Increase In Calcium Accumulation Research Articles

Vapour pressure deficit affects crop water productivity, yield, and quality in tomatoes

A high atmospheric vapour pressure deficit (VPD) hinders calcium absorption in tomatoes (Solanum lycopersicum L.), which severely reduces tomato yield, crop water productivity (WPcrop), and quality. Although reducing the VPD is effective for increasing tomato yield and water productivity, regulating the VPD is costly. Therefore, this study aimed to explore the effects of VPD regulation (high VPD=2.22 kPa, low VPD=0.95 kPa) during different growth stages on tomato WPcrop, yield, and quality to identify a VPD management method that saves costs and enhances the above parameters. The results showed that the yield was significantly positively correlated with fruit calcium absorption. Decreasing the VPD during the fruit expansion stage and increasing it during the flowering stage significantly increased calcium accumulation in the fruits, thereby improving tomato WPcrop, yield, and quality. Additionally, a high VPD during the flowering stage increased stomatal conductance and mesophyll conductance, thereby increasing intercellular and chloroplast CO2 concentrations and enhancing plant photosynthetic capacity, and reduced the stem water potential, leaf water potential, and leaf relative water content, thereby increasing the water potential difference, leaf hydraulic conductance, and calcium absorption. By integrating game theory and the technique for order of preference by similarity to ideal solution method, a comprehensive analysis of tomato appearance, nutrients, and flavour quality revealed that growing plants under a high VPD during the seedling, flowering, and fruit ripening stages and a low VPD during the fruit expansion stage not only enhanced the comprehensive tomato quality value but also maximally reduced the humidification cost. Conclusively, controlling the environmental VPD at 2.22 kPa during the seedling, flowering, and fruit ripening stages and at 0.95 kPa during the fruit expansion stage can effectively increase tomato WPcrop, yield, and quality. This study provides a theoretical basis for the environmental regulation of high-quality, efficient, and water-conserving cultivation of greenhouse tomatoes.

Hyperglycaemia-induced degeneration of bioprosthetic valve tissue and the pivotal role of the extracellular matrix molecule biglycan

Abstract Background/Introduction Bioprosthetic valve degeneration is a growing clinical challenge in our aging society. Especially type 2 diabetes mellitus represents a risk factor for bioprosthetic valve deterioration with likewise worldwide rising prevalence. The proteoglycan biglycan has been shown to be associated with degenerative changes of the valve in diabetic patients even though underlying mechanisms are yet unknown. Purpose The impact of hyperglycaemia on bioprosthetic valve tissue was analysed in an established mouse model of ectopic calcification. Thereby, the role of biglycan in bioprosthetic valve degeneration was assessed in biglycan-deficient mice (Bgn-/0) in comparison to wild type littermates (WT). Methods Hyperglycaemia was induced in six weeks old mice by intraperitoneal injection of streptozotocin (STZ; 55 mg/kg body weight) on five consecutive days. Blood glucose concentration was verified two weeks after treatment and bioprosthetic valve material was implanted subcutaneously. After eight weeks, phenotype of invading cells and extracellular matrix remodelling of implants were analysed with histochemical and immunohistological staining. Furthermore, gene expression of invading cells, circulating cytokines in blood plasma and calcium accumulation in the tissue were quantified. Results STZ treatment significantly increased blood glucose levels in both genotype groups (WT-STZ: 156 mg/dl; WT+STZ: 353 mg/dl; Bgn-/0-STZ: 142 mg/dl; Bgn-/0+STZ: 396 mg/dl). Immunohistological staining identified most of cells invading the bioprosthetic tissue and the surrounded capsule as Mac2-positive and partly positive for vimentin, whereas alpha smooth muscle actin and von Willebrand factor were only detectable sporadically. Movat pentachrome staining exhibited an altered extracellular matrix composition of collagen and proteoglycans due to hyperglycaemia, while the elastin amount remained stable. Gene expression analysis showed an increased expression of the proteoglycan decorin in hyperglycaemic mice (WT-STZ: 0.55; WT+STZ: 1.13; Bgn-/0-STZ: 0.51; Bgn-/0+STZ: 1.70). Circulating cytokines (IL-1β, IL-2, IL-5, IL-10, INF-γ, TNF-α, GM-CSF) were influenced neither by hyperglycaemia nor by the genotype of the mice. Alizarin and von Kossa staining exhibited small to moderate calcium deposits in the bioprosthetic valve tissue. A colorimetric assay showed a significantly increased calcium accumulation in WT with hyperglycaemia versus normoglycemic WT (WT-STZ: 8.96 μg/mg; WT+STZ: 18.54 μg/mg), an observation that was lost in Bgn-/0 (Bgn-/0-STZ: 9.84 μg/mg; Bgn-/0+STZ: 12.97 μg/mg). Conclusion Hyperglycaemia significantly promotes destructive remodelling in bioprosthetic valve material. Biglycan-deficiency limits degenerative processes associated with diabetes, suggesting that biglycan as a component of the extracellular matrix has an adverse effect in diabetes-associated bioprosthetic valve deterioration. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Dr. Rusche grant of the German Heart Foundation and the German Society for Thoracic and Cardiovascular Surgery

Rho-associated protein kinase and cyclophilin a are involved in inorganic phosphate-induced calcification signaling in vascular smooth muscle cells

Arterial calcification, a risk factor of cardiovascular events, develops with differentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells. Cyclophilin A (CypA) is a peptidyl-prolyl isomerase involved in cardiovascular diseases such as atherosclerosis and aortic aneurysms, and rho-associated protein kinase (ROCK) is involved in the pathogenesis of vascular calcification. CypA is secreted in a ROCK activity-dependent manner and works as a mitogen via autocrine or paracrine mechanisms in VSMCs. We examined the involvement of the ROCK-CypA axis in VSMC calcification induced by inorganic phosphate (Pi), a potent cell mineralization initiator. We found that Pi stimulated ROCK activity, CypA secretion, extracellular signal-regulated protein kinase (ERK) 1/2 phosphorylation, and runt-related transcription factor 2 expression, resulting in calcium accumulation in rat aortic smooth muscle cells (RASMCs). The ROCK inhibitor Y-27632 significantly suppressed Pi-induced CypA secretion, ERK1/2 phosphorylation, and calcium accumulation. Recombinant CypA was found to be associated with increased calcium accumulation in RASMCs. Based on these results, we suggest that autocrine CypA is mediated by ROCK activity and is involved in Pi-induced ERK1/2 phosphorylation following calcification signaling in RASMCs.

Nicotinamide treatment in a murine model of familial tumoral calcinosis reduces serum Fgf23 and raises heart calcium

Mutations in the GALNT3 gene result in familial tumoral calcinosis, characterized by persistent hyperphosphatemia and ectopic calcific masses in soft tissues. Since calcific masses often recur after surgical removal, a more permanent solution to the problem is required. Nicotinamide is reported to lower serum phosphate by decreasing sodium-dependent phosphate co-transporters in the gut and kidney. However, its effectiveness in tumoral calcinosis remains unknown. In this study, we investigated nicotinamide as a potential therapy for tumoral calcinosis, using a murine model of the disease—Galnt3 knockout mice. Initially, five different doses of nicotinamide were given to normal heterozygous mice intraperitoneally or orally. Treatment had no effect on serum phosphate levels, but serum levels of a phosphaturic hormone, fibroblast growth factor 23 (Fgf23), decreased in a dose-dependent manner. Subsequently, high-dose nicotinamide (40mM) was tested in Galnt3 knockout mice fed a high phosphate diet. The radiographic data pre- and post-treatment showed that nicotinamide did not reverse the calcification. However, the treatment retarded calcification growth after 4weeks, while in the untreated animals, calcifications increased in size. The therapy did not affect serum phosphate levels, but intact Fgf23 decreased in the treated mice. The treated mice also had increased calcium in the heart. In summary, nicotinamide did not alter serum phosphate levels, likely due to compensatory decrease in Fgf23 to counteract the phosphate lowering effect of nicotinamide. Although increased calcium accumulation in the heart is a concern, the therapy appears to slow down the progression of ectopic calcifications.

Effect of Roscovitine on Intracellular Calcium Dynamics: Differential Enantioselective Responses

Cyclin-dependent kinases (CDKs) inhibitors have emerged as interesting therapeutic candidates. Of these, (S)-roscovitine has been proposed as potential neuroprotective molecule for stroke while (R)-roscovitine is currently entering phase II clinical trials against cancers and phase I clinical tests against glomerulonephritis. In addition, (R)-roscovitine has been suggested as potential antihypertensive and anti-inflammatory drug. Dysfunction of intracellular calcium balance is a common denominator of these diseases, and the two roscovitine enantiomers (S and R) are known to modulate calcium voltage channel activity differentially. Here, we provide a detailed description of short- and long-term responses of roscovitine on intracellular calcium handling in renal epithelial cells. Short-term exposure to (S)-roscovitine induced a cytosolic calcium peak, which was abolished after stores depletion with cyclopiazonic acid (CPA). Instead, (R)-roscovitine caused a calcium peak followed by a small calcium plateau. Cytosolic calcium response was prevented after stores depletion. Bafilomycin, a selective vacuolar H(+)-ATPase inhibitor, abolished the small calcium plateau. Long-term exposure to (R)-roscovitine significantly reduced the basal calcium level compared to control and (S)-roscovitine treated cells. However, both enantiomers increased calcium accumulation in the endoplasmic reticulum (ER). Consistently, cells treated with (R)-roscovitine showed a significant increase in SERCA activity, whereas (S)-roscovitine incubation resulted in a reduced PMCA expression. We also found a tonic decreased ability to release calcium from the ER, likely via IP3 signaling, under treatment with (S)- or (R)-roscovitine. Together our data revealed that (S)-roscovitine and (R)-roscovitine exert distinct enantiospecific effects on intracellular calcium signaling in renal epithelial cells. This distinct pharmacological profile can be relevant for roscovitine clinical use.

Neuroprotective effects of SNX-185 in an In Vitro model of TBI with a second insult

Second insults following traumatic brain injury (TBI), such as ischemia and hypoxia, significantly worsen outcome in patients and in experimental models of TBI. Following TBI there is a pathological increase in intracellular calcium, triggering cellular mechanisms of dysfunction and death. N-type specific voltage gated calcium channel (VGCC) blockers reduce cell death in both in vitro mechanical strain injury (MSI) and in vivo models of TBI, but they have not been previously explored in a model of TBI followed by a second insult. In the following studies, cortical neurons and astrocytes experienced MSI followed by incubation in 20% CO2. Cultures were treated with the N-type VGCC blocker, ω-conopeptide SNX-185 (1 μM), 5-minutes post-injury and intracellular calcium accumulation was assessed at 3, 6 and 24 h. Neuronal viability was assessed 24 h after MSI. Increasing incubator CO2 to 20% significantly increased calcium accumulation and cell death regardless of MSI severity. Treatment with 1 μM of SNX-185 significantly reduced the accumulation of calcium 3 hours following injury and increased the number of viable neurons 24 h post-injury and incubation in 20% CO2. In vitro models provide a critical tool for identifying roles of cell specific mechanisms involved in neuronal dysfunction and death following injury. These data demonstrate the potential of N-type VGCC blockers in reducing the damaging effects of TBI and second insults.

280 OSTEOGENIC ACTIVITY OF IN HOUSE-PRODUCED PORCINE BMP2 ON ADIPOSE-DERIVED STEM CELLS

In our laboratory, we extensively study the possibility of using adipose-derived stem cells (ASC) for maxillofacial bone regeneration. This includes also the tissue repair of large critical-size osteotomies requiring the use of tridimensional scaffolds. Bone regeneration in scaffolds can be greatly enhanced by the use of specific growth factors such as BMP2. In the present study, we compared the activity of commercially available human BMP2 (hBMP2) with in house-produced porcine BMP2 (pBMP2). The latter was synthesised using the BMP2 coding sequence from mRNA obtained from porcine ASC cell cultures. The coding sequence of the mature protein was cloned into a pET-21 plasmid and produced in E. coli as inclusion bodies. The activity of pBMP2 and hBMP2 was tested on ASC isolated from male pigs at passage 4 and at approximately 80% confluence in 48-well plates. Cells were treated in triplicate with hBMP2 or pBMP2 at 0.5, 5, 50, 500, or 1000 ng mL–1, adipogenic medium (AM), osteogenic medium (OM), or normal DMEM medium supplemented with acetic acid (used to resuspend BMP2 as the control) for 5 or 17 days. Cells were harvested for Alizarin Red S (AR) quantification and expression of osteogenic genes. For the AR analysis, cells were fixed with formalin and treated with AR. The AR was then extracted by acetic acid and neutralized with ammonium hydroxide before spectrophotometer reading at an absorbance of 420 nm. Data were analysed using GLM of SAS (SAS Institute Inc., Cary, NC, USA) with treatment, time, concentration, and all interactions as main effects. Using an inverted robotic stage microscope, images of the entire well for each replicate were taken every 2 to 3 days. Images revealed formation of osteogenic nodules in OM and characteristic large cells filled with lipid droplets in AM. No evident nodule formation was observed in the other treated cells at any time point. The AR was higher than control in both hBMP2 and pBMP2 at 0.5, 50, and 1000 ng mL–1 but not at 5 and 500 ng mL–1. There was no overall difference between hBMP2 and pBMP2 but the former had the highest AR value at 5 days in cells treated with 0.5 ng mL–1 and pBMP2 at 17 days with 1000 ng mL–1. Interestingly, both had higher values compared to OM, particularly at 5 days. We also observed an increase of AR due to time in cells treated with acetic acid (control). Overall, the data appear to indicate an increase in calcium accumulation in cells treated with both hBMP2 and pBMP2, with an early increase in the former and a late and larger increase in the latter. This might indicate a larger but slower activity of pBMP2 compared with hBMP2. The lack of formation of osteogenic nodules by both BMP2 might indicate an insufficiency of BMP2 to induce osteogenesis in porcine ASC. This last observation, together with the lack of increased AR accumulation compared with control at the 5 and 50 ng mL–1 doses, suggests the need for a more accurate analysis of BMP2 activity by measuring expression of BMP2-related genes. Finally, the data provide preliminary support for the equivalency of activity of pBMP2 and hBMP2 for in vivo bone regeneration.

KlMID1, a relevant key player between endoplasmic reticulum homeostasis and mitochondrial dysfunction in Kluyveromyces lactis

The interplay between calcium metabolism and glycosylation in yeast is largely unknown. In order to clarify this relationship, the effect of a mutation in the KlOCH1 gene, encoding the Golgi α-1,6-mannosyltransferase, on calcium homeostasis was studied in the yeast Kluyveromyces lactis. In particular, the role of the KlMID1 gene, encoding one of the components of the plasma membrane calcium channel (Cch1-Mid1), was investigated. Almost complete suppression of the phenotypes occurring in the mutant strain, ranging from oxidative stress to cell wall alteration, was observed by increased dosage of KlMID1. In addition, the N-glycosylation mutant showed increased calcium accumulation and decreased transcription of KlMID1 and KlCCH1. Moreover, the calcium alterations included an increased expressional profile for the KlPMC1 gene, encoding the vacuolar calcium ion pump. Furthermore, perturbation of endoplasmic reticulum (ER) homeostasis was observed in Kloch1-1 cells. Similarly, down-modulation of calcium signalling genes as well as altered mitochondrial functionality were induced in wild-type cells after treatment with DTT. However, no mitochondrial alteration occurred in the treated cells when KlMID1 was overexpressed. Our results suggest that the ER stress taking place in Kloch1-1 cells appears to be the primary cause of the KlMID1 down-modulation and its resulting effects on the expression of calcium homeostasis genes.

Differentiation of mesenchymal stem cells in heparin-containing hydrogels via coculture with osteoblasts

The therapeutic potency of delivered mesenchymal stem cells (MSCs) in tissue engineering applications may be improved by priming cells toward a differentiated state via coculture with native, differentiated cells prior to implantation; however, there is a lack of understanding in what may be the most efficacious method. The objective of this study was to investigate the role of negatively-charged heparin in priming hydrogel-encapsulated MSCs toward the osteoblastic lineage during coculture with a monolayer of osteoblasts in the absence of dexamethasone. MSCs encapsulated with higher amounts of heparin and cocultured with osteoblasts exhibited an over 36-fold increase in alkaline phosphatase activity and 13-fold increase in calcium accumulation by day 21, compared to MSCs cocultured with MSCs at the same heparin content. Moreover, hydrogels with higher amounts of heparin and cocultured with osteoblasts exhibited enhanced mineralization on the edges, suggesting that heparin may be important in sequestering osteoblast-secreted soluble factors, particularly on the surfaces of hydrogels. The ability of heparin to selectively interact with soluble positively-charged proteins from the surroundings was confirmed through protein labeling and microscopy. These results suggest that heparin-containing hydrogels as part of a coculture system can be utilized as a versatile platform to study and enhance priming of MSCs toward various cell types for a wide variety of regenerative medicine-based therapies.

Serotonin increases the functional activity of capsaicin-sensitive rat trigeminal nociceptors via peripheral serotonin receptors

Peripheral serotonin (5HT) has been implicated in migraine and temporomandibular pain disorders in humans and animal models and yet the mechanism(s) by which 5HT evokes pain remains unclear. Trigeminal pain can be triggered by activation of the transient receptor potential V1 channel (TRPV1), expressed by a subset of nociceptive trigeminal ganglia (TG) neurons and gated by capsaicin, noxious heat, and other noxious stimuli. As 5HT is released in the periphery during inflammation and evokes thermal hyperalgesia, and TRPV1 is essential for thermal hyperalgesia, we hypothesized that 5HT increases the activity of capsaicin-sensitive trigeminal neurons and that this increase can be attenuated by pharmacologically targeting peripheral 5HT receptors. TG cultures were pretreated with 5HT (10nM–100μM), sumatriptan (5HT1B/1D agonist), ketanserin (5HT2A antagonist), granisetron (5HT3 antagonist), or vehicle prior to capsaicin (30–50nM). Single-cell accumulation of intracellular calcium was recorded or calcitonin gene-related peptide (CGRP) release was measured following each treatment. In addition, using in situ hybridization and immunohistochemistry, we detected the colocalization of 5HT1B, 5HT1D, 5HT2A, and 5HT3A, but not 5HT2C mRNA with TRPV1 in TG cells. 5HT pretreatment evoked a significant increase in calcium accumulation in capsaicin-sensitive trigeminal neurons and enhanced capsaicin-evoked CGRP release, but had no significant effect when given alone. Sumatriptan, ketanserin, and granisetron treatment attenuated calcium accumulation and 5HT enhancement of capsaicin-evoked CGRP release. Together these results indicate that 5HT increases the activity of capsaicin-sensitive peripheral nociceptors, which can be attenuated by pharmacologically targeting peripheral 5HT receptors, thereby providing a mechanistic basis for peripheral craniofacial pain therapy.

Survival in end stage renal disease: calcium carbonate vs. sevelamer

There is concern regarding long-term excess calcium intake in end-stage renal disease populations. Because calcium carbonate is an over-the-counter (OTC) medication, few studies have been able to track its use. The Veterans Health Administration (VA) tracks national pharmacy data for both OTC and prescription drugs. We thus compared survival in incident dialysis patients on sevelamer and calcium carbonate phosphate binders. This was a retrospective cohort study of veterans initiating haemodialysis using existing VA databases. Patients were divided into calcium only (n = 769) and sevelamer only (n = 608) groups, then followed for up to 2 years until FY03 end. Survival was modelled using Cox regression adjusting for age, gender, race, marital status, service-connected disability, region, diabetes, hypertension and Charlson index. Stability of findings was examined using propensity score analysis. Sevelamer only vs. calcium only subjects were younger (respective mean ages 59.6 and 63.0, P < 0.001) with fewer comorbidities (Charlson index 3.8 and 4.5, P < 0.001). By study end, 24% of sevelamer and 30% of calcium subjects had died. Comparing sevelamer to calcium, the unadjusted hazard ratio for death was 0.62 (95% CI 0.50-0.76); the adjusted hazard ratio was 0.67 (CI 0.54-0.84). Propensity score analysis revealed similar results, with a hazard ratio of 0.65 (CI 0.54-0.80). In a national incident dialysis cohort, sevelamer treatment was associated with improved survival compared with calcium carbonate. Further research should investigate whether the worse survival with calcium is a long-term consequence of increased calcium accumulation.

In planta regulation of the Arabidopsis Ca2+/H+ antiporter CAX1

Vacuolar localized Ca(2+)/H(+) exchangers such as Arabidopsis thaliana cation exchanger 1 (CAX1) play important roles in Ca(2+) homeostasis. When expressed in yeast, CAX1 is regulated via an N-terminal autoinhibitory domain. In yeast expression assays, a 36 amino acid N-terminal truncation of CAX1, termed sCAX1, and variants with specific mutations in this N-terminus, show CAX1-mediated Ca(2+)/H(+) antiport activity. Furthermore, transgenic plants expressing sCAX1 display increased Ca(2+) accumulation and heightened activity of vacuolar Ca(2+)/H(+) antiport. Here the properties of N-terminal CAX1 variants in plants and yeast expression systems are compared and contrasted to determine if autoinhibition of CAX1 is occurring in planta. Initially, using ionome analysis, it has been demonstrated that only yeast cells expressing activated CAX1 transporters have altered total calcium content and fluctuations in zinc and nickel. Tobacco plants expressing activated CAX1 variants displayed hypersensitivity to ion imbalances, increased calcium accumulation, heightened concentrations of other mineral nutrients such as potassium, magnesium and manganese, and increased activity of tonoplast-enriched Ca(2+)/H(+) transport. Despite high in planta gene expression, CAX1 and N-terminal variants of CAX1 which were not active in yeast, displayed none of the aforementioned phenotypes. Although several plant transporters appear to contain N-terminal autoinhibitory domains, this work is the first to document clearly N-terminal-dependent regulation of a Ca(2+) transporter in transgenic plants. Engineering the autoinhibitory domain thus provides a strategy to enhance transport function to affect agronomic traits.

Long‐term loading inhibits ERK1/2 phosphorylation and increases FGFR3 expression in MC3T3‐E1 osteoblast cells

Bone tissue homeostasis relies upon the ability of cells to detect and interpret extracellular signals that direct changes in tissue architecture. This study utilized a four-point bending model to create both fluid shear and strain forces (loading) during the time-dependent progression of MC3T3-E1 preosteoblasts along the osteogenic lineage. Loading was shown to increase cell number, alkaline phosphatase (ALP) activity, collagen synthesis, and the mRNA expression levels of Runx2, osteocalcin (OC), osteopontin, and cyclo-oxygenase-2. However, mineralization in these cultures was inhibited, despite an increase in calcium accumulation, suggesting that loading may inhibit mineralization in order to increase matrix deposition. Loading also increased fibroblast growth factor receptor-3 (FGFR3) expression coincident with an inhibition of FGFR1, FGFR4, FGF1, and extracellular signal-related kinase (ERK)1/2 phosphorylation. To examine whether these loading-induced changes in cell phenotype and FGFR expression could be attributed to the inhibition of ERK1/2 phosphorylation, cells were grown for 25 days in the presence of the MEK1/2 inhibitor, U0126. Significant increases in the expression of FGFR3, ALP, and OC were observed, as well as the inhibition of FGFR1, FGFR4, and FGF1. However, U0126 also increased matrix mineralization, demonstrating that inhibition of ERK1/2 phosphorylation cannot fully account for the changes observed in response to loading. In conclusion, this study demonstrates that preosteoblasts are mechanoresponsive, and that long-term loading, whilst increasing proliferation and differentiation of preosteoblasts, inhibits matrix mineralization. In addition, the increase in FGFR3 expression suggests that it may have a role in osteoblast differentiation.

Intensity-related differences in collagen post-translational modification in MC3T3-E1 osteoblasts after exposure to low- and high-intensity pulsed ultrasound

Low-intensity pulsed ultrasound (LIPUS) has distinct effects on biologic mineralization at intensities of <100 mW/cm 2. Intensity-dependent differences in the pattern of accelerated mineralization may be due to different alterations in regulation of collagenous matrix formation. However, little is known about the influence of LIPUS on collagen metabolism in the context of mineralization processes. Therefore, we attempted to evaluate differential effects of two intensities of pulsed ultrasound (30 vs. 120 mW/cm 2) on collagen post-translational modification and mineralization in osteoblastic MC3T3-E1 cells. Murine osteoblastic MC3T3-E1 cells were exposed to pulsed ultrasound (1.5-MHz, 200-ms burst sine wave at 1.0-kHz frequency, either 30 or 120 mW/cm 2 SATA, for 20 min/day from Day 14 to Day 35 postconfluence). Expression patterns of lysyl oxidase (LO), procollagen-lysine, 2-oxyglutarate, 5-dioxigenase 1 (PLOD1, LH1), and 2 (PLOD2, LH2) was examined using quantitative PCR. Quantitative analysis of reducible immature cross-links (dihydroxylysinonorleucine, hydroxylysinonorleucine, and lysinonorleucine) and nonreducible mature cross-links (pyridinoline and deoxypyridinoline) as well as analysis of the maturation of immature to mature cross-links were performed. Exposure to 30 mW/cm 2 LIPUS upregulated LH2 mRNA expression and enzyme activity compared to controls. It was associated with increased relative amounts of telopeptidyl hydroxylysine (Hyl)-derived cross-links beginning on Day 14, upregulated LO mRNA expression, increased total reducible and nonreducible cross-links, and increased ratios of newly formed nonreducible to reducible cross-links. Similarities in the pattern of cross-link formation and calcium deposition in matrices between 30 mW/cm 2 LIPUS-treated MC3T3-E1 cultures and bone suggest that 30 mW/cm 2 LIPUS may promote the maturation of collagenous matrix as a scaffold for calcification. In contrast, exposure to 120 mW/cm 2 ultrasound increased calcium accumulation compared to control at Day 35, but increases were delayed until Day 25. No differences in the extent and pattern of cross-links were observed compared to controls. These results suggest that the promotion of mineralization induced by 120 mW/cm 2 may be attributed to other factors involved in mineralization process rather than cross-link pattern. Our results demonstrated the existence of differential effects of lower versus higher intensities of ultrasound on mineralization processes in vitro.

The transcription factor SOX9 regulates cell cycle and differentiation genes in chondrocytic CFK2 cells.

SOX9 is a transcription factor that is essential for chondrocyte differentiation and cartilage formation. We stably overexpressed SOX9 cDNA in the rat chondrocytic cell line CFK2. Compared with the vector control, a greater proportion of SOX9-transfected cells accumulated in the G0/G1 phase. This was associated with an increase in mRNA and protein expression of p21(cip1), an inhibitor of cyclin-dependent kinase activity. SOX9 enhanced p21(cip1) promoter activity in a luciferase reporter assay. CFK2 cells overexpressing SOX9 became more elongated and adhesive and demonstrated a shift in cytoplasmic F-actin distribution. N-cadherin mRNA levels were elevated in the SOX9-transfected cells, and SOX9 enhanced N-cadherin promoter activity. By electrophoretic mobility shift assay, nuclear extracts of SOX9-transfected CFK2 cells specifically bound an oligonucleotide comprising an N-cadherin promoter region containing a consensus SOX9-binding motif. The transcriptional activity of SOX9 depended upon nuclear localization signals required for SOX9 nuclear entry. Differentiation of transfected CFK2 cells was accelerated as evidenced by more rapid accumulation of alkaline phosphatase activity, increased production of proteoglycans, and increased calcium accumulation, and this was associated with decreased ERK1 expression. These studies demonstrate that SOX9 alters the rate of cell cycle progression of chondrocytes and their differentiation by enhancing or inhibiting the expression of selected genes, including p21(cip1) and ERK1, and that N-cadherin is an additional direct target of this transcriptional regulator.

N-3 Polyunsaturated Fatty Acids and Bone Metabolism

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), n-3 polyunsaturated fatty acid (n-3PUFA), have been shown to have beneficial effects on what? The beneficial effects of n-3PUFA, especially EPA on bone metabolism in vitro and in vivo were examined in this study.Osteoblasts are bone forming cells, that produce prostaglandin E2, an endogenous potent bone resorbing factor. To clarify the effects of EPA on bone metabolism, the effects of EPA on MC3T3-E1 cells, osteoblast-like cells were studied. The addition of MC3T3-E1 cells to EPA, but not DHA, caused increased calcium accumulation and Von-Kossa staining. In vivo experiments, the effect of EPA on strength of the femur in ovariectomized and low calcium/vitamin D3 fed rats were studied. EPA abolished the effects of ovariectomy with consequent recovery of the bone strength.Preliminary epidemiology studies indicate EPA ingestion possibly useful for preventing loss of bone weight and strength with aging. These findings and epidemiological studies, demonstrate EPA and marine oil to possibly have beneficial effects for preventing and improving age-related osteoporosis

Antimony trichloride induces DNA damage and apoptosis in mammalian cells

Antimony compounds are widely used in various manufacturing and semiconducting industries. Previously, it has been shown that antimony trichloride (SbCl 3) elevates sister chromatid exchange (SCE) rates in V79 cells after a 28-h incubation. However, only limited data on its genotoxic effects are available so far. The present results demonstrate that a 4-h exposure to >50 μM SbCl 3 could induce micronuclei (MN) formation in cultured Chinese hamster ovary (CHO-K1) cells, human bronchial epithelial (BES-6) cells and human fibroblasts (HF). The order of sensitivity to SbCl 3 determined by Sulforhodamine B (SRB)-staining survival assay is HF>BES-6 cells>CHO-K1 cells, with LD 50 values in these cells being ≈40, 80 and 180 μM, respectively. Apoptosis and DNA fragmentation was not found in cells immediately following 4-h SbCl 3 treatment. However, DNA fragmentation was detected in CHO-K1 cells after 4-h SbCl 3 treatment and a 16 h or more post incubation in fresh medium by 1.5% agarose gel electrophoresis. The delayed apoptosis was also observed under microscopic examination in HF, BES-6 and CHO-K1 cells after similar treatment protocol. In addition, an increase in calcium accumulation appeared in CHO-K1 cells and HF immediately after a 4-h SbCl 3 treatment, or after a 24-h post incubation in fresh medium. The present results provide important genotoxic and cytotoxic information characterizing the cellular changes induced by short-term SbCl 3 exposure in rodent and human cells.

Effects of isoproterenol and suphan on the concentration of free calcium ions in isolated cardiomyocytes: a comparative study

Isoproterenol and suphan, two cardioactive drugs with different mechanisms of action, are studiedin vitro for their effects on calcium homeostasis in myocardial cells. Isoproterenol lowers the basal Ca2+ level in resting cardiomyocytes and potentiates its rise in these cells after their induction. Suphan stimulates reversible elevation of the diastolic Ca2+ concentration, causing increased calcium accumulation in the sarcoplasmic reticulum of cardiomyocytes. In anin vitro model of hypoxia, the Ca response to isoproterenol is significantly reduced, whereas that to dibutyryl cAMP is retained. The effect of suphan on the Ca2+ content of cardiomyocytes exposed to “chemical” hypoxia is 30–50% higher than its effect on the Ca2+ content of intact cells.

Magnesium cardioplegia enhances mRNA levels and the maximal velocity of cytochrome oxidase I in the senescent myocardium during global ischemia.

The aged myocardium accumulates significantly more cytosolic calcium [Ca2+]i during ischemia, and functional recovery is more severely compromised as compared with the mature heart. Cardioplegia ameliorates these phenomena. The mechanism by which increased calcium accumulation reduces functional recovery in the senescent myocardium is unknown, but it has been suggested that futile calcium cycling in the mitochondria leading to depletion of ATP stores during normothermic global ischemia may be involved. To investigate the effect of cardioplegia on mitochondrial calcium ([Ca2+]mt) accumulation and the expression of cytochrome oxidase I (COX I) during global ischemia, mitochondria were isolated from mature (age, 15 to 20 weeks) and aged (age > 130 weeks) rabbit hearts after Langendorff perfusion. Five perfused heart groups were investigated: 30 minutes of global ischemia without treatment (control), with potassium (K, 20 mmol/L), magnesium (Mg, 20 mmol/L), or potassium and magnesium (K/Mg) cardioplegia. No significant difference in [Ca2+]mt was evident in mature hearts with any protocol. In aged hearts, [Ca2+]mt was increased in global ischemia but was ameliorated with Mg and K/Mg cardioplegia. COX I mRNA levels in aged hearts were lower in both control and global ischemia but were increased with cardioplegia. Maximal velocities for COX I were significantly increased with Mg cardioplegia both in the mature and the aged myocardium. K and/or Mg cardioplegia ameliorates [Ca2+]mt accumulation in aged hearts during normothermic global ischemia and increases COX I mRNA levels to a level not significantly different from that found in mature hearts.

P28. Phylloquinone (vitamin K 1) content of bone in young adults

Low-intensity pulsed ultrasound (LIPUS) has distinct effects on biologic mineralization at intensities of <100 mW/cm2. Intensity-dependent differences in the pattern of accelerated mineralization may be due to different alterations in regulation of collagenous matrix formation. However, little is known about the influence of LIPUS on collagen metabolism in the context of mineralization processes. Therefore, we attempted to evaluate differential effects of two intensities of pulsed ultrasound (30 vs. 120 mW/cm2) on collagen post-translational modification and mineralization in osteoblastic MC3T3-E1 cells. Murine osteoblastic MC3T3-E1 cells were exposed to pulsed ultrasound (1.5-MHz, 200-ms burst sine wave at 1.0-kHz frequency, either 30 or 120 mW/cm2 SATA, for 20 min/day from Day 14 to Day 35 postconfluence). Expression patterns of lysyl oxidase (LO), procollagen-lysine, 2-oxyglutarate, 5-dioxigenase 1 (PLOD1, LH1), and 2 (PLOD2, LH2) was examined using quantitative PCR. Quantitative analysis of reducible immature cross-links (dihydroxylysinonorleucine, hydroxylysinonorleucine, and lysinonorleucine) and nonreducible mature cross-links (pyridinoline and deoxypyridinoline) as well as analysis of the maturation of immature to mature cross-links were performed. Exposure to 30 mW/cm2 LIPUS upregulated LH2 mRNA expression and enzyme activity compared to controls. It was associated with increased relative amounts of telopeptidyl hydroxylysine (Hyl)-derived cross-links beginning on Day 14, upregulated LO mRNA expression, increased total reducible and nonreducible cross-links, and increased ratios of newly formed nonreducible to reducible cross-links. Similarities in the pattern of cross-link formation and calcium deposition in matrices between 30 mW/cm2 LIPUS-treated MC3T3-E1 cultures and bone suggest that 30 mW/cm2 LIPUS may promote the maturation of collagenous matrix as a scaffold for calcification. In contrast, exposure to 120 mW/cm2 ultrasound increased calcium accumulation compared to control at Day 35, but increases were delayed until Day 25. No differences in the extent and pattern of cross-links were observed compared to controls. These results suggest that the promotion of mineralization induced by 120 mW/cm2 may be attributed to other factors involved in mineralization process rather than cross-link pattern. Our results demonstrated the existence of differential effects of lower versus higher intensities of ultrasound on mineralization processes in vitro.