Protein patterns of the Brassica rapa ovules and seeds under altered gravity

Electrophoretic investigation of protein patterns of Brassica rapa L. ovules and seeds from plants grown under clinorotation and in the laboratory control was carried out. Ovules at different stages (7 and 18 days after pollination) and mature seeds were analyzed. Polymorphism of seed storage proteins of B. rapa was taken into consideration in analysis of changes in ovule protein patterns under clinorotation. The appearance of a protein component in the region of about 43 kDa was detected in protein patterns of 7-day-old and 18-day-old ovules in the clinostat variants. Under altered gravity, in 18-day-old ovules, the appearance of a protein in the region of about 70 kDa was also revealed. The appearance of the protein component with the similar mobility (about 43 kDa) in ovules of different age from plants grown at clinorotation suggests that synthesis of this protein may be associated with the plant response to altered gravity. However, the investigation of the nature of this protein and its role requires further research to rule out its appearance because of genotypic differences between ovules of the control and experimental variants.     

Effect of changes in gravity on the quantity and localization of fibrillarin in the nucleolus of cress root meristem

For the first time, in altered gravity, with antifibrillarin antibodies and immunogold microscopy fibrillarin that is one of the most important proteins of rRNA processing was localized. The quantitative study of the density of gold particles in the nuclelous, under clinorotation both the transition zone FC-DFC and the dense fibrillar component were less labeled as compared to the control. Obtained data allow us to suppose the lowering of the rRNA processing level in the nucleoli under the influence of altered gravity.     

Calcium balance changes in tip growing plant cells under clinorotation

Calcium is known to play an important role in the regulation of cellular processes as a secondary messenger in signal transduction to a specific response in all eucaryotic cells. Its messenger role is realized by transient changes in the Ca2+ cytosolic concentration induced by variety of external stimuli such as light, hormones and gravity. Recent findings claim the modifications in a calcium balance in plant cells in microgravity and under clinorotation reproducing partially the microgravity effect. Based on these data, the hypothesis is proposed that Ca2+ level changes can trigger the rearrangements in cell metabolism occurring in the conditions of altered gravity. However, the methods used in previous works permit only to determine the relative quantity of intracellular membrane-bound calcium and to observe the localization of free Ca2+. Therefore, it is of essential interest to measure the concentration of free calcium ions ([Ca2+]i) in plant cells under the influence of altered gravity. In this paper results from measurements of the [Ca2+]i in plant cells under clinorotation will be reviewed and discussed in an effort to examine their effectiveness, causes and consequences.     

Effect of clinorotation on the intensity of lipid peroxidation and superoxide dismutase activity in pea chloroplasts

The lipid peroxidation intensity and the antioxidant enzyme superoxide dismutase (SOD) activity were studied in chloroplasts of Pisum sativum under clinorotation (for 7 and 14 days) for elucidating mechanisms of biological effect of altered gravity. In comparison with the control, increased LP levels in chloroplasts were established during both the terms of clinorotation. The SOD activity rose in the chloroplasts of plants clinorotated for 7 days, which has a significant protective effect. However, under a longer clinorotation (for 14 days) the SOD activity fell, being, however, higher than in the control samples. In accordance with the Selye oxidative stress theory, plants clinorotated for 7 days are in the phase of growing stability, while plants clinorotated for 14 days are in the phase of decreasing stability.     

Subnucleolar location of fibrillarin and NopA64 in Lepidium sativum root meristematic cells is changed in altered gravity

Summary. Fibrillarin and the plant nucleolin homolog NopA64 are two important nucleolar proteins involved in pre-rRNA processing. In order to determine the effects of the altered gravity environment on the nucleolus, we have investigated the location of fibrillarin and NopA64 in nucleolar subcomponents of cress (Lepidium sativum L.) root meristematic cells grown under clinorotation, which reproduces an important feature of microgravity, namely, the absence of the orienting action of a gravity vector, and compared it to the location in control cells grown in normal 1 g conditions. Prior to these experiments, we report here the characterization of cress fibrillarin as a 41 kDa protein which can be isolated from meristematic cells in three nuclear fractions, namely, the soluble ribonucleoprotein fraction, the chromatin fraction, and the nuclear-matrix fraction. Furthermore, as reported for other species, the location of both fibrillarin and NopA64 in the cress cell nucleolus was in zones known to contain complex ribonucleoprotein particles involved in early pre-rRNA processing, i.e., processomes. Under altered gravity, a decrease in the quantity of both fibrillarin and NopA64 compared to controls was observed in the transition zone between fibrillar centers and the dense fibrillar component, as well as in the bulk of the dense fibrillar component. These data suggest that altered (reduced) gravity results in a lowered level of functional activity in the nucleolus. Correspondence and reprints: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas, Ramiro de Maeztu 9, 28040 Madrid, Spain.     

Clinorotation affects morphology and ethylene production in soybean seedlings

The microgravity environment of spaceflight influences growth, morphology and metabolism in etiolated germinating soybean. To determine if clinorotation will similarly impact these processes, we conducted ground-based studies in conjunction with two space experiment opportunities. Soybean (Glycine max [L.] Merr.) seeds were planted within BRIC (Biological Research In Canister) canisters and grown for seven days at 20 degrees C under clinorotation (1 rpm) conditions or in a stationary upright mode. Gas samples were taken daily and plants were harvested after seven days for measurement of growth and morphology. Compared to the stationary upright controls, plants exposed to clinorotation exhibited increased root length (125% greater) and fresh weight (42% greater), whereas shoot length and fresh weight decreased by 33% and 16% respectively. Plants grown under clinorotation produced twice as much ethylene as the stationary controls. Seedlings treated with triiodo benzoic acid (TIBA), an auxin transport inhibitor, under clinorotation produced 50% less ethylene than the untreated control subjected to the same gravity treatment, whereas a treatment with 2,4-D increased ethylene by five-fold in the clinorotated plants. These data suggest that slow clinorotation influences biomass partitioning and ethylene production in etiolated soybean plants.     

The influence of altered gravity on carbohydrate metabolism in excised wheat leaves

We developed a system to study the influence of altered gravity on carbohydrate metabolism in excised wheat leaves by means of clinorotation. The use of excised leaves in our clinostat studies offered a number of advantages over the use of whole plants, most important of which were minimization of exogenous mechanical stress and a greater amount of carbohydrate accumulation during the time of treatment. We found that horizontal clinorotation of excised wheat leaves resulted in significant reductions in the accumulation of fructose, sucrose, starch and fructan relative to control, vertically clinorotated leaves. Photosynthesis, dark respiration and the extractable activities of ADP glucose pyrophosphorylase (EC 2.7.7.27), sucrose phosphate synthase (EC 2.4.4.14), sucrose sucrose fructosyltransferase (EC 2.4.1.99), and fructan hydrolase (EC 3.2.1.80) were unchanged due to altered gravity treatment.     

The effects of prolonged gravity vector randomization on differentiation of precursour cells in vitro

It was shown that proliferative rate of human mesenchymal stem cells (hMSCs) decreased during gravity vector randomization (clinorotation). Clinorotated hMSCs exhibited changes in expression of some external cell markers and adhesion molecules. Marked suppression of osteogenic differentiation of hMSC as to formation of bone nodules and calcium deposits was observed. F-actin stress fibers of cytoskeleton were altered in clinorotated cultures. Prolonged clinorotation of embryoid bodies (EBs) resulted in a delay of embryonic stem cells (ESCs) differentiation into cardiomyocytes. Percentage of beating cardiomyocytes in each EB was significantly reduced. Thus, long-term gravity vector randomization decreases the differentiation processes of precursor cells in vitro.     

Peculiarities [correction of Pfculiarities] of lipid accumulation in Brassica rapa L. embryos on different stages development under altered gravity

Accumulation of lipid inclusions in Brassica rapa embryos generated under slow horizontal clinorotation and in the laboratory control were analyzed by histochemical methods. The research of lipid accumulation was carried out on consecutive stages of the embryo development, from the moment of two-cellular proembryo formation up to the stages of their full differentiation (21-22-day-old embryos). Accumulation of lipid drops was revealed for the first time at early stages of embryogenesis in this species, beginning from 3-day-old embryos (ball-like stage of embryo development) under clinorotation and in the laboratory control. The quantity of lipid inclusion was estimated by morphometrical analysis. Statistically significant differences between the clinorotation and laboratory control variants in quantity of lipid drops per cell were revealed from 6-day-old embryos (heart-shaped stage). Especially pronounced differences were noted in differentiated embryos (beginning from 12-day-old embryos) under horizontal clinorotation in comparison with the laboratory control. The registered differences testify about influence of altered gravity conditions on lipid accumulation in Brassica rapa embryos.     

Further ultrastructural characterization of the intranuclear ring-shaped bodies of the plant Lacandonia schismatica

Ring-shaped bodies are found in the nucleus of Lacandonia schismatica, a rare plant with the sexual organs inverted. They are 0.5-microm-diameter structures that present an electron-dense external ring surrounding a central core. Ultrastructural studies indicate that these bodies contain RNA. The external ring is labeled with antibodies against small nuclear ribonucleoproteins, suggesting that they may be involved in pre-mRNA metabolism. In the present work we further characterized these intranuclear ring-shaped structures by serial-sectioning analysis. Moreover, we tested the presence of additional molecular elements related to pre-mRNA metabolism, such as SR proteins and poly(A)(+) RNA, using immunoelectron microscopy and ultrastructural in situ hybridization. Our results show that these nuclear bodies are spherical. They contain SR proteins involved in splicing and postsplicing events and little to no poly(A)(+) RNA. We also found similar nuclear bodies in other plant and animal species. Therefore, ring-shaped bodies in L. schismatica are spherical, highly compartmentalized nuclear structures that may be involved in pre-mRNA metabolism.     

羟基喜树碱诱导肝癌细胞凋亡前后细胞核蛋白质组变化的定量分析

对羟基喜树碱诱导肝癌细胞凋亡前后的细胞核蛋白质组进行定量研究,获得凋亡前后细胞核蛋白质组中差异蛋白表达量相对变化的信息,为在亚细胞定量蛋白质组水平上深入探讨羟基喜树碱的作用机理提供实验依据。分离提取羟基喜树碱诱导肝癌细胞凋亡前后的细胞核并鉴定其纯度,用含稳定同位素的化学试剂c-ICAT标记细胞凋亡前后的细胞核蛋白,对细胞核标记蛋白进行消化和纯化,利用基于多维色谱-线性离子阱/静电场轨道阱质谱联用技术的鸟枪（Shotgun）法策略及c-ICAT定量策略分析鉴定蛋白质,获得同一种肝癌细胞核蛋白质在凋亡前后细胞中的表达量变化比值。分析鉴定了在细胞凋亡前后的表达量差异有显著统计学意义（P〈0.05）的肝癌细胞核蛋白42个,其中,12个蛋白的表达量在羟基喜树碱诱导细胞凋亡后下调,30个蛋白的表达量在凋亡细胞中上调。这些差异蛋白的分子功能主要与细胞增殖、凋亡和分化、能量代谢、核酸代谢以及细胞骨架相关。     

TNF-alpha-dependent activation of NF-kappa B in human osteoblastic HOS-TE85 cells is repressed in vector-averaged gravity using clinostat rotation

Effects of vector-averaged gravity on tumor necrosis factor (TNF)-alpha-dependent activation of nuclear factor kappa B (NF-kappa B) in human osteoblastic HOS-TE85 cells were investigated by culturing the cells using clinostat rotation (clinorotation). Cell cultures were rotated for 72 h at 40 rpm in a clinostat. At the end of clinorotation, the cells were treated with TNF-alpha for 30 min under stationary conditions. Electrophoretic mobility shift assays revealed that TNF-alpha-dependent activation of NF-kappa B was markedly reduced in the clinorotated cells when compared with the cells in control stationary cultures or after horizontal rotation (motional controls). The NF-kappa B-dependent transactivation was also impaired in the clinorotated cells, as evidenced by a transient transfection assay with a reporter plasmid containing multimerized NF-kappa B sites. Consistent with these findings, the TNF-alpha-dependent induction of endogenous NF-kappa B-responsive genes p105, I kappa B-alpha, and IL-8, was significantly attenuate in clinorotated cells. These results demonstrate that vector-averaged gravity inhibits the responsiveness of osteoblasts to TNF-alpha by repressing NF-kappa B activation.     

Vector-averaged gravity does not alter acetylcholine receptor single channel properties.

To examine the physiological sensitivity of membrane receptors to altered gravity, we examined the single channel properties of the acetylcholine receptor (AChR), in co-cultures of Xenopus myocytes and neurons, to vector-averaged gravity in the clinostat. This experimental paradigm produces an environment in which, from the cell's perspective, the gravitational vector is "nulled" by continuous averaging. In that respect, the clinostat simulates one aspect of space microgravity where the gravity force is greatly reduced. After clinorotation, the AChR channel mean open-time and conductance were statistically not different from control values but showed a rotation-dependent trend that suggests a process of cellular adaptation to clinorotation. These findings therefore suggest that the ACHR channel function may not be affected in the microgravity of space despite changes in the receptor's cellular organization.     

NUCLEAR PORE ANCHOR, the Arabidopsis homolog of Tpr/Mlp1/Mlp2/megator, is involved in mRNA export and SUMO homeostasis and affects diverse aspects of plant development

Vertebrate Tpr and its yeast homologs Mlp1/Mlp2, long coiled-coil proteins of nuclear pore inner basket filaments, are involved in mRNA export, telomere organization, spindle pole assembly, and unspliced RNA retention. We identified Arabidopsis thaliana NUCLEAR PORE ANCHOR (NUA) encoding a 237-kD protein with similarity to Tpr. NUA is located at the inner surface of the nuclear envelope in interphase and in the vicinity of the spindle in prometaphase. Four T-DNA insertion lines were characterized, which comprise an allelic series of increasing severity for several correlating phenotypes, such as early flowering under short days and long days, increased abundance of SUMO conjugates, altered expression of several flowering regulators, and nuclear accumulation of poly(A)+ RNA. nua mutants phenocopy mutants of EARLY IN SHORT DAYS4 (ESD4), an Arabidopsis SUMO protease concentrated at the nuclear periphery. nua esd4 double mutants resemble nua and esd4 single mutants, suggesting that the two proteins act in the same pathway or complex, supported by yeast two-hybrid interaction. Our data indicate that NUA is a component of nuclear pore-associated steps of sumoylation and mRNA export in plants and that defects in these processes affect the signaling events of flowering time regulation and additional developmental processes.     

Current perspectives of the <Emphasis Type="Italic">Escherichia coli</Emphasis> RNA degradosome

Many biological processes in the cell are linked to RNA metabolism and therefore have implications for a wide range of biotechnological applications. The processing and degradation of RNA plays an important role in RNA metabolism with often the same enzymes being involved in both processes. In this review, we highlight the dynamic nature of the structural components of the Escherichia coli RNA degradosome which is a large multiprotein complex that plays an important role in RNA degradation. The activities of the individual components of the degradosome are also discussed. The RNA degradosome forms part of the bacterial cytoskeleton and associated proteins, such as molecular chaperones, may aid in the compartmentalization of enzymatic activities and cytoskeletal organization. An enhanced understanding of the components of the RNA degradosome in other bacterial species will certainly aid in their evaluation as potential antimicrobial agents.     

The effect of clinorotation on vestibular compensation in upside-down swimming catfish.

Upside-down swimming catfish Synodontis nigriventris can keep upside-down swimming posture stably under pseudo-microgravity generated by clinostat. When the vestibular organ is unilaterally ablated, the operated S. nigriventris shows disturbed swimming postures under the clinorotation condition. However, about 1 month after the operation, unilateral vestibular organ-ablated S. nigriventris shows stable upside-down swimming posture under the condition (vestibular compensation). In contrast, a closely related upside-up swimming catfish Synodontis multipunctatus belonging to same Synodontis family can not keep stable swimming postures under the clinorotation conditions. In this study, we examined the effect of continuous clinorotation on vestibular compensation in intact and unilateral vestibular organ-ablated Synodontis nigriventris and Synodontis multipunctatus. After the exposure to continuous clinorotation, the postures of the catfish were observed under microgravity provided by parabolic flights of an aircraft. Unilateral vestibular organ-ablated S. nigriventris which had been exposed to continuous clinorotation showed stable swimming postures and did not show dorsal light reaction (DLR) under microgravity. This postural control pattern of the operated catfish was similar to that of intact catfish. Intact and unilateral vestibular organ-ablated S. multipunctatus showed DLR during microgravity. Our results confirmed that S. nigriventris has a novel balance sensation which is not affected by microgravity. DLR seems not to play an important role in postural control. It remains unclear that the continuous clinorotation effects on vestibular compensation because we could not keep used unilateral vestibular organ-ablated fish alive under continuous clinorotation for uninterrupted 25 days. This study suggests that space flight experiments are required to explore whether gravity information is essential for vestibular compensation.     

Starch metabolism in germinating soybean cotyledons is sensitive to clinorotation and centrifugation

Soybean (Glycine max [L.] Merr. cv. McCall) seedlings germinated and grew for 6d under the altered gravity conditions of horizontal clinorotation and centrifugation. Both of these conditions resulted in decreased growth relative to the control (vertically rotated) plants. Starch concentration in the cotyledons was lower in the clinorotated plants and was higher in the centrifuged plants compared to the controls. The opposite relationship was noted for total lipid concentration. Of the six starch metabolic enzyme activities measured, only ADP glucose pyrophosphorylase was affected by the gravity treatments; being lower in the cotyledons of the horizontally rotated plants and higher in the cotyledons of the centrifuged plants relative to the control values.