scholarly journals Coupling of proadipocyte growth arrest and differentiation. I. Induction by heparinized medium containing human plasma.

1982 ◽  
Vol 94 (2) ◽  
pp. 394-399 ◽  
Author(s):  
B R Krawisz ◽  
R E Scott
Keyword(s):  
Human Plasma ◽  
Adipocyte Differentiation ◽  
Growth Arrest ◽  
Culture Conditions ◽  
Cell Contact ◽  
Low Density ◽  
Prolonged Culture ◽  
Extensive Cell ◽  
High Concentrations

The differentiation of proadipocytes in vitro typically required prolonged culture of cells as a high density in high concentrations of serum and added hormones. With such culture conditions it is difficult to design experiments to determine the mechanisms that control the differentiation process. We now describe the rapid and parasynchronous growth arrest and differentiation of low density murine proadipocytes in heparinized medium containing only human plasma. When low density cells are cultured under these conditions, growth arrest at a distinct state in the G1 phase of the cell cycle occurs within 2 d and the differentiation of 80-100% of the cell population occurs within 4 d thereafter. The factors in human plasma which promote growth arrest and differentiation are heat labile and can be separated by barium adsorption. In the following paper we have used these methods to show that there are five separate phases which regulate the coupling of proadipocyte growth arrest and differentiation. The data reported in this paper establish that: (a) high cell density and extensive cell-to-cell contact are not required for adipocyte differentiation, (b) prolonged culture is not required for adipocyte differentiation, and (c) high concentrations of serum and/or added hormones are not required for adipocyte differentiation.

Observations on the in vitro Effects of Chylomicra, Low-Density Lipoproteins and Phospholipids on Human Plasma Euglobulin Lysis

1963 ◽  
Vol 09 (01) ◽  
pp. 164-174 ◽  
Author(s):  
Albert R Pappenhagen ◽  
J. L Koppel ◽  
John H Olwin
Keyword(s):  
Human Plasma ◽  
Phosphatidyl Ethanolamine ◽  
Phosphatidyl Inositol ◽  
Plasma Lipoproteins ◽  
Low Density ◽  
Inhibitory Effects ◽  
Soybean Phospholipids ◽  
In Vitro Effects ◽  
Complete Precipitation

SummaryData have been presented on the in vitro effects of human chylomicra, low-density human plasma lipoproteins, and partially purified preparations of various phospholipids on human plasma euglobulin lysis. Euglobulin lysis was found to be accelerated by preparations of mixed soybean phospholipids (aso-lectin), cephalin, phosphatidyl inositol, phophatidyl serine and phosphatidyl ethanolamine. In contrast, it was found to be inhibited by preparations of human chylomicra, low-density human plasma liproproteins and lecithin. Inhibition of euglobulin lysis produced by any of these three agents could be diminished or completely overcome by the simultaneous presence of suitable levels of any one of the accelerating agents. In all cases studied, both inhibitory and accelerating effects were observed to be concentration-dependent. Evidence has been obtained to suggest that in the case of the accelerating agents the observed increased rate of euglobulin lysis is not a direct effect on lysis itself, but rather is due to more complete precipitation of plasminogen in the presence of these substances. On the other hand, it appears that the inhibitory effects observed are not related to the extent of plasminogen precipitation, but are actually true inhibitions of euglobulin lysis. The possible clinical significance of some of these observations has been briefly discussed.

scholarly journals A KINETIC ANALYSIS OF MYOGENESIS IN VITRO

1972 ◽  
Vol 52 (1) ◽  
pp. 52-65 ◽  
Author(s):  
Michael C. O'Neill ◽  
Frank E. Stockdale
Keyword(s):  
Cell Cycle ◽  
Cell Fusion ◽  
Tritiated Thymidine ◽  
Culture Conditions ◽  
Complete Fusion ◽  
Low Density ◽  
A Cell ◽  
Muscle Cultures ◽  
S Period

Conditions which yielded reproducible growth kinetics with extensive, relatively synchronous differentiation are described for chick muscle cultures. The effects of cell density and medium changes on the timing of cell fusion were examined. Low-density cultures which received a change of medium at 24 hr after plating show the highest rate of cell fusion, increasing from 15 to 80% fused cells in a 10 hr period. These optimal culture conditions were employed to reexamine two questions from the earlier literature on muscle culture: (a) can cells which normally would fuse at the end of one cell cycle be forced to go through another cell cycle before fusion; and (b) how soon after its final S period can a cell complete fusion? In answer to the first question, it was found that if the medium is changed, many cells which would otherwise fuse can be made to undergo another cell cycle before fusion. In the second case, radioautographs were made from cultures incubated with tritiated thymidine for various times at the beginning of the fusion period. These show labeled nuclei in myotubes as early as 3 hr after the beginning of the incubation period. This indicates that cells can fuse as early as the beginning of the G1 period, and suggests that there is not an obligatory exit from the cell cycle or a prolonged G1 period before cell fusion and differentiation during myogenesis.

Studies of the ceruloplasmin-lactoferrin complex

2002 ◽  
Vol 80 (1) ◽  
pp. 35-39 ◽  
Author(s):  
Maria O Pulina ◽  
Elena T Zakharova ◽  
Alexei V Sokolov ◽  
Mikhail M Shavlovski ◽  
Mikhail G Bass ◽  
...  
Keyword(s):  
Breast Milk ◽  
Human Plasma ◽  
Bacterial Lipopolysaccharide ◽  
Human Lactoferrin ◽  
Present Evidence ◽  
High Concentrations

We have previously shown that iron-containing human lactoferrin (LF) purified from breast milk is able to form both in vitro and in vivo a complex with ceruloplasmin (CP), the copper-containing protein of human plasma. Here we present evidence that the CP–LF complex is dissociated by high concentrations of NaCl, CaCl2, or EDTA, or by decreasing the pH to 4.7. In addition, DNA, bacterial lipopolysaccharide, and heparin can displace CP from its complex with LF. Antibodies to either of the two proteins also cause dissociation of the complex.Key words: lactoferrin, ceruloplasmin, ferroxidase.

scholarly journals Association of the Endotoxin Antagonist E5564 with High-Density Lipoproteins In Vitro: Dependence on Low-Density and Triglyceride-Rich Lipoprotein Concentrations

2003 ◽  
Vol 47 (9) ◽  
pp. 2796-2803 ◽  
Author(s):  
Kishor M. Wasan ◽  
Olena Sivak ◽  
Richard A. Cote ◽  
Aaron I. MacInnes ◽  
Kathy D. Boulanger ◽  
...  
Keyword(s):  
Human Plasma ◽  
Human Subjects ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
High Density ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Distribution Profile ◽  
Transfer Activity

ABSTRACT The objective of this study was to determine the distribution profile of the novel endotoxin antagonist E5564 in plasma obtained from fasted human subjects with various lipid concentrations. Radiolabeled E5564 at 1 μM was incubated in fasted plasma from seven human subjects with various total cholesterol (TC) and triglyceride (TG) concentrations for 0.5 to 6 h at 37°C. Following these incubations, plasma samples were separated into their lipoprotein and lipoprotein-deficient fractions by ultracentrifugation and were assayed for E5564 radioactivity. TC, TG, and protein concentrations in each fraction were determined by enzymatic assays. Lipoprotein surface charge within control and phosphatidylinositol-treated plasma and E5564’s influence on cholesteryl ester transfer protein (CETP) transfer activity were also determined. We observed that the majority of E5564 was recovered in the high-density lipoprotein (HDL) fraction. We further observed that incubation in plasma with increased levels of TG-rich lipoprotein (TRL) lipid (TC and TG) concentrations resulted in a significant increase in the percentage of E5564 recovered in the TRL fraction. In further experiments, E5564 was preincubated in human TRL. Then, these mixtures were incubated in hypolipidemic human plasma for 0.5 and 6 h at 37°C. Preincubation of E5564 in purified TRL prior to incubation in human plasma resulted in a significant decrease in the percentage of drug recovered in the HDL fraction and an increase in the percentage of drug recovered in the TRL and low-density lipoprotein fractions. These findings suggest that the majority of the drug binds to HDLs. Preincubation of E5564 in TRL prior to incubation in normolipidemic plasma significantly decreased the percentage of drug recovered in the HDL fraction. Modifications to the lipoprotein negative charge did not alter the E5564 concentration in the HDL fraction. In addition, E5564 does not influence CETP-mediated transfer activity. Information from these studies could be used to help identify the possible components of lipoproteins which influence the interaction of E5564 with specific lipoprotein particles.

Role of GADD153 (growth arrest- and DNA damage-inducible gene 153) in vascular smooth muscle cell apoptosis

2001 ◽  
Vol 100 (3) ◽  
pp. 275-281 ◽  
Author(s):  
Michiya IGASE ◽  
Takafumi OKURA ◽  
Michitsugu NAKAMURA ◽  
Yasunori TAKATA ◽  
Yutaka KITAMI ◽  
...  
Keyword(s):  
Dna Damage ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Molecular Mechanisms ◽  
Growth Arrest ◽  
Cell Contact ◽  
Inducible Gene

GADD153 (growth arrest- and DNA damage-inducible gene 153) is expressed at very low levels in growing cells, but is markedly induced in response to a variety of cellular stresses, including glucose deprivation, exposure to genotoxic agents and other growth-arresting situations. Forced expression of GADD153 induces cell cycle arrest in many types of cells. It is also reported that GADD153 is directly associated with apoptosis. Recently we have reported that platelet-derived growth factor (PDGF)-BB induces apoptosis in cultured vascular smooth muscle cells (VSMC), but only when 100% confluency is reached. These results suggested that cell–cell contact inhibition (cell growth arrest) may be a critical factor for induction of VSMC apoptosis by PDGF-BB. In the present study, we explored the role of GADD153, one of a number of growth-arrest-related gene products, in the molecular mechanisms of VSMC apoptosis in vitro and in vivo. GADD153 was markedly induced at both the mRNA and protein levels, in parallel with the induction of VSMC apoptosis, after treatment with PDGF-BB. Moreover, overexpression of GADD153 in VSMC significantly reduced cell viability and induced apoptosis. In the carotid artery balloon injury model in rats, GADD153 protein was expressed in apoptotic VSMC which were positively stained by in situ DNA labelling. These results demonstrate an important role for GADD153 in the molecular mechanisms of VSMC apoptosis.

The use of metformin to enhance radiation effects on M1 macrophage subtype polarization in bone marrow-derived macrophage in glioblastoma tumor environment.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e13513-e13513
Author(s):  
Fei Wang ◽  
Nan Zhao ◽  
Chi Lin ◽  
Chi Zhang
Keyword(s):  
Bone Marrow ◽  
Tumor Microenvironment ◽  
Radiation Effects ◽  
Culture Conditions ◽  
Mouse Bone Marrow ◽  
M1 Macrophage ◽  
Tumor Environment ◽  
In Trans ◽  
High Concentrations

e13513 Background: Given the clinical relevance of tumor-associated macrophage (TAM) with its pro-tumor role and as a cell type compromising a large portion in glioblastoma (GBM), reversing the imbalance of TAM polarization in the tumor environment has emerged as a promising novel field for GBM treatment. Radiation therapy (RT) is the standard treatment for GBM patients after surgery, which has been shown to transiently induce M1 polarization of macrophage (M1Ø). Recent studies suggested that metformin could also promote M1Ø in tumor microenvironment. We thus postulate that metformin may enhance and sustain the M1-inducing effect of radiation in GBM. Methods: We first examined the polarization effect of metformin (0.1mM, 1mM and 2mM) on mouse bone marrow-derived macrophage (BMDM) cultured in GBM tumor environment, including media conditioned by GBM cells in monolayer culture or tumor spheres as well as in trans-well co-culturing system. We irradiated GBM cells with different doses (2 Gy, 8 Gy, and 20 Gy) after the treatment of Metformin at various time points; then we used conditioned media to treat BMDM either cultured alone or co-cultured with GBM cells in trans-well system for 24 or 48 hours. A separate set of experiment was conducted by first irradiating GBM cells and then co-culturing them with BMDM at 24 or 48 hours after radiation with metformin added at the start of co-culture. Percentage of various subtypes of BMDM was calculated after flow cytometry. Results: High concentrations of metformin (1mM and 2mM) significantly increased M1Ø and inhibited M2Ø in all culture conditions. Co-culture with irradiated GBM cells or treatment with medium conditioned by irradiated GBM cells could temporally induce M1Ø polarization in BMDM, with the effects being RT dose-dependent. Metformin at high concentrations further promoted M1Ø and suppressed M2Ø polarization in those conditions mimicking tumor microenvironment. This enhancing effect was sustained for at least 48 hours. Conclusions: Metformin at mili-molar concentrations significantly enhances the effects of radiation on M1Ø polarization in BMDM in vitro.

Induction of deep layer cortical neurons in vitro

Development ◽  
1997 ◽  
Vol 124 (4) ◽  
pp. 915-923 ◽  
Author(s):  
A.P. Bohner ◽  
R.M. Akers ◽  
S.K. McConnell
Keyword(s):  
Cortical Neurons ◽  
Short Range ◽  
Deep Layer ◽  
Ventricular Zone ◽  
Cell Contact ◽  
Low Density ◽  
Environmental Signals ◽  
Multipotent Progenitors ◽  
Short Time

Transplantation studies suggest that the laminar fates of cerebral cortical neurons are determined by environmental signals encountered just before mitosis. In ferret, E29 progenitor cells normally produce neurons of layers 5 and 6. When transplanted during S-phase into an older ventricular zone, E29 progenitors produce neurons that change their fates and migrate to layer 2/3; however, cells transplanted later in the cell cycle migrate to their normal deep-layer positions even in an older environment (McConnell and Kaznowski, 1991). Here we utilize three culture systems to investigate the nature of the environmental signals involved in laminar specification. E29 cells were first cultured at low density to ascertain whether cell contact and/or short-range cues are required for deep layer specification. Neurons transplanted after a short time in low-density culture failed to adopt their normal fates and migrated instead to the upper layers. When crude cell contacts were restored by pelleting E29 cells together, most transplanted neurons cells became specified to their normal deep layer fates. Finally, E29 cells were transplanted after being cultured in explants that maintained the architecture of the cerebral wall. Explants allowed normal deep layer specification to occur, as transplanted cells migrated to layers 5 and 6. These results suggest that short-range cues induce multipotent progenitors to produce deep layer neurons.

Assessment of the Culture Requirements for Optimal In Vitro Growth and Survival of Multiple Myeloma (MM) Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3513-3513
Author(s):  
Mihaela Zlei ◽  
Sabine Egert ◽  
Christine Bayer ◽  
Dagmar Wider ◽  
Julia Schüler ◽  
...  
Keyword(s):  
Growth Factors ◽  
Cell Viability ◽  
Plasma Cells ◽  
Fold Increase ◽  
Cell Number ◽  
Culture Conditions ◽  
Cell Contact ◽  
Protective Function ◽  
Growth And Survival

Abstract Since MM expands preferentially in the bone marrow (BM) and the close myeloma and stroma interaction is well recognized, we analyzed whether various soluble factors and different stroma can enhance in vitro MM growth. The expansion and protective function of various growth factors (A. 10ng/ml of each IL-6, VEGF, IGF, +/−super (s) IL-6; B. combination A. plus 10ng/ml of each VEGF, HGF, and IL13, and C. 10ng/ml IL-6, IGF, SDF1, Galectin and IL-1) and different stroma (BM stroma from healthy donors [BMSCs], a mouse stroma line [M210B4], and osteoclasts [OC]) were used for MM cell lines (CL: L363, U266, RPMI 8226). Cell number, viability, percentage and number of CD138+ cells were assessed at day 3 and 6 after culture. The most beneficial culture settings were used to establish their relevance for CD138+ enriched BM cells from MM patients (pts), propagated for up to 14 days in culture. MM cell growth proved to be induced through a cell contact mediated mechanism, this being cytokine and stroma dependent. The most beneficial cytokine combination for CL survival and proliferation was combination B, which could substitute the stroma support. Compared to the control, consisting of culture medium only (RPMI 1640+10%FCS), we detected a median 3.1- (combination B) vs. 1.9-fold increase (BMSCs) at day 3, and 1.24- vs. 1.2-fold increase in cell numbers at day 6. BMSCs proved to be most supportive, especially when used together with combination B (1.8- vs. 1.2-fold increase with BMSCs used alone on day 6 of culture), suggesting that BMSCs provide growth factors acting synergistically with IL-6+VEGF+IGF+sIL-6. The cell viability, albeit not cell expansion potential, was best preserved by OC. With proliferation of myeloma CL, a decrease in the percentage of CD138+ cells was observed, consistent with the reported phenotypic shift of plasma cells (PC) from CD138+ to CD138− during cytokine stimulation. Especially OC favoured the expansion of CD138- cells, suggesting that MM clones require defined conditions for their growth and survival. Culture conditions B and C, with or without BMSCs, could sustain primary BM cells from MM pts for up to 14 days. With the inherent variation of different MM pt samples, the combination of 5 factors (C) was more beneficial for these cells. An additional support for cell viability was provided by BMSCs, especially with longer culture periods (beyond 5 days). The different growth and survival requirements for CL seemed to also apply for CD138+ cells from MM pts, which demonstrated to be remarkably heterogeneous as determined by FACS analysis. We identified at least four different PC populations which coexisted before culture in the same specimen. The phenotype of these cells differed in terms of CD45, CD138, CD38, CD56, CD126, CD221, CD19 and CD28, both in their presence on the cell surface and intensity of expression. Taken together, our data indicate that various culture conditions show substantial differences in their ability to preserve myeloma cells, aimed to reproduce the BM microenvironment where the malignancy develops unconstrained. The availability of in vitro systems will allow testing of novel anti-MM agents, alongside with the development of human MM models in mice.

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