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1.  Regulation and function of potassium channels in aldosterone-sensitive distal nephron 
Purpose of review
K channels in the aldosterone-sensitive distal nephron (ASDN) participate in generating cell membrane potential and in mediating K secretion. The aim of the review is to provide an overview of the recent development regarding physiological function of the K channels and the novel factors which modulate the K channels of the ASDN.
Recent findings
Genetic studies and transgenic mouse models have revealed the physiological function of basolateral K channels including inwardly rectifying K channel (Kir) and Ca2+-activated big-conductance K channels in mediating salt transport in the ASDN. A recent study shows that intersectin is required for mediating with-no-lysine kinase (WNK)-induced endocytosis. Moreover, a clathrin adaptor, autosomal recessive hypercholesterolemia (ARH), and an aging-suppression protein, Klothe, have been shown to regulate the endocytosis of renal outer medullary potassium (ROMK) channel. Also, serum-glucocorticoids-induced kinase I (SGK1) reversed the inhibitory effect of WNK4 on ROMK through the phosphorylation of WNK4. However, Src-family protein tyrosine kinase (SFK) abolished the effect of SGK1 on WNK4 and restored the WNK4-induced inhibition of ROMK.
Basolateral K channels including big-conductance K channel and Kir4.1/5.1 play an important role in regulating Na and Mg2+ transport in the ASDN. Apical K channels are not only responsible for mediating K excretion but they are also involved in regulating transepithelial Mg2+ absorption. New factors and mechanisms by which hormones and dietary K intake regulate apical K secretory channels expand the current knowledge regarding renal K handling.
PMCID: PMC4426959  PMID: 20601877
big-conductance K; c-Src; renal outer medullary potassium channel; voltage-gated K channel; with-no-lysine kinase
2.  Effect of the angiotensin II receptor blocker valsartan on cardiac hypertrophy and myocardial histone deacetylase expression in rats with aortic constriction 
The aim of the present study was to observe the myocardial expression of members of the histone deacetylase (HDAC) family (HDAC2, HDAC5 and HDAC9) in rats with or without myocardial hypertrophy (MH) in the presence and absence of the angiotensin II receptor blocker valsartan. Adult male Wistar rats were randomly divided into three groups (n=6/group): Sham-operated control rats, treated with distilled water (1 ml/day) through gavage; rats with MH (established through aortic constriction), treated with distilled water (1 ml/day) through gavage; and MH + valsartan rats, treated with 20 mg/kg/day valsartan through gavage. Treatments commenced one day after surgery and continued for eight weeks. Body weight (BW), heart weight (HW) and plasma atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) levels were determined, and the myocardial expression of HDAC2, HDAC5 and HDAC9 was analyzed through a reverse transcription semi-quantitative polymerase chain reaction. The BWs of the rats in the three groups were similar at baseline; however, after eight weeks the BW of the rats in the MH + valsartan group was significantly reduced compared with that of the MH rats. Furthermore, the HW/BW ratio and plasma ANP and BNP levels were increased, the myocardial HDAC2 expression was significantly upregulated and the HDAC5 and HDAC9 expression was significantly downregulated in the MH rats compared with those in the control rats; however, these changes were significantly attenuated by valsartan. Modulation of myocardial HDAC5, HDAC9 and HDAC2 expression may therefore be one of the anti-hypertrophic mechanisms of valsartan in this rat MH model.
PMCID: PMC4473348  PMID: 26136964
histone deacetylases; angiotensin II receptor blockers; cardiac hypertrophy
3.  Impact of phosphomimetic and non-phosphorylatable mutations of phospholemman on L-type calcium channels gating in HEK 293T cells 
Phospholemman (PLM) is an important phosphorylation substrate for protein kinases A and C in the heart. Until now, the association between PLM phosphorylation status and L-type calcium channels (LTCCs) gating has not been fully understood. We investigated the kinetics of LTCCs in HEK 293T cells expressing phosphomimetic or nonphosphorylatable PLM mutants.
The LTCCs gating was measured in HEK 293T cells transfected with LTCC and wild-type (WT) PLM, phosphomimetic or nonphosphorylatable PLM mutants: 6263AA, 6869AA, AAAA, 6263DD, 6869DD or DDDD.
WT PLM significantly slowed LTCCs activation and deactivation while enhanced voltage-dependent inactivation (VDI). PLM mutants 6869DD and DDDD significantly increased the peak of the currents. 6263DD accelerated channel activation, while 6263AA slowed it more than WT PLM. 6869DD significantly enhanced PLM-induced increase of VDI. AAAA slowed the channel activation more than 6263AA, and DDDD accelerated the channel VDI more than 6869DD.
Our results demonstrate that phosphomimetic PLM could stimulate LTCCs and alter their dynamics, while PLM nonphosphorylatable mutant produced the opposite effects.
PMCID: PMC4369820  PMID: 25656605
phospholemman; phosphorylation sites mutation; L-type calcium channels; activation; voltage-dependent inactivation; deactivation
4.  WNK4 inhibits Ca2+-activated big-conductance potassium channels (BK) via mitogen-activated protein kinase-dependent pathway 
Biochimica et biophysica acta  2013;1833(10):2101-2110.
We used the perforated whole-cell recording technique to examine the effect of With-No-Lysine Kinase 4 (WNK4) on the Ca2+ activated big-conductance K channels (BK) in HEK293T cells transfected with BK–α subunit (BK-α). Expression of WNK4 inhibited BK channels and decreased the outward K currents. Coexpression of SGK1 abolished the inhibitory effect of WNK4 on BK channels and restored the outward K currents. Expression of WNK4S1169D//1196D, in which both SGK1-phosphorylation sites (serine 1169 and 1196) were mutated to aspartate, had no effect on BK channels. Moreover, coexpression of SGK1 had no additional effect on K currents in the cells transfected with BKα + WNK4 S1169D//1196D, suggesting that SGK1 reversed WNK4-induced inhibition of BK channels by stimulating WNK4 phosphorylation. Expression of WNK4 but not WNK4 S1169D//1196D increased the phosphorylation of ERK and p38 mitogen-activated protein kinase (MAPK); an effect was abolished by coexpression of SGK1. The role of ERK and p38 MAPK in mediating the effect of WNK4 on BK channels was further suggested by the finding that inhibition of ERK and P38 MAPK completely abolished the inhibitory effect of WNK4 on BK channels. In contrast, inhibition of MAPK failed to abolish the inhibitory effect of WNK4 on ROMK channels in both HEK cells and Xenopus oocytes. Expression of dominant negative dynaminK44A (DynK44A) or treatment of the cells with dynasore, a dynamin inhibitor, not only increased K currents but also largely abolished the inhibitory effect of WNK4 on BK channels. However, inhibition of MAPK still increased the outward K currents in the cells transfected with BKα+WNK4 and treated with dynasore. Similar results were obtained in experiments performed in the native tissue in which inhibition of ERK and p38 MAPK increased BK channel activity in the cortical collecting duct (CCD) treated with dynasore. We concluded that WNK4 inhibited BK channels by stimulating ERK and p38 MAPK and that activation of MAPK by WNK4 may inhibit BK channels partially via a mechanism other than stimulating endocytosis.
PMCID: PMC3715553  PMID: 23673010
ERK; P38 MAPK; ROMK; K secretion; cortical collecting duct
5.  Correction: Continuous Regional Arterial Infusion with Fluorouracil and Octreotide Attenuates Severe Acute Pancreatitis in a Canine Model 
PLoS ONE  2013;8(10):10.1371/annotation/9e189146-9c58-44d0-8bff-347bde7018e9.
PMCID: PMC3794072
6.  Resveratrol protects rabbit ventricular myocytes against oxidative stress-induced arrhythmogenic activity and Ca2+ overload 
Acta Pharmacologica Sinica  2013;34(9):1164-1173.
To investigate whether resveratrol suppressed oxidative stress-induced arrhythmogenic activity and Ca2+ overload in ventricular myocytes and to explore the underlying mechanisms.
Hydrogen peroxide (H2O2, 200 μmol/L)) was used to induce oxidative stress in rabbit ventricular myocytes. Cell shortening and calcium transients were simultaneously recorded to detect arrhythmogenic activity and to measure intracellular Ca2+ ([Ca2+]i). Ca2+/calmodulin-dependent protein kinases II (CaMKII) activity was measured using a CaMKII kit or Western blotting analysis. Voltage-activated Na+ and Ca2+ currents were examined using whole-cell recording in myocytes.
H2O2 markedly prolonged Ca2+ transient duration (CaTD), and induced early afterdepolarization (EAD)-like and delayed afterdepolarization (DAD)-like arrhythmogenic activity in myocytes paced at 0.16 Hz or 0.5 Hz. Application of resveratrol (30 or 50 μmol/L) dose-dependently suppressed H2O2-induced EAD-like arrhythmogenic activity and attenuated CaTD prolongation. Co-treatment with resveratrol (50 μmol/L) effectively prevented both EAD-like and DAD-like arrhythmogenic activity induced by H2O2. In addition, resveratrol markedly blunted H2O2-induced diastolic [Ca2+]i accumulation and prevented the myocytes from developing hypercontracture. In whole-cell recording studies, H2O2 significantly enhanced the late Na+ current (INa,L) and L-type Ca2+ current (ICa,L) in myocytes, which were dramatically suppressed or prevented by resveratrol. Furthermore, H2O2-induced ROS production and CaMKII activation were significantly prevented by resveratrol.
Resveratrol protects ventricular myocytes against oxidative stress-induced arrhythmogenic activity and Ca2+ overload through inhibition of INa,L/ICa,L, reduction of ROS generation, and prevention of CaMKII activation.
PMCID: PMC4003166  PMID: 23912472
resveratrol; cardioprotective agents; oxidative stress; cardiac arrhythmias; Ca2+ overload; CaMKII; late sodium current; L-type calcium current
7.  Process optimization and evaluation of novel baicalin solid nanocrystals 
The objective of this study was to prepare baicalin solid nanocrystals (BCN-SNS) to enhance oral bioavailability of baicalin. A Box–Behnken design approach was used for process optimization. The physicochemical properties and pharmacokinetics of the optimal BCN-SNS were investigated. Multiple linear regression analysis for process optimization revealed that the fine BCN-SNS was obtained wherein the optimal values of homogenization pressure (bar), homogenization cycles (cycles), amount of TPGS to drug (w/w), and amount of MCCS to drug (w/w) were 850 bar, 25 cycles, 10%, and 10%, respectively. Transmission electron microscopy and scanning electron microscopy results indicated that no significant aggregation or crystal growth could be observed in the redispersed freeze-dried BCN-SNS. Differential scanning calorimetry and X-ray diffraction results showed that BCN remained in a crystalline state. Dissolution velocity of the freeze-dried BCN-SNS powder was distinctly superior compared to those of the crude powder and physical mixture. The bioavailability of BCN in rats was increased remarkably after oral administration of BCN-SNS (P < 0.05), compared with those of BCN or the physical mixture. The SNS might be a good choice for oral administration of poorly soluble BCN, due to an improvement of the bioavailability and dissolution velocity of BCN-SNS.
PMCID: PMC3746734  PMID: 23976849
baicalin; solid nanocrystals; optimization; in vivo/vitro evaluation
8.  miR-221/222 Targets Adiponectin Receptor 1 to Promote the Epithelial-to-Mesenchymal Transition in Breast Cancer 
PLoS ONE  2013;8(6):e66502.
The epithelial-to-mesenchymal transition (EMT) is a highly conserved physiological program involved in development and tissue repair; however, its aberrant activation has been implicated in accelerating the progression of a variety of cancers. In breast cancer, the microRNAs (miRNAs) miR-221 and miR-222 (miR-221/222) are differentially expressed in the clinically more aggressive basal-like subtype compared to luminal subtype of breast cancer and upregulation of miR-221/222 induces the EMT by targeting the 3′ untranslated region (3′UTR) of the GATA family transcriptional repressor TRPS1 (tricho-rhino-phalangeal syndrome type 1). The complete mechanism through which miR-221/222 promotes the EMT, however, is not fully understood. We identified adiponectin receptor 1 (ADIPOR1), a receptor for the adipocytokine adiponectin, as a direct target of miR-221/222. ADIPOR1 is expressed at higher levels in the luminal compared to the basal-like subtype of breast cancer cell lines, which can be reduced by miR-221/222 targeting of its 3’UTR. In addition, miR-221/222 were negatively correlated with ADIPOR1 expression across breast cancer cell lines and tumors. ADIPOR1 depletion by siRNA in MCF10A cells induced the EMT and increased cell invasion. Depletion of ADIPOR1 by siRNA induced activation of the canonical nuclear factor-kappaB (NF-κB) and subsequent phosphorylation of signal transducer and activator of transcription 3 (STAT3) in an interleukin 6 (IL6)-dependent manner. Finally, overexpression of ADIPOR1 in the basal-like cell line, MDA-MB-231, attenuated cell invasion and promoted the mesenchymal-to-epithelial transition (MET). We conclude that ADIPOR1 negatively regulates EMT in breast cancer and provides an additional node by which miR-221/222 induces the EMT. These results suggest that ADIPOR1 may play an important role in breast cancer progression and metastasis, and could potentially offer an alternative therapeutic strategy for basal-like breast cancer patients.
PMCID: PMC3679042  PMID: 23776679
9.  WNT signaling underlies the pathogenesis of neuropathic pain in rodents 
The Journal of Clinical Investigation  2013;123(5):2268-2286.
Treating neuropathic pain is a major clinical challenge, and the underlying mechanisms of neuropathic pain remain elusive. We hypothesized that neuropathic pain–inducing nerve injury may elicit neuronal alterations that recapitulate events that occur during development. Here, we report that WNT signaling, which is important in developmental processes of the nervous system, plays a critical role in neuropathic pain after sciatic nerve injury and bone cancer in rodents. Nerve injury and bone cancer caused a rapid-onset and long-lasting expression of WNTs, as well as activation of WNT/frizzled/β-catenin signaling in the primary sensory neurons, the spinal dorsal horn neurons, and astrocytes. Spinal blockade of WNT signaling pathways inhibited the production and persistence of neuropathic pain and the accompanying neurochemical alterations without affecting normal pain sensitivity and locomotor activity. WNT signaling activation stimulated production of the proinflammatory cytokines IL-18 and TNF-α and regulated the NR2B glutamate receptor and Ca2+-dependent signals through the β-catenin pathway in the spinal cord. These findings indicate a critical mechanism underlying the pathogenesis of neuropathic pain and suggest that targeting the WNT signaling pathway may be an effective approach for treating neuropathic pain, including bone cancer pain.
PMCID: PMC3635721  PMID: 23585476
10.  Cyp2c44-epoxygenase is essential for preventing the renal sodium absorption during increasing dietary potassium (K)-intake 
Hypertension  2011;59(2):339-347.
The aim of this study is to test whether the Cyp2c44 epoxygenase dependent metabolism of arachidonic acid (AA) prevents the hypertensive effect of a high K intake (HK) by inhibiting the ENaC activity. A HK intake elevated Cyp2c44 mRNA expression and 11,12-epoxyeicosatrienoic acid (11,12-EET) levels in the cortical collecting duct (CCD) in Cyp2c44(+/+) mice(wt). However, a HK intake failed to increase 11,12-EET formation in the CCDs of Cyp2c44(-/-) mice. Moreover, increasing K intake enhanced AA-induced inhibition of ENaC in the wt but not in Cyp2c44(-/-) mice. In contrast, 11,12-EET, a Cyp2c44 metabolite, inhibited ENaC in the wt and Cyp2c44(-/-) mice. The notion that Cyp2c44 is the epoxygenase responsible for mediating the inhibitory effects of AA on ENaC is further suggested by the observation that inhibiting Cyp-epoxygenase increased the whole-cell Na currents in principal cells of wt but not in Cyp2c44(-/-) mice. Feeding mice with a HK diet raised the systemic blood pressures of Cyp2c44(-/-) mice but was without an effect on wt mice. Moreover, application of amiloride abolished the HK-induced hypertension in Cyp2c44(-/-) mice. The HK-induced hypertension of Cyp2c44(-/-) mice was accompanied by decreasing 24-hr urinary Na excretion and increasing the plasma Na concentration, the effects were absent in wt mice. In contrast, disruption of the Cyp2c44 gene did not alter K excretion. We conclude that Cyp2c44 epoxygenase mediates the inhibitory effect of AA on ENaC and that Cyp2c44 functions as a HK-inducible anti-hypertensive enzyme responsible for inhibiting ENaC activity and Na absorption in the aldosterone-sensitive distal nephron (ASDN).
PMCID: PMC3382086  PMID: 22184322
11,12-EET; Arachidonic acid; ENaC; hypertension; kidney
11.  K+ Channel Mutations in Adrenal Aldosterone-Producing Adenomas and Hereditary Hypertension 
Science (New York, N.Y.)  2011;331(6018):768-772.
Endocrine tumors such as aldosterone-producing adrenal adenomas (APAs), a cause of severe hypertension, feature constitutive hormone production and unrestrained cell proliferation; the mechanisms linking these events are unknown. We identify two recurrent somatic mutations in and near the selectivity filter of the potassium (K+) channel KCNJ5 that are present in 8 of 22 human APAs studied. Both produce increased sodium (Na+) conductance and cell depolarization, which in adrenal glomerulosa cells produces calcium (Ca2+) entry, the signal for aldosterone production and cell proliferation. Similarly, we identify an inherited KCNJ5 mutation that produces increased Na+ conductance in a Mendelian form of severe aldosteronism and massive bilateral adrenal hyperplasia. These findings explain pathogenesis in a subset of patients with severe hypertension and implicate loss of K+ channel selectivity in constitutive cell proliferation and hormone production.
PMCID: PMC3371087  PMID: 21311022
12.  Continuous Regional Arterial Infusion with Fluorouracil and Octreotide Attenuates Severe Acute Pancreatitis in a Canine Model 
PLoS ONE  2012;7(5):e37347.
To investigate the therapeutic effects of fluorouracil (5-Fu) and octreotide (Oct) continuous regional arterial infusion (CRAI,) alone or in combination, was administered in a canine model of severe acute pancreatitis (SAP).
Materials and Methods
The animals were divided into five groups; group A (Sham), group B (SAP), group C (SAP and 5-Fu), group D (SAP and Oct), and group E (SAP and 5-Fu + Oct). Levels of amylase, α-tumor necrosis factor (TNF-α), blood urea nitrogen (BUN), creatinine, thromboxane B2 and 6-keto- prostaglandin F1α were measured both before and after the induction of SAP. Pathologic examination of the pancreas and kidneys was performed after termination of the study.
Pathologic changes noted in the pancreas in SAP significantly improved following CRAI with either single or combined administration of 5-Fu and Oct, where combination therapy demonstrated the lowest injury score. All treatment groups had significantly lower levels of serum TNF-α and amylase activity (P<0.05), though only groups D and E had a lower BUN level as compared to group B. The plasma thromboxane B2 level increased in SAP, but the ratio of thromboxane B2/6-keto- prostaglandin F1α decreased in the treatment groups, with the combination therapy (group E) demonstrating the lowest ratio as compared to the other 3 experimental groups (P<0.05).
The findings in the present study demonstrate an attenuation of SAP in a canine model following CRAI administration with 5-Fu or Oct, alone or in combination.
PMCID: PMC3360032  PMID: 22655040
13.  Somatic mutations in p85α promote tumorigenesis through class IA PI3K activation 
Cancer cell  2009;16(6):463-474.
Members of the mammalian phosphoinositide-3-OH kinase (PI3K) family of proteins are critical regulators of various cellular process including cell survival, growth, proliferation and motility. Oncogenic activating mutations in the p110α catalytic subunit of the heterodimeric p110/p85 PI3K enzyme are frequent in human cancers. Here we show the presence of frequent mutations in p85α in colon cancer, a majority of which occurs in the inter-Src homology-2 (iSH2) domain. These mutations uncouple and retain p85α's p110-stabilizing activity, while abrogating its p110-inhibitory activity. The p85α mutants promote cell survival, Akt activation, anchorage independent cell growth, and oncogenesis in a p110-dependent manner.
Somatic mutations in the catalytic p110α subunit of PI3K are common in cancers. In this study, we show the occurrence of frequent mutations in the regulatory p85α subunit of PI3K in human cancers. Our data demonstrate an alternate mechanism for PI3K-pathway activation and oncogenesis resulting from the impaired regulation of p110 activity by mutant p85α. Further, p85α mutations are likely to be useful as diagnostic markers for identification of p110-dependent tumors that may not carry an activating p110α mutation, but are candidates for targeted treatment with PI3K pathway inhibitors that are in development.
PMCID: PMC2804903  PMID: 19962665
14.  Spinal Matrix Metalloproteinase-9 Contributes to Physical Dependence on Morphine in Mice 
The Journal of Neuroscience  2010;30(22):7613-7623.
Preventing and reversing opioid dependence continues to be a clinical challenge and underlying mechanisms of opioid actions remain elusive. We report that matrix metalloproteinase-9 (MMP-9) in the spinal cord contributes to development of physical dependence on morphine. Chronic morphine exposure and naloxone-precipitated withdrawal increase activity of spinal MMP-9. Spinal inhibition or targeted mutation of MMP-9 suppresses behavioral signs of morphine withdrawal and the associated neurochemical alterations. The increased MMP-9 activity is mainly distributed in the superficial dorsal horn and colocalized primarily with neurons and small numbers of astrocytes and microglia. Morphine exposure and withdrawal increase phosphorylation of NR1 and NR2B receptors, ERK1/2, calmodulin-dependent kinase II, and cAMP response element binding proteins; and such phosphorylation is suppressed by either spinal inhibition or targeted mutation of MMP-9. Further, spinal administration of exogenous MMP-9 induces morphine withdrawal-like behavioral signs and mechanical allodynia, activates NR1 and NR2 receptors, and downregulates integrin-β1, while a function-neutralizing antibody against integrin-β1 suppresses MMP-9-induced phosphorylation of NR1 and NR2B. Morphine withdrawal-induced MMP-9 activity is also reduced by an nNOS inhibitor. Thus, we hypothesize that spinal MMP-9 may contribute to the development of morphine dependence primarily through neuronal activation and interaction with NR1 and NR2B receptors via integrin-β1 and NO pathways. The other gelatinase, MMP-2, is not involved in morphine dependence. Inhibiting spinal MMP-9 or MMP-2 reduces chronic and/or acute morphine tolerance. This study suggests a novel therapeutic approach for preventing, minimizing, or reversing opioid dependence and tolerance.
PMCID: PMC3842477  PMID: 20519536
15.  Statistical techniques to construct assays for identifying likely responders to a treatment under evaluation from cell line genomic data 
BMC Cancer  2010;10:586.
Developing the right drugs for the right patients has become a mantra of drug development. In practice, it is very difficult to identify subsets of patients who will respond to a drug under evaluation. Most of the time, no single diagnostic will be available, and more complex decision rules will be required to define a sensitive population, using, for instance, mRNA expression, protein expression or DNA copy number. Moreover, diagnostic development will often begin with in-vitro cell-line data and a high-dimensional exploratory platform, only later to be transferred to a diagnostic assay for use with patient samples. In this manuscript, we present a novel approach to developing robust genomic predictors that are not only capable of generalizing from in-vitro to patient, but are also amenable to clinically validated assays such as qRT-PCR.
Using our approach, we constructed a predictor of sensitivity to dacetuzumab, an investigational drug for CD40-expressing malignancies such as lymphoma using genomic measurements of cell lines treated with dacetuzumab. Additionally, we evaluated several state-of-the-art prediction methods by independently pairing the feature selection and classification components of the predictor. In this way, we constructed several predictors that we validated on an independent DLBCL patient dataset. Similar analyses were performed on genomic measurements of breast cancer cell lines and patients to construct a predictor of estrogen receptor (ER) status.
The best dacetuzumab sensitivity predictors involved ten or fewer genes and accurately classified lymphoma patients by their survival and known prognostic subtypes. The best ER status classifiers involved one or two genes and led to accurate ER status predictions more than 85% of the time. The novel method we proposed performed as well or better than other methods evaluated.
We demonstrated the feasibility of combining feature selection techniques with classification methods to develop assays using cell line genomic measurements that performed well in patient data. In both case studies, we constructed parsimonious models that generalized well from cell lines to patients.
PMCID: PMC2984428  PMID: 20979617
16.  The Study on the Entrapment Efficiency and In Vitro Release of Puerarin Submicron Emulsion 
AAPS PharmSciTech  2009;10(2):376-383.
The entrapment efficiency (EE) and release in vitro are very important physicochemical characteristics of puerarin submicron emulsion (SME). In this paper, the performance of ultrafiltration (UF), ultracentrifugation (UC), and microdialysis (MD) for determining the EE of SME were evaluated, respectively. The release study in vitro of puerarin from SME was studied by using MD and pressure UF technology. The EE of SME was 86.5%, 72.8%, and 55.8% as determined by MD, UF, and UC, respectively. MD was not suitable for EE measurements of puerarin submicron oil droplet, which could only determine the total EE of submicron oil droplet and liposomes micelles, but it could be applied to determine the amount of free drug in SMEs. Although UC was the fastest and simplest to use, its results were the least reliable. UF was still the relatively accurate method for EE determination of puerarin SME. The release of puerarin SME could be evaluated by using MD and pressure UF, but MD seemed to be more suitable for the release study of puerarin emulsion. The drug release from puerarin SME at three drug concentrations was initially rapid, but reached a plateau value within 30 min. Drug release of puerarin from the SME occurred via burst release.
PMCID: PMC2690779  PMID: 19381837
drug release; entrapment efficiency; microdialysis; pressure ultrafiltration technology; submicron emulsion
17.  Web GIS in practice VIII: HTML5 and the canvas element for interactive online mapping 
HTML5 is being developed as the next major revision of HTML (Hypertext Markup Language), the core markup language of the World Wide Web. It aims at reducing the need for proprietary, plug-in-based rich Internet application (RIA) technologies such as Adobe Flash. The canvas element is part of HTML5 and is used to draw graphics using scripting (e.g., JavaScript). This paper introduces Cartagen, an open-source, vector-based, client-side framework for rendering plug-in-free, offline-capable, interactive maps in native HTML5 on a wide range of Web browsers and mobile phones. Cartagen was developed at MIT Media Lab's Design Ecology group. Potential applications of the technology as an enabler for participatory online mapping include mapping real-time air pollution, citizen reporting, and disaster response, among many other possibilities.
PMCID: PMC2838837  PMID: 20199681
18.  Poly[[tetra­aquadi-μ3-oxalato-μ2-oxalato-diprasedymium(III)] dihydrate] 
In the title compound, {[Pr2(C2O4)3(H2O)4]·2H2O}n, the three-dimensional network structure has the PrIII ion coordinated by nine O atoms in a distorted tricapped trigonal-prismatic geometry. The coordinated and uncoordinated water mol­ecules inter­act with the carboxyl­ate O atoms to consolidate the network via O—H⋯O hydrogen bonds.
PMCID: PMC2980182  PMID: 21579975
19.  The Role of the Exocyst in Matrix Metalloproteinase Secretion and Actin Dynamics during Tumor Cell Invadopodia Formation 
Molecular Biology of the Cell  2009;20(16):3763-3771.
Invadopodia are actin-rich membrane protrusions formed by tumor cells that degrade the extracellular matrix for invasion. Invadopodia formation involves membrane protrusions driven by Arp2/3-mediated actin polymerization and secretion of matrix metalloproteinases (MMPs) at the focal degrading sites. The exocyst mediates the tethering of post-Golgi secretory vesicles at the plasma membrane for exocytosis and has recently been implicated in regulating actin dynamics during cell migration. Here, we report that the exocyst plays a pivotal role in invadopodial activity. With RNAi knockdown of the exocyst component Exo70 or Sec8, MDA-MB-231 cells expressing constitutively active c-Src failed to form invadopodia. On the other hand, overexpression of Exo70 promoted invadopodia formation. Disrupting the exocyst function by siEXO70 or siSEC8 treatment or by expression of a dominant negative fragment of Exo70 inhibited the secretion of MMPs. We have also found that the exocyst interacts with the Arp2/3 complex in cells with high invasion potential; blocking the exocyst-Arp2/3 interaction inhibited Arp2/3-mediated actin polymerization and invadopodia formation. Together, our results suggest that the exocyst plays important roles in cell invasion by mediating the secretion of MMPs at focal degrading sites and regulating Arp2/3-mediated actin dynamics.
PMCID: PMC2777935  PMID: 19535457
20.  Analytical methods for inferring functional effects of single base pair substitutions in human cancers 
Human Genetics  2009;126(4):481-498.
Cancer is a genetic disease that results from a variety of genomic alterations. Identification of some of these causal genetic events has enabled the development of targeted therapeutics and spurred efforts to discover the key genes that drive cancer formation. Rapidly improving sequencing and genotyping technology continues to generate increasingly large datasets that require analytical methods to identify functional alterations that deserve additional investigation. This review examines statistical and computational approaches for the identification of functional changes among sets of single-nucleotide substitutions. Frequency-based methods identify the most highly mutated genes in large-scale cancer sequencing efforts while bioinformatics approaches are effective for independent evaluation of both non-synonymous mutations and polymorphisms. We also review current knowledge and tools that can be utilized for analysis of alterations in non-protein-coding genomic sequence.
PMCID: PMC2762536  PMID: 19434427
21.  Hydrogen Peroxide Stimulates the Ca2+-activated Big-Conductance K Channels (BK) Through cGMP Signaling Pathway in Cultured Human Endothelial Cells 
We used the whole cell patch-clamp technique to examine the effect of hydrogen peroxide (H2O2) on the Ca2+-activated BK channels in human endothelial cells. We confirmed the previous finding that a 200 pS BK channel activity was detected when the cell membrane potential was clamped at 50 mV. Application of H2O2 or adding glucose oxidase (GO) stimulated BK channels. The stimulatory effect of H2O2 and GO was absent in cells treated with ebselen, a scavenger of reactive oxygen species (ROS). To determine whether the stimulatory effect of H2O2 and GO on BK channels is the result of increasing NO production in the endothelial cells, we examined the effect of H2O2 and GO on BK channels in the presence of 0.1 mM L-NAME which inhibits NO synthase (NOS). Inhibition of NOS completely abolished the stimulatory effect of H2O2 on BK channels. In contrast, treatment of endothelial cells with D-NAME did not block the effect of H2O2 on BK channels. Moreover, inhibiting soluble guanylate cyclase (sGC) with ODQ mimicked the effect of L-NAME and abolished the effect of H2O2. Addition of 8-bromo-cGMP stimulated BK channels and further application of H2O2 did not increase BK channel activity in the presence of cGMP analog. The notion that the effect of H2O2 on BK channels was the result of stimulating NO-cGMP pathway is further indicated by the observation that inhibition of PKG with KT5823 also abolished the stimulatory effect of H2O2 on BK channels. We conclude that H2O2 stimulates the Ca2+ BK channels through NO/sGC/cGMP pathway in cultured human endothelial cells.
PMCID: PMC2730122  PMID: 18769038
Hydrogen peroxide; Nitric oxide; Endothelial cell; cGMP; K channel
22.  Role of gp91phox-Containing NADPH Oxidase in Mediating the Effect of K Restriction on ROMK Channels and Renal K Excretion 
Previous study has demonstrated that superoxide and the related products are involved in mediating the effect of low K intake on renal K secretion and ROMK channel activity in the cortical collecting duct (CCD). This study investigated the role of gp91phox-containing NADPH oxidase (NOXII) in mediating the effect of low K intake on renal K excretion and ROMK channel activity in gp91(-/-) mice. K depletion increased superoxide levels, phosphorylation of c-Jun, expression of c-Src, and tyrosine phosphorylation of ROMK in renal cortex and outer medulla in wild-type (WT) mice. In contrast, tempol treatment in WT mice abolished whereas deletion of gp91 significantly attenuated the effect of low K intake on superoxide production, c-Jun phosphorylation, c-Src expression, and tyrosine phosphorylation of ROMK. Patch-clamp experiments demonstrated that low K intake decreased mean product of channel number (N) and open probability (P) (NPo) of ROMK channels from 1.1 to 0.4 in the CCD. However, the effect of low K intake on ROMK channel activity was significantly attenuated in the CCD from gp91(-/-) mice and completely abolished by tempol treatment. Immunocytochemical staining also was used to examine the ROMK distribution in WT, gp91(-/-), and WT mice with tempol treatment in response to K restriction. K restriction decreased apical staining of ROMK in WT mice. In contrast, a sharp apical ROMK staining was observed in the tempol-treated WT or gp91(-/-) mice. Metabolic cage study further showed that urinary K loss is significantly higher in gp91(-/-) mice than in WT mice. It is concluded that superoxide anions play a key role in suppressing K secretion during K restriction and that NOXII is involved in mediating the effect of low K intake on renal K secretion and ROMK channel activity.
PMCID: PMC2702222  PMID: 17538186
23.  Process Optimization, Characterization and Pharmacokinetic Evaluation in Rats of Ursodeoxycholic Acid–Phospholipid Complex 
AAPS PharmSciTech  2008;9(1):322-329.
The purpose of this research was to study whether the bioavailability of ursodeoxycholic acid could be improved by administering ursodeoxycholic acid–phospholipid complex (UDCA–PLC) orally to rats. A central composite design approach was used for process optimization in order to obtain the acceptable UDCA–PLC. The physicochemical properties of the complex obtained by optimal parameters were investigated by means of scanning electron microscopy and X-ray diffraction. The pharmacokinetic parameters and bioavailability studies were conducted in rats of UDCA after oral administration of UDCA–PLC and UDCA tablet. Multiple linear regression analysis for process optimization revealed that the acceptable UDCA–PLC was obtained wherein the optimal values of X1, X2 and X3 were 3, 60°C and 3 h, respectively. The XRD studies of UDCA–PLC obtained by the optimal parameters demonstrated that UDCA and phospholipids in the UDCA–PLC were combined by non-covalent bonds, not form new compounds. But pharmacokinetic parameters of the complex in rats were Tmax 1.6 h, Cmax 0.1346 μg/ml, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text{AUC}}_{0 - \infty } $$\end{document}11.437 μg·h/ml, respectively. The relative bioavailability of UDCA of UDCA–PLC was increased by 241%,compared with the reference ursodeoxycholic acid tablet.
PMCID: PMC2976878  PMID: 18446498
characterization; optimization; pharmacokinetics; phospholipid complex; ursodeoxycholic acid
24.  1-{2-[(2-hydroxybenzylidene)-amino]-ethyl}-3-methyl-3H-imidazolium hexafluorophosphate 
The title Schiff base compound, C13H16N3O+·PF6 −, was derived from the condensation of 2-hydroxy­benaldehyde with the ionic liquid 1-(2-amino­ethyl)-3-methyl­imidazolium hexa­fluoro­phosphate in an ethanol solution. The asymmetric unit comprises one cation and two PF6 − anions. The dihedral angle between the aromatic and imidazole rings is 15.2 (2)°. An intra­molecular O—H⋯N hydrogen bond is found which generates an S(6) ring motif.
PMCID: PMC2959942  PMID: 21581337
25.  3,3′-Dibromo-5,5′-bis­[(S)-l-menth­yloxy]-4,4′-(hexane-1,6-diyldiimino)difuran-2(5H)-one 
The title compound, C34H54Br2N2O6, was obtained by the Michael addition–elimination reaction of (5S)-5-(l-menthyl­oxy)-3,4-dibromo­furan-2(5H)-one with 1,6-hexa­nediamine in the presence of triethyl­amine. The crystal structure contains two chiral five-membered furan­one rings, in twist and envelope conformations, and two six-membered cyclo­hexane rings in chair conformations.
PMCID: PMC2962195  PMID: 21203330

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