Oxygen as an important factor modulating in vitro MeHgCl toxicity associated with mitochondrial genes in hiPSCs.

Mitochondria are energy factories of cells and important targets for methylmercury chloride (MgHgCl). Methylmercury (MeHg) is a well-known environmental toxicant that bioaccumulates in fish and shellfish. It readily crosses the placental barrier, making it a threat to correct fetal development. Despite being comprehensively investigated for years, this compound has not been assessed for its in vitro mitochondrial toxicity under different oxygen conditions. In this study, human induced pluripotent stem cells (hiPSCs) were used to evaluate the dependence of the expression of genes associated with pluripotency and mitochondria on atmospheric (21% O2) and low (5% O2) oxygen concentrations upon MeHgCl treatment. We showed that the toxicity of MeHgCl was strongly related to an increased mtDNA copy number and downregulation of the expression of an mtDNA replication and damage repair-associated gene POLG1 (Mitochondrial Polymerase Gamma Catalytic Subunit) in both tested oxygen conditions. In addition, the viability and mitochondrial membrane potential of hiPSCs were significantly lowered by MeHgCl regardless of the oxygen concentration. However, reactive oxygen species accumulation significantly increased only under atmospheric oxygen conditions; what was associated with increased expression of TFAM (Transcription Factor A, Mitochondrial) and NRF1 (Nuclear Respiratory Factor 1) and downregulation of PARK2 (Parkin RBR E3 Ubiquitin Protein Ligase). Taken together, our results demonstrated that MeHgCl could induce in vitro toxicity in hiPSCs through altering mitochondria-associated genes in an oxygen level-dependent manner. Thus, our work suggests that oxygen should be considered a factor was modulating the in vitro toxicity of environmental pollutants. Typical atmospheric conditions of in vitro culture significantly lower the predictive value of studies of such toxicity.

Characterization of nanofluids using multifractal analysis of a liquid droplet trace.

The article presents an innovative approach to the analysis of nanofluids using a nonlinear multifractal algorithm. The conducted research concerned nanofluids prepared from SiO2 nanoparticles (~ 0.01 g) suspended in 100 ml of demineralized water and in 100 ml of 99.5% isopropanol. Subsequently, the nanofluids were subjected to conventional characterization methods such as: determination of the contact angle, determination of zeta potential, pH, and particle size analysis. The obtained results show that the prepared nanofluid is stable in terms of agglomeration over time (nanofluid suspension) and properly prepared in terms of dissolving and dispersing powder particles. The authors, analyzing the results of the presented methods for characterizing nanofluids, proposed a multifractal analysis, which allows detailed local descriptions of complex scaling behaviour, using a spectrum of singularity exponents. Nonlinear analyzes show that the use of multifractal algorithm for nanofluids can improve the process of fluid quality analysis and its preparation based on the multifractal spectrum.

Species-specific models in toxicology: in vitro epithelial barriers.

Species-specific in vitro epithelial barriers represent interesting predictive tools for risk assessment evaluation in toxicological studies. Moreover, these models could be applied either as stand-alone methods for the study of absorption, bioavailability, excretion, transport, effects of xenobiotics, or through an Integrated Testing Strategy. The aim of this review is to give a comprehensive overview of in vitro species-specific epithelial barrier models from bovine, dog and swine. Bovine mammary epithelial barrier as a fundamental instrument for the evaluation of the toxicant excretion, the blood brain barrier as a useful first approach in toxicological and pharmacological studies, the porcine intestinal barrier, the canine skin barrier, and finally the pulmonary barrier from bovine and swine species are described in this review.

Mitochondrial biogenesis and neural differentiation of human iPSC is modulated by idebenone in a developmental stage-dependent manner.

Idebenone, the synthetic analog of coenzyme Q10 can improve electron transport in mitochondria. Therefore, it is used in the treatment of Alzheimer's disease and other cognitive impairments. However, the mechanism of its action on neurodevelopment is still to be elucidated. Here we demonstrate that the cellular response of human induced pluripotent stem cells (hiPSC) to idebenone depends on the stage of neural differentiation. When: neural stem cells (NSC), early neural progenitors (eNP) and advanced neural progenitors (NP) have been studied a significant stimulation of mitochondrial biogenesis was observed only at the eNP stage of development. This coexists with the enhancement of cell viability and increase in total cell number. In addition, we report novel idebenone properties in a possible regulation of neural stem cells fate decision: only eNP stage responded with up-regulation of both neuronal (MAP2), astrocytic (GFAP) markers, while at NSC and NP stages significant down-regulation of MAP2 expression was observed, promoting astrocyte differentiation. Thus, idebenone targets specific stages of hiPSC differentiation and may influence the neural stem cell fate decision.

Sensitivity of hiPSC-derived neural stem cells (NSC) to Pyrroloquinoline quinone depends on their developmental stage.

Pyrroloquinoline quinone (PQQ) is a factor influencing on the mitochondrial biogenesis. In this study the PQQ effect on viability, total cell number, antioxidant capacity, mitochondrial biogenesis and differentiation potential was investigated in human induced Pluripotent Stem Cells (iPSC) - derived: neural stem cells (NSC), early neural progenitors (eNP) and neural progenitors (NP). Here we demonstrated that sensitivity to PQQ is dependent upon its dose and neural stage of development. Induction of the mitochondrial biogenesis by PQQ at three stages of neural differentiation was evaluated at mtDNA, mRNA and protein level. Changes in NRF1, TFAM and PPARGC1A gene expression were observed at all developmental stages, but only at eNP were correlated with the statistically significant increase in the mtDNA copy numbers and enhancement of SDHA, COX-1 protein level. Thus, the "developmental window" of eNP for PQQ-evoked mitochondrial biogenesis is proposed. This effect was independent of high antioxidant capacity of PQQ, which was confirmed in all tested cell populations, regardless of the stage of hiPSC neural differentiation. Furthermore, a strong induction of GFAP, with down regulation of MAP2 gene expression upon PQQ treatment was observed. This indicates a possibility of shifting the balance of cell differentiation in the favor of astroglia, but more research is needed at this point.

Effect of nuclear matrix attachment regions on transgene expression in tobacco plants.

Matrix attachment regions (MARs) are thought to participate in the organization and segregation of independent chromosomal loop domains. Although there are several reports on the action of natural MARs in the context of heterologous genes in transgenic plants, in our study we tested a synthetic MAR (sMAR) with the special property of unpairing when under superhelical strain, for its effect on reporter gene expression in tobacco plants. The synthetic MAR was a multimer of a short sequence from the MAR 3' end of the immunoglobulin heavy chain (IgH) enhancer. This sMAR sequence was used to flank the beta-glucuronidase (GUS) reporter gene within the T-DNA of the binary vector pBI121. Vectors with or without the sMARs were then used to transform tobacco plants by Agrobacterium tumefaciens. Transgenic plants containing the sMAR sequences flanking the GUS gene exhibited higher levels of transgene expression compared with transgenic plants which lacked the sMARs. This effect was observed independently of the position of the sMAR at the 5' side of the reporter gene. However, variation of the detected transgene expression was significant in all transformed plant populations, irrespective of the construct used.

Expression of the plum pox coat protein gene in transgenic Nicotiana tabacum plants.

Plant expression vector pBI 121 containing the gene encoding coat protein of Plum Pox Virus of the Skierniewice isolate (CP PPV-S) was prepared (clone pCM1). The construct was used for transformation of Nicotiana tabacum plants using an Agrobacterium tumefaciens based system. About 82% of kanamycin resistant plant lines contained a transgene (the sequence of CP PPV-S) but only 81% of them actively expressed the PPV-S coat protein gene as measured by RT-PCR.

Nucleotide sequence of the coat protein gene of the Skierniewice isolate of plum pox virus (PPV).

The coat protein (CP) gene of the Skierniewice isolate of plum pox virus (PPV-S) has been amplified using the reverse transcription--polymerase chain reaction (RT-PCR), cloned and sequenced. The nucleotide sequence of the gene and the deduced amino-acid sequence of PPV-S CP were compared with those of other PPV strains. The nucleotide sequence showed very high homology to most of the published sequences. The motif: Asp-Ala-Gly (DAG), important for the aphid transmissibility, was present in the amino-acid sequence. Our isolate did not react in ELISA with monoclonal antibodies MAb06 supposed to be specific for PPV-D.

Conserved signals in the 5' flanking region of eukaryotic nuclear tRNA genes.

The statistical analysis of 5' flanking regions of eukaryotic tRNA genes was done. The analysis of nucleotides in the sequence of fungi and invertebrates showed a high content of A and T in the flanking regions versus coding regions where G and C dominate. In contrast to these results in vertebrates sequences the preferences of any nucleotide in flanking regions was not observed. The analysis of tetrads showed five conserved signals: TTGT, (T/A)(T/A)ATA, A(C/T)(C/A)A in the tRNA genes of fungi, (A/T)TGA of invertebrates and (A/T)GAG of vertebrates. The analysis of 3' flanking regions did not show any conserved signals except well known poly-T tracks.

A nuclear tRNA(UGASer) gene from the wheat Triticum vulgare var. Aria.

The nucleotide sequence of a wheat nuclear tRNA(UGASer) gene from Triticum vulgare var. Aria has been determined. It has a typical intragenic promoter with boxA and boxB elements and a putative termination signal 12 nucleotides downstream from the last tRNA-coding nucleotide. The region upstream from the coding segment contains a G + C-rich sequence with a symmetrical element. The sequence described is the first nuclear tRNA gene from a monocotyledonous plant.

Iodo-Gen-mediated radioiodination of nucleic acids.

Iodo-Gen (1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril), widely used as an oxidizing agent for iodination of proteins, can also be used for iodination of nucleic acids. Optimal conditions were determined for efficient labeling of RNA and DNA with 125I. The proposed procedure for radioiodination of nucleic acids is more beneficial than the methods utilizing TlCl3 because of the milder reaction conditions, the simplicity and completeness of separation of reaction products from the oxidizing agents, and the absence of a toxic catalyst. Using the standard procedure for Iodo-Gen-mediated iodination a specific radioactivity of up to 1.3 X 10(9) dpm/micrograms RNA can be achieved. The proposed procedure is also suitable for radioiodination of DNA.

Is Tb3+ fluorescence enhancement only due to binding to single stranded polynucleotides.

Enhancement of Tb3+ fluorescence upon binding to double-stranded ribo- and deoxyribo-duplexes was investigated. It was observed that certain double stranded ribopolynucleotides completely quenched the Tb3+ fluorescence and others did not. It is concluded that the nature of the base in the duplex is critical for this enhancement. - Polydeoxyduplexes also showed enhancement of Tb3+ fluorescence, but much higher terbium concentrations were necessary to obtain similar fluorescence signals, indicative of unspecific effects. CD spectra evidence considerable conformational changes of these duplexes, in particular poly(dG-C) . poly(dG-C( which assumes the Z-form in 0.1 nM Tb3+.

Gel mapping of wheat and lupin tRNAs.

Transfer RNAs were isolated from plants representing mono- and dicotyledons: wheat embryos and lupin seeds. The two tRNA preparations were compared by polyacrylamide-gel-electrophoresis mapping. Transfer RNAs extracted from the separated spots were tested for acceptance of 11 amino acids. Comparison of electrophoretic mobility of respective tRNA species indicates the overall similarity of tRNA populations in the two plants studied. Especially, isoaccepting tRNAs for glycine, tyrosine and valine and some of isoacceptors of tRNAArg, tRNAAsp, tRNALeu, tRNALys and tRNAPhe occupy identical or closely similar positions on both polyacrylamide-gel maps. However, some tRNA isoacceptors from one population have no counterparts in the second one, which may indicate differences in their primary structures.

Valyl-tRNA and leucyl-tRNA synthetases in wheat germ and seedlings.

Valyl- and leucyl-tRNA synthetases (Val-RS and Leu-RS) isolated from wheat germ and seedlings were separated by chromatography on hydroxylapatite into organellar (Val-RS I and Leu-RS I) and cytoplasmic (Val-RS II and Leu-RS II) enzymes; the enzyme extracted from isolated chloroplasts and mitochondria corresponded to the RS I fractions. It was proved by RPC-5 chromatography of tRNA Val that Val-RS I and Val-RS II recognized all five isoacceptor tRNA Val species both from wheat germ and leaves, as well as tRNA Val from E. coli. However, out of the six isoacceptor tRNA Leu species, Leu-RS II aminoacylated two cytoplasmic species only, while Leu-RS I, the remaining four organellar tRNA Leu fractions. Both leucyl-tRNA synthetases charged E. coli tRNA, Leu-RS I more effectively than Leu-RS II. The absence of fraction RS I in cytosol seems to indicate that valyl- and leucyl-tRNA synthetases (coded for by the nuclear genome) were modified to the organellar forms after (or during) passage into organelles.

Nucleotide sequence of the anticodon region of barley embryo phenylalanine transfer RNA.

Highly purified tRNAPhe from barley embryos was completely digested with pancreatic ribonuclease and T1 ribonuclease. The digestion products were separated using DEAE-cellulose chromatography. The Y base-containing fragment of the anticodon region of tRNAPhe has the following nucleotide sequence: Cpm2(2)GppsipCpApGpApCmpUpGmpApApYpAppsipCpUpGp, i.e. the same as in the anticodon region of wheat germ and pea tRNAPhe.