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Janet Rubin

Publications and source records attributed to Janet Rubin.

At least 19 recordsLinked to original sources
PubMed2006

IGF-I secretion by prostate carcinoma cells does not alter tumor-bone cell interactions in vitro or in vivo.

BACKGROUND: IGF-I is an important growth and differentiative factor for osteoblasts and may have a role in defining prostate cancer risk and skeletal metastases. METHODS: Conditioned media (CM) from human prostate cancer (PC), C4-2 and C4-2B, which produce osteoblastic lesions, and PC-3, which causes osteolysis, was added to MC3T3-E1 bone cultures. SCID mice were injected intratibially with these engineered cells. Tumor bearing tibiae were analyzed by microCT and pQCT. RESULTS: CM from PC cells increased osteoblast proliferation and differentiation and was unaltered by the type of PC cell, IGF-I antibodies, or exogenous IGF-I and IGFBP2. Study of intratibial PC tumors in SCID mice showed that C4-2 cells grew slowly preserving bone structure, while PC-3 tumors caused rapid osteolysis. Overexpression of IGF-I did not change either tumor progression or skeletal response. CONCLUSIONS: IGF-I is neither necessary nor sufficient for the osteoblastic response to PC metastases.

Animals↗
PubMed2006

Transcriptional regulation of the osterix (Osx, Sp7) promoter by tumor necrosis factor identifies disparate effects of mitogen-activated protein kinase and NF kappa B pathways.

Osteoblast (OB) differentiation is suppressed by tumor necrosis factor-alpha (TNF-alpha), an inflammatory stimulus that is elevated in arthritis and menopause. Because OB differentiation requires the expression of the transcription factor osterix (Osx), we investigated TNF effects on Osx. TNF inhibited Osx mRNA in pre-osteoblastic cells without affecting Osx mRNA half-life. Inhibition was independent of new protein synthesis. Analysis of the Osx promoter revealed two transcription start sites that direct the expression of an abundant mRNA (Osx1) and an alternatively spliced mRNA (Osx2). Promoter fragments driving the expression of luciferase were constructed to identify TNF regulatory sequences. Two independent promoters were identified upstream of each transcription start site. TNF potently inhibited transcription of both promoters. Deletion and mutational analysis identified a TNF-responsive region proximal to the Osx2 start site that retained responsiveness when inserted upstream of a heterologous promoter. The TNF response region was a major binding site for nuclear proteins, although TNF did not change binding at the site. The roles of MAPK and NFkappaB were investigated as signal mediators of TNF. Inhibitors of MEK1 and ERK1, but not of JNK or p38 kinase, abrogated TNF inhibition of Osx mRNA and promoter activity. TNF action was not prevented by blockade of NFkappaB nuclear entry. The forced expression of high levels of NFkappaB uncovered a proximal promoter enhancer; however, this site was not activated by TNF. The inhibitory effect of TNF on Osx expression may decrease OB differentiation in arthritis and osteoporosis.

3T3 Cells↗
PubMed2006

Response to mechanical strain in an immortalized pre-osteoblast cell is dependent on ERK1/2.

Mechanical strain inhibits osteoclastogenesis by regulating osteoblast functions: We have shown that strain inhibits receptor activator of NF-kappaB ligand (RANKL) expression and increases endothelial nitric oxide synthase (eNOS) and nitric oxide levels through ERK1/2 signaling in primary bone stromal cells. The primary stromal culture system, while contributing greatly to understanding of how the microenvironment regulates bone remodeling is limited in use for biochemical assays and studies of other osteoprogenitor cell responses to mechanical strain: Stromal cells proliferate poorly and lose aspects of the strain response after a relatively short time in culture. In this study, we used the established mouse osteoblast cell line, conditionally immortalized murine calvarial (CIMC-4), harvested from mouse calvariae conditionally immortalized by insertion of the gene coding for a temperature-sensitive mutant of SV40 large T antigen (TAg) and support osteoclastogenesis. Mechanical strain (0.5-2%, 10 cycles per min, equibiaxial) caused magnitude-dependent decreases in RANKL expression to less than 50% those of unstrained cultures. Overnight strains of 2% also increased osterix (OSX) and RUNX2 expression by nearly twofold as measured by RT-PCR. Importantly, the ERK1/2 inhibitor, PD98059, completely abrogated the strain effects bringing RANKL, OSX, and RUNX2 gene expression completely back to control levels. These data indicate that the strain effects on CIMC-4 cells require activation of ERK1/2 pathway. Therefore, the CIMC-4 cell line is a useful alternative in vitro model which effectively recapitulates aspects of the primary stromal cells and adds an extended capacity to study osteoblast control of bone remodeling in a mechanically active environment.

Alkaline Phosphatase↗
PubMed2005

Molecular pathways mediating mechanical signaling in bone.

Bone tissue has the capacity to adapt to its functional environment such that its morphology is "optimized" for the mechanical demand. The adaptive nature of the skeleton poses an interesting set of biological questions (e.g., how does bone sense mechanical signals, what cells are the sensing system, what are the mechanical signals that drive the system, what receptors are responsible for transducing the mechanical signal, what are the molecular responses to the mechanical stimuli). Studies of the characteristics of the mechanical environment at the cellular level, the forces that bone cells recognize, and the integrated cellular responses are providing new information at an accelerating speed. This review first considers the mechanical factors that are generated by loading in the skeleton, including strain, stress and pressure. Mechanosensitive cells placed to recognize these forces in the skeleton, osteoblasts, osteoclasts, osteocytes and cells of the vasculature are reviewed. The identity of the mechanoreceptor(s) is approached, with consideration of ion channels, integrins, connexins, the lipid membrane including caveolar and non-caveolar lipid rafts and the possibility that altering cell shape at the membrane or cytoskeleton alters integral signaling protein associations. The distal intracellular signaling systems on-line after the mechanoreceptor is activated are reviewed, including those emanating from G-proteins (e.g., intracellular calcium shifts), MAPKs, and nitric oxide. The ability to harness mechanical signals to improve bone health through devices and exercise is broached. Increased appreciation of the importance of the mechanical environment in regulating and determining the structural efficacy of the skeleton makes this an exciting time for further exploration of this area.

Animals↗
PubMed2005

Mechanical inhibition of RANKL expression is regulated by H-Ras-GTPase.

Mechanical input is known to regulate bone remodeling, yet the molecular events involved in mechanical signal transduction are poorly understood. We here investigate proximal events leading to the ERK1/2 activation that is required for mechanical repression of RANKL (receptor activator of NF-kappaB ligand) expression, the factor that controls local recruitment of osteoclasts. Using primary murine bone stromal cells we show that dynamic mechanical strain via substrate deformation activates Ras-GTPase, in particular the H-Ras isoform. Pharmacological inhibition of H-Ras function prevents strain activation of H-Ras as well as the downstream mechanical repression of RANKL. Furthermore, small interfering RNA silencing of H-Ras, but not K-Ras, abrogates mechanical strain repression of RANKL. H-Ras-specific inhibition of mechanorepression of RANKL was also demonstrated in a murine pre-osteoblast cell line (CIMC-4). The requirement of cholesterol for H-Ras activation was probed; cholesterol depletion of rafts using methyl-betacyclodextrin prevented mechanical H-Ras activation. That the mechanical repression of RANKL requires activation of H-Ras, a specific isoform of Ras-GTP that is known to reside in the lipid raft microdomain, suggests that spatial arrangements are critical for generation of specific downstream events in response to mechanical signals. By partitioning signals this way, cells may be able to generate different downstream responses through seemingly similar signaling cascades.

Animals↗
PubMed2005

Effects of titanium particle size on osteoblast functions in vitro and in vivo.

The formation of titanium (Ti)-wear particles during the lifetime of an implant is believed to be a major component of loosening due to debris-induced changes in bone cell function. Radiographic evidence indicates a loss of fixation at the implant-bone interface, and we believe that the accumulation of Ti particles may act on the bone-remodeling process and impact both long- and short-term implant-fixation strengths. To determine the effects of various sizes of the Ti particles on osteoblast function in vivo, we measured the loss of integration strength around Ti-pin implants inserted into a rat tibia in conjunction with Ti particles from one of four size-groups. Implant integration is mediated primarily by osteoblast adhesion/focal contact pattern, viability, proliferation and differentiation, and osteoclast recruitment at the implant site in vivo. This study demonstrates the significant attenuation of osteoblast function concurrent with increased expression of receptor activator of nuclear factor kappaB ligand (RANKL), a dominant signal for osteoclast recruitment, which is regulated differentially, depending on the size of the Ti particle. Zymography studies have also demonstrated increased activities of matrix metalloproteinases (MMP) 2 and 9 in cells exposed to larger Ti particles. In summary, all particles have adverse effects on osteoblast function, resulting in decreased bone formation and integration, but different mechanisms are elicited by particles of different sizes.

Animals↗
PubMed2004

The p55 TNF receptor mediates TNF inhibition of osteoblast differentiation independently of apoptosis.

After menopause, increased tumor necrosis factor-alpha (TNF-alpha) stimulates bone resorption while inhibiting differentiation of new bone-forming osteoblasts (OB). TNF receptors, p55 and p75, signal similar intracellular pathways, but only p55 activates apoptosis. To evaluate the relationship between the TNF receptor mediating inhibition of OB differentiation and the role of apoptosis, marrow stromal cells (MSC) were cultured from mice deficient in either or both receptors. Cells grown in ascorbate and beta-glycerophosphate produce alkaline phosphatase and osteocalcin and mineralize matrix. Treatment of wild-type or p55(+/+)/p75(-/-) MSC with murine TNF (binds p55 and p75) or human TNF (binds only p55) inhibited OB differentiation. TNF did not inhibit OB differentiation in p55(-/-) MSC. Expression of p75 modestly attenuated sensitivity to TNF. To determine the role of apoptosis, changes in total DNA, cell viability, caspase 3, and percentage of annexin V-positive cells were measured in MSC and preosteoblastic MC3T3 cells. TNF treatment that reduced differentiation by 50% did not decrease cell viability or increase apoptosis, as determined by alamar blue reduction, trypan blue exclusion, and percentage of annexin V-positive cells. TNF increased caspase 3 activity 1.5-fold in MC3T3 and insignificantly in MSC cells compared with > 4-fold after 4 h actinomycin D. Treatment of MSC or MC3T3 cells with three caspase inhibitors failed to reverse the inhibitory effect of TNF on OB differentiation despite inhibition of caspase activity. These results suggest that the p55 receptor is essential, and p75 dispensable, for TNF inhibition of OB differentiation through a mechanism that does not require apoptosis.

3T3 Cells↗
PubMed2004

Regulation of RANKL promoter activity is associated with histone remodeling in murine bone stromal cells.

Receptor activator of NFkappa-B ligand (RANKL) is essential for osteoclast formation, function, and survival. Although RANKL mRNA and protein levels are modulated by 1,25(OH)2D3 and other osteoactive factors, regulatory mechanisms remain unclear. In this study, we show that 2 kb or 2 kb plus exon 1 of a RANKL promoter sequence conferred neither 1,25(OH)2D3 response nor tissue specificity. The histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (SB), however, strongly increased RANKL promoter activity. A series of 5'-deleted RANKL promoter constructs from 2,020 to 110 bp showed fourfold increased activity after TSA treatment. TSA also dose dependently enhanced endogenous RANKL mRNA expression with 50 microM of TSA treatment causing equivalent RANKL expression to that seen with 1 nM 1,25(OH)2D3. Using a chromatin immunoprecipitation (ChIP) assay we showed that TSA significantly enhanced association of both acetylated histone H3 and H4 on the RANKL promoter, with H4 > H3. A similar increase in acetylated histone H4 on the RANKL gene locus was seen after 1,25(OH)2D3 treatment, but ChIP assay did not reveal localization of VDR/RXR heterodimers on the putative VDRE of the RANKL promoter. To explore the role of H4 acetylation of 1,25(OH)2D3 stimulated RANKL, we added both TSA and 1,25(OH)2D3 together. While the combination further increased acetylation of H4 on the RANKL locus, surprisingly, TSA inhibited 1,25(OH)2D3-induced RANKL mRNA expression by 70% at all doses of 1 ,25(OH)2D3 studied. These results suggest that TSA increases of endogenous expression of RANKL involve enhanced acetylation of histones on the proximal RANKL promoter. Preventing deacetylation, however, blocks 1,25(OH)2D3 action on this gene. Chromatin remodeling is therefore involved in RANKL expression.

Acetylation↗
PubMed2004

Integration of the NfkappaB p65 subunit into the vitamin D receptor transcriptional complex: identification of p65 domains that inhibit 1,25-dihydroxyvitamin D3-stimulated transcription.

Resistance to the action of vitamin D (D) occurs in response to tumor necrosis factor-alpha (TNF-alpha), an effect mediated by nuclear factor kappa B (NfkappaB). To determine the mechanism of NfkappaB inhibition of D-stimulated transcription, chromatin immunoprecipitation assays (CHIP) were done in osteoblastic ROS 17/2.8 cells that had been treated with TNF-alpha or transfected with the p65 subunit of NfkappaB. These treatments caused stable incorporation of p65 into the transcription complex bound to the vitamin D response element (VDRE) of the osteocalcin promoter. Deletion analysis of p65 functional domains revealed that the p65 N-terminus and a midmolecular region were both required for the inhibitory action of p65. Pull-down assays were done using an immobilized glutathione S-transferase (GST)-VDR fusion protein to study the effect of p65 on VDR binding to steroid coactivator-1 (SRC-1), a major D-dependent coactivator. p65 inhibited VDR-SRC-1 binding in a dose-dependent manner. Mutations of p65 that abrogated the inhibitory effect on D-stimulated transcription also failed to inhibit VDR-SRC-1 binding. The inhibitory effect of p65 on VDR transactivation was not due to recruitment of a histone deacetylase (HDAC), since inhibition was not relieved by the HDAC inhibitors sodium butyrate or trichostatin A. Overexpression of SRC-1 or the general coactivators, Creb binding protein or SRC-3, also failed to relieve p65 inhibition of transcription. In addition, Chip assays revealed that TNF-alpha treatment prevented D recruitment of SRC-1 to the transcription complex. These results show that TNF-alpha inhibition of vitamin D-action includes stable integration of p65 in the VDR transcription complex. Once anchored to proteins within the complex, p65 disrupts VDR binding to SRC-1, thus decreasing the efficiency of D-stimulated gene transcription.

Calcitriol↗
PubMed2004

SEM and TEM study of the hierarchical structure of C57BL/6J and C3H/HeJ mice trabecular bone.

Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to study the hierarchical structure of trabecular bone from C57BL/6J (low bone mass) and C3H/HeJ mice (high bone mass). Bone was harvested from two different anatomical locations: femoral metaphysis and L5 vertebra. This investigation focused on three structural scales: the mesostructural (porous network of trabecular struts), the microstructural (collagen fibril arrangements in trabecular packets), and the nanostructural (collagen fibril and apatite crystals) levels. At the mesostructural level, no distinct differences were found in the trabecular structure of femoral metaphysis but thinner trabecular struts were observed in L5 vertebra for C57BL/6J mice strain. At the microstructural level, the collagen fibrils forming the rotated, twisted, and orthogonal plywood arrangements were distinguished as well as atypical arrangements. At the nanostructural level, the shape and size of apatite crystals, and their arrangement with respect to collagen fibrils were studied. In spite of very different bone mass densities, both mice strains had similar structures at the nanostructural and microstructural levels.

Animals↗
PubMed2004

Congenic mice with low serum IGF-I have increased body fat, reduced bone mineral density, and an altered osteoblast differentiation program.

Targeted gene studies have demonstrated the importance of insulin-like growth factor-I (IGF-I) for osteoblast (OB) differentiation and the acquisition of peak bone mineral density (BMD). The skeletal response to allelic differences in IGF-I expression can also be measured in vivo, using congenic mice. We created a congenic strain with reduced (approximately 20%) circulating IGF-I (C3H.B6-6T [6T]) by backcrossing a small genomic region (30 cM) of Chromosome 6 (Chr6) from C3H/HeJ (C3H) onto a C57Bl/6J (B6) background. 6T female mice have lower serum IGF-I (P<0.001 vs. B6) but similar growth hormone (GH) and serum IGF binding protein (IGFBP) concentrations as B6. At 16 weeks of age, congenics have greater body fat (P<0.02 vs. B6) despite less total body weight, and exhibit smaller femoral cross-sectional size (P=0.001), reduced cortical thickness (P<0.001) and lower trabecular BV/TV (P<0.05) than B6. 6T mice also have suppressed serum leptin (P<0.01), but compared to B6 have similar markers of bone resorption (i.e., urine CTx and serum TRAP 5B). At 8 weeks of age, skeletal IGF-I mRNA from long bones was reduced by 40% (P<0.05) as were liver mRNA transcripts (i.e., 50%, P<0.01). Osteoblast progenitors from the bone marrow of 6T mice formed less colony forming unit fibroblasts by crystal violet staining than B6 (P<0.007) and had significantly reduced alkaline phosphatase-positive colonies than B6(P<0.0001). In addition, staining of bone marrow with oil red O revealed greater numbers of adipocytes in 6T than B6. Several candidate genes in the Chr6 QTL were excluded by lack of strain-related expression differences in bone, but genes positively regulating adipocyte differentiation including Alox 5 and PPAR-gamma require further study as either "pathway" or candidate genes. In summary, allelic differences in a QTL on Chr6 result in altered IGF-I gene expression, changes in OB lineage allocation, and reduced peak bone mass. Congenic mice are useful models not only for mapping genes related to bone mass but also for elucidating the biology underlying various skeletal phenotypes associated with more subtle manipulation of the mouse genome.

Adipocytes↗
PubMed2004

Veterans Administration support for medical research: opinions of the endangered species of physician-scientists.

Over the past three decades the Veterans Affairs (VA) Research program has evolved into a powerful, peer-reviewed funding mechanism for basic and translational research that has resulted in numerous important contributions to medical science and improvements in patient care. Continuity in VA Merit Review funding has fostered and nurtured the scientific careers of a large number of physician-scientists who have remained devoted to the mission of performing creative and innovative research that affects the patient care mission of the VA. VA medical research policies have undergone a major overhaul in the past year. Although many of these changes (de-emphasizing bench research and revamping the peer review process) have recently been reversed, the future direction of VA research remains in flux. The goal of this manuscript is to demonstrate the importance of the Merit Review medical research funding mechanism not just to the VA, but to the entire nation's health care system. To achieve this goal, the opinions of 65 established VA medical investigators were obtained regarding the past success and future direction of VA research. The conclusions reached include the following. 1) Merit Review research funding has been essential to the training, recruitment, and retention of productive VA physician-scientists. 2) The VA research program has contributed both basic and clinical innovations that have led to improvements in medical care. Contributions of VA researchers to excellence in many aspects of patient care at VA hospitals have been extraordinary. 3) Development of initiatives that entice outstanding Ph.D.'s to develop their careers in the VA has been crucial to the success of the program. 4) The VA research program has fostered a mutually beneficial relationship with affiliated medical schools. 5) Better methods to quantify VA research contributions and outcomes are essential for future program development.

Biomedical Research↗
PubMed2003

Nitric oxide regulates receptor activator of nuclear factor-kappaB ligand and osteoprotegerin expression in bone marrow stromal cells.

Bone remodeling reflects an equilibrium between bone resorption and formation. The local expression of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) in bone determines the entry of monoblastic precursors into the osteoclast lineage and subsequent bone resorption. Nitric oxide (NO) inhibits osteoclastic bone resorption in vitro and regulates bone remodeling in vivo. An interaction of NO with RANKL and OPG has not been studied. Here, we show that treatment of ST-2 murine stromal cells with the NO donor sodium nitroprusside (100 microm) for 24 h inhibited 1,25 dihydroxyvitamin D(3)-induced RANKL mRNA to less than 33 +/- 7% of control level, whereas OPG mRNA increased to 204 +/- 19% of control. NOR-4 replicated these NO effects. The effects of NO were dose dependent and associated with changes in protein levels: RANKL protein decreased and OPG protein increased after treatment with NO. PTH-induced RANKL expression in primary stromal cells was inhibited by sodium nitroprusside, indicating that the NO effect did not require vitamin D. NO donor did not change the stability of RANKL or OPG mRNAs, suggesting that NO affected transcription. Finally, cGMP, which can function as a second messenger for NO, did not reproduce the NO effect, nor did inhibition of endogenous guanylate cyclase prevent the NO effect on these osteoactive genes. The effect of NO to decrease the RANKL/OPG equilibrium should lead to decreased recruitment of osteoclasts and positive bone formation. Thus, drugs and conditions that cause local increase in NO formation in bone may have positive effects on bone remodeling.

Animals↗
PubMed2003

Mechanical strain differentially regulates endothelial nitric-oxide synthase and receptor activator of nuclear kappa B ligand expression via ERK1/2 MAPK.

Exercise promotes positive bone remodeling through controlling cellular processes in bone. Nitric oxide (NO), generated from endothelial nitric-oxide synthase (eNOS), prevents resorption, whereas receptor activator of nuclear kappa B ligand (RANKL) promotes resorption through regulating osteoclast activity. Here we show that mechanical strain differentially regulates eNOS and RANKL expression from osteoprogenitor stromal cells in a magnitude-dependent fashion. Strain (0.25-2%) induction of eNOS expression was magnitude-dependent, reaching a plateau at 218 +/- 36% of control eNOS. This was accompanied by increases in eNOS protein and a doubling of NO production. Concurrently, 0.25% strain inhibited RANKL expression with increasing response up to 1% strain (44 +/- 3% of control RANKL). These differential responses to mechanical input were blocked when an ERK1/2 inhibitor was present during strain application. Inhibition of NO generation did not prevent strain-activated ERK1/2. To confirm the role of ERK1/2, cells were treated with an adenovirus encoding a constitutively activated MEK; Ad.caMEK significantly increased eNOS expression and NO production by more than 4-fold and decreased RANKL expression by half. In contrast, inhibition of strain-activated c-Jun kinase failed to prevent strain effects on either eNOS or RANKL. Our data suggest that physiologic levels of mechanical strain utilize ERK1/2 kinase to coordinately regulate eNOS and RANKL in a manner leading to positive bone remodeling.

Adenoviridae↗
PubMed2003

Detection of endothelial nitric oxide release with the 2,3-diaminonapthalene assay.

The reliable measurement of nitric oxide (NO) production by endothelial cells in vitro has become an important tool for investigating mechanisms of endothelial dysfunction. This study evaluates measuring NO production by cultured porcine pulmonary artery endothelial cells (PAEC) using the assay based on the fluorometric detection of 1-(H)-naphthotriazole, the fluorescent product of the reaction between nitrite (NO2-) and 2,3-diaminonapthalene (DAN). To stimulate NO production, PAEC were treated for 60 min with agonists known to stimulate endothelial NO production. The DAN assay was unable to detect NO production from agonist-stimulated PAEC. In contrast, chemiluminescence analysis, which detects NO, NO2-, and nitrate (NO3-) (collectively referred to as NO(x)), detected significant increases in NO(x) from stimulated PAEC. Nitrate reductase-mediated reduction of NO3-to NO2- in media from stimulated PAEC enhanced the ability of the DAN assay to detect NO release from PAEC. These results provide the first direct comparison of the sensitivity of these two commonly employed assays. Our findings emphasize that NO3-reduction may be required to enable the DAN assay to detect small amounts of NO produced by cultured endothelial cells.

2-Naphthylamine↗
PubMed2003

Nitric oxide donors inhibit luciferase expression in a promoter-independent fashion.

Nitric oxide (NO) is an important molecule with diverse bio-messenger functions including regulation of gene expression. Transcriptional studies using sensitive luciferase reporter systems have suggested that NO inhibits the promoter activity of a variety of genes. Here we report that NO donors (sodium nitroprusside, 2',2'-(hydroxynitrosohydrazono)bis-ethanimine, and (+/-)-(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-yl-nicotinamide) decrease luciferase activity in a promoter-independent fashion in both viral and eukaryotic promoters, with a reduction to nearly 50% in the presence of 100 microm NO donor. Addition of an SV40 enhancer downstream of the luciferase coding region shifted NO donor inhibition to the right, with inhibition at approximately 300 microm. In contrast, when studied in a chloramphenicol acetyltransferase reporter, two promoters indicating inhibition by NO were unaffected. The decrease in luciferase activity was not caused by NO suppression of the luciferase enzyme. Real-time PCR data showed that luciferase mRNA half-life decreased by nearly half in the presence of NO donor (from 75 to 45 min). The SV40 enhancer prolonged luciferase mRNA half-life and somewhat blunted the NO effect. Our data suggest that exogenous NO inhibits luciferase activity in a dose-dependent manner through decreasing luciferase mRNA stability. Thus, the use of luciferase reporter systems to study transcriptional regulation by NO should be attempted with caution.

Animals↗
PubMed2003

TEM analysis of the nanostructure of normal and osteoporotic human trabecular bone.

Transmission electron microscopy (TEM) was used to investigate the crystal-collagen interactions in normal and osteoporotic human trabecular bone at the nanostructural level. More specifically, two-dimensional TEM observations were used to infer the three-dimensional information on the shape, the size, the orientation, and the alignment of apatite crystals in collagen fibrils in normal and osteoporotic bone. We found that crystals were of platelet shape with irregular edges and that there was no substantial difference in crystal length or crystal thickness between normal and osteoporotic trabecular bone. The crystal arrangement in cross-sectioned fibrils did not neatly conform to the parallel arrangement of crystals seen in longitudinally-sectioned fibrils. Instead, the crystal arrangement in both normal and osteoporotic trabecular bone took on more of a random, undulated arrangement, with certain localized areas demonstrating circular oriented patterns. The TEM imaging was done using bright fields only. Thus, the results presented are within the limitations of this approach.

Adult↗