Automorphogenesis of maize roots under simulated microgravity conditions

Plant seedlings show exaggerated growth responses on a three-dimensional clinostat. Such an automorphogenesis appears to be one of major factors which govern the life cycle of higher plants under a microgravity environment. On the three-dimensional clinostat, maize roots exhibited curvatures in three different portions; 1) the basal region just protruding from the coleorhiza, 2) the region between the mature and the elongation zone, and 3) the elongation zone, several mm from the tip. Even non-clinostatted control roots showed some degree of curvature. The curvature occurred at random without any dorsiventrality. There was no difference in the osmotic concentration of the cell sap between the convex and the concave halves of any region. However, the convex, rapidly expanding side exhibited a higher extensibility of the cell wall in some regions, which appears to be a cause of the curvature. In order to understand the role of gravity in regulation of plant growth and development, we should clarify a series of events by which an automorphogenesis is induced under simulated microgravity conditions.     

Neonatal rat heart cells cultured in simulated microgravity

Summary In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two-dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by nonmyocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA- designed High-Aspect Ratio Vessel (HARV) bioreactors provide a low shear environment that allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells cultured in HARVs adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARVs using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar; however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissuelike organizations of cardiac cells in vitro.     

槲皮素对模拟微重力条件下体外培养大鼠心肌细胞骨架的影响

采用细胞免疫双荧光染色观察离体培养的大鼠心肌细胞微丝和微管分布 ,探讨模拟微重力条件下槲皮素对心肌细胞骨架分布的影响。结果表明 :模拟微重力条件下心肌细胞微丝、微管在近胞核区的分布增多 ;模拟微重力处理的同时加入槲皮素 ,则使近胞核处微丝、微管分布明显减少 ,微丝束的粗细与对照组无异。提示模拟微重力可显著影响心肌细胞微丝、微管的分布 ,槲皮素可对抗该效应而发挥其心肌细胞保护作用 [动物学报49(1) :98～ 10 3 ,2 0 0 3]。     

Microarray analysis of genes differentially expressed in hepG2 cells cultured in simulated microgravity: Preliminary report

Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.     

Automorphosis of maize shoots under simulated microgravity on a three-dimensional clinostat

Seedlings of maize ( Zea mays L. cv. Golden Cross Bantam T-51) were grown under microgravity conditions simulated by a three-dimensional clinostat. On the clinostat, maize shoots exhibited curvatures in three different portions: (1) the basal transition zone connecting roots and mesocotyls, (2) the coleoptile node located between mesocotyls and coleoptiles, and (3) the elongating region of the coleoptiles. Even non-clinostatted control shoots showed some degree of curvature away from the caryopsis in the transition zone and bending toward the caryopsis in the coleoptile node. Clinostat rotation greatly stimulated these curvatures. Control coleoptiles elongated almost straightly, whereas coleoptiles on the clinostat bent either away from or toward the caryopsis depending on the timing of rotation. The curvature in all three portions became larger with time, both in control and clinostatted seedlings. There was no difference in the osmotic concentration of the cell sap between the convex and the concave halves of any portion. However, in coleoptile nodes and coleoptiles, the faster-expanding convex side exhibited a higher extensibility of the cell wall than the opposite side, and this appears to be a cause of the curvature. Thus, changes in the cell wall metabolism may be involved in automorphosis, which governs the life cycle of plants under a microgravity environment.     

Differential expression of specific cellular defense proteins in rat hypothalamus under simulated microgravity induced conditions: Comparative proteomics

Microgravity severely halts the structural and functional cerebral capacity of astronauts especially affecting their brains due to the stress produced by cephalic fluid shift. We employed a rat tail suspension model to substantiate simulated microgravity (SM) in brain. In this study, comparative mass spectrometry was applied in order to demonstrate the differential expression of 17 specific cellular defense proteins. Gamma‐enolase, peptidyl‐prolyl cis‐trans isomerase A, glial fibrillary acidic protein, heat shock protein HSP 90‐alpha, 10 kDa heat shock protein, mitochondrial, heat shock cognate 71 kDa protein, superoxide dismutase 1 and dihydropyrimidinase‐related protein 2 were found to be upregulated by HPLC/ESI‐TOF. Furthermore, five differentially expressed proteins including 60 kDa heat shock protein, mitochondrial, heat shock protein HSP 90‐beta, peroxiredoxin‐2, stress‐induced‐phosphoprotein, and UCHL‐1 were found to be upregulated by HPLC/ESI‐Q‐TOF MS. In addition, downregulated proteins include cytochrome C, superoxide dismutase 2, somatic, and excitatory amino acid transporter 1 and protein DJ‐1. Validity of MS results was successfully performed by Western blot analysis of DJ‐1 protein. This study will not only help to understand the neurochemical responses produced under microgravity but also will give future direction to cure the proteomic losses and their after effects in astronauts.     

Induction of three-dimensional assembly of human liver cells by simulated microgravity

Summary The establishment of long-term cultures of functional primary human liver cells (PHLC) is formidable. Developed at NASA, the Rotary Cell Culture System (RCCS) allows the creation of the unique microgravity environment of low shear force, high-mass transfer, and 3-dimensional cell culture of dissimilar cell types. The aim of our study was to establish long-term hepatocyte cultures in simulated microgravity. PHLC were harvested from human livers by collagenase perfusion and were cultured in RCCS. PHLC aggregates were readily formed and increased up to 1 cm long. The expansion of PHLC in bioreactors was further evaluated with microcarriers and biodegradable scaffolds. While microcarriers were not conducive to formation of spheroids, PHLC cultured with biodegradable scaffolds formed aggregates up to 3 cm long. Analyses of PHLC spheroids revealed tissue-like structures composed of hepatocytes, biliary epithelial cells, and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes surrounded by complex stromal structures and reticulin fibers, bile canaliculi with multiple microvilli, and tight cellular junctions. Albumin mRNA was expressed throughout the 60-d culture. A simulated microgravity environment is conducive to maintaining long-term cultures of functional hepatocytes. This model system will assist in developing improved protocols for autologous hepatocyte transplantation, gene therapy, and liver assist devices, and facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo.     

A countermeasure to ameliorate immune dysfunction in in vitro simulated microgravity environment: role of cellularnucleotide nutrition

Considerable evidence suggests that space travelers are immunosuppressed, presumably by microgravity environmental stresses, putting them at risk for adverse effects, such as opportunistic infections, poor wound healing, and cancer. The purpose of this study was to examine the role and mechanisms of nucleotide (NT) supplementation as a countermeasure to obviate immunosuppression during space travel. The in vitro rotary cell culture system, a bioreactor (BIO), was used to simulate the effect of microgravity and to isolate the neuroendocrine effects inherent to in vitro models. The splenocytes from normal mice were cultured in BIO and control tissue culture (TC) flasks with and without phytohemagglutinin (PHA) for mitogen assays. The culture medium was then supplemented with various concentrations of a nucleosides-nucleotides mixture (NS + NT), inosine, and uridine. Cytokines interleukin (IL)-1beta, IL-2, IL-3, tumor necrosis factor-alpha, and interferon (IFN)-gamma were measured from the supernatant by enzyme-linked immunosorbent assay. In the PHA-stimulated cultures the cellular proliferation in the BIO was significantly decreased as compared with the TC flask cells. BIO-cultured cells in the presence of NS + NT maintained mitogen responses similar to the control TC flask cells. The maintenance of the mitogen response in BIO was observed by the supplementation of uridine and not of inosine. These results are in agreement with our earlier results from unit gravity experiments that showed that pyrimidines are more effective in pleiogenic immunoprotection to hosts. Cytokines IL-1beta, IL-2, and IFN-gamma in the BIO supernatants of cells cultured in the presence of NS + NT had a significantly higher response than the control vessel. Thus, supplemental NT, especially pyrimidines, can confer immune protection and enhance cytokine responses during space travel.     

Protein pattern of Xenopus laevis embryos grown in simulated microgravity

Numerous studies indicate that microgravity affects cell growth and differentiation in many living organisms, and various processes are modified when cells are placed under conditions of weightlessness. However, until now, there is no coherent explanation for these observations, and little information is available concerning the biomolecules involved. Our aim has been to investigate the protein pattern of Xenopus laevis embryos exposed to simulated microgravity during the first 6 days of development. A proteomic approach was applied to compare the protein profiles of Xenopus embryos developed in simulated microgravity and in normal conditions. Attention was focused on embryos that do not present visible malformations in order to investigate if weightlessness has effects at protein level in the absence of macroscopic alterations. The data presented strongly suggest that some of the major components of the cytoskeleton vary in such conditions. Three major findings are described for the first time: (i) the expression of important factors involved in the organization and stabilization of the cytoskeleton, such as Arp (actin-related protein) 3 and stathmin, is heavily affected by microgravity; (ii) the amount of the two major cytoskeletal proteins, actin and tubulin, do not change in such conditions; however, (iii) an increase in the tyrosine nitration of these two proteins can be detected. The data suggest that, in the absence of morphological alterations, simulated microgravity affects the intracellular movement system of cells by altering cytoskeletal proteins heavily involved in the regulation of cytoskeleton remodelling.     

模拟微重力对肺动脉和胸主动脉的影响

目的 :通过对模拟微重力 (SM)、肺动脉 (PA)和胸主动脉 (TA)局部调节机理研究 ,为大小循环动脉对SM适应机理和SM后立位耐力降低机理研究积累资料。方法 :XXH 2 0 0 0型小循环心功能检测仪检测人体头低位 6°卧床(HDT) 7d心肺循环功能变化。 - 30°尾部悬吊 (TS)大鼠模拟微重力 (microgravity ,M)的生理效应 ,测量 7d、1 4dPA和TA的反应性。结果 :人体HDT初期每搏PA(hz)、静脉 (hc)容量和左心前负荷 (hc/hz)均显著增加 ,96～ 1 4 4h大小循环均出现超调现象 ,前者出现时间早、幅度大。 7d尾部悬吊大鼠 (TS7)与对照组 (CON)比PA舒张反应显著增强 ,TS1 4有降低趋势。TS7TA与CON比舒张反应显著增强 ,TS1 4轻度升高。TS7PA收缩反应与CON比轻度降低 ,TS1 4显著降低。TS1 4TA收缩反应显著降低。去VECPA对KCl、苯肾上腺素和硝普钠的反应在所有组间无差异。结论 :SM对大小循环动脉影响不同 ,可能是SM时局部调节功能降低的重要表现 ,主要由于动脉血管内皮细胞功能变化 ,对立位耐力降低可能有贡献     

Cultivation of fall armyworm ovary cells in simulated microgravity

Summary A methodology is presented to culture Fall Armyworm Ovary cells in simulated microgravity using a novel bioreactor developed by NASA, the High-Aspect Ratio Vessel. In this vessel, the growth and metabolic profile for these insect cells were profoundly different than those obtained in shaker-flask culture. Specifically, stationary phase in the NASA vessel was extended from 24 h to at least 7 d while cell concentration and viability remained in excess of 1 × 10⁷ viable cells/ml and 90%, respectively. Measurements of glucose utilization, lactate production, ammonia production, and pH change indicate that simulated microgravity had a twofold effect on cell metabolism. Fewer nutrients were consumed and fewer wastes were produced in stationary phase by as much as a factor of 4 over that achieved in shaker culture. Those nutrients that were consumed in the NASA vessel were directed along different metabolic pathways as evidenced by an extreme shift in glucose utilization from consumption to production in lag phase and a decrease in yield coefficients by one half in stationary phase. These changes reflect a reduction in hydrodynamic forces from over 1 dyne/cm² in shaker culture to under 0.5 dyne/cm² in the NASA vessel. These results suggest that cultivation of insect cells in simulated microgravity may reduce production costs of cell-derived biologicals by extending production time and reducing medium requirements.     

Regulation of hormone biosynthesis in cultured islet cells from anglerfish

Summary The effects of glucose and arginine on islet hormone biosynthesis were investigated using primary cell cultures prepared from islets of the anglerfish (Lophius americanus). After dispersion under sterile conditions, islet cells were maintained at 23° C in medium containing RPMI 1640 with Hanks' buffer, pH 7.5, modified by the adjustment of glucose (to 0.56 or 5.6 mM) and arginine (to 0.1, 1.15, or 10 mM) with the addition of 10% fetal bovine serum (dialyzed, heat inactivated) and penicillin/streptomycin. After 48 h, media were replaced by incorporation media containing [¹⁴C]isoleucine and [³H]tryptophan and incubated for an additional 8 h under otherwise identical conditions. Culture samples (cells plus media) were extracted, desalted, and gel filtered to identify and quantitate [¹⁴C]insulin, [³H]glucagon(s) plus [³H]somatostatin-28, and [³H]somatostatin-14 were In some experiments, [¹⁴C]insulin, [³H]glucagon(s), [³H]somatostatin-28, and [³H]somatostatin-14 were separated by high performance liquid chromatography. Raising the medium glucose from 0.56 (control) to 5.6 mM resulted in an augmentation in incorporation of [¹⁴C]isoleucine into insulin and an augmentation of [³H]tryptophan into glucagon(s) and somatostatin-14, but no change in incorporation of [³H]tryptophan into somatostatin-28. Raising the concentration of arginine from 0.1 to 1.15 or 10 mM resulted in a dose-dependent inhibition of labeled amino acid incorporation into all hormones except somatostatin-28. The results demonstrate the usefulness of the culture system for studying the modulation of hormone biosynthesis in anglerfish islet cells. This work was supported by Grants AM 16921 and AM 26378 from the National Institutes of Health, Bethesda, MD.     

Energy metabolism pathways in rat muscle under conditions of simulated microgravity

Evidence from rats flown in space suggests that there is a decrease in the ability of the soleus muscle to oxidize long chain fatty acids during space flight. The observation suggests that a shift in the pathways involved in muscle fuel utilization in the absence of load on the muscle has occurred. It is also possible that the reduction is part of a general down-sizing of metabolic capacity since energy needs of inactive muscle are necessarily less. The rodent hind limb suspension model has proved to be a useful ground based model for studying the musculo-skeletal systems changes that occur with space flight. Microarray technology permits the screening of a large number of the enzymes of the relevant pathways thereby permitting a distinction to be made between a shift fuel utilization pattern or a general decrease in metabolic activity. The soleus muscle was isolated from 5 control and 5 hindlimb suspended rats (21 days) and the Affymetrix system for assessing gene expression used to determine the impact of hindlimb unloading on fuel pathways within the muscle of each animal. RESULTS: Suspended rats failed to gain weight at the same rate as the controls (337 +/- 5 g vs 318 +/- 6 g, p < 0.05) and muscle mass from the soleus was reduced (135 +/- 3 mg vs 48 +/- 4 mg, p < 0.05). There was a consistent decrease (p < 0.05) in gene expression of proteins involved in fatty acid oxidation in the suspended group whereas glycolytic activity was increased (p < 0.05). Gene expressions of individual key regulatory enzymes reflected these changes. Carnitine palmitoyltransferase I and II were decreased (p < 0.05) whereas expression of hexokinase, phosphofructokinase and pyruvate kinase were increased (p < 0.05). CONCLUSION: Disuse atrophy is associated with a change in mRNA levels of enzymes involved in fuel metabolism indicative of a shift in substrate utilization away from fat towards glucose.     

茶多酚和艾司洛尔对高AngⅡ状态下血管内皮细胞内皮素分泌的影响

目的：探讨茶多酚和艾司洛尔能否对模拟失重所致的高AngⅡ状态的损害提供保护。方法：将培养的血管内皮细胞分为空白对照组、单纯血管紧张素Ⅱ组、血管紧张素Ⅱ＋茶多酚组、血管紧张素Ⅱ＋艾司洛尔组共4组,用放免法测定在不同时间内皮细胞分泌内皮素含量的变化。结果：AngⅡ可明显促进血管内皮细胞分泌内皮素,茶多酚和艾司洛尔能明显抑制高AngⅡ致血管内皮细胞分泌内皮素的作用（P〈0．01）,茶多酚的作用较艾司洛尔的更强（P〈0．01）。结论：茶多酚和艾司洛尔可减轻高AngⅡ状态的损害作用。     

模拟微重力对热应激时人血浆中单胺类递质含量的影响

目的和方法采用高效液相色谱-电化学检测法,分析7d-6°头低位卧床模拟微重力,40～60℃热应激及其复合因素作用下人外周血中单胺类递质的含量,研究模拟微重力对热应激时人外周血中单胺类递质含量的影响,以探讨航天热应激的人体反应机理.结果①模拟微重力单一因素暴露后,5-HT,NE,E含量与对照组相比均无显著变化(P＞0.05);②热应激单一因素暴露40℃,50℃,60℃热应激时,与对照组相比,5-HT含量均显著降低(P＜0.01),而NE,E含量,在40℃热应激时无显著变化(P＞0.05),50℃,60℃热应激时均显著增加(P＜0.001);③热应激与模拟微重力复合因素暴露时,与对照组相比,5-HT含量显著降低(P＜0.01),而NE,E含量均无显著变化(P＞0.05).与热应激单一因素暴露组相比,50℃,60℃热应激时,NE,E含量均显著降低(P＜0.001),5-HT的含量则无显著变化(P＞0.05).结论模拟微重力可使热诱导的NE,E含量显著降低,而对热应激引起的5-HT含量降低无显著影响.模拟微重力引起热应激时NE,E含量显著降低的变化,可能在航天热应激的人体反应机理中起重要的作用.