This paper deals with the interactions of chlorogenic, caffeic, and quinic acids and p-quinone with myoglobin. The myoglobin derivatives formed have been characterized in terms of physicochemical properties and susceptibility to proteolysis. The results show that the free amino group and tryptophan contents of the myoglobin-phenol derivatives decrease with the increasing extent to which the protein becomes derivatized. Furthermore, the solubility of myoglobin-phenol derivatives decreases in the pH range 3.5-6.5 as compared to solubility of the native protein. The reaction also influences the hydrophilic-hydrophobic character of the protein. The isoelectric point of the derivatized myoglobin is shifted to a lower pH value, and formation of high molecular fractions is also documented. This paper also demonstrates the influence of the protein derivatization with plant phenols on susceptibility to digestion by trypsin, alpha-chymotrypsin, and pepsin, determined in vitro. The enzymatic digestion of the derivatized proteins is adversely affected.
The isolation and propagation of primary human corneal stromal keratocytes (CSK) are crucial for cellular research and corneal tissue engineering. However, this delicate cell type easily transforms into stromal fibroblasts (SF) and scar inducing myofibroblasts (Myo‐SF). Current protocols mainly rely on xenogeneic fetal bovine serum (FBS). Human platelet lysate (hPL) could be a viable, potentially autologous, alternative. We found high cell survival with both supplements in CSK and SF. Cell numbers and Ki67+ ratios increased with higher fractions of hPL and FBS in CSK and SF. We detected a loss in CSK marker expression (Col8A2, ALDH3A1 and LUM) with increasing fractions of FBS and hPL in CSK and SF. The expression of the Myo‐SF marker SMA increased with higher amounts of FBS but decreased with incremental hPL substitution in both cell types, implying an antifibrotic effect of hPL. Immunohistochemistry confirmed the RT‐PCR findings. bFGF and HGF were only found in hPL and could be responsible for suppressing the Myo‐SF conversion. Considering all findings, we propose 0.5% hPL as a suitable substitution in CSK culture, as this xeno‐free component efficiently preserved CSK characteristics, with non‐inferiority in terms of cell viability, cell number and proliferation in comparison to the established 0.5% FBS protocol.
We evaluated the small molecules (AFM) caffeine, curcumin and pirfenidone to find non-toxic concentrations reducing the transformation of activated human corneal stromal keratocytes (aCSK) to scar-inducing myofibroblasts (MYO-SF). CSK were isolated from 16 human corneas unsuitable for transplantation and expanded for three passages in control medium (0.5% FBS). Then, aCSK were exposed to concentrations of caffeine of 0–500 μM, curcumin of 0–200 μM, pirfenidone of 0–2.2 nM and the profibrotic cytokine TGF-β1 (10 ng/mL) for 48 h. Alterations in viability and gene expression were evaluated by cell viability staining (FDA/PI), real-time polymerase chain reaction (RT-PCR) and immunocytochemistry. We found that all AFMs reduced cell counts at high concentrations. The highest concentrations with no toxic effect were 100 µM of caffeine, 20 µM of curcumin and 1.1 nM of pirfenidone. The addition of TGF-β1 to the control medium effectively transformed aCSK into myofibroblasts (MYO-SF), indicated by a 10-fold increase in α-smooth muscle actin (SMA) expression, a 39% decrease in lumican (LUM) expression and a 98% decrease in ALDH3A1 expression (p < 0.001). The concentrations of 100 µM of caffeine, 20/50 µM of curcumin and 1.1 nM of pirfenidone each significantly reduced SMA expression under TGF-β1 stimulation (p ≤ 0.024). LUM and ALDH3A1 expression remained low under TGF-β1 stimulation, independently of AFM supplementation. Immunocytochemistry showed that 100 µM of caffeine, 20 µM of curcumin and 1.1 nM of pirfenidone reduce the conversion rate of aCSK to SMA+ MYO-SF. In conclusion, in aCSK, 100 µM of caffeine, 20 µM of curcumin and 1.1 nM of pirfenidone significantly reduced SMA expression and MYO-SF conversion under TGF-β1 stimulation, with no influence on cell counts. However, the AFMs were unable to protect aCSK from characteristic marker loss.
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