Leupeptin-sensitive proteases involved in cell survival after X-ray irradiation in human RSa cells

Proteases have received attention as important cellular components responsible for stress response in human cells. However, little is known about the role of proteases in the early steps of cell response after X-ray irradiation. In the present study, we first searched for proteases whose activity levels are changed soon after X-ray irradiation in human RSa cells with a high sensitivity to X-ray cell-killing. RSa cells showed an increased level of fibrinolytic protease activity within 10 min after irradiation with X-ray (up to 3 Gy). The induced protease activity was proved to be inhibited by leupeptin. We next examined whether this protease inducibility is related to the X-ray susceptibility of cells. Treatment of RSa cells with leupeptin prior to X-ray irradiation resulted in lowered colony survival and an increased ratio of G(2)/M-arrested cells and apoptotic cells. These results suggest that leupeptin-sensitive proteases are involved in the resistance of human RSa cells to X-ray cell-killing.     

Identification of 5'-adenylylimidodiphosphate-hydrolyzing enzyme activity in rabbit taste bud cells using X-ray microanalysis

X-ray microanalysis has been used to characterize the enzyme activity hydrolyzing the ATP analogue 5'-adenylylimidodiphosphate (AMP-PNP) in taste bud cells. Rabbit foliate papillae fixed with paraformaldehyde and glutaraldehyde were incubated cytochemically with AMP-PNP as the substrate and lead ion as capture agent. The reaction product which appeared on the microvilli of taste bud cells was examined using an energy dispersive X-ray microanalyzer connected to an analytical electron microscope. The X-ray spectrum thus obtained was compared with that obtained from the product obtained from the demonstration of ATPase activity. Comparison of the phosphorus/lead ratios in the two products showed that twice as much phosphorus was released from an AMP-PNP molecule by the activity in question compared with that released from an ATP molecule by ATPase activity. This indicates that the enzyme hydrolyzes AMP-PNP into AMP and imidodiphosphate and that the enzyme is adenylate cyclase or ATP pyrophosphohydrolase, which possesses a similar hydrolytic property, but not ATPase or alkaline phosphatase, which hydrolyzes AMP-PNP into ADP-NH2 and orthophosphate. This paper provides an example of the use of X-ray microanalysis as a tool for enzyme distinction. The method is applicable to a variety of enzymes and tissues.     

Identification of 5'-Adenylylimidodiphosphate-Hydrolyzing Enzyme Activity in Rabbit Taste Bud Cells Using X-Ray Microanalysis

X-ray microanalysis has been used to characterize the enzyme activity hydrolyzing the ATP analogue 5'-adenylylimidodiphosphate (AMP-PNP) in taste bud cells. Rabbit foliate papillae fixed with paraformaldehyde and glutaraldehyde were incubated cytochemically with AMP-PNP as the substrate and lead ion as capture agent. The reaction product which appeared on the microvilli of taste bud cells was examined using an energy dispersive X-ray microanalyzer connected to an analytical electron microscope. The X-ray spectrum thus obtained was compared with that obtained from the product obtained from the demonstration of ATPase activity. Comparison of the phosphorus/lead ratios in the two products showed that twice as much phosphorus was released from an AMP-PNP molecule by the activity in question compared with that released from an ATP molecule by ATPase activity. This indicates that the enzyme hydrolyzes AMP-PNP into AMP and imidodiphosphate and that the enzyme is adenylate cyclase or ATP pyrophosphohydrolase, which possesses a similar hydrolytic property, but not ATPase or alkaline phosphatase, which hydrolyzes AMP-PNP into ADP-NHj and orthophosphate. This paper provides an example of the use of X-ray microanalysis as a tool for enzyme distinction. The method is applicable to a variety of enzymes and tissues.     

Bone mineral density in patients with breast cancer in the perimenopausal period

Dual-energy X-ray absorptiometry was used to examine 54 patients with breast cancer. A clinical comparison group comprised 50 healthy women. All the examinees were aged 45 to 55 years. Bone mineral density was measured in the lumbar spine, proximal femur, and ultradistal antibrachium. The X-ray densitometric values of bone tissues in perimenopausal women with breast cancer were not found to be abnormal, which led to the conclusion that there were no significant bone metabolic disturbances. Along with this it was established that the women at this age had considerably reduced bone mineral density in the epimetaphyseal (ultradistal) forearm with evolving osteopenia. In this connection, it is expedient to identify an ultradistal portion of the antibrachium as an object of X-ray densitometric examination for the early diagnosis of impaired bone mineralization in women in the 45-to-55-year-old age group.     

Contrast enhancement of speckle patterns from blood in synchrotron X-ray imaging

Hemodynamics has been a very important factor in understanding and diagnosing various vascular diseases. Recently, the X-ray particle image velocimetry (X-ray PIV) method using speckle patterns of blood has been introduced as a new quantitative visualization method for blood flows without any seeding tracer or contrast agents. In this study, the peculiar optical characteristics of blood on the synchrotron X-ray imaging method, which were not presented in previous studies, were investigated in depth and systematically. The experimental conditions required for X-ray PIV application were found to be the distance between the sample and the scintillator (～40 cm), the thickening of the blood sample (>0.3 mm), and hematocrit (20.0–80.0%). In addition, we verified that the X-ray PIV method is reliable as an advanced flow velocimetry by comparing the flow rate evaluated from the X-ray PIV result and the input flow rate supplied from a syringe pump with an error of less than 1%. Through this study, based on the understanding of contrast enhancement mechanisms of speckle patterns from blood, we could establish a trustworthy flow visualization method that can be used effectively in hemodynamic studies.     

Increase in recombination rate in Arabidopsis thaliana plants sharing gaseous environment with X-ray and UVC-irradiated plants depends on production of radicals

X-ray and UVC are the two physical agents that damage DNA directly, with both agents capable of inducing double-strand breaks. Some of our recent work has demonstrated that local exposure to UVC results in a systemic increase in recombination frequency, suggesting that information about exposure can be passed from damaged to non-damaged tissue. Indeed, we recently showed that plants sharing the same enclosed environment with UVC-irradiated plants exhibit similar increase in homologous recombination frequency as irradiated plants. Here, we further tested whether yet another DNA-damaging agent, X-ray, is capable of increasing recombination rate (RR) in neighboring plants grown in a Petri dish. To test this, we grew plants exposed to X-ray or UVC irradiation in an enclosed environment next to non-exposed plants. We found that both X-ray and UVC-irradiated plants and neighboring plants exhibited comparable increases in the levels of strand breaks and the RR. We further showed that pre-exposure of plants to radical scavenger DMSO substantially alleviates the radiation-induced increase in RR and prevents formation of bystander signal. Our results suggest that the increase in RR in bystander plants can also be triggered by X-ray and that radicals may play some role in initiation or maintenance of this signal.     

Measurement of lung water dynamics and lung aeration in the perinatal period by use of digital X-ray fluoroscopy

A method has been developed for the measurement of lung water dynamics and regional aeration of the lung in anesthetized newborn lambs by use of X-ray fluoroscopy, video recording, and digital image processing. After cesarean section and before the first breath fetal lambs under halothane-oxygen were placed on an X-ray table and connected to a volume-cycled respirator. X-ray fluoroscopy commenced before the initiation of respiration, and the images were recorded on video tape. X-ray transmission through the thorax increased as the lung was aerated. The enhanced transmission was compared with the values obtained from a calibration water wedge from which an equivalent path length through water can be estimated. In testing this method, it was demonstrated that X-ray transmission was linearly related to the wet lung weight-to-body weight ratio and to the product of the wet-to-dry weight ratio multiplied by anatomic thickness of frozen lung blocks. Calibrated values were also linearly related to this product and to the actual measured height of the fluid and tissue in the fluid-filled lung.     

Behavior of hard X-ray emission in discharges with current disruptions in the DAMAVAND and TVD tokamaks

Results are presented from studies of the behavior of hard X-ray emission in discharges with current disruptions in the DAMAVAND and TVD tokamaks. The current disruptions are caused by either an MHD instability or the instability related to the vertical displacement of the plasma column. Experiments were conducted at a fixed value of the safety factor at the plasma boundary (q _a ? 2.3). Experimental data show that, during a disruption caused by an MHD instability, hard X-ray emission is suppressed by this instability if the amplitude of the magnetic field fluctuations exceeds a certain level. If the disruption is caused by the instability related to the vertical displacement of the plasma column, then hard X-ray emission is observed at the instant of disruption. The experimental results show that the physical processes resulting in the generation and suppression of runaway electron beams are almost identical in large and small tokamaks.     

Monitoring and validating active site redox states in protein crystals

High resolution protein crystallography using synchrotron radiation is one of the most powerful tools in modern biology. Improvements in resolution have arisen from the use of X-ray beamlines with higher brightness and flux and the development of advanced detectors. However, it is increasingly recognised that the benefits brought by these advances have an associated cost, namely deleterious effects of X-ray radiation on the sample (radiation damage). In particular, X-ray induced reduction and damage to redox centres has been shown to occur much more rapidly than other radiation damage effects, such as loss of resolution or damage to disulphide bridges. Selection of an appropriate combination of in-situ single crystal spectroscopies during crystallographic experiments, such as UV-visible absorption and X-ray absorption spectroscopy (XAFS), allows for effective monitoring of redox states in protein crystals in parallel with structure determination. Such approaches are also essential in cases where catalytic intermediate species are generated by exposure to the X-ray beam. In this article, we provide a number of examples in which multiple single crystal spectroscopies have been key to understanding the redox status of Fe and Cu centres in crystal structures. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.     

Structure of porcine heart cytoplasmic malate dehydrogenase: combining X-ray diffraction and chemical sequence data in structural studies

The amino acid sequence of cytoplasmic malate dehydrogenase (sMDH) has been determined by a combination of X-ray crystallographic and chemical sequencing methods. The initial molecular model incorporated an "X-ray amino acid sequence" that was derived primarily from an evaluation of a multiple isomorphous replacement phased electron density map calculated at 2.5-A resolution. Following restrained least-squares crystallographic refinement, difference electron density maps were calculated from model phases, and attempts were made to upgrade the X-ray amino acid sequence. The method used to find the positions of peptides in the X-ray structure was similar to those used for studying protein homology and was shown to be successful for large fragments. For sMDH, X-ray methods by themselves were insufficient to derive a complete amino acid sequence, even with partial chemical sequence data. However, for this relatively large molecule at medium resolution, the electron density maps were of considerable help in determining the linear position of peptide fragments. The N-acetylated polypeptide chain of sMDH has 331 amino acids and has been crystallographically refined to an R factor of 19% for 2.5-A resolution diffraction data.     

Liquid sample delivery techniques for serial femtosecond crystallography

X-ray free-electron lasers overcome the problem of radiation damage in protein crystallography and allow structure determination from micro- and nanocrystals at room temperature. To ensure that consecutive X-ray pulses do not probe previously exposed crystals, the sample needs to be replaced with the X-ray repetition rate, which ranges from 120 Hz at warm linac-based free-electron lasers to 1 MHz at superconducting linacs. Liquid injectors are therefore an essential part of a serial femtosecond crystallography experiment at an X-ray free-electron laser. Here, we compare different techniques of injecting microcrystals in solution into the pulsed X-ray beam in vacuum. Sample waste due to mismatch of the liquid flow rate to the X-ray repetition rate can be addressed through various techniques.     

Repopulation in murine skin after X-ray treatments with multiple fractions per day

The kinetics of repopulation of clonogens in skin after fractionated X-ray exposures was studied in a series of experiments using a top-up design. The feet of mice were exposed to small X-ray doses (1.5 or 2 Gy), given two or three times a day on consecutive days with a minimum interfraction interval of 8 h. A single top-up dose of d(4)-Be neutrons was then given at various intervals after the last X-ray fraction, typically on Days 1,4,8, 15, and 19. The acute skin reaction produced was scored an analyzed by both a standard 23-day averaging and a 7-day averaging procedure. Either method gave similar results and led to the same conclusions. The amount of top-up dose needed to produce a fixed skin reaction was used as a measure of the net effect of the X-ray treatments. This net effect is a result of the initial reduction in skin clonogens due to X rays, and their repopulation before the top-up dose was given. Repopulation was not detected during any of these courses of fractionated treatment, up to an overall time of at least 12 and possibly 16 days. On completion of X-ray schedules lasting 6-16 days, repopulation started 4 days later. In contrast, this delay lengthened to approximately 8 days for shorter overall treatment times of 3-4 days. Once repopulation started, it proceeded rapidly over 11 days so that by 15 days after the cessation of X rays, the skin was restored almost to its normal state with respect to radiosensitivity. The residual damage from Day 15 to Day 19 postirradiation was 3-13% of a full-effect level. The rate of repopulation can be expressed as a clonogen doubling time (Tclon), assuming that an average skin reaction of 1.5 is equivalent to a clonogen surviving fraction of 1.7 x 10(-5). Tclon varied inversely with the amount of initial damage inflicted by the X rays, with the shortest values (1-1.3 days) seen following X-ray doses that gave an initial damage level of 60-80% of full effect. These data are consistent with a hypothesis that damage is "sensed" only 10-12 days after the first X-ray fraction, which provides the stimulus for repopulation of the target cells in the basal layer, the keratinoblasts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Manganese in the shell of Centropyxis (Rhizopodea: Protozoa)

Summary The proteinaceous shells of Centropyxis hirsuta contain a relatively high concentration of manganese in an amorphous state. The concentrations of manganese fill the alveoli which are characteristic of the shell structure. Observations based on cultured animals and subsequently examined by X-ray diffraction, a solid state energy dispersive X-ray analyser, and an analytical electron microscope, lead to the conclusions that manganese is selectively absorbed by Centropyxis hirsuta and deposited in the shell.     

Formation of an anisotropic electron velocity distribution function and production of multicharged ions in the micropinch discharge plasma

Analysis of the results of polarimetric measurements of X-ray line radiation of multicharged ions in a Z-pinch discharge indicates that the formation of an anisotropic electron velocity distribution in the neck of the current channel and the generation of highly charged ions are separated in time. The generation of a fast electron beam in the longitudinal ohmic electric field in the stage of plasma compression in the neck results in the polarization of X-ray bremsstrahlung continuum. In the stage of expansion of the hot dense micropinch plasma, the radial electric field prevails, due to which X-ray line radiation of multicharged ions becomes linearly polarized.     

Intracellular localization of lead in tissues of the mute swan

The aim of this study was to examine the ultrastructure of kidney, liver, blood and intestine of a lead-poisoned bird and compare these with tissues of a healthy bird. From these results it was hoped to reveal that any abnormalities observed were due to lead poisoning and consequently suitable sections were also selected for X-ray analysis. Renal intranuclear inclusions were present in the lead-poisoned swan. X-ray analysis clearly demonstrated that these granules consist of an amorphous mass of lead probably as lead phosphate. Large numbers of electron dense granules were observed in the liver of the lead-poisoned bird and occasionally in the healthy swans liver. X-ray analysis demonstrated that these granules contain iron. Some of the red blood cells of the lead-poisoned swan have opaque granules associated with the plasma membrane, these were not observed in the healthy swan.     

Different expression of cell surface fibronectin in spontaneously and X-ray transformed cells

The presence of cell surface fibronectin was examined by indirect immunofluorescence in 8 groups of related rat fibroblast lines expressing an in vitro transformed phenotype. The transformed cells were selected for anchorage independent growth either after X-ray treatment (X-ray transformed cells) or from control cultures (spontaneously transformed cells). All transformed fibroblasts of the latter class showed reduced expression of fibronectin at the cell surface, whereas most of the X-ray transformed derivatives exhibited a fibronectin-positive phenotype, like the untransformed parents. Moreover, from the fibronectin-negative spontaneously transformed cells, ouabain-resistant variants were isolated, the majority of which had regained the capacity to form an extracellular matrix of fibronectin. These results emphasize the variability in the properties of transformed cells and suggest that the properties of in vitro transformed cells may depend on the cause of transformation.     

Structure of the coat protein in Pf1 bacteriophage determined by solid-state NMR spectroscopy

The atomic resolution structure of Pf1 coat protein determined by solid-state NMR spectroscopy of magnetically aligned filamentous bacteriophage particles in solution is compared to the structures previously determined by X-ray fiber and neutron diffraction, the structure of its membrane-bound form, and the structure of fd coat protein. These structural comparisons provide insights into several biological properties, differences between class I and class II filamentous bacteriophages, and the assembly process. The six N-terminal amino acid residues adopt an unusual "double hook" conformation on the outside of the bacteriophage particle. The solid-state NMR results indicate that at 30 degrees C, some of the coat protein subunits assume a single, fully structured conformation, and some have a few mobile residues that provide a break between two helical segments, in agreement with structural models from X-ray fiber and neutron diffraction, respectively. The atomic resolution structure determined by solid-state NMR for residues 7-14 and 18-46, which excludes the N-terminal double hook and the break between the helical segments, but encompasses more than 80% of the backbone including the distinct kink at residue 29, agrees with that determined by X-ray fiber diffraction with an RMSD value of 2.0 A. The symmetry and distance constraints determined by X-ray fiber and neutron diffraction enable the construction of an accurate model of the bacteriophage particle from the coordinates of the coat protein monomers.     

Evaluation of the findings of computed tomography of the lungs in patients with nonspecific pneumothorax in late periods after surgical treatment

By using the results of examination of 54 patients operated on for nonspecific spontaneous pneumothorax (NSP), the authors conclude that X-ray computed tomography (CT) is of high value. In addition to external respiratory function that identifies pulmonary functional changes, it is recommended that a comprehensive examination should include X-ray CT with parenchymal densitometry of the upper, middle and lower lung to reveal lung tissue structural changes. The examination of patients should be performed as soon as possible when the disease is detected and in the late postoperative period for early surgical correction of an emphysematous process in the lung.     

New capacities of roentgen diagnosis in detecting small intestinal pathology

The paper based on the data of more than 200 studies of the small bowel with the new agent Entero-VU specially designed for X-ray study of the bowel gives an objective evaluation of this agent. The authors of the paper analyze the capacities of Entero-VU in diagnosing 38 cases of Crohn's disease, tuberculosis and tumors of the small bowel. Moreover, as a separate section the paper provides an assessment of the agent in mapping the X-ray standards of the small bowel. In the authors' opinion, the use of this agent as though fills the existing gap in the X-ray study of the small bowel.     

New capacities of X-ray diagnosis in detection of small intestine pathology

The paper based on the data of more than 200 studies of the small bowel with the new agent Entero-VU specially designed for X-ray study of the bowel gives an objective evaluation of this agent. The authors of the paper analyze the capacities of Entero-VU in diagnosing 38 cases of Crohn's disease, tuberculosis and tumors of the small bowel. Moreover, as a separate section the paper provides an assessment of the agent in mapping the X-ray standards of the small bowel. In the authors' opinion, the use of this agent as though fills the existing gap in the X-ray study of the small bowel.