International Journal of Molecular Medicine

Published by Spandidos Publications
Online ISSN: 1791-244X
Publications
Article
Rheumatoid arthritis (RA) is a T cell-mediated autoimmune disease, but target antigens (autoantigens) responsible for T cell activation remain unclear. Type II collagen (CII) is a candidate autoantigen that is largely confined to the articular cartilage. To investigate whether CII is an important antigen in patients with RA, we examined peripheral blood T cell reactivity to CII in HLA-DRB1*0101 and DRB1*0405-positive RA patients. Reactivities to candidate T cell epitopes of CII were also examined. Peripheral blood T cell reactivity to CII and CII peptides (256-271, 429-442, 593-610, 1064-1081) were detected by measurement of IL-2, IFN-gamma, and IL-4 in culture supernatant of PBMC after in vitro antigen stimulation. Cytokine concentration was measured by ELISA. In DRB1*0101-positive patients, T cell reactivity to CII as detected by measurement of IL-2 production in culture supernatant, was present in 4 out of 9 patients. IL-2 production upon stimulation with CII 256-271 peptide was found in all of these 4 patients. In DRB1*0405-positive patients, high frequency of positive T cell response to CII was detected in 9 out of 11 patients. IFN-gamma production was also detected in 4 out of 6 patients producing IL-2 by stimulation with CII. T cell response to CII 256-271 and/or CII 1064-1081 was detected in these patients. In DRB1*0101-positive RA patients, CII 256-271 peptide might function as a T cell epitope, whereas either CII 256-271 or CII 1064-1081 peptide may be a major T cell epitope in DRB1*0405-positive RA patients. In DRB1*0405-positive RA patients, CII reactive T cells might play a crucial role in the development of RA through IFN-gamma production.
 
Cr(VI) induces a concentration-dependent loss of cell viability in L-02 hepatocytes. L-02 hepatocytes were incubated with different concentrations of Cr(VI) (0, 2, 8, 32, 128, 512 µM) for 24 h and then processed for MTT assay to detect cell viability. The Cr(VI) concentration required for 50% inhibition of cell viability (IC 50 ) was 38.97 µM. * P<0.05, ** P<0.01 compared with the control group.
Cr(VI) enhances ROS accumulation in L-02 hepatocytes. (A and B) Cr(VI) induces ROS accumulation. The hepatocytes were treated with PBS or 16, 32 µM Cr(VI) for 24 h. The MRCC I inhibitor, ROT (5 µM, 24 h) was used as a positive treatment control. Treated cells were incubated with the oxidant-sensitive fluorogenic probe CM-H 2 DCFDA. (A) Fluorescence was detected by fluorescence microscope. (B) The amount of ROS production, which was considered to be directly proportional to the fluorescence intensity, was also quantitated by flow cytometer. The values were expressed as mean ± SD of three independent experiments. * P<0.05, ** P<0.01, compared to the control. (C) The alteration of antioxidative proteins induced by Cr(VI) is not involved in promoting ROS accumulation. Whole cell lysates were analyzed by western blotting to examine the expression levels of SOD, catalase and Trx. Representative images of at least three independent experiments are shown. 
Cr(VI) targets and inhibits MRCC I. (A) Cr(VI) significantly inhibits MRCC I activity. L-02 hepatocytes were treated with Cr(VI) (16, 32 µM) for 24 h. The activities of MRCC I, II, III and IV were measured with the Mitochondrial Respiratory Chain Complex Enzyme Activity kits. All columns display the mean ± SD from three independent experiments. * P<0.05, ** P<0.01, compared to control. (B) Inhibition of MRCC I activity is involved in mediating ROS accumulation induced by Cr(VI). L-02 hepatocytes were treated with 32 µM Cr(VI) for the indicated time (30, 60, 120 min). The activities of MRCC I and II were measured by Mitochondrial Respiratory Chain Complex Enzyme Activity kit. All columns display the mean ± SD from three independent experiments. * P<0.05, ** P<0.01, compared to control. (C) Cr(VI) decreases the protein expression levels of MRCC I. The hepatocytes were exposed to Cr(VI) (16, 32 µM) for 24 h and then were processed for western blotting analysis to examine the protein levels of MRCC I-IV. (D) Cr(VI) reduction occurs at MRCC I. The mitochondria isolated from hepatocytes were pretreated with MRCC I substrates 10 mM glutamate/10 mM malate (Glu/Mal), MRCC II substrate 10 mM succinate (Suc), MRCC III substrate 5 µM coenzyme Q (CoQ), or MRCC IV substrate 2 mM vitamin C (Vit C) for 10 min prior to 32 µM Cr(VI) treatment. The Cr(VI) reduction rate was measured using a spectrophotometer at different time points (5, 30, 60 min). 
Cr(VI) induces caspase-3 activation. (A) Cr(VI) increases caspase-3 activity. L-02 hepatocytes were treated with Cr(VI) (16, 32 µM) for 24 h and then harvested and lysed with lysis buffer. The samples were incubated with specific colorigenic substrate Ac-DEVD-pNA and the alternative activity of caspase-3 was described as the cleavage of the colorimetric substrate by measuring the absorbance at 405 nm. ROT (5 µM, 24 h) was used to interpret that Cr(VI) function as MRCC I inhibitor. (B) Cr(VI) upregulates the expression of cleaved caspase-3. Whole cell lysates were analyzed by western blotting to detect the expression of full-length and cleaved caspase-3. (C) Cr(VI) induces apoptosis in a dose-dependent manner. After Cr(VI) treatment, L-02 hepatocytes were harvested and stained with Annexin V and PI, and analyzed with flow cytometry. The horizontal and vertical axes represent labeling with Annexin V and PI, respectively. LR represents early apoptotic cells (Annexin V-positive, as reflected in the lower-right-hand quadrant), UR represents late apoptotic cells (positive for both Annexin V and PI, depicted in the upper-right-hand quadrant). (D) Cr(VI) decreases the expression of heat shock protein. Western blotting was performed to determine the expression levels of HSP70 and HSP90. 
Cr(VI)-induced caspase-3 activation is dependent on ROS. (A) The specificity of NAC was confirmed. The hepatocytes were exposed to Cr(VI) (16, 32 µM) in the presence of 10 mM NAC for 24 h. The amount of ROS production was quantitated by flow cytometer. (B) ROS mediate the decrease of HSP and the activation of caspase-3 induced by Cr(VI). The whole cell lysates were analyzed by western blotting to examine the protein levels of caspase-3 (full-length and cleaved), HSP70, and HSP90. 
Article
Hexavalent chromium [Cr(VI)], which is used for various industrial applications, such as leather tanning and chroming, can cause a number of human diseases including inflammation and cancer. Cr(VI) exposure leads to severe damage to the liver, but the mechanisms involved in Cr(VI)-mediated toxicity in the liver are unclear. The present study provides evidence that Cr(VI) enhances reactive oxygen species (ROS) accumulation by inhibiting the mitochondrial respiratory chain complex (MRCC) I. Cr(VI) did not affect the expression levels of antioxidative proteins such as superoxide dismutase (SOD), catalase and thioredoxin (Trx), indicating that the antioxidative system was not involved in Cr(VI)-induced ROS accumulation. We found that ROS mediated caspase-3 activation partially depends on the downregulation of the heat shock protein (HSP) 70 and 90. In order to confirm our hypothesis that ROS plays a key role in Cr(VI)-mediated cytotoxicity, we used N-acetylcysteine (NAC) to inhibit the accumulation of ROS. NAC successfully blocked the inhibition of HSP70 and HSP90 as well as the activation of caspase-3, suggesting that ROS is essential in Cr(VI)-induced caspase-3 activation. By applying different MRCC substrates as electron donors, we also confirmed that Cr(VI) could accept the electrons leaked from MRCC I and the reduction occurs at MRCC I. In conclusion, the present study demonstrates that Cr(VI) induces ROS-dependent caspase-3 activation by inhibiting MRCC I activity, and MRCC I has been identified as a new target and a new mechanism for the apoptosis-inducing activity displayed by Cr(VI).
 
Article
Quercetin 7-rhamnoside (Q7R) is one of the main flavonoid components of Hypericum japonicum. However, whether Q7R is one of the active ingredients responsible for the hepatopreventive effects of Hypericum japonicum has not yet been ascertained. Thus, the aim of the present study was to elucidate whether Q7R attenuates apoptosis induced by glycochenodeoxycholic acid (GCDC) in vitro, and to elucidate the mechanisms involved. L-02 human normal liver cells were pre-incubated with 0, 50, 100 and 200 µM Q7R for 30 min and then exposed to 100 µM GCDC for the indicated periods of time. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was performed to examine cell viability. Apoptosis was evaluated by Hoechst 33258 staining and Annexin V-FITC/PI double staining. Intracellular reactive oxygen species (ROS) were detected by flow cytometry using the oxidation-sensitive fluorescent probe, DCFH-DA. The assay for glutathione (GSH) was performed using a GSH detection kit. Intracellular Ca2+ concentration was evaluated using a confocal laser scanning microscope with Fluo-3 as the Ca2+ probe and mitochondrial membrane potential (Δψm) was measured by rhodamine 123 (Rh123) fluorescence. Q7R attenuated the GCDC-induced reduction in cell viability and the high apoptotic rate. Moreover, Q7R protected the L-02 cells from ROS overproduction, GSH depletion, intracellular Ca2+ accumulation and Δψm decrease induced by GCDC. These results suggest that Q7R attenuates L-02 cell injury induced by GCDC, possibly by inhibiting the overproduction of ROS, GSH depletion, intracellular Ca2+ accumulation and Δψm decrease, thereby minimizing L-02 cell apoptosis.
 
Article
Tetraspanins are cell-surface glycoproteins and have received attention recently as both suppressors and promoters of metastasis. CO-029 is a member of the tetraspanin family and is implicated to be a metastasis-promoting tetraspanin in some cancers. However, the role of CO-029 in gastric cancer remains unexplored. The present study aimed to investigate the expression of CO-029 in gastric cancer tissues and to determine whether CO-029 is involved in the effects of epidermal growth factor (EGF) on gastric cancer cell proliferation and invasion. We collected clinical samples and found that the expression of CO-029 was increased both at the mRNA level and protein level in gastric cancer tissues in comparison to normal and tumor-adjacent tissues, as demonstrated by RT-qPCR and western blot analysis, respectively. Furthermore, we performed an in vitro experiment using AGS cells and observed that EGF promoted AGS cell proliferation and enhanced the invasion ability of the AGS cells, as shown by MTT assay and cell invasion assay, respectively. To the best of our knowledge, our results reveal for the first time, that CO-029 expression was affected by EGF in a concentration- time-dependent manner. The knockdown of CO-029 attenuated the effects of EGF on gastric cancer cell proliferation and invasion. These findings suggest that CO-029 is an oncogene in human gastric cancer and that CO-029 at least partially mediates the effects of EGF on gastric cancer cell proliferation and invasion. Our data may provide a novel target for therapeutic intervention in human gastric cancer.
 
Article
Several aliphatic dioic acids were recently reported to stimulate insulin release in isolated rat pancreatic islets incubated at close-to-physiological D-glucose concentrations. In order to gain insight into the mode of action of these acids in pancreatic islet B-cells, the oxidation of [1,12-14C]dodecanedioic acid (5.0 mM) was now measured in rat islets. Expressed as pmol of [1, 12-14C]dodecanedioic acid equivalent, the production of 14CO2 was close to 1.0 pmol/islet per 120 min, representing about 8% of that attributable to the oxidation of D-[U-14C]-glucose (8.3 mM). The dioic acid and the hexose failed to exert any significant reciprocal effect upon their respective oxidation rate. These findings support the view that the insulinotropic action of dodecanedioic acid, and presumably other aliphatic dioic acids, is causally linked to their capacity to act as nutrients in pancreatic islet cells.
 
Article
Tumoral pancreatic islet cells of the RINm5F line were incubated, in groups of 25x106 cells each, for 120 min at 37 degrees C in media (5. 0 ml) containing either alpha-D-[1,2-13C]glucose pentaacetate (1.7 mM) or both D-[1,2-13C]glucose (1.7 mM) and acetate (8.5 mM). In both cases, the amounts of 13C-enriched metabolites (D-glucose, L-lactate and acetate) and non-enriched metabolites (acetate) recovered in the incubation medium after incubation were close to the initial amount of esterified or non-esterified D-[1, 2-13C]glucose and acetate, respectively. The 13C-enriched metabolites corresponded mainly to double-labelled D-[1, 2-13C]glucose, L-[2,3-13C]lactate and [1,2-13C]acetate. The output of L-[2,3-13C]lactate and [1,2-13C]acetate was about 3-4 times lower in the cells exposed to alpha-D-[1,2-13C]glucose pentaacetate than in those incubated with unesterified D-[1,2-13C]glucose. These findings indicate that, despite extensive hydrolysis of alpha-D-[1, 2-13C]glucose pentaacetate in the RINm5F cells, the hexose moiety of the ester is less efficiently metabolized than unesterified D-[1, 2-13C]glucose tested at the same molar concentration (1.7 mM) in the presence of 8.5 mM acetate. Thus, a higher utilization of the hexose moiety of alpha-D-glucose pentaacetate than that of unesterified D-glucose, as previously documented in isolated pancreatic islets, represents a far-from-universal situation.
 
Article
The metabolism of [1,3-(13)C]glycerol-1,2,3-tris(methylsuccinate) and glycerol-1,2,3-tris(methyl[2,3-(13)C] succinate) was examined in hepatocytes prepared from hereditarily diabetic Goto-Kakizaki rats. Over 120 min incubation in the presence of one of the two (13)C-labelled esters (2.5 mM), the output of (13)C-enriched glucose averaged 57.1 +/- 18.5 and 54.1 +/- 22.7 nmol per 10(6) cells, when expressed as [1,3-(13)C]glycerol and [2,3-(13)C] succinate equivalent, respectively. In the case of [1,3-(13)C]glycerol-1,2,3-tris(methyl-succinate), the molecules of glucose were symmetrically labelled. In the case of glycerol-1,2,3-tris(methyl[2,3-(13)C] succinate), however, both the single-labelled and double-labelled isotopomers of glucose contained more (13)C atoms in their C(6)-C(5)-C(4) than C(1)-C(2)-C(3) moiety. These findings indicate that glycerol-1,2,3-tris(methylsuccinate), recently proposed as a novel insulinotropic tool for the treatment of non-insulin-dependent diabetes mellitus, is efficiently metabolized in hepatocytes from diabetic rats, the high rate of gluconeogenesis coinciding with channelling of D-glyceraldehyde-3-phosphate between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase.
 
Article
There are statistical data indicating that diabetes is a risk factor for Parkinson's disease (PD). Methylglyoxal (MG), a biologically reactive byproduct of glucose metabolism, the levels of which have been shown to be increase in diabetes, reacts with dopamine to form 1-acetyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (ADTIQ); this formation may provide further insight into the connection between PD and diabetes. In this study, we investigated the role of ADTIQ in these two diseases to determine in an aim to enhance our understanding of the link between PD and diabetes. To this end, a cell model of hyperglycemia and a rat model of diabetes were established. In the cell model of hyperglycemia, compared with the control group, the elevated glucose levels promoted free hydroxyl radical formation (p<0.01). An ADTIQ assay was successfully developed and ADTIQ levels were detected and quantified. The levels of its precursors, MG and dopamine (DA), were determined in both the cell model of hyperglycemia and the rat model of diabetes. The proteins related to glucose metabolism were also assayed. Compared with the control group, ADTIQ and MG levels were significantly elevated not only in the cell model of hyperglycemia, but also in the brains of rats with diabetes (p<0.01). Seven key enzymes from the glycolytic pathway were found to be significantly more abundant in the brains of rats with diabetes. Moreover, it was found that adenosine triphosphate (ATP) synthase and superoxide dismutase (SOD) expression levels were markedly decreased in the rats with diabetes compared with the control group. Therefore, ADTIQ expression levels were found to be elevated under hyperglycemic conditions. The results reported herein demonstrate that ADTIQ, which is derived from MG, the levels of which areincreased in diabetes, may serve as a neurotoxin to dopaminergic neurons, eventually leading to PD.
 
Article
Propyl gallate (PG) as a synthetic antioxidant is widely used in processed food and medicinal preparations. It also exerts a variety of effects on tissue and cell functions. In the present study, we investigated the effects of L-buthionine sulfoximine (BSO, an inhibitor of GSH synthesis), diethyldithiocarbamate (DDC, an inhibitor of Cu/Zn-SOD) or 3-amino-1,2,4-triazole (AT, an inhibitor of catalase) on PG-treated HeLa cells in relation to cell growth, reactive oxygen species (ROS) and glutathione (GSH). Treatment with PG induced growth inhibition, the loss of mitochondrial membrane potential [MMP (DeltaPsim)] and apoptosis in HeLa cells. ROS levels including O2.- were increased or decreased in PG-treated HeLa cells depending on the incubation times. PG caused depletion in GSH content in HeLa cells. While BSO enhanced the growth inhibition of PG-treated HeLa cells at 4 h, DDC and AT did not. All the agents down-regulated MMP (DeltaPsim) levels in PG-treated cells. Although BSO, DDC or AT slightly increased ROS or O2.- levels in PG-treated cells at 1 h, these enhancements of ROS did not intensify apoptosis in these cells. In addition, BSO, DDC or AT slightly reduced GSH level in PG-treated HeLa cells at 1 h, but this reduction did not affect cell death of HeLa. Furthermore, PG induced a G1 phase arrest of the cell cycle. BSO, DDC or AT significantly inhibited the G1 phase arrest in PG-treated cells. Conclusively, the changes of ROS and GSH levels by BSO, DDC or AT in PG-treated HeLa cells did not strongly affect the cell growth and death.
 
Article
Vitamin D exerts profound effects on airway epithelial cells. Thymic stromal lymphopoietin (TSLP) derived from airway epithelial cells plays a role in the innate and antigen‑specific adaptive immune responses. However, the effect of vitamin D on TSLP expression in airway epithelial cells is unclear. In this study, 16-HBE human bronchial epithelial (HBE) cells were cultured with various concentrations of 25-hydroxyvitamin D3 (25 D3) and 1,25-dihydroxyvitamin D3 (1,25 D3). The expression of TSLP in the 16-HBE human bronchial epithelial cell line was analyzed by PCR and enzyme-linked immunosorbent assay (ELISA). We found that the 16-HBE cells converted inactive 25 D3 to active 1,25 D3 and that TSLP mRNA and protein expression levels were significantly increased, peaking at 2 or 12 h in the cells exposed to 500 nM 25 D3 and 50 nM 1,25 D3 respectively. Since vitamin D3 upregulated protein 1 (VDUP1) plays a multifunctional role in a variety of cellular responses, we hypothesized that VDUP1 is involved in the induction of TSLP production by 25 D3. The results showed that the mRNA and protein levels of VDUP1 were significantly upregulated by vitamin D. Furthermore, the silencing of VDUP1 by small interfering RNA (siRNA) significantly inhibited the 25 D3- and 1,25 D3-mediated induction of TSLP expression. To characterize the metabolic properties of vitamin D in airway epithelial biology, we used the chemical inhibitor of 1α-hydroxylase, itraconazole. The results revealed that itraconazole (10-6 M) reduced the 25 D3- but not the 1,25 D3-induced TSLP expression in 16-HBE cells. Based on these data, it can be concluded that vitamin D increases TSLP expression in 16-HBE cells through the VDUP1 pathway, which suggests a novel mechanism by which vitamin D alters immune function in the lungs.
 
Article
Anaplastic carcinoma of thyroid is uncommon and is associated with a poor prognosis. an effective treatment of this carcinoma has not been found. We have examined the inhibition of promotion by 1, 25(OH)2D3 and its analogue without hypercalcemia, 22-oxacalcitriol (OCT) in the thyroid anaplastic carcinoma cell line. TTA-1, thyroid anaplastic carcinoma cell line, and TAC-1, thyroid anaplastic carcinoma cells. TPC-1,2,3,4, which are all papillary carcinoma of thyroid were used as control. Tumor growth was measured by MTT assay. 1,25(OH)2D3 additive cell growth was observed in diphasic pattern in 3/4 papillary carcinoma cell lines. OCT was more effective, showing dose-dependent inhibition of cell growth in anaplastic carcinoma cell lines, than that in papillary carcinoma cells. Conclusively, OCT might be useful for the inhibition of growth in anaplastic thyroid cancer.
 
Article
Vitamin D is essential for optimal calcium absorption needed for maintaining normal bone mineral density (BMD). Consequently, vitamin D-deficiency leads to poorly mineralized bone with diminished strength and load bearing capacity. Surprisingly, several animal and clinical studies have identified suppressive effects of high dose vitamin D supplementation on bone formation. These data suggest that while vitamin D is necessary for basal bone homeostasis, excessive concentrations may be detrimental to the skeleton. To further examine the direct effects of high dose vitamin D on the function of osteoblasts we differentiated primary osteoblast precursors and MC3T3 preosteoblastic cells, in the presence of supraphysiological doses of the active metabolite, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In vitro osteoblast mineralization was potently suppressed by high dose 1,25(OH)2D3. To investigate the mechanism we used a bioassay to examine nuclear factor-κB (NF-κB) activation in MC3T3 cells. Although NF-κB agonists are generally potent inhibitors of osteoblast differentiation, surprisingly, 1,25(OH)2D3 dose-dependently suppressed, rather than stimulated, NF-κB activation. Interestingly, 1,25(OH)2D3 also suppressed Smad activation induced by the osteoblast commitment and differentiation factors transforming growth factor-β (TGF-β) and bone morphogenetic protein 2 (BMP2), which may account for the inhibitory activities of 1,25(OH)2D3 on mineralization. Our data suggest that vitamin D has complex pleiotropic effects on osteoblast signal transduction. As the net balance of high dose 1,25(OH)2D3 appears to be an inhibitory action on osteoblasts, our data suggest that the therapeutic value of vitamin D to maximize bone mass through indirect actions on calcium absorption may need to be carefully balanced with potential inhibitory direct effects on mineralizing cells. Our data suggest that indiscriminate over-dosing may be detrimental to bone formation and optimal concentrations need to be established for humans in vivo.
 
Dose response of the anti-adipogenic effects of 1,25(OH)2D3. (A) The inhibitory effects of the various concentrations of 1,25(OH)2D3 on the intracellular lipid droplet formation. Lipid droplets were stained with Oil Red O on day 6 of adipogenic differentiation and examined under light microscopy. (B) Effects of the various concentrations of 1,25(OH)2D3 on the expressions of adipocyte-specific marker proteins on day 6 of adipogenic differentiation. β-actin was used as a loading control for total cellular proteins. Each experiment was repeated independently at least twice, and the representative results are shown. Pre, undifferentiated preadipocytes. 
Time-course response of the anti-adipogenic effects of 1,25(OH)2D3. (A) Time-course response for the mRNA expression levels of the adipocytespecific marker genes in the absence (open circle) or presence of 10 nM 1,25(OH)2D3 (closed circle). mRNA levels were expressed as the ratio to that of 0 day. (B) Time-course response for the levels of the adipocyte-specific marker proteins in the absence (-) or presence (+) of 10 nM 1,25(OH)2D3. The data represent the mean ± standard deviation (SD) of triplicate assays. Each experiment was repeated independently at least twice, and the representative results are shown. ** p<0.01, *** p<0.001 compared with untreated cells on the same differentiation day. 
Effects of 1,25(OH)2D3 on WNT signaling during adipogenesis. (A-C and E) Time-course response for the mRNA expression levels of genes involved in WNT signaling in the absence (open circle) or presence of 10 nM 1,25(OH)2D3 (closed circle). mRNA levels were expressed as the ratio to that of 0 day. (D) Timecourse response for the protein level of DVL2 in the absence (-) or presence (+) of 10 nM 1,25(OH)2D3. (F) Time-course response for the protein level of GSK3β, for total and phosphorylated forms, in the absence (-) or presence (+) of 10 nM 1,25(OH)2D3. The data represent the mean ± standard deviation (SD) of triplicate assays. Each experiment was repeated independently at least twice, and the representative results are shown. ** p<0.01, *** p<0.001 compared with untreated cells on the same differentiation day. 
Effects of 1,25(OH)2D3 on β-catenin levels during adipogenesis. (A) Time-course response for the nuclear levels of β-catenin protein in the absence (-) or presence (+) of 10 nM 1,25(OH)2D3. TBP was used as a loading control for nuclear proteins. (B) Time-course response for the mRNA expression levels of β-catenin in the absence (open circle) or presence of 10 nM 1,25(OH)2D3 (closed circle). The data represent the mean ± standard deviation (SD) of triplicate assays. Each experiment was repeated independently at least twice, and the representative results are shown. *** p<0.001 compared with untreated adipocytes on the same differentiation day. 
Article
1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D, was found to have anti-adipogenic activity, however, its mechanism of action has not been fully elucidated. In this study, 3T3-L1 preadipocytes were differentiated in the presence and absence of 1,25(OH)2D3, and the expression of the genes and proteins of the wingless-type MMTV integration site (WNT)/β-catenin pathway were analyzed. While the expression of the members of the WNT/β-catenin pathway were significantly downregulated during the adipogenesis of untreated 3T3-L1 cells, 1,25(OH)2D3 treatment was found to maintain the WNT/β-catenin pathway. Among the members of the WNT/β-catenin pathway, the levels of WNT10B and disheveled (DVL)2 as well as the phosphorylation of glycogen synthase kinase (GSK)3β were maintained by 1,25(OH)2D3 treatment. The levels of nuclear β-catenin, which were downregulated during adipogenesis, were also maintained by 1,25(OH)2D3 treatment. The results of this study suggested that the anti-adipogenic effect of 1,25(OH)2D3 was mediated by the maintenance of the WNT/β-catenin pathway, which was normally downregulated during adipogenesis.
 
Article
1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] plays an anticancer role in multiple types of cancer and potentiates the cytotoxic effects of several common chemotherapeutic agents. The hypercalcemia caused by 1,25(OH)2D3 alone or resistance to cisplatin weaken the anticancer effects of vitamin D. Thus, in this study, we aimed to investigate the synergistic effects of 1,25(OH)2D3 and cisplatin on the apoptosis and cell cycle progression of gastric cancer cells. BGC-823 human gastric cancer cells were treated with 1,25(OH)2D3 or cisplatin alone, or a combination of both agents. Cell apoptosis was assessed by TUNEL assay and flow cytometry. The expression of the apoptosis-related proteins, poly(ADP-ribose) polymerase (PARP), Bax, Bcl-2, caspase-3 and caspase-8, was examined using immunoblot analysis. ERK and AKT phosphorylation were examined by immunoblot analysis. The cell cycle distribution was determined by propidium iodide staining and flow cytometric analysis. p21 and p27 protein expression was also examined using immunoblot analysis. Our results revealed that co-treatment with 1,25(OH)2D3 enhanced cisplatin-induced apoptosis and upregulated the expression of Bax, and promoted the cleavage of PARP and caspase-3. The phosphorylation levels of ERK and AKT were reduced following combined treatment with 1,25(OH)2D3 and cisplatin. The percentage of cells in the G0/G1 phase was greater in the cells treated with the combined treatment than in those treated with either 1,25(OH)2D3 or cisplatin alone. p21 and p27 expression was upregulated following co-treatment with both agents. The results of this study suggest that 1,25(OH)2D3 potentiates cisplatin-mediated cell growth inhibition and cell apoptosis, which involves the upregulation of Bax, a decrease in ERK and AKT phosphorylation levels, and increased p21 and p27 levels.
 
Article
Icariin, the main active compound of the traditional Chinese medicine, Epimedium, is commonly used for the clinical treatment of osteoporosis. However, the precise molecular mechanism of the therapeutic effect of icariin has not been elucidated. The aim of this study was to examine the effect of icariin on cell viability, alkaline phosphatase (ALP) activity, the amount of calcified nodules, and to delineate the molecular mechanism of icariin-enhanced bone formation by investigating the expression of bone morphogenic protein‑2 (BMP-2), Smad4, Cbfa1/Runx2, osteoprotegerin (OPG), receptor activator of nuclear factor κ-B ligand (RANKL) and the OPG/RANKL ratio in the hFOB 1.19 human osteoblastic cell line. We found that icariin significantly increased the cell viability, the activity of ALP and the amount of calcified nodules in the hFOB 1.19 cells. Furthermore, we observed that icariin upregulated the expression of BMP-2, Smad4, Cbfa1/Runx2, OPG, RANKL and the OPG/RANKL ratio. Our results indicate that icariin can modulate the process of bone formation via the BMP-2/Smad4 signal transduction pathway in hFOB 1.19 cells.
 
Article
Dihydropyridine (DHP) type Ca2+ channel blocker (CCB) is effective in treatment of hepatitis in man. L-type Ca2+ channel is a target of DHP-CCB, and basic studies suggest that L-type Ca2+ channel alpha1D-subunit (Cav 1.3) seems to be a target of drug development for the treatment of hepatitis. Mouse hepatitis model is useful to study the effect of DHP-CCB on hepatitis. In order to use mouse hepatitis model to screen DHP-CCB specific for Cav 1.3, Cav 1.3 expressed in the mouse liver should have enough structural homology with that of human Cav 1.3. cDNA of the Cav 1.3 was cloned from mouse brain by reverse transcription polymerase chain reaction. The primary structure of the mouse Cav 1.3 comprises an open reading frame of 6540 bp encoding 2180 amino acids. Liver transcript lacked 60 and 45 bases from 1497 to 1556, and from 3949 to 3993 of the sequence, respectively, due to results of an alternative splicing. The present results indicated that mouse Cav 1.3 exhibited 96% homology with human Cav 1.3 and was expressed in the liver. Thus, mouse hepatitis model seemed to be useful to screen DHP-CCB specific for Cav 1.3.
 
Article
TAS-102 is a new oral anti-tumor drug preparation, composed of a 1:0.5 mixture (on a molar basis) of alpha,alpha,alpha-tri-fluorothymidine (FTD) and thymidine phosphorylase inhibitor (TPI). TAS-102 is currently undergoing clinical trials, and has been demonstrated to have at least 2 mechanisms; inhibition of thymidylate synthase (TS) and incorporation into DNA. 5-FU is widely used in the treatment of solid tumor, but the inherent or acquired resistance of certain tumors to 5-FU therapy is a major clinical problem. In the present study, we investigated FTD in vitro and in vivo comparing with 5-FU and using FU-resistant cells. There was no relationship between FTD and 5-FU growth inhibition effect in vitro. A different sensitivity pattern was observed by the log-mean graph. We next investigated the anti-tumor activity of TAS-102 in a FU-resistant xenograft model. Comparative efficacy was observed between FU-resistant cell and its parent cell. We also studied the influence of TAS-102 on liver metastasis in a mouse model of human colorectal cancer, because liver metastasis of colorectal cancer is associated with patient survival. Human cancer DNA was detected by PCR, and TAS-102 markedly inhibited the number of liver metastasis. A novel angiogenic factor, platelet-derived endothelial cell growth factor (PD-ECGF), was shown to be identical to a previously characterized intracellular enzyme, thymidine phosphorylase, TAS-102 can be expected to have not only anti-tumor cytocidal effects but also antiangiogenesis activity and may inhibit liver metastasis. Our findings suggested that TAS-102 is a promising candidate for clinical use and can be expected to decrease minimal residual disease.
 
Article
TAS-102 is a combination drug consisting of alpha,alpha,alpha-trifluorothymidine (FTD) and thymidine phosphorylase inhibitor (TPI). FTD is converted to F3TMP by thymidine kinase and inhibits the thymidylate synthetase (TS) activity by binding to TS. In addition, FTD triphosphate form, F3TTP is incorporated into DNA, which leads to cytocidal effects. Therefore, the incorporation of FTD into DNA is expected to be an important factor, discriminating it from 5-FU showing TS inhibitory activity as their main mechanism of action. To assess a clinically more effective regimen protocol, the intracellular metabolism and the incorporation of FTD into DNA were investigated using human cancer cell lines in vitro and in vivo. FTD was incorporated into DNA in a time-dependent manner, but not in a concentration-dependent manner. FTD was the most efficiently incorporated into DNA after treatment with a several-micro molar level of FTD for around 8 h. The intracellular F3TTP was rapidly eliminated from tumor cells, as soon as FTD was washed out from the culture medium, whereas the FTD incorporated into DNA was retained by 80% or more even at 24 h after a washing-out procedure. When TAS-102 was administered into tumor-bearing mice once daily or three times daily at 3-h intervals at a dose of 150 mg/kg/day for one or 3 consecutive days, incorporation of FTD into tumor DNA by divided dosing significantly higher than that of single dosing. Based on our findings, the antitumor effects of TAS-102 against 3 different human cancer cell xenografts into mice were examined. The divided daily dosing resulted in enhancement of the antitumor effects of TAS-102 without any additional side effects. It is concluded that multiple daily dosing may result in better clinical benefits of TAS-102, when compared with single daily dosing and TAS-102 is a promising candidate for not only FU-sensitive but also FU-resistant cancer patients.
 
Article
The deletion mutation of exon 4 in surfactant protein C (SP-C), a lung surfactant protein, has been identified in parent-child cases of familial interstitial pneumonia. It has been shown that this mutation induces endoplasmic reticulum (ER) stress. Synoviolin is an E3 ubiquitin ligase that is localized to the ER and is an important factor in the degradation of ER-related proteins. It has been demonstrated that synoviolin is involved in liver fibrosis. In the present study, we investigated the involvement of synoviolin in the pathogenesis of interstitial pneumonia caused by the exon 4 deletion in the SP-C gene. We transfected wild-type and exon 4-deleted SP-C genes into A549 human lung adenocarcinoma cells and measured the secretion of collagen, which is a representative extracellular matrix protein involved in fibrosis. Secreted collagen levels were increased in the culture medium in SP-C mutants compared to the wild-type cells. Furthermore, the transcription of mRNAs coding for factors associated with fibrosis was increased. Subsequently, to assess the involvement of synoviolin, we constructed plasmids with a luciferase gene under the control of the synoviolin promoter. The A549 cells were transfected with the construct along with the exon 4-deleted SP-C plasmid for use in the luciferase assay. We found a 1.6-fold increase in luciferase activaty in the cells carrying exon 4 deleted SP-C, as well as an increase in intrinsic synoviolin expression at the mRNA and protein levels. Collagen secretion was decreased by the addition of LS-102, a synoviolin inhibitor, to the A549 culture medium following transfection with wild-type and exon 4-deleted SP-C. These results demonstrate that synoviolin is involved in the onset of interstitial pneumonia induced by exon 4-deleted SP-C, which suggests that synoviolin inhibitors may be used in the treatment of the disease.
 
Article
PI-103, the first synthetic multitargeted compound which simultaneously inhibits PI3Kalpha and mammalian target of rapamycin (mTOR) shows high antitumor activity in glioma xenografts. In the present study, clear antitumor activity was observed with PI-103 treatment in two gefitinib-resistant non-small cell lung cancer (NSCLC) cell lines, A549 and H460, by simultaneously inhibiting p70s6k phosporylation and Akt phosphorylation in response to mTOR inhibition. In addition, H460 cells with activating mutations of PIK3CA were more sensitive to PI-103 than A549 cells with wild-type PIK3CA. PI-103 was found to inhibit growth by causing G0-G1 arrest in A549 and H460 cells. Western blotting showed that PI-103 induced down-regulation of cyclin D1 and E1 and simultaneously up-regulated p21 and p27, associated with arrest in the G0-G1 phase of the cell cycle. Furthermore, p53, the tumor suppressor which transcriptionally regulates p21, was also upregulated with PI-103 treatment. Collectively, our results suggest that multitargeted intervention is the most effective tumor therapy, and the cooperative blockade of PI3Kalpha and mTOR with PI-103 shows promise for treating gefitinib-resistant NSCLC.
 
Article
We isolated 23 marine actinomycetes from seawater samples. Of these, strain SY-103 exhibited the strongest cytotoxic activity on human leukemic cell lines. Biochemical tests and 16S rDNA sequencing of this strain allowed us to identify SY-103 as a strain of the genus Streptomyces. In the present study, the pure cytotoxic compound (PCC) from Streptomyces sp. SY-103 metabolites was purified by reverse-phase HPLC and the biochemical mechanisms of PCC-induced apoptosis in cultured human leukemic cell lines were investigated. The exposure of cells to PCC resulted in growth inhibition and induction of apoptosis, which was associated with the proteolytic activation of caspase-3 and down-regulation of anti-apoptotic Bcl-2 protein. However, PCC-induced caspase-3 activation and apoptosis were significantly attenuated in Bcl-2 overexpressing U937 cells. z-DEVD-fmk, a caspase-3 specific inhibitor, blocked caspase-3 activation and increased the survival rate of PCC-treated U937 cells. The activity of Akt was also inhibited in PCC-treated cells, and phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor, LY294002, sensitized the cells to PCC-induced apoptosis indicating that the down-regulation of the Akt signaling pathway plays a key role in PCC-induced apoptosis. Our findings imply that some of the biological functions of Bcl-2 and PI3K/Akt are attributed to their ability to inhibit PCC-induced apoptosis; therefore, it is suggested that this compound is a promising anti-cancer agent for leukemia cells.
 
TaqMan low-density array screening of differentially expressed microRNAs in plasma. We compared miRNA expression levels in plasma from current smokers without COPD (n=3) with those from current smokers with COPD (n=3). Nine miRNAs (miR-29b, miR-483-5p, miR-152, miR-629, miR-26b, miR-101, miR-106b, miR-532-5p and miR-133b) were significantly downregulated in plasma from COPD patients. 
Expression of plasma miR-106b levels in patients with COPD and controls. (A) Expression of miR-106b was determined by qRT-PCR. The plasma miR-106b level in ex-smokers with COPD was significantly downregulated compared with that in current smokers without COPD (P= 0.0050). The plasma miR-106b level in current smokers with COPD was significantly lower than that in current smokers without COPD (P= 0.0309). Among the COPD subjects, the level of miR-106b in ex-smokers was significantly lower than that in current smokers (P= 0.0449). (B) The receiver operating characteristic curve showed that the cut-off level of plasma miR-106b in all COPD patients at diagnosis was 0.4005; the sensitivity was 75.00% (95% CI, 58.80 - 87.31) and the specificity was 90.00% (95% CI, 55.50 - 99.75). 
Relationship of plasma miR-106b expression in COPD patients and smoking history. (A) Correlation between the levels of plasma miR-106b and duration of disease since diagnosis in ex-smokers with COPD. Pearson ' s correlation coefficient (r) is provided. (B) Correlation between the levels of plasma miR-106b and duration of smoking in current smokers with COPD. 
Article
Chronic obstructive pulmonary disease (COPD) is characterized by both chronic inflammation in the airway and systemic inflammation; however, the molecular mechanism of COPD has not been fully elucidated. By measuring microRNA (miRNA) expression in the plasma of COPD subjects, we aimed to identify the clinical relevance of plasma miRNA levels in these patients. Blood samples were obtained from COPD patients and age-matched normal controls. We initially produced plasma miRNA expression profiles using TaqMan low-density array screening. For further validation, individual qRT-PCRs were performed in 40 COPD patients and 20 healthy subjects. TaqMan low-density array screening showed that 9 miRNAs (miR-29b, miR-483-5p, miR-152, miR-629, miR-26b, miR-101, miR-106b, miR-532-5p and miR-133b) were significantly downregulated in the plasma from COPD patients when compared with normal smokers. Among these miRNAs, we focused on miR-106b. A reduction in the plasma miR-106b levels was evident in COPD ex-smokers and COPD current smokers compared with levels in smokers. There was a negative correlation between the plasma miR-106b level and the duration of disease since diagnosis in COPD ex-smokers and the duration of smoking in COPD current smokers. These findings support the concept that progressive reduction in the plasma miR-106b level may reflect persistent and systemic changes even after the discontinuation of smoking in COPD patients. miR-106b may therefore play an important role in the pathogenesis of COPD.
 
Article
FGFR2 is an oncogene amplified in diffuse-type gastric cancer, and WDR11 is a tumor suppressor gene disrupted in glial tumor. WDR11-FGFR2 locus on human chromosome 10q26 is one of cancer-related recombination hot spots. In this study, we investigated recombination and nucleotide substitution around the WDR11-FGFR2 locus during evolution by using bioinformatics. Inter-chromosomal comparison revealed that the human BAG3-FGFR2-TACC2 region was paralogous to the human BAG4-FGFR1-TACC1 region. Inter-specific comparison on the BAG3-FGFR2-TACC2 region revealed that HTPAPL-WDR11-FGFR2 locus containing species-specific insertion or deletion was one of evolutionary recombination hot spots. Between human and mouse, coding-region nucleotide substitution rate and amino-acid substitution rate were significantly lower in the HTPAPL-WDR11-FGFR2 locus than in the surrounding locus (P<0.0001). The HTPAPL-WDR11-FGFR2 locus was more susceptible to recombination than to nucleotide substitution. Detailed comparison of human and mouse genomes could identify evolutionary recombination hot spots overlooked during gross comparison of human and mouse genomes. Because DNA double-strand break is the initial step in various types of recombination including chromosomal translocation, rearrangement, deletion, gene amplification, retroviral integration and retrotransposition, it is reasonable that the HTPAPL-WDR11-FGFR2 locus is the recombination hot spot during evolution as well as during carcinogenesis. Therefore, comparative genomics might be applicable to identification of recombination hot spots and genes related to cancer.
 
Article
WNTs are a family of secreted-type glycoproteins implicated in embryogenesis and carcinogenesis. We have previously cloned and characterized human WNT2B/WNT13, WNT3, WNT3A, WNT5B, WNT6, WNT7B, WNT8A, WNT8B, WNT10A, WNT10B, WNT11, WNT14, and WNT14B/WNT15. WNT14B gene is clustered with WNT3 gene in human chromosome 17q21, and mRNA expression of WNT14B is significantly up-regulated by retinoic acid during the early phase of neuronal differentiation in human NT2 cells. Here, we identified mouse Wnt14b gene fragments in mouse genome draft sequence AL596108.5 by using bioinformatics, and isolated mouse Wnt14b cDNAs by using cDNA-PCR. Mouse Wnt14b was found to encode a 359-amino-acid WNT family protein with the N-terminal signal peptide, an N-linked glycosylation site, and 24 conserved cysteine residues. Mouse Wnt14b showed 92.5% total-amino-acid identity with human WNT14B, and 64.2% total-amino-acid identity with human WNT14. Mouse Wnt14b gene, consisting of 4 exons, was clustered with mouse Wnt3 gene in mouse chromosome 11. Mouse Wnt14b mRNA was relatively highly expressed in 17-day embryo, and also expressed in adult brain, kidney, liver, 7-day embryo, and 11-day embryo. This is the first report on molecular cloning and characterization of mouse Wnt14b.
 
Article
Heme oxygenase (HO)-1, the rate-limiting enzyme in heme catabolism, can be induced in response to various oxidative stimuli, and its induction is thought to be critical in the cellular defense against oxidative tissue injuries. Carbon tetrachloride (CCl(4)) treatment of rats causes lipid peroxidation of cell membranes and produces massive hepatic injury. We previously demonstrated that HO-1 induction following CCl(4) treatment is an essential part of the cellular defense against the CCl(4)-inducible toxic changes. As recombinant human interleukin-11 (rhIL-11) has been shown to induce HO-1 in cultured hepatoma cells, we examined the effect of rhIL-11 in vivo in rats on the CCl(4)-induced tissue injury. rhIL-11 treatment of animals by itself markedly induced HO-1 mRNA and its functional protein principally in the liver. rhIL-11 treatment (150 microg/kg) of the CCl(4)-administered (1 ml/kg) animals led to a further increase in HO-1 mRNA, while it markedly suppressed CCl(4)-induced serum alanine transaminase, hepatic malondialdehyde formation, tumor necrosis factor-alpha mRNA, nitric oxide synthase mRNA, nuclear factor-kappaB DNA-binding activity, as well as inflammatory changes of hepatocytes. In contrast, inhibition of HO activity by tin-mesoporphyrin, a competitive specific inhibitor of HO, entirely abolished the cytoprotective effect of rhIL-11. These findings thus demonstrate that rhIL-11 confers significant protection against CCl(4)-induced hepatic injury by virtue of its liver-specific HO-1 induction.
 
P7 peptides counteract resistance of HT-29 cells to CPT-11 induced by basic fibroblast growth factor (bFGF). (A) Cells were treated with CPT-11 at increasing concentrations (from 15 to 180 µM) for 48 h. (B) Following pre-treatment with bFGF (20 ng/ml) alone, or bFGF (20 ng/ml) plus P7 peptides (4 µM) for 4 h, the starved cells were treated with CPT-11 (60 µM) for 48 h. Cell viability was measured by MTT assay. Data are presented as the means ± SD of 3 independent experiments performed in triplicate. * P<0.01 vs. CPT-11 group; ** P<0.01 vs. CPT-11 plus bFGF group. 
Effects of P7 peptides on the inhibition of CPT-11-induced apoptosis by basic fibroblast growth factor (bFGF) in HT-29 cells. (A) Following pretreatment with bFGF (20 ng/ml) alone, or bFGF (20 ng/ml) plus P7 peptides (4 µM) for 4 h, the starved cells were treated with CPT-11 (60 µM) for 48 h. Cells were stained with Alexa Fluor 488 Annexin V and PI prior to analysis by flow cytometry. The lower right quadrant indicates early-stage apoptosis and the upper right quadrant indicates late-stage apoptosis. (B) Comparison of percentages of apoptotic cells. Results are expressed as a mean percentage of cells ± SD of 3 independent experiments. ** P<0.001. 
Effects of P7 peptides on the PI3K/Akt signaling pathway in HT-29 cells. (A) Starved cells were pre-treated with basic fibroblast growth factor (bFGF) (20 ng/ml) alone, or bFGF (20 ng/ml) plus P7 (4 µM) for 4 h prior to stimulation with CPT-11 (60 µM). The phosphorylated and total levels of Akt were determined by western blot analysis. (B) Density ratios of phosphorylated proteins to total proteins are presented as the means ± SD of 3 independent experiments. * P<0.01; ** P<0.001. 
P7 peptides suppress the internalization of basic fibroblast growth factor (bFGF). (A) Starved HT-29 cells were treated with 20 ng/ml biot-bFGF for 4 h (lanes 3 and 4), or with P7 (4 µM) for 5 min before stimulated by biot-bFGF (lanes 5 and 6). Control cells were untreated with biotinylated (biot)-bFGF or P7 peptides (lanes 1 and 2). Nuclear and cytoplasm extracts were incubated in the presence of streptavidin beads. Proteins bound on streptavidin beads were analyzed by western blot analysis using anti-bFGF antibody. (B) Density ratios of biot-bFGF in nucleus or cytoplasm in bFGF group or bFGF plus P7 group with the control group are presented as the means ± SD of 3 independent experiments. * P<0.001 vs. control group; ** P<0.001 vs. bFGF group. 
Article
The low survival rate of patients with colorectal cancer (CRC) is mainly due to the drug resistance of tumor cells to chemotherapeutic agents. It has been reported that basic fibroblast growth factor (bFGF) is an essential factor involved in the epigenetic mechanisms of drug resistance, which provides a novel potential target for improving the sensitivity of tumor cells to chemotherapeutic agents. In this study, we first demonstrate that a novel bFGF antagonist, peptide P7, previously isolated by phage display technology, reversed bFGF-induced resistance to irinotecan hydrochloride (CPT-11), and counteracted the anti-apoptotic effects of bFGF on CPT-11-treated HT-29 cells. Further experiments indicated that the inhibition of Akt activation, the suppression of bFGF internalization, the increase in the Bax to Bcl-2 ratio and the downregulation of cytokeratin 8 (CK8) by P7 may contribute to the counteracting of the anti-apoptotic effects of bFGF, and further reversal of bFGF-induced resistance to CPT-11. Our results suggest that peptide P7 may have therapeutic potential in CRC as a sensitizer to chemotherapeutic agents by targeting bFGF.
 
Article
Porphyra yezoensis (P. yezoensis) is the most noteworthy red alga and is mainly consumed in China, Japan and Korea. In the present study, the effects of a P. yezoensis peptide (PY‑PE) on cell proliferation and the associated signaling pathways were examined in IEC‑6 rat intestinal epithelial cells. First, the MTS assay showed that PY‑PE induced cell proliferation in a dose‑dependent manner. Subsequently, the mechanism behind the proliferative activity induced by PY‑PE was determined. The insulin‑like growth factor‑I receptor (IGF‑IR) signaling pathway was the main focus as it plays an important role in the regulation of cell growth and proliferation. PY‑PE increased the protein and mRNA expression of IGF‑IR, insulin receptor substrate‑1, Shc and PY‑99. In addition, PY‑PE stimulated extracellular signal‑regulated kinase phosphorylation and phosphatidylinositol 3‑kinase/Akt activation but inhibited p38 and c‑Jun N‑terminal kinase phosphorylation. Furthermore, PY‑PE treatment increased protein and mRNA expression levels of activator protein‑1, which regulates cell proliferation and survival, in the nuclear fraction. These results have significant implications for understanding the role of cell proliferation signaling pathways in intestinal epithelial cells.
 
Analysis of surface phenotype of Vα24 + Vß11 + NKT cells in cord blood and adult blood. Vα24 + cells were enriched from fresh cord blood and adult peripheral blood by immunomagnetic bead separation, and further subjected to flow cytometrical analysis. (A) Vα24 + Vß11 + cells were gated in the lymphocyte fraction focused on forward scatter and side scatter. (B) CD3 and CD161 expression of Vα24 + Vß11 + gated NKT cells. (C) CD4 and CD8 expression of the CB Vα24 + Vß11 + NKT cells. The data are representative of 10 independent experiments (A-C). Proportion of CD4 + (D), DN (E) or CD8 + (F) NKT cells in cord blood (CB) (n=10) and adult peripheral blood (PB) (n=8) Vα24 + Vß11 + NKT cells. The numbers indicate the mean percentages of positive cells of each phenotype in Vα24 + Vß11 + NKT cells.  
Cytokine production and IL-12Rß1/IL-18Rα expression in CB CD4 + Vα24 + NKT cells. Immunomagnetic bead-separated Vα24 + cells were cultured with α-GalCer and IL-2 for 14 days, which resulted in the preferential expansion of CD4 + Vα24 + Vß11 + NKT cells with >95% purity. Intracellular staining for IL-13 and IFN-γ in fresh (A) and cultured CB CD4 + Vα24 + Vß11 + NKT cells (B) after stimulation by ionomysin/PMA for 4 h. Expression of IL-12Rß1 (C) and IL-18Rα (D) on cultured CD4 + Vα24 + Vß11 + NKT cells. The data are representative of 4 independent experiments (A-D).  
IFN-γ/IL-13 secretion by CD4 + Vα24 + NKT cells. Cultured CD4 + Vα24 + Vß11 + NKT cells were washed and incubated in the presence of IL-2 or IL-12 with or without IL-18. The culture supernatants were harvested 7 days later and analyzed by ELISA. The data are shown as the mean ± SD from 5 separate experiments.  
Article
Natural killer T (NKT) cells, exhibiting both T-cell and NK-cell markers, are known to regulate immune responses by secreting T-helper (Th) 1 and Th2 cytokines. We analyzed NKT cells in cord blood (CB) for phenotypical and functional characteristics and regulatory mechanisms that control Th1 and Th2 determination. Human CB V alpha 24+V beta 11+ NKT cells were predominantly the CD4+ single positive (SP) phenotype (approximately 96%), in contrast to adult peripheral blood V alpha 24+V beta 11+ NKT cells which are composed of a dominant population of the CD4-CD8-double negative (DN) phenotype and a minor population of the CD4+ SP phenotype. The CB CD4+ V alpha 24+ NKT cells, following stimulation with the primary culture, gained the capacity to secrete interferon (IFN)-gamma, a Th1 cytokine, and interleukin (IL)-13, a Th2 cytokine. The combination of IL-18 and IL-12 induced IFN-gamma production in CB CD4+ V alpha 24+ NKT cells, while IL-18 in combination with IL-2 induced IL-13 production in these cells. Thus, IL-18 regulates the determination of the Th1 or Th2 immune response by human CD4+ V alpha 24+ NKT cells through different cytokine combinations.
 
C20:1ω9 relative weight content (upper panel) and C20:1ω9/ C18:1ω9 ratio (lower panel) in liver triglycerides (horizontally hatched columns), adipose tissue lipids (vertically hatched columns) and plasma triglycerides (open columns) of fed or fasted female control (C) rats, fed female STZ rats, and both fed and fasted female or male GK rats. Mean values (± SEM) refer to the number of individual observations indicated in parentheses at the bottom of the figure. 
Article
Considering the proposed preventive effect of nervonic acid on obesity- and diabetes-related coronary risk factors, the content of its precursors (oleic, 11-eicosenoic and 13-docosenoic acids) was measured in liver and plasma phospholipids and triglycerides, brain and spleen phospholipids, and adipose tissue lipids of fed or overnight fasted control and hereditarily diabetic Goto-Kakizaki female rats, as well as fed streptozotocin-induced diabetic female rats. In liver and brain phospholipids, the 11-eicosenoate/oleate ratio was significantly higher in diabetic rats than in control animals. Such was not the case in either spleen phospholipids or liver triglycerides and adipose tissue lipids. The increase in the liver phospholipid 11-eicosenoate/oleate ratio found in female diabetic rats represents a mirror image of the situation recently documented, in the same animal models of diabetes, in male rats. These contrasting findings may be relevant to the higher coronary heart disease risk prevailing in female, as compared to male, diabetic subjects.
 
Article
Beta-catenin serves not only as a structural component of the E-cadherin-mediated cell-cell adhesion system, but also as a signaling molecule of the Wnt/wingless pathway. Mutations of beta-catenin and aberrant expression of its protein have been identified in a number of different types of human malignancies. To determine the role of beta-catenin in solid and cystic tumor (SCT) of the pancreas, a rare neoplasm usually observed in young females, we examined three primary tumors and one corresponding liver metastatic tumor presented in pediatric patients. Single strand conformation polymorphism and neucleotide sequencing analysis confirmed a TCT right curved arrow TTT somatic mutation at codon 37 changing serine to phenylalanine in one of the primary tumors and its corresponding metastatic tumor. Immunohistochemical analysis displayed abnormally strong nuclear and widespread cytoplasmic expression of beta-catenin in these tumors with the mutation. Our observations suggest that, in some SCTs of the pancreas presented in the pediatric age group, mutated beta-catenin has an important oncogenic effect.
 
Article
One of the most aggressive human malignancies, anaplastic thyroid carcinoma (ATC), has an extremely poor prognosis that may be explained by its genomic instability. We hypothesized that the very rapid cell turnover observed in ATC might accelerate telomere shortening and chromosomal instability associated with tumor cell malignancy. To compare and measure chromosomal aberrations and telomere shortening in the anaplastic thyroid cancer cell line OCUT-1, we applied quantitative fluorescence in situ hybridization (Q-FISH) techniques. In all 15 metaphases studied, telomere length estimates from Q-FISH of chromosomes in ATC were shorter than those of a fibroblast cell line derived from the stroma adjacent to the carcinoma. OCUT-1 cells display several chromosomal abnormalities, but have a near-normal chromosome complement of 46, XX, making it easy to analyze the karyotype. The karyotype showed 50, XX, +7, +11, der(11)t(3;11)(q23;q23)x2, del(12)(p11.2p12), +20, +1mar. We analyzed carefully the abnormalities in karyotype of OCUT-1 associated with telomere shortening on each chromosome and expression of subtelomeres. Telomere lengths in the q-arms of the abnormal chromosome del(12)(p11.2p12) were shorter than the average length in the q-arms of the normal chromosome 12 in OCUT-1. Subtelomeres on the abnormal chromosome der(11)t(3;11)(q23;q23)x2 also showed loss of signals on 11p, but there was no loss of signals in the cytogenetically normal trisomies 7 and 20 or the abnormal chromosome del(12)(p11.2p12). Subtelomeres of 3q had eight signals, one pair remaining in place on 3q and another pair on the abnormal 11p. Our findings suggest that telomere shortening and subtelomere loss are correlated with genetic instability in this anaplastic thyroid carcinoma cell line.
 
Article
Histone deacetylases (HDACs) play a central role in the modification of chromatin structure and thus in the regulation of transcription and cellular differentiation. Based on structural and functional similarities, mammalian histone deacetylases may be grouped into four categories: class I HDACs, which are homologs of the yeast histone deacetylase RPD3; class II HDACs, which share a significant degree of homology with the yeast histone deacetylase HDA1; class III HDACs, which are closely related to the yeast SIR2 protein; and the most recently described class IV of HDACs, which comprises HDAC11-related enzymes. We have isolated and characterized the human HDAC11 genomic sequence, which spans a region of 24,074 bp and has a single genomic locus. Determination of the exon-intron splice junctions established that HDAC11 is encoded by 9 exons ranging in size from 43 bp (exon 4) to 867 bp (exon 9). Characterization of the 5' flanking genomic region, which precedes the HDAC11 open reading frame, revealed a TATA and CCAAT box-less promoter that contains a 1-kb CpG island. The 1,733-bp human HDAC11 mRNA encodes a 347 aa protein with a predictive molecular weight of 39.1 kDa and an isoelectric point of 6.88. Fluorescence in situ hybridization analysis localized the human HDAC11 gene to chromosome 3p25, a region characterized by frequent gains and losses of chromosomal material in a number of various types of cancer.
 
Cell-associated 111 In radioactivity as a function of time after interrupted incubation of SKOV-3 cells with 111 In-benzyl-DTPA-Z HER2:342 . The cell-associated radioactivity at time zero after the interrupted incubation was considered as 100%. Data are the mean ± SD (n=3). Error bars might not be seen because they are smaller than point symbols.  
Specificity of 111 In-benzyl-DOTA-Z HER2:342 uptake in vivo 4 h pi (mice bearing LS174T xenografts). One group of animals (blocked) was pre-injected with 475 μg Z HER2:342 to saturate HER2 receptors 1 h before injection of radiolabeled conjugate. All animals were injected with 1 μg 111 In-benzyl-DOTA-Z HER2:342 . Data are the mean ± SD (n=4).  
Comparison of the biodistribution of 111 In-benzyl-DOTA-Z HER2:342 and 111 In-benzyl-DTPA-Z HER2:342 in nude mice bearing LS174T xenografts 4 h pi. All animals were injected with ~1 μg of radiolabeled conjugate. Data are the mean ± SD (n=4).  
Planar γ-camera imaging of HER2 expression in SKOV-3 xenografts in Balb/c nude mice using 111 In-benzyl-DOTA-Z HER2:342 (4 h pi). Tumors (hind legs) were clearly visualized. Schematic animal outlines are superimposed over images to facilitate interpretation. Arrows indicate the positions of the kidneys (K) or tumors (T).  
Tumor-to-organ ratio 4 h after injection of 111 In-benzyl-DOTA- Z HER2:342 and 111 In-benzyl-DTPA-Z HER2:342 in nude mice bearing LS174T xenografts. Data are the mean ± SD (n=4).  
Article
Imaging of expression of human epidermal growth factor receptor type 2 (HER2) in breast carcinomas may help to select patients eligible for trastuzumab therapy. The Affibody molecule Z(HER2:342) is a small (7-kDa) non-immunoglobulin affinity protein, which binds to HER2 with a picomolar affinity. Previously, a benzyl-DTPA conjugate of Z(HER2:342) was labeled with 111In and demonstrated good targeting in murine xenografts. We considered that the use of the macrocyclic chelator DOTA could increase the label stability and enhance a choice of nuclides, which could be used as a label for Z(HER2:342). The goal of this study was the preparation and pre-clinical evaluation of the indium-111- labeled DOTA-derivative of Z(HER2:342). Isothiocyanate-benzyl-DOTA was coupled to recombinant Z(HER2:342), and the conjugate was efficiently labeled with 111In at 60 degrees C. The specificity of 111In-benzyl-DOTA-Z(HER2:342) binding to HER2 was confirmed in vitro using HER2-expressing breast carcinoma BT474 and ovarian carcinoma SKOV-3 cell lines. Biodistribution of 111In-benzyl-DOTA-Z(HER2:342) was performed in nude mice bearing LS174T xenografts and compared directly with the biodistribution of 111In-benzyl-DTPA-Z(HER2:342). In vivo, 111In-benzyl-DOTA-Z(HER2:342) demonstrated quick clearance from blood and non-specific organs except the kidneys. Four hours post injection (pi), the tumor uptake of 111In-benzyl-DOTA-Z(HER2:342) (4.4+/-1.0% IA/g) was specific and the tumor-to-blood ratio was 23. The use of benzyl-DTPA provided higher tumor-to-blood and tumor-to-liver ratios. gamma-camera imaging showed clear visualization of HER2-expressing xenografts using 111In-benzyl-DOTA-Z(HER2:342). 111In-benzyl-DOTA-Z(HER2:342) has a potential for imaging of HER2 expression in malignant tumors.
 
Article
Glioblastoma multiforme (GBM) is the most prevalent, highly malignant, invasive and difficult-to-treat primary brain tumor in adults. At the genetic level, it is characterized by a high degree of chromosomal instability and aneuploidy. It has been shown that defects in the mitotic spindle checkpoint could lead to the development of aneuploidy as well as tumorigenesis. Additional proteins regulating sister chromatid cohesion could also be involved in maintaining the fidelity of chromosome segregation. One such protein is the precocious dissociation of sisters 5A (Pds5A), also known as sister chromatid cohesion protein 112. It is a nuclear protein, expressed from the S right through to the mitotic phase. It is highly conserved from yeast to man and plays a role in the establishment, maintenance and dissolution of sister chromatid cohesion. The mutation of Pds5A orthologs in lower organisms results in chromosome missegregation, aneuploidy and DNA repair defects. It is considered that such defects can cause either cell death or contribute to the development of cancer cells. Indeed, altered expression levels of Pds5A have been observed in tumors of the breast, kidney, oesophagus, stomach, liver and colon. Malignant gliomas, however, have not been analysed so far. Herein, we report on the cloning of Rattus norvegicus Pds5A and on the analysis of its expression pattern in rat tissue. We also show that Pds5A is significantly overexpressed at both the mRNA and protein level and that this overexpression correlates positively with the WHO grade of human gliomas. However, functional assays show that the siRNA-mediated knockdown of Pds5A affects sister chromatid cohesion but does not influence mitotic checkpoint function or the proliferation and survival of GBM cells. Although the mechanism by which Pds5A functions in GBM cells remains unclear, its overexpression in high grade gliomas implies that it could play a pivotal role during the development and progression of astrocytic tumors.
 
Article
Mutations in the KCNQ1 gene account for more than 90% of the individuals with Jervell and Lange-Nielsen syndrome (JLNS). In this study, we identified and characterized two novel KCNQ1 mutations that caused JLNS. A 6-year-old deaf girl suffering from recurrent syncope had a documented electrocardiogram with polymorphic ventricular fibrillation since the age of 4 years. The baseline electrocardiogram showed a significantly prolonged corrected QT interval (524 msec). Genetic analysis revealed that the proband carried two heterozygous mutations of T2C and 1149insT in the KCNQ1 gene on separate alleles. Patch-clamp analysis demonstrated that the T2C mutation resulted in significant reduction in the slowly activated delayed rectifier current (IKs). Furthermore, western blot analysis and confocal imaging revealed that the T2C mutation produced a truncated protein with trafficking defects. In contrast, the 1149insT mutation failed to generate any measurable current, consistent with no protein expression in both the cell membrane and cytoplasm. Moreover, co-expression of the T2C and 1149insT mutations significantly reduced the peak tail current density to 8.27% of the wild-type (WT) current value, while co-transfected WT channels with either T2C or 1149insT mutant channels produced comparable current and channel kinetics to that of WT channels. Our study demonstrates that the compound heterozygous mutations T2C and 1149insT cause the 'loss-of-function' of the IKs that may account for the clinical phenotype of the proband. Multiple mechanisms have been involved in the pathogenesis of 'loss-of-function' of IKs.
 
Histopathology and the expression of miR-1187 in the liver of ALF mice. Histological changes in livers at different time points post D-GalN/LPS induction (H&E, ×400). (a) There was no obvious abnormality in the liver at 0 h (saline control). (b) Damaged lobules of the liver were observed at 5 h. (c) More severe liver damage was observed at 7 h. (d) The expression of hepatic miR-1187 was then detected using the LAN-based microarray at 0, 5, 7 h post D-GalN/LPS treatment (n=5 for each time point). (e) Based on the microarray data, the change of the signal was quantified. (f) Predicted interactions between miR-1187 and the 3′UTR of caspase-8 mRNA by TargetScan. miR-1187 has binding sites at position 192–198 of caspase-8 mRNA 3′UTR highlighted in bold italic print. (g) qRT-PCR was used to verify the hepatic miR-1187 expression. Data represent the mean ± SD of five independent experiments. **P<0.001, ***P<0.001.
The expression of miR-1187 and caspase-8 mRNA in the BNLCL2 cells with different kinds of treatment. (a) miR-1187 expression in BNLCL2 cells transfected with a miR-1187 mimic or a non-specific mimic (NSM) at different concentration for 24 h. (b) miR-1187 expression in BNLCL2 cells transfected with miR-1187 mimic or NSM at the concentration of 50 nM for 48 h. (c) The expression of miR-1187 in the BNLCL2 cells induced by D-GalN/TNF. (d) The expression of caspase-8 mRNA in the BNLCL2 cells induced by D-GalN/TNF with or without 12 h miR-1187 (D/T+1187 mimic) or NSM (D/T + NSM) transfection. Data represent the mean ± SD of 3 independent experiments. **P<0.01.
The regulatory role of miR-1187 in caspase-8 was detected by Western blot analysis. (a) The level of cleaved caspase-8 protein in BNLCL2 cells induced by D-GalN/TNF with or without miR-1187 mimic transfection (D/T+1187 mimic) was detected by Western blotting. (b) The signal of Western blotting was quantified using gray-scale analysis software and data was normalized to β-actin. Data represent the mean ± SD of 3 independent experiments. *P<0.05.
Apoptosis of BNLCL2 cells with different treatments were analyzed. (a) Apoptosis of BNCL2 cells was quantified by Annexin-V/PI labelled flow cytometric analysis in which Annexin-V-FITC was used to label apoptotic cells and PI was used to label necrotic cells in the treatment of the apoptosis of the cells treated with D-GalN/TNF for 12, 36 and 48 h (D/T 12, 24, 36, 48 h) or the cells transfected with miR-1187 mimic for 12 h then treated with D-GalN/TNF for another 12, 24, 36 and 48 h (mimic + D/T 12, 24, 36, 48 h) was detected. The mock treatment was used as a negative control. (b) Based on the flow cytometry data, the apoptosis rates were calculated. Data represent the mean ± SD of 8 independent experiments. *P<0.05.
Article
In the current study, we aimed at elucidating the regulatory mechanisms through which microR-1187 (miR-1187) participates in hepatocyte apoptosis in acute liver failure (ALF). An ALF model was induced with D-galactosamine (D-GalN) plus lipopolysaccharide (LPS) in BALB/c mice. The hepatic miRNA expression profile was detected by microarray analysis and verified by quantitative real-time PCR (qRT-PCR). The possible underlying mechanism was investigated in vitro using an embryonic murine hepatocyte cell line (BNLCL2) and miR-1187 mimic. Caspase-8 protein was detected by Western blotting and cell apoptosis was assayed by flow cytometry. Hepatic miR-1187 was down-regulated in ALF mice based on microarray data (P<0.001) and verified by qRT-PCR (P<0.01). Target scan revealed that caspase-8 was the putative target of miR-1187. In an in vitro study, miR-1187 showed the highest up-regulation in BNLCL2 cells transfected with the miR-1187 mimic at a 50 nM concentration for 12 h compared with cells transfected with the non-specific mimic (P<0.001). miR-1187 was down-regulated (P<0.01) but caspase-8 mRNA (P<0.01) as well as protein (P<0.05) were up-regulated in the BNLCL2 cells treated with D-GalN/TNF. Furthermore, overexpressed miR-1187 reduced caspase-8 expression at both the mRNA and protein levels significantly (P<0.01 and P<0.05 respectively), and significantly attenuated the apoptotic rate of BNLCL2 cells (P<0.05). We show that miR-1187 regulates hepatocyte apoptosis by targeting caspase-8. miR-1187 may serve as a potential therapeutic target for the treatment of ALF.
 
Article
11beta-Hydroxysteroid dehydrogenase type 1 and type 2 (11betaHSD1 and 11betaHSD2) isozymes catalize the conversion of inactive glucocorticoids (e.g. cortisone) to their active forms (e.g. cortisol) and vice versa, respectively. Reverse transcription-polymerase chain reaction allowed the detection of 11betaHSD1 and 11betaHSD2 mRNAs in the human adrenal cortex, liver, kidneys, as well as in six aldosterone-secreting adenomas. 11betaHSD1 and 11betaHSD2 activity, as evaluated by the capacity of the microsomal fraction to convert [3H]cortisone to [3H]cortisol and vice versa, was detected in both human adrenal cortex and aldosteronomas, although it was less elevated than in liver and kidneys. Aldosteronomas possessed more intense 11betaHSD1 activity and less intense 11betaHSD2 activity than the normal adrenal cortex. The hypothesis is advanced that the elevated local concentration of steroid-hormone intermediates occurring in aldosteronomas up-regulates 11betaHSD1 and down-regulates 11betaHSD2, thereby contributing to their enhanced steroidogenic function.
 
Article
In B-cell chronic lymphocytic leukemia (CLL), Rai stage, immunoglobulin gene mutational status, chromosomal abnormalities, CD38 and ZAP-70 expression were used as prognostic markers. In this study, to understand the molecular basis of chromosomal abnormalities leading to tumor progression, 90 CLL patients were grouped into poor prognosis (with 11q deletion and trisomy 12) and good prognosis (with normal karyotype and 13q deletion) and their clinical outcome was assessed. Gene expression profiles of 35 CLL samples with poor outcome (11q deletion, n=9; trisomy 12, n=5) and good outcome (13q deletion, n=13; normal karyotype, n=8) were analyzed using oligonucleotide microarray. Significance analysis of microarray (SAM) identified 27 differentially expressed genes between these two subgroups with significant overexpression of ATF5 and underexpression of CDC16, PCDH8, SLAM, MNDA and ATF2 in CLL patients with poor outcome. ATF5 gene expression in CLL was further studied because of its role in the regulation of cell cycle progression/differentiation and apoptosis. The overexpression of ATF5 was confirmed by real-time PCR using 39 CLL samples from the poor and good outcome groups. ATF5 was significantly (p<0.001) overexpressed in the poor outcome group. Furthermore, ATF5 expression was significantly higher in the 11q deletion as well as trisomy 12 group alone compared to the 13q deletion and normal karyotype groups. ATF5 overexpression was also associated with significantly (p=0.04) shorter time to treatment. Similarly, expression of five underexpressed genes also correlated with longer time to treatment. Thus, this report demonstrates that ATF5 may be one of the key genes involved in increased proliferation and survival in 11q deletion or trisomy 12, whereas CD16, CD86, SLAM, MNDA and ATF2 may be involved in the decreased proliferation of CLL cells with 13q deletion or normal karyotype.
 
Article
The lymph node metastatic (LNM) spread of tumor cells is a frequent event in the initial process of cancer dissemination and is a powerful independent prognostic indicator in gastric adenocarcinoma (GAC). High density genomic arrays were conducted to identify molecular markers associated with lymph node metastasis in GAC. In the genome-wide profile, large copy number gains involving chromosomes 1p, 3q, 8q, 9q, 11q, 16p, 19p, and 20q (log2 ratio >0.25) (>40% of patients) were more prevalent than copy number losses. The most notable finding was copy number gains at the long arm of chromosome 11, which occurred in 75.0% of lymphatic metastasis GAC cases, and the delineated minimal common region was 11q24.2-q12.1. More specifically, 2 amplified (>1 log2 ratio) loci on the 11q13.3 region were detected in 12.5% of the cases. The first locus, covers a region of ~7.7 Mbp, and comprises the representative oncogene of cyclin D1 (CCNDI). This finding occurred in 12.5% of the cases. Additionally, an oral cancer overexpressed 1 (ORAOV1) gene was identified as a probable target within the 11q13 amplicon, which previously was not assumed to play a pathogenic role in GACs (12.5%). A second locus spanning 7.8 Mbp on 11q13.3 without associated genes also showed high-level amplifications in 12.5% of the GACs. This study indicates that the long arm of chromosome 11 harbors protooncogenes that are associated with lymphatic metastasis formation and the ORAOV1 gene at the 11q13.3 region could be a potential target and serve as an indicator for the presence of occult metastases in GAC.
 
Article
A loss of the DNA copy number at chromosomal region 11q23-24 as detected by comparative genomic hybridization (CGH) is a marker of poor prognosis in patients with endometrial cancer. Malignant tumors display genetic instability, which is classified into two types: microsatellite instability (MIN) and chromosomal instability (CIN). In the present study, we examined whether there is a relation between loss of 11q23-24 and genetic instability in endometrial adenocarcinoma. Loss of 11q23-24 was detected in 4 of 70 endometrial cancers by fluorescence in situ hybridization (FISH), and DNA aneuploidy was detected by laser scanning cytometry (LSC) in 14 tumors. All tumors with 11q23-24 loss were aneuploid, and three of them were considered to have CIN. These findings suggest that 11q23-24 contains gene(s) necessary for normal chromosome replication and cell division.
 
Article
Oral adsorbent, AST-120 removes uremic toxins (such as indoxyl sulfate) and retards the progression of chronic renal failure (CRF). However, its mechanism of action has not been precisely clarified. Since indoxyl sulfate elicits renal tubular nuclear factor-kappaB (NF-kappaB) activation in vitro, the present experiments were conducted to elucidate the involvement of NF-kappaB in the beneficial effects of AST-120 using rats with 3/4 nephrectomy, a model of early-stage CRF. Daily administration of AST-120 was started at 6 weeks after 3/4 nephrectomy and continued for 18 weeks. Sham-operated rats, untreated CRF rats and AST-120-treated CRF rats were compared for NF-kappaB DNA-binding activity, gene expression and renal histology. Systolic blood pressure was increased in CRF rats, and this increase was not affected by AST-120. Blood urea nitrogen, serum creatinine and urinary protein were increased in CRF rats. Although AST-120 attenuated these increases, it did not do so to a statistically significant extent. Indoxyl sulfate, which was accumulated in serum of CRF rats, was significantly eliminated by AST-120. Renal cortical NF-kappaB DNA-binding activity was increased in CRF rats. AST-120 significantly inhibited this increase. Monocyte/macrophage infiltration and increased monocyte chemoattractant protein-1 (MCP-1) mRNA observed in CRF rats were attenuated by AST-120. Furthermore, AST-120 significantly blocked renal fibrosis with concomitant inhibition of transforming growth factor beta1 (TGF-beta1) gene expression. It appeared that AST-120 reduced NF-kappaB activation and possibly the activity of NF-kappaB-dependent pathways of interstitial inflammation including MCP-1 expression and macrophage infiltration. The anti-inflammatory effect of AST-120 mediated via inhibition of NF-kappaB is a possible mechanism by which AST-120 retards the progression of renal fibrosis in CRF.
 
Article
Hematopoietic stem cells (HSC) can be identified by the expression of the CD34 molecule. CD34+ cells are found in bone marrow (BM), umbilical cord blood (UCB) and in mobilized peripheral blood (PB). CD34+ cells express P-glycoprotein (Pgp), a product of the multidrug resistance (MDR) gene. Pgp activity can be measured by the efflux of the dye Rhodamine 123 (Rho 123) and can be blocked by verapamil. Transport activity in HSC suggests that Pgp could have a functional role in stem cell differentiation. This study compared the number of CD34+ cells with Pgp activity measured by efflux of Rho 123 in the hematopoietic population obtained from different sources. Samples were analysed for their content of CD34+ cells, and BM had a significantly higher amount of CD34+ cells compared to UCB, mobilized PB and normal PB. When the frequency of Rholow cells was studied among the CD34+ population, an enrichment of cells with Pgp activity was observed. The frequency in BM was significantly lower than that in UCB and mobilized PB. The low retention of Rho 123 could be modified by verapamil, indicating that the measurements reflected dye efflux due to Pgp activity. Although UCB and mobilized PB had a lower number of CD34+ cells compared to BM, the total number of CD34+ cells with Pgp activity was similar in the three tissues. The different profiles may indicate the existence of subpopulations of stem cells or different stages of cellular differentiation detected by the extrusion of the dye Rho 123.
 
FP-CIT uptake in both contralateral and ipsilateral putamen and caudate. The dopaminergic system is more involved in ART and MT patients in both the contralateral and ipsilateral caudate in comparison with MT patients (A and C). TDT patients present a higher 123 I-FP-CIT uptake in the contralateral and ipsilateral putamen in comparison with ART patients (B and D). 
Sample images of 123 I-FP-CIT SPECT: (A) a TDT patient, (B) an ART patient and (C) an MT patient. 
Article
The various associations of motor and non-motor symptoms, the onset of motor complications, the cognitive disorder's appearance and other factors make Parkinson's disease (PD) a heterogeneous syndrome with multiple phenotypes. The necessity of discriminating between different forms of PD could have a role in understanding the pathophysiology of extrapyramidal signs with clinical implications. The aim of this study was to evaluate if there is a relationship between the clinical motor phenotypes of PD and the scintigraphic pattern of 123I-FP-CIT single photon emission computed tomography (SPECT). We examined 47 patients with early idiopathic PD (25 males; 22 females; mean age 58±2 years) and subdivided them in different clinical forms on the basis of dominance of resting tremor (n=20), bradykinesia plus rigidity (n=20) and the presence of both clinical signs [mixed type (MT, n=7)]. We correlated this status with the semi-quantitative analysis of SPECT with 123I-FP-CIT. Tremor type patients showed a lower reduction of 123I-FP-CIT uptake compared to akinetic-rigid type patients in contralateral caudate (P=0.0139) and putamen (P=0.0028) nuclei. 123I-FP-CIT uptake was higher in the ipsilateral caudate (P=0.0050) and putamen (P=0.0012) of tremor type patients compared to akinetic-rigid type patients. Comparisons of the striatal uptake in the tremor type and akinetic-rigid type patients with the MT patients revealed significant differences only in the ipsilateral and contralateral caudate. Our data indicate that in akinetic-rigid patients the dopaminergic system is more involved compared to that in the tremor type patients and that this difference is present from the initial stage of the disease. Moreover, our results suggest that PD phenotypes could be related not only to the dopaminergic involvement but also to other systems.
 
Article
Van der Woude syndrome (VWS) is an autosomal dominant disorder and the most common cleft syndrome characterized by cleft lip and palate with lip pits. Very recently, mutations in the interferon regulatory factor 6 gene (IRF6) were identified to cause VWS in patients of northern European descent. We describe a Thai family with VWS. The proband, an 8-month-old boy, had bilateral complete cleft lip and palate, and two conical elevations with lip pits on his lower lip. Four other family members had various manifestations of the clefts and lower lip pits. Mutation analysis of the proband and his mother for the entire coding region of IRF6 identified a novel mutation, 1234del(C), in its exon 9. The deletion is expected to result in some amino acid changes followed by truncation at amino acid 435. This observation supports that IRF6 is the gene responsible for VWS across different populations and that haploinsufficiency of the gene disturbs development of the lip and palate.
 
Article
The D-glucose analog 6-deoxy-6-¿123Iĭodo-D-glucose (6-DIG) was recently proposed as a potential tracer for the in vivo characterization of D-glucose transport in distinct cell types. In this study, the validity of such a proposal was investigated in both control and streptozotocin-induced diabetic rats. 6-DIG was injected intravenously in either control or diabetic rats. The fate of 6-DIG was assessed by scintigraphy of the injected animals, blood and urine sampling, and measurement of tissue radioactivity at the time of sacrifice, 140 min after 6-DIG injection. The half-life for 6-DIG in plasma and its accumulation in kidney and urinary bladder indicated that it was mainly eliminated from the body by glomerular filtration. The urinary elimination of 6-DIG was accelerated, however, in the polyuric diabetic rats. Bile formation also apparently contributed to the clearance of 6-DIG. Its uptake by liver, heart and muscles yielded values lower than blood concentration. The usefulness of 6-DIG as a tracer for D-glucose transport in selected organs in the perspective of clinical application, e.g. by single photon emission computed tomography, requires further investigations.
 
Article
microRNA-124 (miR-124) plays an important role in regulating growth, invasiveness, stem-like traits, differentiation and apoptosis of glioblastoma cells. PPP1R3L, an inhibitory member of the apoptosis-stimulating protein of p53 family (IASPP), is also able to affect growth, cell cycle progression, metastasis and apoptosis of various types of cancer. To investigate the regulation of PPP1R13L expression by miR-124 and their effects on proliferation, cell cycle transition and invasion in glioblastoma cells, U251 and U373 glioblastoma cells were transfected with miR-124 mimics, its negative control (NC) or an inhibitor. We found that miR-124 was downregulated in glioblastoma tissues, and inversely regulated PPP1R13L expression in U251 and U373 glioblastoma cells. PPP1R13L was found to be a direct target of miR-124 in glioblastoma cells. Overexpression of miR-124 inhibited proliferation, G1/S transition and invasiveness in glioblastoma cells. miR-124 downregulation-mediated malignant progression of glioblastoma was partly attributed to increased PPP1R13L expression. Consequently, our findings provide a molecular basis for the role of miR-124/PPP1R13L in the progression of human glioblastoma and suggest a novel target for the treatment of glioblastoma.
 
Article
D-mannoheptulose was recently proposed as a tool to label preferentially insulin-producing cells in the pancreatic gland in the perspective of the non-invasive imaging of the endocrine pancreas. In such a perspective, we have now synthesized 1-deoxy-1-[125I]iodo-D-mannoheptulose ([125I]MH) and examined its uptake by different rat cell types. No phosphorylation of [125I]MH by bovine heart hexokinase could be detected. The apparent distribution space of [125I]MH largely exceeded that of [U-14C]sucrose, considered as an extracellular marker, in erythrocytes, parotid cells, hepatocytes, pancreatic pieces and isolated pancreatic islets. Relative to the mean intracellular distribution space of 3HOH, that of [125I]MH was not significantly different in pancreatic pieces from either normal rats or streptozotocin-induced diabetic animals (STZ rats). In pancreatic islets, the uptake of [125I]MH was decreased at low temperature, but failed to be significantly affected by cytochalasin B. Sixty min after the intravenous injection of [125I]MH, the radioactive content of selected organs displayed the following hierarchy: muscle<pancreas<liver<parotid<kidney<plasma. In this respect, there was no obvious difference between control and STZ rats. Taken as a whole, these findings suggest that 1-deoxy-1-[125I]iodo-D-mannoheptulose does not display the same specificity towards GLUT2, as that previously documented in the case of D-[3H]- mannoheptulose.
 
Article
The endothelial cell-specific microRNA (miRNA), miR-126, is considered a master regulator of physiological angiogenesis. Transplanted mesenchymal stem cells (MSCs) release soluble factors contributing to neoangiogenesis and cardiac repair. Therefore, we hypothesized that the over-expression of miR-126 may prolong MSC survival and enhance the cell secretome, thereby improving post-infarction angiogenesis and cardiac function. In this study, MSCs harvested from male C57BL/6 mouse bone marrow were infected in vitro with miR-126 (MSCmiR-126) by using recombinant lentiviral vectors; the control cells were either non-transfected or transduced with mock vectors (MSCnull). The results showed an increased secretion of angiogenic factors and a higher resistance against hypoxia in MSCmiR-126 compared with the control cells. The expression of the Notch ligand Delta-like (Dll)-4 in the MSCmiR-126 group was also increased. For in vivo experiments, MSCmiR-126 cultures were intramyocardially injected into the infarct region of the hearts of female C57BL/6 mice (an acute myocardial infarction model) who had undergone ligation of the left anterior descending coronary artery. The survival of MSCmiR-126 cultures, determined by Sry expression, was increased at 7 days after transplantation. MSCmiR-126-treated animals showed significantly improved cardiac function as assessed by echocardiography 2 weeks later. The expression levels of angiogenic factors and Dll-4 in the infarcted myocardium were further increased by MSCmiR-126 compared with MSCs or MSCnull cultures. Furthermore, fluorescent microsphere and histological studies revealed that myocardial blood flow and microvessel density were significantly increased in the MSCmiR-126-transplanted animals. In addition, we found increased immature vessel proliferation following the transplantation of MSCmiR-126 cultures in which the expression of Dll-4 had been knocked down. However, blood vessels with lumen were barely detected, which indicated that Dll-4 plays a key role in tubulogenesis. We conclude that the transplantation of MSCs overexpressing miR-126 can further enhance functional angiogenesis in the ischemic myocardium possibly by the secretion of angiogenic factors and the activation of Dll-4, thus increasing MSC survival. Therefore, MSCs modified with miR-126 may represent a novel and efficient therapeutic approach for ischemic angiogenesis and the improvement of cardiac function.
 
Article
We report the emerging role of microRNA (miRNA) deregulation associated with activation of an oncogene SOX4 (a member of the SRY-related HMG-box) in esophageal carcinoma. Paired esophageal cancer and adjacent non-tumor tissues were obtained from 42 patients who underwent primary surgical resection for esophageal cancer. Experiments such as real-time PCR, western blot analysis, luciferase-reporter assay, cell proliferation and colony formation assays, in vitro migration and invasion assays, and a wound-healing assay were performed to determine the effects of miR-129-2. We found that SOX4 expression was elevated (P<0.005) in esophageal tumors (n=42) when compared with its expression in the controls (n=42). Compared with the normal esophageal tissues, the expression of miR-129-2 was downregulated in 27 of 31 primary esophageal tumors, while the expression of SOX4 was upregulated (P<0.001). Restoration of miR-129-2 by transfection with an miRNA expression plasmid led to a decrease in SOX4 expression, which was accompanied by reduced migration and proliferation of the cancer cells. These results suggest that aberrant expression of SOX4 is associated with repression of miR-129-2, and restoration of miR-129-2 suppresses the migration and proliferation of esophageal cancer cells. Our results demonstrated that the deregulation of miR-129-2 leads to aberrant SOX4 expression, presenting a new paradigm in which the restoration of miRNA suppresses its oncogenic target in esophageal cancer.
 
Article
We studied 692 Swedish children and adolescents (aged 9-10 or 15-16 years, respectively), in order to evaluate the effect of the methylenetetrahydrofolate reductase (MTHFR) 677C>T, 1298A>C, and 1793G>A polymorphisms on total plasma homocysteine concentrations (tHcy). Genotyping was performed with Pyrosequencing technology. The MTHFR 677C>T polymorphism was associated with increased tHcy concentrations in both the children and the adolescents (P<0.001 for both age groups) in both genders. The effect of MTHFR 1298A>C was studied separately in subjects with the 677CC and 677CT genotypes, and the 1298C allele was found to be associated with higher tHcy levels both when children were stratified according to 677C>T genotypes, and when using haplotype analyses and diplotype reconstructions. The 1793A allele was in complete linkage disequilibrium with the 1298C allele. It was still possible to show that the 1793A allele was associated with lower tHcy levels, statistically significant in the adolescents. In conclusion, a haplotype-based approach was slightly superior in explaining the genetic interaction on tHcy plasma levels in children and adolescents than a simple genotype based approach (R2 adj 0.44 vs. 0.40). The major genetic impact on tHcy concentrations is attributable to the MTHFR 677C>T polymorphism. The common 1298A>C polymorphism had a minor elevating effect on tHcy, whereas the 1793G>A polymorphism had a lowering effect on tHcy.
 
Top-cited authors
Yi-Qing Yang
  • Shanghai Jiao Tong University
Yung Hyun Choi
Kim Gi Young
Wen G Jiang
  • Cardiff University
Jerzy Jankun
  • University of Toledo