The diageotropica mutant of tomato lacks high specific activity auxin binding sites.

Tomato plants homozygous for the diageotropica (dgt) mutation exhibit morphological and physiological abnormalities which suggest that they are unable to respond to the plant growth hormone auxin (indole-3-acetic acid). The photoaffinity auxin analog [3H]5N3-IAA specifically labels a polypeptide doublet of 40 and 42 kilodaltons in membrane preparations from stems of the parental variety, VFN8, but not from stems of plants containing the dgt mutation. In roots of the mutant plants, however, labeling is indistinguishable from that in VFN8. These data suggest that the two polypeptides are part of a physiologically important auxin receptor system, which is altered in a tissue-specific manner in the mutant.

Skeletal muscle satellite cells.

Evidence now suggests that satellite cells constitute a class of myogenic cells that differ distinctly from other embryonic myoblasts. Satellite cells arise from somites and first appear as a distinct myoblast type well before birth. Satellite cells from different muscles cannot be functionally distinguished from one another and are able to provide nuclei to all fibers without regard to phenotype. Thus, it is difficult to ascribe any significant function to establishing or stabilizing fiber type, even during regeneration. Within a muscle, satellite cells exhibit marked heterogeneity with respect to their proliferative behavior. The satellite cell population on a fiber can be partitioned into those that function as stem cells and those which are readily available for fusion. Recent studies have shown that the cells are not simply spindle shaped, but are very diverse in their morphology and have multiple branches emanating from the poles of the cells. This finding is consistent with other studies indicating that the cells have the capacity for extensive migration within, and perhaps between, muscles. Complexity of cell shape usually reflects increased cytoplasmic volume and organelles including a well developed Golgi, and is usually associated with growing postnatal muscle or muscles undergoing some form of induced adaptive change or repair. The appearance of activated satellite cells suggests some function of the cells in the adaptive process through elaboration and secretion of a product. Significant advances have been made in determining the potential secretion products that satellite cells make. The manner in which satellite cell proliferative and fusion behavior is controlled has also been studied. There seems to be little doubt that cellcell coupling is not how satellite cells and myofibers communicate. Rather satellite cell regulation is through a number of potential growth factors that arise from a number of sources. Critical to the understanding of this form of control is to determine which of the many growth factors that can alter satellite cell behavior in vitro are at work in vivo. Little work has been done to determine what controls are at work after a regeneration response has been initiated. It seems likely that, after injury, growth factors are liberated through proteolytic activity and initiate an activation process whereby cells enter into a proliferative phase. After myofibers are formed, it also seems likely that satellite cell behavior is regulated through diffusible factors arising from the fibers rather than continuous control by circulating factors.(ABSTRACT TRUNCATED AT 400 WORDS)

The role of psychoneuroendocrine factors on spaceflight-induced immunological alterations.

This paper summarizes previous in-flight infections and novel conditions of spaceflight that may suppress immune function. Granulocytosis, monocytosis, and lymphopenia are routinely observed following short duration orbital flights. Subtle changes within the monocyte and T cell populations can also be noted by flow cytometric analysis. The similarity between the immunological changes observed after spaceflight and other diverse environmental stressors suggest that most of these alterations may be neuroendocrine-mediated. Available data support the hypothesis that spaceflight and other environmental stressors modulate normal immune regulation via stress hormones, other than exclusively glucocorticoids. It will be essential to simultaneously collect in-flight endocrine, immunologic, and infectious illness data to determine the clinical significance of these results. Additional research that delineates the neuroendocrine mechanisms of stress-induced changes in normal immune regulation will allow clinicians in the future to initiate prophylactic immunomodulator therapy to restore immune competence altered by the stress of long-duration spaceflight and therefore reduce morbidity from infectious illness, autoimmune disease, or malignancy.

Post-translational control of collagen fibrillogenesis in mineralizing cultures of chick osteoblasts.

Cultured osteoblasts from chick embryo calvaria were used as a model system to investigate the post-translational extracellular mechanisms controlling the macroassembly of collagen fibrils. The results of these studies demonstrated that cultured osteoblasts secreted a collagenous extracellular matrix that assembled and mineralized in a defined temporal and spatial sequence. The assembly of collagen occurred in a polarized fashion, such that successive orthogonal arrays of fibrils formed between successive cell layers proceeding from the culture surface toward the media. Mineralization followed in the same manner, being observed first in the deepest and oldest fibril layers. Collagen fibrillogenesis, the kinetics of cross-link formation, and collagen stability in the extracellular matrix of the cultures were examined over a 30 day culture period. Between days 8 and 12 in culture, collagen fibril diameters increased from < 30 nm to an average of 30-45 nm. Thereafter, diameters ranged in size from 20 to 200 nm. Quantitation of the collagen cross-linking residues, hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP), showed that these mature cross-links increased from undetectable levels to concentrations found in normal chick bone. Analysis of the kinetics of their formation by pulse-chase labeling the cultures with [3H]lysine showed a doubling time of approximately 5 days. The relationships between cross-link formation, fibrillogenesis, and collagen stability were examined in cultures treated with beta-aminopropionitrile (beta-APN), a potent inhibitor of lysyl oxidase and cross-link formation. In beta-APN-treated cultures, total collagen synthesis was increased twofold, with no change in mRNA levels for type I collagen, whereas the amount of collagen accumulated in the cell layer was decreased by 50% and mineral deposition was reduced. The rate of collagen retention in the matrix was assessed by pulse-chase analysis of [3H]proline over a 16 day period in control and beta-APN-treated cultures. In control cultures, about 20% of the labeled collagen was lost from the cell layers over a 16 day period compared with > 80% in the presence of beta-APN. The beta-APN-treated cultures also showed a wider diversity of fibril diameters with a median in the > 45-60 nm range. In summary, these data suggest that cross-linking and assembly of collagen fibrils secreted by osteoblasts in vitro occur in a fashion similar to that found in vivo. The rate of cross-link formation is relatively constant and may be correlated with increasing collagen mass.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytoplasmic pH influences cytoplasmic calcium in MC3T3-E1 osteoblast cells.

We found that the cytoplasmic concentration of calcium (Cai) of MC3T3-E1 osteoblasts was influenced by the type of pH buffer we used in the perfusing medium, suggesting that intracellular pH (pHi) might influence Cai. To study this effect, the Cai and pHi were monitored as we applied various experimental conditions known to change pHi. Exposure to NH4Cl caused a transient increase in both pHi and Cai without a change in extracellular pH (pHo). Decreasing pHo and pHi by lowering the bicarbonate concentration of the medium decreased Cai, and increasing pHi by the removal of 5% CO2 increased Cai. Clamping pHi to known values with 10 microM nigericin, a potassium proton ionophore, also influenced Cai: acid pHi lowered Cai, whereas alkaline pHi increased it. The rise in Cai appears to be very sensitive to the extracellular concentration of calcium, suggesting the existence of a pH-sensitive calcium influx mechanism. We conclude that physiologic changes in pH could modulate Cai by controlling the influx of calcium ions and could change the time course of the Cai transient associated with hormonal activation.

Skeletal unloading induces selective resistance to the anabolic actions of growth hormone on bone.

Loss of skeletal weight bearing or physical unloading of bone in the growing animal inhibits bone formation and induces a bone mineral deficit. To determine whether the inhibition of bone formation induced by skeletal unloading in the growing animal is a consequence of diminished sensitivity to growth hormone (GH) we studied the effects of skeletal unloading in young hypophysectomized rats treated with GH (0, 50, 500 micrograms/100 g body weight/day). Skeletal unloading reduced serum osteocalcin, impaired uptake of 3H-proline into bone, decreased proximal tibial mass, and diminished periosteal bone formation at the tibiofibular junction. When compared with animals receiving excipient alone, GH administration increased bone mass in all animals. The responses in serum osteocalcin, uptake of 3H-proline and 45Ca into the proximal tibia, and proximal tibial mass in non-weight bearing animals were equal to those in weight bearing animals. The responses in trabecular bone volume in the proximal tibia and bone formation at the tibiofibular junction to GH, however, were reduced significantly by skeletal unloading. Bone unloading prevented completely the increase in metaphyseal trabecular bone normally induced by GH and severely dampened the stimulatory effect (158% vs. 313%, p < 0.002) of GH on periosteal bone formation. These results suggest that while GH can stimulate the overall accumulation of bone mineral in both weight bearing and non-weight bearing animals, skeletal unloading selectively impairs the response of trabecular bone and periosteal bone formation to the anabolic actions of GH.

Reproduction and the renin-angiotensin system.

A unique aspect of the circulating renin-angiotensin system and the many independent tissue renin-angiotensin systems is their interactions at multiple levels with reproduction. These interactions, which have received relatively little attention, include effects of estrogens and possibly androgens on hepatic and renal angiotensinogen mRNA; effects of androgens on the Ren-2 gene and salivary renin in mice; the prorenin surge that occurs with but outlasts the LH surge during the menstrual cycle; the inhibitory effects of estrogens on thirst and water intake; the tissue renin-angiotensin systems in the brain, the anterior pituitary, and the ovaries and testes, that is, in all the components of the hypothalamo-pituitary-gonadal axis; the presence of some components of the renin-angiotensin system in the uterus and the fetoplacental unit; and the possible relation of renin and angiotensin to ovulation and fetal well-being. These interactions are described and their significance considered in this short review.

Human osteoblast-like cells respond to mechanical strain with increased bone matrix protein production independent of hormonal regulation.

Exposure of osteosarcoma cell lines to chronic intermittent strain increases the activity of mechano-sensitive cation (SA-cat) channels. The impact of mechano-transduction on osteoblast function has not been well studied. We analyzed the expression and production of bone matrix proteins in human osteoblast-like osteosarcoma cells, OHS-4, in response to chronic intermittent mechanical strain. The OHS-4 cells exhibit type I collagen production, 1,25-Dihydroxyvitamin D-inducible osteocalcin, and mineralization of the extracellular matrix. The matrix protein message level was determined from total RNA isolated from cells exposed to 1-4 days of chronic intermittent strain. Northern analysis for type I collagen indicated that strain increased collagen message after 48 h. Immunofluorescent labeling of type I collagen demonstrated that secretion was also enhanced with mechanical strain. Osteopontin message levels were increased several-fold by the application of mechanical load in the absence of vitamin D, and the two stimuli together produced an additive effect. Osteocalcin secretion was also increased with cyclic strain. Osteocalcin levels were not detectable in vitamin D-untreated control cells. However, after 4 days of induced load, significant levels of osteocalcin were observed in the medium. With vitamin D present, osteocalcin levels were 4 times higher in the medium of strained cells compared to nonstrained controls. We conclude that mechanical strain of osteoblast-like cells is sufficient to increase the transcription and secretion of matrix proteins via mechano-transduction without hormonal induction.

Elevated 1,25-dihydroxyvitamin D levels in patients with chronic obstructive pulmonary disease treated with prednisone.

Glucocorticoid administration is a well established cause of osteopenia. Mechanisms underlying the deleterious effect of glucocorticoids on bone may include direct inhibition of bone formation as well as indirect effects through changes in intestinal calcium absorption, renal calcium excretion, and/or levels of the calciotropic hormones. To further examine the potential role of the calciotropic hormones we measured serum levels of PTH and 1,25 dihydroxyvitamin D [1,25(OH)2D], as well as serum and urine levels of calcium and vertebral bone density in patients with chronic obstructive pulmonary disease being managed with or without prednisone. Patients treated with prednisone had lower spinal bone density (53 vs. 106 mg/cm3) and higher serum calcium (2.40 vs. 2.33 mmol/l), urine calcium (6.9 vs. 2.7 mmol/24h), and 1,25(OH)2D levels (147 vs. 95 pmol/L). Compared to the patients not treated with glucocorticoids. PTH levels also tended to be higher (33 vs. 26 microliters-eq/ml), but the difference was not significant. Serum and urine calcium levels correlated positively with 1,25(OH)2D levels, but none of these measurements correlated with PTH levels. Our results suggest that prednisone treatment alters the regulation of 1,25(OH)2D production, and this may contribute to the loss of bone mineral induced by prednisone.

Altered cell function in microgravity.

Physiological changes in humans during spaceflight upon return to earth have been attributed to systemic adaptation, response to stress, and lack of normal exercise. Studies from the Skylab, SL-3, and D-1 missions have demonstrated that significant physiological alterations are seen in single cell prokaryotes and eukaryotes, as well as in animal tissues. Basic cellular functions such as electrolyte concentration, cell growth rate, glucose utilization, bone formation, response to growth stimulation, and exocytosis are modified in microgravity. Many of the physiological changes seen in humans, vertebrate and simple organisms in spaceflight may originate from dysfunction of basic biological mechanisms caused by microgravity. Aging humans share many of the symptoms seen in astronauts during spaceflight. These include reduced cardiac function, loss of bone and reduced immune response and orthostatic hypotension. It is possible that some of physiological adaptations seen in aging may share common physiological basis with those changes seen in spaceflight. Since microgravity affects prokaryotic and eukaryotic cell function at a subcellular and molecular level, space offers us an opportunity to learn more about basic biological mechanisms which are essential to life.

The habits of roots: what's up down under?

NASA: Defining interactions of roots with the surrounding soil environment has been the focus of many recent investigations. As a result of these efforts, we are gaining an appreciation of the varied and often surprising strategies whereby roots adjust to and condition their soil environment for optimal growth and development. This article summarizes current knowledge of the often complex interactions between roots and biotic and abiotic factors within the soil. These interactions are interpreted in terms of modifications in the development or the physiology of the root.^ieng

The August Krogh principle applies to plants.

NASA: The Krogh principle refers to the use of a large number of animals to study the large number of physiological problems, rather than limiting study to a particular organism for all problems. There may be organisms that are more suited to study of a particular problem than others. This same principle applies to plants. The authors are concerned with the recent trend in plant biology of using Arabidopsis thaliana as the "organism of choice." Arabidopsis is an excellent organism for molecular genetic research, but other plants are superior models for other research areas of plant biology. The authors present examples of the successful use of the Krogh principle in plant cell biology research, emphasizing the particular characteristics of the selected research organisms that make them the appropriate choice.^ieng

Evidence of a sensory processing unit in the mammalian macula.

We cut serial sections through the medial part of the rat vestibular macula for transmission electron microscopic (TEM) examination, computer-assisted 3-D reconstruction, and compartmental modeling. The ultrastructural research showed that many primary vestibular neurons have an unmyelinated segment, often branched, that extends between the heminode (putative site of the spike initiation zone) and the expanded terminal(s) (calyx, calyces). These segments, termed the neuron branches, and the calyces frequently have spine-like processes of various dimensions with bouton endings that morphologically are afferent, efferent, or reciprocal to other macular neural elements. The major questions posed by this study were whether small details of morphology, such as the size and location of neuronal processes or synapses, could influence the output of a vestibular afferent, and whether a knowledge of morphological details could guide the selection of values for simulation parameters. The conclusions from our simulations are (1) values of 5.0 k omega cm2 for membrane resistivity and 1.0 nS for synaptic conductance yield simulations that best match published physiological results; (2) process morphology has little effect on orthodromic spread of depolarization from the head (bouton) to the spike initiation zone (SIZ); (3) process morphology has no effect on antidromic spread of depolarization to the process head; (4) synapses do not sum linearly; (5) synapses are electrically close to the SIZ; and (6) all whole-cell simulations should be run with an active SIZ.

High resolution and image processing of otoconia matrix.

This study was designed to investigate patterns of fibrils organization in histochemically stained otoconia. Transmission electron microscope and video imaging were used. These data indicate that otoconia of the chick (Gallus domesticus) inner ear may have central cores in vivo. The data also show that the ultrastructural organization of fibrils fixed with aldehydes and histochemical stains follows trajectories that conform to the hexagonal shape of otoconia. These changes in direction may contribute to the formation of a central core. The existence of central cores is important for the in vivo buoyancy of otoconia. Packing of fibrils is tighter after phosphotungstic acid (PTA) stained otoconia than with other histochemical stains, which usually produce looser packing of fibrils and seemingly larger central core. TEM of tilted and untilted material showed that turning of fibrils occurs at the points where the face angles of otoconia form and where central cores exist. Video image processing of the images allowed reconstructing a template which, if assumed to repeat and change trajectories, would fit the pattern of fibrils seen in fixed otoconia. Since it is highly unlikely that aldehyde primary fixation or PTA stain caused such drastic change in the direction of fibrils, the template derived from these results may closely approximate patterns of otoconia fibrils packing in vivo. However, if the above is correct, the perfect crystallographic diffraction pattern of unfixed otoconia do not correspond to patterns of fixed fibrils.

Structural relations between collagen and mineral in bone as determined by high voltage electron microscopic tomography.

Aspects of the ultrastructural interaction between collagen and mineral crystals in embryonic chick bone have been examined by the novel technique of high voltage electron microscopic tomography to obtain three-dimensional information concerning extracellular calcification in this tissue. Newly mineralizing osteoid along periosteal surfaces of mid-diaphyseal regions from normal chick tibiae was embedded, cut into 0.25 microns thick sections, and documented at 1.0 MV in the Albany AEI-EM7 high voltage electron microscope. The areas of the tissue studied contained electron dense mineral crystals associated with collagen fibrils, some marked by crystals disposed along their cylindrically shaped lengths. Tomographic reconstructions of one site with two mineralizing fibrils were computed from a 5 degrees tilt series of micrographs over a +/- 60 degrees range. Reconstructions showed that the mineral crystals were platelets of irregular shape. Their sizes were variable, measured here up to 80 x 30 x 8 nm in length, width, and thickness, respectively. The longest crystal dimension, corresponding to the c-axis crystallographically, was generally parallel to the collagen fibril long axis. Individual crystals were oriented parallel to one another in each fibril examined. They were also parallel in the neighboring but apparently spatially separate fibrils. Crystals were periodically (approximately 67 nm repeat distance) arranged along the fibrils and their location appeared to correspond to collagen hole and overlap zones defined by geometrical imaging techniques. The crystals appeared to be continuously distributed along a fibril, their size and number increasing in a tapered fashion from a relatively narrow tip containing smaller and infrequent crystals to wider regions having more densely packed and larger crystals. Defined for the first time by direct visual 3D imaging, these data describe the size, shape, location, orientation, and development of early crystals in normal bone collagen. The results suggest that platelet-shaped crystals are arranged in channels or grooves which are formed by collagen hole zones in register and that crystal sizes may exceed the dimensions of hole zones. Such data agree with those from mineral-matrix interaction in normally calcifying avian tendon obtained by similar high voltage tomographic means, but in addition they indicate a possible gradual and continuous deposition of crystals in collagen of bone unlike tendon and imply that individual collagen fibrils in local regions of osteoid are organized such that they all may be aligned in a coherent manner.

Co-transplantation of plasmid-transfected myoblasts and myotubes into rat brains enables high levels of gene expression long-term.

We have previously proposed the use of primary muscle cells as a "platform," or "vehicle" for intracerebral transgene expression. Brain grafts of minced muscle, or cultured muscle cells persisted in rat brains for at least 6 mo without any decrease in graft size, or tumor formation. Stable, but moderate levels of intracerebral transgene expression were obtained by transplanting plasmid-transfected myotubes in culture. In the present study, high and stable levels of intracerebral transgene expression were achieved by the co-transplantation of plasmid-transfected myoblasts and myotubes in culture. Approximately 5 X 10(5) myoblasts and myotubes were transfected with 10 micrograms pRSVL plasmid DNA, and 30 micrograms Lipofectin (BRL), respectively. They were mixed together (total cell number was 1 million), and stereotactically injected into the caudate nucleus of an adult rat brain. The activity of luciferase, the product of transgene expression, was stable for at least 4 mo, and much higher than the levels in myotube grafts, or co-grafts of myoblasts and minced muscle. Presumably, the myotubes served as a framework on which the myoblasts can form myotubes. The sections of brains transplanted with co-graft of myoblasts, and myotubes transfected with pRSVLac-Z were stained immunofluorescently for beta-galactosidase activity. The muscle grafts contained beta-galactosidase positive myofibers 4 mo after transplantation. Such high and stable levels of in vivo expression after postnatal gene transfer have rarely been achieved. Primary muscle cells are useful vehicle for transgene expression in brains, and potentially valuable for gene therapy of degenerative neurological disorders.

Peak force and maximal shortening velocity of soleus fibers after non-weight-bearing and resistance exercise.

This study examined the effectiveness of resistance exercise as a countermeasure to non-weight-bearing-induced alterations in the absolute peak force, normalized peak force (force/fiber cross-sectional area), peak stiffness, and maximal shortening velocity (Vo) of single permeabilized type I soleus muscle fibers. Adult rats were subjected to the following treatments: normal weight bearing (WB), non-weight bearing (NWB), or NWB with exercise treatments (NWB+Ex). The hindlimbs of the NWB and NWB+Ex rats were suspended for 14 days via tail harnesses. Four times each day, the NWB+Ex rats were removed from suspension and performed 10 climbs (approximately 15 cm each) up a steep grid with a 500-g mass (approximately 1.5 times body mass) attached to their tail harness. NWB was associated with significant reductions in type I fiber diameter, absolute force, normalized force, and stiffness. Exercise treatments during NWB attenuated the decline in fiber diameter and absolute force by almost 60% while maintaining normalized force and stiffness at WB levels. Type I fiber Vo increased by 33% with NWB and remained at this elevated level despite the exercise treatments. We conclude that in comparison to intermittent weight bearing only (J.J. Widrick, J.J. Bangart, M. Karhanek, and R.H. Fitts. J. Appl. Physiol. 80: 981-987, 1996), resistance exercise was more effective in attenuating alterations in type I soleus fiber absolute force, normalized force, and stiffness but was less effective in restoring type I fiber Vo to WB levels.

Subnormal norepinephrine release relates to presyncope in astronauts after spaceflight.

Postflight orthostatic intolerance is experienced by virtually all astronauts but differs greatly in degree of severity. We studied cardiovascular responses to upright posture in 40 astronauts before and after spaceflights lasting up to 16 days. We separated individuals according to their ability to remain standing without assistance for 10 min on landing day. Astronauts who could not remain standing on landing day had significantly smaller increases in plasma norepinephrine levels with standing than did those who could remain standing (105 +/- 41 vs. 340 +/- 62 pg/ml; P = 0.05). In addition, they had significantly lower standing peripheral vascular resistance (23 +/- 3 vs. 34 +/- 3 mmHg.1l-1).min; P = 0.02) and greater decreases in systolic (-28 +/- 4 vs. -11 +/- 3 mmHg; P = 0.002) and diastolic (-14 +/- 7 vs. 3 +/- 2 mmHg; P = 0.0003) pressures. The presyncopal group also had significantly lower supine (16 +/- 1 vs. 21 +/- 2 mmHg.1l-1).min; P = 0.04) and standing (23 +/- 2 vs. 32 +/- 2 mmHg.1l-1).min; P = 0.038) vascular resistance, supine (66 +/- 2 vs. 73 +/- 2 mmHg; P = 0.008) and standing (69 +/- 4 vs. 77 +/- 2 mmHg; P = 0.007) diastolic pressure, and supine (109 +/- 3 vs. 114 +/- 2 mmHg; P = 0.05) and standing (99 +/- 4 vs. 108 +/- 3 mmHg; P = 0.006) systolic pressures before flight. This is the first study to clearly document these differences among presyncopal and nonpresyncopal astronauts after spaceflight and also offer the possibility of preflight prediction of postflight susceptibility. These results clearly point to hypoadrenergic responsiveness, possibly centrally mediated, as a contributing factor in postflight orthostatic intolerance. They may provide insights into autonomic dysfunction in Earthbound patients.

Functional support of glutamate as a vestibular hair cell transmitter in an amniote.

Although hair cells in the cochlea and in the vestibular endorgans of anamniotes are thought to release glutamate or a similar compound as their transmitter, there is little evidence in amniotes (which, unlike anamniotes, possess both type I and II hair cells) as to the nature of the hair cell transmitters in the vestibular labyrinth. We have recorded extracellularly from single semicircular canal afferents in the turtle labyrinth maintained in vitro and have bath-applied a number of transmitter agonists and antagonists to relate the effects of these substances to the actions of the endogenous transmitter substances. Both glutamate and aspartate strongly excite the afferents while GABA and carbachol have negligible or weak effects. In contrast to its lack of effect on afferent activity in some anamniotes, N-methyl-D-aspartate (NMDA) was also found to excite these afferents. Kynurenic acid reversibly reduced the resting firing rates of the afferents and the increases in firing due to the application of glutamate and aspartate. These findings provide preliminary support for the hypothesis that glutamate (or a related compound) is also a vestibular hair cell transmitter in amniotes.

Microgravity induces prostaglandin E2 and interleukin-6 production in normal rat osteoblasts: role in bone demineralization.

It has been suggested that microgravity alters bone metabolism. Evidence for this phenomenon includes the negative calcium balance and decreased bone density in astronauts, as well as, inhibition of bone formation in rats flown for 2 to 3 weeks. However, the specific mechanisms that modulate these changes in microgravity are unknown. The purpose of this study was to clarify the mechanism of microgravity-induced bone demineralization using normal rat osteoblasts obtained from femur marrow cultures. The osteoblasts were cultured for 5 days during a Shuttle-Spacelab flight (STS-65). After collection of the culture medium, the cellular DNA and RNA were fixed on board. Enzyme-immunoassay of the culture medium for prostaglandin E2 (PGE2) indicated that microgravity induced a 4.5- to 136-fold increase in flight samples as compared to the ground control cultures. This increase of PGE2 production was consistent with a 3.3- to 9.5-fold elevation of inducible prostaglandin G/H synthase-2 (PGHS-2) mRNA, quantitated by reverse transcription-polymerase chain reaction (RT-PCR). The mRNA induction for the constitutive isozyme PGHS-1 was less than that for PGHS-2. The interleukin-6 (IL-6) mRNA was also increased (6.4- to 9.3-fold) in microgravity as compared to the ground controls. Since PGE2 and IL-6 are both known to play a role in osteoclast formation and bone resorption, these data provide molecular mechanisms that contribute to our understanding of microgravity-induced alterations in the bone resorption process.