[Long-term treatment of morphologically verified myocarditis: successes and probable errors. Case report].

Diagnosis and treatment of myocarditis can be challenging, including determining indications for heart transplantation. We present a 6-year medical history of a 54 years old patient with severe morphologically verified viral-negative lymphocytic myocarditis and systemic manifestations (onset of hemorrhagic vasculitis) combined with moderate coronary atherosclerosis, which regressed according to repeated coronary angiography. For 5 years, the patient received immunosuppressive therapy with methylprednisolone and azathioprine with a significant improvement. Repeated relapses of atrial fibrillation required correction of basic therapy and plasmapheresis. The disease was complicated by thyrotoxicosis and multi-organ dysfunction; the autopsy showed persistent myocarditis activity. The myocarditis is a chronic condition and requires a review of the treatment strategy at each stage.

Phytoestrogens decorated nanocapsules for therapeutic methionine γ-lyase targeted delivery.

The main task of targeted therapy is the selective destruction of cancer cells without affecting normal ones. For these purposes, small molecules and antibodies are used that target specific receptors and proteins or block signaling pathways in tumor cells. The natural phytoestrogens daidzein (Dz) and genistein (Gn) possess binding capacity to estrogen receptors (ER). Methionine γ-lyase (MGL) is promising in two strategies of antitumor therapy: for the elimination of l-methionine, which is necessary for the proliferation of tumor cells, and for the production of cytotoxic dialkyl thiosulfinates in situ. For delivery of MGL-loaded nanocapsules (nanoreactors) to the surface of cancer cells a technique for Dz or Gn incorporation into the shell of polyionic vesicles (PICsomes) was developed. The nanoreactors were characterized by dynamic light scattering and transmission electron microscopy. The enzyme retained its catalytic efficiency inside the decorated PICsomes. The binding of Dz/Gn-nanoreactors to the surface of ER + MCF7 breast adenocarcinoma cells was demonstrated. For the first time an influence of enzyme-loaded PICsomes and their individual components on embryos development was evaluated. The high rate of blastocysts formation (>80%) was observed for all tested components and nanoreactors themselves. A strong inhibitory effect on the early embryonic development of MGL-loaded PICsomes in the presence of S-alkyl-l-cysteine sulfoxide substrates was showed. This proves that the substrates can freely penetrate through the polymer shell of the polyionic vesicle and are cleaved by MGL to form cytotoxic thiosulfinates. The data obtained for phytoestrogens decorated PICsomes may be applied in enzyme therapy of malignant tumors.

Daidzein-directed methionine γ-lyase in enzyme prodrug therapy against breast cancer.

Thiosulfinates in situ formed by "pharmacological pair" C115H methionine γ-lyase/S-(allyl/alkyl)-l-cysteine sulfoxides possess cytotoxic activity against various malignant cell lines. To investigate in vivo antitumor activity of thiosulfinates generated directly at the surface of tumor cells, a chemical conjugate between Clostridium novyi C115H methionine γ-lyase (C115H MGL) and isoflavone daidzein was prepared. The binding of conjugate (C115H-Dz) to various breast cancer cell lines was demonstrated, as well as its cytotoxicity in the presence of S-(allyl/alkyl)-l-cysteine sulfoxides. The most promising among thiosulfinates was dipropyl thiosulfinate (IC50 < 0.53 µM). The pharmacokinetic parameters of C115H MGL and C115H-Dz were obtained. Plasma half-lives of the enzyme and conjugated enzyme were 4.4 and 7.2 h, respectively. In vivo antitumor effect of pharmacological pairs on SKBR-3 xenografts was demonstrated. Treatment of tumor-bearing mice with a pair of C115H-Dz/propiin inhibited tumor growth by 85%.

Conjugates of methionine γ-lyase with polysialic acid: Two approaches to antitumor therapy.

The methionine dependence is a well known phenomenon in metabolism of cancer cells. Methionine γ-lyase (EC 4.4.1.11, MGL) catalyzes the γ-elimination reaction of L-methionine and thus could effectively inhibit the growth of malignant cells. Recently we have demonstrated that the mutant form of the enzyme C115H MGL can be used as a component of the pharmacological pair enzyme/S-(allyl/alkyl)-L-cysteine sulfoxides to yield thiosulfinates in situ. Thiosulfinates were shown to be toxic to various cancer cell lines. Therefore the application of the enzyme in enzyme pro-drug therapy may be promising. The conjugates of MGL and C115H MGL with polysialic acid were obtained and their kinetic and pharmacokinetic parameters were determined. The formation of polysialic shell around the enzyme was confirmed by atomic force microscopy. The half-life of conjugated enzymes increased 3-6 times compared to the native enzyme. The cytotoxic effect of conjugated MGL against methionine dependent cancer cell lines was increased two times compared to the values for the native enzymes. The anticancer efficiency of thiosulfinates produced by pharmacological pair C115H MGL/S-(allyl/alkyl)-L-cysteine sulfoxides was demonstrated in vitro. The results indicate that the conjugates of MGL with polysialic acid could be new antitumor drugs.

Role of NUDIX Hydrolases in NAD and ADP-Ribose Metabolism in Mammals.

Proteins of the NUDIX hydrolase (NUDT) superfamily that cleave organic pyrophosphates are found in all classes of organisms, from archaea and bacteria to higher eukaryotes. In mammals, NUDTs exhibit a wide range of functions and are characterized by different substrate specificity and intracellular localization. They control the concentration of various metabolites in the cell, including key regulatory molecules such as nicotinamide adenine dinucleotide (NAD), ADP-ribose, and their derivatives. In this review, we discuss the role of NUDT proteins in the metabolism of NAD and ADP-ribose in human and animal cells.

[Daytime sleepiness and cognitive disorders in elderly patients]. / Dnevnaia sonlivost' i kognitivnye rasstroistva v pozhilom vozraste.

Data on the contribution of daytime sleepiness to cognitive impairment in elderly patients are presented. Currently, diagnostic methods of daytime sleepiness, which include diaries, neurological, clinical and instrumental tools, are used. It has been shown that daytime sleepiness correlates with different types of dementia that suggests the involvement of common neurochemical mechanisms. Multiple studies report that daytime sleepiness increases the risk of dementia in elderly people. Future research of daytime sleepiness could be focused on its clinical and paraclinical presentations to facilitate the prognosis of dementia.

The Catalytic Mechanisms of the Reactions between Tryptophan Indole-Lyase and Nonstandard Substrates: The Role of the Ionic State of the Catalytic Group Accepting the Cα Proton of the Substrate.

In the reaction between tryptophan indole-lyase (TIL) and a substrate containing a bad leaving group (L-serine), general acid catalysis is required for the group's elimination. During this stage, the proton originally bound to the Cα atom of the substrate is transferred to the leaving group, which is eliminated as a water molecule. As a result, the basic group that had accepted the Cα proton at the previous stage has to be involved in the catalytic stage following the elimination in its basic form. On the other hand, when the substrate contains a good leaving group (ß-chloro-L-alanine), general acid catalysis is not needed at the elimination stage and cannot be implemented, because there are no functional groups in enzymes whose acidity is strong enough to protonate the elimination of a base as weak as Cl- anion. Consequently, the group that had accepted the Cα proton does not lose its additional proton during the elimination stage and should take part in the subsequent stage in its acidic (not basic) form. To shed light on the mechanistic consequences of the changes in the ionic state of this group, we have considered the pH dependencies of the main kinetic parameters for the reactions of TIL with L-serine and ß-chloro-L-alanine and the kinetic isotope effects brought about by replacement of the ordinary water used as a solvent with 2H2O. We have found that in the reaction between TIL and ß-chloro-L-alanine, the aminoacrylate hydrolysis stage is sensitive to the solvent isotope effect, while in the reaction with L-serine it is not. We have concluded that in the first reaction, the functional group containing an additional proton fulfills a definite catalytic function, whereas in the reaction with L-serine, when the additional proton is absent, the mechanism of hydrolysis of the aminoacrylate intermediate should be fundamentally different. Possible mechanisms were considered.

Antibacterial Effect of Thiosulfinates on Multiresistant Strains of Bacteria Isolated from Patients with Cystic Fibrosis.

The multiresistance of A. ruhlandii 155B, B. cenocepacia 122, and P. aeruginosa 48B strains isolated from patients with cystic fibrosis was established. The antibacterial effect of allicin, dimethyl thiosulfinate, and dipropyl thiosulfinate on multidrug-resistant strains was shown. Thiosulfinates can have both bacteriostatic and bactericidal effects depending on the microorganism and the concentration. The studied thiosulfinates may be candidates for the development of alternative antibiotic drugs to treat infections caused by multidrug-resistant pathogens.

The Regulatory Role of NAD in Human and Animal Cells.

Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form NADP are the major coenzymes in the redox reactions of various essential metabolic pathways. NAD+ also serves as a substrate for several families of regulatory proteins, such as protein deacetylases (sirtuins), ADP-ribosyltransferases, and poly(ADP-ribose) polymerases, that control vital cell processes including gene expression, DNA repair, apoptosis, mitochondrial biogenesis, unfolded protein response, and many others. NAD+ is also a precursor for calcium-mobilizing secondary messengers. Proper regulation of these NAD-dependent metabolic and signaling pathways depends on how efficiently cells can maintain their NAD levels. Generally, mammalian cells regulate their NAD supply through biosynthesis from the precursors delivered with the diet: nicotinamide and nicotinic acid (vitamin B3), as well as nicotinamide riboside and nicotinic acid riboside. Administration of NAD precursors has been demonstrated to restore NAD levels in tissues (i.e., to produce beneficial therapeutic effects) in preclinical models of various diseases, such as neurodegenerative disorders, obesity, diabetes, and metabolic syndrome.

Sulfoxides, Analogues of L-Methionine and L-Cysteine As Pro-Drugs against Gram-Positive and Gram-Negative Bacteria.

The problem of resistance to antibiotics requires the development of new classes of broad-spectrum antimicrobial drugs. The concept of pro-drugs allows researchers to look for new approaches to obtain effective drugs with improved pharmacokinetic and pharmacodynamic properties. Thiosulfinates, formed enzymatically from amino acid sulfoxides upon crushing cells of genus Allium plants, are known as antimicrobial compounds. The instability and high reactivity of thiosulfinates complicate their use as individual antimicrobial compounds. We propose a pharmacologically complementary pair: an amino acid sulfoxide pro-drug and vitamin B6 - dependent methionine γ-lyase, which metabolizes it in the patient's body. The enzyme catalyzes the γ- and ß-elimination reactions of sulfoxides, analogues of L-methionine and L-cysteine, which leads to the formation of thiosulfinates. In the present work, we cloned the enzyme gene from Clostridium sporogenes. Ionic and tautomeric forms of the internal aldimine were determined by lognormal deconvolution of the holoenzyme spectrum and the catalytic parameters of the recombinant enzyme in the γ- and ß-elimination reactions of amino acids, and some sulfoxides of amino acids were obtained. For the first time, the possibility of usage of the enzyme for effective conversion of sulfoxides was established and the antimicrobial activity of thiosulfinates against Gram-negative and Gram-positive bacteria in situ was shown.

Kinetic Parameters and Cytotoxic Activity of Recombinant Methionine γ-Lyase from Clostridium tetani, Clostridium sporogenes, Porphyromonas gingivalis and Citrobacter freundii.

The steady-state kinetic parameters of pyridoxal 5'-phosphate-dependent recombinant methionine γ -lyase from three pathogenic bacteria, Clostridium tetani, Clostridium sporogenes, and Porphyromonas gingivalis, were determined in ß- and γ-elimination reactions. The enzyme from C. sporogenes is characterized by the highest catalytic efficiency in the γ-elimination reaction of L-methionine. It was demonstrated that the enzyme from these three sources exists as a tetramer. The N-terminal poly-histidine fragment of three recombinant enzymes influences their catalytic activity and facilitates the aggregation of monomers to yield dimeric forms under denaturing conditions. The cytotoxicity of methionine γ-lyase from C. sporogenes and C. tetani in comparison with Citrobacter freundii was evaluated using K562, PC-3, LnCap, MCF7, SKOV-3, and L5178y tumor cell lines. K562 (IC50=0.4-1.3 U/ml), PC-3 (IC50=0.1-0.4 U/ml), and MCF7 (IC50=0.04-3.2 U/ml) turned out to be the most sensitive cell lines.

Aspartic acid 214 in Citrobacter freundii tyrosine phenol-lyase ensures sufficient C--H-acidity of the external aldimine intermediate and proper orientation of the cofactor at the active site.

In the X-ray structure of tyrosine phenol-lyase (TPL) Asp214 is located at H-bonding distance from the N1 atom of the cofactor. This residue has been replaced with Ala and Asn and the properties of the mutant enzymes have been studied. The substitutions result in a decrease in the cofactor affinity of about four orders of magnitude. D214A and D214N TPLs do not catalyze the decomposition of l-Tyr and 3-fluoro-l-Tyr. They decompose substrates, containing better leaving groups with rates reduced by one or two orders of magnitude. Lognormal resolution of the spectra of the mutant enzymes revealed that the N1 atom of the cofactor is deprotonated. Spectral characteristics of internal and external aldimines of the mutant TPLs and the data on their interaction with quasisubstrates demonstrate that replacements of Asp214 lead to alteration of active site conformations. The mutant enzymes do not form noticeable amounts of a quinonoid upon interaction with inhibitors, but catalyze isotope exchange of C-alpha-proton of a number of amino acids for deuterium in (2)H(2)O. The k(ex) values for the isotope exchange of l-phenylalanine and 3-fluoro-l-tyrosine are close to the k(cat) values for reacting substrates. Thus, for the mutant TPLs the stage of C-alpha-proton abstraction may be considered as a rate-limiting for the whole reaction.

Role of arginine 226 in the mechanism of tryptophan indole-lyase from Proteus vulgaris.

In the spatial structure of tryptophanase from Proteus vulgaris the guanidinium group of arginine 226 forms a salt bridge with the 3;-oxygen atom of the coenzyme. The replacement of arginine 226 with alanine using site-directed mutagenesis reduced the affinity of the coenzyme for the protein by one order of magnitude compared to the wild-type enzyme. The catalytic activity of the mutant enzyme in the reaction with L-tryptophan was reduced 10(5)-fold compared to the wild-type enzyme. The rates of the reactions with some other substrates decreased 10(3)-10(4)-fold. The mutant enzyme catalyzed exchange of the C-alpha-proton in complexes with some inhibitors with rates reduced 10(2)-fold compared to the wild-type enzyme. Absorption and circular dichroism spectra of the mutant enzyme and the enzyme-inhibitor complexes demonstrate that the replacement of arginine 226 with alanine does not significantly affect the tautomeric equilibrium of the internal aldimine, but it leads to an alteration of the optimal conformation of the coenzyme-substrate intermediates.

Tryptophan indole-lyase from Proteus vulgaris: kinetic and spectral properties.

An efficient method for purification of recombinant tryptophanase from Proteus vulgaris was developed. Catalytic properties of the enzyme in reactions with L-tryptophan and some other substrates as well as competitive inhibition by various amino acids in the reaction with S-o-nitrophenyl-L-cysteine were studied. Absorption and circular dichroism spectra of holotryptophanase and its complexes with characteristic inhibitors modeling the structure of the principal reaction intermediates were examined. Kinetic and spectral properties of two tryptophanases which markedly differ in their primary structures are compared. It was found that although the spectral properties of the holoenzymes and their complexes with amino acid inhibitors are different, the principal kinetic properties of the enzymes from Proteus vulgaris and Escherichia coli are analogous. This indicates structural similarity of their active sites.

[The determination of total concentration and activity of antioxidants in foodstuffs]. / Opredelenie summarnoi kontsentratsii i aktivnosti antioksidantov v pishchevykh produktakh.

We developed a new method for the analysis of active antioxidants that is based on their reactions with the ABTS+. cation radical obtained by oxidation of ABTS, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt. The feasibility of this method was confirmed by electrochemical and kinetic studies of model antioxidants. ABTS+. was shown to react rapidly with active and slowly with weak antioxidants, which allows it to be used as a model radical for the quantitative determination of the total content of natural antioxidants (antioxidant equivalent) in natural extracts and wines. Another analytical method based on the competitive oxidation of Pyrogallol Red (a detecting molecule) and the examined antioxidants by radicals derived from peroxynitrite was used for measuring the relative activity of antioxidants. A combination of both methods helped measure the total concentration of antioxidants and their average specific activities (per molecule of active compound) in extracts from grape, olive, and tomato and concentrates of various popular beverages (wines, beers, and juices), as well as in the commercial concentrated food product Kréto-A, made from grape, red wine, tomato, and olive. Red wine and red grape juice were shown to be the most rich in antioxidants (up to 20 mM), with their activity being similar to that of polyphenols. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 6; see also http://www.maik.ru.

[A new class of small RNP (alpha-RNP) containing antisense RNA in K-562 cells. I. Their characteristics and changes during erythroid differentiation]. / Novyi klass malykh RNP (al'fa-RNO), soderzhashchokh antismyslovuiu RNK, v kletkakh linii K-562. I. Kharakteristika i izmeneniia pri differentsirovke po éritroidnomu puti.

A new class of small RNP (alpha-RNP) has been detected and identified in nuclei and cytoplasm of A-562 erythroid leukemia cell line; these RNPs have a characteristic spectrum of proteins containing conservative and specific components and a special RNA component, which contains a small antisense component (alpha-RNA), a homolog of short dispersed Alu repeats. alpha-RNP is highly stable, tightly associated with chromatin in the nucleus, and is found in the free state in cytoplasm. The composition of nuclear and cytoplasmic alpha-RNP differ and have a specific pattern of changes in response to dimethylsulfoxide, an agent causing differentiation.

[Interserovar antitoxic immunity to Vibrio cholerae toxins]. / Mezhserovarnyi antitoksicheskii immunitet k toksinam kholernykh vibrionov.

The immunochemical affinity of V. cholerae enterotoxins, serovars Inaba and Ogawa, has been shown in animal experiments on cross antitoxic immunity in the small intestine, the passive hemagglutination test and the toxin neutralization test. However, antitoxic interaction with both enterotoxins is characteristic only for antibodies to V. cholerae of serovar Inaba, while in animals immune to Ogawa toxin the choleragenic effect of enterotoxins produced by V. cholerae of both serovars in retained. The possible mechanisms of one-sided cross interserovar antitoxic immunity in cholera are discussed.