An anticoagulant serine protease from the wasp venom of Vespa magnifica

Wasp is an important venomous animal that can induce human fatalities. Coagulopathy is a clinical symptom after massive wasp stings, but the reason leading to the envenomation manifestation is still not known. In this paper, a toxin protein is purified and characterized by Sephadex G-75 gel filtration, CM-Sephadex C-25 cationic exchange and fast protein liquid chromatography (FPLC) from the venom of the wasp, Vespa magnifica (Smith). This protein, named magnvesin. contains serine protease-like activity and inhibits blood coagulation. The cDNA encoding magnvesin is cloned from the venom sac cDNA library of the wasp. The deduced protein from the cDNA is composed of 305 amino acid residues. Magnvesin shares 52% identity with allergen serine protease from the wasp Polistes dominulus. Magnvesin exerted its anti-coagulant function by hydrolyzing coagulant factors TF, VII, VIII, IX and X. (c) 2008 Elsevier Ltd. All rights reserved.

A phospholipase A1 platelet activator from the wasp venom of Vespa magnifica (Smith)

Wasp is an impor tant venomous animal that can induce human fatalities. Aortic thrombosis and cerebral infarction are major clinical symptoms after massive wasp stings but the reason leading to the envenomation manifestation is still not known. In this paper, a toxin protein is purified and characterized by Sephadex G-75 gel filtration, CM-Sephadex C-25 cationic exchange and fast protein liquid chromatography (FPLC) from the venom of the wasp, Vespa magnifica (Smith). This protein, named magnifin, contains phospholipase-like activity and induces platelet aggregation. The cDNA encoding magnifin is cloned from the venom sac cDNA library of the wasp. The predicted protein was deduced from the cDNA with a sequence composed of 337 amino acid residues. Magnifin is very similar to other phospholipase A(1) (PLA(1)), especially to other wasp allergen PLA(1). Magnifin can activate platelet aggregation and induce thrombosis in vivo. The current results proved that PLA(1) in wasp venom could be contributable to aortic thrombosis after massive wasp stings. (c) 2007 Elsevier Ltd. All rights reserved.

Effects of testosterone undecanoate as a male contraceptive candidate on rat immunological features

Testosterone undecanoate (TU) is under phase III clinical trial as a hormonal male contraceptive in China. Sex hormones can modulate the immune system. Female hormonal contraceptives may affect SIV/HIV-1 transmission. To evaluate the safety of TU and to understand whether long-term use of TU for a male contraceptive affects users' immunological features, adult male rats were treated for a 32-week TU-treated phase at the dose of 20 mg TU/kg body weight and a 24-week recovery phase. The reproductive and immunological parameters of 4-6 rats in each subgroup were examined at the stated time point. The mean sperm count and viability in the treated rats were significantly suppressed (p < 0.01). In the TU-treated group: the mean blood leukocyte and lymphocyte counts; the proliferation indexes of T cells from peripheral blood mononuclear cells (PBMC) and spleen; and, of B cells from spleen, as well as the mean counts of blood T, NK, and B cells decreased in comparison with those of control group. These decreases were not significant (p > 0.01). Similarly, the mean serum IgM, IgG, and IgA levels and complement activity in TU-treated rats were lower than those in control group (p > 0.01), and the changes in the antibody levels of the examined genital secretions were not significant (p > 0.01). The changes in the thickness of urethra epithelium, and in secretory component (SC) expression in genitals were not observed in the treated group. These results demonstrated that long-term supraphysiological TU injection did not obviously affect the examined rat immunological parameters.

何首乌炮制前后生物活性和化学成分的变化研究

何首乌为常用中药，由何首乌及含何首乌的中成药制剂所引起的不良反应也时见报道，科学阐明不良反应的物质基础并提出解决方案对何首乌的使用十分重要。本论文研究了何首乌炮制前后KM小鼠肝脏毒性基因表达谱、生物活性及化学成分的变化。所获结果支持何首乌炮制的目的是减毒、改性（改变药效），何首乌生、熟异治的观点。制首乌对抑郁症的效果显著优于生首乌，这与本草所记载的何首乌炮制后补肝肾、益精血，归肝、肾经一致。 主要结果如下： 1、 生、制首乌的毒理基因芯片研究结果 何首乌的不良反应主要表现在肝损害方面。本研究建立了生何首乌和制何首乌不同剂量的肝毒性作用模型，体重指标统计发现生何首乌各剂量组平均体重显著下降，中剂量组（10 g/kg.d）体重下降20 %，高剂量组（20 g/kg.d）体重下降42%，50%动物死亡，提示动物机体能量代谢障碍；基因芯片研究结果表明何首乌是CYP450的抑制剂，生何首乌相对于制何首乌CYP3A4、CYP4A5显著下调，导致毒性成分在体内的吸收增加，服用大剂量的生何首乌后产生明显的肝毒性；主要对以下六条Pathway产生影响：①PPAR signaling pathway，主要毒性靶基因有RXRB CYP7a1、Acadl、Apoa2、Cyp4a、 FABP2 、MAPKKK5等基因。②Calcium signaling pathway，主要毒性靶基因有CAMK2B、CACNA1F、S100A1、 F2R、Ryr1、Slc8a2、Camk4 ③Neuroactive ligand-receptor interaction，主要毒性靶基因有Chrm4、 Ntsr2 、 GABRR1、 GRIK3、F2R等基因。④Wnt signaling pathway，主要毒性靶基因有Daam2、Rac1 等基因。⑤Complement and coagulation cascades，主要毒性靶基因有F2R、Serpina1b、Cfi 、FGA等基因。⑥Oxidative hosphorylation，主要毒性靶基因有Atp5e、NDUFA1等基因。生何首乌毒性明显强于制首乌，且生何首乌水煎液的毒性大于生何乌首丙酮提取物的毒性，这一结果表明，何首乌主要的毒性成分很可能并不仅仅是传统所认为的以大黄素为代表的蒽醌类化合物，而是何首乌中大量存在的有效组分二苯乙烯苷与大黄素相互作用的结果，这一研究结果与前述的何首乌对肝药酶的影响是一致的。后续生、制首乌的化学成分差异研究表明，炮制后二苯乙烯苷含量明显降低：生首乌为5.512 %、清蒸制首乌为3.811 %、豆制首乌为3.538 %,大黄素的含量炮制后显著升高,生首乌为0.094 %、清蒸制首乌为0.119 %、豆制首乌为0.126 %。 2 生、制首乌药效差异研究结果 本文采用慢性中等强度不可预知应激刺激模型(chronic unpredictable mild stress, CUMS)和动物行为绝望实验法，研究生、制首乌抗抑郁活性的差异，制首乌（5 g/kg.d）与模型组相比有显著差异（P< 0.01），生首乌制首乌（5g/kg.d）与模型组相比无显著差异，这一结果表明制首乌抗抑郁活性显著优于生首乌。 本文比较了生、制首乌对四氧嘧啶糖尿病模型小鼠血糖的影响的差异，生首乌（5 g/kg.d）与模型组相比有显著差异（P< 0.01），制首乌（5 g/kg.d）与模型组相比无显著差异，这一结果表明生首乌降糖活性优于制首乌。这一结果与历代中医古书中生首乌治疗消渴症（糖尿病）的记载一致。 3生、制首乌化学成分差异的研究结果 本文选用HPLC-DAD指纹图谱技术结合药效成分含量测定来研究生、制首乌化学成分的差异。炮制后，何首乌中的主要化学成分并未消失，只是其含量发生了改变。炮制后二苯乙烯苷含量明显降低：生首乌为5.512 %、清蒸制首乌为3.811 %、豆制首乌为3.538 %,大黄素的含量炮制后显著升高,生首乌为0.094 %、清蒸制首乌为0.119 %、豆制首乌为0.126 %。 综上所述，炮制前后何首乌中二苯乙烯苷和大黄素含量比的变化可能是何首乌炮制减毒、改性的物质基础。 根据上述结果我们建立了生、制首乌的质量控制新模式。 In recent years, some adverse drug reactions (ADR) about some traditional Chinese medicine were reported at times. As a Chinese medicine most in use, the ADRs of Radix Polygoni multiflori (RPM) and the medicines containing the RPM were also mentioned. The resolution of the problems caused by the ADRs is very important for the use of the RPM as a medicine. The process (or preparation) is a significant feature for the clinical use of the Chinese medicine and an important technology for the safe use and good effect of the Chinese medicine. By processing, the toxicity of the Chinese medicine can be reduced, its properties can be changed and curative effect can be enhanced at the same time. The changes of the gene expression profiles for KM mice hepatotoxic effects, and the change of the biological activity and the chemical composition after being processed of the RPm were studied in the present dissertation. The RPm heatotoxicity mechanism and the toxicity target genes were explained on the gene level for the first time. With the antidepressant activity, and the hypoglycemic effect as the target, the differences on the pharmacodynamics between the processed RPm and unprocessed RPm, for the first time, were investigated. The results obtained show that the antidepressant activity of the processed RPM is far higher than the ones of unprocessed RPm. As we know, the results were reported for the first time. The quality control systems (QCS) for the processed and the unprocessed RPm were founded. The HPLC-DAD was used in the systems founded on the basis of the toxicology and the pharmacodynamics experiments. As we know, the OCSs were reported for the first time. The above-mentioned experimental results confirm that the unique process theory of the traditional Chinese medicine (TCM) used for the process of the Radix Polygoni multiflori (RPm) is correct, i.e after being processed the toxicity of the RPm decreases and its Pharmacodynamic effects change. It is known to author that there have been no similar reports in the literatures up to now. The main experimental results are summarized as follows: 1 The results on the mice toxicology gene chip for the unprocessed and processed RPm The KM mice hepatotoxic model caused by the RPm at the different dosages was established in the present study. The results obtained show that the mouse average body weight obviously decreased in the groups at the different dosages of the unprocessed RPm: the 10 g/kg.d .group decreased 20%; 20 g/kg.d. group decreased 42%, and 50% mice died at 20 g/kg.d. group. The main experimental results on the mice toxicology gene chip The RPm is the CYP450 inhibitor. As compared with the processd RPm, the CYP3A4, CYP4A5 of the unprocessed RPm demonstrate the marked downregulation, which leads to the increase of the poison absorbtion into the body with the result that the unprocessed RPm yields the marked hepatotoxication. The hepatotoxication was produced because the following 6 pathways were affected: ①PPAR signaling pathway, the chief toxicity target genes are RXRB, CYP7a1, Acadl, Apoa2, Cyp4a, FABP2 and MAPKKK5 etc. ②Calcium signaling pathway, the chief toxicity target genes are CAMK2B, CACNA1F, S100A1, F2R, Ryr1,Slc8a2 and Camk4 etc. ③Neuroactive ligand-receptor interaction, the chief toxicity target genes are Chrm4, Ntsr2, GABRR1, GRIK3 and F2R etc. ④Wnt signaling pathway, the chief toxicity target genes are Daam2, Rac1 etc. ⑤Complement and coagulation cascades, the chief toxicity target genes are F2R, Serpina1b, Cfi and FGA etc. ⑥Oxidative phosphorylation, the chief toxicity target genes are Atp5e, NDUFA1 etc. The above experimental results, for the first time , demonstrate on the gene level that the unprocessed Rpm toxicity is far stronger than the processed RPm one, and the toxicity of the water decoction of the unprocessed RPm is greater than the one of its acetone extracts, which shows that the chief toxicity components of the RPm are probably not only the anthraquinones, for example, the emodin, but the complex compounds produced by the interaction between the emondin and the stilbene glucoside which is the largest component of the unprocessed RPm. The result is accordance with the above effect of the RPm on the hepatic drugenzyme. Aftter being processed, in fact, the content of the stibene glucoside in the RPm markedly decreases. 2. The results on the pharmacodynamic differences between the unprocessed and processed RPm The results obtained show that the effects of processing on RPm pharmacodynamic behaviour received in the Chinese Material Medica are correct. It is known to author that this is the first experimental result in the research materials now available. The chief results are as follows: For the treatment of the antidepressant, the curative effect of the processed RPm is far better than the one of the unprocessed RPm. By contrast with the above results, the hypoblycemic effect of the unprocessed RPm is better than the one of the processed RPm. 3. The results on the Chemical Composition The results obtained by using HPLC-DAD fingerprint and by the determination of effective component content show that the main chemical components in the RPm after being processed do not disappear, but their contents change. The contents of the stilbene glucoside (SG) and emodin in the different samples were determined as follows: SG contents 5.512 % for the unprocessed RPm 3.811 % for the processed RPm (Steamed) 3.588 % for the processed RPm (black soybean) Emodin contents 0.094 % for the unprocessed RPm 0.119 % for the processed RPm (Steamed) 0.126 % for the processed RPm (black soybean) The combination of above experimental results on the toxicity, the pharmacodynamics and the chemical composition indicates that the changes of the content ratio of SG/emodin may be the substance base of the toxicity decrease and pharmacodynamic changes of the RPM by the processing.

Metabolic profiling studies on the toxicological effects of realgar in rats by H-1 NMR spectroscopy

The toxicological effects of realgar after intragastrical administration (1 g/kg body weight) were investigated over a 21 day period in male Wistar rats using metabonomic analysis of H-1 NMR spectra of urine, serum and liver tissue aqueous extracts. Liver and kidney histopathology examination and serum clinical chemistry analyses were also performed. H-1 NMR spectra and pattern recognition analyses from realgar treated animals showed increased excretion of urinary Kreb's cycle intermediates, increased levels of ketone bodies in urine and serum, and decreased levels of hepatic glucose and glycogen, as well as hypoglycemia and hyperlipoidemia, suggesting the Perturbation of energy metabolism. Elevated levels of choline containing metabolites and betaine in serum and liver tissue aqueous extracts and increased serum creatine indicated altered transmethylation. Decreased urinary levels of trimethylamine-N-oxide, phenylacetylglycine and hippurate suggested the effects on the gut microflora environment by realgar.

NMR-based metabonomic study on the subacute toxicity of aristolochic acid in rats

The subacute toxicity of aristolochic acid (AA) was investigated by H-1 NMR spectroscopic and pattern recognition (PR)-based metabonomic methods. Model toxins were used to enable comparisons of the urinary profiles from rats treated with known toxicants and AA at various time intervals. Urinary H-1 NMR spectra were data-processed and analyzed by pattern recognition method. The result of visual comparison of the spectra showed that AA caused a renal proximal tubular and papillary lesion and a slight hepatic impair. Pattern recognition analysis indicated that the renal proximal tubule lesion was the main damage induced by AA, and the renal toxicity induced by AA was a progressive course with the accumulation of dosage by monitoring the toxicological processes from onset, development and part-recovery. These results were also supported by the conventional clinical biochemical parameters.

EFFECTS OF METAL-IONS ON THE CONFORMATION OF POLAR HEADGROUP OF SPHINGOMYELIN BILAYER

With the wide application of rare earth in agriculture, medicament, especially the application of Gd-DTPA as nuclear magnetic resonance image reagent in clinical practice([1]), the studies on the toxicology in biological body, as well as the study on the use as informative probes instead of divalent calcium ion in biological and biochemical research have attracted intensive concern([2]). Phospholipids bilayers have served as a model of biomembrane in the last two decades. The effects of metal ions on the conformation of polar headgroup of dipalmitoylphosphatidylcholine (DPPC) bilayers have been reported([3]). Sphingomyelin is major component of several biological tissues such as brain and nerve cells and has identical polar headgroup to DPPC. The interaction of metal ions with sphingomyelin bilayer remains nonrevealed. This note presents the results of the study on this aspect.