A novel method for cloning of coding sequences of highly toxic proteins

Background

During standard gene cloning, the recombinant protein appearing in bacteria as the result of expression leakage very often inhibits cell proliferation leading to blocking of the cloning procedure. Although different approaches can reduce transgene basal expression, the recombinant proteins, which even in trace amounts inhibit bacterial growth, can completely prevent the cloning process.

Soft songs in male ortolan buntings are used in an aggressive context but are not an aggressive signal

Many bird species produce low‐amplitude acoustic signals that have been poorly studied in comparison with loud, broadcast songs used for mate attraction and repelling rivals. In some birds, these soft signals were found to be emitted in an antagonistic context and were the most reliable predictor of a subsequent physical attack. The function of this signal is poorly understood, and several hypotheses have been proposed to explain the mechanisms and possible functions of such low‐amplitude signals. The subject of this study is the ortolan bunting, a small passerine species that produces soft songs during territorial defence. In this study, we aim to study whether the soft songs of the ortolan bunting are a signal of increased aggressiveness by testing if they meet the context, prediction and response criteria of aggressive signals. We simulated stranger male intrusion into a focal male territory with three different playback experiments. We found no significant differences in the male responses to the taxidermic model regardless of whether they were or were not producing soft songs in response. The males responded more strongly to loud songs than to soft songs during the simulated intrusions, and the males did not treat soft songs as a predictor of conflict escalation. Although soft songs clearly appeared during territorial encounters and were not present during spontaneous singing before the intrusions, our results did not support the hypothesis that soft songs indicate aggressive character. We suggest that soft songs in the ortolan bunting are intentionally used by birds to modify their intentions or target‐specific individuals within a close range.     

Interaction of the antitumor antibiotic mitomycin C with Z-DNA

Mitomycin C (MC), an antitumor antibiotic, alkylated Z-DNAs such as poly(dG-dC)/Co(NH3)3+(6), poly(dG-m5dC)/Mg2+ and brominated poly(dG-dC) upon reductive activation. Computer-generated energy-minimized molecular models indicated that monofunctional alkylation of Z-DNA at the N2-position of guanine by MC did not distort Z-DNA geometry, but bifunctional alkylation, leading to interstrand crosslinks between two N2-positions of guanine was sterically unfavorable. The above three Z-DNA's were exposed both to monofunctionally and bifunctionally activated MC in separate experiments and the resulting covalent MC-polynucleotide complexes were examined for conformation and for covalent MC-adducts, by circular dichroism (CD) spectroscopy and HPLC analysis of nuclease digests, respectively. Monofunctionally activated MC alkylated all three polynucleotides in their Z-forms, resulting in the same monofunctional N2-guanine adduct as that known to be formed with B-DNA. Upon bifunctional activation of MC, poly(dG-dC/Co(NH3)3+(6) reverted to the B-form and bifunctional (cross-link) adducts were detected, identical again with those formed with B-DNA. Poly(dG-m5dC), however, remained in the Z-form after the alkylation and only a monofunctional adduct could be detected. It was concluded that Z-DNA is subject to monofunctional alkylation by MC but cannot be cross-linked. The latter process occurs only when the Z-DNA is labile enough [as is in the case of poly(dG-dC)] to have some B-form in equilibrium at the site of the first formed monolinked adduct; the cross-linking then occurs at such local B-sites, pulling the overall B in equilibrium Z equilibrium irreversibly to the left. These results are in accord with the predictions from the above modeling. The irreversible "lock" by the MC cross-link on B-DNA may be exploited for probing Z-DNA intermediacy in various DNA functions.     

The Discovery of a Reciprocal Relationship between Tyrosine-Kinase Signaling and Cullin Neddylation

While neddylation is known to activate cullin (CUL)-RING ubiquitin ligases (CRLs), its role in regulating T cell signaling is poorly understood. Using the investigational NEDD8 activating enzyme (NAE) inhibitor, MLN4924, we found that neddylation negatively regulates T cell receptor (TCR) signaling, as its inhibition increases IL-2 production, T cell proliferation and Treg development in vitro. We also discovered that loss of CUL neddylation occurs upon TCR signaling, and CRLs negatively regulate IL-2 production. Additionally, we found that tyrosine kinase signaling leads to CUL deneddylation in multiple cell types. These studies indicate that CUL neddylation is a global regulatory mechanism for tyrosine kinase signaling.     

Novel 5-HT7R antagonists,arylsulfonamide derivatives of (aryloxy)propyl piperidines: Add-on effect to the antidepressant activity of SSRI and DRI,and pro-cognitive profile

A novel series of arylsulfonamide derivatives of (aryloxy)propyl piperidines was designed to obtain potent 5-HT₇R antagonists. Among the compounds evaluated herein, 3-chloro-N-{1-[3-(1,1-biphenyl-2-yloxy)2-hydroxypropyl]piperidin-4-yl}benzenesulfonamide (25) exhibited antagonistic properties at 5-HT₇R and showed selectivity over selected serotoninergic and dopaminergic receptors, as well as over serotonin, noradrenaline and dopamine transporters. Compound 25 demonstrated significant antidepressant-like activity in the forced swim test (0.625–2.5 mg/kg, i.p.) and in the tail suspension test (1.25 mg/kg, i.p.), augmented the antidepressant effect of inactive doses of escitalopram (selective serotonin reuptake inhibitor) and bupropion (dopamine reuptake inhibitor) in the FST in mice, and similarly to SB-269970, exerted pro-cognitive properties in the novel object recognition task in cognitively unimpaired conditions in rats (0.3 mg/kg, i.p.). Such an extended pharmacological profile, especially the augmentation effect of the identified 5-HT₇R antagonist on SSRI activity, seems promising regarding the complexity of affective disorders and potentially improved outcomes, including mnemonic performance.     

The effects of percutaneous transluminal coronary intervention on biomarkers of oxidative stress in the erythrocytes of elderly male patients

Objectives: Oxidative stress plays a key role in the pathogenesis of coronary artery disease. The aim of this study was to compare the effects of percutaneous transluminal coronary angioplasty (PTCA) and elective coronary angiography (EC) on erythrocytic antioxidant defense in elderly male patients.

Methods: Twenty-three stable angina pectoris (SAP) patients undergoing PTCA and 18 patients with ischemic symptoms scheduled to undergo diagnostic EC were included in the study. The concentrations of malondialdehyde (MDA) and reduced glutathione (GSH) and the activities of Zn,Cu-superoxide dismutase (SOD-1), catalase (CAT), and cytosolic glutathione peroxidase (GSH-Px) were examined in the erythrocytes before, immediately after and 2 weeks following PTCA or EC.

Results: The MDA concentrations were significantly higher and SOD-1, CAT, and GSH-Px activities were significantly lower in the PTCA group than in the EC group at baseline. Two weeks after treatment, the activities of the enzymes significantly increased in both groups, whereas the MDA concentrations decreased only in the PTCA patients.

Conclusions: The results confirm that an advanced state of atherosclerosis is related to greater levels of oxidative stress. The study indicates that both procedures may induce antioxidant defenses; however, PTCA exclusively induces a long-term reduction in lipid peroxidation.     

Cu(II)-Binding N-Terminal Sequences of Human Proteins

Proteins anchor copper(II) ions mainly by imidazole from histidine residues located in different positions in the primary protein structures. However, the motifs with histidine in the first three N-terminal positions (His¹, His², and His³) show unique Cu(II)-binding properties, such as availability from the surface of the protein, high flexibility, and high Cu(II) exchangeability with other ligands. It makes such sequences beneficial for the fast exchange of Cu(II) between ligands. Furthermore, sequences with His¹ and His², thus, non-saturating the Cu(II) coordination sphere, are redox-active and may play a role in Cu(II) reduction to Cu(I). All human protein sequences deposited in UniProt Knowledgebase were browsed for those containing His¹, His², or His³. Proteolytically modified sequences (with the removal of a propeptide or Met residue) were taken for the analysis. Finally, the sequences were sorted out according to the subcellular localization of the proteins to match the respective sequences with the probability of interaction with divalent copper.     

Zinc and Iron Concentration and SOD Activity in Human Semen and Seminal Plasma

The aim of the present study was to measure zinc (Zn) and iron (Fe) concentration in human semen and superoxide dismutase (SOD) activity in seminal plasma and correlate the results with sperm quality. Semen samples were obtained from men (N = 168) undergoing routine infertility evaluation. The study design included two groups based on the ejaculate parameters. Group I (n = 39) consisted of males with normal ejaculate (normozoospermia), and group II (n = 129) consisted of males with pathological spermiogram. Seminal Zn and Fe were measured in 162 samples (group I, n = 38; group II, n = 124) and SOD activity in 149 samples (group I, n = 37; group II, n = 112). Correlations were found between SOD activity and Fe and Zn concentration, and between Fe and Zn concentration. SOD activity was negatively associated with volume of semen and positively associated with rapid progressive motility, nonprogressive motility, and concentration. Negative correlation was stated between Fe concentration and normal morphology. Mean SOD activity in seminal plasma of semen from men of group I was higher than in seminal plasma of semen from men of group II. Fe concentration was higher in teratozoospermic males than in males with normal morphology of spermatozoa in group II. Our results suggest that Fe may influence spermatozoa morphology.     

Identification of the chromosome complement and the spontaneous 1R/1V translocations in allotetraploid <Emphasis Type="Italic">Secale cereale</Emphasis> × <Emphasis Type="Italic">Dasypyrum villosum</Emphasis> hybrids through cytogenetic approaches

Genome modifications that occur at the initial interspecific hybridization event are dynamic and can be consolidated during the process of stabilization in successive generations of allopolyploids. This study identifies the number and chromosomal location of ribosomal DNA (rDNA) sites between Secale cereale, Dasypyrum villosum, and their allotetraploid S. cereale × D. villosum hybrids. For the first time, we show the advantages of FISH to reveal chromosome rearrangements in the tetraploid Secale × Dasypyrum hybrids. Based on the specific hybridization patterns of ribosomal 5S, 35S DNA and rye species-specific pSc200 DNA probes, a set of genotypes with numerous Secale/Dasypyrum translocations of 1R/1V chromosomes were identified in successive generations of allotetraploid S. cereale × D. villosum hybrids. In addition we analyse rye chromosome pairs using FISH with chromosome-specific DNA sequences on S. cereale × D. villosum hybrids.