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Interplays between nitric oxide and reactive oxygen species in cryptogein signalling.
Get PDFThe cellular messenger nitric oxide (NO) has many functions in plants. In this study, we investigated its interplays with Reactive Oxygen Species (ROS) in the defense responses triggered by the elicitin cryptogein produced by the oomycete Phytophthora cryptogea. The production of NO induced by cryptogein in tobacco cell suspensions was partly regulated through a ROS-dependent pathway involving the NADPH oxidase NtRBOHD. In turn, NO down-regulated the level of H2O2 derived from NtRBOHD activity. Both NO and ROS synthesis appeared to be under the control of two redundant isoforms of histone deacetylases of type 2 acting as negative regulators of cell death. Occurrence of an interplay between NO and ROS was further supported by the finding that cryptogein triggered a fast production of peroxynitrite (ONOO─) resulting from the coupling reaction of superoxide (O2•─) with NO. We provided evidence that ROS, but not NO, negatively regulate the intensity of activity of the protein kinase NtOSAK, a member of the SnRK2 protein kinase family. Furthermore, using a micro-array approach, we next identified fifteen genes early induced by cryptogein via NO. Interestingly, only a part of these genes was also modulated by ROS derived from NtRBOHD activity and encoded proteins showing sequence identity to ubiquitin ligases. Expression of those genes appeared to be negatively regulated by ONOO─, suggesting that ONOO─ mitigate the effects of NO and ROS in cell response to cryptogein. Finally, we provided evidence that NO required NtRBOHD activity for inducing cell death, thus confirming previous assumption that ROS channel NO through cell death pathways
Contribution of a halogen bond to binding of halogenated ligands by protein kinases (Rola wiązania halogenowego w oddziaływaniu halogenowanych ligandów z kinazami białkowymi)
Get PDFThis article presents a brief overview of recent studies on the phenomena of halogen bonding, most of which have been one for the last 15 years in the collaboration with Prof. David Shugar. All these investigations concern reliable estimation of the thermodynamic contribution of a halogen bond to protein-ligand interaction
Total and high molecular weight adiponectin levels in the rat model of post-myocardial infarction heart failure.
Get PDFAdiponectin is a protein secreted primarily by adipose tissue. It has been suggested that adiponectin plays a protective role in the early phase following myocardial infarction. Our primary aim was to investigate the effects of post-myocardial infarction heart failure well-characterized by left ventricular hemodynamic parameters on the total and high molecular weight adiponectin concentrations in plasma, fat and cardiac tissue. Eight weeks after myocardial infarction or sham operation, total and high molecular weight adiponectin concentrations in plasma, fat, and cardiac tissues were assayed in rats. In addition, hemodynamic parameters and expression of the genes encoding atrial natriuretic peptide and brain natriuretic peptide in left ventricle were evaluated. Atrial natriuretic peptide and brain natriuretic peptide mRNA levels in left ventricle tissue were higher in rats with myocardial infarction-induced heart failure compared with the controls. Similarly, total adiponectin concentration was increased in left ventricle (but not in right ventricle) in rats with postmyocardial infarction heart failure. In contrast, adiponectin levels in plasma and cardiac adipose tissue in rats with postmyocardial infarction heart failure were lower than in sham-operated animals. Furthermore, there were no significant differences in levels of high molecular weight adiponectin in plasma, cardiac tissue or adipose tissue between these two groups. We conclude that in the rat model of post-myocardial infarction heart failure, adiponectin level is increased in left ventricle tissue. This is accompanied by decreased adiponectin levels in plasma and cardiac adipose tissue
Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex
Get PDFThe minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical 'wings' of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain
Keratinous waste decomposition and peptide production by keratinase from Geobacillus stearothermophilus AD-11.
Get PDFA keratinolytic proteinase was cloned from thermophilic bacterium Geobacillus stearothermophilus AD-11 and was expressed in Escherichia coli BL21(DE3). Recombinant keratinolytic proteinase (RecGEOker) with an estimated molecular weight of 57 kDa was purified and keratinase activity was measured. RecGEOker showed optimal activity at pH 9 and 60 °C. Recombinant keratinolytic proteinase showed the highest substrate specificity toward keratin from wool > collagen > sodium caseinate > gelatin > and BSA in descending order. RecGEOker is applicable for efficient keratin waste biodegradation and can replace conventional non-biological hydrolysis processes. High-value small peptides obtained from enzymatic biodegradation by RecGEOker are suitable for industrial application in white and/or green biotechnology for use as major additives in various products
Coordination properties of dithiobutylamine (DTBA), a newly introduced protein disulfide reducing agent
No full textThe acid-base properties and metal-binding abilities of (2S)-2-amino-1,4-dimercaptobutane, otherwise termed dithiobutylamine (DTBA), which is a newly introduced reagent useful for reducing protein and peptide disulfides, were studied in solution using potentiometry, (1)H NMR spectroscopy, spectropolarimetry, and UV-vis spectroscopy. The list of metal ions studied here includes Zn(II), Cd(II), Ni(II), Co(II), and Cu(I). We found that DTBA forms specific and very stable polynuclear and mononuclear complexes with all of these metal ions using both of its sulfur donors. DTBA forms complexes more stable than those of the commonly used disulfide reducing agent DTT, giving it more interference capacity in studies of metal binding in thiol-containing biomolecules. The ability of DTBA to strongly bind metal ions is reflected in its limited properties as a thiol protectant in their presence, which is manifested through slower disulfide reduction kinetics. We found that this effect correlated with the stabilities of the complexes. Additionally, the reducing properties of DTBA toward MMTS-modified papain (MMTS = S-methylmethanethiosulfonate) were also significantly affected by the investigated metal ions. In this case, however, electrostatic interactions and stereospecific effects, rather than metal-binding abilities, were found to be responsible for the reduced protective properties of DTBA. Despite its limitations, a high affinity toward metal ions makes DTBA an attractive agent in competition studies with metalloproteins
Multiple myeloma-associated hDIS3 mutations cause perturbations in cellular RNA metabolism and suggest hDIS3 PIN domain as a potential drug target
Get PDFhDIS3 is a mainly nuclear, catalytic subunit of the human exosome complex, containing exonucleolytic (RNB) and endonucleolytic (PIN) active domains. Mutations in hDIS3 have been found in ∼10% of patients with multiple myeloma (MM). Here, we show that these mutations interfere with hDIS3 exonucleolytic activity. Yeast harboring corresponding mutations in DIS3 show growth inhibition and changes in nuclear RNA metabolism typical for exosome dysfunction. Construction of a conditional DIS3 knockout in the chicken DT40 cell line revealed that DIS3 is essential for cell survival, indicating that its function cannot be replaced by other exosome-associated nucleases: hDIS3L and hRRP6. Moreover, HEK293-derived cells, in which depletion of endogenous wild-type hDIS3 was complemented with exogenously expressed MM hDIS3 mutants, proliferate at a slower rate and exhibit aberrant RNA metabolism. Importantly, MM mutations are synthetically lethal with the hDIS3 PIN domain catalytic mutation both in yeast and human cells. Since mutations in PIN domain alone have little effect on cell physiology, our results predict the hDIS3 PIN domain as a potential drug target for MM patients with hDIS3 mutations. It is an interesting example of intramolecular synthetic lethality with putative therapeutic potential in humans
In Silico Identification of Plant miRNAs in Mammalian Breast Milk Exosomes – A Small Step Forward?
Get PDFMicroRNAs (miRNAs) are a class of small RNA molecules that regulate gene expression by inhibiting the protein translation or targeting the mRNA cleavage. They play many important roles in living organism cells; however, the knowledge on miRNAs functions has become more extensive upon their identification in biological fluids and recent reports on plant-origin miRNAs abundance in human plasma and serum. Considering these findings, we performed a rigorous bioinformatics analysis of publicly available, raw data from high-throughput sequencing studies on miRNAs composition in human and
porcine breast milk exosomes to identify the fraction of food-derived miRNAs. Several processing and filtering steps were
applied to increase the accuracy, and to avoid false positives. Through aforementioned analysis, 35 and 17 miRNA species,
belonging to 25 and 11 MIR families, were identified, respectively. In the human samples the highest abundance levels
yielded the ath-miR166a, pab-miR951, ptc-miR472a and bdi-miR168, while in the porcine breast milk exosomes, the zma-
miR168a, zma-miR156a and ath-miR166a have been identified in the largest amounts. The consensus prediction and
annotation of potential human targets for select plant miRNAs suggest that the aforementioned molecules may interact
with mRNAs coding several transcription factors, protein receptors, transporters and immune-related proteins, thus
potentially influencing human organism. Taken together, the presented analysis shows proof of abundant plant miRNAs in
mammal breast milk exosomes, pointing at the same time to the new possibilities arising from this discovery
Effects of various squalene epoxides on coenzyme Q and cholesterol synthesis
Get PDF2,3-Oxidosqualene is an intermediate in cholesterol biosynthesis and 2,3:22,23-dioxidosqualene act as the substrate for an alternative pathway that produces 24(S),25-epoxycholesterol which effects cholesterol homeostasis.In light of our previous findings concerning the biological effects of certain epoxidated all-transpolyisoprenes,the effects of squalene carrying epoxy moieties on the second and third isoprene residues were investigated here. In cultures ofHepG2 cells bothmonoepoxides of squalene and one of their hydrolytic products inhibited cholesterol synthesis and stimulated the synthesis of coenzyme Q (CoQ). Upon prolonged treatment the cholesterol content of these cells and its labeling with [3H]mevalonate were reduced, while the amount and labeling of CoQ increased. Injection of the squalene monoepoxides into mice once daily for 6 days elevated the level of CoQ in their blood, but did not change the cholesterol level. The same effects were observed upon treatment of apoE-deficient mice and diabetic GK-rats. This treatment increased the hepatic level of CoQ10 in mice, but the amount of CoQ9, which is the major form, was unaffected. The presence of the active compounds
in the blood was supported by the finding that cholesterol synthesis in the white blood cells was inhibited. Since the ratio of CoQ9/CoQ10 varies depending on the experimental conditions, the cells were titrated with substrate and inhibitors, leading to the conclusion that the intracellular isopentenyl-PP pool is a regulator of
this ratio. Our present findings indicate that oxidosqualenes may be useful for stimulating both the synthesis and level of CoQ both in vitro and in vivo
Electrochemical immunosensor for detection of antibodies against influenza A virus H5N1 in hen serum.
Get PDFThis paper describes the development of an immunosensor for detection of anti-hemagglutinin antibodies. Its preparation consists of successive modification steps of glassy carbon electrodes: (i) creation of COOH groups, (ii) covalent immobilization of protein A with EDC/NHS coupling reaction, (iii) covering with anti-His IgG monoclonal antibody, (iv) immobilization of the recombinant His-tagged hemagglutinin (His6-H5 HA), (v) filling free space with BSA. The interactions between two variants of recombinant HA (short and long) from highly pathogenic avian influenza virus H5N1 and the anti-H5 HA monoclonal antibody (Mab 6-9-1) have been explored with electrochemical impedance spectroscopy (EIS). The impedimetric immunosensor displayed a very good detection limit (LOD) of 2.1 pg/mL, the quantification limit (LOQ) of 6.3 pg/mL and a dynamic range from 4 pg/mL to 20 pg/mL. In addition, this analytical device was applied for detection of antibodies against His6-H5 HA in serum of vaccinated hen using serial 10-fold dilutions of serum. The immunosensor proposed was able to detect antibody in hen serum diluted up to 7 × 10(7)-fold. The sensitivity of immunosensor was about four orders of magnitude much better than ELISA