A normalization method of the volume and geometry of tooth for X-band in vivo EPR dosimetry.

The accuracy of in vivo EPR tooth dosimetry may be influenced by the volume and geometry variations in teeth, especially when there is considerable non-uniform sensitivity distribution in the active detection area of the cavity. To solve this problem, the present research proposed a normalization method specifically for X-band EPR in vivo tooth dosimetry. The volume and geometry of the measured tooth were reconstructed by digital image processing with images of the tooth impression slices, which were obtained by a custom-made impression module. The sensitivity distribution in the active detection area was established based on experiments with a point sample. Consequently, a composite normalization process that could calibrate the evaluated dose effectively was carried out by taking into account the influences not only from tooth volume and geometry but also from the non-uniform distribution of sensitivity. The effect and practicability of the method were evaluated by incisor samples. Results showed that the standard deviation could be reduced a maximum of 54.8% approximately after the composite normalization, an improvement compared to results from solely tooth volume. The correlation coefficient of the dose-response curve could be improved from 0.731 to 0.986. The preliminary method provides an approach potentially useful on site after radiation accidents when dealing with the influence of variations in the tooth volume and geometry for X-band EPR in vivo dose estimations.

Study of new practical ESR dosimeter based on carbonated hydroxyapatite and its dosimetric properties.

The development of new dosimeters with good dosimetric properties is important for quality control in radiation applications. A new practical electron spin resonance (ESR) dosimeter based on carbonated hydroxyapatite that simulated the composition and structure of tooth enamel was specially synthesized. The synthesized material was investigated by transmission electron microscope, X-ray diffraction, fourier transform infrared spectroscopy and X-ray photo electron spectroscopy to confirm to the main composition of carbonated hydroxyapatite with CO32- successfully doped into the crystal lattice through optimizing the synthesis process of C/P molar ratio, pH value dynamical adjustment, annealing temperature and time. The dosimetric properties were systematically investigated by ESR spectroscopy. The results indicated that the radiation induced signal had a good dose response within a relatively wide dose range. The dose response was linear in the dose range of 0-400 Gy with a correlation coefficient of 0.9999 and had dose accumulative effect in the experimental dose range of 0-100 Gy. In a wider dose range up to 30 kGy, the dose response also presented linear feature in double-logarithmic coordinate system with a correlation coefficient of 0.9970. The dose detection limit was about 0.34Gy with a given probability of 95% confidence level depending upon a rigid calculation algorithm. The signal was extremely stable in the observation time of 360 days with a variation coefficient of 3.8%. The radiation sensitivity of the material showed no remarkable variation against photon energy from 662 KeV to 1.25 MeV and dose rate from 0.86 Gy/min to 12.17 Gy/min. The material showed more sensitive in lower photon energy range below 662 keV, which hint additional calibration may need when using in special photon energy condition. The preliminary results suggested that this newly developed dosimeter was potential to become a practical dosimeter that would expand the application fields of ESR dosimetry.

Effect of the tooth surface water on the accuracy of dose reconstructions in the X-band in vivo EPR dosimetry.

The X-band in vivo EPR tooth dosimetry is promising as a tool for the initial triage after a large-scale radiation accident. The dielectric losses caused by water on the tooth surface (WTS) are one of the major sources of inaccuracies in this method. The effect was studied by theoretical simulation calculations and experiments with water films of various thicknesses on teeth. The results demonstrate the possibility of sufficiently accurate measurements of the radiation-induced signal of the tooth enamel provided that the thickness of the water film on the tooth is below 60 µm. The sensitivity of the cavity decreases with increasing thickness of the water layer. The interference of WTS can be diminished by normalization of the radiation-induced signal to the signal of a reference sample permanently present in the cavity.

The Application and Distribution of Magnetic Field Modulation in the Detection Apertures of X-band EPR Cavities for In Vivo Tooth Dosimetry.

In vivo electron paramagnetic resonance tooth dosimetry could be a practical and ideal tool for quick mass triage of victims in the rescue following a disaster event involving irradiation radiation. Magnetic field modulation is an important issue to improve the sensitivity of X-band in vivo tooth dosimetry. We designed a couple of trapezoidal modulation coil sets fixed on the magnet poles that could be used to apply sufficient magnet field modulation into the detection aperture of the resonant cavity. Measurements of irradiated teeth with such coil sets demonstrated significant radiation-induced signals. The modulation generation efficiencies and magnetic field distributions in apertures with different cavity geometries were analytically calculated, simulated by a finite element method and evaluated by measurements of a free radical point sample to study the influences caused by the geometries of the apertures and other factors.

Calibration ruler for CW-EPR distance measurement using diradical molecule of rigid structure.

Many experimental factors and uncontrollable factors may introduce errors in the distance measurement by continuous wave electron paramagnetic resonance. To deal with this problem, several C60 nitroxide diradical adducts with rigid structure and definite molecular dimension were used as distance calibration rulers. Based on the improvement of distance calculation program via adding simulation programs of experimental spectra and dipolar broadening function, respectively, the distance calibration method was developed under different conditions such as different solvent, solution concentration, measuring temperature, and microwave power. As a result, stable distance calibration rulers were established within the range of 8-13 Å. The distance calibration effect was evaluated resulting in a corresponding distance measurement precision of 0.84 Å. The results suggested that the influence of non-dipolar spectral broadening factors could be overcome, and the established experimental and calculation methods were suitable to a wide range of situations. The developed method will ensure more accurate and objective distance measurement in biomacromolecular analysis.

Tat peptide-mediated soluble expression of the membrane protein LSECtin-CRD in Escherichia coli.

The human liver and lymph node sinusoidal endothelial cell C-type lectin (hLSECtin), a type II integral membrane protein, containing a Ca(2+)-dependent carbohydrate recognition domain (CRD), has a well-established biological activity, yet its three-dimensional structure is unknown due to low expression yields and aggregation into inclusion bodies. Previous study has demonstrated that the HIV-1 virus-encoded Tat peptide ('YGRKKRRQRRR') can increase the yields and the solubility of heterologous proteins. However, whether the Tat peptide could promote the high-yield and soluble expression of membrane proteins in Escherichia coli is not known. Therefore, the prokaryotic expression vector pET28b-Tat-hLSECtin-CRD (using pET28b and pET28b-hLSECtin-CRD as controls) was constructed, and transformed into E. coli BL21 (DE3) cells and induced with isopropyl-ß-d-thiogalactoside (IPTG) followed with identifying by SDS-PAGE and Western blot. Subsequently, the bacterial subcellular structure, in which overexpressed the heterologous proteins Tat-hLSECtin-CRD and Tat-free hLSECtin-CRD, was analyzed by transmission electron microscope (TEM) respectively, and the mannose-binding activity of Tat-hLSECtin-CRD was also determined. Expectedly, the solubility of Tat-LSECtin-CRD significantly increased compared to Tat-free LSECtin-CRD (**p < 0.01) with prolonged time, and the Tat-LSECtin-CRD had a significant mannose-binding activity. The subcellular structure analysis indicated that the bacterial cells overexpressed Tat-hLSECtin-CRD exhibited denser region compared with controls, while dot denser region aggregated in the two ends of bacterial cells overexpressed Tat-free hLSECtin-CRD. This study provided a novel method for improving the soluble expression of membrane proteins in prokaryotic systems by fusion with the Tat peptide, which may be potentially expanded to the expression of other membrane proteins.

Mobility study of individual residue sites in the carbohydrate recognition domain of LSECtin using SDSL-EPR technique.

Conformational changes in proteins profoundly influence their functional profiles. With site-directed spin labeling (SDSL)-electron paramagnetic resonance (EPR) spectroscopy, we investigated the mobility features of individual residue sites in the carbohydrate recognition domain (CRD) of LSECtin, a type II integral membrane protein. The mobility of six different residue sites scatting around the Ca(2+)-1-binding site were investigated by comparing their EPR spectra rotational correlation time τ(c) in order to obtain the information of conformational changes of relevant region. The results showed that the overall mobility of LSECtin-CRD increased after addition of Ca(2+) and N-acetylglucosamine, but different sites in the CRD exhibited different mobility features, suggesting that these sites may have different functional profiles. The preliminary observations thus demonstrated that SDSL-EPR spectroscopy is not only an effective technique to reveal the mobility of single residue sites in LSECtin-CRD but also that the functions of single residue sites may be indicated by their conformational dynamics.

[Application of ESR imaging technique in studying of skin-penetration properties of nitroxide free radical].

A set of L-band electron spin resonance imaging (ESRI) equipment suitable for biological species was developed and an ESRI experiment model for viable skin samples was established. The mechanic process of nitroxide free radical TEMPO (2,2, 6, 6-tetramethyl-1-piperidinyloxy) penetrating through skin sample and the spin density distribution of TEMPO after it interacted with skin sample were detected by the developed ESRI method. Skin samples were extracted from mice back. The experimental samples were prepared by cutting the skin pieces into square shape of 2 x 2 cm2 and then the samples were divided into three groups by treating them with three different methods: Method A, simple treatment by simply cutting the hair; method B, 8% Na2S depilation treatment for 10 min; method C, 8% Na2S depilation and then 5% pancreatic digestion treatment for 2 hours. The liposoluble solvent DMSO (dimethyl sulfoxide) and distilled water were used as two kinds of solvent for the TEMPO liquor. The results indicated that the skin-penetration properties of TEMPO were significantly different among samples treated with different methods and the surface cornifin of skin offered remarkable resistance to TEMPO. The TEMPO liquor of water could hardly penetrate through skins, whereas about 20%-30% of the original TEMPO compounds that solved in liposoluble solvent DMSO could penetrate through the skin sample treated with method C after 16 hours of interaction. Furthermore, the penetration rate of TEMPO through the skin tissue was a strong time dependent process. The preliminary application results suggested that ESRI technique could provide an effective and applicable method for dynamically researching skin-penetration properties of some special kinds of materials such as paramagnetic compounds.

[The effects of electromagnetic pulse on fluidity and lipid peroxidation of mitochondrial membrane].

OBJECTIVE: To study the effects of intense electromagnetic pulse(EMP) on the biological effects of mitochondrial membrane. METHOD: Rat liver mitochondrial suspension was exposed to EMP at 60 kV/m level. The changes of membrane lipid fluidity and membrane protein mobility were detected by ESR and spin label technique. Malondialdehyde(MDA) was detected by spectrophotometer. RESULTS: The mobility of membrane protein decreased significantly(P < 0.05). Correlation time (tau c) of control group was (0.501 +/- 0.077) x 10(-9)s, and tau c of EMP group was (0.594 +/- 0.049) x 10(-9)s, indicating that the mobility of protein was restricted. The fluidity of mitochondrial membrane increased significantly(P < 0.05) at the same time. Order parameter(S) of mitochondrial membrane lipid in control group was 0.63 +/- 0.01, while S of EMP group was 0.61 +/- 0.01(P < 0.05). MDA decreased significantly. CONCLUSION: The mobility and lipid peroxidation of mitochondrial membrane may be disturbed after EMP exposure.

[Comparing the Oxygen Free Radicals and Oxygen Consumption of Leukocytes in Leukemia Patients and Healthy Persons]

Using the electron spin resonance (ESR) and probe technique, the species of reactive oxygen free radicals and the oxygen consumption were observed in the metabolic process of oxygen of leukocytes from leukemia patients and healthy persons. Results showed that weak ESR spectrum of hydroxyl radical ((*)OH) signal was detected in the leukemia patient's leukocytes, there was no significant difference in oxygen consumption with or without PMA stimulation; superoxide anion (O(2)) in normal leukocytes was detected, and oxygen consumption increased markedly compared to the normal respiration without PMA stimulation (P < 0.001). There was no significant difference in oxygen consumption between leukemic and normal leukocytes without PMA stimulation; however, it was higher in normal leukocyte than in leukemic leukoeytes after PMA stimulation (P < 0.001). These observations suggest that dysfunction of respiratory burst is existed in leukemic leukocytes, and their oxygen consumption is markedly lower than that of normal leukocytes.