Modelling of genetically engineered microorganisms introduction in closed artificial microcosms.

The possibility of introducing genetically engineered microorganisms (GEM) into simple biotic cycles of laboratory water microcosms was investigated. The survival of the recombinant strain Escherichia coli Z905 (Apr, Lux+) in microcosms depends on the type of model ecosystems. During the absence of algae blooming in the model ecosystem, the part of plasmid-containing cells E. coli decreased fast, and the structure of the plasmid was also modified. In conditions of algae blooming (Ankistrodesmus sp.) an almost total maintenance of plasmid-containing cells was observed in E. coli population. A mathematics model of GEM's behavior in water ecosystems with different level of complexity has been formulated. Mechanisms causing the difference in luminescent exhibition of different species are discussed, and attempts are made to forecast the GEM's behavior in water ecosystems.     

Population dynamics of transgenic microorganisms in the different microecosystem conditions.

The role of key environmental factors in adaptation of spore-forming and non-spore-forming transgenic microorganisms (TM) have been studied in model ecosystems. Model TM Escherichia coli Z905 (bearing plasmid genes of bacterial luminescence Ap (r) Lux+) has been found to have a higher adaptation potential than TM Bacillus subtilis 2335/105 (bearing genes of human alpha 2-interferon Km (r) Inf+), planned for employment as a living vaccine under varying environmental conditions. Effects of abiotic factors on migration of natural and recombinant plasmids between microorganisms under model ecosystem conditions has been estimated. The transgenic microorganisms with low copy number survived better under introduction conditions in the microcosms studied. This trend has been shown to be independent of the microcosm type and its complexity. Grant numbers: 99-04-96017, 25, 00-07-9011.     

Experimental evaluation of the processes resulting from the introduction of the transgenic microorganism Escherichia coli Z905/pPHL7 (lux+) into aquatic microcosms.

The processes resulting from the introduction of the tranagenic microorganism (TM) E. coli Z905/pPHL7 into aquatic microcosms have been modeled experimentally. It has been shown that the TM E. coli is able to adapt to a long co-existence with indigenous heterotrophic microflora in variously structured microcosms. In more complex microcosms the numerical dynamics of the introduced E. coli Z905/pPHL7 population is more stable. In the TM populations staying in the microcosms for a prolonged time, changes are recorded in the phenotypic expression of plasmid genes (ampicillin resistance and the luminescence level) and chromosome genes (morphological and physiological traits). However, in our study microcosms, the recombinant plasmid persisted in the TM cells for 6 years after the introduction, and as the population adapts to the conditions of the microcosms, the efficiency of the cloned gene expression in the cells is restored. In the microcosms with high microalgal counts (10(7) cells/ml), cells with a high threshold of sensitivity to ampicillin dominate in the population of the TM E. coli Z905/pPHL7.     

Expression of lux-genes as an indicator of metabolic activity of cells in model ecosystem studies.

Quick response to different impacts and easy measurement make the luminescent systems of luminous bacteria an object convenient for application in various fields. Cloning of gene luminescence in different organisms is currently used to study both the survival of microbial cells and the effect of different factors on their metabolic activity, including the environment. A primary test-object in estimating bacteriological contamination of water bodies, Escherichia coli, can be conveniently used as an indicator of bactericidal properties of aquatic ecosystems. The application of Escherichia coli Z905/pPHL7 (lux+) as a marker microorganism can facilitate monitoring the microbiological status of closed biocenoses, including systems with higher organisms. The investigation of various parameters of microecosystems (carbon nutrition type, concentrations of inorganic ions and toxic compounds) shows that the recombinant strain E. coli Z905/pPHL7 can be effectively used as a marker.     

Survival and alteration of the plasmid-containing microorganism Escherichia coli Z905/pPHL7 introduced into manmade closed aquatic microcosms.

It has been demonstrated that the transgenic microorganism Escherichia coli Z905/pPHL7 (AprLux+) can exist for a long time at an elevated concentration of mineral salts. The microorganism was introduced into microcosms with sterile brackish water (salinity variable from 21 to 22 g l-1) taken from Lake Shira (Khakasia, Russia). The survival of the microorganism was estimated both by measuring the growth of the colonies on solid nutrient media and by the bioluminescence exhibited by the transgenic strain in samples from the microcosms and in the enrichment culture with the added selective factor-ampicillin (50 micrograms/ml). In the enrichment culture, the bioluminescent signal was registered through the 160-day experiment. It has been shown that in the closed microcosms with brackish water the E. coli strain becomes heterogeneous in its ampicillin resistance. The populations of the transgenic strain were mainly represented by isolates able to persist in the medium containing 50 micrograms/ml, but there were also the cells (about 10%) with the threshold of ampicillin resistance not more than 0.05 micrograms/ml. Thus, it was shown that in the microcosms with brackish water and in the absence of the selective factor the transgenic strain survives and retails the recombinant plasmid.     

Mathematical modeling of response of ecosystems with different structure to external impact.

A mathematical model was used to study the response of ecosystems of different structures to external impact. The response was measured as a sensitivity coefficient: the magnitude of the system's response vs. the change of the factor in the inflow. The formula has been obtained to calculate the sensitivity coefficient for ecosystems containing different numbers of trophic links. The derived sensitivity coefficients demonstrate that the degree of compensation for the external impact can differ depending on the type of system regulation and the length of the trophic chain. E. g. the sensitivity coefficient decreases with complexity of trophic links in an ecosystem for top-down controlled systems and impact of degree of openness on sensitivity e.g. closed ecosystems show higher sensitivity then fully open ecosystem to impacts also bottom-up control system show less sensitivity then top-down. Grant numbers: N99-04-96017, N25.     

System analysis of links interactions and development of ecosystems of different types.

The anthropogenic impact on the Earth's ecosystems are leading to dramatic changes in ecosystem functioning and even to destruction of them. System analysis and the use of heuristic modeling can be an effective means to determine the main biological interactions and key factors that are of high importance for understanding the development of ecosystems. Cycling of limiting substances, induced by the external free energy flux, and trophic links interaction is the basis of the mathematical modeling studies presented in this paper. Mathematical models describe the dynamics of simplified ecosystems having different characteristics: 1) different degrees of biotic turnover closure (from open to completely closed); 2) different numbers of trophic links (including both "top-down", "bottom-up" regulation types); 3) different intensities of input-output flows of the limiting nutrient and its total amount in the system. Adaptive values of the changes of lower hierarchical levels (populational, trophic chain level) are to be estimated by integrity indices for total system functioning (e.g. NPP, total photosynthesis). The approach developed can be used for evaluating the contributions of lower hierarchical levels to the functioning of the higher hierarchical levels of the system. This approach may have value for determining biomanipulation management and their assessment.     

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.     

Population dynamics of transgenic strain Escherichia coli Z905/pPHL7 in freshwater and saline lake water microcosms with differing microbial community structures.

Populations of Escherichia coli Z905/pPHL7, a transgenic microorganism, were heterogenic in the expression of plasmid genes when adapting to the conditions of water microcosms of various mineralization levels and structure of microbial community. This TM has formed two subpopulations (ampicillin-resistant and ampicillin-sensitive) in every microcosm. Irrespective of mineralization level of a microcosm, when E. coli Z905/pPHL7 alone was introduced, the ampicillin-resistant subpopulation prevailed, while introduction of the TM together with indigenous bacteria led to the dominance of the ampicillin-sensitive subpopulation. A high level of lux gene expression maintained longer in the freshwater microcosms than in sterile saline lake water microcosms. A horizontal gene transfer has been revealed between the jointly introduced TM and Micrococcus sp. 9/pSH1 in microcosms with the Lake Shira sterile water.     

On monitoring the bacterial component as an indicator of the state of small man-made ecosystems.

High reproduction rates make the bacterial component of ecosystems a good indicator of the state of the system on the whole. This determines the necessity to develop rapid monitoring of the functional state of the bacterial component of small ecosystems. Information about substrate concentration in the population is indicative of the state of the bacterial culture. Conventional methods of monitoring the concentration of integral substrate in the system take time much longer than the changes in the ecosystem. The paper presents theoretical foundations for the logical sequence "catalase activity--intracellular substrate concentration--estimate of substrate consumed by bacteria" for experimental verification and as a consequence of development of the integral method of monitoring the bacterial population on the basis of determining bacterial catalase activity. Grant numbers: 04-96017, N25.     

Principles of biological adaptation of organisms in artificial ecosystems to changes of environmental factors.

Studying material transformations and biotic cycling in artificial ecosystems (AES), we need to know the principles of biological adaptation of active organisms to change in the environment. Microorganisms in AES for water purification are the most active transforming organisms and consumers of the organic substances contained in wastes. Utilization of organic substances is directly connected with the energy fluxes used by AES. According to energy criteria, the energy fluxes used by a biological system tend to reach maximum values under stable conditions. Unutilized substrate concentration decreases as a result of biological adaptations. After a dramatic change in environmental factors, for example, after a sharp increase in the flow rate of organic substances, the biological system is not able to react quickly. The concentration of unutilized substrate increases and the energy flux used by the biological system decreases. The structure of the microbial community also changes, with a decrease in biological diversity. The efficiency of energy use by simple terrestrial ecosystems depends on the energetic intensity and interactions between plants and rhizospheric microorganisms.     

Closed Artificial ecosystems as a means of ecosystem studies for Earth and space needs.

Closed Artificial ecosystems (CAES) have good prospects for wide use as new means for quantitative studies of different types of both natural ecosystems and man-made ones. The paper deals with the discussion of three points of CAES applications. The first one is of importance for theoretical ecology development and is connected with bringing together "holistic" and "merological" approaches in ecosystems studies. Using CAES, we can combine both approaches, taking into account the biotic turnover of limiting substrates which few in number even for complicated natural ecosystems. The second CAES use concerns the development of "ecosystems health" concept and application of a key-factor-approach for the indication and measurement of healthy unhealthy state and functioning of ecosystems or their links. The third use is more of an applied nature, oriented to the intensification of bioremediation or biodepollution processes in different types of ecosystems, including the global biosphere. Grant numbers: N 99-04-96017, N25.     

The gravireaction of Ceratodon protonemata treated with gibberellic acid.

Moss protonemata exhibit negative gravitropism and the amyloplasts of the apical cell seem to play a key role in protonemal gravisensitivity. However, the mechanisms of this process are still poorly understood. Previously, we have shown that Ceratodon protonemata grown on agar-medium demonstrated greater gravicurvature than protonemata grown on medium with 11 mM glucose. In this study, we have examined whether gibberellic acid (GA), which promotes alpha-amylase expression, influences graviresponse of C. purpureus protonemata (strains WT-4 and WT-U) and how this event interacts with exogenous soluble sugars. After gravistimulation the WT-4 strain curved about twice as fast as the WT-U strain. However, responses of both strains to added substances were similar. High concentration of glucose (0.11 M) caused a decrease in protonema curvature, while the same concentration of sucrose did not significantly change the angles of curvature compared with controls. GA at 0.1 mM and higher concentrations inhibited gravitropism, and caused some apical cells to swell. The possible involvement of the carbohydrates in gravitropism is discussed.     

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.     

Induction of chromosomal damage in CHO-K1 cells and their repair-deficient mutant XRS5 by X-ray and particle irradiation.

The cytogenetic effects of X-rays and Au ions were investigated in repair-proficient CHO-K1 cells and their radiosensitive mutant strain xrs5, which shows a defect in the rejoining of DNA double-strand breaks. Both cell lines were synchronized by mitotic shake off, irradiated in G1-phase with either 250 kV X-rays or 780 MeV/u Au ions (LET: 1150 keV/micrometer) and chromosome aberrations were analyzed in first post-irradiation metaphases. Isoeffective doses of X-rays for the induction of aberrant cells and aberrations per cell were about 14 times lower for xrs5 than for CHO-K1 cells. After high LET radiation the difference in the cytogenetic response of both cell lines was drastically diminished. Furthermore, the analysis of the aberration types induced by sparsely and densely ionizing radiation showed for both cell lines specific changes in the spectrum of aberration types as LET increases. The experimental results are discussed with respect to the different types of lesions induced by sparsely and densely ionizing radiation.     

Early development of fern gametophytes in microgravity.

Dormant spores of the fern Ceratopteris richardii were flown on Shuttle mission STS-93 to evaluate the effects of micro-g on their development and on their pattern of gene expression. Prior to flight the spores were sterilized and sown into one of two environments: (1) Microscope slides in a video-microscopy module; and (2) Petri dishes. All spores were then stored in darkness until use. Spore germination was initiated on orbit after exposure to light. For the spores on microscope slides, cell level changes were recorded through the clear spore coat of the spores by video microscopy. After their exposure to light, spores in petri dishes were frozen in orbit at four different time points during which on earth gravity fixes the polarity of their development. Spores were then stored frozen in Biological Research in Canister units until recovery on earth. The RNAs from these cells and from 1-g control cells were extracted and analyzed on earth after flight to assay changes in gene expression. Video microscopy results revealed that the germinated spores developed normally in microgravity, although the polarity of their development, which is guided by gravity on earth, was random in space. Differential Display-PCR analyses of RNA extracted from space-flown cells showed that there was about a 5% change in the pattern of gene expression between cells developing in micro-g compared to those developing on earth.     

Kinetic characteristics of the theoretical ecosystems with different extent of openness.

In this paper, the influence of the extent of openness of ecosystem that is defined by the dilution rate, which characterizes the extent of flowage of the pond, on the intensity of the biotic circulation in ecosystems with different regulation types, number of trophic links and extent of closing has been investigated. We considered open systems, we took into account the return of the limiting substances, such as nitrogen and phosphorous, into the cycle by degradation of detritus and products of vital functions of consumers. It was shown by the numerical calculations that the increase of the dilution rate in without recycle ecosystems leads to increase of the net primary production up to the maximum value corresponding to the two-link trophic chain (biogenic substance and producer) and then, to gradually decrease. The residual concentration of biogenic limiting substances monotone increases. Net primary production and residual concentration of biogenic limiting substances in systems with recycle with even number of links behaves similarly to that in without recycle ecosystems. In the systems with recycle with the odd number of links that values lies on the stable level. We showed that in wide range of the dilution rate the recycling of the ecosystem can highly increase the net primary production and reduce residual concentration of biogenic limiting substances. The influence of the dilution rate on numbers of links that may exist in the system was analyzed.     

Expression of p53-regulated genes in human cultured lymphoblastoid TSCE5 and WTK1 cell lines after spaceflight in a frozen state

The 53 kDa tumor suppressor protein p53 is generally thought to contribute to the genetic stability of cells and to protect cells from DNA damage through the activity of p53-centered signal transduction pathways. To clarify the effect of space radiation on the expression of p53-dependent regulated genes, gene expression profiles were compared between two human cultured lymphoblastoid cell lines: one line (TSCE5) has a wild-type p53 gene status, and the other line (WTK1) has a mutated p53 gene status. Frozen human lymphoblastoid cells were stored in a freezer in the International Space Station (ISS) for 133 days. Gene expression was analyzed using DNA chips after culturing the space samples for 6 h on the ground after their return from space. Ground control samples were also cultured for 6 h after being stored in a frozen state on the ground for the same time period that the frozen cells were in space. p53-Dependent gene expression was calculated from the ratio of the gene expression values in wild-type p53 cells and in mutated p53 cells. The expression of 50 p53-dependent genes was up-regulated, and the expression of 94 p53-dependent genes was down-regulated after spaceflight. These expression data identified genes which could be useful in advancing studies in basic space radiation biology. The biological meaning of these results is discussed from the aspect of gene functions in the up- and down-regulated genes after exposure to low doses of space radiation.     

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.