High dose of glucose promotes chondrogenesis via PKCα and MAPK signaling pathways in chick mesenchymal cells

We investigated the molecular mechanism of the glucose effect on the regulation of chondrogenesis. Exposure of chick wing bud mesenchymal cells to high concentrations of glucose stimulated chondrogenesis 2–fold to 2.5-fold without affecting cell proliferation. Glucose increased protein levels and the membrane translocation of protein kinase C alpha (PKC), leading to a reduction of extracellular signal-regulated kinase (ERK) phosphorylation. Phosphorylation of p38 was also increased in a PKC-independent manner by glucose treatment. Glucose also increased cell adhesion molecules such as fibronectin, integrin 1, and N-cadherin at early stages and then decreased these adhesion molecules at later stages of chondrogenesis. These alterations in protein level of adhesion molecules and in the phosphorylation of mitogen-activated protein kinases by glucose were blocked by inhibition of PKC or p38 but were synergistically increased by the inhibition of ERK. Therefore, high doses of glucose induce the down-regulation of ERK activity via PKC and the up-regulation of p38 and result in the stimulation of chondrogenesis of chick mesenchymal cells through modulating the expression of adhesion molecules.This work was supported by the Korea Research Foundation (KRF-2000-DP0352)     

The impact of pH and calcium on the uptake of fluoride by tea plants (Camellia sinensis L.)

BACKGROUND AND AIMS: Tea plants (Camellia sinensis L.) accumulate large amounts of fluoride (F) from soils containing normal F concentrations. The present experiments examined the effects of pH and Ca on F uptake by this accumulating plant species. METHODS: The effect of pH was assessed in two experiments, one using uptake solutions with different pHs, and the other using lime, as CaO, applied to the soil. The effect of Ca was examined by analysing F concentrations in plants supplied with varying amounts of Ca, as Ca(NO3)2, either in uptake solutions or through the soil. KEY RESULTS: F uptake was highest at solution pH 5.5, and significantly lower at pH 4.0. In the soil experiment, leaf F decreased linearly with the amounts of lime, which raised the soil pH progressively from 4.32 to 4.91, 5.43, 5.89 and, finally, 6.55. Liming increased the water-soluble F content of the soil. Including Ca in the uptake solution or adding Ca to soil significantly decreased leaf F concentrations. The distribution pattern of F in tea plants was not altered by Ca treatment, with most F being allocated to leaves. The activity of F- in the uptake solution was unaffected and water-soluble F in the soil was sometimes increased by added Ca. CONCLUSIONS: F uptake by tea plants, which are inherently able to accumulate large quantities of F, was affected both by pH and by Ca levels in the medium. The reduced F uptake following Ca application appeared not to be due simply to the precipitation of CaF2 in solution and soil or to the complexing of Ca and F in roots, although these factors cannot be dismissed. It was more likely due to the effect of Ca on the properties of cell wall or membrane permeability in the solution experiments, and to alteration of F speciations and their quantities in soil solutions following Ca application.     

Characteristics of the killing mechanism of human natural killer cells against hepatocellular carcinoma cell lines HepG2 and Hep3B

Purpose Unlike normal hepatocytes, most hepatocellular carcinomas (HCCs) are quite resistant to death receptor-mediated apoptosis when the cell surface death receptor is cross linked with either agonistic antibodies or soluble death ligand proteins in vitro. The resistance might play an essential role in the escape from the host immune surveillance; however, it has not been directly demonstrated that HCCs are actually resistant to natural killer (NK) cell-mediated death. Therefore, this study investigated the molecular mechanism of NK cell-mediated cytotoxicity against the HCCs, HepG2, and Hep3B, using two distinct cytotoxic assays: a 4-h ⁵¹Cr-release assay and a 2-h [³H] thymidine release assay which selectively measures the extent of necrotic and apoptotic target cell death, respectively.Methods Most of the target cells exhibited marked morphologic changes when they were co-incubated with the NK cells, and the NK cytotoxicity against these HCCs was comparable to that against K562, a NK-sensitive leukemia cell line, when the cytotoxicity was assessed by a 4-h ⁵¹Cr release assay.Results The NK cells also induced significant apoptotic cell death in the Hep3B targets, but not in the HepG2 targets, when the cytotoxicity was assessed by a 2-h [³H]-thymidine release assay. In agreement with these results, procaspase-3 was activated in the Hep3B targets, but not in the HepG2 targets. Interestingly, mildly fixed NK cells had no detectable activity in the 4-h ⁵¹Cr release assay against both HepG2 and Hep3B targets, while they were similarly effective as the untreated NK cells in the 2-h [³H]-thymidine release assay, suggesting that the level of apoptotic cell death of the Hep3B targets is granule independent and might be primarily mediated by the death ligands of the NK cells.Conclusion This study found that a tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)/TRAIL receptor interaction is involved in the NK cell-mediated apoptotic death of the Hep3B targets, but a Fas/Fas ligand (FasL) interaction is not.     

Proteomic analyzes of copper metabolism in an in vitro model of Wilson disease using surface enhanced laser desorption/ionization-time of flight-mass spectrometry

In Wilson disease, mutations in the ATP7B-gene lead to hepatic accumulation of copper that becomes toxic when the hepatic binding capacity is exceeded, leading to oxidative stress and acute liver failure. Several proteins are probably involved in dealing with the excess copper and oxidative stress. As a first step towards biomarker discovery and analyzes of copper metabolism in Wilson disease patients we characterized copper-induced changes in protein expression in cell lysates and culture media from an in vitro copper-overload model using surface enhanced laser desorption/ionization (SELDI) proteomics technology. HepG2 cells were cultured for 48 h with a physiological (0.5 microM) or a pathological (100 microM) copper concentration. Samples were applied to weak cation exchange (WCX) proteinchip arrays and chips were analyzed by time of flight (TOF)-mass spectrometry. Copper-coated IMAC chips were used to detect copper-binding proteins in cell lysate of copper depleted cells using buffers with increasing imidazole concentrations. Data from the 2 to 50 kDa range indicate that high extra-cellular copper substantially altered both intra-cellular protein expression as well as the composition of the secretome. In the lysate 15 proteins were found up-regulated, while 6 proteins were down-regulated. In culture media 21 proteins were increased while 4 proteins were decreased in abundance. Copper-coated protein chips revealed the presence of 18 high-affinity copper-binding proteins. Further identification is necessary to determine the exact cellular roles of the discovered proteins.     

Mechanism of taxol-induced apoptosis in human SKOV3 ovarian carcinoma cells

Taxol is extensively used clinically for chemotherapy of patients with ovarian, breast, and lung cancer. Although taxol induces apoptosis of cancer cells, its exact mechanism of action is not yet known. To determine the mechanism of action of taxol in ovarian cancer, we tested the effects of the drug, on the human ovarian carcinoma cell line, SKOV3. We observed that taxol-induced apoptosis of these cells by phosphatidylserine (PS) externalization and DNA fragmentation. While treatment of cells with taxol resulted in bcl-2 phosphorylation and mitochondrial depolarization, cytochrome c was not released and pro-caspase-3 was not activated. Treatment of SKOV3 cells with taxol, however, resulted in the translocation of AIF from the mitochondria to the nucleus via the cytosol. Taken together, these findings suggest that in SKOV3 cells, taxol induces caspase-independent AIF-dependent apoptosis.     

Phosphorylation of p70s6 kinase is implicated in androgen-induced levator ani muscle anabolism in castrated rats

Androgens are known to increase muscle mass, strength and muscle protein synthesis. However, the molecular mechanisms by which androgens regulate skeletal muscle development remain poorly understood. The ribosomal protein kinase p70^s6k is a regulator of ribosome biogenesis and plays an important role in the regulation of growth-related protein synthesis. The phosphorylation of p70^s6k has been implicated in load-induced skeletal muscle hypertrophy. In the current study, we determined the effect of DHT on the phosphorylation of p70^s6k in the androgen-sensitive levator ani muscle of castrated rats. DHT induced a rapid increase in the phosphorylation of p70^s6k, which was detectable within 6 h after a single injection. Interestingly, DHT-induced phosphorylation of p70^s6k occurred only in androgen-sensitive muscles, but not prostate and seminal vesicle. Co-administration of flutamide, an AR antagonist, inhibited DHT-induced p70^s6k phosphorylation. While serum IGF-I levels were not changed by DHT treatment, IGF-I gene expression levels increased and the mRNA levels of IGFBP3 and IGFBP5 were suppressed in the LA muscle after DHT replacement in castrated rats. These results suggest that the phosphorylation of p70^s6k, likely via the IGF-I pathway, may play an important role in androgen-induced skeletal muscle hypertrophy.     

High-resolution imaging reveals a limit in spatial resolution of blood flow measurements by microspheres

Density of 15-microm microspheres after left atrial application is the standard measure of regional perfusion. In the heart, substantial differences in microsphere density are seen at spatial resolutions <5 ml, implying perfusion heterogeneity. Microsphere deposition imaging permits a superior evaluation of the distribution pattern. Therefore, fluorescent microspheres (FMS) were applied, FMS deposition in the canine heart was imaged by epifluorescence microscopy in vitro, and the patterns were observed compared with MR images of iron oxide microspheres (IMS) obtained in vivo and in vitro. FMS deposition in myocardial slices revealed the following: 1) a nonrandom distribution, with sequentially applied FMS of different color stacked within the same vessel, 2) general FMS clustering, and 3) rather large areas devoid of FMS (n = 3). This pattern was also seen in reconstructed three-dimensional images (<1 nl resolution) of FMS distribution (n = 4). Surprisingly, the deposition pattern of sequentially applied FMS remained virtually identical over 3 days. Augmenting flow by intracoronary adenosine (>2 microM) enhanced local microsphere density, but did not alter the deposition pattern (n = 3). The nonrandom, temporally stable pattern was quantitatively confirmed by a three-dimensional intermicrosphere distance analysis of sequentially applied FMS. T2-weighted short-axis MR images (2-microl resolution) of IMS revealed similar patterns in vivo and in vitro (n = 6), as seen with FMS. The observed temporally stable microsphere patterns are not consistent with the notion that microsphere deposition is solely governed by blood flow. We propose that at high spatial resolution (<2 microl) structural aspects of the vascular network dominate microsphere distribution, resulting in the organized patterns observed.     

Progressive apoptotic cell death triggered by transient oxidative insult in H9c2 rat ventricular cells: a novel pattern of apoptosis and the mechanisms

Many pathophysiological processes are associated with oxidative stress and progressive cell death. Oxidative stress is an apoptotic inducer that is known to cause rapid cell death. Here we show that a brief oxidative insult (5-min exposure to 400 microM H(2)O(2)), although it did not kill H9c2 rat ventricular cells during the exposure, triggered an intracellular death cascade leading to delayed time-dependent cell death starting from 1 h after the insult had been withdrawn, and this post-H(2)O(2) cell death cumulated gradually, reaching a maximum level 8 h after H(2)O(2) withdrawal. By comparison, sustained exposure to H(2)O(2) caused complete cell death within a narrow time frame (2 h). The time-dependent post-H(2)O(2) cell death was typical of apoptosis, both morphologically (cell shrinkage and nuclear condensation) and biochemically (DNA fragmentation, extracellular exposure of phosphatidylserines, and caspase-3 activation). A dichlorofluorescein fluorescent signal showed a time-dependent endogenous increase of reactive oxygen species (ROS) production, which was almost abolished by inhibition of the mitochondrial electron transport chain. Application of antioxidants (vitamin E or DTT) before H(2)O(2) addition or after H(2)O(2) withdrawal prevented the H(2)O(2)-triggered progressive ROS production and apoptosis. Sequential appearance of events associated with activation of the mitochondrial death pathway was found, including progressive dissipation of mitochondrial membrane potential, cytochrome c release, and late activation of caspase-3. In conclusion, transient oxidative stress triggers an intrinsic program leading to self-sustained apoptosis in H9c2 cells via cumulative production of mitochondrial ROS and subsequent activation of the mitochondrial death pathway. This pattern of apoptosis may contribute to the progressive and long-lasting cell loss in some degenerative diseases.