Cell culture

About: Cell culture is a research topic. Over the lifetime, 133361 publications have been published within this topic receiving 5364150 citations. The topic is also known as: cell culture techniques.

Cellular APOBEC3G restricts HIV-1 infection in resting CD4+ T cells

Abstract: In contrast to activated CD4+ T cells, resting human CD4+ T cells circulating in blood are highly resistant to infection with human immunodeficiency virus (HIV). Whether the inability of HIV to infect these resting CD4+ T cells is due to the lack of a key factor, or alternatively reflects the presence of an efficient mechanism for defence against HIV, is not clear. Here we show that the anti-retroviral deoxycytidine deaminase APOBEC3G strongly protects unstimulated peripheral blood CD4+ T cells against HIV-1 infection. In activated CD4+ T cells, cytoplasmic APOBEC3G resides in an enzymatically inactive, high-molecular-mass (HMM) ribonucleoprotein complex that converts to an enzymatically active low-molecular-mass (LMM) form after treatment with RNase. In contrast, LMM APOBEC3G predominates in unstimulated CD4+ T cells, where HIV-1 replication is blocked and reverse transcription is impaired. Mitogen activation induces the recruitment of LMM APOBEC3G into the HMM complex, and this correlates with a sharp increase in permissivity for HIV infection in these stimulated cells. Notably, when APOBEC3G-specific small interfering RNAs are introduced into unstimulated CD4+ T cells, the early replication block encountered by HIV-1 is greatly relieved. Thus, LMM APOBEC3G functions as a potent post-entry restriction factor for HIV-1 in unstimulated CD4+ T cells. Surprisingly, sequencing of the reverse transcripts slowly formed in unstimulated CD4+ T cells reveals only low levels of dG dA hypermutation, raising the possibility that the APOBEC3G-restricting activity may not be strictly dependent on deoxycytidine deamination

The plasminogen system and cell surfaces: evidence for plasminogen and urokinase receptors on the same cell type

Abstract: The capacity of cells to interact with the plasminogen activator, urokinase, and the zymogen, plasminogen, was assessed using the promyeloid leukemic U937 cell line and the diploid fetal lung GM1380 fibroblast cell line. Urokinase bound to both cell lines in a time-dependent, specific, and saturable manner (Kd = 0.8-2.0 nM). An active catalytic site was not required for urokinase binding to the cells, and 55,000-mol-wt urokinase was selectively recognized. Plasminogen also bound to the two cell lines in a specific and saturable manner. This interaction occurred with a Kd of 0.8-0.9 microM and was of very high capacity (1.6-3.1 X 10(7) molecules bound/cell). The interaction of plasminogen with both cell types was partially sensitive to trypsinization of the cells and required an unoccupied high affinity lysine-binding site in the ligand. When plasminogen was added to the GM1380 cells, a line with high intrinsic plasminogen activator activity, the bound ligand was comprised of both plasminogen and plasmin. Urokinase, in catalytically active or inactive form, enhanced plasminogen binding to the two cell lines by 1.4-3.3-fold. Plasmin was the predominant form of the bound ligand when active urokinase was added, and preformed plasmin can also bind directly to the cells. Plasmin on the cell surface was also protected from its primary inhibitor, alpha 2-antiplasmin. These results indicate that the two cell lines possess specific binding sites for plasminogen and urokinase, and a family of widely distributed cellular receptors for these components may be considered. Endogenous or exogenous plasminogen activators can generate plasmin on cell surfaces, and such activation may provide a mechanism for arming cell surfaces with the broad proteolytic activity of this enzyme.

Myocyte hypertrophy in neonatal rat heart cultures and its regulation by serum and by catecholamines.

Abstract: The role of hormones and other humoral factors in the regulation of myocardial hypertrophy has been difficult to evaluate. We asked whether myocardial cell hypertrophy could be demonstrated in cultures from the day-old rat ventricle and evaluated the effect of serum concentration and catecholamines on the growth process. Two single-cell preparations were used: serum-supplemented, bromodeoxyuridine-treated cultures and serum-free cultures with transferrin and insulin. Both preparations were characterized by myocardial cell predominance (about 75--80% of total cells) and constant cell numbers. Myocardial cell size was documented by photomicroscopy and quantified by volume (microscopic diameter of suspended cells), surface area (planimetry of attached cells), and total cell protein concentration (Lowry method and cell counts). Growth was also evaluated in pure nonmyocardial cell cultures. In cultures with 5% (vol/vol) serum, myocardial cell size increased 2- to 3-fold over 11 days in culture. Final volume, surface area, and protein concentration were about 3000 micrometer3/cell, 5000 micrometer2/cell, and 1500 pg/cell, respectively. Serum had a dose-related effect on myocardial cell hypertrophy; myocardial cell size increased about 4-fold when serum concentration was increased from 0% to 5% or 10%. Cells maintained in serum-free medium with transferrin and insulin (each 10 microgram/ml) did not hypertrophy, but did remain responsive to the growth-promoting activity of serum. Chronic exposure to isoproterenol or norepinephrine (1 microM) significantly stimulated myocardial cell hypertrophy. This stimulation was dose-related, was not blocked by equimolar propranolol, was not associated with a sustained chronotropic effect, and was more pronounced in the serum-free preparation. In pure cultures of nonproliferating (bromodeoxyuridine-treated) nonmyocardial cells, cell size also increased with time in culture, but variation in serum concentration and addition of norepinephrine had no significant effect on cell size. Myocardial cell hypertrophy occurs in culture and is regulated by variations in the culture medium, including serum, with its contained hormones and growth factors, and catecholamines. The culture preparation can be used to explore the regulation of myocardial cell hypertrophy by nonhemodynamic factors.