Ultrastructural Alterations in Hela Cells Induced by Spider Venom

1967 ◽  
Vol 25 ◽  
pp. 20-21
Author(s):  
Dale E. McClendon ◽  
Paul N. Morgan ◽  
Bernard L. Soloff
Keyword(s):  
Growth Medium ◽  
Mammalian Cells ◽  
Carcinoma Cells ◽  
Venom Protein ◽  
Sterile Saline ◽  
Carbon Coated ◽  
Available Information ◽  
Loxosceles Reclusa ◽  
Essential Growth ◽  
Solution Cultures

It has been observed that minute amounts of venom from the brown recluse spider, Loxosceles reclusa, are capable of producing cytotoxic changes in cultures of certain mammalian cells (Morgan and Felton, 1965). Since there is little available information concerning the effect of venoms on susceptible cells, we have attempted to characterize, at the electron microscope level, the cytotoxic changes produced by the venom of this spider.Cultures of human epithelial carcinoma cells, strain HeLa, were initiated on sterile, carbon coated coverslips contained in Leighton tubes. Each culture was seeded with approximately 1x105 cells contained in 1.5 ml of a modified Eagle's minimum essential growth medium prepared in Hank's balanced salt solution. Cultures were incubated at 36° C. for three days prior to the addition of venom. The venom was collected from female brown recluse spiders and diluted in sterile saline. Protein determinations on the venom-were made according to the spectrophotometric method of Waddell (1956). Approximately 10 μg venom protein per ml of fresh medium was added to each culture after discarding the old growth medium. Control cultures were treated similarly, except that no venom was added. All cultures were reincubated at 36° C.

Activation of the JAK-STAT pathway by reactive oxygen species

1998 ◽  
Vol 275 (6) ◽  
pp. C1640-C1652 ◽  
Author(s):  
Amy R. Simon ◽  
Usha Rai ◽  
Barry L. Fanburg ◽  
Brent H. Cochran
Keyword(s):  
Reactive Oxygen Species ◽  
Mammalian Cells ◽  
Reactive Oxygen ◽  
Buthionine Sulfoximine ◽  
Early Response ◽  
Carcinoma Cells ◽  
Oxygen Species ◽  
Intracellular Ros ◽  
Diphenylene Iodonium ◽  
Stat Pathway

Reactive oxygen species (ROS) play an important role in the pathogenesis of many human diseases, including the acute respiratory distress syndrome, Parkinson’s disease, pulmonary fibrosis, and Alzheimer’s disease. In mammalian cells, several genes known to be induced during the immediate early response to growth factors, including the protooncogenes c- fos and c- myc, have also been shown to be induced by ROS. We show that members of the STAT family of transcription factors, including STAT1 and STAT3, are activated in fibroblasts and A-431 carcinoma cells in response to H2O2. This activation occurs within 5 min, can be inhibited by antioxidants, and does not require protein synthesis. STAT activation in these cell lines is oxidant specific and does not occur in response to superoxide- or nitric oxide-generating stimuli. Buthionine sulfoximine, which depletes intracellular glutathione, also activates the STAT pathway. Moreover, H2O2stimulates the activity of the known STAT kinases JAK2 and TYK2. Activation of STATs by platelet-derived growth factor (PDGF) is significantly inhibited by N-acetyl-l-cysteine and diphenylene iodonium, indicating that ROS production contributes to STAT activation in response to PDGF. These findings indicate that the JAK-STAT pathway responds to intracellular ROS and that PDGF uses ROS as a second messenger to regulate STAT activation.

scholarly journals α2-macroglobulin used to isolate intracellular endopeptidases from mammalian cells in culture

1988 ◽  
Vol 255 (2) ◽  
pp. 437-443 ◽  
Author(s):  
L A Slot ◽  
K B Hendil
Keyword(s):  
Proteinase Inhibitor ◽  
Mammalian Cells ◽  
Human Fibroblasts ◽  
Polyacrylamide Gel Electrophoresis ◽  
Carcinoma Cells ◽  
Human Carcinoma ◽  
Cell Extracts ◽  
Α2 Macroglobulin ◽  
Unspecific Binding ◽  
Calpain Ii

Extracts of cell cultures labelled with [3H]leucine were incubated with human alpha 2-macroglobulin (alpha 2M), a plasma proteinase inhibitor. The proteinase-alpha 2M complexes were then precipitated with immobilized monoclonal antibodies to alpha 2M and analysed by SDS/polyacrylamide-gel electrophoresis. Parallel experiments were done with methylamine-inactivated alpha 2M to check for unspecific binding of cell proteins to alpha 2M. Several 3H-labelled cell proteins bound to active, but not to inactivated, alpha 2M. Such proteins are likely to be proteinases. Putative endopeptidases of subunit Mr 112000, 78,000, 53,000, and in some experiments 88,000 and 16,000, were trapped by alpha 2M in supernatant fractions from IMR90 human fibroblasts, EBTr bovine fibroblasts and HeLa human carcinoma cells. No additional proteins were trapped in the presence of ATP. The Mr-78,000 endopeptidase was identified as calpain II by immunoblotting. At pH 5.3 putative endopeptidases of subunit Mr 80,000, 53,000 and 28,000-32,000 were trapped from IMR90-fibroblast extracts. Immunoblotting showed that both cathepsin B and cathepsin D were present in the Mr-28,000-32,000 electrophoretic bands. The use of alpha 2M and immobilized antibody to alpha 2M thus allows a rapid enrichment of endopeptidases from cell extracts. Some potentials and limitations of the method are discussed.

Serum-free growth medium for the cultivation of a wide spectrum of mammalian cells in stirred bioreactors

1988 ◽  
Vol 1 (4) ◽  
pp. 319-329 ◽  
Author(s):  
Volker J�ger ◽  
J�rgen Lehmann ◽  
Peter Friedl
Keyword(s):  
Growth Medium ◽  
Mammalian Cells ◽  
Wide Spectrum ◽  
Serum Free ◽  
Free Growth ◽  
Stirred Bioreactors

Growth of mammalian cells on carbon-coated plastic substrata

1990 ◽  
Vol 18 (5) ◽  
pp. 1017-1017
Author(s):  
C. BRADLEY ◽  
J. BERRIMAN ◽  
J. WHISH ◽  
W.J.D. WHISH
Keyword(s):  
Mammalian Cells ◽  
Carbon Coated

scholarly journals Mechanism of oligonucleotide uptake by cells: involvement of specific receptors?

1989 ◽  
Vol 86 (17) ◽  
pp. 6454-6458 ◽  
Author(s):  
L A Yakubov ◽  
E A Deeva ◽  
V F Zarytova ◽  
E M Ivanova ◽  
A S Ryte ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mouse Fibroblast ◽  
Carcinoma Cells ◽  
Dna And Rna ◽  
Chondroitin Sulfates ◽  
Double Stranded Dna ◽  
L929 Mouse Fibroblast ◽  
Specific Receptors ◽  
L929 Mouse ◽  
Oligonucleotide Derivatives

We have investigated the interaction of oligonucleotides and their alkylating derivatives with mammalian cells. In experiments with L929 mouse fibroblast and Krebs 2 ascites carcinoma cells, it was found that cellular uptake of oligodeoxynucleotide derivatives is achieved by an endocytosis mechanism. Uptake is considerably more efficient at low oligomer concentration (less than 1 microM), because at this concentration a significant percentage of the total oligomer pool is absorbed on the cell surface and internalized by a more efficient absorptive endocytosis process. Two modified proteins were detected in mouse fibroblasts that were treated with the alkylating oligonucleotide derivatives. The binding of the oligomers to the proteins is inhibited by other oligodeoxynucleotides, single- and double-stranded DNA, and RNA. The polyanions heparin and chondroitin sulfates A and B do not inhibit binding. These observations suggest the involvement of specific receptor proteins in binding of oligomers to mammalian cells.

scholarly journals The p110αand p110βIsoforms of Class I Phosphatidylinositol 3-Kinase Are Involved in Toll-Like Receptor 5 Signaling in Epithelial Cells

2010 ◽  
Vol 2010 ◽  
pp. 1-12 ◽  
Author(s):  
Sabine M. Ivison ◽  
Mohammed A. S. Khan ◽  
Nicholas R. Graham ◽  
Leila A. Shobab ◽  
Yu Yao ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
P38 Map Kinase ◽  
Carcinoma Cells ◽  
Model Systems ◽  
Toll Like Receptor ◽  
Phosphatidylinositol 3 Kinase ◽  
Proinflammatory Response ◽  
Colon Carcinoma Cells ◽  
Toll Like Receptor 5 ◽  
Phosphatidylinositol 3

Background. Bacterial flagellin triggers inflammation in mammalian cells via Toll-like receptor (TLR) 5. Release of the chemokine IL-8 in response to flagellin involves NF-κB, p38 MAP kinase, and phosphatidylinositol 3-kinase (PI3K). However, PI3K has been reported to be either pro- or anti-inflammatory in different model systems. We hypothesized that this could be due to different activities of the p110αandβisoforms of PI3K.Results. PI3K and Akt were rapidly activated in Caco-2 colon carcinoma cells by flagellin. Using a plasmid-based shRNA delivery system and novel p110 isoform-specific inhibitors, we found that flagellin-induced IL-8 production was dependent on both p110αand p110β. However in the mouse, inhibition of p110βbut not p110αreduced the increase of serum IL-6 levels induced by intraperitoneal injection of flagellin.Conclusions. These data demonstrate that the p110αandβisoforms of class IA PI3K are both required for the proinflammatory response to flagellin.

scholarly journals The SLC2A14 gene: genomic locus, tissue expression, splice variants, and subcellular localization of the protein

2016 ◽  
Vol 94 (4) ◽  
pp. 331-335 ◽  
Author(s):  
Mandana Amir Shaghaghi ◽  
Brent Murphy ◽  
Peter Eck
Keyword(s):  
Mammalian Cells ◽  
Glucose Transporter ◽  
Splice Variants ◽  
Tissue Expression ◽  
Protein Isoforms ◽  
Human Testis ◽  
Genomic Locus ◽  
Transporter Family ◽  
Disease Associations ◽  
Available Information

The SLC2A14 gene encodes for GLUT14, an orphan member of the facilitated membrane glucose transporter family, which was originally described to be exclusively expressed in human testis. However, genetic variations in SLC2A14 are associated with chronic diseases such as Alzheimer’s disease and Inflammatory Bowel Disease, which cannot be explained by a strictly testicular expression. Therefore we analyzed available information on the SLC2A14 gene to update knowledge of the locus and its encoded products. This report presents an expanded SLC2A14 gene locus and a more diverse tissue expression, concurring with the existing evidence for disease associations. The exon utilization is tissue specific, with major expression in testis. When the 2 major testicular protein isoforms were expressed in mammalian cells, they located to the plasmalemma membrane, providing early evidence that GLUT14 could function as a membrane transporter.

Distyrylbenzene oligoelectrolyte (DSBN+) for human cervical carcinoma cells imaging

2013 ◽  
Vol 1569 ◽  
pp. 207-211 ◽  
Author(s):  
Roza Pawlowska ◽  
Paulina Gwozdzinska ◽  
Logan Garner ◽  
Arkadiusz Chworos
Keyword(s):  
Cervical Carcinoma ◽  
Mammalian Cells ◽  
Photophysical Properties ◽  
Carcinoma Cells ◽  
Water Soluble ◽  
Membrane Selectivity ◽  
Human Cervical Carcinoma ◽  
Human Cell Cultures ◽  
Organelle Membrane ◽  
Human Cervical Carcinoma Cells

ABSTRACTIn the presented study, a new application for distyrylbenzene oligoelectrolyte, named DSBN+, as a marker for bioimaging is presented. DSBN+ is a water-soluble, conjugated oligoelectrolyte (COE) with novel photophysical and solvatochromatic properties. Previous studies have shown that this compound spontaneously inserts into bilayer membranes in both synthetic and microbial living systems and can facilitate visualization of cell membranes through fluorescence imaging. In the presented research, we seek to further study and exploit the multifunctional nature of DSBN+ in terms of membrane interactions and photophysical properties for visualization of membranous structures of more complex mammalian cells, namely a human cervical carcinoma (HeLa) cell line. Obtained results confirm the possibility of applying DSBN+ as a fluorescent dye for bioimaging of membranes in human cell cultures systems, both in live-cell imaging and in the studies required formaldehyde fixation. Due to the defined structure of this conjugated oligoelectrolyte we suspect that it will display organelle membrane selectivity, but this has to be further investigated.

scholarly journals Fatty Acyl Availability Modulates Cardiolipin Composition and Alters Mitochondrial Function in HeLa Cells

2020 ◽  
Author(s):  
Gregor Oemer ◽  
Marie-Luise Edenhofer ◽  
Katharina Lackner ◽  
Geraldine Leman ◽  
Jakob Koch ◽  
...  
Keyword(s):  
Cell Culture ◽  
Growth Medium ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Citrate Synthase ◽  
Building Blocks ◽  
Molecular Assembly ◽  
Fatty Acyl ◽  
Membrane Composition ◽  
The Impact

The molecular assembly of cells depends not only on their balance between anabolism and catabolism, but to a large degree also on the building blocks available in the environment. For cultivated mammalian cells, this is largely determined by the composition of the growth medium used. Here we study the impact of medium lipids on mitochondrial membrane architecture and function by combining LC-MS/MS lipidomics and functional tests with lipid supplementation experiments in an otherwise serum- and lipid-free cell culture model. We demonstrate that the composition of mitochondrial cardiolipins (CL) strongly depends on the lipid environment in cultured cells and prefers the incorporation of essential linoleic acid over other fatty acids. Simultaneously, the mitochondrial respiratory complex I activity was altered, whereas the matrix-localized enzyme citrate synthase was unaffected. This suggests a link between membrane composition and respiratory capacity. In summary, we find a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium. Thus, this underlines the importance of considering these factors when using and establishing cell culture models in biomedical research.

Pulmonary Hypertension Syndrome in Young Chickens Challenged with Frozen and Autoclaved Cultures of Enterococcus faecalis

2002 ◽  
Vol 227 (9) ◽  
pp. 812-816 ◽  
Author(s):  
J. D. Tankson ◽  
J. P. Thaxton ◽  
Y. Vizzier-Thaxton
Keyword(s):  
Pulmonary Hypertension ◽  
Enterococcus Faecalis ◽  
Growth Medium ◽  
Ascites Fluid ◽  
Pulmonary Hypertension Syndrome ◽  
Sterile Saline ◽  
Unknown Substance ◽  
Live Culture ◽  
Similar Incidence

Enterococcus faecalis, when administered in a growth medium or sterile saline, will cause pulmonary hypertension syndrome (PHS) in chickens. The objective of this study was to determine if frozen and/or autoclaved cultures of E. faecalis retain ability to evoke PHS. In Trial 1, chicks were inoculated with 3.6 × 107 E. faecalis (IA) in tryptic soy broth (TSB) from either a live culture or one that had been autoclaved (120°C for 20 min). Controls received TSB. Autoclaved and live cultures produced the same degree of PHS in a majority of the birds. Trial 2 used the same protocol, except a frozen (–70°C for 60 min) culture of E. faecalis was compared with the control. The results agreed with those of Trial 1, i.e., the frozen culture also produced PHS. Trial 3 was conducted to determine if E. faecalis caused PHS by producing and releasing some unknown substance into the supernatant. Incidence of PHS was based on percentage of birds exhibiting ascites fluid at 24 hr after challenge. Controls received sterile, frozen, or autoclaved TSB. As compared with controls, those birds that received challenge with E. faecalis alone, supernatant alone, and E. faecalis plus supernatant from live cultures exhibited similar incidence of ascites, whereas birds that received E. faecalis plus supernatant and supernatant alone from cultures that had been either frozen or autoclaved exhibited elevated incidence of ascites as compared with controls. Also, with frozen and autoclaved cultures, those birds that received only pelleted E. faecalis exhibited incidence of ascites that did not differ from controls. Apparently, E. faecalis produces PHS in chicks by producing and releasing an unknown toxin.

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