在小鼠受精卵中PKC、PKB通过p21～（CIP1/WAF1）蛋白影响G2/M转换
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摘要
前言
细胞周期是细胞增殖、分化的一个重要过程，它的启动与进程受到严格调控，参与调控的因子不仅包含起正调控作用的细胞周期素(cyclins)、细胞周期蛋白依赖性激酶(cyclin-dependent kinase，CDK)、还有起负调控作用的CDK抑制因子(cyclin-dependent kinase inhibitor，CKI)。CKI是近年来分离得到的一类很重要的细胞周期调控蛋白，这些蛋白在体外通过与CDK、cyclin或cyclin-CDK复合物的结合，抑制CDK的活性，进而抑制细胞周期的进程。p21蛋白作为其中重要的一员，在细胞周期调控中所起的作用引起广泛的关注，是目前已知的具有最广泛激酶抑制活性的细胞周期抑制蛋白。可抑制各种cyclin-CDK复合物，如cyclinD1-CDK4，cyclinE-CDK2和cyclinA-CDK2，但对cyclinB相关的复合物抑制活性较弱。p21蛋白可在G1和G2／M两个调控点上对细胞周期进行调控。目前研究多集中在G1期，过表达的p21蛋白可通过结合CDK2／cyclinA、E而抑制其活性，使细胞阻滞在G1期。在G2期，p21蛋白可通过降低cdc2活性，阻滞G2期的进程。但研究表明p21结合cdc2的能力不如其他CDKs，其介导的G2／M期的调控是由于p21蛋白抑制CDK2／cyclinA复合物的活性，下调cdc25磷酸酶活性，阻止cdc2 Tyr-15的去磷酸化，降低MPF活性。
蛋白激酶C(protein kinase C，PKC)和蛋白激酶B(protein kinase B，PKB)同属于丝／苏氨酸蛋白激酶，广泛参与细胞的多种生命过程，包括生长、分化、细胞周期调控、细胞凋亡等。其中PKC、PKB与细胞周期之间的研究正成为热点。近年来的研究表明，PKC、PKB都可通过调节细胞周期蛋白依赖性激酶抑制蛋白p21调控细胞周期的进程。为了研究在小鼠受精卵中PKC、PKB是否也通过p21蛋白参与调控G2／M转换，本实验应用了PMA和LY 294002作为PKC、PKB的抑制剂，研究它们对小鼠受精卵中p21蛋白表达和定位，进一步探讨可能的调控机制。
实验材料与方法
一、实验材料
1.实验动物昆明系小鼠由中国医科大学实验动物部提供
2.主要试剂
孕马血清促性腺激素(PMSG)购自天津华孚高新技术生物中心
人绒毛促性腺激素(hCG)购自上海生物生化制药厂
小鼠抗pZI单克隆抗体购自北京中山生物技术公司
碱磷酶标记羊抗鼠二抗购自SantaC二公司
透明质酸酶、PMA、和牛血清白蛋白(BSA)等为美国Sigma公司产品
LY294002购于sigrna公司
TritonX一100购于BOEHRINGER MANNHEIN GmbH
FITC(异硫氰酸荧光素)标记的兔抗鼠I药抗体购于北京中山生物技
术公司
HEPES购于E.MetekD~stadt
二、实验方法
1.小鼠超数排卵和受精卵的收集
根据HoganB采用的方法进行小鼠卵的超排和收集。取4一5周龄成熟
雌性昆明系小白鼠，腹腔注射PMSG(孕马血清促性腺激素)10IU，46一48
小时后腹腔注射hCG(人绒毛膜促性腺激素)1 OIU，将注射hCG后的雌鼠与
8周以上的成熟雄鼠合笼交配，次日检察阴栓，将查到阴栓的雌鼠处死，取
输卵管于MZ培养液中，解剖镜下撕开壶腹，释放细胞团，然后用300林岁nil
透明质酸酶消化去除颗粒细胞，口控吸管将卵细胞在M:中反复清洗，然后
置于孵箱中，根据时间点收集G2期细胞。
2.Westem印迹
将处理组及对照组小鼠一细胞G2期受精卵各200个取出，转移到Ep-
pendoff管中，5000甲m离心4分钟，弃去上清，加入ro川粉碎缓冲液，充分
振荡混匀，在液氮中经过2一3次的冻融循环，迫使卵细胞裂解，加人等量的
样品缓冲液沸水中煮5分钟，用于Westem印迹分析。样品蛋白经12%SDS
一聚丙烯酞胺凝胶电泳分离后，转印到硝酸纤维素膜上，与第一抗体4℃孵
育过夜，经TBS洗涤后，再与第二抗体室温孵育2小时，TTBS充分冲洗后，
显色观察。
3.相差显微镜下观察受精卵分裂率
受精后20小时相差显微镜下观察PMA及LY 29取犯2处理后小鼠受精
卵分裂情况，计算处理组及对照组小鼠受精卵分裂率。
4.间接免疫荧光法
将对照组及处理组受精卵各20个移人新鲜配制的4%多聚甲醛液中，
固定30面n(37℃)，然后在清洗液滴中(含0 .1%BSA的PBS)清洗三次后，
用含0.2%Triton一xloo的PBS处理巧而n(37℃)以增加细胞膜透性，用清
洗液充分清洗后把卵移人封闭液滴(含1%BSA的PBs)中，37℃下处理lh。
鼠单克隆P21抗体室温孵育1h或4℃过夜。用清洗液洗卵3遍以充分去除
未结合的抗体，然后移人二抗(FITC标记)中，室温避光孵育30min，用清洗
液洗3次，去除未结合的二抗。同时，用不加一抗的受精卵作为对照，其他
处理过程与实验组相同。
实验结果
1.PMA对小鼠受精卵分裂的影响(图1)
取小鼠G2期受精卵细胞(受精后17h)，加人4Onm0FL PMA，处理2h，
相差显微镜下观察小鼠受精卵分裂情况。对照组的分裂率为66.1%，而处
理组的分裂率为16.8%。可见用40~FL PMA处理受精卵2h可明显抑
制小鼠受精卵的分裂。
2.LY294002对小鼠受精卵分裂的影响(图2)
取小鼠受精后17h卵细胞加入10林m亦LLY29粼刃2，处理时间为
3Omin，于受精后20h观察小鼠受精卵分裂情况。对照组的分裂率为79.
7%，处理组的分裂率为10
Introduction
Protein kinase C ( PKC ) is a number of serine/ threonine kinase family which play central regulatory roles in a multitude of cellular processes, including cell proliferation, cell cycle progression, differentiation, apoptosis. Increasing evidence from studies using in vitro and in vivo systems suggests that PKC is a key regulator of critical cell cycle transitions, including cell cycle entry and exit and the Gl and G2 checkpoint. PKC - mediated control of these transitions can be negative or positive, depending on the timing of PKC activation during the cell cycle and on the specific PKC isozymes involved. PKC signaling has been implicated in the control of the G2/M transition mostly by the activated MPF (maturation promoting factor) Otherwise, PKC controls the G2/M transition by the cyclin - dependent kinase inhibitor (CKI). p21 is a key target of PKC -mediated cell cycle modulation. It may play a major role in G2/M transition via a mechanism involving blockade of cdk2/cyclinA activity, inhibition of cdc2
5 upregulation, accumulation of phosphorylation on cdc2, and suppression of cdc2/cyclin B activity. In this study, we showed that the protein levels of the cyclin - dependent kinase inhibitor p21 rapidly increased in the one - cell staged fertilized eggs treated with PMA. The cleavage from one - cell stage of the fertilized eggs into two - cell stage was inhibited.
Protein kinase B is newly found as a serine/threonine protein kinase , It could have widely spread functions in cell cycle regulation. The activation of protein kinase B/AKT is thought to be a critical step in the phosphoinositide 3 -kinase pathway which regulates cell growth and differentiation. In this study,we showed that the PDK inhibitor LY294002 regulated the cellular localization of
Materials
1. KUNMING mice were supplied from the Department of Laboratory Animals, China Medical University
2. Pregnant mare serum gonadotropin(PMSG) and human chorionic gona-dotropin ( hCG) were obtained from Tianjin Huafu Biological Products Research Institute and Shanghai Products Research Insititute, respectively.
Anti - P21 mouse monoclonal antibody from Beijing Zhongshan Biotechnology
Anti - mouse or anti - rabbit IgG secondary antibody from Santa Cruz Biotechnology
LY294002 from Sigma Biotechnology TritonX -100 from BOEHRINGER MANNHEIN GmbH Fluorescein isothiocyanate ( FITC) conjugated anti - mouse IgG antibody was purchased from Beijing Zhongshan Biotechnology HEPES from E. Metck Darmstadt
Methods
Superovulation and collection of eggs
For superovulation, female KUNMING mice 4-5 week old were injected with pregnant mare serum gonadotropin ( PMSG) , and after 46 - 48 hours with human chorionic ginadotropin. ( hCG). One - cell fertilized eggs were collected on the next day from oviduct of females. Then one cell eggs were transferred to M16 for culture in incubator with an atmosphere of 5% CO2. According to the time point , collected G2 phase eggs .
Western Blot Analysis
We treated 200 fertilized eggs in a series of steps including lysing them in l0ul of lysis buffer(50mM Tris - Hcl pH 7. 5 , 250mM NaCl, 5mM EDTA, ImM DTT ,0. 1% Triton ,50mM sodium orthovanadate, lOOug/mg PMSF and TPCK, 50ug/ml TLCK, 1 ug/ml leupeptin pepstatin and aprotinin) , frozing them below - 70 , and then thawing them at room temperature for 10 min. We did the same steps for three times, so we could get the extraction using the steps
mentioned.
Laemmli sample buffer was added to the extracted protein,which were then boiled for 5 min. The protein samples were separated by 12% SDS - PAGE and transferred to nitrocellulose membrane. The membranes were blocked in Tris -buffered saline (TBS; 20 mM Tris -HCl, 137 mM NaCl, pH 7.6) containing 5% non - fat dried milk (TBS/milk) for 30 min at 37 . Later the Membranes were incubated for 2 h at room temperature or overnight at 4 with p21 primary antibody in TBS/milk with 0. 1% Tween 20 , followed by washing the membrane in TBSt/milk for 5 min three times. Blots were then incubated for 1 h at
细胞周期是细胞增殖、分化的一个重要过程，它的启动与进程受到严格调控，参与调控的因子不仅包含起正调控作用的细胞周期素(cyclins)、细胞周期蛋白依赖性激酶(cyclin-dependent kinase，CDK)、还有起负调控作用的CDK抑制因子(cyclin-dependent kinase inhibitor，CKI)。CKI是近年来分离得到的一类很重要的细胞周期调控蛋白，这些蛋白在体外通过与CDK、cyclin或cyclin-CDK复合物的结合，抑制CDK的活性，进而抑制细胞周期的进程。p21蛋白作为其中重要的一员，在细胞周期调控中所起的作用引起广泛的关注，是目前已知的具有最广泛激酶抑制活性的细胞周期抑制蛋白。可抑制各种cyclin-CDK复合物，如cyclinD1-CDK4，cyclinE-CDK2和cyclinA-CDK2，但对cyclinB相关的复合物抑制活性较弱。p21蛋白可在G1和G2／M两个调控点上对细胞周期进行调控。目前研究多集中在G1期，过表达的p21蛋白可通过结合CDK2／cyclinA、E而抑制其活性，使细胞阻滞在G1期。在G2期，p21蛋白可通过降低cdc2活性，阻滞G2期的进程。但研究表明p21结合cdc2的能力不如其他CDKs，其介导的G2／M期的调控是由于p21蛋白抑制CDK2／cyclinA复合物的活性，下调cdc25磷酸酶活性，阻止cdc2 Tyr-15的去磷酸化，降低MPF活性。
蛋白激酶C(protein kinase C，PKC)和蛋白激酶B(protein kinase B，PKB)同属于丝／苏氨酸蛋白激酶，广泛参与细胞的多种生命过程，包括生长、分化、细胞周期调控、细胞凋亡等。其中PKC、PKB与细胞周期之间的研究正成为热点。近年来的研究表明，PKC、PKB都可通过调节细胞周期蛋白依赖性激酶抑制蛋白p21调控细胞周期的进程。为了研究在小鼠受精卵中PKC、PKB是否也通过p21蛋白参与调控G2／M转换，本实验应用了PMA和LY 294002作为PKC、PKB的抑制剂，研究它们对小鼠受精卵中p21蛋白表达和定位，进一步探讨可能的调控机制。
实验材料与方法
一、实验材料
1.实验动物昆明系小鼠由中国医科大学实验动物部提供
2.主要试剂
孕马血清促性腺激素(PMSG)购自天津华孚高新技术生物中心
人绒毛促性腺激素(hCG)购自上海生物生化制药厂
小鼠抗pZI单克隆抗体购自北京中山生物技术公司
碱磷酶标记羊抗鼠二抗购自SantaC二公司
透明质酸酶、PMA、和牛血清白蛋白(BSA)等为美国Sigma公司产品
LY294002购于sigrna公司
TritonX一100购于BOEHRINGER MANNHEIN GmbH
FITC(异硫氰酸荧光素)标记的兔抗鼠I药抗体购于北京中山生物技
术公司
HEPES购于E.MetekD~stadt
二、实验方法
1.小鼠超数排卵和受精卵的收集
根据HoganB采用的方法进行小鼠卵的超排和收集。取4一5周龄成熟
雌性昆明系小白鼠，腹腔注射PMSG(孕马血清促性腺激素)10IU，46一48
小时后腹腔注射hCG(人绒毛膜促性腺激素)1 OIU，将注射hCG后的雌鼠与
8周以上的成熟雄鼠合笼交配，次日检察阴栓，将查到阴栓的雌鼠处死，取
输卵管于MZ培养液中，解剖镜下撕开壶腹，释放细胞团，然后用300林岁nil
透明质酸酶消化去除颗粒细胞，口控吸管将卵细胞在M:中反复清洗，然后
置于孵箱中，根据时间点收集G2期细胞。
2.Westem印迹
将处理组及对照组小鼠一细胞G2期受精卵各200个取出，转移到Ep-
pendoff管中，5000甲m离心4分钟，弃去上清，加入ro川粉碎缓冲液，充分
振荡混匀，在液氮中经过2一3次的冻融循环，迫使卵细胞裂解，加人等量的
样品缓冲液沸水中煮5分钟，用于Westem印迹分析。样品蛋白经12%SDS
一聚丙烯酞胺凝胶电泳分离后，转印到硝酸纤维素膜上，与第一抗体4℃孵
育过夜，经TBS洗涤后，再与第二抗体室温孵育2小时，TTBS充分冲洗后，
显色观察。
3.相差显微镜下观察受精卵分裂率
受精后20小时相差显微镜下观察PMA及LY 29取犯2处理后小鼠受精
卵分裂情况，计算处理组及对照组小鼠受精卵分裂率。
4.间接免疫荧光法
将对照组及处理组受精卵各20个移人新鲜配制的4%多聚甲醛液中，
固定30面n(37℃)，然后在清洗液滴中(含0 .1%BSA的PBS)清洗三次后，
用含0.2%Triton一xloo的PBS处理巧而n(37℃)以增加细胞膜透性，用清
洗液充分清洗后把卵移人封闭液滴(含1%BSA的PBs)中，37℃下处理lh。
鼠单克隆P21抗体室温孵育1h或4℃过夜。用清洗液洗卵3遍以充分去除
未结合的抗体，然后移人二抗(FITC标记)中，室温避光孵育30min，用清洗
液洗3次，去除未结合的二抗。同时，用不加一抗的受精卵作为对照，其他
处理过程与实验组相同。
实验结果
1.PMA对小鼠受精卵分裂的影响(图1)
取小鼠G2期受精卵细胞(受精后17h)，加人4Onm0FL PMA，处理2h，
相差显微镜下观察小鼠受精卵分裂情况。对照组的分裂率为66.1%，而处
理组的分裂率为16.8%。可见用40~FL PMA处理受精卵2h可明显抑
制小鼠受精卵的分裂。
2.LY294002对小鼠受精卵分裂的影响(图2)
取小鼠受精后17h卵细胞加入10林m亦LLY29粼刃2，处理时间为
3Omin，于受精后20h观察小鼠受精卵分裂情况。对照组的分裂率为79.
7%，处理组的分裂率为10
Introduction
Protein kinase C ( PKC ) is a number of serine/ threonine kinase family which play central regulatory roles in a multitude of cellular processes, including cell proliferation, cell cycle progression, differentiation, apoptosis. Increasing evidence from studies using in vitro and in vivo systems suggests that PKC is a key regulator of critical cell cycle transitions, including cell cycle entry and exit and the Gl and G2 checkpoint. PKC - mediated control of these transitions can be negative or positive, depending on the timing of PKC activation during the cell cycle and on the specific PKC isozymes involved. PKC signaling has been implicated in the control of the G2/M transition mostly by the activated MPF (maturation promoting factor) Otherwise, PKC controls the G2/M transition by the cyclin - dependent kinase inhibitor (CKI). p21 is a key target of PKC -mediated cell cycle modulation. It may play a major role in G2/M transition via a mechanism involving blockade of cdk2/cyclinA activity, inhibition of cdc2
5 upregulation, accumulation of phosphorylation on cdc2, and suppression of cdc2/cyclin B activity. In this study, we showed that the protein levels of the cyclin - dependent kinase inhibitor p21 rapidly increased in the one - cell staged fertilized eggs treated with PMA. The cleavage from one - cell stage of the fertilized eggs into two - cell stage was inhibited.
Protein kinase B is newly found as a serine/threonine protein kinase , It could have widely spread functions in cell cycle regulation. The activation of protein kinase B/AKT is thought to be a critical step in the phosphoinositide 3 -kinase pathway which regulates cell growth and differentiation. In this study,we showed that the PDK inhibitor LY294002 regulated the cellular localization of
Materials
1. KUNMING mice were supplied from the Department of Laboratory Animals, China Medical University
2. Pregnant mare serum gonadotropin(PMSG) and human chorionic gona-dotropin ( hCG) were obtained from Tianjin Huafu Biological Products Research Institute and Shanghai Products Research Insititute, respectively.
Anti - P21 mouse monoclonal antibody from Beijing Zhongshan Biotechnology
Anti - mouse or anti - rabbit IgG secondary antibody from Santa Cruz Biotechnology
LY294002 from Sigma Biotechnology TritonX -100 from BOEHRINGER MANNHEIN GmbH Fluorescein isothiocyanate ( FITC) conjugated anti - mouse IgG antibody was purchased from Beijing Zhongshan Biotechnology HEPES from E. Metck Darmstadt
Methods
Superovulation and collection of eggs
For superovulation, female KUNMING mice 4-5 week old were injected with pregnant mare serum gonadotropin ( PMSG) , and after 46 - 48 hours with human chorionic ginadotropin. ( hCG). One - cell fertilized eggs were collected on the next day from oviduct of females. Then one cell eggs were transferred to M16 for culture in incubator with an atmosphere of 5% CO2. According to the time point , collected G2 phase eggs .
Western Blot Analysis
We treated 200 fertilized eggs in a series of steps including lysing them in l0ul of lysis buffer(50mM Tris - Hcl pH 7. 5 , 250mM NaCl, 5mM EDTA, ImM DTT ,0. 1% Triton ,50mM sodium orthovanadate, lOOug/mg PMSF and TPCK, 50ug/ml TLCK, 1 ug/ml leupeptin pepstatin and aprotinin) , frozing them below - 70 , and then thawing them at room temperature for 10 min. We did the same steps for three times, so we could get the extraction using the steps
mentioned.
Laemmli sample buffer was added to the extracted protein,which were then boiled for 5 min. The protein samples were separated by 12% SDS - PAGE and transferred to nitrocellulose membrane. The membranes were blocked in Tris -buffered saline (TBS; 20 mM Tris -HCl, 137 mM NaCl, pH 7.6) containing 5% non - fat dried milk (TBS/milk) for 30 min at 37 . Later the Membranes were incubated for 2 h at room temperature or overnight at 4 with p21 primary antibody in TBS/milk with 0. 1% Tween 20 , followed by washing the membrane in TBSt/milk for 5 min three times. Blots were then incubated for 1 h at
引文
1. Nishizuka Y The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature 308,693-698(1984)
2. Doree, M . Control of M - phase by maturation - promoting factor. Curr. Opin. Cell Biol. 1990;2:269-273
3. Nurse, P. Universal control mechanism regulating onset of M - phase. Nature. 1990;344:503-508
4. Kishimoto, T. Cell reproduction: induction of M-phase events by cyclin dependent cdc2 kinase.Int. J. Dev. Biol. 1994;38:185 - 191
5. Gautier, J Solomon, M. J. Booher, R.N. Bazan, J. F Cdc25 is a specific tyrosine phosphatese that directly activates p34cdc2. Cell. 1991;67:197-211
6. Tchou, W. W, W. N. Rom, and K. M. Tchou-Wong Novel form of p21 protein in phorbol ester - induced G2/M arrest . J Biol Chem 1996;271:29556 - 29560
7. Niculescu, A. B. ,3rd,X. Chen,M. Smeets,L. Hengst, C. Prives, and S I. Reed Effects of p21 at both the G1/S and the G2/M cell cycle transitions : pRb is a critical determinant in blocking DNA replication and in preventing endoreduplication. Mol Cell Biol 1998;18:629-643
8. Guadagno, T. M. , and J. W. Newprot Cdk2 kinase is required for entry into mitosis as a positive regulator of Cdc2 - cyclin B kinase activity. Cell 1996;84:73 - 82
9. Pauken CM, Capco DG. The expression and stage specific localization of protein kinase C isotypes during mouse preimplanatarion development. Dev Biol, 2000,233(2):411-421
10. Raz T, Eliyahu E, Yesodi V, Shalgi R Profile of protein kinase C isozymes and their possible role in mammalian egg activation. FEBS Lett. 1998;431(3):415 - 18
11．师秀艳，付伟，赵咏梅，刘莹，刘奕，于秉治 小鼠受精卵早期发育过程中PKC对cdc2和cdc25C活性的影响 中国生物化学与分子生物学报
2002；18(6)：746-749
12. Zhou BP, Liao Y , Xia W, Spohn B, Lee MH, Hung MC. Cytoplasmic localization of p21 by Akt - induced phosphorylation in HER - 2/neu - overexpressing cells Nat Cell Biol 2001;3(3):245-52
13. Hogan B, Costantini F, Lacy E. Manipulating the mouse embryo : a laboratorv manual New York : Cold spring laboratory. 1986;89-108
14．范衡宇，佟超，李满玉，孙青原 用激光共聚焦显微术在小鼠卵母细胞中检测蛋白激酶C生物化学与生物物理进展2001；28(6)900-903
15．陈菲，于爱鸣，冯晨，付伟 蛋白激酶B在小鼠1-细胞期受精卵中活性及表达变化中国生物化学与分子生物学报2003；19(4)542-545
16. Rohlff C, Blagosklonny M V, et al Prostate cancer cell growth inhibition by tamoxifen is associated with inhibition of protein kinase C and induction of p21~(wafl/cipl). Prostate,1998;37(1):51-99
17. Toyoda M, Gotoh N, Handa H, et al. Biochem Biophys Res Commun, 1998;250(2):430 - 435
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9. Pauken CM, Capco DG. The expression and stage specific localization of protein kinase C isotypes during mouse preimplanatarion development. Dev Biol, 2000,233(2):411-421
10. Raz T, Eliyahu E, Yesodi V, Shalgi R Profile of protein kinase C isozymes and their possible role in mammalian egg activation. FEBS Lett. 1998;431(3):415 - 18
11．师秀艳，付伟，赵咏梅，刘莹，刘奕，于秉治 小鼠受精卵早期发育过程中PKC对cdc2和cdc25C活性的影响 中国生物化学与分子生物学报
2002；18(6)：746-749
12. Zhou BP, Liao Y , Xia W, Spohn B, Lee MH, Hung MC. Cytoplasmic localization of p21 by Akt - induced phosphorylation in HER - 2/neu - overexpressing cells Nat Cell Biol 2001;3(3):245-52
13. Hogan B, Costantini F, Lacy E. Manipulating the mouse embryo : a laboratorv manual New York : Cold spring laboratory. 1986;89-108
14．范衡宇，佟超，李满玉，孙青原 用激光共聚焦显微术在小鼠卵母细胞中检测蛋白激酶C生物化学与生物物理进展2001；28(6)900-903
15．陈菲，于爱鸣，冯晨，付伟 蛋白激酶B在小鼠1-细胞期受精卵中活性及表达变化中国生物化学与分子生物学报2003；19(4)542-545
16. Rohlff C, Blagosklonny M V, et al Prostate cancer cell growth inhibition by tamoxifen is associated with inhibition of protein kinase C and induction of p21~(wafl/cipl). Prostate,1998;37(1):51-99
17. Toyoda M, Gotoh N, Handa H, et al. Biochem Biophys Res Commun, 1998;250(2):430 - 435