Enhanced inhibition of RNA synthesis by amanitins in in vitro cultured cells

1976 ◽  
Vol 32 (1) ◽  
pp. 45-46 ◽  
Author(s):  
Laura Foá-Tomasi ◽  
F. Costanzo ◽  
Gabriella Campadelli-Fiume
Keyword(s):  
Rna Synthesis ◽  
Cultured Cells

scholarly journals Mechanistic Consequences of hnRNP C Binding to Both RNA Termini of Poliovirus Negative-Strand RNA Intermediates

2010 ◽  
Vol 84 (9) ◽  
pp. 4229-4242 ◽  
Author(s):  
Kenneth J. Ertel ◽  
Jo Ellen Brunner ◽  
Bert L. Semler
Keyword(s):  
Rna Synthesis ◽  
Cultured Cells ◽  
Wild Type Virus ◽  
Positive Strand ◽  
Negative Strand ◽  
Virus Rna ◽  
Hnrnp C ◽  
C Proteins ◽  
Positive Strand Rna

ABSTRACT The poliovirus 3′ noncoding region (3′ NCR) is necessary for efficient virus replication. A poliovirus mutant, PVΔ3′NCR, with a deletion of the entire 3′ NCR, yielded a virus that was capable of synthesizing viral RNA, albeit with a replication defect caused by deficient positive-strand RNA synthesis compared to wild-type virus. We detected multiple ribonucleoprotein (RNP) complexes in extracts from poliovirus-infected HeLa cells formed with a probe corresponding to the 5′ end of poliovirus negative-strand RNA (the complement of the genomic 3′ NCR), and the levels of these RNP complexes increased during the course of viral infection. Previous studies have identified RNP complexes formed with the 3′ end of poliovirus negative-strand RNA, including one that contains a 36-kDa protein later identified as heterogeneous nuclear ribonucleoprotein C (hnRNP C). We report here that the 5′ end of poliovirus negative-strand RNA is capable of interacting with endogenous hnRNP C, as well as with poliovirus nonstructural proteins. Further, we demonstrate that the addition of recombinant purified hnRNP C proteins can stimulate virus RNA synthesis in vitro and that depletion of hnRNP C proteins in cultured cells results in decreased virus yields and a correspondingly diminished accumulation of positive-strand RNAs. We propose that the association of hnRNP C with poliovirus negative-strand termini acts to stabilize or otherwise promote efficient positive-strand RNA synthesis.

scholarly journals STUDIES ON THE MECHANISM OF ACTION OF PEDERINE

1968 ◽  
Vol 36 (3) ◽  
pp. 485-496 ◽  
Author(s):  
Agnese Brega ◽  
Arturo Falaschi ◽  
Luigi De Carli ◽  
Mario Pavan
Keyword(s):  
Protein Synthesis ◽  
Dna Synthesis ◽  
Cytotoxic Effect ◽  
Rna Synthesis ◽  
Cultured Cells ◽  
Specific Inhibitor ◽  
Cytological Examination ◽  
Effect Analysis ◽  
Inhibitor Of Protein Synthesis

Pederine, a drug extracted from the coleopter Paederus fuscipes, inhibits the growth of in vitro cultured cell lines at concentrations of the order of 1.5 nanogram/ml. Cytological examination shows a generalized cytotoxic effect. Analysis of macromolecular syntheses by the use of radioactive precursors shows that pederine causes an almost immediate block of protein and DNA synthesis, without affecting RNA synthesis. The effects on the synthesis of the two types of macromolecules remain nearly simultaneous even at the lowest active concentrations of pederine. Studies with cell-free systems show that the drug inhibits protein synthesis, whereas it is ineffective on the DNA-polymerizing activity. It seems, therefore, that the drug acts primarily on the amino acid-polymerizing system, and that the effect on DNA is secondary. This idea is strengthened by the observation that puromycin, a specific inhibitor of protein synthesis, also affects promptly DNA synthesis of in vitro cultured cells. Other authors have shown the same phenomenon with a number of inhibitors of protein synthesis; the properties of pederine support, therefore, the view that continuous protein synthesis is necessary for the maintenance of DNA replication in higher organisms.

scholarly journals Virion Disassembly Is Required for Apoptosis Induced by Reovirus

2002 ◽  
Vol 76 (4) ◽  
pp. 1632-1641 ◽  
Author(s):  
Jodi L. Connolly ◽  
Terence S. Dermody
Keyword(s):  
Viral Replication ◽  
Cellular Response ◽  
Rna Synthesis ◽  
Cultured Cells ◽  
Wild Type Virus ◽  
Temperature Sensitive ◽  
Synthesis Inhibitor ◽  
Viral Attachment ◽  
Induced Apoptosis

ABSTRACT Reovirus infection leads to apoptosis in cultured cells and in vivo. Binding of viral attachment protein ς1 to both sialic acid and junction adhesion molecule is required for induction of apoptosis. However, it is not known whether viral engagement of receptors is sufficient to elicit this cellular response. To determine whether steps in reovirus replication subsequent to viral attachment are required for reovirus-induced apoptosis, we used inhibitors of viral disassembly and RNA synthesis, viral disassembly intermediates, temperature-sensitive (ts) reovirus mutants, and reovirus particles deficient in genomic double-stranded RNA (dsRNA). We found that reovirus-induced apoptosis is abolished in the presence of the viral disassembly inhibitors ammonium chloride and E64. Infectious subvirion particles (ISVPs), which are intermediates in reovirus disassembly that can be generated in vitro by protease treatment, are capable of inducing apoptosis in the presence or absence of these inhibitors. Treatment of cells with the viral RNA synthesis inhibitor ribavirin does not diminish the capacity of reovirus to induce apoptosis, and reovirus ts mutants arrested at defined steps in viral replication produce apoptosis with efficiency similar to that of wild-type virus. Furthermore, reovirus particles lacking dsRNA are capable of inducing apoptosis. Finally, we found that viral attachment and disassembly must occur within the same cellular compartment for reovirus to elicit an apoptotic response. These results demonstrate that disassembly of reovirus virions to form ISVPs, but not viral transcription or subsequent steps in viral replication, is required for reovirus to induce apoptosis.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

Preparation of cultured astrocytes for x-ray microanalysis

1989 ◽  
Vol 47 ◽  
pp. 988-989
Author(s):  
N.K.R. Smith ◽  
K.E. Hunter ◽  
P. Mobley ◽  
L.P. Felpel
Keyword(s):  
Cultured Cells ◽  
Elemental Content ◽  
Elemental Distribution ◽  
Sprague Dawley ◽  
X Ray ◽  
Cultured Astrocytes ◽  
Freeze Dried ◽  
Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

IN VITRO METHODS FOR THE STUDY OF THE ADENOHYPOPHYSIAL FUNCTIONS TO SECRETE GONADOTROPHIN

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S27-S40 ◽  
Author(s):  
T. Kobayashi ◽  
T. Kigawa ◽  
M. Mizuno ◽  
T. Watanabe
Keyword(s):  
Cell Culture ◽  
Organ Culture ◽  
Cultured Cells ◽  
Cell Sheet ◽  
Short Term ◽  
Hypothalamic Extract ◽  
Numerous Cell ◽  
Single Cell Culture ◽  
Almost All

ABSTRACT There are several in vitro methods to analyse the function of the adenohypophysis or the mechanisms of its regulation. The present paper deals with single cell culture, organ culture and short term incubation techniques by which the morphology and gonadotrophin-secreting function of the adenohypophysis were studied. In trypsin-dispersed cell culture, the adenohypophysial cells showed extensive propagation to form numerous cell colonies and finally develop into a confluent monolayer cell sheet covering completely the surface of culture vessels. Almost all of the cultured cells, however, became chromophobic, at least at the end of the first week of cultivation, when gonadotrophin was detectable neither in the culture medium nor in the cells themselves. After the addition of the hypothalamic extract, gonadotrophin became detectable again, and basophilic or PAS-positive granules also reappeared within the cells, suggesting that the gonadotrophs were stimulated by the extract to produce gonadotrophin. In organ culture and short term incubation, the incorporation of [3H] leucine into the adenohypophysial cells in relation to the addition of hypothalamic extract was examined. It was obvious that the ability to incorporate [3H] leucine into the gonadotrophs in vitro was highly dependent upon the presence of the hypothalamic extract.

Part 5. How to insonate cultured cells in vitro

Choonpa Igaku ◽  
2017 ◽  
Vol 44 (2) ◽  
pp. 153-155
Author(s):  
Takashi KONDO ◽  
Nobuki KUDO
Keyword(s):  
Cultured Cells

Bone Invasive Properties of Oral Squamous Cell Carcinoma and its Interactions with Alveolar Bone Cells: An In Vitro Study

2019 ◽  
Vol 19 (8) ◽  
pp. 631-640 ◽  
Author(s):  
Omel Baneen Qallandar ◽  
Faeza Ebrahimi ◽  
Farhadul Islam ◽  
Riajul Wahab ◽  
Bin Qiao ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Oral Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Cancer Cells ◽  
Alveolar Bone ◽  
Cultured Cells ◽  
Bone Cells ◽  
Bone Invasion ◽  
Alveolar Bone Cells

Background: Co-culture of cancer cells with alveolar bone cells could modulate bone invasion and destructions. However, the mechanisms of interaction between oral squamous cell carcinoma (OSCC) and bone cells remain unclear. Objective: The aim of this study is to analyse the direct and indirect effects of OSCC cells in the stimulation of osteolytic activity and bone invasion. Methods: Direct co-culture was achieved by culturing OSCC (TCA8113) with a primary alveolar bone cell line. In the indirect co-culture, the supernatant of TCA8113 cells was collected to culture the alveolar bone cells. To assess the bone invasion properties, in vitro assays were performed. Results: The proliferation of co-cultured cancer cells was significantly (p<0.05) higher in comparison to the monolayer control cells. However, the proliferation rates were not significantly different between direct and indirect co-cultured cells with indirect co-cultured cells proliferated slightly more than the direct co-cultured cells. Invasion and migration capacities of co-cultured OSCC and alveolar bone cells enhanced significantly (p<0.05) when compared to that of control monolayer counterparts. Most importantly, we noted that OSCC cells directly co-cultured with alveolar bone cells stimulated pronounced bone collagen destruction. In addition, stem cells and epithelialmesenchymal transition markers have shown significant changes in their expression in co-cultured cells. Conclusion: In conclusion, the findings of this study highlight the importance of the interaction of alveolar bone cells and OSCC cells in co-culture setting in the pathogenesis of bone invasion. This may help in the development of potential future biotherapies for bone invasion in OSCC.

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