The Different Behavior of Diphtheria Toxin, Modeccin and Ricin in HeLa Cells Infected with Trypanosoma cruzi

ABSTRACT. We have studied the action of diphtheria toxin, modeccin and ricin on HeLa cells infected by Trypanosoma cruzi . Parasitized HeLa cells were resistant to diphtheria toxin and modeccin, whereas non-parasitized cells from the same cultures and control cultures showed cytopathological alterations. Protein synthesis, assayed by the incorporation of labelled methionine, diminished in toxin-treated control cultures but remained unaltered in the infected ones, compared to synthesis by untreated infected cells. Ricin, on the other hand, is a toxin that enters the cytoplasm by endocytosis. It has greater cytopathological effects in parasitized cells than in non-parasitized ones from the same cultures or uninfected control cells. Protein synthesis was inhibited in infected cultures treated with ricin.     

Partitioning of the Golgi Apparatus during Mitosis in Living HeLa Cells

The Golgi apparatus of HeLa cells was fluorescently tagged with a green fluorescent protein (GFP), localized by attachment to the NH₂-terminal retention signal of N-acetylglucosaminyltransferase I (NAGT I). The location was confirmed by immunogold and immunofluorescence microscopy using a variety of Golgi markers. The behavior of the fluorescent Golgi marker was observed in fixed and living mitotic cells using confocal microscopy. By metaphase, cells contained a constant number of Golgi fragments dispersed throughout the cytoplasm. Conventional and cryoimmunoelectron microscopy showed that the NAGT I–GFP chimera (NAGFP)-positive fragments were tubulo-vesicular mitotic Golgi clusters. Mitotic conversion of Golgi stacks into mitotic clusters had surprisingly little effect on the polarity of Golgi membrane markers at the level of fluorescence microscopy. In living cells, there was little self-directed movement of the clusters in the period from metaphase to early telophase. In late telophase, the Golgi ribbon began to be reformed by a dynamic process of congregation and tubulation of the newly inherited Golgi fragments. The accuracy of partitioning the NAGFP-tagged Golgi was found to exceed that expected for a stochastic partitioning process. The results provide direct evidence for mitotic clusters as the unit of partitioning and suggest that precise regulation of the number, position, and compartmentation of mitotic membranes is a critical feature for the ordered inheritance of the Golgi apparatus.     

Repair Replication of HeLa Cell Deoxyribonucleic Acid in Cells Infected with Newcastle Disease Virus or Mengovirus or Treated with Puromycin

We examined repair replication of HeLa cell deoxyribonucleic acid (DNA) in cells infected with mengovirus or Newcastle disease virus or treated with puromycin. Cellular DNA was damaged by ultraviolet light and then pulse-labeled with (3)H-thymidine. Autoradiographic analysis of non-S-phase DNA synthesis (repair replication) showed that there was no inhibition of this process at a time when overall cellular DNA synthesis was severely inhibited by either virus infection or puromycin treatment.     

Inhibition of Cleavage of Large Poliovirus-Specific Precursor Proteins in Infected HeLa Cells by Inhibitors of Proteolytic Enzymes

Inhibitors of chymotrypsin interfere with the post-translational cleavage of large poliovirus-specific polypeptides in the molecular weight range of 100,000 to 250,000 in infected HeLa cells.     

Cells Persistently Infected with Newcastle Disease Virus: II. Ribonucleic Acid and Protein Synthesis in Cells Infected with Mutants Isolated from Persistently Infected L Cells

A comparison of the replication patterns in L cells and in chick embryo (CE) cell cultures was carried out with the Herts strain of Newcastle disease virus (NDV(o)) and with a mutant (NDV(pi)) isolated from persistently infected L cells. A significant amount of virus progeny, 11 plaque-forming units (PFU)/cell, was synthesized in L cells infected with NDV(o), but the infectivity remained cell-associated and disappeared without being detectable in the medium. In contrast, in L cells infected with NDV(pi), progeny virus (30 PFU/cell) was released efficiently upon maturation. It is suggested that the term "covert" rather than "abortive" be used to describe the infection of L cells with NDV(o). In both L and CE cells, the latent period of NDV(pi) was 2 to 4 hr longer than for NDV(o). The delay in synthesis of viral ribonucleic acid (RNA) in the case of NDV(pi) coincided with the delay in the inhibition of host RNA and protein synthesis. Although both NDV(o) and NDV(pi) produced more progeny and more severe cell damage in CE cells than in L cells, the shut-off of host functions was significantly less efficient in CE cells than in L cells. Paradoxically, no detectable interferon was produced in CE cells by either of the viruses, whereas in L cells most of the interferon appeared in the medium after more than 90% of host protein synthesis was inhibited. These results suggest that the absence of induction of interferon synthesis in CE cells infected with NDV is not related to the general shut-off of host cell synthetic mechanisms but rather to the failure of some more specific event to occur. In spite of the fact that NDV(pi) RNA synthesis commenced 2 to 4 hr later than that of NDV(o), interferon was first detected in the medium 8 hr after infection with both viruses. This finding suggests that there is no relation between viral RNA synthesis and the induction of interferon synthesis.     

冰片提取物对沙眼衣原体感染HeLa细胞CT703及CT259表达的影响

目的:探究冰片提取物对沙眼衣原体感染后的He La细胞模型CT703和CT259表达的影响。方法:将成功建立的40例感染L2血清型沙眼衣原体的人宫颈癌上皮(He La)细胞模型随机分成A、B两组,分别添加冰片提取物和等剂量生理盐水。观察感染后He La细胞模型的包涵体数目及大小、RNA抽提结果完整性以及CT70与CT259的表达变化。结果:染色后的40例受沙眼衣原体感染的He La细胞模型体内均发现包涵体,在同一时间点B组细胞内包涵体比A组大,且数目比A组多(P0.05);两组提取的总RNA的OD值均在1.8~2.0之间,通过RNA凝胶电泳结果可清楚发现28S、18S及5S条带;同一时间点CT259和CT703扩增产物的平均灰度值比较,A组感染后He La细胞模型样本基因表达量低于B组,差异具有统计学意义(P0.05)。结论:冰片提取物能够有效降低经沙眼衣原体感染的He La细胞模型中CT703与CT259基因表达量。     

Intranuclear Inclusions in Leaf Mesophyll Cells of Davidia involucrata

The ultrastructure of intranuclear inclusions in leaf mesophyll cells of Davidia involucrata was investigated with electron microscopy. Intranuclear inclusions occur generally in the cells of young and mature leaves. They consist of numerous bundles aggregated by several fibres (diameter about 10 nm), sometimes a few of bundles turn to tubules enveloped by fibres. Authors suggested that it is a new subtype (F2) of intranuclear fibrillar inclusion.     

HeLa细胞中与组织型转谷氨酰胺酶相互作用蛋白质的筛选

组织型转谷氨酰胺酶 (tissuetransglutaminase ,tTG ,TGII)是转谷氨酰胺酶家族成员之一 ,多数细胞凋亡过程中均有tTG表达水平的升高。为研究tTG在细胞凋亡过程中发挥作用的机制 ,利用Gal 4酵母双杂交系统筛选了HeLa细胞中与tTG相互作用的蛋白质 ,获得了 17个阳性酵母克隆。序列测定显示其中 1个克隆所含cDNA序列编码TIA 1相关蛋白 (TIA 1 relatedprotein ,TIAR )C端 12 9个氨基酸残基序列 ,GST下拉 (pull down)实验也证实tTG与TIAR能相互作用 ,而且这种相互作用需要Ca2 参与作用。这些结果提示tTG可能通过其Ca2 依赖的转谷氨酰胺活性对TIAR进行修饰从而影响TIAR的功能 ,可能在细胞凋亡中发挥着一定的作用。     

Electrolyte Metabolism in HeLa Cells

Methods have been developed to study cellular Na, K, and Cl concentrations in HeLa cells. Cell [Na] and [K] are functions of the age of the culture. As the culture grows [K], expressed in mmols/liter cell H₂O, rises from an initial value of 121 to a peak of 206 at about 4 days, and thereafter falls until it has almost returned to the initial value by the 9th day. [Na] falls as [K] rises, but there is no fixed relationship between the cellular concentrations of the two cations. There is, however, a correlation between generation time and cellular [K]. Measurements of net K uptake and net Na extrusion were carried out during 1 hour incubation at 37°C of low K cells. Both net K uptake and net Na extrusion took place against chemical concentration gradients, so that at least one transport system must be active; if the Cl distribution is passive both net K uptake and net Na extrusion are active. Studies with inhibitors of respiration and glycolysis lead to the conclusion that respiration is not required for these net transports, which appear to derive their energy from glycolytic sources.     

Mitochondrial Ribosomes in HeLa Cells

HeLa cell mitochondria contain 60S ribosomes which seem to consist of subunits of 45S and 35S particles. The 16S and 12S RNA components are coded by mitochondrial DNA.     

Occurrence of the Crabtree effect in HeLa cells

The occurrence of a Crabtree effect in HeLa cells was detected. Some properties of pyruvate kinase (PK) were also evaluated. Hexose phosphate, triose-phosphate and phosphoenolpyruvate (PEP) significantly decreased the oxygen consumption of digitonin-permeabilized HeLa cells, which were oxidizing succinate. The Crabtree effect promoted by PEP was concentration-dependent and was lowered by an increase of ADP concentration, suggesting a participation of PK. The dependence of fructose-1,6-bisphosphate (FDP) by HeLa cell PK was observed. The PK of HeLa cells was inhibited by L -alanine only in the absence of FDP, while in the presence of the metabolite, an increase in the activity was observed. PK was also inhibited in the presence of L -histidine and L -leucine, while L -serine promoted activation. L -Cysteine and L -phenylalanine also inhibited the PK of HeLa cells. This, together with the sigmoidal character in relation to substrate concentration, suggests the presence of the K-type of PK in HeLa cells. © 1998 John Wiley & Sons, Ltd.     

Biosynthesis of Arginine in L Cells Infected with Chlamydiae

Three members of the genus Chlamydia were examined for their ability to synthesize arginine, an ability their L cell (mouse fibroblasts) hosts lacked. C. psittaci (strain 6BC) multiplied in arginine-free medium 199 without significant decrease in titer and incroporated (14)C-glutamate and (14)C-ornithine into the arginine fraction of its protein. In arginine-free media, C. trachomatis (strain mouse pneumonitis) and C. psittaci (strain meningopneumonitis) grew to only 1 to 10% of the titer obtained in arginine-containing media. The decreased ability of these two strains to multiply in arginine-free media was paralleled by a decreased ability of infected host cells to incorporate (14)C-glutamate into protein arginine. These results suggest that chlamydiae either synthesize arginine themselves, or, in some unknown manner, cause their host cells to do so.     

Growth in Agarose of Human Cells Infected with Cytomegalovirus

After infection by human cytomegalovirus (CMV), human diploid fibroblasts could grow in agarose medium for several generations. Clones of infected cells grew for weeks, although in every case they ultimately underwent lysis owing to the cytopathic effect of the virus. Virus was inoculated at high dilution and after UV irradiation in an effort to derive cells infected with noninfectious defective particles still capable of inducing cell stimulation. Dilute or irradiated virus occasionally yielded large colonies of replicating cells, although permanent transformation was not observed. One clone derived from UV-CMV-infected cells was passaged four times before undergoing lysis. During these passages the cells exhibited alterations in morphology and orientation.     

Intranuclear Inclusions in the Ameboid Cells of Protostelium zonatum*

SYNOPSIS. Two morphologically distinct types of intranuclear inclusions are found in ameboid cells of the protostelid mycetozoan Protostelium zonatum. One type of inclusion is a coiled tubular structure which in cross section appears as cisternae and oval to elliptical vesicles 40–60 nm in diameter. These tubular and vesicular structures are formed by a unit membrane that is connected directly with the inner nuclear membrane. The other type of inclusion is a membrane-bound structure that contains amorphous and/or fibrous material. These inclusions usually are present at several locations in a nucleus. No similar structures occur in the cytoplasm.     

Ribonucleic Acid Synthesis in Cells Infected with Influenza Virus

Virus-specific ribonucleic acid (RNA), synthesized in influenza virus-infected cells from 3.5 to 7.5 hr after infection, was studied. After velocity centrifugation in sucrose, three peaks of virus-specific RNA could be identified: 34S, 18S, and 11S. These RNA species are predominantly single-stranded and consist of 90% viral (plus) and 10% complementary (minus) RNA strands. Most (75%) of the complementary RNA is single-stranded, i.e., not part of RNA duplexes or replicative intermediates. The 34S RNA species is an aggregate of 18S and 14S RNA species. Both 18S and 11S RNA species are relatively heterogenous compared to 18S ribosomal RNA, and these species probably contain different RNA molecules having closely related sedimentation coefficients.