棉酚对海胆精子鞭毛轴丝及力蛋白(Dynein)ATP酶的抑制作用

本工作观测了不同浓度棉酚(10—80μmol/L)对海胆精子鞭毛轴丝及力蛋白ATP酶的影响,发现棉酚对之具有剂量依赖方式的抑制作用。 实验对比了几种ATP酶抑制剂对轴丝ATP酶的作用,结果表明它们之间差别很大。使酶活力下降一半所需的各种抑制剂浓度分别为:钒酸钠17μmol/L;棉酚36μmol/L;槲皮酮450μmol/L,DCCD[双环已基碳化二亚胺(Dicyclohexylcarbodiimide)]1,600μmol/L;乌本苷>2,000μmol/L。 本文对棉酚的杀精机理,抑制强度及其控制生育的意义进行了讨论。     

大鼠及羊精子在附睾成熟过程中ATP酶活力及其对棉酚敏感性的变化

大鼠及羊精子在附睾成熟过程中ATP酶活力发生明显下降,酶活力的变化形式存在着种间差异。大鼠附睾体及附睾尾精子的ATP酶相对活力分别为附睾头的55.7％及59.6％,而羊则为92.1％及59.8％。 大鼠及羊附睾各区域精子的ATP酶对棉酚抑制作用的敏感程度不同。在5μmol/L的低棉酚的浓度下,大鼠附睾头、体及尾部精子的ATP酶活力分别降至对照组的40.8％、62.7％及81.4％;当棉酚浓度增至40μmol/L时,羊附睾头、体、尾部精子的ATP酶活力才分别降至对照组的63.8％、83.7％及90.7％。作者提出如能使药物作用于附睾精子的敏感区域以干扰其成熟,可能是一条有发展前景的抗生育新途径。     

磷脂酶A2激活对中性粒细胞趋化和粘附的作用

外源性磷脂酶A2(Ⅰ型PLA2)和钙离子载体(A23187)可明显促进中性粒细胞对TNF,FMLP的趋化及同玻璃球的粘附.PLA2抑制剂二溴苯乙酮(PBPB)和PLA2抗体对PLA2诱发的趋化和粘附具有浓度相关的抑制作用,而对A23187的相同作用并无影响.提示PBPB和PLA2抗体可能通过直接同PLA2作用而抑制PMN趋化和粘附,A23187诱导的促趋化及粘附作用可能不同于PLA2.     

胞浆型磷脂酶A2介导弱氧化修饰低密度脂蛋白诱导的人脐静脉内皮细胞凋亡

探讨弱氧化修饰低密度脂蛋白(MM-LDL)能否诱导人脐静脉内皮细胞(HUVECs)凋亡以及胞浆型磷脂酶A2(cPLA2)在此过程中的作用.MTT法测定细胞存活率;相差显微镜、荧光显微镜和流式细胞仪检测细胞凋亡;3H-花生四烯酸(3H-AA)预标法测定PLA2活性;蛋白质印迹检测cPLA2磷酸化;激光共聚焦显微镜检测单个细胞内钙离子浓度的变化.结果表明,MM-LDL(100～300 mg/L)作用后的HUVECs呈现凋亡典型的形态特征,凋亡率随MM-LDL浓度的增加而上升.MM-LDL能引起胞内钙离子浓度增加,cPLA2的活化及磷酸化.15 μmol/L AACOCF3和5 mmol/L EGTA在抑制cPLA2活性的同时,部分抑制MM-LDL诱导的HUVECs凋亡.加入外源性AA(50 μmol/L)能逆转AACOCF3引起的凋亡抑制.结果提示,cPLA2参与了MM-LDL诱导HUVECs凋亡的信号传递.     

棉酚对几种动物精子ATP酶抑制作用的比较研究

本文比较了棉酚对几种动物精子ATP酶的抑制作用。实验所测为精子细胞的总ATP酶活力;采用的棉酚浓度为10μM至80μM。20μM时精子酶的相对活力为海胆7604％,大鼠74％,家兔51.6％;40μM时酶的相对活力百分数下降至海胆52.5,大鼠35和家兔29.7;达80μM时酶的相对活力进一步下降至海胆26.7,而大鼠及家兔仅余13.2。结果表明(1)棉酚对精于ATP酶的抑制作用是依赖浓度的变化;(2)各种动物精子ATP酶活力的下降情况显示,大鼠及家兔(哺乳动物)精子ATP酶对棉酚的敏感性比海胆(无脊椎动物)要高些。这种差异性可能对棉酚在临床上的应用具有一定的参考价值。     

白介素-2对心肌细胞[Ca~(2 )]_i的作用及其信号转导途径

为研究白介素 2 (interleukin 2 ,IL 2 )对心肌细胞内钙浓度 ([Ca2 ]i)的影响及其信号转导途径 ,实验采用酶解法分离成年大鼠心室肌细胞 ,以Fura 2 /AM为钙探针 ,用细胞内双波长钙荧光系统检测细胞 [Ca2 ]i 的变化。结果发现 :(1)IL 2 (0 5～ 2 0 0U/ml)浓度依赖性地降低单个心室肌细胞内钙瞬态 ,IL 2 (2 0 0U/ml)对咖啡因诱导的肌浆网内储钙的释放无影响 ;(2 )纳洛酮 (naloxone ,Nal) (10 -8mol/L)和nor binaltorphimine (nor BNI,10 -8mol/L)可阻断IL 2对心肌细胞钙瞬态的作用 ,而纳曲吲哚 (naltrindole ,NTI) (10 -6mol/L)不能阻断此作用 ;(3)κ阿片受体激动剂U5 0 488H (10 -6mol/L)降低心肌细胞钙瞬态 ,nor BNI (10 -8mol/L)可阻断此作用 ;(4 ) 5mg/L百日咳毒素 (PTX)预处理可取消IL 2降低心肌细胞钙瞬态的作用 ,而酪氨酸激酶抑制剂genistein (10 -4 mol/L)不能取消IL 2的作用 ;(5 )U7312 2预处理可阻断IL 2的作用。研究结果表明 ,IL 2降低心肌细胞钙瞬态的作用 ,是通过心肌细胞上κ阿片受体介导的 ,其下游途径包括PTX敏感的G蛋白和磷脂酶C。     

棉酚对Jurkat T细胞增殖与凋亡的影响

研究棉酚(gossypol)对Jurkat T细胞增殖和凋亡的影响及可能机制.将不同浓度的棉酚作用于Jurkat T细胞,用MTS比色法检测细胞存活率;以膜联蛋白V-PE染色分析细胞凋亡;用Hoechst33342染色观察核形态:用荧光染料Mitocapture结合流式细胞术和激光共焦显微镜检测线粒体跨膜电位变化.结果显示棉酚作用Jurkat T细胞24 h、48 h、72 h,其IC50值分别为77.2μmol/L、57.3 μmol/L、23.3 μmol/L,对细胞的抑制作用与药物存在时间-剂量依赖关系;终浓度为8、16、32 μmol/L的棉酚处理24 h后的细胞凋亡率分别为5.30%、15.20%、51.19%,对照组凋亡率为3.43%;高浓度棉酚作用后大量细胞核呈现染色质固缩、核碎裂和致密浓染等凋亡特征;随着浓度增加细胞线粒体跨膜电位明显降低.研究表明棉酚能有效抑制Jurkat T细胞增殖和诱导其发生凋亡,并呈现出时间-剂量依赖关系,其诱导凋亡的作用可能依赖于线粒体途径.     

γ-生育三烯酚联合亚砷酸对急性早幼粒细胞NB4生长的抑制作用及机制

目的:研究γ-生育三烯酚联合亚砷酸对急性早幼粒细胞NB4生长的抑制作用及可能的分子机制。方法:采用CCK-8、细胞周期实验检测细胞增殖、利用激光共聚焦显微镜、Annexin V/PI染色、Caspase活性检测、Western Blot等方法测定细胞凋亡。采用1μmol/L ATO及不同浓度(0、15、30、45μmol/L)的γ-生育三烯酚处理NB4细胞24、48和72小时,通过CCK-8检测细胞的增殖情况,流式细胞术、激光共聚焦显微镜检测细胞周期和凋亡情况,检测Caspase3,8,9的活性,Western Blot检测细胞中c-caspase-3、Bcl-2和survivin的蛋白表达。结果:γ-生育三烯酚联合亚砷酸显著抑制NB4细胞增殖(P0.01),且随着作用时间延长和γ-生育三烯酚浓度的增加,其增殖抑制作用增强;细胞周期阻滞在S期,S期的比例由38.21%±2.99上升到50.31%±5.03;γ-生育三烯酚联合亚砷酸诱导NB4细胞凋亡,1μmol/L ATO联合15、30μmol/L的γ-生育三烯酚处理48h后,细胞活率分别为82.27%±3.16、66.97%±3.17、12.63%±2.66;1μmol/L ATO联合30μmol/L的γ-生育三烯酚处理后,NB4细胞caspase-3,-8,-9的活性均较ATO单独用药组显著增高,c-caspase-3表达增高而Bcl-2和survivin蛋白的表达无明显变化。结论:生育三烯酚联合亚砷酸对急性早幼粒细胞NB4的生长具有抑制作用,此作用可能与抑制增殖并诱导凋亡相关,其作用靶点可能与促进Cas-pase诱导的凋亡有关。     

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。     

棉酚对DNA酶某些性质的影响

DNase用棉酚处理后,经聚丙烯酸胺凝胶平板电泳与对照相比,电泳度无明显差异,均呈单一区带,表明棉酚对DNase所带电荷及分子量无影响。棉酚可改变DNase在225nm和280nm波长处的吸收,对DNase的荧光有粹灭作用,表明棉酚破坏了DNase的空间构象。棉酚与DNase的浓度(W/V)为1:1以上时,酶活性受到显著影响,呈现出反竞争性抑制作用。     

Gossypol-induced free radical toxicity to isolated islet cells

1. The cytotoxicity of the polyphenolic potential male antifertility agent gossypol was investigated on isolated mouse islets cells. 2. Gossypol shared many properties with the diabetogenic agent alloxan. 3. Gossypol (0.1-1.0 mmol/l) induced a concentration-dependent increase of Trypan Blue uptake by the cells, indicating an increase of membrane permeability to the dye. 4. Trypan Blue uptake induced by 0.5 mmol/l gossypol was inhibited by concomitant incubation of the cells with enzymatic (200 mg/l superoxide dismutase, 200 mg/l catalase, 3 mmol/l cytochrome-c), or low-molecular weight (50 mmol/l D-mannitol) scavengers of oxygen radicals, and the metal chelator diethylenetriaminepentacetic acid (DTPA) (50 mumol/l). 5. The results support the hypothesis that gossypol is B-cytotoxic by generation of noxious free radicals and that when proposing gossypol as a male antifertility agent, studies to exclude gossypol as a diabetogenic agent should first be performed in vivo.     

Oxygen radical formation induced by gossypol in rat liver microsomes and human sperm

Gossypol, a polyphenolic compound found in cotton plants, has many potential uses, including use as a male antifertility drug and spermicide. Gossypol affects a variety of cell processes and many of these effects may be explained by a common underlying mechanism. Here we report that gossypol promotes the formation of oxygen radicals when incubated with rat liver microsomes and human sperm suggesting that oxygen radical production may be the underlying basis of its biological activity.     

Binding of gossypol to purified tubulin and inhibition of its assembly into microtubules

Gossypol is a polyphenolic pigment, which is employed as a male antifertility drug. It inhibits, among other reported effects, the growth of cultured mammalian cells, spermiogenesis, flagellar motility in Trypanosoma and sperm, dynein ATPase and the lactate dehydrogenase X (LDH-X) isozyme. We have characterized the non-covalent binding of gossypol to purified calf brain tubulin in 10 mM phosphate buffer, 0.1 mM GTP pH 7.0 at 25 degrees C. Equilibrium measurements were performed by difference spectroscopy. A peak at 435 nm was produced by the perturbation of gossypol light absorption upon binding to tubulin. The experimental isotherm was fitted by 1.96 +/- 0.06 gossypol binding sites per tubulin molecule, with identical apparent equilibrium binding constants of (7.5 +/- 1.1) X 10(4) M-1. The complex formed could be separated from free gossypol by gel chromatography. Binding of gossypol was independent of the presence of 0.1 mM GTP in the buffer. Gossypol did not affect the binding of ligands to the colchicine site. Gossypol interacted with vinblastine but apparently did not bind to the vinblastine sites of tubulin. Gossypol did not displace anilinonaphthalene sulphonate (ANS) bound to tubulin, but caused a strong (fivefold) quenching of its fluorescence. This indicated that gossypol probably binds in the vicinity of the ANS site of tubulin. Gossypol inhibited in vitro microtubule assembly at the same concentration range employed in the binding studies. An increase in the critical protein concentration required for polymerisation was observed, most simply interpreted by a stoichiometric mechanism. Gossypol did not induce any noticeable distortion of the microtubules observed under the electron microscope. This compound constitutes a new tubulin ligand and an inhibitor of microtubule assembly in vitro.     

Gossypol Increases Expression of the Pro-apoptotic BH3-only Protein NOXA through a Novel Mechanism Involving Phospholipase A2, Cytoplasmic Calcium,and Endoplasmic Reticulum Stress

Gossypol is a putative BH3 mimetic proposed to inhibit BCL2 and BCLXL based on cell-free assays. We demonstrated previously that gossypol failed to directly inhibit BCL2 in cells or induce apoptosis in chronic lymphocytic leukemia (CLL) cells or platelets, which require BCL2 or BCLXL, respectively, for survival. Here, we demonstrate that gossypol rapidly increased activity of phospholipase A2 (PLA2), which led to an increase in cytoplasmic calcium, endoplasmic reticulum (ER) stress, and up-regulation of the BH3-only protein NOXA. Pretreatment with the PLA2 inhibitor, aristolochic acid, abrogated the increase in calcium, ER stress, and NOXA. Calcium chelation also abrogated the gossypol-induced increase in calcium, ER stress, and NOXA, but not the increase in PLA2 activity, indicating that PLA2 is upstream of these events. In addition, incubating cells with the two products of PLA2 (lysophosphatidic acid and arachidonic acid) mimicked treatment with gossypol. NOXA is a pro-apoptotic protein that functions by binding the BCL2 family proteins MCL1 and BFL1. The BCL2 inhibitor ABT-199 is currently in clinical trials for CLL. Resistance to ABT-199 can occur from up-regulation of other BCL2 family proteins, and this resistance can be mimicked by culturing CLL cells on CD154⁺ stroma cells. We report here that AT-101, a derivative of gossypol in clinical trials, overcomes stroma-mediated resistance to ABT-199 in primary CLL cells, suggesting that a combination of these drugs may be efficacious in the clinic.     

The structural and functional contribution of N-terminal region and His-47 on Taiwan cobra phospholipase A2.

Modification of His-47 and removal of the N-terminal octapeptide caused a different effect on the structure of Naja naja atra (Taiwan cobra) phospholipase A2 (PLA2). Unlike native enzyme, Ca2+ induced an alteration in the structural flexibility of His-modified PLA2. Moreover, the spatial positions of Trp residues in His-modified PLA2 were not properly rearranged toward lipid-water interface in the presence of Ca2+. CD spectra and fluorescence measurement showed that the dynamic properties of Trp residues and the gross conformation of N-terminally truncated PLA2 were totally different from native enzyme. Although a precipitous drop in the enzymatic activity was observed with modified PLA2, His-modified PLA2 and N-terminally truncated PLA2 retained cytotoxicity on inducing necrotic death of human neuroblastoma SK-N-SH cells. Our data suggest that structural perturbations elicited by the chemical modification cause a dissociation of enzymatic activity and cytotoxicity of PLA2.     

The (+)- and (−)-gossypols potently inhibit human and rat 11β-hydroxysteroid dehydrogenase type 2

Gossypol has been proven to be a very effective male contraceptive. However, clinical trials showed that the major side effect of gossypol was hypokalemia. Gossypol occurs naturally as enantiomeric mixtures of (+)-gossypol and (−)-gossypol. The (−)-gossypol is found to be the active component of antifertility. 11β-Hydroxysteroid dehydrogenase 2 (11βHSD2) has been demonstrated to be a mineralocorticoid receptor (MR) protector by inactivating active glucocorticoids including corticosterone (CORT) in rats, and therefore mutation or suppression of 11βHSD2 causes hypokalemia and hypertension. In the present study, the potency of gossypol enantiomers was tested for the inhibition of 11βHSD1 and 2 in rat and human. Both (+) and (−)-gossypols showed a potent inhibition of 11βHSD2 with the half maximal inhibitory concentration (IC₅₀) of 0.61 and 1.33 μM for (+) and (−)-gossypols, respectively in rats and 1.05 and 1.90 μM for (+) and (−)-gossypols, respectively in human. The potency of gossypol to inhibit 11βHSD1 was far less; the IC₅₀ was ≥100 μM for racemic gossypol. The gossypol-induced hypokalemia is likely associated with its potent inhibition of kidney 11βHSD2.     

微孔比色法在猪胰腺PLA_2制备中的应用

微孔比色法采用合成的磷脂类似物２－硫代十六酰乙基磷酸胆碱作底物,在多孔聚苯乙烯板的小孔中反应,并用酶联免疫检测器连续测定和记录吸收值．同时应用此法及滴定法检测酶活力,从猪胰腺中制备了一种分子量低（１４．３ｋＤ）,对热、酸稳定,活性依赖Ｃａ２＋的ＰＬＡ２．两种方法检测结果具有可比性,而微孔比色法同时可测多个样品,有节约样品,灵敏度较高等优点．微孔比色法特别适用于大量的样品测定,如拮抗剂筛选、临床样品及制备酶时层析级分的检测等．     

Clinical study of gossypol as a male contraceptive

Animal experiments conducted in the late 1960s and early 1970s in various institutes of China showed that the effective antifertility agent in crude cottonseed oil was gossypol. Gossypol is a yellow substance which occurs in various parts of the cotton plant; its chemical structure is naphthol phenol. At the Capital Hospital gossypol was tested on 172 volunteers selected from hospital employees and workers from a nearby factory. All of the volunteers were under age 50, married and healthy with at least 1 child. Examinations required before treatment were general physical examination, a blood and urine analysis, an electrocardiogram, serum potassium concentration and serum analysis. 2 stages of gossypol treatment were required in the clinical study: the initial stage, the loading period, is of about 60 days. A daily dose of 20 mg gossypol was given successively for 60 days causing the sperms to become immotile, reduced in number or totally absent. A sperm count below 4 million/ml semen was considered to indicate infertility. The dosage in the 2nd stage, the maintenance period, was reduced to 1/3 of the original dose to maintain infertility. The volunteers were followed up every 2-3 months. Fatigue, decrease of libido and impaired appetite were the 3 main complaints. Serum glutamic-pyruvic transaminase (SGPT) activities showed that the transient symptoms of fatigue and loss of appetite were not related to disturbance of liver function. The mechanism of hypokalemia induced by gossypol is probably of renal origin. Infertility induced by gossypol in contraceptive doses over a relatively short period of administration was reversed about 3 months after stopping treatment. Gossypol used as a male antifertility agent has been found to be very effective and relatively safe.     

Inactivation of intestinal brush-border enzymes by gossypol

Gossypol, a pigment found in cottonseed that has recently been shown to have antifertility properties, inhibited the activity of 3 intestinal brush border enzymes in a concentration-dependent manner. Suspensions of rat intestinal mucosa were incubated with various concentrations of gossypol for 45 minutes and then washed. At a concentration of 6 mg per gm mucosa, gossypol inhibited the activities of alkaline phosphatase, maltase, and sucrase by 57, 73, and 77%, respectively. Gossypol is a bifunctional agent, capable of cross-linking amino acid side chains, and its action on brush-border enzymes may be due to this mechanism. Recent investigations have demonstrated that rats fed a diet of 10-15 mg of gossypol/day/kg of body weight exhibit reduced fertility. This study suggests that a partial inhibition of brush-border enzymes may occur at doses used to cause infertility. Such a side effect should be considered in studies and treatments utilizing a gossypol diet.     

Inhibition of testicular LDH-X from laboratory animals and man by gossypol and its isomers

The inhibitory effect of (+)-, (-)-, (+/-)-gossypol and (+/-)-gossypol acetic acid upon testicular cytosolic LDH-X was measured in vitro. Gossypol acetic acid (0-100 mumol/l) inhibited LDH-X prepared from the testes of the mouse greater than rabbit greater than human greater than rat greater than hamster. There was no relationship between inhibition and in-vivo antifertility activity. LDH activity measured in vitro in serum of men and hamsters was unaffected by gossypol. Gossypol and its isomers were non-competitive inhibitors of human and hamster LDH-X with respect to the coenzyme NADH, competitive inhibitors of human LDH-X and noncompetitive-competitive inhibitors of hamster LDH-X with respect to the substrate alpha-ketobutyrate. Co-incubation with human serum albumin or poly-L-lysine but not lysine protected human and hamster LDH-X from gossypol.