Identification and origin of Nε-homocysteinyl-lysine isopeptide in humans and mice

Homocysteine (Hcy) metabolites, Hcy-thiolactone and N-Hcy-proteins, have been linked to the pathology of human cardiovascular and neurodegenerative diseases. Hcy-thiolactone is generated in an error-editing reaction in protein biosynthesis when Hcy is selected in place of methionine by methionyl-tRNA synthetase. N-Hcy-protein, in which Hcy is linked via isopeptide bond to ε-amino group of a protein lysine residue, forms in a post-translational reaction of Hcy-thiolactone with proteins. Here, we identify a novel metabolite, Nε-Hcy-Lys, in human and mouse plasma, and show that this metabolite is elevated in genetic (cystathionine β-synthase deficiency in humans and mice, methylenetetrahydrofolate reductase deficiency in mice) or dietary (high Met diet in mice) deficiencies in Hcy metabolism. We also show that Nε-Hcy-Lys is generated by proteolytic degradation of N-Hcy-protein in mouse liver extracts. Our data indicate that free Nε-Hcy-Lys is an important pathology-related component of Hcy metabolism in humans and mice.     

Possible Association between Suicide Committed under Influence of Ethanol and a Variant in the AUTS2 Gene

Background

rs6943555 in AUTS2 has been shown to modulate ethanol consumption. We hypothesized that rs6943555 might be associated with completed suicide.

Methods

We genotyped rs6943555 in 625 completed suicides and 3861 controls using real-time TaqMan Allelic Discrimination Assay. All individuals were Polish Caucasians.

Results

We detected an association between suicide and rs6943555 A allele (OR = 1.17, P = 0.018 for allelic comparison, OR = 1.24, P = 0.013 for dominant, and OR = 1.18, P = 0.020 for co-dominant model of inheritance). The association remained significant after adjusting for age and gender (co-dominant: P = 0.002 and dominant model: P = 0.001). After stratifying suicides according to blood ethanol concentration (BAC≤ 20 mg/dl and BAC > 20 mg/dl) the association remained significant only for cases who committed suicide under influence of alcohol (co-dominant: OR  =  1.37, P = 0.004 and dominant model: OR = 1.45, P = 0.006). To validate this finding we genotyped another cohort of 132 cases. We reproduced the association between rs6943555 A allele and suicide under influence of ethanol (allelic comparison: OR = 1.55, P = 0.023; co-dominant : OR = 1.54, P = 0.031; dominant model: OR = 1.84, P = 0.015). Analyzing pooled suicides with BAC >20 mg/dl (N = 300) we found the association of rs6943555 A allele not only vs. controls (allelic OR = 1.41, P = 0.00029) but also vs. cases with BAC ≤ 20 mg/dl (N = 449, allelic OR = 1.33, P = 0.019).

Conclusions

In our study rs6943555 A allele is associated with suicide committed after drinking ethanol shortly before death. The rs6943555 A allele may be linked to adverse emotional reaction to ethanol, which could explain the association with lower consumption in general population as well as the predisposition to suicide under influence of ethanol.     

Cloning, Expression, and Purification of the Uropathogenic Escherichia coli Invasin DraD

In this study we presented a very efficient expression system, based on pET30LIC/Ek vector, for producing DraD invasin of the uropathogenic Escherichia coli and a one-step chromatography purification procedure for obtaining pure recombinant protein (DraD-C-His₆). This protein has a molecular weight of 14,818 and calculated pI of 6.6. It contains a polyhistidine tag at the C-terminus (13 additional amino acids) that allowed single-step isolation by Ni affinity chromatography. Also, we obtained specific antibodies against DraD invasin to develop tools for characterizing the expression and biological function of this protein. The amount and quality of DraD-C-His₆ fusion protein purified from E. coli overexpression system seems to be fully appropriate for crystallographic studies (soluble form), and for establishing role of the protein in bacterium (cultured cell line interaction and in the internalization process) and for obtaining rabbit polyclonal antisera (insoluble form).     

The Relationships between Polymorphisms in Genes Encoding the Growth Factors TGF-β1, PDGFB,EGF, bFGF and VEGF-A and the Restenosis Process in Patients with Stable Coronary Artery Disease Treated with Bare Metal Stent

Background

Neointima forming after stent implantation consists of vascular smooth muscle cells (VSMCs) in 90%. Growth factors TGF-β1, PDGFB, EGF, bFGF and VEGF-A play an important role in VSMC proliferation and migration to the tunica intima after arterial wall injury. The aim of this paper was an analysis of functional polymorphisms in genes encoding TGF-β1, PDGFB, EGF, bFGF and VEGF-A in relation to in-stent restenosis (ISR).

Materials and Methods

265 patients with a stable coronary artery disease (SCAD) hospitalized in our center in the years 2007–2011 were included in the study. All patients underwent stent implantation at admission to the hospital and had another coronary angiography performed due to recurrence of the ailments or a positive result of the test assessing the coronary flow reserve. Angiographically significant ISR was defined as stenosis >50% in the stented coronary artery segment. The patients were divided into two groups–with angiographically significant ISR (n = 53) and without significant ISR (n = 212). Additionally, the assessment of late lumen loss (LLL) in vessel was performed. EGF rs4444903 polymorphism was genotyped using the PCR-RFLP method whilst rs1800470 (TGFB1), rs2285094 (PDGFB) rs308395 (bFGF) and rs699947 (VEGF-A) were determined using the TaqMan method.

Results

Angiographically significant ISR was significantly less frequently observed in the group of patients with the A/A genotype of rs1800470 polymorphism (TGFB1) versus patients with A/G and G/G genotypes. In the multivariable analysis, LLL was significantly lower in patients with the A/A genotype of rs1800470 (TGFB1) versus those with the A/G and G/G genotypes and higher in patients with the A/A genotype of the VEGF-A polymorphism versus the A/C and C/C genotypes. The C/C genotype of rs2285094 (PDGFB) was associated with greater LLL compared to C/T heterozygotes and T/T homozygotes.

Conclusions

The polymorphisms rs1800470, rs2285094 and rs6999447 of the TGFB1, PDGFB and VEGF-A genes, respectively, are associated with LLL in patients with SCAD treated by PCI with a metal stent implantation.     

Interactions of vasopressin and oxytocin receptors with vasopressin analogues substituted in position 2 with 3,3′‐diphenylalanine – a molecular docking study

Vasopressin and oxytocin receptors belong to the superfamily of G protein‐coupled receptors and play an important role in many physiological functions. They are also involved in a number of pathological conditions being important drug targets. In this work, four vasopressin analogues substituted at position 2 with 3,3′‐diphenylalanine have been docked into partially flexible vasopressin and oxytocin receptors. The bulky residue at position 2 acts as a structural restraint much stronger in the oxytocin receptor (OTR) than in the vasopressin V2 receptor (V2R), resulting in a different location of the analogues in these receptors. This explains the different, either agonistic or antagonistic, activities of the analogues in V2R and OTR, respectively. In all complexes, the conserved polar residues serve as anchor points for the ligand both in OTR and V2R. Strong interactions of the C‐terminus of analogue II ([Mpa¹,d ‐Dpa²,Val⁴,d ‐Arg⁸]VP) with extracellular loop 3 may be responsible for its highest activity at V2R. It also appears that V2R adapts more readily to the docking analogues by conformational changes in the aromatic side chains triggering receptor activation. A weak activity at V1a vasopressin receptor appears to be caused by weak receptor–ligand interactions. Results of this study may facilitate a rational design of new analogues with the highest activity/selectivity at vasopressin and OTRs. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Changes in plasma thiol levels induced by different phases of treatment in breast cancer; the role of commercial extract from black chokeberry

Different low-molecular-weight thiols, including glutathione, cysteine, and cysteinylglycine are physiological free radical scavengers. On the other hand, homocysteine may play a role as an oxidant. The aim of our present study was to establish in vitro the effects of the commercial extract of Aronia melanocarpa (Aronox^?) on the amount of selected low-molecular-weight thiols and the activity of antioxidative enzymes (superoxide dismutase, glutathione peroxidase, and glutathione reductase) in plasma obtained from patients with invasive breast cancer during different phases of treatment [before or after the surgery and patients after different phases of chemotherapy (doxorubicin and cyclophosphamide)] and from healthy subjects. Patients were hospitalized in Department of Oncological Surgery and Department of Chemotherapy, Medical University of Lodz, Poland. The level of low-molecular-weight thiols was determined by high-performance liquid chromatography. We observed that in the presence of the Aronia extract changes in amount of thiols in plasma from breast cancer patients (at all tested groups) were significantly reduced. Our results showed that tested commercial extract reduced modifications of antioxidative enzymes activity in plasma from patients during different phases of treatment, but this effect was not statistical significant. Our results suggest that the Aronia extract supplementation in breast cancer patients has a beneficial effect on thiols concentration in plasma. Plasma, as reported in this work, could be used as an experimental model to evaluate the beneficial action of plant supplements, including phenolic extracts on thiols or other molecules during different phases of treatment.     

Reorientation of cytochrome c2 upon interaction with oppositely charged macromolecules probed by SR EPR: implications for the role of dipole moment to facilitate collisions in proper configuration for electron transfer

The reaction of water-soluble cytochrome c (c(2)) with its physiological redox partners is facilitated by electrostatic attractions between the two protein surfaces. Using spin-labeled cytochrome c(2) from Rhodobacter capsulatus and pulse electron paramagnetic resonance (EPR) measurements we compared spatial orientation of cytochrome c(2) upon its binding to surfaces of opposite charge. We observed that cytochrome c(2) can use its negatively charged "back" side when exposed to interact with positively charged surfaces (DEAE resin) which is the opposite to the use of its positively charged "front" side in physiological interaction with negatively charged binding domain of cytochrome bc(1). The later orientation is also adopted upon non-physiological binding of cytochrome c(2) to negatively charged carboxymethyl cellulose resin. These results directly demonstrate how the electric dipolar nature of cytochrome c(2) influences its orientation in interactions with charged surfaces, which may facilitate collisions with other redox proteins in a proper orientation to support physiologically-competent electron transfer. Saturation recovery EPR provides an attractive tool for monitoring spatial orientation of proteins in their interaction with surfaces in liquid phase. It is particularly valuable for metalloproteins engaged in redox reactions as a means to monitor the geometry and dynamics of formation of protein complexes in measurements that are independent of electron transfer processes.     

Efficient apoptosis and necrosis induction by proteasome inhibitor: bortezomib in the DLD-1 human colon cancer cell line

The inhibition of the 26S proteasome evokes endoplasmic reticulum stress, which has been shown to be implicated in the antitumoral effects of proteasome inhibitors. The cellular and molecular effects of the proteasome inhibitor—bortezomib—on human colon cancer cells are as yet poorly characterized. Bortezomib selectively induces apoptosis in some cancer cells. However, the nature of its selectivity remains unknown. Previously, we demonstrated that, in contrast to normal fibroblasts, bortezomib treatment evoked strong effect on apoptosis of breast cancer cells incubated in hypoxic and normoxic conditions. The study presented here provides novel information on the cellular effects of bortezomib in DLD-1 colon cancer cells line. We observe twofold higher percentage of apoptotic cells incubated for 48 h with 25 and 50 nmol/l of bortezomib in hypoxic conditions and four-, fivefold increase in normoxic conditions in comparison to control cells, incubated without bortezomib. It is of interest that bortezomib evokes strong effect on necrosis of DLD-1 colon cancer cell line. We observe the sixfold increase in necrosis of DLD-1 cells incubated with 25 or 50 nmol/l of bortezomib for 48 h in hypoxia and fourfold increase in normoxic conditions in comparison to adequate controls. We suggest that bortezomib may be candidates for further evaluation as chemotherapeutic agents for human colon cancer.     

Exogenous melatonin expediently modifies proteome of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) embryo during seed germination

Melatonin (N-acetyl-5-methoxytryptamine) has a great potential for plant biostimulation. Its role in plant physiology is intensively explored, and its important function in plant stress defence frequently underlined. Melatonin is particularly effective when applied as an additional factor of seed priming. In the presented research, hydroconditioning was chosen experimentally as optimal for maize (Zea mays L.) seeds. The following seed variants were compared: controlled non-treated, hydroprimed with water and hydroprimed with melatonin solutions 50 and 500 μM. To identify modifications in proteome of maize seeds caused by the applied hydroconditioning techniques, protein extracts of germinated seed embryos (24 h, 25 °C) were separated by 2D-PAGE. Next, obtained maps of proteomes were compared (statistically and graphically) using PDQuest software, and characteristic spots of proteins were analysed qualitatively by mass-spectrometric techniques and identified in the Mascot protein databases. Research helped to identify hydropriming-associated proteins, and for the first time those which were expressed only in the presence of melatonin. Study confirmed that suitably selected pre-sowing treatment with melatonin, by embryo proteome modification, effectively prepares plants to adverse environmental conditions. In melatonin treated seeds during the initial state of embryos growth, even under optimal conditions, additional antioxidative, detoxifying, anti-stresses proteins were synthesized. Moreover, the supply of energy from seed storage substances was pretty intensified. The presented results partially explain how melatonin acts in plant stress defence, and why plants with higher melatonin content have exhibited increased capacity for stress tolerance.     

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.