International Journal of Molecular Sciences

2622 KiB

Open AccessArticle

The Peroxisome Proliferator-Activated Receptor (PPAR) α Agonist Fenofibrate Suppresses Chemically Induced Lung Alveolar Proliferative Lesions in Male Obese Hyperlipidemic Mice

by Toshiya Kuno, Kazuya Hata, Manabu Takamatsu, Akira Hara, Yoshinobu Hirose, Satoru Takahashi, Katsumi Imaida and Takuji Tanaka

Int. J. Mol. Sci. 2014, 15(5), 9160-9172; https://doi.org/10.3390/ijms15059160 - 22 May 2014

Cited by 9 | Viewed by 6787

Abstract

Activation of peroxisome proliferator-activated receptor (PPAR) α disrupts growth-related activities in a variety of human cancers. This study was designed to determine whether fenofibrate, a PPARα agonist, can suppress 4-nitroquinoline 1-oxide (4-NQO)-induced proliferative lesions in the lung of obese hyperlipidemic mice. Male Tsumura [...] Read more.

Activation of peroxisome proliferator-activated receptor (PPAR) α disrupts growth-related activities in a variety of human cancers. This study was designed to determine whether fenofibrate, a PPARα agonist, can suppress 4-nitroquinoline 1-oxide (4-NQO)-induced proliferative lesions in the lung of obese hyperlipidemic mice. Male Tsumura Suzuki Obese Diabetic mice were subcutaneously injected with 4-NQO to induce lung proliferative lesions, including adenocarcinomas. They were then fed a diet containing 0.01% or 0.05% fenofibrate for 29 weeks, starting 1 week after 4-NQO administration. At week 30, the incidence and multiplicity (number of lesions/mouse) of pulmonary proliferative lesions were lower in mice treated with 4-NQO and both doses of fenofibrate compared with those in mice treated with 4-NQO alone. The incidence and multiplicity of lesions were significantly lower in mice treated with 4-NQO and 0.05% fenofibrate compared with those in mice treated with 4-NQO alone (p < 0.05). Both doses of fenofibrate significantly reduced the proliferative activity of the lesions in 4-NQO-treated mice (p < 0.05). Fenofibrate also significantly reduced the serum insulin and insulin-like growth factor (IGF)-1 levels, and decreased the immunohistochemical expression of IGF-1 receptor (IGF-1R), phosphorylated Akt, and phosphorylated Erk1/2 in lung adenocarcinomas. Our results indicate that fenofibrate can prevent the development of 4-NQO-induced proliferative lesions in the lung by modulating the insulin-IGF axis. Full article

(This article belongs to the Section Biochemistry)

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Graphical abstract

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Histopathologic features of representative pulmonary proliferative lesions (hematoxylin and eosin stain). (A) bronchioloalveolar hyperplasia; (B) adenoma; (C) adenocarcinoma. There are numerous hyperplastic alveolar cells with an alveolar structure in bronchioloalveolar hyperplasia while tumor cells without an alveolar structure are present in adenoma. The central area of the adenocarcinoma is necrotic. Full article ">
Histopathology (A,E,I,M,Q) and immunohistochemistry (insulin-like growth factor 1 receptor (IGF-1R), phosphorylated (p)-Akt, and p-extracellular signal-related kinase (Erk) 1/2) of bronchioloalveolar hyperplasia (B–D,N,O,P,R–T), adenoma (F–H), and adenocarcinoma (J–L) induced by 4-nitroquinoline 1-oxide (4-NQO) administration in Tsumura Suzuki Obese Diabetic (TSOD) mice. The adenomas M–P and Q–T are developed in a mouse treated with 4-NQO followed by dietary exposure to 0.01% and 0.05% fenofibrate, respectively. Alveolar hyperplasia (A–D), adenoma (E–H), and adenocarcinoma (I–L) were positively stained with IGF-1R (B,F,J), p-Akt (C,G,K), and p-Erk1/2 (D,H,L) antibodies, respectively. Note the weakly positive or negative reactions against IGF-1R, p-Akt, and p-Erk1/2 of adenoma in mice that received 4-NQO and fenofibrate. (A,E,I,M,Q): H&E stain; (B,F,J,N,R): IGF-1R immunohistochemistry; (C,G,K,O,S): p-Akt immunohistochemistry; and (D,H,L,P,T): p-Erk1/2 immunohistochemistry. Full article ">
Immunohistochemical staining of PPARα (A–C) and PPARγ (D–F) in TSOD mice treated with 4-NQO. Representative slides show a strong increase in PPARα expression in the nuclei of lung proliferative lesions: (A,D): bronchioloalveolar hyperplasia; (B,E): adenoma; and (C,F): adenocarcinoma. Full article ">
Ki-67 labeling index and immunohistochemistry of 4-NQO-induced pulmonary proliferative lesions from mice treated with or without fenofibrate. (A) Representative features of Ki-67 immunohistochemistry in a mouse treated with 4-NQO alone (upper; group 1) or 4-NQO followed by 0.01% fenofibrate (lower; group 2). Bars = 20 μm; (B) Dietary administration of 0.01% fenofibrate significantly lowered the Ki-67 labeling index in group 2 compared with mice treated with 4-NQO alone (group 1) (p < 0.05). Full article ">

335 KiB

Open AccessArticle

Functional Polymorphisms of the ABCG2 Gene Are Associated with Gout Disease in the Chinese Han Male Population

by Danqiu Zhou, Yunqing Liu, Xinju Zhang, Xiaoye Gu, Hua Wang, Xinhua Luo, Jin Zhang, Hejian Zou and Ming Guan

Int. J. Mol. Sci. 2014, 15(5), 9149-9159; https://doi.org/10.3390/ijms15059149 - 22 May 2014

Cited by 43 | Viewed by 8687

Abstract

Background: Gout is a common type of arthritis that is characterized by hyperuricemia, tophi and joint inflammation. Genetic variations in the ABCG2 gene have been reported to influence serum uric acid levels and to participate in the pathogenesis of gout, but no further [...] Read more.

Background: Gout is a common type of arthritis that is characterized by hyperuricemia, tophi and joint inflammation. Genetic variations in the ABCG2 gene have been reported to influence serum uric acid levels and to participate in the pathogenesis of gout, but no further data have been reported in the Han Chinese population. Methods: Peripheral blood DNA was isolated from 352 male patients with gout and 350 gout-free normal male controls. High-resolution melting analysis and Sanger sequencing were performed to identify the genetic polymorphisms V12M, Q141K and Q126X in the ABCG2 gene. Genotype and haplotype analyses were utilized to determine the disease odds ratios (ORs). A prediction model for gout risk using ABCG2 protein function was established based on the genotype combination of Q126X and Q141K. Results: For Q141K, the A allele frequency was 49.6% in the gout patients and 30.9% in the controls (OR 2.20, 95% confidence interval (CI): 1.77–2.74, p = 8.99 × 10⁻¹³). Regarding Q126X, the T allele frequency was 4.7% in the gout patients and 1.7% in the controls (OR 2.91, 95% CI: 1.49–5.68, p = 1.57 × 10⁻³). The A allele frequency for V12M was lower (18.3%) in the gout patients than in the controls (29%) (OR 0.55, 95% CI 0.43–0.71, p = 2.55 × 10⁻⁶). In the order of V12M, Q126X and Q141K, the GCA and GTC haplotypes indicated increased disease risk (OR = 2.30 and 2.71, respectively). Patients with mild to severe ABCG2 dysfunction accounted for 78.4% of gout cases. Conclusion: The ABCG2 126X and 141K alleles are associated with an increased risk of gout, whereas 12M has a protective effect on gout susceptibility in the Han Chinese population. ABCG2 dysfunction can be used to evaluate gout risk. Full article

(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)

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366 KiB

Open AccessArticle

Aerobic Degradation of Trichloroethylene by Co-Metabolism Using Phenol and Gasoline as Growth Substrates

by Yan Li, Bing Li, Cui-Ping Wang, Jun-Zhao Fan and Hong-Wen Sun

Int. J. Mol. Sci. 2014, 15(5), 9134-9148; https://doi.org/10.3390/ijms15059134 - 22 May 2014

Cited by 43 | Viewed by 7999

Abstract

Trichloroethylene (TCE) is a common groundwater contaminant of toxic and carcinogenic concern. Aerobic co-metabolic processes are the predominant pathways for TCE complete degradation. In this study, Pseudomonas fluorescens was studied as the active microorganism to degrade TCE under aerobic condition by co-metabolic degradation [...] Read more.

Trichloroethylene (TCE) is a common groundwater contaminant of toxic and carcinogenic concern. Aerobic co-metabolic processes are the predominant pathways for TCE complete degradation. In this study, Pseudomonas fluorescens was studied as the active microorganism to degrade TCE under aerobic condition by co-metabolic degradation using phenol and gasoline as growth substrates. Operating conditions influencing TCE degradation efficiency were optimized. TCE co-metabolic degradation rate reached the maximum of 80% under the optimized conditions of degradation time of 3 days, initial OD₆₀₀ of microorganism culture of 0.14 (1.26 × 10⁷ cell/mL), initial phenol concentration of 100 mg/L, initial TCE concentration of 0.1 mg/L, pH of 6.0, and salinity of 0.1%. The modified transformation capacity and transformation yield were 20 μg (TCE)/mg (biomass) and 5.1 μg (TCE)/mg (phenol), respectively. Addition of nutrient broth promoted TCE degradation with phenol as growth substrate. It was revealed that catechol 1,2-dioxygenase played an important role in TCE co-metabolism. The dechlorination of TCE was complete, and less chlorinated products were not detected at the end of the experiment. TCE could also be co-metabolized in the presence of gasoline; however, the degradation rate was not high (28%). When phenol was introduced into the system of TCE and gasoline, TCE and gasoline could be removed at substantial rates (up to 59% and 69%, respectively). This study provides a promising approach for the removal of combined pollution of TCE and gasoline. Full article

(This article belongs to the Special Issue Biodegradability of Materials)

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548 KiB

Open AccessReview

Brain Metastasis-Initiating Cells: Survival of the Fittest

by Mohini Singh, Branavan Manoranjan, Sujeivan Mahendram, Nicole McFarlane, Chitra Venugopal and Sheila K. Singh

Int. J. Mol. Sci. 2014, 15(5), 9117-9133; https://doi.org/10.3390/ijms15059117 - 22 May 2014

Cited by 23 | Viewed by 9482

Abstract

Brain metastases (BMs) are the most common brain tumor in adults, developing in about 10% of adult cancer patients. It is not the incidence of BM that is alarming, but the poor patient prognosis. Even with aggressive treatments, median patient survival is only [...] Read more.

Brain metastases (BMs) are the most common brain tumor in adults, developing in about 10% of adult cancer patients. It is not the incidence of BM that is alarming, but the poor patient prognosis. Even with aggressive treatments, median patient survival is only months. Despite the high rate of BM-associated mortality, very little research is conducted in this area. Lack of research and staggeringly low patient survival is indicative that a novel approach to BMs and their treatment is needed. The ability of a small subset of primary tumor cells to produce macrometastases is reminiscent of brain tumor-initiating cells (BTICs) or cancer stem cells (CSCs) hypothesized to form primary brain tumors. BTICs are considered stem cell-like due to their self-renewal and differentiation properties. Similar to the subset of cells forming metastases, BTICs are most often a rare subpopulation. Based on the functional definition of a TIC, cells capable of forming a BM could be considered to be brain metastasis-initiating cells (BMICs). These putative BMICs would not only have the ability to initiate tumor growth in a secondary niche, but also the machinery to escape the primary tumor, migrate through the circulation, and invade the neural niche. Full article

(This article belongs to the Special Issue Brain Metastasis 2014)

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2188 KiB

Open AccessArticle

Chemical Characterization and Antitumor Activities of Polysaccharide Extracted from Ganoderma lucidum

by Zengenni Liang, Youjin Yi, Yutong Guo, Rencai Wang, Qiulong Hu and Xingyao Xiong

Int. J. Mol. Sci. 2014, 15(5), 9103-9116; https://doi.org/10.3390/ijms15059103 - 22 May 2014

Cited by 68 | Viewed by 8918

Abstract

Ganoderma lucidum polysaccharide (GLP) is a biologically active substance reported to possess anti-tumor ability. Nonetheless, the mechanisms of GLP-stimulated apoptosis are still unclear. This study aims to determine the inhibitory and apoptosis-inducing effects of GLP on HCT-116 cells. We found that GLP reduced [...] Read more.

Ganoderma lucidum polysaccharide (GLP) is a biologically active substance reported to possess anti-tumor ability. Nonetheless, the mechanisms of GLP-stimulated apoptosis are still unclear. This study aims to determine the inhibitory and apoptosis-inducing effects of GLP on HCT-116 cells. We found that GLP reduced cell viability on HCT-116 cells in a time- and dose-dependent manner, which in turn, induced cell apoptosis. The observed apoptosis was characterized by morphological changes, DNA fragmentation, mitochondrial membrane potential decrease, S phase population increase, and caspase-3 and -9 activation. Furthermore, inhibition of c-Jun N-terminal kinase (JNK) by SP600125 led to a dramatic decrease of the GLP-induced apoptosis. Western blot analysis unveiled that GLP up-regulated the expression of Bax/Bcl-2, caspase-3 and poly (ADP-ribose) polymerase (PARP). These results demonstrate that apoptosis stimulated by GLP in human colorectal cancer cells is associated with activation of mitochondrial and mitogen-activated protein kinase (MAPK) pathways. Full article

(This article belongs to the Section Biochemistry)

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The UV spectra of polysaccharide fractions of Ganoderma lucidum. Full article ">
Infrared spectroscopy of Ganoderma lucidum polysaccharide (GLP). Full article ">
Cytotoxicity of GLP on HCT-116 cells: (A) GLP suppressed the cell viability of HCT-116. Inhibitory rate was measured by MTT method. Starch-incubated cells were applied as control. Data represent means ± SD of three independent experiments; and (B) Morphological changes in HCT-116 cells. After treatment with GLP, exfoliation of HCT-116 cells and naked areas were observed and captured under an inverted microscope (×100). The arrows (↑) show naked areas without cells. Full article ">
Effects of GLP on apoptosis in HCT-116 cells. (A) Apoptotic cells measured by Hoechst 33,258 staining after treatment with GLP for 48 h under a fluorescence microscope (×200). The arrows show cell fragments; (B) HCT-116 cells were exposed to the indicated concentrations of GLP for 24 h. DNA was isolated and examined on 1.2% agarose gel; (C) Mitochondrial membrane potential (ΔΨm) was monitored by microscopy and photographed at each concentration of GLP; and (D) quantitative evaluation. Data are presented as mean ± SD. a, p < 0.01 compared with control. b, c and d, p < 0.01 compared with 1.25, 2.5 and 5 mg/mL GLP treatment, respectively. Full article ">
Flow cytometry analysis of GLP-treated HCT-116 cells. Cells were incubated with GLP at various concentrations (1.25–10 mg/mL) for 24 h and then were harvested for quantifying apoptosis and cell cycle phase by flow cytometry (Propidium Iodide (PI) staining). Apoptosis and S phase cell populations of GLP-treated group are significantly higher than in the control. The blue peak = apoptosis; the first red peak = G1; the second red peak = G2; hatched peak = S. Full article ">
Effects of mitogen-activated protein kinase (MAPK) inhibitors on GLP-induced HCT-116 cell death. Cells were treated in the absence or presence of different MAPK-specific inhibitors, 1 h prior to the addition of GLP, and then incubated in 5 mg/mL GLP for 12 h. Cells were harvested to determine the percentage of viable cells as described in the Experimental section. A, p < 0.05 compared with only 5 mg/mL GLP treatment group. Data expressed as mean ± SD. Full article ">
GLP induces cell apoptosis via caspase-dependent mitochondrial pathways. (A) Activity of caspases in GLP treated HCT-116 cells. A, p < 0.05, and a, p < 0.01 compared with control. b, p < 0.01 compared with 2.5 mg/mL GLP treatment; (B) Western blot analyses of mitochondria pathway-related proteins. All bands were compared with the β-actin band and indicated as down-regulation (−) or up-regulation (+); and (C) Statistical analysis for western blot analysis. Full article ">
GLP induces cell apoptosis via caspase-dependent mitochondrial pathways. (A) Activity of caspases in GLP treated HCT-116 cells. A, p < 0.05, and a, p < 0.01 compared with control. b, p < 0.01 compared with 2.5 mg/mL GLP treatment; (B) Western blot analyses of mitochondria pathway-related proteins. All bands were compared with the β-actin band and indicated as down-regulation (−) or up-regulation (+); and (C) Statistical analysis for western blot analysis. Full article ">

1067 KiB

Open AccessArticle

Green Conversion of Agroindustrial Wastes into Chitin and Chitosan by Rhizopus arrhizus and Cunninghamella elegans Strains

by Lúcia Raquel Ramos Berger, Thayza Christina Montenegro Stamford, Thatiana Montenegro Stamford-Arnaud, Sergio Roberto Cabral De Alcântara, Antonio Cardoso Da Silva, Adamares Marques Da Silva, Aline Elesbão Do Nascimento and Galba Maria De Campos-Takaki

Int. J. Mol. Sci. 2014, 15(5), 9082-9102; https://doi.org/10.3390/ijms15059082 - 21 May 2014

Cited by 46 | Viewed by 7817

Abstract

This article sets out a method for producing chitin and chitosan by Cunninghamella elegans and Rhizopus arrhizus strains using a green metabolic conversion of agroindustrial wastes (corn steep liquor and molasses). The physicochemical characteristics of the biopolymers and antimicrobial activity are described. Chitin [...] Read more.

This article sets out a method for producing chitin and chitosan by Cunninghamella elegans and Rhizopus arrhizus strains using a green metabolic conversion of agroindustrial wastes (corn steep liquor and molasses). The physicochemical characteristics of the biopolymers and antimicrobial activity are described. Chitin and chitosan were extracted by alkali-acid treatment, and characterized by infrared spectroscopy, viscosity and X-ray diffraction. The effectiveness of chitosan from C. elegans and R. arrhizus in inhibiting the growth of Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica, Escherichia coli and Yersinia enterocolitica were evaluated by determining the minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC). The highest production of biomass (24.60 g/L), chitin (83.20 mg/g) and chitosan (49.31 mg/g) was obtained by R. arrhizus. Chitin and chitosan from both fungi showed a similar degree of deacetylation, respectively of 25% and 82%, crystallinity indices of 33.80% and 32.80% for chitin, and 20.30% and 17.80% for chitosan. Both chitin and chitosan presented similar viscosimetry of 3.79–3.40 cP and low molecular weight of 5.08 × 10³ and 4.68 × 10³ g/mol. They both showed identical MIC and MBC for all bacteria assayed. These results suggest that: agricultural wastes can be produced in an environmentally friendly way; chitin and chitosan can be produced economically; and that chitosan has antimicrobial potential against pathogenic bacteria. Full article

(This article belongs to the Section Green Chemistry)

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Pareto charts showing the effect of the independent variables, corn steep liquor and molasses, on the biomass production by R. arrhizus (A) and C. elegans (B). Full article ">
Pareto charts showing the effect of the independent variables, corn steep liquor and molasses, on the chitin yield by R. arrhizus (A) and C. elegans (B). Full article ">
Pareto charts showing the effect of the independent variables, corn steep liquor and molasses, on the chitosan yield by R. arrhizus (A) and C. elegans (B). Full article ">
Surface response to chitosan production by Rhizopus arrhizus related to interaction of molasses and corn steep liquor. Full article ">
Surface response to chitosan production by Cunninghamella elegans related to interaction of molasses and corn steep liquor substrates. Full article ">
Infrared absorption spectra: (A) Chitosan produced by microbiological R. arrhizus Assay 7; (B) Commercial chitosan (Sigma Aldrich Corp., St. Louis, MO, USA); (C) Chitin produced by microbiological R. arrhizus Assay 2; (D) Commercial chitin (Sigma). Full article ">
X-ray diffractograms of chitin (A,B) and chitosan (C,D) obtained from C. elegans and R. arrhizus biomass, respectively. Full article ">
Scanning electron microscopy (SEM) photographs of chitin (A) and chitosan (B) produced by R. arrhizus and chitosan (C) and chitin (D) produced by C. elegans, in Assays 1 and 2, at 500× and 1000× magnification. The measurement bar = 50 μM. Full article ">

2224 KiB

Open AccessArticle

A pH and Redox Dual Responsive 4-Arm Poly(ethylene glycol)-block-poly(disulfide histamine) Copolymer for Non-Viral Gene Transfection in Vitro and in Vivo

by Kangkang An, Peng Zhao, Chao Lin and Hongwei Liu

Int. J. Mol. Sci. 2014, 15(5), 9067-9081; https://doi.org/10.3390/ijms15059067 - 21 May 2014

Cited by 18 | Viewed by 8568

Abstract

A novel 4-arm poly(ethylene glycol)-b-poly(disulfide histamine) copolymer was synthesized by Michael addition reaction of poly(ethylene glycol) (PEG) vinyl sulfone and amine-capped poly(disulfide histamine) oligomer, being denoted as 4-arm PEG-SSPHIS. This copolymer was able to condense DNA into nanoscale polyplexes (<200 nm in average [...] Read more.

A novel 4-arm poly(ethylene glycol)-b-poly(disulfide histamine) copolymer was synthesized by Michael addition reaction of poly(ethylene glycol) (PEG) vinyl sulfone and amine-capped poly(disulfide histamine) oligomer, being denoted as 4-arm PEG-SSPHIS. This copolymer was able to condense DNA into nanoscale polyplexes (<200 nm in average diameter) with almost neutral surface charge (+(5–10) mV). Besides, these polyplexes were colloidal stable within 4 h in HEPES buffer saline at pH 7.4 (physiological environment), but rapidly dissociated to liberate DNA in the presence of 10 mM glutathione (intracellular reducing environment). The polyplexes also revealed pH-responsive surface charges which markedly increased with reducing pH values from 7.4–6.3 (tumor microenvironment). In vitro transfection experiments showed that polyplexes of 4-arm PEG-SSPHIS were capable of exerting enhanced transfection efficacy in MCF-7 and HepG2 cancer cells under acidic conditions (pH 6.3–7.0). Moreover, intravenous administration of the polyplexes to nude mice bearing HepG2-tumor yielded high transgene expression largely in tumor rather other normal organs. Importantly, this copolymer and its polyplexes had low cytotoxicity against the cells in vitro and caused no death of the mice. The results of this study indicate that 4-arm PEG-SSPHIS has high potential as a dual responsive gene delivery vector for cancer gene therapy. Full article

(This article belongs to the Special Issue Biodegradable Materials)

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(a) Synthesis of poly(disulfide histamine) oligomer via Michael addition reaction of cystamine bisacrylamide and an excess amount of histamine; (b) Preparation of 4-arm PEG-b-poly(disulfide histamine) copolymer (denoted as 4-arm PEG-SSPHIS). Full article ">
(a) 1H NMR spectra (D2O, 300 MHz) of 4-arm PEG-SSPHIS; (b) GPC chromatogram of 4-arm PEG-SSPHIS, 4-arm PEG and SSPHIS oligomer. Full article ">
(a) Argarose gel retardation assay on binding behavior of 4-arm PEG-SSPHIS and plasmid DNA with (−GSH) or without (+GSH) of glutathione (GSH); (b) Particle size of the polyplexes of 4-arm PEG-SSPHIS at different polymer/DNA ratios as a function of incubation time in the presence of 130 mM NaCl; (c) Typical TEM image of polyplexes of 4-arm PEG-SSPHIS at the mass ratio of 12/1 (scale bar: 100 nm); (d) Zeta potential of the polyplexes of 4-arm PEG-SSPHIS at different polymer/DNA ratios; (e) Effect of pH values on size and zeta potential of the polyplexes of 4-arm PEG-SSPHIS at the mass ratio of 24/1. Full article ">
(a) Cytotoxicity of 4-arm PEG-SSPHIS copolymer (and 25 kDa PEI as a control) against COS-7 cells at different polymer concentrations; (b) Transfection efficiency and cytotoxicity of 4-arm PEG-SSPHIS in COS-7 cells at 48 h after 4 h co-incubation with the polyplexes of 4-arm PEG-SSPHIS at the mass ratio of 24/1 (2 μg DNA) in the absence of serum and then 44 h-post transfection. Polyplexes of 25 kDa PEI at an optimal ratio of 1/1 was used as a control; (c,d) Transfection efficiency (c) and cytotoxicity (d) of 4-arm PEG-SSPHIS in COS-7 and NIH 3T3 cells at 48 h after 4 h co-incubation with the polyplexes of 4-arm PEG-SSPHIS at the mass ratio of 24/1 (8 μg DNA) in the presence of serum and then 44 h-post transfection. Polyplexes of 25 kDa PEI at an optimal ratio of 1/1 was used as a control. Full article ">
(a) Transfection efficiency and cytotoxicity of 4-arm PEG-SSPHIS in MCF-7 cells as a function of DNA dose (2–8 μg); (b,c) Transfection efficiency and cytotoxicity of 4-arm PEG-SSPHIS using 8 μg DNA against MCF-7 (b) and HepG2 (c) cells as a function of pH values. The cells were detected at 48 h after 4 h transfection with the polyplexes of 4-arm PEG-SSPHIS in the absence of serum and 44 h-post transfection. Polyplexes of 25 kDa PEI at the mass ratio of 1/1 was used as a control. * p < 0.05, ** p < 0.01, *** p < 0.001; (d) Typical images of GFP+ MCF-7 cells after transfection at varying pH values (scale bar: 50 nm); (e) Effect of FBS on the transfection efficiency of 4-arm PEG-SSPHIS with 4 μg of DNA; (f) Effect of 100 μM chloroquine (CQ) on the transfection efficiency of 4-arm PEG-SSPHIS with 4 μg of DNA. ** p < 0.01. All the polyplexes were prepared at the mass ratio of 12/1. Full article ">
Luciferase expression in the tumor and organs of nude mice after intravenous injection of 4-arm PEG-SSPHIS-based polyplexes (30 μg DNA, at mass ratio of 12/1). The expression was presented as RLU/mg protein (n = 4). A formulation of PEI at an optimal mass ratio of 1/1 was used as a control. * p < 0.05, *** p < 0.001. Full article ">
Schematic illustration of pH-targeting gene delivery towards tumor cells with 4-arm PEG-SSPHIS copolymer as a pH and redox dual responsive gene delivery vector: (a) gene binding of 4-arm PEG-SSPHIS copolymer to form the polyplexes with almost neutral surface; (b) increased surface charge upon the polyplexes in acidic tumor microenvironment via protonation of imidazole groups; (c) enhanced cellular uptake of the polyplexes to tumor cells; (d) intracellular gene release after disulfide cleavage. Full article ">

868 KiB

Open AccessArticle

Detection of a Specific Biomarker for Epstein-Barr Virus Using a Polymer-Based Genosensor

by Renata P. A. Balvedi, Ana C. H. Castro, João M. Madurro and Ana G. Brito-Madurro

Int. J. Mol. Sci. 2014, 15(5), 9051-9066; https://doi.org/10.3390/ijms15059051 - 21 May 2014

Cited by 25 | Viewed by 6298

Abstract

This paper describes methodology for direct and indirect detections of a specific oligonucleotide for Epstein-Barr virus (EBV) using electrochemical techniques. The sequence of oligonucleotide probe (EBV1) revealed a high sequence identity (100%) with the EBV genome. For the development of the genosensor, EBV1 [...] Read more.

This paper describes methodology for direct and indirect detections of a specific oligonucleotide for Epstein-Barr virus (EBV) using electrochemical techniques. The sequence of oligonucleotide probe (EBV1) revealed a high sequence identity (100%) with the EBV genome. For the development of the genosensor, EBV1 was grafted to the platform sensitized with poly(4-aminothiophenol). After that, the hybridization reaction was carried out with the complementary target (EBV2) on the modified electrode surface using ethidium bromide as DNA intercalator. The oxidation peak currents of ethidium bromide increased linearly with the values of the concentration of the complementary sequences in the range from 3.78 to 756 µmol·L⁻¹. In nonstringent experimental conditions, this genosensor can detect 17.32 nmol·L⁻¹ (three independent experiments) of oligonucleotide target, discriminating between complementary and non-complementary oligonucleotides, as well as differentiating one-base mismatch, as required for detection of genetic diseases caused by point mutations. The biosensor also displayed high specificity to the EBV target with elimination of interference from mix (alanine, glucose, uric acid, ascorbic acid, bovine serum albumin (BSA), glutamate and glycine) and good stability (120 days). In addition, it was possible to observe differences between hybridized and non-hybridized surfaces through atomic force microscopy. Full article

(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)

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(A) Cyclic voltammogram in 4-aminotiophenol (4-ATP) solution (15 mmol·L−1) onto graphite electrode. Supporting electrolyte: H2SO4 0.5 mol·L−1; Scan rate 50 mV·s−1; 100 scans. The arrows indicate the influence of the current response with the increasing of the number of scans; and (B) Cyclic voltammograms of bare graphite electrode (a) and graphite electrode functionalized with poly(4-ATP) (b). Supporting electrolyte: H2SO4 0.5 mol·L−1; Scan rate: 50 mV·s−1. Full article ">
Cyclic voltammograms of bare graphite and graphite electrode modified with poly(4-ATP): (A) in aqueous solution containing K4Fe(CN)6/K3Fe(CN)6 (5.00 mmol·L−1) and KCl (0.10 mol·L−1); (B) in aqueous solution containing Ru(NH3)6Cl2 (5.00 mmol·L−1) and KCl (0.10 mol·L−1). Bare graphite electrode (a) and modified graphite electrode with 4-ATP (b) Scan rate: 50 mV·s−1. Full article ">
Differential pulse voltammograms of graphite electrode modified with poly(4-ATP) prepared in pH 0.5 (baseline-corrected), 100 scans, containing [EBV1, (oligonucleotide probe) 126 μmol·L−1]: before hybridization (a) and after 20 min of incubation with complementary target (EBV2, 378 μmol·L−1) (b). Electrolyte: phosphate buffer (0.10 mol·L−1), pH 7.4. Modulation amplitude: 0.05 mV. Pulse interval: 0.2 s. Scan rate 5 mV·s−1. Full article ">
Differential pulse voltammograms of ethidium bromide (1 × 10−6 mol·L−1) onto graphite electrode modified with poly(4-ATP) prepared in pH 0.5 (baseline-corrected), 100 scans, containing EBV1/probe (126 μmol·L−1) before hybridization (a) and after hybridization with: non-complementary target (189 μmol·L−1) (b); oligonucleotide containing one-base mismatch EBV2Mis1 (378 μmol·L−1) (c); and complementary target (EBV2, 378 μmol·L−1) (d). Electrolyte: phosphate buffer (0.10 mol·L−1), pH 7.4. Modulation amplitude: 25 mV. Pulse interval: 0.2 s; Scan rate 20 mV·s−1. Inset: Bar chart of differential pulse voltammograms responses using the oxidation signal from ethidium bromide. Full article ">
Selectivity coefficient for graphite electrode/poly(4-ATP)/EBV1 in the detection of complementary target in absence or presence of the interfering compounds: uric acid (UA) 1 mg·dL−1; ascorbic acid (AA) 3.6 mg·dL−1; glycine (Gly) 1 mmol·L−1; alanine (Ala) 1 mmol·L−1; glucose (Glu) 1 mmol·L−1; bovine serum albumin (BSA) 5 g·dL−1; glutamate (Glut) 1 mmol·L−1 and mixture. Electrolyte: phosphate buffer (0.10 mol·L−1), pH 7.4. Modulation amplitude: 25 mV. Pulse interval: 0.2 s; Scan rate 20 mV·s−1. Ethidium bromide (1 × 10−6 mol·L−1) was used as indicator of the hybridization. Full article ">
Electrochemical response for the oxidation signal of ethidium bromide (1 × 10−6 mol·L−1) obtained after the hybridization of modified electrode containing the probe EBV1 (126 μmol·L−1) with different concentrations of EBV2 (0, 0.0010, 0.010, 0.10, 1.89, 3.78, 37.8, 378 and 756 μmol·L−1). Inset shows linear range of current peak vs. concentration of EBV2. Full article ">
Storage stability profile of genosensor at 8 °C. The biosensors were stored in refrigerators when not in use. Full article ">
Atomic force microscopy (AFM) images of (A) graphite; (B) graphite/poly (4-ATP); (C) graphite/poly(4-ATP)/EBV1; and (D) graphite/poly(4-ATP)/EBV1:EBV2. Full article ">

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Open AccessArticle

Over-Expression of Catalase in Myeloid Cells Confers Acute Protection Following Myocardial Infarction

by E. Bernadette Cabigas, Inthirai Somasuntharam, Milton E. Brown, Pao Lin Che, Karl D. Pendergrass, Bryce Chiang, W. Robert Taylor and Michael E. Davis

Int. J. Mol. Sci. 2014, 15(5), 9036-9050; https://doi.org/10.3390/ijms15059036 - 21 May 2014

Cited by 10 | Viewed by 6366

Abstract

Cardiovascular disease is the leading cause of death in the United States and new treatment options are greatly needed. Oxidative stress is increased following myocardial infarction and levels of antioxidants decrease, causing imbalance that leads to dysfunction. Therapy involving catalase, the endogenous scavenger [...] Read more.

Cardiovascular disease is the leading cause of death in the United States and new treatment options are greatly needed. Oxidative stress is increased following myocardial infarction and levels of antioxidants decrease, causing imbalance that leads to dysfunction. Therapy involving catalase, the endogenous scavenger of hydrogen peroxide (H₂O₂), has been met with mixed results. When over-expressed in cardiomyocytes from birth, catalase improves function following injury. When expressed in the same cells in an inducible manner, catalase showed a time-dependent response with no acute benefit, but a chronic benefit due to altered remodeling. In myeloid cells, catalase over-expression reduced angiogenesis during hindlimb ischemia and prevented monocyte migration. In the present study, due to the large inflammatory response following infarction, we examined myeloid-specific catalase over-expression on post-infarct healing. We found a significant increase in catalase levels following infarction that led to a decrease in H₂O₂ levels, leading to improved acute function. This increase in function could be attributed to reduced infarct size and improved angiogenesis. Despite these initial improvements, there was no improvement in chronic function, likely due to increased fibrosis. These data combined with what has been previously shown underscore the need for temporal, cell-specific catalase delivery as a potential therapeutic option. Full article

(This article belongs to the Special Issue Oxidative Stress in Cardiovascular Disease 2015)

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3835 KiB

Open AccessArticle

Mangiferin Facilitates Islet Regeneration and β-Cell Proliferation through Upregulation of Cell Cycle and β-Cell Regeneration Regulators

by Hai-Lian Wang, Chun-Yang Li, Bin Zhang, Yuan-De Liu, Bang-Min Lu, Zheng Shi, Na An, Liang-Kai Zhao, Jing-Jing Zhang, Jin-Ku Bao and Yi Wang

Int. J. Mol. Sci. 2014, 15(5), 9016-9035; https://doi.org/10.3390/ijms15059016 - 20 May 2014

Cited by 47 | Viewed by 7922

Abstract

Mangiferin, a xanthonoid found in plants including mangoes and iris unguicularis, was suggested in previous studies to have anti-hyperglycemic function, though the underlying mechanisms are largely unknown. This study was designed to determine the therapeutic effect of mangiferin by the regeneration of β-cells [...] Read more.

Mangiferin, a xanthonoid found in plants including mangoes and iris unguicularis, was suggested in previous studies to have anti-hyperglycemic function, though the underlying mechanisms are largely unknown. This study was designed to determine the therapeutic effect of mangiferin by the regeneration of β-cells in mice following 70% partial pancreatectomy (PPx), and to explore the mechanisms of mangiferin-induced β-cell proliferation. For this purpose, adult C57BL/6J mice after 7–14 days post-PPx, or a sham operation were subjected to mangiferin (30 and 90 mg/kg body weight) or control solvent injection. Mangiferin-treated mice exhibited an improved glycemia and glucose tolerance, increased serum insulin levels, enhanced β-cell hyperplasia, elevated β-cell proliferation and reduced β-cell apoptosis. Further dissection at the molecular level showed several key regulators of cell cycle, such as cyclin D1, D2 and cyclin-dependent kinase 4 (Cdk4) were significantly up-regulated in mangiferin-treated mice. In addition, critical genes related to β-cell regeneration, such as pancreatic and duodenal homeobox 1 (PDX-1), neurogenin 3 (Ngn3), glucose transporter 2 (GLUT-2), Forkhead box protein O1 (Foxo-1), and glucokinase (GCK), were found to be promoted by mangiferin at both the mRNA and protein expression level. Thus, mangiferin administration markedly facilitates β-cell proliferation and islet regeneration, likely by regulating essential genes in the cell cycle and the process of islet regeneration. These effects therefore suggest that mangiferin bears a therapeutic potential in preventing and/or treating the diabetes. Full article

(This article belongs to the Special Issue Nutritional Control of Metabolism)

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Open AccessArticle

Effect of a Novel Quaternary Ammonium Methacrylate Polymer (QAMP) on Adhesion and Antibacterial Properties of Dental Adhesives

by Yasmine M. Pupo, Paulo Vitor Farago, Jessica M. Nadal, Luzia C. Simão, Luís Antônio Esmerino, Osnara M. M. Gomes and João Carlos Gomes

Int. J. Mol. Sci. 2014, 15(5), 8998-9015; https://doi.org/10.3390/ijms15058998 - 20 May 2014

Cited by 23 | Viewed by 8694

Abstract

This study investigated the resin–dentin bond strength (μTBS), degree of conversion (DC), and antibacterial potential of an innovative adhesive system containing a quaternary ammonium methacrylate polymer (QAMP) using in situ and in vitro assays. Forty-two human third molars were flattened until the dentin [...] Read more.

This study investigated the resin–dentin bond strength (μTBS), degree of conversion (DC), and antibacterial potential of an innovative adhesive system containing a quaternary ammonium methacrylate polymer (QAMP) using in situ and in vitro assays. Forty-two human third molars were flattened until the dentin was exposed and were randomly distributed into three groups of self-etching adhesive systems: Clearfil™ SE Bond containing 5% QAMP (experimental group), Clearfil™ Protect Bond (positive control) and Clearfil™ SE Bond (negative control). After light curing, three 1 mm-increments of composite resin were bonded to each dentin surface. A total of thirty of these bonded teeth (10 teeth per group) was sectioned to obtain stick-shaped specimens and tested under tensile stress immediately, and after 6 and 12 months of storage in distilled water. Twelve bonded teeth (4 teeth per group) were longitudinally sectioned in a mesio-to-distal direction to obtain resin-bonded dentin slabs. In situ DC was evaluated by micro-Raman spectroscopy. In vitro DC of thin films of each adhesive system was measured using Fourier transform infrared spectroscopy. In vitro susceptibility tests of these three adhesive systems were performed by the minimum inhibitory/minimum bactericidal concentration (MIC/MBC) assays against Streptococcus mutans, Lactobacillus casei, and Actinomyces naeslundii. No statistically significant difference in μTBS was observed between Clearfil™ SE Bond containing 5% QAMP and Clearfil™ SE Bond (p > 0.05) immediately, and after 6 and 12 months of water storage. However Clearfil™ Protect Bond showed a significant reduction of μTBS after 12 months of storage (p = 0.039). In addition, QAMP provided no significant change in DC after incorporating into Clearfil™ SE Bond (p > 0.05). Clearfil™ SE Bond containing 5% QAMP demonstrated MIC/MBC values similar to the positive control against L. casei and A. naeslundii and higher than the negative control for all evaluated bacterial strains. The use of QAMP in an adhesive system demonstrated effective bond strength, a suitable degree of conversion, and adequate antibacterial effects against oral bacteria, and may be useful as a new approach to provide long-lasting results for dental adhesives. Full article

(This article belongs to the Special Issue Antimicrobial Polymers)

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Graphical abstract

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Chemical structure of the quaternary ammonium methacrylate polymer (QAMP) [Brazil patent application number 10 2012 0266 4] [<a href="#b28-ijms-15-08998" class="html-bibr">28</a>]. Full article ">
Raman spectra of QAMP and raw materials (Eudragit™ E100 and n-octyl bromide). (a1) Raman band at 3030 cm−1 as a broadening of C–H alkyl stretching bands; (a2) A novel band attributed to the symmetric stretching vibration of octyl dimethyl ammonium substituent was verified at 726 cm−1; (b) The C–Br stretching vibration presented in n-octyl bromide between 560 and 650 cm−1 disappeared in QAMP; and (c) Raman bands associated with dimethylamino groups of Eudragit™ E100, previously observed at 2772 and 2821 cm−1 also disappeared. Full article ">
Micro-Raman spectras of uncured and cured adhesive systems Clearfil™ SE Bond containing 5% QAMP, Clearfil™ Protect Bond, Clearfil™ SE Bond. The reference band is shown at 1609 cm−1 and the reactive band related to C=C group is indicated at 1639 cm−1 (arrows). This band decreases during polymerization and was used for calculating the degree of conversion. Full article ">
Micro-Raman spectras of uncured and cured adhesive systems Clearfil™ SE Bond containing 5% QAMP, Clearfil™ Protect Bond, Clearfil™ SE Bond. The reference band is shown at 1609 cm−1 and the reactive band related to C=C group is indicated at 1639 cm−1 (arrows). This band decreases during polymerization and was used for calculating the degree of conversion. Full article ">

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Open AccessArticle

Transition from Cyclosporine-Induced Renal Dysfunction to Nephrotoxicity in an in Vivo Rat Model

by José Sereno, Paulo Rodrigues-Santos, Helena Vala, Petronila Rocha-Pereira, Rui Alves, João Fernandes, Alice Santos-Silva, Eugénia Carvalho, Frederico Teixeira and Flávio Reis

Int. J. Mol. Sci. 2014, 15(5), 8979-8997; https://doi.org/10.3390/ijms15058979 - 20 May 2014

Cited by 24 | Viewed by 8496

Abstract

Cyclosporin A (CsA), a calcineurin inhibitor, remain the cornerstone of immunosuppressive regimens, regardless of nephrotoxicity, which depends on the duration of drug exposure. The mechanisms and biomarkers underlying the transition from CsA-induced renal dysfunction to nephrotoxicity deserve better elucidation, and would help clinical [...] Read more.

Cyclosporin A (CsA), a calcineurin inhibitor, remain the cornerstone of immunosuppressive regimens, regardless of nephrotoxicity, which depends on the duration of drug exposure. The mechanisms and biomarkers underlying the transition from CsA-induced renal dysfunction to nephrotoxicity deserve better elucidation, and would help clinical decisions. This study aimed to clarify these issues, using a rat model of short- and long-term CsA (5 mg/kg bw/day) treatments (3 and 9 weeks, respectively). Renal function was assessed on serum and urine; kidney tissue was used for histopathological characterization and gene and/or protein expression of markers of proliferation, fibrosis and inflammation. In the short-term, creatinine and blood urea nitrogen (BUN) levels increased and clearances decreased, accompanied by glomerular filtration rate (GFR) reduction, but without kidney lesions; at that stage, CsA exposure induced proliferating cell nuclear antigen (PCNA), transforming growth factor beta 1 (TGF-β1), factor nuclear kappa B (NF-κβ) and Tumor Protein P53 (TP53) kidney mRNA up-regulation. In the long-term treatment, renal dysfunction data was accompanied by glomerular and tubulointerstitial lesions, with remarkable kidney mRNA up-regulation of the mammalian target of rapamycin (mTOR) and the antigen identified by monoclonal antibody Ki-67 (Mki67), accompanied by mTOR protein overexpression. Transition from CsA-induced renal dysfunction to nephrotoxicity is accompanied by modification of molecular mechanisms and biomarkers, being mTOR one of the key players for kidney lesion evolution, thus suggesting, by mean of molecular evidences, that early CsA replacement by mTOR inhibitors is indeed the better therapeutic choice to prevent chronic allograft nephropathy. Full article

(This article belongs to the Special Issue Renal Toxicology—Epidemiology and Mechanisms)

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Serum, urine and kidney markers of renal function. Creatinine serum levels (A) and clearance (B); blood urea nitrogen (BUN) levels (C) and clearance (D); kidney lipid peroxidation evaluated by the malondyaldehyde content (E); malondyaldehyde clearance (F) throughout the short- and long-term Cyclosporin A (CsA) treatments. Values are mean ± SEM. * p < 0.05 and ** p < 0.01 vs. the Control group. Full article ">
Kidney lesions. Representative photomicrographs of kidney histological sections stained with periodic acid of Schiff (PAS), for Control (A,F,K) and CsA (B,G,L) groups in the long-term CsA treatment model; (A) represents a normal kidney arteriole from the control group and (B) an arteriolosclerosis lesion present in all the rats treated with CsA, indicated by the two arrows; (F,G) represent a normal capsule and a thickening Bowman’s capsule (black arrow) from the CsA group, respectively; (K,L) show a normal tubules and tubular calcification (black arrows) in control and CsA-treated rats, respectively; (C,H,M) represent the index of each kidney lesion for the Control and CsA groups; Representative photomicrographs of kidney histomorphologic sections with Masson’s trichrome staining for Control (D,I,N) and CsA (E,J,O) groups in the long-term CsA treatment model, showing the pattern of collagen deposition (blue color); (D,I,N) show normal vascular, glomerular and tubulointerstitial regions of the control rats; (E) presents an arteriolosclerosis lesion and collagen deposition around vessels (black arrows) CsA-treated rats; (J,O) show collagen deposition around Bowman’s capsule and tubules (black arrows) and tubule-interstitial fibrosis (black arrows) from the CsA-treated group, respectively. Each bar represents 25 μm. Full article ">
Kidney gene (mRNA) expression of markers of proliferation. PCNA (A); TGF-β1 (B); TP53 (C); NF-κB (D); mTOR (E) and Mki67 (F) for the control and CsA treatments, in the short- and long-term models. Values are mean of % of the control ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the Control group of each model; ## p < 0.01 and ### p < 0.001 vs. the CsA-acute model. Mki67, antigen identified by monoclonal antibody Ki-67; mTOR, mammalian target of rapamycin; NF-κB1, nuclear factor kappa B; PCNA, proliferating cell nuclear antigen; TGF-β1, transforming growth factor beta 1; TP53, tumor protein p53. Full article ">
Kidney gene (mRNA) expression of markers of inflammation and angiogenesis. COX-1 (A); VEGF (B); CRP (C); COX-2 (D); TNF-α (E) and IL-1β (F) for the control and CsA treatments, in the short- and long-term models. Values are mean of % of the control ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the Control group of each model; # p < 0.05 and ## p < 0.01 vs. the CsA-acute model. COX-1, ciclooxigenase-1; COX-2, ciclooxigenase-2; CRP, C-reactive protein; IL-1β, interleukin 1 beta; TNF-α, tumor necrosis factor alpha. Full article ">
Kidney mTOR protein expression. Immunohistochemistry pictures from control (A1,B1) and CsA-treated (A2,B2) rats, after short- and long-term treatments (3 and 9 weeks, respectively), and staining area and intensity in the glomerular (A3,B3) and tubule-interstitial region (A4,B4). Each bar represents 50 μm. Full article ">
Kidney mTOR protein expression. Immunohistochemistry pictures from control (A1,B1) and CsA-treated (A2,B2) rats, after short- and long-term treatments (3 and 9 weeks, respectively), and staining area and intensity in the glomerular (A3,B3) and tubule-interstitial region (A4,B4). Each bar represents 50 μm. Full article ">

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Open AccessArticle

Genes and Gene Networks Involved in Sodium Fluoride-Elicited Cell Death Accompanying Endoplasmic Reticulum Stress in Oral Epithelial Cells

by Yoshiaki Tabuchi, Tatsuya Yunoki, Nobuhiko Hoshi, Nobuo Suzuki and Takashi Kondo

Int. J. Mol. Sci. 2014, 15(5), 8959-8978; https://doi.org/10.3390/ijms15058959 - 20 May 2014

Cited by 21 | Viewed by 7166

Abstract

Here, to understand the molecular mechanisms underlying cell death induced by sodium fluoride (NaF), we analyzed gene expression patterns in rat oral epithelial ROE2 cells exposed to NaF using global-scale microarrays and bioinformatics tools. A relatively high concentration of NaF (2 mM) induced [...] Read more.

Here, to understand the molecular mechanisms underlying cell death induced by sodium fluoride (NaF), we analyzed gene expression patterns in rat oral epithelial ROE2 cells exposed to NaF using global-scale microarrays and bioinformatics tools. A relatively high concentration of NaF (2 mM) induced cell death concomitant with decreases in mitochondrial membrane potential, chromatin condensation and caspase-3 activation. Using 980 probe sets, we identified 432 up-regulated and 548 down-regulated genes, that were differentially expressed by >2.5-fold in the cells treated with 2 mM of NaF and categorized them into 4 groups by K-means clustering. Ingenuity^® pathway analysis revealed several gene networks from gene clusters. The gene networks Up-I and Up-II included many up-regulated genes that were mainly associated with the biological function of induction or prevention of cell death, respectively, such as Atf3, Ddit3 and Fos (for Up-I) and Atf4 and Hspa5 (for Up-II). Interestingly, knockdown of Ddit3 and Hspa5 significantly increased and decreased the number of viable cells, respectively. Moreover, several endoplasmic reticulum (ER) stress-related genes including, Ddit3, Atf4 and Hapa5, were observed in these gene networks. These findings will provide further insight into the molecular mechanisms of NaF-induced cell death accompanying ER stress in oral epithelial cells. Full article

(This article belongs to the Section Molecular Toxicology)

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Effects of sodium fluoride (NaF) on cell number and cell viability in rat oral epithelial ROE2 cells. Cells were incubated with NaF at concentrations of 0 to 4 mM for 24 h. (A) Cell number was counted using a hematocytometer. The data represent the means ± standard deviations (N = 4); and (B) Cell viability was monitored using a WST-8 assay. Cells treated with 0 mM NaF served as a control (100%). The data represent the means ± standard deviations (N = 8). * p < 0.05 vs. NaF (0 mM) (Student’s t-test). Full article ">
Effects of NaF on protein content and chromatin condensation in ROE2 cells. Cells were incubated with NaF at concentrations of 0 to 4 mM for 24 h. (A) The protein contents in cells were estimated by using a Pierce® bicinchoninic acid (BCA) Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA); and (B) Chromatin condensation was measured using a Nuclear-ID Green Chromatin Condensation Kit (Enzo Life Sciences Inc., Farmingdale, NY, USA). The data represent the means ± standard deviations (N = 3).* p < 0.05 vs. NaF (0 mM) (Student’s t-test). Full article ">
Effects of NaF on mitochondrial membrane potential (MMP) in ROE2 cells. Cells were cultured with or without 2 mM of NaF for 6 h. MMP was measured by JC-1, an indicator of mitochondrial function. (A,D) monomeric JC-1 green fluorescence; (B,E) aggregate JC-1 red fluorescence; and (C,F) merge images. (A–C) control cells (Ctr.); and (D–F) NaF-treated cells. Ctr., control. Scale Bar, 200 μm. Full article ">
Effects of NaF on caspase-3 cleavage in ROE2 cells. Cells were cultured with or without 2 mM of NaF for 3 to 24 h. Western blot analysis was performed with anti-caspase-3 antibody, which reacted with procaspase-3 and cleaved caspase-3 (A); anti-cleaved caspase-3 antibody (B) and anti-Gapdh antibody. Gapdh served as a loading control. Ctr., control. Full article ">
Cluster analysis of genes that were differentially expressed. ROE2 cells were cultured with or without 2 mM of NaF for 3 to 12 h. K-means clustering of the probe sets that were differentially expressed by a factor of 2.5 or more was carried out using the GeneSpring® GX software (Agilent Technologies Inc., Santa Clara, CA, USA). Expression levels are shown. Non-treated cells served as a control (Ctr.). The figures in the parentheses indicate probe sets numbers. (A) cluster Up-I; (B) cluster Up-II; (C) cluster Down-I; and (D) cluster Down-II. Full article ">
A gene network Up-I. Genes that were up-regulated in the cluster Up-I were analyzed by the Ingenuity® Pathway analysis software. The network is displayed graphically as nodes (genes) and edges (the biological relationships between the nodes). Nodes and edges are displayed as various shapes and labels that present the functional class of genes and the nature of the relationship between the nodes, respectively. Full article ">
A gene network Up-II. Genes that were up-regulated in the cluster Up-II were analyzed by Ingenuity® Pathway analysis software. For an explanation of the symbols and letters, see <a href="#f6-ijms-15-08959" class="html-fig">Figure 6</a>. Full article ">
Verification of microarray results with real-time quantitative polymerase chain reaction (qPCR). Cells were incubated with or without NaF (2 mM) for 0 to 12 h. Real-time qPCR was performed. (A) Atf3; (B) Atf4; (C) Ddit3; (D) Fos; and (E) Hspa5. The data represent means ± standard deviations from 4 different experiments. Each expression level was normalized by Gapdh. Open circles, 0 mM NaF; closed circles, 2 mM NaF. * p < 0.05 vs. each NaF (0 mM) (Student’s t-test). Full article ">
Effects of NaF on protein expressions for Hspa5 and Ddit3 in ROE2 cells. Cells were cultured with or without 2 mM of NaF for 3 to 24 h. Western blot analysis was performed with the specific primary antibodies for Hspa5 (A) and Ddit3 (B). Gapdh served as a loading control. Ctr., control (non-treated cells). Full article ">

384 KiB

Open AccessArticle

Synthesis and Bioactivity of 5-Substituted-2-furoyl Diacylhydazide Derivatives with Aliphatic Chain

by Zining Cui, Xinghai Li, Fang Tian and Xiaojing Yan

Int. J. Mol. Sci. 2014, 15(5), 8941-8958; https://doi.org/10.3390/ijms15058941 - 20 May 2014

Cited by 12 | Viewed by 5248

Abstract

A series of 5-substituted-2-furoyl diacylhydazide derivatives with aliphatic chain were designed and synthesized. Their structures were characterized by IR, ¹H NMR, elemental analysis, and X-ray single crystal diffraction. The anti-tumor bioassay revealed that some title compounds exhibited promising activity against the selected [...] Read more.

A series of 5-substituted-2-furoyl diacylhydazide derivatives with aliphatic chain were designed and synthesized. Their structures were characterized by IR, ¹H NMR, elemental analysis, and X-ray single crystal diffraction. The anti-tumor bioassay revealed that some title compounds exhibited promising activity against the selected cancer cell lines, especially against the human promyelocytic leukemic cells (HL-60). Their fungicidal tests indicated that most of the title compounds showed significant anti-fungal activity. The preliminary structure-activity relationship showed that the aliphatic chain length and differences in the R² group had obvious effects on the anti-tumor and anti-fungal activities. The bioassay results demonstrated that the title compounds hold great promise as novel lead compounds for further drug discovery. Full article

(This article belongs to the Section Materials Science)

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Molecular structure of compound III-3-2, showing 30% probability ellipsoids; H atoms are shown as small spheres of arbitrary radii. Full article ">
Design strategy for title compounds. Full article ">
General synthesis procedure for title compounds III. III-1-1: R1 = CH3, R2 = 2-Cl; III-2-1: R1 = CH3, R2 = 3-Cl; III-3-1: R1 = CH3, R2 = 4-Cl; III-4-1: R1 = CH3, R2 = 2-F; III-5-1: R1 = CH3, R2 = 3-F; III-6-1: R1 = CH3, R2 = 4-F; III-7-1: R1 = CH3, R2 = 2,4-di-F; III-8-1: R1 = CH3, R2 = 2,6-di-F; III-9-1: R1 = CH3, R2 = 2-NO2; III-10-1: R1 = CH3, R2 = 3-NO2; III-11-1: R1 = CH3, R2 = 4-NO2; III-12-1: R1 = CH3, R2 = H; III-13-1: R1 = CH3, R2 = 4-CH3; III-14-1: R1 = CH3, R2 = 4-OCH3; III-15-1: R1 = CH3, R2 = 4-Br; III-3-2: R1 = CH2CH3, R2 = 4-Cl; III-3-3: R1 = n-C3H7, R2 = 4-Cl; III-3-4: R1 = i-C3H7, R2 = 4-Cl; III-3-5: R1 = n-C5H11, R2 = 4-Cl; III-3-6: R1 = n-C6H13, R2 = 4-Cl; III-3-7: R1 = n-C7H15, R2 = 4-Cl; III-3-8: R1 = n-C8H17, R2 = 4-Cl; III-3-9: R1 = n-C9H19, R2 = 4-Cl. Full article ">
General synthesis procedure for title compounds III. III-1-1: R1 = CH3, R2 = 2-Cl; III-2-1: R1 = CH3, R2 = 3-Cl; III-3-1: R1 = CH3, R2 = 4-Cl; III-4-1: R1 = CH3, R2 = 2-F; III-5-1: R1 = CH3, R2 = 3-F; III-6-1: R1 = CH3, R2 = 4-F; III-7-1: R1 = CH3, R2 = 2,4-di-F; III-8-1: R1 = CH3, R2 = 2,6-di-F; III-9-1: R1 = CH3, R2 = 2-NO2; III-10-1: R1 = CH3, R2 = 3-NO2; III-11-1: R1 = CH3, R2 = 4-NO2; III-12-1: R1 = CH3, R2 = H; III-13-1: R1 = CH3, R2 = 4-CH3; III-14-1: R1 = CH3, R2 = 4-OCH3; III-15-1: R1 = CH3, R2 = 4-Br; III-3-2: R1 = CH2CH3, R2 = 4-Cl; III-3-3: R1 = n-C3H7, R2 = 4-Cl; III-3-4: R1 = i-C3H7, R2 = 4-Cl; III-3-5: R1 = n-C5H11, R2 = 4-Cl; III-3-6: R1 = n-C6H13, R2 = 4-Cl; III-3-7: R1 = n-C7H15, R2 = 4-Cl; III-3-8: R1 = n-C8H17, R2 = 4-Cl; III-3-9: R1 = n-C9H19, R2 = 4-Cl. Full article ">

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Open AccessArticle

Tagging SNPs in the MTHFR Gene and Risk of Ischemic Stroke in a Chinese Population

by Bao-Sheng Zhou, Guo-Yun Bu, Mu Li, Bin-Ge Chang and Yi-Pin Zhou

Int. J. Mol. Sci. 2014, 15(5), 8931-8940; https://doi.org/10.3390/ijms15058931 - 20 May 2014

Cited by 43 | Viewed by 7005

Abstract

Stroke is currently the leading cause of functional impairments worldwide. Folate supplementation is inversely associated with risk of ischemic stroke. Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme involved in folate metabolism. The aim of this study is to examine whether genetic variants in [...] Read more.

Stroke is currently the leading cause of functional impairments worldwide. Folate supplementation is inversely associated with risk of ischemic stroke. Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme involved in folate metabolism. The aim of this study is to examine whether genetic variants in MTHFR gene are associated with the risk of ischemic stroke and fasting total serum homocysteine (tHcy) level. We genotyped nine tag SNPs in the MTHFR gene in a case-control study, including 543 ischemic stroke cases and 655 healthy controls in China. We found that subjects with the rs1801133 TT genotype and rs1801131 CC genotype had significant increased risks of ischemic stroke (adjusted odds ratio (OR) = 1.82, 95% confidence interval (CI): 1.27–2.61, p = 0.004; adjusted OR = 1.99, 95% CI: 1.12–3.56, p = 0.01) compared with subjects with the major alleles. Haplotype analysis also found that carriers of the MTHFR CTTCGA haplotype (rs12121543-rs13306553-rs9651118-rs1801133-rs2274976-rs1801131) had a significant reduced risk of ischemic stroke (adjusted OR = 0.53, 95% CI: 0.35–0.82) compared with those with the CTTTGA haplotype. Besides, the MTHFR rs1801133 and rs9651118 were significantly associated with serum levels of tHcy in healthy controls (p < 0.0001 and p = 0.02). These findings suggest that variants in the MTHFR gene may influence the risk of ischemic stroke and serum tHcy. Full article

(This article belongs to the Special Issue Human Single Nucleotide Polymorphisms and Disease Diagnostics)

596 KiB

Open AccessHypothesis

Cancer Stem Cell Theory and the Warburg Effect, Two Sides of the Same Coin?

by Nicola Pacini and Fabio Borziani

Int. J. Mol. Sci. 2014, 15(5), 8893-8930; https://doi.org/10.3390/ijms15058893 - 19 May 2014

Cited by 56 | Viewed by 16776

Abstract

Over the last 100 years, many studies have been performed to determine the biochemical and histopathological phenomena that mark the origin of neoplasms. At the end of the last century, the leading paradigm, which is currently well rooted, considered the origin of neoplasms [...] Read more.

Over the last 100 years, many studies have been performed to determine the biochemical and histopathological phenomena that mark the origin of neoplasms. At the end of the last century, the leading paradigm, which is currently well rooted, considered the origin of neoplasms to be a set of genetic and/or epigenetic mutations, stochastic and independent in a single cell, or rather, a stochastic monoclonal pattern. However, in the last 20 years, two important areas of research have underlined numerous limitations and incongruities of this pattern, the hypothesis of the so-called cancer stem cell theory and a revaluation of several alterations in metabolic networks that are typical of the neoplastic cell, the so-called Warburg effect. Even if this specific “metabolic sign” has been known for more than 85 years, only in the last few years has it been given more attention; therefore, the so-called Warburg hypothesis has been used in multiple and independent surveys. Based on an accurate analysis of a series of considerations and of biophysical thermodynamic events in the literature, we will demonstrate a homogeneous pattern of the cancer stem cell theory, of the Warburg hypothesis and of the stochastic monoclonal pattern; this pattern could contribute considerably as the first basis of the development of a new uniform theory on the origin of neoplasms. Thus, a new possible epistemological paradigm is represented; this paradigm considers the Warburg effect as a specific “metabolic sign” reflecting the stem origin of the neoplastic cell, where, in this specific metabolic order, an essential reason for the genetic instability that is intrinsic to the neoplastic cell is defined. Full article

(This article belongs to the Special Issue Advances in the Research of Melatonin 2014)

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Some important features of the cancer stem cell (CSC) theory are highlighted, the neoplastic tissue, as the normal tissue, is carried and renewed by its stem cell population, which self-renews; CSCs, similar to other stem cells, exist in niches with a suitable microenvironment of low pO2; similar to normal tissue, the neoplastic tissue interactions with cells in the microenvironment are fundamental; and traditional cytostatic drugs, such as alkylating agents, are active in the neoplastic bunk but less in CSCs. From this consideration will result a potential mechanism of metastasization. (Modified from Curtin and Lorenzi [<a href="#b24-ijms-15-08893" class="html-bibr">24</a>]) Full article ">
Figure 2 briefly summarizes our model in which, at the origin of a neoplasia, there are not only changes, genetic or epigenetic, but also triple synchronous changes in three systems, metabolic, genetic and epigenetic. We propose an integrative model that is based on cooperative and multiple changes in these three systems. Very probably, a change in one of these cellular systems is not sufficient cause but serves to prepare the cell. From all of these facts and from the growing evidence supporting the CSC theory, we can hypothesize that the transformation process, which foresees this triple progressive change in the three basis systems, occurs in stem cells that preside over self renewal (ASC) or in somatic cells that, by the lost of the correct metabolome and by the change in numerous genetic and epigenetic systems, proceed to a process of somatic rescheduling (iPSC). Certainly, the energetic homeostasis, mitochondrial function and the metabolome as a whole play roles of equal importance in genetic or epigenetic phenomena. From this perspective, more care and attention should be given to other well-know factors. Full article ">

222 KiB

Open AccessReview

Marine Microbial Metagenomics: From Individual to the Environment

by Ching-Hung Tseng and Sen-Lin Tang

Int. J. Mol. Sci. 2014, 15(5), 8878-8892; https://doi.org/10.3390/ijms15058878 - 19 May 2014

Cited by 31 | Viewed by 8944

Abstract

Microbes are the most abundant biological entities on earth, therefore, studying them is important for understanding their roles in global ecology. The science of metagenomics is a relatively young field of research that has enjoyed significant effort since its inception in 1998. Studies [...] Read more.

Microbes are the most abundant biological entities on earth, therefore, studying them is important for understanding their roles in global ecology. The science of metagenomics is a relatively young field of research that has enjoyed significant effort since its inception in 1998. Studies using next-generation sequencing techniques on single genomes and collections of genomes have not only led to novel insights into microbial genomics, but also revealed a close association between environmental niches and genome evolution. Herein, we review studies investigating microbial genomics (largely in the marine ecosystem) at the individual and community levels to summarize our current understanding of microbial ecology in the environment. Full article

(This article belongs to the Special Issue Metagenomics: a Powerful Lens Viewing the Microbial World)

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355 KiB

Open AccessArticle

The Cytoprotective Effect of Sulfuretin against tert-Butyl Hydroperoxide-Induced Hepatotoxicity through Nrf2/ARE and JNK/ERK MAPK-Mediated Heme Oxygenase-1 Expression

by Dong-Sung Lee, Kyoung-Su Kim, Wonmin Ko, Bin Li, Gil-Saeng Jeong, Jun-Hyeog Jang, Hyuncheol Oh and Youn-Chul Kim

Int. J. Mol. Sci. 2014, 15(5), 8863-8877; https://doi.org/10.3390/ijms15058863 - 19 May 2014

Cited by 48 | Viewed by 7666

Abstract

Sulfuretin is one of the major flavonoid components in Rhus verniciflua Stokes (Anacardiaceae) isolates. In this study, we investigated the protective effects of sulfuretin against tert-butyl hydroperoxide (t-BHP)-induced oxidative injury. The results indicated that the addition of sulfuretin before t [...] Read more.

Sulfuretin is one of the major flavonoid components in Rhus verniciflua Stokes (Anacardiaceae) isolates. In this study, we investigated the protective effects of sulfuretin against tert-butyl hydroperoxide (t-BHP)-induced oxidative injury. The results indicated that the addition of sulfuretin before t-BHP treatment significantly inhibited cytotoxicity and reactive oxygen species (ROS) production in human liver-derived HepG2 cells. Sulfuretin up-regulated the activity of the antioxidant enzyme heme oxygenase (HO)-1 via nuclear factor E2-related factor 2 (Nrf2) translocation into the nucleus and increased the promoter activity of the antioxidant response element (ARE). Moreover, sulfuretin exposure enhanced the phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2), which are members of the mitogen-activated protein kinase (MAPK) family. Furthermore, cell treatment with a JNK inhibitor (SP600125) and ERK inhibitor (PD98059) reduced sulfuretin-induced HO-1 expression and decreased its protective effects. Taken together, these results suggest that the protective effect of sulfuretin against t-BHP-induced oxidative damage in human liver-derived HepG2 cells is attributable to its ability to scavenge ROS and up-regulate the activity of HO-1 through the Nrf2/ARE and JNK/ERK signaling pathways. Therefore, sulfuretin could be advantageous as a bioactive source for the prevention of oxidative injury. Full article

(This article belongs to the Section Biochemistry)

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Chemical structure of sulfuretin (A) and effect of sulfuretin on the cell viability (B). Human liver-derived HepG2 cells were incubated for 24 h with various concentrations of sulfuretin (5–80 μM). Cell viability was determined by using MTT assay, as described in the Experimental section. Data shown represent the mean values of three experiments ± SD. * p < 0.05 vs. control. Full article ">
Protective effects of sulfuretin on t-butyl hydroperoxide-induced oxidative toxicity (A) and inhibition of ROS generation (B) in human liver-derived HepG2 cells. Cells were treated with sulfuretin and subsequently incubated for 12 h with t-butyl hydroperoxide (50 μM). Cell viability and ROS generation were determined as described in the Experimental Section. Data shown represent the mean values of three experiments ± SD. * p < 0.05 vs. t-BHP (50 μM) treatment. Full article ">
Effects of sulfuretin on heme oxygenase (HO)-1 mRNA (A) and protein (B,C) expression in human liver-derived HepG2 cells; (A,B) Cells were incubated with sulfuretin for 12 h; and (C) Cells were incubated with 40 μM of sulfuretin for the indicated periods. Data shown represent the mean values of three experiments ± SD. Expression of HO-1 was determined by western blot analysis, and representative blots from three independent experiments with similar results. * p < 0.05 vs. control. Full article ">
Effects of HO-1 Induction by sulfuretin on t-butyl hydroperoxide-induced oxidative toxicity (A) and ROS generation (B) in human liver-derived HepG2 cells. Cells were treated with various concentrations of sulfuretin and 50 μM tin protoporphyrin (SnPP), and were subsequently exposed to t-butyl hydroperoxide (50 μM) for 12 h. Cell viability and ROS generation were determined as described in the Experimental Section. Data shown represent the mean values of three experiments ± SD. * p < 0.05 vs. t-BHP (50 μM) treatment, ** p < 0.05 vs. same treatment plus SnPP. Full article ">
Effects of sulfuretin on Nrf2 nuclear translocation (A), antioxidant response element (ARE) activation (B), and transfection with Nrf2 siRNA (C) in human liver-derived HepG2 cells. (A) Cells were treated with 40 μM sulfuretin for 0–120 min. The nuclei were fractionated from the cytosol using PER Mammalian Protein Extraction Buffer as described in the Experimental section; (B) Quiescent cells transiently transfected with ARE-luciferase or control vector were incubated for 1 h with the indicated concentrations of sulfuretin in the presence of 5% fetal bovine serum (FBS). Cell lysates were assayed for the fold induction of luciferase activity by normalizing the transfection efficiency and dividing the values of each experiment relative to the control; and (C) Cells were transiently transfected with Nrf2 siRNA, and then treated with 40 μM of sulfuretin for 12 h. Nrf2 and HO-1 protein were detected by western blot analysis, and representative blots from three independent experiments with similar results. Data shown represent the mean values of three experiments ± SD. * p < 0.05 vs. control. Full article ">
Effects of sulfuretin on MAPKs activation in human liver-derived HepG2 cells. Cells were treated with 40 μM sulfuretin for the indicated times (A–C). Cells were incubated with 40 μM of sulfuretin for the indicated periods. Activation of p38 (A); JNK (B) and ERK1/2 (C) were determined by western blot analysis. Membranes were stripped and re-probed for the total amount of each MAPK antibody as a control, and representative blots from three independent experiments with similar results. Full article ">
Effects of sulfuretin-induced mitogen-activated protein kinases (MAPKs) activation on HO-1 expression (A) and t-BHP-induced cytotoxicity (B) in human liver-derived HepG2 cells. Cells were incubated with 40 μM of sulfuretin for 12 h in the presence or absence of SP600125 (25 μM), SB203580 (20 μM), or PD98059 (10 μM). Then, cells subsequently were exposed to 50 μM of t-BHP for 12 h. Representative blots from three independent experiments with similar results. Data shown represent the mean values of three experiments ± SD. * p < 0.05 vs. t-BHP (50 μM) treatment. Full article ">

298 KiB

Open AccessArticle

Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane

by Bantong Xue, Jinlong Guo, Youxiong Que, Zhiwei Fu, Luguang Wu and Liping Xu

Int. J. Mol. Sci. 2014, 15(5), 8846-8862; https://doi.org/10.3390/ijms15058846 - 19 May 2014

Cited by 23 | Viewed by 7869

Abstract

Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or [...] Read more.

Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential “single copy” genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3—high copy number group, TST-1 and PRR-1—medium copy number group, P4H-1, APRT-2 and CYC-2—low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane. Full article

(This article belongs to the Section Biochemistry)

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1470 KiB

Open AccessArticle

Molecular Characterization of α- and β-Thalassaemia among Malay Patients

by Nur Fatihah Mohd Yatim, Masitah Abd. Rahim, Kavitha Menon, Faisal Muti Al-Hassan, Rahimah Ahmad, Anita Bhajan Manocha, Mohamed Saleem and Badrul Hisham Yahaya

Int. J. Mol. Sci. 2014, 15(5), 8835-8845; https://doi.org/10.3390/ijms15058835 - 19 May 2014

Cited by 20 | Viewed by 8188

Abstract

Both α- and β-thalassaemia syndromes are public health problems in the multi-ethnic population of Malaysia. To molecularly characterise the α- and β-thalassaemia deletions and mutations among Malays from Penang, Gap-PCR and multiplexed amplification refractory mutation systems were used to study 13 α-thalassaemia determinants [...] Read more.

Both α- and β-thalassaemia syndromes are public health problems in the multi-ethnic population of Malaysia. To molecularly characterise the α- and β-thalassaemia deletions and mutations among Malays from Penang, Gap-PCR and multiplexed amplification refractory mutation systems were used to study 13 α-thalassaemia determinants and 20 β-thalassaemia mutations in 28 and 40 unrelated Malays, respectively. Four α-thalassaemia deletions and mutations were demonstrated. −−^SEA deletion and α^CSα accounted for more than 70% of the α-thalassaemia alleles. Out of the 20 β-thalassaemia alleles studied, nine different β-thalassaemia mutations were identified of which β^E accounted for more than 40%. We concluded that the highest prevalence of (α- and β-thalassaemia alleles in the Malays from Penang are −−^SEA deletion and β^E mutation, respectively. Full article

(This article belongs to the Special Issue Human Single Nucleotide Polymorphisms and Disease Diagnostics)

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Multiplex-Gap PCR genotype analysis of the α-globin gene cluster on agarose gel electrophoresis. Lanes 1 and 12 (left to right) show DNA marker ladders; 2350 bp band on lane 2 depicts the internal control and 1800 bp band indicates the presence of normal α2 globin gene. Lanes 3–5 are positive controls for heterozygous −α3.7 deletion, −α4.2 deletion, and −−SEA deletion; Lanes 6–10 are patients where lane 6 shows compound heterozygosity for −α3.7 and −−SEA deletions; Lanes 7 and 8 “none of the deletion tested present”; Lane 9 −α3.7 deletion; Lane 10 −−SEA deletion; and Lane 11 is non-template control. Full article ">
Multiplex ARMS PCR genotype analysis of α globin gene cluster on agarose gel electrophoresis. Lane 1 ladder; Lanes 2–4 are positive controls for Cd 59 (G > A), Cd 125 (T > C), and term Cd TAA > CAA (Hb CS) with their respective bands; Lane 5 non-template control; Lanes 6 and 7 Hb CS; and Lane 8 Cd 59 (G > A); 930 bp bands on Lanes 2–4 and 6–8 are internal control bands amplifying a segment of 3′ UTR of LIS1 gene. Full article ">
Capillary electrophoresis (CE) electropherogram for multiplex ARMS-A of β globin gene cluster. Lanes 2–5 are the positive controls for 41/41 (−TTCT), IVS 1-5 (G > C), Cd 26 (G > A), and Cd 17 (A > T) respectively. Lanes 6, 9, 11, and 12 show Cd 26 (G > A); Lane 10 shows “none of the mutations tested present”; and Lanes 7 and 8 show the presence of IVS 1-5 (G > C). Full article ">

621 KiB

Open AccessArticle

Antibody-Conjugated Paramagnetic Nanobeads: Kinetics of Bead-Cell Binding

by Shahid Waseem, Michael A. Allen, Stefan Schreier, Rachanee Udomsangpetch and Sebastian C. Bhakdi

Int. J. Mol. Sci. 2014, 15(5), 8821-8834; https://doi.org/10.3390/ijms15058821 - 19 May 2014

Cited by 11 | Viewed by 6771

Abstract

Specific labelling of target cell surfaces using antibody-conjugated paramagnetic nanobeads is essential for efficient magnetic cell separation. However, studies examining parameters determining the kinetics of bead-cell binding are scarce. The present study determines the binding rates for specific and unspecific binding of 150 [...] Read more.

Specific labelling of target cell surfaces using antibody-conjugated paramagnetic nanobeads is essential for efficient magnetic cell separation. However, studies examining parameters determining the kinetics of bead-cell binding are scarce. The present study determines the binding rates for specific and unspecific binding of 150 nm paramagnetic nanobeads to highly purified target and non-target cells. Beads bound to cells were enumerated spectrophotometrically. Results show that the initial bead-cell binding rate and saturation levels depend on initial bead concentration and fit curves of the form A(1 − exp(−kt)). Unspecific binding within conventional experimental time-spans (up to 60 min) was not detectable photometrically. For CD3-positive cells, the probability of specific binding was found to be around 80 times larger than that of unspecific binding. Full article

(This article belongs to the Section Materials Science)

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Characterization of paramagnetic nanobeads. (A) TEM micrograph of antibody-conjugated magnetic nanobeads. The multi-domain iron oxide cores from two nanobeads are visible. Bar size is 100 nm; (B) XRD pattern of antibody-conjugated magnetic nanobeads; (C) Size distribution of antibody-conjugated magnetic nanobeads is shown by a dynamic light scattering (DLS) graph. Ten replicates, as shown by peaks of different colours, were analysed. Full article ">
Flow cytometric analysis of untouched CD14- and CD3-positive cells separated by buffer optimized high gradient magnetic separation (HGMS) (negative selection). Plots show CD14- and CD3-positive cells before and after magnetic separation. CD14-positive cells were enriched from 17% to 95% and CD3-positive cells from 62% to 96%. Full article ">
Standard curves for the number of magnetic beads with (solid line, dots) or without peripheral blood mononuclear cells (PBMC) (dashed line, crosses). For both standard curves R2 = 0.99. In this and later figures, data points are the mean of triplicate values. Vertical lines (sometimes smaller than the plotted points) indicate the range of values. Full article ">
Time-dependent binding of anti-CD3 conjugated magnetic nanobeads to untouched CD3-positive cells. In this figure and the next, data are fitted to saturation-type curves of the form A(1 − exp(−kt)) where t is the incubation time. Solid black and grey line and round dots: 32 μg beads/100 μL, 106 cells/100 μL, R2 = 0.95 and 0.99 (cells from two different donors); black dashed line and square dots: 32 μg beads/100 μL, 2 × 106 cells/100 μL, R2 = 0.99; dotted line and squares: 16 μg beads/100 μL, 106 cells/100 μL, R2 = 0.95; grey dashed line and square dots: 8 μg beads/100 μL, 5 × 105 cells/100 μL, R2 = 0.98. Full article ">
Time-dependent binding of anti-CD14 conjugated magnetic nanobeads to untouched CD14-positive cells. Solid line: 32 μg beads/100 μL, R2 = 0.98; dashed line and circles, dotted line and square dots: 16 μg beads/100 μL, R2 = 0.98 and 0.99. Concentration of target cells: 1 × 106/100 μL. The experiments are from samples from three different blood donors. Full article ">
Maximum saturation level for target cells was determined by using multi-step incubation, as described in the Experimental Section. Solid and open circles represent the number of beads per untouched CD14- and CD3-positive cell, respectively. Full article ">
Time-dependent unspecific binding of anti-CD14 conjugated magnetic nanobeads to untouched CD3-positive cells. Concentration of nanobeads: 16 μg/100 μL; concentration of target cells: 106/100 μL. Full article ">
Initial rate of bead-cell binding as a function of initial bead concentration. Solid line and dots: CD3; dashed line and circles: CD14. Full article ">

750 KiB

Open AccessCommunication

Synthesis, Preliminary Bioevaluation and Computational Analysis of Caffeic Acid Analogues

by Zhiqian Liu, Jianjun Fu, Lei Shan, Qingyan Sun and Weidong Zhang

Int. J. Mol. Sci. 2014, 15(5), 8808-8820; https://doi.org/10.3390/ijms15058808 - 16 May 2014

Cited by 8 | Viewed by 7084

Abstract

A series of caffeic acid amides were designed, synthesized and evaluated for anti-inflammatory activity. Most of them exhibited promising anti-inflammatory activity against nitric oxide (NO) generation in murine macrophage RAW264.7 cells. A 3D pharmacophore model was created based on the biological results for [...] Read more.

A series of caffeic acid amides were designed, synthesized and evaluated for anti-inflammatory activity. Most of them exhibited promising anti-inflammatory activity against nitric oxide (NO) generation in murine macrophage RAW264.7 cells. A 3D pharmacophore model was created based on the biological results for further structural optimization. Moreover, predication of the potential targets was also carried out by the PharmMapper server. These amide analogues represent a promising class of anti-inflammatory scaffold for further exploration and target identification. Full article

(This article belongs to the Special Issue Molecular Science for Drug Development and Biomedicine)

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1093 KiB

Open AccessArticle

Biological Evaluation and 3D-QSAR Studies of Curcumin Analogues as Aldehyde Dehydrogenase 1 Inhibitors

by Hui Wang, Zhiyun Du, Changyuan Zhang, Zhikai Tang, Yan He, Qiuyan Zhang, Jun Zhao and Xi Zheng

Int. J. Mol. Sci. 2014, 15(5), 8795-8807; https://doi.org/10.3390/ijms15058795 - 16 May 2014

Cited by 7 | Viewed by 6339

Abstract

Aldehyde dehydrogenase 1 (ALDH1) is reported as a biomarker for identifying some cancer stem cells, and down-regulation or inhibition of the enzyme can be effective in anti-drug resistance and a potent therapeutic for some tumours. In this paper, the inhibitory activity, mechanism mode, [...] Read more.

Aldehyde dehydrogenase 1 (ALDH1) is reported as a biomarker for identifying some cancer stem cells, and down-regulation or inhibition of the enzyme can be effective in anti-drug resistance and a potent therapeutic for some tumours. In this paper, the inhibitory activity, mechanism mode, molecular docking and 3D-QSAR (three-dimensional quantitative structure activity relationship) of curcumin analogues (CAs) against ALDH1 were studied. Results demonstrated that curcumin and CAs possessed potent inhibitory activity against ALDH1, and the CAs compound with ortho di-hydroxyl groups showed the most potent inhibitory activity. This study indicates that CAs may represent a new class of ALDH1 inhibitor. Full article

(This article belongs to the Section Physical Chemistry, Theoretical and Computational Chemistry)

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2405 KiB

Open AccessArticle

In Vitro Treatment of Melanoma Brain Metastasis by Simultaneously Targeting the MAPK and PI3K Signaling Pathways

by Inderjit Daphu, Sindre Horn, Daniel Stieber, Jobin K. Varughese, Endy Spriet, Hege Avsnes Dale, Kai Ove Skaftnesmo, Rolf Bjerkvig and Frits Thorsen

Int. J. Mol. Sci. 2014, 15(5), 8773-8794; https://doi.org/10.3390/ijms15058773 - 16 May 2014

Cited by 25 | Viewed by 10918

Abstract

Malignant melanoma is the most lethal form of skin cancer, with a high propensity to metastasize to the brain. More than 60% of melanomas have the BRAF^V600E mutation, which activates the mitogen-activated protein kinase (MAPK) pathway [1]. In addition, increased PI3K (phosphoinositide [...] Read more.

Malignant melanoma is the most lethal form of skin cancer, with a high propensity to metastasize to the brain. More than 60% of melanomas have the BRAF^V600E mutation, which activates the mitogen-activated protein kinase (MAPK) pathway [1]. In addition, increased PI3K (phosphoinositide 3-kinase) pathway activity has been demonstrated, through the loss of activity of the tumor suppressor gene, PTEN [2]. Here, we treated two melanoma brain metastasis cell lines, H1_DL2, harboring a BRAF^V600E mutation and PTEN loss, and H3, harboring WT (wild-type) BRAF and PTEN loss, with the MAPK (BRAF) inhibitor vemurafenib and the PI3K pathway associated mTOR inhibitor temsirolimus. Combined use of the drugs inhibited tumor cell growth and proliferation in vitro in H1_DL2 cells, compared to single drug treatment. Treatment was less effective in the H3 cells. Furthermore, a strong inhibitory effect on the viability of H1_DL2 cells, when grown as 3D multicellular spheroids, was seen. The treatment inhibited the expression of pERK1/2 and reduced the expression of pAKT and p-mTOR in H1_DL2 cells, confirming that the MAPK and PI3K pathways were inhibited after drug treatment. Microarray experiments followed by principal component analysis (PCA) mapping showed distinct gene clustering after treatment, and cell cycle checkpoint regulators were affected. Global gene analysis indicated that functions related to cell survival and invasion were influenced by combined treatment. In conclusion, we demonstrate for the first time that combined therapy with vemurafenib and temsirolimus is effective on melanoma brain metastasis cells in vitro. The presented results highlight the potential of combined treatment to overcome treatment resistance that may develop after vemurafenib treatment of melanomas. Full article

(This article belongs to the Special Issue Brain Metastasis 2014)

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Cell proliferation and survival of H1_DL2 and H3 melanoma brain metastasis cells grown as monolayer cultures, after treatment with vemurafenib and temsirolimus. (A,B) Treatment of H1_DL2 melanoma cells, harboring the BRAFV600E mutation. (A) H1_DL2 cells treated with increasing drug concentrations for 72 h (n = 6, mean ± SD); (B) A more detailed analysis of the cell survival shown in (A), after drug treatment with 0, 0.05, 5 or 10 μM of vemurafenib, temsirolimus or a combination therapy. Scatter dot plot (n = 6, mean ± SD). *** p < 0.001; **** p < 0.0001; (C,D) Treatment of H3 melanoma cells, harboring wild-type (WT) BRAF. (C) H3 cells treated with increasing drug concentrations for 72 h (n = 6, mean ± SD); and (D) A more detailed analysis of the cell survival shown in (C), after drug treatment with 0, 0.05, 5 or 10 μM vemurafenib, temsirolimus or combined therapy. Scatter dot plot (n = 6, mean ± SD). n.s.: not significant; * p < 0.05; ** p < 0.01; **** p < 0.0001. Full article ">
Wound healing assay of H1_DL2 melanoma brain metastasis cells after treatment with vemurafenib and temsirolimus. (A) Untreated H1_DL2 cells at 0 h; (B) Untreated cells 60 h after initiating a scratch wound; (C) Representative picture 60 h after initiating a scratch wound and starting the treatment with 5 μM of temsirolimus; (D) Representative picture 60 h after initiating a scratch wound and starting the treatment with 5 μM of vemurafenib; (E) Representative picture 48 h after initiating a scratch wound and starting combined treatment (5 μM temsirolimus and 5 μM vemurafenib); and (F) Representative picture 60 h after initiating a scratch wound and starting combined treatment (5 μM temsirolimus and 5 μM vemurafenib). Scale bar = 200 μm. Full article ">
Multicellular spheroid growth of H1_DL2 cells after treatment with vemurafenib and temsirolimus. (A, left) Spheroid diameters measured zero and eight days after treatment with 10 μM of temsirolimus, 10 μM of vemurafenib or the combined treatment (5 μM temsirolimus + 5 μM vemurafenib) (n = 6, mean ± SD). n.s.: not significant; * p < 0.05, **** p < 0.0001. (A, right) The percentage of dead cells within the spheroids after treatment for three days with 10 μM of temsirolimus, 10 μM of vemurafenib or the combined treatment (5 μM temsirolimus + 5 μM vemurafenib); (B) Maximum intensity projection images of the red channel (dead cells) of confocal image stacks of spheroids treated with 10 μM of temsirolimus, 10 μM of vemurafenib or the combined treatment (5 μM temsirolimus + 5 μM vemurafenib); (C) The surface volume of the green channel (live cells) of the same spheroids as in (B); and (D) An overlay of the red and green volume surfaces of the same spheroids as in (B). Scale bar = 100 μm. neg. control = negative control. Full article ">
Western blot of H1_DL2 cells after drug treatment with vemurafenib and temsirolimus. Western blot of phosphorylated ERK1 and phosphorylated ERK2 (pERK1/2), phosphorylated mTOR (p-mTOR) and phosphorylated AKT (pAKT), in H1_DL2 cells treated with 15 μM of vemurafenib (Vem), 25 μM of temsirolimus (Tems), or the combined treatment (Combi; 10 μM vemurafenib + 10 μM temsirolimus). The K16 cell line (A) served as positive control for pMAPK; and the 293T cell line (B) was a positive control for pAKT and p-mTOR. GAPDH protein levels were assessed as a loading control. Full article ">
Principal component analysis (PCA) of H1_DL2 cells after drug treatment with vemurafenib and temsirolimus. PCA analysis of all 20 samples resulted in four distinct clusters for each of the respective treatments: Untreated samples (blue), vemurafenib-treated cells (15 μM; green), temsirolimus-treated cells (25 μM; purple) and the combination treatment (5 μM each; red) (n = 5 for all treatments). Full article ">
Graphical representation of two of the top score networks identified by Ingenuity Pathway Analysis (IPA). Canonical pathways from Ingenuity for combination treatment (5 μM vemurafenib and 5 μM temsirolimus) compared to untreated cells. Molecular relationships between genes downregulated (green) or upregulated (red) after treatment are shown. (A) The canonical pathway “Cell cycle: G1/S checkpoint regulation”; and (B) The canonical pathway “Cell cycle: G2/M DNA damage checkpoint regulation”. Full article ">
Ingenuity function analysis. (A) Bar plot of the activation z-scores of selected downstream functions in combination treated samples (5 μM vemurafenib and 5 μM temsirolimus) compared to untreated cells. Functions with z-scores greater than two are predicted as being “activated” in this comparison, while those with scores less than −2 are “inhibited”. All columns were statistically significant; p < 0.005. Column numbers correspond to the list of downstream functions shown in <a href="#t3-ijms-15-08773" class="html-table">Table 3</a>; and (B) A detailed presentation of bar plots of activation z-scores, showing the most activated downstream functions for each of the three treatments; 25 μM temsirolimus vs. untreated cells, 15 μM vemurafenib vs. untreated cells or 5 μM vemurafenib + 5 μM temsirolimus vs. untreated cells. All columns were statistically significant; p < 0.005. Full article ">

976 KiB

Open AccessReview

Colonization and Infection of the Skin by S. aureus: Immune System Evasion and the Response to Cationic Antimicrobial Peptides

by Sunhyo Ryu, Peter I. Song, Chang Ho Seo, Hyeonsook Cheong and Yoonkyung Park

Int. J. Mol. Sci. 2014, 15(5), 8753-8772; https://doi.org/10.3390/ijms15058753 - 16 May 2014

Cited by 111 | Viewed by 18189

Abstract

Staphylococcus aureus (S. aureus) is a widespread cutaneous pathogen responsible for the great majority of bacterial skin infections in humans. The incidence of skin infections by S. aureus reflects in part the competition between host cutaneous immune defenses and S. aureus [...] Read more.

Staphylococcus aureus (S. aureus) is a widespread cutaneous pathogen responsible for the great majority of bacterial skin infections in humans. The incidence of skin infections by S. aureus reflects in part the competition between host cutaneous immune defenses and S. aureus virulence factors. As part of the innate immune system in the skin, cationic antimicrobial peptides (CAMPs) such as the β-defensins and cathelicidin contribute to host cutaneous defense, which prevents harmful microorganisms, like S. aureus, from crossing epithelial barriers. Conversely, S. aureus utilizes evasive mechanisms against host defenses to promote its colonization and infection of the skin. In this review, we focus on host-pathogen interactions during colonization and infection of the skin by S. aureus and methicillin-resistant Staphylococcus aureus (MRSA). We will discuss the peptides (defensins, cathelicidins, RNase7, dermcidin) and other mediators (toll-like receptor, IL-1 and IL-17) that comprise the host defense against S. aureus skin infection, as well as the various mechanisms by which S. aureus evades host defenses. It is anticipated that greater understanding of these mechanisms will enable development of more sustainable antimicrobial compounds and new therapeutic approaches to the treatment of S. aureus skin infection and colonization. Full article

(This article belongs to the Special Issue Molecular Science for Drug Development and Biomedicine)

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Toll-like receptor-mediated cutaneous immune response against S. aureus. Toll-like receptor 2 (TLR2) and nucleotide-binding oligomerization domain containing 2 (NOD2), which are expressed by keratinocytes, respectively recognize S. aureus lipopeptides/lipoteichoic acid and muramyl dipeptide. Both TLR2 and NOD2 signaling triggers the activation of nuclear factor-κB (NF-κB), which leads to the production of AMPs, cytokines, chemokines, adhesion molecules and granulopoesis factors, all of which contribute to the cutaneous host defense against S. aureus. Full article ">
IL-1- and IL-17-mediated cutaneous immune response against S. aureus. Infection of the skin by S. aureus leads to the production of IL-1α, IL-1β and IL-17, which in turn triggers activation of nuclear factor-κB (NF-κB). These signaling pathways lead to the production of AMPs, cytokines, chemokines, adhesion molecules and granulopoesis factors, which recruit neutrophils from the circulation to the site of S. aureus infection in the skin. The recruited neutrophils form an abscess that helps control and limit the spread of the infection, and is ultimately required for bacterial clearance. IL-1R1, interleukin-1 receptor 1; IL-17R, interleukin-17 receptor. Full article ">
Strategies by which S. aureus evades CAMPs. S. aureus counteracts CAMPs by secreting trapping molecules and proteases that inactivate CAMPs and by modifying the cell membrane hydrophobicity or net charge [<a href="#b108-ijms-15-08753" class="html-bibr">108</a>]. Full article ">

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Open AccessArticle

Molecular Recognition of Agonist and Antagonist for Peroxisome Proliferator-Activated Receptor-α Studied by Molecular Dynamics Simulations

by Mengyuan Liu, Lushan Wang, Xian Zhao and Xun Sun

Int. J. Mol. Sci. 2014, 15(5), 8743-8752; https://doi.org/10.3390/ijms15058743 - 15 May 2014

Cited by 5 | Viewed by 5535

Abstract

Peroxisome proliferator activated receptor-α (PPAR-α) is a ligand-activated transcription factor which plays important roles in lipid and glucose metabolism. The aim of this work is to find residues which selectively recognize PPAR-α agonists and antagonists. To achieve this aim, PPAR-α/13M and PPAR-α/471 complexes [...] Read more.

Peroxisome proliferator activated receptor-α (PPAR-α) is a ligand-activated transcription factor which plays important roles in lipid and glucose metabolism. The aim of this work is to find residues which selectively recognize PPAR-α agonists and antagonists. To achieve this aim, PPAR-α/13M and PPAR-α/471 complexes were subjected to perform molecular dynamics simulations. This research suggests that several key residues only participate in agonist recognition, while some other key residues only contribute to antagonist recognition. It is hoped that such work is useful for medicinal chemists to design novel PPAR-α agonists and antagonists. Full article

(This article belongs to the Section Physical Chemistry, Theoretical and Computational Chemistry)

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Graphical abstract

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Full article ">
The overall structures of peroxisome proliferator activated receptor-α (PPAR-α)/ligand complexes. (A) PPAR-α/13M complex; and (B) PPAR-α/471 complex. PPAR-α backbone is shown in ribbon (Helix: white; Strand: yellow; Coil: blue). Agonist and antagonist are shown in sphere (Carbon atom: purple; Oxygen atom: red; Nitrogen atom: blue; Fluorine atom: green). Full article ">
The active site of PPAR-α. (A) PPAR-α/13M complex; and (B) PPAR-α/471 complex. Residues in PPAR-α are only shown with backbone atoms. Agonist and antagonist are shown in stick with purple carbon atoms. The arm I region is shown in stick with blue atoms. The arm II region is shown in stick with orange atoms. The entrance region is shown in stick with green atoms. For the sake of clarity, only the polar hydrogen atoms are displayed. Full article ">
Chemical structures of PPAR-α agonist 13M and antagonist 471. Full article ">
The root mean square deviation (RMSD) of Cα atoms for different systems. 13M: PPAR-α/13M complex; 471: PPAR-α/471 complex. Full article ">
Snapshots of PPAR-α/ligand complexes at 50 ns (A) PPAR-α/13M complex; and (B) PPAR-α/471 complex. Agonist and antagonist are shown in stick with purple carbon atoms, while residues of PPAR-α are shown in stick with green carbon atoms. The hydrogen bonds are shown in black lines (For the sake of clarity, only the polar hydrogen atoms are displayed). Full article ">
The average total interaction energies of agonist and antagonist with residues in (A) Arm I region; (B) Arm II region; and (C) Entrance region. Full article ">

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Open AccessReview

Role of Mitochondria in Nonalcoholic Fatty Liver Disease

by Fatiha Nassir and Jamal A. Ibdah

Int. J. Mol. Sci. 2014, 15(5), 8713-8742; https://doi.org/10.3390/ijms15058713 - 15 May 2014

Cited by 274 | Viewed by 17480

Abstract

Nonalcoholic fatty liver disease (NAFLD) affects about 30% of the general population in the United States and includes a spectrum of disease that includes simple steatosis, non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. Significant insight has been gained into our understanding of the pathogenesis [...] Read more.

Nonalcoholic fatty liver disease (NAFLD) affects about 30% of the general population in the United States and includes a spectrum of disease that includes simple steatosis, non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. Significant insight has been gained into our understanding of the pathogenesis of NALFD; however the key metabolic aberrations underlying lipid accumulation in hepatocytes and the progression of NAFLD remain to be elucidated. Accumulating and emerging evidence indicate that hepatic mitochondria play a critical role in the development and pathogenesis of steatosis and NAFLD. Here, we review studies that document a link between the pathogenesis of NAFLD and hepatic mitochondrial dysfunction with particular focus on new insights into the role of impaired fatty acid oxidation, the transcription factor peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), and sirtuins in development and progression of NAFLD. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Human Liver Diseases)

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The “multiple parallel-hits” hypothesis of NAFLD: Insulin resistance leads to increased uptake and synthesis of FFAs in the liver, which sensitizes the liver to a series of hits causing liver injury and progression from simple steatosis to NASH [<a href="#b26-ijms-15-08713" class="html-bibr">26</a>]. Full article ">
Hepatic β-oxidation: Mitochondrial β-oxidation involves four individual reactions (1–4) to generate NADH or FADH2, which are then oxidized to H2O by the mitochondrial respiratory chain. The mitochondrial respiratory chain consists of four respiratory complexes (I–IV) involved in the conversion of NADH and FADH2 into oxidized cofactors NAD and FAD. Leakage of electrons at complexes I and II results in the formation of superoxide (O2 −) which is then transformed to H2O2 by superoxide dismutase (SOD1) in the intermediate space and by SOD2 in the matrix to H2O2. Both H2O2 and O2 − generated are reactive oxygen species (ROS). Mitochondrial antioxidant enzymes (SOD and glutathione peroxidase GPX) play a role in scavenging mitochondrial ROS. Adapted with permission from [<a href="#b61-ijms-15-08713" class="html-bibr">61</a>]. Copyright 1999–2014 John Wiley & Sons, Inc.) Full article ">
Role of the mitochondria in NAFLD. Impairment of mitochondrial function by genetic factors, aging, and overnutrition causes insulin resistance and mitochondrial dysfunction. Defective mitochondrial β-oxidation causes fatty liver and increases lipid toxic metabolites which may in turn causes insulin resistance, thus creating a vicious cycle between insulin resistance and mitochondrial dysfunction. Improved mitochondrial function by weight loss through caloric restriction and/or exercise improves insulin resistance. Regulators of mitochondrial biogenesis and function include genes such as peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and sirtuins. AMP-activated protein kinase (AMPK), fatty acid oxidation (FAO), mitochondrial respiratory chain (MRC), long chain fatty acid CoA (LCFA), Adenosine triphosphate (ATP), reactive oxygen species (ROS). Full article ">

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Open AccessArticle

Expression and Effects of High-Mobility Group Box 1 in Cervical Cancer

by Xiaoao Pang, Yao Zhang, Heng Wei, Jing Zhang, Qingshuang Luo, Chenglin Huang and Shulan Zhang

Int. J. Mol. Sci. 2014, 15(5), 8699-8712; https://doi.org/10.3390/ijms15058699 - 15 May 2014

Cited by 37 | Viewed by 8781

Abstract

We investigated the significance of high- mobility group box1 (HMGB1) and T-cell-mediated immunity and prognostic value in cervical cancer. HMGB1, forkhead/winged helix transcription factor p3 (Foxp3), IL-2, and IL-10 protein expression was analyzed in 100 cervical tissue samples including cervical cancer, cervical intraepithelial [...] Read more.

We investigated the significance of high- mobility group box1 (HMGB1) and T-cell-mediated immunity and prognostic value in cervical cancer. HMGB1, forkhead/winged helix transcription factor p3 (Foxp3), IL-2, and IL-10 protein expression was analyzed in 100 cervical tissue samples including cervical cancer, cervical intraepithelial neoplasia (CIN), and healthy control samples using immunohistochemistry. Serum squamous cell carcinoma antigen (SCC-Ag) was immunoradiometrically measured in 32 serum samples from 37 cases of squamous cervical cancer. HMGB1 and SCC-Ag were then correlated to clinicopathological characteristics. HMGB1 expression tends to increase as cervical cancer progresses and it was found to be significantly correlated to FIGO stage and lymph node metastasis. These findings suggest that HMGB1 may be a useful prognostic indicator of cervical carcinoma. In addition, there were significant positive relationships between HMGB1 and FOXP3 or IL-10 expression (both p < 0.05). In contrast, HMGB1 and IL-2 expression was negatively correlated (p < 0.05). HMGB1 expression may activate Tregs or facilitate Th2 polarization to promote immune evasion of cervical cancer. Elevated HMGB1 protein in cervical carcinoma samples was associated with a high recurrence of HPV infection in univariate analysis (p < 0.05). HMGB1 expression and levels of SCC-Ag were directly correlated in SCC (p < 0.05). Thus, HMGB1 may be a useful biomarker for patient prognosis and cervical cancer prediction and treatment. Full article

(This article belongs to the Special Issue Molecular Classification of Human Cancer and Its Role in Tailored Cancer Therapy)

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367 KiB

Open AccessReview

Signaling Pathways in Cartilage Repair

by Erminia Mariani, Lia Pulsatelli and Andrea Facchini

Int. J. Mol. Sci. 2014, 15(5), 8667-8698; https://doi.org/10.3390/ijms15058667 - 15 May 2014

Cited by 135 | Viewed by 15251

Abstract

In adult healthy cartilage, chondrocytes are in a quiescent phase characterized by a fine balance between anabolic and catabolic activities. In ageing, degenerative joint diseases and traumatic injuries of cartilage, a loss of homeostatic conditions and an up-regulation of catabolic pathways occur. Since [...] Read more.

In adult healthy cartilage, chondrocytes are in a quiescent phase characterized by a fine balance between anabolic and catabolic activities. In ageing, degenerative joint diseases and traumatic injuries of cartilage, a loss of homeostatic conditions and an up-regulation of catabolic pathways occur. Since cartilage differentiation and maintenance of homeostasis are finely tuned by a complex network of signaling molecules and biophysical factors, shedding light on these mechanisms appears to be extremely relevant for both the identification of pathogenic key factors, as specific therapeutic targets, and the development of biological approaches for cartilage regeneration. This review will focus on the main signaling pathways that can activate cellular and molecular processes, regulating the functional behavior of cartilage in both physiological and pathological conditions. These networks may be relevant in the crosstalk among joint compartments and increased knowledge in this field may lead to the development of more effective strategies for inducing cartilage repair. Full article

(This article belongs to the Special Issue Signal Transduction of Tissue Repair)

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Open AccessArticle

Geminal Brønsted Acid Ionic Liquids as Catalysts for the Mannich Reaction in Water

by Leqin He, Shenjun Qin, Tao Chang, Yuzhuang Sun and Jiquan Zhao

Int. J. Mol. Sci. 2014, 15(5), 8656-8666; https://doi.org/10.3390/ijms15058656 - 15 May 2014

Cited by 24 | Viewed by 7145

Abstract

Quaternary ammonium geminal Brønsted acid ionic liquids (GBAILs) based on zwitterionic 1,2-bis[N-methyl-N-(3-sulfopropyl)-alkylammonium]ethane (where the carbon number of the alkyl chain is 4, 8, 10, 12, 14, 16, or 18) and p-toluenesulfonic acid monohydrate were synthesized. The catalytic ionic [...] Read more.

Quaternary ammonium geminal Brønsted acid ionic liquids (GBAILs) based on zwitterionic 1,2-bis[N-methyl-N-(3-sulfopropyl)-alkylammonium]ethane (where the carbon number of the alkyl chain is 4, 8, 10, 12, 14, 16, or 18) and p-toluenesulfonic acid monohydrate were synthesized. The catalytic ionic liquids were applied in three-component Mannich reactions with an aldehyde, ketone, and amine at 25 °C in water. The effects of the type and amount of catalyst and reaction time as well as the scope of the reaction were investigated. Results showed that GBAIL-C₁₄ has excellent catalytic activity and fair reusability. The catalytic procedure was simple, and the catalyst could be recycled seven times via a simple separation process without noticeable decreases in catalytic activity. Full article

(This article belongs to the Special Issue Ionic Liquids 2014 & Selected Papers from ILMAT 2013)

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Int. J. Mol. Sci., Volume 15, Issue 5 (May 2014) – 121 articles , Pages 7049-9172

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