International Journal of Molecular Sciences

1796 KiB

Open AccessArticle

Necrostatin-1 Attenuates Ischemia Injury Induced Cell Death in Rat Tubular Cell Line NRK-52E through Decreased Drp1 Expression

by Li Zhang, Fen Jiang, Yuanhan Chen, Jialun Luo, Shuangxin Liu, Bin Zhang, Zhiming Ye, Wenjian Wang, Xinling Liang and Wei Shi

Int. J. Mol. Sci. 2013, 14(12), 24742-24754; https://doi.org/10.3390/ijms141224742 - 18 Dec 2013

Cited by 41 | Viewed by 8210

Abstract

Necrostatin-1 (Nec-1) inhibits necroptosis and is usually regarded as having no effect on other cell deaths. Here, this study explored whether the addition of Nec-1 has an effect on cell death induced by simulated ischemia injury in rat tubular cell line NRK-52E. In [...] Read more.

Necrostatin-1 (Nec-1) inhibits necroptosis and is usually regarded as having no effect on other cell deaths. Here, this study explored whether the addition of Nec-1 has an effect on cell death induced by simulated ischemia injury in rat tubular cell line NRK-52E. In addition, we also investigated the mechanism of Nec-1 attenuates cell death in this renal ischemia model. The NRK-52E cells were incubated with TNF-α + antimycinA (TA) for 24 h with or without Nec-1. Cell death was observed under fluorescent microscope and quantified by flow cytometry. Cell viabilities were detected by MTT assay. The protein expression of dynamin-related protein 1 (Drp1) was detected by Western blotting and immunofluorescence assay. Increased cell death in simulated ischemia injury of NRK-52E cells were markedly attenuated in the Nec-1 pretreated ischemia injury group. Meanwhile, cell viability was significantly improved after using Nec-1. In addition, we also observed that the protein expression of Drp1, a mediator of mitochondrial fission, was significantly increased in simulated ischemia injury group. Increased Drp1 expression in the ischemia injury group can be abolished by Nec-1 or Drp1-knock down, accompanied with decreased cell death and improved cell viabilities. These results suggest that Nec-1 may inhibit cell death induced by simulated ischemia injury in the rat tubular cell line NRK-52E through decreased Drp1 expression. Full article

(This article belongs to the Collection Programmed Cell Death and Apoptosis)

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384 KiB

Open AccessReview

βArrestins in Cardiac G Protein-Coupled Receptor Signaling and Function: Partners in Crime or “Good Cop, Bad Cop”?

by Anastasios Lymperopoulos and Shmuel Negussie

Int. J. Mol. Sci. 2013, 14(12), 24726-24741; https://doi.org/10.3390/ijms141224726 - 18 Dec 2013

Cited by 27 | Viewed by 7981

Abstract

βarrestin (βarr)-1 and -2 (βarrs) (or Arrestin-2 and -3, respectively) are universal G protein-coupled receptor (GPCR) adapter proteins expressed abundantly in extra-retinal tissues, including the myocardium. Both were discovered in the lab of the 2012 Nobel Prize in Chemistry co-laureate Robert Lefkowitz, initially [...] Read more.

βarrestin (βarr)-1 and -2 (βarrs) (or Arrestin-2 and -3, respectively) are universal G protein-coupled receptor (GPCR) adapter proteins expressed abundantly in extra-retinal tissues, including the myocardium. Both were discovered in the lab of the 2012 Nobel Prize in Chemistry co-laureate Robert Lefkowitz, initially as terminators of signaling from the β-adrenergic receptor (βAR), a process known as functional desensitization. They are now known to switch GPCR signaling from G protein-dependent to G protein-independent, which, in the case of βARs and angiotensin II type 1 receptor (AT₁R), might be beneficial, e.g., anti-apoptotic, for the heart. However, the specific role(s) of each βarr isoform in cardiac GPCR signaling and function (or dysfunction in disease), remain unknown. The current consensus is that, whereas both βarr isoforms can desensitize and internalize cardiac GPCRs, they play quite different (even opposing in certain instances) roles in the G protein-independent signaling pathways they initiate in the cardiovascular system, including in the myocardium. The present review will discuss the current knowledge in the field of βarrs and their roles in GPCR signaling and function in the heart, focusing on the three most important, for cardiac physiology, GPCR types (β₁AR, β₂AR & AT₁R), and will also highlight important questions that currently remain unanswered. Full article

(This article belongs to the Collection G Protein-Coupled Receptor Signaling and Regulation)

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373 KiB

Open AccessReview

Multidrug Resistance and Cancer Stem Cells in Neuroblastoma and Hepatoblastoma

by Anna Alisi, William C. Cho, Franco Locatelli and Doriana Fruci

Int. J. Mol. Sci. 2013, 14(12), 24706-24725; https://doi.org/10.3390/ijms141224706 - 18 Dec 2013

Cited by 89 | Viewed by 9127

Abstract

Chemotherapy is one of the major modalities in treating cancers. However, its effectiveness is limited by the acquisition of multidrug resistance (MDR). Several mechanisms could explain the up-regulation of MDR genes/proteins in cancer after chemotherapy. It is known that cancer stem cells (CSCs) [...] Read more.

Chemotherapy is one of the major modalities in treating cancers. However, its effectiveness is limited by the acquisition of multidrug resistance (MDR). Several mechanisms could explain the up-regulation of MDR genes/proteins in cancer after chemotherapy. It is known that cancer stem cells (CSCs) play a role as master regulators. Therefore, understanding the mechanisms that regulate some traits of CSCs may help design efficient strategies to overcome chemoresistance. Different CSC phenotypes have been identified, including those found in some pediatric malignancies. As solid tumors in children significantly differ from those observed in adults, this review aims at providing an overview of the mechanistic relationship between MDR and CSCs in common solid tumors, and, in particular, focuses on clinical as well as experimental evidence of the relations between CSCs and MDR in neuroblastoma and hepatoblastoma. Finally, some novel approaches, such as concomitant targeting of multiple key transcription factors governing the stemness of CSCs, as well as nanoparticle-based approaches will also be briefly addressed. Full article

(This article belongs to the Section Biochemistry)

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Open AccessArticle

Importance of H-Abstraction in the Final Step of Nitrosoalkane Formation in the Mechanism-Based Inactivation of Cytochrome P450 by Amine-Containing Drugs

by Hajime Hirao, Nandun M. Thellamurege, Pratanphorn Chuanprasit and Kai Xu

Int. J. Mol. Sci. 2013, 14(12), 24692-24705; https://doi.org/10.3390/ijms141224692 - 18 Dec 2013

Cited by 23 | Viewed by 8155

Abstract

The metabolism of amine-containing drugs by cytochrome P450 enzymes (P450s) is prone to form a nitrosoalkane metabolic intermediate (MI), which subsequently coordinates to the heme iron of a P450, to produce a metabolic-intermediate complex (MIC). This type of P450 inhibition, referred to as [...] Read more.

The metabolism of amine-containing drugs by cytochrome P450 enzymes (P450s) is prone to form a nitrosoalkane metabolic intermediate (MI), which subsequently coordinates to the heme iron of a P450, to produce a metabolic-intermediate complex (MIC). This type of P450 inhibition, referred to as mechanism-based inactivation (MBI), presents a serious concern in drug discovery processes. We applied density functional theory (DFT) to the reaction between N-methylhydroxylamine (NMH) and the compound I reactive species of P450, in an effort to elucidate the mechanism of the putative final step of the MI formation in the alkylamine metabolism. Our DFT calculations show that H-abstraction from the hydroxyl group of NMH is the most favorable pathway via which the nitrosoalkane intermediate is produced spontaneously. H-abstraction from the N–H bond was slightly less favorable. In contrast, N-oxidation and H-abstraction from the C–H bond of the methyl group had much higher energy barriers. Hence, if the conversion of NMH to nitrosoalkane is catalyzed by a P450, the reaction should proceed preferentially via H-abstraction, either from the O–H bond or from the N–H bond. Our theoretical analysis of the interaction between the MI and pentacoordinate heme moieties provided further insights into the coordination bond in the MIC. Full article

(This article belongs to the Collection Computational, Structural and Spectroscopic Studies of Enzyme Mechanisms, Inhibition and Dynamics)

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Open AccessArticle

Synthesis, Characterisation, and Evaluation of a Cross-Linked Disulphide Amide-Anhydride-Containing Polymer Based on Cysteine for Colonic Drug Delivery

by Vuanghao Lim, Kok Khiang Peh and Shariza Sahudin

Int. J. Mol. Sci. 2013, 14(12), 24670-24691; https://doi.org/10.3390/ijms141224670 - 18 Dec 2013

Cited by 14 | Viewed by 6746

Abstract

The use of disulphide polymers, a low redox potential responsive delivery, is one strategy for targeting drugs to the colon so that they are specifically released there. The objective of this study was to synthesise a new cross-linked disulphide-containing polymer based on the [...] Read more.

The use of disulphide polymers, a low redox potential responsive delivery, is one strategy for targeting drugs to the colon so that they are specifically released there. The objective of this study was to synthesise a new cross-linked disulphide-containing polymer based on the amino acid cysteine as a colon drug delivery system and to evaluate the efficiency of the polymers for colon targeted drug delivery under the condition of a low redox potential. The disulphide cross-linked polymers were synthesised via air oxidation of 1,2-ethanedithiol and 3-mercapto-N-2-(3-mercaptopropionamide)-3-mercapto propionic anhydride (trithiol monomers) using different ratio combinations. Four types of polymers were synthesised: P10, P11, P151, and P15. All compounds synthesised were characterised by NMR, IR, LC-MS, CHNS analysis, Raman spectrometry, SEM-EDX, and elemental mapping. The synthesised polymers were evaluated in chemical reduction studies that were performed in zinc/acetic acid solution. The suitability of each polymer for use in colon-targeted drug delivery was investigated in vitro using simulated conditions. Chemical reduction studies showed that all polymers were reduced after 0.5–1.0 h, but different polymers had different thiol concentrations. The bacterial degradation studies showed that the polymers were biodegraded in the anaerobic colonic bacterial medium. Degradation was most pronounced for polymer P15. This result complements the general consensus that biodegradability depends on the swellability of polymers in an aqueous environment. Overall, these results suggest that the cross-linked disulphide-containing polymers described herein could be used as coatings for drugs delivered to the colon. Full article

(This article belongs to the Section Biochemistry)

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Open AccessArticle

Human Interleukin 23 Receptor Induces Cell Apoptosis in Mammalian Cells by Intrinsic Mitochondrial Pathway Associated with the Down-Regulation of RAS/Mitogen-Activated Protein Kinase and Signal Transducers and Activators of Transcription factor 3 Signaling Pathways

by Wei-Ye Shi, Chang-Yan Che and Li Liu

Int. J. Mol. Sci. 2013, 14(12), 24656-24669; https://doi.org/10.3390/ijms141224656 - 18 Dec 2013

Cited by 9 | Viewed by 6549

Abstract

The composition of IL-23R complex is similar to that of the IL-12 receptor (IL-12R) complex with a shared IL-12R-β1 chain. The IL-12R-β1 heterodimerizes with IL-23R and IL-12R-β2 to form IL-23R and IL-12R complexes, respectively. The IL-12R-β2 has been shown to function as a [...] Read more.

The composition of IL-23R complex is similar to that of the IL-12 receptor (IL-12R) complex with a shared IL-12R-β1 chain. The IL-12R-β1 heterodimerizes with IL-23R and IL-12R-β2 to form IL-23R and IL-12R complexes, respectively. The IL-12R-β2 has been shown to function as a tumor suppressor gene and apoptotic inducer. However, whether IL-23R also functions in cell apoptosis is currently unknown. In this study, we demonstrate for the first time that overexpression of IL-23R markedly induces cell apoptosis in both 293ET and HeLa cells. The activations of caspase 3 and caspase 9 are induced by IL-23R. Mechanistic study reveals that IL-23R markedly inhibits RAS/MAPK and STAT3 but not STAT1 and PI-3K/Akt signaling pathways in both 293ET and HeLa cells. Overexpression of IL-23R significantly up-regulates IL-12Rβ1 expression but not IL-23α and IL-12β expressions in both cell lines. Therefore, our data strongly indicates that IL-23R is able to induce cell apoptosis by activating the intrinsic mitochondrial pathways associated with the inhibition in RAS/MAPK and STAT3 activations in mammalian cells. Full article

(This article belongs to the Collection Programmed Cell Death and Apoptosis)

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648 KiB

Open AccessArticle

Expression Pattern of Class B Gene PAP3 in Flower Development of Pepper

by Xin Li, Chen Liu, Fengjiao Da, Ning Ma and Huolin Shen

Int. J. Mol. Sci. 2013, 14(12), 24643-24655; https://doi.org/10.3390/ijms141224643 - 18 Dec 2013

Cited by 2 | Viewed by 6392

Abstract

Class B gene APETALA3 (AP3) plays a key role in the development of petals and stamens. Here, we investigated the expression pattern of PAP3 gene (genbank accession number: HM104635) in the buds of cytoplasmic male sterility line 121A and its near-isogenic [...] Read more.

Class B gene APETALA3 (AP3) plays a key role in the development of petals and stamens. Here, we investigated the expression pattern of PAP3 gene (genbank accession number: HM104635) in the buds of cytoplasmic male sterility line 121A and its near-isogenic restorer line 121C at four developmental stages and analyzed the possible association between Class B genes and cytoplasmic male sterility of pepper. Semi-quantitative PCR and quantitative real-time RT-PCR (qRT-PCR) as well as RNA in situ hybridization showed increased expression of PAP3 at late phase of anther development and its higher expression in restorer line compared with sterility line indicating PAP3’s role at late developmental stage of anther and suppressed expression in sterility line. RNA in situ hybridization showed Class B gene features: high abundance in stamen and petal; lower expression in pistil; no expression in sepal. Results of transient expression in onion epidermal cells also showed PAP3 localized in the nucleus, which is consistent with the expression pattern of transcription factors of MADS-box gene family. Full article

(This article belongs to the Section Biochemistry)

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Graphical abstract

2809 KiB

Open AccessReview

Preparation of Magnetic Carbon Nanotubes (Mag-CNTs) for Biomedical and Biotechnological Applications

by Andrea Masotti and Andrea Caporali

Int. J. Mol. Sci. 2013, 14(12), 24619-24642; https://doi.org/10.3390/ijms141224619 - 18 Dec 2013

Cited by 94 | Viewed by 14312

Abstract

Carbon nanotubes (CNTs) have been widely studied for their potential applications in many fields from nanotechnology to biomedicine. The preparation of magnetic CNTs (Mag-CNTs) opens new avenues in nanobiotechnology and biomedical applications as a consequence of their multiple properties embedded within the same [...] Read more.

Carbon nanotubes (CNTs) have been widely studied for their potential applications in many fields from nanotechnology to biomedicine. The preparation of magnetic CNTs (Mag-CNTs) opens new avenues in nanobiotechnology and biomedical applications as a consequence of their multiple properties embedded within the same moiety. Several preparation techniques have been developed during the last few years to obtain magnetic CNTs: grafting or filling nanotubes with magnetic ferrofluids or attachment of magnetic nanoparticles to CNTs or their polymeric coating. These strategies allow the generation of novel versatile systems that can be employed in many biotechnological or biomedical fields. Here, we review and discuss the most recent papers dealing with the preparation of magnetic CNTs and their application in biomedical and biotechnological fields. Full article

(This article belongs to the Special Issue Magnetic Nanoparticles)

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Open AccessArticle

The Antidiabetic Drug Metformin Inhibits the Proliferation of Bladder Cancer Cells in Vitro and in Vivo

by Tao Zhang, Peng Guo, Yinan Zhang, Hui Xiong, Xiao Yu, Shan Xu, Xinyang Wang, Dalin He and Xunbo Jin

Int. J. Mol. Sci. 2013, 14(12), 24603-24618; https://doi.org/10.3390/ijms141224603 - 18 Dec 2013

Cited by 71 | Viewed by 9651

Abstract

Recent studies suggest that metformin, a widely used antidiabetic agent, may reduce cancer risk and improve prognosis of certain malignancies. However, the mechanisms for the anti-cancer effects of metformin remain uncertain. In this study, we investigated the effects of metformin on human bladder [...] Read more.

Recent studies suggest that metformin, a widely used antidiabetic agent, may reduce cancer risk and improve prognosis of certain malignancies. However, the mechanisms for the anti-cancer effects of metformin remain uncertain. In this study, we investigated the effects of metformin on human bladder cancer cells and the underlying mechanisms. Metformin significantly inhibited the proliferation and colony formation of 5637 and T24 cells in vitro; specifically, metformin induced an apparent cell cycle arrest in G0/G1 phases, accompanied by a strong decrease of cyclin D1, cyclin-dependent kinase 4 (CDK4), E2F1 and an increase of p21^waf^-¹. Further experiments revealed that metformin activated AMP-activated protein kinase (AMPK) and suppressed mammalian target of rapamycin (mTOR), the central regulator of protein synthesis and cell growth. Moreover, daily treatment of metformin led to a substantial inhibition of tumor growth in a xenograft model with concomitant decrease in the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and p-mTOR. The in vitro and in vivo results demonstrate that metformin efficiently suppresses the proliferation of bladder cancer cells and suggest that metformin may be a potential therapeutic agent for the treatment of bladder cancer. Full article

(This article belongs to the Special Issue Molecular Bases of Cancer Research)

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Open AccessEssay

Characterization of a κ-Carrageenase from Marine Cellulophaga lytica strain N5-2 and Analysis of Its Degradation Products

by Ziang Yao, Feifei Wang, Zheng Gao, Liming Jin and Haige Wu

Int. J. Mol. Sci. 2013, 14(12), 24592-24602; https://doi.org/10.3390/ijms141224592 - 17 Dec 2013

Cited by 45 | Viewed by 7239

Abstract

A carrageenan-degrading marine Cellulophaga lytica strain N5-2 was isolated from the sediment of carrageenan production base. A κ-carrageenase (EC 3.2.1.83) with high activity was purified to electrophoretic homogeneity from the culture supernatant by a procedure of ammonium sulfate precipitation, dialyzing and gel filtration [...] Read more.

A carrageenan-degrading marine Cellulophaga lytica strain N5-2 was isolated from the sediment of carrageenan production base. A κ-carrageenase (EC 3.2.1.83) with high activity was purified to electrophoretic homogeneity from the culture supernatant by a procedure of ammonium sulfate precipitation, dialyzing and gel filtration on SephadexG-200 and SephadexG-75. The purified enzyme was verified as a single protein on SDS-PAGE, and whose molecular weight was 40.8 kDa. The κ-carrageenase yielded a high activity of 1170 U/mg protein. For κ-carrageenase activity, the optimum temperature and pH were 35 °C and pH 7.0, respectively. The enzyme was stable at 40 °C for at least 2.5 h. The enzyme against κ-carrageenan gave a K_m value of 1.647 mg/mL and a V_max value of 8.7 μmol/min/mg when the reaction was carried out at 35 °C and pH 7.0. The degradation products of the k-carrageenase were analyzed by thin layer chromatography (TLC), high performance liquid chromatography (HPLC), electrospray ionization time-of-flight mass spectroscopy (ESI-TOF-MS) and ¹³C-NMR spectroscopy, and the results indicated that the enzyme was specific of the β-1,4 linkage and hydrolyzed κ-carrageenan into κ-neocarraoctaose-sulfate and κ-neocarrahexaose-sulfate first, and then broke κ-neocarraoctaose-sulfate into κ-neocarrabiose-sulfate and κ-neocarrahexaose-sulfate. Full article

(This article belongs to the Section Biochemistry)

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Open AccessArticle

A Simple Strategy for Development of Single Nucleotide Polymorphisms from Non-Model Species and Its Application in Panax

by Ming Rui Li, Xin Feng Wang, Cui Zhang, Hua Ying Wang, Feng Xue Shi, Hong Xing Xiao and Lin Feng Li

Int. J. Mol. Sci. 2013, 14(12), 24581-24591; https://doi.org/10.3390/ijms141224581 - 17 Dec 2013

Cited by 7 | Viewed by 5795

Abstract

Single nucleotide polymorphisms (SNPs) are widely employed in the studies of population genetics, molecular breeding and conservation genetics. In this study, we explored a simple route to develop SNPs from non-model species based on screening the library of single copy nuclear genes (SCNGs). [...] Read more.

Single nucleotide polymorphisms (SNPs) are widely employed in the studies of population genetics, molecular breeding and conservation genetics. In this study, we explored a simple route to develop SNPs from non-model species based on screening the library of single copy nuclear genes (SCNGs). Through application of this strategy in Panax, we identified 160 and 171 SNPs from P. quinquefolium and P. ginseng, respectively. Our results demonstrated that both P. ginseng and P. quinquefolium possessed a high level of nucleotide diversity. The number of haplotype per locus ranged from 1 to 12 for P. ginseng and from 1 to 9 for P. quinquefolium, respectively. The nucleotide diversity of total sites (π_T) varied between 0.000 and 0.023 for P. ginseng and 0.000 and 0.035 for P. quinquefolium, respectively. These findings suggested that this approach is well suited for SNP discovery in non-model organisms and is easily employed in standard genetics laboratory studies. Full article

(This article belongs to the Section Biochemistry)

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Open AccessArticle

Towards Lipidomics of Low-Abundant Species for Exploring Tumor Heterogeneity Guided by High-Resolution Mass Spectrometry Imaging

by Jonathan Cimino, David Calligaris, Johann Far, Delphine Debois, Silvia Blacher, Nor Eddine Sounni, Agnès Noel and Edwin De Pauw

Int. J. Mol. Sci. 2013, 14(12), 24560-24580; https://doi.org/10.3390/ijms141224560 - 17 Dec 2013

Cited by 24 | Viewed by 9387

Abstract

Many studies have evidenced the main role of lipids in physiological and also pathological processes such as cancer, diabetes or neurodegenerative diseases. The identification and the in situ localization of specific low-abundant lipid species involved in cancer biology are still challenging for both [...] Read more.

Many studies have evidenced the main role of lipids in physiological and also pathological processes such as cancer, diabetes or neurodegenerative diseases. The identification and the in situ localization of specific low-abundant lipid species involved in cancer biology are still challenging for both fundamental studies and lipid marker discovery. In this paper, we report the identification and the localization of specific isobaric minor phospholipids in human breast cancer xenografts by FTICR MALDI imaging supported by histochemistry. These potential candidates can be further confirmed by liquid chromatography coupled with electrospray mass spectrometry (LC-ESI-MS) after extraction from the region of interest defined by MALDI imaging. Finally, this study highlights the importance of characterizing the heterogeneous distribution of low-abundant lipid species, relevant in complex histological samples for biological purposes. Full article

(This article belongs to the Special Issue Fourier Transform Mass Spectrometry in Molecular Sciences)

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518 KiB

Open AccessArticle

EGFR Mutations in Surgically Resected Fresh Specimens from 697 Consecutive Chinese Patients with Non-Small Cell Lung Cancer and Their Relationships with Clinical Features

by Yuanyang Lai, Zhipei Zhang, Jianzhong Li, Dong Sun, Yong'an Zhou, Tao Jiang, Yong Han, Lijun Huang, Yifang Zhu, Xiaofei Li and Xiaolong Yan

Int. J. Mol. Sci. 2013, 14(12), 24549-24559; https://doi.org/10.3390/ijms141224549 - 17 Dec 2013

Cited by 44 | Viewed by 7862

Abstract

We aimed to reveal the true status of epidermal growth factor receptor (EGFR) mutations in Chinese patients with non-small cell lung cancer (NSCLC) after lung resections. EGFR mutations of surgically resected fresh tumor samples from 697 Chinese NSCLC patients were analyzed [...] Read more.

We aimed to reveal the true status of epidermal growth factor receptor (EGFR) mutations in Chinese patients with non-small cell lung cancer (NSCLC) after lung resections. EGFR mutations of surgically resected fresh tumor samples from 697 Chinese NSCLC patients were analyzed by Amplification Refractory Mutation System (ARMS). Correlations between EGFR mutation hotspots and clinical features were also explored. Of the 697 NSCLC patients, 235 (33.7%) patients had tyrosine kinase inhibitor (TKIs) sensitive EGFR mutations in 41 (14.5%) of the 282 squamous carcinomas, 155 (52.9%) of the 293 adenocarcinomas, 34 (39.5%) of the 86 adenosquamous carcinomas, one (9.1%) of the 11 large-cell carcinomas, 2 (11.1%) of the 18 sarcomatoid carcinomas, and 2 (28.6%) of the 7 mucoepidermoid carcinomas. TKIs sensitive EGFR mutations were more frequently found in female patients (p < 0.001), non-smokers (p = 0.047) and adenocarcinomas (p < 0.001). The rates of exon 19 deletion mutation (19-del), exon 21 L858R point mutation (L858R), exon 21 L861Q point mutation (L861Q), exon 18 G719X point mutations (G719X, including G719C, G719S, G719A) were 43.4%, 48.1%, 1.7% and 6.8%, respectively. Exon 20 T790M point mutation (T790M) was detected in 3 squamous carcinomas and 3 adenocarcinomas and exon 20 insertion mutation (20-ins) was detected in 2 patients with adenocarcinoma. Our results show the rates of EGFR mutations are higher in all types of NSCLC in Chinese patients. 19-del and L858R are two of the more frequent mutations. EGFR mutation detection should be performed as a routine postoperative examination in Chinese NSCLC patients. Full article

(This article belongs to the Special Issue Advances in Cancer Diagnosis)

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Open AccessArticle

Cloud Point Extraction of Parabens Using Non-Ionic Surfactant with Cylodextrin Functionalized Ionic Liquid as a Modifier

by Md Saleh Noorashikin, Muggundha Raoov, Sharifah Mohamad and Mhd Radzi Abas

Int. J. Mol. Sci. 2013, 14(12), 24531-24548; https://doi.org/10.3390/ijms141224531 - 17 Dec 2013

Cited by 25 | Viewed by 7484

Abstract

A cloud point extraction (CPE) process using non-ionic surfactant (DC193C) to extract selected paraben compounds from water samples was investigated using reversed phase high performance liquid chromatography (RP-HPLC). The CPE process with the presence of β-cyclodextrin (βCD) functionalized ionic liquid as a modifier [...] Read more.

A cloud point extraction (CPE) process using non-ionic surfactant (DC193C) to extract selected paraben compounds from water samples was investigated using reversed phase high performance liquid chromatography (RP-HPLC). The CPE process with the presence of β-cyclodextrin (βCD) functionalized ionic liquid as a modifier (CPE-DC193C-βCD-IL) is a new extraction technique that has been applied on the optimization of parameters, i.e., pH, βCD-IL concentration and phase volume ratio. This CPE-DC193C-βCD-IL method is facilitated at 30 °C, showing great losses of water content in the surfactant-rich phase, resulting in a high pre-concentration factor and high distribution coefficient. The developed method CPE-DC193C-βCD-IL did show enhanced properties compared to the CPE method without the modifier (CPE-DC193C). The developed method of CPE-DC193C-βCD-IL gives an excellent performance on the detection of parabens from water samples with the limit of detection falling in the range of 0.013–0.038 µg mL⁻¹. Finally, the inclusion complex formation, hydrogen bonding, and π–π interaction between the βCD-IL, benzyl paraben (ArP), and DC 193C were proven using ¹H NMR and 2D NOESY spectroscopy. Full article

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

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Open AccessArticle

CYP 2D6 Binding Affinity Predictions Using Multiple Ligand and Protein Conformations

by Lovorka Perić-Hassler, Eva Stjernschantz, Chris Oostenbrink and Daan P. Geerke

Int. J. Mol. Sci. 2013, 14(12), 24514-24530; https://doi.org/10.3390/ijms141224514 - 17 Dec 2013

Cited by 29 | Viewed by 7209

Abstract

Because of the large flexibility and malleability of Cytochrome P450 enzymes (CYPs), in silico prediction of CYP binding affinities to drugs and other xenobiotic compounds is a true challenge. In the current work, we use an iterative linear interaction energy (LIE) approach to [...] Read more.

Because of the large flexibility and malleability of Cytochrome P450 enzymes (CYPs), in silico prediction of CYP binding affinities to drugs and other xenobiotic compounds is a true challenge. In the current work, we use an iterative linear interaction energy (LIE) approach to compute CYP binding affinities from molecular dynamics (MD) simulation. In order to improve sampling of conformational space, we combine results from simulations starting with different relevant protein-ligand geometries. For calculated binding free energies of a set of thiourea compounds binding to the flexible CYP 2D6 isoform, improved correlation with experiment was obtained by combining results of MD runs starting from distinct protein conformations and ligand-binding orientations. This accuracy was obtained from relatively short MD simulations, which makes our approach computationally attractive for automated calculations of ligand-binding affinities to flexible proteins such as CYPs. Full article

(This article belongs to the Special Issue Xenobiotic Metabolism)

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Graphical abstract

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Open AccessArticle

Identification and Biochemical Characterization of Protein Phosphatase 5 from the Cantharidin-Producing Blister Beetle, Epicauta chinensis

by Xi'en Chen, Shumin Lü and Yalin Zhang

Int. J. Mol. Sci. 2013, 14(12), 24501-24513; https://doi.org/10.3390/ijms141224501 - 16 Dec 2013

Cited by 4 | Viewed by 5898

Abstract

Protein phosphatase 5 (PP5) is a unique member of serine/threonine phosphatases which has been recognized in regulation of diverse cellular processes. A cDNA fragment encoding PP5 (EcPP5) was cloned and characterized from the cantharidin-producing blister beetle, E. chinensis. EcPP5 contains [...] Read more.

Protein phosphatase 5 (PP5) is a unique member of serine/threonine phosphatases which has been recognized in regulation of diverse cellular processes. A cDNA fragment encoding PP5 (EcPP5) was cloned and characterized from the cantharidin-producing blister beetle, E. chinensis. EcPP5 contains an open reading frame of 1500 bp that encodes a protein of 56.89 kDa. The deduced amino acid sequence shares 88% and 68% identities to the PP5 of Tribolium castaneum and humans, respectively. Analysis of the primary sequence shows that EcPP5 has three TPR (tetratricopeptide repeat) motifs at its N-terminal region and contains a highly conserved C-terminal catalytic domain. RT-PCR reveals that EcPP5 is expressed in all developmental stages and in different tissues. The recombinant EcPP5 (rEcPP5) was produced in Escherichia coli and purified to homogeneity. The purified protein exhibited phosphatase activity towards pNPP (p-nitrophenyl phosphate) and phosphopeptides, and its activity can be enhanced by arachidonic acid. In vitro inhibition study revealed that protein phosphatase inhibitors, okadaic acid, cantharidin, norcantharidin and endothall, inhibited its activity. Further, protein phosphatase activity of total soluble protein extract from E. chinensis adults could be impeded by these inhibitors suggesting there might be some mechanism to protect this beetle from being damaged by its self-produced cantharidin. Full article

(This article belongs to the Section Biochemistry)

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Open AccessReview

Bioresorbable Drug-Eluting Magnesium-Alloy Scaffold for Treatment of Coronary Artery Disease

by Carlos M. Campos, Takashi Muramatsu, Javaid Iqbal, Yao-Jun Zhang, Yoshinobu Onuma, Hector M. Garcia-Garcia, Michael Haude, Pedro A. Lemos, Boris Warnack and Patrick W. Serruys

Int. J. Mol. Sci. 2013, 14(12), 24492-24500; https://doi.org/10.3390/ijms141224492 - 16 Dec 2013

Cited by 113 | Viewed by 9606

Abstract

The introduction of metallic drug-eluting stents has reduced the risk of restenosis and widened the indications of percutaneous coronary intervention in treatment of coronary artery disease. However, this medical device can induce hypersensitive reaction that interferes with the endothelialization and healing process resulting [...] Read more.

The introduction of metallic drug-eluting stents has reduced the risk of restenosis and widened the indications of percutaneous coronary intervention in treatment of coronary artery disease. However, this medical device can induce hypersensitive reaction that interferes with the endothelialization and healing process resulting in late persistent or acquired malapposition of the permanent metallic implant. Delayed endotheliaization and malapposition may lead to late and very late stent thrombosis. Bioresorbable scaffolds (BRS) have been introduced to potentially overcome these limitations, as they provide temporary scaffolding and then disappear, liberating the treated vessel from its cage. Magnesium is an essential mineral needed for a variety of physiological functions in the human body and its bioresorbable alloy has the strength-to-weight ratio comparable with that of strong aluminum alloys and alloy steels. The aim of this review is to present the new developments in Magnesium BRS technology, to describe its clinical application and to discuss the future prospects of this innovative therapy. Full article

(This article belongs to the Special Issue Biodegradable Magnesium Alloys and Implants)

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Atorvastatin Attenuates Bleomycin-Induced Pulmonary Fibrosis via Suppressing iNOS Expression and the CTGF (CCN2)/ERK Signaling Pathway

by Bo Zhu, Ai-Qun Ma, Lan Yang and Xiao-Min Dang

Int. J. Mol. Sci. 2013, 14(12), 24476-24491; https://doi.org/10.3390/ijms141224476 - 16 Dec 2013

Cited by 56 | Viewed by 7787

Abstract

Pulmonary fibrosis is a progressive and fatal lung disorder with high mortality rate. To date, despite the fact that extensive research trials are ongoing, pulmonary fibrosis continues to have a poor response to available medical therapy. Statins, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, known for [...] Read more.

Pulmonary fibrosis is a progressive and fatal lung disorder with high mortality rate. To date, despite the fact that extensive research trials are ongoing, pulmonary fibrosis continues to have a poor response to available medical therapy. Statins, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, known for its broad pharmacological activities, remains a remedy against multiple diseases. The present study investigated the antifibrotic potential of atorvastatin against bleomycin-induced lung fibrosis and to further explore the possible underlying mechanisms. Our results showed that atorvastatin administration significantly ameliorated the bleomycin mediated histological alterations and blocked collagen deposition with parallel reduction in the hydroxyproline level. Atorvastatin reduced malondialdehyde (MDA) level and lung indices. Atorvastatin also markedly decreased the expression of inducible nitric oxide synthase (iNOS) in lung tissues and, thus, prevented nitric oxide (NO) release in response to bleomycin challenge. Furthermore, atorvastatin exhibited target down-regulation of connective tissue growth factor (CTGF (CCN2)) and phosphorylation extracellular regulated protein kinases (p-ERK) expression. Taken together, atorvastatin significantly ameliorated bleomycin-induced pulmonary fibrosis in rats, via the inhibition of iNOS expression and the CTGF (CCN2)/ERK signaling pathway. The present study provides evidence that atorvastatin may be a potential therapeutic reagent for the treatment of lung fibrosis. Full article

(This article belongs to the Section Biochemistry)

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Open AccessReview

Oxidative Stress and Neurodegenerative Disorders

by Jie Li, Wuliji O, Wei Li, Zhi-Gang Jiang and Hossein A. Ghanbari

Int. J. Mol. Sci. 2013, 14(12), 24438-24475; https://doi.org/10.3390/ijms141224438 - 16 Dec 2013

Cited by 334 | Viewed by 22577

Abstract

Living cells continually generate reactive oxygen species (ROS) through the respiratory chain during energetic metabolism. ROS at low or moderate concentration can play important physiological roles. However, an excessive amount of ROS under oxidative stress would be extremely deleterious. The central nervous system [...] Read more.

Living cells continually generate reactive oxygen species (ROS) through the respiratory chain during energetic metabolism. ROS at low or moderate concentration can play important physiological roles. However, an excessive amount of ROS under oxidative stress would be extremely deleterious. The central nervous system (CNS) is particularly vulnerable to oxidative stress due to its high oxygen consumption, weakly antioxidative systems and the terminal-differentiation characteristic of neurons. Thus, oxidative stress elicits various neurodegenerative diseases. In addition, chemotherapy could result in severe side effects on the CNS and peripheral nervous system (PNS) of cancer patients, and a growing body of evidence demonstrates the involvement of ROS in drug-induced neurotoxicities as well. Therefore, development of antioxidants as neuroprotective drugs is a potentially beneficial strategy for clinical therapy. In this review, we summarize the source, balance maintenance and physiologic functions of ROS, oxidative stress and its toxic mechanisms underlying a number of neurodegenerative diseases, and the possible involvement of ROS in chemotherapy-induced toxicity to the CNS and PNS. We ultimately assess the value for antioxidants as neuroprotective drugs and provide our comments on the unmet needs. Full article

(This article belongs to the Special Issue Molecular Research in Neurotoxicology)

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Open AccessArticle

Maternal Methyl Donors Supplementation during Lactation Prevents the Hyperhomocysteinemia Induced by a High-Fat-Sucrose Intake by Dams

by Paul Cordero, Fermin I. Milagro, Javier Campion and J. Alfredo Martinez

Int. J. Mol. Sci. 2013, 14(12), 24422-24437; https://doi.org/10.3390/ijms141224422 - 16 Dec 2013

Cited by 38 | Viewed by 7483

Abstract

Maternal perinatal nutrition may program offspring metabolic features. Epigenetic regulation is one of the candidate mechanisms that may be affected by maternal dietary methyl donors intake as potential controllers of plasma homocysteine levels. Thirty-two Wistar pregnant rats were randomly assigned into four dietary [...] Read more.

Maternal perinatal nutrition may program offspring metabolic features. Epigenetic regulation is one of the candidate mechanisms that may be affected by maternal dietary methyl donors intake as potential controllers of plasma homocysteine levels. Thirty-two Wistar pregnant rats were randomly assigned into four dietary groups during lactation: control, control supplemented with methyl donors, high-fat-sucrose and high-fat-sucrose supplemented with methyl donors. Physiological outcomes in the offspring were measured, including hepatic mRNA expression and global DNA methylation after weaning. The newborns whose mothers were fed the obesogenic diet were heavier longer and with a higher adiposity and intrahepatic fat content. Interestingly, increased levels of plasma homocysteine induced by the maternal high-fat-sucrose dietary intake were prevented in both sexes by maternal methyl donors supplementation. Total hepatic DNA methylation decreased in females due to maternal methyl donors administration, while Dnmt3a hepatic mRNA levels decreased accompanying the high-fat-sucrose consumption. Furthermore, a negative association between Dnmt3a liver mRNA levels and plasma homocysteine concentrations was found. Maternal high-fat-sucrose diet during lactation could program offspring obesity features, while methyl donors supplementation prevented the onset of high hyperhomocysteinemia. Maternal dietary intake also affected hepatic DNA methylation metabolism, which could be linked with the regulation of the methionine-homocysteine cycle. Full article

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

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Open AccessEditorial

Biological Functional Relevance of Asymmetric Dimethylarginine (ADMA) in Cardiovascular Disease

by Sara Franceschelli, Alessio Ferrone, Mirko Pesce, Graziano Riccioni and Lorenza Speranza

Int. J. Mol. Sci. 2013, 14(12), 24412-24421; https://doi.org/10.3390/ijms141224412 - 16 Dec 2013

Cited by 37 | Viewed by 8846

Abstract

There is growing evidence that increased levels of the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) may contribute to endothelial dysfunction. Studies in animal models as well as in humans have suggested that the increase in ADMA occurs at a time when vascular [...] Read more.

There is growing evidence that increased levels of the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) may contribute to endothelial dysfunction. Studies in animal models as well as in humans have suggested that the increase in ADMA occurs at a time when vascular disease has not yet become clinically evident. ADMA competitively inhibits NO elaboration by displacing L-arginine from NO synthase. In a concentration-dependent manner, it thereby interferes not only with endothelium-dependent, NO-mediated vasodilation, but also with other biological functions exerted by NO. The upshot may be a pro-atherogenic state. Recently, several studies have investigated the effect of various therapeutical interventions on ADMA plasma concentrations. [...] Full article

(This article belongs to the Special Issue ADMA and Nitrergic System)

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Open AccessArticle

Cbl-b Enhances Sensitivity to 5-Fluorouracil via EGFR- and Mitochondria-Mediated Pathways in Gastric Cancer Cells

by Dan Feng, Yanju Ma, Jing Liu, Ling Xu, Ye Zhang, Jinglei Qu, Yunpeng Liu and Xiujuan Qu

Int. J. Mol. Sci. 2013, 14(12), 24399-24411; https://doi.org/10.3390/ijms141224399 - 16 Dec 2013

Cited by 21 | Viewed by 7049

Abstract

5-Fluorouracil (5-FU) is an essential component of anticancer chemotherapy against gastric cancer. However, the response rate of single drug is still limited. The ubiquitin ligase Cbl-b is a negative regulator of growth factor receptor signaling and is involved in the suppression of cancer [...] Read more.

5-Fluorouracil (5-FU) is an essential component of anticancer chemotherapy against gastric cancer. However, the response rate of single drug is still limited. The ubiquitin ligase Cbl-b is a negative regulator of growth factor receptor signaling and is involved in the suppression of cancer cell proliferation. However, whether Cbl-b could affect 5-FU sensitivity remains unclear. The present study showed that Cbl-b knockdown caused higher proliferation concomitant with the decrease of apoptosis induced by 5-FU treatment in gastric cancer cell. Further mechanism investigation demonstrated that Cbl-b knockdown caused significant increase of phosphorylation of EGFR, ERK and Akt, decrease of mitochondrial membrane potential, and increase of expression ratio of Bcl-2/Bax. These results suggest that Cbl-b enhances sensitivity to 5-FU via EGFR- and mitochondria-mediated pathways in gastric cancer cells. Full article

(This article belongs to the Section Biochemistry)

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Open AccessArticle

Cardiac Ablation of Rheb1 Induces Impaired Heart Growth, Endoplasmic Reticulum-Associated Apoptosis and Heart Failure in Infant Mice

by Yunshan Cao, Lichan Tao, Shutong Shen, Junjie Xiao, Hang Wu, Beibei Li, Xiangqi Wu, Wen Luo, Qi Xiao, Xiaoshan Hu, Hailang Liu, Junwei Nie, Shuangshuang Lu, Baiyin Yuan, Zhonglin Han, Bo Xiao, Zhongzhou Yang and Xinli Li

Int. J. Mol. Sci. 2013, 14(12), 24380-24398; https://doi.org/10.3390/ijms141224380 - 13 Dec 2013

Cited by 14 | Viewed by 7645

Abstract

Ras homologue enriched in brain 1 (Rheb1) plays an important role in a variety of cellular processes. In this study, we investigate the role of Rheb1 in the post-natal heart. We found that deletion of the gene responsible for production of Rheb1 from [...] Read more.

Ras homologue enriched in brain 1 (Rheb1) plays an important role in a variety of cellular processes. In this study, we investigate the role of Rheb1 in the post-natal heart. We found that deletion of the gene responsible for production of Rheb1 from cardiomyocytes of post-natal mice resulted in malignant arrhythmias, heart failure, and premature death of these mice. In addition, heart growth impairment, aberrant metabolism relative gene expression, and increased cardiomyocyte apoptosis were observed in Rheb1-knockout mice prior to the development of heart failure and arrhythmias. Also, protein kinase B (PKB/Akt) signaling was enhanced in Rheb1-knockout mice, and removal of phosphatase and tensin homolog (Pten) significantly prolonged the survival of Rheb1-knockouts. Furthermore, signaling via the mammalian target of rapamycin complex 1 (mTORC1) was abolished and C/EBP homologous protein (CHOP) and phosphorylation levels of c-Jun N-terminal kinase (JNK) were increased in Rheb1 mutant mice. In conclusion, this study demonstrates that Rheb1 is important for maintaining cardiac function in post-natal mice via regulation of mTORC1 activity and stress on the endoplasmic reticulum. Moreover, activation of Akt signaling helps to improve the survival of mice with advanced heart failure. Thus, this study provides direct evidence that Rheb1 performs multiple important functions in the heart of the post-natal mouse. Enhancing Akt activity improves the survival of infant mice with advanced heart failure. Full article

(This article belongs to the Section Biochemistry)

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Improved Bonding of Partially Osteomyelitic Bone to Titanium Pins Owing to Biomimetic Coating of Apatite

by Hirotaka Mutsuzaki, Yu Sogo, Ayako Oyane and Atsuo Ito

Int. J. Mol. Sci. 2013, 14(12), 24366-24379; https://doi.org/10.3390/ijms141224366 - 13 Dec 2013

Cited by 13 | Viewed by 6316 | Correction

Abstract

Increased fixation strength of the bone-pin interface is important for inhibiting pin loosening after external fixation. In a previous study, an apatite (Ap) layer was formed on anodically oxidized titanium (Ti) pins by immersing them in an infusion fluid-based supersaturated calcium phosphate solution [...] Read more.

Increased fixation strength of the bone-pin interface is important for inhibiting pin loosening after external fixation. In a previous study, an apatite (Ap) layer was formed on anodically oxidized titanium (Ti) pins by immersing them in an infusion fluid-based supersaturated calcium phosphate solution at 37 °C for 48 h. In the present study, an Ap layer was also successfully formed using a one-step method at 25 °C for 48 h in an infusion fluid-based supersaturated calcium phosphate solution, which is clinically useful due to the immersion temperature. After percutaneous implantation in a proximal tibial metaphysis for four weeks in rabbits (n = 20), the Ti pin coated with the Ap layer showed significantly increased extraction torque compared with that of an uncoated Ti screw even with partial osteomyelitis present, owing to dense bone formation on the Ap layer in the cortical and medullary cavity regions. When the infection status was changed from “no osteomyelitis” to “partial osteomyelitis,” the extraction torque in the Ap group with “partial osteomyelitis” was almost identical to that for “no osteomyelitis” cases. These results suggest that the Ap layer formed by the room temperature process could effectively improve the fixation strength of the Ti pin for external fixation clinically even with partial osteomyelitis present. Full article

(This article belongs to the Special Issue Biologic Coatings for Orthopaedic Implant)

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Open AccessArticle

Effect of Exogenous Factors on Bacteriocin Production from Lactobacillus paracasei J23 by Using a Resting Cell System

by Huaxi Yi, Xue Han, Yanyan Yang, Wenli Liu, Hui Liu, Yingchun Zhang, Kai Sun, Lanwei Zhang and Fang Ma

Int. J. Mol. Sci. 2013, 14(12), 24355-24365; https://doi.org/10.3390/ijms141224355 - 13 Dec 2013

Cited by 19 | Viewed by 6824

Abstract

A resting cell system was developed for bacteriocin Lac-B23 production from Lactobacillus paracasei J23. The resting cell medium contained (g/L): Glucose 20, Sodium acetate 5.0, MnSO₄ 0.25 MgSO₄ 0.5, Ammoniumhydrogencitrate 1.0, KH₂PO₄ 1.0. The resting cell incubation time [...] Read more.

A resting cell system was developed for bacteriocin Lac-B23 production from Lactobacillus paracasei J23. The resting cell medium contained (g/L): Glucose 20, Sodium acetate 5.0, MnSO₄ 0.25 MgSO₄ 0.5, Ammoniumhydrogencitrate 1.0, KH₂PO₄ 1.0. The resting cell incubation time and temperature were 20 h and 37 °C and the effects of exogenous factors, including amino acids, glycerol, pyruvic acid, and α-ketoglutaric acid were investigated. Cys and Gly could stimulate the production of bacteriocin, while no stimulus effect was observed for Glu, Tyr and Ala. Glycerol and pyruvic acid increased bacteriocin production and the optimum concentrations were 1% and 30 g/L, respectively. Bacteriocin could act as an inducer of its own biosynthesis. These findings are of importance for the further study of bacteriocin biosynthesis regulation and for the improvement of bacteriocin production yields. Full article

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Selection of Reliable Reference Genes for Gene Expression Studies in the Biofuel Plant Jatropha curcas Using Real-Time Quantitative PCR

by Lu Zhang, Liang-Liang He, Qian-Tang Fu and Zeng-Fu Xu

Int. J. Mol. Sci. 2013, 14(12), 24338-24354; https://doi.org/10.3390/ijms141224338 - 13 Dec 2013

Cited by 65 | Viewed by 13010

Abstract

Jatropha curcas is a promising renewable feedstock for biodiesel and bio-jet fuel production. To study gene expression in Jatropha in different tissues throughout development and under stress conditions, we examined a total of 11 typical candidate reference genes using real-time quantitative polymerase chain [...] Read more.

Jatropha curcas is a promising renewable feedstock for biodiesel and bio-jet fuel production. To study gene expression in Jatropha in different tissues throughout development and under stress conditions, we examined a total of 11 typical candidate reference genes using real-time quantitative polymerase chain reaction (RT-qPCR) analysis, which is widely used for validating transcript levels in gene expression studies. The expression stability of these candidate reference genes was assessed across a total of 20 samples, including various tissues at vegetative and reproductive stages and under desiccation and cold stress treatments. The results obtained using software qBase^PLUS showed that the top-ranked reference genes differed across the sample subsets. The combination of actin, GAPDH, and EF1α would be appropriate as a reference panel for normalizing gene expression data across samples at different developmental stages; the combination of actin, GAPDH, and TUB5 should be used as a reference panel for normalizing gene expression data across samples under various abiotic stress treatments. With regard to different developmental stages, we recommend the use of actin and TUB8 for normalization at the vegetative stage and GAPDH and EF1α for normalization at the reproductive stage. For abiotic stress treatments, we recommend the use of TUB5 and TUB8 for normalization under desiccation stress and GAPDH and actin for normalization under cold stress. These results are valuable for future research on gene expression during development or under abiotic stress in Jatropha. To our knowledge, this is the first report on the stability of reference genes in Jatropha. Full article

(This article belongs to the Section Biochemistry)

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Comparability of in Vitro Tests for Bioactive Nanoparticles: A Common Assay to Detect Reactive Oxygen Species as an Example

by Matthias Roesslein, Cordula Hirsch, Jean-Pierre Kaiser, Harald F. Krug and Peter Wick

Int. J. Mol. Sci. 2013, 14(12), 24320-24337; https://doi.org/10.3390/ijms141224320 - 13 Dec 2013

Cited by 78 | Viewed by 9557

Abstract

The release of reactive oxygen species (ROS) during the electron transport of mitochondrial aerobic respiration is the major source of ROS. However, contact between cells and nanoparticles (NPs) can also induce release of ROS, leading to an imbalance towards the pro-oxidative state. At [...] Read more.

The release of reactive oxygen species (ROS) during the electron transport of mitochondrial aerobic respiration is the major source of ROS. However, contact between cells and nanoparticles (NPs) can also induce release of ROS, leading to an imbalance towards the pro-oxidative state. At low levels of ROS production, cells initiate a protective response to guarantee their survival, but an excess of ROS can damage cellular compounds such as membranes and various organelles, or directly cause genotoxicity. Thus an elevated level of ROS is an important indicator of cellular stress and an accurate recording of this parameter would be very informative. ROS can be measured by various assays, but all known assays measuring and quantifying ROS possess certain weaknesses. The problems and challenges of quantitatively detecting ROS in vitro using the 2',7'-dichlorodihydrofluorescein (DCF) assay is discussed as an example. In addition, we debate the difficulties in finding a suitable and stable chemical reaction control for the DCF assay (or other ROS-detecting assays). As a conclusion, we believe that using 3-morpholinosydnonimine hydrochloride (Sin-1) as a ROS inducer in the DCF assay is feasible only qualitatively. However, a quantitative measurement of the absolute amount of ROS produced and a quantitative comparison between experiments is (at the moment) impossible. Full article

(This article belongs to the Special Issue Bioactive Nanoparticles 2014)

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The Preparation of Capsaicin-Chitosan Microspheres (CCMS) Enteric Coated Tablets

by Jian Chen, Gui-Dong Huang, Si-Rong Tan, Jiao Guo and Zheng-Quan Su

Int. J. Mol. Sci. 2013, 14(12), 24305-24319; https://doi.org/10.3390/ijms141224305 - 13 Dec 2013

Cited by 19 | Viewed by 9407

Abstract

This study aimed to research the preparation and content determination of capsaicin-chitosan microspheres (CCMS) enteric coated tablets. The core tablets were prepared with the method of wet granulation. Nine formulae were designed to determine the optimal formula of the core tablet. Eudragit L100 [...] Read more.

This study aimed to research the preparation and content determination of capsaicin-chitosan microspheres (CCMS) enteric coated tablets. The core tablets were prepared with the method of wet granulation. Nine formulae were designed to determine the optimal formula of the core tablet. Eudragit L100 was used to prepare the CCMS enteric-coated tablets. The effect of enteric coated formulation variables such as content of talc (10%, 25% and 40%), plasticisers (TEC and DBS), dosage of plasticiser (10%, 20% and 30%) and coating weight (2%, 3% and 5%) were evaluated for drug release characteristics. The in vitro release was studied using 0.1 N HCl and pH 6.8 phosphate buffer. Enteric coated tablets without ruptures or swelling behaviour over 2 h in 0.1 N HCl indicated that these tablets showed acid resistance. The accumulated release rate in phosphate buffer (pH 6.8) revealed that the prepared tablets were able to sustain drug release into the intestine and a first-order release was obtained for capsaicin. This research is the first report of the preparation and content determination of CCMS enteric coated tablets. The sustained release behavior of enteric coated formulations in pH 6.8 phosphate buffer demonstrated that it would be a potential drug delivery platform for sustained delivery of gastric irritant drugs. Full article

(This article belongs to the Special Issue Bioactive Nanoparticles 2014)

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Effect of the GLP-1 Analog Exendin-4 and Oxaliplatin on Intrahepatic Cholangiocarcinoma Cell Line and Mouse Model

by Ben-Dong Chen, Wen-Chao Zhao, Qing-An Jia, Wen-Yan Zhou, Yang Bu, Zuo-Zheng Wang, Feng Wang, Wu-Jun Wu and Qi Wang

Int. J. Mol. Sci. 2013, 14(12), 24293-24304; https://doi.org/10.3390/ijms141224293 - 13 Dec 2013

Cited by 15 | Viewed by 6958

Abstract

The influence of Glucagon-like peptide-1 (GLP-1) and Exendin-4 on development of intrahepatic cholangiocarcinoma (ICC) is evaluated in the study. In vitro tests, including acute toxicity test, cell colony formation assays, cells proliferation and apoptosis, transwell assay, were performed. An ICC in situ tumor [...] Read more.

The influence of Glucagon-like peptide-1 (GLP-1) and Exendin-4 on development of intrahepatic cholangiocarcinoma (ICC) is evaluated in the study. In vitro tests, including acute toxicity test, cell colony formation assays, cells proliferation and apoptosis, transwell assay, were performed. An ICC in situ tumor animal model was established. Then, animals were randomly divided into four groups (n = 6): control, Exendin-4 treatment, oxaliplatin treatment and Exendin-4-oxaliplatin treatment. Animals in the Exendin-4 treatment and Exendin-4-oxaliplatin treatment groups received a subcutaneous injection of Exendin-4 (100 μg/kg/day) for 1 week, and then received oxaliplatin (10 mg/kg/week) by tail vein injection. Animals in the control group received PBS. Immunohistochemistry tests were used for PCNA, Ki67, Caspase 3 expression in tumor tissue. Results show that that, after incubation of human cholangiocarcinoma cell lines, HuCCTI and GLP-1, or HuCCTI and Exendin-4, colony formation number was sharply decreased. However, GLP-1, HuCCTI or Exendin-4 did not affect the colony of normal cells. Combination treatment with oxaliplatin and Exendin-4 can significantly inhibit tumor cells’ proliferation and promote apoptosis. The combined effect is stronger than that of oxaliplatin or Exendin-4. Combination treatment with oxaliplatin and Exendin4 can significantly decrease Ki67 and PCNA proteins’ expression in subcutaneous tumors of nude mice. The inhibitory effect of Combination treatment with oxaliplatin and Exendin4 is clearly stronger than that of oxaliplatin. In addition, Combination treatment with oxaliplatin and Exendin4 can significantly increase Caspase3 protein positive expression. In short, these results show that combination treatment with oxaliplatin and Exendin4 can inhibit tumor cells’ proliferation, and promote apoptosis. Full article

(This article belongs to the Section Biochemistry)

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Effect of Ion Concentration Changes in the Limited Extracellular Spaces on Sarcolemmal Ion Transport and Ca²⁺ Turnover in a Model of Human Ventricular Cardiomyocyte

by Dana Hrabcová, Michal Pásek, Jiří Šimurda and Georges Christé

Int. J. Mol. Sci. 2013, 14(12), 24271-24292; https://doi.org/10.3390/ijms141224271 - 13 Dec 2013

Cited by 11 | Viewed by 7379

Abstract

We have developed a computer model of human cardiac ventricular myocyte (CVM), including t-tubular and cleft spaces with the aim of evaluating the impact of accumulation-depletion of ions in restricted extracellular spaces on transmembrane ion transport and ionic homeostasis in human CVM. The [...] Read more.

We have developed a computer model of human cardiac ventricular myocyte (CVM), including t-tubular and cleft spaces with the aim of evaluating the impact of accumulation-depletion of ions in restricted extracellular spaces on transmembrane ion transport and ionic homeostasis in human CVM. The model was based on available data from human CVMs. Under steady state, the effect of ion concentration changes in extracellular spaces on [Ca²⁺]_i-transient was explored as a function of critical fractions of ion transporters in t-tubular membrane (not documented for human CVM). Depletion of Ca²⁺ and accumulation of K⁺ occurring in extracellular spaces slightly affected the transmembrane Ca²⁺ flux, but not the action potential duration (APD₉₀). The [Ca²⁺]_i-transient was reduced (by 2%–9%), depending on the stimulation frequency, the rate of ion exchange between t-tubules and clefts and fractions of ion-transfer proteins in the t-tubular membrane. Under non-steady state, the responses of the model to changes of stimulation frequency were analyzed. A sudden increase of frequency (1–2.5 Hz) caused a temporal decrease of [Ca²⁺] in both extracellular spaces, a reduction of [Ca²⁺]_i-transient (by 15%) and APD₉₀ (by 13 ms). The results reveal different effects of activity-related ion concentration changes in human cardiac t-tubules (steady-state effects) and intercellular clefts (transient effects) in the modulation of membrane ion transport and Ca²⁺ turnover. Full article

(This article belongs to the Special Issue Computational Modelling of Biological Membranes)

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Int. J. Mol. Sci., Volume 14, Issue 12 (December 2013) – 101 articles , Pages 23212-24754

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