Electron microscopic study of swine embryonic kidney cells infected with the tick-borne encephalitis virus

Electron microscopic studies of morphological lesions in pig embryo kidney cells (PEK) infected with tick-borne encephalitis (TBE) virus as well as morphology and features of TBE morphogenesis after treatment with actinomycin D, cycloheximide and hypertonic NaC1 concentrations in the medium were carried out. Most marked morphological lesions were observed in the cells after combined effect of high NaC1 concentrations in the medium and inhibitors of protein synthesis. After all kinds of treatment, smooth-contour membrane structures were observed in TBE-infected cells. Their number increased considerably with increasing ionic strength of the medium and subsequent return to normal accompanied by treatment with actinomycin D and cycloheximide. No "budding" particles were found in any case and after any treatment. Features of TBE virus morphogenesis are discussed.     

Inhibition of adenovirus multiplication by metabolic inhibitors

Summary Under conditions of one-step growth the multiplication of adenovirus 19 in HeLa cells was inhibited by 7 inhibitors of DNA synthesis (see Table 1), by cycloheximide, streptovitacin A, actinomycin D and mitomycin D. The inhibition involved the formation of infectious virus and of capsid proteins. The viral CPE was not inhibited by inhibitors of DNA synthesis; cycloheximide, streptovitacin and actinomycin inhibited the CPE with viral multiplicities up to 2. With medium doses of IUdR and BUdR the formation of infectious virus was inhibited with almost normal formation of capsid proteins. A selective formation of group-specific CF antigen was also observed under a number of conditions. The inhibitory effect of several drugs was reversible on removal. A reversion of the inhibition mediated by amethopterin, fluoruracil and FUdR was achieved only by addition of thymidine. No reversion was seen after removal of actinomycin. Deoxycytidine had an antagonistic effect against the inhibition mediated by cytosine arabinoside, when added 8 to 22 hours after the drug.The time during the replication cycle until which the inhibitors are fully effective was found to be 8 to 10 hours for inhibitors of DNA synthesis, 12 hours for actinomycin and 14 hours for cycloheximide and streptovitacin. This inhibition time was largely independent of inhibitor doses and viral multiplicity. When inhibitors were added during the exponential rise of viral activity, cycloheximide and streptovitacin stopped the further rise. Cycloheximide and actinomycin also stopped the progression of viral CPE. Apparently continuous protein synthesis is required for both viral maturation and viral CPE. Eor cycloheximide two inhibitor-sensitive periods (early proteins from 6 hpi, structural proteins from 14 hpi) were distinguished, while the period of synthesis of viral DNA lasted from 8 to 18 hours.Aided by a grant from the Deutsche Forschungsgemeinschaft.     

Protein synthesis in Japanese encephalitis virus-infected cells

We studied the effects of actinomycin D, cycloheximide, and puromycin on virus-specified protein synthesis in Japanese encephalitis (JE) virus-infected chick embryo and LLC-MK₂ (rhesus monkey kidney) cells. To prove that the radioactively labeled proteins we had previously identified in infected chick cells were virus-specified, we showed that they electrophoretically comigrated with radioactively labeled proteins from infected, but not from uninfected, LLC-MK₂ cells. We found that the maximum value for the ratio of protein synthesis in infected chick cells to uninfected cells occurred when the cells were treated with actinomycin D and were pulse-inhibited with cycloheximide. Alone, actinomycin D treatment decreased the background radioactivity of high molecular weight in electropherograms of infected chick cells and allowed virus-specified proteins to be prominent. Cycloheximide pulse-inhibition of infected, actinomycin D-treated cells decreased total cellular protein synthesis and slightly decreased the background radioactivity in electropherograms without changing the distribution of radioactivity among virus-specified proteins. Neither drug treatment decreased the yield of infectious virus. These results differ in some respects from the related results of Trent and Qureshi (1971). In contrast to our results with cycloheximide, pulse-inhibition of infected chick embryo cells with puromycin inhibited the synthesis of polypeptides NV-5, NV-4, and NV-1 (virus-specified nonstructural, nonglycosylated proteins) to a greater extent than that of V-3, NV-3, NV-2, and V-2 (virus-specified glycosylated and/or structural proteins). It also generally inhibited the synthesis of large proteins relative to small ones.We then studied the effects of puromycin and cycloheximide in LLC-MK₂ cells. In contrast to our results in chick embryo cells, pulse-inhibition of infected LLC-MK₂ cells with either drug (in the continuous presence of actinomycin D) did not alter the pattern of virus-specified proteins in electropherograms from that obtained without pulse-inhibition. Treatment with continuous levels of either drug (in the presence of actinomycin D) did, however, alter the protein pattern by differentially inhibiting the synthesis of nonstructural, nonglycosylated proteins. By labeling infected cells with one of eight different amino acids, we were unable to find an unusually enriched (or lowered) amino acid content that was common to all nonstructural, nonglycosylated proteins. A possible explanation for the differential inhibition is that the virus directs the formation of functionally different polyribosomes, messengers, or initiation factors which vary in their susceptibility to low levels of inhibitors of protein synthesis.     

Inhibition of protein synthesis by vaccinia virus. II. Studies on the role of virus-induced RNA synthesis

Cytoplasmic RNA synthesis can be detected in vaccinia virus-infected HeLa cells in the presence of 2 micrograms/ml but not 20 micrograms/ml of actinomycin D. When RNA synthesis is observed protein synthesis is inhibited in infected, treated cells. We had previously noted that such a correlation may also be observed in infected, cycloheximide-treated cells. If actinomycin D (20 micrograms/ml) is added to these cells at various times after infection and treatment, the inhibition of protein synthesis seen upon removal of cycloheximide does not continue beyond the point to which it had developed before the actinomycin D was added. These results indicate that the inhibition of protein synthesis can be correlated with the amount of cytoplasmic RNA synthesized in infected cells and that this RNA synthesis and the subsequent inhibition of protein synthesis can be prevented by sufficiently high concentrations of actinomycin D. The cytoplasmic RNA which is synthesized does not appear to consist of double-stranded RNA nor of extensive self complementary regions. The cytoplasmic RNA synthesized in infected, cycloheximide treated cells appears to consist of early virus mRNA which can function as mRNA in vitro in a cell-free system derived from normal cells. An examination of the phosphorylation of ribosomal proteins shows six additional phosphoproteins in infected cells, two of which may be observed in infected cycloheximide-treated cells, suggesting that phosphorylation of ribosomal proteins cannot be directly correlated with the inhibition of overall protein synthesis seen in infected cycloheximide-treated cells.     

Studies on Cellular Autophagocytosis: The Relationship of Autophagocytosis to Protein Synthesis and to Energy Metabolism in Rat Liver and Flounder Kidney Tubules in Vitro

The purpose of the present study was to elucidate the metabolic requirements of autophagocytosis. Two model systems were used for this purpose: a) glucagon-induced autophagocytosis in the rat liver, and b) the wave of autophagocytosis which occurs when isolated flounder kidney tubules are incubated in vitro. In the rat liver, protein synthesis was inhibited by the administration of cycloheximide (1.5 mg/kg) to rats 2 hours prior to glucagon injection. In flounder kidney tubules, protein synthesis was inhibited at least 90% by adding cycloheximide, actinomycin D, pactamycin and puromycin to the medium. In both systems the inhibition of protein synthesis failed to inhibit the formation of autophagic vacuoles or their subsequent transformation into autolysosomes, as depicted from electron microscopic histochemical preparations. In flounder kidney tubules no differences were found in the levels of p-nitrophenylphosphates, β-DL-glycerophosphatase, N-acetyl-β-D-glucosaminidase, arylsulphatase, β-D-galactosidase or acid proteinase when tubules were incubated up to 5 hours in the presence or absence of protein synthesis inhibitors. When ethionine was administered to rats 2 hours before glucagon injection, a decrease of approximately 75% in the ATP levels was observed. After ethionine administration, glucagon failed to induce the formation of autophagic vacuoles. The incubation of flounder kidney tubules in the presence of cyanide or in a nitrogen atmosphere decreased the ATP levels to less than 10% of controls and blocked autophagy. On the other hand, cyanide had little effect on acid hydrolase levels at 1 hour of incubation. A wide variety of other inhibitors were also shown to block autophagy. These results further support the hypothesis that, in the formation of antophagic vacuoles, preexisting enzyme and membrane pools are utilized. On the other hand, the esotropy-exotropy membrane conformational changes occurring in the formation of autophagic vacuoles seem to be energy dependent and can therefore be blocked by lowering intracellular ATP levels.     

Effect of pH, carbon source and K+ on the Na+-inhibited germ tube formation of Candida albicans.

The effect of pH, carbon source and K+ on the Na+ -inhibited germ tube formation of the pathogenic fungus Candida albicans was examined in the arginine-phosphate modified (APM) medium. All C. albicans cells formed germ tubes in APM medium at pH 5.0-9.0. Na+ inhibited germ tube formation in a concentration dependent manner ranging from 0.2 to 1.0 M, and was further influenced by the pH of the medium. The inhibitory effect of Na+ was lowest at pH 8.0, and germ tube formation ceased at 1.0 M Na+ for any pH (4.0-9.0). At pH > or = 6.0, non-germ tube-forming cells did not show yeast growth; whereas at pH < or = 5.0, Na+ inhibited only germ tube formation but did not inhibit yeast growth. The inhibitory effect of Na+ was stronger in glucose medium than in galactose medium as carbon source. K+, at 0-0.8 M, had almost no effect on germ tube formation. However, in the presence of Na+, a very low concentration of K+ (0.5 mM) was able to release the cells from Na+ arrest and produced an increase in the rate as well as the percentage of germ tube formation. Intracellular Na+/K+ ratios increased with the increase in extracellular Na+ concentration, whereas the ratios decreased and remained within nontoxic levels when the extracellular K+ concentration was increased.     

In vitro effect of fibrinogen on Candida albicans germ tube formation

Pseudohyphae formation by Candida albicans blastoconidia, as seen in vaginal smears, is a phenotypical change commonly assumed to mean fungal invasiveness, i.e. not mere colonization. C. albicans forms germ tubes in vitro in the presence of serum. In our search for inhibitory components of germ tube formation, we decided to study fibrinogen. The inhibition of germ tube formation by clinical isolates of C. albicans was evaluated in the presence of serial concentrations of fraction I, type IV and fraction I, type Is of fibrinogen from bovine plasma. Fibrinogen showed a dose-dependent, pH-independent inhibitory effect on the germ tube formation by C. albicans.     

Growth inhibition of Candida albicans by rabbit alveolar macrophages.

Normal rabbit alveolar macrophages were infected in vitro with Candida albicans. Early after infection, germ tube formation of phagocytized C. albicans was inhibited in contrast to extracellular (nonphagocytized) C. albicans. Over and 8-h period, plate counts of C. albicans incubated with alveolar macrophages revealed a decrease in colony-forming units in contrast to C. albicans alone. In addition, an assay was developed which specifically measured C. albicans [3H]leucine incorporation in the presence of alveolar macrophages. Using this assay, we observed a 71 to 93% inhibition of macromolecular synthesis in C. albicans when incubated with alveolar macrophages. Autoradiographic studies showed that the inhibition of leucine incorporation was restricted to the ingested Candida.     

Studies on the mechanisms of vaccina virus cytopathic effects. I. Inhibition of protein synthesis in infected cells is associated with virus-induced RNA synthesis.

The mechanism of vaccinia virus-induced inhibition of protein synthesis was studied in LLC-MK2, HeLa and L cells. Removal of cycloheximide (300 microgram/ml) from cells infected at a multiplicity of infection (m.o.i.) of 300 particles/cell at 4 h after infection resulted in the resumption of both host and virus protein synthesis in LLC-MK2 cells, but not in HeLa and L cells. In order to determine whether virus-induced RNA synthesis, which occurs in infected cells in the presence of cycloheximide, is related to the inhibition of protein synthesis, (cut-off), the rate of virus-induced RNA synthesis in the presence of cycloheximide was measured in all three cell types. In L cells and HeLa cells the rate of virus-induced RNA synthesis increased with time, whereas in LLC-MK2 cells it remained constant for at least 4 h. However, when higher multiplicities (900 and 2700 particles/cell) were used to infect LLC-MK2 cells, the rate of RNA synthesis in the presence of cycloheximide did increase with time and was greater at the higher multiplicity. Under these conditions there was a direct relationship between the extent of virus RNA synthesis and the degree of cut-off after the removal of cycloheximide. In HeLa and L cells infected at 300 particles/cell, the longer the exposure to cycloheximide, the greater was the cut-off observed upon removal of the drug. As was the case the LLC-MK2 cells, there was a direct relationship between the rate of RNA synthesis and the degree of inhibition of protein synthesis. Since virus-induced RNA synthesis occurs in the presence of cycloheximide, the effects of actinomycin D and cordycepin on host polypeptide synthesis were tested. Inhibition of host cell protein synthesis was virtually abolished when HeLA cells were infected in the presence of cordycepin (50 and 25 microgram/ml) or actinomycin D (20 microgram/ml). These results indicate that, as the rate of virus-induced RNA synthesis increased, regardless of the type of cell used, protein synthesis was inhibited at earlier times and to a greater extent. These observations are consistent with the hypothesis that the cut-off phenomenon is related to the synthesis of an early virus-induced RNA(s).     

Metabolic inhibitors distinguish cytolytic activity of CD4 and CD8 clones

The effect of various metabolic inhibitors on the expression of cytolytic activity of CD4 (TH1) and CD8 (CTL) clones was studied. The cytolytic activity of CD4 clones, but not CD8 clones, was sensitive to the RNA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide. Conversely, cholera toxin (CT) inhibited cytolytic activity of CD8, but not CD4 clones. Both mitomycin C, a DNA synthesis inhibitor, and cyclosporin A (CsA) failed to inhibit the cytolytic activity of either CD4 or CD8 clones. Although pretreatment with CsA or CT did not inhibit the cytolytic activity of CD4 clones, lymphokine (interleukin 2, IL2, interferon-gamma, IFN-gamma, and tumor necrosis factor, TNF) production was strongly inhibited. Similarly, pretreatment of a CD8 clone with actinomycin D or CsA inhibited lymphokine production without affecting cytolytic activity. The production of mRNA for TNF and IFN-gamma by concanavalin A-activated CD4 clones was also inhibited by CsA and CT. Moreover, perforin-specific mRNA was not detected in activated CD4 clones. Collectively, these observations demonstrated that de novo synthesis of RNA and protein is required for expression of cytolytic activity of CD4 clones, yet production of TNF, INF-gamma, IL 2 and perforin is not involved. In contrast, the cytolytic machinery of CD8 clones is present prior to activation and is quickly expressed following activation even when de novo synthesis of RNA, protein and lymphokines is blocked.     

The presumptive identification of Candida albicans with germ tube induced by high temperature

For direct identification of Candida albicans from other Candida species, the chlamydospore formation and the mycelial transition induced by high temperature and by sera were examined in 198 Candida isolates. The germ tubes of C. albicans developed early at 30 min in high temperature-induction, but at 60 min in serum-induction. C. albicans generated germ tubes well at concentrations lower than 2 x 10(7) cells/ml, but the germ tube formation was markedly restrained at concentrations higher than 4 x 10(7) cells/ml. In a serum-free, yeast extract-peptone-dextrose (YEPD) medium, C. albicans grew as a yeast form at 30 degrees C and as a mycelial form at 35-42 degrees C. Mycelial development was maximal at 37 degrees C in serum and at 39 degrees C in YEPD. Germ tubes were formed within 30 min in YEPD at 39 degrees C, but after 60 min in serum at 37 degrees C. Our examination showed that the 39 degrees C-induced germ tube formation tests were very reliable (sensitivity 100%, specificity 100%) at discerning C. albicans from other Candida species. These results suggest that the high temperature-induced germ tube formation testing could be a useful identification method of C. albicans in clinical laboratories.     

Analysis of the activity of DNA, RNA, and protein synthesis inhibitors on Xenopus embryo development

The teratogenic and growth-inhibiting potential of DNA, RNA, and protein synthesis inhibitors was explored using the Frog Embryo Teratogenesis Assay: Xenopus (FETAX). Endpoints measured in 96-h static tests were survival, malformation, ability to swim, skin pigmentation, stage of development, and growth. The DNA synthesis inhibitors hydroxyurea, cytosine arabinoside, and ethidium bromide proved to be teratogenic by the severity of malformations induced. Hydroxyurea gave an LC50 of 1.82 mg/ml, an EC50 (malformation) of 0.43 mg/ml, while the values for cytosine arabinsode were 5.41 and 0.76, respectively. The values for ethidium bromide were 0.05 and 0.035. The RNA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide were more embryolethal than teratogenic but significantly inhibited growth as determined by head-tail length measurements. Actinomycin D caused severe malformations, while cycloheximide caused relatively minor abnormalities. The LC50 for actinomycin D was 1.89 mg/ml, while the EC50 (malformation) was 2.17 mg/ml. For cycloheximide, the values were 1.59 and 1.19, respectively. FETAX advantages include rapid data collection, the ability to measure stage-dependent effects, and the ability to use a large number of embryos to obtain excellent dose-response curves with narrow confidence limits. Disadvantages include lack of a metabolic activation system, absence of a placental relationship, and the inability to detect specific abnormalities such as limb defects in 96 h.     

Cell cycle dependence for activation of Epstein-Barr virus by inhibitors of protein synthesis or medium deficient in arginine.

Activation of Epstein-Barr (EB) virus by inhibitors of protein synthesis (cycloheximide or puromycin) or medium deficient in arginine was studied in synchronized nonproducer Raji cells and producer P3HR-1 cells. The highest percentage of virus-activated cells was observed when treatment with inhibitors of protein synthesis or medium deficient in arginine was started at 1 hr after initiation of the S phase and terminated at 6 hr. The time for starting the treatment (S-1 period) corresponded to the previously reported time for incorporation of iododeoxyuridine to effect virus activation and the time of replication of the resident virus genome. The time for termination of the treatment corresponded to the time of completion of the S phase in untreated cells. Virus activation by inhibitors of protein synthesis or arginine-deficient medium in P3HR-1 cells resulted in the synthesis of both the early antigen (EA) and virus structural antigen (VCA) complexes, while virus-activated Raji cells synthesized only EA. Cell DNA synthesis was inhibited >80% in the presence of cycloheximide or medium deficient in arginine. The remaining low levels of DNA synthesis were required for virus activation since the addition of arabinofuranosylcytosine (ara-C) or hydroxyurea (HU) to cells during the period of treatment with cycloheximide or medium deficient in arginine prevented activation of virus. The time for termination of treatment with cycloheximide or arginine-deficient medium to obtain maximum levels of virus activation corresponded to the time of completion of the cells' requirement for continued DNA synthesis. Synthesis of EA occurred approximately 5 hr after removal of the cycloheximide or addition of arginine, and the addition of ara-C or HU at any time during this 5-hr period did not prevent the synthesis of EA. A mechanism is proposed which suggests that activation of EB virus by drugs or following spontaneously occurring events is due to factors which prevent the proper reassociation of the cell DNA and plasmid-like viral DNA.     

State and function of liver polysomes during recovery from ischemia

The reestablishment of blood supply to the liver lobes which have suffered ischemia is followed by an increase in protein synthesis only if ischemia did not last longer than 60 minutes. The increase is accompanied by the recovery of a normal polyribosomal pattern. Treatment of the rats with actinomycin D does not prevent this restoration. In the early phase of the reestablishment of circulation there is a sudden formation of monomers. These monosomes respond well to polyuridylic acid (poly-U) in vitro. The monosome formation can be prevented by treatment of the animal with cycloheximide. Treatment with cycloheximide also prevents the disappearance of the polysomal shoulder during ischemia. The results support the hypothesis that the lowering of the polysomal shoulder and the formation of monosomes are not the effect of ribonuclease action, but the consequence of an interference with recycling of ribosomes on mRNA. The recovery of a normal polysomal pattern after ischemia does not depend on the synthesis of new mRNA.     

The toxic plant proteins ricin and abrin induce apoptotic changes in mammalian lymphoid tissues and intestine

The toxins ricin and abrin are potent inhibitors of protein synthesis. Apoptosis has been shown to be induced in some cells by cycloheximide and actinomycin D whereas the process is prevented in other cells by the same agents, both inhibitors of protein synthesis. We were interested to find whether ricin and abrin caused any apoptotic changes in rapidly dividing tissues where we believed that these toxins concentrate. Rats were injected intramuscularly with toxin and killed at time intervals, tissues being removed and examined by light and electron microscopy. Apoptotic-like bodies were abundant in para-aortic lymph nodes, Peyer's patches and ideal crypts of ricin or abrin intoxicated rats. Abrin was found to cause markedly more pronounced changes in these tissues, when compared with a similar dose of ricin. Prior to this, these toxins have been reported as causing necrosis in animal tissues.     

Synthesis of the precursor to avian RNA tumor virus internal structural proteins early after infection.

The synthesis of the 76,000-dalton precursor (Pr 76) of the avian RNA tumor virus internal structural proteins was studied as a function of time after infection of chick embryo fibroblasts (CEF) with avian myeloblastosis virus (AMV). During the course of infection, cells were pulse-labeled with [³⁵S]methionine, lysed, and the labeled viral polypeptide precursor was precipitated with antibody against detergent-lysed AMV. Pr 76 was detected by SDS gel electrophoresis of immune precipitates.The earliest time at which Pr 76 synthesis could be detected was 3 hr after infection. Pr 76 synthesis remained low (about 1% of the level of synthesis several days after infection) and constant from 3 until 7 hr after infection. Between 7 and 9 hr after infection, Pr 76 synthesis increased by fivefold.Cells treated with cycloheximide during the first 8 or 12 hr after infection showed an 85–90% inhibition of Pr 76 synthesis and virus production measured late during infection. This finding does not necessarily imply that early viral protein synthesis is required for a productive infection, because cycloheximide also inhibited chick embryo fibroblast DNA synthesis.If cells were treated prior to and during infection with actinomycin D or cytosine arabinoside, precursor synthesis was still observed early (3 to 7 hr after infection). The amount of precursor synthesized early in the presence of inhibitors was similar to that synthesized in the absence of inhibitors, suggesting that the incoming RNA served as a messenger RNA for Pr 76.     

Degree of lung maturity determines the direction of the interleukin-1- induced effect on the expression of surfactant proteins

Intra-amniotic interleukin (IL)-1 increases surfactant components in immature fetal lung, whereas high IL-1 after birth is associated with surfactant dysfunction. Our aim was to investigate whether the fetal age influences the responsiveness of surfactant proteins (SPs) to IL-1. Rabbit lung explants from fetuses at 19, 22, 27, and 30 d of gestation and 1-d-old newborns were cultured in serum-free medium in the presence of recombinant human (rh) IL-1alpha or vehicle. The influence of IL-1alpha on SP-A, -B, and -C messenger RNA (mRNA) content was dependent on the conceptional age. In very immature lung on Day 19, rhIL-1alpha (570 ng/ml for 20 h) increased SP-A, -B, and -C mRNA by 860+/-15%, 314+/-108%, and 64+/-17%, respectively. The increase in SP-A mRNA was evident within 4 to 6 h. IL-1alpha increased the SP-A concentration in alveolar epithelial cells and in the culture medium within 20 h. In contrast, at 27 to 30 d of gestation and in newborns, IL-1alpha decreased SP-C, -B, and -A mRNA by means of 64 to 67%, 48 to 59%, and 12 to 15%, respectively. SP-B protein decreased by 45 to 60%. The decrease in mRNA became evident within 8 to 12 h and was dependent on IL-1 concentration. On Day 27, IL-1alpha accelerated the degradation of SP-B mRNA in the presence of actinomycin D. IL-1 did not increase the degradation rate of SP-A mRNA unless both actinomycin D and cycloheximide were added to the explants. The present findings may explain some of the contrasting associations between inflammatory cytokines and lung diseases during the perinatal period. The determinants of the direction of the IL-1 effect on the expression of SPs remain to be identified.