The association between exposure determined by radiofrequency personal exposimeters and human exposure: A simulation study

The selection of an adequate exposure assessment approach is imperative for the quality of epidemiological studies. The use of personal exposimeters turned out to be a reasonable approach to determine exposure profiles, however, certain limitations regarding the absolute values delivered by the devices have to be considered. Apart from the limited dynamic range, it has to be taken into account that these devices give only an approximation of the exposure due to the influence of the body of the person carrying the exposimeter, the receiver characteristics of the exposimeter, as well as the dependence of the measured value on frequency band, channel, slot configuration, and communication traffic. In this study, the relationship between the field strength measured close to the human body at the location of the exposimeter and the exposure, that is, the field strength at the location of the human body without the human body present, is investigated by numerical means using the Visible Human model as an anatomical phantom. Two different scenarios were chosen: (1) For FM, GSM, and UMTS an urban outdoor scenario was examined that included a transmitting antenna mounted on the roof of one of four buildings at a street crossing, (2) For WLAN an indoor scenario was investigated. For GSM the average degree of underestimation by the exposimeter (relation of the average field levels at the location of the exposimeter to the field level averaged over the volume of the human body without the body present) was 0.76, and for UMTS 0.87; for FM no underestimation was found, the ratio was 1. In the case of WLAN the degree of underestimation was more pronounced, the ratio was 0.64. This study clearly suggests that a careful evaluation of correction factors for different scenarios is needed prior to the definition of the study protocol. It has to be noted that the reference scenario used in this study does not allow for final conclusions on general correction factors. Bioelectromagnetics 31:535–545, 2010. © 2010 Wiley‐Liss, Inc.     

Subcellular localization, oligomerization, and ATP-binding of Caenorhabditis elegans CED-4.

Caspase family cell death proteases are activated during apoptosis through the oligomerization of caspase-binding "adapter" proteins. In the nematode Caenorhabditis elegans one adapter protein, CED-4, exists. Here we report an analysis of CED-4 protein expressed in insect Sf9 cells by infection with recombinant baculovirus. During expression, CED-4 assumed a perinuclear spherical or reticular localization where it was partly resistant to extraction with nonionic detergents. Both purified FLAG-CED-4 and GST-FLAG-CED-4 proteins were present in solution as large complexes. FLAG-CED-4 complexes were estimated by gel filtration to have a molecular weight of approximately 500 kDa to >1.2 MDa, while GST-FLAG-CED-4 complexes appeared somewhat smaller. Unlike its mammalian homologue Apaf-1, CED-4 exhibited a marked preference for ATP over dATP in filter binding studies and in competition experiments. ATP hydrolysis was required neither for complex stability nor for binding of CED-3. These features are likely to be relevant for CED-4's function as a caspase adapter.     

Structure and architecture of the maize genome

Maize (Zea mays or corn) plays many varied and important roles in society. It is not only an important experimental model plant, but also a major livestock feed crop and a significant source of industrial products such as sweeteners and ethanol. In this study we report the systematic analysis of contiguous sequences of the maize genome. We selected 100 random regions averaging 144 kb in size, representing about 0.6% of the genome, and generated a high-quality dataset for sequence analysis. This sampling contains 330 annotated genes, 91% of which are supported by expressed sequence tag data from maize and other cereal species. Genes averaged 4 kb in size with five exons, although the largest was over 59 kb with 31 exons. Gene density varied over a wide range from 0.5 to 10.7 genes per 100 kb and genes did not appear to cluster significantly. The total repetitive element content we observed (66%) was slightly higher than previous whole-genome estimates (58%-63%) and consisted almost exclusively of retroelements. The vast majority of genes can be aligned to at least one sequence read derived from gene-enrichment procedures, but only about 30% are fully covered. Our results indicate that much of the increase in genome size of maize relative to rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana) is attributable to an increase in number of both repetitive elements and genes.     

A novel type of replicative enzyme harbouring ATPase,primase and DNA polymerase activity

Although DNA replication is a process common in all domains of life, primase and replicative DNA polymerase appear to have evolved independently in the bacterial domain versus the archaeal/eukaryal branch of life. Here, we report on a new type of replication protein that constitutes the first member of the DNA polymerase family E. The protein ORF904, encoded by the plasmid pRN1 from the thermoacidophile archaeon Sulfolobus islandicus, is a highly compact multifunctional enzyme with ATPase, primase and DNA polymerase activity. Recombinant purified ORF904 hydrolyses ATP in a DNA-dependent manner. Deoxynucleotides are preferentially used for the synthesis of primers approximately 8 nucleotides long. The DNA polymerase activity of ORF904 synthesizes replication products of up to several thousand nucleotides in length. The primase and DNA polymerase activity are located in the N-terminal half of the protein, which does not show homology to any known DNA polymerase or primase. ORF904 constitutes a new type of replication enzyme, which could have evolved independently from the eubacterial and archaeal/eukaryal proteins of DNA replication.     

Highly pathogenic avian influenza viruses do not inhibit interferon synthesis in infected chickens but can override the interferon-induced antiviral state

From infection studies with cultured chicken cells and experimental mammalian hosts, it is well known that influenza viruses use the nonstructural protein 1 (NS1) to suppress the synthesis of interferon (IFN). However, our current knowledge regarding the in vivo role of virus-encoded NS1 in chickens is much more limited. Here, we report that highly pathogenic avian influenza viruses of subtypes H5N1 and H7N7 lacking fully functional NS1 genes were attenuated in 5-week-old chickens. Surprisingly, in diseased birds infected with NS1 mutants, the IFN levels were not higher than in diseased birds infected with wild-type virus, suggesting that NS1 cannot suppress IFN gene expression in at least one cell population of infected chickens that produces large amounts of the cytokine in vivo. To address the question of why influenza viruses are highly pathogenic in chickens although they strongly activate the innate immune system, we determined whether recombinant chicken alpha interferon (IFN-α) can inhibit the growth of highly pathogenic avian influenza viruses in cultured chicken cells and whether it can ameliorate virus-induced disease in 5-week-old birds. We found that IFN treatment failed to confer substantial protection against challenge with highly pathogenic viruses, although it was effective against viruses with low pathogenic potential. Taken together, our data demonstrate that preventing the synthesis of IFN is not the primary role of the viral NS1 protein during infection of chickens. Our results further suggest that virus-induced IFN does not contribute substantially to resistance of chickens against highly pathogenic influenza viruses.     

Quantitative polymerase chain reaction as a reliable method to determine functional lentiviral titer after ex vivo gene transfer in human mesenchymal stem cells

BACKGROUND: Human mesenchymal stem cells (hMSCs) are a promising target for ex vivo gene therapy and lentiviruses are excellent gene transfer vehicles in hMSCs since they achieve high transduction rates with long-term gene expression. Nevertheless, senescence of hMSCs may limit therapeutic applications due to time-consuming cell selection and viral titration. Here, we describe a fast and reliable method to determine functional lentiviral titer by quantitative polymerase chain reaction (qPCR) after highly efficient ex vivo gene transfer in hMSCs. METHODS: Lentivirus production was tested with different types of packaging systems. Using p24 ELISA remaining viral particles were detected in the cell culture supernatant. The lentiviral gene transfer efficiency was quantified by FACS analysis. Lentiviral titers were determined by qPCR of expressed transgenes. RESULTS: Third-generation self-inactivating vectors showed highly efficient gene transfer in hMSCs. No viral antigen was detected in the cell culture supernatant after four media changes, suggesting the absence of infectious particles after 4 days. We observed a linear correlation between virus dilution and level of transgene expression by qPCR analysis, therefore allowing viral titering by quantification of transgene expression. Finally, we demonstrated that transduced hMSCs retained their stem cell character by differentiation towards adipogenic, osteogenic and chondrogenic lineages. CONCLUSIONS: Quantification of transgene copy numbers by qPCR is a fast and reliable method to determine functional lentiviral titer after ex vivo gene transfer in hMSCs.     

Developmental changes of nerve growth factor and its mRNA in the rat hippocampus: Comparison with choline acetyltransferase

Previous experiments have demonstrated that in the septo-hippocampal system choline acetyltransferase (ChAT) is induced by nerve growth factor (NGF) (Gnahn et al. (1983) Dev. Brain Res. 9, 45-52) and that hippocampal NGF and mRNANGF levels are correlated with the density of cholinergic innervation (Korsching et al. (1985) EMBO J. 4, 1389-1393). In the present investigation we have compared the developmental changes of ChAT, NGF, and mRNANGF levels in this system. During the postnatal development of the hippocampus the time courses of NGF and ChAT were well correlated including the most rapid increase between P12 and P14. This increase in hippocampal NGF was preceded by a corresponding increase in mRNANGF. The developmental changes in hippocampal NGF levels were also closely reflected by corresponding changes in the septum. This, together with previous observations (Korsching et al., 1985) that the adult septum, in spite of relatively high NGF levels, does not contain measurable quantities of mRNANGF, suggests that the NGF levels in the septum are determined by the quantity of NGF transported retrogradely from the field of innervation rather than by local synthesis. During the prenatal period hippocampal NGF levels were relatively high, whereas the mRNANGF was below the level of detection. Since the ingrowth of septal fibers, and with that also the removal of NGF by retrograde transport, begins around birth, the relatively high prenatal NGF levels probably result from an accumulation produced by a small copy number of mRNANGF prior to the removal of NGF by retrograde axonal transport. It is concluded that the correlation of the developmental changes in NGF and mRNANGF with the ChAT activity in the hippocampus further supports the concept of a physiological role of NGF in the central nervous system.     

Holistic differential analysis of embryo-induced alterations in the proteome of bovine endometrium in the preattachment period

During the peri-implantation period, molecular signaling between embryo and endometrium (layer of tissue lining the uterus lumen) is supposed to be crucial for the maintenance of pregnancy. To investigate embryo-induced alterations in the proteome of bovine endometrium in the preattachment period (day 18), we used monozygotic cattle twins (generated by embryo splitting) as a model eliminating genetic variability as a source for proteome differences. One of the twins was pregnant after the transfer of two in vitro produced blastocysts, while the corresponding twin received a sham-transfer and served as a nonpregnant control. The two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) analysis of the endometrium samples of three twin pairs (pregnant/nonpregnant) revealed four proteins with significantly higher abundance (p < 10(-9)) in each sample derived from the pregnant animals: Rho GDP dissociation inhibitor beta; 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD); soluble NADP(+)-dependent isocitrate dehydrogenase 1; and acyl-CoA-binding protein. To verify the accuracy of the 2-D DIGE quantification, the abundances of 20 alpha-HSD were quantified by a targeted cleavable isotope-coded affinity tag (ICAT) approach. The mass spectrometry-based ICAT quantification matched perfectly the results obtained by 2-D DIGE quantification, demonstrating the accuracy of our data. These results demonstrate that our model (monozygotic twins) in combination with the appropriate analytical tools is particularly suitable for the detection of the proteins involved in the embryo-maternal interactions.     

The size of mitochondrial DNA from a cytoplasmic petite mutant of Saccharomyces cerevisiae

Summary Mitochondrial DNA has been isolated from a cytoplasmic petite mutant of Saccharomyces cerevisiae which has retained only about 2% of the mitochondrial wild type genome. The denatured DNA was analyzed by agarose gel electrophoresis and a homogeneous, single band of DNA was found. Petite and wild type mitochondrial DNAs exhibited similar gel electrophoretic mobilities. Using denatured DNA from the E. coli phages T4 and T3 for comparison a molecular weight of 55×10⁶ daltons has been calculated for the double-stranded petite mitochondrial DNA. On the basis of this observation most of the mitochondrial DNA of this petite mutant appeared to consist of a polymer of about 50 repeats to account for a size similar to that of the wild type molecule. Thus a regulatory mechanism might exist which keeps constant the physical size of the mitochondrial DNA molecule in spite of the elimination of large fractions of the wild type genome.Dedicated to Dr. Dr. h. c. Peter Michaelis on the occasion of his 75th birthday