Generate Dose Response Research Articles

Differential apoptotic response to ionizing radiation in subpopulations of human white blood cells

The purpose of this paper is to characterize the apoptotic response of various subpopulations of human white blood cells after in vitro exposure to ionizing radiation using the modified neutral comet assay (MNCA). White blood cells, isolated from human whole blood, were fractionated into granulocytes and mononuclear cells which were further separated into B-cells, natural killer (NK) cells, and CD4 + and CD8 + T-cells. The separated fractions were exposed to low doses of X-rays and then MNCA was used to measure the apoptotic fraction (AF) at different time points in irradiated and unirradiated aliquots of sorted cultures. The spontaneous AF in unirradiated control cells was the most critical determinant of whether an apoptotic response could be detected in irradiated cells. When cultured in isolation granulocytes and B-cells had the highest background AF, with NK cells having the next highest. CD4 + and CD8 + T-cells had a low, stable, spontaneous AF which gave them the highest signal-to-noise ratio. Although B-cells demonstrated the highest radiation-induced apoptotic response to 1 Gy of X-rays, CD8 + T-cells were the most radiation-responsive lymphocytes due to their low spontaneous AF. By generating dose response curves for CD4 + and CD8 + T-cells, the sensitivity of the MNCA for detecting apoptosis in these two cell types was also examined.

A sensitive standardised micro-gel well diffusion assay for the determination of antimicrobial activity

We have developed a highly sensitive micro-gel well diffusion assay for the determination of antimicrobial activity. In essence, the normal radial diffusion type assay was adapted to perform it in a microtiter plate. We compared our micro-gel well diffusion assay to a radial diffusion assay and a microtiter broth dilution method, using gramicidin S as model antibiotic, and Micrococcus luteus as the indicator organism. The micro-gel well diffusion assay was as sensitive as the microtiter broth dilution method, and approximately twice as sensitive as the radial diffusion method. Data analysis to calculate minimum inhibitory concentration, 50% microbial growth inhibition and maximum inhibitory concentration was refined by generating dose–response curves with the software package Prism ® 3.0 (Graphpad Software Inc.). The minimum inhibitory concentrations, determined by the three methods, were significantly different ( P<0.001), highlighting the limitations involved in comparing data obtained from different methods.

Genetically Engineered Obelin as a Bioluminescent Label in an Assay for a Peptide

The marine polypObelia longissimaproduces a protein, obelin, which emits light in a calcium-dependent manner. This photoprotein consists of a stable complex of its apoprotein, a chromophore, and oxygen. In the presence of calcium ions, the protein undergoes a change in conformation that allows it to catalyze the oxidation of the chromophore, coelenterazine, to coelenteramide with the release of light and CO2. Photoproteins are attractive as labels in analytical applications because the bioluminescent signal that they produce is the result of a chemical reaction and, therefore, has virtually no background. Thus, bioluminescence allows for extremely sensitive detection. In that regard, the feasibility of using obelin as a label has been explored with the development of a competitive immunoassay for the determination of a small peptide analyte. To attach the obelin label in a controlled manner to the octapeptide, a fusion protein was produced using recombinant DNA techniques. The protein consisted of the C-terminus of the peptide fused to the N-terminus of obelin. The octapeptide–obelin fusion protein retained the bioluminescence properties of the native protein, and was subsequently used to generate dose–response curves for the free octapeptide.

Regional dose response to pulmonary irradiation using a manual method

Purpose: To better understand the dose dependence of radiation therapy (RT)-induced changes in regional lung perfusion and tissue density, using a manual method to reduce inaccuracies that might be present in previously described automated methods. Materials and methods: Patients who were to receive RT for tumors in and around the thorax, wherein portions of healthy lung would be incidentally irradiated, were prospectively studied. Changes in regional perfusion and tissue density were assessed by comparison of pre- and post-RT single photon emission computed tomography (SPECT), lung perfusion scans and computed tomography (CT) scans, respectively. The three-dimensional dose distribution was calculated on the pre-RT CT scan and correlated to the other scans via image registration. Study volumes were defined by hand and individually visualized on pre- and post-RT scans. The manually generated dose response data were compared to data generated using automated methods. The relationship between CT density and SPECT perfusion was also determined. Results: Thirteen patients with lung cancer were evaluated for changes in tissue density and 11 patients were evaluated for changes in regional perfusion at 12 months post-RT. In general, density increases with increasing regional dose, with marked changes at >60 Gy. Regional perfusion decreases with increasing regional dose. In the low dose regions, relative perfusion increases by 35% on average. Manually measured dose responses correlated well with those determined automatically. The relationship between regional perfusion and CT density indicates a wide range of perfusion over a narrow range of CT density, with markedly reduced perfusion at CT densities of >−600 and <−900 H. Conclusions: The manually generated CT density dose response data broadly agree with data previously generated using automated methods. The manually generated perfusion dose response data are in fairly good agreement with automated data, lending credibility to the accuracy of the automated methods. Regional perfusion is markedly diminished where CT density is outside the range of normal lung tissue.

MultiPROBE nL Complements Drug Discovery Assay Miniaturization

The Packard MultiPROBE® nL is designed to enable the MultiPROBE Automated Liquid Handling System to aspirate and dispense nanoliter volumes. Several features add confidence to small volume transfers. A preview of nanoliter dispensing can be seen on a video camera monitor. In addition to the standard wash station, syringe and ultrasonic flushes can be run at the start of a program to prevent dirt or air obstructions. The MultiPROBE nL can transfer ionic, nonionic, and solutions containing organic solvents such as DMSO directly from master to assay plates and into high-density plate arrays. Additionally, the MultiPROBE nL increases the efficiency of generating dose response curves for secondary screening by eliminating a dilution step. IC50 values obtained after compound preparation with the instrument are consistent with those values previously determined using an MultiPROBE 208.

Q-band ESR studies of fossil tooth enamel: Implications for spectrum deconvolution and dating

Q-band ESR spectra of eight samples of powdered fossil tooth enamel from the Florisbad archaeological site in South Africa have been measured. On one of these, spectrum deconvolution of the integrated spectrum has been performed. The results are consistent with a model that describes the ESR signal used in dating studies as a superposition of an axialsymmetrically split signal and two isotropic Gaussian signals. A narrow additional signal with higher radiation sensitivity than the main signal has been observed and this line may affect the dose determination in dating if peak-to-peak amplitudes are used to generate dose response curves.

The response of the major crop and pasture pest, the red-legged earth mite (Halotydeus destructor) to pesticides: dose-response curves and evidence for tolerance

We describe an assay for testing the response of the adult red- legged earth mite, Halotydeus destructor, to pesticides. This assay is used to generate dose response curves for an organophosphate (omethoate) and an organochlorine (endosulphan) commonly used to control these mites, as well as a synthetic pyrethroid (alfamethrin). Comparisons of the populations from five sites around Victoria indicated tolerance to omethoate. A heritable basis for the tolerance was demonstrated by comparing the stocks after they had been reared under laboratory conditions for a generation. No tolerance of endosulphan was detected, although this chemical was not used heavily at any of the sites. Some tolerance of alfamethrin was also suggested. These results suggest that alternative methods for controlling this pest will eventually need to be considered.

Impairment of endothelial-dependent pulmonary vasorelaxation after mesenteric ischemia/reperfusion

A major hemodynamic feature of acute lung injury is pulmonary hypertension caused by pulmonary vasoconstriction. Impairment of the mechanisms of pulmonary vasorelaxation may contribute to this pulmonary vasoconstriction. This study examined the effect of mesenteric ischemia/reperfusion (I/R) on lung neutrophil accumulation and endothelial-dependent and -independent cyclic 3'-5' guanosine monophosphate-mediated pulmonary vasorelaxation in rats. Rats were studied after 1 hour of superior mesenteric artery occlusion and 2 hours of reperfusion. Lung neutrophil accumulation was determined by myeloperoxidase assay (MPO). The following mechanisms of pulmonary vasorelaxation were studied in isolated pulmonary artery rings by generating dose response curves (10(-9) to 10(-6)mol/L): (1) receptor-dependent, endothelial-dependent relaxation (response to acetylcholine), (2) receptor-independent, endothelial-dependent relaxation (response to the calcium ionophore, A23187), and (3) endothelial-independent relaxation (response to sodium nitroprusside [SNP]). Lung MPO activity was significantly increased from 2.4 +/- 0.2 units/gm lung weight in controls to 10.3 +/- 0.4 after mesenteric I/R (p < 0.05). The vasorelaxation response to SNP was not different after mesenteric I/R, but vasorelaxation by both acetylcholine and A23187 were significantly impaired. Endothelial-dependent pulmonary vasorelaxation is significantly impaired after mesenteric I/R. Such impairment of pulmonary vasorelaxation may help tip the net balance of pulmonary vasomotor tone toward vasoconstriction and contribute to the pulmonary hypertension seen in acute lung injury.

Role of endothelium in conversion of angiotensin I to angiotensin II in rabbit aorta.

Rabbit aortic rings with either an intact endothelium or a disrupted endothelium were used to generate dose response curves to angiotensin I (AI) in the presence (ED50 = 3 X 10(-7) M) and absence (ED50 = 1.7 X 10(-8) M) of 10 micrograms/ml teprotide, a converting-enzyme inhibitor. Treatment with teprotide did not alter responses to angiotensin II (AII). Comparable dose-dependent responses were obtained with AII regardless of endothelial integrity. Contraction velocities in response to angiotensin I (10(-7) M) and AII (10(-7) M) were also measured. Angiotensin II produced a significantly greater contraction velocity (p less than 0.001) than that produced by AI. The amount of conversion to AII by both intact rabbit aortic rings and rings following removal of the endothelium was determined using 125I-AI and 125I-AII. Waters C18 SEP-PAK columns were used to separate AI and AII. During the first 3 to 4 minutes after the addition of AI, contraction velocity measurements and conversion were greater in intact rings than rings without endothelium. Conversion of AI to AII in endothelial-disrupted rings was the same as in intact rings by 5 minutes after the addition of AI. Conversion of AI to AII was inhibited by 30 micrograms/ml teprotide at all times measured, and there was no evidence of an alternate route of metabolism. Angiotensin I contraction velocity measurements after 10 micrograms/ml teprotide also demonstrated impaired efficiency of conversion of AI to AII. Thus, it was established that a lack of endothelium attenuated the rate of conversion of AI to AII initially, and formation of AII with or without endothelium was blocked by teprotide.(ABSTRACT TRUNCATED AT 250 WORDS)

Protection against both lethal and behavioral effects of soman.

This work developed two drug mixtures which alone had no effect on performance of a criterion behavior but when given as a pretreatment would protect against organophosphate-induced lethality and incapacitation. Candidate drugs (alone and together) were given to rats trained to respond on a two-component Fixed Ratio 10 - Extinction (FR10-EXT) schedule. After generating dose response curves for each cholinolytic drug, mixtures of atropine (A) + mecamylamine (M) + pyridostigmine (Py) or physostigmine (Ph) were prepared and a combination of doses that produced no effects on operant performance was determined (Mix I:A = .78, M = .78, Py = .056 mg/kg; Mix II:A = .78, M = .78, Ph = .026 mg/kg). Both pretreatment mixtures provided equivalent protection against the lethal effects of the organophosphate soman; however only Mix II was capable of reversing soman-induced physical incapacitation (PI) as assessed by performance on an accelerating rotarod or FR10 responding. Pretreatment of animals with Mix II resulted in significantly higher levels of brain acetylcholinesterase (AChE) than Mix I pretreated subjects 4 hrs after 1.3 LD50 soman, although peripheral AChE levels were not different. The results indicate organophosphate-induced PI can be attenuated by pretreatment with tertiary carbamates which protect significant amounts of brain AChE from irreversible inhibition.

The effects of fractionated doses of fast neutrons or photons on the canine brain: Evaluation by computerized tomography and evoked response recording

The use of fast neutrons in the treatment of cancer necessitates a knowledge of the normal tissue responses. This study was designed to compare the late effects of fractionated doses of fast neutrons with fractionated doses of photons on canine brains by evoked response recording and viewing computerized tomograms (CT). Adult male beagles were irradiated to the entire brain (four fractions per week) with fast neutrons to total doses of 13.33 Gy (1333 rad), 20 Gy (2000 rad), 30 Gy (3000 rad) or 45 Gy (4500 rad) or with photons (four fractions per week) to total doses of 40 Gy (4000 rad), 60 Gy (6000 rad) or 90 Gy (9000 rad). A relative biological effectiveness (RBE) of 4 was obtained for normal brain tissue assessed by mortality and onset of neurologic symptoms. Every three months post-irradiation, visual and sensory evoked responses were recorded. Changes over time appeared to be minimal; however, computerized tomographs showed marked brain shrinkage. A method of quantitating cerebrospinal fluid and parenchyma) volumes from scans is described and future use of these CT ratios to generate dose response curves and RBE values is postulated.

Radioimmunoassay study of immunologic changes associated with the conversion of rabbit testicular proacrosin to acrosin.

This study was made to determine the degree of immunologic similarity or dissimilarity between rabbit proacrosin and acrosin and thereby to elucidate the extent of molecular change that occurs when proacrosin is converted to acrosin. A highly sensitive and specific radioimmunoassay for rabbit acrosin was developed and used to quantify the immunologic reactions. Testicular proacrosin was purified, but not to homogeneity, by sequential acid extraction, gel filtration, cation exchange and Concanavalin A chromatography. Acrosin immunogen was purified to homogeneity by subjecting activated proacrosin to affinity chromatography on CH-Sepharose benzamidine. A monospecific antiserum against acrosin was obtained from female rabbits following immunization with 500 µg doses of TLCK (tosyl-lysine-chloromethyl-ketone) treated acrosin 6 weeks apart. A labeled antigen (specific activity 40 µCi/µg) for the radioimmunoassays was obtained by iodination of TLCK treated acrosin by the chloramine T method. All subsequent immunologic tests were performed in the presence of 0.05 M benzamidine. Qualitative indications of immunologic dissimilarity between proacrosin and acrosin were obtained in Ouchterlony tests where proacrosin gave no precipitin band while acrosin gave a strong band against the antiacrosin serum. For the radioimmunoassays, serial dilutions of proacrosin, representing progressive stages in the activation, were used to generate dose response curves with the antiacrosin serum. Calculations based on the 50% displacement values obtained from these curves showed that the cross reaction between unactivated proacrosin and antiacrosin antibody was only 1.8% of that obtained with fully activated proacrosin (acrosin) and the same antibody. The results indicate that a major conformational change occurs when proacrosin is activated to acrosin.

Techniques of assessing air pollution damage to paints

In this study, research was concerned primarily with the development of test methods that are sufficiently sensitive to detect the initial degradation of selected exterior coatings exposed to various pollutant‐containing environments. The test methods employed in generating dose response data included erosion rates, attenuated total reflectance (ATR), gloss and sheen, surface roughness, tensile strength and scanning election microscopy. Five commercially important paint systems were selected for characterizations including an oil house paint, latex coating, an alkyd industrial maintenance coating, a coil coating and an automotive refinish lacquer. The properties of the coating systems were determined periodically consequent to ‘short term’ exposure at four exterior locations and to various pollutant‐containing environments under controlled, but accelerated conditions in an Atlas Xenon Arc Weather‐Ometer® Model 600WR12. The test sites for the former ‘short term’ exterior study were located at Leeds (north central), North Dakota; Los Angeles, California; Chicago (Research Center), Illinois and Valparaiso, Indiana. These sites represent a ‘clean’ rural environment, a high O3 environment, a high SO2 environment, and a relatively high O3 environment plus a moderate SO2 environment, respectively. In the accelerated laboratory exposure study, five enenvironmental pollutant conditions were employed in the Weather‐Ometer including a zero pollutant, 0·1ppm and 1·0ppm SO2 and 0·1ppm or 1·0ppm O3 level. The zero pollutant condition represents the control (clean air) with 0·1 ppm of each pollutant type considered representative of the levels frequently reached in polluted cities. The 1·0ppm levels represent a highly polluted (industrial) site.