Metallothionein and cellular energy allocation in the estuarine mysid shrimp Neomysis integer exposed to cadmium at different salinities

In the present study the induction of metallothioneins (MTs) and effects on the cellular energy allocation (CEA) in euryhaline crustacean Neomysis integer exposed to Cd at different salinities were studied. N. integer was exposed to the same sub-lethal concentration of free cadmium ion (1/5 of the cadmium activity of the reported 96 h LC₅₀ value) in hypo-osmotic (7.2 μg Cd/L at 5 psu), isosmotic (23.0 μg Cd/L at 16 psu) and hyper-osmotic media (38.1 μg Cd/L at 25 psu) for 7 days. By using the free Cd concentration as the basis for conducting the exposures, the effect of salinity on cadmium speciation was eliminated and therefore the true effect of salinity as an abiotic factor on the MT induction and CEA could be studied.MT content was quantified by differential pulse voltammetry (DPV); this is the first time that this method is applied to assays with N. integer. No significant differences in MT levels between the control and Cd-exposed groups were observed. The measured MT levels (ranging from 32.3 to 75.7 μg/mg_{(cytosolic protein)}) probably represent the constitutive MT levels responsible for binding essential metals. The differences in MT levels observed at the different salinities indicate a possible relationship between some physiological process (possibly osmoregulation) other than detoxification and MT induction. A decrease in salinity caused an increase in MT level in N. integer, and this was significantly correlated to the CEA values (Spearman correlation coefficient r = − 0.56, p = 0.016). Our results indicate that salinity can change the energy status and MT content of N. integer. These findings should be taken into account when using these biomarkers - and especially MTs - in field studies and environmental monitoring.     

Calculating the geometry and Raman spectrum of physiological bis(<Emphasis Type="SmallCaps">l</Emphasis>-histidinato)copper(II): an assessment of DFT functionals for aqueous and isolated systems

Reliable density functional theory (DFT) calculations can be performed in conjuction with spectroscopic measurements to elucidate the structural properties of physiologically important bis(amino acidato)copper(II) compounds in solutions. They can provide insight into the influence of intermolecular interactions on the molecular geometry in the crystal lattice or solution when compared with a DFT gas-phase minimum. Our previous paper [Markovi? et al. (2014) Eur J Inorg Chem 198] reported the DFT-determined geometries and Raman spectra for different conformers of physiological bis(l-histidinato)copper(II) with 20 explicit water molecules, as calculated using the B3LYP functional. The present study examined the reliability of those B3LYP results by applying the M06 functional instead, as it should better account for noncovalent interactions. The water molecules were positioned more compactly around the complex by M06 than by B3LYP. The accuracies of the two functionals when compared to relevant experimental data showed that M06 was better at reproducing in-plane Cu?N bond lengths but B3LYP gave more accurate axial Cu?O distances. Both functionals reproduced the experimental Raman spectrum at pH 8 to similar levels of accuracy and provided precise information on the Cu(II) coordination mode and conformation in aqueous solution. Additionally, we assessed several DFT and DFT-D functionals (BP86, B3LYP, B3LYP-D, M06, M06 L, wB97XD, mPW2PLYPD) by using them to model the geometries of experimental bis(l-histidinato)copper(II) crystalline conformations as isolated systems, and then benchmarking the results against those from high-level second-order pertubation Møller–Plesset (MP2) calculations. Although this assessment resulted in an equivocal conclusion because the MP2 results for the isolated complex were inconsistent with the corresponding DFT outcomes, it does provide new information on future benchmark options.     

Comparison of different procedures for the lipid extraction from HL-60 cells: a MALDI-TOF mass spectrometric study

A human leukaemia cell line--HL-60--can be differentiated into neutrophils or macrophages and both differentiation processes are accompanied by changes of the lipid composition. Various methods were described for the extraction of lipids from cellular systems, but only two of them were applied to the HL-60 cell line so far. In this study we compared five selected extraction methods for the lipid extraction from HL-60 cells with regard to their qualitative analysis by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS): chloroform/methanol at volume ratios 2:1 and 1:2, isopropanol/ chloroform, isopropanol/hexane and butanol. In addition, the cholesterol and phospholipid concentrations in organic extracts were measured by colorimetric assays. Results can be summarized as follows: For the analysis of polar phospholipids obtained from HL-60 cells by MALDI-TOF MS, a chlorofom/methanol (1:2) or isopropanol/chloroform mixture or butanol can be applied as extraction systems On the other hand, if one would like to analyze changes in triacylglycerols, then chloroform/methanol (2:1) would be the method of choice.     

Cnidarian and bilaterian promoters can direct GFP expression in transfected hydra

Complete sexual development is not easily amenable to experimentation in hydra. Therefore, the analysis of gene function and gene regulation requires the introduction of exogenous DNA in a large number of cells of the hydra polyps and the significant expression of reporter constructs in these cells. We present here the procedure whereby we coupled DNA injection into the gastric cavity to electroporation of the whole animal in order to efficiently transfect hydra polyps. We could detect GFP fluorescence in both endodermal and ectodermal cell layers of live animals and in epithelial as well as interstitial cell types of dissociated hydra. In addition, we could confirm GFP protein expression by showing colocalisation between GFP fluorescence and anti-GFP immunofluorescence. Finally, when a FLAG epitope was inserted in-frame with the GFP coding sequence, GFP fluorescence also colocalised with anti-FLAG immunofluorescence. This GFP expression in hydra cells was directed by various promoters, either homologous, like the hydra homeobox cnox-2 gene promoter, or heterologous, like the two nematode ribosomal protein S5 and L28 gene promoters, and the chicken beta-actin gene promoter. This strategy provides new tools for dissecting developmental molecular mechanisms in hydra; more specifically, the genetic regulations that take place in endodermal cells at the time budding or regeneration is initiated.     

Comparison of digital rectal examination and prostate specific antigen in early detection of prostate cancer

This study compares the value of digital rectal examination (DRE) and prostate specific antigen (PSA) determination in the detection of prostate cancer. 1,000 men aged > or = 50 from the Osijek surroundings were examined. The subjects with prostatitis were excluded from the study. The subjects with elevated concentration of total prostate specific antigen and/or digital rectal examination suspect of carcinoma underwent prostate biopsy. The rate of prostate cancer detection showed to be 3.3% for PSA > 4 ng/ml, 2% for abnormal finding of DRE, and 3.7% for combination of the two methods. Out of 35 patients with prostate cancer detected, 19 had suspect DRE finding and 32 had PSA exceeding 4 ng/ml. Thus, PSA pointed to the diagnosis of prostate cancer in 91.4%, and abnormal finding of DRE in 54.2% of cases, the difference being statistically significant. The positive predictive value was 48.7% for abnormal finding of DRE, 47% for PSA > 4 ng/ml, and 80.0% for the combination of both. Although PSA determination detected a considerable proportion of tumors missed on DRE, the former alone was found to be insufficient as a screening method because of its inadequate sensitivity. When combined with digital rectal examination, the probability of prostate cancer detection increased considerably.     

Prokaryotic-style frameshifting in a plant translation system: conservation of an unusual single-tRNA slippage event

Ribosomal frameshifting signals are found in mobile genetic elements, viruses and cellular genes of prokaryotes and eukaryotes. Typically they comprise a slippery sequence, X XXY YYZ, where the frameshift occurs, and a stimulatory mRNA element. Here we studied the influence of host translational environment and the identity of slippery sequence-decoding tRNAs on the frameshift mechanism. By expressing candidate signals in Escherichia coli, and in wheatgerm extracts depleted of endogenous tRNAs and supplemented with prokaryotic or eukaryotic tRNA populations, we show that when decoding AAG in the ribosomal A-site, E.coli tRNA(Lys) promotes a highly unusual single-tRNA slippage event in both prokaryotic and eukaryotic ribosomes. This event does not appear to require slippage of the adjacent P-site tRNA, although its identity is influential. Conversely, asparaginyl-tRNA promoted a dual slippage event in either system. Thus, the tRNAs themselves are the main determinants in the selection of single- or dual-tRNA slippage mechanisms. We also show for the first time that prokaryotic tRNA(Asn) is not inherently 'unslippery' and induces efficient frameshifting when in the context of a eukaryotic translation system.     

The western and eastern roots of the Saami--the story of genetic "outliers" told by mitochondrial DNA and Y chromosomes

The Saami are regarded as extreme genetic outliers among European populations. In this study, a high-resolution phylogenetic analysis of Saami genetic heritage was undertaken in a comprehensive context, through use of maternally inherited mitochondrial DNA (mtDNA) and paternally inherited Y-chromosomal variation. DNA variants present in the Saami were compared with those found in Europe and Siberia, through use of both new and previously published data from 445 Saami and 17,096 western Eurasian and Siberian mtDNA samples, as well as 127 Saami and 2,840 western Eurasian and Siberian Y-chromosome samples. It was shown that the “Saami motif” variant of mtDNA haplogroup U5b is present in a large area outside Scandinavia. A detailed phylogeographic analysis of one of the predominant Saami mtDNA haplogroups, U5b1b, which also includes the lineages of the “Saami motif,” was undertaken in 31 populations. The results indicate that the origin of U5b1b, as for the other predominant Saami haplogroup, V, is most likely in western, rather than eastern, Europe. Furthermore, an additional haplogroup (H1) spread among the Saami was virtually absent in 781 Samoyed and Ob-Ugric Siberians but was present in western and central European populations. The Y-chromosomal variety in the Saami is also consistent with their European ancestry. It suggests that the large genetic separation of the Saami from other Europeans is best explained by assuming that the Saami are descendants of a narrow, distinctive subset of Europeans. In particular, no evidence of a significant directional gene flow from extant aboriginal Siberian populations into the haploid gene pools of the Saami was found.     

Protective role of glutathione reductase in paraquat induced neurotoxicity

Paraquat (PQ), a widely used herbicide is a well-known free radical producing agent. The mechanistic pathways of PQ neurotoxicity were examined by assessing oxidative/nitrosative stress markers. Focus was on the role of glutathione (GSH) cycle and to examine whether the pre-treatment with enzyme glutathione reductase (GR) could protect the vulnerable brain regions (VBRs) against harmful oxidative effect of PQ. The study was conducted on Wistar rats, randomly divided in five groups: intact-control group, (n=8) and four experimental groups (n=24). All tested compounds were administered intrastriatally (i.s.) in one single dose. The following parameters of oxidative status were measured in the striatum, hippocampus and cortex, at 30min, 24h and 7days post treatment: superoxide anion radical (O(2)(-)), nitrate (NO(3)(-)), malondialdehyde (MDA), superoxide dismutase (SOD), total GSH (tGSH) and its oxidized, disulfide form (GSSG) and glutathione peroxidase (GPx). Results obtained from the intact and the sham operated groups were not statistically different, confirming that invasive i.s. route of administration would not influence the reliability of results. Also, similar pattern of changes were observed between ipsi- and contra- lateral side of examined VBRs, indicating rapid spatial spreading of oxidative stress. Mortality of the animals (10%), within 24h, along with symptoms of Parkinsonism, after awakening from anesthesia for 2-3h, were observed in the PQ group, only. Increased levels of O(2)(-), NO(3)(-) and MDA, increased ratio of GSSG/GSH and considerably high activity of GPx were measured at 30min after the treatment. Cytotoxic effect of PQ was documented by drastic drop of all measured parameters and extremely high peak of the ratio GSSG/GSH at 24th hrs after the PQ i.s. injection. In the GR+PQ group, markedly low activity of GPx and low content of NO(3)(-) (in striatum and cortex) were measured during whole experiment, while increase value was observed only for O(2)(-), at 7th days. We concluded that oxidative/nitrosative stress and excitotoxicity are the most important events since the early stage of PQ induced neurotoxicity. Based on the ratio GSSG/GSH, the oxidation of GSH to GSSG is probably dominant way of GHS depletion and main reason for reduced antioxidative defense against PQ harmful oxidative effect. The GR pre-treatment resulted in the absence of Parkinson's disease-like symptoms and mortality of the rats. Additionally, oxidative/nitrosative stress did not developed, as well as almost diminished metabolism of the VBRs at 24th hours (as has been documented in the PQ group) did not occurred in the GR+PQ, suggesting a neuroprotective role for the GR in PQ induced neurotoxicity.     

Determination of combined sibship indices “gray zone” using 15 STR loci for central Bosnian human population

In our previous population studies of Bosnia and Herzegovina human population, we have used autosomal STR, Y-STR, and X-STR loci, as well as Y-chromosome NRY biallelic markers. All obtained results were included in Bosnian referent database. In order of future development of applied population molecular genetics researches of Bosnia and Herzegovina human population, we have examined the effectiveness of 15 STR loci system in determination of sibship by using 15 STR loci and calculating different cut-off points of combined sibship indices (CSI) and distribution of sharing alleles. From the perspective of its application, it is very difficult and complicated to establish strict CSI cut-off values for determination of the doubtless sibship. High statistically significant difference between the means of CSI values and in distribution of alleles sharing in siblings and non-siblings was noticed (P < 0.0001). After constructing the “gray zone”, only one false positive result was found in three CSI cut-off levels with the highest percent of determined sibship/non-sibship at the CSI = 0.067, confirming its practical benefit. Concerning the distribution of sharing alleles, it is recommended as an informative estimator for its usage within Bosnia and Herzegovina human population.