Conditions Of Incubation Time Research Articles

An Optimized Approach to Recover Secreted Proteins from Fibroblast Conditioned-Media for Secretomic Analysis.

The proteins secreted by a particular type of cell, the secretome, play important roles in the regulation of many physiological processes via paracrine/autocrine mechanisms, and they are of increasing interest to help understanding rare diseases and to identify potential biomarkers and therapeutic targets. To facilitate ongoing research involving secreted proteins, we revisited cell culture protocols and whole secreted protein enrichment protocols. A reliable method for culturing and precipitating secreted protein from patient-derived fibroblast conditioned-medium was established. The method is based on the optimization of cell confluency and incubation time conditions. The well-established carrier-based TCA-DOC protein precipitation method was consistently found to give higher protein recovery yield. According to our results, we therefore propose that protein enrichment should be performed by TCA-DOC precipitation method after 48 h at 95% of confluence in a serum-deprived culture medium. Given the importance of secreted proteins as a source to elucidate the pathogenesis of rare diseases, especially neurological disorders, this approach may help to discover novel candidate biomarkers with potential clinical significance.

Discrepancy between ELISPOT IFN-gamma secretion and binding of A2/peptide multimers to TCR reveals interclonal dissociation of CTL effector function from TCR-peptide/MHC complexes half-life.

Activation of CD8(+) cytolytic T lymphocytes (CTLs) by antigen is triggered by the interaction of clonotypic alphabeta T cell receptors (TCRs) with antigenic peptides bound to MHC class I molecules (pMHC complexes). Fluorescent multimeric pMHC complexes have been shown to specifically stain antigen-specific CTLs by directly binding the TCR. In tumor-infiltrating lymphocytes from a melanoma patient we found a high frequency of tyrosinase(368-376) peptide-specific cells as detected by IFN-gamma ELISPOT, without detectable staining with the corresponding A2/peptide multimers. Surprisingly, these T cells were able to lyse tyrosinase(368-376) peptide-pulsed target cells as efficiently as other specific T cells that were stained by multimers. Analysis of the staining patterns under different conditions of incubation time and temperature revealed that these results were explained by major differences in TCR-multimeric ligand interaction kinetics among the clones. Whereas no direct quantitative correlation between antigenic peptide concentration required for CTL effector functions and equilibrium multimer binding was observed interclonally, the latter was profoundly affected by the kinetics of TCR-ligand interaction. More importantly, our data indicate that similar levels of T cell activation can be achieved by independent CD8(+) T cell clonotypes displaying different TCR/pMHC complex dissociation rates.

Adenylate cyclase of human articular chondrocytes. Responsiveness to prostaglandins and other hormones.

Adenylate cyclase [ATP pyrophosphate lyase (cyclizing), EC 4.6.1.1] was shown to be present in cultured human articular chondrocytes. Optimal conditions of incubation time, protein and substrate concentrations and pH were determined in whole cell lysates. Maximal activity occurred at pH 8.5 with no decrease in activity up to pH 10.0. Adenylate cyclase activity of particulate membrane preparations was enhanced by the addition of crude cytosol preparations. The prostaglandins E1, E2, F1 alpha, F2 alpha, D2, B1, B2, A1 and A2, as well as adrenaline and isoprenaline, stimulated adenylate cyclase derived from either adult or foetal chondrocytes. No significant stimulation was observed in the presence of human calcitonin or glucagon. Bovine parathyroid hormone always significantly stimulated the adenylate cyclase derived from foetal chondrocytes, but not from adult chondrocytes. Preincubation of the chondrocytes in culture with indomethacin and with or without supernatant medium from cultured mononuclear cells increased the responsiveness of the adenylate cyclase to prostaglandin E1.

Reduction in [ 3H]-corticosterone binding to cytoplasmic receptors in the brain of diabetic rats

The binding of [1, 2, 6, 7- 3H]-corticosterone was studied in brain cytosol from normal and streptozotocin-diabetic male rats. The experiments were performed under conditions of incubation time (4h), temperature (0–4°C), time after adrenalectomy (6 days) and corticosterone concentrations (1.2 × 10 −8 and 1.15 × 10 −9M) previously established for determining binding activity in the brain of normal rats. The binding of [ 3H]-corticosterone was found invariably lower in cytosol of the brain from diabetic rats, studied under three different conditions: in non-adrenalectomized animals, in adrenalectomized using a non-saturating corticosterone concentration, and in adrenalectomized plus a saturating steroid concentration. These results support previous contentions that the diminished sensitivity to the negative feedback for steroids which is present in diabetics, may be related to a reduction in binding capacity for corticoids in the central nervous system

Oxygen inhibition of acetylene reduction (nitrogen fixation) in soil: Effect of glucose and oxygen concentrations

Sandy loam soil was amended with different concentrations of glucose and was incubated at different pO 2 levels. Under many conditions of incubation time and treatment, N 2 ase activity as determined by 1-h aerobic C 2H 2 reduction assay (flushed with Ar:O 2, 4:1 before assay) was significantly less than that determined by means of ambient assay (carried out at the pO 2 of incubation without flushing with Ar:O 2, 4:1 before assay). The difference between the N 2ase activity in aerobic assay and that in ambient assay increased with decreasing glucose and O 2 concentrations imposed during incubation. The inhition in aerobic assays was analogous to O 2-induced shut-off of N 2ase and amounted to 75 per cent inhibition after incubation at 0.06 atm pO 2 of samples amended with 0.75% glucose (w/w). Similar O 2 inhibition was observed after amendment with mannitol and with lactate. Times of incubation were chosen such that development of anaerobic N 2ase activity was either absent or too low to account for the observed effects of O 2 during assay. It was shown that 0.05 atm pC 2H 2 was adequate for routine 1-h assays of the soil system employed. Individual soil samples could be subjected to repeated 1-h assays (with removal of C 2H 2 and C 2H 4 by evacuation after each assay) thus avoiding side-effects of long exposure to C 2H 2.