OP Cells Research Articles

Design, synthesis, and biological evaluation of pyrido[2,3-d]pyrimidin-7(8H)-one derivatives as potent USP1 inhibitors

USP1 has emerged as a novel and potential target for drug discovery in single therapeutic agents or combination with chemotherapy and molecular targeted therapy. In this study, based on the disclosed structure of ML323 and KSQ-4279, we designed and synthesized a series of pyrido[2,3-d]pyrimidin-7(8H)-one derivatives as potent USP1 inhibitors by cyclization strategy and the systematic structure−activity relationship exploration was conducted. The representative compounds 1k, 1m and 2d displayed excellent USP1/UAF inhibition and exhibited strong antiproliferation effect in NCI–H1299 cells. Further flow cytometry analysis revealed that they could arrest breast cancer cells MDA-MB-436 in the S phase. Inhibition mechanism study of compound 1m indicated these derivatives acted as reversible and noncompetitive USP1 inhibitors. Of note, the combination of compound 1m with PARP inhibitor olaparib generated enhanced cell killing in olaparib-resistant MDA-MB-436/OP cells, and compound 1m exhibited excellent oral pharmacokinetic properties in mice. Overall, our efforts may provide a reliable basis for the development of novel USP1 inhibitor as a single therapeutic agent and in combination with PARP inhibitors.

Osteogenic capacity of mesenchymal stem cells from patients with osteoporotic hip fractures in vivo

ABSTRACT Purpose Human mesenchymal stem cells (MSCs) have the ability to differentiate into bone-forming osteoblasts. The aim of this study was to elucidate if MSCs from patients with OP show a senescent phenotype and explore their bone-forming ability in vivo. Materials and methods MSCs from patients with OP and controls with osteoarthritis (OA) were implanted into the subcutaneous tissue of immunodeficient mice for histological analysis and expression of human genes by RT-PCR. The expression of senescence-associated phenotype (SASP) genes, as well as p16, p21, and galactosidase, was studied in cultures of MSCs. Results In vivo bone formation was evaluated in 103 implants (47 OP, 56 OA). New bone was observed in 45% of the implants with OP cells and 46% of those with OA cells (p = 0.99). The expression of several bone-related genes (collagen, osteocalcin, alkaline phosphatase, sialoprotein) was also similar in both groups. There were no differences between groups in SASP gene expression, p16, and p21 expression, or in senescence-associated galactosidase activity. Conclusion Senescence markers and the osteogenic capacity in vivo of MSCs from patients with OP are not inferior to that of cells from controls of similar age with OA. This supports the interest of future studies to evaluate the potential use of autologous MSCs from OP patients in bone regeneration procedures.

The active role of osteoporosis in the interaction between osteoblasts and bone metastases

IntroductionTo minimize the severity of bone metastases and to delay their onset, it is important to analyze the underlying biological mechanisms. The present study focused on the link between OP and metastatic cells, with particular attention to osteoblast behavior. MethodsOsteoblasts (OB) were isolated from the trabecular bone of iliac crest of healthy (SHAM) and ovariectomized (OVX) adult female rats and co-cultured with MRMT-1 rat breast carcinoma cells as conditioned medium (CM) or alone (CTR) for 24h, 7 and 14days and tested for cell viability, morphology and synthetic activity, i.e. C-terminal procollagen type I, alkaline phosphatase, osteoprotegerin, receptor activator for nuclear factor KB ligand and interleukin-8. ResultsOsteoblast morphology showed a reduced organization in the OVX group, in particular in the CM condition. Conversely, the analysis of cell viability revealed significantly higher values in the OVXCM group with respect to the SHAMCM group at all experimental times, whereas the OVXCTR group had significantly lower values at 7 and 14days in comparison to those of the SHAM group. ALP release was significantly lower in the CM condition than that of CTR at all timepoints, and so was procollagen type I at 7 and 14days. The RANKL/OPG ratio showed significantly higher values in OVX osteoblasts in comparison with those of the SHAM group, both in CTR and in CM conditions at each experimental time. Finally, OVXCM showed significantly higher values of IL-8 than those of SHAMCM at 7 and 14days. ConclusionsThe results clearly indicate an influence of the metastatic cells on the osteoblastic physiology at different levels: morphology, viability, release of typical proteins, and also IL-8 as a proinflammatory cytokine, especially marked by osteoporosis. Further investigations might highlight the relationship between osteoblasts and breast cancer cells, which might be useful to improve common drugs used against osteoporosis and bone metastases, by enhancing the bone deposition/tumor progression ratio.

Effects of stromal cells on sentitivity to imatinib in Sup-B15 Philadelphia chromosome positive acute lymphoblastic leukemia cells

目的探讨基质细胞OP9能否影响Ph+急性淋巴细胞白血病细胞株Sup-B15细胞对伊马替尼的敏感性以及作用机制。方法实验分为Sup-B15细胞组及基质细胞OP9与Sup-B15共培养组（即Sup-B15/OP9组）。采用CCK-8法测定细胞增殖抑制率；流式细胞术检测Annexin V/7-AAD标记的细胞凋亡率、CD34+CD38−细胞比例；荧光定量PCR检测细胞ALDH1、CD144、β-catenin mRNA水平；Western blot法检测细胞CD133、CD144、β-catenin蛋白表达水平；免疫共沉淀方法检测结合于CD144的β-catenin水平。结果10~45 µmol/L伊马替尼对Sup-B15、Sup-B15/OP9细胞均有增殖抑制作用，其IC50值分别为26.3和35.8 µmol/L，两组差异有统计学意义（P<0.05）。30 µmol/L伊马替尼作用于Sup-B15、Sup-B15/OP9细胞24 h，细胞总凋亡率分别为（14.24±2.11）%和（3.45±0.68）%，两组差异有统计学意义（P<0.05）。与Sup-B15组相比，Sup-B15/OP9组CD34+CD38−细胞比例增高[（3.42±0.28）%对（2.16±0.15）%，P<0.05]，ALDH1 mRNA水平增高（0.097±0.012对0.046±0.010，P<0.05），CD133蛋白表达水平升高；CD144 mRNA水平增高（0.103±0.015对0.010±0.003，P<0.05），CD144蛋白表达水平亦明显增高；β-catenin mRNA水平无明显变化（P>0.05），其蛋白总量明显上升，转位到细胞核的β-catenin蛋白明显增加，结合于CD144的β-catenin蛋白增加。结论与OP9细胞共培养后，Sup-B15细胞对伊马替尼敏感性下降，该作用可能与上调CD144表达、稳定CD144 /β-catenin信号通路、增加β-catenin核转位有关。

Surgeon graft less viable endothelial cells than the eye bank cell count suggests

AbstractPurpose it is universally admitted that a very early important postoperative decrease in ECD occurs after all types of corneal graft, generally attributed to surgical traumatism. This presumed cell loss is calculated between eye bank ECD (ebECD) and post op cell count done in recipients by specular microscopy (SM). Aim: to redefine the very early postop cell loss.Methods 1/Prospective series of consecutive penetrating keratoplasty for all‐comer indications with post‐op follow‐up by SM (SP3000‐P) performed as soon as possible and at least at D15, M1 and M3. This series provided reasonable quantification of the gap between ebECD and very early postop ECD in patients. 2/Ten pairs of corneas with comparable left/right ebECD were randomized: one cornea was grafted, the other was stored similarly and its viable ECD was measured after triple labelling with Hoechst/Ethidium/calcein‐AM (IOVS2011 Pipparelli) and quantification with CorneaJ (Cornea2014 Bernard). Graft and vECD measurement were done simultaneously. Relationship between vECD and early postop ECD were studied.Results For series 1, a gap of 623 (98‐1479) cells/mm2 (median (range)) between ebECD and early postop was measured. For series 2, vECD and early postop ECD of paired corneas were well correlated (r=0.7, P&lt;0.05), with a difference of only 143 (5‐359) cells between them.Conclusion These 2 complementary series demonstrated that ebECD provided to surgeons overestimate the number of viable CEC grafted to patients. Taking account only the vECD at the end of storage (just before graft) and the postop ECD measured as early as possible, we show that the presumed initial very important cell loss is fictive and that the model of postop ECD decrease should be revisited.

DESIGN AND SIMULATION OF INDIUM GALLIUM NITRIDE MULTIJUNCTION TANDEM SOLAR CELLS

As our global energy expenditure increases exponent ially, it is apparent that renewable energy solutio n must be utilized. Solar PV technology is the best way to utilize the unlimited solar energy. The InGaN is a recently developed no vel solar cell material for its promising tunable band gap of 0.7 eV to 3.4 eV for the realization of high efficiency tandem solar cel ls in space and terrestrial applications. In this work, various numerical simul ations were performed using Analysis of Microelectr onic and Photonic Structure (AMPS) simulator to explore the possibility of high er efficiency of InGaN based solar cells. At these aim three different types of InGaN based solar cells (single junction, double ju nction and triple junction) were designed and optim ized. Numerical simulations were done with different band gap of InGaN material and found that maximum efficiency occurs around 1. 2 eV to 1.5 eV, it has been optimized at 1.34 eV for (single junction solar cel l) with maximum conversion efficiency of 25.02%. Th e single junction solar cell were simulated and optimized for optimum thickness of p-layer and n-layer. Doping concentration and ba ck contact material of the designed cells were investigated and found that 1◊1 0 16 cm -3 of doping concentration for both p and n type material and Nical as back contact with ΦbL of 1.3 eV are best fitted for higher conversion ef ficiency. From tunable band gap of In xGa 1-xN material, selection has been done at 1.61 eV, 1.44 eV and 1.21 eV for the t op, middle and bottom cells respectively for the ta ndem triple junction and double junction (1.61 eV, 1.21 eV) solar cells. The best c onversion efficiency of the single junction, double junction and triple junction solar cells are 25.019%, 35.45% and 42.34% respectively. Effect of tunnel junction for the tandem cells also investigated and found that required thickness for tunnel junction is around 25 nm with doping concentration, N A and N D of 1◊10 19 and 1◊10 16 respectively were found in this analysis. Finally, the temperature co efficient (TC) of the above proposed cells were sim ulated to investigate the thermal stability of the proposed cells. It has been found that the TC of InGaN cells is about -0.04%/°C, whic h indicate the higher stability of the proposed cells.

Paper 101: Use of Tranaxemic Acid vs. Post Op Cell Salvage in Reducing Blood Loss After Total Knee Replacement Surgery

Paper 101: Use of Tranaxemic Acid vs. Post Op Cell Salvage in Reducing Blood Loss After Total Knee Replacement Surgery

Purified Mesenchymal Stem Cells Are an Efficient Source for iPS Cell Induction

BackgroundInduced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by the forced expression of defined transcription factors. Although most somatic cells are capable of acquiring pluripotency with minimal gene transduction, the poor efficiency of cell reprogramming and the uneven quality of iPS cells are still important problems. In particular, the choice of cell type most suitable for inducing high-quality iPS cells remains unclear.Methodology/Principal FindingsHere, we generated iPS cells from PDGFRα+ Sca-1+ (PαS) adult mouse mesenchymal stem cells (MSCs) and PDGFRα− Sca-1− osteo-progenitors (OP cells), and compared the induction efficiency and quality of individual iPS clones. MSCs had a higher reprogramming efficiency compared with OP cells and Tail Tip Fibroblasts (TTFs). The iPS cells induced from MSCs by Oct3/4, Sox2, and Klf4 appeared to be the closest equivalent to ES cells by DNA microarray gene profile and germline-transmission efficiency.Conclusions/SignificanceOur findings suggest that a purified source of undifferentiated cells from adult tissue can produce high-quality iPS cells. In this context, prospectively enriched MSCs are a promising candidate for the efficient generation of high-quality iPS cells.

Plasma human papillomavirus (HPV) DNA as a potential tool for tumor detection and monitoring response in HPV-related oropharyngeal carcinoma (OP).

5529 Background: There is a rising incidence of HPV-related OP, which has a distinct prognosis. Previous studies in other viral-related cancers such as nasopharyngeal carcinoma have shown that the level of circulating instigating virus DNA in the blood can be used to monitor treatment response and tumor relapse. The main objective of this study is to develop a similar assay for HPV(+) OP. Methods: We designed a semiquantitative PCR approach to detect circulating HPV DNA using the L1 Gp5/6 degenerate primer as well as HPV16/18 E6 and E7 nested primers. We used this approach to detect HPV DNA in culture media of SCC90, a HPV(+) OP cell line as well as plasma samples of SCC90 bearing SCID mice. Control mice were those implanted with non-HPV tumors. HPV DNA was also assessed in pretreatment plasma samples of 32 HPV(+) OP patients and 4 HPV(-) control patients with other head and neck squamous cell carcinoma. HPV status within the tumor was confirmed by both p16 staining and HPV16/18 PCR (N = 30) or in-situ hybridization (n = 2). Plasma HPV DNA levels were also quantified by a fluorescent probe multiplex real-time PCR (QRTPCR) assay, targeting the HPV16/18 E6 region, in 6 patients with HPV(+) OP and serial plasma samples obtained during radiotherapy (RT). Results: Cell-free HPV DNA was detected in the culture media of SCC90 and the plasma of SCC90 bearing mice but not in control mice. It was detected in pretreatment plasma of 84% HPV(+) patients and in none of the HPV(-) controls using the L1 primers and conventional PCR. However using less sensitive primer sets (nested E6 and E7), plasma HPV DNA was positive in only 50% of patients with detectable E6/E7 DNA in the tumors. QRTPCR was able to detect as low as 10 copies of HPV DNA in each reaction. In 6 patients with serial plasma samples during RT, QRTPCR measurement showed a rapid decline in HPV DNA level, which became undetectable at RT completion. Conclusions: The xenograft studies indicate that plasma HPV DNA is released directly from HPV-related tumors. The high sensitivity of this method allows detection of HPV DNA in plasma even with low viral load. Plasma HPV DNA may be a valuable tool for monitoring therapeutic response and disease progression in HPV(+) OP. No significant financial relationships to disclose.

Transgenic mice expressing a cameleon fluorescent Ca2+ indicator in astrocytes and Schwann cells allow study of glial cell Ca2+ signals in situ and in vivo

Glial cell Ca2+ signals play a key role in glial–neuronal and glial–glial network communication. Numerous studies have thus far utilized cell-permeant and injected Ca2+ indicator dyes to investigate glial Ca2+ signals in vitro and in situ. Genetically encoded fluorescent Ca2+ indicators have emerged as novel probes for investigating cellular Ca2+ signals. We have expressed one such indicator protein, the YC 3.60 cameleon, under the control of the S100β promoter and directed its expression predominantly in astrocytes and Schwann cells. Expression of YC 3.60 extended into the entire cellular cytoplasmic compartment and the fine terminal processes of protoplasmic astrocytes and Schwann cell Cajal bands. In the brain, all the cells known to express S100β in the adult or during development, expressed YC 3.60. While expression was most extensive in astrocytes, other glial cell types that express S100β, such as NG2 and CNP-positive oligodendrocyte progenitor cells (OP cells), microglia, and some of the large motor neurons in the brain stem, also contained YC 3.60 fluorescence. Using a variety of known in situ and in vivo assays, we found that stimuli known to elicit Ca2+ signals in astrocytes caused substantial and rapid Ca2+ signals in the YC 3.60-expressing astrocytes. In addition, forepaw stimulation while imaging astrocytes through a cranial window in the somatosensory cortex in live mice, revealed robust evoked and spontaneous Ca2+ signals. These results, for the first time, show that genetically encoded reporter is capable of recording activity-dependent Ca2+ signals in the astrocyte processes, and networks.

Phenotypic and genetic differences between opaque and translucent colonies of Vibrio alginolyticus

Many pathogens undergo phase variation between rugose and smooth colony morphology or between opaque and translucent colony morphology, which is mainly due to the variation in the surface polysaccharides. In this study, Vibrio alginolyticus ZJ-51 displayed phase variation between opaque, rugose colonies (Op) and translucent, smooth colonies (Tr). Unlike the vibrios reported previously, Tr cells of ZJ-51 enhanced biofilm formation and motility, but they did not differ from Op cells in the quantity of surface polysaccharides produced. Real time PCR was used to analyze the expression of the genes involved in polysaccharide biosynthesis, flagellar synthesis, and the AI-2 quorum-sensing system. The results revealed that the K-antigen capsule gene cluster (which consists of homologs to the cpsA-K in Vibrio parahaemolyticus) and O-antigen polysaccharide gene cluster (which contains homologs to the wza-wzb-wzc) were significantly more transcribed in Tr cells. The AI-2 quorum-sensing genes showed enhanced expression in the Tr variant which also exhibited greater expression of genes associated with polar flagellar biosynthesis. These results suggest that colony phase variation might affect the virulence and survival ability in the stressful environment inhabited by V. alginolyticus.

Generation of laser-polarized xenon using fiber-coupled laser-diode arrays narrowed with integrated volume holographic gratings

Volume holographic gratings (VHGs) can be exploited to narrow the spectral output of high-power laser-diode arrays (LDAs) by nearly an order of magnitude, permitting more efficient generation of laser-polarized noble gases for various applications. A ∼3-fold improvement in 129Xe nuclear spin polarization, P Xe, (compared to a conventional LDA) was achieved with the VHG-LDA’s center wavelength tuned to a wing of the Rb D 1 line. Additionally, an anomalous dependence of P Xe on the xenon density within the OP cell is reported—including high P Xe values (>10%) at high xenon partial pressures (∼1000 torr).

15d-PGJ2 induces apoptosis of mouse oligodendrocyte precursor cells

BackgroundProstaglandin (PG) production is associated with inflammation, a major feature in multiple sclerosis (MS) that is characterized by the loss of myelinating oligodendrocytes in the CNS. While PGs have been shown to have relevance in MS, it has not been determined whether PGs have a direct effect on cells within the oligodendrocyte lineage.MethodsUndifferentiated or differentiated mouse oligodendrocyte precursor (mOP) cells were treated with PGE2, PGF2α, PGD2 or 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2). Cell growth and survival following treatment were examined using cytotoxicity assays and apoptosis criteria. The membrane receptors for PGD2 and the nuclear receptor peroxisome proliferator-activated receptor (PPAR)γ, as well as reactive oxygen species (ROS) in the death mechanism were examined.ResultsPGE2 and PGF2α had minimal effects on the growth and survival of mOP cells. In contrast, PGD2 and 15d-PGJ2 induced apoptosis of undifferentiated mOP cells at relatively low micromolar concentrations. 15d-PGJ2 was less toxic to differentiated mOP cells. Apoptosis was independent of membrane receptors for PGD2 and the nuclear receptor PPARγ. The cytotoxicity of 15d-PGJ2 was associated with the production of ROS and was inversely related to intracellular glutathione (GSH) levels. However, the cytotoxicity of 15d-PGJ2 was not decreased by the free radical scavengers ascorbic acid or α-tocopherol.ConclusionTaken together, these results demonstrated that 15d-PGJ2 is toxic to early stage OP cells, suggesting that 15d-PGJ2 may represent a deleterious factor in the natural remyelination process in MS.

Synergistic induction of cyclin D1 in oligodendrocyte progenitor cells by IGF‐I and FGF‐2 requires differential stimulation of multiple signaling pathways

D-type cyclins are direct targets of extracellular signals and critical regulators of G(1) progression. Our previous data demonstrated that IGF-I and FGF-2 synergize to enhance cyclin D1 expression, cyclin E/cdk2 complex activation, and S-phase entry in OP cells. Here, we provide a mechanistic explanation for how two growth factor signaling pathways converge on a major cell cycle regulator. IGF-I and FGF-2 differentially activate signaling pathways to coordinately promote cyclin D1 expression. We show that the p44/p42 MAPK signaling pathway is essential for FGF-2 induction of cyclin D1 mRNA. In contrast, blocking the PI3-Kinase pathway results in loss of IGF-I/FGF-2 synergistic induction of cyclin D1 protein levels. Moreover, the presence of IGF-I significantly enhances nuclear localization of cyclin D1, which also requires PI3K signaling. GSK-3beta, a downstream target of the PI3K/Akt pathway, is phosphorylated in the presence of IGF-I in OPs. Consistent with a known role for GSK-3beta in cyclin D1 degradation, we show that proteasome inhibition in OPs exposed to FGF-2 increased cyclin D1 levels, equivalent to levels seen in IGF-I/FGF-2 treated cells. Thus, we provide a model for cyclin D1 coordinate regulation where FGF-2 stimulation of the MAPK pathway promotes cyclin D1 mRNA expression while IGF-I activation of the PI3K pathway inhibits proteasome degradation of cyclin D1 and enhances nuclear localization of cyclin D1.

Long-term culture in dexamethasone unmasks an abnormal phenotype in osteoblasts isolated from osteoporotic subjects

We have shown that osteoblastic cells derived from trabecular bone explants of osteoporotic subjects (OP cells) exhibited an altered alkaline phosphatase (ALP) response to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] compared to control (CON) cells. Our hypothesis that OP cells have other intrinsic abnormalities was investigated using our cell models representing two different stages of differentiation. OP and CON cells were cultured in the absence (-DEX) or presence (+DEX) of 10 nM dexamethasone (DEX) in 10% fetal calf serum (FCS) prior to exposure to serum-free medium containing 1 nM of PTH and/or 17-beta estradiol (E2). Both OP and CON cells responded to DEX with a two-fold increase in basal ALP activity. While E2 or PTH+E2 had no effect on OP cells, both treatments inhibited ALP activity in CON cells (p<0.05). OP and CON cells grown in DEX also expressed PTH-stimulated adenylate cyclase (AC) activities higher than those of (-DEX) cells. OP+DEX cells, however, exhibited activities which were 8-fold higher than those of CON+DEX cells (p<0.001). In OP+DEX cells, E2 stimulated basal AC activity (p<0.05) but did not affect PTH-stimulated activity. In contrast, in CON+DEX cells, E2 had no effect on basal activity but inhibited PTH-stimulated AC activity (p<0.001). Osteocalcin production was 4-fold lower in OP+DEX cells compared to OP-DEX and CON cells (p<0.05) while osteocalcin mRNA levels were significantly lower in OP+DEX and CON+/-DEX cells compared to OP-DEX cells (p<0.05). E2 did not affect osteocalcin protein or mRNA levels in either OP or CON cells. No differences in mRNA levels were found for estrogen receptor-alpha (ER-a) in OP+/-DEX cells whereas these levels were significantly higher in CON+DEX compared to CON-DEX cells (p<0.05). These results indicate that DEX amplified the differences between OP and CON cells and confirm the presence of intrinsic osteoblastic abnormalities in patients with osteoporosis that persist in culture.

29. RhBMP-6 exposed osteoprogenitor cell implantation time: does it make a difference in achieving spinal fusion?

HYPOTHESIS: We have used the New Zealand white (NZW) rabbit posterolateral intertransverse fusion model to explore a cell-based therapy aimed at improving spinal fusion rates.Our purpose was to optimize the spinal fusion rate of rhBMP-6 exposed osteoprogenitor cells (rhBMP-6 OP cells) by delaying their implantation and developing a new method of delivery to the fusion site.

Regulation of Kv1 subunit expression in oligodendrocyte progenitor cells and their role in G 1 /S phase progression of the cell cycle

Proliferative oligodendrocyte progenitor cells (OPs) express large, delayed outward-rectifying K(+) currents (I(K)), whereas nondividing immature and mature oligodendrocytes display much smaller I(K). Here, we show that up-regulation of I(K) occurs in G(1) phase of the cell cycle in purified cultured OPs and is the result of an RNA synthesis-dependent, selective increase of the K(+) channel subunit proteins Kv1.3 and Kv1.5. In oligodendrocyte cells acutely isolated from developing rat brain, a decrease of cyclin D expression is observed as these cells mature along their lineage. This is accompanied by a decrease in Kv1.3 and Kv1.5 subunit expression, suggesting a role for these subunits in the proliferative potential of OPs in situ. I(K) expressed in OPs in subventricular zone and developing white matter in acutely isolated slice preparations were selectively blocked by antagonists of Kv1.3, illustrating the functional presence of this subunit in situ. Interestingly, Kv1.3 block inhibited S-phase entry of both purified OPs in culture and in tissue slice cultures. Thus, we employ both in vitro and in situ experimental approaches to show that (i) RNA-dependent synthesis of Kv1.3 and Kv1.5 subunit proteins occurs in G(1) phase of the OP cell cycle and is responsible for the observed increase in I(K), and (ii) currents through Kv1.3-containing channels play a crucial role in G(1)/S transition of proliferating OPs.

Early cell adhesion events differ between osteoporotic and non-osteoporotic osteoblasts

In osteoporosis, the regenerative capacity of bone is compromised, which may involve altered osteoblast (OB) activity. This could be attributed to an inappropriate synthesis and assembly of an extracellular matrix (ECM), altered cell adhesion to the ECM, or be due to inappropriate downstream activation of adhesion-mediated signaling cascades through proteins such as focal adhesion kinase (FAK). The purpose of our study was to compare early adhesion-mediated events using previously described and characterized clinically derived OBs obtained from human patients undergoing major joint arthroplasty for osteoporosis or osteoarthritis. The presence or absence of osteoporosis was established with a radiographic index. Using light microscopy and crystal violet staining, we show that OB cells derived from sites of osteoporosis do not attach and spread as well as non-osteoporotic (OP) OB cells. OP cells initially have a more rounded morphology, and show significantly less ( P<0.001) attachment to serum-coated tissue culture plastic over a 24 h time period. Immunofluorescent labeling after 24 h of attachment showed that OP OB focal adhesions (FAs) and stress fibers were less defined, and that the OP cells were smaller and had a more motile phenotype. When normalized protein lysates were Western blotted for phosphotyrosine (PY) a band corresponding to pp125FAK was identified. FAK tyrosine phosphorylation was evident at 6 h in both the OP and non-OP OBs, but decreased or was absent through 24 h in OP OBs. These results suggest early adhesion-mediated events, such as cell adhesion, attachment, and FAK signaling via PY may be altered in OP OBs.

Octylphenol inhibits testosterone biosynthesis by cultured precursor and immature Leydig cells from rat testes

4-tert-octyphenol (OP) is a surfactant additive widely used in the manufacture of a variety of detergents and plastic products. OP has been reported to mimic the actions of estrogen in many cellular systems. The present studies evaluated the direct effects of OP on human chorionic gonadotropin (hCG)-stimulated testosterone biosynthesis by cultured precursor and immature Leydig cells from 23-day old (prepubertal) rats. Exposure to increasing OP concentrations (1 to 2000 nM) progressively decreased hCG-stimulated testosterone formation in both precursor and immature Leydig cells at higher OP concentrations (100 or 500 to 2000 nM). Testosterone levels were reduced ∼ 30 to 70% below control at the highest concentration in both cell types. Similar reductions in testosterone associated with OP exposure were observed in cells stimulated with 1 mM 8-Br-cAMP, suggesting that the main actions of OP occur after the generation of cAMP. Increasing concentrations of 17β-estradiol (1 to 1000 nM) had no effect on hCG-stimulated testosterone formation in both precursor and immature Leydig cells and the inclusion of 100 nM ICI 182,780, a pure estrogen antagonist, in precursor and immature Leydig cells exposed to OP and hCG, did not alter the inhibition by higher OP concentrations of testosterone formation in both cell types. These results suggest that OP is a hormonally active agent, but that some of its actions are distinct from those of 17β-estradiol and are not mediated through the estrogen receptor α or β pathway. To further localize the potential site(s) of action of OP, cultured precursor and immature Leydig cells were exposed to increasing concentrations of OP and hCG for 24 h. Next, fresh media containing 1 μM 22(R)-hydroxycholesterol, 1 μM pregnenolone, 1 μM progesterone, or 1 μM androstenedione was added, and the conversion of each substrate to testosterone was determined after incubation for 4 h. The conversion of androstenedione to testosterone was unaffected by exposure to OP, suggesting that the 17β-hydroxysteroid dehydrogenase step is not inhibited. However, the conversion of 22(R)-hydroxycholesterol, pregnenolone and progesterone all were inhibited by prior exposure to OP and hCG. This finding suggests that the 17α-hydroxylase/c17–20-lyase step, which converts progesterone to androstenedione, is inhibited by OP, and that the cholesterol side-chain cleavage and 3β-hydroxysteroid dehydrogenase -isomerase steps, which convert cholesterol to pregnenolone and pregnenolone to progesterone, respectively, are other potential sites of OP action. Because concomitant exposure to the antioxidants α-tocopherol or ascorbate did not alter the inhibition of testosterone formation by higher OP concentrations, it does not appear that OP is acting as a pseudosubstrate for the generation of free radicals, which can damage P450 enzymes.

Permeability transition pore regulates both mitochondrial membrane potential and agonist-evoked Ca2+signals in oligodendrocyte progenitors

In this study, we investigated the importance of mitochondrial permeability transition pore (PTP) in agonist-evoked cytosolic Ca2+([Ca2+]c) signals in oligodendrocyte progenitor cells (OP cells). We measured transmembrane potential across the mitochondrial inner membrane (ΔΨm) and [Ca2+]cin the immediate vicinity simultaneously using tetramethylrhodamine ethyl ester (TMRE) and calcium green respectively. Stimulation of OP cells with methacholine evoked robust [Ca2+]csignals in approximately 80% of cells which were either oscillatory or showed a peak followed by a plateau. Elevations in [Ca2+]cinduced by supramaximal concentrations of the agonist (> 200 μM) were accompanied by changes in ΔΨmin 33–42% of the total mitochondria investigated. The mitochondria that responded either depolarized (26–29%), hyperpolarized (7–13%) or showed no change (58–67%). Thus, of the responsive mitochondria, most (70%) depolarized during agonist-evoked [Ca2+]csignals. Blockade of PTP with cyclosporin A (CSA) reduced the number of mitochondria that depolarized with a corresponding increase in the number that hyperpolarized. In addition, CSA or its analogue methyl valine-4- CSA (MeVal-CSA), reduced the frequency of agonist-evoked global [Ca2+]coscillations. In resting cells, CSA (63%) and MeVal-CSA (77%) hyperpolarized a majority of the mitochondria suggesting that PTP is constitutively active and may show flickering openings. Such hyperpolarizations were not mimicked by either cyclosporine H or verapamil and were inhibited by Ru360, which blocks the mitochondrial uniporter. This observation suggested that in resting cells, Ca2+ions might redistribute between cytosol and mitochondrial matrix through the uniporter and the PTP. Taken together, these data suggest that PTP may play an important role in regulating ΔΨmand local [Ca2+]csignals during agonist stimulation in OP cells.