Green Fluorescence Intensity Research Articles

Rifampicin induces clathrin-dependent endocytosis and ubiquitin-proteasome degradation of MRP2 via oxidative stress-activated PKC-ERK/JNK/p38 and PI3K signaling pathways in HepG2 cells.

It was reported that antituberculosis medicines could induce liver damage via oxidative stress. In this study, we investigated the effects of rifampicin (RFP) on the membrane expression of multidrug resistance-associated protein 2 (MRP2) and the relationship between oxidative stress and RFP-induced endocytosis of MRP2 in HepG2 cells. We found that RFP (12.5–50 μM) dose-dependently decreased the expression and membrane localization of MRP2 in HepG2 cells without changing the messenger RNA level. RFP (50 μM) induced oxidative stress responses that further activated the PKC-ERK/JNK/p38 (protein kinase C-extracellular signal-regulated kinase/c-JUN N-terminal kinase/p38) and PI3K (phosphoinositide 3-kinase) signaling pathways in HepG2 cells. Pretreatment with glutathione reduced ethyl ester (2 mM) not only reversed the changes in oxidative stress indicators and signaling molecules but also diminished RFP-induced reduction in green fluorescence intensity of MRP2. We conducted co-immunoprecipitation assays and revealed that a direct interaction existed among MRP2, clathrin, and adaptor protein 2 (AP2) in HepG2 cells, and their expression was clearly affected by the changes in intracellular redox levels. Knockdown of clathrin or AP2 with small interfering RNA attenuated RFP-induced decreases of membrane and total MRP2. We further demonstrated that RFP markedly increased the ubiquitin–proteasome degradation of MRP2 in HepG2 cells, which was mediated by the E3 ubiquitin ligase GP78, but not HRD1 or TEB4. In conclusion, this study demonstrates that RFP-induced oxidative stress activates the PKC-ERK/JNK/p38 and PI3K signaling pathways that leads to clathrin-dependent endocytosis and ubiquitination of MRP2 in HepG2 cells, which provides new insight into the mechanism of RFP-induced cholestasis.

Independent modulation of individual genomic component transcription and a cis-acting element related to high transcriptional activity in a multipartite DNA virus

BackgroundThe genome of Banana bunchy top virus (BBTV) consists of at least six circular, single-stranded DNA components of ~ 1 kb in length. Some BBTV isolates may also carry satellite DNA molecules that are not essential for BBTV infection. The relation between multipartite DNA virus replication and their transcriptional levels and the underlying mechanism remain unclear.ResultsTo understand the coordinated replication and transcription of the multiple genomic components, the absolute amounts of each BBTV DNA component were measured by real-time PCR (qPCR), and their transcriptional levels were determined by RNAseq and reverse transcription-qPCR (qRT-PCR). Significant differences were found in the absolute amounts of individual BBTV genomic components. Transcriptional levels of each BBTV genomic component obtained from the RNAseq data matched closely to those obtained from qRT-PCR, but did not correspond to the absolute amount of each DNA component. The ratio of transcript over DNA copies ranged from 46.21 to 1059.44%, which was possibly regulated by the promoter region in the intergenic region of each component. To further determine this speculation, the promoter region of the DNA-S, −M or -N was constructed to the upstream of green fluorescent protein (GFP) gene for transient expression by agrobacterium-mediated transformation method. The qRT-PCR showed the highest transcriptional activity was promoted by DNA-N promoter, about 386.58% activity comparing with CaMV 35S promoter. Confocal microscopy observation showed that the intensity of green fluorescence was corresponding to that of qRT-PCR.ConclusionsOur data clearly showed that BBTV was able to control the transcriptional level of each DNA component independently by through the promoter sequences in the intergenic region. Moreover, a cis-acting element from DNA-N component had a high transcriptional activity.

Pantoea agglomerans YS19 poly(A) polymerase I gene possesses the indole-sensing sequence in the promoter region.

Pantoea agglomerans YS19 is a predominant diazotrophic endophyte with multiple growth-promoting effects on its host plant that was isolated from rice. Indole is confirmed to induce many changes of physiological and biochemical characteristics in bacteria. Although YS19 cannot produce indole, it can sense indole in the environment and be regulated by indole. Here, using gfp as a reporter gene, we constructed a series of recombinant plasmids containing the promoter region of the poly(A) polymerase I gene (pcnB) fused with gfp, and compared the green fluorescence intensity at different concentrations of exogenous indole by a flow cytometer. In this research, we confirmed that exogenous indole significantly inhibited the expression of pcnB by its promoter; the regulation sequence sensitive to indole in the promoter region of the pcnB gene (In-pcnB) was between -129 and -88 bp. In-pcnB is widely distributed and strictly conserved in the same genus. These results suggest novel roles of In-pcnB in P. agglomerans YS19, showing its special relation to the indole regulatory pathway.

Intermittent Hypoxia Prevents Myocardial Mitochondrial Ca2+ Overload and Cell Death during Ischemia/Reperfusion: The Role of Reactive Oxygen Species.

It has been documented that reactive oxygen species (ROS) contribute to oxidative stress, leading to diseases such as ischemic heart disease. Recently, increasing evidence has indicated that short-term intermittent hypoxia (IH), similar to ischemia preconditioning, could yield cardioprotection. However, the underlying mechanism for the IH-induced cardioprotective effect remains unclear. The aim of this study was to determine whether IH exposure can enhance antioxidant capacity, which contributes to cardioprotection against oxidative stress and ischemia/reperfusion (I/R) injury in cardiomyocytes. Primary rat neonatal cardiomyocytes were cultured in IH condition with an oscillating O2 concentration between 20% and 5% every 30 min. An MTT assay was conducted to examine the cell viability. Annexin V-FITC and SYTOX green fluorescent intensity and caspase 3 activity were detected to analyze the cell death. Fluorescent images for DCFDA, Fura-2, Rhod-2, and TMRM were acquired to analyze the ROS, cytosol Ca2+, mitochondrial Ca2+, and mitochondrial membrane potential, respectively. RT-PCR, immunocytofluorescence staining, and antioxidant activity assay were conducted to detect the expression of antioxidant enzymes. Our results show that IH induced slight increases of O2−· and protected cardiomyocytes against H2O2- and I/R-induced cell death. Moreover, H2O2-induced Ca2+ imbalance and mitochondrial membrane depolarization were attenuated by IH, which also reduced the I/R-induced Ca2+ overload. Furthermore, treatment with IH increased the expression of Cu/Zn SOD and Mn SOD, the total antioxidant capacity, and the activity of catalase. Blockade of the IH-increased ROS production abolished the protective effects of IH on the Ca2+ homeostasis and antioxidant defense capacity. Taken together, our findings suggest that IH protected the cardiomyocytes against H2O2- and I/R-induced oxidative stress and cell death through maintaining Ca2+ homeostasis as well as the mitochondrial membrane potential, and upregulation of antioxidant enzymes.

Acute nociceptive stimuli rapidly induce the activity of serotonin and noradrenalin neurons in the brain stem of awake mice

Nociception is an important type of perception that has major influence on daily human life. There are some descending pathways related to pain management and modulation, which are collectively known as the descending antinociceptive system (DAS). Noradrenalin (NA) in the locus coeruleus (LC) and serotonin (5-HT) in the rostral ventromedial medulla (RVM) are components of the DAS. Most 5-HT neurons in the dorsal raphe (DR) have ascending projections rather than descending projections, and they project to the thalamus that modulates nociception. Both the DAS and the DR are believed to be involved in pain-emotion symptoms. In this study, we utilized a fiber photometry system to specifically examine the activity of LC NA neurons and RVM/DR 5-HT neurons using mice carrying tetracycline-controlled transactivator transgene (tTA) under the control of either a dopamine β-hydroxylase promoter or a tryptophan hydroxylase-2 promoter and site-specific infection of an adeno-associated virus carrying a TetO G-CaMP6gene. After confirmation of specific expression of G-CaMP6 in the target populations, changes in green fluorescent signal intensity were recorded in awake mice upon exposure to acute nociceptive stimulation consisting of a pinch and application of heat (55°C) to the tail. Both stimuli resulted in rapid and transient (<15s) increases in the activity of LC NA neurons and RVM/DR 5-HT neurons while the control stimuli did not induce any changes. The present results clearly indicate that acute nociceptive stimuli increase the activity of LC NA neurons and RVM/DR 5HT neurons and suggest a possible therapeutic target for pain treatment.

Developing myelin specific promoters for schwannoma gene therapy

BackgroundSchwannomas are peripheral nerve sheath tumors composed entirely of Schwann-lineage cells that cause pain and sensory-motor dysfunction through compression of peripheral nerves, the spinal cord, and/or the brain stem. Treatment of schwannoma is largely limited to resection which itself has limited value. The goal of this study is to establish a technique to identify the most efficient and tissue-specific promoter for use in a schwannoma gene therapy construct. New methodThis work involves transfection of schwannoma cells with adeno-associated viral vector plasmids expressing GFP under different myelin cell specific promoters. The transfected cells were evaluated for green fluorescence intensity in vitro, and in vivo after implantation into sciatic nerves of nude mice. ResultsOur data demonstrate that myelin protein zero (MPZ, P0) and peripheral myelin protein 22 (PMP22) promoters produce greater GFP expression in schwannoma cell lines than myelin basic protein (MBP) promoter. In vitro, P0 promoter activity in schwannoma cell lines was shown to be less active than the cytomegalovirus and chicken β-actin (CBA) promoter. However, we did not observe any significant difference between the activity of the CBA and P0 promoters in a xenograft schwannoma model. Comparison with existing methods(s)We show here the influence of the peripheral nerve microenvironment on promoter efficacy in expressing transgenes using simple transfection by lipofection followed by prompt implantation of the transfected cells into the sciatic nerve of nude mice. ConclusionsWe demonstrate that of the myelin specific promoters evaluated, P0 is optimal for driving expression of transgenes in schwannoma cells.

Temperature and plasmon dependent Y6O5F8:Ho3+, Yb3+ up-conversion phosphors

Optical materials composed of an Y5.7−xHoxYb0.3O5F8 (x = 0.006–0.06) solid solution were prepared through a solid-state reaction using excess NH4F flux at 950 °C for 2 h. X-ray diffraction patterns were obtained from samples of Y5.7−xHoxYb0.3O5F8 prepared using an Y(Ho,Yb)O3/2:NH4F molar ratio of 1:2. The effective up-conversion green emission spectra and dependence of the emission intensity on the pump power (in the range of 77–118 mW) for the Y5.67Ho0.03Yb0.3O5F8 phosphor were investigated during excitation with a diode laser with a wavelength of 980 nm. Temperature-dependent green up-conversion emissions and desired Commission Internationale de l’Eclairage (CIE) values, including emissions from green to yellow wavelength regions, were realized with the phosphors at temperatures from 25 to 175 °C under 980-nm excitation. The green-fluorescence intensity of the Y5.67Ho0.03Yb0.3O5F8 phosphor was considerably enhanced by surface plasmon resonance of Au nanoparticles.

T-cell mitochondrial dysfunction and lymphopenia in DOCK2-deficient patients

This study delineates the mitochondrial defects in DOCK2-deficient T cells contributing to the T cell lymphopenia characteristic of this primary immunodeficiency.

Effects of albumin overload on mitochondrial membrane potential and ultrastructural changes in renal tubular epithelial cells

Objective To investigate effects of albumin overload on mitochondrial membrane potential and ultrastructural changes in renal tubular epithelial cells(HK-2). Methods Vitro-cultured HK-2 cells were treated with different concentrations of albumin.The mitochondrial membrane potential in HK-2 cells was detected by fluorescence probe JC-1, and the electron microscopy was used to detect the mitochondrial ultrastructure of cells. Results JC-1-detected red/green ratio was 2.34±0.21 at 0 g/L of albumin treatment, 0.83±0.09 at 4 g/L, 0.41±0.07 at 8 g/L, 0.38±0.08 at 16 g/L albumin overload, which showed a gradually decreased red and green fluorescence intensity ratio along with increased albumin overload(F=162.794, P<0.001), and with significant differences in the ratio among groups(all P<0.05). Albumin overload-induced mitochondrial abnormality in transmission electron microscope included mitochondrial swelling, disordered cristae arrangement, cristae lysis and cavitation, and the increase of bilayer membrane structures in HK-2 cells. Conclusions Albumin overload can induce the decline of mitochondrial membrane potential, mitochondrial damage and autophagy in renal tubular epithelial cells, which suggest that mitochondrial damage and autophagy may be one of the mechanisms of albumin overload-induced renal tubular epithelial cell injury. Key words: Albuminuria; Kidney tubules, proximal; Mitochondria; Autophagy

Developmental changes in the intensity and distribution pattern of green fluorescence in coral larvae and juveniles

Developmental changes in the intensity and distribution pattern of green fluorescence in coral larvae and juveniles

Construction of a glucaric acid biosensor for screening myo-inositol oxygenase variants

Glucaric acid (GA), a top value-added chemical from biomass, has been widely used for prevention and control of diseases and the production of polymer materials. In GA biosynthesis pathway, the conversion of inositol to glucuronic acid that catalyzed by myo-inositol oxygenase is the limiting step. It is necessary to improve MIOX activity. In the present study, we constructed a high-throughput screening system through combing the concentration of GA with the green fluorescent protein fluorescence intensity. By applying this screening system, three positive variants (K59V/R60A, R171S and D276A) screened from the mutant library. In comparison, the recombinant strain Escherichia coli BL21(DE3)/MU-R171S accumulated more GA, 136.5% of that of the parent strain.

Influence of nicotinic acid adenine dinucleotide phosphate on autophagy in hypoxic cardiomyocytes of rats and its mechanism

Objective: To investigate influence of nicotinic acid adenine dinucleotide phosphate (NAADP) on autophagy in hypoxic cardiomyocytes of rats and its mechanism. Methods: Five neonatal Sprague-Dawley rats were collected and sacrificed to harvest the hearts, and primary cardiomyocytes were separated for the following experiments. (1) Primary cardiomyocytes were collected and divided into normoxia group, hypoxia 9 h group, and hypoxia 9 h+ NAADP group according to random number table, with 5 wells in each group. Cells in normoxia group were cultured routinely in the constant temperature incubator at 37 ℃ for 9 hours. Cells in hypoxia 9 h group and hypoxia 9 h+ NAADP group were cultured in hypoxic incubator with volume fraction 94% nitrogen, 5% carbon dioxide, and 1% oxygen for 9 hours. Before hypoxia, cells in hypoxia 9 h+ NAADP group were dealt with final amount-of-substance concentration 10 μmol/L NAADP. Cell counting kit 8 was used to measure cell viability. (2) Primary cardiomyocytes were collected and divided into normoxia group, hypoxia 9 h group, hypoxia 9 h+ NAADP group, hypoxia 9 h+ tran-Ned-19 group, and hypoxia 9 h+ trans-Ned-19+ NAADP group according to the random number table, with 2 wells in each group. Cells in normoxia group were cultured routinely in the constant temperature incubator at 37 ℃ for 9 hours. And cells in the other 4 groups were cultured in hypoxic incubator as that in experiment (1) Before hypoxia, cells in hypoxia 9 h+ NAADP group were dealt with amount-of-substance concentration 10 μmol/L NAADP, cells in hypoxia 9 h+ tran-Ned-19 group were dealt with amount-of-substance concentration 1 μmol/L trans-Ned-19, and cells in hypoxia 9 h+ trans-Ned-19 + NAADP group were dealt with amount-of-substance concentration 10 μmol/L NAADP and 1 μmol/L trans-Ned-19. Protein expressions of microtubule associated protein 1 light chain 3-Ⅱ and P62 were detected by Western blotting. (3) Primary cardiomyocytes were collected and grouped as those in experiment (1). The lysosomal acidity was determined by immunofluorescence method. Data were processed with one-way analysis of variance and least-significant difference test. Results: (1) The cell viability in normoxia group was 1.114±0.024, which was significantly higher than 0.685±0.079 of cells in hypoxia 9 h group (P<0.01). The cell viability of hypoxia 9 h+ NAADP group was 0.886±0.061, which was obviously higher than that of cells in hypoxia 9 h group (P<0.05). (2) Expressions of microtubule-associated protein 1 light chain 3-Ⅱ and P62 of cells in hypoxia 9 h group were significantly higher than those of cells in normoxia group (P<0.01). Compared with those in hypoxia 9 h group, expression of P62 in hypoxia 9 h+ NAADP group was significantly decreased (P<0.01), while expression of microtubule-associated protein 1 light chain 3-Ⅱ did not change significantly (P>0.05). There were no significantly statistical difference in expressions of microtubule-associated protein 1 light chain 3-Ⅱ and P62 between hypoxia 9 h group and hypoxia 9 h+ trans-Ned-19 group (P>0.05). Compared with those of cells in hypoxia 9 h+ NAADP group, expression of P62 of cells in hypoxia 9 h+ trans-Ned-19+ NAADP group was obviously increased (P<0.01), while expression of microtubule-associated protein 1 light chain 3-Ⅱ did not change significantly (P>0.05). (3) The intensity of green fluorescence of cells in normoxia group was strong and co-localized well with red fluorescence, and internal environment of lysosome was with stronger acidity. The intensity of green fluorescence in cells of hypoxia 9 h group was significantly lower than that of cells in normoxia group, and acidity of internal environment of lysosome was weakened. The intensity of green fluorescence and acidity of internal environment of lysosome in hypoxia 9 h+ NAADP were significantly stronger than those of cells in hypoxia 9 h group, but significantly lower than those of cells in normoxia group. Conclusions: NAADP can improve myocardial cell viability through acidifying internal environment of lysosome of cardiomyocyte after hypoxia, promoting degradation of autophagosomes, reducing autophagic lysosomal accumulation, and repairing damaged autophagic flow.

A Turn-On Detection of DNA Sequences by Means of Fluorescence of DNA-Templated Silver Nanoclusters via Unique Interactions of a Hydrated Ionic Liquid.

Nucleic acid stability and structure, which are crucial to the properties of fluorescent DNA-templated silver nanoclusters (DNA-Ag NCs), significantly change in ionic liquids. In this work, our purpose was to study DNA-Ag NCs in a buffer containing the hydrated ionic liquid of choline dihydrogen phosphate (choline dhp) to improve fluorescence for application in DNA detection. Due to the stabilisation of an i-motif structure by the choline cation, a unique fluorescence emission—that was not seen in an aqueous buffer—was observed in choline dhp and remained stable for more than 30 days. A DNA-Ag NCs probe was designed to have greater fluorescence intensity in choline dhp in the presence of a target DNA. A turn-on sensing platform in choline dhp was built for the detection of the BRCA1 gene, which is related to familial breast and ovarian cancers. This platform showed better sensitivity and selectivity in distinguishing a target sequence from a mutant sequence in choline dhp than in the aqueous buffer. Our study provides new evidence regarding the effects of structure on properties of fluorescent DNA-Ag NCs and expands the applications of fluorescent DNA-Ag NCs in an ionic liquid because of improved sensitivity and selectivity.

Flow cytometric analysis of Xenopus laevis and X. tropicalis blood cells using acridine orange

Automated blood cell counters can distinguish cells based on their size and the presence or absence of a nucleus. However, most vertebrates have nucleated blood cells that cannot be counted automatically. We established an alternative automatic method for counting peripheral blood cells by staining cells with the fluorescent dye acridine orange (AO) and analysing cell populations using flow cytometry (FCM). As promising new animal models, we chose Xenopus laevis and three inbred strains of X. tropicalis. We compared the haematological phenotypes, including blood cell types, cell sizes, cellular structure, and erythrocyte lifespans/turnover rate among X. laevis and the three inbred strains of X. tropicalis. Each cell type from X. laevis was sorted according to six parameters: forward- and side-scattered light emission, AO red and green fluorescence intensity, and cellular red and green fluorescence. Remarkably, the erythrocyte count was the highest in the Golden line, suggesting that genetic factors were associated with the blood cells. Furthermore, immature erythrocytes in anaemic X. laevis could be separated from normal blood cells based on red fluorescence intensity. These results show that FCM with AO staining allows for an accurate analysis of peripheral blood cells from various species.

Effect of over-expressed phosphatidylinositol-5 gene on epithelial-mesenchymal transition of lung adenocarcinoma and clinical significance

Objective To observer the effect of phosphatidylinositol-5 (GPC5) on epithelial-mesenchymal transition (EMT) in lung adenocarcinoma cells and to investigate the clinical correlation between the expression of GPC5 and EMT key proteins (E-cadherin, Twist1, Vimentin, N-cadherin) in human lung adenocarcinoma tissues. Methods Luciferase labeled lentivirus-induced GPC5 over-expression was used for in vitro studies in A549 cell line (OE-GPC5) and a negative control group (NC). Western blotting was used to compare the expression level differences in EMT related proteins between the two groups. Immunofluorescent assay was used to detect the expression of E-cadherin and Vimentin in the two groups under the intervention of EMT-induced factor transforming growth factor-β (TGF-β) (10 ng/ml, 36 h). The mRNA expression levels of GPC5, E-cadherin and Vimentin were detected by real-time quantitative PCR in 134 cases of lung adenocarcinoma tissues. The correlation analysis of clinical expression was performed by Pearson method. Results The expression level of E-cadherin in the A549 cell line was up-regulated by over-expressed GPC5 (P=0.008), and the expression levels of Vimentin (P=0.012), N-cadherin (P=0.001) and Twist1 (P=0.001) were down-regulated. The immunofluorescent assay indicated that the E-cadherin green fluorescence intensity in OE-GPC5 group was significantly stronger than in NC group (P=0.020), and the Vimentin green fluorescence intensity in OE-GPC5 group was significantly weaker than in NC group (P=0.001). Pearson correlation analysis showed that the mRNA expression of GPC5 was positively correlated with that of E-cadherin in human lung adenocarcinoma tissues (P=0.007), and negatively with that of Twist1 (P=0.023). Conclusion GPC5 may play a role of tumor suppressor gene by reversing the EMT process of lung adenocarcinoma. Key words: Phosphatidylinositol-5; Epithelial-mesenchymal transition; Lung adenocarcinoma

Surfactants Improve Live Cell Imaging of Human Pancreatic Islets.

Newport Green is a zinc-specific fluorescent dye developed to monitor cellular zinc transport. In pancreatic islets with zinc-rich β-cells, Newport Green is expected to be useful as an islet-specific indicator for live imaging. However, the low penetration of Newport Green into islets hinders clear detection. The aim of this study was to develop a practical method of live islet imaging by using surfactants to enhance the penetration efficiency. Surfactants (F127, Tween 20, and Triton X-100) were co-incubated with Newport Green for fluorescent imaging of live isolated human islet and nonislet tissues. Toxicity, enhancement of Newport Green fluorescence, and effects on specificity to islets were examined. Newport Green fluorescent intensity was increased after co-incubation with all surfactants tested (0.2-3.2 mM); however, surfactants were toxic to islets at high concentrations. Within the nontoxic range, high specificity to islets was observed when co-incubated with Tween 20 at 0.2-0.4 mM, compared with F127 and Triton X-100. This optimized range successfully distinguished islets from nonislet tissues using statistically calculated cutoff value of Newport Green fluorescent intensity. Surfactants, particularly Tween 20 in the optimized range, effectively and selectively enhanced Newport Green fluorescence in live islets without increasing islet toxicity.

Effect of dl-3-n-Butylphthalide on apoptosis of retinal müller cells induced by hydrogen peroxide

Objective To observe the protective effect of dl-3-n-Butylphthalide (NBP) on apoptosis of retinal Muller cells induced by hydrogen peroxide (H2O2). Methods Human retinal Muller cells cultured in vitro were divided into normal control group, model group (H2O2 group) and experimental group (H2O2+NBP group). The cells in the H2O2 group and H2O2+NBP group were cultured with 200 μmol/L H2O2 for 2 h. Then the culture solution of the H2O2 group replace with complete medium and the H2O2+NBP group replace with complete medium containing 1 μmol/L NBP. The normal control group was a conventional cultured cells. Muller cells were identified by immunofluorescence staining. Hematoxylin-eosin (HE) staining was used to observe the apoptosis morphological changes. MTT assay was used to detect the activity of of retinal Muller cells after after 24 h and 48 h of NBP intervention. Hoechst33258 staining was used to observe the apoptosis. LIVE/DEAD ® cell activity/cytotoxicity kit was used to detect cell viability. Dichlorofluorescein diacetate (DCFH-DA) + endoplasmic reticulum (ER) red fluorescent probe (ER-Tracker Red) double staining was used to observe the expression level of reactive oxygen species (ROS) in ER of cells. One-way ANOVA combined with Dunnett statistical method were used for data analysis. Results HE staining showed that the number of cells in H2O2+NBP group was higher than that in H2O2 group. MTT assay showed that after 24 h and 48 h of NBP intervention, the differences in cell viability between the normal control group and the H2O2 group, the H2O2 group and the H2O2+NBP group were statistically significant (t=28.96, 3.658, 47.58, 20.33; P<0.001, 0.022). The results of Hoechst33258 showed that the nuclear nucleus of a few cells in the H2O2+NBP group was crescent-shaped and the nuclear fragmentation was reduced, and the blue fluorescence of the remaining cells was uniform. The LIVE/DEAD ® cell activity/cytotoxicity kit showed that the number of dead cells with red fluorescence in the H2O2 group increased significantly, and the number of viable cells with green fluorescence decreased significantly. In the H2O2+NBP group, the number of viable cells with green fluorescence increased, and the number of dead cells with red fluorescence decreased. The double staining results of DCFH-DA+ER-Tracker Red showed that the green fluorescence intensity of H2O2 group was significantly enhanced; the green fluorescence intensity of H2O2+NBP group was lower than that of H2O2 group. Conclusion NBP alleviates H2O2-induced apoptosis of human retinal Muller cells by inhibiting ROS production. Key words: Butylphthalide; Oxidative stress; Apoptosis; Hydrogen peroxide; Muller cells

Functional haplotypes of ARID4A affect promoter activity and semen quality of bulls

The AT-rich interaction domain 4 A (ARID4A) has an important role in regulating Sertoli cell function and male fertility. Its molecular mechanisms, however, remain largely unknown. In this study, two single nucleotide polymorphisms (SNPs) (g.53 G > T, ss 1966531596, and g.826 G > A, rs 210809648) were identified in the promoter region of ARID4A in 215 Chinese Holstein bulls using polymerase chain reaction (PCR)-restriction fragment length polymorphism and created restriction site-PCR. Results revealed that bulls with g.53 G > T-GG and g.826 G > A-G G genotype exhibited higher sperm deformity rate than those with g.53 G > T-TT and g.826 G > A-AA genotype (P < 0.01). Furthermore, three haplotypes (H1 (GG), H3 (TG), H4 (TA)) and six haplotype combinations (H1H1, H1H3, H1H4, H3H3, H3H4, H4H4) were obtained. The bulls with H4H4 exhibited lower sperm deformity rate than those with H1H1 and H1H3 (P < 0.05). In addition, results of bioinformatics analysis revealed that ARID4A has two promoters and that two SNPs of ARID4A are located in transcription factor binding sites. Compared with g.53 G > T-G and g.826 G > A-G allele, there was a greater fluorescence intensity in g.53 G > T-T and g.826 G > A-A allele by transient transfection in MLTC-1 cells and the luciferase report assay. qRT-PCR indicated the ARID4A expression was greater in bull spermatozoa with H4H4 haplotype combination than those with H1H1 haplotype combination (P < 0.05). Results of the present study indicate that g.53 G > T and g.826 G > A are functional mutations that are involved in regulation of ARID4A gene expression by affecting promoter activity and thus semen quality of Chinese Holstein bulls.

X线诱导人鼻咽鳞状细胞癌CNE1细胞株MDR-1及P-gp的表达研究

Objective To examine the expressions of multidrug resistance gene (MDR)-1 and P-glycoprotein (P-gp) in nasopharyngeal squamous cell carcinoma CNE1 cell line before and after X-ray exposure. Methods CNE1 cells were exposed to X-ray. After the irradiation, the CNE1 cells were cultured for 24 hours and tested. The mRNA expressions of MDR-1 in CNE1 cells were measured by semi-quantitative real-time polymerase chain reaction (RT-PCR) before and after X-ray exposure, and the protein expressions of P-gp in CNE1 cells were detected by Western blotting. The protein expressions of P-gp in CNE1 cells were observed by confocal microscope before and after X-ray exposure. Results The results of RT-PCR showed that the absorbance (A) values of MDR-1 mRNA in CNE1 cells were 0.17±0.01 and 0.34±0.03 before and after irradiation, and the difference was statistically significant (t=16.541, P<0.001). The results of Western blotting showed that the A values of P-gp protein in CNE1 cells were 0.02±0.01 and 0.04±0.01, and the difference was statistically significant (t=4.612, P=0.016). The green fluorescence intensity of P-gp protein in CNE1 cells after X-ray irradiation was higher than that before X-ray irradiation by confocal microscope. Conclusion X-ray irradiation can cause the high expressions of MDR-1 and P-gp in CNE1 cells, suggesting that X-ray irradiation can induce the occurrence of multidrug resistance in CNE1 cells. Key words: Nasopharyngeal neoplasms; CNE1 cells; Drug resistance, multiple; X-ray irradiation

Spectral conversion from green to red in Yb3+/Ho3+:Sr2GdF7 glass ceramics via Ce3+ doping

Ce3+ co-doped Sr2GdF7:Yb3+/Ho3+ transparent oxyfluoride glass ceramics were successfully fabricated through melt-quenching technique, which were further confirmed by intense diffraction peaks of X-ray diffraction, transmission electron microscopy, selected area electron diffraction, obvious Stark splitting of emission spectra and prolonged lifetimes of Ho3+ after crystallization. Under 980 nm excitation, green upconversion emission is dominant in Sr2GdF7:Yb3+/Ho3+ glass ceramics and is about 10,000 times stronger than that in precursor glass. Furthermore, upconversion emission tuning from green to red in Sr2GdF7:Yb3+/Ho3+ glass ceramics was obtained via codoping Ce3+ ions with different concentrations. Impressively, red to green fluorescence intensity ratio increases from 0.1 to 91, which is caused by two efficient cross-relaxation processes derived from Ho3+ and Ce3+ ions. The current study on the multicolor tuning behavior of Ce3+ doped Sr2GdF7:Yb3+/Ho3+ glass ceramics offers a potential application in color displays.