Hydroxylamine oxidoreductase [EC 1.7.3.4] of Nitrosomonas europaea was purified to an electrophoretically homogeneous state and some of its properties were studied. The molecular weight of the enzyme as determined by gel filtration on Sephadex G150 and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate is 175,000-180,000, while the minimum molecular weight per heme determined from the dry weight and heme content is 17,500. The enzyme is a C-type cytochrome; its reduced form shows absorption peaks at 418 (gamma peak), 521 (beta peak), 553 (alpha peak), and 460 nm (due to an unidentified chromophore). Although the alpha peak at 553 nm has a shoulder at 559 nm, the enzyme does not posses protoheme or a cytochrome b subunit. It seems likely that the enzyme molecule possess heme c molecules in different states. The enzyme reacts rapidly with various eukaryotic cytochromes c, but does not react with "bacterial-type" cytochromes c. Although the enzyme does not react with cytochrome c-552 (N. europaea), another C-type cytochrome of the organism, cytochrome c-554 (N. europaea) acts as an electron acceptor for the enzyme.

Directed evolution can rapidly achieve dramatic improvements in the properties of a protein or bestow entirely new functions on it. We have discovered a strong correlation between the probability of nding a productive mutation at a particular position of a protein and a chemical shift perturbation in Nuclear Magnetic Resonance spectra upon addition of an inhibitor for the chemical reaction it promotes. In a proof-of-concept study we converted myoglobin, a non-enzymatic protein, into the most active Kemp eliminase reported to date using only three mutations. The observed levels of catalytic e ciency are on par with the levels shown by natural enzymes. This simple approach, that requires no a priori structural or bioinformatic knowledge, is widely applicable and will unleash the full potential of directed evolution. Full TextDirected evolution is a powerful tool for improving existing properties and imparting completely new functionalities onto proteins. [1][2][3][4] Nonetheless, even in small proteins its potential is inherently limited by the astronomical number of possible amino acid sequences. Sampling the complete sequence space of a 100-residue protein would require testing of 20 100 combinations, which is currently beyond any existing experimental approach. Fortunately, in practice, selective modi cation of relatively few residues is su cient for e cient improvement, functional enhancement and repurposing of existing proteins. 5 Moreover, computational methods have been developed to predict the location, and, in certain cases, identities of potentially productive mutations. [6][7][8][9] Importantly, all current approaches for prediction of hot spots and productive mutations rely heavily on structural information and/or bioinformatics, which is not always available for proteins of interest. Moreover, they offer limited ability to identify bene cial mutations far from the active site, even though such changes may dramatically improve the catalytic properties of an enzyme. 10 Here we show that mutagenic hot spots in enzymes can be identi ed using Nuclear Magnetic Resonance (NMR) spectroscopy. In a proof-of-concept study we converted myoglobin, a non-enzymatic oxygen storage protein, into a highly e cient Kemp eliminase using only three mutations. The observed levels of catalytic e ciency (k cat /K M of 2.8 x 10 6 M -1 s -1 and k cat /k uncat > 10 8 ) are the highest reported for any designed protein and are on par with the levels shown by natural enzymes for the reactions they are evolved to catalyze. Given the simplicity of this experimental approach, which requires no a priori structural or bioinformatic knowledge, we expect it to be widely applicable and to unleash the full potential of directed enzyme evolution.Recent paradigm shifting advances in understanding the fundamental principles that drive enzyme evolution point to a major role of global conformational selection for productive arrangements of functional groups to perfect transition state stabilization, as well as steric and electrostatic interactio...

Introduction The use of veno-venous extracorporeal membrane oxygenation (VV-ECMO) in trauma patients has been controversial, but VV-ECMO plays a crucial role when the lungs are extensively damaged and when conventional management has failed. VV-ECMO provides adequate tissue oxygenation and an opportunity for lung recovery. However, VV-ECMO remains contraindicated in patients with a risk of bleeding because of systemic anticoagulation during the treatment. The most important point is controlling the bleeding from severe trauma. Case A 32-year-old male experienced blunt trauma due to a traffic accident. He presented with bilateral hemopneumothorax and bilateral flail chest. We performed emergency thoracotomy for active bleeding and established circulatory stability. After surgery, the oxygenation deteriorated under mechanical ventilation, so we decided to establish VV-ECMO. However, bleeding from the bilateral lung contusions increased after VV-ECMO was established, and the patient was switched to heparin-free ECMO. After conversion, we could control the bronchial bleeding, especially the lung hematomas, and the oxygenation recovered. The patient was discharged without significant complications. VV-ECMO and mechanical ventilation were stopped on days 10 and 11, respectively. He was discharged from the ICU on day 15. Conclusion When we consider the use of ECMO for patients with uncontrollable, severe bleeding caused by blunt trauma, it may be necessary to use a higher flow setting for heparin-free ECMO than typically used for patients without trauma to prevent thrombosis.

Abstract. The n-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), found in fish oil, exert a number of beneficial effects, and they are used in the treatment of hyperlipidemia. In recent years, EPA and DHA have been found to affect cancer cell proliferation. In the present study, PC3 cells, which are androgen-independent prostate cancer cells that resemble castration-resistant prostate cancer cells, were used to investigate a possible novel treatment for castration-resistant prostate cancer. The PC3 cells were cultured and incubated with various concentrations of EPA or DHA. Cancer proliferation was confirmed by trypan blue microscopy. Invasion and migration assays were used in the upper chamber in PC3 cells, and serum-free medium and various concentrations of EPA or DHA were placed in the lower chamber in serum-containing medium. EPA and DHA decreased PC3 cell proliferation, invasion and migration. The effect of EPA on PC3 cells was dose-dependent and significant differences were observed at concentrations of 100 and 200 µg/ml. The effect of DHA on PC3 cells was similar to that of EPA. In the migration assay, EPA exerted almost no effects at 25 µg/ml, but migration was reduced at 50 µg/ml. Similar to EPA, DHA exerted almost no effects at 25 µg/ml, but further reduction was observed at the 50 µg/ml concentration. In the invasion assay, EPA at 25 µg/ml was not significantly different from the control, but suppressed invasion at 50 µg/ml. DHA decreased invasion compared with the control at 25 µg/ml, whereas invasion was significantly reduced at a DHA concentration of 50 µg/ml. In conclusion, it was demonstrated that EPA and DHA were effective in decreasing the proliferation, invasion and migration of prostate PC3 cancer cells. However, the detailed underlying mechanisms have not yet been fully elucidated. IntroductionEicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are n-3 fatty acids that have several beneficial effects, including decreasing the plasma triglyceride concentration, lipoprotein metabolism (1,2), inhibiting platelet aggregation by collagen (3), and improving the reduced elasticity of the arterial walls through the endothelium-dependent relaxation response in rabbits (4). For these reasons, DHA and EPA are clinically used in Japan. In recent years, n-3 fatty acids have been shown to decrease the proliferation of cancer cells in colon, breast and liver cancer, as well as neuroblastoma. In addition, n-3 fatty acids may be used in cancer cachexia; the combination of chemotherapy and n-3 fatty acids was helpful in the curative as well as the palliative clinical setting (5), but a systematic review did not evaluate the balance of their cost-effectiveness against their utility (6). A number of studies have investigated cancer cell growth; however, only a limited number of studies have examined invasion and metastasis in prostate cancer, which exhibits an increasing prevalence. In the present study, the PC3 prostate cancer cell line was used, which is an androgen-independen...

The effects of cross-linked hyaluronate hydrogel and liquid sodium hyaluronate on post-surgical adhesion reformation were examined using a rabbit model. Primary adhesions in the ileocaecal region of Japanese white rabbits were induced by mechanical and chemical irritants during laparotomy. After 1 month the primary adhesions were lysed by microsurgery and cross-linked hyaluronate hydrogel or liquid sodium hyaluronate was applied to the lysed lesions. After 10-14 days the area of adhesion reformation was measured to assess any inhibitory effect of the test materials. Rabbits treated with cross-linked hyaluronate hydrogel showed a significant reduction in adhesion reformation area compared with liquid sodium hyaluronate or physiological saline treatment, and the area reduced to (mean +/- standard deviation) 0.6 +/- 1.95% of the original lesion. In a separate study, histological evaluation of rabbits treated with cross-linked hyaluronate hydrogel revealed a better healing pattern and a lower inflammatory response compared with controls. All these findings suggest cross-linked hyaluronate hydrogel may be a valuable anti-adhesion material to prevent post-surgical adhesion in abdominal or pelvic surgery.

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The degeneration of intervertebral disc (IVD) is a major cause of low back pain. However, there is no satisfactory preventive treatment for degenerative disc disease (DDD). In this study, we examined the effects of a novel cross-linked hyaluronate hydrogel and cross-linked chondroitin sulfate (CS) hydrogel on a rabbit model of IVD injury. We injected 300 µl of phosphate buffer saline, 1% sodium hyaluronate, cross-linked hyaluronate hydrogel, or cross-linked CS hydrogel into the injured IVDs.One, three or six months after treatment, the whole spinal columns were dissected and magnetic resonance (MR) images of the IVDs were examined. It was noted that the IVD, which was injected with cross-linked hyaluronate hydrogel or cross-linked CS hydrogel mostly retained the normal signal intensity of the MR images. These IVDs exhibited a higher degree of staining with safranin-O than the control discs or 1% sodium hyaluronate-injected discs, suggesting that the intradiscal application of cross-linked hyaluronate hydrogel or cross-linked CS hydrogel probably inhibits the degenerative cascade of the DDD. The intradiscal administration of these drugs is safe, easy and costs less. In the near future, these intradiscal injections may become the standard therapy for the treatment of DDD instead of the spine surgeries.

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