Enzymatic synthesis of halogen derivatives of L-phenylalanine and phenylpyruvic acid stereoselectively labeled with hydrogen isotopes in the side chain.

Halogenated, labeled with deuterium, tritium or doubly labeled with deuterium and tritium in the 3S position of the side chain isotopomers of L-phenylalanine and phenylpyruvic acid were synthesized. Isotopomers of halogenated L-phenylalanine were obtained by addition of ammonia from isotopically enriched buffer solution to the halogenated derivative of (E)-cinnamic acid catalyzed by phenylalanine ammonia lyase. Isotopomers of halogenated phenylpyruvic acid were obtained enzymatically by conversion of the appropriate isotopomer of halogenated L-phenylalanine in the presence of phenylalanine dehydrogenase. As a source of deuterium was used deuterated water, as a source of tritium was used a solution of highly diluted tritiated water. The labeling takes place in good yields and with high deuterium atom% abundance.

Ultra-Fast Impedimetric Immunoassay for Detection of Streptococcus agalactiae Using Carbon Electrode with Nanodiamonds Film.

This publication presents the results of work on the development of a quick and cheap electrochemical immunosensor for the diagnosis of infections with the pathogen Streptococcus agalactiae. The research was carried out on the basis of the modification of the well-known glassy carbon (GC) electrodes. The surface of the GC (glassy carbon) electrode was covered with a film made of nanodiamonds, which increased the number of sites for the attachment of anti-Streptococcus agalactiae antibodies. The GC surface was activated with EDC/NHS (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-Hydroxysuccinimide). Determination of electrode characteristics after each modification step, performed using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS).

Highly selective impedimetric determination of Haemophilus influenzae protein D using maze-like boron-doped carbon nanowall electrodes.

This study reports a novel impedimetric immunosensor for protein D detection in purified and bacterial (Haemophilus influenzae, Hi) samples. The detection was based on antigen recognition by anti-protein D antibodies (apD) immobilised at the maze-like boron-doped carbon nanowall electrodes (B:CNW). The B:CNW electrodes were synthesised, and their surface was characterised by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) methods. The sensor was prepared in a two-step procedure: apD were covalently linked on the previously modified B:CNW electrodes using diazonium salt. Modification steps were controlled by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measurements. The immunosensor exhibited excellent electrochemical performance, stability, satisfactory sensitivities, and linear ranges for antigen detection. Protein D was detected down to 2.39 × 102fg/mL with a linear range extending from 3.37 × 10-11to 3.37 × 10-3µg/mL (in purified sample). Next, Hi's LOD was 5.20 × 102CFU/mL with a linear range of 8.39 × 101-8.39 × 103CFU/mL. Selectivity studies showed no reaction with negative samples as Streptococcus pyogenes, Streptococcus pneumoniae or Bordetella parapertussis bacteria. Therefore, the new approach is suitable for rapid and quantitative detection of Hi, and is a good candidate for further tests on clinical samples.

Antibody Modified Gold Electrode as an Impedimetric Biosensor for the Detection of Streptococcus pyogenes.

Streptococcus pyogenes is a known cause of a wide spectrum of diseases, from mild and acute to severe invasive infections. This paper concerns the development of a novel impedimetric biosensor for the detection of the mentioned human pathogen. The proposed biosensor is a gold disk electrode modified with commercially available antibodies attached to the surface of the electrode by carbodiimide chemistry. The conducted tests confirmed the specificity of the antibodies used, which was also demonstrated by the results obtained during the detection of S. pyogenes using electrochemical impedance spectroscopy. The developed sensor successfully detected the presence of S. pyogenes in the sample and the detection limit was calculated as 9.3 cfu/mL. The results obtained show a wide linear range for verified concentrations of this pathogen in a sample from 4.2 × 102 to 4.2 × 106 cfu/mL. Furthermore, the optimal experimentally determined time required to perform pathogen detection in the sample was estimated as 3 min, and the test did not lead to the degradation of the sample.

The chemo- enzymatic synthesis of labeled l-amino acids and some of their derivatives.

This review compiles the combined chemical and enzymatic synthesis of aromatic l-amino acids (l-phenylalanine, l-tyrosine, l-DOPA, l-tryptophan, and their derivatives and precursors) specifically labeled with carbon and hydrogen isotopes, which were elaborated in our research group by the past 20 years. These compounds could be then employed to characterize the mechanisms of enzymatic reactions via kinetic and solvent isotope effects methods.

Isotopic effects in mechanistic studies of biotransformations of fluorine derivatives of L-alanine catalysed by L-alanine dehydrogenase.

Synthesis of 3-fluoro-[2-2H]-L-alanine (3-F-[2H]-L-Ala) in reductive amination of 3-fluoropyruvic acid catalysed by L-alanine dehydrogenase (AlaDH) was described. Fluorine derivative was used to study oxidative deamination catalysed by AlaDH applied kinetic (for 3-F-L-Ala in H2O - KIE's on Vmax: 1.1; on Vmax/KM: 1.2; for 3-F-L-Ala in 2H2O - on Vmax: 1.4; on Vmax/KM: 2.1) and solvent isotope effect methods (for 3-F-L-Ala - SIE's on Vmax: 1.0; on Vmax/KM: 0.87; for 3-F-[2-2H]-L-Ala - on Vmax: 1.4; on Vmax/KM: 1.5). Studies explain some details of reaction mechanism.

Kinetic and solvent isotope effects on biotransformation of aromatic amino acids and their derivatives.

Aromatic amino acids such as l-phenylalanine, l-tryptophan, 3',4'-dihydroxy-l-phenylalanine (l-DOPA), and their derivatives 3',4'-dihydroxyphenylacelaldehyde (DOPAL) and 3',4'-dihydroxyphenylethanol (DOPET), play an essential role in human metabolic processes. Incorrect or slow biotransformation of these compounds leads to some metabolic dysfunctions and in some cases to some neurodegenerative diseases. Therefore, studies of the biotransformation mechanisms of these metabolites draw biochemists' and medical researchers' attention. This study investigates the mechanisms of biotransformation of the aforementioned compounds using kinetic (KIE) and solvent (SIE) isotope effect methods. The overview presents the results and the numerical values of KIE and SIE methods, obtained in the study of biotransformation of l-phenylalanine, 5'-chloro-l-tryptophan, and l-DOPA, catalyzed by the enzymes from the lyases group (phenylalanine ammonia lyase, tryptophan indole-lyase, and tyrosine decarboxylase). Deuterium KIE was also determined during the deamination of 2'-chloro-l-phenylalanine in the presence of the enzyme l-phenylalanine dehydrogenase, as well as in the conversion of DOPAL into DOPET catalyzed by the enzyme alcohol dehydrogenase. The values of KIE and SIE have been determined using a noncompetitive spectrophotometric and a competitive (combined with internal radioactivity standard) radiometric methods.

Syntheses of halogen derivatives of L-tryptophan, L-tyrosine and L-phenylalanine labeled with hydrogen isotopes.

Halogenated, labeled with tritium and doubly with deuterium and tritium, derivatives of L-tryptophan, i.e. 5'-bromo-[2-(3)H]-, 5'-bromo-[2-(2)H/(3)H]-, 5'-fluoro-[2-(3)H]-5'-fluoro-[2-(2)H/(3)H]-, 6'-fluoro-[2-(3)H]-, 6'-fluoro-[2-(2)H/(3)H]-L-tryptophan, as well as, L-tyrosine, i.e. 3'-fluoro-[2-(3)H]-, 3'-fluoro-[2-(2)H/(3)H]-, 3'-chloro-[2-(3)H]-, and 3'-chloro-[2-(2)H/(3)H]-L-tyrosine, and also L-phenylalanine, i.e. 2'-fluoro-[(3S)-(3)H]-, 2'-fluoro-[(3S)-(2)H/(3) H]-, 2'-chloro-[(3S)-(3)H]-, 2'-chloro-[(3S)-(2)H/(3)H]-, 4'-chloro-[(3S)-(3)H]-, and 4'-chloro-[(3S)-(2)H/(3)H]-L-phenylalanine were synthesized using enzymatic methods. Isotopomers of L-tryptophan were synthesized by coupling of halogenated indoles with S-methyl-L-cysteine carried out in deuteriated or tritiated incubation media. Labeled halogenated derivatives of L-tyrosine were obtained by the enzymatically supported exchange between halogenated L-tyrosine and isotopic water. Labeled halogenated isotopologues of L-Phe were synthesized by the enzymatic addition of ammonia to halogenated cinnamic acid. As a source of hydrogen tritiated water (HTO) and heavy water (D2O) with addition of HTO were used.

[Comparison of basic electrical features of ICD defibrillating leads implanted to right ventricular apex or right ventricular outflow track and septum]. / Porównanee podstawowych parametrów elektrycznych elektrod defibrylujacych wszczepianych do wierzcholka oraz do okolic drogi odplywu prawej komory serca.

UNLABELLED: Automatic implantable cardioverter-defibrillators (ICDs) are nowadays an essential tool for reducing mortality due to sudden cardiac death. Technological advances in the miniaturization of devices and lead fixation, and the development of surgical techniques has led to more frequent implantation of the defibrillation leads outside the right ventricular apex (RVA), especially in those patients requiring cardiac pacing, as data from large clinical trials showed that chronic RVA pacing is harmful, especially in heart failure subjects, who are an important target for the ICD. Very few studies have been published comparing the electrical characteristics of leads placed in the RVA versus those implanted outside the RVA, mainly to right ventricular outflow tract of the heart (RVA), hence any subsequent analysis of this issue seems to be a valuable addition to the available information in this topic. The aim of this study was to compare the electrical parameters of ICD leads implanted into the right ventricular apex (RVA), to those placed in one of the alternative sites: the right ventricular outflow tract (RVOT), or the area of the interventricular septum (RVS). MATERIAL AND METHODS: Retrospective analysis of medical data from a single centre (teaching hospital), which included 132 patients with ICD implanted in 2010-2011, both in primary and secondary prevention of sudden cardiac death. We compared the most important electrical parameters of the ICD system, as the resistance of the pacing system, resistance of high-voltage coil, the amplitude of the sensed beats and pacing threshold. In addition, we compared the time of implantation, X-ray fluoroscopy time and X-ray exposure. RESULTS: There were no statistically significant differences between the two analysed groups in terms of pacing-system resistance (601.012 vs. 602.7omega, p = 0,499), high-energy coil resistance (63.7omega vs. 67.22, p = 0,201), amplitude of sensed R-waves (14,6mV vs. 15.3mV, p = 0, 710) and the pacing threshold energy (0,368 microJ vs. 0.259 microJ, p = 0,803). Also the duration of implantation (123, 3 min vs. 123, 9 min, p = 0,940), fluoroscopy time (11,0 minutes vs. 8,6 minutes, p = 0,06) and dose exposure (1594, 5cGy/cm2 vs. 2094, 4cGy/cm2, p = 0,069) were comparable in both groups. CONCLUSIONS: Implantation of ICD leads to the RVOT/RVS is a safe procedure, and the basic electrical parameters of such systems are comparable to ICDs with lead implanted to the RVA.

Kinetic and solvent deuterium isotope effects in the oxidation of putrescine catalysed by enzyme diamine oxidase.

In this study, the kinetic isotope effects and solvent isotope effects in the reaction of the deamination of [(1R)-(2)H ] putrescine--catalysed by enzyme diamine oxidase (EC 1.4.3.6)--were determined using a non-competitive spectroscopic method. Putrescine, stereospecifically labelled with deuterium, was obtained by enzymatic decarboxylation of l-ornithine that was carried out in a fully deuteriated incubation medium.

[Post transplant lymphoprolipherative disorder (PTLD) in patients after kidney transplantation--clinical manifestation, treatment and prognosis]. / Potransplantacyjna choroba limfoproliferacyjna (PTLD) u pacjentów po przeszczepie nerki--obraz kliniczny, leczenie i rokowanie.

Post transplant lymphoprolipherative disorder (PTLD) is a very severe, life threatening complication after organ transplantation. In adults after kidney transplantation the frequency of this complication is 0.3-3%; whereas in children 0.4-10%. Epstein-Barr virus infection discovered in 90% of patients, as a result of immunosuppression, plays an important role in the PTLD etiology. There is a coincidence between heavy immunosuppression, especially its cumulative doses, and the increased risk of lymphoprolipherative disorder. The clinical manifestation of PTLD may present as: mononucleosis like syndrome (fever, pharyngitis, tonsillar enlargement, lymphadenopathy), or as a rapid progressive course presenting with severe acidosis, lymphadenopathy, dysfunction of graft and other internal organs, where infiltration may mimic tumour like appearance. The present paper discusses methods and effectiveness of PTLD treatment, which aims to restore effectiveness of the immunological system, elimination of neoplastic cells and EBV.

The influence of cyclomaltooligosaccharides (cyclodextrins) on the enzymatic decomposition of l-phenylalanine catalyzed by phenylalanine ammonia-lyase.

Cyclomaltohexaose (α-cyclodextrin) and cyclomaltoheptaose (ß-cyclodextrin) as well as their four methyl ether derivatives, that is, hexakis(2,3-di-O-methyl)cyclomaltohexaose, hexakis(2,3,6-tri-O-methyl)cyclomaltohexaose, heptakis(2,3-di-O-methyl)cyclomaltoheptaose, and heptakis(2,3,6-tri-O-methyl)cyclomaltoheptaose were investigated as the additives in the course of enzymatic decomposition of l-phenylalanine catalyzed by phenylalanine ammonia-lyase. Only a few of those additives behaved like classical inhibitors of the enzymatic reaction under investigation because the values of the Michaelis constants that were obtained, as well as the maximum velocity values depended mostly atypically on the concentrations of those additives. In most cases cyclodextrins caused mixed inhibition, both competitive and noncompetitive, but they also acted as activators for selected concentrations. This atypical behaviour of cyclodextrins is caused by three different and independent effects. The inhibitory effect of cyclodextrins is connected with the decrease of substrate concentration and unfavourable influence on the flexibility of the enzyme molecules. On the other hand, the activating effect is connected with the decrease of product concentration (the product is an inhibitor of the enzymatic reaction under investigation). All these effects are caused by the ability of the cyclodextrins to form inclusion complexes.