Fibroblast removal

An increased rate of metabolic clearance has been observed after removal of sialic acid from human, low-density lipoprotein in vivo.472 Sialic acid controls the receptor-mediated uptake of this lipoprotein by fibroblasts. Removal of sialic acid residues accelerates the rate of internalization of the lipoprotein and, subsequently, the regulation of the metabolism of cellular cholesterol.473... 

After mRNA splicing, the tropoelastin mRNA is translated at the surface of the rough endoplasmic reticulum (RER) in a variety of cells smooth muscle cells, endothelial and microvascular cells, chondrocytes and fibroblasts. The approximately 70 kDa precursor protein (depending on isoform) is synthesized with an N-terminal 26-amino-acid signal peptide. This nascent polypeptide chain is transported into the lumen of the RER, where the signal peptide is removed cotranslationally [9]. 

For the in vitro test, the fibroblasts are allowed to form a half-confluent monolayer within 24 h. Different concentrations of the test chemical are then incubated for 1 h with two sets of cells in parallel (typically on 96-well plates, 104 cells per well, passage number <100). After the incubation with the test substances, one set is irradiated with a nontoxic dose of UVA light (5 J/cm2), while the other set is kept in the dark. Twenty hours after irradiation, cell viability is evaluated by measuring the uptake of NR for 3 h. After the end of the absorption process, excess NR is removed and the cells are treated with an NR desorption solution (ethanol/acetic acid) to extract the dye taken up by the cells. Subsequently, the optical density of the NR solution is measured at 540 nm. As positive control, a test with chlorpromazine is performed. 

S. cerevisiae aurora/IPllp, C. elegans aurora/AIR-2, and mammalian aurora B (also called AIM-1) phosphorylate H3 at Ser-10 and Ser-28 during mitosis [39 1] (Fig. 5). Protein phosphatase 1 removes the phosphate at these sites [39,42]. INCENP is bound to aurora B and is essential for the proper targeting of aurora B on the chromosomes [43-45]. INCENP and aurora B (AIM-1) are overexpressed in a variety of human cancers, including colorectal cancer [43,46]. Overexpression of aurora B (AIM-1) in CHE diploid fibroblasts leads to chromosomal instability, suggesting that aurora B overexpression may play a role in carcinogenesis [46]. INCENP/aurora B and H3 phosphorylation appear to be involved in assembly of mitotic chromosomes, but not mitotic chromosome compaction [44,47]. 

Incubation of fibroblasts with tunieamycin resulted in a dose-de-pendent inhibition of binding, and internalization of low-density lipoprotein. Upon removal of the inhibiting medium, this effect is reversible within 3 days. It was hypothesized that the expression of LDL receptors on the surface of fibroblasts is dependent on intact N-glyco-sylation,480 which may be necessary for proper orientation and function of LDL-receptors on the fibroblast cell-surface. 

Adhesion of mononuclear cells from human bone marrow aspirates (BM-MNC) to tissue culture plastic and removal of nonadherent cells during the first days of culture selects for a population of proliferating spindle-shaped fibroblast-like non-... 

Fibroblasts are cultured under standard conditions in F10+ medium with 15% fetal calf serum. The level of free NeuAc is greatly influenced by culture conditions. Do not use Chang medium (leads to very high control values). The final culture is done in a 175-cm2 culture flask for 7 days, the last 3 days with 5% fetal calf serum. Medium is removed, cells are washed with phosphate-buffered saline (PBS), trypsinized, centrifuged at 100 xg, and washed with PBS. The resultant pellets are frozen before use, in 1.5-ml sample vials at -80°C. 

Grow the fibroblasts in Ham F-10 medium with 10% fetal calf serum (FCS). Harvest the cells, seed at a density of 30,000 cells and grow to near confluency on cover slips in 6-well plates in Ham F-10 medium with 10% FCS. Remove the medium and wash the cells three times with 2 ml Hanks balanced salt solution. Fix the cells with 3 ml fixative. Seal the plate with tape and store in the refrigerator at 4 C until staining. 

Fibroblasts are grown under standard conditions in Ham FI0 medium supplemented with 10% FCS. Cells are harvested by trypsinization, taken up in medium, counted, and reseeded in 6-well plates at a density of 200,000 cells per well (2.5 ml cell suspension, 80,000 cells per milliliter). A total of six wells on three different plates is needed per cell line (two wells per plate for duplicate assays). For each assay, one control and one NPC cell line with classical biochemical phenotype is included. Incubate for 3 days in Ham F-10 with 10% FCS, remove the culture medium and grow the cells in... 

Human skin fibroblasts are cultured from skin biopsy samples. The dermis is cut into small pieces (0.5 mm on each side) and placed into a dish in DMEM containing 10% (v/v) FCS and 1% (v/v) antibiotic-antimycotic solution. When these primary cultures are confluent they are split and cells between passage three and six are used for experiments. For the cholesterol efflux assay, cells are grown in 24-well plates to 60-80% confluence and are labeled with [1,2-3H]-cholesterol (1 pCi/well) for 24 h. Cells are then washed with DMEM and incubated for 4 h at 37°C with DMEM containing BSA (0.2%, v/v) and either 0 (negative control) or 5-30 pg/ml apoA-I. The efflux medium is collected and centrifuged to remove cell debris. Cells are solubilized in 0.1 mol/1 NaOH and the radioactivity in the efflux media and in the cell lysates is determined by scintillation counting [11, 30, 75]. 

The metabolism of HDL probably involves interaction with both hepatic and peripheral cells, as well as with other lipoproteins. HDL may remove cholesterol from tissues, the "scavenger hypothesis (11,12). The cholesterol may then be esterifed by the action of lecithin cholesterol acyl transferase. HDL may provide cholesterol to the liver for bile acid synthesis (13) and some HDL may be catabolized by the liver in the process. HDL has not been found to interfere with the binding of LDL in cultured human fibroblasts (6). However, in cultured human arterial cells, porcine or rat hepatocytes, and rat adrenal gland, there appears to be some competition of HDL with LDL binding sites, suggesting the presence of a "lipoprotein-binding" site (14). 

Approximately 2 x 105 normal and FH homozygous fibroblasts were seeded in 60 mm petri dishes and grown for 6 days in MEM containing 10% fetal bovine serum. Subsequently, the medium was removed, monolayers washed and 2 ml of fresh MEM containing 5% LPDS, 1% FCS and 2 uCi each of the above isotopes added and incubated for 48 hrs. Subsequently, medium was removed, the mono-layers washed twice with 5 ml of PBS and 2% BSA and thrice with PBS. The entire monolayer was solubilized in 1 ml of 1 N NaOH. Suitable aliquots of the cell extracts were used for protein and radioactivity measurements. 

Table VII. In a typical experiment, normal and mutant fibroblasts (one 150 cm2 flask, each containing approximately 2 x 10 cells) were incubated for 6 days in 20 ml medium containing 10% fetal calf serum and antibiotics. On the sixth day, medium was removed, the confluent monolayers washed five times with warm PBS, and further incubated for 48 hrs in 10 ml medium containing 1% fetal calf serum, antibiotics, and 5 p Ci of [ HJ-D-galactose. Subsequently, the medium was removed, the monolayers washed with PBS, harvested, and centrifuged at 500 x g for 5 min at 4°. The cell pellet was washed thrice with 40 vol of ice cold PBS. The washed cell pellets were suspended in a small volume of water, sonicated and a suitable aliquot withdrawn for total protein and radioactivity measurements. The remainder of the cell pellets were subjected to solvent extraction for the purposes of isolation of GSLs according to previously described procedures (30).

---