scholarly journals Comparison of Extracellular and Intracellular Potency of Botulinum Neurotoxins

2006 ◽  
Vol 74 (10) ◽  
pp. 5617-5624 ◽  
Author(s):  
Fang Cai ◽  
Carrie B. Adrion ◽  
James E. Keller
Keyword(s):  
Culture Medium ◽  
Reaction Model ◽  
Cultured Neurons ◽  
Total Inhibition ◽  
Botulinum Neurotoxins ◽  
A Cell ◽  
Proteolytic Activities ◽  
Living Neurons ◽  
Subsequent Addition

ABSTRACT Levels of botulinum neurotoxin (BoNT) proteolytic activity were compared using a cell-free assay and living neurons to measure extracellular and intracellular enzymatic activity. Within the cell-free reaction model, BoNT serotypes A and E (BoNT/A and BoNT/E, respectively) were reversibly inhibited by chelating Zn2+ with N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN). BoNT/E required relatively long incubation with TPEN to achieve total inhibition, whereas BoNT/A was inhibited immediately upon mixing. When naïve Zn2+-containing BoNTs were applied to cultured neurons, the cellular action of each BoNT was rapidly inhibited by subsequent addition of TPEN, which is membrane permeable. Excess Zn2+ added to the culture medium several hours after poisoning fully restored intracellular toxin activity. Unlike TPEN, EDTA irreversibly inhibited both BoNT/A and -E within the cell-free in vitro reaction. Excess Zn2+ did not reactivate the EDTA-treated toxins. However, application of EDTA-treated BoNT/A or -E to cultured neurons demonstrated normal toxin action in terms of both blocking neurotransmission and SNAP-25 proteolysis. Different concentrations of EDTA produced toxin preparations with incrementally reduced in vitro proteolytic activities, which, when applied to living neurons showed undiminished cellular potency. This suggests that EDTA renders the BoNT proteolytic domain conformationally inactive when tested with the cell-free reaction, but this change is corrected during entry into neurons. The effect of EDTA is unrelated to Zn2+ because TPEN could be applied to living cells before or after poisoning to produce rapid and reversible inhibition of both BoNTs. Therefore, bound Zn2+ is not required for toxin entry into neurons, and removal of Zn2+ from cytosolic BoNTs does not irreversibly alter toxin structure or function. We conclude that EDTA directly alters both BoNTs in a manner that is independent of Zn2+.

In Vitro Removal of Ochratoxin A by Wine Lactic Acid Bacteria

2007 ◽  
Vol 70 (9) ◽  
pp. 2155-2160 ◽  
Author(s):  
VINCENZO DEL PRETE ◽  
HECTOR RODRIGUEZ ◽  
ALFONSO V. CARRASCOSA ◽  
BLANCA de las RIVAS ◽  
EMILIA GARCIA-MORUNO ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Ochratoxin A ◽  
High Performance ◽  
Culture Medium ◽  
Degradation Products ◽  
Cell Binding ◽  
A Cell ◽  
In Vitro Interaction

A study was carried out to determine the in vitro interaction between ochratoxin A (OTA) and wine lactic acid bacteria (LAB). Fifteen strains belonging to five relevant oenological LAB species were grown in liquid synthetic culture medium containing OTA. The portion of OTA removed during the bacterial growth was 8 to 28%. The OTA removed from the supernatants was partially recovered (31 to 57%) from the bacterial pellet. Cell-free extracts of three representative strains were produced by disrupting cells in a French pressure cell. The ability of crude cell-free extracts to degrade OTA was studied. OTA was not degraded by cell-free extracts of wine LAB strains, and no degradation products of OTA were detected in the high-performance liquid chromatograms of the methanol extract of the bacterial pellet. On the basis of these results, we conclude that OTA removal by wine LAB is a cell-binding phenomenon. The chemistry and the molecular basis of OTA binding to wine LAB remains unknown.

In Vitro Biological Activity of Adriamycin

1970 ◽  
Vol 56 (3) ◽  
pp. 137-148 ◽  
Author(s):  
Rosella Silvestrini ◽  
Carmela Gambarucci ◽  
Teresa Dasdia
Keyword(s):  
Biological Activity ◽  
Cellular Proliferation ◽  
Therapeutic Index ◽  
Dna And Rna ◽  
Total Inhibition ◽  
A Cell ◽  
Nuclear Structures ◽  
Higher Doses

Adriamycin is an antibiotic, isolated from cultures of a mutant of Streptomyces peucetius, var. caesius, with a chemical structure very similar to daunomycin but with a higher therapeutic index in experimental tumors. The biological activity of this antibiotic has been studied in vitro on the HeLa cell strain. Adriamycin quickly penetrates into the cells and fixes to the nuclear structures with a marked localization at the level of the perinucleolar chromatin. It causes a marked and immediate disturbance of the mitotic process, viz. pre-prophasic inhibition at the low doses and mitotic block at the higher doses. Even the synthesis of DNA and RNA, evaluated autoradiographically as incorporation of 3H-thymidine and 3H-uridine, appear markedly inhibited. The viability of the cells, tested both as regards capacity to give rise to colonies and as regards proliferative activity of a cell population, was seriously reduced, in a degree proportional to the period of treatment and to the concentration of the antibiotic, until total inhibition. In comparison with daunomycin, adriamycin exerts an immediate antimitotic and anti-metabolic effect which, at equivalent doses, is slightly lower than that of daunomycin. The long-term antiproliferative activity on cellular proliferation is however, identical for the two antibiotics.

Silicate and Calcium Ions Releasing Biomaterials for Bone Reconstruction

2011 ◽  
Vol 493-494 ◽  
pp. 561-565
Author(s):  
Jin Nakamura ◽  
Akiko Obata ◽  
Julian R. Jones ◽  
Toshihiro Kasuga
Keyword(s):  
Calcium Ions ◽  
Culture Medium ◽  
Fetal Bovine Serum ◽  
Poly Lactic Acid ◽  
Cell Culture Medium ◽  
A Cell ◽  
Fetal Bovine ◽  
Mouse Osteoblast ◽  
Phosphate Deposits

Siloxane-containing vaterite (SiV) / poly (lactic acid) hybrid (SiPVH) beads with the releasability of silicate and calcium ions were prepared with an electrospraying method. According to the increase in the silicon content of the SiV, the amount of silicate ion released from the resulting beads also increased. When the beads were soaked in a cell culture medium, proteins derived from fetal bovine serum were adsorbed on their surfaces. Cell adhesion tests were also performed on the beads with using mouse osteoblast-like cell line (MC3T3-E1) in vitro. After 5 days of culturing, the cells adhered and spread well to cover the surface of the beads. In the localized area, agglomerated cells were observed to combine with cauliflower-shaped calcium phosphate deposits.

Heterogeneous glycosylation of cationic peanut peroxidase

1994 ◽  
Vol 72 (9-10) ◽  
pp. 411-417 ◽  
Author(s):  
Lianglu Wan ◽  
Mark Gijzen ◽  
Robert B. Van Huystee
Keyword(s):  
Ion Exchange ◽  
Culture Medium ◽  
Size Exclusion ◽  
Binding Property ◽  
In Vitro Experiments ◽  
Complex Type ◽  
A Cell ◽  
Glycan Heterogeneity ◽  
Peanut Peroxidase

Cationic peanut peroxidase (CPrx) from a cell suspension culture is N-glycosylated at Asn60, Asn144, and Asn185. All three N-glycans are complex type and galactose rich, and show heterogeneity in length and ConA (concanavalin A) binding property. The glycan heterogeneity causes a polymorphism of the enzyme. Based on its behavior on ConA columns, CPrx can be grouped into two fractions: nonbinding (CPrx−) and binding (CPrx+) types. A synchronously cosecreted β-galactosidase has been discovered in the culture medium; there are two isozymes of 60 kDa (pI 7.3) and 66 kDa (pI 7.6). This β-galactosidase has been partially purified by a combination of ion-exchange and size-exclusion chromatographies and preparative isoelectrofocusing. In vitro experiments indicate that the cosecreted β-galactosidase is able to convert peroxidase from CPrx− to CPrx+ and may, to some extent, contribute to the glycan heterogeneity of peroxidase in the cell culture.Key words: peroxidase, peanut, glycan, heterogeneity, β-galactosidase.

Differential Effects of Colchicine on Cardiac Cell Viability in an in vitro Model Simulating Myocardial Infarction

Cardiology ◽  
2016 ◽  
Vol 134 (1) ◽  
pp. 57-64 ◽  
Author(s):  
Gilad Margolis ◽  
Einat Hertzberg-Bigelman ◽  
Ran Levy ◽  
Jeremy Ben-Shoshan ◽  
Gad Keren ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Cell Line ◽  
Cardiac Cells ◽  
Sham Operation ◽  
H9c2 Cells ◽  
Myocardial Apoptosis ◽  
A Cell ◽  
Vitro Model ◽  
Subsequent Addition

Objectives: We aimed to examine the effects of colchicine, currently in clinical trials for acute myocardial infarction (AMI), on the viability of cardiac cells using a cell line model of AMI. Methods: HL-1, a murine cardiomyocyte cell line, and H9C2, a rat cardiomyoblast cell line, were incubated with TNFα or sera derived from rats that underwent AMI or sham operation followed by addition of colchicine. In another experiment, HL-1/H9C2 cells were exposed to anoxia with or without subsequent addition of colchicine. Cell morphology and viability were assessed by light microscopy, flow cytometry and Western blot analyses for apoptotic markers. Results: Cellular viability was similar in both sera; however, exposing both cell lines to anoxia reduced their viability. Adding colchicine to anoxic H9C2, but not to anoxic HL-1, further increased their mortality, at least in part via enhanced apoptosis. Under any condition, colchicine induced detachment of H9C2 cells from their culture plates. This phenomenon did not apply to HL-1 cells. Conclusions: Colchicine enhanced cardiomyoblast mortality under in vitro conditions mimicking AMI and reduced their adherence capability. HL-1 was not affected by colchicine; nevertheless, no salvage effect was observed. We thus conclude that colchicine may not inhibit myocardial apoptosis following AMI.

scholarly journals Ciliostatic, hemagglutinating, and proteolytic activities in a cell extract of Mycoplasma pneumoniae

1980 ◽  
Vol 29 (3) ◽  
pp. 1111-1116
Author(s):  
D K Chandler ◽  
M F Barile
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Mycoplasma Pneumoniae ◽  
Sodium Dodecyl ◽  
Cell Extract ◽  
Ciliary Activity ◽  
Organ Cultures ◽  
Freeze Thaw ◽  
A Cell ◽  
Proteolytic Activities

An extract of Mycoplasma pneumoniae, prepared from glass-grown organisms by extraction with 2 M NaCl, followed by freeze-thaw, ultracentrifugation, dialysis, and lyophilization, yielded approximately 20% of the total mycoplasmal protein. The extract contained at least 20 protein bands on sodium dodecyl sulfate-polyacrylamide gels and 2 to 5% carbohydrate and inhibited 70 to 100% of the ciliary activity of hamster tracheal organ cultures (ciliostasis). The extent of ciliostasis was dependent on the concentration of the extract. The extract also produced hemagglutination of human O-positive erythrocytes and showed proteolytic activity with a synthetic tetrapeptide substrate, S-2222. These in vitro tissue-damaging activities may be associated with the virulence of the mycoplasmas and with the pathogenesis of M. pneumoniae disease.

scholarly journals Retrovirally transduced antisense sequences stably suppress P210BCR-ABL expression and inhibit the proliferation of BCR/ABL-containing cell lines

Blood ◽  
1993 ◽  
Vol 81 (2) ◽  
pp. 502-509 ◽  
Author(s):  
P Martiat ◽  
P Lewalle ◽  
AS Taj ◽  
M Philippe ◽  
Y Larondelle ◽  
...  
Keyword(s):  
Culture Medium ◽  
Philadelphia Chromosome ◽  
K562 Cells ◽  
Cellular Growth ◽  
Growth Properties ◽  
Bone Marrow Cultures ◽  
A Cell ◽  
Oncogenic Protein ◽  
B10 Cells

Abstract There is now strong evidence that the BCR-ABL gene product of the Philadelphia chromosome (P210) plays a crucial role in the pathogenesis of chronic myeloid leukemia (CML). We have previously shown that introduction of antisense oligonucleotides into K562 cells could transiently block the expression of P210 and specifically inhibit cellular growth in culture. In this report, we describe the use of a retroviral vector to introduce selected antisense and sense sequences, first into murine B10 cells, previously rendered interleukin-3 (IL-3) independent by transfection of BCR-ABL sequences, and second into K562 cells. The antisense transcripts generated under the control of MoMLV promoter specifically killed B10 cells in the absence of IL-3 and inhibited P210 expression almost completely. In K562 cells, the antisense sequences led to a dramatic reduction of P210 expression and increased their doubling time by more than twofold. This effect was not reversed by the addition of exogenous IL-3 to the culture medium. Control HeLa or HL60 cells infected with the same constructs did not show any change in proliferation rate, despite abrogation of the normal BCR gene products. Rather unexpectedly, P210 suppression was not lethal in K562 cells, showing that such a cell line does not rely entirely on the expression of P210 for surviving, but depends on it as far as growth properties are concerned. We conclude that this approach can successfully achieve stable suppression of the oncogenic protein P210 and may be used to study further the mechanisms by which P210 is transforming cells. The effect on fresh CML cells in bone marrow cultures remains to be assessed before we can tell whether this technique may be used for selective suppression of leukemic hematopoiesis in vitro.

Characterization of fullerene colloidal suspension in a cell culture medium for in vitro toxicity assessment

2010 ◽  
Vol 6 (7) ◽  
pp. 1238 ◽  
Author(s):  
Haruhisa Kato ◽  
Naohide Shinohara ◽  
Ayako Nakamura ◽  
Masanori Horie ◽  
Katsuhide Fujita ◽  
...  
Keyword(s):  
Cell Culture ◽  
Culture Medium ◽  
Colloidal Suspension ◽  
Toxicity Assessment ◽  
In Vitro Toxicity ◽  
Cell Culture Medium ◽  
A Cell ◽  
In Vitro Toxicity Assessment

scholarly journals Retrovirally transduced antisense sequences stably suppress P210BCR-ABL expression and inhibit the proliferation of BCR/ABL-containing cell lines

Blood ◽  
1993 ◽  
Vol 81 (2) ◽  
pp. 502-509
Author(s):  
P Martiat ◽  
P Lewalle ◽  
AS Taj ◽  
M Philippe ◽  
Y Larondelle ◽  
...  
Keyword(s):  
Culture Medium ◽  
Philadelphia Chromosome ◽  
K562 Cells ◽  
Cellular Growth ◽  
Growth Properties ◽  
Bone Marrow Cultures ◽  
A Cell ◽  
Oncogenic Protein ◽  
B10 Cells

There is now strong evidence that the BCR-ABL gene product of the Philadelphia chromosome (P210) plays a crucial role in the pathogenesis of chronic myeloid leukemia (CML). We have previously shown that introduction of antisense oligonucleotides into K562 cells could transiently block the expression of P210 and specifically inhibit cellular growth in culture. In this report, we describe the use of a retroviral vector to introduce selected antisense and sense sequences, first into murine B10 cells, previously rendered interleukin-3 (IL-3) independent by transfection of BCR-ABL sequences, and second into K562 cells. The antisense transcripts generated under the control of MoMLV promoter specifically killed B10 cells in the absence of IL-3 and inhibited P210 expression almost completely. In K562 cells, the antisense sequences led to a dramatic reduction of P210 expression and increased their doubling time by more than twofold. This effect was not reversed by the addition of exogenous IL-3 to the culture medium. Control HeLa or HL60 cells infected with the same constructs did not show any change in proliferation rate, despite abrogation of the normal BCR gene products. Rather unexpectedly, P210 suppression was not lethal in K562 cells, showing that such a cell line does not rely entirely on the expression of P210 for surviving, but depends on it as far as growth properties are concerned. We conclude that this approach can successfully achieve stable suppression of the oncogenic protein P210 and may be used to study further the mechanisms by which P210 is transforming cells. The effect on fresh CML cells in bone marrow cultures remains to be assessed before we can tell whether this technique may be used for selective suppression of leukemic hematopoiesis in vitro.

scholarly journals Noninvasive Superoxide Monitoring ofIn VitroNeuronal Differentiation Using a Cell-Based Biosensor

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Georgia Moschopoulou ◽  
Spyridon Kintzios
Keyword(s):  
Superoxide Dismutase ◽  
Membrane Potential ◽  
Cell Membrane ◽  
Neuronal Differentiation ◽  
Superoxide Anion ◽  
Culture Medium ◽  
Ultrasensitive Detection ◽  
A Cell

Membrane-engineered cells bearing superoxide dismutase (SOD) molecules on their surface offer the capability of ultrarapid and ultrasensitive detection of the superoxide anion (O2-) through the measurement of changes of their cell membrane potential. We herewith report the application of this technology for the noninvasive determination of superoxide levels during thein vitrodifferentiation of PC12 cells. We were able to detect changes inO2-accumulation in the culture medium, which were closely associated with the progress of neuronal differentiation.

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