Modification of Daxx by small ubiquitin-related modifier-1

Small ubiquitin-related modifier-1 (SUMO-1) is a protein that is covalently modified to various cellular proteins and protects cells against both anti-Fas and TNF-induced cell death. Previously, we reported that the C-terminus of Daxx interacted with Ubc9, an E2 type SUMO-1 conjugating enzyme, as well as with SUMO-1. In BOSC23 cells expressing FLAG-Daxx together with HA-SUMO-1, 110 and 130kDa Daxx appeared and the 130kDa band bound to both anti-HA and anti-FLAG antibodies. This means that Daxx can be covalently modified by SUMO-1. Substitution of K630 and K631 abrogated the modification of Daxx by SUMO-1, implying that K630 and K631 were essential for sumoylation. Daxx (K630, 631A) and Daxx (K634, 636, 637A) in which the putative C-terminal nuclear localization signals (NLSs) were disrupted appeared in the nucleus, suggesting that the C-terminal NLS was not functional. Daxx (K630, 631A), the sumoylation defective mutant, was able to interact with PML and co-localized with PML in the PML oncogenic domains (PODs). Thus, our data show that sumoylation status of Daxx does not affect its presence in PODs.     

Enhancing biomass and fatty acid productivity of <Emphasis Type="Italic">Tetraselmis</Emphasis> sp. in bubble column photobioreactors by modifying light quality using light filters

Some artificial light sources able to emit photons at specific wavelengths, such as LEDs, are useful for studying the effects of light quality on microalgal growth and production of fatty acids; however, they should not be used for outdoor cultivation of microalgae to produce bioenergy. Instead, various light filters capable of selectively transmitting red, blue, and red+blue light regions in solar radiation were used to cover 0.4 L bubble column photobioreactors to cultivate Tetraselmis sp. KCTC12236BP and investigate the influence of light quality on microalgal growth and fatty acid production. Biomass and fatty acid productivities in red light (0.10 ± 0.05 g/L/day and 11.8 ± 0.5 mg/L/day, respectively) were 7 ~ 53% and 9 ~ 61% higher than other colored lights based on the same number of supplied photons, respectively. The composition of fatty acids did not change significantly in response to transmitted light qualities of the filter. The ratio of saturated to unsaturated fatty acids was 3:7, and their contents were 12% in all groups, which corresponds with the results of LEDs. Plotting biomass and fatty acid productivity over the red photon fraction in supplied light revealed that increased productivities were closely correlated with red photon fraction in the filtered light. Overall, the results presented herein indicate that enhanced production of algal fatty acid could be achieved by application of light filters in outdoor settings without artificial lights.     

Biochemical properties of penicillin amidohydrolase from Micrococcus luteus.

Some biochemical properties of whole-cell penicillin amidohydrolase from Micrococcus luteus have been studied. This whole-cell enzyme showed its maximal activity at 36 degrees C at pH 7.5. It was found that the activation energy of this enzyme was 8.03 kcal (ca. 33.6 kJ) per mol, and this amidohydrolase showed first-order decay at 36 degrees C. The penicillin amidohydrolase was deactivated rapidly at temperatures above 50 degrees C during storage or preincubation for 24 h. The Michaelis constant, Km, for penicillin G was determined as 2.26 mM, and the substrate inhibition constant, Kis, was 155 mM. The whole-cell penicillin amidohydrolase from M. luteus was capable of hydrolyzing penicillin G, penicillin V, ampicillin, and cephalexin, but not cephalosporin C and cloxacillin. This whole-cell enzyme also had synthetic activity for semisynthetic penicillins or cephalosporins from D-(--)-alpha-phenylglycine methyl ester and 6-alpha-aminopenicillanic acid or 7-amino-3-deacetoxycephalosporanic acid.     

Purification and partial characterization of a novel glucanhydrolase from Lipomyces starkeyi KSM 22 and its use for inhibition of insoluble glucan formation

A novel glucanhydrolase from a mutant of Lipomyces starkeyi ATCC 74054 was purified. The single protein (100 kDa) showed either dextranolytic or amylolytic activity. We referred to the glucanhydrolase as a DXAMase. The DXAMase was produced in a starch medium and it was 3.75-fold more active for hydrolysis of the purified insoluble-glucan of Streptococcus mutans than Penicillium funiculosum dextranase. Aggregation of S. mutans cells with dextran and adherence to glass were eliminated by incubating with the DXAMase. The addition of DXAMase (0.1 IU/ml) to the mutansucrase reaction digest with sucrose reduced the formation of insoluble-glucan about 80%. Also the DXAMase (0.5 IU/ml) removed 80% of the pre-formed sucrose-dependent adherent film. These in vitro properties of L. starkeyi KSM 22 DXAMase are desirable for its application as a dental plaque control agent.     

Hypoxia-inducible factor-2α regulates Fas-mediated chondrocyte apoptosis during osteoarthritic cartilage destruction

Apoptosis of articular chondrocytes is associated with the pathogenesis of osteoarthritis (OA). Recently, we demonstrated that hypoxia-inducible factor (HIF)-2α, encoded by Epas1, causes OA cartilage destruction by regulating the expression of various matrix-degrading enzymes. Here, we investigated the involvement of HIF-2α in chondrocyte apoptosis and OA cartilage destruction. HIF-2α levels in human and mouse OA chondrocytes were markedly elevated in association with increased apoptosis of articular chondrocytes. Overexpression or knockdown of HIF-2α alone did not cause chondrocyte apoptosis. However, HIF-2α expression markedly increased chondrocyte apoptosis in the presence of an agonistic anti-Fas (CD95) antibody. HIF-2α enhanced Fas expression and potentiated downstream signaling pathways, increasing the activity of initiator and executioner caspases. Overexpression of HIF-2α in mouse cartilage tissue, either by intra-articular injection of Epas1 adenovirus (Ad-Epas1) or in the context of chondrocyte-specific Epas1 transgenic mice, increased chondrocyte apoptosis and cartilage destruction. In contrast, chondrocyte-specific knockout of Epas1 in mice suppressed DMM (destabilization of the medial meniscus)-induced chondrocyte apoptosis and inhibited OA cartilage destruction. Moreover, Fas-deficient mice exhibited diminished chondrocyte apoptosis and OA cartilage destruction in response to Ad-Epas1 injection or DMM surgery. Taken together, our results demonstrate that HIF-2α potentiates Fas-mediated chondrocyte apoptosis, which is associated with OA cartilage destruction.