Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids–in silico predictions and in vitro evidence

Amplification of DNA in vivo occurs in intracellular environments characterized by macromolecular crowding (MMC). In vitro Polymerase-chain-reaction (PCR), however, is non-crowded, requires thermal cycling for melting of DNA strands, primer-template hybridization and enzymatic primer-extension. The temperature-optima for primer-annealing and extension are strikingly disparate which predicts primers to dissociate from template during extension thereby compromising PCR efficiency. We hypothesized that MMC is not only important for the extension phase in vivo but also during PCR by stabilizing nucleotide hybrids. Novel atomistic Molecular Dynamics simulations elucidated that MMC stabilizes hydrogen-bonding between complementary nucleotides. Real-time PCR under MMC confirmed that melting-temperatures of complementary DNA–DNA and DNA–RNA hybrids increased by up to 8°C with high specificity and high duplex-preservation after extension (71% versus 37% non-crowded). MMC enhanced DNA hybrid-helicity, and drove specificity of duplex formation preferring matching versus mismatched sequences, including hair-pin-forming DNA- single-strands

C-phycocyanin protects cellular mitochondrial membrane potential from oxalate-induced ROS.

<p>Integrity of mitochondrial membrane potential in MDCK cells investigated by a mitochondrial potential-sensitive dye, JC-1, showing normal stippled spots of bright red fluorescence (G1_a–c). The numbers (viz. a–c) in the panels represents the red, green and merged channel fields observed respectively. Oxalate-exposed MDCK cells observed later (G2_a–c) shows the absence of characteristic red fluorescence. Cells pre-treated with CP and oxalate showing the re-appearance of red fluorescence spots following normalization of mitochondrial potential (G3_a–c). This confirms that CP could protect against mitochondrial membrane oxalate induced-ROS and does not itself affect the mitochondrial potential. Cells treated with CP alone show a normal picture (G4_a–c). G1: Control; G2: Oxalate; G3: Oxalate+CP; G4: CP alone.</p

Oxalate-induced ATP depletion restored by C-phycocyanin pre-treatment in cells.

<p>ATP levels in cultured MDCK cells were significantly lowered when incubated with oxalate alone (G2) than control group (G1) (P = 0.02). ATP depletion was significantly restored when cells were pre-treated with CP under oxalate-induced conditions (G3) (P = 0.012). *G2 compared with G1; G3 compared with G2. P<0.05 were considered significant. G1: Control; G2: Oxalate; G3: Oxalate+CP; G4: CP alone.</p

C-phycocyanin protects cells from free radical-mediated lipid peroxidation (LPO) induced by oxalate.

<p>To test the extent of free radical-mediated cellular damage, some oxidative stress markers have been developed and recognized suggestive of lipid peroxidation (LPO); for instance, HEL (hexanoyl-lysine adduct) is one the typical aldehyde products used as a marker of LPO. Thus, LPO was assessed by Western blotting for HEL in the lysates of MDCK cells from control (G1), oxalate alone (G2), oxalate+CP (G3) and CP alone (G4). Band intensity of HEL is prominent in oxalate-alone-treated cells (G2) at 72 kDa and 50 kDa compared to control MDCK cells (G1), whereas oxalate+CP cells (G3) showed decreased HEL band intensity relative to oxalate-treated cells (G2). The densitometric analyses were performed using a ImageJ (NIH) software. Densitometry data of 72 kDa and 50 kDa were normalized using internal control actin. Values represent mean±SD derived from immunoblots of all the groups from 3 experiments. The <i>P</i> values for HEL 72 kDa and 50 kDa of all the groups were as follows: HEL 72 kDa: G1 vs G2 (*P = 0.017) and G2 vs G3 (*P = 0.05); HEL 50 kDa: G1 vs G2 (*P = 0.013) and G2 vs G3 (*P = 0.009). P<0.05 were considered significant (*). G1: Control; G2: Oxalate; G3: Oxalate+CP; G4: CP alone.</p

Concentrations of C-phycocyanin in the presence of oxalate in MDCK cells.

<p>Different concentrations of CP (5, 10, 20, 50 mM) in the presence of oxalate (0.1 mM) in MDCK cell line showing the effective dosage of CP at 20 mM concentration at 24 and 48 hours.</p

C-phycocyanin quenches oxalate-induced ROS production in MDCK cells.

<p>(<b>A</b>) MDCK cells treated with oxalate were observed for ROS generation with a non-fluorescent dye DCFDA, which converts to a fluorescent DCF compound during oxidative stress (panel G2). This is compared with panel G1 (normal conditions). Panel G3 shows reduced fluorescence due to reduced generation of ROS upon CP pre-treatment, which resembles normal cells. Panel G4 confirms that CP alone by itself is not cytotoxic. (<b>B</b>) Quantitative estimation of DCF fluorescence, oxalate-induced MDCK cells (G2) shows exaggerated fluorescence as compared to control, and reduced florescence was observed in the CP pre-treated cells (G3). Values are expressed as mean±S.D. P<0.05 were considered significant. *G2 compared with G1; G3 compared with G2. G1: Control; G2: Oxalate; G3: Oxalate+CP; G4: CP alone.</p