The role of cellulases in the mechanism of changes of cell walls of Funaria hygrometrica moss protonema at clinostating.

Using electroncytochemical and biochemical methods, differences between the cytochemical reaction intensity and activity of the cellulosolytic enzymes in Funaria hygrometrica moss cells grown for 30 days in the horizontal clinostat (2 rev/min) and in control have been studied. It has been shown that on clinostating the precipitate amount and size increases with the cellulase activity enhancement in the periplasmic space and protonema cell walls, when compared to control. Using biochemical methods it has been found that the activity of both endo-1,4-beta-glucanase and exo-1,4-beta-glucanase was higher under these conditions. A decrease of cellulose total content, its crystalline form, and pectic substances as well as an increase of hemicellulose content have been revealed in the clinostated material compared to control. Data obtained are discussed regarding the possible mechanism of cellulase activation and synthesis inhibition and cellulose crystallization in plant cell walls at clinostating.     

Long clinostation influence on the ultrastructure of Funaria hygrometrica moss protonema cells

Changes in the ultrastructure of protonema cells of Funaria hygrometrica, cultivated during 20 days on a horizontal clinostat (2 rev/min), were determined by the electron microscopy method. About 20% of the cells were almost identical to those in the control, 20% were destructive cells, and in 60% ultrastructure changes were observed. The heterogeneity of the reaction demonstrated the evidence of sensitive cells on the clinostation process. Changes affected the ultrastructure of plastids, wall of the cell, and the form of the nucleus as well. Starch disappeared from chloroplasts practically completely, thylakoids swelled, granas frequently disappeared from plastids. Peroxisomes number in cells increased appreciably, width of cell walls decreased by almost half their size. Ca⁺⁺-binding sites were revealed in cytoplasma of cells. Electronocytochemical exposure of ATPases activity with the presence of Mg⁺⁺ and Ca⁺⁺ ions showed that Mg²⁺-ATPase activity localization in clinosted cells was not too different from the control, while Ca²⁺-ATPase location presented differences in plasmalemma and Ca-sites. These changes are perhaps connected with the membranes permeability breaking and affect the plant cells adaptation to the influence of hypogravitation.     

Long clinostation influence on the localization of free and weakly bound calcium in cell walls of Funaria hygrometrica moss protonema cells.

The pyroantimonate method was used to study the localization of free and weakly bound calcium in cells of moss protonema of Funaria hygrometrica Hedw. cultivated on a clinostat (2rev/min). Electroncytochemical study of control cells cultivated at 1 g revealed that granular precipitate marked chloroplasts, mitochondria, Golgi apparatus, lipid drops, nucleoplasma, nucleolus, nucleus membranes, cell walls and endoplasmic reticulum. In mitochondria the precipitate was revealed in stroma, in chloroplast it was found on thylakoids and envelope membranes. The cultivation of protonema on clinostat led to the intensification in cytochemical reaction product deposit. A considerable intensification of the reaction was noted in endomembranes, vacuoles, periplasmic space and cell walls. At the same time analysis of pectinase localization was made using the electroncytochemical method. A high reaction intensity in walls in comparison to that in control was found out to be a distinctive peculiarity of the cells cultivated on clinostat. It testifies to the fact that increasing of free calcium concentrations under conditions of clinostation is connected with pectinic substances hydrolysis and breaking of methoxy groups of pectins. Data obtained are discussed in relation to problems of possible mechanisms of disturbance in calcium balance of plant cells and the role of cell walls in gomeostasis of cell grown under conditions of simulated weightlessness.     

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.     

Correlation between cell death and induction of non-rejoining PCC breaks by carbon-ion beams.

We have shown a correlation between cell death and induction of non-rejoining chromatin breaks in two normal human cells and three human tumor cell lines irradiated by carbon-ion beams and X rays. Non-rejoining chromatin breaks were measured by counting the number of remaining chromatin fragments detected by the premature chromosome condensation (PCC) technique. Carbon-ion beams were accelerated by the Heavy Ion Medical Accelerator in Chiba (HIMAC). The cells were irradiated by two different mono-LET beams (LET = 13 keV/micrometer and 77 keV/micrometer ) and 200 kV X rays. The RBE values of cell death for carbon-ion beams relative to X rays were 1.1 to 1.4 for 13 keV/micrometer beams and 2.5 to 2.9 for 77 keV/micrometer beams. The induction rate of non-rejoining PCC breaks per cell per Gy was found to be highest for the 77 keV/micrometer beams for all of the cell lines.The results found in this study show that there is a good correlation between cell death and induction of non-rejoining PCC breaks for these human cell lines.     

Biological effects due to weak magnetic field on plants.

Throughout the evolution process, Earth's magnetic field (MF, about 50 microT) was a natural component of the environment for living organisms. Biological objects, flying on planned long-term interplanetary missions, would experience much weaker magnetic fields, since galactic MF is known to be 0.1-1 nT. However, the role of weak magnetic fields and their influence on functioning of biological organisms are still insufficiently understood, and is actively studied. Numerous experiments with seedlings of different plant species placed in weak magnetic field have shown that the growth of their primary roots is inhibited during early germination stages in comparison with control. The proliferative activity and cell reproduction in meristem of plant roots are reduced in weak magnetic field. Cell reproductive cycle slows down due to the expansion of G1 phase in many plant species (and of G2 phase in flax and lentil roots), while other phases of cell cycle remain relatively stable. In plant cells exposed to weak magnetic field, the functional activity of genome at early pre-replicate period is shown to decrease. Weak magnetic field causes intensification of protein synthesis and disintegration in plant roots. At ultrastructural level, changes in distribution of condensed chromatin and nucleolus compactization in nuclei, noticeable accumulation of lipid bodies, development of a lytic compartment (vacuoles, cytosegresomes and paramural bodies), and reduction of phytoferritin in plastids in meristem cells were observed in pea roots exposed to weak magnetic field. Mitochondria were found to be very sensitive to weak magnetic field: their size and relative volume in cells increase, matrix becomes electron-transparent, and cristae reduce. Cytochemical studies indicate that cells of plant roots exposed to weak magnetic field show Ca2+ over-saturation in all organelles and in cytoplasm unlike the control ones. The data presented suggest that prolonged exposures of plants to weak magnetic field may cause different biological effects at the cellular, tissue and organ levels. They may be functionally related to systems that regulate plant metabolism including the intracellular Ca2+ homeostasis. However, our understanding of very complex fundamental mechanisms and sites of interactions between weak magnetic fields and biological systems is still incomplete and still deserve strong research efforts.     

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.     

The role of calcium ions in cytological effects of hypogravity

Electron-cytochemical and biochemical methods made it possible to reveal certain differences in ATPase activity stimulation by calcium ions in root apex cells of pea seedlings and moss protonema $\text{Funaria}$$\text{hygrometrica}$ grown under stationary and slow clinostatic (2 rev/min) conditions. It was showed that under clinostatic conditions in comparison with the control variant the ATPase activity decreases in plasmalemma. The protein content in the plasmalemma fraction was also twice as low under these conditions. The root apex cells of the pea seedlings grown under spaceflight conditions were found to contain high concentrations of membrane-bound calcium. The data obtained are discussed in relation to problems of possible mechanisms of disturbance in calcium balance and the system of active calcium ion transport through plasmalemma under hypogravity.     

LET dependence of cell death, mutation induction and chromatin damage in human cells irradiated with accelerated carbon ions.

We investigated the LET dependence of cell death, mutation induction and chromatin break induction in human embryo (HE) cells irradiated by accelerated carbon-ion beams. The results showed that cell death, mutation induction and induction of non-rejoining chromatin breaks detected by the premature chromosome condensation (PCC) technique had the same LET dependence. Carbon ions of 110 to 124keV/micrometer were the most effective at all endpoints. However, the number of initially induced chromatin breaks was independent of LET. About 10 to 15 chromatin breaks per Gy per cell were induced in the LET range of 22 to 230 keV/micrometer. The deletion pattern of exons in the HPRT locus, analyzed by the polymerase chain reaction (PCR), was LET-specific. Almost all of the mutants induced by 124 keV/micrometer beams showed deletion of the entire gene, while all mutants induced by 230keV/micrometer carbon-ion beams showed no deletion. These results suggest that the difference in the density distribution of carbon-ion track and secondary electron with various LET is responsible for the LET dependency of biological effects.     

Induction of DNA double-strand breaks in mammalian cells and yeast.

Induction of DNA double-strand breaks (dsb) and their distribution are dependent on the energy deposition pattern within the cell nucleus (physical structure) and the ultrastructure of the chromosomes and its variation by the cell cycle and gene activities (biological structure). For electron radiation very similar RBE-values are observed for mammalian and yeast cells (AlK, 1.5 keV, 15 keV/micrometer: 2.6 in mammalian cells and 2.2 in yeast; CK 0.278 keV, 23 keV/micrometer: approx. 2.5 in mammalian cells and 3.8 in yeast). In contrast, the RBE-values for the induction of dsb of 4He2+ and light ions in the LET range from about 100 keV/micrometer up to 1000 keV/micrometer are significantly higher for yeast cells compared to mammalian cells. For example, the RBE-value of alpha-particles (120 keV/micrometer) is about 1.2 for mammalian cells whereas for yeast the RBE-value is about 2.5. The yeast chromatin has less condensed fibres compared with mammalian cells. Since a single CK photoelectron can induce only one dsb, the different condensation of the mammalian and yeast chromatin has no influence. However, particles may induce more than one dsb when traversing a chromatin fibre. The probability for the induction of closely neighboured dsb is higher the more condensed the chromatin fibres are. Since small DNA fragments (50 bp up to several kbp) are lost by standard methods of lysis, the underestimation of dsb yields increases with fibre condensation, which is in accordance with the observes dsb yields in mammalian cells and yeast. In order to obtain relevant yields of dsb (and corresponding RBE-values) the measurement of all DNA fragments down to about 50 bp are needed.     

Functional, regulatory and indicator features of microorganisms in man-made ecosystems.

Functional, regulatory and indicator features of microorganisms in development and functioning of the systems and sustaining stability of three man-made ecosystem types has been studied. 1) The functional (metabolic) feature was studied in aquatic ecosystems of biological treatment of sewage waters for the reducer component. 2) The regulatory feature of bacteria for plants (producer component) was studied in simple terrestrial systems "wheat plants-rhizospheric microorganisms-artificial soil" where the behavior of the system varied with activity of the microbial component. For example with atmospheric carbon dioxide content elevated microbes promote intensification of photosynthesis processes, without binding the carbon in the plant biomass. 3) The indicator feature for the humans (consumer component) was studied in Life Support Systems (LSS). High sensitivity of human microflora to system conditions allowed its use as an indicator of the state of both system components and the entire ecosystem. Grant numbers: N99-04-96017, N15.     

Present results of the joint radiobiological ESA/IBMP experiments “seeds” aboard Cosmos 2044 and 2229 - correlation of micro-dosimetric data and damage endpoints in Arabidopsis thaliana plants

Experimental data obtained from two Cosmos missions (2044, 2229) performed with dry plant seeds of Arabidopsis thaliana in a Biostack configuration are compared. Biological stress and genetic risk are induced in dry seeds by cosmic radiation, especially the high energetic, densely ionizing component of eavy ons (HI). Subdivision of impacting HI particles into LET-groups (15–35; 35–100; >100 keV/μm) showed the contribution of each LET group to the induction of different biological damage endpoints (survival, cell transformation, lethal plant development). An attempt is presented to comprehend the influence of the spatial energy deposition as a biophysical HI track parameter.     

A bioreactor system for the nitrogen loop in a Controlled Ecological Life Support System.

As space missions become longer in duration, the need to recycle waste into useful compounds rises dramatically. This problem can be addressed by the development of Controlled Ecological Life Support Systems (CELSS) (i.e., Engineered Closed/Controlled Eco-Systems (ECCES)), consisting of human and plant modules. One of the waste streams leaving the human module is urine. In addition to the reclamation of water from urine, recovery of the nitrogen is important because it is an essential nutrient for the plant module. A 3-step biological process for the recycling of nitrogenous waste (urea) is proposed. A packed-bed bioreactor system for this purpose was modeled, and the issues of reaction step segregation, reactor type and volume, support particle size, and pressure drop were addressed. Based on minimization of volume, a bioreactor system consisting of a plug flow immobilized urease reactor, a completely mixed flow immobilized cell reactor to convert ammonia to nitrite, and a plug flow immobilized cell reactor to produce nitrate from nitrite is recommended. It is apparent that this 3-step bioprocess meets the requirements for space applications.     

The effect of space radiation of the nervous system.

The long-term effects of irradiation by accelerated heavy ions on the structure and function of the nervous system have not been studied extensively. Although the adult brain is relatively resistant to low LET radiation, cellular studies indicate that individual heavy ions can produce serious membrane lesions and multiple chromatin breaks. Capillary hemorrhages may follow high LET particle irradiation of the developing brain as high RBE effects. Evidence has been accumulating that the glial system and blood-brain barrier (BBB) are relatively sensitive to injury by ionizing radiation. While DNA repair is active in neural systems, it may be assumed that a significant portion of this molecular process is misrepair. Since the expression of cell lethality usually requires cell division, and nerve cells have an extremely low rate of division, it is possible that some of the characteristic changes of premature aging may represent a delayed effect of chromatin misrepair in brain. Altered microcirculation, decreased local metabolism, entanglement and reduction in synaptic density, premature loss of neurons, myelin degeneration, and glial proliferation are late signs of such injuries. HZE particles are very efficient in producing carcinogenic cell transformation, reaching a peak for iron particles. The promotion of viral transformation is also efficient up to an energy transfer of approximately 300 keV/micron. The RBE for carcinogenesis in nerve tissues remains unknown. On the basis of available information concerning HZE particle flux in interplanetary space, only general estimates of the magnitude of the effects of long-term spaceflight on some nervous system parameters may be constructed.     

Plant cell in the process of the adaptation to simulated microgravity.

Analysis of structural-and-functional rearrangements in the organelles of meristematic, differentiating and differentiated cells of pea root under microgravity demonstrated certain consistencies in their manifestation, namely: a) heterogeneity of the organelles in a cell population with respect to the degree of the rearrangements; b) coincidence of a spatial succession in development; c) increased reactivity under changes in functional load during cell growth and differentiation; d) enhanced activity when a cell loses its specific functions (replacement of functions). It is assumed that microgravity does not prevent the development of certain adaptative reactions of organisms at the cellular level.     

Effects of altered gravity on plant cell processes: results of recent space and clinostatic experiments.

Space and clinostatic experiments revealed that plant cell structure and metabolism rearrangements depend on taxonomical position and physiological state of objects, growth phase and real or simulated microgravity influence duration. It was shown that clinostat conditions reproduce only a part of microgravity biological effects. It is established that various responses occur in microgravity: 1) rearrangements of cytoplasmic organelles ultrastructure and calcium balance; 2) physical-chemical properties of the plasmalemma are changed; 3) enzymes activity is often enhanced. These events provoke the acceleration of growth and differentiation of cells and their aging as a result; at the same time some responses can be considered as cell adaptation to microgravity.     

Biological effects of weightlessness and clinostatic conditions registered in cells of root meristem and cap of higher plants.

Research in cellular reproduction, differentiation and vital activity, i.e. processes underlying the development and functioning of organisms, plants included, is essential for solving fundamental and applied problems of space biology. Detailed anatomical analysis of roots of higher plants grown on board the Salyut 6 orbital research station show that under conditions of weightlessness for defined duration mitosis, cytokinesis and tissue differentiation in plant vegetative organs occur essentially normally. At the same time, certain rearrangements in the structural organization of cellular organelles--mainly the plastid apparatus, mitochondria, Golgi apparatus and nucleus--are established in the root meristem and cap of the experimental plants. This is evidence for considerable changes in cellular metabolism. The structural changes in the subcellular level arising under spaceflight conditions are partially absent in clinostat experiments designed to simulate weightlessness. Various clinostatic conditions have different influences on the cell structural and functional organization than does space flight. It is suggested that alterations of cellular metabolism under weightlessness and clinostatic conditions occur within existing genetic programs.     

重力对植物细胞生长的效应

从地基研究植物向重性和在神舟八号卫星微重力条件下培养植物细胞出发,探讨重力变化对植物细胞发生作用时产生的生物学效应.已有结果显示,在植物向重性反应和处于失重状态时,重力方向和大小变化对细胞壁代谢具有一定影响.推断细胞形状的维持是由细胞壁的刚性与细胞内膨压平衡所致,当细胞膨压大于细胞壁刚性导致上述平衡打破时就会引起细胞体积增大.因此,重力的变化可能会通过影响植物细胞壁刚性与细胞内膨压的平衡影响细胞生长.      

Weightlessness acts on human breast cancer cell line MCF-7.

Because cells are sensitive to mechanical forces, weightlessness might act on stress-dependent cell changes. Human breast cancer cells MCF-7, flown in space in a Photon capsule, were fixed after 1.5, 22 and 48 h in orbit. Cells subjected to weightlessness were compared to 1 g in-flight and ground controls. Post-flight, fluorescent labeling was performed to visualize cell proliferation (Ki-67), three cytoskeleton components and chromatin structure. Confocal microscopy and image analysis were used to quantify cycling cells and mitosis, modifications of the cytokeratin network and chromatin structure. Several main phenomena were observed in weightlessness: The perinuclear cytokeratin network and chromatin structure were looser; More cells were cycling and mitosis was prolonged. Finally, cell proliferation was reduced as a consequence of a cell-cycle blockade; Microtubules were altered in many cells. The results reported in the first point are in agreement with basic predictions of cellular tensegrity. The prolongation of mitosis can be explained by an alteration of microtubules. We discuss here the different mechanisms involved in weightlessness alteration of microtubules: i) alteration of their self-organization by reaction-diffusion processes, and a mathematical model is proposed, ii) activation or deactivation of microtubules stabilizing proteins, acting on both microtubule and microfilament networks in cell cortex.