Markedly altered membrane transport and intracellular binding of vincristine in multidrug-resistant Chinese hamster cells selected for resistance to vinca alkaloids

Studies of a multidrug-resistant variant (DC-3F/VCRd-5L) of Chinese hamster lung cells selected for resistance to vinca alkaloids revealed marked alterations in transport and intracellular binding of [3H]vincristine compared to parental DC-3F cells. Influx of [3H]vincristine in DC-3F cells appears to be an equilibrating, but mediated, process. Although saturation kinetics for [3H]vincristine influx were not demonstrated, an extremely high temperature-dependence (Q10 27-37 degrees C = 5-6) and trans-inhibition of influx following preloading of cells with nonradioactive vincristine argue in favor of a carrier-mediated process. Efflux of [3H]vincristine from parental cells conformed to first-order kinetics (t1/2 37 degrees = 3.6 +/- 0.4) and exhibited a lower temperature-dependence (Q10 27-37 degrees C = 3-3.5) than influx. In variant vs. parental cells, influx of [3H]vincristine was reduced 24-fold and efflux was increased two-fold, accounting for the large (approximately 48-fold) reduction in steady-state level of exchangeable drug accumulating in variant cells. Otherwise, transport of [3H]vincristine in these cells showed characteristics similar to parental DC-3F cells. Also, the rate and amount of intracellular binding of [3H]vincristine in variant cells was almost 40-fold lower than in parental cells. These alterations in influx and efflux of [3H]vincristine and its intracellular binding appear to account, at least to a major extent, for the high level of resistance (2,750-fold) of this variant to vinca alkaloids. In contrast, cross-resistance of this variant to daunomycin (178-fold) could be explained only minimally by a transport alteration. Only a two-fold increase in efflux of [3H]daunomycin was demonstrated in variant vs. parental cells along with some decrease in intracellular binding. Influx of [3H]daunomycin was unaltered. In view of these results, we conclude that these two agents most likely do not share the same route for entry in these cells but might share the same efflux route.     

Carbon dioxide enhances the development of the ethylene forming enzyme in tobacco leaf discs

Since CO₂ is known to stimulate ethylene production by promoting the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene, the effect of CO₂ on the activity and the development of the ethylene forming enzyme (EFE) was studied in tobacco (Nicotiana tabacum L. cv Havana 425 and Xanthi) leaf discs. In addition to previous observations that EFE activity is dependent on CO₂ concentration and is saturable with 2% CO₂, present data show two saturation curves at 2% and 10% CO₂. Promotion of EFE development was dependent also on CO₂ concentration (saturated at 2% CO₂) and duration (maximum at 24 in the dark), and was abolished by 20 micromolar cycloheximide. Application of exogenous ethylene (20 microliters per liter) or light treatment further increased the CO₂-enhanced development of EFE, implying that these two factors can also affect EFE development via interaction with CO₂. The results suggest that CO₂ exerts its stimulatory effect on the conversion of ACC to ethylene by enhancing not only the activity but also the synthesis of EFE in leaf discs.     

Identification of the pleiotropic sacQ gene of Bacillus subtilis.

The sacQ gene of Bacillus subtilis, a pleiotropic gene affecting the expression of a number of secreted gene products, has been identified as a small 46-amino-acid polypeptide. The increased expression of this polypeptide in strains carrying the sacQ36 allele, or in strains carrying the sacQ gene on a high copy plasmid, appears to be responsible for the phenotype of higher levels of proteases seen in these strains. A deletion of the sacQ gene had no apparent phenotype, indicating that it is not an essential gene.     

荧光显微术在当代植物细胞生物学研究中的应用

自从八十年前第一次在显微镜下观察到生物组织经紫外线照射后发射荧光的现象以来,荧光显微术不断获得进步,现已发展成细胞生物学中一个重要的研究手段。高度的灵敏性和专一性、制样与观察程序的简便、尤其是适宜于活细胞研究等特点,是它所具有的独特长处。荧光显微术特别是免疫荧光技术在现代医学生物学研究中的应用是一个     

Specific binding of three neurotoxins with phospholipase A2 activity to synaptosomal membrane preparations from the guinea pig brain

Snake presynaptic toxins such as crotoxin, β-bungarotoxin and taipoxin block neuromuscular transmission through inhibiting the release of acetylcholine by their phospholipase A₂ activities. On the other hand, many other phospholipase A₂s show little neurotoxicity. It is likely that the difference lies in whether high affinity binding to nerve cell membranes exists or not. To test this idea, crotoxin, β-bungarotoxin and taipoxin were first radioactively labeled with Na(¹²⁵I) without loss of their neurotoxicity. Using the radioactive toxins we have found that each of the three showed specific binding to synaptosomal membranes from guinea pig brain. In contrast, we could not detect specific binding of a non-neurotoxic pancreatic phospholipase A₂. Crotoxin and taipoxin, but not β-bungarotoxin, also bound specifically to membrane preparation from other tissues. The binding of each toxin was not greatly affected by the other two toxins. The photoaffinity labeling technique has been used to obtain further information about the components which bind crotoxin. For this purpose, (¹²⁵I) crotoxin was derivatized with N-hydroxysuccinimidyl-4-azidobenzoate. Autoradiographic analysis of the membranes following photoirradiation in the presence of the modified crotoxin revealed that an 85K dalton component was preferentially covalently conjugated with the crotoxin analogue in a specific manner.     

蚕豆叶肉原生质体电融合的研究

本文研究了蚕豆叶肉原生质体经透明质酸酶、核糖核酸酶、神经氨酸酶、碱性磷酸酶、胰蛋白酶、脂肪酶六种水解酶和SDS、Triton X-100、CTMAB三种表面活性剂以及秋水仙素、细胞松驰素B处理后的电融合过程。结果表明:胰蛋白酶处理后的原生质体融合率明显下降;碱性磷酸酶、脂肪酶以及核糖核酸酶、透明质酸酶、神经氨酸酶处理的原生质体电融合率均有不同程度的上升。Triton X-100和CTMAB促进原生质体的电融合,但较高浓度(0.01%)的SDS起抑制作用。秋水仙素和细胞松驰素B处理的原生质体其电融合率有较大幅度的增高。     

B淋巴细胞在多向造血祖细胞生长中的地位

小鼠骨髓细胞在体外培养中,加入用流式自由电泳法分离所得的高纯度正常B淋巴细胞,可使多向祖细胞(CFU-mix)集落增加至5倍;加入小鼠B淋巴瘤细胞株的条件培基(M_(12.4.1)-CM)时,CFU-mix数也可增加至4倍。单集落形态学分析结果表明M_(12.4.1)-CM可加强CFU-GEMm及p-BFU-E等早期造血祖细胞的增殖与分化。小鼠高纯度B细胞样品在体外培养中加入1000 rad照射的骨髓细胞可出现CFU-mix集落,如果再加入适量的小鼠肺条件培基,则CFU-mix数量比对照大15倍,其集落性质为CFU-GEMm,GMm及p-BFU-E。在此培养中加不同稀释度抗小鼠IgM血清,结果CFU-mix的产率与抗IgM血清的浓度成直线反比关系,当加入1:10抗小鼠IgM血清时,CFU-mix为0。作者假设在一定培养条件下,IgM阳性的部分B细胞可返祖转化为CFU-mix。     

Effect of alkylation of tryptophan residues on the enzymatic and pharmacological properties of snake venom phospholipase A2

Snake venom phospholipases A2 show a remarkable degree of amino acid sequence homology yet differ markedly in enzymatic and pharmacological activities. The basic phospholipase A2 from Naja nigricollis venom has much greater lethal potency, cardiotoxicity, hemolytic and anticoagulant activity than the acidic or neutral enzymes from Naja naja atra or Hemachatus haemachatus venoms, respectively, even though it has lower enzymatic activity than the latter two enzymes. Previous studies in which we selectively modified lysine and free carboxyl groups suggested that the pharmacological and enzymatic active sites are not identical. Tryptophan residues have been suggested as being involved in substrate binding although some phospholipases have no tryptophan. We investigated the effect of alkylating the tryptophans in N. nigricollis, N. n. atra, and H. haemachatus phospholipases A2 with 2-hydroxy-5-nitrobenzyl bromide. Chemical modification caused decreases in enzymatic activity, although the extent of inactivation varied with the enzyme and with the substrate (lecithin micelles, egg yolk, heart homogenates). The specificity of the enzymes for individual phospholipid substrates was not affected. Alkylation of the tryptophans also caused decreases in lethal, hemolytic, anticoagulant, and cardiotoxic potencies, which were similar to the extents of decrease in enzymatic activity. Our results suggest that tryptophans are not specifically associated with either the enzymatic or the pharmacological active site nor are essential for either activity.     

The synergistic lethal interaction of cis-diamminedichloroplatinum and natural nucleosides is related to increased DNA cross-links

Human tumor cells were treated in vitro with combinations of cis- or trans-dichlodiammineplatinum (DDP) and natural nucleosides (thymidine, uridine, cytidine and adenosine). Effects were measured by inhibition of colony-formation (cell survival) and DNA alkaline elution (DNA cross-links). No increments in cell lethality or DNA cross-links were elicited by any combination of trans-DDP and nucleosides. In contrast, every combination of cis-DDP and nucleoside was eminently synergistic with 5- and 10-fold increases in cell lethality over the predicted sum of each agent alone. These increments in cell kill correlated linearly with increases in DNA crosslinks suggesting that the nucleosides interact with cis-DDP to enhance its cytotoxic crosslinking mode of action.     

Suppression of defective-sporulation phenotypes by mutations in the major sigma factor gene (rpoD) of Bacillus subtilis

Summary Mutations (crsA47 and crsA4) in the major sigma factor gene (rpoD) of Bacillus subtilis RNA polymerase have been found to be powerful intergenic suppressors of spoOB, spoOE, spoOF, spoOK and spoIIG mutations. The crsA47 suppressor restores sporulation of spoOE, spoOF, spoOK and spoIIG mutants to levels near those of wild type bacteria and substantially improves the sporulation of a spoOB strain. The crsA mutations are shown to prevent the induction by aliphatic alcohols of SpoO phenocopies in wild type B. subtilis cells.