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At least 325 records · Page 18

Extracellular calcium (Ca2+(o))-sensing receptor in a murine bone marrow-derived stromal cell line (ST2): potential mediator of the actions of Ca2+(o) on the function of ST2 cells

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+(o)) homeostasis by mediating the actions of Ca2+(o) on parathyroid gland and kidney. Bone marrow stromal cells support the formation of osteoclasts from their progenitors as well as the growth of hematopoietic stem cells by secreting humoral factors and through cell to cell contact. Stromal cells also have the capacity to differentiate into bone-forming osteoblasts. Bone resorption by osteoclasts probably produces substantial local increases in Ca2+(o) that could provide a signal for stromal cells in the immediate vicinity, leading us to determine whether such stromal cells express the CaR. In this study, we used the murine bone marrow-derived, stromal cell line, ST2. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in ST2 cells. We also identified CaR transcripts in ST2 cells by Northern analysis using a CaR-specific probe and by RT-PCR with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of ST2 cells to high Ca2+(o) (4.8 mM) or to the polycationic CaR agonists, neomycin (300 microM) or gadolinium (100 microM), stimulated both chemotaxis and DNA synthesis in ST2 cells. Therefore, taken together, our data strongly suggest that the bone marrow-derived stromal cell line, ST2, possesses both CaR protein and messenger RNA that are very similar if not identical to those in parathyroid and kidney. Furthermore, as ST2 cells have the potential to differentiate into osteoblasts, the CaR in stromal cells could participate in bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local, osteoclast-mediated release of Ca2+(o) and, thereafter, initiating bone formation after their differentiation into osteoblasts.

NASA Discipline Musculoskeletal↗

Localized infusion of IGF-I results in skeletal muscle hypertrophy in rats

Insulin-like growth factor I (IGF-I) peptide levels have been shown to increase in overloaded skeletal muscles (G. R. Adams and F. Haddad. J. Appl. Physiol. 81: 2509-2516, 1996). In that study, the increase in IGF-I was found to precede measurable increases in muscle protein and was correlated with an increase in muscle DNA content. The present study was undertaken to test the hypothesis that direct IGF-I infusion would result in an increase in muscle DNA as well as in various measurements of muscle size. Either 0.9% saline or nonsystemic doses of IGF-I were infused directly into a non-weight-bearing muscle of rats, the tibialis anterior (TA), via a fenestrated catheter attached to a subcutaneous miniosmotic pump. Saline infusion had no effect on the mass, protein content, or DNA content of TA muscles. Local IGF-I infusion had no effect on body or heart weight. The absolute weight of the infused TA muscles was approximately 9% greater (P < 0.05) than that of the contralateral TA muscles. IGF-I infusion resulted in significant increases in the total protein and DNA content of TA muscles (P < 0.05). As a result of these coordinated changes, the DNA-to-protein ratio of the hypertrophied TA was similar to that of the contralateral muscles. These results suggest that IGF-I may be acting to directly stimulate processes such as protein synthesis and satellite cell proliferation, which result in skeletal muscle hypertrophy.

Non-NASA Center↗

Immune alterations in male and female mice after 2-deoxy-D-glucose administration

Administration of 2-deoxy-D-glucose (2-DG) induces acute cellular glucoprivation. In the current study, we examined differences in immune parameters after 2-DG administration in both sexes. Male and female BDF1 mice were injected three times, 48 h apart, either with a saline solution (control group) or with 2-DG in saline (500 mg/kg). Two hours after the last injection, blood and spleens were collected. Plasma levels of interleukin-1beta, and interferon-gamma levels were measured. Additionally, the levels of the specific leukocyte antigens CD3, CD4, CD8, T cell receptor (TCR) alpha/beta, I-Ad, and H-2Ld/H-2Db were evaluated by flow cytometry on both blood and spleen cells. The blastogenic response of leukocytes from both tissues to mitogens was assessed. Levels of glucose, corticosterone, testosterone, progesterone, 17beta-estradiol, follicle-stimulating hormone, and luteinizing hormone were also determined. Increases in the percentage of cells bearing TCR alpha/beta and I-Ad in the blood and H-2Ld/H-2Db in the spleen were observed in the 2-DG-treated group for both sexes. In contrast, higher corticosterone and IL-1beta plasma concentrations, as well as higher percentages of splenocytes bearing TCR alpha/beta and I-Ad, and lower mitogen-induced proliferation of mature T splenocytes (79%) were observed in female but not in male mice injected with 2-DG compared with those injected with saline (p < 0.05). Taken together, these results suggest that female mice are more sensitive than male mice to immune alterations induced by 2-DG administration.

Non-NASA Center↗

Calbindin and parvalbumin are early markers of non-mitotically regenerating hair cells in the bullfrog vestibular otolith organs

Earlier studies have demonstrated hair cell regeneration in the absence of cell proliferation, and suggested that supporting cells could phenotypically convert into hair cells following hair cell loss. Because calcium-binding proteins are involved in gene up-regulation, cell growth, and cell differentiation, we wished to determine if these proteins were up-regulated in scar formations and regenerating hair cells following gentamicin treatment. Calbindin and parvalbumin immunolabeling was examined in control or gentamicin-treated (GT) bullfrog saccular and utricular explants cultured for 3 days in amphibian culture medium or amphibian culture medium supplemented with aphidicolin, a blocker of nuclear DNA replication in eukaryotic cells. In control cultures, calbindin and parvalbumin immunolabeled the hair bundles and, less intensely, the cell bodies of mature hair cells. In GT or mitotically-blocked GT (MBGT) cultures, calbindin and parvalbumin immunolabeling was also seen in the hair bundles, cuticular plates, and cell bodies of hair cells with immature hair bundles. Thus, these antigens were useful markers for both normal and regenerating hair cells. Supporting cell immunolabeling was not seen in control cultures nor in the majority of supporting cells in GT cultures. In MBGT cultures, calbindin and parvalbumin immunolabeling was up-regulated in the cytosol of single supporting cells participating in scar formations and in supporting cells with hair cell-like characteristics. These data provide further evidence that non-mitotic hair cell regeneration in cultures can be accomplished by the conversion of supporting cells into hair cells.

Non-NASA Center↗

Antarctic isolation: immune and viral studies

Stressful environmental conditions are a major determinant of immune reactivity. This effect is pronounced in Australian National Antarctic Research Expedition populations exposed to prolonged periods of isolation in the Antarctic. Alterations of T cell function, including depression of cutaneous delayed-type hypersensitivity responses and a peak 48.9% reduction of T cell proliferation to the mitogen phytohaemagglutinin, were documented during a 9-month period of isolation. T cell dysfunction was mediated by changes within the peripheral blood mononuclear cell compartment, including a paradoxical atypical monocytosis associated with altered production of inflammatory cytokines. There was a striking reduction in the production by peripheral blood mononuclear cells of the predominant pro-inflammatory monokine TNF-alpha and changes were also detected in the production of IL-1, IL-2, IL-6, IL-1ra and IL-10. Prolonged Antarctic isolation is also associated with altered latent herpesvirus homeostasis, including increased herpesvirus shedding and expansion of the polyclonal latent Epstein-Barr virus-infected B cell population. These findings have important long-term health implications.

NASA Discipline Environmental Health↗

Cloning and characterization of the murine homolog of the sno proto-oncogene reveals a novel splice variant

The cellular function(s) of the SNO protein remain undefined. To gain a better understanding of possible developmental roles of this cellular proto-oncogene, we have cloned two murine sno cDNAs and have investigated their expression patterns in embryonic and postnatal tissues. A single major transcript of 7.5 kb is detected in multiple tissues by Northern blot. However, reverse transcriptase polymerase chain reaction (RT-PCR) and RNAse protection assays revealed a novel splice variant in every tissue examined. Two isoforms, termed sno N and sno-dE3 (dE3, deletion within exon 3), were identified. The sno-dE3 isoform employs a novel 5' splice site located within the coding region of the third exon and deletes potential kinase recognition motifs. Transcripts of both sno isoforms accumulate ubiquitously but are most abundant in the developing central nervous system. The in situ hybridization patterns of sno expression during murine development suggest potential roles in tissues with a high degree of cellular proliferation. Expression in terminally differentiated tissues such as muscle and neurons indicates that SNO may have multiple functional activities.

Non-NASA Center↗

Immunohistochemical evidence of rapid extracellular matrix remodeling after iron-particle irradiation of mouse mammary gland

High-LET radiation has unique physical and biological properties compared to sparsely ionizing radiation. Recent studies demonstrate that sparsely ionizing radiation rapidly alters the pattern of extracellular matrix expression in several tissues, but little is known about the effect of heavy-ion radiation. This study investigates densely ionizing radiation-induced changes in extracellular matrix localization in the mammary glands of adult female BALB/c mice after whole-body irradiation with 0.8 Gy 600 MeV iron particles. The basement membrane and interstitial extracellular matrix proteins of the mammary gland stroma were mapped with respect to time postirradiation using immunofluorescence. Collagen III was induced in the adipose stroma within 1 day, continued to increase through day 9 and was resolved by day 14. Immunoreactive tenascin was induced in the epithelium by day 1, was evident at the epithelial-stromal interface by day 5-9 and persisted as a condensed layer beneath the basement membrane through day 14. These findings parallel similar changes induced by gamma irradiation but demonstrate different onset and chronicity. In contrast, the integrity of epithelial basement membrane, which was unaffected by sparsely ionizing radiation, was disrupted by iron-particle irradiation. Laminin immunoreactivity was mildly irregular at 1 h postirradiation and showed discontinuities and thickening from days 1 to 9. Continuity was restored by day 14. Thus high-LET radiation, like sparsely ionizing radiation, induces rapid-remodeling of the stromal extracellular matrix but also appears to alter the integrity of the epithelial basement membrane, which is an important regulator of epithelial cell proliferation and differentiation.

NASA Discipline Radiation Health↗

Role of calcium in growth inhibition induced by a novel cell surface sialoglycopeptide

Our laboratory has purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) from intact bovine cerebral cortex cells. Evidence presented here demonstrates that sensitivity to CeReS-18-induced growth inhibition in BALB-c 3T3 cells is influenced by calcium, such that a decrease in the calcium concentration in the growth medium results in an increase in sensitivity to CeReS-18. Calcium did not alter CeReS-18 binding to its cell surface receptor and CeReS-18 does not bind calcium directly. Addition of calcium, but not magnesium, to CeReS-18-inhibited 3T3 cells results in reentry into the cell cycle. A greater than 3-hour exposure to increased calcium is required for escape from CeReS-18-induced growth inhibition. The calcium ionophore ionomycin could partially mimic the effect of increasing extracellular calcium, but thapsigargin was ineffective in inducing escape from growth inhibition. Increasing extracellular calcium 10-fold resulted in an approximately 7-fold increase in total cell-associated 45Ca+2, while free intracellular calcium only increased approximately 30%. However, addition of CeReS-18 did not affect total cell-associated calcium or the increase in total cell-associated calcium observed with an increase in extracellular calcium. Serum addition induced mobilization of intracellular calcium and influx across the plasma membrane in 3T3 cells, and pretreatment of 3T3 cells with CeReS-18 appeared to inhibit these calcium mobilization events. These results suggest that a calcium-sensitive step exists in the recovery from CeReS-18-induced growth inhibition. CeReS-18 may inhibit cell proliferation through a novel mechanism involving altering the intracellular calcium mobilization/regulation necessary for cell cycle progression.

Non-NASA Center↗

Immune responses in humans after 60 days of confinement

A confinement experiment in a normobaric diving chamber was undertaken to better understand the effect of confinement and isolation on human psychology and physiology. Pre- and postconfinement blood samples were obtained from four test subjects and control donors to analyze immune responses. No modification in the levels of CD2+, CD3+, CD4+, CD8+, CD19+, and CD56+ cells was observed after confinement. Mitogen-induced T-lymphocyte proliferation and interleukin-2 receptor expression were not altered significantly. Whole blood interferon-alpha and gamma-induction and plasma cortisol levels were also unchanged, as was natural killer cell activity. These data suggest that in humans, no specific components of the immune response are affected by a 2-month isolation and confinement of a small group.

NASA Discipline Number 18-10↗

In vitro chick pre-cardiac explant tissue differentiation during spaceflight on SpaceHab-02

Chick precardiac tissue explants were cultured on the 8-day mission of STS-60, space shuttle Discovery. Development of in vitro cultures of precardiac chick tissue from embryo stages 5 though 8 (H-H) were initiated during orbit and were terminated after approximately fifteen hours of 37 degree C culture. Transmission electron microscopy and tritiated thymidine studies were performed postflight. No significant differences in cell proliferation were observed between flight and ground controls. Electron-microscopic studies revealed stage 8 explants were capable of differentiation during flight in a pattern which matched ground control tissues. As anticipated, stage 7 explant tissues had differentiated to a lesser extent compared to stage 8 tissues. Interestingly, stage 7 precardiac explant flight tissue differentiation was less than ground control tissue. This difference in differentiation between flight and ground cultures was enhanced in stage 6 tissues, as high levels of myofibril organization were only seen in ground controls. Other cellular components such as Golgi apparatus, junctional complexes, and mitochondria were present and appeared normal and healthy.

NASA Program NSCORT↗

The effect of space and parabolic flight on macrophage hematopoiesis and function

We used weak electric fields to monitor macrophage spreading in microgravity. Using this technique, we demonstrated that bone marrow-derived macrophages responded to microgravity within 8 s. We also showed that microgravity differentially altered two processes associated with bone marrow-derived macrophage development. Spaceflight enhanced cellular proliferation and inhibited differentiation. These data indicate that the space/microgravity environment significantly affects macrophages.

NASA Discipline Cell Biology↗

Metaphase yields from staphylococcal enterotoxin A stimulated peripheral blood lymphocytes of unirradiated and irradiated aged rhesus monkeys

The mitogen phytohemagglutinin (PHA) works well in both human and cynomolgus monkey (Macaca fascicularis) lymphocyte cultures to stimulate T cell proliferation. T cells from rhesus monkeys (Macaca mulatta) are less responsive than human cells, producing few metaphases when thousands are required, e.g. in biological dosimetry studies. We show that staphylococcal enterotoxin A (SEA), one of the most potent mitogens known, at a concentration of 0.5 microgram/ml stimulated peripheral lymphocytes to grow with a mitotic index (MI) averaging 0.13 metaphases/cell in old, irradiated rhesus macaques. This was significantly greater (p < 0.001) than that produced by PHA (MI < 0.01) in lymphocytes from the same animals. Whole blood was cultured for 96, 120 and 144 h for five irradiated individuals and for two controls. All cells cultured with SEA produced a high MI with a peak response at 120 h whereas the same cultures showed low MI for each PHA stimulated culture.

Non-NASA Center↗

cAMP levels in fast- and slow-twitch skeletal muscle after an acute bout of aerobic exercise

The present study examined whether exercise duration was associated with elevated and/or sustained elevations of postexercise adenosine 3',5'-cyclic monophosphate (cAMP) by measuring cAMP levels in skeletal muscle for up to 4 h after acute exercise bouts of durations that are known to either produce (60 min) or not produce (10 min) mitochondrial proliferation after chronic training. Treadmill-acclimatized, but untrained, rats were run at 22 m/min for 0 (control), 10, or 60 min and were killed at various postexercise (0, 0.5, 1, 2, and 4 h) time points. Fast-twitch white and red (quadriceps) and slow-twitch (soleus) muscles were quickly excised, frozen in liquid nitrogen, and assayed for cAMP with a commercial kit. Unexpectedly, cAMP contents in all three muscles were similar to control (nonexercise) at most (21 of 30) time points after a single 10- or 60-min run. Values at 9 of 30 time points were significantly different from control (P < 0.05); i.e., 3 time points were significantly higher than control and 6 were significantly less than control. These data suggest that the cAMP concentration of untrained skeletal muscle after a single bout of endurance-type exercise is not, by itself, associated with exercise duration.

NASA Discipline Musculoskeletal↗

Decreased heterotopic osteogenesis in vitamin-D-deficient, but normocalcemic guinea pigs

The effect of vitamin D deficiency unhampered by hypocalcemia on de novo bone formation was studied in guinea pigs. Heterotopic induction of osteogenesis was evaluated 4 weeks after intramuscular transplantation of allogenic urinary bladder transitional epithelium from vitamin-D-repleted (+D) donors into +D and -D recipients. In -D recipients the frequency of osteogenesis and the amount of induced bone were significantly diminished; induced bone was less mature, scantly cellular woven bone poorly repopulated with bone marrow. No effect of vitamin D deficiency on orthotopic bone growth and on mineralization of orthotopic and heterotopically induced bone was observed. It is proposed that in addition to inducing factors (BMPs, growth factors) which may be responsible for transformation of mesenchymal cells to osteoprogenitor cells, normal concentrations of 1,25-(OH)2D3 may be required for proliferation and further differentiation of these cells into osteoblasts and for expression of genes engaged in extracellular matrix formation and maturation.

NASA Discipline Musculoskeletal↗

Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei

A casein kinase II (CK II)-like protein kinase was identified and partially isolated from a purified envelope-matrix fraction of pea (Pisum sativum L.) nuclei. When [gamma-32P]ATP was directly added to the envelope-matrix preparation, the three most heavily labeled protein bands had molecular masses near 71, 48, and 46 kDa. Protein kinases were removed from the preparation by sequential extraction with Triton X-100, EGTA, 0.3 M NaCl, and a pH 10.5 buffer, but an active kinase still remained bound to the remaining lamina-matrix fraction after these treatments. This kinase had properties resembling CK II kinases previously characterized from animal and plant sources: it preferred casein as an artificial substrate, could use GTP as efficiently as ATP as the phosphoryl donor, was stimulated by spermine, was calcium independent, and had a catalytic subunit of 36 kDa. Some animal and plant CK II kinases have regulatory subunits near 29 kDa, and a lamina-matrix-bound protein of this molecular mass was recognized on immunoblot by anti-Drosophila CK II polyclonal antibodies. Also found associated with the envelope-matrix fraction of pea nuclei were p34cdc2-like and Ca(2+)-dependent protein kinases, but their properties could not account for the protein kinase activity bound to the lamina. The 71-kDa substrate of the CK II-like kinase was lamin A-like, both in its molecular mass and in its cross-reactivity with anti-intermediate filament antibodies. Lamin phosphorylation is considered a crucial early step in the entry of cells into mitosis, so lamina-bound CK II kinases may be important control points for cellular proliferation.

NASA Discipline Plant Biology↗

Regulation of an H-ras-related transcript by parathyroid hormone in rat osteosarcoma cells

The rat osteosarcoma cell line UMR 106-01 is a commonly used model system for the study of osteoblast function. However, it also expresses a phenotype characteristic of transformed cells. To test whether the latter could be accounted for by aberrant oncogene expression, we probed Northern blots of UMR and other osteoblastic cells with a panel of oncogene probes. These blots, when probed with a cDNA specific for v-H-ras, revealed a 7.0-kilobase (kb) H-ras-related transcript (designated HRRT) in UMR 106-01 cells that was not expressed in other osteoblastic cells. Osteoblast-enriched calvarial cells expressed the typical 1.1-kb H-ras mRNA, which was absent in UMR cells. Additionally, Western blots of lysates of UMR cells documented the presence of three proteins immunologically related to H-rasp21. To determine whether HRRT represented a recombinant retrovirus product, Northern blots were probed with a cDNA specific for the highly conserved gag-pol region of Moloney murine leukemia virus. These blots showed parallel cross-reactivity with an apparently identical transcript of 7.0 kb. The 7.0-kb transcripts detected by both v-H-ras and gag-pol probes declined to the same extent after treatment with concentrations of PTH known to inhibit proliferation of these cells. PTH regulated the abundance of HRRT in a time- and dose-dependent manner, with greatest repression of the transcript after 8 h of treatment with 10(-8) M PTH. The decrease in HRRT could not be completely accounted for by changes in transcriptional activity, as determined by nuclear run-on assays.(ABSTRACT TRUNCATED AT 250 WORDS).

Non-NASA Center↗

Experiments with suspended cells on the Space Shuttle

Spaceflight experiments since 1981 have demonstrated that certain cell functions are altered by micro-g. Biophysical models suggest that cell membranes and organelles should not be affected directly by gravity, however, the chemical microenvironment surrounding the cell and molecular transport could be altered by reduced gravity. Most experiments have used suspended live cells in small chambers without stirring or medium exchange. Flight results include increased attachment of anchorage-dependent human cells to collagen coated microcarriers, reduced secretion of growth hormone from pituitary cells, decreased mitogenic response of lymphocytes, increased Interferon-alpha by lymphocytes, increased Interleukin-1 and Tumor Necrosis Factor secretion by macrophages. Related experiments on cells immediately postflight and on procaryotic cells have shown significant changes in secretory capacity, cell proliferation, differentiation and development. Postulated mechanism include altered cell-cell interactions, altered calcium ion transport, effects on cell cytoskeleton, transport of transmitters and interactions with receptors. The discussion includes use of new molecular methods, considerations for cell environmental control and a preview of several experiments planned for the Shuttle and Spacelab flights to study the basic effects of microgravity on cellular physiology and potential interactions of spaceflight with radiation damage and cellular repair mechanisms.

Review↗

Imaging of the hip joint. Computed tomography versus magnetic resonance imaging

The authors reviewed the applications and limitations of computed tomography (CT) and magnetic resonance (MR) imaging in the assessment of the most common hip disorders. Magnetic resonance imaging is the most sensitive technique in detecting osteonecrosis of the femoral head. Magnetic resonance reflects the histologic changes associated with osteonecrosis very well, which may ultimately help to improve staging. Computed tomography can more accurately identify subchondral fractures than MR imaging and thus remains important for staging. In congenital dysplasia of the hip, the position of the nonossified femoral head in children less than six months of age can only be inferred by indirect signs on CT. Magnetic resonance imaging demonstrates the cartilaginous femoral head directly without ionizing radiation. Computed tomography remains the imaging modality of choice for evaluating fractures of the hip joint. In some patients, MR imaging demonstrates the fracture even when it is not apparent on radiography. In neoplasm, CT provides better assessment of calcification, ossification, and periosteal reaction than MR imaging. Magnetic resonance imaging, however, represents the most accurate imaging modality for evaluating intramedullary and soft-tissue extent of the tumor and identifying involvement of neurovascular bundles. Magnetic resonance imaging can also be used to monitor response to chemotherapy. In osteoarthrosis and rheumatoid arthritis of the hip, both CT and MR provide more detailed assessment of the severity of disease than conventional radiography because of their tomographic nature. Magnetic resonance imaging is unique in evaluating cartilage degeneration and loss, and in demonstrating soft-tissue alterations such as inflammatory synovial proliferation.

Review↗