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The RNase H-like superfamily: new members, comparative structural analysis and evolutionary classification.
Get PDFRibonuclease H-like (RNHL) superfamily, also called the retroviral integrase superfamily, groups together numerous enzymes involved in nucleic acid metabolism and implicated in many biological processes, including replication, homologous recombination, DNA repair, transposition and RNA interference. The RNHL superfamily proteins show extensive divergence of sequences and structures. We conducted database searches to identify members of the RNHL superfamily (including those previously unknown), yielding >60 000 unique domain sequences. Our analysis led to the identification of new RNHL superfamily members, such as RRXRR (PF14239), DUF460 (PF04312, COG2433), DUF3010 (PF11215), DUF429 (PF04250 and COG2410, COG4328, COG4923), DUF1092 (PF06485), COG5558, OrfB_IS605 (PF01385, COG0675) and Peptidase_A17 (PF05380). Based on the clustering analysis we grouped all identified RNHL domain sequences into 152 families. Phylogenetic studies revealed relationships between these families, and suggested a possible history of the evolution of RNHL fold and its active site. Our results revealed clear division of the RNHL superfamily into exonucleases and endonucleases. Structural analyses of features characteristic for particular groups revealed a correlation between the orientation of the C-terminal helix with the exonuclease/endonuclease function and the architecture of the active site. Our analysis provides a comprehensive picture of sequence-structure-function relationships in the RNHL superfamily that may guide functional studies of the previously uncharacterized protein families
Influenza prevention and treatment by passive immunization.
Get PDFPassive immunity is defined as a particular antigen resistance provided by external antibodies. It can be either naturally or artificially acquired. Natural passive immunization occurs during pregnancy and breast-feeding in mammals and during hatching in birds. Maternal antibodies are passed through the placenta and milk in mammals and through the egg yolk in birds. Artificial passive immunity is acquired by injection of either serum from immunized (or infected) individuals or antibody preparations. Many independent research groups worked on selection, verification and detailed characterization of polyclonal and monoclonal antibodies against the influenza virus. Numerous antibody preparations were tested in a variety of in vitro and in vivo experiments for their efficacy to neutralize the virus. Here, we describe types of antibodies tested in such experiments and their viral targets, review approaches resulting in identification of broadly neutralizing antibodies and discuss methods used to demonstrate their protective effects. Finally, we shortly discuss the phenomenon of maternal antibody transfer as a way of effective care for young individuals and as an interfering factor in early vaccination
Pentetic acid (DPTA) Cu(II) monolayer deposited on gold electrode—The base of biosensors for electrochemical screening of kinase JAK2 and potential inhibitor interactions
Get PDFHere, the new biosensor destined for screening of interactions between kinase JAK2 and compounds which may act as inhibitors was presented. The Cu(II) complex of pentetic acid thiol ligand was applied for immobilization of kinase JAK2 on the gold electrode surface through his-tagged chemistry. The base of the biosensor response was the change of the electrochemical properties of the Cu(II) redox centres upon formation of the kinase JAK2–potential inhibitor complex. The increasing inhibitor concentration caused the decrease of reduction/oxidation Cu(II) current observed with Osteryoung square wave voltammetry. The biosensor usability was checked using known inhibitors, berberines–isoquinolone alkaloids, as well as caffeic acid—a control compound which has no affinity to kinase JAK2. The possible parameters suitable for estimation of the strength of the interactions between kinase JAK2 covalently attached to the Cu(II) complex of pentetic acid thiol ligand deposited on the gold electrode surface and potential inhibitors present in the solution were presented
A Protein Data Bank Survey Reveals Shortening of Intermolecular Hydrogen Bonds in Ligand-Protein Complexes When a Halogenated Ligand Is an H-Bond Donor.
Get PDFHalogen bonding in ligand-protein complexes is currently widely exploited, e.g. in drug design or supramolecular chemistry. But little attention has been directed to other effects that may result from replacement of a hydrogen by a strongly electronegative halogen. Analysis of almost 30000 hydrogen bonds between protein and ligand demonstrates that the length of a hydrogen bond depends on the type of donor-acceptor pair. Interestingly, lengths of hydrogen bonds between a protein and a halogenated ligand are visibly shorter than those estimated for the same family of proteins in complexes with non-halogenated ligands. Taking into account the effect of halogenation on hydrogen bonding is thus important when evaluating structural and/or energetic parameters of ligand-protein complexes. All these observations are consistent with the concept that halogenation increases the acidity of the proximal amino/imino/hydroxyl groups and thus makes them better, i.e. stronger, H-bond donors
Hypophosphatemia and sudden infant death syndrome (SIDS)--is ATP the link?
No full textIn their recent letter Van Kempen and co-workers presented an intriguing hypothesis linking hypophosphatemia and sudden infant death syndrome (SIDS) (1), founded on the work by Siren and Siren on the critical diaphragm failure being the cause of SIDS incidences (2,3). Van Kempen and co-workers argued that both etiology and symptoms of SIDS can be explained by low concentrations in erythrocytes of phosphate metabolites, mainly 2,3-biphosphoglycerate (2,3-BPG, also known as 2,3-diphosphoglycerate, 2,3-DPG), which in effect would impair muscle function (especially diaphragm) and lead to respiratory failure as Siren and Siren originally stated. We find the hypothesis interesting and definitely worth further investigation; however, we would assign the main role to ATP, not to 2,3-BPG
Dual catalytic role of the metal ion in nickel-assisted peptide bond hydrolysis
Get PDFIn our previous research we demonstrated the sequence specific peptide bond hydrolysis of the R1-(Ser/Thr)-
Xaa-His-Zaa-R2 in the presence of Ni(II) ions. The molecular mechanism of this reaction includes an N–O acyl
shift of the R1 group from the Ser/Thr amine to the side chain hydroxyl group of this amino acid. The proposed
role of the Ni(II) ion is to establish favorable geometry of the reacting groups. In this work we aimed to find
out whether the crucial step of this reaction – the formation of the intermediate ester – is reversible. For this purpose we synthesized the test peptide Ac-QAASSHEQA-am, isolated and purified its intermediate ester under acidic conditions, and reacted it, alone, or in the presence of Ni(II) or Cu(II) ions at pH 8.2. We found
that in the absence of either metal ion the ester was quickly and quantitatively (irreversibly) rearranged to the
original peptide. Such reaction was prevented by either metal ion. Using Cu(II) ions as CD spectroscopic probe
we showed that themetal binding structures of the ester and the final amine are practically identical. Molecular
calculations of Ni(II) complexes indicated the presence of steric strain in the substrate, distorting the complex
structure from planarity, and the absence of steric strain in the reaction products. These results demonstrated
the dual catalytic role of the Ni(II) ion in this mechanism. Ni(II) facilitates the acyl shift by setting the peptide geometry, and prevents the reversal of the acyl shift, by stabilizing subsequent reaction products
Influence of synbiotics delivered in ovo on immune organs development and structure.
Get PDFPrebiotics and probiotics applied alone or together (synbiotics) can influence the intestinal microbiota and modulate the immune response. We analyzed the impact of in ovo administration of synbiotics on immune system development in Ross (broiler) and Green-legged Partridgelike (GP, dual-purpose fowl) chickens. For in ovo delivery on the 12th day of the eggs incubation, two strains of lactic acid bacteria (LAB) were used, i.e. Lactococcus lactis subsp. lactis IBB SL1 (S1) and Lactococcus lactis subsp. cremoris IBB SC1 (S2), combined with raffinose family oligosaccharides (RFO) prebiotic. Other treatments included in ovo delivery of commercial synbiotic (S3), RFO prebiotics alone (P) and physiological saline (C). Immune system development was analyzed by relative weight (indices) and histology of the lymphatic organs (bursa of Fabricius, thymus and spleen) at two time points (3rd and 6th week of life). The results indicate that the development of the lymphatic organs was significantly affected by in ovo treatment. The bursa and bursa to spleen index was higher in P and S2 groups of broilers (P < 0.05) when compared to S3. In GP at the 3rd week of age, the spleen index was significantly higher in S2 (P < 0.05). The histological image of the thymus displayed an increase of thymocytes in the cortex in all synbiotic-treated groups (S1, S2, S3). In ovo delivery of synbiotics is an efficient mode of immune system stimulation in chickens but its efficiency depends on chicken genotype
Probabilistic approach to predicting substrate specificity of methyltransferases.
Get PDFWe present a general probabilistic framework for predicting the substrate specificity of enzymes. We designed this approach to be easily applicable to different organisms and enzymes. Therefore, our predictive models do not rely on species-specific properties and use mostly sequence-derived data. Maximum Likelihood optimization is used to fine-tune model parameters and the Akaike Information Criterion is employed to overcome the issue of correlated variables. As a proof-of-principle, we apply our approach to predicting general substrate specificity of yeast methyltransferases (MTases). As input, we use several physico-chemical and biological properties of MTases: structural fold, isoelectric point, expression pattern and cellular localization. Our method accurately predicts whether a yeast MTase methylates a protein, RNA or another molecule. Among our experimentally tested predictions, 89% were confirmed, including the surprising prediction that YOR021C is the first known MTase with a SPOUT fold that methylates a substrate other than RNA (protein). Our approach not only allows for highly accurate prediction of functional specificity of MTases, but also provides insight into general rules governing MTase substrate specificity
An immunosensor based on antibody binding fragments attached to gold nanoparticles for the detection of peptides derived from avian influenza hemagglutinin h5.
Get PDFThis paper concerns the development of an immunosensor for detection of peptides derived from avian influenza hemagglutinin H5. Its preparation consists of successive gold electrode modification steps: (i) modification with 1,6-hexanedithiol and gold colloidal nanoparticles; (ii) immobilization of antibody-binding fragments (Fab') of anti-hemagglutinin H5 monoclonal antibodies Mab 6-9-1 via S-Au covalent bonds; and (iii) covering the remaining free space on the electrode surfaces with bovine serum albumin. The interactions between Fab' fragments and hemagglutinin (HA) variants have been explored with electrochemical impedance spectroscopy (EIS) in the presence of [Fe(CN)6]3-/4- as an electroactive marker. The immunosensor was able to recognize three different His-tagged variants of recombinant hemagglutinin from H5N1 viruses: H1 subunit (17-340 residues) of A/swan/Poland/305-135V08/2006, the long HA (17-530 residues) A/Bar-headed Goose/Qinghai/12/2005 and H1 subunit (1-345 residues) of A/Vietnam/1194/2004. The strongest response has been observed for the long variant with detection limit of 2.2 pg/mL and dynamic range from 4.0 to 20.0 pg/mL
TMAO, a carnitine-derived metabolite, prolongs the hypertensive effect of Ang II in rats
Get PDFBackground
Recent evidence suggests that an elevated plasma trimethylamine N-oxide (TMAO) level is associated with an increased risk of adverse cardiovascular events in humans; however, the mechanism is not clear. The aims of this study were to establish plasma TMAO level in rats as well as to evaluate the effect of TMAO on arterial blood pressure (BP) and hemodynamic effects of angiotensin II (Ang II).
Methods
12-week-old, Sprague-Dawley rats were implanted with telemetry transmitters and continuous recordings of heart rate, systolic (SBP) and diastolic (DBP) arterial blood pressures were made for 7 days before and 14 days during osmotic minipump-driven subcutaneous infusion of either: saline (controls), TMAO, low-dose Ang II or Ang II+TMAO. Plasma TMAO concentration was evaluated using liquid chromatography coupled with triple-quadrupole mass spectrometry.
Results
Plasma TMAO concentration in controls was 0.57 μmol/L, while in TMAO infused rats it was 58 μmol/L. Neither saline nor TMAO infusion affected SBP and DBP. Infusion of Ang II significantly increased SBP and DBP for the first 5 days of infusion only. In contrast, infusion of Ang II+TMAO produced hypertensive response which lasted until the end of the experiment. TMAO infusions did not affect body weight and motor activity.
Conclusions
We showed that physiological plasma TMAO concentration in rats was approximately ten times lower than that reported in humans. Furthermore, the new finding of the study is that TMAO does not affect BP in normotensive animals. However, it prolongs the hypertensive effect of Ang II