Combined small angle X-ray solution scattering with atomic force microscopy for characterizing radiation damage on biological macromolecules.

BACKGROUND: Synchrotron radiation facilities are pillars of modern structural biology. Small-Angle X-ray scattering performed at synchrotron sources is often used to characterize the shape of biological macromolecules. A major challenge with high-energy X-ray beam on such macromolecules is the perturbation of sample due to radiation damage. RESULTS: By employing atomic force microscopy, another common technique to determine the shape of biological macromolecules when deposited on flat substrates, we present a protocol to evaluate and characterize consequences of radiation damage. It requires the acquisition of images of irradiated samples at the single molecule level in a timely manner while using minimal amounts of protein. The protocol has been tested on two different molecular systems: a large globular tetremeric enzyme (ß-Amylase) and a rod-shape plant virus (tobacco mosaic virus). Radiation damage on the globular enzyme leads to an apparent increase in molecular sizes whereas the effect on the long virus is a breakage into smaller pieces resulting in a decrease of the average long-axis radius. CONCLUSIONS: These results show that radiation damage can appear in different forms and strongly support the need to check the effect of radiation damage at synchrotron sources using the presented protocol.

Novel human radiation exposure biomarker panel applicable for population triage.

PURPOSE: To identify a panel of radiation-responsive plasma proteins that could be used in a point-of-care biologic dosimeter to detect clinically significant levels of ionizing radiation exposure. METHODS AND MATERIALS: Patients undergoing preparation for hematopoietic cell transplantation using radiation therapy (RT) with either total lymphoid irradiation or fractionated total body irradiation were eligible. Plasma was examined from patients with potentially confounding conditions and from normal individuals. Each plasma sample was analyzed for a panel of 17 proteins before RT was begun and at several time points after RT exposure. Paired and unpaired t tests between the dose and control groups were performed. Conditional inference trees were constructed based on panels of proteins to compare the non-RT group with the RT group. RESULTS: A total of 151 patients (62 RT, 41 infection, 48 trauma) were enrolled on the study, and the plasma from an additional 24 healthy control individuals was analyzed. In comparison with to control individuals, tenascin-C was upregulated and clusterin was downregulated in patients receiving RT. Salivary amylase was strongly radiation responsive, with upregulation in total body irradiation patients and slight downregulation in total lymphoid irradiation patients compared with control individuals. A panel consisting of these 3 proteins accurately distinguished between irradiated patients and healthy control individuals within 3 days after exposure: 97% accuracy, 0.5% false negative rate, 2% false positive rate. The accuracy was diminished when patients with trauma, infection, or both were included (accuracy, 74%-84%; false positive rate, 14%-33%, false negative rate: 8%-40%). CONCLUSIONS: A panel of 3 proteins accurately distinguishes unirradiated healthy donors from those exposed to RT (0.8-9.6 Gy) within 3 days of exposure. These findings have significant implications in terms of triaging individuals in the case of nuclear or other radiologic events.

Effects of radiation and alpha-tocopherol on saliva flow rate, amylase activity, total protein and electrolyte levels in oral cavity cancer.

OBJECTIVE: The objective of the present study was to evaluate early and late effects of radiation and a-tocopherol on the secretion rate of saliva and on selected saliva salivary parameters in oral cavity cancer patients. PATIENTS & METHODS: Eighty-nine histologically confirmed oral cavity cancer patients (OCC) were enrolled in the study. Resting whole saliva was collected before, during and at the end of the radiation therapy (RT) and simultaneous supplementation with alpha - tocopherol to the radiation treated patients (RT + AT). RESULTS: Salivary flow rate, pH, amylase activity, total protein, sodium and potassium were analyzed. Increased pH, potassium and decreased flow rate, amylase activity, protein content and sodium were observed in 6 weeks of radiation treated patients when compared to OCC patients. A significant improvement of those parameters was observed on alpha - tocopherol supplementation in RT + AT patients. CONCLUSION: Supplementation with alpha - tocopherol improves the salivary flow rate thereby, maintains salivary parameters.

Effect of defocused infrared diode laser on salivary flow rate and some salivary parameters of rats.

This study aims to investigate whether infrared diode low-level laser therapy (LLLT) increased salivary flow rate and altered pH value, protein concentration, and peroxidase and amylase activities in saliva of rats. Wistar rats were used and divided into three groups. Experimental groups (A and B) had their parotid, submandibular and sublingual glands submitted to diode laser, 808-nm wavelength, on two consecutive days. The dose results were 4 and 8 J/cm(2), respectively. A red guide light was used to visualize the irradiated area. Group C was irradiated only with red pilot beam and served as control. The saliva samples were collected after each irradiation step (first and second collection days) and 1 week after the first irradiation (seventh day). Statistical analysis was performed, and differences were observed according to different days of salivary collection. The results showed that salivary flow rate for groups A and B was higher on the seventh day if it is compared to data obtained for the first day (p < 0.05). LLLT applications on salivary glands are a therapy procedure that requires further studies.

[The non-contact effect of substances containing benzene rings and heterocycles on biological systems]. / Nekontaktnoe deistvie soedinenii s benzol'nymi kol'tsami i geterotsiklami na biosistemy.

It was found that some substances containing benzolic rings and heterocyclic structures have a noncontact effect on biosystems. Some results of experiments dealing with the noncontact effect on enzyme molecules, cells, and uni- and multicellular organisms are presented. Factors influencing the efficiency of the noncontact effect were revealed.

Radiation-induced apoptosis in relation to acute impairment of rat salivary gland function.

PURPOSE: To find an answer to the question: Are the acute radiation effects on salivary gland function, as seen in earlier studies, causally related to radiation-induced apoptosis? MATERIALS AND METHODS: Rat parotid and submandibular glands were X-irradiated with doses up to 25 Gy and morphological damage assayed up to 6 days after irradiation. Damage to the different cell types in the glands was assessed after H & E staining. Apoptotic appearance was judged by compacted chromatin and fragmentation of cells into lobulated masses. RESULTS: In about 3% of the cells aberrant nuclei were observed after doses as low as 2 Gy and around 7.5 and 24 h after irradiation. About half of these aberrant nuclei had an apoptotic appearance. After a dose of about 5 Gy no dose-response for apoptotic cells was found, as evidenced by a plateau in the dose-effect curve. At 6 days after 2 Gy, no signs of radiation-induced apoptosis was apparent and for most cell types a value close to zero was observed. CONCLUSIONS: Radiation studies on salivary function in the rat show the typical response with respect to dose (5-15 Gy) and time (1-3 days). This differs from reported findings with light microscopy. Therefore, the extent of apoptosis induced by radiation cannot explain the observed gland malfunction. Alternative mechanisms are proposed.

The alterations in the activity of amylase and its salivary isoenzyme in the serum of patients with ovarian carcinoma, submitted to radiotherapy.

Total activity of alpha-amylase and its salivary isoenzyme in the serum of patients with ovarian carcinoma of various types were evaluated before radiotherapy, in the middle of radiotherapy period, in the last day and 2 months after radiotherapy. Before radiotherapy the activity of these enzymes were significantly higher in patients with ovarian serous cystadenocarcinoma. It was found that irradiation resulted in a decrease of both total amylase activity and its salivary isoenzyme in the serum. The proportional participation of salivary isoenzymes in total amylase activity was normalized. It is suggested that the assay of salivary alpha-amylase activity may be useful in the evaluation of radiotherapy effectiveness.

Immunohistochemical and quantitative changes in salivary EGF, amylase and haptocorrin following radiotherapy for oral cancer.

Epidermal growth factor (EGF), amylase and haptocorrin are molecules produced in the salivary glands. The aim of the present study was to determine immunohistochemical and quantitative alterations in EGF as compared with haptocorrin and amylase following radiotherapy for oral cancer. Changes in the salivary secretion of EGF are of interest because of the importance of EGF in mucosal regeneration. Immunohistochemical studies on normal tissue from parotid and submandibular glands have demonstrated EGF in the serous acini with a tendency to single cell expression in the parotid gland. Amylase has been found in the serous acini of both the submandibular and parotid glands. Haptocorrin was localized in the duct system of both glands. In the submandibular glands with radiotherapy induced sialoadenitis only very few acini with weak or no staining for EGF and amylase were demonstrated, while no changes were observed in the staining for haptocorrin. Analysis on stimulated whole saliva samples collected from 20 healthy individuals and from 20 patients prior to, and 1, 2 and 3 weeks following radiotherapy showed significant reduction in salivary contents of EGF and amylase after treatment as expressed per g protein (p < 0.0002). The salivary content of haptocorrin increased significantly after treatment (p < 0.002). These alterations may be explained by the different cellular sites of the molecules studied, the serous acini being more sensitive to ionising radiation than the duct system. The concentration of EGF in saliva before treatment was significantly higher in patients than in the control group (p < 0.02), which may indicate that the tumors induce increased secretion of salivary EGF, or alternatively that the oral tumors contribute with EGF to the saliva. In conclusion we have demonstrated a reduction in the mitogenic peptide EGF both immunohistochemically and quantitatively following irradiation for oral cancer, results which may contribute to the understanding of the clinical signs of mucositis.

The preparation of an autologous saliva for use with patients undergoing therapeutic radiation for head and neck cancer.

PURPOSE: At the present time there is no general agreement about how to prevent the symptoms and clinical signs that accompany therapeutic irradiation for head and neck cancer. Because saliva is the principal protector of the oral tissues, it is logical to assume that many of these changes are due to the radiation-induced damage to the salivary glands. We have observed that the flow and composition of saliva is normal in most patients before their irradiation. Theoretically, it should, therefore, be possible to collect their saliva before they commence their course of radiation, store it in a "saliva bank," and give it back to them when they undergo radiation. The key to the use of such an autologous saliva is the fabrication of a technique that disinfects or sterilizes the saliva yet preserves its protective properties. The objective of this study was to prepare an autologous saliva that would be used by patients during their irradiation for head and neck cancer. MATERIALS AND METHODS: Stimulated saliva was obtained from healthy subjects; none of the subjects consumed any medications. The saliva was treated by a variety of techniques. Included among them were heat, radiation, filtration, centrifugation, and an antibacterial agent. The samples were analyzed for total protein, amylase, viscosity, and sterility; individual salivary proteins were assessed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. RESULTS: The results showed that beta radiation (> 2.5 kGy) and lyophilization + chlorhexidine (0.03% to 0.12%) could be used to prepare a sterile autologous saliva that retained most of its protective properties.

[Measurements of amylase isoenzymes in sera and saliva of patients after radiotherapy because of larynx carcinoma]. / Aktywnosc izoenzymów amylazy sliny i osocza krwi pacjentów poddanych radioterapii z powodu nowotworów krtani.

Serum and salivary alpha-amylase were measured for controls and patients with laryngeal carcinoma before, and after localised irradiation including salivary glands. Additionally amylase isoenzymes in sera were measured using mini-column method. A significant increase in amylasemia was observed after irradiation. Alpha-amylase activity in saliva was decreased after irradiation but differences were not statistically significant due to the significant decrease of protein in saliva of irradiated group. An increase of salivary isoenzyme S activity was observed while pancreatic isoenzyme activity was not altered. This method allows easy differentiation of hyperamylasemia due to irradiation of parotid gland and disorders of the pancreas. Alpha-amylase activity measurements may detect metabolic changes in salivary glands after irradiation.

[The effect of ionizing radiation on enzymes. X. The effect of gamma irradiation on pancreatic amylase in pancreatin]. / Ucinek ionizujícího zárení na enzymy. X. Vliv zárení gama na pankreatickou amylázu v pankreatinu.

The present paper examined the effect of ionizing radiation (source, 60Co) within a range of doses from 10 to 120 kGy on amylolytic efficacy of pancreatin of two types: pancreatin obtained by isolation from an extract of the pancreas (sample 1) and pancreatin containing parts of the pancreatic tissue, but with higher amylolytic efficacy (sample 2). Efficacy was expressed in F.I.P. units. As shown in the chart, a percentual decrease in efficacy is higher in the more active sample 2. Graphical representation is in good agreement with the statistical evaluation of the significance between the decreases in efficacy in both samples irradiated with doses of 30 and 120 kGy, if the median test was used and probability was calculated with the use of Fisher's test. This is not, however, the case of irradiation with a dose of 10 kGy. The residual, graphically corrected efficacy after irradiation with a sterilizing dose of 25 kGy was 84.1% (sample 1) and 80.3% (sample 2). With the maximal dose used, the residual efficacy was 43.7% (sample 1) and 36.7% (sample 2).

Dose-dependency of radiation on enzyme production in Trichoderma reesei.

Effect of irradiation dose on the production of cellulase and amylase related enzymes in Trichoderma reesei was studied, in which post-irradiation time response pattern was measured. The damage of the cells irradiated with certain irradiation doses (1.40 +/- 0.20 x 10(5), 2.20 +/- 0.10 x 10(5), 3.00 +/- 0.50 x 10(5) and 3.50 +/- 0.20 x 10(5) rad) was rapidly recovered. The increased enzyme production in the culture of the irradiated cells resulted from the recovery of radiation damage after irradiation. The function of cell growth was not affected by irradiation below dose of 5 x 10(5) rad, though the function of enzyme synthesis was drastically affected.

[Radiation sterilization of pancreatin preparation].

The enzymatic activities (in detail, amylase-, lipase- and protease-activity) of gamma-irradiated pancreatin powder preparation decrease to 50-95% at doses range of 50 kR-8 MR. The dose-inactivation relation-ships vary depending on every different production lot. In the case of higher moisture content preparation as irradiation sample, amylase activities are more stable, the other side, protease activities are less stable. On the variation of these activities by gamma-irradiation, there are no difference between room temperature and lower temperature (-70 degrees C) as irradiation condition. The D10 values of contaminating bacteria and fungus (contamination number: about 1 X 10(3) cells/g and 9 X 10(2) spores/g) of a pancreatin preparation are 74 C/kg (280 kR) and 60 C/kg(230 kR). Then, these values are corresponded to 645 C/kg (2.5 MR) and 540 C/kg (2.1 MR) as perfect sterilization.