Biochemical Markers of Bone Tissue Metabolism in Cosmonauts after a Prolonged Spaceflight

Parameters of calcium homeostasis and its hormonal regulation, including biochemical markers of bone metabolism, were measured in the blood serum of Russian cosmonauts after prolonged flights on the International Space Station during the period from 2000 to 2003. The duration of the spaceflights was 129–196 days. Flight factors had an impact on calcium and bone tissue metabolism after a flight. Increased levels of osteogenesis and resorption markers were detected in the blood of the cosmonauts in the early rehabilitation period after a spaceflight. The prevalence of resorption over the formation of new bone tissue was observed in the early rehabilitation period, when the hormonal system maintaining calcium homeostasis was activated.     

Animal housing influences the response of bone to spaceflight in juvenile rats.

The rat has been used extensively as an animal model to study the effects of spaceflight on bone metabolism. The results of these studies have been inconsistent. On some missions, bone formation at the periosteal bone surface of weight-bearing bones is impaired and on others it is not, suggesting that experimental conditions may be an important determinant of bone responsiveness to spaceflight. To determine whether animal housing can affect the response of bone to spaceflight, we studied young growing (juvenile) rats group housed in the animal enclosure module and singly housed in the research animal holding facility under otherwise identical flight conditions (Spacelab Life Science 1). Spaceflight reduced periosteal bone formation by 30% (P < 0.001) and bone mass by 7% in single-housed animals but had little or no effect on formation (-6%) or mass (-3%) in group-housed animals. Group housing reduced the response of bone to spaceflight by as much as 80%. The data suggest that housing can dramatically affect the skeletal response of juvenile rats to spaceflight. These observations explain many of the discrepancies in previous flight studies and emphasize the need to study more closely the effects of housing (physical-social interaction) on the response of bone to the weightlessness of spaceflight.     

The effects of vitamin D deficiency on proteoglycan and hyaluronate constituents of chick bone

The effect of vitamin D deficiency on proteoglycan and hyaluronate constituents of cortical diaphyseal chick bone was studied. Proteoglycans in rachitic bone showed no significant change with respect to their size, composition, or amount relative to other extractable macromolecular components. In contrast, bone hyaluronate levels were raised in chicks fed on diets that were either vitamin D-deficient or depleted in calcium or phosphate, a 7-fold increase being seen in hypocalcaemic vitamin D-deficient chicks. This increase in hyaluronate was not directly related either to the absence of vitamin D or to abnormal levels of blood calcium or phosphate per se; hyaluronate levels are probably regulated by another factor, not yet identified, that is responsive to changes in vitamin D and mineral metabolism.     

Effects of spaceflight and PEG-IL-2 on rat physiological and immunological responses.

Sprague-Dawley rats were subjected to two 8-day spaceflights on the space shuttle. Rats housed in the National Aeronautics and Space Administration's animal enclosure were injected (iv or sc) with pegylated interleukin-2 (PEG-IL-2) or a placebo. We tested the hypothesis that PEG-IL-2 would ameliorate some of the effects of spaceflight. We measured body and organ weights; blood cell differentials; plasma corticosterone; colony-forming units (macrophage and granulocyte macrophage); lymphocyte mitogenic, superantigenic, and interferon-gamma responses; bone marrow cell and peritoneal macrophage cytokine secretion; and bone strength and mass. Few immunological parameters were affected by spaceflight. However, some spaceflight effects were observed in each flight. Specifically, peritoneal macrophage spontaneous secretion of tumor necrosis factor-alpha occurred in the first but not in the second flight. A significant monocytopenia and lymphocytopenia were detected in the second but not in the first flight. The second mission produced bone changes more consistent with past spaceflight investigations. PEG-IL-2 did not appear to be beneficial; however, this was mostly due to the lack of spaceflight effects. These studies reflect the difficulty in reproducing experimental models by using current space shuttle conditions.     

Bone surface structure in osteopetrotic grey lethal (gl/gl) and microphthalmic (mi/mi) mutant mice as revealed by scanning electron microscopy

The surface structure of bone from two genetically distinct osteopetrotic strains of mice, grey lethal (gl) and microphthalmic (mi) has been examined by scanning electron microscopy. Although both conditions produce a classical osteopetrolic phenotype the means by which this is achieved is quite different. gl mice appear to retain woven bone and show no evidence of resorption: mi mice show evidence of imperfect remodelling. These results are in accordance with what is already known of osteoclast structure and function in these mutations.     

Postmetamorphic Growth and Metabolism of Long-Spined Black Sea Urchin (Diadema antillarum) Reared in the Laboratory

Postmetamorphic growth and metabolism measurements were obtained on two cohorts of laboratory-reared Diadema antillarum. The cohorts grew linearly from less than 1 mm to over 43 mm. Daily growth averaged 0.097 and 0.11 mm d^-1, respectively, for the two cohorts, and was found to differ significantly. Urchin metabolism was examined by a series of simultaneous measurements of oxygen consumption and ammonium excretion over 16 days on starved juveniles ranging 16.5 to 18.3 mm. Metabolic activity under conditions of starvation was used as a test of the viability of urchins reared in the laboratory with cultured food resources. Catabolic activity differed from the first week of starvation compared to the second. Metabolic response included: (1) a 2.2-fold increase in oxygen consumption rate; (2) 50% decline in ammonium excretion rate; and (3) a 5.1-fold increase in oxygen to nitrogen ratio. These measurements are consistent with a shift from almost pure protein catabolism during the first seven days of starvation to a lipid : protein catabolic ratio of 1 : 1 after the first week. Growth and metabolism experiments of this type are seen as a first step towards optimizing laboratory culture techniques of this species.     

Postmetamorphic Growth and Metabolism of Long-Spined Black Sea Urchin (Diadema antillarum) Reared in the Laboratory

Postmetamorphic growth and metabolism measurements were obtained on two cohorts of laboratory-reared Diadema antillarum. The cohorts grew linearly from less than 1 mm to over 43 mm. Daily growth averaged 0.097 and 0.11 mm d^?1, respectively, for the two cohorts, and was found to differ significantly. Urchin metabolism was examined by a series of simultaneous measurements of oxygen consumption and ammonium excretion over 16 days on starved juveniles ranging 16.5 to 18.3 mm. Metabolic activity under conditions of starvation was used as a test of the viability of urchins reared in the laboratory with cultured food resources. Catabolic activity differed from the first week of starvation compared to the second. Metabolic response included: (1) a 2.2-fold increase in oxygen consumption rate; (2) 50% decline in ammonium excretion rate; and (3) a 5.1-fold increase in oxygen to nitrogen ratio. These measurements are consistent with a shift from almost pure protein catabolism during the first seven days of starvation to a lipid : protein catabolic ratio of 1 : 1 after the first week. Growth and metabolism experiments of this type are seen as a first step towards optimizing laboratory culture techniques of this species.     

The biophysical limitations in physiological transport and exchange in plants grown in microgravity

This paper describes how changes in the physical behavior of fluids and gases in microgravity can limit the physiological transport and exchange in higher plants. These types of effects are termed indirect effects of microgravity because they are not due to gravity interacting with the mass of the plant body itself. The impact of limiting gravity-dependent transport phenomena has been analyzed by the use of mathematical modeling to simulate and compare biophysical transport in the 1g and spaceflight environments. These data clearly show that the microgravity environment induces significant limitations on basic physiological and biochemical processes within the aerial and rootzone portions of the plant. Furthermore, this mathematical model provides a solid foundation for explaining the physiological effects that have been noted in past spaceflight experiments.     

Identification of a glyphosate-resistant mutant of rice 5-enolpyruvylshikimate 3-phosphate synthase using a directed evolution strategy

5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) is a key enzyme in the shikimate pathway and is targeted by the wide-spectrum herbicide glyphosate. Here, we describe the use of a selection system based on directed evolution to select glyphosate-resistant mutants of EPSPS. Using this system, the rice (Oryza sativa) EPSPS gene, mutagenized by Error-Prone polymerase chain reaction, was introduced into an EPSPS-deficient Escherichia coli strain, AB2829, and transformants were selected on minimal medium by functional complementation. Three mutants with high glyphosate resistance were identified in three independent glyphosate selection experiments. Each mutant contained a C(317)-->T transition within the EPSPS coding sequence, causing a change of proline-106 to leucine (P106L) in the protein sequence. Glyphosate resistance assays indicated a 3-fold increase in glyphosate resistance of E. coli expressing the P106L mutant. Affinity of the P106L mutant for glyphosate and phosphoenolpyruvate was decreased about 70-fold and 4.6-fold, respectively, compared to wild-type EPSPS. Analysis based on a kinetic model demonstrates that the P106L mutant has a high glyphosate resistance while retaining relatively high catalytic efficiency at low phosphoenolpyruvate concentrations. A mathematical model derived from the Michaelis-Menten equation was used to characterize the effect of expression level and selection conditions on kinetic (Ki and Km) variation of the mutants. This prediction suggests that the expression level is an important aspect of the selection system. Furthermore, glyphosate resistance of the P106L mutant was confirmed in transgenic tobacco (Nicotiana tabacum), demonstrating the potential for using the P106L mutant in transgenic crops.     

钙敏感受体在骨代谢中的研究进展

钙敏感受体感受细胞外的钙离子水平,调控一系列激素的释放以维持机体的钙稳态。钙稳态的调节过程与骨代谢相偶联,钙敏感受体通过直接或间接对破骨和成骨细胞的调控,动员或者抑制骨钙入血。虽然钙敏感受体已被证实调控骨代谢,但是详尽的调控机制仍在不断探究中。目前认为细胞外的高钙水平会激活钙敏感受体,抑制甲状旁腺激素分泌并促进降钙素释放,进而破骨细胞被抑制,成骨细胞动员,增加了骨质合成。本文就近年来关于钙敏感受体调控骨代谢的研究进展作一综述,为促进钙敏感受体及相关作用因子治疗骨代谢疾病的研究提供参考。     

Calcium metabolism and its regulation in man during adaptation to microgravitation

This work generalizes the results of studies of calcium metabolism in the participants of long-term space flights of 30 to 438 days on the Salyut and Mir orbital stations during 1978–1998. The results of pre- and postflight examination of 44 cosmonauts (18 subjects participated twice in long-term space flights) were analyzed. After space flights of medium (of 3 to 6 months) and long (of 6 to 14 months) duration, the total blood calcium content was increased, mainly due to its ionized fraction; the blood level of parathyroid hormone was significantly increased and the level of calcitonin was decreased. The content of osteocalcin was increased after space flights. Calcium kinetics was studied using stable isotopes in three cosmonauts before, during, and after the 115-day flight. During the flight, intestinal absorption of calcium and its gastrointestinal excretion were decreased, whereas its renal excretion was increased. Early postflight intestinal absorption was, on average, lower than during the flight, whereas intestinal excretion increased. Both renal and intestinal excretion of calcium were not normalized 3.5 to 4.5 months after the glight. The mathematical models used for evaluating the rates of main calcium flows revealed increased bone tissue resorption that resulted in the negative bone balance during the flight. The conclusion about the decreased rate of bone tissue remodeling and its increased resorption was confirmed by biochemical data, including endocrine markers.     

The calcium requirement and factors causing calcium loss

Studies carried out under strictly controlled conditions during different calcium intakes in adult males have shown that the average calcium balance was only slightly positive (+22 mg/day) during a calcium intake of 800 mg/day, the recommended dietary calcium intake, not taking into consideration dermal losses of calcium. During this calcium intake, the calcium balances were negative in 34% of the subjects studied. Increasing the calcium intake to 1200 mg/day resulted in a significant increase of the calcium balance; further increases to different intake levels up to 2300 mg/day did not improve the calcium balance further. Increasing the phosphorus intake up to 2000 mg/day as well as increasing the protein intake from 1 g/kg body weight to 2 g/kg, given as meat, did not have an adverse effect on calcium metabolism. A variety of drugs, notably aluminum-containing antacids, induced calcium loss. Increasing the calcium intake more than 10-fold from 200 to 2500 mg/day did not lower the blood pressure in a large number of normotensive patients and in a small number of hypertensive patients studied.     

Calcium phosphate transfection optimization for serum-free suspension culture

Aim of this study was to identify optimal conditions for suspension transfection in the absence of serum. Transfection parameters for suspension culture can be very different to ones in adherent cells. Most transfection protocols have been developed and optimizedfor adherent culture. Using green fluorescent protein (GFP) as reporter, FCS was eliminated from the transfection process by altering critical parameters and by substituting serum with albumin. Using standard phosphate and calcium concentrations for transfection in the absence of serum resulted in titers of only 1% of those observed in the presence of serum. A reduction of the calcium concentration from 250 mM to 100 mM, yielded a 25-fold increase in the expression of the recombinant protein compared to the serum-free standard conditions. Altering the phosphate concentration, 1.4 mM in the transfection buffer, did not improve the protein expression. Interestingly, reduction of DNA quantity by half to a concentration of 0.5 μg per milliliter of culture volume resulted in a two-fold increase of protein production. Addition of albumin to serum-free medium protected the cells against the toxicity of the calcium phosphate transfection particles (CaPi) yielding higher protein expression. All the experiments were executed in a shaken multi-well system, allowing high multiplicity parameter screening to speed up optimizations. The culture system is inexpensive, simple and efficient, minimizing costs for labor and consumables. This revised version was published online in July 2006 with corrections to the Cover Date.     

Lack of effect of spaceflight on bone mass and bone formation in group-housed rats

As part ofan experiment to study the role of corticosteroids in bone changesduring spaceflight, male Sprague-Dawley rats (6 wk old, 165 g bodyweight) were placed in orbit for 17 days, in groups of six, inanimal-enclosure modules (AEMs) aboard the space shuttle Columbia(STS-78). Control rats were group housed in a similar manner inground-based AEMs or standard vivarium cages. Adrenal hypertrophyoccurred in flight rats, but bone histomorphometric analyses revealed alack of significant changes in bone mass and bone formation in theseanimals. Cancellous bone volume and osteoblast surface in the proximaltibial metaphysis were nearly the same in flight and ground-based rats.Normal levels of cancellous bone mass and bone formation were alsodetected in the lumbar vertebrae and femoral necks of flight rats. Inthe tibial diaphysis, periosteal bone formation rate was found to beidentical in flight and ground-based rats. The results indicate that,under conditions of group housing in AEMs, spaceflight has minimaleffects on bone mass and bone formation in rapidly growingrats. These findings emphasize the need to investigate theimportance of rat age, strain, and especially housing conditions forstudies of the skeletal effects of spaceflight.

     

Alterations in extracellular calcium concentration differentially influence prostaglandin and thromboxane synthesis in epithelial cells from the toad urinary bladder

The effects of alterations in extracellular calcium concentration on prostaglandin (PGE) and thromboxane (TXB₂) syntheses were studied in isolated epithelial cells from the urinary bladder of the toad, Bufo marinus. In epithelial cells prepared using collagenase, basal iPGE synthesis was greater than iTXB₂ synthesis. Increasing extracellular calcium from zero to 1 mm increased iPGE synthesis and decreased iTXB₂ synthesis equivalently such that total conversion of endogenous arachidonate to these two metabolites was unaltered. Vasopressin stimulated iPGE and iTXB₂ syntheses when the incubation buffer contained 1 mm calcium but had no effect in the presence of 0.4 μm calcium. In contrast, using an EDTA isolation method, basal iPGE and iTXB₂ syntheses were equal in the presence of zero calcium. Increasing extracellular calcium concentration to 1 mm caused a greater enhancement in iTXB₂ synthesis compared to iPGE. Increasing extracellular calcium to 2 mm was associated with a decline in iPGE and iTXB₂ syntheses back to the levels observed with no calcium added to the medium. The effect of increasing the calcium concentration was greater in phosphate than in bicarbonate buffer. In a Tris buffer the effect of altered calcium was almost completely abrogated. These studies demonstrate that the choice of buffer and alterations in extracellular calcium concentration differentially alter basal arachidonic acid metabolism to prostaglandins and thromboxane in isolated toad urinary bladder cells. The results suggest that there may exist several endogenous pools of arachidonic acid which are differentially influenced by calcium. Furthermore, the pool sensitive to vasopressin has an absolute requirement for calcium.     

Calcium metabolism, serum thyroid-stimulating hormone, prolactin, and growth hormone in spontaneously hypercholesterolemic rats

We have shown previously that spontaneously hypercholesterolemic (SHC) rats exhibit abnormal bone metabolism with advanced bone resorption, which develops with age. In this study, we measured serum levels of growth hormone, thyroid-stimulating hormone, and prolactin in addition to several parameters of calcium metabolism and renal function in young (6-week) and old (24-week) SHC rats and compared these with age-matched Sprague-Dawley rats. In young SHC rats, urinary excretion of hydroxyproline and serum levels of calcium were significantly elevated and excretion of protein into urine and urea nitrogen in the serum were normal, suggesting that calcium metabolism was abnormal without kidney dysfunction at this age. Serum growth hormone and thyroid-stimulating hormone levels were markedly higher (20- to 30-fold and 4- to 5-fold, respectively) in young and old SHC rats, whereas serum prolactin levels were similar. A high level of serum thyroid-stimulating hormone was associated with elevated levels of thyroxine and triiodothyronine in young SHC rats, but not old ones. These results demonstrate that the rat exhibits abnormalities in endocrine function as well as calcium metabolism preceding the occurrence of renal dysfunction.     

Fluid flow induced PGE2 release by bone cells is reduced by glycocalyx degradation whereas calcium signals are not

It has been hypothesized that bone cells have a hyaluronic acid (HA) rich glycocalyx (cell coat or pericellular matrix) and that this contributes to bone cell mechanotransduction via fluid flow. The glycocalyx of bone cells of the MC3T3-E1 osteoblastic cell line and the MLO-Y4 osteocytic cell line were characterized. Alcian blue staining and lectin binding experiments suggested that these cells have a glycocalyx rich in HA. Sulphated proteoglycans were not detected. Staining with hyaluronic acid binding protein and degradation by hyaluronidase confirmed that HA was a major component of the glycocalyx. We subjected cells, with and without hyaluronidase treatment, to oscillating fluid flow under standardized in vitro conditions. There was no effect of glycocalyx degradation on the intracellular calcium signal, in either cell type, in terms of the percentage of cells responding (40-80%) or the magnitude of the response (2-5 times baseline). However, a 4-fold fluid flow induced increase in PGE2 was eliminated by hyaluronidase pre-treatment in MLO-Y4 cells. We conclude that under these conditions the calcium and PGE2 responses occur via different pathways. An intact glycocalyx is not necessary in order to initiate a calcium signal in response to oscillating fluid flow. However, in osteocyte-like cells the PGE2 pathway is more dependent on mechanical signals transmitted through the glycocalyx.     

Physiological Factors Responsible for the Development of Osteopenia under Conditions of Mechanical Unloading

A summary of results of investigations by the author and a brief review of some literature data on human bone tissue deprived of mechanical loading (spaceflight, hypokinesia) is given. The direction and markedness of changes in bone mass—the bone mineral density and the bone mineral content—in different skeletal segments depend on their position relative to the gravity vector. A theoretically expected bone mass reduction was revealed in the trabecular structures of the bones of the lower part of the skeleton (local osteopenia). In the upper part of the skeleton, an increase in the bone mineral content is observed, which is considered as a secondary response and is due to redistribution of body fluids cephalad. The main cause of osteopenia is mechanical unloading. Arguments are presented that osteocyte osteolysis, delayed osteoblast histogenesis, and osteoclast resorption provoked by rearrangement in the hierarchy of the systems of volume regulation, ion regulation, and the endocrine regulation of calcium homeostasis are the main mechanisms of osteopenia.     

Modulation of neuronal excitability by intracellular calcium buffering: from spiking to bursting

We have investigated the detailed regulation of neuronal firing pattern by the cytosolic calcium buffering capacity using a combination of mathematical modeling and patch-clamp recording in acute slice. Theoretical results show that a high calcium buffer concentration alters the characteristic regular firing of cerebellar granule cells and that a transition to various modes of oscillations occurs, including bursting. Using bifurcation analysis, we show that this transition from spiking to bursting is a consequence of the major slowdown of calcium dynamics. Patch-clamp recordings on cerebellar granule cells loaded with a high concentration of the fast calcium buffer BAPTA (15 mM) reveal dramatic alterations in their excitability as compared to cells loaded with 0.15 mM BAPTA. In high calcium buffering conditions, granule cells exhibit all bursting behaviors predicted by the model whereas bursting is never observed in low buffering. These results suggest that cytosolic calcium buffering capacity can tightly modulate neuronal firing patterns leading to generation of complex patterns and therefore that calcium-binding proteins may play a critical role in the non-synaptic plasticity and information processing in the central nervous system.