scholarly journals Cytoplasmic transfer of chloramphenicol resistance in human tissue culture cells.

1975 ◽  
Vol 67 (1) ◽  
pp. 174-188 ◽  
Author(s):  
D C Wallace ◽  
C L Bunn ◽  
J M Eisenstadt
Keyword(s):  
Mass Culture ◽  
Cytoplasmic Inheritance ◽  
Hypoxanthine Phosphoribosyl Transferase ◽  
Chloramphenicol Resistance ◽  
Culture Cells ◽  
Permanent Cell ◽  
Fusion Products ◽  
Hela Strain

The cytoplasmic inheritance of human chloramphenicol (cap) resistance has been demonstrated by removing the nuclei of cells of the CAP-resistant HeLa strain 296-1 (enucleation) and fusing them to a CAP-sensitive HeLa strain lacking nuclear thymidine kinase. Plating the fusion products in bromodeoxyuridine and CAP resulted in the growth of about 150 colonies/10(6) parent cells plated. Permanent cell lines (cybrids) grown from such fusions have been designated HEB. A recloned HEB cybrid (HEB7A) has also been enucleated and fused to hypoxanthine phosphoribosyl transferase (HPRT)-deficient HeLa cells (S3AG1) and HPRT-deficient lymphocytes (WAL-2A). Cybrids were selected in thioguanine and CAP. In the fusion of enucleated (en) HEB7A to S3AG1, 1,200 colonies/10(6) parents were observed. Fusion of enHEB7A to WAL-2A was done in mass culture and cybrids were obtained on three separate occasions. In every case the parental controls were negative. All isolates tested from the above fusions have the CAP-resistant characteristics, in vivo and in vitro, of the enucleated parent and the nuclear characteristics of the CAP-sensitive parent, such as chromosome number, morphology, and specific isozyme and chromosome markers. Therefore, it can be concluded that CAP resistance is coded in the cytoplasm and not in the nucleus of 296-1 cells. Furthermore, this resistance can be transferred to cells of widely different origin and differentiated state. These studies represent the first genetic evidence of cytoplasmic inheritance in human cells.

In vitro and in vivo pathologic effects of Vibrio parahaemolyticus on human epithelial cells

1984 ◽  
Vol 30 (3) ◽  
pp. 381-388 ◽  
Author(s):  
B. R. Merrell ◽  
R. I. Walker ◽  
S. W. Joseph
Keyword(s):  
Epithelial Cells ◽  
Epithelial Tissue ◽  
Ileal Mucosa ◽  
Cytotoxic Factor ◽  
Culture Cells ◽  
Vibrio Parahemolyticus ◽  
Buccal Epithelial Cells ◽  
Culture Supernate

The initial interaction and adherence of Vibrio parahemolyticus to epithelial tissue culture cells, human buccal epithelial cells, and the ileal mucosa of mice were studied. Using scanning electron microscopy, adherent bacteria were observed only on degenerating human embryonic intestinal, HeLa, and buccal cells; healthy normal cells were devoid of bacteria. Sheared V. parahaemolyticus, i.e., lacking flagella, did not adhere to either normal or degenerating tissue cells. Neither ultraviolet-inactivated organisms nor cell-free culture supernate affected the epithelial cells. Similar findings were observed on the mucosa of the ileum in mice inoculated with V. parahaemolyticus. It appears that V. parahaemolyticus possesses a cytotoxic factor which alters epithelial cells. This factor appears to be closely associated with viable organisms and may be a functional element in the adherence process of flagellated V. parahaemolyticus to mammalian epithelial cells.

scholarly journals ELECTRON MICROSCOPY OF PLASMA-CELL TUMORS OF THE MOUSE

1961 ◽  
Vol 9 (2) ◽  
pp. 369-381 ◽  
Author(s):  
D. F. Parsons ◽  
M. A. Bender ◽  
E. B. Darden ◽  
Guthrie T. Pratt ◽  
D. L. Lindsley
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Complex Structure ◽  
Granular Body ◽  
Virus Particles ◽  
Culture Cells ◽  
Large Numbers ◽  
Tumor Agent

The X5563 tumor has been grown in tissue culture. Cells similar to those of the original tumor migrated from the explant and attached to the glass walls of the culture vessels. Electron microscopy showed that large numbers of particles, similar in morphology to virus particles, were associated with these cells after 7 days of culture. The two principal types of particles found in the tumor in vivo appear to be present in vitro. Many more of these particles, however, were larger and showed a more complex structure. Whereas the particles were mainly localized inside endoplasmic reticulum or the Golgi zone in the tumors in vivo, in the tissue culture the majority of the particles were associated with the plasma membrane and were found outside of the cells. The relation of the particles to the granular body is discussed as well as a possible relation to the mammary tumor agent.

scholarly journals THE MECHANISM OF ACTION OF COLCHICINE

1967 ◽  
Vol 34 (2) ◽  
pp. 525-533 ◽  
Author(s):  
G. G. Borisy ◽  
E. W. Taylor
Keyword(s):  
Soluble Fraction ◽  
Noncovalent Complex ◽  
Binding Activity ◽  
Chromatographic Behavior ◽  
Mitotic Apparatus ◽  
Vitro Assay ◽  
Culture Cells ◽  
Antimitotic Activity

The majority of the colchicine-3H bound by tissue culture cells (KB or Hela) was found to be present as a noncovalent complex with a macromolecule which appears in the soluble fraction after homogenization. Similar binding was demonstrated in vitro and was confined to a component of the soluble fraction. The binding-equilibrium constant and the kinetic constants were essentially the same in vivo and in vitro. Bound radioactivity was reisolated and shown to be present in a molecule with the same chromatographic behavior and specific antimitotic activity as colchicine. In vitro assay of binding activity of a variety of cells and tissues showed a correlation with the presence of microtubules. High binding activity was given by dividing cells, mitotic apparatus, cilia, sperm tails, and brain tissue. Binding to extracts of slime mold or to purified muscle proteins was very low or undetectable. The binding site had a sedimentation constant of 6S and it is suggested that the protein is a subunit of microtubules.

scholarly journals Bioreactors as physiologicallike in vitro models

2020 ◽  
Author(s):  
Sara Mantero ◽  
Federica Boschetti
Keyword(s):  
Culture Conditions ◽  
In Vitro Models ◽  
In Vivo Models ◽  
In Vitro Development ◽  
Culture Cells ◽  
Vitro Development ◽  
Tissue Models ◽  
Mechanical Stretching

Bioreactors are powerful tools for in vitro development of engineered substitutes through controlled biological, physical, and mechanical culture conditions: bioreactor technology allows a closer in vitro replication of native tissues. One of bioreactors applications is the design of in vitro 3D tissue models as a bridge between 2D and in vivo models, allowing the application of 3R (replacement, reduction, refinement) principle. To this aim, bioreactors can be used to culture cells seeded on engineered scaffolds under in vivo-like conditions. Another key use of bioreactors is for perfusion decellularization of tissues and organs to be used as scaffolds. This contribution describes a dynamic stretching. bioreactor, imposing a mechanical stretching to the cultured constructs, allowing the development of skeletal muscle engineered constructs, and a decellularization bioreactor, designed for decellularization of blood vessels.

scholarly journals Esterases in Serum-Containing Growth Media Counteract Chloramphenicol Acetyltransferase Activity In Vitro

1999 ◽  
Vol 43 (3) ◽  
pp. 655-660 ◽  
Author(s):  
Charles D. Sohaskey ◽  
Alan G. Barbour
Keyword(s):  
Human Serum ◽  
Horse Serum ◽  
Chloramphenicol Acetyltransferase ◽  
Rabbit Serum ◽  
Growth Media ◽  
Chloramphenicol Resistance ◽  
E Coli ◽  
Cell Extracts

ABSTRACT The spirochete Borrelia burgdorferi was unexpectedly found to be as susceptible to diacetyl chloramphenicol, the product of the enzyme chloramphenicol acetyltransferase, as it was to chloramphenicol itself. The susceptibilities of Escherichia coli and Bacillus subtilis, as well as that ofB. burgdorferi, to diacetyl chloramphenicol were then assayed in different media. All three species were susceptible to diacetyl chloramphenicol when growth media were supplemented with rabbit serum or, to a lesser extent, human serum. Susceptibility ofE. coli and B. subtilis to diacetyl chloramphenicol was not observed in the absence of serum, when horse serum was used, or when the rabbit or human serum was heated first. In the presence of 10% rabbit serum, a strain of E. colibearing the chloramphenicol acetyltransferase (cat) gene had a fourfold-lower resistance to chloramphenicol than in the absence of serum. A plate bioassay for chloramphenicol activity showed the conversion by rabbit, mouse, and human sera but not bacterial cell extracts or heated serum of diacetyl chloramphenicol to an inhibitory compound. Deacetylation of acetyl chloramphenicol by serum components was demonstrated by using fluorescent substrates and thin-layer chromatography. These studies indicate that esterases of serum can convert diacetyl chloramphenicol back to an active antibiotic, and thus, in vitro findings may not accurately reflect the level of chloramphenicol resistance by cat-bearing bacteria in vivo.

scholarly journals MiRP3 acts as an accessory subunit with the BK potassium channel

2008 ◽  
Vol 295 (2) ◽  
pp. F380-F387 ◽  
Author(s):  
Daniel I. Levy ◽  
Sherry Wanderling ◽  
Daniel Biemesderfer ◽  
Steve A. N. Goldstein
Keyword(s):  
Potassium Channel ◽  
Mixed Complex ◽  
Intercalated Cells ◽  
Voltage Gated ◽  
Calcium Dependent ◽  
Culture Cells ◽  
Urinary Potassium ◽  
Α Subunits

MinK-related peptides (MiRPs) are single-span membrane proteins that assemble with specific voltage-gated K+ (Kv) channel α-subunits to establish gating kinetics, unitary conductance, expression level, and pharmacology of the mixed complex. MiRP3 (encoded by the KCNE4 gene) has been shown to alter the behavior of some Kv α-subunits in vitro but its natural partners and physiologic functions are unknown. Seeking in vivo partners for MiRP3, immunohistochemistry was used to localize its expression to a unique subcellular site, the apical membrane of renal intercalated cells, where one potassium channel type has been recorded, the calcium- and voltage-gated channel BK. Overlapping staining of these two proteins was found in rabbit intercalated cells, and MiRP3 and BK subunits expressed in tissue culture cells were found to form detergent-stable complexes. Electrophysiologic and biochemical evaluation showed MiRP3 to act on BK to reduce current density in two fashions: shifting the current-voltage relationship to more depolarized voltages in a calcium-dependent fashion (∼10 mV at normal intracellular calcium levels) and accelerating degradation of MiRP3-BK complexes. The findings suggest a role for MiRP3 modulation of BK-dependent urinary potassium excretion.

scholarly journals The use of naturally occurring hybrid variants of chloramphenicol acetyltransferase to investigate subunit contacts

1981 ◽  
Vol 193 (2) ◽  
pp. 541-552 ◽  
Author(s):  
L C Packman ◽  
W V Shaw
Keyword(s):  
Chloramphenicol Acetyltransferase ◽  
Alpha Subunit ◽  
Type I ◽  
Chloramphenicol Resistance ◽  
Type Iii ◽  
Naturally Occurring ◽  
Alpha Beta ◽  
Beta 2

1. Hybrids of the tetrameric enzyme chloramphenicol acetyltransferase (EC 2.3.1.28) were formed in vivo in a strain of Escherichia coli which harbours two different plasmids, each of which normally confers chloramphenicol resistance and specifies an easily distinguished enzyme variant (type I or type III) which is composed of identical subunits. Cell-free extracts of the dual-plasmid strain were found to contain five species of active enzyme, two of which were the homomeric enzymes corresponding to the naturally occurring tetramers of the type-I (beta 4) and type-III (alpha 4) enzymes. The other three variants were judged to be the heteromeric hybrid variants (alpha 3 beta, alpha 2 beta 2, alpha beta 3). 2. The alpha 3 beta and alpha 2 beta 2 hybrids of chloramphenicol acetyltransferase were purified to homogeneity by combining the techniques of affinity and ion-exchange chromatography. The alpha beta 3 variant was not recovered and may be unstable in vitro. 3. The unique lysine residues that could not be modified with methyl acetimidate in each of the native homomeric enzymes were also investigated in the heteromeric tetramers. 4. Lysine-136 remains buried in each beta subunit of the parental (type I) enzyme and in each of the hybrid tetramers. Lysine-38 of each alpha subunit is similarly unreactive in the native type-III chloramphenicol acetyltransferase (alpha 4), but in the alpha 2 beta 2 hybird lysine-38 of each alpha subunit is fully exposed to solvent. Another lysine residue, fully reactive in the alpha 4 enzyme, was observed to be inaccessible to modification in the symmetrical hybrid. The results obtained for the alpha 3 beta enzyme suggest that lysine-38 in two subunits and a different lysine group (that identified in the alpha 2 beta 2 enzyme) in the third alpha subunit are buried. 5. A tentative model for the subunit interactions of chloramphenicol acetyltransferase is proposed on the basis of the results described.

scholarly journals Actin Bound to the Heterogeneous Nuclear Ribonucleoprotein Hrp36 Is Associated with Balbiani Ring mRNA from the Gene to Polysomes

2001 ◽  
Vol 153 (1) ◽  
pp. 229-236 ◽  
Author(s):  
Piergiorgio Percipalle ◽  
Jian Zhao ◽  
Brian Pope ◽  
Alan Weeds ◽  
Uno Lindberg ◽  
...  
Keyword(s):  
Nucleocytoplasmic Transport ◽  
Balbiani Ring ◽  
Culture Cells ◽  
Cytoplasmic Actin ◽  
Hnrnp Proteins ◽  
Messenger Ribonucleoprotein ◽  
Hnrnp Protein ◽  
Nuclear Ribonucleoprotein

In the salivary glands of the dipteran Chironomus tentans, a specific messenger ribonucleoprotein (mRNP) particle, the Balbiani ring (BR) granule, can be visualized during its assembly on the gene and during its nucleocytoplasmic transport. We now show with immunoelectron microscopy that actin becomes associated with the BR particle concomitantly with transcription and is present in the particle in the nucleoplasm. DNase I affinity chromatography experiments with extracts from tissue culture cells indicate that both nuclear and cytoplasmic actin are bound to the heterogeneous RNP (hnRNP) protein hrp36, but not to the hnRNP proteins hrp23 and hrp45. The interaction is likely to be direct as purified actin binds to recombinant hrp36 in vitro. Furthermore, it is demonstrated by cross linking that nuclear as well as cytoplasmic actin are bound to hrp36 in vivo. It is known that hrp36 is added cotranscriptionally along the BR mRNA molecule and accompanies the RNA through the nuclear pores and into polysomes. We conclude that actin is likely to be bound to the BR transcript via hrp36 during the transfer of the mRNA from the gene all the way into polysomes.

scholarly journals dDP Is Needed for Normal Cell Proliferation

2005 ◽  
Vol 25 (8) ◽  
pp. 3027-3039 ◽  
Author(s):  
Maxim V. Frolov ◽  
Nam-Sung Moon ◽  
Nicholas J. Dyson
Keyword(s):  
Cell Proliferation ◽  
Normal Cell ◽  
Binding Activity ◽  
S Phase ◽  
Dna Binding Activity ◽  
Culture Cells ◽  
Complete Inactivation ◽  
Mutant Cells

ABSTRACT To gain insight into the essential functions of E2F, we have examined the phenotypes caused by complete inactivation of E2F and DP family members in Drosophila. Our results show that dDP requires dE2F1 and dE2F2 for DNA-binding activity in vitro and in vivo. In tissue culture cells and in mutant animals, the levels of dE2F and dDP proteins are strongly interdependent. In the absence of dDP, the levels of dE2F1 and dE2F2 decline dramatically, and vice versa. Accordingly, the cell cycle and transcriptional phenotypes caused by targeting dDP mimic the effects of targeting both dE2F1 and dE2F2 and are indistinguishable from the effects of inactivating all three proteins. Although trans-heterozygous dDP mutant animals develop to late pupal stages, the analysis of somatic mutant clones shows that dDP mutant cells are at a severe proliferative disadvantage when compared directly with wild-type neighbors. Strikingly, the timing of S-phase entry or exit is not delayed in dDP mutant clones, nor is the accumulation of cyclin A or cyclin B. However, the maximal level of bromodeoxyuridine incorporation is reduced in dDP mutant clones, and RNA interference experiments show that dDP-depleted cells are prone to stall in S phase. In addition, dDP mutant clones contain reduced numbers of mitotic cells, indicating that dDP mutant cells have a defect in G2/M-phase progression. Thus, dDP is not essential for developmental control of the G1-to-S transition, but it is required for normal cell proliferation, for optimal DNA synthesis, and for efficient G2/M progression.

scholarly journals Analysis of the Dynein-Dynactin Interaction In Vitro and In Vivo

2003 ◽  
Vol 14 (12) ◽  
pp. 5089-5097 ◽  
Author(s):  
Stephen J. King ◽  
Christa L. Brown ◽  
Kerstin C. Maier ◽  
Nicholas J. Quintyne ◽  
Trina A. Schroer
Keyword(s):  
Cytoplasmic Dynein ◽  
Binding Assays ◽  
Microtubule Organization ◽  
Binding Domains ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Dynein Intermediate Chain ◽  
Transient Overexpression

Cytoplasmic dynein and dynactin are megadalton-sized multisubunit molecules that function together as a cytoskeletal motor. In the present study, we explore the mechanism of dynein-dynactin binding in vitro and then extend our findings to an in vivo context. Solution binding assays were used to define binding domains in the dynein intermediate chain (IC) and dynactin p150Glued subunit. Transient overexpression of a series of fragments of the dynein IC was used to determine the importance of this subunit for dynein function in mammalian tissue culture cells. Our results suggest that a functional dynein-dynactin interaction is required for proper microtubule organization and for the transport and localization of centrosomal components and endomembrane compartments. The dynein IC fragments have different effects on endomembrane localization, suggesting that different endomembranes may bind dynein via distinct mechanisms.

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