TATA Binding Proteins Can Recognize Nontraditional DNA Sequences

We demonstrate an accurate, quantitative, and label-free optical technology for high-throughput studies of receptor-ligand interactions, and apply it to TATA binding protein (TBP) interactions with oligonucleotides. We present a simple method to prepare single-stranded and double-stranded DNA microarrays with comparable surface density, ensuring an accurate comparison of TBP activity with both types of DNA. In particular, we find that TBP binds tightly to single-stranded DNA, especially to stretches of polythymine (poly-T), as well as to the traditional TATA box. We further investigate the correlation of TBP activity with various lengths of DNA and find that the number of TBPs bound to DNA increases >7-fold as the oligomer length increases from 9 to 40. Finally, we perform a full human genome analysis and discover that 35.5% of human promoters have poly-T stretches. In summary, we report, for the first time to our knowledge, the activity of TBP with poly-T stretches by presenting an elegant stepwise analysis of multiple techniques: discovery by a novel quantitative detection of microarrays, confirmation by a traditional gel electrophoresis, and a full genome prediction with computational analyses.     

N-acetyl-L-cysteine abrogates fibrogenic properties of fibroblasts isolated from Dupuytren's disease by blunting TGF-beta signalling

Dupuytren's disease, a benign fibroproliferative disorder of the palmar fascia, represents an ideal model to study tissue fibrosis. Transforming growth factor-beta1 (TGF-beta1) and its downstream Smad signalling system is well established as a key player during fibrogenesis. Thus, targeting this basic pathomechanism seems suitable to establish new treatment strategies. One such promising treatment involves the substance N-acetyl-L-cysteine (NAC), shown to have antifibrotic properties in hepatic stellate cells and rat fibroblasts. In order to investigate antifibrotic effects of N-acetyl-L-cysteine (NAC), fibroblasts were isolated from surgically resected fibrotic palmar tissues (Dupuytren fibroblasts, DF) and exposed to different concentrations of NAC and recombinant TGF-beta1. Fibroblasts isolated from tendon pulleys served as controls (control fibroblasts, CF). Smad signalling was investigated by a Smad binding element driven reporter gene analysis. Both cell types express TGF-beta1, indicating autocrine signalling in DF and CF. This was confirmed by comparing reporter gene activity from LacZ and Smad7 adenovirus infected cells. NAC treatment resulted in abrogation of Smad mediated signalling comparable to ectopically overexpressed Smad7, even when the cells were stimulated with recombinant TGF-beta1 or ectopically expressed a constitutively active TGF-beta receptor type I. Additionally, NAC dose-dependently decreased expression of three major indicators of impaired fibrotic matrix turnover, namely alpha-smooth muscle actin (alpha-SMA), alpha 1 type I procollagen (Col1A1), and plasminogen activator inhibitor-type I (PAI-1). Our results suggest that TGF-beta signalling and subsequent expression of fibrogenesis related proteins in Dupuytren's disease is abrogated by NAC thus providing a basis for a therapeutic strategy in Dupuytren's disease and other fibroproliferative disorders.     

Review of in vivo static and ELF electric fields studies performed at Gazi Biophysics Department

In vivo effects of Static Electric and ELF Magnetic and Electric fields have been carried out for more than 20 years in the Bioelectromagnetic Laboratory at the Biophysics Department of the Medical Faculty of Gazi University. In this article, the results of in vivo ELF Electric field studies are presented as a review. Static and 50 Hz ELF (Extremely Low Frequency) Electric (E) fields effects on free radical synthesis, antioxidant enzyme level, and collagen synthesis were analyzed on tissues of guinea pigs, such as brain, liver, lung, kidney, spleen, testis, and plasma. Animals were exposed to static and ELF electric fields with intensities ranging from 0.3 kV/m to 1.9 kV/m in vertical and horizontal directions. Exposure periods were 1, 3, 5, 7, and 10 days. Electric fields were generated from a specially designed parallel plate capacitor system. The results indicate that the effects of electric fields on the tissues studied depend significantly on the type and magnitude of electric field and exposure period.     

TLR-2-activated B cells suppress Helicobacter-induced preneoplastic gastric immunopathology by inducing T regulatory-1 cells

B cells regulate autoimmune pathologies and chronic inflammatory conditions such as autoimmune encephalomyelitis and inflammatory bowel disease. The potential counterregulatory role of B cells in balancing pathogen-specific immune responses and the associated immunopathology is less well understood owing to the lack of appropriate persistent infection models. In this paper, we show that B cells have the ability to negatively regulate adaptive immune responses to bacterial pathogens. Using mouse models of infection with Helicobacter felis, a close relative of the human gastrointestinal pathogen H. pylori, we found that B cells activated by Helicobacter TLR-2 ligands induce IL-10-producing CD4(+)CD25(+) T regulatory-1 (Tr-1)-like cells in vitro and in vivo. Tr-1 conversion depends on TCR signaling and a direct T-/B-interaction through CD40/CD40L and CD80/CD28. B cell-induced Tr-1 cells acquire suppressive activity in vitro and suppress excessive gastric Helicobacter-associated immunopathology in vivo. Adoptive cotransfer of MyD88-proficient B cells and Tr-1 cells restores a normal gastric mucosal architecture in MyD88(-/-) and IL-10(-/-) mice in a manner that depends on T cellular, but not B cellular, IL-10 production. Our findings describe a novel mechanism of B cell-dependent Tr-1 cell generation and function in a clinically relevant disease model. In conclusion, we demonstrate that the B cell/Tr-1 cell axis is essential for balancing the control of Helicobacter infection with the prevention of excessive Th1-driven gastric immunopathology, promoting gastric mucosal homeostasis on the one hand and facilitating Helicobacter persistence on the other.     

Extremely low-frequency magnetic fields of transformers and possible biological and health effects

Physiological processes in organisms can be influenced by extremely low-frequency (ELF) electromagnetic energy. Biological effect studies have great importance; as well as measurement studies since they provide information on the real exposure situations. In this study, the leakage magnetic fields around a transformer were measured in an apartment building in Küçükçekmece, Istanbul, and the measurement results were evaluated with respect to the international exposure standards. The transformer station was on the bottom floor of a three-floor building. It was found that people living and working in the building were exposed to ELF magnetic fields higher than the threshold magnetic field value of the International Agency for Research on Cancer (IARC). Many people living in this building reported health complaints such as immunological problems of their children. There were child-workers working in the textile factories located in the building. Safe distances or areas for these people should be recommended. Protective measures could be implemented to minimize these exposures. Further residential exposure studies are needed to demonstrate the exposure levels of ELF magnetic fields. Precautions should, therefore, be taken either to reduce leakage or minimize the exposed fields. Shielding techniques should be used to minimize the leakage magnetic fields in such cases.     

Scoring the collective effects of SNPs: association of minor alleles with complex traits in model organisms

It has long been assumed that most parts of a genome and most genetic variations or SNPs are non-functional with regard to reproductive fitness.However,the collective effects of SNPs have yet to be examined by experimental science.We here developed a novel approach to examine the relationship between traits and the total amount of SNPs in panels of genetic reference populations.We identified the minor alleles(MAs)in each panel and the MA content(MAC)that each inbred strain carried for a set of SNPs with genotypes determined in these panels.MAC was nearly linearly linked to quantitative variations in numerous traits in model organisms,including life span,tumor susceptibility,learning and memory,sensitivity to alcohol and anti-psychotic drugs,and two correlated traits poor reproductive fitness and strong immunity.These results suggest that the collective effects of SNPs are functional and do affect reproductive fitness.     

Mobile Phone Radiation Alters Proliferation of Hepatocarcinoma Cells

This study investigated the effects of intermittent exposure (15 min on, 15 min off for 1, 2, 3, or 4 h, at a specific absorption rate of 2 W/kg) to enhanced data rates for global system for mobile communication evolution-modulated radiofrequency radiation (RFR) at 900- and 1,800-MHz frequencies on the viability of the Hepatocarcinoma cells (Hep G2). Hep G2 cell proliferation was measured by a colorimetric assay based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells. Cell injury was evaluated by analyzing the levels of lactate dehydrogenase (LDH) and glucose released from lysed cells into the culture medium. Morphological observation of the nuclei was carried out by 4′,6-diamidino-2-phenylindole (DAPI) staining using fluorescence microscopy. In addition, TUNEL assay was performed to confirm apoptotic cell death. It was observed that cell viability, correlated with the LDH and glucose levels, changed according to the frequency and duration of RFR exposure. Four-hour exposure produced more pronounced effects than the other exposure durations. 1,800-MHz RFR had a larger impact on cell viability and Hep G2 injury than the RFR at 900 MHz. Morphological observations also supported the biochemical results indicating that most of the cells showed irregular nuclei pattern determined by using the DAPI staining, as well as TUNEL assay which shows DNA damage especially in the cells after 4 h of exposure to 1,800-MHz RFR. Our results indicate that the applications of 900- and 1,800-MHz (2 W/kg) RFR cause to decrease in the proliferation of the Hep G2 cells after 4 h of exposure. Further studies will be conducted on other frequency bands of RFR and longer duration of exposure.