated to respond to new chemical signals. However, the overall change in specificity depended on the target compound. A Tar variant that could detect cysteic acid still showed a strong sensitivity to aspartate, indicating that the new receptor had a broadened specificity relative to wild-type Tar. Tar variants that responded to phenylalanine or N-methyl aspartate, or that had an increased sensitivity to glutamate showed a strong decrease in their response to aspartate. In at least some of the cases, the maximal level of sensitivity that was obtained could not be attributed solely to substitutions within the binding pocket. The new tar alleles and the techniques described here provide a new approach for exploring the relationship between ligand binding and signal transduction by chemoreceptors and for engineering new receptors for applications in biotechnology.

Some thiol-derived Maillard reaction products (MRPs) may exert antioxidant activity, depending on the reaction conditions as well as on the sugar and the sulphydryl compound. Recently, we reported that MRPs derived from glucose or fructose with cysteine (CSH) or glutathione (GSH) mixtures greatly inhibited polyphenoloxidases (PPOs), oxidoreductases responsible for discoloration of fresh or minimally processed fruits and vegetables. Glucose and GSH were shown to be the most active in producing inhibitory MRPs. Therefore, we examined the way in which the nature of the reactants affected their synthesis, in order to establish a structure-activity relationship for the inhibitory products. Various aqueous (0.083 M, 0.125 M, or 0.25 M) mixtures of a sugar (hexose, pentose, or diholoside) with either a CSH-related compound (CSH, GSH, N-acetyl-cysteine, cysteamine, cysteic acid, methyl-cysteine, cysteine methyl ester), an amino acid (gamma-glutamic acid, glycine, methionine), or other sulfur compound (thiourea, 1,4-dithiothreitol, 2-mercaptoethanol) were heated at 103 degrees C for 14 h. Soluble MRPs were compared for their ability to inhibit apple PPO activity. In the presence of CSH, the rated sugars (same molar concentration) ranked as to inhibitory effect were pentoses > sucrose > hexoses > or = maltose. In the presence of glucose, the simultaneous presence of an amino group, a carboxyl group, and a free thiol group on the same molecule seemed essential for the production of highly inhibitory compounds.