Crystal structure of the Holliday junction migration motor protein RuvB from Thermus thermophilus HB8

We report here the crystal structure of the RuvB motor protein from Thermus thermophilus HB8, which drives branch migration of the Holliday junction during homologous recombination. RuvB has a crescent-like architecture consisting of three consecutive domains, the first two of which are involved in ATP binding and hydrolysis. DNA is likely to interact with a large basic cleft, which encompasses the ATP-binding pocket and domain boundaries, whereas the junction-recognition protein RuvA may bind a flexible beta-hairpin protruding from the N-terminal domain. The structures of two subunits, related by a noncrystallographic pseudo-2-fold axis, imply that conformational changes of motor protein coupled with ATP hydrolysis may reflect motility essential for its translocation around double-stranded DNA.     

Bioactivation of loxoprofen to a pharmacologically active metabolite and its disposition kinetics in human skin

Loxoprofen (LX) is a prodrug‐type non‐steroidal anti‐inflammatory drug which is used not only as an oral drug but also as a transdermal formulation. As a pharmacologically active metabolite, the trans‐alcohol form of LX (trans‐OH form) is generated after oral administration to humans. The objectives of this study were to evaluate the generation of the trans‐OH form in human in vitro skin and to identify the predominant enzyme for its generation. In the permeation and metabolism study using human in vitro skin, both the permeation of LX and the formation of the trans‐OH form increased in a time‐ and dose‐dependent manner after the application of LX gel to the skin. In addition, the characteristics of permeation and metabolism of both LX and the trans‐OH form were examined by a mathematical pharmacokinetic model. The K_m value was calculated to be 10.3 mm in the human in vitro skin. The predominant enzyme which generates the trans‐OH form in human whole skin was identified to be carbonyl reductase 1 (CBR1) by immunodepletion using the anti‐human CBR1 antibody. The results of the enzyme kinetic study using the recombinant human CBR1 protein demonstrated that the K_m and V_max values were 7.30 mm and 402 nmol/min/mg protein, respectively. In addition, it was found that no unknown metabolites were generated in the human in vitro skin. This is the first report in which LX is bioactivated to the trans‐OH form in human skin by CBR1. Copyright © 2015 John Wiley & Sons, Ltd.     

Cloning of serum RNA associated with hepatitis C infection suggesting heterogeneity of the agent(s) responsible for the infection

Fifty-six lambda gt11-random-primed-cDNA recombinants of which translation products react with antibodies in the serum drawn from patients with hepatitis C (blood-borne non-A, non-B hepatitis) were cloned from serum pooled from donors presumably infected with hepatitis C. The specificity of these clones for hepaitits C infection was determined using 3 test panels. Of these 29 clones were determined to be specific for Japanese hepatitis C infection. However one of the 29 clones was positive for 1 out of 5 normals in an American test panel while 12 clones were positive for the American panel as well. The remaining 28 clones reacted well with serum from transfusion associated chronic hepatitis C comparing to the sporadic cases in the Japanese panel. When they were tested with normal donors, another clone reacted with a distinct donor group with which the other clones did not react. These results may suggest the presence of heterogeneity in Japanese hepatitis C.     

Effect of the calcium antagonist,diltiazem, on beta-agonist-induced reduction of beta-adrenergic responsiveness in the guinea pig lung

This study was designed to clarify the mechanism of tolerance that occurs during prolonged administration of a beta-agonist in relation to membrane phospholipid degradation and to elucidate the effect of diltiazem, a calcium antagonist. Guinea pigs were divided into 3 groups: (1) control—physiological saline (0.5 ml) was injected once a day for 7 successive days; (2) metaproterenol (Mp)—Mp was injected intraperitoneally (10 mg/kg/day) for 7 successive days; (3) Mp + diltiazem—diltiazem was injected intraperitoneally (20 mg/kg/day) 30 min before Mp injection for 7 successive days. The number of beta-adrenoceptors and the 10⁻⁵ M (−)-isoproterenol-stimulated adenylate cyclase activity were significantly decreased in the metaproterenol group. Diltiazem reduced these decreases. Phospholipase activity was increased and phosphatidylcholine and phosphatidylethanolamine levels were decreased in the metaproterenol group. Diltiazem also reduced these changes. These results suggest that the degradation of membrane phospholipids by phospholipase may be involved in a decrease in beta-adrenergic response caused by successive administration of metaproterenol. Diltiazem protects membrane phospholipids from phospholipase attack, which in turn maintains beta-adrenergic responsiveness. Part of this study was presented at the Annual Meeting of the American Thoracic Society, May 12, 1987, New Orleans, Louisiana     

Vascular Wall Imaging of Unruptured Cerebral Aneurysms with a Hybrid of Opposite-Contrast MR Angiography

BACKGROUND AND PURPOSE:Inflammation and degeneration of the intracranial saccular aneurysm wall play a major role in aneurysm formation, development and subsequent rupture. The aim of this study was to characterize the walls of unruptured intracranial aneurysms by using a hybrid of opposite-contrast MRA at 3T.MATERIALS AND METHODS:Fourteen consecutive patients with 17 unruptured intracranial aneurysms who initially underwent clipping surgery were prospectively evaluated. All aneurysms were scanned preoperatively by using a hybrid of opposite-contrast MRA in 3T high-resolution MR imaging. We classified intraoperative findings of atherosclerotic plaques in the aneurysms into 3 grades: grade A (major plaques), grade B (minor plaques), and grade C (no plaques). The contrast ratio of the high-intensity area was also measured relative to the background low-intensity area inside the carotid artery.RESULTS:Findings from preoperative plaque imaging of the aneurysm corresponded to the intraoperative findings in 15 of 16 aneurysms (excluding 1 that was impossible to visualize in its entirety due to anatomic reasons). Overall sensitivity and specificity of the hybrid of opposite-contrast MRA were 88.9% and 100%, respectively. During the operation, 4 aneurysms were classified as grade A; 5, as grade B; and 7, as grade C. The means of the contrast ratio for grades A, B, and C were 0.72 ± 0.03, 0.34 ± 0.30, and −0.02 ± 0.09, respectively.CONCLUSIONS:The hybrid of opposite-contrast MRA can detect visible atherosclerotic plaques in the unruptured aneurysm wall, and the contrast ratio in intracranial aneurysms correlated with their presence and extent. A study including a larger series is needed to validate the diagnostic potential of this imaging technique.

Intracranial aneurysms are common vascular lesions, often consisting of a saccular dilation of a cerebral artery vessel. The prevalence of intracranial aneurysms in the general population is estimated between 2.5% and 5%.^1,2 Aneurysmal rupture occurs with a 1% risk per year, depending on the size, location, and morphometry of the aneurysm, and leads to subarachnoid hemorrhage with associated high morbidity and mortality rates.^1,2 Intracranial aneurysms with an estimated high risk of rupture undergo management via a surgical or endovascular approach, depending on the specific risks of treatment.^3,4 Therefore, it is important to accurately assess the risk of aneurysmal rupture.The pathogenesis of intracranial aneurysms and their natural history are not well-understood. Histopathologic studies have shown that the infiltration of inflammatory cells and the degeneration of the aneurysm wall with atherosclerosis correlates with the formation, development, and rupture risk of cerebral aneurysms.^5–9 However, characterization of the aneurysm wall is limited by imaging data quality and the need to harvest surgical specimens.In this regard, the characteristics of high-field-strength MR imaging, which has a favorable SNR and changes in relaxation time and susceptibility, can depict the intracranial vessel walls and their pathologies, including small vessels with atherosclerosis.^10,11The hybrid of opposite-contrast MR angiography (HOP-MRA) used in this study is a modern technique that combines the advantages of 3D TOF MRA and flow-sensitive black-blood (FSBB) MRA.¹² The clinical efficacy of this technique was established to improve the visualization of peripheral vessels.^13,14 Theoretically, tissue with shorter T1 and T2* introduces high signal in FSBB of HOP-MRA, which demonstrates atherosclerotic plaques, including fat, as high-signal-intensity areas and demonstrates the blood space as low-signal-intensity areas in intracranial aneurysms.¹² The strength of this technique is the dual-echo 3D gradient-echo sequence, which enables a shorter imaging time and minimization of misregistration. The present study investigated the utility of HOP-MRA at 3T for the characterization of visible atherosclerotic plaques in intracranial aneurysms by using subtraction between TOF and FSBB imaging.     

Infection mechanism of biofilm‐forming Staphylococcus aureus on indwelling foreign materials in mice

Indwelling foreign‐body infections are a critical medical problem, especially in immunocompromised patients. To examine the pathogenicity of biofilm‐forming bacteria settling on foreign materials, mice implanted with plastic discs were infected with Staphylococcus aureus. After opening a wide subcutaneous pocket on the dorsal side of mice with or without temporal leukocytopenia, a plastic sheet was placed in the left subcutaneous space; subsequently, bacteria in a planktonic state were dispersed over the subcutaneous space. Bacterial numbers were examined 7 days after inoculation. In subcutaneous tissue on the right, S. aureus was found only in leukocytopenic mice. Meanwhile, bacteria were detected on the plastic and neighbouring tissue in both leukocytopenic and normal mice; however, colony‐forming analysis indicated that leukocytopenic mice possessed significantly more bacteria. Tissue reaction against bacteria was pathologically examined. Invading S. aureus induced severe inflammation. In transient leukocytopenic mice, bacterial microcolonies formed on the plastic as well as in the developed necrotic tissue – both of which were shielded from inflammatory cell infiltration – result in bacteraemia. These results indicate that biofilm‐forming S. aureus settling on indwelling foreign material are tolerant against host immunity and assault neighbouring tissue, which may lead to chronic wound infection.