Radiation-induced chromosomal instability in human mammary epithelial cells.

Karyotypes of human cells surviving X- and alpha-irradiation have been studied. Human mammary epithelial cells of the immortal, non-tumorigenic cell line H184B5 F5-1 M/10 were irradiated and surviving clones isolated and expanded in culture. Cytogenetic analysis was performed using dedicated software with an image analyzer. We have found that both high- and low-LET radiation induced chromosomal instability in long-term cultures, but with different characteristics. Complex chromosomal rearrangements were observed after X-rays, while chromosome loss predominated after alpha-particles. Deletions were observed in both cases. In clones derived from cells exposed to alpha-particles, some cells showed extensive chromosome breaking and double minutes. Genomic instability was correlated to delayed reproductive death and neoplastic transformation. These results indicate that chromosomal instability is a radiation-quality-dependent effect which could determine late genetic effects, and should therefore be carefully considered in the evaluation of risk for space missions.     

太空环境对肿瘤细胞生理特性的影响

将3种肿瘤细胞搭载于“神舟4号”的卫星返回舱内,经过7天太空飞行,回收后对存活细胞进行单克隆化,观察细胞形态,并测定了细胞周期、黏附力及细胞因子表达.结果显示,经太空飞行,小鼠黑色素瘤B16细胞的周期发生改变,G1期细胞明显增多(p<0.05),并表现多种细胞形态;人肺鳞癌细胞L78对血管内皮细胞黏附力明显减弱,但经传代培养其黏附力恢复且超过对照组细胞;Caski细胞IL-2、IL-8、TNF和TGF的表达均明显增加,而L78细胞上述4种细胞因子的表达均显著下降.结论,太空环境可影响肿瘤细胞的某些生理特性,但可否影响肿瘤细胞的免疫原性,仍需做进一步的实验.     

Characterizing parameters of Jatropha curcas cell cultures for microgravity studies

Jatropha (Jatropha curcas) is a tropical perennial species identified as a potential biofuel crop. The oil is of excellent quality and it has been successfully tested as biodiesel and in jet fuel mixes. However, studies on breeding and genetic improvement of jatropha are limited. Space offers a unique environment for experiments aiming at the assessment of mutations and differential gene expression of crops and in vitro cultures of plants are convenient for studies of genetic variation as affected by microgravity. However, before microgravity studies can be successfully performed, pre-flight experiments are necessary to characterize plant material and validate flight hardware environmental conditions. Such preliminary studies set the ground for subsequent spaceflight experiments. The objectives of this study were to compare the in vitro growth of cultures from three explant sources (cotyledon, leaf, and stem sections) of three jatropha accessions (Brazil, India, and Tanzania) outside and inside the petriGAP, a modified group activation pack (GAP) flight hardware to fit petri dishes. In vitro jatropha cell cultures were established in petri dishes containing a modified MS medium and maintained in a plant growth chamber at 25 ± 2 °C in the dark. Parameters evaluated were surface area of the explant tissue (A), fresh weight (FW), and dry weight (DW) for a period of 12 weeks. Growth was observed for cultures from all accessions at week 12, including subsequent plantlet regeneration. For all accessions differences in A, FW and DW were observed for inside vs. outside the PetriGAPs. Growth parameters were affected by accession (genotype), explant type, and environment. The type of explant influenced the type of cell growth and subsequent plantlet regeneration capacity. However, overall cell growth showed no abnormalities. The present study demonstrated that jatropha in vitro cell cultures are suitable for growth inside PetriGAPs for a period of 12 weeks. The parameters evaluated in this study provide the basic ground work and pre-flight assessment needed to justify a model for microgravity studies with jatropha in vitro cell cultures. Future studies should focus on results of experiments performed with jatropha in vitro cultures in microgravity.     

Gravity effects on connective tissue biosynthesis by cultured mesenchymal cells.

Quantitative and qualitative aspects of collagen synthesis under microgravity, normal gravity and hypergravity conditions were investigated during the spacelab D-2 mission by incubating human fibroblast cultures with [3H]-proline for 0, 4, 7, 10 and 20 hours. Quantitative analysis revealed an increase of collagen synthesis under microgravity conditions, being 40% higher than 1g controls. Hypergravity samples at 1.44g, 6.6g and 10g showed a decrease in collagen synthesis with increasing g, being down to about 15% at 10g. The relative proportion of collagen from total protein synthesized, the secretion of collagen by the cells, proline hydroxylation of individual collagen alpha-chains and the relative proportions of collagens I, III and V synthesized were not affected at any of the applied conditions.     

Temporal regulation of global gene expression and cellular morphology in Xenopus kidney cells in response to clinorotation.

Here, we report changes gene expression and morphology of the renal epithelial cell line, A6, which was derived from Xenopus laevis adult kidney that had been induced by long-term culturing with a three-dimensional clinostat. An oligo microarray analysis on the A6 cells showed that mRNA levels for 52 out of 8091 genes were significantly altered in response to clinorotation. On day 5, there was no dramatic change in expression level, but by day 8 and day 10, either upregulation or downregulation of gene expression became evident. By day 15, the expression levels of 18 out of 52 genes had returned to the original levels, while the remaining 34 genes maintained the altered levels of expression. Quantitative analyses of gene expression by real-time PCR confirmed that changes in the mRNA levels of selected genes were found only under clinorotation and not under hypergravity (7 g) or ground control. Morphological changes including loss of dome-like structures and disorganization of both E-cadherin adherence junctions and cortical actin were also observed after 10 days of culturing with clinorotation. These results revealed that the expression of selected genes was altered specifically in A6 cells cultured under clinorotation.     

O2 evolution and cyclic electron flow around photosystem I in long-term ground batch culture of Euglena gracilis

Based on the purpose of better exploring the function of green producers in the closed aquatic biological life support system, the condition of dynamic O₂ evolution and performance of cyclic electron flow around photosystem I (CEF-PSI) in long-term ground batch culture of Euglena gracilis were studied, the relationship between linear electron flow (LEF) and CEF-PSI was revealed, the function of CEF-PSI was investigated. Excellent consistency in O₂ evolution pattern was observed in cultures grown in both closed and open containers, O₂ evolution was strictly suppressed in phase 1, but the rate of it increased significantly in phase 2. CEF-PSI was proposed to be active during the whole course of cultivation, even in the declining phase 3, it still operated at the extent of 47–55%. It is suggested that the relationship between LEF and CEF-PSI is not only competition but also reciprocity. CEF-PSI was proposed to contribute to the considerable growth in phase 1; it was also suggested to play an important protective role against photosystem II (PSII) photoinhibition at the greatly enhanced level (approximately 80–95%) on the 2nd day. Our results in this research suggest that E. gracilis had very particular photosynthetic characteristics, the strict O₂ evolution suppression in the initial culture phase might be a special light acclimation behavior, and CEF-PSI could be an important mechanism involved in this kind of adaptation to the changeable light environment.     

基于策略的遥感卫星管控方法研究

针对遥感卫星管理控制中业务应用与底层调度紧耦合导致的操作复杂、人工干预多等问题，提出一种基于策略的遥感卫星管控方法。在搭建策略管控模型的基础上，采用分层控制闭环结构，构建卫星控制闭环；提出卫星集群分域管理方法，并设置策略部署机制；详细论述了基于策略的遥感卫星管控实现方法；以海上失联舰船搜救任务为典型案例进行仿真，验证该方法可以完成异构卫星集群基于事件驱动的闭环控制。基于策略的管理方法能够有效降低遥感卫星管控业务的复杂度，实现遥感卫星集群的自动控制。     

Adaptations of the vestibular system to short and long-term exposures to altered gravity.

Long-term space flight creates unique environmental conditions to which the vestibular system must adapt for optimal survival of a given organism. The development and maintenance of vestibular connections are controlled by environmental gravitational stimulation as well as genetically controlled molecular interactions. This paper describes the effects of hypergravity on axonal growth and dendritic morphology, respectively. Two aspects of this vestibular adaptation are examined: (1) How does long-term exposure to hypergravity affect the development of vestibular axons? (2) How does short-term exposure to extremely rapid changes in gravity, such as those that occur during shuttle launch and landing, affect dendrites of the vestibulocerebellar system? To study the effects of longterm exposures to altered gravity, embryonic rats that developed in hypergravity were compared to microgravity-exposed and control rats. Examination of the vestibular projections from epithelia devoted to linear and angular acceleration revealed that the terminal fields segregate differently in rat embryos that gestated in each of the gravitational environments.To study the effects of short-term exposures to altered gravity, mice were exposed briefly to strong vestibular stimuli and the vestibulocerebellum was examined for any resulting morphological changes. My data show that these stimuli cause intense vestibular excitation of cerebellar Purkinje cells, which induce up-regulation of clathrin-mediated endocytosis and other morphological changes that are comparable to those seen in long-term depression. This system provides a basis for studying how the vestibular environment can modify cerebellar function, allowing animals to adapt to new environments.     

Molecular alterations in tumorigenic human bronchial and breast epithelial cells induced by high LET radiation.

Carcinogenesis is a multi-stage process with sequence of genetic events governing the phenotypic expression of a series of transformation steps leading to the development of metastatic cancer. In the present study, immortalized human bronchial (BEP2D) and breast (MCF-10F) cells were irradiated with graded doses of either 150 keV/micrometer alpha particles or 1 GeV/nucleon 56Fe ions. Transformed cells developed through a series of successive steps before becoming tumorigenic in nude mice. Cell fusion studies indicated that radiation-induced tumorigenic phenotype in BEP2D cells could be completely suppressed by fusion with non-tumorigenic BEP2D cells. The differential expressions of known genes between tumorigenic bronchial and breast cells induced by alpha particles and their respective control cultures were compared using cDNA expression array. Among the 11 genes identified to be differentially expressed in BEP2D cells, three (DCC, DNA-PK and p21(CIP1)) were shown to be consistently down-regulated by 2 to 4 fold in all the 5 tumor cell lines examined. In contrast, their expressions in the fusion cell lines were comparable to control BEP2D cells. Similarly, expression levels of a series of genes were found to be altered in a step-wise manner among tumorigenic MCF-10F cells. The results are highly suggestive that functional alterations of these genes may be causally related to the carcinogenic process.     

Comparative characteristic of mitochondria ultrastructural organization in Chlorella cells under altered gravity conditions.

Results from experiments that used cells from the unicellular alga Chlorella vulgaris (strain Larg-1) grown on a clinostat, demonstrated the occurrence of rearrangements in cellular organelles, including changes in the mitochondrial ultrastructure compared to controls. Changes in mitochondrial structure were observed in auto- and heterotrophic regimes of cells grown in altered gravity conditions, especially in long-term experiments. The mitochondrial rearrangements become apparent during cell proliferation, which resulted in an increase in the relative volume of mitochondria per cell: up to 2.7 +/- 0.3% in short-term clino-rotation (2.2 +/- 0.1% in the control) and up to 5.3 +/- 0.4% and 5.1 +/- 0.4% in long-term clinorotation (2.3 +/- 0.2% in the control). The size of the mitochondria and their cristae increased in cells grown under long-time clinorotation. In addition, hypertrophied organelles, not typical for this strain, were observed. These changes in the cells were accompanied by increased electron density of the matrix and a well-ordered topography of the cristae. To examine the separation of oxidative phosphorylation and respiration, an inhibitory agent 2,4-dinitrophenol (2,4-DNP) was applied to cells which resulted in insignificant volume changes of the mitochondria (2.5 +/- 0.4% versus 2.1 +/- 0.2% in the control). The increase of mitochondrial size with regularly arranged cristae, with more condensed matrix and extension of cristae areas of clino-rotated cells, may demonstrate higher functional activity of the mitochondria under altered gravity conditions. Changes observed early in clinorotated cells, in particular the increased level of respiration, adenylate content (especially ATP) and more intensive electron-cytochemical reactions of Mg2(+)-ATPase and succinate [correction of succinat] dehydrogenase (SDH) in mitochondria (including hypertrophic organelles), also suggest increased activity of mitochondria from cells grown under altered gravity conditions compared to controls.     

Effect of clinostat rotation on differentiation of embryonic bone in vitro.

We have investigated the effect of changes in the gravity vector on osteoblast behaviour, using the clinostat set at 8 rpm. Two sources of osteoblasts were used: secondary cultures of fetal rat bone cells, and the rat osteosarcoma line 17/2.8 (ROS). Cell number was determined by incubation with 3-(4,dimethyl-2yl)-2,3 diphenyl) tetrazolium bromide (MTT) and measurement of optical density at 570 nm (OD). Alkaline phosphatase activity was detected by standard cytochemical methods. Dividing cells were localised by labelling dividing nuclei with Bromodeoxyuridine (BrdU), detected by immunofluorescence. Cell culture was initiated at densities between 1-4x10(4) cells ml-1. Growth rates in all cultures during the first 48 hours exposure to clinostat rotation were less than in stationary controls. After 3 days, ROS cell numbers were 35% lower, and calvarial cells 39% lower than their respective controls. Alkaline phosphatase activity in calvarial control cultures was uniformly present in characteristically polygonal cells, but after culture in the clinostat the enzyme was present sporadically, and the cells were cuboid. There was also no BrdU uptake in nuclei, but it was present in cell cytoplasms. We conclude that the clinostat decreases cell numbers and cell division. Both cell shape and the distribution of alkaline phosphatase activity in calvarial cell cultures were also affected. This implies that changes in the gravity vector can affect osteoblasts directly, without interaction with other cell types.     

Effects of spaceflight (STS-87) on tropisms and plastid positioning in protonemata of the moss Ceratodon purpureus.

Apical cells of moss protonemata represent a single-celled system that perceives and reacts to light (positive and negative phototropism) and to gravity (negative gravitropism). Phototropism completely overrides gravitropism when apical cells are laterally irradiated with relatively high red light intensities, but below a defined light intensity threshold gravitropism competes with the phototropic reaction. A 16 day-long exposure to microgravity conditions demonstrated that gravitropism is allowed when protonemata are laterally illuminated with light intensities below 140 nmol m-2s-1. Protonemata that were grown in darkness in microgravity expressed an endogenous tendency to grow in arcs so that the overall culture morphology resembled a clockwise spiral. However this phenomenon only was observed in cultures that had reached a critical age and/or size. Organelle positioning in dark-grown apical cells was significantly altered in microgravity. Gravisensing most likely involves the sedimentation of starch-filled amyloplasts in a well-defined area of the tip cell. Amyloplasts that at 1-g are sedimented were clustered at the apical part of the sedimentation zone in microgravity. Clustering observed in microgravity or during clino-rotation significantly differs from sedimentation-induced plastid aggregations after inversion of tip cells at 1-g.     

Chromosomal changes in cultured human epithelial cells transformed by low- and high-LET radiation.

For a better assessment of radiation risk in space, an understanding of the responses of human cells, especially the epithelial cells, to low- and high-LET radiation is essential. In our laboratory, we have successfully developed techniques to study the neoplastic transformation of two human epithelial cell systems by ionizing radiation. These cell systems are human mammary epithelial cells (H184B5) and human epidermal keratinocytes (HEK). Both cell lines are immortal, anchorage dependent for growth, and nontumorigenic in athymic nude mice. Neoplastic transformation was achieved by irradiating cells successively. Our results showed that radiogenic cell transformation is a multistep process and that a single exposure of ionizing radiation can cause only one step of transformation. It requires, therefore, multihits to make human epithelial cells fully tumorigenic. Using a simple karyotyping method, we did chromosome analysis with cells cloned at various stages of transformation. We found no consistent large terminal deletion of chromosomes in radiation-induced transformants. Some changes of total number of chromosomes, however, were observed in the transformed cells. These transformants provide an unique opportunity for further genetic studies at a molecular level.     

Effects of space flight exposure on cell growth,tumorigenicity and gene expression in cancer cells

It is well recognized that harsh outer space environment, consisting of microgravity and radiation, poses significant health risks for human cells. To investigate potential effects of the space environment exposure on cancer cells we examined the biological changes in Caski cells carried by the “Shen Zhou IV” spaceship. After exposure for 7 days in spaceflight, 1440 survival subclonal cell lines were established and 4 cell lines were screened. 44F10 and 17E3 were selected because of their increased cell proliferation and tumorigenesis, while 48A9 and 31F2 had slower cytological events. Experiments with cell proliferation assay, flow cytometry, soft agar assay, tumorigenesis assay and DNA microarray analysis have shown that selected cell lines presented multiple biological changes in cell morphology, cell growth, tumorigenicity and gene expression. These results suggest that space environment exposure can make significant biological impact on cancer cells and provide an entry point to find the immunological target of tumorigenesis.     

Residual chromatin breaks as biodosimetry for cell killing by carbon ions

We have studied the relationship between cell killing and the induction of residual chromatin breaks on various human cell lines and primary cultured cells obtained by biopsy from patients irradiated with either X-rays or heavy-ion beams to identify potential bio-marker of radiosensitivity for radiation-induced cell killing. The carbon-ion beams were accelerated with the Heavy Ion Medical Accelerator in Chiba (HIMAC). Six primary cultures obtained by biopsy from 6 patients with carcinoma of the cervix were irradiated with two different mono-LET beams (LET= 13 keV/μm, 76 keV/μm) and 200kV X rays. Residual chromatin breaks were measured by counting the number of non-rejoining chromatin fragments detected by the premature chromosome condensation (PCC) technique after a 24 hour postirradiation incubation period. The induction rate of residual chromatin breaks per cell per Gy was the highest for 76 keV/μm beams on all of the cells. Our results indicated that cell which was more sensitive to the cell killing was similarly more susceptible to induction of residual chromatin breaks. Furthermore there is a good correlation between these two end points in various cell lines and primary cultured cells. This suggests that the detection of residual chromatin breaks by the PCC technique may be useful as a predictive assay of tumor response to cancer radiotherapy.     

An alternative approach to solar system exploration providing safety of human mission to Mars.

For systematic human Mars exploration, meeting crew safety requirements, it seems perspective to assemble into a spacecraft: an electrical rocket, a well-shielded long-term life support system, and a manipulator-robots operating in combined "presence effect" and "master-slave" mode. The electrical spacecraft would carry humans to the orbit of Mars, providing short distance (and low signal time delay) between operator and robot-manipulators, which are landed on the surface of the planet. Long-term hybrid biological and physical/chemical LSS could provide environment supporting human health and well being. Robot-manipulators operating in "presence effect" and "master-slave" mode exclude necessity of human landing on Martian surface decreasing the level of risk for crew. Since crewmen would not have direct contact with the Martian environment then the problem of mutual biological protection is essentially reduced. Lightweight robot-manipulators, without heavy life support systems and without the necessity of returning to the mother vessel, could be sent as scouts to different places on the planet surface, scanning the most interesting for exobiological research site. Some approximate estimations of electric spacecraft, long-term hybrid LSS, radiation protection and mission parameters are conducted and discussed.     

Repair and misrepair of heavy-ion-induced chromosomal damage.

The premature chromosome condensation (PCC) technique was used to investigate chromosomal damage, repair, and misrepair in the G phase of a human/hamster hybrid cell line that contains a single human chromosome. Plateau-phase cell cultures were exposed to either x-rays or a 425 MeV/u beam of neon ions near the Bragg peak where the LET is 183 kev/micrometers. An in situ hybridization technique coupled to fluorescent staining of PCC spreads confirmed the linearity of the dose response for initial chromatin breakage in the human chromosome to high doses (1600 cGy x-ray or 1062 cGy Ne). On Giemsa-stained slides, initial chromatin breakage in the total genome and the rejoining kinetics of these breaks were determined. As a measure of chromosomal misrepair, ring PCC aberrations were also scored. Ne ions were about 1.5 x more effective per unit dose compared to x-rays at producing the initially measured chromatin breakage. 90% of the x-ray-induced breaks rejoined in cells incubated at 37 degrees C after exposure. In contrast, only 50% of Ne-ion-induced breaks rejoined. In the irradiated G1 cells, ring PCC aberrations increased with time apparently by first order kinetics after either x-ray or Ne exposures. However, far fewer rings formed in Ne-irradiated cells after a dose giving a comparable initial number of chromatin breaks. Following x-ray exposures, the yield of rings formed after long repair times (6 to 9 hrs) fit a quadratic dose-response curve. These results indicate quantitative and qualitative differences in the chromosomal lesions induced by low- and high-LET radiations.     

Results of analysis of human impact on environment using the time series of vegetation satellite images around large industrial centers

The paper shows the efficiency of an application of the vegetation index image time series to determine long-term vegetation dynamics. The influence of large industrial centers of Siberia on the near-by vegetation is demonstrated. The analysis of the data shows that the influence of industrial waste is stronger in the Siberian North. These regions are characterized by critical conditions for vegetation existence. In the south of the Krasnoyarsk region, human impact is also important, but the possibility of vegetation self-rehabilitation is higher. The present-day economic situation in Russia is unique, with a temporary abrupt fall of industrial production and its following increase. Thus, we managed to analyze the degree of human impact on the environment within a relatively short-time interval.     

Direct utilization of human liquid wastes by plants in a closed ecosystem.

Model experiments in phytotrons have shown that urea is able to cover 70% of the demand in nitrogen of the conveyer cultivated wheat. At the same time wheat plants can directly utilize human liquid wastes. In this article by human liquid wastes the authors mean human urine only. In a long-term experiment on "man-higher plants" system with two crewmen, plants covered 63 m2, with wheat planted to--39.6 m2. For 103 days, complete human urine (total amount--210.7 l) was supplied into the nutrient solution for wheat. In a month and a half NaCl supply into the nutrient solution stabilized at 0.9-1.65 g/l. This salination had no marked effect on wheat production. The experiment revealed the realistic feasibility to directly involve liquid wastes into the biological turnover of the life support system. The closure of the system, in terms of water, increased by 15.7% and the supply of nutrients for wheat plants into the system was decreased.     

Biosphere 2: a prototype project for a permanent and evolving life system for Mars base.

As part of the ground-based preparation for creating long-term life systems needed for space habitation and settlement, Space Biospheres Ventures (SBV) is undertaking the Biosphere 2 project near Oracle, Arizona. Biosphere 2, currently under construction, is scheduled to commence its operations in 1991 with a two-year closure period with a crew of eight people. Biosphere 2 is a facility which will be essentialy materially-closed to exchange with the outside environment. It is open to information and energy flow. Biosphere 2 is designed to achieve a complex life-support system by the integration of seven areas or "biomes"--rainforest, savannah, desert, marsh, ocean, intensive agriculture and human habitat. Unique bioregenerative technologies, such as soil bed reactors for air purification, aquatic waste processing systems, real-time analytic systems and complex computer monitoring and control systems are being developed for the Biosphere 2 project. Its operation should afford valuable insight into the functioning of complex life systems necessary for long-term habitation in space. It will serve as an experimental ground-based prototype and testbed for the stable, permanent life systems needed for human exploration of Mars.