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The geographical distribution of Bacillus anthracis strains and isolates bearing some of the same genetic markers as the Amerithrax Ames isolate was examined and evaluated. At least one mechanism for the horizontal movement of genetic markers was shown amongst isolates and closely related species and the effect of such mixing was demonstrated on phenotype. The results provided potential mechanisms by which attempts to attribute isolates of Bacillus anthracis to certain geographical and isolate sources may be disrupted.

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The existence of specific bioeffects due to high peak power microwaves and their potential health hazards are among the most debated but least explored problems in microwave biology. The present study attempted to reveal such effects by comparing the bioeffects of short trains of extremely high power microwave pulses (EHPP, 1 micros width, 250-350 kW/g, 9.2 GHz) with those of relatively low power pulses (LPP, 0.5-10 s width, 3-30 W/g, 9.2 GHz). EHPP train duration and average power were made equal to those of an LPP; therefore both exposure modalities produced the same temperature rise. Bioeffects were studied in isolated, spontaneously beating slices of the frog heart. In most cases, a single EHPP train or LPP immediately decreased the inter-beat interval (IBI). The effect was proportional to microwave heating, fully reversible, and easily reproducible. The magnitude and time course of EHPP- and LPP-induced changes always were the same. No delayed or irreversible effects of irradiation were observed. The same effect could be repeated in a single preparation numerous times with no signs of adaptation, sensitization, lasting functional alteration, or damage. A qualitatively different effect, namely, a temporary arrest of preparation beats, could be observed when microwave heating exceeded physiologically tolerable limits. This effect also did not depend on whether the critical temperature rise was produced by LPP or EHPP exposure. Within the studied limits, we found no indications of EHPP-specific bioeffects. EHPP- and LPP-induced changes in the pacemaker rhythm of isolated frog heart preparation were identical and could be entirely attributed to microwave heating.

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Sustained whole-body exposure of anesthetized rats to 35-GHz radio frequency radiation produces localized hyperthermia and hypotension, leading to circulatory failure and death. The physiological mechanism underlying the induction of circulatory failure by 35-GHz microwave (MW) heating is currently unknown. We hypothesized that oxidative stress may play a role in the pathophysiology of MW-induced circulatory failure and examined this question by probing organs for 3-nitrotyrosine (3-NT), a marker of oxidative stress. Animals exposed to low durations of MW that increased colonic temperature but were insufficient to produce hypotension showed a 5- to 12-fold increase in 3-NT accumulation in lung, liver, and plasma proteins relative to the levels observed in control rats that were not exposed to MW. 3-NT accumulation in rats exposed to MW of sufficient duration to induce circulatory shock returned to low, baseline levels. Leukocytes obtained from peripheral blood showed significant accumulation of 3-NT only at exposure levels associated with circulatory shock. 3-NT was also found in the villus tips and vasculature of intestine and within the distal tubule of the kidney but not in the irradiated skin of rats with MW-induced circulatory failure. The relationship between accumulation in liver, lung, and plasma proteins and exposure duration suggests either that nitro adducts are formed in the first 20 min of exposure and are then cleared or that synthesis of nitro adducts decreases after the first 20 min of exposure. Taken together, these findings suggest that oxidative stress occurs in many organs during MW heating. Because nitration occurs after microwave exposures that are not associated with circulatory collapse, systemic oxidative stress, as evidenced by tissue accumulation of 3-NT, is not correlated with circulatory failure in this model of shock.

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Bacillus anthracis has been recognized as a highly likely biological warfare or terrorist agent. We have designed culture techniques to rapidly isolate and identify "live" anthrax from suspected environmental release. A special medium (3AT medium) allows for discrimination between closely related bacilli and non-pathogenic strains. Nitrate was found to be a primary factor influencing spore formation in Bacillus anthracis. Nitrate reduction in anthrax is not an adaptation to saprophytic environmental existence, but it is a signal to enhance environmental survival upon the death of the anthrax host, which can be mimicked in culture.

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Systematic evolution of ligands by exponential enrichment (SELEX) was used to select and PCR amplify DNA sequences (aptamers) capable of binding to and detecting nonpathogenic Sterne strain Bacillus anthracis spores. A simplified affinity separation approach was employed, in which autoclaved anthrax spores were used as the separation matrix. An aptamer-magnetic bead-electrochemiluminescence (AM-ECL) sandwich assay scheme was devised for detecting anthrax spores. Using a low SELEX DNA to spore ratio (154 ng DNA/10(6) spores), at least three distinct populations of single-stranded DNA aptamers, having varied affinities for anthrax spores, were noted by the AM-ECL assay. Results reflect detection of spore components with a dynamic range equivalent to < 10- > 6 x 10(6) anthrax spores. In the low DNA to spore ratio experiments, aptamers could be liberated from spore pellets by heating at 96 degrees C for 5 min after each round of SELEX. When a much higher DNA to spore ratio (10,256 ng DNA/10(6) spores) was used for SELEX development, a higher affinity set of aptamers was selected that could not be heat-eluted even at 99 degrees C for 5 min following round four of SELEX. However, high affinity spore surface bound aptamers were detectable via their 5'-biotinylated tails using labeled avidin and could be eluted in deionized water. Aptamers have potential for use as inexpensive, in vitro-generated receptors for biosensors in biological warfare detection and other areas.

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In the presence of 3-amino-L-tyrosine (3-AT), abundant brown pigment forms in human HL-60 cells, but not in a variety of other cell lines, which are reported to be lower in mean myeloperoxidase (MPO) content than HL-60. Cells were assessed for peroxidase activity with an ABTS-based colorimetric assay and compared to values obtained with known amounts of human myeloperoxidase. HL-60 cells were estimated to contain the equivalent of 37.1 ng myeloperoxidase/10(6) cells versus 26.1 and 5.0 ng/10(6) cells for human K562 and murine RAW 264.7 cell lines, respectively. HL-60 cells exhibited a nearly 60% inhibition of proliferation and > 70% reduction in cell viability after 4 d of culture in the presence of 100 microg 3-AT per ml. Higher concentrations of 3-AT (up to 400 microg/ml) for 4 d reduced HL-60 proliferation by 80% and decreased viability to 1-3%. Comparable levels of cytotoxicity were achieved in KG-1 cells after 7 d with 200 or 400 microg 3-AT per ml. K562 cells exhibited a 40% reduction in cell number after 7 d with 400 microg 3-AT per ml, but concentrations less than 400 microg/ml did not significantly affect K562 proliferation. K562 viability remained unchanged with doses of 3-AT up to 400 microg/ml. RAW 264.7 cells exhibited unchanged viability and proliferation in the presence of 3-AT at concentrations up to 400 microg 3-AT per ml. K562, KG-1, and RAW 264.7 cells exhibited no evidence of brown pigment formation in the presence of 3-AT and medium containing 10% fetal bovine serum. However, RAW 264.7 cells that were converted to protein-free medium and exposed to 3-AT exhibited intense brown pigment in some cell nuclei. A high percentage of HL-60 cells treated with 3-AT exhibited membrane blebbing, pyknosis, and nuclear fragmentation, which was not observed among other 3-AT-treated cell lines. A mechanism involving toxic intermediates of peroxidase-mediated "aminomelanin" formation is hypothesized.

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Thermoluminescent dosimetry has been the industry standard for ionizing radiation dosimetry because it is inexpensive, sensitive, and accurate. No such system exists for radio frequency radiation. This paper describes the state of the art of efforts toward developing such a system. Thermochemiluminescent (TCL) dosimetry, first reported in 1991, is a first step toward achieving this goal. However, it has had problems in the production of TCL materials and in conversion of the luminescent signal into specific absorption rate (SAR). The former problem has been solved by the development of a genetically engineered Escherichia coli bacterium (JM 109/plC20RNR1.1), described herein, that produces the TCL material in a fermentation process. The latter problem stems from the difficulty in determining the structure of the currently best TCL material diazoluminomelanin. A theoretical approach for the solution of this problem has been achieved by combining equations for delayed fluorescence, temperature determination by TCL, and the free energy equation for equilibrium reactions. It has led to an explanation for the stable display of steady-state energy disposition, illustrated by TCL, in phantoms without the expected disruption by thermal conduction or convection, at frequencies ranging from 2.06 GHz to 35 GHz.

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We studied insulin, fasting glucose, and sex hormone-binding globulin (SHBG) in Air Force veterans exposed to Agent Orange and its contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), during the Vietnam War. The index subjects were veterans of Operation Ranch Hand, the unit responsible for aerial herbicide spraying in Vietnam from 1962-1971. Other Air Force veterans who served in Southeast Asia during the same period but were not involved with spraying herbicides served as comparisons. We assigned each Ranch Hand veteran based on his dioxin level to one of three exposure categories, named background, low, and high. Among nondiabetic veterans, we found the mean of the logarithm of insulin significantly increased in the high dioxin category. Additionally, in nondiabetic veterans the relation between SHBG and insulin interacted significantly with dioxin category on the log scale within strata defined by age and percent body fat. Among young (age, < or = 53 yr), lean (percent body fat, < or = 25%) nondiabetic veterans in the high category, the slope relating the logarithm of SHBG and the logarithm of insulin was significantly decreased. These findings suggest a compensatory metabolic relationship between dioxin and insulin regulation.

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Intense flashes of light were observed in sodium bicarbonate and hydrogen peroxide solutions when they were exposed to pulsed microwave radiation, and the response was greatly enhanced by a microwave-absorbing, biosynthesized polymer, diazoluminomelanin. A FPS-7B radar transmitter, operating at 1.25 GHz provided pulses of 5.73 +/- 0.09 micros in duration at 10.00 +/- 0.03 pulses/s with 2.07 +/- 0.08 MW forward power (mean +/- standard deviation), induced the effect but only when the appropriate chemical interaction was present. This phenomenon involves acoustic wave generation, bubble formation, pulsed luminescence, ionized gas ejection, and electrical discharge. The use of pulsed microwave radiation to generate highly focused energy deposition opens up the possibility of a variety of biomedical applications, including targeting killing of microbes or eukaryotic cells. The full range of microwave intensities and frequencies that induce these effects has yet to be explored and, therefore, the health and safety implications of generating the phenomena in living tissues remain an open question.

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Electrochemiluminescence (ECL) studies of the chemiluminescent (CL) polymer diazoluminomelanin (DALM) biosynthesized in nitrate reductase transfected Escherichia coli JM109 bacteria revealed noteworthy anodic ECL and even more intense cathodic ECL. Bacterial DALM (BD) ECL was also assessed in the presence of 100 ppm of 33 different metal and non-metal ions which revealed specific anodic, but not cathodic, enhancements of BD ECL with Ag+, Hg2+ and Ru3+. The precursors and intermediate polymers which comprise DALM, such as luminol, 3-amino-L-tyrosine (3-AT), aminomelanin (AM) and diazomelanin (DM) were screened for ECL enhancement against the same set of elemental ions. Significant anodic ECL enhancements were observed for luminol with Hg2+ in the presence of tripropylamine (TPA), but not for any other DALM component in combination with other elemental ions, either anodically or cathodically. Comparison of BD with luminol in the presence and absence of TPA and Hg2+ revealed very different ECL activity patterns and suggested different mechanisms for BD and luminol ECL.

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Development of new emitter systems capable of producing high-peak-power electromagnetic pulses with very fast rise times and narrow pulse widths is continuing. Such directed energy weapons systems will be used in the future to defeat electronically vulnerable targets. Human exposures to these pulses can be expected during testing and operations. Development of these technologies for radar and communications purposes has the potential for wider environmental exposure, as well. Current IEEE C95.1-1991 human exposure guidelines do not specifically address these types of pulses, though limits are stated for pulsed emissions. The process for developing standards includes an evaluation of the relevant bioeffects data base. A recommendation has been made that human exposure to ultrashort electromagnetic pulses that engender electromagnetic transients, called precursor waves, should be avoided. Studies that purport to show the potential for tissue damage induced by such pulses were described. The studies cited in support of the recommendation were not relevant to the issues of tissue damage by propagated pulses. A number of investigations are cited in this review that directly address the biological effects of electromagnetic pulses. These studies have not shown evidence of tissue damage as a result of exposure to high-peak-power pulsed microwaves. It is our opinion that the current guidelines are sufficiently protective for human exposure to these pulses.

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Nitrate reductase (NR) gene fragments (1.1 kb and 800 bp) from the barley plant were incorporated into pSV2neo and transfected by electroporation into a variety of cell lines of different functionality. Only transfected murine macrophage cell lines demonstrated appreciably enhanced NO2- production (i.e., NR activity) both in the presence and absence of exogenous nitrate (NO3-). Addition of NO3- caused the greatest increase in NO2- production when macrophages were primed with interferon-gamma (INF-gamma) and lipopolysaccharide (LPS). Transfection of RAW 264.7 murine macrophages led to isolation of several novel neomycin-resistant subpopulations designated NR10(1), NR10(2) (both containing the 1.1 kb NR fragment) and NR800(5) (containing the 800 bp NR fragment). Similarly transfected nonleukocytic and leukocytic stem cell lines showed no significant NO2- production. Outside of the macrophage cell lines, only the murine T cell line EL-4 showed evidence of mild nitrite production enhancement. The mechanism of enhanced NO2- formation in NR transfected murine macrophages is unknown. However, study of these novel cells may lead to greater understanding of the expression of a plant NR in mammalian cells and highly controlled production of a cytotoxic molecule (NO2-) in macrophages.

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The U.S. Air Force is concerned with the health and safety of personnel working with emitters of nonionizing electromagnetic radiation. Therefore, an understanding of basic mechanisms of interaction of this radiation with biosystems is essential. A nonresonance, nonclassical mechanism exists for interactions with biosystems under low energy (isothermal) field conditions. This mechanism is slow, or delayed, luminescence. Such a mechanism can form the basis for very sensitive biosensors naturally or artificially connected to metabolic and synthetic cellular processes.

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Chemical and bacterial synthesis of a thermochemiluminescent polymer known as diazoluminomelanin (DALM) has been previously reported. This paper focuses on the intracellular synthesis of aminomelanin (AM) in mammalian cell lines and subsequent DALM synthesis from this core molecule. B16 melanoma cells, HL-60 myeloid leukemia cells, and RAW 264.7 macrophages show AM and DALM production. Macroscopic image analysis of HL-60 cell lysates containing DALM using the Quantitative Luminescence Imaging System (QLIS) showed increased chemiluminescence (CL) with increased microwave power input and increased temperature. This work represents a first step toward the goal of microscopic radiofrequency dosimetry of individual DALM-loaded cells using image analysis.

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This paper describes use of a novel substituted melanin which is useful in detection of differentiating leukemia cells and their membranes. Comparisons of luminol-(5-amino-2,3-dihydro-1,4-phthalazinedione) and diazoluminomelanin (DALM)-mediated chemiluminescence (CL) were made with various types of differentiated and undifferentiated HL-60 whole cells, cell lysates, and membrane fractions. Luminol had a greater CL response than DALM with HL-60 promyelocytic stem cells and differentiated macrophage-like or neutrophil-like whole cell and cell lysate preparations. However, DALM showed markedly greater CL than luminol for membrane fractions derived from each cell type. The greatest luminol-dependent CL was observed for cell types high in myeloperoxidase (MPO). The greatest DALM-mediated CL was seen with cell types that are high in MPO or strong producers of superoxide (O2-) anions. In some cases, significant differences in CL could also be distinguished on the basis of inducing agent used [i.e. dimethylsulfoxide, all-trans retinoic acid or 12-o-tetradecanoylphorbol-13-acetate]. Both luminol- and DALM-dependent CL were strongly inhibited by preincubation of cellular preparations with 3-amino-L-tyrosine (a component of DALM). Taken together, these data suggest that the reaction mechanism of luminol favors interaction with cytoplasmic MPO whereas that of DALM favors membrane interactions. Thus, both reagents may be of use in assays to detect differentiating leukocytes or their cellular components.

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The purpose of this work was to synthesize a water-soluble derivative of 5-amino-2, 3-dihydro-1, 4-phthalazinedione (luminol) that generated sustained high level luminescence under physiologic conditions without the necessity of a catalyst. The derivative was made by a diazotization reaction with luminol and 3-amino-L-tyrosine. The resulting orange-brown anionic polymer has been given the trivial name of diazoluminomelanin (DALM). It was water soluble above and insoluble at or below pH 5.0. DALM luminesced when treated with hydrogen peroxide without the presence of a catalyst at pHs ranging from 6.5 to 12.0. Microgram quantities produced high levels of chemiluminescence for longer than 52 hr. Dried polymer generated a long-term stable electron spin resonance spectrum. The long-term chemiluminescence of DALM at pH 6.8-7.4 makes it a potentially useful reagent for detecting free radicals and peroxides in cellular and biochemical preparations.

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Peroxidation of luminol (5-amino-2,3-dihydrophthalazine-1,4-dione) catalyzed by human green hemoprotein (GHP) and bovine methemoglobin (MetHb) in gels composed of cross-linked bovine serum albumin was examined. The chemiluminescence (CL) was followed with a low-light intensity video camera and imaging system attached to a circularly polarized microwave guide (2450 MHz) for heating the samples from 24 degrees C to 37 degrees C. Steady-state CL was maintained in the gels for 10 min. The intensity of the CL varied with temperature. When combined with MetHb in the same gel, GHP inhibited CL of MetHb from 83.6% to 98.2% over a fiftyfold concentration range of GHP. Although MetHb/GHP combination gels were inhibited, they generated a 6.12-fold CL per degree C change compared to a 0.19-fold per degree C change for MetHb gels and a 0.31-fold per degree C change for GHP gels. The data suggest an interaction between GHP and MetHb that inhibits the CL reaction, is not interfered with by large amounts of albumin, and is partially reversed by heating.

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It has been suggested that feeder cells and 2-mercaptoethanol enhance the survival and growth of murine lymphocytes in culture by increasing cysteine availability. We previously reported that although feeder cells produce thiols, they support lymphocyte growth at densities too low for measurable thiol production. This suggested that increasing the availability of cysteine might not be the major mechanism of feeder cell action. In the present study, [35S] cystine was used to directly monitor cyst(e)ine uptake in lymphocyte-feeder cell co-cultures. The results demonstrate that feeder cells substantially increase cyst(e)ine uptake by lymphocytes, even in the absence of detectable free thiols. Data are presented which suggest an explanation for this unexpected observation.

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