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.

A simple one pot purification of bacterial amylase from fermented broth based on affinity toward starch-functionalized magnetic nanoparticle.

Surface-functionalized adsorbant particles in combination with magnetic separation techniques have received considerable attention in recent years. Selective manipulation on such magnetic nanoparticles permits separation with high affinity in the presence of other suspended solids. Amylase is used extensively in food and allied industries. Purification of amylase from bacterial sources is a matter of concern because most of the industrial need for amylase is met by microbial sources. Here we report a simple, cost-effective, one-pot purification technique for bacterial amylase directly from fermented broth of Bacillus megaterium utilizing starch-coated superparamagnetic iron oxide nanoparticles (SPION). SPION was prepared by co-precipitation method and then functionalized by starch coating. The synthesized nanoparticles were characterized by transmission electron microscopy (TEM), a superconducting quantum interference device (SQUID, zeta potential, and ultraviolet-visible (UV-vis) and Fourier-transform infrared (FTIR) spectroscopy. The starch-coated nanoparticles efficiently purified amylase from bacterial fermented broth with 93.22% recovery and 12.57-fold purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the molecular mass of the purified amylase was 67 kD, and native gel showed the retention of amylase activity even after purification. Optimum pH and temperature of the purified amylase were 7 and 50°C, respectively, and it was stable over a range of 20°C to 50°C. Hence, an improved one-pot bacterial amylase purification method was developed using starch-coated SPION.

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.

[Analysis of hydrolytic enzyme activities on sludge aerobic/anoxic digestion after ultrasonic pretreatment].

In order to evaluate the function of sludge aerobic/anoxic digestibility by ultrasonic pretreatment. The SS, VSS and hydrolytic enzyme activities (amylase, glucosidase, protease, phosphatase) were measured before and after ultrasonic pretreatment (28 kHz, 0.15 kW x L(-1), 10 min). The results showed that the performances of aerobic/anoxic were greatly improved after ultrasonic pretreatment, the removal efficiency of VSS went to 44.3%, 7.8% better than of traditional aerobic/anoxic digestion. The variational trend of sludge hydrolytic enzyme activities increased firstly and then fell off during 13d digestion, the maximum of amylase activity and glucosidase activity in ultrasonic sludge, appeared in the 5 d, amylase activity was 0.104 micromol x g(-1) and glucosidase activity was 0.637 (micromol x g(-1). The maximum of intracellular protease activity and extracellular proteases activity in ultrasonic sludge, appeared in the 7 d, intracellular protease activity was 23.68 micromol x g(-1), higher than extracellular proteases activity, and it was playing a leading role in sludge digestion. The acid phosphatase activity of ultrasonic sludge was higher than the control sludge, and the alkaline phosphatase was sensitive to environment. So the alkaline phosphatase activity reduced when the internal properties of sludge was changed.

Integrity of proteins in human saliva after sterilization by gamma irradiation.

Microbial contamination of whole human saliva is unwanted for certain in vitro applications, e.g., when utilizing it as a growth substratum for biofilm experiments. The aim of this investigation was to test gamma irradiation for its suitability to sterilize saliva and to investigate the treatment's influence on the composition and integrity of salivary proteins in comparison to filter sterilization. For inhibition of bacterial growth by gamma irradiation, a sterility assurance level of 10(-6) was determined to be reached at a dose of 3.5 kGy. At this dose, the integrity of proteins, as measured by fluorescence, circular dichroism, and gel electrophoretic banding pattern, and the enzymatic activities of salivary amylase and lysozyme were virtually unchanged. Filtration reduced the total protein concentration to about half of its original value and decreased lysozyme activity to about 10%. It can be concluded that irradiation is suitable for sterilizing whole saliva in its native form.

Effect of same-dose single or dual field irradiation on damage to miniature pig parotid glands.

AIM: To evaluate the effect of single or dual field irradiation (IR) with the same dose on damage to miniature pig parotid glands. METHODOLOGY: Sixteen miniature pigs were divided into two IR groups (n=6) and a control group (n=4). The irradiation groups were subjected to 20 Gy X-radiation to one parotid gland using single-field or dual-field modality by linear accelerator. The dose-volume distributions between two IR groups were compared. Saliva from parotid glands and blood were collected at 0, 4, 8 and 16 weeks after irradiation. Parotid glands were removed at 16 weeks to evaluate tissue morphology. RESULTS: The irradiation dose volume distributions were significantly different between single and dual field irradiation groups (t=4.177, P=0.002), although dose volume histogramin (DVH) indicated the equal maximal dose in parotid glands. Saliva flow rates from IR side decreased dramatically at all time points in IR groups, especially in dual field irradiation group. The radiation caused changes of white blood cell count in blood, lactate dehydrogenase and amylase in serum, calcium, potassium and amylase in saliva. Morphologically, more severe radiation damage was found in irradiated parotid glands from dual field irradiation group than that from single field irradiation group. CONCLUSION: Data from this large animal model demonstrated that the radiation damage from the dual field irradiation was more severe than that of the single field irradiation at the same dose, suggesting that dose-volume distribution is an important factor in evaluation of the radiobiology of parotid glands.

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.

Measuring short-term gamma-irradiation effects on mouse salivary gland function using a new saliva collection device.

A restraining device was designed specifically for the collection of whole saliva from mice without using anesthesia. As the procedure does not involve surgical cannulation of the salivary glands, saliva can be collected from the same mouse at different times. The time between the injection of a secretory stimulant (pilocarpine) and the appearance of saliva in the mouth (lag time) was 100.5 +/-8.5 s (mean+/-S.E.M., n=10) for control mice. The volume of saliva collected in the first 5 min was three times greater than that collected between 15 and 20 min. The average flow rate for a collection period of 15 min was 16.7 +/-1.8 microl/min (n=10). The flow rate was decreased 50% (P<0.005) whereas the lag time was increased more than 300% (P<0.05) at 24 h after irradiation. The concentrations of a 23.5-kDa protein and a mucin were decreased after irradiation whereas there was no significant effect on the concentration of amylase or peroxidase.

Effect of 24 hours light on circadian rhythms of secretory enzymes and morphology of rat von Ebner's glands.

Von Ebner's glands of the rat are minor salivary serous glands in the posterior portion of the tongue. They secrete two digestive enzymes, lingual lipase and amylase. In this investigation, circadian rhythm in feeding was established under a normal 12 h light/12 h dark cycle, with the rats eating primarily during the dark period. At lights on, the size of the acinar cells and the area of the inclusive secretory granules, and the amount of digestive enzyme activity (lingual lipase and amylase) remaining in the gland was significantly less than in the mid-afternoon, after very little daylight food consumption. However, after 7 days of continuous light the circadian rhythm was altered: the food consumption during the normal night-time hours (5 p.m. to 8 a.m.) went from 88% of total 24 h food consumption to 45%, and during normal daylight hours (8 a.m. to 5 p.m.) from 12% to 55%. These changes were correlated with histometric findings of a near reversal of the areas of acinar cells and secretory granules of a.m. and p.m. samples under continuous light. Lingual lipase activity in the glands went from 35% under 12 h light to 61% under continuous light in the a.m. and from 65% to 39% in the p.m. Amylase activity also showed nearly a reversal in activity remaining in the gland, from 36% at 12 h light to 58% at 24 h light in the a.m. and 64% to 41% for the p.m. samples. These results indicate that the von Ebner's glands of the rat have a circadian rhythm of secretion and storage of secretory proteins that is subject to light entrainment similar to that seen in other exocrine glands such as the parotid and pancreas.

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.

[The action of gamma irradiation on terrilytin immobilized on cellulose-fiber materials]. / Deistvie gamma-oblucheniia na terrilitin, immobilizovannyi na tselliuloznykh voloknistnykh materialakh.

The effect of gamma-radiation on terrilytin, a proteolytic enzyme immobilized on modified and nonmodified cellulose materials was studied by EPR. Dialdehyde cellulose and graft copolymer of cellulose and polyacrylic acid were used as the modified cellulose materials. Dependence of the native and immobilized terrilytin activity and the content of free radicals in the irradiated samples on the irradiation dose was observed. It was shown that immobilization of the enzyme led to increasing of its stability to the effect of the ionizing radiation. This was due to transfer of the free valency from terrilytin to the carrying polymer which prevented radiation and chemical destruction of the enzyme. The proteolytic activity of native terrilytin subjected to gamma-irradiation markedly decreased because of intramolecular and intermolecular interactions during reactions of the terrilytin free radicals, since in this case there was no polymer as an acceptor of the enzyme free valency.