Chun Mao Lin

Structure-activity relationship of coumarin derivatives on xanthine oxidase-inhibiting and free radical-scavenging activities.

We employed 1,1-diphenyl-2-picrylhydrazyl hydrate (DPPH)- and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-electron spin resonance (ESR) to study the effects of suppression of reactive oxygen species (ROS) by eight selected coumarin derivatives under oxidative conditions. Esculetin was the most potent radical scavenger among the eight tested compounds. Our results suggest that the number of hydroxyl groups on the ring structure of coumarins is correlated with the effects of ROS suppression. We also investigated the effect of the derivatives on the inhibition of xanthine oxidase (XO) activity, and the structure-activity relationships (SARs) of these derivatives against XO activity were further examined using computer-aided molecular modeling. All determined derivatives competitively inhibited XO. The results of the structure-based molecular modeling exhibited interactions between coumarins and the molybdopterin region of XO. The carbonyl pointed toward the Arg880, and the ester O atom formed hydrogen bonds with Thr1010. Esculetin, which bears two hydroxyl moieties on its benzene rings, had the highest affinity toward the binding site of XO, and this was mainly due to the interaction of 6-hydroxyl with the E802 residue of XO. The hypoxanthine/XO reaction in the DMPO-ESR technique was used to assess the combined effect on enzyme inhibition and ROS suppression by these coumarins, and the results showed that esculetin was the most potent agent among the tested compounds. We further evaluated the effects of the test compounds on living cells, and esculetin was still the most potent agent at protecting cells against ROS-mediated Abeta-damage among the tested coumarins.

Glucose biosensor based on glucose oxidase immobilized at gold nanoparticles decorated graphene-carbon nanotubes

Biopolymer pectin stabilized gold nanoparticles were prepared at graphene and multiwalled carbon nanotubes (GR-MWNTs/AuNPs) and employed for the determination of glucose. The formation of GR-MWNTs/AuNPs was confirmed by scanning electron microscopy, X-ray diffraction, UV-vis and FTIR spectroscopy methods. Glucose oxidase (GOx) was successfully immobilized on GR-MWNTs/AuNPs film and direct electron transfer of GOx was investigated. GOx exhibits highly enhanced redox peaks with formal potential of -0.40 V (vs. Ag/AgCl). The amount of electroactive GOx and electron transfer rate constant were found to be 10.5 × 10(-10) mol cm(-2) and 3.36 s(-1), respectively, which were significantly larger than the previous reports. The fabricated amperometric glucose biosensor sensitively detects glucose and showed two linear ranges: (1) 10 μM - 2 mM with LOD of 4.1 μM, (2) 2 mM - 5.2 mM with LOD of 0.95 mM. The comparison of the biosensor performance with reported sensors reveals the significant improvement in overall sensor performance. Moreover, the biosensor exhibited appreciable stability, repeatability, reproducibility and practicality. The other advantages of the fabricated biosensor are simple and green fabrication approach, roughed and stable electrode surface, fast in sensing and highly reproducible.

Evodiamine Stabilizes Topoisomerase I-DNA Cleavable Complex to Inhibit Topoisomerase I Activity

Evodiamine (EVO), an alkaloidal compound isolated from Evodia rutaecarpa (Juss.), has been reported to affect many physiological functions. Topoisomerase inhibitors have been developed in a variety of clinical applications. In the present study, we report the topoisomerase I (TopI) inhibitory activity of EVO, which may have properties that lead to improved therapeutic benefits. EVO is able to inhibit supercoiled plasmid DNA relaxation catalyzed by TopI. Upon treatment 0~10 μM EVO TopI was depleted in MCF-7 breast cancer cells in a concentration-dependent and time-dependent manner in 0~120 min. A K-SDS precipitation assay was performed to measure the extent of Top I-trapped chromosomal DNA. The ability of EVO to cause the formation of a TopI-DNA complex increased in a concentration-dependent manner, in that the DNA trapped increased by 24.2% in cells treated with 30 μM. The results suggest that EVO inhibits TopI by stabilizing the enzyme and DNA covalent complex.

Structural Characterization and Antioxidative Activity of Low-Molecular-Weights Beta-1,3-Glucan from the Residue of Extracted Ganoderma lucidum Fruiting Bodies

The major cell wall constituent of Ganoderma lucidum (G. lucidum) is β-1,3-glucan. This study examined the polysaccharide from the residues of alkaline-extracted fruiting bodies using high-performance anion-exchange chromatography (HPAEC), and it employed nuclear magnetic resonance (NMR) and mass spectrometry (MS) to confirm the structures. We have successfully isolated low-molecular-weight β-1,3-glucan (LMG), in high yields, from the waste residue of extracted fruiting bodies of G. lucidum. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay evaluated the capability of LMG to suppress H2O2-induced cell death in RAW264.7 cells, identifying that LMG protected cells from H2O2-induced damage. LMG treatment decreased H2O2-induced intracellular reactive oxygen species (ROS) production. LMG also influenced sphingomyelinase (SMase) activity, stimulated by cell death to induce ceramide formation, and then increase cell ROS production. Estimation of the activities of neutral and acid SMases in vitro showed that LMG suppressed the activities of both neutral and acid SMases in a concentration-dependent manner. These results suggest that LMG, a water-soluble β-1,3-glucan recycled from extracted residue of G. lucidum, possesses antioxidant capability against H2O2-induced cell death by attenuating intracellular ROS and inhibiting SMase activity.

A potent sphingomyelinase inhibitor from Cordyceps mycelia contributes its cytoprotective effect against oxidative stress in macrophages.

A novel water-soluble polysaccharide fraction, CME-1, with a molecular mass of 27.6 kDa and containing mannose and galactose in a respective ratio of 4:6, was prepared from Cordyceps sinensis mycelia and identified by NMR and GC-MS. In the current study, we examined whether CME-1 has anti-inflammatory effects in RAW264.7 cells. The ability of CME-1 to inhibit H2O2-induced cell death in RAW264.7 cells was assessed by using an MTT assay and annexin V/propidium iodide double staining; we found that CME-1 protected cells against H2O2-induced injury. H2O2-induced intracellular oxidative stress and mitochondrial membrane depolarization were also diminished with CME-1 treatment. We evaluated the hydroxyl radical scavenging ability of CME-1 by using the DMPO-electron spin resonance technique, which indicated that CME-1 acts as an intracellular antioxidant in a concentration-dependent manner through a mechanism other than its scavenging activity. Activities of both neutral and acid sphingomyelinases (SMases) were assessed in vitro, and results showed that the CME-1 inhibited activities of both neutral and acid SMases in a concentration-dependent manner. CME-1 reduced H2O2 treatment-elevated C16- and C18-ceramide levels measured by LC/MS/MS in RAW264.7 cells. Results suggest that CME-1 protects RAW264.7 cells against oxidative stress through inhibition of SMase activity and reduction of C16- and C18-ceramide levels.

Development and biological evaluation of C60 fulleropyrrolidine-thalidomide dyad as a new anti-inflammation agent

Research studies in the field of C(60) fullerene derivatives have significantly increased due to the broad range of biological activities that were found for these compounds. We designed and prepared a new C(60) fullerene hybrid bearing thalidomide as a potential double-action anti-inflammatory agent, capable of simultaneous inhibition of LPS-induced NO and TNF-alpha production. The C(60) fulleropyrrolidine-thalidomide dyad, CLT, was an effective agent to suppress the release of NO and TNF-alpha by the LPS-stimulated macrophages RAW 264.7. Ten micromolars of CLT effectively inhibited LPS-induced NO and TNF-alpha production by 47.3+/-4.2% and 70.2+/-4% with respected to the control, respectively. Furthermore, preliminary biochemical investigation revealed that CLT was a potent agent to suppress both LPS-induced intracellular ROS production and iNOS expression, and CLT also inhibited the phosphorylation of ERK which is an important protein kinase involved in the activation of TNF-alpha synthesis in LPS-activated macrophages. We believed that the studies herein would hold promise for future development of a new generation of potent anti-inflammatory agents.

Hydrophilic Ester-Bearing Chlorogenic Acid Binds to a Novel Domain to Inhibit Xanthine Oxidase

Caffeic acid is a xanthine oxidase (XO) inhibitor that binds to the molybdopterin region of its active site. Caffeic acid phenethyl ester (CAPE) has higher hydrophobicity and exhibits stronger inhibition potency toward XO. Chlorogenic acid is a quinyl ester of caffeic acid that has increased hydrophilicity and also shows stronger XO inhibitory activity compared with caffeic acid. Caffeic acid and CAPE showed competitive inhibition against XO, whereas chlorogenic acid displayed mixed-type inhibition, implying that it binds to sites other than the active site. Structure-based molecular modeling was performed to account for the different binding characteristics of the hydrophobic and hydrophilic esters of caffeic acid. Chlorogenic acid showed weak binding to the molybdopterin region of XO, while it more strongly bound the flavin adenine dinucleotide region than it did the molybdopterin region. These results provide the basis for interactions of caffeic acid analogues with XO via various binding domains.

Suppression effect of seminal vesicle autoantigen on platelet-activating factor-induced mouse sperm capacitation

Mammalian sperm gain the ability to fertilize an egg successfully by the capacitation process. An unregulated capacitation process causes sperm to undergo a spontaneous acrosome reaction (AR) and resulting in loss of their fertilization activity. Thus, functional sperm activation is tightly regulated by a capacitation and suppression (decapacitation) mechanism. Factors, such as platelet‐activating factor (PAF) present in both sperm and the female genital tract, are able to stimulate sperm capacitation. Seminal plasma is thought to have the ability to suppress sperm capacitation; however, the regulatory mechanisms of seminal plasma protein on sperm capacitation are not well understood. Recently, we demonstrated that seminal vesicle autoantigen (SVA), a major seminal vesicle secretory protein, is able to suppress mouse sperm capacitation. To further study the suppression spectra of SVA on sperm capacitation, we investigated the effect of SVA on PAF‐induced mouse sperm capacitation‐related signals. Here, we demonstrate that SVA decreases the [Ca2+]i to suppress the PAFs effects on [Ca2+]i, the cAMP level, protein tyrosine phosphorylation, and capacitation. The inhibition of PAF‐induced protein tyrosine phosphorylation and capacitation by SVA can be reversed by cAMP agonists. Characterization of the interactions of SVA with PAF by TLC overlay and tryptophan fluorescence spectrum analyses indicates that SVA is capable of binding PAF with an apparent dissociation constant Kd > 50 µM. Together with these results, we demonstrate that SVA deceases [Ca2+]i and cross‐talks with PAF‐induced intracellular signals to regulate mouse sperm capacitation. J. Cell. Biochem. 100: 941–951, 2007.

Inhibition of AMPK-associated autophagy enhances caffeic acid phenethyl ester-induced cell death in C6 glioma cells

An increasing number of studies show that AMP-activated protein kinase (AMPK) activation can inhibit apoptosis. To clarify the antitumor mechanism of caffeic acid phenethyl ester (CAPE) and achieve increased therapeutic efficiency, we investigated the potential roles of AMPK and autophagy in CAPE treatment against C6 glioma cells. The roles of AMPK and autophagy inhibition in CAPEs cytotoxic action were investigated. Phosphorylation of AMPK and mitogen-activated protein kinases (MAPKs) were observed in tumor cells following CAPE treatment. A combination of CAPE and the AMPK inhibitor, compound C, resulted in augmented cell death. Similar effects of compound C were observed in response to changes in the mitochondrial membrane potential ( ΔΨ(m)). Small interfering RNA-mediated AMPK downregulation increased CAPE-induced cell death. The results suggest that AMPK activation plays a role in diminishing apoptosis. CAPE treatment induced an increase in LC3 conversion as represented by the LC3-II/LC3-I ratio. Enlarged lysosomes and autophagosomes were present according to electron microscopy. The autophagy inhibitor, 3-MA, caused increased CAPE cytotoxicity, which suggests that autophagy induction protected glioma cells from CAPE. The combination of CAPE with autophagy and AMPK inhibitors markedly enhanced the cytotoxicity toward C6 glioma cells. Accordingly, CAPE-triggered activation of AMPK and the autophagic response protected tumor cells from apoptotic death. This provides new insights for combined therapy to enhance the therapeutic potential of cancer treatments.

Immobilizing topoisomerase I on a surface plasmon resonance biosensor chip to screen for inhibitors

BackgroundThe topoisomerase I (TopI) reaction intermediate consists of an enzyme covalently linked to a nicked DNA molecule, known as a TopI-DNA complex, that can be trapped by inhibitors and results in failure of re-ligation. Attempts at new derivative designs for TopI inhibition are enthusiastically being pursued, and TopI inhibitors were developed for a variety of applications. Surface plasmon resonance (SPR) was recently used in TopI-inhibition studies. However, most such immobilized small molecules or short-sequence nucleotides are used as ligands onto sensor chips, and TopI was used as the analyte that flowed through the sensor chip.MethodsWe established a sensor chip on which the TopI protein is immobilized to evaluate TopI inhibition by SPR. Camptothecin (CPT) targeting the DNA-TopI complex was used as a representative inhibitor to validate this label-free method.ResultsPurified recombinant human TopI was covalently coupled to the sensor chip for the SPR assay. The binding of anti-human (h)TopI antibodies and plasmid pUC19, respectively, to the immobilized hTopI was observed with dose-dependent increases in resonance units (RU) suggesting that the immobilized hTopI retains its DNA-binding activity. Neither CPT nor evodiamine alone in the analyte flowing through the sensor chip showed a significant increase in RU. The combination of pUC19 and TopI inhibitors as the analyte flowing through the sensor chip caused increases in RU. This confirms its reliability for binding kinetic studies of DNA-TopI binders for interaction and for primary screening of TopI inhibitors.ConclusionsTopI immobilized on the chip retained its bioactivities of DNA binding and catalysis of intermediates of the DNA-TopI complex. This provides DNA-TopI binders for interaction and primary screening with a label-free method. In addition, this biochip can also ensure the reliability of binding kinetic studies of TopI.