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Fwu-Shan Sheu

Publications and source records attributed to Fwu-Shan Sheu.

18 recordsLinked to original sources
PubMed2007

Role of P-glycoprotein in the intestinal absorption of glabridin, an active flavonoid from the root of Glycyrrhiza glabra.

Glabridin is a major constituent of the root of Glycyrrhiza glabra, which is commonly used in the treatment of cardiovascular and central nervous system diseases. This study aimed to investigate the role of P-glycoprotein (PgP/MDR1) in the intestinal absorption of glabridin. The systemic bioavailability of glabridin was approximately 7.5% in rats, but increased when combined with verapamil. In single-pass perfused rat ileum with mesenteric vein cannulation, the permeability coefficient of glabridin based on drug disappearance in luminal perfusates (P(lumen)) was approximately 7-fold higher than that based on drug appearance in the blood (P(blood)). Glabridin was mainly metabolized by glucuronidation, and the metabolic capacity of intestine microsomes was 1/15 to 1/20 of that in liver microsomes. Polarized transport of glabridin was found in Caco-2 and MDCKII monolayers. Addition of verapamil in both apical (AP) and basolateral (BL) sides abolished the polarized transport of glabridin across Caco-2 cells. Incubation of verapamil significantly altered the intracellular accumulation and efflux of glabridin in Caco-2 cells. The transport of glabridin in the BL-AP direction was significantly higher in MDCKII cells overexpressing PgP/MDR1 than in the control cells. Glabridin inhibited PgP-mediated transport of digoxin with an IC(50) value of 2.56 microM, but stimulated PgP/MDR1 ATPase activity with a K(m) of 25.1 microM. The plasma AUC(0-24h) of glabridin in mdr1a(-/-) mice was 3.8-fold higher than that in wild-type mice. These findings indicate that glabridin is a substrate for PgP and that both PgP/MDR1-mediated efflux and first-pass metabolism contribute to the low oral bioavailability of glabridin.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2006

Microelectrode array biochip: tool for in vitro drug screening based on the detection of a drug effect on dopamine release from PC12 cells.

Novel, yet simple detection techniques of drug effect, including the effect of a vesicular monoamine transporter inhibitor (reserpine), a dopamine precursor (L-dopa), and a dopamine transporter inhibitor (nomifensine), on dopamine release from dopaminergic PC12 cells were developed based on a microelectrode array (MEA) biochip. Upon multi-injections of KCl solution into the culture of PC12 cells attached on a MEA biochip, the K+-stimulated dopamine release was temporally and amperometrically recorded by biochip microelectrodes. Two parameters in the recorded amperometric spectra were defined in this study: the peak current of the first KCl injection (Max1), and the steady current after the fourth KCl injection (St4). Statistically significant effects of L-dopa and reserpine were demonstrated by comparing both Max1 and St4 of the second detections in drugs with those of the control without drug treatment. The values of both Max1 and St4 in the first detections were normalized as 1. In contrast, the statistically significant effect of nomifensine was detected by comparing the ratios of St4 to Max1 in the first detections in drug with those of the control. The reason for using different analytical methods for measurements between L-dopa/reserpine and nomifensine lies in the different mechanisms of action on PC12 cells among these drugs. The novel analytical methods developed use the same detection setup and parameters, and the data analysis for the effect of drugs becomes simple. The methods hence may provide a high-throughput in vitro drug screening approach for dopamine-related psychiatric disorders.

Animals↗
PubMed2006

St. John's wort attenuates irinotecan-induced diarrhea via down-regulation of intestinal pro-inflammatory cytokines and inhibition of intestinal epithelial apoptosis.

Diarrhea is a common dose-limiting toxicity associated with cancer chemotherapy, in particular for drugs such as irinotecan (CPT-11), 5-fluouracil, oxaliplatin, capecitabine and raltitrexed. St. John's wort (Hypericum perforatum, SJW) has anti-inflammatory activity, and our preliminary study in the rat and a pilot study in cancer patients found that treatment of SJW alleviated irinotecan-induced diarrhea. In the present study, we investigated whether SJW modulated various pro-inflammatory cytokines including interleukins (IL-1beta, IL-2, IL-6), interferon (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) and intestinal epithelium apoptosis in rats. The rats were treated with irinotecan at 60 mg/kg for 4 days in combination with oral SJW or SJW-free control vehicle at 400 mg/kg for 8 days. Diarrhea, tissue damage, body weight loss, various cytokines including IL-1beta, IL-2, IL-6, IFN-gamma and TNF-alpha and intestinal epithelial apoptosis were monitored over 11 days. Our studies demonstrated that combined SJW markedly reduced CPT-11-induced diarrhea and intestinal lesions. The production of pro-inflammatory cytokines such as IL-1beta, IFN-gamma and TNF-alpha was significantly up-regulated in intestine. In the mean time, combined SJW significantly suppressed the intestinal epithelial apoptosis induced by CPT-11 over days 5-11. In particular, combination of SJW significantly inhibited the expression of TNF-alpha mRNA in the intestine over days 5-11. In conclusion, inhibition of pro-inflammatory cytokines and intestinal epithelium apoptosis partly explained the protective effect of SJW against the intestinal toxicities induced by irinotecan. Further studies are warranted to explore the potential for STW as an agent in combination with chemotherapeutic drugs to lower their dose-limiting toxicities.

Animals↗
PubMed2006

A mechanistic study on reduced toxicity of irinotecan by coadministered thalidomide, a tumor necrosis factor-alpha inhibitor.

Dose-limiting diarrhea and myelosuppression compromise the success of irinotecan (7-ethyl-10-[4-[1-piperidino]-1-piperidino]carbonyloxycamptothecin) (CPT-11)-based chemotherapy. A recent pilot study indicates that thalidomide attenuates the toxicity of CPT-11 in cancer patients. This study aimed to investigate whether coadministered thalidomide modulated the toxicities of CPT-11 and the underlying mechanisms using several in vivo and in vitro models. Diarrhea, intestinal lesions, cytokine expression, and intestinal epithelial apoptosis were monitored. Coadministered thalidomide (100 mg/kg i.p. for 8 days) significantly attenuated body weight loss, myelosuppression, diarrhea, and intestinal histological lesions caused by CPT-11 (60 mg/kg i.v. for 4 days). This was accompanied by inhibition of tumor necrosis factor-alpha, interleukins 1 and 6 and interferon-gamma, and intestinal epithelial apoptosis. Coadministered thalidomide also significantly increased the systemic exposure of CPT-11 but decreased that of SN-38 (7-ethyl-10-hydroxycampothecin). It significantly reduced the biliary excretion and cecal exposure of CPT-11, SN-38, and SN-38 glucuronide. Thalidomide hydrolytic products inhibited hydrolysis of CPT-11 in rat liver microsomes but not in primary rat hepatocytes. In addition, thalidomide and its major hydrolytic products, such as phthaloyl glutamic acid (PGA), increased the intracellular accumulation of CPT-11 and SN-38 in primary rat hepatocytes. They also significantly decreased the transport of CPT-11 and SN-38 in Caco-2 and parental MDCKII cells. Thalidomide and PGA also significantly inhibited P-glycoprotein (PgP/MDR1), multidrug resistance-associated protein (MRP1)- and MRP2-mediated CPT-11 and SN-38 transport in MDCKII cells. These results provide insights into the pharmacodynamic and pharmacokinetic mechanisms for the protective effects of thalidomide against CPT-11-induced intestinal toxicity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

Small interfering RNA-mediated silencing of cytochrome P450 3A4 gene.

RNA interference (RNAi) is a specific and powerful tool used to manipulate gene expression and study gene function. The cytochrome P450 3A4 (CYP3A4) can metabolize more than 50% of drugs. In the present study, we investigated whether vector-expressed small interfering RNAs (siRNAs) altered the CYP3A4 expression and function using the Chinese hamster cell line (V79) overexpressing CYP3A4 (CHL-3A4). Three different siRNA oligonucleotides (3A4I, 3A4II, and 3A4III) were designed and tested for their ability to interfere with CYP3A4 gene expression. Our study demonstrated that transient transfection of CHL-3A4 cells with the 3A4III siRNAs, but not 3A4I and II, significantly reduced CYP3A4 mRNA levels by 65% and protein expression levels by 75%. All these siRNAs did not affect the expression of CYP3A5 at both mRNA and protein levels in V79 cells overexpressing CYP3A5. Transfection of CHL-3A4 cells with 3A4III siRNAs significantly diminished the cytotoxicity of two CYP3A4 substrate drugs, cyclophosphamide and ifosfamide, in CHL-3A4 cells, with the IC50 increased from 55 to 210 microM to >1000 microM. Nifedipine at 5.78, 14.44, and 28.88 microM was significantly (P < 0.01) depleted by approximately 100, 40, and 22%, respectively, in S9 fractions from CHL-3A4 cells compared with parental CHL-pIC19h cells. In addition, transfection of the CHL-3A4 cells with vectors expressing the 3A4III siRNAs almost completely inhibited CYP3A4-mediated nifedipine metabolism. This study demonstrated, for the first time, the specific suppression of CYP3A4 expression and function using vector-based RNAi technique. The use of RNAi is a promising tool for the study of cytochrome P450 family function.

Animals↗
PubMed2006

Involvement of the GC-rich sequence and specific proteins (Sp1/Sp3) in the basal transcription activity of neurogranin gene.

Neurogranin (Ng), a neuronal protein implicated in learning and memory, contains a TATA-less promoter. Analysis of 5'-deletion mutations and site-directed mutations of the mouse Ng promoter revealed that a 258bp 5'-flanking sequence (+3 to +260) conferred the basal transcription activity, and that the GC-rich sequence (+22 to +33) served as an important determinant of the promoter activity. Transient transfection of the Sp1 expression plasmid transactivated the reporter activity in neuroblastoma N2A cells while knocking down of endogenous Sp1 expression resulted in a 2.5-fold reduction of the reporter activity in HEK 293 cells. Exogenous expression of Sp3 in HEK 293 cells, however, repressed the reporter activity by 50%. Nevertheless, by gel shift assays, Sp1 and Sp3 were not found to be responsible for the protein-DNA complexes formed by the GC-rich sequence. Moreover, a nuclear factor from the mouse brain tissues was discovered to bind to multiple AT-rich regions in Ng promoter.

Base Composition↗
PubMed2006

Differential mechanisms underlying the modulation of delayed-rectifier K+ channel in mouse neocortical neurons by nitric oxide.

The modulatory effects of nitric oxide (NO) on voltage-dependent K+ channels are intricate. In our present study, the augmentation and reduction of K+ currents by NO donor S-nitro-N-acetylpenicillamine (SNAP) and pure dissolved NO was observed in dissociated neurons from mice neocortex with both whole cell and cell-attached patch clamp. By using a specific electrochemical sensor, the critical concentrations of NO that increased or reduced the channel activities were accurately quantified. Low concentrations of SNAP (20 microM) or NO solution (0.1 microM) enhanced whole cell delayed rectifier K+ -current (IK) and left the fast inactivating A current (IA) unchanged. However, high concentrations of SNAP (100 microM) and NO (0.5 microM) reduced both IK and IA currents. In cell-attached experiments, a significant increase in channel open probability (NP0) was observed when using low concentrations of SNAP or NO. High concentrations of SNAP or NO dramatically decreased NP0. The increase in channel activities by low concentrations of SNAP was abolished in the presence of either inhibitors of soluble guaylate cyclase or inhibitors of cGMP-dependent protein kinase G, suggesting a link to the NO-cGMP signaling cascade. The reduction of channel activities by high concentrations of SNAP was reversed by the reducing agent dithiothreitol, implying a redox reaction mechanism. Thus both NO-cGMP signaling and a redox mechanism are involved in the modulation of IK channel activity for neuron excitability.

Animals↗
PubMed2006

Hypochlorous acid induces apoptosis of cultured cortical neurons through activation of calpains and rupture of lysosomes.

3-Chlorotyrosine, a bio-marker of hypochlorous acid (HOCl) in vivo, was reported to be substantially elevated in the Alzheimer's disease (AD) brains. Thus, HOCl might be implicated in the development of AD. However, its effect and mechanism on neuronal cell death have not been investigated. Here, we report for the first time that HOCl treatment induces an apoptotic-necrotic continuum of concentration-dependent cell death in cultured cortical neurons. Neurotoxicity caused by an intermediate concentration of HOCl (250 microm) exhibited several biochemical markers of apoptosis in the absence of caspase activation. However, the involvement of calpains was demonstrated by data showing that calpain inhibitors protect cortical neurons from apoptosis and the formation of 145/150 kDa alpha-fodrin fragments. Moreover, an increase in cytosolic Ca2+ concentration was associated with HOCl neurotoxicity and Ca2+ channel antagonists, and Ca2+ chelators prevented cleavage of alpha-fodrin and the induction of apoptosis. Finally, we found that calpain activation ruptured lysosomes. Stabilization of lysosomes by calpain inhibitors or imidazoline drugs, as well as inhibition of cathepsin protease activities, rescued cells from HOCl-induced neurotoxicity. Our results showed for the first time that HOCl induces apoptosis in cortical neurons, and that the cell death process involves calpain activation and rupture of lysosomes.

Adenosine Triphosphate↗
PubMed2005

Imaging the development of an ischemic core following photochemically induced cortical infarction in rats using Laser Speckle Contrast Analysis (LASCA).

Laser Speckle Contrast Analysis (LASCA) has been used to image the development of an ischemic core following photochemically induced infarction in rats up to 1 h post-lesion. Using LASCA, we have been able to image a central ischemic core which had little or no perfusion surrounded by a penumbral region with reduced perfusion. In addition, we have shown the existence of a surrounding region of hyperemic tissue. A potential feature of this imaging approach is its capability to track cerebral blood flow (CBF) changes in the region within and outside the ischemic core besides demonstrating the real-time progression of the ischemic core into the penumbral region. We have demonstrated the continuous disruption of CBF to the ischemic core that eventually affected the blood supply to the surrounding regions. The penumbral flow is shown to exhibit a sudden increase post-ischemic induction followed by a slow decline to the final baseline level. Interestingly, we observed an interaction (P < 0.03) between penumbral flow peak and the time effects of increase in pixel area from the infarct region to the surrounding penumbral region. Using a paired-sample t test, we observed that the mean pixel area was larger for the infarct region than for the penumbral region (P < 0.004) during the time interval between the induction of ischemia and the time point of peak flow in the penumbral region.

Algorithms↗
PubMed2005

Dissociation of cortical regions modulated by both working memory load and sleep deprivation and by sleep deprivation alone.

Working memory is an important mental capacity that is compromised following sleep deprivation (SD). To understand how working memory load interacts with state to influence brain activation in load-sensitive regions, and the extent to which SD-related changes are common across different loads, we used fMRI to study twelve healthy subjects following 24 h of SD using a verbal n-back task with three load levels. Performance decline was observed by way of reduced accuracy and slower response times following SD. The left prefrontal region and thalamus showed load dependent activity modulation that interacted with state. The right parietal and anterior medial frontal regions showed load dependent changes in activity as well as an effect of state. The anterior cingulate and occipital regions showed activation that displayed state effects that were independent of working memory load. These findings represent a step toward identifying how different brain regions exhibit varying vulnerability to the deleterious effects of SD on working memory.

Adult↗
PubMed2005

Preparation and characterization of aligned carbon nanotube-ruthenium oxide nanocomposites for supercapacitors.

A novel type of ruthenium oxide (RuO(2))-modified multi-walled carbon nanotube (MWNT) nanocomposite electrode (RuO(2)/MWNT) for supercapacitors has been prepared. The nanocomposites were formed by depositing Ru by magnetic-sputtering in an Ar/O(2) atmosphere onto MWNTs, which were synthesized on Ta plates by chemical vapor deposition. Cyclic voltammetry, chronopotentiometry, and electrochemical impedance measurements were applied to investigate the performance of the RuO(2)/MWNT nanocomposite electrodes. The capacitance of the MWNT electrodes in 1.0 M H(2)SO(4) is significantly increased from 0.35 to 16.94 mF cm(-2) by modification with RuO(2). The RuO(2) film on the surface of the nanotubes is composed of small crystal grains with tilted bundle-like microstructures, as observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The results demonstrate a promising route to prepare RuO(2)/MWNT-based double-layer supercapacitors.

Electrochemistry↗
PubMed2004

Neurogranin expression in stably transfected N2A cell line affects cytosolic calcium level by nitric oxide stimulation.

To test a cellular effect of rodent neurogranin (Ng) oxidation as compared to Ng phosphorylation, we develop a cell model capable of stable expression of Ng using the Tet-On system, and determine whether Ng oxidation regulates intracellular calcium level. Our results show that Ng oxidation by nitric oxide donor induces an increase of [Ca(2+)](i) in Ng-expressed cells as compared to the control cells without expressing Ng. These results suggest that Ng oxidation plays a significant role in intracellular Ca(2+) homeostasis, essential for the activated signaling networks in learning and memory.

Animals↗
PubMed2004

Biosensing properties of diamond and carbon nanotubes.

The biochemical properties of boron-doped diamond (BDD), carbon nanofiber, fullerene, and multiwalled carbon nanotube (MWCNT) electrodes have been investigated comparatively. Physiochemical factors which affect the biosensing properties such as surface hydrophobicities, effective surface area, and intrinsic material properties are studied. Voltammetric responses of the as-grown thin film electrode and surface-modified electrode to biomolecules such as L-ascorbic acid (L-AA), dopamine (DA), and uric acid are examined. As-grown MWCNT electrodes exhibit selective voltammetric responses to the different biomolecules and faster electron-transfer kinetics compared to BDD. The selective response is due to the considerably lower anodic potential of L-AA on MWCNT (-48 mVvs Ag/AgCl compared to 575 mV on BDD). This electrocatalytic response can be replicated on a nonselective carbon nanofiber electrode by coating it with gold nanoparticles. BDD has no intrinsic selective response to L-AA, and surface modification by anodic polarization is necessary for resolving L-AA and DA.

Ascorbic Acid↗
PubMed2004

Do mitochondria make nitric oxide? no?

Several papers have claimed that mitochondria contain nitric oxide synthase (NOS) and make nitric oxide (NO*) in amounts sufficient to affect mitochondrial respiration. However, we found that the addition of L-arginine or the NOS inhibitor L-NMMA to intact rat liver mitochondria did not have any effect on the respiratory rate in both State 3 and State 4. We did not detect mitochondrial NO* production by the oxymyoglobin oxidation assay, or electrochemically using an NO* electrode. An apparent NO* production detected by the Griess assay was identified as an artifact. NO* generated by eNOS added to the mitochondria could easily be detected, although succinate-supplemented mitochondria appeared to consume NO*. Our data show that NO* production by normal rat liver mitochondria cannot be detected in our laboratory, even though the levels of production claimed in the literature should easily have been measured by the techniques used. The implications for the putative mitochondrial NOS are discussed.

Animals↗
PubMed2004

Induction of transient ion channel-like pores in a cancer cell by antibiotic peptide.

The anticancer activity of anti-bacterial cecropins makes them potentially useful as peptide anti-cancer drugs. We used the cell-attached patch to study the effect of cecropin B (CB; having one hydrophobic and one amphipathic alpha-helix) and its derivative, cecropin B3 (CB3; having two hydrophobic alpha-helices) on the membrane of Ags cancer cells. Application of 10-60 microM CB onto the membrane of the cancer cell produces short outward currents. Comparative study with CB3, which induces no outward currents, shows that the amphipathic group of CB is necessary for the pore formation. The results provide a rationale to study the cell-killing activity of antimicrobial peptides at the single cancer cell level.

Anti-Bacterial Agents↗
PubMed2003

Structural and dynamic characterization of a neuron-specific protein kinase C substrate, neurogranin.

Neurogranin/RC3 is a neuron-specific, Ca(2+)-sensitive calmodulin binding protein and a specific protein kinase C substrate. Neurogranin may function to regulate calmodulin levels at specific sites in neurons through phosphorylation at serine residue within its IQ motif, oxidation outside the IQ motif, or changes in local cellular Ca(2+) concentration. To gain insight into the functional role of neurogranin in the regulation of calmodulin-dependent activities, we investigated the structure and dynamics of a full-length rat neurogranin protein with 78 amino acids using triple resonance NMR techniques. In the absence of calmodulin or PKC, neurogranin exists in an unfolded form as evidenced by high backbone mobility and the absence of long-range nuclear Overhauser effect (NOE). Analyses of the chemical shifts (13)C(alpha), (13)C(beta), and (1)H(alpha) reveal the presence of a local alpha-helical structure for the region between residues G25-A42. Three-bond (1)H(N)-(1)H(alpha) coupling constants support the finding that the sequence between residues G25 and A42 populates a non-native helical structure in the unfolded neurogranin. Homonuclear NOE results are consistent with the conclusions drawn from chemical shifts and coupling constants. (15)N relaxation data indicate motional restrictions on a nanosecond time scale in the region from D15 to S48. Spectral densities and order parameters data further confirm that the unfolded neurogranin exists in conformation with residual secondary structures. The medium mobility of the nascent helical region may help to reduce the entropy loss when neurogranin binds to its targets, but the complex between neurogranin and calmodulin is not stable enough for structural determination by NMR. Calmodulin titration of neurogranin indicates that residues D15-G52 of neurogranin undergo significant structural changes upon binding to calmodulin.

Animals↗
PubMed2003

Nanostructured platinum-lipid bilayer composite as biosensor.

The present work describes the preparation of supported bilayer lipid membrane (s-BLM) doped with metal nanoparticles for the design of biosensors. Platinum (Pt) nanoparticles were deposited through s-BLM to build a hybrid device of nanoscale electrode array by potential cycling in 1 mM K(2)PtCl(6) solution containing 0.1 M KCl. The properties of Pt nanoparticle-doped s-BLM composite were then characterized by cyclic voltammetry, electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM). Our results showed that Pt nanoparticles grew in voids of the s-BLMs, through which the underlying glassy carbon (GC) electrode was connected, with maximum length extended out of the lipid membrane around 40 nm. Doping of Pt nanoparticles through s-BLM increased the membrane capacitance and decreased the membrane resistance of s-BLM. Pt nanoparticles array in s-BLM electrocatalyzed the reduction of oxygen (O(2)) in phosphate buffer solution (PBS). Practical application of Pt nanoparticle-doped s-BLM for the construction of glucose biosensor was also demonstrated in terms of its dose-response curve, stability and reproducibility. Thus, lipid membrane doped with Pt nanoparticles is a novel electrode system at nanoscale that can penetrate through the insulating membrane to probe molecular recognition and catalytic events at the lipid membrane-solution interface.

Biosensing Techniques↗