Johnathan L. Kiel

Fluorescence Assay Based on Aptamer-Quantum Dot Binding to Bacillus thuringiensis Spores

A novel assay was developed for the detection of Bacillus thuringiensis (BT) spores. The assay is based on the fluorescence observed after binding an aptamer-quantum dot conjugate to BT spores. The in vitro selection and amplification technique called SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used in order to identify the DNA aptamer sequence specific for BT. The 60 base aptamer was then coupled to fluorescent zinc sulfide-capped, cadmium selenide quantum dots (QD). The assay is semi-quantitative, specific and can detect BT at concentrations of about 1,000 colony forming units/ml.

Oxidative stress precedes circulatory failure induced by 35-GHz microwave heating.

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.

Gene Expression Changes in the Skin of Rats Induced by Prolonged 35 GHz Millimeter-Wave Exposure

Abstract Millenbaugh, N. J., Roth, C., Sypniewska, R., Chan, V., Eggers, J. S., Kiel, J. L., Blystone, R. V. and Mason, P. A. Gene Expression Changes in the Skin of Rats Induced by Prolonged 35 GHz Millimeter-Wave Exposure. Radiat. Res. 169, 288–300 (2008). To better understand the cellular and molecular responses to overexposure to millimeter waves, alterations in the gene expression profile and histology of skin after exposure to 35 GHz radiofrequency radiation were investigated. Rats were subjected to sham exposure, to 42°C environmental heat, or to 35 GHz millimeter waves at 75 mW/cm2. Skin samples were collected at 6 and 24 h after exposure for Affymetrix GeneChip analysis. The skin was harvested from a separate group of rats at 3–6 h or 24–48 h after exposure for histopathology analysis. Microscopic findings observed in the dermis of rats exposed to 35 GHz millimeter waves included aggregation of neutrophils in vessels, degeneration of stromal cells, and breakdown of collagen. Changes were detected in 56 genes at 6 h and 58 genes at 24 h in the millimeter-wave-exposed rats. Genes associated with regulation of transcription, protein folding, oxidative stress, immune response, and tissue matrix turnover were affected at both times. At 24 h, more genes related to extracellular matrix structure and chemokine activity were altered. Up-regulation of Hspa1a, Timp1, S100a9, Ccl2 and Angptl4 at 24 h by 35 GHz millimeter-wave exposure was confirmed by real-time RT-PCR. These results obtained from histopathology, microarrays and RT-PCR indicate that prolonged exposure to 35 GHz millimeter waves causes thermally related stress and injury in skin while triggering repair processes involving inflammation and tissue matrix recovery.

Comparison Of Blood Pressure And Thermal Responses In Rats Exposed To Millimeter Wave Energy Or Environmental Heat

ABSTRACT Electromagnetic fields at millimeter wave lengths are being developed for commercial and military use at power levels that can cause temperature increases in the skin. Previous work suggests that sustained exposure to millimeter waves causes greater heating of skin, leading to faster induction of circulatory failure than exposure to environmental heat (EH). We tested this hypothesis in three separate experiments by comparing temperature changes in skin, subcutis, and colon, and the time to reach circulatory collapse (mean arterial blood pressure, 20 mmHg) in male Sprague-Dawley rats exposed to the following conditions that produced similar rates of body core heating within each experiment: (1) EH at 42°C, 35 GHz at 75 mW/cm2, or 94 GHz at 75 mW/cm2 under ketamine and xylazine anesthesia; (2) EH at 43°C, 35 GHz at 90 mW/cm2, or 94 GHz at 90 mW/cm2 under ketamine and xylazine anesthesia; and (3) EH at 42°C, 35 GHz at 90 mW/cm2, or 94 GHz at 75 mW/cm2 under isoflurane anesthesia. In all three experiments, the rate and amount of temperature increase at the subcutis and skin surface differed significantly in the rank order of 94 GHz more than 35 GHz more than EH. The time to reach circulatory collapse was significantly less only for rats exposed to 94 GHz at 90 mW/cm2, the group with the greatest rate of skin and subcutis heating of all groups in this study, compared with both the 35 GHz at 90 mW/cm2 and the EH at 43°C groups. These data indicate that body core heating is the major determinant of induction of hemodynamic collapse, and the influence of heating of the skin and subcutis becomes significant only when a certain threshold rate of heating of these tissues is exceeded.ABBREVIATIONS-MMW - millimeter wave, EH - environmental heat, MAP - mean arterial pressure, HR - heart rate, TC - colonic temperature, TSQ - subcutaneous temperature, Tsurf - skin surface temperature

Diazoluminomelanin: A Synthetic Electron and Nonradiative Transfer Biopolymer

The green hemoprotein (GHP) of erythrocytes is a type-b cytochrome with no known function except for the incidental finding of its ferroactivation of phosphoenolpyruvate carboxykinase which is not present in erythrocytes (Chee and Lardy, 1981). We have previously postulated that GHP can transfer superoxide to methemoglobin, converting it to oxyhemoglobin (Kiel et al., 1988). Although no artificial substrate has been available for measuring the electron transfers mediated by GHP, previous data has indicated that the peroxidations putatively mediated by GHP and hemoglobin in erythrocytes can be inhibited by 3-amino-L-tyrosine (Kiel and Erwin., 1986; Kiel et al., 1988). This derivatized tyrosine is an inhibitor of peroxidases (Kiel, 1988) and inhibits the oxidative burst (superoxide and hydrogen peroxide production) of mouse peritoneal macrophages (Lefkowitz et al., 1988). The latter is associated with an electron transport chain containing a type-b cytochrome (Rossi et al., 1986). Furthermore, when lactoperoxidase is reduced by thiol, its binding of tyrosine derivatives becomes significantly enhanced (Pommier and Cahnmann, 1979). Therefore, a potential existed for the development of a tyrosihe-derived substrate that specifically bound to GHP.

Thermochemiluminescent assay of porcine, rat, and human erythrocytes for antioxidative deficiencies.

The thermal induction of chemiluminescence of luminol-horseradish peroxidase-labeled erythrocytes from pigs, rats, and man was studied. The luminescent responses of rat, porcine, and human erythrocytes to heating were linear in respect to logs of counts per minute versus temperature. Landrace-Duroc crossbred pigs with a history of malignant hyperthermia (porcine stress syndrome) and Poland-China-miniature pigs inbred for malignant hyperthermia (MH) yielded erythrocytes with high-level thermochemiluminescence (TCL). Sprague-Dawley rat erythrocytes were intermediate in their production of TCL. Normal human and MH-resistant miniature swine erythrocytes produced low-level TCL. However, pretreatment of human erythrocytes with 1-chloro-2,4-dinitrobenzene (CDNB) resulted in high-level TCL. Furthermore, halothane enhanced the TCL of CDNB-treated human erythrocytes and Landrace-Duroc porcine erythrocytes that were not treated with CDNB. Red blood cells from pigs susceptible to the porcine stress syndrome demonstrated a TCL response very similar to CDNB-treated erythrocytes.

A quantitative luminescence imaging system for biochemical diagnostics

A prototype quantitative low‐light‐level imaging system, capable of providing calibrated gray‐scale imagery of radio‐frequency (rf) stimulated chemiluminescence distributions in biological samples, has been developed by these authors. The quantitative luminescence imaging system (QLIS) represents a significant advance in the experimental instrumentation used for the study of such reactions, in that the system allows time‐resolved and spatially resolved quantitative analyses to be performed. Measurements of chemiluminescence have traditionally been made by using individual photon counting photomultiplier tubes, integrating over the spatial volume and over time. The QLIS provides imagery at video rates, in which individual or integrated multiple frames can be calibrated to give a quantified image of the two‐dimensional distribution of chemiluminescence in the sample. The system has a temporal resolution of 1/30th of a second, spatial resolution of 0.25 mm, a 40‐mm‐diam linear field of view, and a radiometri...

Gel State Chemiluminescence: An Artificial Electron Transport System

Crosslinked glucose oxidase (GO) and horseradish peroxidase (HRP) can mimic macrophage oxidative function in vivo in respect to tumor cells as targets.1–3 Both the flavoprotein of NADPH oxidase of macrophages and GO contain flavin adenine dinucleotide as their prosthetic group. HRP has a noncovalently bound hematoporphyrin IX prosthetic group as does the cytochrome b559 (green hemoprotein) of NADPH oxidase. Therefore, we anticipated a similarity in biochemical mechanism of action and chemi-luminescence (CL) between the semi-synthetic crosslinked GO and HRP and the macrophage NADPH oxidase.

Microwave radiation effects on the thermally driven oxidase of erythrocytes

Sheep red blood cells (SRBCs) were labelled with a concanavalin A-luminol-bovine serum albumin conjugate specific for the transmembrane anion transport protein (Band 3) and exposed to 2450 MHz continuous wave microwave radiation at an average specific absorption rate of 91 W/kg for 10 min. The temperature was held constant at 25, 37, 40, 42 or 45 degrees C with an airflow heat exchange system. Following exposure to microwave or air heating, the decrease in residual base-activated chemiluminescence (CL) of the SRBCs was measured as an indication of infield oxidase activity. Air heating resulted in a significant decrease in residual CL at temperatures above 37 degrees C (74 per cent decrease at 45 degrees C). Microwave radiation inhibited the decline in residual CL above 37 degrees C. At 45 degrees C the inhibition was 40 per cent. The results suggest microwave radiation either reversibly altered the thermodynamics of oxygen binding to haemoglobin or failed to energize a significant portion of the haemoglobin molecules in each sample to the thermal threshold of haemoglobin autoxidation.

A sensitive method for the determination of cytolytic activity

Abstract A new method is described for the determination of the cytolytic activity of extremely low levels of stable as well as very labile cytotoxins. The method involves the application of the cytotoxin to a column of immunobilized erythrocytes or other suitable cells and a continuous monitoring of the column eluate for the presence of hemoglobin or other cell constituents. The cytotoxic activity of horseradish peroxidase at concentrations as low as 10 −12 , m can be measured with this technique. The column hemolytic assay is compared with a static (batch) hemolytic assay with respect to sensitivity and reproducibility. Furthermore, a method is described to determine the true rates of lysis, i.e., the number of cells lysed per minute.