Mechanism of cystine reaccumulation by cystinotic fibroblasts in vitro

1990 ◽  
Vol 10 (2) ◽  
pp. 225-229 ◽  
Author(s):  
Susan Forster ◽  
Lynne Scarlett ◽  
John B. Lloyd
Keyword(s):  
Plasma Membrane ◽  
Culture Medium ◽  
Human Fibroblasts ◽  
Competitive Inhibitor ◽  
Free Medium ◽  
Lysosome Membrane ◽  
Putative Mechanism

It is well established that when cystine-depleted cystinotic cells are cultured in cystine-containing medium, they reaccumulate cystine within their lysosomes more rapidly than when cultured in cystine-free medium. This has been a puzzling result, since the lysosome membrane of cystinotic cells is impermeable to cystine. To probe the mechanism of cystine reaccumulation, we have measured reaccumulation in the presence of colchicine, an inhibitor of pinocytosis, or of glutamate, a competitive inhibitor of cystine transport into human fibroblasts. Colchicine had no effect, thus eliminating pinocytosis as a putative mechanism for cystine translocation from the culture medium to the lysosomes. Glutamate, however, strongly inhibited cystine reaccumulation. It is concluded that the true mechanism is as follows. 1. Exogenous cystine crosses the plasma membrane on the cystine-glutamate porter. 2. Cystine is reduced in the cytoplasm by GSH. 3. The cysteine that is generated enters the lysosome, where it becomes cystine by participating in the reduction of cystine residues during intralysosomal proteolysis, or by autoxidation.

scholarly journals Effect of Lignocaine Concentration on Human Fibroblasts Growth in Eyes Undergoing Trabeculectomy: An in vitro Study

2018 ◽  
Vol 3 (3) ◽  
pp. 1-10 ◽  
Author(s):  
Madhuravasal Krishnan Janani ◽  
Venkatakrishnan Jaichandran ◽  
Hajib Narahari Rao Madhavan ◽  
Lingam Vijaya ◽  
Ronnie Jacob George ◽  
...  
Keyword(s):  
Culture Medium ◽  
In Vitro Model ◽  
Morphological Changes ◽  
Human Fibroblasts ◽  
Annexin V ◽  
In Vitro Study ◽  
Tenon’S Capsule ◽  
Tenon's Capsule ◽  
Vitro Model

Purpose: To evaluate the effect of lignocaine on growth and apoptosis indication of primary human Tenon’s capsule fibroblast (HTFs) in an in vitro model. Patients and Methods: Tenon’s capsule tissue obtained from patients undergoing trabeculectomy were grown in cell culture medium. The effect of different concentrations of lignocaine (0.5, 1.0, 1.5, and 2%) on the morphology and growth of the fibroblasts was studied using microscopy, cell viability, and proliferation assay, and apoptosis was detected using the FITC Annexin V Apoptosis Kit. Results: Morphological changes similar to those of apoptotic cells, including cytoplasmic vacuolation, shrinkage, and rounding were visualized in the cells treated with concentrations greater than 1.0% (i.e., 1.5, 2.0%). Though proliferation inhibition was found with all four concentrations (0.5–2.0%), the viability of cells decreased from 1.0% lignocaine. Conclusion: 0.5% lignocaine prevents proliferation of fibroblasts without causing apoptosis in vitro.

Fasciola hepatica in vitro: increased susceptibility to fasciolicides in a defined serum-free medium

Parasitology ◽  
1987 ◽  
Vol 95 (1) ◽  
pp. 165-171 ◽  
Author(s):  
D. C. Jenkins ◽  
P. Topley ◽  
E. B. Rapson
Keyword(s):  
Culture Medium ◽  
Fasciola Hepatica ◽  
Liver Microsomes ◽  
Metabolic Activation ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Highly Active ◽  
Mammalian Liver

SUMMARYThe cidal properties of some phenolic, halogenated diphenyl, salicylanilide, benzimidazole and diaminophenoxyalkane anthelmintics, against 6-week-old worms of Fasciola hepatica were assessed in vitro. In a conventional fluke culture medium containing RPMI 1640, supplemented with serum with or without rabbit erythrocytes or pink-ghosts, only the halogenated diphenyl and salicylanilide compounds showed activity at concentrations equal to or less than 100 μm. However, when basal, serum and cell-free RPMI 1640 was used, all compounds other than diamphenethide were highly active, their minimum lethal concentrations being some 25–125 times lower under these conditions. The inclusion of rabbit liver microsomes in the basal culture medium resulted in diamphenethide exhibiting cidal activity equivalent to that seen when its free-amine active metabolite was assayed. The possibility that the activity of many of these compounds was masked in vitro because of their serum binding properties is discussed. Recommendations are made that in vitro screens for new fasciolicides should be carried out in serum-free medium and that additional replicates containing mammalian liver microsomes and liver cytosolic extracts be included as means for the metabolic activation of certain otherwise undetectable prodrugs.

scholarly journals Propagation of edible honeysuckle (Lonicera edulis Turcz) in in vitro conditions

2018 ◽  
Vol 5 (2) ◽  
pp. 18-26
Author(s):  
Ya. Zapolsky ◽  
T. Medvedeva ◽  
T. Natalchuk ◽  
M. Bublyk
Keyword(s):  
Culture Medium ◽  
Positive Impact ◽  
Mercury Chloride ◽  
Free Medium ◽  
Aseptic Culture ◽  
Effi Ciency ◽  
Comparison Results ◽  
The Impact

Aim. To propagate edible honeysuckle (Lonicera edulis Turcz) in in vitro conditions; to study the impact of sterilization agents on honeysuckle explants; to investigate the impact of the culture medium composition on the coeffi cient of propagation and rooting; to study the capability to adapt to in vivo conditions. Methods. Laboratory, mathematical, estimation and comparison. Results. The impact of sterilizing substances on obtain- ing the aseptic culture of edible honeysuckle in in vivo conditions was studied. The experiments were con- ducted on the following species: Alicia, Spokusa, Chaika, Nimfa, Doch Velikana, Karina. Lisoformin 3000 and mercury chloride were used as sterilizing agents. In the variant with Lisoformin 3000 it was studied in three exposures – 5, 7, and 10 minutes. In terms of explant regeneration effi ciency after sterilization with Lisofor- min 3000, three groups of edible honeysuckle species were isolated: 1 – with high regeneration capacity (94– 96 %) – Alicia, Karina and Spokusa; 2 – medium capacity (86–87 %) – Chaika and Doch Velikana, 3 – low capacity (80 %) – Nimfa. The experiments aimed at studying the impact of culture medium components on the propagation effi ciency determined the increase in the latter in case of rotating media with different quantitative and qualitative composition. Permanent application of uniform media leads to a sharp decrease in the prolif- eration coeffi cient in all the investigated species. Both hormone-free medium and the medium with growth regulators are effi cient for rooting. High indices of rooting were achieved in both variants. The use of auxins promoted the formation of a larger amount of plant roots (from 3.09 in Spokusa to 4.21 in Alicia) which in its turn impacted the survivability of plants in in vivo conditions. Conclusions. It was established that Lisoformin 3000 in the concentration of 3 % and at the exposure duration of 5 min ensured optimal effi ciency of steriliza- tion and regeneration of edible honeysuckle explants and did not decrease their propagation coeffi cients. With corresponding concentrations and sterilization duration, this preparation may be recommended for obtaining the aseptic culture of honeysuckle. It was demonstrated that the rotation of media, rich and poor in macro- and microsalts was effi cient for obtaining high indices of proliferation: the plants had a larger amount of tillering even in case of using not high concentrations of cytokinin. The introduction of rhizogenesis inducer, IBA, (1 mg/l) into the culture medium did not increase the percentage of rooted plants compared to hormone-free medium, but stimulated the formation of a larger amount of roots, which had further positive impact on the adaptation properties.

scholarly journals Pineapple (Ananas comosus) cv. pérola ex vitro growth and mycorrhizal colonization affected by in vitro sucrose concentration

2014 ◽  
Vol 36 (3) ◽  
pp. 766-770 ◽  
Author(s):  
Alceu Kunze ◽  
Paulo Emílio Lovato ◽  
Murilo Dalla Costa ◽  
Lírio Luiz Dal Vesco
Keyword(s):  
Root Biomass ◽  
Culture Medium ◽  
Plant Biomass ◽  
Sucrose Concentration ◽  
Mycorrhizal Colonization ◽  
Ananas Comosus ◽  
Free Medium ◽  
Rhizophagus Clarus ◽  
Ex Vitro Growth

The aim of this study was to evaluate the effect of sucrose concentration in the culture medium on growth and on the establishment of mycorrhizas during the acclimatization of pineapple cv. Pérola. The plantlets were micropropagated in MS culture medium with 0, 10, 20 and 30 g L-1 of sucrose and then they were acclimatized during 12 weeks under greenhouse conditions, in a sandy soil - compost mixture, uninoculated or inoculated with a Rhizophagus clarus isolate. Plantlets from the culture medium with 20 g and 30 g of sucrose L-1 showed higher shoot and root biomass than those from sugar-free medium. Mycorrhizal colonization was lower in plantlets micropropagated in sucrose-free medium, but the intensity of arbuscules did not differ among treatments. In the 12-week period of acclimatization, mycorrhizal colonization had no effect on plant biomass.

Porcine oocyte maturation in vitro under different conditions

2006 ◽  
Vol 3 (2) ◽  
pp. 95-100
Author(s):  
Hu Jun-He ◽  
Yang Chun-Rong ◽  
Dou Zhong-Ying
Keyword(s):  
Oocyte Maturation ◽  
Culture Medium ◽  
Follicle Stimulating Hormone ◽  
Human Chorionic Gonadotrophin ◽  
Serum Free Medium ◽  
Free Medium ◽  
Maturation In Vitro ◽  
Porcine Oocyte ◽  
Pregnant Mare Serum Gonadotrophin

AbstractThe effects of hormone additions at various stages and different basic media, with or without serum, on porcine oocyte maturation in vitro were studied. The results showed that the rate of maturation was not significantly different with three different stages of hormone supplement; the rate of maturation on modified TCM199 medium (54.01%) was higher than that on TCM199 (46.16%) and (47.14%), but these differences were not significant; and the rate of maturation on serum-free medium (67.10%) was significantly higher than that on medium plus serum (52.22%). Therefore, modifed tissue culture medium 199 (mTCM199)+10I U/ml pregnant mare serum gonadotrophin (PMSG)+10I U/ml human chorionic gonadotrophin (hCG)+2.5 IU/ml follicle stimulating hormone (FSH) was a suitable medium for culture of porcine oocytes in vitro, and the rate of maturation was 67.10%.

scholarly journals Induction of cystine transport activity in mouse peritoneal macrophages.

1987 ◽  
Vol 165 (3) ◽  
pp. 628-640 ◽  
Author(s):  
H Watanabe ◽  
S Bannai
Keyword(s):  
Amino Acids ◽  
Culture Medium ◽  
Glutamate Uptake ◽  
Human Fibroblasts ◽  
Peritoneal Macrophages ◽  
Transport Activity ◽  
Drastic Increase ◽  
Uptake Activity ◽  
Mouse Peritoneal Macrophages

Uptake of cystine was investigated in mouse peritoneal macrophages. The rates of the uptake of cystine in resident macrophages or macrophages elicited by some irritants were very low, but a drastic increase was observed when the cells were cultured in vitro. This increase was time-dependent and required protein synthesis. In macrophages elicited by thioglycollate broth, the rate of the uptake of cystine increased by about 40-fold after 16 h in culture. Contrary to the uptake of cystine, the rates of uptake of some neutral amino acids did not change markedly during culture. We characterized the induced activity of the cystine uptake in macrophages elicited by thioglycollate broth. Cystine was taken up in an Na+-independent and pH-sensitive manner, and the uptake was potently inhibited by extracellular glutamate and the analogous anionic amino acids, but not by aspartate. The activity of the glutamate uptake was also induced during the culture in a way similar to that of cystine uptake, and the uptake of glutamate was potently inhibited by cystine. From these results we concluded that the uptake of cystine and glutamate in macrophages was mostly mediated by a single transport system similar to the ones previously reported in human fibroblasts and some other cells. As a consequence of the induction of the activity of the cystine uptake, glutathione levels in macrophages doubled during culture, and a thiol compound, presumably cysteine, was released into the culture medium and accumulated there. When the macrophages were cultured hypoxically, the induction of the cystine uptake activity was markedly depressed, suggesting an involvement of oxygen in the induction.

scholarly journals A quantitative study of pinocytosis and intracellular proteolysis in rat peritoneal macrophages

1977 ◽  
Vol 168 (3) ◽  
pp. 365-372 ◽  
Author(s):  
M K Pratten ◽  
K E Williams ◽  
J B Lloyd
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Culture Medium ◽  
Peritoneal Macrophages ◽  
The Other ◽  
High Rate ◽  
Intracellular Proteolysis ◽  
Pinocytic Vesicles ◽  
Stimulation Of

A method for the culture of rat peritoneal macrophages in vitro is described, in which pinocytic uptake of colloidal [198 Au]gold, 125I--labelled poly(vinylpyrrolidone) and [14C]sucrose proceeds at contant and fairly reproducible rates for several hours. The rat of uptake of colloidal [198 Au]gold, which wxhibited some inter-batch variation, was approx. 100 times that of the other two substrates. Colloidal gold did not affect the rate of uptake of 125I-labelled poly(vinylpyrrolidone) and therefore its own high rate of uptake could not be attributed to a stimulation of the formation of pinocytic vesicles. It conclude that uptake of collodial gold is highly dependent on adsorption on binding sites on the plasma membrane. Uptake of formaldehyde-treated 125I-labelled bovine serum albumin was followed by the release of [125I]iodo-L-tyrosine into the culture medium and took place at a rate intermediate between those of collodial [198Au]gold and the other two non-digestible substrates, 125I-labelled poly(vinylpyrrolidone) and [14C]sucrose.

scholarly journals THE REQUIREMENT FOR HYDROCORTISONE IN ANTIBODY-FORMING TISSUE CULTIVATED IN SERUM-FREE MEDIUM

1964 ◽  
Vol 119 (6) ◽  
pp. 1027-1049 ◽  
Author(s):  
Charles T. Ambrose
Keyword(s):  
Culture Medium ◽  
Essential Amino Acids ◽  
Secondary Response ◽  
Rabbit Serum ◽  
Normal Rabbit Serum ◽  
Secondary Antibody ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free

It was previously reported from this laboratory that the secondary antibody response can regularly be elicited in vitro from fragments of rabbit lymph node node cultured in Eagle's medium supplemented with normal rabbit serum. Evidence is now presented that physiological levels of hydrocortisone (0.01 to 1.0 µM) can substitute for serum in the culture medium. However, with the omission of serum, serine (0.1 mM) must be included among Eagle's "essential" amino acids for consistent optimal antibody production. In some experiments the addition of insulin (0.5 unit/ml) and vitamin B12 (0.5 µg/ml) has further enhanced the secondary response in this serum-free medium.

scholarly journals Differential Growth Characteristics and Streptomycin Susceptibility of Virulent and Avirulent Mycobacterium tuberculosis Strains in a Novel Fibroblast-Mycobacterium Microcolony Assay

1998 ◽  
Vol 66 (11) ◽  
pp. 5132-5139 ◽  
Author(s):  
Thomas F. Byrd ◽  
Gary M. Green ◽  
Sharon E. Fowlston ◽  
C. Rick Lyons
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Culture Medium ◽  
Agar Medium ◽  
Human Fibroblasts ◽  
Differential Growth ◽  
Vitro Assay ◽  
Virulence Determinant ◽  
Virulent Strains ◽  
Cell Spread

ABSTRACT The ability to spread from cell to cell may be an important virulence determinant of Mycobacterium tuberculosis. An in vitro assay was developed to characterize this ability among four strains of M. tuberculosis: the attenuated strain H37Ra, the virulent strains H37Rv and Erdman, and a virulent clinical isolate (Stew). Confluent monolayers of human skin fibroblasts were infected with these strains and overlaid with agar-medium. M. tuberculosis infection developed over 21 days as microcolonies originating within the plane of the fibroblasts. Microcolonies of the virulent strains had an elongated appearance and exhibited extensive cording. The cords appeared to invade adjacent cells within the plane of the monolayer. Microcolony diameter of the Erdman strain was significantly larger than that of the other virulent strains, indicating that virulent strains can have distinguishing phenotypes in this assay. In contrast, avirulent H37Ra microcolonies were rounded and noncorded. H37Ra microcolonies were significantly smaller than those of the virulent strains. Microcolony diameter of the virulent strains was not reduced by the extracellularly acting antibiotic streptomycin at concentrations of up to 5.0 μg/ml. In contrast, H37Ra microcolony size was reduced at concentrations as low as 0.5 μg/ml. Growth of all strains was similarly inhibited by 1.0 μg of streptomycin per ml in fibroblast-conditioned tissue culture medium alone. When fibroblasts were infected with the M. tuberculosis strains without an agar overlay, with and without streptomycin, numbers of CFU mirrored the changes observed in the microcolony assay. There was a statistically significant decrease in H37Ra CFU compared to virulent strains after treatment with streptomycin. These differences between H37Ra and virulent strains in human fibroblasts suggest that H37Ra may be lacking a virulence determinant involved in cell-to-cell spread ofM. tuberculosis.

In Vitro Benchmark of Cytokine Removal by Dialyzers with Various Permeability Profiles

2017 ◽  
Vol 40 (11) ◽  
pp. 615-621 ◽  
Author(s):  
Michael Hulko ◽  
Rose Speidel ◽  
Julia Gauss ◽  
Markus Storr ◽  
Bernd Krause
Keyword(s):  
Cell Activation ◽  
Culture Medium ◽  
Human Fibroblasts ◽  
Cardiovascular Complications ◽  
Enzyme Linked Immunosorbent Assay ◽  
In Vitro Test ◽  
Removal Capacity ◽  
Tnf Α ◽  
Vitro Test

Purpose Removal of cytokines is relevant for dialysis patients as they are suspected to promote cardiovascular complications. The objective of this study was to benchmark membranes with different permeability profiles under standardized in vitro test conditions using miniaturized devices with respect to their ability to remove cytokines from human serum and to lower cell activating potential. Methods In vitro dialysis was used to dialyze cytokine enriched serum in 3 independent experiments per tested membrane. IL-6 in the serum and dialysate was measured at defined times by enzyme-linked immunosorbent assay. IL-8, IL-1β, IL-6 and TNF-α in dialysate were measured by immunoassay. Dialysate samples were subjected to cultured tubular epithelial cells or human fibroblasts to study cell activation via IL-6 generation. Dialysate samples were added to human whole blood with subsequent analysis of granulocyte and monocyte activation by detection of CD11b. Results IL-6 decreased in serum and increased in dialysate during in vitro dialysis. IL-8, IL-1β, and TNF-α were identified in dialysate. Dialysate added to cell cultures increased IL-6 concentration in culture medium or increased expression of CD11b. High cut-off membranes showed the strongest transfer of cytokines, albumin and total proteins from serum to dialysate and led to strongest cell activation. This effect was lower for medium cutoff membranes and lowest for conventional high-flux membranes. Conclusions This study demonstrated an in vitro test by which membranes were benchmarked with respect to cytokine and cell activation removal capacity. Cell activation levels could be influenced by the choice of membrane by altering cytokine concentration levels.

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