Genetic polymorphisms and possible gene-gene interactions in metabolic and DNA repair genes: effects on DNA damage

We investigated in a central European population, the association between genetic polymorphisms in several genes coding for xenobiotic metabolizing enzymes (CYP1A1, CYP2E1, EPHX1, GSTP1, GSTM1 and GSTT1) and in DNA repair genes (XPD, XPG, XPC and XRCC1) and the levels of single-strand breaks (SSBs) and SSB endonuclease III sensitive sites (endoIII sites) in peripheral blood lymphocytes. No significant differences in the mean levels of SSBs and endoIII sites after stratification for main confounders and occupational exposure were observed in the studied population. Significantly higher levels of SSBs were observed in individuals bearing the wild-type alleles (AA) (0.75+/-0.51SSB/10(9)Da) and heterozygous (AC) genotypes (0.67+/-0.49SSB/10(9)Da) compared to those with homozygous XPD (CC) genotype (0.43+/-0.28SSB/10(9)Da, P=0.033). A moderate increase in the levels of SSBs was also found in individuals with the homozygous XPG exon 15 wild type (GG) and heterozygous (GC) genotypes in comparison to those with the homozygous (CC) genotype (P=0.066) and in individuals with low activity EPHX1 genotype in comparison to those with high activity genotype. Nevertheless, these differences were not statistically significant. No other significant association was found. When gene-gene interactions were evaluated, a combination of EPHX1 activity genotypes with that of either XPD or XPG significantly (P=0.003 and 0.016, respectively) modulated SSB levels resulting in a three-fold difference between the "protective" and the "adverse" genotype-combinations. Almost three-fold differences in SSB levels were found between the "protective" and the "adverse" genotype-combinations of EPHX1 activity genotype and GSTM1 or GSTT1 genotypes, respectively. In conclusion, our results suggest a relation between markers of genotoxicity and polymorphisms in genes coding for xenobiotic metabolizing and DNA repair enzymes as well as a modulating effect of combinations of these polymorphisms.     

On the separation of net ecosystem exchange into assimilation and ecosystem respiration: review and improved algorithm

This paper discusses the advantages and disadvantages of the different methods that separate net ecosystem exchange (NEE) into its major components, gross ecosystem carbon uptake (GEP) and ecosystem respiration (R_eco). In particular, we analyse the effect of the extrapolation of night‐time values of ecosystem respiration into the daytime; this is usually done with a temperature response function that is derived from long‐term data sets. For this analysis, we used 16 one‐year‐long data sets of carbon dioxide exchange measurements from European and US‐American eddy covariance networks. These sites span from the boreal to Mediterranean climates, and include deciduous and evergreen forest, scrubland and crop ecosystems. We show that the temperature sensitivity of R_eco, derived from long‐term (annual) data sets, does not reflect the short‐term temperature sensitivity that is effective when extrapolating from night‐ to daytime. Specifically, in summer active ecosystems the long‐term temperature sensitivity exceeds the short‐term sensitivity. Thus, in those ecosystems, the application of a long‐term temperature sensitivity to the extrapolation of respiration from night to day leads to a systematic overestimation of ecosystem respiration from half‐hourly to annual time‐scales, which can reach >25% for an annual budget and which consequently affects estimates of GEP. Conversely, in summer passive (Mediterranean) ecosystems, the long‐term temperature sensitivity is lower than the short‐term temperature sensitivity resulting in underestimation of annual sums of respiration. We introduce a new generic algorithm that derives a short‐term temperature sensitivity of R_eco from eddy covariance data that applies this to the extrapolation from night‐ to daytime, and that further performs a filling of data gaps that exploits both, the covariance between fluxes and meteorological drivers and the temporal structure of the fluxes. While this algorithm should give less biased estimates of GEP and R_eco, we discuss the remaining biases and recommend that eddy covariance measurements are still backed by ancillary flux measurements that can reduce the uncertainties inherent in the eddy covariance data.     

Performance of optimized McRAPD in identification of 9 yeast species frequently isolated from patient samples: potential for automation

Background  

Rapid, easy, economical and accurate species identification of yeasts isolated from clinical samples remains an important challenge for routine microbiological laboratories, because susceptibility to antifungal agents, probability to develop resistance and ability to cause disease vary in different species. To overcome the drawbacks of the currently available techniques we have recently proposed an innovative approach to yeast species identification based on RAPD genotyping and termed McRAPD (Melting curve of RAPD). Here we have evaluated its performance on a broader spectrum of clinically relevant yeast species and also examined the potential of automated and semi-automated interpretation of McRAPD data for yeast species identification.     

Karyotypically normal and abnormal human embryonic stem cell lines derived from PGD-analyzed embryos

Although a normal karyotype is generally a requirement for stem cell lines, new applications are likely to emerge for stem cells with defined chromosomal aneuploidies. We therefore investigated the use of embryos found to be aneuploid on biopsy followed by preimplantation genetic diagnosis (PGD) with fluorescent in situ hybridization (FISH), and developmentally arrested embryos for stem cell derivation. Eleven stem cell lines were obtained from 41 embryos in 36 cultures, with higher success rate achieved from PGD-analyzed, developmentally advanced embryos (45%) than from clinically unsuitable non-PGD embryos (13%). The resulting stem cell lines were karyotyped, and surprisingly, six of the nine lines from aneuploid embryos as well as both lines from non-PGD embryos were karyotypically normal. Three lines from PGD embryos were aneuploid exhibiting trisomy 5, trisomy 16, and an isochromosome 13, respectively. None of the aneuploid lines presented the same anomally as the original PGD analysis. Our study has three important implications. First, we confirm the ability to produce stem cell lines from PGD-tested embryos as well as developmentally abnormal embryos, offering specialty stem cell lines for research into the clinically important aneuploidies. Second, we observe that stem cell derivation from apparently aneuploid embryos is often thwarted by underlying mosaicism and emerging dominance of the stem cell line by karyotypically normal cells. The corollary, however, is that regular production of normal stem cell lines from developmentally abnormal embryos ordinarity discarded opens a new source of embryos for stem cells, whether for research or for eventual therapeutic use within the donating families.     

ERK1/2 map kinase metabolic pathway is responsible for phosphorylation of translation initiation factor eIF4E during in vitro maturation of pig oocytes

Eukaryotic initiation factor 4E (eIF4E) plays an important role in mRNA translation by binding the 5'-cap structure of the mRNA and facilitating the recruitment to the mRNA of other translation factors and the 40S ribosomal subunit. eIF4E undergoes regulated phosphorylation on Ser-209 and this phosphorylation is believed to be important for its binding to mRNA and to other initiation factors. The findings showing that the translation initiation factor eIF4E becomes gradually phosphorylated during in vitro maturation (IVM) of pig oocytes with a maximum in metaphase II (M II) stage oocytes have been documented by us recently (Ellederova et al., 2006). The aim of this work was to study in details the metabolic pathways involved in this process. Using inhibitors of cyclin-dependent kinases, Butyrolactone I (BL I) and protein phosphatases, okadaic acid (OA) we show that ERK1/2 MAP kinase pathway is involved in this phosphorylation. We also demonstrate that activation and phosphorylation of ERK1/2 MAP kinase and eIF4E is associated with the activating phosphorylation of Mnk1 kinase, one of the two main kinases phosphorylating eIF4E in somatic cells.     

HDAC inhibitors sodium butyrate and sodium valproate do not affect human ncor1 and ncor2 gene expression in HL-60 cells

AIM. This study was designed to examine whether the class I and class IIa histone deacetylase (HDAC) inhibitors, sodium butyrate and sodium valproate alter the expression of human NCOR1 and/or NCOR2 genes coding for N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone receptors), respectively. METHODS: Human leukemia HL-60 cells were treated for 24 h with 0.5 and 1 mM sodium butyrate, 1 to 3 mM sodium valproate, 1 mcM all-trans retinoic acid (ATRA) or cotreated with 1 mcM ATRA and 0.5 mM sodium butyrate. The acetylation of histones H3 and H4 was analysed by western blotting. The levels of NCOR1 and NCOR2 mRNA were determined by quantitative real-time PCR. Expression of NCF2 gene coding for the NADPH oxidase subunit p67phox was evaluated as a marker of myeloid differentiation. Results. Both butyrate and valproate increased the acetylation of histone H3 at Lys9 and/or Lys14 as well as histone H4 at Lys12. Both HDAC inhibitors caused a significant increase in NCF2 mRNA levels without affecting NCOR1 or NCOR2 mRNA levels. Similarly, ATRA alone or in combination with butyrate induced NCF2 gene expression without any significant influence on the expression of NCOR1 or NCOR2 genes. CONCLUSION: We conclude that inhibitors of class I and class IIa HDACs do not alter the expression of human NCOR1 or NCOR2 genes and that the onset of myeloid differentiation is not accompanied by induction or repression of these genes in HL-60 cells.     

Usefulness of McRAPD for typing and importance of biofilm production in a case of nosocomial ventriculoperitoneal shunt infection caused by <Emphasis Type="Italic">Candida lusitaniae</Emphasis>

A case report of ventriculoperitoneal shunt infection caused by Candida lusitaniae in a 6-year-old patient with cerebral astrocytoma and obstructive hydrocephalus is presented briefly with emphasis on the course of antifungal treatment. Seven isolates recovered subsequently from the cerebrospinal fluid were studied retrospectively. To confirm identity, isolates were typed using pulsed-field gel electrophoresis and melting curve of random amplified polymorphic DNA (McRAPD). Further, the ability to form biofilm and its susceptibility to systemic antifungals were evaluated. Using McRAPD, identity of C. lusitaniae isolates showing slight microevolutionary changes in karyotypes was undoubtedly confirmed; successful application of numerical interpretation of McRAPD for typing is demonstrated here for the first time. The strain was also recognized as a strong biofilm producer. Moreover, minimum biofilm inhibitory concentrations were very high, in contrast to low antifungal minimum inhibitory concentrations of isolates. It can be concluded that McRAPD seems to be a simple and reliable method not only for identification but also for typing of yeasts. A ventriculoperitoneal shunt colonized by C. lusitaniae was revealed as the source of this nosocomial infection, and the ability of the strain to form biofilm on its surface likely caused treatment failure.     

Molecular-genetic approaches to identification and typing of pathogenic Candida yeasts

Currently, invasive candidal infections represent an increasing cause of morbidity and mortality in seriously ill hospitalised patients. Because the accurate diagnosis of candidiasis remains difficult, a fast and reliable assay for characterization of fungal pathogens is critical for the early initiation of adequate antifungal therapy and/or for introduction of preventive measures. As novel molecular genetic techniques are continuously introduced, their role in the management of infectious diseases has also been growing. Today, molecular strategies complement conventional methods and provide more accurate and detailed insight. It can be expected that future technical development will improve their potential furthermore. In this article, we provide a critical review on the value and limitations of molecular tools in pathogenic Candida species identification and strain typing regarding their sensitivity, discriminatory power, reproducibility, cost and ease of performance.     

Mhc-DRB genes of platyrrhine primates

The two infraorders of anthropoid primates, Platyrrhini (New World monkeys) and Catarrhini (Old World monkeys and the hominoids) are estimated to have diverged from a common ancestor 37 million years ago. The major histocompatibility complex class II DRB gene and haplotype polymorphism of the Catarrhini has been characterized in several recent studies. The present study was undertaken to obtain information on the DRB polymorphism of the Platyrrhini. Fifty-five complete exon 2 DRB sequences were obtained from six species of Platyrrhini representing both the Callitrichidae and the Cebidae families. Combined with the results of a parallel contig mapping study, our data indicate that at least three loci (DRB1*03, DRB3, and DRB5) are shared by the Catarrhini and the Platyrrhini. However, the three loci are occupied by functional genes in the former infraorder and mostly by pseudogenes in the latter. Instead of the pseudogenes, the Platyrrhini have evolved a new set of apparently functional genes — DRB11 and DRB*W12 through DRB*W19, which have thus far not been found in the Catarrhini. The DRB*W13, *W14, *W15, *W17, *W18, and *W19 genes seem to be restricted to the Cebidae family, whereas the DRB*W16 locus has so far been documented in the Callitrichidae family only. The DRB alleles of the cotton-top tamarin, and perhaps also those of the common marmoset (both members of the family Callitrichidae), are characterized by low nucleotide diversity, possibly indicating that they diverged from a common ancestral gene relatively recently. Correspondence to: J. Klein.