Construction of an M13 histidine-transducing phage: a single-stranded cloning vehicle with one EcoRI site.

In order to create a ready source of single-stranded DNA for DNA sequence determination by the dideoxy chain-termination method, the promoter-proximal part of the histidine operon, the hisOGD region of Salmonella typhimurium, was cloned onto the single-stranded phage M13. Both orientations of the his DNA were cloned to supply DNA template for sequencing of each strand. Insertion was achieved at an HaeIII site in the intergenic region (IR) of M13, and a single EcoRI site was purposely regenerated at one boundary of the his DNA insert. Infected colonies, not plaques, were selected using the hisD gene as a selective marker. The single RI site and the hisD marker for auxotrophic selection represent improvements on the wild type M13 as a single-stranded vector for cloning other DNA.     

Computational studies of tryptophanyl-tRNA synthetase: activation of ATP by induced-fit

Catalysis of amino acid activation by Bacillus stearothermophilus tryptophanyl-tRNA synthetase involves three allosteric states: (1) Open; (2) closed pre-transition state (PreTS); and (3) closed products (Product). The interconversions of these states entail significant domain motions driven by ligand binding. We explore the application of molecular dynamics simulations to investigate ligand-linked conformational stability changes associated with this catalytic cycle. Multiple molecular dynamics trajectories (5 ns) for 11 distinct liganded and unliganded monomer configurations show that the PreTS conformation is unstable in the absence of ATP, reverting within approximately 600 ps nearly to the Open conformation. In contrast, Open and Product state trajectories were stable, even without ligands, confirming the previous suggestion that catalysis entails destabilization of the protein conformation, driven by ATP-binding energies developed as the PreTS state assembles during induced-fit. The simulations suggest novel mechanistic details associated with both induced-fit (Open-PreTS) and catalysis (PreTS-Product). Notably, Mg2+ -ATP interactions are coupled to interactions between ATP and active-site lysine side-chains via mechanisms that cannot be captured under the molecular mechanics approximations, and which therefore require restraining potentials for stable simulation. Simulations of Mg2+. ATP-bound PreTS complexes with restraining potentials and with a virtual K111A mutant confirm that these coupling interactions are necessary to sustain the PreTS conformation and, in turn, provide a new model for how the PreTS conformation activates ATP for catalysis. These results emphasize the central role of the PreTS state as a high-energy intermediate structure along the catalytic pathway and suggest that Mg2+ and the KMSKS loop function cooperatively during catalysis.     

Radiofrequency radiation does not induce stress response in human T-lymphocytes and rat primary astrocytes

Heat shock proteins (HSPs) are rapidly induced by a variety of stressors, including heat shock, ethanol, heavy metals, UV, and gamma-radiation. Mitogen-activated protein kinases (MAPKs) are also involved in the stress transduction pathways in all eukaryotes. In this study, we attempted to determine whether radiofrequency (RF) radiation is able to induce a non-thermal stress response. Human T-lymphocyte Jurkat cells and rat primary astrocytes were exposed to 1763 MHz of RF radiation at an average specific absorption rate (SAR) of either 2 W/kg or 20 W/kg, for 30 min or 1 h. Temperature was completely controlled at 37 +/- 0.2 degrees C throughout the exposure period. The sham exposures were performed under exactly identical experimental conditions without exposure to RF radiation. We assessed alterations in the expression of HSPs and the activation of MAPKs in the RF-exposed cells. No detectable difference was observed in the expression levels of HSP90, HSP70, and HSP27. The phosphorylation status of MAPKs, extracellular signal-regulated kinases (ERK1/2), c-Jun N-terminal protein kinases (JNK1/2), or p38, did not change significantly. In order to determine whether RF radiation can promote the effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on stress response, cells were exposed to RF radiation coupled with TPA treatment. When TPA alone was applied, the MAPKs were found to be phosphorylated in a dose-dependent manner. However, RF radiation did not result in any enhancement of TPA-induced MAPK phosphorylation. Neither TPA nor RF radiation exerted any detectable effect on the induction of HSPs. These results indicate that 1763 MHz RF radiation alone did not elicit any stress response, nor did it have any effect on TPA-induced MAPK phosphorylation, under our experimental conditions.     

Interaction of radio frequency electromagnetic fields and passive metallic implants--a brief review

During the last decade, use of radio frequency (RF) applications like mobile phones and other wireless devices, has increased remarkably. This has triggered numerous studies related to possible health risks due to the exposure of RF electromagnetic (EM) fields. One safety aspect is the coupling of EM fields with active and passive implants in the human body. While interactions with active implants have been quite extensively researched, only a few studies have focused on passive implants. The present article reviews interaction mechanisms and studies of passive metallic, that is, conductive, implants in common external RF EM fields. It is found that implants have been mostly studied numerically, and experimental studies are rare. Furthermore, the studies cover mostly far-field conditions and only a few have studied implants in near fields. A summary of results indicates that a conductive object in tissues may cause notable local enhancement of the EM field and thus enhanced power absorption. The degree of enhancement depends, for example, on the orientation, the dimensions, the shape, and the location of the implant. However, in most of the cases, the field enhancement has not been strong enough to cause remarkable excess heating (more than 1 degrees C) of tissues.     

Lack of direct DNA damage in human blood leukocytes and lymphocytes after in vitro exposure to high power microwave pulses

Currently, the potential genotoxicity of high power microwave pulses (HPMP) is not clear. Using the alkaline single cell gel electrophoresis assay, also known as the alkaline comet assay, we studied the effects of HPMP (8.8 GHz, 180 ns pulse width, peak power 65 kW, pulse repetition frequency 50 Hz) on DNA of human whole-blood leukocytes and isolated lymphocytes. The cell suspensions were exposed to HPMP for 40 min in a rectangular waveguide. The average SAR calculated from the temperature kinetics was about 1.6 kW/kg (peak SAR was about 300 MW/kg). The steady-state temperature rise in the 50 microl samples exposed to HPMP was 3.5 +/- 0.1 degrees C. In independent experiments, we did not find any statistically significant DNA damage manifested immediately after in vitro HPMP exposure of human blood leukocytes or lymphocytes or after HPMP exposure of leukocytes subsequently incubated at 37 degrees C for 30 min. Our results indicate that HPMP under the given exposure conditions did not induce DNA strand breaks, alkali-labile sites, and incomplete excision repair sites, which could be detected by the alkaline comet assay.     

Molecular heterogeneity among north Indian isolates of Group A Streptococcus

AIM: To monitor molecular heterogeneity among the clinical isolates of group A Streptococcus (GAS) from north India by Vir and emm typing. METHODS AND RESULTS: GAS isolates, 31 from pharyngitis and nine from rheumatic fever (RF)/rheumatic heart disease (RHD) patients were differentiated into 16 Vir types (VT). These isolates were further discriminated into 23 emm types. Most of emm types were Vir type specific, except few (7.5%), which revealed different Vir types within same emm type. The most prevalent emm type found was emm 49 (15%) followed by 7.5% of emm 69, emm 71 and emm 75 which were different from emm type distribution reported from south India. CONCLUSIONS: Analysis of data revealed 40% heterogeneity by Vir typing and 57.5% by emm typing among GAS isolates which is significant in view of small number of isolates studied. SIGNIFICANCE OF IMPACT OF THE STUDY: The molecular study for the first time demonstrates different emm types prevalent and circulating in northern region of India and such data may help in selection of types for vaccine development.     

Dosimetric analysis of the carousel setup for the exposure of rats at 1.62 GHz

The so-called carousel setup has been widely utilized for testing the hypotheses of adverse health effects on the central nervous system (CNS) due to mobile phone exposures in the frequency bands 800-900 MHz. The objectives of this article were to analyze the suitability of the setup for the upper mobile frequency range, i.e., 1.4-2 GHz, and to conduct a detailed experimental and numerical dosimetry for the setup at the IRIDIUM frequency band of 1.62 GHz. The setup consists of a plastic base on which ten rats, restrained in radially positioned tubes, are exposed to the electromagnetic field emanating from a sleeved dipole antenna at the center. Latest generation miniaturized dosimetric E field and temperature probes were used to measure the specific absorption rate (SAR) inside the brain of three rat cadavers of the Lewis strain and two rat cadavers of the Fisher 344 strain. A numerical analysis was conducted on the basis of three numerical rat phantoms with voxel sizes between 1.5 and 0.125 mm3 that are based on high resolution MRI scans of a 300 g male Wistar rat and a 370 g male Sprague-Dawley rat. The average of the assessed SAR values in the brain was 2.8 mW/g per W antenna input power for adult rats with masses between 220 and 350 g and 5.3 mW/g per W antenna input power for a juvenile rat with a mass of 95 g. The strong increase of the SAR in the brain with decreasing animal size was verified by simulations of the absorption in numerical phantoms scaled to sizes between 100 and 500 g with three different scaling methods. The study also demonstrated that current rat phantom models do not provide sufficient spatial resolution to perform absolute SAR assessment for the brain tissue. The variation of the SAR(brain)(av) due to changes in position was assessed to be in the range from +15% to -30%. A study on the dependence of the performance of the carousel setup on the frequency revealed that efficiency, defined as SAR(brain)(av) per W antenna input power, and the ratio between SAR(brain)(av) and SAR(body)(av) are optimal in the mobile communications frequency range, i.e., 0.8-3 GHz.     

HemK2 protein, encoded on human chromosome 21, methylates translation termination factor eRF1

The ubiquitous tripeptide Gly-Gly-Gln in class 1 polypeptide release factors triggers polypeptide release on ribosomes. The Gln residue in both bacterial and yeast release factors is N5-methylated, despite their distinct evolutionary origin. Methylation of eRF1 in yeast is performed by the heterodimeric methyltransferase (MTase) Mtq2p/Trm112p, and requires eRF3 and GTP. Homologues of yeast Mtq2p and Trm112p are found in man, annotated as an N6-DNA-methyltransferase and of unknown function. Here we show that the human proteins methylate human and yeast eRF1.eRF3.GTP in vitro, and that the MTase catalytic subunit can complement the growth defect of yeast strains deleted for mtq2.     

Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells

RNF8 is a nuclear protein having an N-terminal forkhead-associated (FHA) domain and a C-terminal RING-finger (RF) domain. Depletion of RNF8 caused cell growth inhibition and cell cycle arrest at not only S but also G2/M phases. In addition, cell death was frequently observed in RNF8-depleted cells. Analyses of time-lapse microscopy revealed that the cells died in mitosis and interphase. To elucidate the RNF8 function in M phase, the Plk1 content in RNF8-depleted cells was examined. The amount of RNF8 decreased time-dependently, whereas Plk1 reciprocally increased by transfection of RNF8 siRNA. Protein contents of RNF8 and Plk1 among various cell lines were also compared. RNF8 in normal cell lines was much higher than that in many cancer cell lines. Conversely, Plk1 in normal cell lines was lower than in cancer cell lines. These results suggest that RNF8 is downregulated in many cancer cells and inversely correlated with Plk1.     

2.45 GHz radiofrequency fields alter gene expression in cultured human cells

The biological effect of radiofrequency (RF) fields remains controversial. We address this issue by examining whether RF fields can cause changes in gene expression. We used the pulsed RF fields at a frequency of 2.45 GHz that is commonly used in telecommunication to expose cultured human HL-60 cells. We used the serial analysis of gene expression (SAGE) method to measure the RF effect on gene expression at the genome level. We observed that 221 genes altered their expression after a 2-h exposure. The number of affected genes increased to 759 after a 6-h exposure. Functional classification of the affected genes reveals that apoptosis-related genes were among the upregulated ones and the cell cycle genes among the downregulated ones. We observed no significant increase in the expression of heat shock genes. These results indicate that the RF fields at 2.45 GHz can alter gene expression in cultured human cells through non-thermal mechanism.