In-vitro Neurotoxicity of Two Malaysian Krait Species (Bungarus candidus and Bungarus fasciatus) Venoms: Neutralization by Monovalent and Polyvalent Antivenoms from Thailand

Bungarus candidus and Bungarus fasciatus are two species of krait found in Southeast Asia. Envenoming by these snakes is often characterized by neurotoxicity and, without treatment, causes considerable morbidity and mortality. In this study, the in vitro neurotoxicity of each species, and the effectiveness of two monovalent antivenoms and a polyvalent antivenom, against the neurotoxic effects of the venoms, were examined in a skeletal muscle preparation. Both venoms caused concentration-dependent inhibition of indirect twitches, and attenuated responses to exogenous nicotinic receptor agonists, in the chick biventer preparation, with B. candidus venom being more potent than B. fasciatus venom. SDS-PAGE and western blot analysis indicated different profiles between the venoms. Despite these differences, most proteins bands were recognized by all three antivenoms. Antivenom, added prior to the venoms, attenuated the neurotoxic effect of the venoms. Interestingly, the respective monovalent antivenoms did not neutralize the effects of the venom from the other Bungarus species indicating a relative absence of cross-neutralization. Addition of a high concentration of polyvalent antivenom, at the t₉₀ time point after addition of venom, partially reversed the neurotoxicity of B. fasciatus venom but not B. candidus venom. The monovalent antivenoms had no significant effect when added at the t₉₀ time point. This study showed that B. candidus and B. fasciatus venoms display marked in vitro neurotoxicity in the chick biventer preparation and administration of antivenoms at high dose is necessary to prevent or reverse neurotoxicity.     

Wound Infections from Taiwan Cobra (Naja atra) Bites: Determining Bacteriology,Antibiotic Susceptibility,and the Use of Antibiotics-A Cobra BITE Study

Wound necrosis and secondary infection are common complications after Naja atra bites. Clinical tools to evaluate the infection risk after Taiwan cobra bites are lacking. In this Cobra BITE study, we investigated the prevalence of wound infection, bacteriology, and corresponding antibiotic usage in patients presenting with Taiwan cobra snakebites. Patients with wound infection lacking tissue necrosis were included in developing Cobra BITE score utilizing univariate and multiple logistic regression, as patients with wound necrosis require antibiotics for infection treatment. 8,295,497 emergency department visits occurred in the span of this study, with 195 of those patients being diagnosed as having cobra bites. Of these patients, 23 had wound necrosis, and 30 had wound infection, resulting in a wound infection rate of 27.2% (53/195). Enterococcus faecalis and Morganella morganii were the main bacteria identified in the culture report regardless of whether patients’ wounds had necrosis. As per our Cobra BITE score, the three factors predicting secondary wound infection after cobra bites are hospital admission, a white blood cell count (in 103/µL) × by neu-trophil-lymphocyte ratio value of ≥114.23, and the use of antivenin medication. The area under the receiver operating characteristic curve for the Cobra BITE score system was 0.88; ideal sensitivity and specificity were 0.89 and 0.76. This scoring system enables the assessment of wound infections after N. atra bites, and it could be modified and improved in the future for other Naja spp. bites.     

In Vitro Neutralization of the Myotoxicity of Australian Mulga Snake (Pseudechis australis) and Sri Lankan Russell’s Viper (Daboia russelii) Venoms by Australian and Indian Polyvalent Antivenoms

We studied the neutralisation of Sri Lankan Russell’s viper (Daboia russelii) and Australian mulga snake (Pseudechis australis) venom-induced myotoxicity by Indian (Vins and Bharat) and Australian (Seqirus) polyvalent antivenoms, using the in vitro chick biventer skeletal muscle preparation. Prior addition of Bharat or Vins antivenoms abolished D. russelii venom (30 µg/mL)-mediated inhibition of direct twitches, while Australian polyvalent antivenom was not protective. Bharat antivenom prevented, while Vins and Australian polyvalent antivenoms partially prevented, the inhibition of responses to exogenous KCl. Myotoxicity of Mulga venom (10 µg/mL) was fully neutralised by the prior addition of Australian polyvalent antivenom, partially neutralised by Vins antivenom but not by Bharat antivenom. Although the myotoxicity of both venoms was partially prevented by homologous antivenoms when added 5 min after the venom, with an increasing time delay between venom and antivenom, the reversal of myotoxicity gradually decreased. However, antivenoms partially prevented myotoxicity even 60 min after venom. The effect of antivenoms on already initiated myotoxicity was comparable to physical removal of the toxins by washing the bath at similar time points, indicating that the action of the antivenoms on myotoxicity is likely to be due to trapping the toxins or steric hindrance within the circulation, not allowing the toxins to reach target sites in muscles.     

Isolation and Characterization of A2-EPTX-Nsm1a,a Secretory Phospholipase A2 from Malaysian Spitting Cobra (Naja sumatrana) Venom

Phospholipase A₂ (PLA₂) toxins are one of the main toxin families found in snake venom. PLA₂ toxins are associated with various detrimental effects, including neurotoxicity, myotoxicity, hemostatic disturbances, nephrotoxicity, edema, and inflammation. Although Naja sumatrana venom contains substantial quantities of PLA₂ components_, there is limited information on the function and activities of PLA₂ toxins from the venom. In this study, a secretory PLA₂ from the venom of Malaysian N. sumatrana, subsequently named A2-EPTX-Nsm1a, was isolated, purified, and characterized. A2-EPTX-Nsm1a was purified using a mass spectrometry-guided approach and multiple chromatography steps. Based on LC-MSMS, A2-EPTX-Nsm1a was found to show high sequence similarity with PLA₂ from venoms of other Naja species. The PLA₂ activity of A2-EPTX-Nsm1 was inhibited by 4-BPB and EDTA. A2-EPTX-Nsm1a was significantly less cytotoxic in a neuroblastoma cell line (SH-SY5Y) compared to crude venom and did not show a concentration-dependent cytotoxic activity. To our knowledge, this is the first study that characterizes and investigates the cytotoxicity of an Asp49 PLA₂ isolated from Malaysian N. sumatrana venom in a human neuroblastoma cell line.     

Comparative study on in vitro inhibition of grass carp (Ctenopharyngodon idellus) and mouse protein phosphatases by microcystins

Microcystins isolated from toxic cyanobacteria are potent inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A). The inhibitory effects of three structural variants of microcystins (microcystin‐LR, ‐YR, and ‐RR) on protein phosphatases isolated and purified from the liver and kidney of grass carp (Ctenopharyngodon idellus) were investigated using the ³²P radiometric assay. The relationships between percentage inhibition of protein phosphatase activity and microcystin levels followed a typical dose‐dependent sigmoid curve. These results were compared to those obtained from mouse PP2A. The degree and pattern of inhibition of both fish and mouse protein phosphatases by microcystins were similar. Protein phosphatases in crude fish tissue homogenates showed similar inhibition patterns as purified fish PP2A toward microcystins. © 2000 John Wiley & Sons, Inc. Environ Toxicol 15: 71–75, 2000     

胰岛素微粒剂的体外释药、小鼠体内分布与绝对生物利用度

采用放射免疫分析方法测定了胰岛素微粒剂的体外释药性 .用放射性标记技术制备了12 5Ⅰ 胰岛素微粒制剂 ,且以微粒剂中12 5Ⅰ 胰岛素的放射性计数为指标 ,研究了12 5Ⅰ 胰岛素在小鼠体内的生物利用度与组织分布 .研究表明 :微粒剂在不同pH值的磷酸盐缓冲液中 ,释药规律接近一级动力学方程 :ln(Q∞ -Q) =bt +A ,相关系数为rpH 2 5=- 0 .9398、rpH 7 0 =- 0 .9933,但更好地符合自拟释药方程 :Q =Q∞ bt/(1+bt) ,相关系数为rpH 2 5=- 0 .995 2、rpH 7.0 =- 0 .996 9.小鼠口服绝对生物利用度为 11.5 1%± 4.41% .12 5Ⅰ 胰岛素在小鼠脏器的同位素强度分布依次为肝、肾、心、肺、脾 .     

Characterization of nerve growth factors (NGFs) from snake venoms by use of a novel, quantitative bioassay utilizing pheochromocytoma (PC12) cells overexpressing human trkA receptors.

Snake venoms are a very abundant source of nerve growth factors (NGF). NGFs of Elapidae showing 65% sequence homology with mouse or human NGF, while the Viperidae NGF shows N-glycosylation (Asn-21) typical of these mammalian NGFs. Snake NGF-induced neurite outgrowth (neurotropic activity) was measured in the past by using PC12 cell or dorsal root ganglion bioassays.The present study was aimed at comparing, by dose-response experiments, the neurotropic activity of cobra and vipera versus mammalian NGFs, by using a novel bioassay involving PC12 cells genetically engineered to overexpress NGF-trkA receptors of human origin.These cells respond to NGF by differentiation (morphologically expressed as neurite outgrowth) by a process mediated by NGF-trkA receptors. This process was evaluated by two different criteria: (1) elongation of neurites (E), and (2) Percentage of responsive cells (PRC) determined by digital acquisition of data and computer analysis. We found that snake venom NGFs were less potent than mouse NGF, and that cobra NGF was more potent than vipera NGF. These data indicate the following order of NGF activity towards recombinant human trkA receptors: recombinant human NGF>mouse NGF>cobra NGF>vipera NGF. The neurotropic efficacy of these NGFs was found to be similar, reaching 80-90% of maximal activity obtained with all NGF forms. Interestingly, cobra (but not vipera) NGF demonstrated prolonged neurotropic activity compared with mouse NGF.The results of the present study indicate that cobra and vipera venom NGFs represent natural agonists of human trkA-receptor of a lower potency, but of similar efficacy, compared with mammalian NGFs. These compounds are important pharmacological tools to characterize the trkA receptor structure-function relationship, and to develop novel neurotropic drugs.     

Enhancing Initial Release of Peptide from Poly(d,l-lactide-co-glycolide) (PLGA) Microspheres by Addition of a Porosigen and Increasing Drug Load

The objective of this study was to evaluate formulation variables such as drug load and addition of a porosigen in achieving an increased initial release of peptide from poly(d,l-lactide-co-glycolide) (PLGA) microspheres by altering carrier characteristics. Leuprolide acetate-loaded PLGA microspheres were prepared by a solvent-extraction–evaporation process and were characterized for their drug load (HPLC assay), bulk density (tapping method), size distribution (dynamic light scattering), specific surface area (Brunauer–Emmett–Teller [BET] analysis), surface morphology (scanning electron microscopy), in vitro drug release (at 37°C), and in vivo efficacy (suppression of rat serum testosterone). Increasing the drug load, and adding various amounts of calcium chloride to organic and aqueous phases of the emulsion during processing yielded particles with increased porosity, lower bulk density, higher specific surface area, and accordingly higher initial release. In an animal model, these formulations showed a faster onset of testosterone suppression compared to microspheres without higher drug load or calcium chloride. The approaches employed in this study were found to be effective in avoiding the therapeutic lag phase usually observed with microencapsulated macromolecular drugs.     

In Vitro/In Vivo Comparison of Drug Release and Polymer Erosion from Biodegradable P(FAD-SA) Polyanhydrides—A Noninvasive Approach by the Combined Use of Electron Paramagnetic Resonance Spectroscopy and Nuclear Magnetic Resonance Imaging

Purpose. The purpose of this study was to compare drug release and polymer erosion from biodegradable P(FAD-SA) polyanhydrides in vitro and in vivoin real time and with minimal disturbance of the investigated system. Methods. P(FAD-SA) 20:80 and P(FAD-SA) 50:50 polymer tablets were loaded with the spin probe 3-carboxy-2,2,5,5-tetramethyl-pyrrollidine-1-oxyl (PCA) and implanted subcutaneously in the neck of rats or placed in 0.1 M phosphate buffer. 1.1 GHz EPR spectroscopy experiments and 7T MRI studies (Tl and T2 weighted) were performed. Results. A front of water penetration was visible by MRI in vitro in the case of P(FAD-SA) 20:80, but not for P(FAD-SA) 50:50. For both polymers, the thickness of the tablets decreased with time and a insoluble, easy deformable residue remained. Important processes such as edema, deformation of the implant, encapsulation and bioresorption were observable by MRIin vivo. P(FAD-SA) 50:50 was almost entirely absorbed by day 44, whereas an encapsulated residue was found for P(FAD-SA) 20:80 after 65 days. The EPR studies gave direct evidence of a water penetration induced changes of the microenvironment inside the tablet. EPR signals were still detectable in P(FAD-SA) 20:80 implants after 65 days, while the nitroxide was released in vitro within 16 days. Conclusions. Important parameters and processes such as edema, deformation of the tablet, micro viscosity inside the tablet and encapsulation can be monitored in real time by the combined use of the noninvasive techniques MRI and EPR leading to better understanding of the differences between the in vitroandin vivo situation.     

3种5-HT3受体拮抗剂预防肝癌患者介入治疗后恶心呕吐的疗效比较

目的:通过测定中药制剂参芪扶正注射液中Cu、Zn、Mn、Se、Mo、Fe、Cr、Ni、V、Sn、Pb、Cd、Hg、As等14种微量元素的含量,探讨该中药注射液中元素的分布是否存在某种规律性.方法:微波消解法制备供试品,电感耦合等离子体-质谱(ICP-MS)法测定.结果:参芪扶正注射液中Cu、Fe、Zn、Cr、Se的相对含量较高,有毒元素Pb、Cd、Hg、As中As含量接近美国FAD药品与功能性食品要求标准,Pb、Cd、Hg含量均明显低于最高限量.结论:测定结果为探讨中药制剂中微量元素与抗肿瘤作用的关系提供有用的数据.     

Fate and distribution of brevetoxin (PbTx) following lysis of Karenia brevis by algicidal bacteria, including analysis of open A-ring derivatives

Flavobacteriaceae (strain S03) and Cytophaga sp. (strain 41-DBG2) are algicidal bacteria active against the brevetoxin (PbTx)-producing, red tide dinoflagellate, Karenia brevis. Little is known about the fate of PbTx associated with K. brevis cells following attack by such bacteria. The fate and distribution of PbTx in K. brevis cultures exposed to these algicidal strains were thus examined by receptor binding assay and liquid chromatography/mass spectrometry (LC/MS) in three size fractions (>5, 0.22–5, <0.22 μm) over a 2-week time course. In control cultures, brevetoxin concentrations in the >5 μm particulate size fraction correlated with changes in cell density, whereas significant increases in dissolved (i.e., <0.22 μm) toxin were observed in the later stages of culture growth. Exposure of K. brevis to either of the two algicidal bacteria tested caused cell lysis, coinciding with a rapid decline in the >5 μm PbTX size fraction and a simultaneous release of dissolved toxin into the growth medium. Upon cell lysis, dissolved brevetoxin accounted for ca. 60% of total toxin and consisted of 51–82% open A-ring derivatives. Open A-ring PbTx-2 and PbTx-3 derivatives bound with lower affinity (approximately 22- and 57-fold, respectively) to voltage-gated sodium channels and were considerably less cytotoxic (86- and 142-fold, respectively) to N2A cells than their individual parent toxins (i.e., PbTx-2 and PbTx-3). These novel findings of changes in PbTx size-fractioned distribution and overall reduction in K. brevis toxicity following attack by algicidal bacteria improve our understanding of potential trophic transfer routes and the fate of PbTx during red tide events. Moreover, this information will be important to consider when evaluating the potential role of algicidal bacteria in harmful algal bloom (HAB) management strategies involving control of bloom populations.     

In vitro exposure of DE‐71, a penta‐PBDE mixture,on immune endpoints in bottlenose dolphins (Tursiops truncatus) and B6C3F1 mice

Polybrominated diphenyl ethers (PBDEs) are an emerging contaminant of concern with low level exposures demonstrating toxicity in laboratory animals and wildlife, although immunotoxicity studies have been limited. Bottlenose dolphin peripheral blood leukocytes (PBLs) and mouse splenocytes were exposed to environmentally relevant DE‐71 (a penta‐PBDE mixture) concentrations (0–50 µg ml^?1) in vitro. Natural killer (NK) cell activity and lymphocyte (B and T cell) proliferation were evaluated using the parallelogram approach for risk assessment. This study aimed to substantiate results from field studies with dolphins, assess the sensitivities between the mouse model and dolphins, and to evaluate risk using the parallelogram approach. In mouse cells, NK cell activity increased at in vitro doses 0.05, 0.5 and 25 µg DE‐71 ml^?1, whereas proliferation was not modulated. In dolphin cells, NK cell activity and lymphocyte proliferation was not altered after in vitro exposure. In vitro exposure of dolphin PBLs to DE‐71 showed similar results to correlative field studies; NK cell activity in mice was more sensitive to in vitro exposure than dolphins, and the parallelogram approach showed correlation with all three endpoints to predict risk in bottlenose dolphins. Copyright © 2014 John Wiley & Sons, Ltd.     

Biochemical mechanism of modulation of human P-glycoprotein (ABCB1) by curcumin I, II, and III purified from Turmeric powder

P-glycoprotein (Pgp, ABCB1) is an ATP-dependent drug efflux pump linked to development of multidrug resistance (MDR) in cancer cells. Previously [Biochem Pharmacol 2002;64:573-82], we reported that a curcumin mixture could modulate both function and expression of Pgp. This study focuses on the effect of three major curcuminoids--curcumin I, II and III purified from a curcumin mixture--on modulation of Pgp function in a multidrug resistant human cervical carcinoma cell line (KB-V1). The similar IC(50) values for cytotoxicity of curcuminoids of KB-V1, and KB-3-1 (parental drug sensitive cell line) suggest that these curcuminoids may not be substrates for Pgp. Treating the cells with non-toxic doses of curcuminoids increased their sensitivity to vinblastine only in the Pgp expressing drug resistant cell line, KB-V1, and curcumin I retained the drug in KB-V1 cells more effectively than curcumin II and III, respectively. Effects of each curcuminoid on rhodamine123, calcein-AM, and bodipy-FL-vinblastine accumulation confirmed these findings. Curcumin I, II and III increased the accumulation of fluorescent substrates in a dose-dependent manner, and at 15 microM, curcumin I was the most effective. The inhibitory effect in a concentration-dependent manner of curcuminoids on verapamil-stimulated ATPase activity and photoaffinity labeling of Pgp with the [(125)I]-iodoarylazidoprazosin offered additional support; curcumin I was the most potent modulator. Taken together, these results indicate that curcumin I is the most effective MDR modulator among curcuminoids, and may be used in combination with conventional chemotherapeutic drugs to reverse MDR in cancer cells.     

HPLC-DAD/MS法测定抗风湿中成药中非法添加10种抗风湿化学成分

目的：建立检测抗风湿中成药中非法添加10种抗风湿化学成分的HPLC-DAD方法,并用MS/MS进行了化合物的归属分析。方法：用C18色谱柱(4.6mm×250mm,5um),以0.02mol/L乙酸铵(含0.05%乙酸)水溶液为流动相A,以乙腈为流动相B,梯度洗脱,波长240nm,流速1.0 ml/min,进行HPLC定性定量分析;再采用串联四级杆质谱的MRM模式检测,进行化合物归属分析,检测添加在中成药中的对乙酰氨基酚、甲氧苄啶、氨基比林、吡罗昔康、萘普生、双氯芬酸钠、醋酸泼尼松、吲哚美辛、保泰松、布洛芬10种化学成分。结果：10种抗风湿性化学成分液相检测的线性范围宽,相关性好,r≥0.9992,方法精密度的RSD为0.13%~0.45%;回收率在98.0%~100.9%之间;精密度、稳定性均符合要求;检测限为0.05~0.5 ug.ml_-1;定量限为0.1~1.8 ug.ml_-1。结论：本方法专属性强,操作简单,快捷,可作为中成药中非法添加以上10种抗风湿性化学成分的有效检测方法。     

Analysis of amphetamine and methamphetamine contents in seized tablets from Jazan,Saudi Arabia by liquid chromatography-mass spectroscopy (LC-MS/MS) and chemometric techniques

A number of illegal amphetamine tablets were seized from three different cities of Jazan province of southern Saudi Arabia and were analyzed for amphetamine and methamphetamine contents using LC-MS/MS technique. Analyses were performed using a previously reported method taking 0.1 M ammonium formate buffer (85%) and 15% acetonitrile with 0.1% formic acid as mobile phase with a total runtime of 12 min. This method was successfully applied for the routine analysis of amphetamine and methamphetamine in the seized tablets using amphetamine-D5 and methamphetamine-D5 as internal standards. Hierarchical cluster analysis was performed to establish the similarity between samples. The retention times (RT) for internal standard, amphetamine and methamphetamine were observed to be within 6.0–7.1 min. Ten tablet samples from each city were subjected to analysis and the amount of amphetamine in all the samples were found to be in the range of 9.07–14.77 mg, whereas, the amount of methamphetamine ranged from 0.12 to 0.24 mg in each tablet. Hierarchical cluster analysis showed presence of five clusters of samples indicating different characteristics and possible sources of amphetamine tablets. The largest cluster consisted of 15 samples which are expected to be of the same origin. Both amphetamine and methamphetamine are considered to be illegal products and their illegal trade and use is banned in many countries including Saudi Arabia. Therefore, there is an urgent need of strict regulations worldwide to check the illicit trafficking of these psychoactive substances and should be considered on priority.     

Inhibition of leukocyte function and interleukin-2 gene expression by 2-methylarachidonyl-(2'-fluoroethyl)amide, a stable congener of the endogenous cannabinoid receptor ligand anandamide

Arachidonylethanolamide (anandamide, AEA) has been identified as an endogenous ligand for cannabinoid receptors CB1 and CB2. Characterization of the direct cannabimimetic actions of anandamide has been hampered by its short duration of action and rapid degradation in in vivo and in vitro systems to arachidonic acid, a precursor in the biosynthesis of a broad range of biologically active molecules. In the present studies, we utilized 2-methylarachidonyl-(2'-fluoroethyl)amide (F-Me-AEA), an analog of anandamide resistant to enzymatic degradation, to determine whether F-Me-AEA modulated T cell function similar to that of plant-derived cannabinoids. Indeed, F-Me-AEA at low micromolar concentrations exhibited a marked inhibition of phorbol ester plus calcium ionophore (PMA/Io)-induced IL-2 protein secretion and steady state mRNA expression. Likewise, a modest suppression of the mixed lymphocyte response was observed in the presence of F-Me-AEA indicating an alteration in T cell responsiveness to allogeneic MHC class II antigens. F-Me-AEA was also found to modestly inhibit forskolin-stimulated adenylate cyclase activity in thymocytes and splenocytes, a hallmark of cannabinoid receptor agonists. Further characterization of the influence of F-Me-AEA on the cAMP signaling cascade revealed an inhibition of CREB-1/ATF-1 phosphorylation and subsequently, an inhibition of CRE DNA binding activity. Characterization of nuclear binding proteins further revealed that NF-AT and, to a lesser extent, NF-kappaB DNA binding activities were also suppressed. These studies demonstrate that F-Me-AEA modulates T cell function in a similar manner to plant-derived and endogenous cannabinoids and therefore can be utilized as an amidase- and hydrolysis-resistant endogenous cannabinoid.     

苦荞内生真菌产物EE-2抑制HepG2生长及诱导其细胞凋亡

目的研究苦荞内生真菌KQH-2代谢醇提物EE-2对人肝癌细胞的抑制作用及其作用机制。方法采用MTT法考察EE-2对人肝癌细胞HepG2体外增殖的抑制作用,显微镜观察EE-2对HepG2细胞形态的影响,琼脂糖凝胶电泳检测细胞DNA完整性,并利用流式细胞仪检测HepG2细胞凋亡及细胞周期变化情况。结果用EE-2处理过的HepG2细胞,显微镜观察可见细胞脱壁圆缩,出现凋亡小体;细胞核降解。流式细胞仪检测发现,处理组的DNA直方图上有比对照组加强的SubG1峰,且可将HepG2细胞阻滞于G0/G1期。结论苦荞内生真菌KQH-2代谢醇提物EE-2可诱导人肝癌HepG2细胞的凋亡,且具有细胞周期阻滞作用。