大鼠非致瘤四倍体细胞系的建立及细胞遗传学和酶学特征

从大鼠自然诱发的肉瘤细胞中,我们建立了一个四倍体细胞系(4n=84),命名为RC(ratcell)。它具有典型的成纤维细胞外形,能在玻璃表面贴壁生长,但不能生长在琼脂半固体培养基中。该细胞在含15％小牛血清的RPMI 1640培养基中生长良好,至今已连续繁殖112世代,细胞群体倍增时间约为15小时。染色体G-带分析表明,RC为整四倍体细胞,它的1条X染色体在第32至34区为均染区。RC细胞核仁组织者(NORs)活性显然比大鼠二倍体细胞NORs活性的加倍还高(P<0.001)。这个具有非常高NORs活性的RC细胞系对于研究细胞18S+28S rRNA基因转录活性的调控、基因表达与基因剂量关系有一定的意义。RC细胞还有异常高的磷酸酯酶活性,而且它的同工酶谱也与大鼠肌肉细胞明显不同。体内接种实验和扫描电镜的观察表明,RC是非致瘤细胞。RC细胞各号染色体的C-带图样与大鼠二倍体细胞无明显的差异。     

鸡胚视网膜发育中花生凝集素结合糖蛋白的图谱

我们应用微量(1—3微克蛋白)等电点聚焦和SDS双向电泳对鸡胚视网膜发育的第4天(E_4)到第16天(E_(16))总蛋白图谱进行分析,并结合印迹的PNA免疫酶标反应,分析了总蛋白中与PNA结合的糖蛋白图谱的变化。E_4时没有看到PNA结合糖蛋白的任何阳性反应的斑点,E_6时这种糖蛋白的种数突然增加,约占总蛋白数的20%。新的糖蛋白增加的速率在E_6时最快,存其后的发育阶段(E_8—E_(16))慢于总蛋白增加的速率。这种糖蛋白大都是等电点在中性pH范围内,中等大小分子量的糖肽。视神经的总蛋白中含PNA结合糖蛋白数的比例高于同一发育年龄视网膜中的比例,这说明糖蛋白可能主要存在于质膜上。视网膜的总蛋白图谱相似于前脑的,但与视顶盖的更相近。本文对这种能与PNA结合,即含有D-半乳糖残基的糖蛋白在视网膜发育中的意义进行了讨论。     

三七中达马烷型皂甙的热不稳定性及酸水解产物

前人已证明人参和三七中富含的达马烷型人参皂甙在通常酸性水解下甙元即发生变化,而在弱酸(如50％醋酸,0.1N盐酸)条件下则形成次级皂甙。本文报道人参甙(ginseno-sides)和三七甙(notoginsenosides)的水溶液在水浴上加热亦分别形成相应的C-20位去糖基的次级皂甙。联系到人参和三七均有在蒸煮加工后C-20位去糖基皂甙收率增大的趋势,似可认为人参和三七中的这类皂甙有相当一部分是在生药的加工泡制以及提取过程中形成的次级皂甙,而不一定是植物体的原生成分。将人参甙Rb_1单体以酸水解,不仅得到主产物人参二醇(3),还分离到异去氢原人参二醇(5)、达马烷-20(22)-烯-3β,12β,26-三醇(6)、20(R)-达马烷-3β,12β,20,25-四醇(7)以及20(S)-和20(R)-原人参二醇(1、2)的混合物,从而认为这些微量成分与人参二醇一样均为达马烷型人参皂甙在酸性水解条件下C-20位糖基断裂后由真甙元的侧链转化形成的工作产物。     

重楼属植物的免疫血清学研究

以海南重楼(Paris dunniana),花叶重楼(P.marmorata),多叶重楼(P.polyphylla),凌云重楼(P.cronquistii),五指莲(P.axialis),滇重楼(Paris polyphylla var.yunnanensis)等六种重楼属植物的根茎为材料,提取分离其球蛋白组份作为抗原,免疫家兔得到相应的抗血清。通过免疫双扩散,聚丙烯酰胺凝胶电泳、免疫电泳、免疫吸收试验以及酶标免疫等方法,研究了重楼属上述六个种和变种的血清学行为以及他们之间的相互关系。在此基础上,用不加权算术平均对群法(UPGMA)经成聚运算,得到基于平均吸收相似系数(Sabs.mean.)和Jaccard's结合系数的两种表相图,这两种表相图的结果是一致的。花叶重楼与多叶重楼有较大的血清反应相似性,五指莲与滇重楼有最大的血清反应相似性。多型种P.polyphylla在形态上的较大变异与血清反应分析的结果是一致的,而且血清反应相似性分析支持了从形态分析得出的假设:Paris dunniana在重楼属的系统发生中是一比较原始的种。     

~(60)CO-γ射线诱发的德国蜚蠊精母细胞中心体畸变

德国蜚蠊经不同剂量的~(60)Co-γ射线照射,以光镜和电镜观察精母细胞巨大中心体结构和数目的变化,发现:γ射线可诱发各种类型中心体畸变,如棒状、断裂、不分离、超数和微小中心体等;畸变率随剂量增加而升高;畸变类型和辐射剂量有密切关系,低剂量(500—1000r)诱发较多的棒状中心体和中心体断裂、中等剂量(2000r)诱发较多的超数中心体和中心体不分离、高剂量(5000r)诱发较多的微小中心体,看来低剂量γ射线主要影响中心体结构,高剂量则对中心体的生长发育有明显的阻滞作用。本文还讨论了中心体畸变的可能产生机制及在遗传毒理研究中应用的前景。     

The hemolymph coagulation system in invertebrate animals

A hemocyte lysate from horseshoe crab produced a gel, when exposed to Gram-negative bacterial endotoxins. This gelation reaction of the lysate, so-called Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins. Recent biochemical studies on the principle of Limulus test indicate that the hemocytes contain several serine protease zymogens, which constitute a coagulation cascade triggered by endotoxins, and that there is a (1 3)--d-glucan-mediated coagulation pathway which also results in the formation of gel. Up to now, six protein components, designated coagulogen, proclotting enzyme, factor B, factor C, factor G and anti-LPS factor, all of which are closely associated with the endotoxin-mediated coagulation pathway, have been purified and biochemically characterized. Among these components, the complete amino acid sequences of coagulogens isolated from one American and three Asian species of horseshoe crabs have been established. Moreover, the reconstitution experiment using the isolated clotting factors, C, B, proclotting enzyme and coagulogen in the presence of endotoxin, leads to the formation of coagulin get. Based on these results, we propose here a mechanism for the Limulus coagulation cascade.     

Distribution and biosynthesis of aminopeptidase N and dipeptidyl aminopeptidase IV in rat small intestine

The regional, cellular and subcellular distribution patterns of aminopeptidase N and dipeptidyl aminopeptidase IV were examined in rat small intestine. Aminopeptidase N of brush border membrane had maximal activity in the upper and middle intestine, while dipeptidyl aminopeptidase IV had a more uniform distribution profile with relatively high activity in the ileum. Along the villus and crypt cell gradient, the activity of both enzymes was maximally expressed in the mid-villus cells. However there was substantial dipeptidyl aminopeptidase IV activity in the crypt cells. Both enzymes were primarily associated with brush border membranes in all segments, however, in the proximal intestine, a significant amount of dipeptidyl aminopeptidase IV activity was associated with the cytosol fraction. The cytosol and brush border membrane forms of dipeptidyl aminopeptidase IV were immunologically identical and had the same electrophoretic mobility on disc gels. In contrast, the soluble and brush border membrane-bound forms of aminopeptidase N were immunologically distinct. When the total amount of aminopeptidase N and dipeptidyl aminopeptidase IV was determined by competitive radioimmunoassay, there were no regional or cellular differences in specific activity (enzyme activity/mg of enzyme protein) of either enzyme in brush border membrane and homogenate. The specific activity of both enzymes in a purified Golgi membrane fraction as measured by radioimmunoassay was about half that of the brush border membrane fraction. These results suggest that (1) aminopeptidase N and dipeptidyl aminopeptidase IV have different regional, cellular and subcellular distribution patterns; (2) there are enzymatically inactive forms of both enzymes present in a constant proportion to active molecules and that (3) a two-fold activation of precursor enzyme forms occurs during transfer from the Golgi membranes to the brush border membranes.     

Preliminary crystallographic studies of lobster D-glyceraldehyde-3-phosphate dehydrogenase and the modified enzyme carrying the fluorescent derivative

When the active-site carboxymethylated D-glyceraldehyde-3-phosphate dehydrogenase is irradiated with ultraviolet light in the presence of NAD+, a fluorescent NAD derivative that is covalently linked to the enzyme is obtained. A preliminary crystallographic study of this fluorescent derivative, as well as of the native and the carboxymethylated enzymes from Palinurus versicolor, showed that they are isomorphous and belong to space group C2 as reported for the native enzyme from Palinurus vulgaris. The three forms of the enzyme, although they have identical unit cell parameters, differ considerably in their diffraction patterns, indicating marked differences in conformation in spite of the fact that they differ chemically only in a restricted region around the active site.     

Synthesis of ribonucleic acid in normal bone in vitro

1. The incorporation of [2-(14)C]uridine into nucleic acids of bone cells was studied in rat and pig trabecular-bone fragments surviving in vitro. 2. The rapid uptake of uridine into trichloroacetic acid-soluble material, and its subsequent incorporation into a crude nucleic acid fraction of bone or purified RNA extracted from isolated bone cells, was proportional to uridine concentration in the incubation medium over a range 0.5-20.0mum. 3. During continued exposure to radioactive uridine, bulk RNA became labelled in a curvilinear fashion. Radioactivity rapidly entered nuclear RNA, which approached its maximum specific activity by 2hr. of incubation; cytoplasmic RNA, and particularly microsomal RNA, was more slowly labelled. The kinetics of labelling and rapid decline of the nuclear/microsomal specific activity ratio were consistent with a precursor-product relationship. 4. Bulk RNA preparations were resolved by zonal centrifugation in sucrose density gradients into components with approximate sedimentation coefficients 28s, 18s and 4s. 5. Rapidly labelled RNA, predominantly nuclear in location, demonstrated a polydisperse sedimentation pattern that did not conform to the major types of stable cellular RNA. Material of highest specific activity, sedimenting in the 4-18s region and insoluble in 10% (w/v) sodium chloride, rapidly achieved its maximum activity during continued exposure to radioactive precursor and decayed equally rapidly during ;chase' incubation, exhibiting an average half-life of 4.3hr. 6. Ribosomal 28s and 18s RNA were of lower specific activity, which increased linearly for at least 6hr. in the continued presence of radioactive uridine. There was persistent but variable incorporation into ribosomal RNA during ;chase' incubation despite rapid decline in total radioactivity of the acid-soluble pool containing RNA precursors.     

Acid hydrolases of the rat uterus in relation to pregnancy, post-partum involution and collagen breakdown

1. The rat uterus contains acid cathepsin, beta-glucuronidase, beta-galactosidase, acid phosphatase and deoxyribonuclease II at concentrations comparable with those found in liver. Two non-hydrolytic uterine enzymes, cytochrome c oxidase and aspartate aminotransferase, display only 2-6% of the activity found in liver. 2. The concentrations of acid cathepsin and beta-glucuronidase are significantly decreased in pregnancy and increase 3-4-fold during post-partum involution. 3. The concentrations of beta-galactosidase and acid phosphatase are not decreased in pregnancy and increase only 2-3-fold during involution. 4. The concentrations of these four acid hydrolases increase linearly during the first 4 days post partum and reach their peak values at the same time that wet weight and collagen content fall to their lowest point. 5. The concentration of deoxyribonuclease is depressed in pregnancy but does not rise above normal in the post-partum period. 6. Only a small proportion of each hydrolytic activity can be isolated in the mitochondrial-lysosomal fraction of sucrose homogenates of the rat uterus. This proportion increases during involution. However, the extensive mitochondrial rupture occurring during homogenization indicates that the technique is probably too harsh to obtain a true measure of the proportion of lysosomes present in the intact tissue. 7. There are no significant changes in either the concentration or subcellular distribution of the five acid hydrolases in the livers of the experimental rats during pregnancy or involution. In each case the largest proportion of the activity is found in the mitochondrial-lysosomal fraction of liver homogenates. 8. The results are interpreted in terms of the lysosomal theory of intracellular digestion.