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1.  Molecular identification for epigallocatechin-3-gallate-mediated antioxidant intervention on the H2O2-induced oxidative stress in H9c2 rat cardiomyoblasts 
Epigallocatechin-3-gallate (EGCG) has been documented for its beneficial effects protecting oxidative stress to cardiac cells. Previously, we have shown the EGCG-mediated cardiac protection by attenuating reactive oxygen species and cytosolic Ca2+ in cardiac cells during oxidative stress and myocardial ischemia. Here, we aimed to seek a deeper elucidation of the molecular anti-oxidative capabilities of EGCG in an H2O2-induced oxidative stress model of myocardial ischemia injury using H9c2 rat cardiomyoblasts.
Proteomics analysis was used to determine the differential expression of proteins in H9c2 cells cultured in the conditions of control, 400 μM H2O2 exposure for 30 min with and/or without 10 to 20 μM EGCG pre-treatment. In this model, eight proteins associated with energy metabolism, mitochondrial electron transfer, redox regulation, signal transduction, and RNA binding were identified to take part in EGCG-ameliorating H2O2-induced injury in H9c2 cells. H2O2 exposure increased oxidative stress evidenced by increases in reactive oxygen species and cytosolic Ca2+ overload, increases in glycolytic protein, α-enolase, decreases in antioxidant protein, peroxiredoxin-4, as well as decreases in mitochondrial proteins, including aldehyde dehydrogenase-2, ornithine aminotransferase, and succinate dehydrogenase ubiquinone flavoprotein subunit. All of these effects were reversed by EGCG pre-treatment. In addition, EGCG attenuated the H2O2-induced increases of Type II inositol 3, 4-bisphosphate 4-phosphatase and relieved its subsequent inhibition of the downstream signalling for Akt and glycogen synthase kinase-3β (GSK-3β)/cyclin D1 in H9c2 cells. Pre-treatment with EGCG or GSK-3β inhibitor (SB 216763) significantly improved the H2O2-induced suppression on cell viability, phosphorylation of pAkt (S473) and pGSK-3β (S9), and level of cyclin D1 in cells.
Collectively, these findings suggest that EGCG blunts the H2O2-induced oxidative effect on the Akt activity through the modulation of PIP3 synthesis leading to the subsequent inactivation of GSK-3β mediated cardiac cell injury.
PMCID: PMC4070642  PMID: 24913014
EGCG; H9c2; Oxidative stress; Proteomics analysis; Survival pathway
2.  Structural and functional characterization of MERS coronavirus papain-like protease 
A new highly pathogenic human coronavirus (CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and Saudi Arabia and quickly spread to some European countries since September 2012. Until 15 May 2014, it has infected at least 572 people with a fatality rate of about 30% globally. Studies to understand the virus and to develop antiviral drugs or therapy are necessary and urgent. In the present study, MERS-CoV papain-like protease (PLpro) is expressed, and its structural and functional consequences are elucidated.
Circular dichroism and Tyr/Trp fluorescence analyses indicated that the secondary and tertiary structure of MERS-CoV PLpro is well organized and folded. Analytical ultracentrifugation analyses demonstrated that MERS-CoV PLpro is a monomer in solution. The steady-state kinetic and deubiquitination activity assays indicated that MERS-CoV PLpro exhibits potent deubiquitination activity but lower proteolytic activity, compared with SARS-CoV PLpro. A natural mutation, Leu105, is the major reason for this difference.
Overall, MERS-CoV PLpro bound by an endogenous metal ion shows a folded structure and potent proteolytic and deubiquitination activity. These findings provide important insights into the structural and functional properties of coronaviral PLpro family, which is applicable to develop strategies inhibiting PLpro against highly pathogenic coronaviruses.
PMCID: PMC4051379  PMID: 24898546
MERS coronavirus; Papain-like protease; Deubiquitination; Antiviral target
3.  Sphingosine-1-phosphate promotes the differentiation of adipose-derived stem cells into endothelial nitric oxide synthase (eNOS) expressing endothelial-like cells 
Adipose tissue provides a readily available source of autologous stem cells. Adipose-derived stem cells (ASCs) have been proposed as a source for endothelial cell substitutes for lining the luminal surface of tissue engineered bypass grafts. Endothelial nitric oxide synthase (eNOS) is a key protein in endothelial cell function. Currently, endothelial differentiation from ASCs is limited by poor eNOS expression. The goal of this study was to investigate the role of three molecules, sphingosine-1-phosphate (S1P), bradykinin, and prostaglandin-E1 (PGE1) in ASC endothelial differentiation. Endothelial differentiation markers (CD31, vWF and eNOS) were used to evaluate the level of ASCs differentiation capability.
ASCs demonstrated differentiation capability toward to adipose, osteocyte and endothelial like cell phenotypes. Bradykinin, S1P and PGE were used to promote differentiation of ASCs to an endothelial phenotype. Real-time PCR showed that all three molecules induced significantly greater expression of endothelial differentiation markers CD31, vWF and eNOS than untreated cells. Among the three molecules, S1P showed the highest up-regulation on endothelial differentiation markers. Immunostaining confirmed presence of more eNOS in cells treated with S1P than the other groups. Cell growth measurements by MTT assay, cell counting and EdU DNA incorporation suggest that S1P promotes cell growth during ASCs endothelial differentiation. The S1P1 receptor was expressed in ASC-differentiated endothelial cells and S1P induced up-regulation of PI3K.
S1P up-regulates endothelial cell markers including eNOS in ASCs differentiated to endothelial like cells. This up-regulation appears to be mediated by the up-regulation of PI3K via S1P1 receptor. ASCs treated with S1P offer promising use as endothelial cell substitutes for tissue engineered vascular grafts and vascular networks.
PMCID: PMC4064270  PMID: 24898615
4.  Hes-1 SUMOylation by protein inhibitor of activated STAT1 enhances the suppressing effect of Hes-1 on GADD45α expression to increase cell survival 
Hairy and Enhancer of split 1 (Hes-1) is a transcriptional repressor that plays an important role in neuronal differentiation and development, but post-translational modifications of Hes-1 are much less known. In the present study, we aimed to investigate whether Hes-1 could be SUMO-modified and identify the candidate SUMO acceptors on Hes-1. We also wished to examine the role of the SUMO E3 ligase protein inhibitor of activated STAT1 (PIAS1) in SUMOylation of Hes-1 and the molecular mechanism of Hes-1 SUMOylation. Further, we aimed to identify the molecular target of Hes-1 and examine how Hes-1 SUMOylation affects its molecular target to affect cell survival.
In this study, by using HEK293T cells, we have found that Hes-1 could be SUMO-modified and Hes-1 SUMOylation was greatly enhanced by the SUMO E3 ligase PIAS1 at Lys8, Lys27 and Lys39. Furthermore, Hes-1 SUMOylation stabilized the Hes-1 protein and increased the transcriptional suppressing activity of Hes-1 on growth arrest and DNA damage-inducible protein alpha (GADD45α) expression. Overexpression of GADD45α increased, whereas knockdown of GADD45αα expression decreased cell apoptosis. In addition, H2O2 treatment increased the association between PIAS1 and Hes-1 and enhanced the SUMOylation of Hes-1 for endogenous protection. Overexpression of Hes-1 decreased H2O2-induced cell death, but this effect was blocked by transfection of the Hes-1 triple sumo-mutant (Hes-1 3KR). Overexpression of PIAS1 further facilitated the anti-apoptotic effect of Hes-1. Moreover, Hes-1 SUMOylation was independent of Hes-1 phosphorylation and vice versa.
The present results revealed, for the first time, that Hes-1 could be SUMO-modified by PIAS1 and GADD45α is a novel target of Hes-1. Further, Hes-1 SUMOylation mediates cell survival through enhanced suppression of GADD45α expression. These results revealed a novel role of Hes-1 in addition to its involvement in Notch signaling. They also implicate that SUMOylation could be an important posttranslational modification that regulates cell survival.
PMCID: PMC4071220  PMID: 24894488
Hes-1; PIAS1; GADD45α; SUMOylation; Cell survival
6.  Taurine improves the spatial learning and memory ability impaired by sub-chronic manganese exposure 
Excessive manganese exposure induced cognitive deficit. Several lines of evidence have demonstrated that taurine improves cognitive impairment induced by numerous neurotoxins. However, the role of taurine on manganese-induced damages in learning and memory is still elusive. This goal of this study was to investigate the beneficial effect of taurine on learning and memory capacity impairment by manganese exposure in an animal model.
The escape latency in the Morris Water Maze test was significantly longer in the rats injected with manganese than that in the rats received both taurine and manganese. Similarly, the probe trial showed that the annulus crossings were significantly greater in the taurine plus manganese treated rats than those in the manganese-treated rats. However, the blood level of manganese was not altered by the taurine treatment. Interestingly, the exposure of manganese led to a significant increase in the acetylcholinesterase activity and an evidently decrease in the choline acetyltransferase activity, which were partially restored by the addition of taurine. Additionally, we identified 9 differentially expressed proteins between the rat hippocampus treated by manganese and the control or the manganese plus taurine in the proteomic analysis using the 2-dimensional gel electrophoresis followed by the tandem mass spectrometry (MS/MS). Most of these proteins play a role in energy metabolism, oxidative stress, inflammation, and neuron synapse.
In summary, taurine restores the activity of AChE and ChAT, which are critical for the regulation of acetylcholine. We have identified seven differentially expressed proteins specifically induced by manganese and two proteins induced by taurine from the rat hippocampus. Our results support that taurine improves the impaired learning and memory ability caused by excessive exposure of manganese.
PMCID: PMC4045917  PMID: 24885898
7.  Study of Coxsackie B viruses interactions with Coxsackie Adenovirus receptor and Decay-Accelerating Factor using Human CaCo-2 cell line 
Decay Accelerating Factor (DAF) and Coxsackievirus-Adenovirus Receptor (CAR) have been identified as cellular receptors for Coxsackie B viruses (CV-B). The aim of this study is to elucidate the different binding properties of CV-B serotypes and to find out if there are any amino acid changes that could be associated to the different phenotypes.
Twenty clinical CV-B isolates were tested on CaCo-2 cell line using anti-DAF (BRIC216) and anti-CAR (RmcB) antibodies. CV-B3 Nancy prototype strain and a recombinant strain (Rec, CV-B3/B4) were tested in parallel. The P1 genomic region of 12 CV-B isolates from different serotypes was sequenced and the Trans-Epithelial Electrical Resistance (TEER) along with the virus growth cycle was measured.
Infectivity assays revealed clear differences between CV-B isolates with regard to their interactions with DAF and CAR. All tested CV-B isolates showed an absolute requirement for CAR but varied in their binding to DAF. We also reported that for some isolates of CV-B, DAF attachment was not adapted. Genetic analysis of the P1 region detected multiple differences in the deduced amino acid sequences.
Within a given serotype, variations exist in the capacity of virus isolates to bind to specific receptors, and variants with different additional ligands may arise during infection in humans as well as in tissue culture.
PMCID: PMC4035751  PMID: 24885774
CV- B; CaCo-2 cell line; Receptors; Phenotypes; Variants; TEER
8.  Visfatin induces MUC8 and MUC5B expression via p38 MAPK/ROS/NF-κB in human airway epithelial cells 
Among a variety of inflammatory mediators, visfatin is a proinflammatory adipocytokine associated with inflammatory reactions in obesity, metabolic syndrome, chronic inflammatory disease, and autoimmune disease. However, the biological role of visfatin in secretion of major mucins in human airway epithelial cells has not been reported. Therefore, this study was conducted in order to investigate the effect and the brief signaling pathway of visfatin on MUC8 and MUC5B expression in human airway epithelial cells.
Visfatin significantly induced MUC8 and MUC5B expression. Visfatin significantly activated phosphorylation of p38 MAPK. Treatment with SB203580 (p38 MAPK inhibitor) and knockdown of p38 MAPK by siRNA significantly blocked visfatin-induced MUC8 and MUC5B expression.Visfatin significantly increased ROS formation. Treatment with SB203580 significantly attenuated visfatin-induced ROS formation. Treatment with NAC (ROS scavenger) and DPI (NADPH oxidase inhibitor) significantly attenuated visfatin-induced MUC8 and MUC5B expression. However, treatment with NAC and DPI did not attenuate visfatin-activated phosphorylation of p38 MAPK. Visfatin significantly activated the phosphorylation of NF-κB. Treatment with PDTC (NF-κB inhibitor) significantly attenuated visfatin-induced MUC8 and MUC5B expression.
These results suggest that visfatin induces MUC8 and MUC5B expression through p38 MAPK/ROS/NF-κB signaling pathway in human airway epithelial cells.
PMCID: PMC4041053  PMID: 24885580
Visfatin; p38 MAPK; ROS; NF-κB; MUC8; MUC5B; Epithelial cell
9.  Association between risk of oral precancer and genetic variations in microRNA and related processing genes 
MicroRNAs have been implicated in cancer but studies on their role in precancer, such as leukoplakia, are limited. Sequence variations at eight miRNA and four miRNA processing genes were studied in 452 healthy controls and 299 leukoplakia patients to estimate risk of disease.
Genotyping by TaqMan assay followed by statistical analyses showed that variant genotypes at Gemin3 and mir-34b reduced risk of disease [OR = 0.5(0.3–0.9) and OR = 0.7(0.5–0.9) respectively] in overall patients as well as in smokers [OR = 0.58(0.3–1) and OR = 0.68(0.5–0.9) respectively]. Among chewers, only mir29a significantly increased risk of disease [OR = 1.8(1–3)]. Gene-environment interactions using MDR-pt program revealed that mir29a, mir34b, mir423 and Xpo5 modulated risk of disease (p < 0.002) which may be related to change in expression of these genes as observed by Real-Time PCR assays. But association between polymorphisms and gene expressions was not found in our sample set as well as in larger datasets from open access platforms like Genevar and 1000 Genome database.
Variations in microRNAs and their processing genes modulated risk of precancer but further in-depth study is needed to understand mechanism of disease process.
PMCID: PMC4035900  PMID: 24885463
Oral leukoplakia; miRNA; DNA sequence variation; MDR; Gene expression
10.  Silicon substrate as a novel cell culture device for myoblast cells 
Tissue and organ regeneration via transplantation of cell bodies in-situ has become an interesting strategy in regenerative medicine. Developments of cell carriers to systematically deliver cell bodies in the damage site have fall shorten on effectively meet this purpose due to inappropriate release control. Thus, there is still need of novel substrate to achieve targeted cell delivery with appropriate vehicles. In the present study, silicon based photovoltaic (PV) devices are used as a cell culturing substrate for the expansion of myoblast mouse cell (C2C12 cells) that offers an atmosphere for regular cell growth in vitro. The adherence, viability and proliferation of the cells on the silicon surface were examined by direct cell counting and fluorescence microscopy.
It was found that on the silicon surface, cells proliferated over 7 days showing normal morphology, and expressed their biological activities. Cell culture on silicon substrate reveals their attachment and proliferation over the surface of the PV device. After first day of culture, cell viability was 88% and cell survival remained above 86% as compared to the seeding day after the seventh day. Furthermore, the DAPI staining revealed that the initially scattered cells were able to eventually build a cellular monolayer on top of the silicon substrate.
This study explored the biological applications of silicon based PV devices, demonstrating its biocompatibility properties and found useful for culture of cells on porous 2-D surface. The incorporation of silicon substrate has been efficaciously revealed as a potential cell carrier or vehicle in cell growth technology, allowing for their use in cell based gene therapy, tissue engineering, and therapeutic angiogenesis.
PMCID: PMC4035859  PMID: 24885347
Cell culturing; Photovoltaic effect; Silicon substrate; Cell carrier
11.  Intermittent hypoxia-induced protein phosphatase 2A activation reduces PC12 cell proliferation and differentiation 
Intermittent hypoxia (IH) plays a critical role in sleep breathing disorder-associated hippocampus impairments, including neurocognitive deficits, irreversible memory and learning impairments. IH-induced neuronal injury in the hippocampus may result from reduced precursor cell proliferation and the relative numbers of postmitotic differentiated neurons. However, the mechanisms underlying IH-induced reactive oxygen species (ROS) generation effects on cell proliferation and neuronal differentiation remain largely unknown.
ROS generation significantly increased after 1–4 days of IH without increased pheochromocytoma-12 (PC12) cell death, which resulted in increased protein phosphatase 2A (PP2A) mRNA and protein levels. After 3–4 days of IH, extracellular signal-regulated kinases 1/2 (ERK1/2) protein phosphorylation decreased, which could be reversed by superoxide dismutase (SOD), 1,10-phenanthroline (Phe), the PP2A phosphorylation inhibitors, okadaic acid (OKA) and cantharidin, and the ERK phosphorylation activator nicotine (p < 0.05). In particular, the significantly reduced cell proliferation and increased proportions of cells in the G0/G1 phase after 1–4 days of IH (p < 0.05), which resulted in decreased numbers of PC12 cells, could be reversed by treatment with SOD, Phe, PP2A inhibitors and an ERK activator. In addition, the numbers of nerve growth factor (NGF)-induced PC12 cells with neurite outgrowths after 3–4 days of IH were less than those after 4 days of RA, which was also reversed by SOD, Phe, PP2A inhibitors and an ERK activator.
Our results suggest that IH-induced ROS generation increases PP2A activation and subsequently downregulates ERK1/2 activation, which results in inhibition of PC12 cell proliferation through G0/G1 phase arrest and NGF-induced neuronal differentiation.
PMCID: PMC4058715  PMID: 24885237
Oxidative stress; Apoptosis; Cell viability; Cell cycle; Neurite outgrowth
12.  A transgenic approach to study argininosuccinate synthetase gene expression 
Argininosuccinate synthetase (ASS) participates in urea, nitric oxide and arginine production. Besides transcriptional regulation, a post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. To study whether such post-transcriptional regulation underlines particular temporal and spatial ASS expression, and to investigate how human ASS gene behaves in a mouse background, a transgenic mouse system using a modified bacterial artificial chromosome carrying the human ASS gene tagged with EGFP was employed.
Two lines of ASS-EGFP transgenic mice were generated: one with EGFP under transcriptional control similar to that of the endogenous ASS gene, another with EGFP under both transcriptional and post-transcriptional regulation as that of the endogenous ASS mRNA. EGFP expression in the liver, the organ for urea production, and in the intestine and kidney that are responsible for arginine biosynthesis, was examined. Organs taken from embryos E14.5 stage to young adult were examined under a fluorescence microscope either directly or after cryosectioning. The levels of EGFP and endogenous mouse Ass mRNAs were also quantified by S1 nuclease mapping. EGFP fluorescence and EGFP mRNA levels in both the liver and kidney were found to increase progressively from embryonic stage toward birth. In contrast, EGFP expression in the intestine was higher in neonates and started to decline at about 3 weeks after birth. Comparison between the EGFP profiles of the two transgenic lines indicated the developmental and tissue-specific regulation was mainly controlled at the transcriptional level. The ASS transgene was of human origin. EGFP expression in the liver followed essentially the mouse Ass pattern as evidenced by zonation distribution of fluorescence and the level of EGFP mRNA at birth. However, in the small intestine, Ass mRNA level declined sharply at 3 week of age, and yet substantial EGFP mRNA was still detectable at this stage. Thus, the time course of EGFP expression in the transgenic mice resembled that of the human ASS gene.
We demonstrate that the transgenic mouse system reported here has the merit of sensitivity and direct visualization advantage, and is ideal for annotating temporal and spatial expression profiles and the regulation mode of the ASS gene.
PMCID: PMC4025196  PMID: 24884799
Argininosuccinate synthetase; Transgenic mouse; Bacterial artificial chromosome; Green fluorescence protein; Developmental regulation; Tissue-specific regulation; Post-transcriptional regulation
13.  Oral mucosa stem cells alleviates spinal cord injury-induced neurogenic bladder symptoms in rats 
Spinal cord injury (SCI) deteriorates various physical functions, in particular, bladder problems occur as a result of damage to the spinal cord. Stem cell therapy for SCI has been focused as the new strategy to treat the injuries and to restore the lost functions. The oral mucosa cells are considered as the stem cells-like progenitor cells. In the present study, we investigated the effects of oral mucosa stem cells on the SCI-induced neurogenic bladder in relation with apoptotic neuronal cell death and cell proliferation.
The contraction pressure and the contraction time in the urinary bladder were increased after induction of SCI, in contrast, transplantation of the oral mucosa stem cells decreased the contraction pressure and the contraction time in the SCI-induced rats. Induction of SCI initiated apoptosis in the spinal cord tissues, whereas treatment with the oral mucosa stem cells suppressed the SCI-induced apoptosis. Disrupted spinal cord by SCI was improved by transplantation of the oral mucosa stem cells, and new tissues were increased around the damaged tissues. In addition, transplantation of the oral mucosa stem cells suppressed SCI-induced neuronal activation in the voiding centers.
Transplantation of oral mucosa stem cells ameliorates the SCI-induced neurogenic bladder symptoms by inhibiting apoptosis and by enhancing cell proliferation. As the results, SCI-induced neuronal activation in the neuronal voiding centers was suppressed, showing the normalization of voiding function.
PMCID: PMC4028106  PMID: 24884998
Spinal cord injury; Oral mucosa stem cells; Cystometry; Apoptosis; Nerve growth factor; c-Fos
14.  Prospective signs of cleidocranial dysplasia in Cebpb deficiency 
Although runt-related transcription factor 2 (RUNX2) has been considered a determinant of cleidocranial dysplasia (CCD), some CCD patients were free of RUNX2 mutations. CCAAT/enhancer-binding protein beta (Cebpb) is a key factor of Runx2 expression and our previous study has reported two CCD signs including hyperdontia and elongated coronoid process of the mandible in Cebpb deficient mice. Following that, this work aimed to conduct a case-control study of thoracic, zygomatic and masticatory muscular morphology to propose an association between musculoskeletal phenotypes and deficiency of Cebpb, using a sample of Cebpb-/-, Cebpb+/- and Cebpb+/+ adult mice. Somatic skeletons and skulls of mice were inspected with soft x-rays and micro-computed tomography (μCT), respectively. Zygomatic inclination was assessed using methods of coordinate geometry and trigonometric function on anatomic landmarks identified with μCT. Masseter and temporal muscles were collected and weighed. Expression of Cebpb was examined with a reverse transcriptase polymerase chain reaction (RT-PCR) technique.
Cebpb-/- mice displayed hypoplastic clavicles, a narrow thoracic cage, and a downward tilted zygomatic arch (p < 0.001). Although Cebpb+/- mice did not show the phenotypes above (p = 0.357), a larger mass percentage of temporal muscles over masseter muscles was seen in Cebpb+/- littermates (p = 0.012). The mRNA expression of Cebpb was detected in the clavicle, the zygoma, the temporal muscle and the masseter muscle, respectively.
Prospective signs of CCD were identified in mice with Cebpb deficiency. These could provide an additional aetiological factor of CCD. Succeeding investigation into interactions among Cebpb, Runx2 and musculoskeletal development is indicated.
PMCID: PMC4039338  PMID: 24885110
Cebpb; Cleidocranial dysplasia; Clavicle; Thoracic cage; Zygomatic arch; Masseter; Temporal muscle
15.  Synthesis and activity of three new trinuclear platinums with cis-geometry for terminal metal centres 
As compared to cisplatin, trinuclear platinum compounds such as BBR3464 and DH6Cl have an altered spectrum of activity possibly because they form long-range adducts with DNA as against mainly intrastrand 1,2-bifunctional adducts formed by cisplatin and its analogues. Because of the labilizing effect associated with the trans-geometry, the compounds are expected to break down inside the cell thus serving to reduce the number of long-range adducts formed. In contrast, trinuclear platinum complexes with cis-geometry for the terminal metal centres would be less subject to such breakdown and hence may produce a greater number of long-range inter- and intrastrand adducts with the DNA. This paper describes the synthesis and activity against human ovarian tumour models of of three new trinuclear platinum complexes with cis-geometry for terminal platinum centres, coded as QH4, QH7 and QH8. The paper also describes cellular accumulation of platinum, level of drug−DNA binding, and nature of interaction of the compounds with pBR322 plasmid DNA.
Methods of synthesis, elemental analysis, spectral studies and molar conductivity measurements provide support to the suggested structures of the compounds. QH4 and QH8 are found to be more cytotoxic than cisplatin against the parental A2780 cell line; QH8 is more active than cisplatin against the resistant A2780cisR and A2780ZD0473R cell lines as well. The least compound QH7 shows a greater activity against the resistant cell lines than the parental cell line; it is most damaging to pBR322 plasmid DNA and most able to induce changes in DNA conformation. The variations in activity of the compounds, changes in intracellular drug accumulation and levels of Pt−DNA binding with the changes in number of planaramine ligands bound to central platinum and the length of the linking diamines, can be seen (1) to illustrate structure-activity relationships and (2) to highlight that the relationship between antitumour activity and interaction with cellular platinophiles including DNA can be quite complex as the cell death is carried out by downstream processes in the cell cycle where many proteins are involved.
Among the three designed trinuclear platinum complexes with cis-geometry for the terminal metal centres, the most active compound QH8 is found to be more active than cisplatin against the parental A2780 and the resistant A2780cisR and A2780ZD0473R cell lines.
PMCID: PMC4022405  PMID: 24884683
Ovarian cancer; Platinum drug; Trinuclear; A2780; pBR322 plasmid; Drug resistance; Pt−DNA binding
16.  Characterization of a new mouse p53 variant: loss-of-function and gain-of-function 
p53 is a major tumor suppressor that is inactivated in over 50% of human cancer types through either mutation or inactivating interactions with viral or cellular proteins. The uncertainties around the link between p53 status, therapeutic response, and outcome in cancer suggest that additional factors may be involved. p53 isoforms that are generated via the alternative splicing pathway may be promising candidates for further investigation.
In this study, we report one new p53 protein with two internally deleted regions, resulting in one deleted amino acid fragment (from amino acid residues 42 to 89) and one reading frame-shift region (from amino acid residues 90-120) compared to wild-type p53. The functional status of the new p53 protein, which has a defect in its proline-rich and N-terminal DNA-binding domains, was characterized as possessing an intact conformation, exhibiting no transactivation activity, exerting a dominant-negative effect and an interacting with a coactivator with an arginine methyltransferase activity.
Taken together, our findings provide valuable information about the structure and function of p53 for the regulation of transactivation activity and cellular protein-protein interactions. Furthermore, natural p53 isoforms will help us understand the functional roles of the p53 family and potential therapeutics for p53-dependent cancers.
PMCID: PMC4022406  PMID: 24884657
p53; p21; Transactivation; Conformation; Dominant negative effect
17.  Attenuated neuroprotective effect of riboflavin under UV-B irradiation via miR-203/c-Jun signaling pathway in vivo and in vitro 
Riboflavin (RF) or vitamin B2 is known to have neuroprotective effects. In the present study, we report the attenuation of the neuroprotective effects of RF under UV-B irradiation. Preconditioning of UV-B irradiated riboflavin (UV-B-RF) showed attenuated neuroprotective effects compared to that of RF in SH-SY5Y neuroblostoma cell line and primary cortical neurons in vitro and a rat model of cerebral ischemia in vivo.
Results indicated that RF pretreatment significantly inhibited cell death and reduced LDH secretion compared to that of the UV-B-RF pretreatment in primary cortical neuron cultures subjected to oxygen glucose deprivation in vitro and cortical brain tissue subjected to ischemic injury in vivo. Further mechanistic studies using cortical neuron cultures revealed that RF treatment induced increased miR-203 expression which in turn inhibited c-Jun expression and increased neuronal cell survival. Functional assays clearly demonstrated that the UV-B-RF preconditioning failed to sustain the increased expression of miR-203 and the decreased levels of c-Jun, mediating the neuroprotective effects of RF. UV-B irradiation attenuated the neuroprotective effects of RF through modulation of the miR-203/c-Jun signaling pathway.
Thus, the ability of UV-B to serve as a modulator of this neuroprotective signaling pathway warrants further studies into its role as a regulator of other cytoprotective/neuroprotective signaling pathways.
PMCID: PMC4049496  PMID: 24884571
Cerebral ischemia; Riboflavin; UV-B; miR-203; C-jun; Neuroprotection
18.  Valproate pretreatment protects pancreatic β-cells from palmitate-induced ER stress and apoptosis by inhibiting glycogen synthase kinase-3β 
Reduction of pancreatic β-cells mass, major secondary to increased β-cells apoptosis, is increasingly recognized as one of the main contributing factors to the pathogenesis of type 2 diabetes (T2D), and saturated free fatty acid palmitate has been shown to induce endoplasmic reticulum (ER) stress that may contribute to promoting β-cells apoptosis. Recent literature suggests that valproate, a diffusely prescribed drug in the treatment of epilepsy and bipolar disorder, can inhibit glycogen synthase kinase-3β (GSK-3β) activity and has cytoprotective effects in neuronal cells and HepG2 cells. Thus, we hypothesized that valproate may protect INS-1 β-cells from palmitate-induced apoptosis via inhibiting GSK-3β.
Valproate pretreatment remarkable prevented palmitate-mediated cytotoxicity and apoptosis (lipotoxicity) as well as ER distension. Furthermore, palmitate triggered ER stress as evidenced by increased mRNA levels of C/EBP homologous protein (CHOP) and activating transcription factor 4 (ATF4) in a time-dependent fashion. However, valproate not only reduced the mRNA and protein expression of CHOP but also inhibited GSK-3β and caspase-3 activity induced by palmitate, whereas, the mRNA expression of ATF4 was not affected. Interestingly, TDZD-8, a specific GSK-3β inhibitor, also showed the similar effect on lipotoxicity and ER stress as valproate in INS-1 cells. Finally, compared with CHOP knockdown, valproate displayed better cytoprotection against palmitate.
Valproate may protect β-cells from palmitate-induced apoptosis and ER stress via GSK-3β inhibition, independent of ATF4/CHOP pathway. Besides, GSK-3β, rather than CHOP, may be a more promising therapeutic target for T2D.
PMCID: PMC4084580  PMID: 24884462
Valproate; Endoplasmic reticulum stress; Glycogen synthase kinase-3β; Pancreatic β-cells; Apoptosis
19.  Involvement of dopamine D2 receptor in the diurnal changes of tuberoinfundibular dopaminergic neuron activity and prolactin secretion in female rats 
An endogenous dopaminergic (DA) tone acting on D3 receptors has been shown to inhibit tuberoinfundibular (TI) DA neuron activity and stimulate prolactin (PRL) surge in the afternoon of estrogen-primed ovariectomized (OVX+E2) rats. Whether D2 receptor (D2R) is also involved in the regulation of TIDA and PRL rhythms was determined in this study.
Intracerebroventricular (icv) injection of PHNO, a D2R agonist, in the morning inhibited TIDA and midbrain DA neurons’ activities, and stimulated PRL secretion. The effects of PHNO were significantly reversed by co-administration of raclopride, a D2R antagonist. A single injection of raclopride at 1200 h significantly reversed the lowered TIDA neuron activity and the increased serum PRL level at 1500 h. Dopamine D2R mRNA expression in medial basal hypothalamus (MBH) exhibited a diurnal rhythm, i.e., low in the morning and high in the afternoon, which was opposite to that of TIDA neuron activity. The D2R rhythm was abolished in OVX+E2 rats kept under constant lighting but not in OVX rats with regular lighting exposures. Pretreatment with an antisense oligodeoxynucleotides (AODN, 10 μg/3 μl/day, icv) against D2R mRNA for 2 days significantly reduced D2R mRNAs in central DA neurons, and reversed both lowered TIDA neuron activity and increased serum PRL level in the afternoon on day 3. A diurnal rhythm of D2R mRNA expression was also observed in midbrain DA neurons and the rhythm was significantly knocked down by the AODN pretreatment.
We conclude that a diurnal change of D2R mRNA expression in MBH may underlie the diurnal rhythms of TIDA neuron activity and PRL secretion in OVX+E2 rats.
PMCID: PMC4019350  PMID: 24884386
Circadian rhythm; 3, 4-dihydroxyphenylacetic acid (DOPAC); Prolactin; Dorsal medial arcuate nucleus; Median eminence; Dopamine receptor
20.  Phospholipase A/Acyltransferase enzyme activity of H-rev107 inhibits the H-RAS signaling pathway 
H-rev107, also called HRASLS3 or PLA2G16, is a member of the HREV107 type II tumor suppressor gene family. Previous studies showed that H-rev107 exhibits phospholipase A/acyltransferase (PLA/AT) activity and downregulates H-RAS expression. However, the mode of action and the site of inhibition of H-RAS by H-rev107 are still unknown.
Our results indicate that H-rev107 was co-precipitated with H-RAS and downregulated the levels of activated RAS (RAS-GTP) and ELK1-mediated transactivation in epidermal growth factor-stimulated and H-RAS-cotransfected HtTA cervical cancer cells. Furthermore, an acyl-biotin exchange assay demonstrated that H-rev107 reduced H-RAS palmitoylation. H-rev107 has been shown to be a PLA/AT that is involved in phospholipid metabolism. Treating cells with the PLA/AT inhibitor arachidonyl trifluoromethyl ketone (AACOCF3) or methyl arachidonyl fluorophosphate (MAFP) alleviated H-rev107-induced downregulation of the levels of acylated H-RAS. AACOCF3 and MAFP also increased activated RAS and ELK1-mediated transactivation in H-rev107-expressing HtTA cells following their treatment with epidermal growth factor. In contrast, treating cells with the acyl-protein thioesterase inhibitor palmostatin B enhanced H-rev107-mediated downregulation of acylated H-RAS in H-rev107-expressing cells. Palmostatin B had no effect on H-rev107-induced suppression of RAS-GTP levels or ELK1-mediated transactivation. These results suggest that H-rev107 decreases H-RAS activity through its PLA/AT activity to modulate H-RAS acylation.
We made the novel observation that H-rev107 decrease in the steady state levels of H-RAS palmitoylation through the phospholipase A/acyltransferase activity. H-rev107 is likely to suppress activation of the RAS signaling pathway by reducing the levels of palmitoylated H-RAS, which decreases the levels of GTP-bound H-RAS and also the activation of downstream molecules. Our study further suggests that the PLA/AT activity of H-rev107 may play an important role in H-rev107-mediated RAS suppression.
PMCID: PMC4012743  PMID: 24884338
H-rev107; HRASLS3; PLA2G16; H-RAS; Phospholipase A/acyltransferase; Acyl-biotin exchange assay
21.  Detection of vascular endothelial growth factor in colon cancer xenografts using bevacizumab based near infrared fluorophore conjugate 
The aim of this study was to develop the near infrared fluorescence (NIRF)-based imaging agent for the visualization of vascular endothelial growth factor (VEGF) in colon cancer. AlexaFluor 750 conjugating with bevacizumab, and injected intravenously into nude mice bearing VEGF over-expressing HT29 human colorectal cancer. Optical imaging was performed at 15 min, 24 h and 48 h post injection. Immunofluorescences staining of the tumor sections were performed. HT29 colorectal cancer xenografts were clearly visualized with bevacizumab-AlexaFluor 750.
Ex vivo analysis showed 2.1 ± 0.4%, 37.6 ± 6.3% and 38.5 ± 6.2% injected dose/g accumulated in the tumors at 15 min, 24 h and 48 h respectively. Tumor uptake was significantly decreased in pretreated with excess of bevacizumab (p = 0.002). Immunofluorescence analysis showed strong staining of anti-CD 31 antibody around the blood vessels. Anti-VEGF-A and bevacizumab showed heterogeneous expression throughout the tumor.
Current study successfully detected the VEGF expression in HT29 colorectal cancer xenografts, signifying as a potential agent for non-invasive imaging of VEGF expression, which may be applied in clinical practice.
PMCID: PMC4012715  PMID: 24780003
Near infrared fluorescence; VEGF; Optical Imaging; Bevacizumab; Colorectal cancer
22.  SARS-CoV envelope protein palmitoylation or nucleocapid association is not required for promoting virus-like particle production 
Coronavirus membrane (M) proteins are capable of interacting with nucleocapsid (N) and envelope (E) proteins. Severe acute respiratory syndrome coronavirus (SARS-CoV) M co-expression with either N or E is sufficient for producing virus-like particles (VLPs), although at a lower level compared to M, N and E co-expression. Whether E can release from cells or E/N interaction exists so as to contribute to enhanced VLP production is unknown. It also remains to be determined whether E palmitoylation or disulfide bond formation plays a role in SARS-CoV virus assembly.
SARS-CoV N is released from cells through an association with E protein-containing vesicles. Further analysis suggests that domains involved in E/N interaction are largely located in both carboxyl-terminal regions. Changing all three E cysteine residues to alanines did not exert negative effects on E release, E association with N, or E enhancement of VLP production, suggesting that E palmitoylation modification or disulfide bond formation is not required for SARS-CoV virus assembly. We found that removal of the last E carboxyl-terminal residue markedly affected E release, N association, and VLP incorporation, but did not significantly compromise the contribution of E to efficient VLP production.
The independence of the SARS-CoV E enhancement effect on VLP production from its viral packaging capacity suggests a distinct SARS-CoV E role in virus assembly.
PMCID: PMC4014084  PMID: 24766657
23.  Mutations in the non-structural protein region contribute to intra-genotypic evolution of enterovirus 71 
Clinical manifestations of enterovirus 71 (EV71) range from herpangina, hand-foot-and-mouth disease (HFMD), to severe neurological complications. Unlike the situation of switching genotypes seen in EV71 outbreaks during 1998–2008 in Taiwan, genotype B5 was responsible for two large outbreaks in 2008 and 2012, respectively. In China, by contrast, EV71 often persists as a single genotype in the population and causes frequent outbreaks. To investigate genetic changes in viral evolution, complete EV71 genome sequences were used to analyze the intra-genotypic evolution pattern in Taiwan, China, and the Netherlands.
Genotype B5 was predominant in Taiwan’s 2008 outbreak and was re-emergent in 2012. EV71 strains from both outbreaks were phylogenetically segregated into two lineages containing fourteen non-synonymous substitutions predominantly in the non-structural protein coding region. In China, genotype C4 was first seen in 1998 and caused the latest large outbreak in 2008. Unlike shifting genotypes in Taiwan, genotype C4 persisted with progressive drift through time. A majority of non-synonymous mutations occurred in residues located in the non-structural coding region, showing annual increases. Interestingly, genotype B1/B2 in the Netherlands showed another stepwise evolution with dramatic EV71 activity increase in 1986. Phylogeny of the VP1 coding region in 1971–1986 exhibited similar lineage turnover with genotype C4 in China; however, phylogeny of the 3D-encoding region indicated separate lineage appearing after 1983, suggesting that the 3D-encoding region of genotype B2 was derived from an unidentified ancestor that contributed to intra-genotypic evolution in the Netherlands.
Unlike VP1 coding sequences long used for phylogenetic study of enteroviruses due to expected host immune escape, our study emphasizes a dominant role of non-synonymous mutations in non-structural protein regions that contribute to (re-)emergent genotypes in continuous stepwise evolution. Dozens of amino acid substitutions, especially in non-structural proteins, were identified via genetic changes driven through intra-genotypic evolution worldwide. These identified substitutions appeared to increase viral fitness in the population, affording valuable insights not only for viral evolution but also for prevention, control, and vaccine against EV71 infection.
PMCID: PMC4021180  PMID: 24766641
Enterovirus 71; Intra-genotypic evolution
24.  Phosphotriesterase-related protein sensed albuminuria and conferred renal tubular cell activation in membranous nephropathy 
Membranous nephropathy (MN) is a common cause of nephrotic syndrome that may progress to end-stage renal disease (ESRD). The formation of MN involves the in situ formation of subepithelial immune deposits and leads to albuminuria; however, the underlying mechanism of how MN leads to ESRD remains unclear. The aim of this study was to investigate the expression and biological functions of phosphotriesterase-related protein (PTER) in MN.
In the progression of MN, the expression of PTER increased significantly and was mainly expressed in the renal tubular cells. Both mRNA and protein expression levels of PTER were increased in a concentration- and time-dependent manner in the in vitro albuminuria tubular cell model. Silencing the expression of PTER by RNA interference diminished albuminuria-induced inflammatory and pro-fibrotic cytokines production.
Our findings reveal that PTER may sense albuminuria in the progression of MN, induce tubular cell activation and lead to ESRD.
PMCID: PMC4012828  PMID: 24750591
Albuminuria; Nephrotic syndrome; Phosphotriesterase-related protein (PTER)
25.  Animal models of enterovirus 71 infection: applications and limitations 
Human enterovirus 71 (EV71) has emerged as a neuroinvasive virus that is responsible for several outbreaks in the Asia-Pacific region over the past 15 years. Appropriate animal models are needed to understand EV71 neuropathogenesis better and to facilitate the development of effective vaccines and drugs. Non-human primate models have been used to characterize and evaluate the neurovirulence of EV71 after the early outbreaks in late 1990s. However, these models were not suitable for assessing the neurovirulence level of the virus and were associated with ethical and economic difficulties in terms of broad application. Several strategies have been applied to develop mouse models of EV71 infection, including strategies that employ virus adaption and immunodeficient hosts. Although these mouse models do not closely mimic human disease, they have been applied to determine the pathogenesis of and treatment and prevention of the disease. EV71 receptor-transgenic mouse models have recently been developed and have significantly advanced our understanding of the biological features of the virus and the host-parasite interactions. Overall, each of these models has advantages and disadvantages, and these models are differentially suited for studies of EV71 pathogenesis and/or the pre-clinical testing of antiviral drugs and vaccines. In this paper, we review the characteristics, applications and limitation of these EV71 animal models, including non-human primate and mouse models.
PMCID: PMC4013435  PMID: 24742252
Enterovirus 71; Animal models; Pathogenesis

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