Xiangyu Cao

Cholesterol induces pancreatic β cell apoptosis through oxidative stress pathway

Type 2 diabetes is often associated with high blood cholesterol. Here, we investigated the effect of cholesterol loading on MIN6 cells derived from pancreatic β cells. Exposure of MIN6 cells to cholesterol-induced apoptosis in time- and dose-dependent manner. Treatment with methyl-β-cyclodextrin that removes cholesterol from plasma membrane prevented the cells from cholesterol-induced apoptosis. Western blot analysis revealed that the levels of phosphorylated-p38 mitogen-activated protein kinase (P-p38 MAPK) and c-Jun N-terminal kinases (P-JNK) were significantly increased after the cholesterol loading, suggesting that the stress-activated protein kinase signaling was stimulated. A specific p38 inhibitor rescued MIN6 cells from cholesterol-induced apoptosis, while JNK inhibitor failed, suggesting the importance of activation of p38 MAPK signaling in response to cholesterol. The expression of Bip and CHOP, the endoplasmic reticulum (ER) stress markers, remained unaffected, indicating that the ER stress may not be involved in the cytotoxicity of cholesterol on the ΜΙΝ6 cells. The intracellular concentration of reactive oxygen species measured by use of 2′,7′-dichlorofluorescin diacetate was significantly increased after cholesterol loading, demonstrating the induced apoptosis was mediated through oxidative stress. Addition of reduced form of glutathione in the medium rescued MIN6 cells from apoptosis induced by cholesterol loading. Taken together, these results demonstrate that the free cholesterol loading can induce apoptosis of MIN6 cells mediated by oxidative stress and the activation of p38 MAPK signaling.

Gender and urinary pH affect melamine-associated kidney stone formation risk

Objectives: Melamine was known as a new risk for kidney stone due to recent incidences of milk powder contamination in China. Here, we performed a retrospective study to investigate whether age, gender, and urinary pH affect melamine-associated kidney stone risk. Materials and Methods: A retrospective review was performed of 217 children aged less than 3 years old. All children had a history of being fed with Sanlu milk powder contaminated by melamine, and underwent a clinical screening on kidney stone in Shenyang from November 2008 to February 2009. A comparison with the Chi-square was conducted between 83 cases and 125 normal subjects. The difference between childrens gender, age, and urinary pH was evaluated. Results: A total of 208 subjects, 136 boys and 72 girls, were included in the study. Significant association was observed between melamine-associated kidney stone risk and gender [odds ratio (OR), 2.03; 95% confidence interval (CI), 1.11-3.74; P=0.02] and urinary pH (OR, 1.78; 95% CI, 1.01-3.11; P=0.04), respectively. Male children were at about twofold increased melamine-associated kidney stone risk compared with female children. Acidic urine showed about 1.78-fold increased melamine-associated kidney stone risk compared with normal urine. Conclusions: Our investigation results showed an association of gender and urinary pH with melamine-associated kidney stone formation risk.

The membrane topological analysis of 3β-hydroxysteroid-Δ24 reductase (DHCR24) on endoplasmic reticulum

DHCR24 encodes 3β-hydroxysteroid-Δ24 reductase, catalyzing the conversion of desmosterol to cholesterol. Our previous study demonstrated that DHCR24 exerts an anti-apoptotic function as a reactive oxygen species (ROS) scavenger, for which it needs its FAD-binding domain. The membrane topology of DHCR24 on endoplasmic reticulum (ER) and the functional significance of its FAD-binding domain are not completely understood. Based on the structure predicted by bioinformatics, we studied the membrane topology of DHCR24 in murine neuroblastoma cells (N2A), using the fluorescent protease protection (FPP) technique. We showed that full-length DHCR24 is localized to the membrane of ER, whereas the predicted transmembrane (TM) domain-deleted DHCR24 mutation is localized to the cytoplasm. The change of DHCR24 localization suggests that the N-terminal TM domain is essential for the ER membrane targeting of DHCR24. The FPP assay demonstrated the membrane topology of DHCR24 with an N-terminal luminal/C-terminal cytoplasmic orientation. Measurement of intracellular ROS using H(2)DCFDA revealed that the ROS levels of cells infected by plasmids driving expression of full-length DHCR24 or the TM domain-deleted DHCR24 mutation after H(2)O(2) exposure were lower than those of control cells, suggesting that the ER membrane targeting of DHCR24 is not required for its enzymatic ROS scavenging activity. Confocal fluorescence microscopy revealed that the DHCR24-overexpressed cells were protected from apoptosis in response to oxidative stress, which was accompanied by a decrease in DHCR24 content on the ER and activation of caspase-3, suggesting that the anti-apoptotic function of DHCR24 is associated with its cleavage by caspase.

Native low density lipoprotein induces pancreatic β cell apoptosis through generating excess reactive oxygen species

BackgroundThe growing evidences demonstrated hyperlipidemia in obesity and type 2 diabetes is characterized by high levels of free fatty acids, low-density lipoprotein (LDL), triglyceride, and cholesterol.Method and ResultsWe investigated the effect of LDL particles (LDLs) loading on MIN6 cells derived from pancreatic β cells. Exposure of MIN6 cells to LDLs induced apoptosis in dose and time-dependent manner, demonstrated by the TUNEL in situ apoptotic assay. The immunocytochemical analysis and Western blotting revealed that the LDLs-induced apoptosis is associated with the activation of caspase 3 and upregulation of p53. The intracellular concentration of Reactive Oxygen Species (ROS) measured by use of DCFHDA was significantly increased after loading LDLs, demonstrating the induced apoptosis by LDLs loading was mediated through oxidative stress. Addition of reduced form of Glutathione (GSH) in the medium rescued MIN6 cells from apoptosis. The Cellular cholesterol level was increased significantly after LDLs loading, suggesting that the excess cholesterol induced by LDLs loading might contribute to the apoptosis in MIN6s. Agarose electrophoresis demonstrated that the LDLs added to the medium were not oxidized.ConclusionTaken together, these results demonstrate that the LDLs loading can induce apoptosis of MIN6 cells mediated by the excess cellular cholesterol and generation of oxidative stress.

Construction of a green fluorescent protein (GFP)-marked multifunctional pesticide-degrading bacterium for simultaneous degradation of organophosphates and γ-hexachlorocyclohexane.

An autofluorescent whole-cell biocatalyst capable of simultaneously degrading organophosphates (OPs) and γ-hexachlorocyclohexane (γ-HCH) was constructed by display of organophosphorus hydrolase (OPH) and green fluorescent protein (GFP) fusion on the cell surface of a γ-HCH-degrading Sphingobium japonicum UT26 using the truncated ice nucleation protein (INPNC) as an anchoring motif. The surface localization of INPNC-OPH-GFP fusion was verified by cell fractionation, Western blot, proteinase accessibility, and immunofluorescence microscopy. Surface display of macromolecular OPH-GFP fusion (63 kDa) neither inhibits cell growth nor affects cell viability. In sterile and nonsterile soil samples, a mixture of parathion (100 mg kg(-1)) and γ-HCH (10 mg kg(-1)) could be degraded completely within 15 days when inoculated with the engineered UT26, and the strain could be easily monitored by fluorescence during bioremediation. These results indicate that the engineered UT26 is a promising multifunctional bacterium that could be used for the bioremediation of environments contaminated with multiple pesticides.

WITHDRAWN: Studies on the interaction between triphenyltin and bovine serum albumin by fluorescence and CD spectroscopy

The interaction between triphenyltin (TPT) and bovine serum albumin (BSA) in physiological buffer (pH=7.4) was investigated by the fluorescence quenching technique. The results of fluorescence titration revealed that TPT could strongly quench the intrinsic fluorescence of BSA through a static quenching procedure. The apparent binding constants K and number of binding sites n of TPT with BSA were (7.04±0.0057)×10(2) and (0.77±0.016) which were obtained by the fluorescence quenching method. The thermodynamic parameters enthalpy change (ΔH), entropy change (ΔS) were positive, which indicated that the interaction of TPT with BSA was driven mainly by hydrophobic forces. The process of binding was a spontaneous process in which Gibbs free energy change was negative. The distance r between donor (BSA) and acceptor (TPT) was calculated to be 3.05nm based on Forsters non-radiative energy transfer theory. The results of synchronous fluorescence, three-dimensional fluorescence and Circular Dichroism (CD) spectra showed that the triphenyltin induced conformational changes of BSA.

Characterizing the binding interaction of astilbin with bovine serum albumin: a spectroscopic study in combination with molecular docking technology

Astilbin (ASN) is a flavonoid compound isolated from the rhizome of Smilax china L. (Smilacaceae). It has many bioactivities, such as selective immunosuppression, antioxidant, anti-hepatic injury, etc., and is widely used in traditional Chinese medical treatments. The interaction of ASN with bovine serum albumin (BSA) was studied in a physiological buffer (pH = 7.40) using multi-spectroscopic techniques in combination with molecular docking methods. UV-vis absorption measurements proved that a ASN–BSA complex could be formed. Fluorescence data revealed that ASN could strongly quench the intrinsic fluorescence of BSA in terms of a static quenching procedure. The process of binding was spontaneous and the binding occurred mainly through hydrogen bonding and van der Waals forces. The distance r between donor (BSA) and acceptor (ASN) was calculated to be 4.80 nm based on Forsters non-radiative energy transfer theory. The binding constant (Ka = 7.31 × 104 mol L−1) and the number of binding sites (n ≈ 1) at 298 K suggested that ASN only occupied one site in BSA with high affinity. Moreover, the results of molecular docking indicated that ASN was more likely to be located in site I (sub-domain IIA) of BSA. The results of synchronous fluorescence and three-dimensional fluorescence spectra showed that ASN induced conformational changes of BSA. The findings would be beneficial for research on the transportation, distribution and some important bioactivities of ASN in the human body.

Elucidation of major contributors involved in nitrogen removal and transcription level of nitrogen-cycling genes in activated sludge from WWTPs

We investigated nitrogen-cycle bacterial communities in activated sludge from 8 municipal wastewater treatment plants (WWTPs). Redundancy analyses (RDA) showed that temperature was the most significant driving force in shaping microbial community structure, followed by influent NH4+ and total nitrogen (TN). The diversity of ammonia oxidizing and nitrite reducing bacteria were investigated by the construction of amoA, nirS and nirK gene clone libraries. Phylogenetic analysis indicated that Thauera and Mesorhizobium were the predominant nitrite reducing bacteria, and Nitrosomonas was the only detected ammonia oxidizing bacteria in all samples. Quantification of transcription level of nirS and nirK genes indicated that nirS-type nitrite reducing bacteria played the dominant roles in nitrite reduction process. Transcription level of nirS gene positively correlated with influent NH4+ and TN significantly, whereas inversely linked with hydraulic retention time. Temperature had a strong positive correlation to transcription level of amoA gene. Overall, this study deepened our understanding of the major types of ammonia oxidizing and nitrite reducing bacteria in activated sludge of municipal WWTPs. The relationship between transcription level of nitrogen-cycle genes and operational or environmental variables of WWTPs revealed in this work could provide guidance for optimization of operating parameters and improving the performance of nitrogen removal.

Antitumor activity of polysaccharide extracted from Pleurotus ostreatus mycelia against gastric cancer in vitro and in vivo

The present study aimed to determine the antitumor effects of polysaccharides extracted from Pleurotus ostreatus mycelium on gastric cancer in vitro and in vivo. Polysaccharides were extracted from Pleurotus ostreatus mycelium and an antitumor component, known as Pleurotus ostreatus mycelium polysaccharides 2 (POMP2), with a relative molecular weight of 29 kDa, was then sequentially purified using Sephadex G200 size-exclusion chromatography and diethylaminoethyl-52 cellulose ion-exchange chromatography. The MTT method was used to determine the proliferation of BGC-823 cells treated with POMP2; cell migration assay, colony formation assay and in vivo antitumor tests were used to assess the effect of POMP2 on migration, cell survival and the in vivo tumor formation of BGH-823 cells. Results of the MTT assay indicated that POMP2 had a marked inhibitory effect on the BGC-823 human gastric cancer cell line; when administered at a concentration of 400 mg/l for 72 h, the rate of inhibition was 35.6%. In addition, the colony forming capacity of the BGC-823 cells was significantly reduced following treatment with POMP2. A migration assay indicated that the invasive capabilities of the BGC-823 cells were also significantly inhibited by POMP2. Furthermore, in vivo tests of mice engrafted with BGC-823 cancer cells demonstrated that both tumor weight and volume were markedly reduced following two weeks of treatment with POMP2. The results of the present study suggested that the polysaccharide POMP2 may have a potential application as a natural antitumor treatment for gastric cancer.

Spectroscopic and molecular modeling studies on the interactions of fluoranthene with bovine hemoglobin

This study aims to investigate the interaction between fluoranthene (FLA) and Bovine hemoglobin (BHb) by ultraviolet-visible (UV-vis) absorption, fluorescence, synchronous fluorescence, circular dichroism (CD) spectroscopy and molecular docking method. The results showed that the fluorescence intensity of BHb was declined with the increase of FLA concentration. The binding procedure was spontaneous mainly driven by hydrophobic force. The number of binding sites were 0.709 (298 K), and 1.41 (310 K). The binding constants were equal to 4.68 × 103 mol·L-1 at 298 K and 6.17 × 105 mol·L-1 at 310 K. The binding distance between FLA and the tryptophan residue of BHb was 4.50 nm. The results of UV-vis spectra, synchronous fluorescence and CD spectra revealed that FLA could change the conformation of BHb, which might affect the physiological functions of hemoglobin. Moreover, molecular modeling results showed that the fluorescence experimental results were in agreement with the results obtained by molecular docking.