Current advances in research of cytochrome c oxidase

The function of cytochrome c oxidase as a biomolecular nanomachine that transforms energy of redox reaction into protonmotive force across a biological membrane has been subject of intense research, debate, and controversy. The structure of the enzyme has been solved for several organisms; however details of its molecular mechanism of proton pumping still remain elusive. Particularly, the identity of the proton pumping site, the key element of the mechanism, is still open to dispute. The pumping mechanism has been for a long time one of the key unsolved issues of bioenergetics and biochemistry, but with the accelerating progress in this field many important details and principles have emerged. Current advances in cytochrome oxidase research are reviewed here, along with a brief discussion of the most complete proton pumping mechanism proposed to date, and a molecular basis for control of its efficiency.     

Prediction of Associations between microRNAs and Gene Expression in Glioma Biology

Despite progress in the determination of miR interactions, their regulatory role in cancer is only beginning to be unraveled. Utilizing gene expression data from 27 glioblastoma samples we found that the mere knowledge of physical interactions between specific mRNAs and miRs can be used to determine associated regulatory interactions, allowing us to identify 626 associated interactions, involving 128 miRs that putatively modulate the expression of 246 mRNAs. Experimentally determining the expression of miRs, we found an over-representation of over(under)-expressed miRs with various predicted mRNA target sequences. Such significantly associated miRs that putatively bind over-expressed genes strongly tend to have binding sites nearby the 3'UTR of the corresponding mRNAs, suggesting that the presence of the miRs near the translation stop site may be a factor in their regulatory ability. Our analysis predicted a significant association between miR-128 and the protein kinase WEE1, which we subsequently validated experimentally by showing that the over-expression of the naturally under-expressed miR-128 in glioma cells resulted in the inhibition of WEE1 in glioblastoma cells.     

Proton pumping mechanism and catalytic cycle of cytochrome c oxidase: Coulomb pump model with kinetic gating

Using electrostatic calculations, we have examined the dependence of the protonation state of cytochrome c oxidase from bovine heart on its redox state. Based on these calculations, we propose a possible scheme of redox-linked proton pumping. The scheme involves His291 - one of the ligands of the Cu(B) redox center - which plays the role of the proton loading site (PLS) of the pump. The mechanism of pumping is based on ET reaction between two hemes of the enzyme, which is coupled to a transfer of two protons. Upon ET, the first proton (fast reaction) is transferred to the PLS (His291), while subsequent transfer of the second "chemical" proton to the binuclear center (slow reaction) is accompanied by the ejection of the first (pumped) proton. Within the proposed model, we discuss the catalytic cycle of the enzyme.     

An Efficient Approach for the Development of Locus Specific Primers in Bread Wheat (Triticum aestivum L.) and Its Application to Re-Sequencing of Genes Involved in Frost Tolerance

Recent declines in costs accelerated sequencing of many species with large genomes, including hexaploid wheat (Triticum aestivum L.). Although the draft sequence of bread wheat is known, it is still one of the major challenges to developlocus specific primers suitable to be used in marker assisted selection procedures, due to the high homology of the three genomes. In this study we describe an efficient approach for the development of locus specific primers comprising four steps, i.e. (i) identification of genomic and coding sequences (CDS) of candidate genes, (ii) intron- and exon-structure reconstruction, (iii) identification of wheat A, B and D sub-genome sequences and primer development based on sequence differences between the three sub-genomes, and (iv); testing of primers for functionality, correct size and localisation. This approach was applied to single, low and high copy genes involved in frost tolerance in wheat. In summary for 27 of these genes for which sequences were derived from Triticum aestivum, Triticum monococcum and Hordeum vulgare, a set of 119 primer pairs was developed and after testing on Nulli-tetrasomic (NT) lines, a set of 65 primer pairs (54.6%), corresponding to 19 candidate genes, turned out to be specific. Out of these a set of 35 fragments was selected for validation via Sanger''s amplicon re-sequencing. All fragments, with the exception of one, could be assigned to the original reference sequence. The approach presented here showed a much higher specificity in primer development in comparison to techniques used so far in bread wheat and can be applied to other polyploid species with a known draft sequence.     

B-type natriuretic peptide predicts new-onset atrial fibrillation in patients with ST-segment elevation myocardial infarction treated by primary percutaneous coronary intervention

The predictive value of B-type natriuretic peptide (BNP) with respect to the occurrence of new-onset atrial fibrillation (AF) in patients with ST-segment elevation myocardial infarction (STEMI) treated by primary percutaneous coronary intervention (PCI) is unknown. The aim of this study was to evaluate whether BNP has a predictive value for the occurrence of new-onset AF in patients with STEMI treated by primary PCI. In 180 patients with STEMI treated by primary PCI, BNP concentrations were measured 24h after chest pain onset. The Receiver Operating Characteristic analysis was performed to identify the most useful BNP cut-off level for the prediction of AF. The patients were divided into the two groups according to calculated cut-off level: high BNP group (BNP≥720 pg/mL, n=33) and low BNP group (BNP<720 pg/mL, n=147). The incidence of AF was 5.0%, and occurred more frequently in high BNP group (7/33, 21.2%) than in low BNP group (2/147, 1.4%), (p<0.001). Patients with high BNP were older (p=0.017), had more often anterior wall infarction (p=0.015), higher Killip class on admission (p=0.038), higher peak troponin I (p=0.002), lower left ventricular ejection fraction (p=0.029) than patients with low BNP. After multivariate adjustment, BNP was an independent predictor of AF (OR 3.70, 95% CI 1.40-9.77, p=0.008). BNP independently predicts the occurrence of new-onset AF in STEMI patients treated by primary PCI.     

Effects of homocysteine and its related compounds on oxygen consumption of the rat heart tissue homogenate: the role of different gasotransmitters

Accumulating evidence indicates that microRNAs are implicated in tumor initiation and progression through negatively regulating oncogenes or tumor suppressor genes. In the present study, we report that the expression of miR-200a was significantly lower in renal cell carcinoma (RCC) specimens and RCC cell lines. Restoration of miR-200a suppressed cell growth, arrested cell cycle progression, and promoted cell apoptosis in RCC cell lines. We next used qRT-PCR array technology to identify Sirtuin 1 (SIRT1) as one of the downregulated proteins during miR-200a overexpression in 786-O cells. Following a further assay by luciferase reporter system, SIRT1 was validated as a direct target of miR-200a. Moreover, siRNA-mediated knockdown of SIRT1 could partially phenocopy the effects of miR-200a overexpression. In contrast, overexpression of truncated SIRT1 (without an endogenous 3′-UTR) could rescue the effect of miR-200a overexpression on 786-O cells, which suggested that SIRT1 3′-UTR is targeted by miR-200a specifically. These observations provide further evidence for a critical tumor-suppressive role of the miR-200a in RCC in addition to identifying a novel regulatory mechanism, which may contribute to SIRT1 upregulation in RCC.     

Effect of elicitors on xanthone accumulation and biomass production in hairy root cultures of <Emphasis Type="Italic">Gentiana dinarica</Emphasis>

Abandoned metalliferous wastes can be spontaneously colonized by specialized species or ecotypes, and therefore representatives from such populations might be exploited in phytoremediation. Thus, this study was focused on determining the conditions for culture initiation and elaborating the propagation protocol of wild calamine ecotype of Dianthus carthusianorum. The proper propagation medium proved modified MS enriched with 1.0 mg/L 2iP and 0.2 mg/L IAA. The massive majority (93%) of microplantlets were successfully transferred to ex vitro conditions. Micropropagated calamine ecotype of D. carthusianorum has proved to be useful for phytoremediation application. The obtained plants experimentally cultivated on post-flotation wastes generated during the process of zinc-lead ore enrichment were monitored, and compared with specimens of the population obtained as a result of seed sowing. Plants propagated through tissue culture grew better, developed faster and more abundantly bloomed in comparison with the generatively propagated control material. This is one of the few reports concerning the possibility of using in vitro technique for effective production of plant material ready to be used in chemically degraded area.     

Use of blood outgrowth endothelial cells as virus-producing vectors for gene delivery to tumors

Cell-based delivery of therapeutic viruses has potential advantages over systemic viral administration, including attenuated neutralization and improved viral targeting. One of the exciting new areas of investigation is the potential ability of endothelial-lineage cells to deliver genes to the areas of neovascularization. In the present study, we compared two types of endothelial-lineage cells [outgrowth endothelial cells (OECs) and culture-modified mononuclear cells (CMMCs), also known as "endothelial progenitor cells"] for their ability to be infected with adenovirus and to home to the areas of neovascularization. Both cell types were isolated from peripheral blood of healthy human donors and expanded in culture. We demonstrate that OECs are more infectable and home better to tumors expressing VEGF on systemic administration. Furthermore, we used an adenoviral/retroviral chimeric system to convert OECs to retrovirus-producing cells. When injected systemically into tumor-bearing mice, OECs retain their ability to produce retrovirus and infect surrounding tumor cells. Our data demonstrate that OECs could be efficient carriers for viral delivery to areas of tumor neovascularization.