Physiological characterization of gravitaxis in Euglena gracilis and Astasia longa studied on sounding rocket flights.

Euglena gracilis is a photosynthetic, unicellular flagellate found in eutrophic freshwater habitats. The organisms control their vertical position in the water column using gravi- and phototaxis. Recent experiments demonstrated that negative gravitaxis cannot be explained by passive buoyancy but by an active physiological mechanism. During space experiments, the threshold of gravitaxis was determined to be between 0.08 and 0.12 x g. A strong correlation between the applied acceleration and the intracellular cAMP and Ca2+ was observed. The results support the hypothesis, that the cell body of Euglena, which is denser than the surrounding medium exerts a pressure onto the lower membrane and activates mechanosensitive Ca2+ channels. Changes in the membrane potential and the cAMP concentration are most likely subsequent elements in a signal transduction chain, which results in reorientation strokes of the flagellum.     

Paramecium--a model system for studying cellular graviperception.

Experiments under varied gravitational accelerations as well as in density-adjusted media showed that sensation of gravity in protists may be linked to the known principles of mechanosensation. Paramecium, a ciliate with clear graviresponses (gravitaxis and gravikinesis) is an ideal model system to prove this hypothesis since the ciliary activity and thus the swimming behaviour is controlled by the membrane potential. It has also been assumed that the cytoplasmic mass causes a distinct stimulation of the bipolarly distributed mechano-sensitive K+ and Ca2+ ion channels in the plasma membrane in dependence of the spatial orientation of the cell. In order to prove this hypothesis, different channel blockers are currently under investigation. Gadolinium did not inhibit gravitaxis in Paramecium, showing that it does not specifically block gravireceptors. Further studies concentrated on the question of whether second messengers are involved in the gravity signal transduction chain. Exposure to 5 g for up to 10 min led to a significant increase in cAMP.     

Graviperception and gravitaxis in algae.

Photosynthetic flagellates are among the most intensely studied unicellular organisms in the field of graviperception and gravitaxis. While the phenomenon of graviorientation has been known for many decades, only recently was the molecular mechanism unveiled. Earlier hypotheses tried to explain the precise orientation by a passive buoy mechanism assuming the tail end to be heavier than the front. In the photosynthetic flagellate Euglena gracilis, the whole cell body is denser than the surrounding medium, pressing onto the lower cell membrane where it seems to activate mechanosensitive ion channels specific for calcium. The calcium entering the cells during reorientation can be visualized by the fluorescence probe, Calcium Crimson. Cyclic AMP is likewise involved in the molecular pathway. Inhibitors of calcium channels and ionophores impair gravitaxis while caffeine, a blocker of the phosphodiesterase, enhances the precision of orientation.     

Putative graviperception mechanisms of protists.

Many (if not all) free-living cells use the gravity vector for their spatial orientation (gravitaxis). Additional responses may include gravikinesis as well as changes in morphological and physiological parameters. Though using essentially different modes of locomotion, ameboid and ciliated cells seem to rely on common fundamental graviperception mechanisms. Uniquely in the ciliate family Loxodidae a specialized intracellular gravireceptor organelle has been developed, whereas in all other cells common cell structures seem to be responsible for gravisensing. Changes in direction or magnitude of acceleration (from 0 to 5 g) as well as experiments in density-adjusted media strongly indicate that either the whole cytoplasm or dense organelles like nuclei act as statoliths and open directly or via cytoskeletal elements mechano-sensitive ion channels in the cell membrane. A recent spaceflight experiment (S/MM-06) demonstrated that prolonged (9 d) actual weightlessness did not affect the ability of Loxodes to respond to acceleration stimuli. However, prolonged cooling (> or = l4 d, 4-10 degrees C) destroyed the ability for gravitactic orientation of Paramecium. This may reflect a profound effect either on the gravireceptor itself or on the gravity-signal processing. In gravity signalling the ubiquitous second messenger cAMP may be involved in acceleration-stimulus transduction.     

Gravitaxis and graviperception in flagellates.

There is strong evidence that gravitactic orientation in flagellates and ciliates is mediated by an active physiological gravireceptor rather than by passive alignment of the cells in the water column. In flagellates the threshold for graviorientation was found to be at 0.12 x g on a slow rotating centrifuge during the IML-2 mission on the Shuttle Columbia and a subsequent parabolic rocket flight (TEXUS). During the IML-2 mission no adaptation to microgravity was observed over the duration of the space flight, while gravitaxis was lost in a terrestrial closed environmental system over the period of almost two years. Sedimenting statoliths are not likely to be involved in graviperception because of the small size of the cells and their rotation around the longitudinal axis during forward locomotion. Instead the whole cytoplasmic content of the cell, being heavier than the surrounding aqueous medium (1.05 g/ml), exerts a pressure on the lower membrane. This force activates stretch-sensitive calcium specific ion channels which can be inhibited by the addition of gadolinium which therefore abolishes gravitaxis. The channels seem to mainly allow calcium ions to pass since gravitaxis is blocked by the addition of the calcium ionophore A23187 and by vanadate which blocks the Ca-ATPase in the cytoplasmic membrane. Recently, a gene for a mechanosensitive channel, originally sequenced for Saccharomyces, was identified in Euglena by PCR. The increase in intracellular free calcium during reorientation can be visualized by the fluorophore Calcium Crimson using laser excitation and image intensification. This result was confirmed during recent parabolic flights. The gated calcium changes the membrane potential across the membrane which may be the trigger for the reorientation of the flagellum. cAMP plays a role as a secondary messenger. Photosynthetic flagellates are suitable candidates for life support systems since they absorb CO2 and produce oxygen. Preliminary experiments are discussed.     

Gravitaxis and graviperception in Euglena gracilis.

Gravitactic orientation in the flagellate Euglena gracilis is mediated by an active physiological receptor rather than a passive alignment of the cells. During a recent space flight on the American shuttle Columbia the cells were subjected to different accelerations between 0 and 1.5 x g and tracked by computerized real-time image analysis. The dependence of orientation on acceleration followed a sigmoidal curve with a threshold at < or = 0.16 x g and a saturation at about 0.32 x g. No adaptation of the cells to the conditions of weightlessness was observed over the duration of the space mission (12 days). Under terrestrial conditions graviorientation was eliminated when the cells were suspended in a medium the density of which (Ficoll) equaled that of the cell body (1.04 g/ml) and was reversed at higher densities indicating that the whole cytoplasm exerts a pressure on the respective lower membrane. There it probably activates stretch-sensitive calcium specific ion channels since gravitaxis can be affected by gadolinium which is a specific inhibitor of calcium transport in these structures. The sensory transduction chain could involve modulation of the membrane potential since ion channel blockers, ionophores and ATPase inhibitors impair graviperception.     

Gravitaxis in unicellular microorganisms.

Orientation of organisms with respect to the gravitational field of the Earth has been studied for more than 100 years in a number of unicellular microorganisms including flagellates and ciliates. Several hypotheses have been developed how the weak stimulus is perceived. Intracellular statoliths have been found to be involved in gravitaxis of Loxodes, while no specialized organelles have been detected in other ciliates, e.g. Paramecium. Also in the slime mold Physarum no specialized gravireceptors have been identified yet. In the flagellate Euglena gracilis the whole cell body, which is denser than the surrounding medium, seems to act as a statolith pressing onto the lower membrane where it activates mechanosensitive ion channels. Similar results were obtained for the ciliate Paramecium. In contrast to the flagellate Euglena, several ciliates have been found to show gravikinesis, which is defined as a dependence of the swimming velocity on the direction of movement in the gravity field.     

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.     

Graviperception in ciliates: steps in the transduction chain.

Ciliates represent suitable model systems to study the mechanisms of graviperception and signal transduction as they show clear gravity-induced behavioural responses (gravitaxis and gravikinesis). The cytoplasm seems to act as a "statolith" stimulating mechanosensitive ion channels in the cell membrane. In order to test this hypothesis, electrophysiological studies with Stylonychia mytilus were performed, revealing the proposed changes (de- or hyperpolarization) depending on the cell's spatial orientation. The behaviour of Paramecium and Stylonychia was also analyzed during variable acceleration conditions of parabolic flights (5th German Parabolic Flight Campaign, 2003). The corresponding data confirm the relaxation of the graviresponses in microgravity as well as the existence of thresholds of graviresponses, which are found to be in the range of 0.4xg (gravikinesis) and 0.6xg (gravitaxis).     

Comparative studies of the graviresponses of Paramecium and Loxodes.

Gravitactic protozoa offer advantages in studying how the gravity stimulus is perceived on the cellular level. By means of a slow rotating centrifuge microscope in space the acceleration thresholds for gravitaxis of Loxodes striatus and Paramecium biaurelia were determined: < or = 0.15 x g for Loxodes and 0.3 x g for Paramecium, indicating different sensitivities of these species. Neutral-buoyant densities of immobilized cells were determined using media of different densities, revealing densities of 1.03 to 1.035 g/cm3 for Loxodes and 1.04 g/cm3 to 1.045 g/cm3 for Paramecium. Behavioral studies revealed that gravitaxis of Loxodes persisted independent of the density of the medium. In contrast, negative gravitaxis of Paramecium was no longer measurable if the density of the medium approached the density of the cell. The results suggest that in the case of Loxodes gravity is perceived by an intracellular receptor and, in the case of Paramecium by its own mass via the pressure on the lower cell membrane.     

Radiosensitivity to high energy iron ions is influenced by heterozygosity for Atm, Rad9 and Brca1

Loss of function of DNA repair genes has been implicated in the development of many types of cancer. In the last several years, heterozygosity leading to haploinsufficiency for proteins involved in DNA repair was shown to play a role in genomic instability and carcinogenesis after DNA damage is induced, for example by ionizing radiation. Since the effect of heterozygosity for one gene is relatively small, we hypothesize that predisposition to cancer could be a result of the additive effect of heterozygosity for two or more genes critical to pathways that control DNA damage signaling, repair or apoptosis. We investigated the role of heterozygosity for Atm, Rad9 and Brca1 on cell oncogenic transformation and cell survival induced by 1 GeV/n⁵⁶Fe ions. Our results show that cells heterozygous for both Atm and Rad9 or Atm and Brca1 have high survival rates and are more sensitive to transformation by high energy iron ions when compared with wild-type controls or cells haploinsufficient for only one of these proteins. Since mutations or polymorphisms for similar genes exist in a small percentage of the human population, we have identified a radiosensitive sub-population. This finding has several implications. First, the existence of a radiosensitive sub-population may distort the shape of the dose–response relationship. Second, it would not be ethical to put exceptionally radiosensitive individuals into a setting where they may potentially be exposed to substantial doses of radiation.     

Characterization of a novel gravitropic mutant of morning glory, weeping2

In higher plants, gravity is a major environmental cue that governs growth orientation, a phenomenon termed gravitropism. It has been suggested that gravity also affects other aspects of morphogenesis, such as circumnutation and winding movements. Previously, we showed that these aspects of plant growth morphology require amyloplast sedimentation inside gravisensing endodermal cells. However, the molecular mechanism of the graviresponse and its relationship to circumnutation and winding remains obscure. Here, we have characterized a novel shoot gravitropic mutant of morning glory, weeping2 (we2). In the we2 mutant, the gravitropic response of the stem was absent, and hypocotyls exhibited a severely reduced gravitropic response, whereas roots showed normal gravitropism. In agreement with our previous studies, we found that we2 mutant has defects in shoot circumnutation and winding. Histological analysis showed that we2 mutant forms abnormal endodermal cells. We identified a mutation in the morning glory homolog of SHORT-ROOT (PnSHR1) that was genetically linked to the agravitropic phenotype of we2 mutant, and which may underlie the abnormal differentiation of endodermal cells in this plant. These results suggest that the phenotype of we2 mutant is due to a mutation of PnSHR1, and that PnSHR1 regulates gravimorphogenesis, including circumnutation and winding movements, in morning glory.     

Blue light-dependent phosphorylations of cryptochromes are affected by magnetic fields in Arabidopsis

The blue light receptor cryptochrome that could form radical pairs after exposure to blue light was suggested to be a magnetoreceptor based on the proposition that radical pairs were involved in the magnetoreception. But the effects of magnetic fields on the function of cryptochrome are poorly understood. Phosphorylation of cryptochrome in Arabidopsis was closely associated with the function of this photoreceptor. Here, we grew Arabidopsis seedlings in a 500 μT magnetic field and a near-null magnetic field and found that the 500 μT magnetic field enhanced the blue light-dependent phosphorylations of CRY1 and CRY2, and the near-null magnetic field weakened the blue light-dependent phosphorylation of CRY2 but not CRY1. Dephosphorylations of CRY1 and CRY2 in the darkness were slowed down in the 500 μT magnetic field, whereas dephosphorylations of CRY1 and CRY2 were accelerated in the near-null magnetic field. These results suggest that magnetic field with strength higher or weaker than the local geomagnetic field affects the activated states of cryptochromes, which thus modifies the functions of cryptochromes.     

Efficacy of oxygen-supplying capacity of Azolla in a controlled life support system

Azolla shows high growth and propagation rates, strong photosynthetic O₂-releasing ability and high nutritional value. It is suitable as a salad vegetable and can be cultured on a multi-layered wet bed. Hence, it possesses potential as a fresh vegetable, and to release O₂ and absorb CO₂ in a Controlled Ecological Life Support System in space. In this study, we investigated the O₂-providing characteristics of Azolla in a closed chamber under manned, controlled conditions to lay a foundation for use of Azolla as a biological component in ground simulation experiments for space applications. A closed test chamber, representing a Controlled Ecological Life Support System including an Azolla wet-culture device, was built to measure the changes in atmospheric O₂ and CO₂ concentrations inside the chamber in the presence of coexisting Azolla, fish and men. The amount of O₂ consumed by fish was 0.0805–0.0831 L kg⁻¹ h⁻¹ and the level of CO₂ emission was 0.0705–0.0736 L kg⁻¹ h⁻¹; O₂ consumption by the two trial volunteers was 19.71 L h⁻¹ and the volume of respiration-released CO₂ was 18.90 L h⁻¹. Under 7000–8000 Lx artificial light and Azolla wet-culture conditions, human and fish respiration and Azolla photosynthesis were complementary, thus the atmospheric O₂ and CO₂ concentrations inside chamber were maintained in equilibrium. The increase in atmospheric CO₂ concentration in the closed chamber enhanced the net photosynthesis efficiency of the Azolla colony. This study showed that Azolla has strong photosynthetic O₂-releasing ability, which equilibrates the O₂ and CO₂ concentrations inside the chamber in favor of human survival and verifies the potential of Azolla for space applications.     

Calcium influx through stretch-activated channels mediates microfilament reorganization in osteoblasts under simulated weightlessness

We have explored the role of Ca²⁺ signaling in microfilament reorganization of osteoblasts induced by simulated weightlessness using a random positioning machine (RPM). The RPM-induced alterations of cell morphology, microfilament distribution, cell proliferation, cell migration, cytosol free calcium concentration ([Ca²⁺]_i), and protein expression in MG63 osteoblasts were investigated. Simulated weightlessness reduced cell size, disrupted microfilament, inhibited cellular proliferation and migration, and induced an increase in [Ca²⁺]_i in MG63 human osteosarcoma cells. Gadolinium chloride (Gd), an inhibitor for stretch-activated channels, attenuated the increase in [Ca²⁺]_i and microfilament disruption. Further, the expression of calmodulin was significantly increased by simulated weightlessness, and an inhibitor of calmodulin, W-7, aggravated microfilament disruption. Our findings demonstrate that simulated weightlessness induces Ca²⁺ influx through stretch-activated channels, then results in microfilament disruption.     

Involvement of nitric oxide in the mechanism of biochemical alterations induced by simulated microgravity in Microcystis aeruginosa

Simulated microgravity (SMG) can inhibit proliferation and enhance microcystin production of Microcystis aeruginosa. We investigated the role of nitric oxide (NO) in regulating the SMG induced changes of proliferation, photochemical system II photochemical activity, pigment, soluble protein and microcystin production in M. aeruginosa. M. aeruginosa was exposed to 0.1 mM sodium nitroprusside (SNP, NO donor) or 0.02 mM 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO, NO scavenger) alone or in combination with SMG for 48 h. SMG and SNP inhibited the growth of M. aeruginosa while c-PTIO had no effect on cell number. As to yield, the negative effect of SMG was augmented by SNP and suppressed by c-PTIO. The intracellular concentrations of chlorophyll a, carotenoid, phycocyanin, soluble protein and microcystin were increased by SMG after 48 h. The effects of SMG on these metabolic processes could be enhanced by SNP and be partly eliminated by c-PTIO. Moreover, SNP and c-PTIO only functioned in these biochemical processes under SMG, unlike in the regulation of cell proliferation and yield. These results showed that the effects of SMG could be enhanced by adding exogenous NO and be mitigated by scavenging endogenous NO, revealing the involvement of NO in the changes in biochemistry processes induced by SMG in M. aeruginosa.     

A broad climatology of very high latitude substorms

Magnetic data from a newly commissioned Indian Antarctic station Bharati (corrected geomagnetic (CGM) coordinates 74.7°S, 97.2°E) and closely-spaced IMAGE chain observatories (∼100° magnetic meridian in Northern hemisphere) has been analyzed to study the climatology of substorms which were localized poleward of the standard auroral oval. We considered four austral summers (year 2007–2010) when data from Bharati was available. Several very high latitude substorms were observed in this duration when the solar activity remained unexpectedly low for a long time. Various features of very high latitude substorms, e.g., local time dependence, interplanetary state, hemispherical asymmetry and their nightside low latitude signatures are examined. Events studied here, suggested the following properties of substorms occurring at very high latitudes: (1) maximum occurrence was observed near magnetic midnight (21:00–02:00 MLT). (2) In contradiction to earlier reports, many substorms were observed even during negative IMF Bz condition. In addition, majority of substorms occurred during low or moderate solar wind streams. (3) Magnetic signatures were often pronounced in the winter hemisphere. (4) Even if widely used standard AE indices fail to monitor very high latitude substorms, their low latitude signatures are often evident.