Quantitative Distribution of DNA, RNA, Histone and Proteins Other than Histone in Mammalian Cells, Nuclei and a Chromosome at High Resolution Observed by Scanning Transmission Soft X-Ray Microscopy (STXM).

Soft X-ray microscopy was applied to study the quantitative distribution of DNA, RNA, histone, and proteins other than histone (represented by BSA) in mammalian cells, apoptotic nuclei, and a chromosome at spatial resolutions of 100 to 400 nm. The relative distribution of closely related molecules, such as DNA and RNA, was discriminated by the singular value decomposition (SVD) method using aXis2000 software. Quantities of nucleic acids and proteins were evaluated using characteristic absorption properties due to the 1s⁻π * transition of N=C in nucleic acids and amide in proteins, respectively, in the absorption spectra at the nitrogen K absorption edge. The results showed that DNA and histone were located in the nucleus. By contrast, RNA was clearly discriminated and found mainly in the cytoplasm. Interestingly, in a chromosome image, DNA and histone were found in the center, surrounded by RNA and proteins other than histone. The amount of DNA in the chromosome was estimated to be 0.73 pg, and the content of RNA, histone, and proteins other than histone, relative to DNA, was 0.48, 0.28, and 4.04, respectively. The method we present in this study could be a powerful approach for the quantitative molecular mapping of biological samples at high resolution.

Discrimination of DNA and RNA distribution in a mammalian cell by scanning transmission soft X-ray microscopy.

Soft X-ray spectromicroscopy was applied to study the distribution of DNA and RNA in a mammalian cell at the spatial resolution of 400 nm. The relative distribution of DNA and RNA was examined by the SVD (singular value decomposition) method in aXis2000 program using combined full spectra of DNA and RNA at the absorption edge regions of carbon, nitrogen and oxygen. The absorption of nucleic acid was evaluated using 1s-π* transitions in the NEXAFS spectra at the nitrogen K absorption edge and distributed to DNA and RNA according to the relative level obtained above. The present results revealed the usefulness of the SVD method to discriminate closely related molecules such as DNA and RNA.

Optimization of X-ray microplanar beam radiation therapy for deep-seated tumors by a simulation study.

A Monte Carlo simulation was applied to study the energy dependence on the transverse dose distribution of microplanar beam radiation therapy (MRT) for deep-seated tumors. The distribution was found to be the peak (in-beam) dose and the decay from the edge of the beam down to the valley. The area below the same valley dose level (valley region) was decreased with the increase in the energy of X-rays at the same beam separation. To optimize the MRT, we made the following two assumptions: the therapeutic gain may be attributed to the efficient recovery of normal tissue caused by the beam separation; and a key factor for the efficient recovery of normal tissue depends on the area size of the valley region. Based on these assumptions and the results of the simulated dose distribution, we concluded that the optimum X-ray energy was in the range of 100-300 keV depending on the effective peak dose to the target tumors and/or tolerable surface dose. In addition, we proposed parameters to be studied for the optimization of MRT to deep-seated tumors.

Application of XANES profiles to X-ray spectromicroscopy for biomedical specimens: part II. Mapping oxidation state of cysteine in human hair.

Human hair fibers are primarily composed of keratin protein, characterized by a very high content of cysteine, a sulfur-containing amino acid, which ordinarily forms cystine via a disulfide bond. It is known that some cystine residues are converted to cysteic acid during permanent waving or hair coloring, although details of their distribution and extent are still unclear. In this study, by using difference in XANES profiles of cystine and cysteic acid at the S-K absorption edge, the formation of cysteic acid was confirmed for homogenized samples of permed or bleached hair. Furthermore chemical mapping of cysteic acid was performed on hair-section samples with X-ray contact microscopy. The peripheral region, cuticle, in bleached hair showed the highest content of cysteic acid compared with the other parts, while permed hair showed relatively uniform distribution. This finding suggests that perming and bleaching damage hair by different mechanisms.

A narrow microbeam is more effective for tumor growth suppression than a wide microbeam: an in vivo study using implanted human glioma cells.

The tumoricidal mechanisms of microbeam radiation therapy, and the more recently proposed minibeam radiation therapy, for the treatment of brain tumors are as yet unclear. Moreover, from among the various parameters of beam geometry the impact of changing the beam width is unknown. In this study, suppression of tumor growth in human glioma cells implanted in mice was evaluated experimentally using microbeams of two different widths: a conventional narrow beam (20 µm width, 100 µm center-to-center distance) and a wide beam (100 µm width, 500 µm center-to-center distance). The tumor growth ratio was compared and acute cell death was studied histologically. With cross-planar irradiation, tumor growth was significantly suppressed between days 4 and 28 after 20 µm microbeam irradiation, whereas tumor growth was suppressed, and not significantly so, only between days 4 and 18 after 100 µm microbeam irradiation. Immunohistochemistry using TUNEL staining showed no increase in TUNEL-positive cells with either microbeam at 24 and 72 h post-irradiation. The 20 µm microbeam was found to be more tumoricidal than the 100 µm microbeam, and the effect was not related to apoptotic cell death. The underlying mechanism may be functional tissue deterioration rather than direct cellular damage in the beam path.

Application of XANES profiles to X-ray spectromicroscopy for biomedical specimens: Part I. Discrimination of macromolecules with sulfur atoms.

XANES spectra of biomacromolecules such as histone, hemoglobin or bovine serum albumin (BSA) were measured in transmission at the S-K absorption edge for comparison with those of sulfur containing low-molecular weight biomolecules with special reference to the dependence on the chemical environment of sulfur. The spectra of dry histone and hemoglobin exhibited a prominent peak at the same energy as that of cysteine and glutathione (GSH), while the BSA spectrum showed an additional peak at a lower energy, which coincided with that of cystine and glutathione disulfide (GSSG). XANES peaks were found at the same energy even in a mammalian cell dry pellet. Spectra in the hydrated state exhibited similar profiles except for a very slight shift of resonance peaks to the lower energy. These results indicate that XANES profiles could be applicable to the mapping of S-C and S-S bonds in dry/hydrated biological systems using a spectromicroscopic technique. In addition, mass absorption coefficients of GSH and GSSG were determined.

Role of wild-type p53 in apoptotic and non-apoptotic cell death induced by X-irradiation and heat treatment in p53-mutated mouse M10 cells.

The sensitizing effects of wild-type p53 on X-ray-induced cell death and on heat-induced apoptosis in M10, a radiosensitive and Trp53 (mouse p53 gene)-mutated lymphoma cell line which dies through necrosis by X-irradiation, were investigated using three M10 derived transfectants with wild-type TP53 (human p53 gene). Cell death was determined by colony formation and/or dye exclusion test, and apoptosis was detected as the changes in nuclear morphology by Giemsa staining. Expression of wild-type p53 protein increased radiosensitivity of cell death as determined by both clonogenic and dye exclusion assays. This increase in radiosensitivity was attributable largely to apoptosis induction in addition to a small enhancement of necrosis. Interestingly neither pathway to cell death was accompanied by caspase-3 activation. On the other hand, heat-induced caspase-3 dependent apoptotic cell death without transfection was further increased by the transfection of wild-type p53. In conclusion, the introduction of wild-type p53 enhanced apoptotic cell death by X-rays or heat via different mechanisms that do or do not activate caspase-3, respectively. In addition, p53 also enhanced the X-ray-induced necrosis in M10 cells.

Novel assessment of hepatic iron distribution by synchrotron radiation X-ray fluorescence microscopy.

Excess iron deposition in the liver is known to be hepatotoxic and may exacerbate liver injury. However, little is known about iron distribution in the lobule because of the lack of a highly sensitive detection method. The aim of this study is to determine iron distribution in the lobule of human liver by means of synchrotron radiation X-ray fluorescence (SRXRF) microscopy. Mapping of the trace elements was done with use of SRXRF microscopy and compared with the results of staining by Berlin blue and oxidative stress markers. Iron deposits were distributed predominantly in periportal hepatocytes in the normal liver in a decreasing gradient from the periportal area to the perivenous area. This distribution was consistent with the formation of oxidative stress markers, suggesting that hepatocytes in the periportal area may be predominantly primed by iron-induced free radical damage even in normal liver. On the other hand, iron deposits in the periportal area were more intense than those in the centrilobular area in both the liver with chronic hepatitis C and the cirrhotic liver. In conclusion, elemental mapping by SRXRF microscopy was a highly sensitive method for the detection and mapping of elements such as iron and copper in liver sections.

Sarcoptes scabiei var. hominis: three-dimensional structure of a female imago and crusted scabies lesions by X-ray micro-CT.

The three-dimensional structure of scabies mites (Sarcoptes scabiei var. hominis) and keratin layers affected by crusted scabies lesions were obtained using X-ray computed tomography at sub-micrometer and micrometer resolution, respectively (X-ray micro-CT). Clear three-dimensional images including internal structure of scabies mites were obtained. Utilizing reconstructed micro-CT data, the sections of the capitulum (head part), digestive organs, and legs are shown. The reconstructed capitulum shows a jaw-like structure capable of penetrating the keratin layer of the skin. The tip of the forelegs of female scabies mites has a flat disk structure that may be used to grasp the skin surface. The keratin layer of a crusted scabies lesion spontaneously exfoliated from a patient was also reconstructed by the X-ray micro-CT technique. Extracted sections from CT data revealed a network structure of tunnels made by scabies mites with numerous larvae and eggs inside the tunnels.

Induction of DNA double-strand breaks and cellular migration through bystander effects in cells irradiated with the slit-type microplanar beam of the spring-8 synchrotron.

PURPOSE: To determine whether glioma cells irradiated with a microplanar X-ray beam exert bystander effects. METHODS AND MATERIALS: Microplanar beam irradiation of glioma cells in vitro was done using the SPring-8 synchrotron radiation facility. The amount of DNA double-strand breaks (dsbs) was measured by the fluorescence intensity of phosphorylated H2AX or the number of 53BP1 foci. The dose distribution in a cell population exposed to a single microplanar beam was determined by the amount of phosphorylated H2AX-positive cells. Bystander effects were determined by counting the number of 53BP1 foci in nonirradiated cells treated with conditioned medium from cultures of irradiated cells. RESULTS: More DNA dsbs were detected in cells adjacent to an area irradiated by the single beam than in cells in distant, nonirradiated areas as a result of bystander effects caused by scattered X-rays and DNA dsbs. In support of this, more 53BP1 foci were observed in nonirradiated, conditioned medium-treated cells than in control cells (i.e., cells not treated with irradiation or conditioned medium). These results suggest that DNA dsbs were induced in nonirradiated cells by soluble factors in the culture medium. In addition, we observed cellular migration into areas irradiated with peak doses, suggesting that irradiated cells send signals that cause nonirradiated cells to migrate toward damaged cells. CONCLUSIONS: Bystander effects are produced by factors secreted as a result of slit-type microplanar X-ray beam irradiation.

Spectromicroscopic film dosimetry for high-energy microbeam from synchrotron radiation.

A microscope with band-pass filters was used to measure the optical-density distribution of GafChromic films irradiated with multi-slit microbeam X-rays. The planar width was 25 microm, and the center-to-center distance was 200 microm. The peak and valley dose rates in air were found to be 120 and 0.7Gy/s, respectively. In a polymethylmethacrylate phantom, the peak-to-valley dose ratio decreased to 80 at a 1-mm depth. Doses calculated with the PENELOPE code agreed with those around the peak but became smaller in the valley.

Atherosclerotic plaque imaging using phase-contrast X-ray computed tomography.

Reliable, noninvasive imaging modalities to characterize plaque components are clinically desirable for detecting unstable coronary plaques, which cause acute coronary syndrome. Although recent clinical developments in computed tomography (CT) have enabled the visualization of luminal narrowing and calcified plaques in coronary arteries, the identification of noncalcified plaque components remains difficult. Phase-contrast X-ray CT imaging has great potentials to reveal the structures inside biological soft tissues, because its sensitivity to light elements is almost 1,000 times greater than that of absorption-contrast X-ray imaging. Moreover, a specific mass density of tissue can be estimated using phase-contrast X-ray CT. Ex vivo phase-contrast X-ray CT was performed using a synchrotron radiation source (SPring-8, Japan) to investigate atherosclerotic plaque components of apolipoprotein E-deficient mice. Samples were also histologically analyzed. Phase-contrast X-ray CT at a spatial resolution of 10-20 mum revealed atherosclerotic plaque components easily, and thin fibrous caps were detected. The specific mass densities of these plaque components were quantitatively estimated. The mass density of lipid area was significantly lower (1.011 +/- 0.001766 g/ml) than that of smooth muscle area or collagen area (1.057 +/- 0.001407 and 1.080 +/- 0.001794 g/ml, respectively). Moreover, the three-dimensional assessment of plaques could provide their anatomical information. Phase-contrast X-ray CT can estimate the tissue mass density of atherosclerotic plaques and detect lipid-rich areas. It can be a promising noninvasive technique for the investigation of plaque components and detection of unstable coronary plaques.

Threshold level of p53 required for the induction of apoptosis in X-irradiated MOLT-4 cells.

PURPOSE: To determine the threshold level for the initiation of apoptosis by studying the quantitative aspect of p53 response to DNA damage in individual cells, to better understand the process in X-ray-induced p53-dependent apoptosis. METHODS AND MATERIALS: Time-sequential changes in p53 protein level were obtained for X-irradiated MOLT-4 cells using flow cytometry and analyzed. RESULTS: The changes in the cellular frequency distribution pattern of p53 content could be divided into two parts at a certain p53 level. The p53 vs. side-scatter in flow cytometry showed the sequential changes of p53 increase followed by an increase in cell death. On the basis of these results we determined a threshold level of p53 for the initiation of apoptosis. The level was estimated to be (1.08 +/- 0.05) x 10(5) molecules per cell, which was approximately threefold higher than the mean content of control cells. The minimum times for p53 level to reach this threshold level were independent of X-ray dose and 1.4-1.6 h. The times for the signal transduction from the p53 accumulation to disruption of the mitochondrial membrane potential, caspase-3 activation, and cell death were 1.6, 2.1, and 2.8 h, respectively. CONCLUSIONS: The threshold level of p53 for the initiation of apoptosis and the time sequence in the course of apoptotic events were determined in X-irradiated MOLT-4 cells.

Different contributions of the indirect effects of gamma-rays on the cytotoxicity in M10 and XRCC4 transfected M10 cells.

The protective effects of dimethyl sulfoxide (DMSO) against cell killing by (137)Cs gamma-rays were investigated in XRCC4-deficient cell line M10, XRCC4-complemented M10 and the parental mouse leukemia cell line L5178Y. Cell survival was determined by the colony-forming ability. M10 cells were more sensitive to gamma-ray-induced cell death than L5178Y and complemented M10 cells. Cell survival was increased in both M10 and L5178Y in the presence of DMSO. However, estimation of the DMSO-protectable fraction revealed a smaller protectable fraction for M10 cells than for L5178Y cells, indicating that indirect effects contributed in a smaller extent to the cytotoxicity in M10 than that in L5178Y. This effect is due to XRCC4 deficiency, since transfection of XRCC4 cDNA into M10 cells restored the radioprotective effects of DMSO to the level seen in L5178Y. In M10 cells, the killing effects of high LET radiation (Auger electrons from (125)I-antipyrine, carbon ions with an LET of 166 keV microm(-1)) were similar to those of low LET radiation ((137)Cs gamma-rays, characteristic X-rays from (125)I-bovine serum albumin). We discuss that lethal lesions produced by indirect actions in L5178Y and XRCC4-complemented M10 cells may differ, at least in part, from DNA double-strand breaks repairable by non-homologous end joining.

Contribution of indirect action to radiation-induced mammalian cell inactivation: dependence on photon energy and heavy-ion LET.

The contribution of indirect action mediated by OH radicals to cell inactivation by ionizing radiations was evaluated for photons over the energy range from 12.4 keV to 1.25 MeV and for heavy ions over the linear energy transfer (LET) range from 20 keV/microm to 440 keV/microm by applying competition kinetics analysis using the OH radical scavenger DMSO. The maximum level of protection provided by DMSO (the protectable fraction) decreased with decreasing photon energy down to 63% at 12.4 keV. For heavy ions, a protectable fraction of 65% was found for an LET of around 200 keV/microm; above that LET, the value stayed the same. The reaction rate of OH radicals with intracellular molecules responsible for cell inactivation was nearly constant for photon inactivation, while for the heavy ions, the rate increased with increasing LET, suggesting a reaction with the densely produced OH radicals by high-LET ions. Using the protectable fraction, the cell killing was separated into two components, one due to indirect action and the other due to direct action. The inactivation efficiency for indirect action was greater than that for direct action over the photon energy range and the ion LET range tested. A significant contribution of direct action was also found for the increased RBE in the low photon energy region.

Batch grinding kinetics of Ethenzamide particles by fluidized-bed jet-milling.

Ethenzamide solids as a representative active pharmaceutical ingredient (API) were batch-ground by means of a fluidized-bed jet-mill which is a relatively new equipment and promising for production in the pharmaceutical field. Thus, the characteristic grinding mechanism was investigated. As a result, the variation of the residual ratio with grinding time after milling was expressed simply by a mathematical model using only the first Kapur function, and it was consistent with experimental data satisfactorily. As the shape of the function was much different from that of inorganic compound and peculiar to API, a cubic function with respect to particle diameter was defined newly and well fitted to the experimental data. The function was also found to be affected by the operating parameters as the grinding gas pressure, the charge weight of raw material and the linear velocity at the grinding nozzle. According to the assessments of the breakage and the selection functions derived from the first Kapur function, it was found that the grinding mechanism of Ethenzamide particles was related with particle attrition mainly.

Dissolution characteristics of cylindrical particles and tablets.

In this paper, dissolution characteristics of primary-particles and compressed tablets were investigated by experiments using a mathematical model. For the primary-particle, it was found that the dissolution rate increased with a decrease in the particle size. Assuming that primary-particles of size distribution were of cylindrical shape and that the dissolution occurs from the total external surface, an extended Nernst-Noyes-Whitney equation fitted to the experimental data well. As the influences of particle size and shape on thickness of a diffusion-boundary film were found to be quite low, the dissolution rate was considered to be affected by the specific surface area dominantly. Furthermore, the same model was applied to a compressed tablet and fitted to the data well. Though the rate constant obtained were not affected by the properties of primary-particles forming the tablet, it was found to increase with the apparent voidage which occupies the inter-particle volume of tablet diluent among less soluble particles. Consequently, an increase in the apparent voidage is presumed to accelerate penetration of water into the internal voids of the tablet. Thus, the dissolution going, the effective surface area inside the tablet is considered to be extended.

Variation in particle shape of active pharmaceutical ingredients prepared by fluidized-bed jet-milling.

In pharmaceutical industries, most active pharmaceutical ingredients are poorly water soluble, and therefore milling processes are important to obtain fine particles that can be easily dissolved in the body. However, the main purpose of milling is micronization of particles. From the viewpoint of fine particle preparation in the formulation process, milling has not been investigated sufficiently. In this paper, ethenzamide was milled under various operating conditions using a fluidized-bed jet-mill. It was found that not only the particle size but also the particle shape varied with the milling conditions. The relationship between particle shape and milling conditions has been obtained experimentally.

Effect of particle shape of active pharmaceutical ingredients prepared by fluidized-bed jet-milling on cohesiveness.

Milling is a common procedure to improve bioavailability of many active pharmaceutical ingredients (APIs), which typically have low solubility in water. But such micronization can yield an increase in the cohesiveness of particles. Although particle cohesiveness is desirable for tablet strength in the subsequent formulation process, increased particle cohesiveness can lead to operational difficulties in a milling equipment due to compaction of particles inside. In this article, the impact of milling via a fluidized-bed jet-mill on the cohesive strength and interparticle force was studied using Ethenzamide as a pharmaceutical model compound. As a result, the particle shape was found to affect both the tensile strength of powder bed and the interparticle cohesive force. A powder bed, having relatively high void fraction by direct tensile test, shows a positive correlation between the cohesive force and the particle sphericity, while powders with low void fraction by diametral compression test show a positive correlation between the cohesive force and the angularity of the particle.

Effects of single-pulse (< or = 1 ps) X-rays from laser-produced plasmas on mammalian cells.

The effects of low linear energy transfer (LET) radiation on mammalian cells have been studied at dose-rates as high as 10(9) Gy/sec delivered as a single 3-nanosecond pulse, and no increase in cytotoxicity was shown compared with delivery at a conventional dose-rate. There have been no observations on the effects of radiation delivered at even higher dose-rates on the picosecond time-scale. Here we examined, for the first time, the effects on cultured mouse L5178Y cells and its radiosensitive XRCC4-deficient mutant M10 cells of sub-picosecond X-rays emitted from laser-produced plasmas at the ultrahigh dose-rate of 10(12)-10(13) Gy/sec. No increase in the sensitivity to the X-rays was observed compared with gamma-rays at a conventional dose-rate. The increase in the sensitivity of L5178Y cells by labeling with 5-iododeoxyuridine was smaller than those irradiated with gamma-rays at a conventional dose-rate, while the difference was apparently the reverse in M10 cells. The D10 ratio between L5178Y cells and M10 cells produced by the X-rays at temporally dense ionization was the same as that produced by X(gamma)-rays at the conventional dose-rate, while the ratio is greatly reduced in the case of particle radiation. These results suggest that there is no increase in the cytotoxic effects of X-rays at dose-rates as high as 10(13) Gy/sec, and that the increased cytotoxicity of particle radiation is not attributable to temporally dense ionization. It is discussed that the mechanism for the induction of radiation damage responsible for cytotoxicity may be slightly modified at ultrahigh dose-rates.