一种新的从新疆穴居狼蛛中鉴定的细胞裂解肽

蜘蛛粗毒中富含生物活性物质,尤其以多肽类成分为主. 分离鉴定了新疆穴居狼蛛粗毒中一种新的细胞裂解肽,命名为LSTX-A1,其分子量为7 335.33,含有65个氨基酸残基,且羧基端残基酰胺化.研究了LSTX-A1的细胞裂解作用,结果显示,在100 μmol/L浓度引起42% 红细胞溶血.同时LSTX-A1具有抗肿瘤作用,能抑制HeLa细胞的增殖,其IC50剂量为22 μmol/L.     

穴居狼蛛毒中一个抗菌活性多肽的鉴定和纯化

从我国新疆地区产穴居狼蛛的毒液中分离纯化了一种多肽——狼蛛抗菌肽(Lycosin)。穴居狼蛛的粗毒在酸性聚丙烯酰胺凝胶电泳中可分出11条蛋白带,用0.6％的大肠杆菌琼脂胶覆盖在凝胶上进行鉴定表明,电泳迁移率最快的区带有抗菌活性。经鉴定该多肽分子系由43至45个氨基酸残基组成,N-末端为丙氨酸,分子中的碱性和疏水性氨基酸分别占总氨基酸残基数的1/4和1/3。     

浙江蝮蛇毒诱导人白血病Jurkat细胞凋亡的体外实验研究

蛇毒含有多种酶类和不同生理与药理活性蛋白。经研究证实它具有抗肿瘤作用 ,能对多种肿瘤细胞有杀伤和抑制作用。作者为研究浙江蝮蛇毒诱导人白血病 Jurkat细胞凋亡作用并探讨其机理 ,采用 MMT法测定半数抑制浓度( IC50 )及生长曲线 ,用流式细胞仪 ( FCM) AnnexinV FITC/PI法检测细胞凋亡率 ,PI染色法检测细胞周期 ,用流式细胞计 FCM及 Western- blot检测Jurkat细胞 Bcl- 2蛋白表达。结果 :浙江蝮蛇毒处理后 Jurkat细胞生长受到明显抑制 ,且呈剂量依赖关系。 48h IC50 值为 ( 9.78± 0 .38) μg/ml。给浙江蝮蛇毒 5μg/ml能诱导…     

间斑寇蛛粗毒的生物学特性

近几十年来,间斑寇蛛(Latrodectus tredecimguttatus)已引起相关研究工作者的广泛关注,因为阐明其毒液中的特异性蛋白质和多肽活性成分对蜘蛛咬伤治疗具有重要的意义,且其粗毒液中的生物学活性成分在神经生物学和药物研究方面也有着潜在的应用前景。但直到现在,尽管已有不少研究报道了包括α-latrotoxin在内的几种间斑寇蛛毒性蛋白质的生物学性质和结构,但有关毒液生物学特性的系统研究的报道还很少。本研究采用解剖分离毒囊的方法从产于我国新疆的间斑寇蛛提取粗毒,并对其理化和生物学性质进行了系统的分析。定量测定结果表明,粗毒的蛋白质含量为36.99%,对小鼠和蜚蠊的LD50分别为0.39mg/kg和2.32μg/g体重。小鼠经腹腔注射粗毒后,出现呆滞、共济失调、排汗、痉挛、食欲减退、呼吸短促、睁眼困难等中毒症状,而注射生理盐水的对照小鼠活动正常。粗毒具有透明质酸酶、碱性磷酸酶、酸性磷酸酶、脱氧核糖核酸酶、乙酰胆碱酯酶、蛋白水解酶等多种水解酶的活性。10μg/ml该粗毒可以在31min±3.05min内完全抑制电刺激引起的大鼠输精管的收缩反应。6μg/ml粗毒可在25min±2.2min内完全阻断小鼠膈神经-膈肌标本的神经肌肉接头传递。膜片钳电生理实验显示,粗毒(1g/L)对蜚蠊背侧不成对中间(Dorsal unpaired median,DUM)神经元的快瞬时钾电流、钠电流、高电压激活的钙电流和大鼠背根神经节(Dorsal root ganglion,DRG)细胞延迟整流钾电流、TTX-S型钠电流、高电压激活的钙电流均无明显作用。     

蜂肽对人肝癌细胞系细胞及人LAK细胞的影响

为了探索蜂毒应用的前景,我们在细胞体外液体培养试验中,观察了中国农业科学院养蜂研究所提取的蜜蜂毒不同组分BV_1(透明质酸酶)、BV_2(磷脂酶A_2)、BV_5(蜂肽或蜂针素)对人肝癌细胞株H7402的杀伤活性和对正常人的淋巴因子激活的杀伤细胞(LAK)的增殖的影响。三种蜜蜂毒组分均为水溶性,以RPMI1640液溶解,分别加入培养液中(使终浓度分别为2、20、200mg/L),与人肝癌细胞株H7402细胞于37℃共孵育     

敬钊缨毛蛛毒素-Ⅷ的分离纯化与鉴定

从敬钊缨毛蛛（Chilobrachys jingzhao）粗毒中,通过阳离子交换色谱和反相高效液相色谱分离得到一种新的多肽神经毒素,命名为敬钊缨毛蛛毒素-Ⅷ（jingzhaotoxin-Ⅷ,JZTX-Ⅷ）.经MALDI-TOF分析表明该多肽分子质量为4 329.37 Da,结合氨基酸序列分析和RACE的cDNA快速扩增法得到了敬钊缨毛蛛毒素-Ⅷ的氨基酸序列和全长cDNA,序列为LFECSFSCDIKKNGKPCKGSGEKKCSGGWRCKMNFCVKV-COOH,其中6个半胱氨酸形成3对二硫键.敬钊缨毛蛛毒素-Ⅷ能阻断小鼠膈神经-膈肌标本的神经肌肉接头传递,经初步膜片钳实验分析,表明该多肽是一种钙离子通道抑制剂.     

穴居狼蛛毒中磷酸单酯酶和核酸酶的研究

本文分别用对硝基酚磷酸酯、3＇-[32p]标记的RNA和大肠杆菌噬菌体入DNA作底物测定了新疆产穴居狼蛛毒中与核酸代谢有关的酶,发现此毒中含有磷酸单酯酶、核糖核酸酶和脱氧核糖核酸酶。     

人胚细胞提取物抗突变作用的研究

本文应用体外培养人淋巴细胞和小鼠骨髓微核与染色体畸变检测,研究了人胚细胞水提物(胚提物)的遗传毒性和抗突变效应。结果表明,(1)胚提物(3和30μg/ml,下同)可显著抑制培养人淋巴细胞的自发和γ-射线诱发的微核形成;(2)胚提物对培养淋巴细胞的自发染色体畸变无明显影响,但可显著抑制丝裂酶素C(MMC)诱发的染色体畸变; (3)胚提物显著抑制环磷酰胺诱发的小鼠骨髓多染性红细胞微核(PCE-MN)的增加; (4)胚提物的上述抗突变效应呈剂量依赖性增加。因此,作者认为,人胚细胞水提物是一种抗突变剂, 可望用于肿瘤的化学预防,并可用作为肿瘤放疗和化疗的辅助药物,以减少毒付反应和二次肿瘤的发生率。 Abstract:Genetially toxic and antimutagenic effects of the aqueous extracts of human fetal cells(HFCAE) against mutagen-induced chromosomal aberrations and micronucleus formation in human lymphocytes in vitro and bone marrow of mice were studied.HFCAE (3 and 30 μg/ml)significantly inhibited spontaneous and γ-rays-induced micronucleus formation and MMC-induced chromosomal aberrations in human lymphocytes in vitro.HFCAE (3 and 30μg/ml) strongly suppressed also micronucleus formation induced by CP in PCEs of mice.These antimutagenic effects of HFCAE were dose-dependent.Our results suggest that HFCE might be an antimutagen and have potential value in its clinical application.     

广西眼镜蛇蛇毒磷脂酶A_2的分离纯化及其对HSC-T6细胞的作用

目的对广西眼镜蛇毒中磷脂酶A2(PLA2)进行分离纯化,测定其对肝星状细胞HSC-T6的增殖抑制作用。方法采用Sephadex G-50凝胶层析柱、CM-Sepharose CL-6B离子交换柱、Macro-prep High S预装柱结合的方法分离广西眼镜蛇粗毒,经平板法测定各峰的PLA2活性;经SDS-PAGE电泳鉴定终产物纯度并测定分子量,NanoLC-ESI-MS/MS鉴定其组分;CCK-8法测定PLA2对肝星状细胞(HSC-T6)的增殖抑制作用,确定其凋亡的最小毒性浓度。结果 Sephadex G-50凝胶层析柱、CM-Sepharose CL-6B离子交换柱、Macro-prep High S预装柱层析法,得到第Ⅲ峰具PLA2活性,且达到电泳纯,经NanoLCESI-MS/MS鉴定其为PLA2,分子量约为14.06kD;PLA2在0~1μg/ml的浓度下对HSC-T6细胞具有一定的促增殖作用,2μg/ml时细胞数达到最大值,4~16μg/ml时对细胞生长有抑制作用,且随浓度增大细胞数降低。结论采用Sephadex G-50、CM-Sepharose CL-6B、Macro-prep High S预装柱结合的方法对广西眼镜蛇毒进行分离纯化,得到电泳纯且具PLA2活性的磷脂酶A2;广西眼镜蛇毒PLA2对肝星状细胞HSC-T6增殖有抑制作用,PLA2对HSC-T6细胞的最小毒性浓度为2μg/ml。     

五步蛇毒毒中蛋白凝集素的发现及其凝集特性的研究

发现尖吻蝮蛇(Agkistrodon acutus)粗毒对动物细胞有凝集作用。顺序用DEAE—Sephadlex A-50,CM Sephadex C25柱层析,从尖吻蝮蛇毒中分离纯化出一种毒蛋白凝集素。经分子量,等电点和出血活性等实验测定确定该凝集素就是徐询等人早期分离、纯化的出血毒素Ⅲ(AaHⅢ)。这种凝集素对许多动物细胞都有凝集作用,例如脾淋巴细胞、HeLa细胞、人肾成纤维细胞和草鱼(Ctenoph argngodon idellus)。但对组细胞和血小板无作用。和植物凝集素PHA相比AaHⅢ产生细胞的凝集块体积要大些。对淋巴细胞起凝集作用的最低浓度为2.5ug/ml。能使HeLa细胞和淋巴细胞凝集在一起。如预先加肝素和溶菌酶可以抑制对淋巴细胞的凝集怍用。用溶菌酶(2mg/ml)和AaHⅢ(1mg/ml)在37℃′下保温2小时AaHⅢ的出血活性仍然存在。     

Chemical composition and anticancer activity of leaf essential oil of Myrica gale L.

Myrica gale L. (Myricaceae), a native plant from Canada used in traditional medicine, was extracted by hydrodistillation and the oil was collected after 30 and 60 min. The chemical composition of these two extracts was determined using GC-MS analysis. We identified 53 components and myrcene (23.18-12.14%), limonene (11.20-6.75%), alpha-phellandrene (9.90-6.49%) and beta-caryophyllene (9.31-10.97%) were the major components in the 30- and 60-min fractions, respectively, whereas higher caryophyllene oxide content was detected in the 60-min fraction (9.94%) than in the 30-min fraction (3.47%). The anticancer activities of these extracts were assessed against human lung carcinoma cell line A-549 and human colon adenocarcinoma cell line, DLD-1. The 60-min fraction showed higher anticancer activity against both tumor cell lines with an IC50 value of 88 +/- 1 microg/ml. The 30-min fraction had an IC50 value of 184 +/- 4 microg/ml for A-549 and 160 +/- 3 microg/ml for DLD-1. The higher cell growth inhibition induced by the 60-min fraction, as compared to the 30-min fraction, could be due to sesquiterpene enrichment.     

In vitro cytotoxic activity of Salsola oppositifolia Desf. (Amaranthaceae) in a panel of tumour cell lines

The aim of the present study was to evaluate for the first time the in vitro cytotoxic activity of fractions and isolated flavonols from Salsola oppositifolia Desf. (Amaranthaceae). The n-hexane fraction demonstrated an effective cytotoxic activity on the large lung carcinoma and amelanotic melanoma cell lines with IC50 values of 19.1 microg/ml and 24.4 microg/ml, respectively. Also the dichloromethane fraction exhibited cytotoxic activity against COR-L23 (IC50 30.4 microg/ml) and C32 (IC50 33.2 microg/ml) cells, while the EtOAc fraction demonstrated a selective cytotoxic activity against MCF-7 cells (IC50 67.9 microg/ml). The major active constituents of this fraction were isorhamnetin-3-O-glucoside (1) and isorhamnetin-3-O-rutinoside (2), which showed an interesting activity against the cell line MCF-7 with IC50 values of 18.2 and 25.2 microg/ml, respectively. Compound 2 exhibited a strong activity against the hormone-dependent prostate carcinoma LNCaP cell line with an IC50 of 20.5 microg/ml. Constituents of S. oppositifolia were identified by GC-MS and NMR analyses.     

In vitro fertilization and development of llama (Lama glama ) oocytes using epididymal spermatozoa and oviductal cell co-culture

A study was designed to determine the feasibility of developing in vitro maturation, fertilization and culture systems utilizing follicular oocytes and epididymal spermatozoa collected from llamas at slaughter. From a total of 1324 cumulus oocyte complexes (COCs) recovered, 972 were cultured in 50-ul drops of TCM-199 medium with 10% heat inactivated steer serum (DBS) and hormones for 30 h. After maturation, the oocytes were randomly allocated into 4 groups in a 2x2 factorial design: cumulus-enclosed oocytes, 2 ug/ml heparin (Group 1); cumulus-enclosed oocytes, 5 ug/ml heparin (Group 2); denuded oocytes, 2 ug/ml heparin (Group 3); and denuded oocytes, 5 ug/ml heparin (Group 4). Denuded oocytes were obtained for groups 3 and 4 by vortexing. Epididymides were also collected at slaugther and fresh spermatozoa (for each replicate) were obtained by mincing the cauda epididymis with a scalpel blade. A total of 721 oocytes were inseminated with 2-3 x 10(6) epididymal spermatozoa/ml in a 50-ul drop of FERT-TALP medium. After 18 h of in vitro insemination, 234 oocytes were placed in a llama oviductal epithelial cell (LLOEC) co-culture in TCM-199 for 9 d. All cultures were done at 38.5 degrees C under 5% CO(2) in air with high humidity. The rate of fertilization, initial cleavage and development in co-culture were evaluated and compared. Of 192 oocytes examined for signs of fertilization, 56 (29.2%) were penetrated by spermatozoa with 57.1% (32 56 ) of the penetrated oocytes having a male and female pronucleus. There were no differences among treatment groups in total fertilization. However, the frequency of oocytes fertilized normally tended to be higher in the denuded oocytes 67.7% (21 31 ) than the oocytes inseminated with cumulus cells 44.0% (11 25 ) independent of heparin concentration (P<0.06). The total embryo development rate to the 2 cells to blastocyst stage was 32.1% (75 234 ). There was no difference in development rate between groups. From the 234 oocytes co-cultured in LLOEC for 9 d, 15.8% developed into 2 to 16 cells, 5.6% into morulae, 6.0% into early/expanded blastocysts and 4.7% into hatching/hatched blastocysts. The results indicate that an in vitro fertilization system is possible in the llama utilizing slaughterhouse material and that llama oocytes can be fertilized in the presence of heparin and epididymal spermatozoa.     

Evidence for a mediator role of thromboxane A2 in the myotropic action of leukotriene B4 (LTB4) on the guinea-pig lung

The mechanism of action of LTB₄ has been investigated on the guinea-pig lung parenchymal strip. Mepacrine (20 ug/ml), an inhibitor of phospholipase A₂, abolished the action of LTB₄ on parenchymal strips. Eicosatetraynoic acid (10 ug/ml) and BW755C (40 ug/ml) which are inhibitors of cyclooxygenase and lipoxygenase pathways, produced a marked inhibition of the lung strip contraction to LTB₄. Similarly, aspirin (30 ug/ml) and flufenamate (lug/ml) showed a strong inhibition of the contraction of parenchymal strips to LTB₄; these results suggested that cyclooxygenase products mediate the action of LTB₄. The response to LTB₄ was unaffected by 15-hydroperoxyeicosatatraenoic acid (15-HPETE; 1 ug/ml) while L8027 (25 ng/ml) reduced the contraction by 50%, suggesting that thromboxane A₂ rather than prostacyclin was involved. Since parenchymal strips do not appear to be very sensitive to PGF₂α, PGE₂ and the endoperoxides, and since effluents from LTB₄-treated lungs produced contractions of lung strip and rabbit aorta which were reduced after 5 min. at 250, thromboxane A₂ was postulated to mediate the lung effect of LTB₄. The release of thromboxane B₂ (TxB₂) from lungs stimulated with LTB₄ was confirmed by gaschromatography-mass spectrometric (GC-MS) analyses.     

Mutagenesis by simian virus 40. I. detection of mutations in Chinese hamster cell lines using different resistance markers.

The mutagenic action of SV40 in permanent lines of Chinese hamster cells (CHO-K1 and V79) was investigated with the aid of different resistance markers. The markers studied had resistance to 8-azaguanine (25 and 30 mug/ml), aminopterin (3.3--5.5X10(-3) mug/ml), colchicine (6.5 and 7.0X10(-2) mug/ml) and 5-bromodeoxyuridine (50--120 mug/ml), respectively. After virus infection the mutation frequencies were increased by one (azaguanine, aminopterin) and two (colchicine) orders of magnitude as compared with spontaneous mutation frequencies. In contrast, it was not possible to enhance the frequency of mutation to BUdR resistance. On the other hand, the ability to proliferate in HAT medium was induced in three of five BUdR-resistant cell clones by infection with SV40. The resistance induced by SV40 was stable when isolated clones were cultured under non-selective conditions. Mechanisms are proposed that may be responsible for the mutagenic action of SV40.     

Certain characteristics of human lymphocyte migration inhibitory factors (LyMIF) and the effect of ampicillin on their appearance in unstimulated and PHA-stimulated cultures

Supernatants of human blood mononuclear cells stimulated with PHA, contained factors inhibitory for in vitro migration of human lymphocytes and granulocytes. After ultrafiltration of supernatants through Amicon PM-10 some stimulatory activity appeared in the bottom fraction. Sephadex G-100 gel filtration chromatography of supernatants showed three zones of lymphocyte migration inhibitory activity: In the range of molecules of m.w. 35 000-90 000, heat-stable; Factors of m.w. from several to from ten to twenty thousand daltons, heat unstable; Low molecular weight substances, resistant to heat. The possible relationship of these factors to lymphotoxins, soluble lymphocyte T receptors for SRBC, lymphocyte chemotactic factor and prostaglandins is discussed. Ampicillin in doses of 10, 20 and 50 micrograms/ml potentiated both the development of lymphocyte migration inhibitory factors and the production of factors with an opposing effect (stimulating lymphocyte migration).     

Antimicrobial and cytotoxicity activities of the medicinal plant Primula macrophylla

Primula macrophylla (Primulaceae) is reported as to be useful in asthma, restlessness, insomnia and fish poisoning. Antifungal and toxic activities of crude extract, fractions and a pure isolated compound exhibited statistically significant activities. Excellent antifungal activity was found in the crude extract, benzene and ethyl acetate fractions against T. longifusis and against M. canis with different MIC values. Antileishmanial activity (IC(50) = 50ug/mL) was observed as compared to standard drug Amphotericin B, and cytotoxic activity (LD(50) = 47.919microg/mL) was also found in the chloroform fraction. While pure compound 2-phenylchromone (Flavone) isolated from the chloroform fraction showed good activity (IC(50) = 25microg/mL) against Leishmania and cytotoxicity (LD(50) = 2.0116 microg/mL) in Brine Shrimp experiments. From antileishmanial and cytotoxic activity it can be concluded that 2-phenylchromone is the major compound responsible for these activities.     

Antioxidant,Antibacterial and Antischistosomal Activities of Extracts from Grateloupia livida (Harv). Yamada

The present study was designated to evaluate the antioxidant, antibacterial and antischistosomal activities of Grateloupia livida (GL) extracts in vitro. A GL Ethanol extract (EE) was separated into petroleum ether (PE), ethyl acetate (EA), n-butyl alcohol (BuOH) and aqueous (AQ) fractions to fractionate the polar and non-polar compounds in the EE. Extracts antioxidant activities were evaluated in vitro by DPPH radical-scavenging, deoxyribose radical scavenging, and β-carotene bleaching assays, all using butylated hydroxytoluene (BHT) as the reference antioxidant compound. The most effective antioxidant properties were observed in the PE fraction in all three assays. Antimicrobial testing showed that the PE fraction exhibited broad-spectrum antimicrobial activity, with the PE fraction also exhibiting strong activity against the human pathogenic trematode S. japonicum adult worm. In order to investigate the relationships between bioactivity and chemical composition, the chemical composition of the PE fraction was analyzed by gas chromatography-mass spectrometry (GC-MS). In total, 25 components were identified in the PE fraction, most of which have known antioxidant and antimicrobial activities. However, none of the compounds have reported activity against Schistosoma, suggesting that the schistosomicidal activity of the PE fraction may be related to minor constituents present in the extract, or governed by more intricate synergistic or additive relationships. Finally, fractions with the greatest biological activity displayed neither cellular cytotoxicity, at concentrations up to 100 ug/ml, or acute oral toxicity in mice, at doses up to 2000 mg/kg. Based on antioxidant, antimicrobial, antischistosomal activities, and low toxicity, the PE fraction possesses properties useful for food preservation and overall improvement of human health.     

Angiotensin I-converting enzyme localization on cultured fibroblasts by immunofluorescence

Summary Angiotensin I-converting enzyme is responsible for the activation of angiotensin I and the inactivation of bradykinin. It has been localized by immunofluorescence on the endothelium of a variety of tissues and has been considered to be a specific marker for endothelial cells in culture. The present paper demonstrates, by immunofluorescence, the presence of angiotensin I-converting enzyme in monolayer cultures of fibroblasts derived from adult rat lung, bovine calf pulmonary artery, and human foreskin (CF-3 cells). Fluorescent localization of angiotensin I-converting enzyme was observed over the cytoplasm of adult rat lung and bovine calf pulmonary artery fibroblasts and as distinct areas overlying the nuclei of human foreskin fibroblasts. Determination of angiotensin I-converting enzyme activity by fluorimetric assay in parallel studies confirmed the presence of angiotensin I-converting enzyme activity in cultured fibroblasts. Immunofluorescent studies with antibody to Factor VIII demonstrated the presence of Factor VIII on cultured endothelial cells but not on fibroblasts. These results indicate that angiotensin I-converting enzyme is not confined to endothelial cells, and thus may not serve as a specific marker for endothelial cells in culture. Factor VIII may be a more specific marker for these cells. Presented in part at the 31st Annual Meeting of the Histochemical Society, April 11–15, 1980, New Orleans, Louisiana. Wendy Baur and Ms. Jane Aghajanian for expert assistance in the preparation of the cell cultures. This work was supported by Research Grant HL 14456 and Training Grant HL 07053 from the National Institutes of Health, Bethesda, MD.     

Comparison of cytotoxic activities betweenin vitro and field grown plants ofTyphonium flagelliforme (Lodd.) blume

Anin vitro cytotoxicity screening of theTyphonium flagelliforme extracts indicated high cytotoxicity effect on human lung carcinoma NCl-H23 cells and human mammary gland carcinoma T-47D cells, but the extracts were not active on human liver carcinoma HepG2 cells. NCl-H23 cells were more susceptible toT. flagelliforme extracts than T-47D cells. EDP₅₀ values of the hexane fractions of the mature plant and thein vitro plantlet ofT. flagelliforme on NCl-H23 cells were less than 2 μg/mL Extract from the mature plant was relatively more cytotoxic than the one fromin vitro plantlet except for the hexane fraction. The chloroform and butanol fraction of the mature plant had higher cytotoxicity effect than the fraction fromin vitro plantlet on NCl-H23 cells. All the 3 fractions (hexane, chloroform, and butanol) of the mature plant exhibited higher cytotoxicity effects on human mammary gland carcinoma T-47D cells than the 3 fractions ofin vitro plantlet. However, the human liver carcinoma cells were resistant toT. flagelliforme extracts except for higher concentration of hexane fractions of both the mature and thein vitro plants and the chloroform fraction of the mature plant. Micropropagated plantlets ofT. flagelliforme could hence be used as herbal materials for the treatment of human lung and breast cancers.