PK15细胞凋亡过程中角蛋白中间纤维的变化

用放线菌素D诱导上皮型猪肾细胞PK15（Porcrne Kidney-15)发生凋亡,细胞在凋亡诱导过程中经历了从铺展、皱缩变圆至脱落的形态变化。凝胶电泳证明被诱导细胞的DNA发生降解,形成明显的DNAladder。免疫荧光显示凋亡过程中细胞角蛋白网状结构发生改变;应用选择性抽提结合整装电镜技术,观察到凋亡细胞中仍有中间纤维网络存在,这一现象前人未曾报道,免疫印迹反应进一步证明,凋亡细胞中部分Ⅱ型角蛋白发生降解。而Ⅰ型角蛋白则无降解现象。     

CRYOANALYTICAL STUDIES OF FREEZING DAMAGE AND RECOVERY IN FUCUS VESICULOSUS (PHAEOPHYCEAE)

Receptacles of Fucus vesiculosus L. were examined using cryoscanning electron microscopy of rapidly frozen, hydrated tissue to avoid extraction and collapse of the extracellular matrix (ECM) and provide new structural information. The pore of the conceptacle was occluded by the tightly appressed tips of the paraphyses. These were embedded in copious ECM within the conceptacle. The ECM inside the conceptacle and in the medulla differed markedly in texture and with respect to the distribution of S and Cl, as revealed by energy-dispersive X-ray microanalysis. High levels of S were restricted to the conceptacle's ECM, whereas the ECM in the medulla was always S poor and Cl rich. Receptacles frozen naturally at low tide during the winter of 1998 showed extensive damage in the medulla, and this likely accounts for the cessation of reproduction during the winter on the Maine shore. The damaged receptacles recovered and resumed growth during the spring, and they released gametes by May. Abscission of the overwintered receptacles was observed in June to July, coincident with the formation of new receptacles at the tips of newly grown portions of the fronds.     

A TEST OF TWO POSSIBLE MECHANISMS FOR PHOTOTACTIC STEERING IN VOLVOX CARTERI (CHLOROPHYCEAE)

We tested two competing models that could explain how differential flagellar activity leads to phototactic turning in spheroids of Volvox carteri f. weismannia (Powers) Iyengar. In one model, turning results from the flagella of anterior cells in the lighted and shadowed hemispheres beating at different frequencies. In a competing model, turning results from a change in beat direction in these flagella. Both models successfully explain phototactic steering under constant illumination, but they make different predictions when colonies are exposed to abrupt changes in light intensity. If turning is due to control of flagellar beat frequency, both progression and rotation rates will change in the same direction and with similar magnitudes. If spheroid turning is due to a change in flagellar beat direction, a decreased rate of progression will accompany an increased rate of rotation and vice versa. We used video-microscopy to observe the behavior of positively phototactic V. carteri spheroids exposed to 10× step-up and step-down stimuli. After a step-up stimulus, spheroids slow their progression and rotation by equal amounts. No significant changes are reported in these parameters after the reciprocal step-down response. These observations are consistent with the variable flagellar frequency model and inconsistent with the variable flagellar direction model for phototactic turning. Switching the direction of light stimulus by 180° results in reorientation of positively phototactic spheroids. The kinetics of this reorientation did not precisely match the predictions of either model.     

胞内钙与蛋白激酶C对人脐静脉内皮细胞组织因子途径抑制物及组织因子表达的影响

为探讨细胞内钙及蛋白激酶C在血管内皮细胞释放组织因子（TF）,组织因子途径抑制物（TFPI）中的作用,本文采用钙离子载入剂A23187与蛋白激酶C激动剂佛波脂（PMA）刺激原代培养人脐静脉内皮细胞（HUVEC）,观察各实验组内皮细胞冻融液中TF活性及条件培养液中TFPI活性的变化,以一期凝固法测TF活性,二期生色法测定TFPI活性。结果显示：A23187,PMA及A23187＋PMA组TF活性较对照明显升高（P＜0.001）,三组中A23187和A23187＋PMA组活性明显低于PMA组（P＜0．05）,但前两组间无显著性差异（P＞0．05）;A23187组TFPI活性与对照无明显差别,但PMA与A23187＋PMA组TFPI活性较对照及A23187组明显升高（P＜0.01）。结果提示：细胞内钙升高及PKC激活促进血管内皮细胞TF的合成,以PKC为强;PKC促进血管内皮细胞释放TFPI,而钙对此无明显作用。     

APOPTOTIC‐LIKE CELL DEATH PATHWAY IS INDUCED IN UNICELLULAR CHLOROPHYTE CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE) CELLS FOLLOWING UV IRRADIATION: DETECTION AND FUNCTIONAL ANALYSES1

Chlamydomonas reinhardtii (Ehrenberg) cells exhibited cell death process akin to that of apoptosis when exposed to ultraviolet (UV)‐C irradiation (1–100 J/m²). We observed typical hallmarks of apoptosis including cell shrinkage, associated nuclear morphological changes, flipping of phosphatidylserine, and DNA fragmentation detected by the terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling assay and oligonucleosomal DNA laddering assay. Interestingly, fluorescence imaging of DNA changes in UV‐C exposed cells, following PicoGreen staining, revealed that extra‐nuclear DNA disintegrates before that of nuclear changes, where the latter extensively diffuses out of the nuclear compartment, spreading into the whole cell and reaching the periphery of dying cells. Antibodies against a mammalian caspase‐3 shared epitopes with a protein of 28 kDa; whose pattern of expression correlated with the onset of cell death. Moreover, growth experiments indicate that spent medium recovered from UV‐C exposed cells exhibit a protective effect against cell killing of fresh cultures of C. reinhardtii cells by UV irradiation. The protective effect of UV‐spent medium is not a general growth promotional response on normal cells, but rather, is specific to UV‐exposed cells. We propose a model that C. reinhardtii cells exposed to UV elicit apoptotic‐like changes, which in turn lead to an adaptive response in neighboring cells against fresh rounds of UV exposure, thereby promoting survival of the cell population.     

人胎脾交错突细胞对T,B淋巴细胞发育影响的免疫组织化学研究

用免疫酶单重和双重染色研究人胎儿脾连续切片中交错突细胞（ＩＤＣ）与Ｔ,Ｂ淋巴细胞的定位关系及ＨＬＡ－ＤＲ表达。结果表明,Ｓ－１００阳性树突状细胞为ＩＤＣ,多数表达ＨＬＡ－ＤＲ。９－１２周的胎脾中就可见到散在分布的ＩＤＣ。１３－１６周胎脾中ＩＤＣ开始定位于白髓的Ｔ细胞集落内和周缘,及Ｂ细胞集落的周边。在上述区域ＩＤＣ常与Ｔ细胞形成ＩＤＣ－Ｔ细胞聚合体。在脾的发育过程中,ＩＤＣ不仅与Ｔ,Ｂ淋巴细胞在分布上关系密切,而且可与这两类细胞形成突起－胞体、胞体－胞体的连接。提示,胎儿脾中ＩＤＣ与Ｔ,Ｂ细胞的迁移,定位及功能成熟过程有密切联系。     

成纤维细胞表皮生长因子的胞吞和向细胞核转移研究

本文以C3H小鼠胚胎正常成纤维细胞株C3H10T1/2C18(简称NC3H10)及其由氚标记脱氧胸苷(~3H-TdR)恶性转化的细胞株(简称TC 3H 10)为对象,研究了表皮生长因子(EGF)在受体介导下的胞吞和向细胞核转移现象。~(125)I-EGF 与细胞膜上的EGF 受体结合后,胞吞和向细胞核转移呈时间依赖性。两种细胞的EGF 的胞吞和向细胞核转移率接近。但是NC 3 H 10细胞~(125)I-EGF 胞知和向细胞核转移的绝对量明显高于TC 3 H 1 0细胞(p<0.05)。SDS-PAGE 电泳表明向细胞核转移的~(125)I-EGF 是未被降解的完整分子,溶酶体抑制剂NH_4Cl 对~(125)I-EGF 向细胞核转移有明显促进作用(p<0.05)。这些结果表明受体介导下的EGF 胞吞和向细胞核转移可能与EGF 的细胞核内凋节作用密切相关。     

ISOLATION OF A SULFOQUINOVOSYL MONOACYLGLYCEROL FROM BRYOPSIS SP. (CHLOROPHYTA): IDENTIFICATION OF A FACTOR CAUSING A POSSIBLE SPECIES-SPECIFIC ECDYSIS RESPONSE IN GAMBIERDISCUS TOXICUS (DINOPHYCEAE)1,

A bioactive compound that induced ecdysis (thecal loss) in Gambierdiscus toxicus Adachi et Fukuyo (Dinophyceae) cultures was isolated from a host macroalga, Bryopsis sp. (Chlorophyta). The ecdysis factor was identified by spectroscopic methods as 1- O -palmitoyl-3- O -(6'-sulfo-α- d -quinovopyranosyl)- sn -glycerol (PSQG). From our results, PSQG induced ecdysis at a high frequency and appeared not to inhibit the growth of G. toxicus cultures. The induction of ecdysis followed a dose-dependent saturation curve from 4 to 8 μM PSQG. To determine specificity of PSQG, the effects of palmitoyl- l -α-lysophosphatidylcholine (PLPC) were observed. Because PLPC contains a lipophilic palmitoyl moiety and hydrophilic phosphatidyl choline group, the compound possesses a detergent-like amphiphathic property similar to PSQG. Our results demonstrate that PLPC induced ecdysis at a high frequency in G. toxicus cultures and generated a similar dose–response curve as PSQG. The ecdysis activity observed in PSQG and PLPC may correlate with the detergent-like amphiphathic property of both compounds. Although PLPC induced a similar ecdysis response as PSQG, PLPC appeared to inhibit the growth of G. toxicus cultures. Preliminary results on the effects of PSQG on the dinoflagellates Prorocentrum lima (Ehrenberg) Dodge and Coolia monotis Meunier did not parallel the results observed in G. toxicus. This study demonstrated the existence of a factor from Bryopsis sp. that elicited a possible species-specific ecdysis response in G. toxicus cultures. This is the first report of a compound that induced ecdysis in G. toxicus or in any dinoflagellate.     

INCOMPLETENESS OF THE COCCOSPHERE AS A POSSIBLE STIMULUS FOR COCCOLITH FORMATION IN PLEUROCHRYSIS CARTERAE (PRYMNESIOPHYCEAE)1

Pleurochrysis carterae is a marine biflagellate that produces calcified structures called coccoliths. The coccoliths are formed inside the cells and released from the latter after formation. The light dependence of calcium incorporation in this species was studied using ⁴⁵Ca as a tracer. Cells exposed to a repeating cycle of 16 h of light and 8 h of darkness incorporated calcium in extracellular coccoliths at a more or less constant rate throughout a cycle. The cells divided during the dark periods with a concomitant decrease in size. Their size increased during the light periods. Coccolith formation in cells incubated in continuous darkness was greatly reduced and finally ceased. These cells did not divide and did not increase in size. Removal of extracellular coccoliths prior to the calcium incorporation experiments stimulated coccolith formation both in dark-incubated cells and in cells exposed to a repeating light-dark cycle. Cells in the stationary phase of growth ceased producing coccoliths. Calcification could be induced in these cells by removal of the extracellular coccoliths. Based on these findings we suggest that cells of Pleurochrysis carterae tend to produce a complete cover of coccoliths and that the available cell surface is a factor controlling coccolith formation.     

INCOMPLETENESS OF THE COCCOSPHERE AS A POSSIBLE STIMULUS FOR COCCOLITH FORMATION IN PLEUROCHRYSIS CARTERAE(PRYMNESIOPHYCEAE)1

Pleurochrysis carterae is a marine biflagellate that produces calcified structures called coccoliths. The coccoliths are formed inside the cells and released from the latter after formation. The light dependence of calcium incorporation in this species was studied using⁴⁵Ca as a tracer. Cells exposed to a repeating cycle of 16 h of light and 8 h of darkness incorporated calcium in extracellular coccoliths at a more or less constant rate throughout a cycle. The cells divided during the dark periods with a concomitant decrease in size. Their size increased during the light periods Coccolith formation in cells incubated in continuous darkness was greatly reduced and finally ceased. These cells did not divide and did not increase in size. Removal of extracellular coccoliths prior to the calcium incorporation experiments stimulated coccolith formation both in dark-incubated cells and in cells exposed to a repeating light-dark cycle. Cells in the stationary phase of growth ceased producing coccoliths. Calcification could be induced in these cells by removal of the extracellular coccoliths. Based on these findings we suggest that cells of Pleurochrysis carterae tend to produce a complete cover of coccoliths and that the available cell surface is a factor controlling coccolith formation.     

脓毒血症早晚期肝细胞核被膜NTPase活性及poly(A)+mRNA核浆转运的变化

目的和方法 :在盲肠结扎穿孔脓毒血症大鼠模型上 ,研究早晚期脓毒血症肝细胞核被膜核苷三磷酸酶 (NT Pase)活性及 poly(A) mRNA核浆转运的变化。结果 :脓毒血症早期NTPase活性增加而晚期活性降低 ,即早期Vmax与对照组比增加 6 2 % (ATP ,P <0 .0 5 )和 18% (GTP ,P <0 .0 5 ) ;晚期Vmax下降 2 6 % (ATP ,P <0 .0 5 )和5 6 % (GTP ,P <0 .0 5 )。脓毒血症仅晚期NTPase底物亲和力降低 ,即晚期Km与对照组比分别升高 2 6 % (ATP ,P<0 .0 5 )和 37% (GTP ,P <0 .0 5 )。脓毒血症早期 poly(A) mRNA核浆转运速率增加而晚期降低 ,即早期组 10min转运速率较对照组增加 2 7% (ATP ,P <0 .0 5 )和 30 % (GTP ,P <0 .0 5 ) ;晚期组降低 2 1% (P <0 .0 1)和 35 % (P <0 .0 1)。结论 :脓毒血症肝细胞核膜NTPase活性呈早期升高、晚期下降的双相变化与肝细胞核被膜 poly(A) mR NA核浆转运速率的早、晚期变化相平行 ,同时与脓毒血症血糖浓度的早、晚期变化相关。本实验在一定程度上解释了脓毒血症早晚期代谢双相变化发生的分子机理     

多巴胺对血管平滑肌细胞钙激活钾通道的影响及其机制

目的：研究多巴胺对血管平滑肌细胞大电流、钙激活钾（ＢＫｃａ）通道的影响及其信息传递机制。方法用膜片钳细胞贴附式技术,记录细胞能液内灌流多巴胺受体激动剂、阻断剂以及第二信使及相关蛋白激酶拮机剂对猪冠状动脉血管平滑肌细胞ＢＫｃａ 爱道活动的影响。结果：多巴胺增加ＢＫｃａ通道活性（Ｐ〈０．０１）,并可被ＣＡ－１受体阻断剂ＳＣＨ２３３９０完全阻断,但不受β２受体阻断剂普藉洛尔的影响。腺苷酸环化酶抑制剂ＳＯ     

A TEST OF THE GLACIAL REFUGIUM HYPOTHESIS USING PATTERNS OF MITOCHONDRIAL AND NUCLEAR DNA SEQUENCE VARIATION IN ROCK PTARMIGAN (LAGOPUS MUTUS)

The glacial refugium hypothesis (GRH) proposes that glaciers promoted differentiation and generation of intraspecific diversity by isolating populations in ice-free refugia. We tested three predictions of this hypothesis for the evolutionary divergence of rock ptarmigan (Lagopus mutus) during the Wisconsin glaciation of the late Pleistocene. To do this, we examined subspecies distributions, population genetic structure, and phylogenetic relationships in 26 populations across North America and the Bering Sea region. First, we analyzed sequence variation in the mitochondrial control region, in a nuclear intron (Gapdh), and in an internal transcribed spacer (ITS1). Control region sequences of 154 rock ptarmigan revealed strong population and phylogeographic structure. Variation in intron sequences of 114 rock ptarmigan also revealed significant population structure compatible with results for the control region. Rock ptarmigan were invariant for ITS1. Second, we show that five known Nearctic refugia and an Icelandic refugium are concordant with the current distribution of morphologically distinct subspecies; five of these six refugia are geographically concordant with the distribution of closely related control region haplotypes. Third, our estimates of the time since phylogenetic lineages diverged predated the last glacial maximum for all but two lineages. In addition, all lines of evidence suggest that two unknown refugia in the Bering Sea region supported rock ptarmigan during the Wisconsin glaciation. Overall, our results are most consistent with the hypothesis that isolated populations of rock ptarmigan diverged in multiple refugia during the Wisconsin and that geographic variation reflects patterns of recolonization of the Nearctic after the ice receded. The GRH may therefore offer the most plausible explanation for similar biogeographic patterns in a variety of Nearctic vertebrates.