Anti-tumor activity evaluation of novel chrysin-organotin compound in MCF-7 cells.

Chrysin (5,7-dihydroxylflavone, Chry) is a natural flavonoid extracted from plants and propolis. In this work, a novel chrysin-organotin (Chry-Sn) compound with enhanced anticancer activities was synthesized by the reaction of chrysin and triphenyltin chloride, and its potential anticancer effects against cancer cells were measured using various methods. Sulforhodamine B (SRB) results showed that chrysin and Chry-Sn had significant inhibition effects on the proliferation of MCF-7, A549 and HeLa human cancer cell lines in a dose- and time- dependent manner. These results suggested that Chry-Sn possessed enhanced anticancer effects. Hoechst 33258 staining and acridine orange staining results showed apoptosis and nuclei fragments significantly increased after being treated with chrysin and Chry-Sn respectively. Moreover, chrysin and Chry-Sn significantly increased ROS levels in MCF-7 cells. Western blot results showed that chrysin and Chry-Sn activated caspase 3 and induced autophagy by increasing LC3-II level. All results showed collectively that Chry-Sn could be a more promising drug than chrysin in anticancer treatment.

α-Synuclein: A Multifunctional Player in Exocytosis, Endocytosis, and Vesicle Recycling.

α-synuclein (α-Syn) is a presynaptic enriched protein involved in the pathogenesis of Parkinson's disease. However, the physiological roles of α-Syn remain poorly understood. Recent studies have indicated a critical role of α-Syn in the sensing and generation of membrane curvature during vesicular exocytosis and endocytosis. It has been known to modulate the assembly of SNARE complex during exocytosis including vesicle docking, priming and fusion steps. Growing evidence suggests that α-Syn also plays critical roles in the endocytosis of synaptic vesicles. It also modulates the availability of releasable vesicles by promoting synaptic vesicles clustering. Here, we provide an overview of recent progresses in understanding the function of α-Syn in the regulation of exocytosis, endocytosis, and vesicle recycling under physiological and pathological conditions.

Bioactive Components of Chinese Propolis Water Extract on Antitumor Activity and Quality Control.

To understand the material basis of antitumor activity of Chinese propolis water extract (CPWE), we developed a simple and efficient method using macroporous absorptive resin coupled with preparative high performance liquid chromatography and separated and purified eleven chemical components (caffeic acid, ferulic acid, isoferulic acid, 3,4-dimethoxycinnamic acid, pinobanksin, caffeic acid benzyl ester, caffeic acid phenethyl ester, apigenin, pinocembrin, chrysin, and galangin) from CPWE; then we tested the antitumor activities of these eleven components using different human tumor cell lines (MCF-7, MDA-MB-231, HeLa, and A549). Furthermore, cell migration, procaspase 3 level, and reactive oxygen species (ROS) of effective components from CPWE were investigated. Our data showed that antitumor activities of the eleven components from CPWE were different from each other. CPWE and its effective components induced apoptosis by inhibiting tumor cell migration, activating caspase 3, and promoting ROS production. It can be deduced that the antitumor effects of propolis did not depend on a single component, and there must exist "bioactive components," which also provides a new idea for Chinese propolis quality control.

Optimization of a multi-channel puffer system for rapid delivery of solutions during patch-clamp experiments.

In biological experiments, especially in neuroscience research, it is important to manipulate the extracellular environment efficiently. We have developed a micro-puffing system for local drug delivery to single cells in electrophysiological experiments, and validated the kinetic properties of this instrument. Based on our results, the kinetics of the delivery of solutions and the territory controlled by this system are influenced by several factors: (1) inner diameter (I.D.) of the guide tubing; (2) I.D. of the puffing tip; (3) angle of the puffing tip; and (4) gravity or external pressure applied to the solution. The system can fully control a territory of 200 x 600 micrometer2. The minimum delay in response to drug delivery is 10-20 ms. Switching between different solutions takes less than 100 ms. The minimum volume of solution required by the system is 0.2 ml. Taken together, our results provide useful data for designing and using an efficient drug/solution delivery system in electrophysiological experiments.

Monoclonal antibodies developed for detection of an epizootic virus associated with mass mortalities of cultured scallop Chlamys farreri.

Recently, an enveloped, spherical RNA virus was identified as the causative agent of mass mortalities among adult scallop Chlamys farreri, which is cultured on the northern coast of China. Hybridomas were prepared from mice immunized with highly purified virions. Four stable hybridomas secreting monoclonal antibodies (MAbs) of IgG isotype were obtained after screening by means of enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA). The specificity of the MAbs to this virus was confirmed by immunogold electron microscopy (IEM). All the selected MAbs recognized epitopes on the envelope spikes of the virions. Subsequently, the MAbs were used for in situ immunofluorescent detection of the virus in Davidson's fixed tissue sections. The results showed that the fluorescent cells were mostly observed in epithelia of different organs, but not in the epithelium of the digestive diverticulae. Cytopathological changes and focal lesions corresponding to virus-positive cells were clearly recognized in the affected epithelia, revealing a potential role of this virus in pathogenesis.

[Monoclonal antibodies prepared and used for detection of acute virus necrobiotic disease virus in scallop Chlamys farreri by indirect ELISA].

For developing monoclonal antibodies against acute virus necrobiotic disease (AVND) virus, mice of Balb/c strain were immunized with AVND virions which were isolated from the infected scallop Chlamys farreri. The spleen cells from immunized mice were then fused with NS-1 myeloma cells and the hybridomas were screened by means of enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA). Finally, 4 stable MAbs of IgG isotype were obtained. Moreover, the combined position of these 4 MAbs to this virus was examined by immunogold electron microscopy (IEM). The results demonstrate that all 4 MAbs recognized epitopes on the envelope of the virions. Subsequently, a MAb-based ELISA was developed and used for detection of the infection rate and densities of the scallops which were sampled during different seasons from mid-April to mid-October, 2003. The result exhibited that both of the infection rate and infection densities sharply rose in mid-July and reached to the spikes, which right corresponds with their mortality during this period.

[Histopathological research and immunofluorescence of AVND virus infection in cultured scallop Chlamys farreri].

The naturally infected scallops Chlamys farreri sampled during mass mortality in summer of 2003 was detected by means of histopathological and MAb-based immunofluorescence assay (IFA). The results of histological examination demonstrated that a series of histopathological changes including cell swelling, basophilic increase, disorder, partial sloughing and excessive sloughing were always observed in epithelia of many different organs, e.g. mantle, gills, stomach, intestine and kidney. Additionally, the infected tissues were applied for in situ detection of the "acute virus necrobiotic disease" (AVND) virus by means of specific MAb-based IFA, and the result demonstrated that this pathological changes or lesions were perfectly coincident with the positive cells (fluorescencing cells) . The positive cells were denser in some local area of epithelia, and exhibited serious pathological lesions, which would reveal the roles of this virus in pathogenesis and further confirm that the AVND virus is the main causative agent of mass mortalities among cultured scallop Chlamys farreri farmed in northern coast of China.

[Study on application of amphotericin B in the perforated whole-cell patch clamp technique].

OBJECTIVE: Established with amphotericin B perforated patch-clamp technique, to study the electrophysiological properties of calyx synapses. METHODS: In the present experiments, we studied the application of perforated patch clamp technique on the calyx synapses of mice with Amphotericin B. RESULTS: The use of Amphotericin B significantly slowed down the decay of channel currents and the optimum concentration was 400 microg/ml. CONCLUSION: The syudy developed a stable of perforated patch clamp whole cell recording technique, could be more effecitve, more real reaction neurons electrophysiological characteristics of the channel current. Our work might provide the basic information to future users studying the signal transmission and regulation of auditory system of rodents.

Propolis Reduces Phosphatidylcholine-Specific Phospholipase C Activity and Increases Annexin a7 Level in Oxidized-LDL-Stimulated Human Umbilical Vein Endothelial Cells.

To understand the mechanisms underlying the regulating dyslipidemia action of Chinese propolis and Brazilian green propolis, we investigated their effects on phosphatidylcholine-specific phospholipase C (PC-PLC) activity and annexin a7 (ANXA7) level which play crucial roles in the control of the progress of atherosclerosis. Furthermore, active oxygen species (ROS) levels, nuclear factor-KappaB p65 (NF- κ B p65), and mitochondrial membrane potential (MMP) were also investigated in oxidized-LDL- (ox-LDL-) stimulated human umbilical vein endothelial cells (HUVECs). Our data indicated that the treatment of both types of propolis 12.5 µ g/mL significantly increased cell viability and attenuated apoptosis rate, increased ANXA7 level, and decreased PC-PLC activity. Both types of propolis also inhibited ROS generation as well as the subsequent MMP collapse, and NF- κ B p65 activation induced by ox-LDL in HUVECs. Our results also indicated that Chinese propolis and Brazilian green propolis had similar biological activities and prevented ox-LDL induced cellular dysfunction in HUVECs.

Nicotine dynamically modulates dopamine clearance in rat striatum in vivo.

Nicotine binds to nicotinic acetylcholine receptors on dopaminergic terminals to evoke dopamine (DA) release. The clearance of released DA occurs rapidly through reuptake into nerve terminals through the DA transporter (DAT). However, whether nicotine modulates DAT function in vivo is still not well understood. In the present study, we determined the effect of nicotine on DA clearance using in vivo amperometric recording in the striatum of urethane-anesthetized rats. Stable DA release was evoked by electrical stimulation of the medial forebrain bundle (MFB). Subsequently, nicotine or saline was administered with MFB stimulation at 10-min intervals for 60 min. Kinetic analysis revealed that nicotine decreased the amplitude of DA overflow and the maximal DA clearance rate (V(max)) in response to stimulation of 96 pulses at 80 Hz. Surprisingly, nicotine enhanced the maximal DA clearance rate (V(max)) by stimulation of 768 pulses at 80 Hz. Furthermore, we found that this paradoxical effect of nicotine on V(max) depended on the stimulation pattern. These results suggest that nicotine may exert its addictive role by dynamically modulating DAT function in vivo.