Cystine Groups Research Articles

Predicting the stone composition of children preoperatively by Hounsfield unit detection on non-contrast computed tomography

Many studies have been performed on adult patients to reveal the relationship between Hounsfield unit (HU) value and composition of stone, but none have focused on childhood. We aimed to predict stone composition by HU properties in pre-intervention non-contrast computed tomography (NCCT) in children. This could help to orient patients towards more successful interventions. Data of 94 children, whose pre-intervention NCCT and post-interventional stone analysis were available were included. Stones were grouped into three groups: calcium oxalate (CaOx), cystine, and struvite. Besides spot urine PH value, core HU, periphery HU, and Hounsfield density (HUD) values were measured and groups were compared statistically. The mean age of patients was 7±4 (2-17) years and the female/male ratio was 51/43. The mean stone size was 11.7±5 (4-24) mm. There were 50, 38, and 6 patients in the CaOx, cystine, and struvite groups, respectively. The median values for core HU, periphery HU, and mean HU in the CaOx group were significantly higher than the corresponding median values in the cystine and struvite groups. Significant median HUD difference was seen only between the CaOx and cystine groups. No difference was seen between the cystine and struvite groups in terms of HU parameters. To distinguish these groups, mean spot urine PH values were compared and were found to be higher in the struvite group than the cystine group (Table). The retrospective nature and small number of patients in some groups are limitations of this study, which also does not include all stone compositions. Our cystine stone rate was higher than childhood stone composition distribution in the literature. This is because our center is a reference center in a region with high recurrence rates of cystine stones. In fact, high numbers of cystine stones helped us to compare them with calcium stones more accurately and became an advantage for this study. NCCT at diagnosis can provide some information for determination of stone composition. While CaOx stones can be discriminated from cystine and struvite stones using HU parameters, a simple spot urine pH assessment must be added to distinguish cystine stones from struvite stones.

Predicting Urinary Stone Composition Based on Single-energy Noncontrast Computed Tomography: The Challenge of Cystine

To study several measurements from a single-energy noncontrast computed tomography (NCCT) that may distinguish calcium oxalate, uric acid, and cystine stones. Patients with pure urinary stones who had at least 1 single-energy NCCT before the stone composition analysis from January 2008 to December 2012 were enrolled in this study. The analyzed data comprised stone size, volume, core Hounsfield unit (HU), periphery HU, absolute and relative HU differences between core and periphery, and HU density. After these measurements, an NCCT bone window was subjectively evaluated to study the homogeneity of each stone from core to periphery. The Spearman correlation test was used to determine the correlation between HU values and stone size and volume for each group. A total of 113 patients were found with pure urinary stones who also had a corresponding NCCT. There were 36, 47, and 30 patients in the calcium oxalate, uric acid, and cystine groups, respectively. The core HU, periphery HU, absolute and relative HU differences, and HU density were significantly different among the 3 groups (P<.001). Stone size and volume had a positive correlation with core and periphery HUs only for calcium oxalate and cystine stones. The subjective evaluation of the urinary calculi revealed a different pattern for each stone composition. Single-energy NCCT may predict calcium oxalate stones with a high degree of accuracy. There is an overlap in radiographic profiles of cystine and uric acid stones, making a definitive differentiation more challenging.

Effect of free radical oxidation on the structure and function of plasma fibrin-stabilizing factor

Plasma fibrin-stabilizing factor (pFXIII) is a heterosubunit protein, the main function of which is the covalent stabilization of the fibrin matrix. This paper examines the ozone-induced free radical oxidation of plasma fibrin-stabilizing factor at different stages of its enzymatic activation. Electrophoretic data on the accumulation of γ-γ-dimers and α-polymers in the formation of stabilized fibrin suggests that a reduction in the enzymatic activity of FXIIIa is largely dependent on the stage of activation of pFXIII at which the oxidation is carried out. The results of UV, IR, and Raman spectroscopy measurements suggests that the oxidation of the amino acid residues of the polypeptide chains of pFXIII primarily affects cyclic, cysteine, and cystine groups and leads to the formation of oxidation products, being accompanied by the disruption of the protein secondary structure. Dynamic light scattering measurements revealed a loosening of the protein structure in the course of free radical oxidation. Spin label EPR spectroscopy data are suggestive of a change in the pFXIII conformation as a result of oxidative modification. Possible reasons for the different sensitivity of pFXIII to oxidative modification in the process of enzymatic activation are discussed. It is presumed that FXIII-B2 regulatory subunits function as scavenges of reactive oxygen species, thereby protecting FXIII-A2 catalytic subunits from oxidation.

Maillard 반응에 의한 미더덕 체액의 풍미개선

Mideoduck drips were mixed with amino acids (Met, Tau, Gly, Ala, Thr, Cys), thiamine and sugars (Glucose, Ribose) for flavor modification and evaluation using the Maillard reaction. To mask the seafood flavor, onions, spring onions, garlic, ginger, citric orange and green tea were mixed with Mideoduck drips at for 2.5 hr in a stainless still reaction bomb. The glucose/thiamine model reaction system was estimated to be lower than the ribose/thiamine model system, and an extreme case is the ribose/Met model system. Mixed system of glucose, ribose and taurine containing sulfur compounds showed fair results. Among the Mideoduck drips mixed with sugars and amino groups, only thiamine model systems were estimated to be normal. The flavor composition of Mideoduck drips/sugars model system, and long chain fatty acids were composed of 31.32~62.71% total flavor content. The 1,2-benzenedicarboxylic acid dibutylester contents made up more than 20% of the model system in groups A, B and C. From the model system in this study, drip/glucose, drip/ribose, drip/glucose/citric orange, and drip/glucose/glycine/cystine groups showed most intense good flavor.

Blastocyst production by in vitro maturation and development of porcine oocytes in defined media following intracytoplasmic sperm injection

The present study was carried out to establish porcine defined IVP. In Experiments 1 and 2, we investigated the efficacy of additional 0.6 mM cystine and/or 100 microM cysteamine (Cys) to a defined TCM199 maturation medium with regard to the intracellular glutathione (GSH) concentration and the developmental competence of in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The control medium was a modified TCM199 containing 0.05% (w/v) polyvinyl alcohol (PVA). Cys and/or cystine were added to the control medium. The control group and immature oocytes (presumptive germinal vesicle oocytes; GV) were prepared for GSH assay. In Experiment 3, the efficacy of epidermal growth factor (EGF) addition to a modified porcine zygote medium (mPZM) for in vitro culture (IVC) medium was investigated on embryonic development and the mean cell number of blastocysts following ICSI. As a positive or negative control, 0.3% BSA (mPZM-3) or 0.3% PVA (mPZM-4), respectively, was added to the base medium. The defined IVC medium was supplemented with 5 or 10 ng/ml EGF. In Experiment 1, no significant difference was found in the rates of cleavage (31.4-64.3%) and blastocyst formation (6.5-22.9%) among the treatment and control groups. The mean cell numbers per blastocyst ranged from 30 to 48 among the groups without significant differences. However, in Experiment 2, the intracellular GSH concentrations in the oocytes cultured in the medium supplemented with 100 microM Cys (9.6 pmol/oocyte) or Cys + cystine (9.9 pmol/oocyte) were significantly (p < 0.05) higher than the control (2.5 pmol/oocyte) and 0.6 mM cystine (6.5 pmol/oocyte) groups, but not different from the GV group (9.0 pmol/oocyte). The GSH concentration in the cystine group was also significantly (p < 0.05) higher than that in the control group, but not different from the GV group. In Experiment 3, the rates of cleavage and blastocyst formation and the mean cell numbers of blastocysts were not significantly different among the groups. However, the addition of 5 ng/ml EGF into the mPZM-4 resulted in a significantly (p < 0.05) higher blastocyst rate per cleaved embryo than the other two defined groups (mPZM-4 + 5 ng/ml: 48.6%, mPZM-4 and mPZM-4 +10 ng/ml: 23.4% and 23.1%, respectively). The present results indicate that the addition of Cys to a defined medium for in vitro maturation (IVM) of porcine oocytes increases intracellular GSH concentration. Further addition of cystine into the IVM medium containing 100 microM Cys is not necessary and TCM199 plus Cys (100 microM) could be used as a defined IVM medium for porcine oocytes. The addition of 5 ng/ml EGF to a defined IVC medium has enhanced subsequent development after ICSI. This study shows that porcine blastocysts can be produced by defined media throughout the steps of IVP (IVM, ICSI and IVC).

In Vitro and Intracellular Production of Peptide‐Encapsulated Fluorescent Silver Nanoclusters

In Vitro and Intracellular Production of Peptide‐Encapsulated Fluorescent Silver Nanoclusters

A Strategy to Locate Cysteine Residues in Proteins by Specific Chemical Cleavage Followed by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

A simple methodology has been developed to characterize the number and location of free cysteine and cystine groups in peptides and proteins, using chemical modification and matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOF MS). This new approach employs a specific reaction between free sulfhydryls and 2-nitro-5-thiocyanobenzoic acid (NTCB) to selectively cyanylate cysteine thiols. The N-terminal peptide bond of the modified cysteinyl residue can then be cleaved under alkaline conditions to form an amino-terminal peptide and a series of 2-iminothiazolidine-4-carboxylyl peptides which can be mapped to the sequence by MALDI-MS. The number and location of cysteines can be deduced from mass analysis of the peptide mixture resulting from NTCB chemical processing of the original protein of known sequence under nonreducing conditions. Similar experiments are then performed following disulfide bond reduction to further characterize both cysteine and cystine groups. Experimental conditions are described for protein disulfide bond reduction, sulfhydryl cyanylation, and cleavage reactions performed both in solution and on Zetabind membranes.

Peptide mapping of complex proteins at the low-picomole level with capillary electrophoretic separations.

A variety of different peptide-mapping schemes are presented, with emphasis on the development of procedures which can be done with limited quantities (i.e. 5 pmol) of protein. Results are obtained from model proteins which contain disulfide bonds, which must be broken prior to fragmentation of the protein. A reaction involving the simultaneous use of tributylphosphine and 2-methylaziridine to reduce and alkylate the disulfide bonds is employed, due to favorable attributes of these reagents for the scaled-down procedure. The traditional performic acid oxidation reaction to cleave cystine groups is also successfully used with low-picomole quantities of protein. Three different protein digestion reagents are used: trypsin, chymotrypsin, and cyanogen bromide. Each reagent produces a unique mixture of peptides. Capillary electrophoresis is used to separate the peptides, offering high separation efficiencies, short analysis times, and compatibility with small sample sizes. In addition to the conventional use of UV detection for underivatized peptides, laser-induced fluorescence detection is employed in conjunction with an arginine-selective derivatization reaction. This latter procedure for derivatization and detection offers an alternative peptide-mapping mode, in which only the arginine-containing peptides are detected, and is useful in simplifying the peptide maps of large proteins.

Electrochemistry of xanthine oxidase at glassy carbon and mercury electrodes

The electrochemistry of xanthine oxidase has been examined at glassy carbon and mercury electrodes. No response is observed at glassy carbon unless denaturation of the enzyme has occurred in which case a surface-confined chemically reversible process FAD + 2 H + + 2 e − ⇄ FADH 2 is observed (FAD = flavin adenine dinucleotide). In contrast, at mercury electrodes, processes associated with FAD and with enzyme-bound mercuric cysteinate, cysteine and cystine groups are observed, even under conditions where no free FAD is detected at a glassy carbon electrode. Study of these processes as a function of pH, after interaction with Hg 2+ and after conversion of xanthine oxidase to xanthine dehydrogenase, allows a self-consistent reaction scheme to be proposed. Interestingly, at the mercury electrode, no potential is available at which a redox process does not occur and consequently, denaturation of the protein takes place at the electrode surface at all potentials. No molybdenum- or Fe 2S 2-based electrochemistry was detected.

Structural study of the adsorption dynamics of prothrombin fragment 2 at the electrode/solution interface

The dynamics of the adsorption of prothrombin fragment 2 on a HMDE were studied as a function of concentration, by measuring the variation with time of the differential capacity and the charges corresponding to the reduction of the SS bonds of the adsorbed molecules or to their reoxidation. Under our experimental conditions, the adsorption process is diffusion controlled, which allows us to determine the number of molecules adsorbed. As a consequence, an interesting feature is the possibility to determine the bulk protein concentration from the linear part of the variation of the capacitance vs. time. A dynamic study of the adsorption process, by plotting the number of SS bonds reduced or reoxidized, calculated from the voltammetric peak areas, against the double-layer capacitance, shows the existence of two distinct adsorption states. Only a fraction of the total amount of SS bonds is reduced, indicating that only the cystine groups in contact with the mercury are electroactive. The adsorbed protein molecules resist complete unfolding on the surface. At high coverage, where surface reactions take place, a clear difference is observed for fragment 2 relative to fragment 1 and prothrombin, which might be due to γ-carboxyglutamic residues.

Structural study of the adsorption dynamics of prothrombin fragment 2 at the electrode/solution interface

The dynamics of the adsorption of prothrombin fragment 2 on a HMDE were studied as a function of concentration, by measuring the variation with time of the differential capacity and the charges corresponding to the reduction of the SS bonds of the adsorbed molecules or to their reoxidation. Under our experimental conditions, the adsorption process is diffusion controlled, which allows us to determine the number of molecules adsorbed. As a consequence, an interesting feature is the possibility to determine the bulk protein concentration from the linear part of the variation of the capacitance vs. time. A dynamic study of the adsorption process, by plotting the number of SS bonds reduced or reoxidized, calculated from the voltammetric peak areas, against the double-layer capacitance, shows the existence of two distinct adsorption states. Only a fraction of the total amount of SS bonds is reduced, indicating that only the cystine groups in contact with the mercury are electroactive. The adsorbed protein molecules resist complete unfolding on the surface. At high coverage, where surface reactions take place, a clear difference is observed for fragment 2 relative to fragment 1 and prothrombin, which might be due to γ-carboxyglutamic residues.

Cloning and characterization of complementary DNA for human tryptase.

The amino acid sequence of human mast cell tryptase was determined from corresponding cDNA cloned from a lambda ZAP library made with mRNA derived from a human mast cell preparation. Tryptase is the major neutral protease present in human mast cells and serves as a specific marker of mast cells by immunohistologic techniques and as a specific indicator of mast cell activation when detected in biologic fluids. Based on nucleic acid sequence, human tryptase consists of a 244-amino acid catalytic portion of 27,423 D with two putative N-linked carbohydrate binding sites and a 30-amino acid leader sequence of 3,048 D. A His74, Asp120, Ser223 catalytic triad and four cystine groups were identified by analogy to other serine proteases. Regions of amino acid sequence that are highly conserved in serine proteases, in general, were conserved in tryptase. The catalytic portion of human tryptase had an 84% amino acid sequence similarity with that of dog tryptase; their leader sequences had a 67% similarity. Asp217 in the substrate binding pocket of human tryptase is consistent with a specificity for Arg and Lys residues at the site of cleavage (P1), whereas Glu245 is consistent with the known preference of human tryptase for substrates with Arg or Lys also at P3, analogous residues also being present in dog tryptase. Asp244, which is substituted for the Gly found in dog tryptase and in most serine proteases, is present in the putative substrate binding pocket and may confer additional substrate specificity on human tryptase for basic residues. Further studies now can be designed to elucidate these structure-function relationships.

Electroactivity of adsorbed prothrombin at the Hg/solution interface and relation with the nature of the adsorption states

The adsorption rate of prothrombin on a HMDE was studied as a function of concentration, by measuring the variation with time of the differential capacity and the areas of voltammetric peaks corresponding to the reduction of the S-S bonds of the adsorbed molecules. Under our experimental conditions, the adsorption process is diffusion controlled, which allows us to determine the number of molecules adsorbed. A detailed dynamic study of the adsorption process shows the existence of two adsorption states, for which are defined an area and a differential molecular area, associated with two different numbers of S-S groups reduced when one molecule is added in the adlayer. It can be concluded from the results that: o (a) In all the adsorption states, only a fraction of the total S-S bonds is reduced, indicating that only the cystine groups in contact with the mercury are electroactive. This fraction seems to increase with the exposure time of the molecules to the surface and at the low exposure times it is higher as the starting potential is higher. (b) The adsorbed protein molecules resist complete unfolding in the surface. The presence of Ca 2+ in solution causes stabilization of the globular molecular structure. (c) Lateral interactions between adsorbed protein molecules seem to aid exposure of the sulphide groups.

Behavior of O-glycosyl and O-phosphoryl proteins in alkaline solution.

0-Glycosyl and 0-phosphoryl groups, as well as disulfide bonds, are rapidly removed from proteins in alkaline solution primarily via β-elimination. The reaction is initiated by abstraction of the α-hydrogen of an amino acid residue by hydroxide ion. The carbanion undergoes rearrangement expelling the glyco- or phosphoryl group resulting in formation of a dehydroalanyl (from serine) or β-methyldehy-droalanyl (from threonine) residue. The unsaturated derivatives are reactive with internal protein nucleophilic groups and with external nucleophiles. These addition reactions, some leading to crosslinking, result in changed properties of the protein. Several factors may affect rates of β-elimination and addition. For these studies, we used two unique proteins: phosvitin, a well-characterized protein with 120 0-phosphoryl groups and no cystine or 0-glycosyl groups; and a glycopeptide related to the antifreeze protein from Antarctic fish, a well-characterized protein consisting of (Ala-Ala-Thr)n in which all of the threonyl residues are glycosylated. At the dilute concentrations of proteins used, rates of β-elimination and addition were independent of protein concentration but directly dependent upon the hydroxide ion concentration. With phosvitin, rates of β-elimination and addition were quite dependent on ionic strength and the rate of β-elimination was increased 20-fold in the presence of Ca2+. Activation energies for both β-elimination and addition were near 20 kcal/mole. Implications of these reactions for protein chemistry and protein processing will be discussed.