In-vitro antiplatelet effect of melatonin in healthy individuals and patients with type 2 diabetes mellitus.

PURPOSE: The incidence of acute myocardial infarctions (AMI) shows circadian variation typically peaking during morning hours with a decline at night. However, this variation does not occur in patients with diabetes mellitus (DM). The night's decline of AMI may be partially explained by melatonin-related platelet inhibition. Whether this effect is absent in diabetic patients is unknown. The aim was to study the effect of melatonin on in-vitro platelet aggregation in healthy individuals and patients with type 2 DM. METHODS: Platelet aggregation was measured in blood samples from healthy individuals (n = 15) and type 2 DM patients (n = 15) using multiple electrode aggregometry. Adenosine diphosphate (ADP), arachidonic acid (ASPI) and thrombin (TRAP) were used as agonists. Aggregability for each subject was tested after adding melatonin in two concentrations. RESULTS: In healthy individuals, melatonin inhibited platelet aggregation in both higher (10-5 M) and lower concentrations (10-9 M) induced by ADP, ASPI, and TRAP (p < 0.001, p = 0.002, p = 0.029, respectively). In DM patients, melatonin did not affect platelet aggregation in both concentrations induced by ADP, ASPI, and TRAP. Melatonin decreased platelet aggregation induced by ADP, ASPI, and TRAP significantly more in healthy individuals compared to patients with DM. (p = 0.005, p = 0.045 and p = 0.048, respectively). CONCLUSION: Platelet aggregation was inhibited by melatonin in healthy individuals. In-vitro antiplatelet effect of melatonin in type 2 DM patients is significantly attenuated.

Thioredoxin - structural and functional complexity.

Thioredoxins are small globular proteins that proved to be excellent model for investigating the relationship between the structure of protein and their physico-chemical and functional properties. The results from the experiments on thioredoxins offer the basic for the development of the new paradigms in the field of chemistry, biophysics and biology of proteins, with special attention to redox reaction in living cells, protein stability and design. It is a good example of broad class of sulphur-containing redox proteins.

Malate dehydrogenases--structure and function.

Malate dehydrogenases (MDH, L-malate:NAD oxidoreductase, EC 1.1.1.37), catalyze the NAD/NADH-dependent interconversion of the substrates malate and oxaloacetate. This reaction plays a key part in the malate/aspartate shuttle across the mitochondrial membrane, and in the tricarboxylic acid cycle within the mitochondrial matrix. They are homodimeric molecules in most organisms, including all eukaryots and the most bacterial species. The enzymes share a common catalytic mechanism and their kinetic properties are similar, which demonstrates a high degree of structural similarity. The three-dimensional structures and elements essential for catalysis are conserved between mitochondrial and cytoplasmic forms of MDH in eukaryotic cells even though these isoenzymes are only marginally related at the level of primary structure.

Effect of thioredoxin on the sensitivity of bacteria to chemical damage.

The ability of thioredoxin (Trx) to protect cells from chemical damage was determined by comparing the growth of a control strain of Escherichia coli JM101 and isogenic strain transformed with the plasmid pKKTS1 containing the Streptomyces aureofaciens thioredoxin gene, in the presence of the nucleoside analogs arabinosylcytosine, 5-fluorouridine, ftorafur and carcinogen beta-naftylamine. Arabinosylcytosine showed no effect on the growth of either of the two strains. 5-fluorouridine, ftorafur [1-((R,S)-tetrahydrofuran-2-yl)-5-fluorouracil] and beta-naftylamine demonstrated lower inhibitory effects on the growth of the thioredoxin overproducing strain than on the growth of the control strain. These results suggested that Trx could protect the cells from chemical damage under certain metabolic conditions.

Malate dehydrogenase: distribution, function and properties.

Malate dehydrogenase (MDH) (EC 1.1.1.37) catalyzes the conversion of oxaloacetate and malate. This reaction is important in cellular metabolism, and it is coupled with easily detectable cofactor oxidation/reduction. It is a rather ubiquitous enzyme, for which several isoforms have been identified, differing in their subcellular localization and their specificity for the cofactor NAD or NADP. The nucleotide binding characteristics can be altered by a single amino acid change. Multiple amino acid sequence alignments of MDH show that there is a low degree of primary structural similarity, apart from several positions crucial for catalysis, cofactor binding and the subunit interface. Despite the low amino acids sequence identity their 3-dimensional structures are very similar. MDH is a group of multimeric enzymes consisting of identical subunits usually organized as either dimer or tetramers with subunit molecular weights of 30-35 kDa. MDH has been isolated from different sources including archaea, eubacteria, fungi, plant and mammals.

Purification and partial characterization of thioredoxin reductase from Streptomyces aureofaciens.

Thioredoxin reductase (TrxR) is one of a number of flavoproteins that catalyze the transfer of electrons between pyridine nucleotides and a specific disulfide-containing substrate. Thioredoxin reductase from Streptomyces aureofaciens 3239 has been purified to homogeneity by a two-step chromatographic procedure including anion-exchange chromatography and affinity chromatography on 2'5'-ADP-Sepharose 4B. Molar mass determined by chromatography on Superose 12 HR 10/30 and sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed 69 kDa for the native protein and 34.8 kDa for the enzyme subunit. The isoelectric point determined by isoelectric focusing gel electrophoresis was 4.3. TrxR effectively catalyzed the reduction of DTNB in the presence of S. aureofaciens thioredoxin-1. TrxR activity in the presence of S. aureofaciens thioredoxin-2 was only 1/4 of the activity with thioredoxin-1 (1). The activity of pure TrxR decreased drastically in the presence of NADPH, while NADP+ as well as Streptomyces aureofaciens thioredoxin-1 protected the enzyme from inactivation. These results indicate that thioredoxin reductase activity in bacteria could be modulated by the redox status of NADP+/NADPH and thioredoxin pools.

Purification and characterization of the malate dehydrogenase from Streptomyces aureofaciens.

The malate dehydrogenase (MDH) from Streptomyces aureofaciens was purified to homogeneity and its physical and biochemical properties were studied. Its amino-terminal sequence perfectly matched the amino-terminal sequence of the MDH from Streptomyces atratus whose biochemical characteristics have never been determined. The molecular mass of the native enzyme, estimated by size-exclusion chromatography, was 70 kDa. The protein was a homodimer, with a 38-kDa subunit molecular mass. It showed a strong specificity for NADH and was much more efficient for the reduction of oxaloacetate than for the oxidation of malate, with a pH optimum of 8. Unlike MDHs from other sources, it was not inhibited by excess oxaloacetate. This first complete functional characterization of an MDH from Streptomyces shows that the enzyme is very similar in many respects to other bacterial MDHs with the notable exception of a lack of inhibition by excess substrate.

[Fibrillary glomerulopathy]. / Fibrilárne glomerulopatie.

The authors present an account on contemporary knowledge of the diagnosis and differential diagnosis of fibrillar glomerulopathies. The latter are characterized by extracellular localized microfibrils and microtubules resp. in the glomeruli of the kidneys, their diameter being 8-60 nm. They are divided into amyloid and non-amyloid types. The others are classified according to the immunofluorescent finding into immunoglobulin positive and negative ones. The differential diagnosis is important in particular in immunoglobulin positive ones as they are present in serious diseases such as cryoglobulinaemia, monoclonal gammapathy, systemic lupus erythematosus and immunotactoid glomerulopathy.

Purification and partial characterization of two thioredoxins from Streptomyces aureofaciens.

Thioredoxins are low molecular weight proteins, which participate in a wide spectrum of biochemical reactions. Two thioredoxins from Streptomyces aureofaciens 3239 have been purified to homogeneity by a sequence of chromatography steps including chromatography on Sephacryl S-300, Phenyl Sepharose CL 4B and MonoQ HR 5/5. Thioredoxin activity clearly separates into two protein fractions on MonoQ HR 5/5 chromatography. Molecular weights determined by chromatography on Superose 12 HR 10/30 and sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed M(r) approximately 10,500 for thioredoxin 1 (TR1) and M(r) approximately 11,000 for thioredoxin 2 (TR2). The isoelectric points of the two thioredoxins are different pI = 4.7 for TR1 and 5.6 for TR2, respectively. Both were effectively reduced with NADPH in reaction catalyzed by Streptomyces aureofaciens thioredoxin reductase. The specific activity of viewly for discovered TR2 is about 1/4 of the specific activity of TR1. Both thioredoxins activate spinach NADPH-malate dehydrogenase. Activation of this enzyme by TR2 is only half effective than by TR1. The stability of TR1 is high and similar to thioredoxins from other organisms unlike the activity of TR2 which is decreased during purification. The proteins diversed in their contents in exponentially growing mycelium.

Purification and partial characterization of Ca(2+)-dependent ribonucleotide reductase from Streptomyces aureofaciens.

Ribonucleotide reductase (EC 1. 17. 4. 1.) is an essential enzyme providing 2'-deoxy-ribonucleotides for DNA replication. Ribonucleotide reductase from Streptomyces aureofaciens was purified 3365-fold with a yield of 6.5%. After homogenization of cells by ultrasonic homogenizer and DNA removing by 7% (w/v) solution of streptomycin sulphate, the sample was chromatographed on a DEAE-Sepharose CL 6 B, Phenyl-Sepharose CL 4 B, Heparin-Sepharose CL 6 B and a Sephacryl S-200. The specific activity of the purified protein was 1740 pmol per s per mg. Sephacryl S-200 chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that in the presence of calcium ions the enzyme appears to be a dimer with an apparent molecular weight of 125.9 kDa. In the absence of calcium dimer dissociates into a monomer with the apparent molecular weight of 64.3 kDa. On the basis of these results, we suggest that calcium plays a role in the formation of the dimer, which is the biologically active form of ribonucleotide reductase.

PCR cloning and sequencing of the coding portion of the thioredoxin-encoding gene from Streptomyces aureofaciens BMK.

The nucleotide sequence of the PCR-cloned coding portion of the thioredoxin-encoding gene from Streptomyces aureofaciens BMK was determined. The deduced 106-amino-acid sequence was compared with three other thioredoxins.

Arabidopsis thaliana NAPHP thioredoxin reductase. cDNA characterization and expression of the recombinant protein in Escherichia coli.

Using a clone characterized in the course of a random sequencing programme of Arabidopsis thaliana, two cDNAs encoding plant type cytosolic NADPH-dependent thioredoxin reductase (NTR) have been isolated. Their sequence homology with Escherichia coli NRT (the only thioredoxin reductase of known primary structure) is about 45%. In addition, analysis of the sequence of the encoded polypeptide (333 amino acids) reveals that several motifs are conserved in the FAD, central and NADPH binding domains, suggesting a similar folding of the protein. Definitive proof that the clone ATTHIREDB indeed encodes NTR was obtained by expressing the recombinant protein in E. coli cells. It was observed that plant type NTR was strongly overproduced (about 10 mg homogeneous protein could be purified per liter of culture). The recombinant enzyme is homodimeric, each subunit containing an FAD prosthetic group. Recombinant plant type NTR is as effective as E. coli NTR in the DTNB (5,5'-dithiobis nitrobenzoic acid) reduction reaction, but its affinity for thioredoxin substrates was strikingly different. These results are discussed in relation to the primary structures of NADPH thioredoxin reductases.

PCR cloning of a protein-coding part of the thioredoxin gene fromStreptomyces aureofaciens.

The method of two-stage half-specific amplification was described and successfully used in the isolation of the protein-coding part of the thioredoxin gene from Streptomyces aureofaciens BMK. The efficiency of a new PCR modification for the specific amplification of the target DNA fragments (genes) with unknown sequences is compared with the used half-specific PCR. The determined target sequence demonstrates the highest homology with the thioredoxin genes from Corynebacterium nephridii C-1 and Anabaena 7119.

[Thioredoxin-reductase: structure, properties, and function]. / Tioredoksin-reduktaza: struktura, svoistva, funktsii.

Thioredoxin reductase (TR-RED) pertains to the family of pyridine nucleotide disulfide oxidoreductases distinguished by their remarkable structural homology. The enzyme is a constituent component of the thioredoxin complex which is present in all types of organisms and is universal in respect of the numerous physiological functions it performs. The ability of TR-RED to protect the skin from UV-generated free oxygen species has been found. Owing to its ability to control melanin biosynthesis, the enzyme "doses" the suntan.

Influence of the different amino acid substitutions in Escherichia coli thioredoxin on the growth of bacteriophages T7 and f1.

We have constructed three mutants in the thioredoxin (trxA) gene changing its catalytic core between Cys-32 and Cys-35. Oligonucleotide-directed mutagenesis was carried out to replace conservative Gly-33 or Pro-34 by leucine, lysine, glutamine, phenylalanine or tryptophane. The mutants were characterized using an in vivo assay based on the ability of cell (mutants in the chromosomal trxA gene) to support growth of T7 and filamentous f1 phages. The results indicate that the smaller group side-chain in the position 33 and 34 of amino acid residues are indispensable for the growth of phages.

Characteristics of NADPH-dependent thymidylate synthetase purified from Streptomyces aureofaciens.

NADPH-dependent thymidylate synthetase from Streptomyces aureofaciens has been purified to homogenity by a two-step chromatographic procedure including anion-exchange chromatography and affinity chromatography on methotrexate-Sepharose 4B. The enzyme was purified 1025-fold with a 34% yield. Basic characteristics of the enzyme were determined: molecular weight of the enzyme subunit (28,000), pH and temperature optimum, effect of cations, dependency on reducing agents, Km values for dUMP, mTHF, and NADPH (3.78, 21.1, and 38.9 microM, respectively), and inhibition effect of 5-FdUMP. Binding studies revealed the enzyme mechanism to be ordered sequential: dUMP bound before mTHF. S. aureofaciens thymidylate synthetase exhibits an absolute requirement for NADPH for the enzyme activity--a unique feature not displayed by any of the thymidylate synthetases isolated so far. NADPH is not consumed during enzyme reaction, indicating its regulatory role. The properties of S. aureofaciens thymidylate synthetase show that it is a monofunctional bacterial enzyme.

RNA polymerase heterogeneity in Streptomyces aureofaciens: characterization by antibody-linked polymerase assay.

Using antibody-linked polymerase assay we studied the polypeptide composition of DNA-dependent RNA polymerase from Streptomyces aureofaciens and immunological cross-reaction with Escherichia coli RNA polymerase. We identified about 25 'ALPA-reactive' polypeptides which are probably involved in the transcriptional apparatus. We demonstrated that beta' and beta subunits from S. aureofaciens and E. coli are immunologically related and sigma70 (E. coli) shows immunochemical similarity with sigma35 (S. aureofaciens). According to the reconstitution of RNA polymerase holoenzyme and antibody-linked polymerase assay we identified sigma factors responsible for recognition of two promoters.

[Ribonucleotide reductase--transition enzymes from RNA metabolism to DNA metabolism]. / Ribonukleotid-reduktazy--énzymy perekhoda ot metabolizma RNK k metabolizmu DNK.

Ribonucleotide reductases catalyze the reduction of ribonucleotides to deoxyribonucleotides, this reaction being vitally important for all organisms. The enzyme provides a link between RNA and DNA metabolisms. Several forms of the enzyme occur in nature and those differ in their structure and catalytic mechanisms. While the direct reduction of ribonucleotides via a radical mechanism is a general mode of dNTP synthesis in all organisms, the way in which this is achieved varies. The ability of the enzyme to control DNA synthesis is of considerable clinical interest. Selective inhibition of DNA synthesis is desired, for example, in clinical applications, such as restriction of tumour growth or virus replication.

[Thioredoxin: structure, properties, function]. / Tireodoksin: struktura, svoistva, funktsii.

Thioredoxin, a disulfide-containing protein having a highly conservative structure, is present in all types of organisms that are phylogenetically distant from one another. The protein possesses unique capabilities and is universal in respect of physiological functions it performs. In particular, this protein participates in nucleic acid metabolism and thus may protect cells against the lethal influence of gamma-irradiation.