Structure-functional organization of eukaryotic high-affinity copper importer CTR1 determines its ability to transport copper,silver, and cisplatin

It was shown recently, that high affinity Cu(I) importer eukaryotic protein CTR1 can also transport in vitro abiogenic Ag(I) ions and anticancer drug cisplatin. At present, there is no rational explanation how CTR1 can transfer platinum group which is different by coordination properties from highly similar Cu(I) and Ag(I). To understand the phenomenon, we analyzed 25 sequences of chordate CTR1 proteins and found out the conserved patterns of organization of N-terminal extracellular part of CTR1 which is responsible for initial metal binding. Extracellular copper-binding motifs were qualified by their coordination properties. It was shown that the relative position of methionine- and histidine-rich copper-binding motifs predisposes the extracellular CTR1 region to binding of copper, silver, and cisplatin. Relation between the tissuespecific expression of the CTR1 gene, steady-state copper concentration, and silver and platinum accumulation in organs of mice in vivo were analyzed. Significant positive yet incomplete correlation was found to exist between these variables. Basing on structural and functional peculiarities of N-terminal part of CTR1 a hypothesis of coupled transport of copper and cisplatin has been suggested which avoids disagreement between CTR1-mediated cisplatin transport in vitro and irreversible binding of platinum to Met-rich peptides.     

Expression of small RNAs of Mycobacterium tuberculosis in murine models of tuberculosis infection

The determination of the mechanisms contributing to the survival of pathogenic bacteria in the infected organism and the possible ways of their blocking is a promising approach to the development of new methods of affecting these bacteria. Among these mechanisms, the regulation of bacterial metabolism by small RNAs attracts particular interest since it has been found recently to play an important role in the bacterial pathogenesis. We have studied the expression of three most highly expressed small RNAs of Mycobacterium tuberculosis: MTS0997, MTS1338, and MTS2823 during tuberculosis progression in the strains of mice having different genetic resistance to the disease. It has been shown that the maximum expression of these small RNAs occurs at earlier stages of infection.     

Initial stages of interaction of Azospirillum brasilense bacteria with wheat germ roots: adsorption, deformation of root hairs

The initial stages of colonization of wheat roots by cells of Azospirillum brasilense strains 75 and 80 isolated from soils of the Saratov oblast were studied. The adsorption of azospirilla on root hairs of soft spring wheats rapidly increased in the first hours of incubation, going then to a plateau phase. Within the first 15 h of incubation, exponential-phase cells were adsorbed more intensively than stationary-phase cells. Conversely, stationary-phase cells were adsorbed more intensively than exponential-phase cells, if the period of azospirilla incubation with the wheat roots was extended. As the time of incubation increased, the attachment of azospirilla to the wheat roots became stronger. The effect of cell attachment to root hairs was strain-dependent; the number of adsorbed cells of a given strain of azospirilla was greater in the case of host wheat cultivars. The deformation of wheat root hairs was affected by the polysaccharide-containing complexes isolated from the capsular material of azospirilla. The suggestion is made that common receptor systems are involved in the adsorption of azospirilla on roots and in root hair deformation.     

A simple method of prediction of visibility of peptides in mass spectrometry with electrospray ionization

A new method for selection of essential peptides applicable for protein detection and quantification analysis in the targeted positive electrospray mass spectrometry has been proposed. It is based on the prediction of the normalized abundance of the mass spectrometric peaks by using a linear regression model. This method has the following a priori restrictions: first selection of peptides must be arranged so that at pH 2.5 the tested peptides must be presented mainly as the 2⁺ and 3⁺ ions. Only peptides containing C-terminal lysine or arginine residues should be considered. The amino acid composition of the peptide, the peptide concentration, the ratio of the polar surface of peptide to common surface and ratio of the polar volume to the common volume are used as independent variables. Among several considered combinations of variables the best linear regression model had a determination coefficient in leave-one-out cross-validation procedure of 0.54. This model confidently discriminated peptides with high response ability and peptides with low response ability, and therefore it is applicable for selection of the most favorable peptides among peptides selected by means of simple criteria. This simple and fast screening method can be successfully applied to reduce the list of observed peptides.     

HMG1 domains: the victims of circumstance

The method of circular dichroism (CD) was used to compare DNA behavior during its interaction with linker histone H1 and with non-histone chromosomal protein HMG1 at different ionic strength and at different protein content in the system. The role of negatively charged C-terminal fragment of HMG1 was analyzed using recombinant protein HMG1-(A + B), which lacks the C terminal amino acid sequence. The psi-type CD spectra were common for DNA interaction with histone H1, but no spectra of this type were observed in HMG1-DNA systems even at high ionic strength. The CD spectrum of the truncated recombinant protein at high salt concentration somewhat resembled the psi-type spectrum. Two very intense positive bands were located near 215 nm and near 273 nm, and the whole CD spectrum was positive. The role of C-terminal tail of HMG1 in formation of the ordered DNA-protein complexes is discussed.     

TERRA mimicking ssRNAs prevail over the DNA substrate for telomerase in vitro due to interactions with the alternative binding site

Telomerase is a key component of the telomere length maintenance system in the majority of eukaryotes. Telomerase displays maximal activity in stem and cancer cells with high proliferative potential. In humans, telomerase activity is regulated by various mechanisms, including the interaction with telomere ssDNA overhangs that contain a repetitive G‐rich sequence, and with noncoding RNA, Telomeric repeat‐containing RNA (TERRA), that contains the same sequence. So these nucleic acids can compete for telomerase RNA templates in the cell. In this study, we have investigated the ability of different model substrates mimicking telomere DNA overhangs and TERRA RNA to compete for telomerase in vitro through a previously developed telomerase inhibitor assay. We have shown in this study that RNA oligonucleotides are better competitors for telomerase that DNA ones as RNA also use an alternative binding site on telomerase, and the presence of 2′‐OH groups is significant in these interactions. In contrast to DNA, the possibility of forming intramolecular G‐quadruplex structures has a minor effect for RNA binding to telomerase. Taking together our data, we propose that TERRA RNA binds better to telomerase compared with its native substrate – the 3′‐end of telomere DNA overhang. As a result, some specific factor may exist that participates in switching telomerase from TERRA to the 3′‐end of DNA for telomere elongation at the distinct period of a cell cycle in vivo. Copyright © 2015 John Wiley & Sons, Ltd.     

Prevalence of human papillomavirus infection,distribution of viral types and risk factors in cervical samples from human immunodeficiency virus-positive women attending three human immunodeficiency virus-acquired immune deficiency syndrome reference centres in northeastern Brazil

Human immunodeficiency virus (HIV)-positive patients have a greater prevalence ofcoinfection with human papillomavirus (HPV) is of high oncogenic risk. Indeed, thepresence of the virus favours intraepithelial squamous cell lesion progression andmay induce cancer. The aim of this study was to evaluate the prevalence of HPVinfection, distribution of HPV types and risk factors among HIV-positive patients.Cervical samples from 450 HIV-positive patients were analysed with regard to oncoticcytology, colposcopy and HPV presence and type by means of polymerase chain reactionand sequencing. The results were analysed by comparing demographic data and datarelating to HPV and HIV infection. The prevalence of HPV was 47.5%. Among theHPV-positive samples, 59% included viral types of high oncogenic risk. Multivariateanalysis showed an association between HPV infection and the presence of cytologicalalterations (p = 0.003), age greater than or equal to 35 years (p = 0.002), number ofpartners greater than three (p = 0.002), CD4⁺ lymphocyte count <200/mm³ (p = 0.041) and alcohol abuse (p = 0.004). Although high-riskHPV was present in the majority of the lesions studied, the low frequency of HPV 16(3.3%), low occurrence of cervical lesions and preserved immunological state in mostof the HIV-positive patients were factors that may explain the low occurrence ofprecancerous cervical lesions in this population.     

Proteomics profiling of microdissected low- and high-grade prostate tumors identifies Lamin A as a discriminatory biomarker

Proteomics screening methods for the identification of diagnostic and prognostic biomarkers in cancer are still lagging behind DNA- or RNA-based analysis. We used two-dimensional differential gel electrophoresis (2D-DIGE) in combination with laser capture microdissection (LCM) and MALDI-TOF/TOF mass spectrometry to determine differentially abundant proteins and candidate biomarkers in prostate cancer. Paired (benign and tumor) samples were isolated from 23 Gleason Score 6 (GS 6) and 23 Gleason Score 8 and higher (GS 8+) radical prostatectomy specimens and subjected to 2D-DIGE analysis. Minimal fluorescent dye labeling was applied and electrophoresis performed with triple samples (paired benign and tumor; internal control) for each case of tumor. Nineteen differently abundant proteins were identified by mass spectrometry and further validated. One half of them were associated with glycolysis and the Warburg effect; these were upregulated in tumors. The upregulation correlated with tumor dedifferentiation and might be relevant for selection of therapeutic strategies. Among the other proteins, heat shock protein 60 (HSP60) was significantly upregulated in tumor tissue compared to its benign counterpart. Furthermore, lamin A was statistically highly discriminatory between low and high Gleason score tumors and might serve as a new biomarker of tumor differentiation and prognosis.