The relationship between coronary artery calcification and bone metabolic markers in maintenance hemodialysis patients.

BACKGROUND: To study the influencing factors for coronary artery calcification (CAC) in maintenance hemodialysis (MHD) patients and the relationship between CAC and bone metabolism markers and to attempt to find a reliable marker linking vascular calcification and bone metabolism in MHD patients. METHODS: A total of 123 patients were enrolled. CAC was assessed by multislice spiral computed tomography (MSCT), and the CAC score (CACS) was evaluated using the Agaston method. Routine laboratory parameters, including triglycerides (TG), total cholesterol (TC), glucose (Glu), calcium (Ca), phosphorus (P), magnesium (Mg), etc., were measured. Serum markers of bone metabolism, such as alkaline phosphatase(ALP), calcitonin (CT), 25-hydroxy vitamin D [25-(OH)D], intact parathyroid hormone (iPTH), total type I procollagen amino-terminal peptide (tPINP), N-terminal mid-fragment of osteocalcin (N-MID OC), and ß-type I collagen crosslinked carboxyl-terminal peptide (ß-CTX), were also measured. RESULTS: Among 123 MHD patients, 37 patients (30.08%) did not have CAC, and 86 patients (69.92%) had CAC, including 41 patients (47.67%) with mild calcification and 45 patients (52.33%) with moderate to severe calcification. Age, Body Mass Index(BMI), the prevalence of hypertension and diabetes mellitus, TC, Glu, P, and Ca×P in the calcification group were higher than those in the noncalcification group, whereas Mg, iPTH, tPINP, N-MID OC, and ß-CTX were lower than those in the noncalcified group (P < 0.05). Compared with the mild calcification group (0 0.05). A logistic regression model was used to evaluate the influencing factors for CAC. The results showed that age, BMI, TC, Glu, P, and Ca×P were risk factors for CAC and its severity in MHD patients, whereas diabetes mellitus, Mg, and N-MID OC were protective factors for CAC in MHD patients. In addition, N-MID OC was a protective factor for the severity of CAC. After adjusting for the corresponding confounding factors, the results of the risk factors were consistent, and N-MID OC was still an independent protective factor for CAC and its severity. CONCLUSIONS: Elevated serum P and Ca×P were independent risk factors for CAC in MHD patients, and serum Mg may be an independent protective factor for CAC. CAC was closely related to abnormal bone metabolism and bone metabolic markers in MHD patients. Relatively low bone turnover can promote the occurrence and development of CAC. N-MID OC may be a reliable bone metabolic marker linking vascular calcification and bone metabolism in MHD patients.

Characterization of the volatile profile and its estrogenic activity in Kadsura coccinea fruit.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruit of Kadsura coccinea (Lem.) A. C. Smith is an ethnomedicine used to treat abnormal menstruation, menopausal syndrome, and female infertility among the Dong Nationality in China. AIM OF THE STUDY: Our study aimed to identify the volatile oil profiles of the K. coccinea fruit and elucidate their estrogenic activity. MATERIALS AND METHODS: The peel volatile oil (PeO), pulp volatile oil (PuO), and seed volatile oil (SeO) of K. coccinea were extracted using hydrodistillation and qualitatively analyzed using gas chromatography-mass spectrometry (GC-MS). Estrogenic activity was evaluated in vitro using cell assay and in vivo using immature female rats. Serum 17ß-Estradiol (E2) and follicle-stimulating hormone (FSH) levels were detected using ELISA. RESULTS: In total, 46 PeO, 27 PuO, and 42 SeO components representing 89.96%, 90.19%, and 97% of the total composition, respectively, were identified. The compounds with the highest content in PeO, PuO, and SeO were ß-caryophyllene, γ-amorphene, and n-hexadecanoic acid, respectively. PeO induced proliferation of MCF-7 cells with an EC50 of 7.40 µg/mL. Subcutaneous administration of 10 mg/kg PeO significantly increased the weight of the uteri in immature female rats, with no effect on serum E2 and FSH levels. PeO acted as an agonist of ERα and ERß. PuO and SeO showed no estrogenic activity. CONCLUSION: The chemical compositions of PeO, PuO, and SeO of K. coccinea are different. PeO is the main effective fraction for estrogenic activities, providing a new source of phytoestrogen for the treatment of menopausal symptoms.

The effect of influenza A (H1N1) pdm09 virus infection on cytokine production and gene expression in BV2 microglial cells.

Acute influenza infection has been reported to be associated with neurological symptoms such as influenza-associated encephalopathy (IAE). Although the pathophysiology of this condition remain unclear, neuroinflammation and associated alterations in the central nervous system (CNS) are usually induced. Microglia (MGs), CNS-resident macrophages, are generally the first cells to be activated in response to brain infection or damage. We performed reverse transcriptase droplet digital PCR (RT-ddPCR) and luminex assays to investigate virus proliferation and immune reactions in BV2 MGs infected with influenza A(H1N1)pdm09 virus. Furthermore, isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics methods were used to investigate the dynamic change in the protein expression profile in BV2 MGs to gain insight into the CNS response to influenza A (H1N1) pdm09 infection. Our results showed that the influenza A(H1N1)pdm09 virus was replicative and productive in BV2 MG cells, which produced cytokines such as interleukin (IL)-1ß, IL-6, tumour necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1. The expression of osteopontin (OPN) in the influenza A (H1N1) pdm09-infected BV2 MGs was upregulated at 16 and 32 h post-infection (hpi) compared to that in the control group, resulting in aggravated brain damage and inflammation. Our study indicates that OPN signalling might provide new insights into the treatment of CNS injury and neurodegenerative diseases in IAE.

Role of the ATP-dependent chromatin remodeling enzyme Fun30/Smarcad1 in the regulation of mRNA splicing.

The yeast ATP-dependent chromatin remodeling enzyme Fun30 has been shown to regulate heterochromatin silencing, DNA repair, transcription, and chromatin organization. Although chromatin structure has been proposed to influence splice site recognition and regulation, whether ATP-dependent chromatin remodeling enzyme plays a role in regulating splicing is not known. In this study, we find that pre-mRNA splicing efficiency is impaired and the recruitment of spliceosome is compromised in Fun30-depleted cells. In addition, Fun30 is enriched in the gene body of individual intron-containing genes. Moreover, we show that pre-mRNA splicing efficiency is dependent on the chromatin remodeling activity of Fun30. The function of Fun30 in splicing is further supported by the observation that, Smarcad1, the mammalian homolog of Fun30, regulates alternative splicing. Taken together, these results provide evidence for a novel role of Fun30 in regulating splicing.

MicroRNA­132 mediates proliferation and migration of pulmonary smooth muscle cells via targeting PTEN.

Pulmonary arterial hypertension (PAH) is a severe and progressive disease characterized by the remodeling of small pulmonary arteries. The aberrant proliferation of pulmonary arterial smooth muscle cells (PASMCs) is the primary feature of PAH. MicroRNA (miR)­132 has been demonstrated to inhibit the proliferation of vascular smooth muscle cells and repress neointimal formation. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a direct target of miR­132 that has been revealed to be involved in the development of PAH. However, the role of miR­132 in PAH remains unclear. The present study demonstrated that miR­132 expression was upregulated in monocrotaline­induced PAH rats and platelet­derived growth factor­induced PASMCs. In addition, treatment of PASMCs with miR­132 mimics inhibited their proliferation, whereas miR­132 inhibition exhibited the opposite effects. Furthermore, miR­132 mimics promoted cell migration and maintained the PASMC contractile phenotype. Finally, the expression levels of PTEN were significantly decreased in PAH and PASMCs treated with miR­132 mimics. Taken collectively, the data suggested that miR­132 regulated PASMC function via PTEN and that it may be used as a potential target for the treatment of PAH.

Schisandrin B Improves the Renal Function of IgA Nephropathy Rats Through Inhibition of the NF-κB Signalling Pathway.

Schisandrin B (SchB) is an active compound extracted from the Chinese herb Schisandra chinensis and shows excellent anti-inflammatory activity. This study was performed to examine the effects of SchB in a rat model of IgA nephropathy (IgAN). IgAN was established in Sprague-Dawley rats by immunization with lipopolysaccharide (LPS), bovine serum albumin, and carbon tetrachloride. Renal function was evaluated by determining the levels of urinary red blood cells, proteinuria, blood urea nitrogen (BUN), and creatinine (Cr). Renal tissue and protein samples were collected for further analysis. Pre-treatment and treatment with SchB significantly ameliorated renal function of IgAN rats, which was evidenced by decreased levels of proteinuria, hematuria, BUN, and Cr. IgAN rats exhibited increased serum IgA, renal IgA deposition, mesangial cell proliferation, and inflammatory cell infiltration, which were significantly attenuated by intervention with SchB. Moreover, SchB inhibited infiltration of CD3+ and CD11b+ cells, decreased levels of tumour necrosis factor-alpha, interleukin-1ß, and monocyte chemoattractant protein-1 in the kidney, and decreased the numbers of CD3+CD69+ cells in the spleen. Of note, SchB therapy significantly increased cytoplasmic p65 and IκB expression and decreased nuclear p65 levels both in the damaged renal tissue and LPS-stimulated HK-2 cells, indicating a direct inhibitory effect on the NF-κB pathway in IgAN rats. Taken together, our data provide insight into a new application of SchB for the treatment of IgAN and represent a novel mechanism behind these effects.

ZmHAK5 and ZmHAK1 function in K+ uptake and distribution in maize under low K+ conditions.

Potassium (K+ ) is an essential macronutrient for plant growth and development. Transporters from the KT/HAK/KUP family play crucial roles in K+ homeostasis and cell growth in various plant species. However, their physiological roles in maize are still unknown. In this study, we cloned ZmHAK5 and ZmHAK1 and investigated their functions in maize (Zea mays L.). In situ hybridization showed that ZmHAK5 was mainly expressed in roots, especially in the epidermis, cortex, and vascular bundle. ZmHAK5 was characterized as a high-affinity K+ transporter. Loss of function of ZmHAK5 led to defective K+ uptake in maize, under low K+ conditions, whereas ZmHAK5-overexpressing plants showed increased K+ uptake activity and improved growth. ZmHAK1 was upregulated under low K+ stress, as revealed by RT-qPCR. ZmHAK1 mediated K+ uptake when heterologously expressed in yeast, but its transport activity was weaker than that of ZmHAK5. Overexpression of ZmHAK1 in maize significantly affected K+ distribution in shoots, leading to chlorosis in older leaves. These findings indicate that ZmHAK5 and ZmHAK1 play distinct roles in K+ homeostasis in maize, functioning in K+ uptake and K+ distribution, respectively. Genetic manipulation of ZmHAK5 may represent a feasible way to improve K+ utilization efficiency in maize.

Upregulation of long noncoding RNA CCAT1-L promotes epithelial-mesenchymal transition in gastric adenocarcinoma.

OBJECTIVE: In this study, we aimed to investigate the role of a long-chain noncoding RNA, colorectal cancer-associated transcript 1-long (CCAT1-L) in gastric adenocarcinoma. PATIENTS AND METHODS: Expressions of CCAT1-L and c-MYC mRNA and MYC protein in gastric adenocarcinoma tissue and adjacent normal tissues of 60 patients were analyzed using quantitative real-time polymerase chain reaction and Western blot, respectively. The CCAT1-L levels in the normal gastric epithelial cell line, GES1, and human gastric adenocarcinoma cell lines, MGC803, MKN-28, SGC7901, and BGC823 were analyzed by quantitative real-time polymerase chain reaction. CCAT1-L knockdown in MGC803 and MKN28 cells was performed using RNA interference, followed by evaluating cell proliferation, invasion, and migration with soft agar colony formation assay, scratch wound assay, and transwell assay. Twenty BALB/C-nu-nu nude mice were inoculated with gastric tumor xenografts and treated with CCAT1-L small-interfering RNA (siRNA), followed by monitoring survival and tumor growth. Western blot was also used to analyze the expression of epithelial-mesenchymal transition-related proteins, including MYC, RAS, T-ERK, P-ERK, E-cadherin, and vimentin, in gastric adenocarcinoma MKN-28 cells. RESULTS: The expression of CCAT1-L and MYC in tumor tissue was significantly higher than that in adjacent normal tissues (P<0.001). There was a positive correlation between the expression level of CCAT1-L mRNA and c-MYC mRNA (r=0.863, P<0.001). CCAT1-L expression was also significantly higher in gastric adenocarcinoma cell lines than that in normal cell lines (P<0.01). Knockdown of CCAT1-L in MGC803 and MKN-28 cells markedly reduced the cell proliferation, migration, and invasion (P<0.001). CCAT1-L knockdown also evidently inhibited tumor growth and improved survival in nude mice (P<0.001). Expressions of MYC, RAS, and vimentin, and the phosphorylation of ERK protein were dramatically decreased, while the expression of E-cadherin protein was increased by CCAT1-L knockdown in MKN-28 cell. CONCLUSION: CCAT1-L is a pro-oncogenic marker in gastric adenocarcinoma. CCAT1-L knockdown inhibits epithelial-mesenchymal transition of gastric adenocarcinoma cells and thus suppresses the gastric adenocarcinoma metastasis.

Facile electrochemiluminescence sensing platform based on high-quantum-yield gold nanocluster probe for ultrasensitive glutathione detection.

This report outlines a highly sensitive and facile electrochemiluminescence (ECL) sensing platform based on a novel high-quantum-yield Au-nanocluster (AuNC) probe for glutathione (GSH) detection. Owing to the prominent quenching effect of GSH on the ECL of the AuNCs, the proposed ECL nanosensor showed a wide response to GSH in the ranges of 1.0 × 10-9-1.0 × 10-5M and 1.0 × 10-5-1.0 × 10-1M and a low detection limit of 3.2 × 10-10M. In addition, the proposed system exhibited good selectivity for GSH in the presence of other chemical/biological interferences. Moreover, since no further functionalization of AuNC-based sensor interface was necessary, together with the stability, high sensitivity and selectivity of the proposed nanosensor, this convenient approach was able to successfully detect GSH in both of human urine samples and blood samples with excellent recoveries, which indicated its promising application under physiological conditions. Of significant importance is that this study not only helps in gaining a better understanding of the applicability of the ECL properties of AuNCs, but also provides a new avenue for the design and development of ECL sensors based on the novel high-quantum-yield AuNC-based probe and other functional-metal-based NC probes.

Abscisic Acid Modulates Seed Germination via ABA INSENSITIVE5-Mediated PHOSPHATE1.

The phytohormone abscisic acid (ABA) controls many developmental and physiological processes. Here, we report that PHOSPHATE1 (PHO1) participates in ABA-mediated seed germination and early seedling development. The transcription of PHO1 was obviously enhanced during seed germination and early seedling development and repressed by exogenous ABA. The pho1 mutants (pho1-2, pho1-4, and pho1-5) showed ABA-hypersensitive phenotypes, whereas the PHO1-overexpressing lines were ABA-insensitive during seed germination and early seedling development. The expression of PHO1 was repressed in the ABI5-overexpressing line and elevated in the abi5 mutant, and ABI5 can bind to the PHO1 promoter in vitro and in vivo, indicating that ABI5 directly down-regulated PHO1 expression. Disruption of PHO1 abolished the ABA-insensitive germination phenotypes of abi5 mutant, demonstrating that PHO1 was epistatic to ABI5 Together, these data demonstrate that PHO1 is involved in ABA-mediated seed germination and early seedling development and transcriptionally regulated by ABI5.

[Expression of BMP2/Smad1/Runx2 Signal Pathway in Renal Artery of Rat with Vascular Calcification].

OBJECTIVE: To investigate the activation and its role of bone morphogenetic protein 2 (BMP2)/Smad1/Runt-related transcription factor 2 (Runx2) signal pathway in renal artery of rat models with vascular calcification. METHODS: Twenty four male SD rats were randomly divided into control group and calcification group. Rat vascular calcification model was constructed by administration of vitamin D3 plus nicotine. Vascular calcification was confirmed by Von Kossa staining and calcium content was detected by calcium assay. Real time-PCR was applied to detect the expression of BMP2, Smad1, Runx2 mRNA, and immunohistochemistry was used to measure the protein levels of BMP2, Smad1, Runx2, α-smooth muscle actin (α-SMA). RESULTS: Von Kossa staining showed a large number of black granules deposited in renal artery. Calcium content in calcification group was significantly higher than that in normal group. Compared with the control group, the expressions of BMP2, Smad1 and Runx2 mRNA in renal artery were increased in calcification group. The protein levels of BMP2, Smad1 and Runx2 were higher while the expression of α-SMA was lower in calcification group than those in control group. The correlation analysis was found a positivie correlation between the calcium content and BMP2 mRNA (r = 0.655, P < 0.05), Smad1 mRNA (r = 0.735, P < 0.05), Runx2 mRNA (r = 0.734, P < 0.05). CONCLUSION: The expression of BMP2/Smad1/Runx2 signal pathway was strongly correlated with the severity of vascular calcification, which may be involved in the occurrence and development of vascular calcification.

Identification of Novel 14-3-3 Residues That Are Critical for Isoform-specific Interaction with GluN2C to Regulate N-Methyl-D-aspartate (NMDA) Receptor Trafficking.

The 14-3-3 family of proteins is widely distributed in the CNS where they are major regulators of essential neuronal functions. There are seven known mammalian 14-3-3 isoforms (ζ,, τ, ϵ, η, ß, and σ), which generally function as adaptor proteins. Previously, we have demonstrated that 14-3-3ϵ isoform dynamically regulates forward trafficking of GluN2C-containing NMDA receptors (NMDARs) in cerebellar granule neurons, that when expressed on the surface, promotes neuronal survival following NMDA-induced excitotoxicity. Here, we report 14-3-3 isoform-specific binding and functional regulation of GluN2C. In particular, we show that GluN2C C-terminal domain (CTD) binds to all 14-3-3 isoforms except 14-3-3σ, and binding is dependent on GluN2C serine 1096 phosphorylation. Co-expression of 14-3-3 (ζ and ϵ) and GluN1/GluN2C promotes the forward delivery of receptors to the cell surface. We further identify novel residues serine 145, tyrosine 178, and cysteine 189 on α-helices 6, 7, and 8, respectively, within ζ-isoform as part of the GluN2C binding motif and independent of the canonical peptide binding groove. Mutation of these conserved residues abolishes GluN2C binding and has no functional effect on GluN2C trafficking. Reciprocal mutation of alanine 145, histidine 180, and isoleucine 191 on 14-3-3σ isoform promotes GluN2C binding and surface expression. Moreover, inhibiting endogenous 14-3-3 using a high-affinity peptide inhibitor, difopein, greatly diminishes GluN2C surface expression. Together, these findings highlight the isoform-specific structural and functional differences within the 14-3-3 family of proteins, which determine GluN2C binding and its essential role in targeting the receptor to the cell surface to facilitate glutamatergic neurotransmission.

WRKY42 modulates phosphate homeostasis through regulating phosphate translocation and acquisition in Arabidopsis.

The Arabidopsis (Arabidopsis thaliana) WRKY transcription factor family has more than 70 members, and some of them have been reported to play important roles in plant response to biotic and abiotic stresses. This study shows that WRKY42 regulated phosphate homeostasis in Arabidopsis. The WRKY42-overexpressing lines, similar to the phosphate1 (pho1) mutant, were more sensitive to low-inorganic phosphate (Pi) stress and had lower shoot Pi content compared with wild-type plants. The PHO1 expression was repressed in WRKY42-overexpressing lines and enhanced in the wrky42 wrky6 double mutant. The WRKY42 protein bound to the PHO1 promoter under Pi-sufficient condition, and this binding was abrogated during Pi starvation. These data indicate that WRKY42 modulated Pi translocation by regulating PHO1 expression. Furthermore, overexpression of WRKY42 increased root Pi content and Pi uptake, whereas the wrky42 mutant had lower root Pi content and Pi uptake rate compared with wild-type plants. Under Pi-sufficient condition, WRKY42 positively regulated PHOSPHATE TRANSPORTER1;1 (PHT1;1) expression by binding to the PHT1;1 promoter, and this binding was abolished by low-Pi stress. During Pi starvation, the WRKY42 protein was degraded through the 26S proteasome pathway. Our results showed that AtWRKY42 modulated Pi homeostasis by regulating the expression of PHO1 and PHT1;1 to adapt to environmental changes in Pi availability.

The Os-AKT1 channel is critical for K+ uptake in rice roots and is modulated by the rice CBL1-CIPK23 complex.

Potassium (K(+)) is one of the essential nutrient elements for plant growth and development. Plants absorb K(+) ions from the environment via root cell K(+) channels and/or transporters. In this study, the Shaker K(+) channel Os-AKT1 was characterized for its function in K(+) uptake in rice (Oryza sativa) roots, and its regulation by Os-CBL1 (Calcineurin B-Like protein1) and Os-CIPK23 (CBL-Interacting Protein Kinase23) was investigated. As an inward K(+) channel, Os-AKT1 could carry out K(+) uptake and rescue the low-K(+)-sensitive phenotype of Arabidopsis thaliana akt1 mutant plants. Rice Os-akt1 mutant plants showed decreased K(+) uptake and displayed an obvious low-K(+)-sensitive phenotype. Disruption of Os-AKT1 significantly reduced the K(+) content, which resulted in inhibition of plant growth and development. Similar to the AKT1 regulation in Arabidopsis, Os-CBL1 and Os-CIPK23 were identified as the upstream regulators of Os-AKT1 in rice. The Os-CBL1-Os-CIPK23 complex could enhance Os-AKT1-mediated K(+) uptake. A phenotype test confirmed that Os-CIPK23 RNAi lines exhibited similar K(+)-deficient symptoms as the Os-akt1 mutant under low K(+) conditions. These findings demonstrate that Os-AKT1-mediated K(+) uptake in rice roots is modulated by the Os-CBL1-Os-CIPK23 complex.

The ATP-dependent chromatin remodeling enzyme Fun30 represses transcription by sliding promoter-proximal nucleosomes.

The evolutionarily conserved ATP-dependent chromatin remodeling enzyme Fun30 has recently been shown to play important roles in heterochromatin silencing and DNA repair. However, how Fun30 remodels nucleosomes is not clear. Here we report a nucleosome sliding activity of Fun30 and its role in transcriptional repression. We observed that Fun30 repressed the expression of genes involved in amino acid and carbohydrate metabolism, the stress response, and meiosis. In addition, Fun30 was localized at the 5' and 3' ends of genes and within the open reading frames of its targets. Consistent with its role in gene repression, we observed that Fun30 target genes lacked histone modifications often associated with gene activation and showed an increased level of ubiquitinated histone H2B. Furthermore, a genome-wide nucleosome mapping analysis revealed that the length of the nucleosome-free region at the 5' end of a subset of genes was changed in Fun30-depleted cells. In addition, the positions of the -1, +2, and +3 nucleosomes at the 5' end of target genes were shifted significantly, whereas the position of the +1 nucleosome remained largely unchanged in the fun30Δ mutant. Finally, we demonstrated that affinity-purified, single-component Fun30 exhibited a nucleosome sliding activity in an ATP-dependent manner. These results define a role for Fun30 in the regulation of transcription and indicate that Fun30 remodels chromatin at the 5' end of genes by sliding promoter-proximal nucleosomes.

Amelioration of rhabdomyolysis-induced renal mitochondrial injury and apoptosis through suppression of Drp-1 translocation.

INTRODUCTION: Mitochondrial dysfunction plays an important role in acute kidney injury (AKI). Mitochondrial fission regulated by dynamin-related protein 1 (Drp-1) impairs the function of the mitochondria and the survival of cells. This study was conducted to explore the effects of suppression of Drp-1 accumulation in the mitochondria, on mitochondrial function and renal tubular cell apoptosis in rhabdomyolysis (RM)-induced AKI. METHODS: An RM model was induced by intramuscular injection of glycerol in Sprague Dawley rats. Twenty-four and 48 hours after intraperitoneal injections of mitochondrial division inhibitor 1 (Mdivi-1), we observed the functions of the kidney, changes in pathology, expressions of Drp-1 in tubular tissues (by immunohistochemistry and Western blot) and accumulation of Drp-1 and mitofusin 2 in tubular mitochondria (by Western blot). Mitochondrial function (ATP and ROS) and tubular epithelial cell apoptosis (by TUNEL) were also measured. RESULTS: RM induced Drp-1 accumulation, decreased ATP production and increased ROS in mitochondria. With increasing cytochrome c expression, cell apoptosis increased, whereas kidney function decreased. These changes were time-dependent. At different time points, despite not significantly influencing the overall expression of Drp-1, Mdivi-1 suppressed the accumulation of Drp-1, inhibited the insertion of proapoptotic Bax in mitochondria and inhibited the release of cytochrome c, thus ameliorating cell apoptosis. CONCLUSIONS: To conclude, in RM-induced AKI, suppression of Drp-1 accumulation in mitochondria favors the maintenance of mitochondrial function and reduces the apoptosis of tubular cells. Regulation of the mitochondrial fusion-fission balance may offer a novel strategy for the prevention and treatment of RM-induced AKI.

A minority subpopulation of CD133(+) /EGFRvIII(+) /EGFR(-) cells acquires stemness and contributes to gefitinib resistance.

AIMS: To study the contribution of epidermal growth factor receptor variant III (EGFRvIII) to glioblastoma multiforme (GBM) stemness and gefitinib resistance. METHODS: CD133(+) and CD133(-) cells were separated from EGFRvIII(+) clinical specimens of three patients with newly diagnosed GBM. Then, RT-PCR was performed to evaluate EGFRvIII and EGFR expression in CD133(+) and CD133(-) cells. The tumorigenicity and stemness of CD133(+) cells was verified by intracranial implantation of 5 × 10(3) cells into immunodeficient NOD/SCID mice. Finally, cells were evaluated for their sensitivity to EGFR tyrosine kinase inhibition by gefitinib. RESULTS: RT-PCR results showed that the sorted CD133(+) cells expressed EGFRvIII exclusively, while the CD133(-) cells expressed both EGFRvIII and EGFR. At 6-8 weeks postimplantation, CD133(+) /EGFRvIII(+) /EGFR(-) cells formed intracranial tumors. Cell counting kit-8 results showed that the IC50 values of the three isolated EGFRvIII(+) cell lines treated with gefitinib were 14.44, 16.00, and 14.66 µM, respectively, whereas the IC50 value of an isolated EGFRvIII(-) cell line was 8.57 µM. CONCLUSIONS: EGFRvIII contributes to the stemness of cancer stem cells through coexpression with CD133 in GBMs. Furthermore, CD133(+) /EGFRvIII(+) /EGFR(-) cells have the ability to initiate tumor formation and may contribute to gefitinib resistance.

Inhibitory effect of selaginellin on high glucose-induced apoptosis in differentiated PC12 cells: role of NADPH oxidase and LOX-1.

Hyperglycemia clearly plays a key role in the development and progression of diabetic neuropathy. Hyperglycemia induces oxidative stress to generate reactive oxygen species in diabetic neurons resulting in neuronal damage and dysfunction. Apoptosis has been proposed as a possible mechanism for high glucose-induced neural dysfunction and neuronal cell injury. High glucose per se enhances lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) expression via activation of NADPH oxidase/reactive oxygen species pathway in endothelial cells. Selaginellin, a component extracted from Saussurea pulvinata (Hook. et Grev.) Maxim, was assessed for its ability to protect rat pheochromocytoma (PC12) cells against oxidative toxicity induced by high glucose. The differentiated PC12 cells were pretreated with various concentrations (10(-7), 3×10(-7) or 10(-6) M) of selaginellin for 1 h and then co-treated with selaginellin and D-glucose (75 mM) for 72 h. Selaginellin was shown to protect differentiated PC12 cells against high glucose toxicity, as determined by characteristic morphological features, cell viability, and apoptosis as evaluated by Hoechst 33,258 staining assay, annexin V-propidium iodide double staining assay and caspase-3 activity. In addition, the increase in NADPH oxidase activity, mRNA expression of NADPH oxidase subunits (NOX-1 and NOX-2) and LOX-1, and reactive oxygen species production induced by high glucose were significantly inhibited by selaginellin or by anti-LOX-1 antibody. The present study demonstrated that inhibitory effect of selaginellin on high glucose-induced cell injury and apoptosis in differentiated PC12 cells is related to inhibition of LOX-1/NADPH oxidase-reactive oxygen species/caspase-3 signaling pathway.

Biochemical assay for histone H2A.Z replacement by the yeast SWR1 chromatin remodeling complex.

The evolutionarily conserved histone variant H2A.Z has an important role in the regulation of gene expression and the establishment of a buffer to the spread of silent heterochromatin. Saccharomyces cerevisiae Swr1, a Swi2/Snf2-related ATPase, is the catalytic core of a multisubunit chromatin remodeling enzyme, called the SWR1 complex, that efficiently replaces conventional histone H2A in nucleosomes with histone H2A.Z. Swr1 is required for the deposition of histone H2A.Z at stereotypical promoter locations in vivo, and Swr1 and H2A.Z commonly regulate a subset of yeast genes. Here, we describe an integrated nucleosome assembly-histone replacement system whereby histone exchange by chromatin remodeling activities may be analyzed in vitro. The system demonstrates ATP- and SWR1-complex-dependent replacement of histone H2A for histone H2A.Z on a preassembled nucleosome array. This system may also be adapted to analyze dynamic interactions between chromatin remodeling and modifying enzymes, histone chaperones, and nucleosome substrates containing canonical, variant, or covalently modified histones.

Inhibitory effect of calcitonin gene-related peptide on hypoxia-induced rat pulmonary artery smooth muscle cells proliferation: role of ERK1/2 and p27.

Calcitonin gene-related peptide (CGRP) inhibits angiotensin II-induced proliferation of aortic smooth muscle cells via inactivation of extracellular signal-regulated protein kinase 1/2 (ERK1/2). ERK1/2 is necessary for the degradation or down-regulation of the cell cycle inhibitor p27, and is also crucial in mediating proliferation of pulmonary artery smooth muscle cells (PASMCs). Whether ERK1/2/p27 signal pathway is involved in CGRP-mediated pathogenesis of pulmonary hypertension and vascular remodeling remains unknown. Pulmonary hypertension was induced by hypoxia in rats, and capsaicin (50 mg/kg, s.c.) was used to deplete endogenous CGRP. Proliferation of cultured PASMCs was determined by BrdU incorporation method and flow cytometry. The expression/level of CGRP, p27, ERK1/2, c-fos and c-myc was analyzed by radioimmunoassay, immunohistochemistry, real-time PCR or Western blot. Sensory CGRP depletion by capsaicin exacerbated hypoxia-induced pulmonary hypertension in rats, as shown by an increase in right ventricle systolic pressure, mean pulmonary artery pressure and vascular hypertrophy, accompanied with decreased p27 expression and increased expression of phosphorylated ERK1/2, c-fos and c-myc. Exogenous application of CGRP significantly inhibited hypoxia-induced proliferation of PASMCs concomitantly with increased p27 expression and decreased expression of phosphorylated ERK1/2, c-fos and c-myc. These effects of CGRP were abolished in the presence of CGRP(8-37). Knockdown of p27 also reversed the inhibitory effect of CGRP on proliferation of PASMCs and expression of c-fos and c-myc, but not on ERK1/2 phosphorylation. These results suggest that CGRP inhibits hypoxia-induced proliferation of PASMCs via ERK1/2/p27/c-fos/c-myc pathway. Down-regulation of CGRP may contribute to remodeling of pulmonary arteries in hypoxia-induced pulmonary hypertension.