EGF Protects Cells Against Dox-Induced Growth Arrest Through Activating Cyclin D1 Expression.

It has been reported that the antitumor drug doxorubicin (Dox) exerts its toxic effects via GATA-4 depletion and that over-expression of GATA-4 reverses Dox-induced toxicity and apoptosis; however, the precise mechanisms remain unclear. In this study, we observed, for the first time, that EGF protects cells against Dox-mediated growth arrest, G2/M-phase arrest, and apoptosis. Additionally, EGF expression was down-regulated in Dox-treated cells and up-regulated in GATA-4 over-expressing cells. Utilizing real-time PCR and western blotting analysis, we found that the expression of the cell cycle-associated protein cyclin D1 was inhibited in GATA-4-silenced cells and Dox-treated cells and was enhanced in GATA-4 over-expressing cells and EGF-treated cells. Furthermore, EGF treatment reversed the inhibited expression of cyclin D1 that was mediated by GATA-4 RNAi or Dox. Our results indicate that EGF, as a downstream target of Dox, may be involved in Dox-induced toxicity as well as in the protective role of GATA-4 against toxicity induced by Dox via regulating cyclin D1 expression, which elucidates a new molecular mechanism of Dox toxicity with important clinical implications.

[Preparation and pharmacokinetic evaluation of long-acting injectable oily suspensions for ophiopogonis radix polysaccharide MDG-1].

OBJECTIVE: To evaluate in vivo pharmacokinetics of Ophiopogonis Radix polysaccharide MDG-1 oily suspension injection prepared with different prescriptions in rats, and explore the feasibility of the long-acting drug delivery of MDG-1 Injection by using the oily suspension drug release system. METHOD: MDG-1 microparticles were prepared by the anti-solvent precipitation method. Their size and size distribution were characterized. Castor oil with a high viscosity or aluminum stearate were added into soybean oil with a low viscosity, in order to prepare oily media with different viscosities, detect their rheological properties and screen out superior prescriptions for in vivo evaluation. RESULT: The average size of microparticles was 21.81 microm, and the span between them was 2.63. The in vivo evaluation was conducted for prescriptions of mixed oil (soybean oil/castor oil, 2: 3) and soybean oils gelled by 2% and 4% aluminium stearate. Among them, the prescription of soybean gelled by 4% aluminium stearate could significantly reduce C(max) and prolong the apparent t1/2, with the MDG-1 release time of several days. CONCLUSION: It is feasible to achieve the long-acting MDG-1 drug delivery by using oily media with a high viscosity.

The effects of tai chi on the renal and cardiac functions of patients with chronic kidney and cardiovascular diseases.

[Purpose] To assess the effects of Tai Chi on the renal and cardiac functions of patients with chronic kidney disease (CKD) and cardiovascular disease (CVD). [Subjects and Methods] Twenty-one patients with CKD and CVD were randomly divided into control and exercise groups. The exercise group performed Tai Chi training for 30 minutes three to five times a week for 12 weeks, while the control group did not. All patients' renal and cardiac functions and blood lipid parameters were measured at baseline and after 12 weeks. [Results] The 12 weeks Tai Chi intervention improved the estimated glomerular filtration rate (eGFR), left ventricular ejection fraction (LVEF), and the high density lipoprotein (HDL) level, and decreased the serum creatintine (Scr) level, heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), and the total cholesterol (CH), triglyceride (TG) and low density lipoprotein (LDL) levels. The change in eGFR correlated negatively with the changes in CH, TG and LDL, and positively with the change in HDL. In addition, the change in SBP correlated positively with the changes in CH, TG and LDL, and negatively with the change in HDL. [Conclusion] Tai Chi training might improve the renal and cardiac functions of CKD and CVD patients via improved regulation of lipid metabolism.

Insulin induces C2C12 cell proliferation and apoptosis through regulation of cyclin D1 and BAD expression.

Insulin is a secreted peptide hormone identified in human pancreas to promote glucose utilization. Insulin has been observed to induce cell proliferation and myogenesis in C2C12 cells. The precise mechanisms underlying the proliferation of C2C12 cells induced by insulin remain unclear. In this study, we observed for the first time that 10 nM insulin treatment promotes C2C12 cell proliferation. Additionally, 50 and 100 nM insulin treatment induces C2C12 cell apoptosis. By utilizing real-time PCR and Western blotting analysis, we found that the mRNA levels of cyclinD1 and BAD are induced upon 10 and 50 nM/100 nM insulin treatment, respectively. The similar results were observed in C2C12 cells expressing GATA-6 or PPARα. Our results identify for the first time the downstream targets of insulin, cyclin D1, and BAD, elucidate a new molecular mechanism of insulin in promoting cell proliferation and apoptosis.

Insulin induces proliferation and cardiac differentiation of P19CL6 cells in a dose-dependent manner.

Insulin is a peptide hormone produced by beta cells of the pancreas. The roles of insulin in energy metabolism have been well studied, with most of the attention focused on glucose utilization, but the roles of insulin in cell proliferation and differentiation remain unclear. In this study, we observed for the first time that 10 nmol/L insulin treatment induces cell proliferation and cardiac differentiation of P19CL6 cells, whereas 50 and 100 nmol/L insulin treatment induces P19CL6 cell apoptosis and blocks cardiac differentiation of P19CL6 cells. By using real-time polymerase chain reaction (PCR) and Western blotting analysis, we found that the mRNA levels of cyclin D1 and α myosin heavy chain (α-MHC) are induced upon 10 nmol/L insulin stimulation and inhibited upon 50/100 nmol/L insulin treatment, whereas the mRNA levels of BCL-2-antagonist of cell death (BAD) exists a reverse trend. The similar results were observed in P19CL6 cells expressing GATA-6 or peroxisome proliferator-activated receptor α (PPARα). Our results identified the downstream targets of insulin, cyclin D1, BAD, α-MHC, and GATA-4, elucidate a novel molecular mechanism of insulin in promoting cell proliferation and differentiation.

miR-200b targets GATA-4 during cell growth and differentiation.

GATA-4 is an important transcription factor involved in several developmental processes of the heart, such as cardiac myocyte proliferation, differentiation and survival. The precise mechanisms underlying the regulation of GATA-4 remain unclear, this is especially true for the mechanisms that mediate the post-transcriptional regulation of GATA-4. Here, we demonstrate that miR-200b, a member of the miR-200 family, is a critical regulator of GATA-4. Overexpression of miR-200b leads to the downregulation of GATA-4 mRNA and a decrease in GATA-4 protein levels. Moreover, miR-200b not only inhibits cell growth and differentiation but also reverses the growth response mediated by GATA-4, whereas depletion of miR-200b leads to a slight reversal of the anti-growth response achieved by knocking down endogenous GATA-4. More importantly, the cell cycle-associated gene cyclin D1, which is a downstream target of GATA-4, is also regulated by miR-200b. Thus, miR-200b targets GATA-4 to downregulate the expression of cyclin D1 and myosin heavy chain (MHC), thereby regulating cell growth and differentiation.

Toxic effects and transcriptional responses in zebrafish liver cells following perfluorooctanoic acid exposure.

As a typical type of persistent organic pollutant, perfluorooctanoic acid (PFOA) is pervasive in the environment. Multiple studies have found that PFOA has hepatotoxicity, but the mechanism remains poorly understood. In this study, the toxic effects of different concentrations of PFOA on zebrafish liver cells were systematically assessed by recording cell survival, ultrastructural observations, and transcriptome analyses. The results showed that the inhibition of cell viability and the massive accumulation of autophagic vacuoles were observed at 400 µM PFOA, while transcriptomic changes occurred with treatments of 1 and 400 µM PFOA. The transcription levels of 1055 (977 up- and 78 down-regulated genes) and 520 (446 up- and 74 down-regulated genes) genes were significantly changed after treatment with 1 and 400 µM PFOA, respectively. Based on Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis, significant expression changes were observed in autophagy, tight junction, signal transduction, immune system, endocrine system, and metabolism-related pathways, indicating that such processes were greatly affected by PFOA exposure. The findings of this study will provide a scientific basis for the toxic effects and potential toxic mechanisms of PFOA on zebrafish, and provide information for ecological risk assessments.

A GRIP-1-EZH2 switch binding to GATA-4 is linked to the genesis of rhabdomyosarcoma through miR-29a.

Terminal differentiation failure is an important cause of rhabdomyosarcoma genesis, however, little is known about the epigenetic regulation of aberrant myogenic differentiation. Here, we show that GATA-4 recruits polycomb group proteins such as EZH2 to negatively regulate miR-29a in undifferentiated C2C12 myoblast cells, whereas recruitment of GRIP-1 to GATA-4 proteins displaces EZH2, resulting in the activation of miR-29a during myogenic differentiation of C2C12 cells. Moreover, in poorly differentiated rhabdomyosarcoma cells, EZH2 still binds to the miR-29a promoter with GATA-4 to mediate transcriptional repression of miR-29a. Interestingly, once re-differentiation of rhabdomyosarcoma cells toward skeletal muscle, EZH2 was dispelled from miR-29a promoter which is similar to that in myogenic differentiation of C2C12 cells. Eventually, this expression of miR-29a results in limited rhabdomyosarcoma cell proliferation and promotes myogenic differentiation. We thus establish that GATA-4 can function as a molecular switch in the up- and downregulation of miR-29a expression. We also demonstrate that GATA-4 acts as a tumor suppressor in rhabdomyosarcoma partly via miR-29a, which thus provides a potential therapeutic target for rhabdomyosarcoma.

[A study on decreasing the instrument detection limit of atomic fluorescence spectrometry (AFS-930) for Hg].

In the present study, the detection limit of atomic fluorescence spectrometry (AFS-930) was decreased to 2 ng x L(-1) (n=6) based on several optimizing modifications, including that the sub-high voltage of photomultiplier tube and the current of hollow-cathode lamp were elevated to 280 V and to 30 mA, respectively, and the height of atomization cell was set as 10 mm; In addition, the concentration of KBH4 was decreased to 0.5% (KOH 0.2%). With the optimized parameters, a good standard curve of Hg concentration versus intensity of fluorescence (If) could been obtained readily, after that, a 4-ng x L(-1)-Hg water samples was measured accurately with a little relative standard deviation (RSD) of <5%, while for approximately 2-ng x L(-1)-Hg waters the RSD varied within a wide range of 10.9%-27.2%, likely due to the absorption of Hg by polyethylene vessels used in this study and/or due to the contamination by analysis grade reagents used in this study. By using low-absorption polytetrafluoroethylene (PTFE) materials and the guaranteed reagents, the instrument detection limit was further decreased to 1 ng x L(-1) (n=10).

[Residue and Degradation of Roxarsone in the System of Soil-Vegetable].

The field experiment was developed for simulating the residues, transformation and degradation in soil-vegetable system of Roxarsone contained in organic fertilizer. Under the treatment, the yield of Brassica chinensis decreased in low Roxarsone concentration with a decline by 15% to 32% compared with the control group; there had an accumulating role of vegetables to arsenic, and the root was the main part; total content of arsenic in the soil was positively correlated with the dose of the applied Roxarsone; total arsenic in soil first increased and then decreased with the planting time prolonged and peaked at 12.94 mg x kg(-1), while the level of inorganic arsenic in the soil stabilized after 30 d, which accounting for 66.75% to 98.56% of the total arsenic; there existed a positively significant correlation of total arsenic content between the Brassica chinensis and the soil as well as the arsenic enrichment factor of vegetables; the degradation rate of Roxarsone in soil was slow, there was still some Roxarsone remained in soil after 45 d when added the organic fertilizer which containing Roxarsone with the dose higher than 5 000 kg x hm(-2); Roxarsone could significantly increase the number of bacteria in the soil, and low concentration showed an inhibited role in the growth of fungi and actinomyces, while high concentration of Roxarsone promoted the growth.

[Characteristics of arsenic content in the livestock farms' surrounding environment in Shanghai suburbs].

In the suburbs of Shanghai, selected five large farms and surface water, feed, soil and vegetable samples were collected, and then total and inorganic arsenic were detected to survey the current arsenic level of the livestock farms in Shanghai suburban, as well as the usage situation of organic arsenic as feed additives. The results showed that the total arsenic content in water samples ranged from 0. 00 microgL-1 to 23.00 microg L-1 , below the first standard of surface water quality (50 microg L-1 ) ; total arsenic in feed was among 0.40-12.13 mgkg-1 , in which inorganic arsenic was 10.0% -80.0% ; total arsenic in spot-checking feed samples was 0. 16-21.39 mg kg-1 and inorganic arsenic was 0. 003-10. 67 mgkg-1 , and samples exceeding the limits of arsenic in feed accounted for 16. 7% ; total arsenic content in soils ranged from 8.08-18.50 mgkg-1 , in which 22. 2% samples were higher than the first standard of soil environmental quality, and inorganic arsenic accounted for 44. 2% -78. 9% of total arsenic; vegetables' total arsenic was 0. 003-0. 093 mg kg-1, not higher than the maximal residue limit of 0. 50 mg kg -1 on the current national standard; there were some differences in different parts of the same species vegetables on arsenic content: arsenic content in roots were higher than that in the aboveground part, and the bioconcentration factors showed a significantly positive correlation with the arsenic content in vegetables and a negative correlation with the arsenic content in rhizosphere.

Could Pin1 help us conquer essential hypertension at an earlier stage? A promising early-diagnostic biomarker and its therapeutic implications for the disease.

Essential hypertension is a major risk factor for cardiovascular morbidity and mortality, and the early-diagnosis is very important for the prevention of essential hypertension. Previously, we found that Pin1, the only known enzyme isomerizing pSer/pThr-Pro motifs in proteins, may gradually become inactive under conditions of stress such as intracellular acidification and fever. Interestingly, essential hypertension and the dysfunction of Pin1 often synchronously occur with the increasing age. Recent evidence indicates that Pin1 primarily increases the activity of endothelial nitric oxide synthase (eNOS) and the production of nitric oxide (NO) in multiple ways, significantly promoting the relaxation response of blood vessels and preventing the elevation of blood pressure. Further, the inhibition of Pin1 results in significantly increased blood pressure in rats. So, we hypothesized and evaluated the potential of Pin1 to be a new early-diagnostic biomarker as well as a therapeutic drug for essential hypertension. The unique activity of Pin1 and some epidemiological and experimental data evidence that the decreased activity of Pin1 may be closely associated with the development of essential hypertension. The factors that may impact the activity of Pin1 and correlate with the risk of essential hypertension were also discussed. These findings indicate that Pin1 plays a key and permanent role in efficiently preventing the development of essential hypertension, and that Pin1 may be a promising early-diagnostic biomarker as well as an effective therapeutic drug for the early-diagnosis, prevention, and treatment of essential hypertension, potentially decreasing the risk of cardiovascular morbidity and mortality.

Improvement of myocardial lipid accumulation and prevention of PGC-1α induction by fenofibrate.

We recently demonstrated that fenofibrate induces the activities of citrate synthase and NADH oxidase in cardiac mitochondria. To further determine the molecular mechanisms underlying fenofibrate action, 8-week-old mice were administered fenofibrate (100 mg/kg/day) for 7 and 14 days, and the expression of genes involved in cardiac mitochondrial function, such as nuclear respiratory factor 1 transcript variant 2 (NRF-1-L) and 6 (NRF-1-S), mitochondrial outer membrane protein 40 (Tom40), lipoic acid synthetase (Lias), cytochrome b, medium-chain acyl-coenzyme A dehydrogenase (MCAD) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) was determined. Expression of PGC-1α, a key regulator of the entire fatty acid oxidation system, was significantly downregulated after 14 days of fenofibrate administration. Moreover, ventricular triglycerides were also accumulated following 14 days of fenofibrate administration. Thus, fenofibrate functions to improve myocardial lipid accumulation and to prevent PGC-1α induction, which is crucial for understanding the molecular mechanisms underlying fenofibrate action on the heart.

Transcription factor GATA-6 recruits PPARα to cooperatively activate Glut4 gene expression.

Peroxisome proliferator-activated receptor α (PPARα) is a nuclear hormone receptor that regulates energy metabolism, but its precise mechanisms remain unknown. Here, we demonstrate that the PPARα agonist fenofibrate activated expression of the glucose transporter Glut4. Moreover, PPARα was associated with the Glut4 promoter through GATA sites upon fenofibrate stimulation in cardiomyocytes. This occupancy is achieved through an interaction between amino acids 1-136 of PPARα with amino acids 276-443 of the cardiac transcription factor GATA-6. In addition, the interaction of PPARα with GATA-6 activated Glut4 gene expression, improved glucose consumption, and enhanced activity of mitochondrial citrate synthase in C2C12 myoblasts; both mutants of PPARα (1-101 aa) and GATA-6 (227-331 aa) were unable to cooperate in Glut4 activation. Thus, GATA-6 is an important component of the transcription network required for energy metabolism mediated by PPARα, and these findings provide a molecular basis for understanding the role of GATA-6 proteins in muscle development and disease.

[Nitrate contents in autumn vegetables and assessment of nitrate intake in Shanghai].

To analyze and assess the nitrate contents in Autumn vegetables and nitrate intake in Shanghai resident 25 groups and 439 various vegetables were collected and analyzed from the green houses and outdoors in Songjiang, Fengxian, Jinshan and Pudong of Shanghai during Sep.-Nov., 2009. Nitrate contents were analyzed by UV-spectrophotometer. The results showed that the prevalence of severe contamination was 41.46%, the prevalence of heavy and medium contamination was 30.53%, the prevalence of mild contamination was 28.02%; the content of nitrate in vegetables was in the following descent order: leafy vegetables, root and stem vegetables, melons, egg plants, beans, the nitrate contents in different species differed greatly; the nitrate contents in leafy, root and stem vegetables with green-house planting were less than that of outdoor planting in Fengxian and Songjiang except Pudong and Jinshan. According to the assessment of nitrate intake, the average daily intake of local resident is 445.22 mg which exceeds ADI 38.42%, so limited standard and control are urgently needed.

Effects of Doxorubicin and Fenofibrate on the activities of NADH oxidase and citrate synthase in mice.

Doxorubicin (Dox) has widely been used as an anticancer drug, but its use is limited by serious toxicity to the heart, kidney and liver. Mitochondrial dysfunction is one of the potential mechanisms of toxicity but not fully understood. Fenofibrate, one of the peroxisome proliferator-activated receptor-alpha (PPARα) ligands, is involved in lipid metabolism which takes place primarily in the mitochondria, so mitochondrial function may be affected by fenofibrate. Therefore, we investigated the effects of DOX and fenofibrate on activities of both mitochondrial citrate synthase and NADH oxidase, which are marker enzymes in the tricarboxylic acid (TCA) cycle and a measure of the complex I-III-IV activity in electron transport chain, respectively. Dox (15 mg/kg) and/or fenofibrate (100 mg/kg/day) were administered to mice for 3 or 14 days, and the activities of citrate synthase and NADH oxidase were measured. Our study showed that Dox significantly inhibits the activity of citrate synthase while fenofibrate induces the activity. Similar to citrate synthase, NADH oxidase activity was also induced by fenofibrate except in spleen but inhibited by Dox except in the heart and liver. Furthermore, fenofibrate not only protects citrate synthase activity from Dox-induced toxicity in the ventricle but also significantly rescues NADH oxidase activity in the kidney. These results reveal the actions of fenofibrate and Dox on the mitochondria, and the underlying mechanism may be related to the toxicity of Dox, which has clinical implications in the side effects of Dox treatment by modulation of mitochondrial function.

[Arsenic contents in soil, water, and crops in an e-waste disposal area].

In order to study whether disposing electronic wastes and secondary metal smelting could cause an arsenic pollution in the environment or not, Luqiao town, Taizhou City, Zhejiang Province was selected as a study area. The main purpose of this paper was to characterize arsenic contents in the local environment, including waters, sediments, soils and rice, and to assess the potential risk to humans. Additionally, the arsenic spatial distribution property and arsenic uptake-translocation rule in soil-rice system were also studied. The results showed that the average arsenic levels in the surface water and the groundwater were 8.26 microg/L and 18.52 microg/L, respectively, which did not exceed the limiting value of Chinese Environment Standards class III . Whereas,some groundwater exceeded the recommended standard by the WHO for drinking water (10 microg/L). The arsenic (on average 7.11 mg/kg) in paddy soils and arsenic (on average 6.17 mg/kg) in the vegetable garden soils were lower than the value recommended by the National Standard (level I). The average arsenic contents in brown rice and husks were 165.1 microg/kg and 144.2 microg/kg, which was also lower than the Chinese Foods Quality Standard. The arsenic contents between the corresponding soils-rice and husks-brown rice showed significantly positive correlations. By comparison, the arsenic contents of soils and husks collected around electroplating were relatively higher than most of other pollutant sources, indicating the electroplating may lead accumulation of arsenic in the paddy soil-rice system.

[Characteristics of six elements contents in human hairs in the vicinity of metal smelting factories].

To assess the impact of secondary smelting activities to the local resident's well beings, the contents of six elements-Cu, Zn, Pb, Cd, As, Se-in human scalp hairs of residents in suburb Fuyang, Zhejiang Province, were determined by atomic absorption spectrometer (AAS) and by atomic fluorescence spectrometer (AFS-930), respectively. The results showed that hair elemental contents were markedly higher than those in unpolluted area. The highest hair contents of Cu, Zn, Pb and Cd reached 312, 513, 700 and 7.41 microg x g(-1), respectively, As and Se 10.08 and 0.85 microg x g(-1), respectively. Additionally, a significant relationship between Cu, Zn, Pb, Cd and As in human hair was observed, which indicated at a certain degree that the elemental pollution was caused by similar source-small blast furnace smelting. Moreover, it showed that the Cu, Zn, Pb, Cd, and As contents in hair of human who were above 40 years old was twice more than those whose age below 40. There was no apparent difference in average content of Cu, Zn, Cd, Se between male and female, except that Pb and As of male hair was higher than female, however, by statistic analysis showing no significant age and gender dependence.

[Soils salinity content of greenhouse in Shanghai suburb].

Salinity content and characteristic of farmland soil in Shanghai suburb was studied. Result indicates that soils in greenhouse in Shanghai suburb are partially salted. Soils of suburb where melons or vegetables grow in Shanghai city, 88.52% soil is non-salted while 10.37% mildly salted, 0.74% obviously salted and 0.37% badly salted. Anions component of salt salinity in soil are mainly SO4(2-), Cl-, NO3(-) and cations component are mainly Ca2+, Na+, Mg2+, K+. These ions are mostly from fertilizer auxiliary component or fertilizer transformation component besides some original deposition in soil. The formation of soil secondary salted in greenhouse cultivation in suburbs of Shanghai has a close relationship with improper fertilization or employing too much fertilizer. Soil salinity is different with different cultivation mode and utilization time. From high to low, sequence of soil salinity content in 0 - 20 cm cultivation layer of different crop mode is greenhouse vegetable soil, melon soil, vegetable melon rotation soil and hypaethral vegetable soil respectively. In the same region, salinity in greenhouse soil continually increases and accumulates from underlayer to surface along with more utilization years.