Purification and characterization of laccase from the white rot fungus Trametes versicolor

Laccase is one of the ligninolytic enzymes of white rot fungus Trametes versicolor 951022, a strain first isolated in Korea. This laccase was purified 209-fold from culture fluid with a yield of 6.2% using ethanol precipitation, DEAE-Sepharose, Phenyl-Sepharose, and Sephadex G-100 chromatography. T. versicolor 951022 excretes a single monomeric laccase showing a high specific activity of 91,443 U/mg for 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as a substrate. The enzyme has a molecular mass of approximately 97 kDa as determined by SDS-PAGE, which is larger than those of other laccases reported. It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 3.0 and a temperature of 50 degrees C. The Km value of the enzyme for substrate ABTS is 12.8 micrometer and its corresponding Vmax value is 8125.4 U/mg. The specific activity and substrate affinity of this laccase are higher than those of other white rot fungi, therefore, it may be potentially useful for industrial purposes.     

Suppression of ceramide-induced cell death by hepatitis C virus core protein

The hepatitis C virus (HCV) core protein is believed to be one of viral proteins that are capable of preventing virus-infected cell death upon various stimuli. But, the effect of the HCV core protein on apoptosis that is induced by various stimuli is contradictory. We examined the possibility that the HCV core protein affects the ceramide-induced cell death in cells expressing the HCV core protein through the sphingomyelin pathway. Cell death that is induced by C(2)-ceramide and bacterial sphingomyelinase was analyzed in 293 cells that constitutively expressed the HCV core protein and compared with 293 cells that were stably transfected only with the expression vector. The HCV core protein inhibited the cell death that was induced by these reagents. The protective effects of the HCV core protein on ceramide-induced cell death were reflected by the reduced expression of p21(WAF1/Cip1/Sid1) and the sustained expression of the Bcl-2 protein in the HCV core-expressing cells with respect to the vector-transfected cells. These results suggest that the HCV core protein in 293 cells plays a role in the modulation of the apoptotic response that is induced by ceramide. Also, the ability of the HCV core protein to suppress apoptosis might have important implications in understanding the pathogenesis of the HCV infection.     

Growth of Pichia guilliermondii A9, an osmotolerant yeast, in waste brine generated from kimchi production

The possibility of culturing an osmotolerant yeast using waste brine from a kimchi factory as a substrate for the production of single cell protein was investigated. Pichia guilliermondii A9 was selected from 70 isolates of yeast demonstrating substantial growth in the waste brine. The growth of P. guilliermondii A9 in waste brine was not inhibited by NaCl concentrations of up to 10% (w/v). However, it was reduced drastically at concentrations greater than 12% (w/v). Approximately 90% of BOD was removed from the waste brine by culturing of P. guilliermondii A9 for 24 h. The maximum cell yield was 0.69 g of dry cells per liter, containing 40% of protein. When the waste brine was enriched with cabbage juice from waste cabbage, the final cell mass increased proportionally with the amount of added organic material. Salt stressed cells of P. guilliermondii A9 grown in waste brine are shown in scanning electron micrographs. In conclusion, the large amounts of waste brine generated from kimchi production could be used directly for the culture of the osmotolerant yeast P. guilliermondii A9.     

Characterization of xylanase from Lentinus edodes M290 cultured on waste mushroom logs

Extracellular enzymes from Lentinus edodes M290 on normal woods (Quercus mongolica) and waste logs from oak mushroom production were comparatively investigated. Endoglucanase, cellobiohydrolase, beta-glucosidase, and xylanase activities were higher on waste mushroom logs than on normal woods after L. edodes M290 inoculation. Xylanase activity was especially different, with a three times higher activity on waste mushroom logs. When the waste mushroom logs were used as a carbon source, a new 35 kDa protein appeared. After the purification, the optimal pH and temperature for xylanase activity were determined to be 4.0 and 50 degrees C, respectively. More than 50% of the optimal xylanase activity was retained when the temperature was increased from 20 to 60 degrees C, after a 240 min reaction. At 40 degrees C, the xylanase maintained 93% of the optimal activity, after a 240 min reaction. The purified xylanase showed a very high homology to the xylanase family 10 from Aspergillus terreus by LC/MS-MS analysis. The highest Xcorr (1.737) was obtained from the peptide KWI SQGIPIDGIG SQTHLGSGGS WTVK originated from Aspergillus terreus, indicating that the 35 kDa protein was xylanase. This protein showed low homology to a previously reported L. edodes xylanase sequence.     

Simulation of sequential batch reactor (SBR) operation for simultaneous removal of nitrogen and phosphorus

Modeling of the operation of sequential batch reactor (SBR) was performed to find out optimum design parameters for simultaneous removal of nitrogen and phosphorus in a small-scale wastewater treatment plant. The models were set up with material balances on SBR operation and Monod kinetics. The model parameters were obtained to best fit the experimental results in a small scale SBR. The models were useful in optimizing hydraulic retention time (HRT) and successfully simulated operations of SBR in a larger scale. Especially the model predicted well the reactions occurring in the filling period as well as the effect of dilution, and evaluated the performance of SBR process under diverse operating conditions.     

Identification of a novel cell-penetrating peptide from human phosphatidate phosphatase LPIN3

Biomolecules such as proteins, DNA, and RNA are macromolecules and can not cross the cell membrane. However, cell-penetrating peptide (CPP) has been shown to deliver therapeutic biomolecules successfully into cells. The various and widely used CPPs including TAT, VP22, and Antp are mostly non-human originated CPPs, and are limited by their potential toxicity and immunogenicity. We report here on a newly identified novel cell-penetrating sequence (LPIN; RRKRRRRRK) from the nuclear localization sequence (NLS) of human nuclear phosphatase, LPIN3. LPIN-EGFP recombinant protein was concentration- and time-dependently delivered into cells and localized to the nucleus as well as the cytoplasm. It penetrated the cell membrane by lipid raft-mediated endocytosis by binding to heparan sulfate proteoglycan. LPIN-EGFP was successfully delivered into primary mouse splenocytes in vitro and it could be delivered into various tissues including liver, kidney, and intestine in mice after intra-peritoneal injection. This research suggests that LPIN-CPP could be used in a drug delivery system to deliver therapeutic biomolecules including peptides, proteins, DNA, and RNA and without the limitations of non-human originated CPPs such as TAT-CPP.     

Sonic hedgehog signaling regulates Gli3 processing, mesenchymal proliferation, and differentiation during mouse lung organogenesis

Lack of Sonic hedgehog (Shh) signaling, mediated by the Gli proteins, leads to severe pulmonary hypoplasia. However, the precise role of Gli genes in lung development is not well established. We show Shh signaling prevents Gli3 proteolysis to generate its repressor forms (Gli3R) in the developing murine lung. In Shh(-/-) or cyclopamine-treated wild-type (WT) lung, we found that Gli3R level is elevated, and this upregulation appears to contribute to defects in proliferation and differentiation observed in the Shh(-/-) mesenchyme, where Gli3 is normally expressed. In agreement, we found Shh(-/-);Gli3(-/-) lungs exhibit enhanced growth potential. Vasculogenesis is also enhanced; in contrast, bronchial myogenesis remains absent in Shh(-/-);Gli3(-/-) compared with Shh(-/-) lungs. Genes upregulated in Shh(-/-);Gli3(-/-) relative to Shh(-/-) lung include Wnt2 and, surprisingly, Foxf1 whose expression has been reported to be Shh-dependent. Cyclins D1, D2, and D3 antibody labelings also reveal distinct expression patterns in the normal and mutant lungs. We found significant repression of Tbx2 and Tbx3, both linked to inhibition of cellular senescence, in Shh(-/-) and partial derepression in Shh(-/-); Gli3(-/-) lungs, while Tbx4 and Tbx5 expressions are less affected in the mutants. Our findings shed light on the role of Shh signaling on Gli3 processing in lung growth and differentiation by regulating several critical genes.     

Altered cortical synaptic morphology and impaired memory consolidation in forebrain- specific dominant-negative PAK transgenic mice

Molecular and cellular mechanisms for memory consolidation in the cortex are poorly known. To study the relationships between synaptic structure and function in the cortex and consolidation of long-term memory, we have generated transgenic mice in which catalytic activity of PAK, a critical regulator of actin remodeling, is inhibited in the postnatal forebrain. Cortical neurons in these mice displayed fewer dendritic spines and an increased proportion of larger synapses compared to wild-type controls. These alterations in basal synaptic morphology correlated with enhanced mean synaptic strength and impaired bidirectional synaptic modifiability (enhanced LTP and reduced LTD) in the cortex. By contrast, spine morphology and synaptic plasticity were normal in the hippocampus of these mice. Importantly, these mice exhibited specific deficits in the consolidation phase of hippocampus-dependent memory. Thus, our results provide evidence for critical relationships between synaptic morphology and bidirectional modifiability of synaptic strength in the cortex and consolidation of long-term memory.     

Detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase

Failure to detect the intermediate in spite of its existence often leads to the conclusion that two-state transition in the unfolding process of the protein can be justified. In contrast to the previous equilibrium unfolding experiment fitted to a two-state model by circular dichroism and fluorescence spectroscopies, an equilibrium unfolding intermediate of a dimeric ketosteroid isomerase (KSI) could be detected by small angle X-ray scattering (SAXS) and analytical ultracentrifugation. The sizes of KSI were determined to be 18.7A in 0M urea, 17.3A in 5.2M urea, and 25.1A in 7M urea by SAXS. The size of KSI in 5.2M urea was significantly decreased compared with those in 0M and 7M urea, suggesting the existence of a compact intermediate. Sedimentation velocity as obtained by ultracentrifugation confirmed that KSI in 5.2M urea is distinctly different from native and fully-unfolded forms. The sizes measured by pulse field gradient nuclear magnetic resonance (NMR) spectroscopy were consistent with those obtained by SAXS. Discrepancy of equilibrium unfolding studies between size measurement methods and optical spectroscopies might be due to the failure in detecting the intermediate by optical spectroscopic methods. Further characterization of the intermediate using (1)H NMR spectroscopy and Kratky plot supported the existence of a partially-folded form of KSI which is distinct from those of native and fully-unfolded KSIs. Taken together, our results suggest that the formation of a compact intermediate should precede the association of monomers prior to the dimerization process during the folding of KSI.