Previously, we reported the molecular cloning of cDNA for the prophenoloxidase activating factor‐I (PPAF‐I) that encoded a member of the serine proteinase group with a disulfide‐knotted motif at the N‐terminus and a trypsin‐like catalytic domain at the C‐terminus [Lee, S.Y., Cho, M.Y., Hyun, J.H., Lee, K.M., Homma, K.I., Natori, S., Kawabata, S.I., Iwanaga, S. & Lee, B.L. (1998) Eur. J. Biochem. 257, 615–621]. PPAF‐I is directly involved in the activation of pro‐phenoloxidase (pro‐PO) by limited proteolysis and the overall structure is highly similar to that of Drosophila easter serine protease, an essential serine protease zymogen for pattern formation in normal embryonic development. Here, we report purification and molecular cloning of cDNA for another 45‐kDa novel PPAF from the hemocyte lysate of Holotrichia diomphalia larvae. The gene encodes a serine proteinase homologue consisting of 415 amino‐acid residues with a molecular mass of 45 256 Da. The overall structure of the 45‐kDa protein is similar to that of masquerade, a serine proteinase homologue expressed during embryogenesis, larval, and pupal development in Drosophila melanogaster. The 45‐kDa protein contained a trypsin‐like serine proteinase domain at the C‐terminus, except for the substitution of Ser of the active site triad to Gly and had a disulfide‐knotted domain at the N‐terminus. A highly similar 45‐kDa serine proteinase homologue was also cloned from the larval cDNA library of another coleopteran, Tenebrio molitor. By in vitro reconstitution experiments, we found that the purified 45‐kDa serine proteinase homologue, the purified active PPAF‐I and the purified pro‐PO were necessary for expressing phenoloxidase activity in the Holotrichia pro‐PO system. However, incubation of pro‐PO with either PPAF‐I or 45‐kDa protein, no phenoloxidase activity was observed. Interestingly, when the 45‐kDa protein was incubated with PPAF‐I and pro‐PO in the absence, but not in the presence of Ca2+, the 45‐kDa protein was cleaved to a 35‐kDa protein. RNA blot hybridization revealed that expression of the 45‐kDa protein was increased in the Holotrichia hemolymph after Escherichia coli challenge.
Previously, we purified and characterized a pro-phenol-oxidase (pro-PO) of 79 kDa from coleopteran insect, Holotrichia diomphalia larvae [Kwon et al. (1997) Mol. Cells 7, 90Ϫ97]. Here, we describe the identification of two pro-PO-activating factors (PPAF), named PPAF-I and PPAF-II, directly involved in the activation of the isolated pro-PO. When pro-PO was incubated with either PPAF-I or PPAF-II, no phenol oxidase activity was observed. However, incubation of pro-PO with both PPAF-I and PPAF-II specifically exhibited phenol oxidase activity. The purified PPAF-I with a molecular mass of 33 kDa on SDS/PAGE had characteristics of a serine protease. It exhibited amidase activity against fluorogenic peptide substrates, tert-butoxycarbonyl-phenylalanyl-seryl-arginyl-4-methylcoumaryl-7-amide being the best among the substrates examined. The activity was completely inhibited by 0.02 mM p-nitrophenylp′-guanidinobenzoate · HCl and diisopropylflurophosphate. The NH 2 -terminal sequence of PPAF-I had significant sequence similarity to those of serine proteases. On the other hand, the purified PPAF-II had a molecular mass of 40 kDa on SDS/PAGE and 400 kDa determined by gel filtration, indicating an oligomeric protein. The NH 2 -terminal sequence of PPAF-II showed no similarity to known proteins. PPAF-II exhibited no amidase activity against the fluorogenic substrates. Reconstitution experiments and immunoblotting analysis using affinity-purified antibody against pro-PO demonstrated that PPAF-I first cleaves the intact pro-PO to an intermediate of 76 kDa with no phenol oxidase activity, and then, PPAF-I converts the intermediate to the active phenol oxidase of 60 kDa in the presence of PPAF-II. These results indicate that the activation of pro-PO system in hemolymph of H. diomphalia larvae is accomplished by at least two activating factors, a serine protease and a protein cofactor.
No abstract
Glucose homeostasis is maintained by the orchestration of peripheral glucose utilization and hepatic glucose production, mainly by insulin. In this study, we found by utilizing a combined parallel chromatography mass profiling approach that lysophosphatidylcholine (LPC) regulates glucose levels. LPC was found to stimulate glucose uptake in 3T3-L1 adipocytes dose-and time-dependently, and this activity was found to be sensitive to variations in acyl chain lengths and to polar head group types in LPC. Treatment with LPC resulted in a significant increase in the level of GLUT4 at the plasma membranes of 3T3-L1 adipocytes. Moreover, LPC did not affect IRS-1 and AKT2 phosphorylations, and LPC-induced glucose uptake was not influenced by pretreatment with the PI 3-kinase inhibitor LY294002. However, glucose uptake stimulation by LPC was abrogated both by rottlerin (a protein kinase C␦ inhibitor) and by the adenoviral expression of dominant negative protein kinase C␦. In line with its determined cellular functions, LPC was found to lower blood glucose levels in normal mice. Furthermore, LPC improved blood glucose levels in mouse models of type 1 and 2 diabetes. These results suggest that an understanding of the mode of action of LPC may provide a new perspective of glucose homeostasis.
Magnesium alloys are biodegradable metals receiving increasing attention, but the clinical applications of these materials are delayed by concerns over the rapid corrosion rate and gas formation. Unlike corrosion, which weakens mechanical properties, the gas formation issue has received little attention. Therefore, we evaluated the gas formation and biological effects for Mg implants through preclinical (immersed in Earle’s balanced salt solution and in vivo) and clinical studies. The immersion test examined the gas volume and composition. The in vivo study also examined gas volume and histological analysis. The clinical study examined the gas volume and safety after Mg screw metatarsal fixation. Gas was mainly composed of H2, CO and CO2. Maximum volumes of gas formed after 5 days for in vivo and 7 days in clinical study. Within the clinical examination, two superficial wound complications healed with local wound care. Osteolytic lesions in the surrounding metaphysis of the Mg screw insertion developed in all cases and union occurred at 3 months. Mg implants released gas with variable volumes and composition (H2, CO, and CO2), with no long-term toxic effects on the surrounding tissue. The implants enabled bone healing, although complications of wound breakdown and osteolytic lesions developed.
Aggregates of amyloidogenic peptides are involved in the pathogenesis of several degenerative disorders. Herein, an iridium(III) complex, Ir-1, is reported as a chemical tool for oxidizing amyloidogenic peptides upon photoactivation and subsequently modulating their aggregation pathways. Ir-1 was rationally designed based on multiple characteristics, including 1) photoproperties leading to excitation by low-energy radiation; 2) generation of reactive oxygen species responsible for peptide oxidation upon photoactivation under mild conditions; and 3) relatively easy incorporation of a ligand on the IrIII center for specific interactions with amyloidogenic peptides. Biochemical and biophysical investigations illuminate that the oxidation of representative amyloidogenic peptides (i.e., amyloid-β, α-synuclein, and human islet amyloid polypeptide) is promoted by light-activated Ir-1, which alters the conformations and aggregation pathways of the peptides. Additionally, their potential oxidation sites are identified as methionine, histidine, or tyrosine residues. Overall, our studies on Ir-1 demonstrate the feasibility of devising metal complexes as chemical tools suitable for elucidating the nature of amyloidogenic peptides at the molecular level, as well as controlling their aggregation.
We demonstrate a novel fabrication technology of the microneedle array applied to painless drug delivery and minimal invasive blood extraction. The fabrication technology consists of a vertical deep X-ray exposure and a successive inclined deep X-ray exposure with a deep X-ray mask whose pattern has a hollow triangular array. The vertical exposure makes triangular column array with a needle conduit. With the successive inclined exposure, the column array shapes into the microneedle array without deep X-ray mask alignment. Changing the inclined angle and the gap between the mask and PMMA (PolyMethylMetaAcrylic) substrate, different types of microneedle array are fabricated in 750-1000 lm shafts length, 15 o -20 o tapered tips angle, and 190-300 lm bases area. The masks are designed to 400-600 lm triangles length, 70-100 lm conduits diameter, 25-60EA/5 mm 2 arrays density, and various tip shapes such as triangular, rounded, or arrow-like features. In the medical application, the fabricated PMMA microneedle array fulfills the structural requirements such as three-dimensional sharp tapered tip, HAR (High-AspectRatio) shafts, small invasive surface area, and out-of-plane structure. In the skin test, the microneedle array penetrates back of the hand skin with minimum pain and without tip break and blood is drawn after puncturing the skin. Hot embossing process and mold fabrication process are also investigated with silicon and PDMS mold. The processed tetrahedral PMMA structures are fabricated into the microneedle array by the additional deep X-ray exposure. With these processes, the microneedle array can be utilized as the mold base for electroplating process.
Nanostructures converting chemical energy to mechanical work by using benign metabolic fuels,h ave huge implications in biomedical science.Here,weintroduce Au/Ptbased Janus nanostructures,r esembling to "egg-in-nest" morphology (Au/Pt-ENs), showing enhanced motion as aresult of dual enzyme-relay-like catalytic cascade in physiological biomedia, and in turn showing molecular-laden transport to living cells.W ed eveloped dynamic-casting approach using silica yolk-shell nanoreactors:f irst, to install al arge Au-seed fixing the silica-yolk aside while providing the anisotropically confined concave hollown anospace to grow curved Ptdendritic networks.O wing to the intimately interfaced Au and Pt catalytic sites integrated in au nique anisotropic nestlike morphology, Au/Pt-ENse xhibited high diffusion rates and displacements as the result of glucose-converted oxygen concentration gradient. High diffusiophoresis in cell culture media increased the nanomotor-membrane interaction events, in turn facilitated the cell internalization. In addition, the porous network of Au/Pt-ENsf acilitated the drug-molecule cargo loading and delivery to the living cells.
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