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PubMed2003

Expression and location of taurine transporters and channels in the epididymis of infertile c-ros receptor tyrosine kinase-deficient and fertile heterozygous mice.

Defective sperm volume regulation causes infertility in c-ros knockout (KO) mice lacking the initial segment of the epididymis (IS). As taurine is in high concentration in the male tract and acts as an organic osmolyte in somatic cells, the taurine transporter (TauT), taurine content and taurine channel phospholemman (PLM) were examined in the epididymides of these and fertile heterozygous (+/-) mice. TauT and PLM genes were demonstrated by RT-PCR in the caput of +/- and c-ros knockout males. Northern blots revealed one transcript of TauT (8 kb) in all regions with different expression between segments (cauda > corpus > IS > caput) and no differences in expression between genotypes. Western blotting revealed three translation products of TauT in all epididymal regions (107, 69, 49 kDa) with higher expression of the 69 kDa and 49 isoforms in the -/- than +/- caput. Immunohistochemical staining revealed staining of principal cells and stronger staining of apical and clear cells in all epididymal regions. The expression of the PLM transcript (0.75 kb: cauda = corpus > caput > IS) was upregulated in the proximal caput and cauda of KO mice. Tissue taurine was higher in the cauda >corpus>IS congruent with caput in fertile males and significantly higher in the proximal caput of the KO male. By contrast, taurine concentrations in cauda epididymidal fluid and content per 10(6) sperm did not differ between genotypes. TauT and PLM may be involved in taurine regulation in the normal epididymis and the proximal accumulation of taurine in the infertile males.

Animals↗

PubMed2002

L-type calcium channels reduce ROS generation in cerebellar granule cells following kainate exposure.

Cerebellar granule cells (CGC) deprived of serum or trophic factors develop sensitivity to kainate neurotoxicity that is mediated by the alpha-amino-3-hydroxy-5-methyl-isoxazole proprionic acid (AMPA) subtypes of glutamate receptors (GluR). The L-type voltage-gated calcium channel (L-type VGCC) blocker nifedipine increases the potency of kainate 50-fold. Thus, one goal of this laboratory is to determine the underlying protective mechanism triggered by calcium influx through this channel. The cell-permeable heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine effected complete protection against kainate treatment in the presence of nifedipine, as did the iron chelator deferoxamine. The chelatable heavy metal pool decreased approximately 70% immediately following treatment with kainate, but did not change following kainate/nifedipine treatment. Tetramethylrhhodamine ethyl ester (TMRE) fluorescence, an indicator of mitochondrial membrane potential, decreased approximately 70% following kainate treatment but displayed a more modest decrease ( approximately 15%) when CGC were treated with kainate/nifedipine. Reactive oxygen species (ROS) formation decreased in CGC immediately following kainate treatment but was slightly elevated following kainate/nifedipine treatment. Electron microscopic examinations of the CGC indicated severe swelling and distortion of mitochondria immediately following kainate/nifedipine treatment and the appearance of mitochondrial herniations, whorls, and bridges 2 h later, features that were rarely observed following kainate treatment. These results support the hypothesis that calcium entry through L-type VGCCs protects CGC during kainate treatment by lowering the chelatable heavy metal pool and the mitochondrial membrane potential, thereby mitigating the formation of ROS.

Animals↗

PubMed1996

1 alpha,25(OH)2 vitamin D3-mediated stimulation of outward anionic currents in osteoblast-like ROS 17/2.8 cells.

1 alpha,25-dihydroxyvitamin-D3 [1 alpha,25(OH)2D3] is known to potentiate in osteoblast-like rat osteosarcoma cells (ROS 17/2.8) the influx of Ca2+ through voltage-activated L-type Ca2+ channels. In the present study, by means of the whole-cell patch-clamp technique, we found that physiological concentrations of 1 alpha,25(OH)2D3 (5-5000 x 10(-11) M) also stimulate, in a dose-dependent manner, outwardly rectifying anionic currents over the course of 1-5 minutes. These currents, recorded in the presence of 130 mM glutamate or approximately 140 mM C1- in the external solution, were blocked by 200 microM DIDS, a stilbene derivative selective for C1- channels. We conclude that, in addition to the already known effects of 1 alpha,25(OH)2D3 on Ca2+ channels, the hormone also enhances in ROS 17/2.8 cells an outward anion conductance. This is the first report concerning activation of anion channels by 1 alpha,25(OH)2D3.

Animals↗

PubMed1992

Lead intoxication alters basal and parathyroid hormone-regulated cellular calcium homeostasis in rat osteosarcoma (ROS 17/2.8) cells.

The skeleton is the major reservoir of lead and calcium in humans, and plays an important role in systemic calcium regulation. Lead perturbs normal calcium transport and second messenger function, directly or indirectly, in virtually all cells studies so far. Therefore, we and others have postulated that an early and discrete toxic effect of lead is perturbation of one or more loci within the calcium messenger system. To understand further the role of lead on calcium homeostasis in bone, we undertook this study to characterize calcium homeostasis and the effect of lead on calcium homeostasis in rat osteosarcoma (ROS 17/2.8) cells, which exhibit the osteoblast phenotype. ROS cells were incubated in medium containing 45Ca for 20 hours. Monitoring the efflux of 45Ca from the cultures for 210 minutes allowed for the determination of kinetic parameters defining steady state calcium homeostasis. Three distinct intracellular kinetic calcium pools characterized 45Ca homeostasis. Treatment with either 400 ng parathyroid hormone (PTH)/ml culture medium for 1 hour or 25 microM lead for 20 hours increased total cell calcium. Treatment with PTH caused a larger increase of cell calcium in lead-intoxicated cells than either lead intoxication or PTH treatment alone. This increase suggests that lead may perturb normal calcium-mediated PTH responsiveness of the osteoblast. These experiments further establish a kinetic model for the study of calcium homeostasis in osteoblastic bone cells. The studies also advance the hypothesis that lead-induced perturbations of calcium-mediated processes represent an early effect of lead toxicity at the cellular level.

Animals↗

PubMed1995

Microsomal casein kinase II in endoplasmic reticulum- and Golgi apparatus-rich fractions of ROS 17/2.8 osteoblast-like cells: an enzyme that modifies osteopontin.

Osteopontin is an acidic phosphoprotein containing casein kinase II (CKII) phosphorylatable sites and an acidic amino acid cluster. The metabolically 32P-labelings of both serines and threonines in vitro in osteopontin immunoprecipitated from rat osteoblast-like ROS 17/2.8 cells may suggest that casein kinase II catalyzes this modification. The enzyme occurs in microsomal fractions of rat osteoblast-like ROS 17/2.8 cells. Subcellular fractions containing endoplasmic reticulum and Golgi apparatus were isolated by differential centrifugation and were identified according to their ultrastructures and the presence of marker enzymes such as glucose-6-phosphatase and thiamine pyrophosphatase, respectively. both fractions phosphorylated the partially dephosphorylated osteopontin and the specific substrate peptide RRREEETEEE. Endoplasmic reticulum-catalyzed peptide phosphorylation was 2.7 times lower than that of Golgi although both endoplasmic reticulum- and Golgi-catalyzed peptide reactions were 50% inhibited by 20 and 100 ng/ml heparin, respectively. Western blot analysis revealed that both fractions contained osteopontin and microsomal CKII. Furthermore, microsomal CKII was immunogold-labeled in endoplasmic reticulum and Golgi apparatus. Heparin inhibition and utilization of [gamma-32P]GTP as a phosphate donor by both fractions confirmed their capacity to phosphorylate osteopontin. The results suggest that microsomal CKII modifies the acidic matrix proteins during transportation. These matrix phosphoproteins may participate in the mineralization process of hard tissues.

Amino Acid Sequence↗

PubMed1977

[Experimental allergic chorioretinitis. Appearance and behavior of complement-fixing anti-ROS antibodies in the sera following immunization with retinal antigens (author's transl)].

The authors studied the appearance and behavior of complement-fixing anti-ROS antibodies in the sera of GrCh rabbits following one or two intracutaneous injections of retinal antigens emulsified in Freund's complete adjuvant (KFA). Fourteen different antigenic preparations from heterologous, homologous, and autologous retinal components incorporated in KFA were tested and compared. Apart from the supernatant of a centrifuged homogenized preparation of homologous retina, all immunogens generally induced anti-ROS antibodies. The antibody titers (reciprocal values) reached a maximum 4 to 8 weeks post injection, at only low levels. The changes in antibody concentrations showed some relationship to the quality and dose of the immunogens. Anamnestic effects following reinjection reached maximum values within as little as 5 to 14 days. Animals not developing chorioretinitic lesions showed higher antibody titers more frequently than rabbits with clinically confirmed disease. Provided there is sufficient relevance of the humoral reactions observed, the complement-fixing antibodies we found in our experiments are more likely to have protective than pathogenic functions.

Animals↗

PubMed1987

Cell density-dependent vitamin D effects on calcium accumulation in rat osteogenic sarcoma cells (ROS 17/2).

Rat osteogenic sarcoma cells have been used widely as a model system to study actions of 1,25(OH)2D3 and other hormones in osteoblastlike cells. However, some of the pleiotypic manifestations of hormones in these cells vary greatly dependent upon the cell population density and other conditions of culture. Therefore, we have studied the effect of cell density on the relationship between 1,25(OH)2D3 and the initial 45Ca accumulation in ROS 17/2 cells in order to establish conditions suitable for studying the effect of 1,25(OH)2D3 on calcium fluxes in these cells. Cells were grown in the presence and absence of 1,25(OH)2D3 for 48 hours and then incubated for 4 min in the culture medium containing 0.5 microCi/ml of 45CaCl2. In high population density cultures, 0.25-1.0 pg/ml of 1,25(OH)2D3 stimulated the intracellular accumulation of 45Ca (cpm/mg protein), whereas 80 pg/ml or higher concentrations inhibited accumulation of 45Ca. In low density cultures, concentrations less than 80 pg/ml had no effect, 80-120 pg/ml increased the intracellular accumulation, and as much as 200 pg/ml failed to show the inhibitory effect. These results indicate that the ROS 17/2 cell responses to 1,25(OH)2D3 are biphasic--low concentrations stimulating and high concentrations inhibiting 45Ca accumulation. The sensitivity of the cells to 1,25(OH)2D3 increases as the cell population density increases. These observations suggest that the culture density and dose-response relationship must be carefully defined in in vitro studies utilizing osteogenic cell culture systems.

Animals↗

PubMed1995

The bovine rod outer segment guanylate cyclase, ROS-GC, is present in both outer segment and synaptic layers of the retina.

Cyclic-GMP, which plays a pivotal role in visual transduction in the vertebrate retina, is synthesized by guanylate cyclase. The purpose of this study was to localize a rod outer segment-derived particulate guanylate cyclase (ROS-GC) to the retina of several species that have different populations of rods and cones. A rabbit antibody was raised against a synthetic peptide, corresponding to the sequence A107-L125 of bovine ROS-GC. Western blot analysis showed a single immunoreactive band at about 115 kDa with bovine rod outer segments but not with human rod outer segments. Light microscopic immunocytochemistry of tissue sections revealed immunoreactivity in the outer segment layer and in the outer and inner plexiform layers. The rod-rich rat retina showed uniform immunolabeling of outer segments; the cone-containing cat retina showed heavily labeled cone outer segments and lighter labeling of rod outer segments; the cone-rich chicken retina showed a uniformly and intensely labeled outer segment layer. Preincubation of the primary antibody with the peptide completely blocked antibody binding. Electron microscopic immunocytochemistry of the cat retina confirmed the presence of guanylate cyclase in photoreceptor outer segments and demonstrated its association with disk and plasma membranes. These data support a concept in which guanylate cyclase is much more concentrated in the outer segments of cones than rods. The immunolabeling of the plexiform layers suggests that the particulate guanylate cyclase is not unique to the photoreceptor outer segments, and may also play a role in transduction processes of retinal synapses.

Amino Acid Sequence↗

PubMed2005

Agastache rugosa leaf extract inhibits the iNOS expression in ROS 17/2.8 cells activated with TNF-alpha and IL-1beta.

It has been suggested that nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) may act as a mediator of cytokine-induced effects on bone turn-over. NO is also recognized as an important factor in bone remodeling, i.e., participating in osteoblast apoptosis in an arthritic joint. The components of Agastache rugosa are known to have many pharmacological activities. In the present study, we investigated the effects of Agastache rugosa leaf extract (ELAR) on NO production and the iNOS expression in ROS 17/2.8 cells activated by a mixture of inflammatory cytokines including TNF-alpha and IL-1beta. A preincubation with ELAR significantly and concentration-dependently reduced the expression of iNOS protein in ROS 17/2.8 cells activated with the cytokine mixture. Consequently, the NO production was also significantly reduced by ELAR with an IC50 of 0.75 mg/mL. The inhibitory mechanism of iNOS induction by ELAR prevented the activation and translocation of NF-kappaB (p65) to the nucleus from the cytosol fraction. Furthermore, ELAR concentration-dependently reduced the cellular toxicity induced by sodium nitroprusside, an NO-donor. These results suggest that ELAR may be beneficial in NO-mediated inflammatory conditions such as osteoporosis.

Agastache↗

PubMed2003

Further isolation of antioxidative (+)-1-hydroxypinoresinol-1-O-beta-D-glucoside from the rhizome of Salvia miltiorrhiza that acts on peroxynitrite, total ROS and 1,1-diphenyl-2-picrylhydrazyl radical.

A furanofuranoid lignan glycoside, with radical scavenging on peroxynitrite, total reactive oxygen species (ROS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, was isolated from the rhizome of Salvia miltiorrhiza and characterized as (+)-1-hydroxypinoresinol-1-O-beta-D-glucoside based on spectroscopic evidence. The compound exhibited peroxynitrite, total ROS and DPPH radical scavenging activities with IC50 values of 3.23 +/- 0.04, 2.26 +/- 0.07 and 32.3 +/- 0.13 microM, respectively. Penicillamine, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and L-ascorbic acid, acting as positive controls, showed radical scavenging activities with IC50 values of 6.72 +/- 0.25, 1.43 +/- 0.04 and 11.4 +/- 0.07 microM, respectively.

Antioxidants↗

PubMed1998

Loss of the differentiated phenotype precedes apoptosis of ROS 17/2.8 osteoblast-like cells.

Osteoblast cells, recruited from mesenchymal precursors, initiate the final phase of bone remodeling by secreting the protein components of the bone matrix. Upon completion of remodeling, some of these osteoblasts may further differentiate, giving rise to matrix-embedded osteocytes and bone lining cells. The fate of the remaining osteoblasts is unknown, although by analogy with other cell systems, apoptotic cell death may be involved. We induced and characterized the apoptotic process in ROS 17/2.8 osteosarcoma cells by growing and maintaining confluent cultures in low serum medium. At confluence, but prior to apoptosis, the levels of collagen type I, alkaline phosphatase, and osteocalcin mRNAs declined abruptly. Expression of two housekeeping genes (ribosomal protein RPS6 and GAPDH) remained unchanged. Some 72 hours later cells began to show morphological and biochemical features of apoptosis, namely, chromatin condensation, membrane budding, and internucleosomal degradation of genomic DNA. We conclude that serum starvation-induced apoptosis of ROS 17/2.8 cells can serve as a model for investigating the mechanisms of osteoblastic apoptosis.

Animals↗

PubMed2005

Apoptosis induced by glycoprotein (150-kDa) isolated from Solanum nigrum L. is not related to intracellular reactive oxygen species (ROS) in HCT-116 cells.

This study was carried out to investigate the apoptotic effects of glycoprotein [Solanum nigrum L. (SNL) glycoprotein, 150-kDa] isolated from Solanum nigrum L., which has been used as an antipyretic and anticancer agent in folk medicine. With the purified SNL glycoprotein, we evaluated the cytotoxic and apoptotic effects of SNL glycoprotein on HCT-116 cells, DNA fragmentation and nuclear staining assays, respectively. SNL glycoprotein has an apparent cytotoxic and apoptotic effect at a concentration of 40 microg/ml after 4 h. To further verify the apoptotic effect, we investigated the changes in activity of the apoptotic-related proteins [Bid, cytochrome c, caspases and poly(ADP-ribose)polymerase (PARP)] triggered by SNL glycoprotein, using a western blot analysis. The results in this study indicated that SNL glycoprotein has a stimulatory effect on Bid activation, resulting in the release of cytochrome c, the stimulation of caspase-8, -9 and -3 activities, and the cleavage of PARP in HCT-116 cells. However, SNL glycoprotein did not significantly stimulate an increase in levels of intracellular reactive oxygen species (ROS). From the results in this experiment, it is suggested that SNL glycoprotein induces apoptosis through the mitochondrial apoptotic signal pathway in HCT-116 cells, rather than through intracellular ROS.

Antineoplastic Agents, Phytogenic↗

PubMed1999

Comparative study of ROS degradation by IPE and RPE cells in vitro.

BACKGROUND: The aim of this study was to compare the degradation of rod outer segments (ROS) in porcine iris pigment epithelial cells (IPE) and retinal pigment epithelial (RPE) cells by measuring the increase of lipofuscin-like fluorescence. METHODS: We measured the development of autofluorescence of lipofuscin-like material in living cells over a period of 4 weeks using an image-analyzing system comprising a light microscope, a filter set with an appropriate wavelength for the detection of lipofuscin-like autofluorescence and a silicon-intensified target camera connected to a computer. The lipofuscin-like fluorescence was quantified as the mean gray value of pixels over a defined area in the cell. In addition, ultrastructural examination of the cells was performed using transmission electron microscopy. RESULTS: We found that while both cell types had increased autofluorescence over time, the increase of lipofuscin-like fluorescence was significantly higher in IPE cells than in RPE cells. The ultrastructure of both cell types was similar and no accumulation of lipofuscin-like granules was observed. CONCLUSION: These findings suggest that although IPE cells are able to phagocytize ROS, their ability to degrade them may be lower than in RPE cells. The increase of lipofuscin-like fluorescence is not due to the accumulation of lipofuscin-like granules.

Animals↗

PubMed1999

Immunohistochemical investigations on the differentiation marker protein E11 in rat calvaria, calvaria cell culture and the osteoblastic cell line ROS 17/2.8.

Until now, many extracellular matrix proteins, e.g. osteopontin and osteonectin, have been used to determine a cell's osteogenic maturation. The disadvantage in evaluation of these proteins is their relative wide-ranging appearance throughout the osteogenic differentiation process. Thus, the aim of this study was to establish an immunohistochemical setup using E11, a marker that binds selectively to cells of the late osteogenic cell lineage. In addition, the histochemical expression of the bone matrix proteins osteonectin, osteopontin and fibronectin was compared to that of E11 using monoclonal antibodies. For light microscopical detection of osteogenic markers in cultured cells we developed a simple paraffin technique using a fibrin glue as embedding medium. This allows the handling of cultured cells such as a tissue sample and includes the use of stored biological specimens for further immunohistochemical experiments. We used newborn rat calvariae for whole tissue preparations and for isolation and cultivation of bone cells. In addition, we included the rat osteosarcoma cell line ROS 17/2.8 in this study. For the first time, we have localised E11 in osteocytes of rat calvaria preparations at the electron microscopical level. E11 was detected at plasma membranes of osteocytes and their processes, but not at those of osteoblasts. Accompanying experiments with cultured newborn rat calvaria cells and ROS 17/2.8 cells revealed E11 reactivity on a subset of cells. The results obtained confirm the suitability of the differentiation marker E11 as a sensitive instrument for the characterisation of bone cell culture systems.

Animals↗

PubMed2001

ROS generation, lipid peroxidation and antioxidant enzyme activities in the aging brain.

The objective of this study was to determine the specific relationship between brain aging and changes in the level of oxidative stress, lipid peroxidation (LPO) and in the activities of antioxidant enzymes. We used four different age groups (2-3 months, 10-11 months, 16-17 months and 20-21 months) which represented young adults, adults, beginning senescence and senescence, respectively. Basal levels of LPO products measured as malondialdehyde increased gradually with age in mouse brain homogenate. The extent of stimulated LPO products, however, was clearly decreased in the brain of adult mice compared to young mice but increased again in the brain of senescent mice. We could not detect any appreciable age-related changes in the basal as well as in stimulated levels of ROS measured with the fluorescent dyes dichlorofluorescein and dihydrorhodamine123. Nevertheless, there was a significant delay in the time course of ROS-generation in brain cells from old mice. The activities of the antioxidant enzymes CuZn-superoxide dismutase and glutathione reductase increased with age whereas glutathione peroxidase remained unchanged. On the basis of our present findings, we envisage a potential model that integrates several divergent findings described in the literature about the role of oxidative stress in brain aging.

Aging↗

PubMed2005

Aluminum-tolerant Pseudomonas fluorescens: ROS toxicity and enhanced NADPH production.

Aluminum (Al) triggered a marked increase in reactive oxygen species (ROS) such as O(2) (-) and H(2)O(2) in Pseudomonas fluorescens. Although the Al-stressed cells were characterized with higher amounts of oxidized lipids and proteins than controls, NADPH production was markedly increased in these cells. Blue native polyacrylamide gel electrophoresis (BN-PAGE) analyses coupled with activity and Coomassie staining revealed that NADP(+) -dependent isocitrate dehydrogenase (ICDH, E.C. 1.1.1.42) and glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) played a pivotal role in diminishing the oxidative environment promoted by Al. These enzymes were overexpressed in the Al-tolerant microbes and were modulated by the presence of either Al or hydrogen peroxide (H(2)O(2)) or menadione. The activity of superoxide dismutase (SOD, E.C. 1.15.1.1), an enzyme known to combat ROS stress was also increased in the cells cultured in millimolar amounts of Al. Hence, Al-tolerant P. fluorescens invokes an anti-oxidative defense strategy in order to survive.

Aluminum↗

PubMed2006

Curcumin protects PC12 cells against 1-methyl-4-phenylpyridinium ion-induced apoptosis by bcl-2-mitochondria-ROS-iNOS pathway.

The aim of present study is to explore the cytoprotection of curcumin against 1-methyl-4-phenylpridinium ions (MPP(+))-induced apoptosis and the molecular mechanisms underlying in PC12 cells. Our findings indicated that MPP(+) significantly reduced the cell viability and induced apoptosis of PC12 cells. Curcumin protected PC12 cells against MPP(+)-induced cytotoxicity and apoptosis not only by inducing overexpression of Bcl-2, but also reducing the loss of mitochondrial membrane potential (MMP), an increase in intracellular reactive oxygen species (ROS) and overexpression of inducible nitric oxide synthase (iNOS). The selective iNOS inhibitor AG partly blocked MPP(+)-induced apoptosis of PC12 cells. The results of present study suggested that the cytoprotective effects of curcumin might be mediated, at least in part, by the Bcl-2-mitochondria-ROS-iNOS pathway. Because of its non-toxic property, curcumin could be further developed to treat the neurodegenerative diseases which are associated with oxidative stress, such as Parkinson's disease (PD).

1-Methyl-4-phenylpyridinium↗

PubMed2005

Effect of GRP75/mthsp70/PBP74/mortalin overexpression on intracellular ATP level, mitochondrial membrane potential and ROS accumulation following glucose deprivation in PC12 cells.

Glucose regulated protein 75 (GRP75) is an important molecular chaperon belonged to the heat shock protein (HSP) family. To evaluate the effect of GRP75 overexpression on PC12 cells under glucose deprivation, cell viability and mitochondrial function of GRP75-overexpressing PC12 cells and the vector transfected control PC12 cells were monitored during glucose deprivation. Upon exposure to glucose deprivation, GRP75-overexpressing PC12 cells exhibited more moderate cell damage than control PC12 cells. Both of the two groups of cells showed a decreased ATP level following an early increase in the condition of glucose deprivation, and the mitochondrial potential were also reduced in the similar manner in the two groups of cells. Control PC12 cells showed an immediate and rapid increase in ROS accumulation after the onset of GD treatment, and this accumulation was slowed and reduced in GRP75-overexpressing PC12 cells. These findings suggested that GRP75 could inhibit the ROS accumulation, and it may be associated with the cytoprotective effect of GRP75 overexpression upon glucose deprivation.

Adenosine Triphosphate↗