ROMK channels are regulated by internal pH (pH(i)) and extracellular K(+) (K(+)(o)). The mechanisms underlying this regulation were studied in these channels after expression in Xenopus oocytes. Replacement of the COOH-terminal portion of ROMK2 (Kir1.1b) with the corresponding region of the pH-insensitive channel IRK1 (Kir 2.1) produced a chimeric channel (termed C13) with enhanced sensitivity to inhibition by intracellular H(+), increasing the apparent pKa for inhibition by approximately 0.9 pH units. Three amino acid substitutions at the COOH-terminal end of the second transmembrane helix (I159V, L160M, and I163M) accounted for these effects. These substitutions also made the channels more sensitive to reduction in K(+)(o), consistent with coupling between the responses to pH(i) and K(+)(o). The ion selectivity sequence of the activation of the channel by cations was K(+) congruent with Rb(+) > NH(4)(+) >> Na(+), similar to that for ion permeability, suggesting an interaction with the selectivity filter. We tested a model of coupling in which a pH-sensitive gate can close the pore from the inside, preventing access of K(+) from the cytoplasm and increasing sensitivity of the selectivity filter to removal of K(+)(o). We mimicked closure of this gate using positive membrane potentials to elicit block by intracellular cations. With K(+)(o) between 10 and 110 mM, this resulted in a slow, reversible decrease in conductance. However, additional channel constructs, in which inward rectification was maintained but the pH sensor was abolished, failed to respond to voltage under the same conditions. This indicates that blocking access of intracellular K(+) to the selectivity filter cannot account for coupling. The C13 chimera was 10 times more sensitive to extracellular Ba(2+) block than was ROMK2, indicating that changes in the COOH terminus affect ion binding to the outer part of the pore. This effect correlated with the sensitivity to inactivation by H(+). We conclude that decreasing pH(I) increases the sensitivity of ROMK2 channels to K(+)(o) by altering the properties of the selectivity filter.

The emergence of influenza strains that are resistant to commonly used antivirals has highlighted the need to develop new compounds that target viral gene products or host mechanisms that are essential for effective virus replication. Existing assays to identify potential antiviral compounds often use high throughput screening assays that target specific viral replication steps. To broaden the search for antivirals, cell-based replication assays can be performed, but these are often labor intensive and have limited throughput.

The increasing availability of large-scale protein-protein interaction data has made it possible to understand the basic components and organization of cell machinery from the network level. The arising challenge is how to analyze such complex interacting data to reveal the principles of cellular organization, processes and functions. Many studies have shown that clustering protein interaction network is an effective approach for identifying protein complexes or functional modules, which has become a major research topic in systems biology. In this review, recent advances in clustering methods for protein interaction networks will be presented in detail. The predictions of protein functions and interactions based on modules will be covered. Finally, the performance of different clustering methods will be compared and the directions for future research will be discussed.

The effects of a thiazolidinone derivative, 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (or CFTRinh-172), on cystic fibrosis transmembrane conductance regulator (CFTR) gating were studied in excised inside-out membrane patches from Chinese hamster ovary cells transiently expressing wild-type and mutant CFTR. We found that the application of CFTRinh-172 results in an increase of the mean closed time and a decrease of the mean open time of the channel. A hyperbolic relationship between the closing rate and [CFTRinh-172] suggests that CFTRinh-172 does not act as a simple pore blocker. Interestingly, the potency of inhibition increases as the open time of the channel is increased with an IC50 in the low nanomolar range for CFTR channels locked in an open state for tens of seconds. Our studies also provide evidence that CFTRinh-172 can bind to both the open state and the closed state. However, at least one additional step, presumably reflecting inhibitor-induced conformational changes, is required to shut down the conductance after the binding of the inhibitor to the channel. Using the hydrolysis-deficient mutant E1371S as a tool as the closing rate of this mutant is dramatically decreased, we found that CFTRinh-172-dependent inhibition of CFTR channel gating, in two aspects, mimics the inactivation of voltage-dependent cation channels. First, similar to the recovery from inactivation in voltage-gated channels, once CFTR is inhibited by CFTRinh-172, reopening of the channel can be seen upon removal of the inhibitor in the absence of adenosine triphosphate (ATP). Second, ATP induced a biphasic current response on inhibitor-bound closed channels as if the ATP-opened channels "inactivate" despite a continuous presence of ATP. A simplified six-state kinetic scheme can well describe our data, at least qualitatively. Several possible structural mechanisms for the effects of CFTRinh-172 will be discussed.

It has been proved that chronic administration and pre-treatment with atorvastatin could protect brain tissue against ischemic injury. However, little is known regarding the effect of atorvastatin in the acute phase of ischemic stroke. This study investigated the potential neuroprotective effects of atorvastatin and underlying mechanisms in vivo.

Membrane proteins serve as cellular gatekeepers, regulators, and sensors. Prior studies have explored the functional breadth and evolution of proteins and families of particular interest, such as the diversity of transport-associated membrane protein families in prokaryotes and eukaryotes, the composition of integral membrane proteins, and family classification of all human G-protein coupled receptors. However, a comprehensive analysis of the content and evolutionary associations between membrane proteins and families in a diverse set of genomes is lacking. Here, a membrane protein annotation pipeline was developed to define the integral membrane genome and associations between 21,379 proteins from 34 genomes; most, but not all of these proteins belong to 598 defined families. The pipeline was used to provide target input for a structural genomics project that successfully cloned, expressed, and purified 61 of our first 96 selected targets in yeast. Furthermore, the methodology was applied (1) to explore the evolutionary history of the substrate-binding transmembrane domains of the human ABC transporter superfamily, (2) to identify the multidrug resistance-associated membrane proteins in whole genomes, and (3) to identify putative new membrane protein families.

The prognosis of malignant pleural effusion (MPE) was poor, injecting anti-angiogenesis agents in pleural cavity might be to reducing the volume of pleural effusion. The aim of this study is to investigate the therapeutical effect of pleural injection of recombinant human endostain, cisplatin and recombinant human endostain combined with cisplatin to MPE nude mice.

Nicardipine (NC) is the most commonly used antihypertensive drug in neurological patients with hypertension. Although nimodipine (NM) is widely used to treat cerebral vasospasm in patients with aneurysmal subarachnoid hemorrhage, trials exploring its antihypertensive effect after intravenous administration in subjects with intracerebral hemorrhage (ICH) are scarce.

Promiscuous inhibition of the human ether-à-go-go-related gene (hERG) potassium channel by drugs poses a major risk for life threatening arrhythmia and costly drug withdrawals. Current knowledge of this phenomenon is derived from a limited number of known drugs and tool compounds. However, in a diverse, naïve chemical library, it remains unclear which and to what degree chemical motifs or scaffolds might be enriched for hERG inhibition. Here we report electrophysiology measurements of hERG inhibition and computational analyses of >300,000 diverse small molecules. We identify chemical 'communities' with high hERG liability, containing both canonical scaffolds and structurally distinctive molecules. These data enable the development of more effective classifiers to computationally assess hERG risk. The resultant predictive models now accurately classify naïve compound libraries for tendency of hERG inhibition. Together these results provide a more complete reference map of characteristic chemical motifs for hERG liability and advance a systematic approach to rank chemical collections for cardiotoxicity risk.

The high-affinity choline transporter (CHT) is the rate-limiting determinant of acetylcholine (ACh) synthesis, yet the transporter remains a largely undeveloped target for the detection and manipulation of synaptic cholinergic signaling. To expand CHT pharmacology, we pursued a high-throughput screen for novel CHT-targeted small molecules based on the electrogenic properties of transporter-mediated choline transport. In this effort, we identified five novel, structural classes of CHT-specific inhibitors. Chemical diversification and functional analysis of one of these classes identified ML352 as a high-affinity (Ki = 92 nM) and selective CHT inhibitor. At concentrations that fully antagonized CHT in transfected cells and nerve terminal preparations, ML352 exhibited no inhibition of acetylcholinesterase (AChE) or cholineacetyltransferase (ChAT) and also lacked activity at dopamine, serotonin, and norepinephrine transporters, as well as many receptors and ion channels. ML352 exhibited noncompetitive choline uptake inhibition in intact cells and synaptosomes and reduced the apparent density of hemicholinium-3 (HC-3) binding sites in membrane assays, suggesting allosteric transporter interactions. Pharmacokinetic studies revealed limited in vitro metabolism and significant CNS penetration, with features predicting rapid clearance. ML352 represents a novel, potent, and specific tool for the manipulation of CHT, providing a possible platform for the development of cholinergic imaging and therapeutic agents.

Cigarette smoking may contribute to pulmonary hypertension in chronic obstructive pulmonary disease by resulting in pulmonary vascular remodeling that involves pulmonary artery smooth muscle cell proliferation. This study investigated the effects of basic fibroblast growth factor (bFGF) and cyclin D1 on the pulmonary vascular remodeling in smoking-exposed rats. Twenty-four male Wistar rats were randomly divided into four groups. Three tobacco-exposed groups were exposed to the smoke produced by 20 cigarettes for 60 min, twice a day for two, four or eight weeks, and the control group were exposed to fresh air. The expression of bFGF and cyclin D1 in the pulmonary arterial smooth muscle cells were determined using immunohistochemistry. Quantitative polymerase chain reaction was conducted to determine the expression levels of bFGF and cyclin D1 mRNA. In addition, the expression of bFGF and cyclin D1 proteins was evaluated by western blotting. The expression of bFGF and cyclin D1 at the mRNA and protein levels was shown to increase with the duration of smoke exposure (P<0.05). The correlation analysis indicated the expression of bFGF and cyclin D1 was positively associated with the pulmonary vessel wall thickness. The expression of bFGF was positively associated with that of cyclin D1. Collectively, the data demonstrated that the upregulation of bFGF and cyclin D1 occurred in rats subjected to smoke exposure, which may be associated with the abnormal proliferation of the smooth muscle cells in the pulmonary arteries.

Human adipose tissue-derived stem cells (ADSCs) are an attractive multipotent stem cell source with therapeutic applicability across diverse fields for the repair and regeneration of acute and chronically damaged tissues. In recent years, there has been increasing interest in ADSC for tissue engineering applications. However, the mechanisms underlying the regulation of ADSC proliferation are not fully understood. Here we show that 47 transcripts are up-regulated while 23 are down-regulated in ADSC compared to terminally differentiated cells based on global mRNA profiling and microRNA profiling. Among the up-regulated genes, the expression of vascular endothelial growth factor (VEGF) is fine-tuned by miR-199a-5p. Further investigation indicates that VEGF accelerates ADSC proliferation whereas the multipotency of ADSC remains stable in terms of adipogenic, chondrogenic and osteogenic potentials after VEGF treatment, suggesting that VEGF may serve as an excellent supplement for accelerating ADSC proliferation during in vitro expansion.

Adiponectin is an adipocyte-secreted adipokine with pleiotropic actions. Clinical evidence has shown that serum adiponectin levels are increased and that adiponectin can protect pancreatic beta cells against apoptosis, which suggests that adiponectin may play an anti-apoptotic role in pancreatic cancer (PC). Here, we investigated the effects of adiponectin on PC development and elucidated the underlying molecular mechanisms. Adiponectin deficiency markedly attenuated pancreatic tumorigenesis in vivo. We found that adiponectin significantly inhibited the apoptosis of both human and mouse pancreatic cancer cells via adipoR1, but not adipoR2. Furthermore, adiponectin can increase AMP-activated protein kinase (AMPK) phosphorylation and NAD-dependent deacetylase sirtuin-1 (Sirt1) of PC cells. Knockdown of AMPK or Sirt1 can increase the apoptosis in PC cells. AMPK up-regulated Sirt1, and Sirt1 can inversely phosphorylate AMPK. Further studies have shown that Sirt1 can deacetylate peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), which can increase the expression levels of mitochondrial genes. Thus, adiponectin exerts potent anti-apoptotic effects on PC cells via the activation of AMPK/Sirt1/PGC1α signaling. Finally, adiponectin can elevate β-catenin levels. Taken together, these novel findings reveal an unconventional role of adiponectin in promoting pancreatic cancers, and suggest that the effects of adiponectin on tumorigenesis are highly tissue-dependent.

Transcription factor pancreatic and duodenal homeobox-1 (PDX-1) plays an essential role in pancreatic development, β-cell differentiation, maintenance of normal β-cell function and tumorigenesis. PDX-1 expression is tightly controlled through a variety of mechanisms under different cellular contexts. We report here that overexpression of Notch1 intracellular domain (NICD), an activated form of Notch1, enhanced PDX-1 expression in both PDX-1 stable HEK293 cells and mouse insulinoma β-TC-6 cells, while NICD shRNA inhibited the enhancing effect. NICD-enhanced PDX-1 expression was accompanied by increased insulin expression/secretion and cell proliferation in β-TC-6 cells, which was reversed by NICD shRNA. Cre activation-induced specific expression of NICD in islet β cells of transgenic βNICD+/+ mice induced increased expression of PDX-1, insulin and proliferating cell nuclear antigen (PCNA) and decreased expression of p27 with accompanied fasting hyperinsulinemia and hypoglycemia and altered responses to intraperitoneal glucose tolerance test. Systemically delivered NICD shRNA suppressed islet expression of PDX-1 and reversed the hypoglycemia and hyperinsulinemia. Moreover, expression levels of NICD were correlated with those of PDX-1 in human pancreatic neuroendocrine tumor. Thus, Notch1 acts as a positive regulator for PDX-1 expression, cooperates with PDX-1 in the development of insulin overexpression and islet cell neoplasia and represents a potential therapeutic target for islet neoplasia.

Extranodal NK/T-cell lymphoma, nasal type, is an aggressive mature NK-cell/T-cell lymphoma. Using array-based comparative genomic hybridization (array CGH) assays, we screened genomic alterations and potential candidate genes implicated in pathogenesis, progression, and prognosis. Our array CGH analysis detected an average of 83 chromosomal aberrations in 13 cases, ranging from 0 to 387. There were 177 recurrent chromosomal gains and 35 recurrent losses. Eleven gains and 14 losses were detected in more than 30 % of the cases, including gains of 3q26.1, 7q34, and 8q24.3 and losses of 15q24.2, 19q13.32, 5p13.2, and 14q21.1. The most common losses were observed in the 15q24.2 and 19q13.32 regions (9 cases, 69.2 %, respectively). Loss of 8p11.23 was associated with significant poor survival (P = 0.024). Five out of six patients with the loss of 8p11.23 died within 8 months after initial diagnosis with a median survival of 6 months. Several candidate genes were identified in the regions with frequent chromosomal aberrations, including ADAM3A (8p11.23) and GSTT1 (22q11.23). In summary, our studies detected recurrent genetic alterations in NK/T-cell lymphoma, some of which are associated with adverse prognosis. Some candidate genes in these regions may be involved in the pathogenesis and disease progression.

The principal green tea polyphenol, (-)-epigallocatechin-3-O-gallate (EGCg), may provide chemoprotection against conditions ranging from cardiovascular disease to cancer. Binding to plasma proteins stabilizes EGCg during its transport to targeted tissues. This study explored the details EGCg binding to bovine serum albumin. Both fluorescence lifetime and intensity data showed that the hydrophobic pocket between subdomains IIA and IIIA is the binding site for EGCg. Fluorescence and circular dichroism were used to establish the roles of the flavan-3-ol and galloyl moieties of the EGCg in binding and to demonstrate a binding-dependent conformational change in the protein. Competitive binding experiments confirmed the location of binding, and molecular modeling identified protein residues that play key roles in the interaction. This model of EGCg-BSA interactions improves the understanding of the likely physiological fate of this green tea-derived bioactive polyphenol.

The Beclin 1-Vps34 complex, the core component of the class III phosphatidylinositol-3 kinase (PI3K-III), binds Atg14L or UVRAG to control different steps of autophagy. However, the mechanism underlying the control of PI3K-III activity remains elusive. Here we report the identification of NRBF2 as a component in the specific PI3K-III complex and a modulator of PI3K-III activity. Through its microtubule interaction and trafficking (MIT) domain, NRBF2 binds Atg14L directly and enhances Atg14L-linked Vps34 kinase activity and autophagy induction. NRBF2-deficient cells exhibit enhanced vulnerability to endoplasmic reticulum (ER) stress that is reversed by re-introducing exogenous NRBF2. NRBF2-deficient mice develop focal liver necrosis and ductular reaction, accompanied by impaired Atg14L-linked Vps34 activity and autophagy, although the mice show no increased mortality. Our data reveal a key role for NRBF2 in the assembly of the specific Atg14L-Beclin 1-Vps34-Vps15 complex for autophagy induction. Thus, NRBF2 modulates autophagy via regulation of PI3K-III and prevents ER stress-mediated cytotoxicity and liver injury.

Melatonin is an indolamine synthesized in the pineal gland that has a wide range of physiological functions, and it has been under clinical investigation for expanded applications. Increasing evidence demonstrates that melatonin can ameliorate cadmium-induced hepatotoxicity. However, the potentially protective effects of melatonin against cadmium-induced hepatotoxicity and the underlying mechanisms of this protection remain unclear. This study investigates the protective effects of melatonin pretreatment on cadmium-induced hepatotoxicity and elucidates the potential mechanism of melatonin-mediated protection. We exposed HepG2 cells to different concentrations of cadmium chloride (2.5, 5, and 10 μM) for 12 h. We found that Cd stimulated cytotoxicity, disrupted the mitochondrial membrane potential, increased reactive oxygen species production, and decreased mitochondrial mass and mitochondrial DNA content. Consistent with this finding, Cd exposure was associated with decreased Sirtuin 1 (SIRT1) protein expression and activity, thus promoted acetylation of PGC-1 alpha, a key enzyme involved in mitochondrial biogenesis and function, although Cd did not disrupt the interaction between SIRT1 and PGC-1 alpha. However, all cadmium-induced mitochondrial oxidative injuries were efficiently attenuated by melatonin pretreatment. Moreover, Sirtinol and SIRT1 siRNA each blocked the melatonin-mediated elevation in mitochondrial function by inhibiting SIRT1/ PGC-1 alpha signaling. Luzindole, a melatonin receptor antagonist, was found to partially block the ability of melatonin to promote SIRT1/ PGC-1 alpha signaling. In summary, our results indicate that SIRT1 plays an essential role in the ability of moderate melatonin to stimulate PGC-1 alpha and improve mitochondrial biogenesis and function at least partially through melatonin receptors in cadmium-induced hepatotoxicity.

Current technologies for studying ion channels are fundamentally limited because of their inability to functionally link ion channel activity to cellular pathways. Herein, we report the use of label-free cell phenotypic profiling to decode the composition and signaling of an endogenous ATP-sensitive potassium ion channel (KATP) in HepG2C3A, a hepatocellular carcinoma cell line. Label-free cell phenotypic agonist profiling showed that pinacidil triggered characteristically similar dynamic mass redistribution (DMR) signals in A431, A549, HT29 and HepG2C3A, but not in HepG2 cells. Reverse transcriptase PCR, RNAi knockdown, and KATP blocker profiling showed that the pinacidil DMR is due to the activation of SUR2/Kir6.2 KATP channels in HepG2C3A cells. Kinase inhibition and RNAi knockdown showed that the pinacidil activated KATP channels trigger signaling through Rho kinase and Janus kinase-3, and cause actin remodeling. The results are the first demonstration of a label-free methodology to characterize the composition and signaling of an endogenous ATP-sensitive potassium ion channel.

Bisphenol A (BPA) is an environmental endocrine disruptor which has been detected in human bodies. Many studies have implied that BPA exposure is harmful to human health. Previous studies mainly focused on BPA effects on estrogen receptor (ER)-positive cells. Genome-wide impacts of BPA on gene expression in ER-negative cells is unclear. In this study, we performed RNA-seq to characterize BPA-induced cellular and molecular impacts on ER-negative HEK293 cells. The microscopic observation showed that low-dose BPA exposure did not affect cell viability and morphology. Gene expression profiling analysis identified a list of differentially expressed genes in response to BPA exposure in HEK293 cells. These genes were involved in variable important biological processes including ion transport, cysteine metabolic process, apoptosis, DNA damage repair, etc. Notably, BPA up-regulated the expression of ERCC5 encoding a DNA endonuclease for nucleotide-excision repair. Further electrochemical experiment showed that BPA induced significant DNA damage in ER-positive MCF-7 cells but not in ER-negative HEK293 cells. Collectively, our study revealed that ER-negative HEK293 cells employed mechanisms in response to BPA exposure different from ER-positive cells.