A B S T R A C T We have demonstrated that human plasma contains a heparin-dependent inhibitor of thrombin that is distinguishable from antithrombin III (AT III). When a 1:50 dilution of plasma was incubated with .0.01 U/ml heparin and 1 U/ml 1251-thrombin, the labeled thrombin B-chains became incorporated into two complexes ofMr-96,000 and M,-85,000 that were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and f3-mercaptoethanol. Neither complex was detectable at heparin concentrations <0.01 U/ml. When a limiting amount of 1251-thrombin was present, the proportion of radioactivity incorporated into each of the two complexes varied with the heparin concentration. Thus, the Mr-85,000 complex predominated at 0.01-5 U/ml heparin, whereas the Mr96,000 complex predominated at 5-100 U/ml heparin. The Mr, 85,000 complex reacted with antibodies to human AT III and comigrated with the purified thrombin-AT III complex. The Mr-96,000 complex did not react with antibodies to AT III or to al-antitrypsin, and it was detected in normal quantities after incubating 1251-thrombin with plasma immunodepleted of AT III, a2-antiplasmin, a2-macroglobulin, Cl inactivator, al-antichymotrypsin, or inter-a-trypsin inhibitor. The protein that combines with thrombin to form the M,-96,000 complex was estimated to be present at a minimum concentration of 90+26 ,ug/ml (mean±SD) in normal plasma. We conclude that the protein is not identical to any of the known plasma protease inhibitors and that at relatively high heparin concentrations in vitro it reacts with thrombin more rapidly than does AT III.

A 900 base pair segment of the c-myc promoter, containing eight nCTCTn sequences, is required for the induction of c-myc expression by electromagnetic (EM) fields. Similarly, a 70 bp region of the HSP70 promoter, containing three nCTCTn sequences, is required for the induction of HSP70 expression by EM fields. Removal of the 900 base pair segment of the c-myc promoter eliminates the ability of EM fields to induce c-myc expression. Similarly, removal of the 70 bp region of the HSP70 promoter, with its three nCTCTn sequences, eliminates the response to EM fields. The nCTCTn sequences apparently act as electromagnetic field response elements (EMRE). To test if introducing EMREs imparts the ability to respond to applied EM fields, the 900 bp segment of the c-myc promoter (containing eight EMREs) was placed upstream of CAT or luciferase reporter constructs that were otherwise unresponsive to EM fields. EMREs-reporter constructs were transfected into HeLa cells and exposed to 8 microT 60 Hz fields. Protein extracts from EM field-exposed transfectants had significant increases in activity of both CAT and luciferase, compared with identical transfectants that were sham-exposed. Transfectants with CAT or luciferase constructs lacking EMREs remained unresponsive to EM fields, i.e., there was no increase in either CAT or luciferase activity. These data support the idea that EMREs can be used as switches to regulate exogenously introduced genes in gene therapy.

Low frequency (< 300 Hz) electromagnetic (EM) fields induce biological changes that include effects ranging from increased enzyme reaction rates to increased transcript levels for specific genes. The induction of stress gene HSP70 expression by exposure to EM fields provides insight into how EM fields interact with cells and tissues. Insights into the mechanism(s) are also provided by examination of the interaction of EM fields with moving charges and their influence on enzyme reaction rates in cell-free systems. Biological studies with in vitro model systems have focused, in general, on the nature of the signal transduction pathways involved in response to EM fields. It is likely, however, that EM fields also interact directly with electrons in DNA to stimulate biosynthesis. Identification of an EM field-sensitive DNA sequence in the heat shock 70 (HSP70) promoter, points to the application of EM fields in two biomedical applications: cytoprotection and gene therapy. EM field induction of the stress protein hsp70 may also provide a useful biomarker for establishing a science-based safety standard for the design of cell phones and their transmission towers.

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Electromagnetic fields (EMF), in both ELF (extremely low frequency) and radio frequency (RF) ranges, activate the cellular stress response, a protective mechanism that induces the expression of stress response genes, e.g., HSP70, and increased levels of stress proteins, e.g., hsp70. The 20 different stress protein families are evolutionarily conserved and act as 'chaperones' in the cell when they 'help' repair and refold damaged proteins and transport them across cell membranes. Induction of the stress response involves activation of DNA, and despite the large difference in energy between ELF and RF, the same cellular pathways respond in both frequency ranges. Specific DNA sequences on the promoter of the HSP70 stress gene are responsive to EMF, and studies with model biochemical systems suggest that EMF could interact directly with electrons in DNA. While low energy EMF interacts with DNA to induce the stress response, increasing EMF energy in the RF range can lead to breaks in DNA strands. It is clear that in order to protect living cells, EMF safety limits must be changed from the current thermal standard, based on energy, to one based on biological responses that occur long before the threshold for thermal changes. © 2009 Elsevier Ireland Ltd. All rights reserved.Keywords: DNA; Biosynthesis; Electromagnetic fields; ELF; RF Electromagnetic fields (EMF) alter protein synthesisUntil recently, genetic information stored in DNA was considered essentially invulnerable to change as it was passed on from parent to progeny. Mutations, such as those caused by cosmic radiation at the most energetic end of the EM spectrum, were thought to be relatively infrequent. The model of gene regulation was believed to be that the negatively charged DNA was tightly wrapped up in the nucleus with positively charged histones, and that most genes were 'turned off' most of the time. Of course, different regions of the DNA code are being read more or less all the time to replenish essential Abbreviations: EMF, electromagnetic fields; Hz, hertz; ELF, extremely low frequency; RF, radio frequency; MAPK, mitogen activated protein kinase; ERK1\2, extracellular signal regulated kinase; JNK, c-Jun-terminal kinase p38MAPK; SAPK, stress activated protein kinase; NADH, nicotinamide adenine dinucleotide dehydrogenase; ROS, reactive oxygen species.* Corresponding author at: Department of Physiology, Columbia Univer- proteins that have broken down and those needed during cell division. New insights into the structure and function of DNA have resulted from numerous, well-done laboratory studies. The demonstration that EMF induces gene expression and the synthesis of specific proteins [1,2] generated considerable controversy from power companies, government agencies, physicists, and most recently, cell phone companies. Physicists have insisted that the reported results were not possible because there was not enough energy in the power frequency range (ELF) to activate DNA. They were thinking solely of mechanical interaction with a large molec...