Chien Tsu Chen

Molecular modeling of flavonoids that inhibits xanthine oxidase

The inhibition of xanthine oxidase activity by various flavonoids was assessed. All of the tested flavonoids were competitive inhibitors, and from the kinetic analysis suggested that flavonoids bind to the reactive site. To further understand the stereochemistry between these flavonoids and xanthine oxidase, structure-based molecular modeling was performed. Apigenin was the most potent inhibitor which showed the most favorable interaction in the reactive site. The bicyclic benzopyranone ring of apigenin stacked with phenyl of Phe 914, and the phenolic group stretched to the space surrounding with several hydrophobic residues. Quercetin and myricetin composed a 3-hydroxyl group on benzopyranone which resulting in reduction of binding affinity. The phenolic group of genistein positioned in opposite orientation comparison with apigenin, and resulted in a weaker interaction with xanthine oxidase. Isovitexin showed the weakest inhibitory effect among the compounds tested. The bulky group of sugar in isovitexin may hamper its interaction with xanthine oxidase.

Mediating of caspase-independent apoptosis by cadmium through the mitochondria-ROS pathway in MRC-5 fibroblasts

Cadmium (Cd) is an environmental pollutant of global concern with a 10–30‐year biological half‐life in humans. Accumulating evidence suggests that the lung is one of the major target organs of inhaled Cd compounds. Our previous report demonstrated that 100 μM Cd induces MRC‐5 cells, normal human lung fibroblasts, to undergo caspase‐independent apoptosis mediated by mitochondrial membrane depolarization and translocation of apoptosis‐inducing factor (AIF) from mitochondria into the nucleus. Here, using benzyloxycarbonyl‐Val‐Ala‐Asp‐(ome) fluoromethyl ketone (Z‐VAD.fmk) as a tool, we further demonstrated that Cd could induce caspase‐independent apoptosis at concentrations varied from 25 to 150 μM, which was modulated by reactive oxygen species (ROS) scavengers, such as N‐acetylcysteine (NAC), mannitol, and tiron, indicating that ROS play a crucial role in the apoptogenic activity of Cd. Consistent with this notion, the intracellular hydrogen peroxide (H2O2) was 2.9‐fold elevated after 3 h of Cd treatment and diminished rapidly within 1 h as detected by flow cytometry with 2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA) staining. Using inhibitors of the mitochondrial electron transport chain (ETC) (oligomycin A and rotenone for complex I and V, respectively) and mitochondrial permeability transition pore (MPTP) (cyclosporin A and aristolochic acid), we coincidently found the ROS production, mitochondrial membrane depolarization, and apoptotic content were almost completely or partially abolished. As revealed by confocal microscopy staining with chloromethyl‐X‐rosamine (CMXRos) and an anti‐AIF antibody, the collapse of mitochondrial membrane potential induced by Cd (3 h‐treatment) was a prelude to the translocation of caspase‐independent pro‐apoptotic factor, AIF, into the nucleus (after 4 h of Cd treatment). In summary, this study demonstrated that, in MRC‐5 fibroblasts, Cd induced caspase‐independent apoptosis through a mitochondria‐ROS pathway. More importantly, we provide several lines of evidence supporting a role of mitochondrial ETC and MPTP in the regulation of caspase‐independent cell death triggered by Cd.

Mitochondria-mediated caspase-independent apoptosis induced by cadmium in normal human lung cells.

Cadmium, a well‐known environmental hazard, has caused serious health problems in humans and animals. Accumulating evidence suggests the cadmium toxicity is mediated by oxidative stress‐induced cell death. However, the molecular signaling underlying cadmium‐induced apoptosis remains unclear. In this study, we demonstrate here that cadmium induced mixed types of cell death including primary apoptosis (early apoptosis), secondary necrosis (late apoptosis), and necrosis in normal human lung cells, MRC‐5, as revealed by chromatin condensation, phosphatidylserine (PS) externalization, and hypodiploid DNA content. The total apoptotic cells reached a plateau of around 40.0% after 24 h exposure of 100 μM cadmium. Pretreatment with Z‐Val–Ala–Asp–fluoromethylketone (Z‐VAD–fmk), a broad spectrum of caspase inhibitor, could not rescue apoptotic cells from cadmium toxicity. Coincidently, we failed to detect the activation of pro‐caspase‐3 and cleavage of PARP by immunoblot, which implies the apoptogenic activity of cadmium in MRC‐5 cells is caspase‐independent. JC‐1 staining also indicated that mitochondrial depolarization is a prelude to cadmium‐induced apoptosis, which was accompanied by a translocation of caspase‐independent pro‐apoptotic factor apoptosis‐inducing factor (AIF) into the nucleus as revealed by the immunofluorescence assay. In summary, this study demonstrated for the first time that cadmium induced a caspase‐independent apoptotic pathway through mitochondria‐mediated AIF translocation into the nucleus. J. Cell. Biochem. 89: 335–347, 2003.

A New Strategy for Intracellular Delivery of Enzyme Using Mesoporous Silica Nanoparticles: Superoxide Dismutase

We developed mesoporous silica nanoparticle (MSN) as a multifunctional vehicle for enzyme delivery. Enhanced transmembrane delivery of a superoxide dismutase (SOD) enzyme embedded in MSN was demonstrated. Conjugation of the cell-penetrating peptide derived from the human immunodeficiency virus 1 (HIV) transactivator protein (TAT) to mesoporous silica nanoparticle is shown to be an effective way to enhance transmembrane delivery of nanoparticles for intracellular and molecular therapy. Cu,Zn-superoxide dismutase (SOD) is a key antioxidant enzyme that detoxifies intracellular reactive oxygen species, ROS, thereby protecting cells from oxidative damage. In this study, we fused a human Cu,Zn-SOD gene with TAT in a bacterial expression vector to produce a genetic in-frame His-tagged TAT-SOD fusion protein. The His-tagged TAT-SOD fusion protein was expressed in E. coli using IPTG induction and purified using FMSN-Ni-NTA. The purified TAT-SOD was conjugated to FITC-MSN forming FMSN-TAT-SOD. The effectiveness of FMSN-TAT-SOD as an agent against ROS was investigated, which included the level of ROS and apoptosis after free radicals induction and functional recovery after ROS damage. Confocal microscopy on live unfixed cells and flow cytometry analysis showed characteristic nonendosomal distribution of FMSN-TAT-SOD. Results suggested that FMSN-TAT-SOD may provide a strategy for the therapeutic delivery of antioxidant enzymes that protect cells from ROS damage.

Effects of Cadmium on Structure and Enzymatic Activity of Cu,Zn-SOD and Oxidative Status in Neural Cells

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder disease. Ten percent of the ALS patients are congenital (familial ALS), and the other 90% are sporadic ALS (SALS). It has been shown that mutations found in the Cu,Zn‐SOD cause 20% of the familial ALS due to its low enzyme activity. We hypothesized that heavy metals may interfere the structure of Cu,Zn‐SOD protein to suppress its activity in some of the SALS. In this study, we expressed and characterized the recombinant human Cu,Zn‐SOD under various concentrations of Cu2+, Zn2+, and Cd2+. By atomic absorption spectrophotometry, we demonstrated that adding of cadmium significantly increased the content of cadmium ion, but reduced its Zn2+ content and enzyme activity of the Cu,Zn‐SOD protein. The data of circular dichroism spectra demonstrated that the secondary structure of Cu,Zn‐SOD/Cd is different from Cu,Zn‐SOD, but close to apo‐SOD. In addition to the effect of cadmium on Cu,Zn‐SOD, cadmium was also shown to induce neural cell apoptosis. To further investigate the mechanism of neural cell apoptosis induced by cadmium, we used proteomics to analyze the altered protein expressions in neural cells treated with cadmium. The altered proteins include cellular structural proteins, stress‐related and chaperone proteins, proteins involved in reactive oxygen species (ROS), enzyme proteins, and proteins that mediated cell death and survival signaling. Taken together, in this paper, we demonstrate that cadmium decreases the content of Zn2+, changes the conformation of Cu,Zn‐SOD protein to decrease its enzyme activity, and causes oxidative stress‐induced neural cell apoptosis. J. Cell. Biochem. 98: 577–589, 2006.

Three-dimensional finite element analysis of subdural hematoma.

BACKGROUND Head motion, an important factor in acute subdural hematoma (ASDH), can be broken down into translational and rotational elements. We used three-dimensional finite element analysis to examine the thresholds of angular and tangential acceleration required to tear bridging veins in humans during head impact. METHODS The lengths of midsagittal and parasagittal bridging veins were calculated first. To assess the effect of translational and rotational acceleration, the strain of each vein was then computed under three different motions. The threshold of ASDH was expressed in terms of tangential and rotational acceleration. RESULTS Deformation-angle histories of the midsagittal and parasagittal bridging veins showed that veins that drain forward into the superior sinus at a 130-degree angle incurred the greatest stretch strain during occipital impact. In the midsagittal plane, pure rotation induced greater stretch strain on these veins (14.4%) than pure translation (2.5%) or combined translation and rotation motion (10.4%). A tangential acceleration of 3,912.9 G or an angular acceleration of 71.2 krad/s2 seemed to approximate the threshold for ASDH in the human midsagittal plane, whereas 5,010.9 G and 97.4 krad/s2 approximated the threshold in the parasagittal plane. CONCLUSION Impact direction and orientation of bridging veins are both important factors in ASDH. Threshold criteria for ASDH can be expressed in terms of tangential and rotational acceleration.

Inhibitory effects of a rice hull constituent on tumor necrosis factor α, prostaglandin E2, and cyclooxygenase-2 production in lipopolysaccharide-activated mouse macrophages

Abstract: Isovitexin, isolated from rice hull of Oryza sativa, has been characterized as a potent antioxidant. Its antioxidant activity, determined on the basis of inhibition of lipid peroxidation by the Fenton reaction, was comparable with that of α‐tocopherol, a well‐established antioxidant. Isovitexin was able to reduce the amount of hydrogen peroxide production induced by lipopolysaccharide (LPS) in mouse macrophage RAW264.7 cells. In this study, we assessed its effects on the production of tumor necrosis factor α (TNF‐α), prostaglandin E2 (PGE2), and the expression of cyclooxygenase‐2 (COX‐2) in LPS‐activated RAW 264.7 macrophages. Isovitexin inhibited the release of TNF‐α, a proinflammatory cytokine, upon LPS activation with a 50% inhibitory concentration (IC50) of 78.6 μM. Isovitexin markedly reduced LPS‐stimulated PGE2 production in a concentration‐dependent manner, with an IC50 of 80.0 μM. The expression of COX‐2 was also inhibited by isovitexin treatment. Our results suggest that suppression of ROS‐mediated COX‐2 expression by isovitexin is beneficial in reducing inflammation and carcinogenesis.

Finite element analysis of brain contusion: An indirect impact study

The mechanism of brain contusion has been investigated using a series of three-dimensional (3D) finite element analyses. A head injury model was used to simulate forward and backward rotation around the upper cervical vertebra. Intracranial pressure and shear stress responses were calculated and compared. The results obtained with this model support the predictions of cavitation theory that a pressure gradient develops in the brain during indirect impact. Contrecoup pressure-time histories in the parasagittal plane demonstrated that an indirect impact induced a smaller intracranial pressure (−53.7 kPa for backward rotation, and −65.5 kPa for forward rotation) than that caused by a direct impact. In addition, negative pressures induced by indirect impact to the head were not high enough to form cavitation bubbles, which can damage the brain tissue. Simulations predicted that a decrease in skull deformation had a large effect in reducing the intracranial pressure. However, the areas of high shear stress concentration were consistent with those of clinical observations. The findings of this study suggest that shear strain theory appears to better account for the clinical findings in head injury when the head is subjected to an indirect impact.

Identification of neutral and acidic sphingomyelinases in Helicobacter pylori

We demonstrated for the first time the presence of sphingomyelinase (SMase) in Helicobacter pylori. Activation of SMase has been implicated as the cause of elevation of cellular ceramide levels and consequently of apoptosis. The data indicate that there are two classes of SMase, defined by their optimal pHs and cellular locations, existing in H. pylori. One is an Mg2+‐dependent membrane‐bound enzyme with an optimal activity at pH 7, and the other is an Mg2+‐independent cytosolic enzyme with an optimal activity at pH 5. Bisalumin, a bismuth salt, was found to inhibit the activities of both forms of SMase regardless of the presence of Mg2+. By Western blot analysis, the membrane‐bound SMases of H. pylori and Bacillus cereus were shown to be antigenically related and to have a similar denatured molecular mass of 28 kDa.

In vitro diffusion of lidocaine across endotracheal tube cuffs.

PurposeLidocaine diffuses across endotracheal tube cuffs, which may serve as a reservoir for local anesthetic to assist in the prevention of ETT-induced cough while emerging from general anesthesia. However, the rate of diffusion is slow. Two techniques, alkalization and warming, may increase the proportion of uncharged drug available for diffusion. The purpose of this study is to determine the effectiveness of warming alkalization or warming with alkalization on diffusion.MethodsFour preparations of lidocaine 4% were studied. Group (Gr) L-lidocaine (24°C), Gr WL — warmed lidocaine (38°C), Gr AL — alkalized lidocaine (24°C), Gr WAL — warmed, alkalized lidocaine (38°C). Twenty-four Mallinckrodt 8.0 ID (Mallinckrodt Critical Care Division of Mallinckrodt, Inc., Glens Falls, New York) endotracheal tube cuffs were filled with 6 ml of one of the four preparations. They were then placed in a 20 ml water bath at 38°C and samples were drawn from the water bath at intervals for up to 360 min. The lidocaine concentration in each sample was determined by gas chromatography.ResultsThe highest lidocaine concentration was reached in Gr WAL (410.98 ± 8.53 μ·ml−1) after 300 min and then decreased to 376.18 ± 4.59 μ·ml−1 after 360 min. In Gr AL the highest concentration (235.05 ± 2.99 μ·ml−1) was reached after 360 min. Lidocaine concentrations in Gr L and WL after 360 min were 3.19 ± 1.16 μ·ml−1 and 4.32 ± 2.02 μ·ml−1 respectively.ConclusionAlkalization with or without warming, but not warming alone, promotes lidocaine diffusion from endotracheal tube cuff.RésuméObjectifLa lidocaine diffuse, lentement toutefois, au travers des ballonnets des tubes endotrachéaux. Les ballonnets peuvent servir de réservoir à l’anesthésique local et participer à la prévention de la toux induite par le TET pendant la récupération de l’anesthésie générale. L’alcalinisation et le réchauffement peuvent accroître la proportion de médicament susceptible de diffuser. Lobjectif de la présente étude est de déterminer l’efficacité du réchauffement, seul ou avec alcalinisation, sur la diffusion.MéthodeOn a étudié quatre préparations de lidocaïne. Le groupe (Gr) L-lidocaïne (24 °C), Gr LR — lidocaïne réchauffée (38 °C), Gr LA — lidocaïne alcalinisée (24 °C), Gr LRA - lidocaïne réchauffée, alcalinisée (38 °C). Vingtquatre tubes endotrachéaux à ballonnets Mallinckrodt de Dl 8,0 (Mallinckrodt Critical Care Division of Mallinckrodt, Inc., Glens Falls, New York) ont été remplis de 6 ml de l’une des préparations. Placés ensuite dans un bain d’eau de 20 ml à 38 °C, on en a ensuite retiré des échantillons à des intervalles pouvant aller jusqu’à 360 min. La concentration de lidocaïne a été déterminée par Chromatographie en phase gazeuse.RésultatsLa plus forte concentration de lidocaïne a été atteinte dans le Gr LRA, 410,98 ± 8,53 μ·ml−1, après 300 min, puis elle a baissé à 376,18 ± 4,59 μ·ml−1 après 360 min. Dans le Gr LA, la concentration maximale était de 235,05 ± 2,99 μ·ml−1 après 360 min. Dans les Gr L et LR, la lidocaïne était, après 360 min de 3,19 ± 1,16 μ·ml−1 et de 4,32 ± 2,02 μ·ml−1 respectivement.ConclusionLalcalinisation avec ou sans réchauffement, mais non le réchauffement employé seul, favorise la diffusion de la lidocaïne au travers du ballonnet du tube endotrachéal.