Tryptose phosphate broth confers to chick embryo cells resistance to the inhibitory effect of chloramphenicol on growth

Tryptose phosphate broth (10% V/V) prevents the inhibitory effect of chloramphenicol (80 ug/ml) on the growth of chick embryo fibroblasts. Cellular cytochrome $\text{c}$ oxidase activity is rapidly lost indicating that chloramphenicol inhibits the mitochondrial protein-synthesizing system in the presence of the broth. To compensate for the apparent decreased mitochondrial ATP production, pyruvate kinase and lactate dehydrogenase activities, 2-deoxyglucose uptake and lactic acid produced in treated cells are greater than those in the control cells during the first few generations. These fermentation values tend to revert to normal during later cell generations.     

Hsp72 inhibits apoptosis upstream of the mitochondria and not through interactions with Apaf-1

Hsp72 protects cells against apoptosis in response to various stresses. By simultaneously measuring cytochrome c localization and nuclear morphology in mouse embryo fibroblasts, we have shown that Hsp72 blocks cytochrome c release from mitochondria in response to cytotoxic stress and that permeabilization of the outer mitochondrial membrane is the critical point in deciding the fate of the cell. Hsp72 did not inhibit apoptosis in mouse embryo fibroblasts once cytochrome c had been released from the mitochondria. Recent reports have claimed that Hsp72 can prevent caspase activation by inhibiting the oligomerization of Apaf-1 in the presence of cytochrome c and dATP. We now show that this apparent function of recombinant Hsp72 is due to the presence of salt in the Hsp72 preparation and that the same response can be achieved by the addition of heat-denatured Hsp72 in the same high salt buffer or by the high salt buffer alone. Hsp72 expressed in a range of different cell lines had no inhibitory effect on cytochrome c-stimulated caspase activity of cytosolic extracts. We conclude that the protective effect of Hsp72 occurs upstream of the mitochondria and not through the inhibition of the apoptosome.     

On the contribution of the mitochondrial genome to the growth of Chinese hamster embryo cells in culture

The present results demonstrate that Chinese hamster embryo cell populations in culture can be adapted to grow in the presence of chloramphenicol. It is shown that tryptose phosphate broth and uridine, one of its components, prevent the growth inhibitory effect of the drug. Study of some respiratory parameters (cytochrome c oxidase, cytochrome spectra, oxygen consumption) indicated that neither the broth nor uridine prevented the inhibitory effect of chloramphenicol on mitoribosomal protein synthesis. The cells grew with mitochondria devoid of a functional respiratory chain. Auxotrophy for pyrimidines appeared to result from the absence of dihydroorotate dehydrogenase, a respiratory chain-linked enzyme that catalyzes the fourth step of de novo pyrimidine biosynthesis. These and other results suggest that the synthesis of orotic acid may be considered as one of the main contributions of mitochondria to the growth of animal cells in culture.     

Hemopoietic colonies on the chorioallantoic membrane of the chick embryo: Induction by embryonic,adherent, non-hemopoietic spleen cells

Granulocytic and erythrocytic colonies developed on the chick embryo chorioallantoic membrane (CAM) following the inoculation of chick embryo spleen cells. Dose response and kinetic experiments showed that the colonies were derived from cell aggregates present in the inoculum. Dissociation and reaggregation studies of the CAM colony-inducing cells (CAM-CIC) indicated that these cells must be present as aggregates in order to form colonies. Results from the morphology and cell marker experiments suggested that the colony-inducing aggregates (CAM-CIA) attract and support the differentiation of primitive host hemopoietic cells. The physical characteristics of the CAM-CIC, which are different from those of the hemopoietic progenitor cells, indicated that they represent a stromal cell population of the chick embryo spleen. Further evidence supporting this notion was provided by the radiation studies which showed that the colony-inducing ability of the CAM-CIC is radioresistant. The above characteristics of the CAM-CIC strongly suggest that they represent the stromal cells of the chick embryo spleen which influence hemopoiesis.     

The 1986 Upjohn award lecture. Interaction of chemicals with hemoproteins: implications for the mechanism of action of porphyrinogenic drugs and nitroglycerin

The ferrochelatase inhibitory activity of a variety of analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) was studied in chick embryo liver cells. The ferrochelatase inhibitory activity of the 4-butyl, 4-pentyl, and 4-hexyl analogues was considered to be due to catalytic activation by cytochrome P-450 leading to heme alkylation and formation of the corresponding N-alkylporphyrins. The relative ferrochelatase inhibitory activity of the DDC analogues has implications for a postulated model of the binding of porphyrins in the ferrochelatase active site. 3-[2-(2,4,6-Trimethylphenyl)thioethyl]-4-methylsydnone (TTMS) was shown to be a potent porphyrinogenic agent and to inhibit ferrochelatase in chick embryo liver cells. A related sydnone, 3-benzyl-4-phenylsydnone did not inhibit ferrochelatase activity. These results supported the idea that the porphyrinogenicity of TTMS was due to catalytic activation by cytochrome P-450 leading to heme alkylation and formation of N-vinylprotoporphyrin which inhibits ferrochelatase. Polychlorinated biphenyls, phenobarbital, nifedipine, and a large number of structurally different chemicals which are porphyrinogenic in chick embryo liver cells inhibit uroporphyrinogen decarboxylase by an unknown mechanism. Thus drug-induced porphyrin biosynthesis in chick embryo liver cell culture appears to be caused by inhibition of either ferrochelatase or uroporphyrinogen decarboxylase. The biotransformation of nitroglycerin by human red blood cells is due to a combination of a sulfhydryl-dependent enzymatic process and an interaction with reduced hemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome c1 of bakers' yeast. II. Synthesis on cytoplasmic robosomes and influence of oxygen and heme on accumulation of the apoprotein.

In the preceding paper (Ross, E., and Schatz, G. (1976) J. Biol. Chem. 251, 1991-1996) yeast cytochrome c1 was characterized as a 31,000 dalton polypeptide with a covalently bound heme group. In order to determine the site of translation of this heme-carrying polypeptide, yeast cells were labeled with [H]leu(be under the following conditions: (a) in the absence of inhibitors, (b) in the presence of acriflavin (an inhibitor of mitochondrial translation), or (c) in the presence of cycloheximide (an inhibitor of cytoplasmic translation). The incorporation of radioactivity into the hemeprotein was measured by immunoprecipitating it from mitochondrial extracts and analyzing it by dodecyl sulfate-polyacrylamide gel electrophoresis. Label was incorporated into the cytochrome c1 apoprotein only in the presence of acriflavin or in the absence of inhibitor, but not in the presence of cycloheximide. Cytochrome c1 is thus a cytoplasmic translation product. This conclusion was further supported by the demonstration that a cytolasmic petite mutant lacking mitochondrial protein synthesis still contained holocytochrome c1 that was indistinguishable from cytochrome c1 of wild type yeast with respect to molecular weight, absorption spectru, the presence of a covalently bound heme group, and antigenic properties. Cytochrome c1 in the mitochondria of the cytoplasmic petite mutant is firmly bound to the membrane, and its concentration approaches that typical of wild type mitochondria. However, its lability to proteolysis appeared to be increased. A mitochondrial translation product may thus be necessary for the correct conformation or orientation of cytochrome c1 in the mitochondrial inner membrane. Accumulation of cytochrome c1 protein in mitochondria is dependent on the abailability of heme. This was shown with a delta-aminolevulinic acid synthetase-deficient yeast mutant which lacks heme and any light-absorbing peaks attributable to cytochromes. Mitochondria from mutant cells grown without added delta-aminolevulinic acid contained at least 20 times less protein immunoprecipitable by cytochrome c1-antisera than mitochondria from cells grown in the presence of the heme precursor. Similarly, the respiration-deficient promitochondria of anaerobically grown wild type cells are almost completely devoid of material cross-reacting with cytochrome c1-antisera. A 105,000 X g supernatant of aerobically grown wild type cells contains a 29,000 dalton polypeptide that is precipitated by cytochrome c1-antiserum but not by nonimmune serum. This polypeptide is also present in high speed supernatants from the heme-deficient mutant or from anaerobically gorwn wild type cells. The possible identity of this polypeptide with soluble apocytochrome c1 is being investigated.     

Fibronectin alters the phenotypic properties of cultured chick embryo chondroblasts.

The state of chick embryo chondroblasts in culture was found to be sensitive to both fibronectin and another substance(s) (activity A) which could be extracted from chick embryo fibroblasts with 1 M urea or from conditioned medium. In the presence of either of these activities at concentrations of 25-150 micrograms/ml, chondroblasts, which normally grow as mixed cultures of floating and adherent cells, all immediately became attached to the tissue culture dish and spread. After several days, the morphology of these typically epithelioid cells became fibroblastic. This did not involve a selection process, since the effect was reversible. The synthetic program of these cells was also dramatically modified: the cultures no longer synthesized the chondroblast-unique type IV sulfated proteoglycan and began synthesizing alpha 2 collagen chains typical of fibroblastic or early limb bud cells. Fibronectin was resolved from activity A by gelatin affinity chromatography or gel filtration. Both activities were trypsin-sensitive. The two activities differed, however, on the basis of how the protein fractions in which they were found migrated in SDS-polyacrylamide gels, their specific activities and their effects on cell morphology and cell growth.     

Protein content and enzyme levels of cultured chick embryo cells treated with camptothecin and actinomycin D.

The alkaloid camptothecin uncouples the growth and adivision of chick embryo cells. At a moderate dose (0.5 microgram/ml) it inhibits the incorporation of thymidine but not of uridine and leucine and the cell protein content increases and reaches twice that of control after 4 days of treatment. Twelve hours after addition of the drug, the activities per cell of the mitochondrial enzymes poly A hydrolase (EC 3.1. 4.21), cytochrome c oxidase (EC 1.9.3.1), and succinate dehydrogenase (EC 1.3.99.1) are greater than that of the control and keep increasing for at least 96 H. The increase in the activities of the mitochondrial enzymes precede that of NADPH-cytochrome c reductase (EC 1.6.2.4) and cytidine triphosphatase (EC 3.6.1.15), which are microsomal and plasma membranes enzymes respectively. Actinomycin D (0.01 microgram/ml) also inhibits the multiplication of the chick cells and the synthesis of DNA. The protein content of the actinomycin D treated cells decreases to 70% of the control by day 2. Nevertheless, the activities of the mitochondrial enzymes increase over that of the control but to a smaller extent that with camptothecin. The activities of the enzymes of the other organelles are not stimulated. Camptothecin at a higher dose (5.0 microgram/ml) induces effects similar to those of actinomycin D.     

The mitochondrial permeability transition regulates cytochrome c release for apoptosis during endoplasmic reticulum stress by remodeling the cristae junction

The role of the mitochondrial permeability transition (MPT) in apoptosis and necrosis is controversial. Here we show that the MPT regulates the release of cytochrome c for apoptosis during endoplasmic reticulum (ER) stress by remodeling the cristae junction (CJ). CEM cells, HCT116 colon cancer cells, and murine embryo fibroblast cells were treated with the ER stressor thapsigargin (THG), which led to cyclophilin D-dependent mitochondrial release of the profusion GTPase optic atrophy 1 (OPA1), which controls CJ integrity, and cytochrome c, leading to apoptosis. Interference RNA knockdown of Bax blocked OPA1 and cytochrome c release after THG treatment but did not prevent the MPT, showing that Bax was essential for the release of cytochrome c by MPT. In isolated mitochondria, MPT led to OPA1 and cytochrome c release independently of voltage-dependent anion channel and the outer membrane, indicating that the MPT is an inner membrane phenomenon. Last, the MPT was regulated by the electron transport chain but not mitochondrial reactive oxygen species, since THG-induced cell death was not blocked by antioxidants and did not occur in cells lacking mitochondrial DNA. Our results show that the MPT regulates CJ remodeling for cytochrome c-dependent apoptosis induced by ER stress and that mitochondrial electron transport is indispensable for this process.     

The Persistence in the Synthesis of Type H Proteoglycan and Type II Collagen by Chondrocytes Cultured in the Presence of Chick Embryo Extract1

The effect of chick embryo extract on the phenotypic expression of differentiated chondrocytes has been studied in consideration of the fact that these cells are well characterized by certain specific cell products, such as type H proteochondroitin sulfate and type II collagen. In this study, we utilized floating chondrocytes derived from chick embryonic sterna, which can be cultured in suspension with no apparent change in the type of cell products for at least a period of eight weeks, as described in a previous paper (1). In the presence of chick embryo extract in the medium, the floating chondrocytes became attached to the bottom of the culture dish, and the attached cells took on a fibroblast-like appearance. Biochemical analyses of the proteochondroitin sulfate and collagen synthesized by the attached cells revealed that if the culture medium was renewed everyday, the cells having a fibroblast-like appearance continued to synthesize type H proteochondroitin sulfate and type II collagen. When however, the medium was replaced every other day, the synthesis of both proteochondroitin sulfate and collagen by the attached cells switched from the chondrocyte type to the fibroblast type, i.e. the synthesis of type M proteochondroitin sulfate and type I collagen, with little change in the fibroblast-like appearance. The results show that the morphological features of chondrocytes are not necessarily associated with the biochemical properties of these cells, and further suggest that, in chick embryo extract, there is no modulator capable of acting directly on the chondrocytes to bring about phenotypic changes with respect to the synthesis of collagen and proteoglycans.     

Cytochrome synthesis in synchronous cultures of the yeast, Saccharomyces cerevisiae.

The synthesis of cytochromes aa3, b, and c has been investigated during synchronous growth in the yeast, Saccharomyces cerevisiae. These cytochromes increase in concentration continuously throughout each cell cycle, with an approximate doubling in rate during successive cycles. The rates of cytochrome formation are considerably higher in galactose-grown cultures than in cells grown in glucose. Although cytochrome aa3 increases at a continuous rate, its functional counterpart, cytochrome c oxidase, increases in stepwise fashion, with the increments occurring at the beginning of each new cell cycle. Chloramphenicol, a specific inhibitor of intramitochondrial protein synthesis, inhibits the formation of cytochrome aa3 at all stages of the cell cycle, but does not inhibit cytochrome c. Chloramphenicol exhibits a somewhat intermediate effect on cytochrome b synthesis, with transient inhibition occurring only when the drug is added prior to or during the initial part of the first cell cycle. After this time, chloramphenicol had no effect on the rate of cytochrome b synthesis. The data indicate that under our conditions of cell synchrony mitochondrial membrane formation as reflected by increments in mitochondrial cytochromes occurs by continuous accretion of new material throughout the cell cycle. Intramitochondrially synthesized polypeptide products, responsible for the formation of new cytochrome aa3, appear to be synthesized throughout the cell cycle.     

Induction and characterization of mitochondrial DNA mutants in Chlamydomonas reinhardtii

In addition to lethal minute colony mutations which correspond to loss of mitochondrial DNA, acriflavin induces in Chlamydomonas reinhardtii a low percentage of cells that grow in the light but do not divide under heterotrophic conditions. Two such obligate photoautotrophic mutants were shown to lack the cyanide-sensitive cytochrome pathway of the respiration and to have a reduced cytochrome c oxidase activity. In crosses to wild type, the mutations are transmitted almost exclusively from the mating type minus parent. A same pattern of inheritance is seen for the mitochondrial DNA in crosses between the two interfertile species C. reinhardtii and Chlamydomonas smithii. Both mutants have a deletion in the region of the mitochondrial DNA containing the apocytochrome b gene and possibly the unidentified URFx gene.     

Tumor necrosis factor-alpha increases the steady-state reduction of cytochrome b of the mitochondrial respiratory chain in metabolically inhibited L929 cells

The mechanism of tumor necrosis factor alpha (TNFalpha)-induced cytotoxicity in metabolically inhibited cells is unclear, although some studies have suggested that mitochondrial dysfunction and generation of reactive oxygen species may be involved. Here we studied the effect of TNFalpha on the redox state of mitochondrial cytochromes and its involvement in the generation of reactive oxygen species in metabolically inhibited L929 cells. Treatment with TNFalpha and cycloheximide (TNFalpha/CHX) induced mitochondrial cytochrome c release, increased the steady-state reduction of cytochrome b, and decreased the steady-state reduction of cytochromes cc(1) and aa(3). TNFalpha/CHX treatment also induced lipid peroxidation, intracellular generation of reactive oxygen species, and cell death. Furthermore, as the cells died mitochondrial morphology changed from an orthodox to a hyperdense and condensed and finally to a swollen conformation. Antimycin A, a mitochondrial respiratory chain complex III inhibitor that binds to cytochrome b, blocked the formation of reactive oxygen species, suggesting that the free radicals are generated at the level of cytochrome b. Moreover, antimycin A, when added after 3 h of TNFalpha/CHX treatment, arrested the further release of cytochrome c and the cytotoxic response. We propose that the reduced cytochrome b promotes the formation of reactive oxygen species, lipid peroxidation of the cell membrane, and cell death.     

Glycyrrhizin protection against 3-morpholinosydnonime-induced mitochondrial dysfunction and cell death in lung epithelial cells

The present study was designed to assess the preventive effect of licorice compounds glycyrrhizin and 18beta-glycyrrhetinic acid against mitochondrial damage and cell death in lung epithelial cells exposed to 3-morpholinosydnonime, a donor of nitric oxide and superoxide. Treatment of lung epithelial cells with 3-morpholinosydnonime resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species and depletion of GSH. Treatment of glycyrrhizin and 18beta-glycyrrhetinic acid attenuated the 3-morpholinosydnonime-induced mitochondrial damage, formation of reactive oxygen species and GSH depletion and revealed a maximal inhibitory effect at 10 and 1 muM, respectively; beyond these concentrations the inhibitory effect declined. Melatonin, carboxy-PTIO, rutin and uric acid reduced the 3-morpholinosydnonime-induced cell death. The results show that glycyrrhizin and 18beta-glycyrrhetinic acid seem to prevent the toxic effect of 3-morpholinosydnonime against lung epithelial cells by suppressing the mitochondrial permeability transition that leads to the release of cytochrome c and activation of caspase-3. The preventive effect may be ascribed to the inhibitory action on the formation of reactive oxygen species and depletion of GSH. The findings suggest that licorice compounds seem to prevent the nitrogen species-mediated lung cell damage.     

Chick embryo chorioallantoic membrane model systems to study and visualize human tumor cell metastasis

Since their introduction almost a century ago, chick embryo model systems involving the technique of chorioallantoic grafting have proved invaluable in the in vivo studies of tumor development and angiogenesis and tumor cell dissemination. The ability of the chick embryo’s chorioallantoic membrane (CAM) to efficiently support the growth of inoculated xenogenic tumor cells greatly facilitates analysis of human tumor cell metastasis. During spontaneous metastasis, the highly vascularized CAM sustains rapid tumor formation within several days following cell grafting. The dense capillary network of the CAM also serves as a repository of aggressive tumor cells that escaped from the primary tumor and intravasated into the host vasculature. This spontaneous metastasis setting provides a unique experimental model to study in vivo the intravasation step of the metastatic cascade. During experimental metastasis when tumor cells are inoculated intravenously, the CAM capillary system serves as a place for initial arrest and then, for tumor cell extravasation and colonization. The tissue composition and accessibility of the CAM for experimental interventions makes chick embryo CAM systems attractive models to follow the fate and visualize microscopically the behavior of grafted tumor cells in both spontaneous and experimental metastasis settings.     

Embryo Cell Surfaces—Lectin Binding and Cell Proliferation

The interaction between chick embryo fibroblasts and various lectins has been studied at different stages of embryo development. There is evidence that Robinia lectin, Dolichos lectin, and Conca navalin A decrease cell number and proportion of cells incorporating [³H] thymidine in case of 8and 10day-old chick embryo fibroblasts, whereas they stimulated the proliferation of 16-dayold embryo cells. No effect was noticed in 12-day cells.
These results suggest that some cell surface changes occur during embryo development. The site number of Dolichos lectin remains the same during embryo development, and the affinity constant decreases. The site number of Robinia lectin and Concanavalin A decreases from the 8^th to the 12^th day of development, and slowly increases on the 16–day cells, the affinity constant remaining rather constant.
The results indicate that the age–dependent effect of lectin on embryo cells could not be directly related to the number of lectin–binding sites. Competitive binding experiments revealed that Dolichos receptor sites were distincts from binding sites of Robinia lectin and Concanavalin A, and Robina receptor sites distinct from those of Concanavalin A.
Lectin effects on embryo fibroblasts were very specific as determined by inhibitory assays.     

Detailing the role of Bax translocation, cytochrome c release, and perinuclear clustering of the mitochondria in the killing of HeLa cells by TNF

Induction of cell death in HeLa cells with TNF and cycloheximide (CHX) required an adequate ATP supply and was accompanied by decrease in intracellular pH, translocation of Bax, perinuclear clustering of the mitochondria, and cytochrome c release. The chloride channel inhibitor furosemide prevented the intracellular acidification, the translocation of Bax and the cell death. Cyclosporin A (CyA) or bongkrekic acid (BK) inhibited the induction of the MPT, the release of cytochrome c and the cell death without affecting the perinuclear clustering of the mitochondria or the translocation of Bax. Energy depletion with the ATP synthase inhibitor oligomycin or the uncoupler FCCP in the presence of 2-deoxy-glucose prevented the perinuclear clustering of the mitochondria and the cell killing. However, mitochondrial translocation of Bax was still observed. By contrast, cytochrome c was released in the oligomycin-treated cells but not in the same cells treated with FCCP. The data demonstrate that apoptosis in HeLa cells is ATP dependent and requires the translocation of Bax. The movement of Bax to the mitochondria occurs before and during the perinuclear clustering of these organelles and does not require the presence of ATP. The release of cytochrome c depends on the induction of the mitochondrial permeability transition but not ATP content.     

Factors influencing the assessment of anticoccidial activity in cell culture

A comparative study has been made of the factors influencing the assessment of anticoccidial potency in vitro against Eimeria tenella using established anticoccidials and arprinocid and some of its analogues. Drugs whose potency depended upon medium composition were amprolium, lasalocid and halofuginone. There was a difference in strain sensitivity with robenidine. Host cell type had an important effect on potency of monensin, decoquinate, arprinocid and its analogues. Arprinocid was active in chick liver cell systems but totally inactive in chick kidney cell systems, although its N-oxide was active in both cell types. Arprinocid-containing medium, conditioned by supporting the growth of chick embryo liver cell cultures, had an anticoccidial effect on E. tenella growing in chick kidney cells. It is deduced that the anticoccidial activity of arprinocid in the chick is due to a metabolite.