Critical roles of reactive oxygen species in mitochondrial permeability transition in mediating evodiamine-induced human melanoma A375-S2 cell apoptosis

Previous studies have shown that evodiamine could trigger apoptosis in human malignant melanoma A375-S2 cells within 24 h. To further investigate the biochemical basis of this activity, the roles of reactive oxygen species (ROS) and mitochondrial permeability transition (MPT) were evaluated. Exposure to evodiamine led to a rapid increase in intracellular ROS followed by an onset of mitochondrial depolarization. ROS scavenger rescued the ΔΨm dissipation and cell death induced by evodiamine, whilst MPT inhibitor blocked the second-time ROS formation as well as cell death. Expressions of key proteins in Fas- and mitochondria-mediated pathways were furthermore examined. Both pathways were activated and regulated by ROS and MPT and were converged to a final common pathway involving the activation of caspase-3. These data suggested that a phenomenon termed ROS-induced ROS release (RIRR) was involved in evodiamine-treated A375-S2 cells and greatly contributed to the apoptotic process through both extrinsic and intrinsic pathways.     

Reactive oxygen species and nitric oxide regulate mitochondria-dependent apoptosis and autophagy in evodiamine-treated human cervix carcinoma HeLa cells

The redox environment of the cell is currently thought to be extremely important to control either apoptosis or autophagy. This study reported that reactive oxygen species (ROS) and nitric oxide (NO) generations were induced by evodiamine time-dependently; while they acted in synergy to trigger mitochondria-dependent apoptosis by induction of mitochondrial membrane permeabilization (MMP) through increasing the Bax/Bcl-2 or Bcl-x_L ratio. Autophagy was also stimulated by evodiamine, as demonstrated by the positive autophagosome-specific dye monodansylcadaverine (MDC) staining as well as the expressions of autophagy-related proteins, Beclin 1 and LC3. Pre-treatment with 3-MA, the specific inhibitor for autophagy, dose-dependently decreased cell viability, indicating a survival function of autophagy. Importantly, autophagy was found to be promoted or inhibited by ROS/NO in response to the severity of oxidative stress. These findings could help shed light on the complex regulation of intracellular redox status on the balance of autophagy and apoptosis in anti-cancer therapies.     

Pharmacokinetics Study of Amoxycillin and Clavulanic acid (8:1)-A New Combination in Healthy Chinese Adult Male Volunteers Using the LC–MS/MS Method

New oral granules of amoxicillin and clavulanic acid in 8:1 ratio have recently been developed and approved to conduct clinical trial in China. To date, there has been no report studying the pharmacokinetic characteristics of amoxicillin and clavulanic acid in man. Therefore, it is urgent to investigate the pharmacokinetic properties of amoxicillin and clavulanic acid in man. The aim of the study was to assess the pharmacokinetic properties of amoxicillin and clavulanic acid in 8:1 with different dosage in healthy volunteers and provide support for this drug to obtain marketing authorization in China. A liquid chromatography-tandem mass spectrometry method for determining the concentration of amoxicillin and clavulanic acid in human plasma was developed and applied to this open-label, single- and multiple-dose Pharmacokinetics study. Subjects were randomized to receive a single dose of 1, 2, and 4 pouches of the test granulation of amoxicillin and clavulanic acid in 8:1 ratio (amoxicillin is 250 mg and clavulanic acid is 31.25 mg per pouch). In the single-dose phase, blood samples were collected before dosing and at 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 5, 8, 12, and 24 h after drug administration. In the multiple-dose phase, samples were obtained before drug administration on days 1, 2, 3, and 4 to determine the C_min of amoxicillin and clavulanic acid. In the 4th day, samples were collected from 0.25 to 24 h after drug administration. Profiles of the concentration–time curves of amoxicillin and clavulanic acid were best fitted to two-compartment model. In this group of healthy Chinese subjects, the pharmacokinetics of amoxicillin fitted the linear dynamic feature at doses of 250,500 and 1,000 mg, and not obviously about clavulanic acid at doses of 31.25, 62.5, and 125 mg. The t _1/2 of single dose and multidoses were (1.45 ± 0.12) and (1.44 ± 0.26) h of amoxicillin and (1.24 ± 0.23) and (1.24 ± 0.17) of clavulanic acid, respectively; The AUC_0–24 of single dose and multidoses were (27937.85 ± 4265.59) and (24569.80 ± 3663.63) ng h mL^?1 of amoxicillin and (891.45 ± 194.30) and (679.61 ± 284.05) ng h mL^?1 of clavulanic acid, respectively; The C_max of single dose and multidoses were (8414.58 ± 1416.78) and (7929.17 ± 1291.54) ng mL^?1 of amoxicillin and (349.00 ± 89.54) and (289.00 ± 67.36) ng h mL^?1 of clavulanic acid, respectively. t _1/2, AUC_0–24, and C_max were similar after multiple-dose administration and after single-dose administration, suggesting that amoxicillin and clavulanic acid do not accumulate with multiple-dose administration of 500 and 62.5 mg, respectively.     

A Genome-wide RNAi Screen Reveals a Trio-Regulated Rho GTPase Circuitry Transducing Mitogenic Signals Initiated by G Protein-Coupled Receptors

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Highlights? Human cancers harbor frequent mutations in Gq-linked GPCRs and Gα_q subunits ? A genome-wide RNAi screen revealed that Trio is essential for activating AP-1 via Gα_q ? A network of Trio-regulated Rho GTPases and MAPKs links Gq to the nucleus ? A hard-wired Gq-Trio signaling axis promotes the growth of many human malignancies     

Derivation and characterization of cell cultures from the skin of the Indo-Pacific humpback dolphin Sousa chinensis

The marine mammalian Indo-Pacific humpback dolphin, once widely lived in waters of the Indian to western Pacific oceans, has become an endangered species. The individual number of this dolphin has significantly declined in recent decades, which raises the concern of extinction. Direct concentration on laboratorial conservation of the genetic and cell resources should be paid to this marine species. Here, we report the successful derivation of cell lines form the skin of Indo-Pacific humpback dolphin. The cell cultures displayed the characteristics of fibroblast in morphology and grew rapidly at early passages, but showed obvious growth arrest at higher passages. The karyotype of the cells consisted of 42 autosomes and sex chromosomes X and Y. The immortalized cell lines obtained by forced expression of the SV40 large T-antigen were capable of proliferation at high rate in long-term culture. Immortalization and long-term culture did not cause cytogenetically observable abnormality in the karyotype. The cell type of the primary cultures and immortalized cell lines were further characterized as fibroblasts by the specific expression of vimentin. Gene transfer experiments showed that exogenetic genes could be efficiently delivered into the cells by both plasmid transfection and lentivirus infection. The cells derived from the skin of the Indo-Pacific humpback dolphin may serve as a useful in vitro system for studies on the effects of environmental pollutants and pathogens in habitats on the dolphin animals. More importantly, because of their high proliferation rate and susceptibility to lentivirus, these cells are potential ideal materials for generation of induced pluripotent stem cells.     

MMP13 is a critical target gene during the progression of osteoarthritis

Introduction

Osteoarthritis (OA) is a degenerative joint disease affecting a large population of people. The mechanism of this highly prevalent disease is not fully understood. Currently there is no effective disease-modifying treatment for OA. The purpose of this study was two-fold: 1) to investigate the role of MMP13 in the development of OA; and 2) to evaluate the efficacy of the MMP13 inhibitor CL82198 as a pharmacologic treatment for preventing OA progression.

Methods

To investigate the role of the endogenous Mmp13 gene in OA development, tamoxifen was administered to two-week-old Col2CreER;Mmp13^fx/fx (Mmp13^Col2ER) and Cre-negative control mice for five days. OA was induced by meniscal-ligamentous injury (MLI) when the mice were 10 weeks old and MLI or sham-operated joints were harvested 4, 8, 12, or 16 weeks after surgery. To evaluate the efficacy of CL82198, MLI surgery was performed on 10-week-old wild type mice. CL82198 or saline was administered to the mice daily beginning immediately after the surgery for up to 16 weeks. The joint tissues collected from both experiments were evaluated by cartilage grading, histology/histomorphometry, immunohistochemistry (IHC), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The ability of CL82198 to inhibit MMP13 activity in vitro was confirmed by ELISA.

Results

The OA progression was decelerated in Mmp13^Col2ER mice 8, 12, and 16 weeks post-surgery. Cartilage grading by blinded observers confirmed decreased articular cartilage degeneration in Mmp13^Col2ER mice at 8, 12 and 16 weeks compared to Cre-negative mice. Histomorphometric analysis demonstrated that Mmp13^Col2ER mice had a higher articular cartilage area and thickness at 12 and 16 weeks post-surgery compared to the control mice. Results of IHC revealed greater type II collagen and proteoglycan expression in Mmp13^Col2ER mice. Chondrocyte apoptosis, as determined by TUNEL staining, was higher in control mice compared to Mmp13^Col2ER mice. CL82198 inhibited MMP13 activity in conditioned media from vehicle (> 85%) or bone morphogenetic protein 2 (BMP2)-treated (> 90%) primary murine sternal chondrocytes. Intraperitoneal injection of CL82198 decelerated MLI-induced OA progression, increased type II collagen and proteoglycan levels, and inhibited chondrocyte apoptosis compared to saline treatment as determined by OA grading, histology, histomorphometry, IHC, and TUNEL staining, respectively.

Conclusions

Mmp13 is critical for OA progression and pharmacologic inhibition of MMP13 is an effective strategy to decelerate articular cartilage loss in a murine model of injury-induced knee OA.     

Volumetric-modulated arc therapy with RapidArc®: An evaluation of treatment delivery efficiency

Aim/background

To evaluate how the use of volumetric-modulated arc therapy (VMAT) with RapidArc^® can improve treatment delivery efficiency based on the analysis of the beam-on times and monitor units (MU) needed to deliver therapy for multiple clinical applications in a large patient population.

Materials and methods

A total of 898 treatment courses were delivered in 745 patients treated from October 2008 to March 2013 using RapidArc® treatment plans generated in Eclipse™ TPS. All patients were treated with curative or palliative intent using different techniques including conventional fractionation (83%) and radiosurgery or SBRT (17%), depending on the clinical indications. Treatment delivery was evaluated based on measured beam-on time and recorded MU values delivered on a Varian Trilogy™ linear accelerator.

Results

For conventional fractionation treatments using RapidArc®, the delivery times ranged from 38 s to 4 min and 40 s (average 2 min and 6 s). For radiosurgical treatments the delivery times ranged from 1 min and 42 s to 9 min and 22 s (average 4 min and 4 s). The average number of MU per Gy was 301 for the entire group, with 285 for the conventional group and 317 for the radiosurgical group.

Conclusions

In this study with a large heterogeneous population, treatments using RapidArc® were delivered with substantially less beam-on time and fewer MUs than conventional fractionation. This was highly advantageous, increasing flexibility of the scheduling allowing treatment of radiosurgery patients during the regular daily work schedule. Additionally, reduction of leakage radiation dose was achieved.     

Flow‐injection chemiluminescence determination of diazepam by oxidation with N‐bromosuccinimide

A rapid and sensitive flow‐injection chemiluminescence (FI–CL) method is described for the determination of diazepam based on its reaction with N‐bromosuccinimide (NBS) in alkaline medium in the presence of dichlorofluorescein (DCF) as an effective energy‐transfer agent. Under optimum conditions, the proposed method allowed the measurement of diazepam over the range of 2.0 × 10^?6 to 2.0 × 10^?4 mol/L with a detection limit of 5.0 × 10^?7 mol/L. The relative standard deviation for 11 parallel measurements of 2.0 × 10^?5 mol/L diazepam was 2.1%. The method was applied satisfactorily for the determination of diazepam in pharmaceutical preparations, and the results agree well with those obtained by spectrophotometry. The use of the proposed system for the determination of diazepam in urine and plasma samples was also tested. The possible mechanism of the chemiluminescence reaction is discussed briefly. Copyright © 2012 John Wiley & Sons, Ltd.     

Nucleosides. LIV1 Synthesis and Properties of 3′-Azido- and 2′,3′-Dideoxy-6,7-diphenyllumazine Nucleosides

Abstract

The best approach for the synthesis of1-(3-azido-2,3-dideoxy-β-D-erythro-pento-furanosyl)lumazine (5) and its 6,7-dimethyl- (4) and 6,7-diphenyl derivatives (3) has been found in the interconversion of the corresponding 1-(2-deoxy- β-threo-pentofuranosyl)-lumazines. Monomethoxytritylation at the 5′-position (1 7, 3 4, 4 9) followed by mesylation at the 3′-OH group and subsequent nucleophilic displacement by lithium azide afforded 1 9, 2 9 and 4 7 which were deprotected by acid treatment to give 3–5 in good yields. The syntheses of 1-(2,3-dideoxy-β-D-glycero-pentofuranosyl)-6,7-diphenyllumazine (6) and its 6,7-dimethyl derivative (7) were achieved from 1-(2-deoxy-β-D-erythro-pentofuranosyl)-6,7-diphenyllumazine and the corresponding 6,7-dimethyllumazine (2 6) via their 5′-O-p-toluoyl- (2 0, 3 0), and 3′-deoxy-3′-iodo derivatives (2 4, 3 1) to form, after radical dehalogenation and final deprotection, 6 and 7. The newly synthesized lumazine nucleosides have been characterized by elemental analyses, UV-and NMR spectra.