Structural insight into the interaction between p53 TAD1 and AIMP2-DX2 by NMR.

p53 is the most studied tumor suppressor and a key transcriptional factor, with discrete domains that regulate cellular pathways such as apoptosis, angiogenesis, cell-cycle arrest, DNA repair, and senescence. Previous studies have suggested that AIMP2, and ARS-interacting multifunctional protein 2, promote cell death via the protective interaction with p53 upon DNA damage. Also, oncogenic splicing variant of AIMP2 lacking exon2, AIMP2-DX2, compromises the pro-apoptotic activity and anti-proliferative activities of the AIMP2 by competing with AIMP2 for the binding with p53. However, the molecular mechanism for the interaction of p53 and AIMP2 remains elusive. Using NMR spectroscopy, we studied the structural details of the interaction of transactivation domain 1 (TAD1) of p53 with GST domain of AIMP2, which is also common in AIMP2-DX2. The chemical shift perturbation (CSP) experiments demonstrate that amino acid residues from E17 to E28 of p53, known to bind to MDM2 are also involved in binding to AIMP2-DX2. Structure determination of this region based on the transferred-NOE (trNOE) data revealed that TAD1 of the p53 forms a turn structure with hydrophobic interactions by side chains of F19, L22, W23 and L26, distinct from the structure for MDM2 binding. Also, docking results based on NMR CSP data suggest the binding mode of p53 with AIMP2-DX2 GST domain. These data provide the first structural insight into the binding of the p53 TAD1 on AIMP2 and AIMP2-DX2.

Enhancement of lipid extraction from marine microalga, Scenedesmus associated with high-pressure homogenization process.

Marine microalga, Scenedesmus sp., which is known to be suitable for biodiesel production because of its high lipid content, was subjected to the conventional Folch method of lipid extraction combined with high-pressure homogenization pretreatment process at 1200 psi and 35°C. Algal lipid yield was about 24.9% through this process, whereas only 19.8% lipid can be obtained by following a conventional lipid extraction procedure using the solvent, chloroform:methanol (2:1, v/v). Present approach requires 30 min process time and a moderate working temperature of 35°C as compared to the conventional extraction method which usually requires >5 hrs and 65°C temperature. It was found that this combined extraction process followed second-order reaction kinetics, which means most of the cellular lipids were extracted during initial periods of extraction, mostly within 30 min. In contrast, during the conventional extraction process, the cellular lipids were slowly and continuously extracted for >5 hrs by following first-order kinetics. Confocal and scanning electron microscopy revealed altered texture of algal biomass pretreated with high-pressure homogenization. These results clearly demonstrate that the Folch method coupled with high-pressure homogenization pretreatment can easily destruct the rigid cell walls of microalgae and release the intact lipids, with minimized extraction time and temperature, both of which are essential for maintaining good quality of the lipids for biodiesel production.

The effect of ultrasonificated extracts of Spirulina maxima on the anticancer activity.

The effect of ultrasonic extraction on extraction yields, cytotoxicity, and anticancer activity of Spirulina maxima was investigated in this study. Optimal extraction conditions were determined as 60 kHz frequency at 60°C for 30 min with 120 W intensity, which resulted in 19.3% of extraction yields and 19.1% of cytotoxicity on normal human cells. Yields from conventional water and ethanol extraction were 15.8% at 100°C and 8.3% at 80°C, respectively. It was found that the extracts obtained by ultrasonic extraction process selectively inhibited the digestive-related cancer cell lines, such as human stomach cancer cells, having 89% of the highest inhibition ratio and 4.5 of the highest selectivity. In adding 0.5 mg/mL of the extract, human promyelocytic leukemia cells' cell differentiation was increased 1.72 times over that of the control. Expression level of B cell lymphoma-2 from Hep3B cell was also effectively suppressed by the extract obtained at 60 kHz and 60°C, leading to the inhibition of the early step of carcinogenesis. This work suggests that anticancer activity of the extracts is due to water-soluble polysaccharides rather than proteins and is further supported by the result that the ultrasonification extraction process can efficiently extract relatively intact polysaccharides rather than digesting the proteins in S. maxima by matrix assisted laser desorption ionization-time of flight and high performance size exclusion chromatography chromatogram analyses. Therefore, ultrasonic extraction increases both extraction yield and the biological activity of S. maxima extracts, which might be useful as an alternative natural anticancer agent in the medical and food industries.

Long-term outdoor cultivation by perfusing spent medium for biodiesel production from Chlorella minutissima.

A unique perfusion process was developed to maintain high concentrations of marine alga, Chlorella minutissima. This method is based on recycling cells by continuous feeding with warm spent sea water from nuclear power plants, which has very similar properties as sea water. A temperature of at least 30 degrees C in a 200 L photo-bioreactor was maintained in this system by perfusion of the thermal plume for 80 days in the coldest season. The maximum cell concentration and total lipid content was 8.3 g-dry wt./L and 23.2 %, w/w, respectively, under mixotrophic conditions. Lipid production was found to be due to a partially or non-growth related process, which implies that large amounts of biomass are needed for a high accumulation of lipids within the cells. At perfusion rates greater than 1.5 L/h, the temperature of the medium inside the reactor was around 30 degrees C, which was optimal for cell growth. For this system, a perfusion rate of 2.8 L/h was determined to be optimal for maintaining rapid cell growth and lipid production during outdoor cultivation. It was absolutely necessary to maintain the appropriate perfusion rate so that the medium temperature was optimal for cell growth. In addition, the lipids produced using this process were shown to be feasible for biodiesel production since the lipid composition of C. minutissima grown under these conditions consisted of 17 % (w/w) of C(16) and 47% (w/w) of C(18). The combined results of this study clearly demonstrated that the discharged energy of the thermal plume could be reused to cultivate marine alga by maintaining a relatively constant temperature in an outdoor photo-bioreactor without the need for supplying any extra energy, which could allow for cheap production of biodiesel from waste energy.

Lipid production in Porphyridium cruentum grown under different culture conditions.

Autotrophic growth of Porphyridium cruentum under 18:12 h and 12:12 h light:dark cycles showed the maximum cell concentration of 2.1 g-dry wt./L, whereas the specific growth rate, 0.042 (1/h), at 18:6 h is faster than that of 12:12 h, 0.031 (1/h), respectively. The highest lipid accumulation level, 19.3 (%, w/w), was achieved at 12:12 h cycle. Under dark cultivation condition with 10 g/L of glucose, the lipid accumulation in the cell was 10.9 (%, w/w), whereas the heterotrophic growth with glycerol as the carbon resource showed low level of cell concentration and lipid production, compared to that of glucose. The glucose was decided to be a suitable carbon resource for the heterotrophic growth of P. cruentum. The lipids from P. cruentum seemed be feasible for biodiesel production, because over 30% of the lipid was C16-C(18:1). The cultivation time and temperature were important factors to increase the maximum cell concentration. Extending the cultivation time helps maintain the maximum cell concentration, and higher lipid accumulation was achieved at 25 degrees C, compared to 35 degrees C. The fed-batch cultures showed that, under the light condition, the specific production rate was slightly decreased to 0.4% lipid/g-dry wt./day at the later stage, whereas, under the dark condition, the specific production rate was maintained to be a maximum value of 1.1% lipid/g-dry wt./day, even in the later stage of cultivation. The results indicate that the heterotrophic or 12:12 h cyclic mixotrophic growth of P. cruentum could be used for the production of biodiesel in long-term fed-batch cultivation of P. cruentum.