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Aims Environmental stressors such as traffic noise represent a global threat, accounting for 1.6 million healthy life years lost annually in Western Europe. Therefore, the noise-associated health side effects must be effectively prevented or mitigated. Non-pharmacological interventions such as physical activity or a balanced healthy diet are effective due to the activation of the adenosine monophosphate-activated protein kinase (α1AMPK). Here, we investigated for the first time in a murine model of aircraft noise-induced vascular dysfunction the potential protective role of α1AMPK activated via exercise, intermittent fasting, and pharmacological treatment. Methods and results Wild-type (B6.Cg-Tg(Cdh5-cre)7Mlia/J) mice were exposed to aircraft noise [maximum sound pressure level of 85 dB(A), average sound pressure level of 72 dB(A)] for the last 4 days. The α1AMPK was stimulated by different protocols, including 5-aminoimidazole-4-carboxamide riboside application, voluntary exercise, and intermittent fasting. Four days of aircraft noise exposure produced significant endothelial dysfunction in wild-type mice aorta, mesenteric arteries, and retinal arterioles. This was associated with increased vascular oxidative stress and asymmetric dimethylarginine formation. The α1AMPK activation with all three approaches prevented endothelial dysfunction and vascular oxidative stress development, which was supported by RNA sequencing data. Endothelium-specific α1AMPK knockout markedly aggravated noise-induced vascular damage and caused a loss of mitigation effects by exercise or intermittent fasting. Conclusion Our results demonstrate that endothelial-specific α1AMPK activation by pharmacological stimulation, exercise, and intermittent fasting effectively mitigates noise-induced cardiovascular damage. Future population-based studies need to clinically prove the concept of exercise/fasting-mediated mitigation of transportation noise-associated disease.

Diabetes mellitus is characterized by tissue oxidative damage and impaired microcirculation, as well as worsened erythrocyte properties. Measurements of erythrocyte deformability together with determination of nitric oxide (NO) production and osmotic resistance were used for the characterization of erythrocyte functionality in lean (control) and obese Zucker diabetic fatty (ZDF) rats of two age categories. Obese ZDF rats correspond to prediabetic (younger) and diabetic (older) animals. As antioxidants were suggested to protect erythrocytes, we also investigated the potential effect of quercetin (20 mg/kg/day for 6 weeks). Erythrocyte deformability was determined by the filtration method and NO production using DAF-2DA fluorescence. For erythrocyte osmotic resistance, we used hemolytic assay. Erythrocyte deformability and NO production deteriorated during aging—both were lower in older ZDF rats than in younger ones. Three-way ANOVA indicates improved erythrocyte deformability after quercetin treatment in older obese ZDF rats only, as it was not modified or deteriorated in both (lean and obese) younger and older lean animals. NO production by erythrocytes increased post treatment in all experimental groups. Our study indicates the potential benefit of quercetin treatment on erythrocyte properties in condition of diabetes mellitus. In addition, our results suggest potential age-dependency of quercetin effects in diabetes that deserve additional research.

Matrix metalloproteinases (MMPs) are important in the pathogenesis of numerous diseases. The present study aimed to monitor the activation of MMP-2 and MMP-9 in spontaneously hypertensive rats (SHR) and their normotensive counterparts&mdash, Wistar-Kyoto rats (WKY). The animals were divided according to age (7, 20, and 52 weeks) and phenotype into: WKY-7, WKY-20, WKY-52, SHR-7, SHR-20 and SHR-52 groups. MMP plasma activities were determined by gelatine zymography. We monitored selected parameters of oxidative stress and antioxidant status. N-terminal pro-brain natriuretic peptide (NT-proBNP) was determined as a marker of heart function and neurohumoral activation. SHR-7 showed higher MMP-2 activity compared with WKY-7, while SHR-52 showed lower MMP-2 and MMP-9 activities compared with WKY-52. Examining age-dependent changes in MMP activities, we found a decrease in MMP-2 activity and increase in MMP-9 activity with increasing age in both phenotypes. Parameters of oxidative stress and antioxidant status as well as NT-proBNP levels were not significantly worsened due to aging in SHR. Our results suggest that hypertension is accompanied by varying MMP activation during aging. The results of our study may indicate that MMP-2 inhibition is therapeutically applicable during the development of hypertension, while in developed, stabilized and uncomplicated hypertension, systemic MMP-2 and MMP-9 inhibition may not be desirable.

Nowadays there are numerous pathogens that have created a resistance to commonly used antibiotics and drugs. Therefore research is focused on finding new therapeutic targets and on determination of their 3D structures that could help in designing new effective substances and inhibitors. Thioredoxin system not only plays a crucial role as thiol/disulfide redox controller, it is also essential for certain organisms as the only system ensuring the redox homeostasis. It is the redox-regulating function, which makes thioredoxin and thioredoxin reductase attractive for scientific research, especially in many studies of diseases caused by redox instability. Thanks to these facts, the proteins of thioredoxin system are suitable candidates for new therapeutic purposes. In this review we summarized the basic features of the thioredoxin system, we justified why the proteins of thioredoxin system are appropriate therapeutical targets and we provided overview of the possibilities of their inhibition by several types of inhibitors.

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.

The nuclease reaction of a copper complex with the macrocyclic Schiff base ligand tetrabenzo [b,f,j,n][1,3,9,13] tetraazacyclohexadecine (TAAB) at the cleavage of DNA templates has been investigated and compared with that of the copper phenanthroline complex. The labeled single-stranded GC containing 25-mer oligonucleotide, single-stranded AT 20-mer oligonucleotide and double-stranded AT 20-mer oligonucleotide with the complemented sequence in the presence of ascorbic acid and aerobic conditions were used as the substrates. The AT specificity of Cu(TAAB)2+ for both the single-stranded and double-stranded templates may be explained by the specific formation of intercalated complex and oxygen radicals that nick the DNA.

Chemical nucleases are compounds that are able to bind to the surface of nucleic acids and are involved in the generation of free radicals which cleave nucleic acids. The aim of this study was to investigate the interaction of copper(II) tetrabenzo [b,f,j,n] [1,3,9,13] tetraazacyclohexadecine (Cu(TAAB)2+) with DNA and determine the DNase-like activity of this complex. Dichroic properties, electronic absorption and electrophoretical techniques were used to study the reaction of the complex with DNA. Cu(TAAB)2+ affected the dichroic properties and u.v. absorption of calf thymus DNA under anaerobic conditions. The anaerobic melting point of DNA was not affected in the presence of Cu(TAAB)2+. The hyperchromicity of DNA at 260 nm in the presence of Cu(TAAB)2+ with both ascorbic acid and oxygen was determined and compared with the known chemical nuclease Cu(phenantroline)2. Plasmid DNA in the presence of ascorbic acid and oxygen was effectively cleaved by Cu(TAAB)2+ in a concentration dependent manner. It is concluded that the Cu(TAAB)2+ complex is a potential chemical nuclease.

Thioredoxin reductases are homodimeric flavoenzymes that catalyze the transfer of electrons from NADPH to oxidized thioredoxin substrate. Bacterial thioredoxin reductases represent a promising target for the development of new antibiotics. Recombinant thioredoxin reductase TrxB from Streptomyces coelicolor was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from cryocooled crystals to 2.4 Å resolution using a synchrotron-radiation source. The crystals belonged to the primitive monoclinic space group P2(1), with unit-cell parameters a = 82.9, b = 60.6, c = 135.4 Å, α = γ = 90.0, β = 96.5°.

Purified malate dehydrogenase (MDH) of Streptomyces aureofaciens was crystallized either in the absence or in the presence of NADH or NADPH coenzymes by hanging-drop vapour-diffusion method. An X-ray study has shown, that MDH crystals belong to space group C222(1) with unit-cell parameters a = 53.2 A, b = 104.6 A, c = 520.0 A, alpha = beta = gamma = 90( degrees ), MDH-NADH crystals to space group C2 with unit-cell parameters a = 51.5 A, b = 51.5 A, c = 256 A, alpha = beta = gamma = 90( degrees ), and MDH-NADPH crystals to space group C222(1) with unit-cell parameters a = 72, A b = 72 A, c = 520 A, alpha = beta = gamma = 90( degrees ). The crystal of native MDH diffracted to 2.1 A resolution.

The physico-chemical features of the NADPH-thioredoxin reductase (TRR) and two thioredoxins from Streptomyces aureofaciens (A14) are reported. The activity of pure S. aureofaciens thioredoxin reductase decreased drastically in the presence of NADPH or NADH while NADP(+), NAD(+), as well as S. aureofaciens thioredoxin-1 (TR1) activated the enzyme activity significantly. TR1 fully protected the enzyme from inactivation and also promoted its complete reactivation. S. aureofaciens thioredoxin-2 (TR2) did not protect thioredoxin reductase from NADPH inactivation. The results indicate that although the two thioredoxins from S. aureofaciens have similar biochemical properties, their essential oxidoreductase activities are not interchangeable.

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.

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.

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.

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.

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.

An RNA polymerase-binding 167 bp Hinfl fragment from a low-copy Streptomyces plasmid pSA 2201 has been shown to have promoter activity in vivo using a promoter-probe vector. This promoter (A1) is probably involved in expression of the genes responsible for the production of an antibiotic compound, found to be located on this plasmid. A 2600 nucleotides (nt) long transcript starting from this promoter has been identified by Northern hybridization analysis. The transcription start point has been determined using high-resolution S1 mapping and confirmed by in vitro transcription analysis with purified S. aureofaciens 2201 RNA polymerase. The A1 promoter shows no homology in the −10 and −35 consensus sequence of the typical bacterial promoters, and expression from this promoter is temporally dependent on the phase of growth having the maximum transcription activity in the stationary phase.

Deoxyribonucleotides are formed de novo by direct reduction of the corresponding ribonucleotides. The reduction of ribonucleotides is a stepwise process in which transfer of electrones from NADPH takes place first to thioredoxin and from it to ribonucleotides. The first step is catalysed by thioredoxin reductase and the second by ribonucleotide reductase.