A programmable DNA origami nanospring that reveals force-induced adjacent binding of myosin VI heads.

Mechanosensitive biological nanomachines such as motor proteins and ion channels regulate diverse cellular behaviour. Combined optical trapping with single-molecule fluorescence imaging provides a powerful methodology to clearly characterize the mechanoresponse, structural dynamics and stability of such nanomachines. However, this system requires complicated experimental geometry, preparation and optics, and is limited by low data-acquisition efficiency. Here we develop a programmable DNA origami nanospring that overcomes these issues. We apply our nanospring to human myosin VI, a mechanosensory motor protein, and demonstrate nanometre-precision single-molecule fluorescence imaging of the individual motor domains (heads) under force. We observe force-induced transitions of myosin VI heads from non-adjacent to adjacent binding, which correspond to adapted roles for low-load and high-load transport, respectively. Our technique extends single-molecule studies under force and clarifies the effect of force on biological processes.

Impaired intrinsic chiral inversion activity of ibuprofen in rats with adjuvant-induced arthritis.

1. The effects of adjuvant-induced arthritis on the chiral inversion of 'profens', a type of non-steroidal anti-inflammatory drug, have hardly been investigated. The authors investigated the effects of adjuvant-induced arthritis on the chiral inversion of ibuprofen using freshly isolated rat hepatocytes. 2. S- or R-ibuprofen was incubated with hepatocytes isolated from control and adjuvant-induced arthritis rats in the absence of the serum. In the hepatocyte system the chiral inversion rate constant of R- to S-ibuprofen and the metabolic rate constants of both enantiomers in adjuvant-induced arthritis rats were significantly decreased to about 64-80% of the corresponding values in control rats. In contrast, the addition of serum from each group to the corresponding hepatocyte medium resulted in no significant differences in these rate constants between control and adjuvant-induced arthritis rats. 3. With regard to chiral inversion enzymes, adjuvant-induced arthritis decreased the messenger RNA levels of acyl-coenzyme A synthetase (ACS) isoforms, but not 2-arylpropionyl-CoA epimerase, compared with control rats. 4. Chiral inversion of R- to S-ibuprofen was inhibited by triacsin C, a specific inhibitor of ACS1. 5. The results suggest that adjuvant-induced arthritis induces down-regulation of ACS enzymes involved in chiral inversion of R- to S-ibuprofen in rats.

Prediction of metabolic clearance of diclofenac in adjuvant-induced arthritis rats using a substrate depletion assay.

1. The purpose of this study was to evaluate drug clearance measured by the metabolic intrinsic clearance (CL(int)) in a substrate depletion assay in comparison with the in vivo clearance (CL(tot)) observed in adjuvant-induced arthritis (AA) rats. 2. After intravenous administration of diclofenac as a model drug, CL(tot) was 2.8-fold higher in AA rats than in control rats. In two different substrate depletion assays with liver microsomes for glucuronidation and hydroxylation, the CL(int) values for glucuronidation was significantly decreased in AA rats to 60% of the value in control rats, whereas the CL(int) values for hydroxylation were similar. The unbound fraction of diclofenac in plasma (f(u, plasma)) was significantly higher (2.8-fold) in AA rats than in control rats. 3. Hepatic clearance predicted from the CL(int) values for both biotransformation pathways and f(u, plasma) was higher in AA rats than in control rats, with good consistency between predicted and observed values. The same results were obtained for experiments using hepatocytes. 4. The plasma protein-binding activities, rather than metabolic clearance, in both types of rats would be a determining factor in the pharmacokinetic behaviour differences between control and AA rats. 5. In summary, substrate depletion assays with liver microsomes and hepatocytes in combination with protein binding assessment can help to predict changes in pharmacokinetics under AA conditions.

Analysis of progressive ophthalmic lesion in a patient with subacute sclerosing panencephalitis.

PURPOSE: To evaluate the progressive lesions affecting the visual system in a patient with subacute sclerosing panencephalitis (SSPE). METHODS: The authors observed a 15-year-old boy with SSPE. Since the diagnosis was made before the appearance of ocular manifestations, the authors recorded the progressive ocular lesions using various ophthalmic examinations. RESULTS: The patient showed no ophthalmic abnormalities until he developed a left homonymous hemianopia with sudden bilateral disturbed visual acuity. Severe progressive macular lesions including a pigment epithelial window defect by fluorescein angiography, a marked decrease in foveal thickness by optical coherence tomography, and an extensive disorder mainly specific to cone cells in the central retina by electroretinography were demonstrated. Novel findings such as a transient relative afferent pupillary defect and an anterior uveitis were also observed. CONCLUSIONS: Analyses over a long period of time showed progressive ophthalmic findings in a patient with SSPE.

A comparison of catalytic site intermediates of cytochrome c oxidase and peroxidases.

Compounds I and II of peroxidases such as horseradish peroxidase and cytochrome c peroxidase are relatively well understood catalytic intermediates in terms of their structures and redox states of iron, heme, and associated radical species. The intermediates involved in the oxygen reduction chemistry of the cytochrome c oxidase superfamily are more complicated because of the need for four reducing equivalents and because of the linkage of the oxygen chemistry with vectorial proton translocations. Nevertheless, two of these intermediates, the peroxy and ferryl forms, have characteristics that can in many ways be considered to be counterparts of peroxidase compounds I and II. We explore the primary factors that minimize the generation of unwanted reactive oxygen species products and ensure that the principal enzymological function becomes either that of a peroxidase or an oxidase. These comparisons can provide insights into the nature of biological oxygen reduction chemistry and guidance for the engineering of biomimetic synthetic materials.

Decreased PXR and CAR inhibit transporter and CYP mRNA Levels in the liver and intestine of mice with collagen-induced arthritis.

Nuclear receptors, such as pregnane X receptor (PXR) and constitutive androstane receptor (CAR), regulate the transcription of transporters and cytochrome P450s (CYPs). We investigated whether quantitative and functional changes in PXR and CAR affected the transporters and CYPs in a mouse model of chronic arthritis. The mRNA levels of PXR were significantly decreased in the intestine of mice with collagen-induced arthritis (CIA) compared with control mice. The mRNA levels of CAR were significantly decreased in both the liver and intestine of CIA mice. The mRNA levels of Mdr1a/1b, Mrp3, BCRP and Cyp2b10 were decreased in the liver of CIA mice, while little change in the mRNA levels was observed for Cyp3a11 in the liver and the transporters in the intestine. Taken together, the present results reveal that the effects of CAR mRNA suppression on the regulation of transporters and CYPs differ between the liver and intestine in chronic arthritis.

Transformation of Acinetobacter sp. BD413 with DNA from commercially available genetically modified potato and papaya.

AIM: To estimate the likelihood of transfer of kanamycin-resistance gene (nptII) from commercially available genetically modified (GM) plants. METHODS AND RESULTS: Acinetobacter sp. BD413 carrying a plasmid containing an inactivated nptII gene was treated with DNA derived from GM potato and GM papaya. Kanamycin-resistant transformants were obtained at a frequency of 10-30 microg(-1) DNA. Calculation of the results suggested that 6-9 x 10(4) molecules of genomic DNA from GM plants were needed to obtain one transformant. However, such transformation events were not detectable in the absence of the plasmid in the host strain. CONCLUSIONS: Acinetobacter sp. BD413 was transformed with DNA derived from GM potato and GM papaya, in the presence of an inactivated nptII gene on a plasmid. However, the frequency of such events in the natural environment on wild-type strains, while evidently low, remains unknown. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results may help to evaluate potential risks associated with the use of antibiotic-resistance determinants as genetic markers in GM plants. Complete risk assessment must consider factors other than transformation frequency alone, including the natural background of antibiotic resistance present in bacterial populations, and the spectrum and clinical use of the antimicrobial agents in question.

Usefulness of hepatocytes for evaluating the genetic polymorphism of CYP2D6 substrates.

The usefulness of human hepatocytes for assessing CYP2D6-related genetic polymorphisms was investigated. Propranolol and propafenone, which undergo phase I and II biotransformations, were used as model substrates alongside metoprolol, which is only metabolized via oxidative pathways. The contributions of CYP2D6 to the primary metabolisms of the substrates were estimated from the quinidine-mediated inhibition of their depletion rate constants in human hepatocytes and liver microsomes. The contributions in hepatocytes were 19.2% for propranolol at 0.05 microM and 36.7--76.3% for propafenone at 0.05--1.0 microM, and smaller than the contribution in microsomes, unlike the case for metoprolol. The differences between microsomes and hepatocytes were attributable to conjugate formation. The CYP2D6 contributions in hepatocytes reflected the in vivo data. The relevance of the concentration-dependent involvement of CYP2D6 in propafenone metabolism in hepatocytes to the in vivo polymorphic profile and the applicability of hepatocytes for evaluating these polymorphisms are discussed.

Advanced glycation end product (age) inhibitors and their therapeutic implications in diseases.

Nonenzymatic modification of proteins by reducing sugars, a process that is also known as the Maillard reaction, leads to the formation of advanced glycation end products (AGEs) in vivo. There is a growing body of evidence that formation and accumulation of AGEs progress during normal aging, and at an extremely accelerated rate under diabetes, and are thus involved in the pathogenesis of various diseases such as diabetic vascular complications and neurodegenerative diseases. Therefore, inhibition of AGE formation may be a promising target for therapeutic intervention in AGE-related disorders. In this review, we discuss several types of AGE inhibitors and their therapeutic implications in diseases.

Modification of photosystem I reaction center by the extraction and exchange of chlorophylls and quinones.

The photosystem (PS) I photosynthetic reaction center was modified thorough the selective extraction and exchange of chlorophylls and quinones. Extraction of lyophilized photosystem I complex with diethyl ether depleted more than 90% chlorophyll (Chl) molecules bound to the complex, preserving the photochemical electron transfer activity from the primary electron donor P700 to the acceptor chlorophyll A(0). The treatment extracted all the carotenoids and the secondary acceptor phylloquinone (A(1)), and produced a PS I reaction center that contains nine molecules of Chls including P700 and A(0), and three Fe-S clusters (F(X), F(A) and F(B)). The ether-extracted PS I complex showed fast electron transfer from P700 to A(0) as it is, and to FeS clusters if phylloquinone or an appropriate artificial quinone was reconstituted as A(1). The ether-extracted PS I enabled accurate detection of the primary photoreactions with little disturbance from the absorbance changes of the bulk pigments. The quinone reconstitution created the new reactions between the artificial cofactors and the intrinsic components with altered energy gaps. We review the studies done in the ether-extracted PS I complex including chlorophyll forms of the core moiety of PS I, fluorescence of P700, reaction rate between A(0) and reconstituted A(1), and the fast electron transfer from P700 to A(0). Natural exchange of chlorophyll a to 710-740 nm absorbing chlorophyll d in PS I of the newly found cyanobacteria-like organism Acaryochloris marina was also reviewed. Based on the results of exchange studies in different systems, designs of photosynthetic reaction centers are discussed.

[Anti-allergic drugs inhibit the proliferation of bovine lens epithelial cells in culture].

PURPOSE: To examine the inhibitory effects of tranilast, ketotifen fumarate, and disodium cromoglycate which are used clinically as anti-allergic agents, on the growth of bovine lens epithelial cells (LE) in culture. METHODS: LE was grown in Dulbecco's modified Eagle's medium(DMEM) with 10% fetal calf serum and growth was measured with 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide(MTT) and 5-bromo-2'deoxy-uridine(BrdU). Production of collagen, transforming growth factor-beta 1(TGF-beta 1), and basic-fibroblast growth factor(b-FGF) were measured with corresponding enzyme-linked immunosorbent assay(ELISA). Apoptotic cell death was detected by TdT-mediated dUTP-biotin nick end labelling method(TUNEL technique) and the DNA ladder method. RESULTS: Both ketotifen and tranilast inhibited the growth of LE, and half-inhibitory concentrations were 200 microM and 1,000 microM, respectively. Disodium cromoglycate did not inhibit LE proliferation significantly. Ketotifen and tranilast decreased the synthesis of collagen but had no obvious effect on TGF-beta 1 and b-FGF production. Apoptotic cell death was detected in LE treated with ketotifen or tranilast. CONCLUSION: Ketotifen and tranilast may be clinically useful for the prevention of aftercataract. Apoptotic cell death may be involved in the process.

[Clinical features of idiopathic macular holes-differences between sexes and stages].

PURPOSE: The purpose of this study was to evaluate the differences in the clinical features of idiopathic macular holes between sexes and stages. METHODS: Five hundred and twenty-six eyes of 480 patients with stage 3 or 4 idiopathic macular hole that had undergone vitrectomy were observed consecutively in this study. The each stage ratio, bilaterality, and affected eye were examined and the differences in age, hole duration, hole size, visual acuity, refractive power, axial length, and corneal refractive power were evaluated. RESULTS: Twenty-six % of the cases were stage 4 in males and 31% in females. There were no significant differences in bilaterality or affected eye between the sexes. Younger age and larger size were found in females of stage 3. Larger size was found in stage 4. More myopic eye and longer axial length were found in males of stage 4. There were no significant differences in hole duration and visual acuity between sexes or stages. CONCLUSIONS: In females the onset of macular hole occurred at a younger age than in males, size of the hole was larger from an earlier stage, and refractive power was less myopic. More myopic eye and longer axial length were found in stage 4, especially in males. This fact might be related to the existence of posterior vitreous detachment. We concluded that there were some differences in the mechanism of the onset and the progression of idiopathic macular hole between males and females.

Pharmacokinetics of epinastine and a possible mechanism for double peaks in oral plasma concentration profiles.

The pharmacokinetics of epinastine (EPN), an anti-allergic agent, was investigated in rats. The plasma concentration-time profile of EPN after intravenous (i.v.) administration was triexponential. After oral administration of EPN (7.5 and 20 mg/kg), the drug was rapidly absorbed, and Cmax was reached 2 h after dosing. A minor secondary peak was observed in EPN plasma concentration-time profiles at both doses. The bioavailability of EPN after oral dosing was 41 and 40%. The kinetic parameters (T 1/2, AUC and MRT) for unlabeled EPN were much smaller than those for 14C-EPN, which has already been reported. The total biliary excretion of EPN at a 7.5 mg/kg dose was 15.5% of the dose, but the percentage of conjugates in bile was extremely low and about 11% of the total biliary excretion. The increase in the plasma concentration in bile duct-linked rats after oral administration of EPN (20 mg/kg) was not observed, indicating that a secondary increase in drug concentration based on enterohepatic circulation was ruled out. When the gastrointestinal (GI)-transit of phenol red (PR) after oral administration of EPN (20 mg/kg) was estimated, the GI-transit of PR was significantly delayed, and at 3-4 h after dosing half of the PR dose reached the jejunum. The remaining EPN in the small intestine after oral administration (7.5 mg/kg) reached peak levels 2 h after dosing, but then partly increased again at 4 h. As a result, it was clarified that the double peaks observed after oral doses are mainly due to the delayed absorption of a part of EPN, based on the reduction in gastric motility caused by the drug.

Three-dimensional STEM for observing nanostructures.

A new scanning transmission electron microscope has been developed for three-dimensional (3D) observations of nanostructures. Using double spherical fulcra, accurate eucentric rotation was achieved. Cylindrical specimens for 3D-observation were prepared by a microsampling technique using a focused ion beam. Copper via-holes of a semiconductor memory device and ZnO particles were observed by the 3D-STEM from different directions, and 3D-data of the ZnO particles were successfully reconstructed in a topography mode.

Oral delivery of synthetic eel calcitonin, elcatonin, in rats.

This study was designed to develop an oral dosage form of elcatonin (EC), a hypocalcemic peptide. The EC absorption was estimated by the reduction in plasma calcium concentrations. When EC was orally coadministered with nitroso-N-acetyl-D,L-penicillamine (SNAP, 4.0 mg) and 0.02% Carbopol solution or with taurocholate (20 mM) and 0.02% Carbopol solution, the lowering effect was increased compared with that after EC alone, but the F values (0.32 and 0.30%) were extremely small. The oral administration of the mucoadhesive emulsion, which was prepared by coating the W/O/W emulsion with 0.1% Carbopol, enhanced the calcium lowering effect, with the F value of 0.43%. The strong mucoadhesion of the mucoadhesive emulsion to the gastrointestinal mucosa was observed. A capsule containing EC (500 microg), taurocholate (6 mg) and lyophilized Carbopol (3.5 mg) administered orally gave a sustained but comparatively small calcium lowering effect. In the in vitro enzymatic hydrolysis experiment, EC was more rapidly hydrolyzed in the intestinal fluid than in the mucosal extract. The combination of 20 mM taurocholate with 0.02% Carbopol showed the greatest inhibitory effect in both fluid and extract. These data indicated that EC was effectively absorbed through the intestinal wall, but the peptide was dominantly degraded by proteolytic enzymes in the GI tract. These results will offer a potential approach to the oral delivery of EC.

Capillary electrophoresis of sialic acid-containing glycoprotein. Effect of the heterogeneity of carbohydrate chains on glycoform separation using an alpha1-acid glycoprotein as a model.

alpha1-Acid glycoprotein (AGP) showed multiple peaks on separation using capillary electrophoresis in a chemically modified capillary with dimethylpolysiloxane at slightly acidic conditions. We analyzed glycoforms of AGP species after separation by ion-exchange chromatography, Con A affinity chromatography, and Cu(II)-chelating affinity chromatography. The AGP species thus obtained were digested with N-glycosidase F, and the released carbohydrate chains were analyzed by high-performance liquid chromatography after labeling with 3-aminobenzoic acid. The results afforded basic information on the contribution of carbohydrate chains to the separation mechanism of glycoforms of AGP by capillary electrophoresis. In addition, we describe an easy method for AGP analysis in serum samples using the electrokinetic injection.

Variables That Influence Visual Acuity After Macular Hole Surgery.

Purpose: To evaluate the variables that influence visual acuity and visual improvement after macular hole surgery.Methods: Our study included 421 eyes in which macular holes were successfully closed after surgery and followed up at least 1 year after the last surgery. Surgical techniques were conventional methods (Group 1: 350 eyes) with retinal pigment scalping of the macular hole basis added in the refractory cases (Group 2: 71 eyes). The variables used for the multiple regression were gender, age, preoperative visual acuity, hole stage, duration of symptoms, hole size, and axial length.Results: The variables that most influenced postoperative visual acuity were as follows: Group 1: gender (r = -0.011, P =.016), age (r = -0.17, P =.005), preoperative visual acuity (r = 0.51, P <.0001), duration of symptoms (r = -0.015, P <.0001), and axial length (r = -0.090, P =.045). Group 2: age (r = -0.18, P =.047), and preoperative visual acuity (r = 0.47, P <.0001).Conclusions: The variables that influenced visual acuity and visual improvement after macular hole surgery were common. In Group 1: gender, age, preoperative visual acuity, duration of symptoms, and axial length; in Group 2: age and preoperative visual acuity.

Incidence of Reopening and Variables That Influence Reopening After Macular Hole Surgery.

Purpose: To evaluate the incidence and variables of reopening of macular holes after macular hole surgery.Methods: Our study included 467 eyes in which macular holes were successfully closed after surgery. Surgical techniques consisted of conventional methods (358 eyes) and scalping methods (109 eyes) with retinal pigment scalping of the macular hole basis added in such cases: reoperation, hole size (more than 0.4 disc diameter), duration of symptoms (more than 2 years). Long term incidence of reopening was predicted by life table method. After we compared reopened cases with non-reopened cases, the variables of gender, stage, biocular occurrence, age, duration of symptoms, hole size, preoperative visual acuity, refraction axial length ratio, and intraoperative retinal tears were used for the multiple regression.Results: Reopening was found in 20 eyes (5.6%) treated by conventional methods and in 10 eyes (9.2%) treated by scalping methods. Survival ratio was 87% for the conventional methods in 6 years and 79% for the scalping methods in 5 years. The variables influencing reopening were as follows: conventional methods: gender (r = 0.065, P =.19), biocular occurrence (r = 0.12, P =.026), and refraction axial length ratio (r = -0.11, P =.045); scalping methods: hole size (r = 0.14, P =.25).Conclusions: Incidence of reopening in scalping methods was high. The variables that influenced reopening after macular hole surgery were biocular occurrence and refraction axial length ratio in conventional methods. The shape of the eye may be related to reopening.

Transdermal absorption of bupranolol in rabbit skin in vitro and in vivo.

This study was designed to clarify the percutaneous penetration of bupranolol (BP), a beta-adrenoceptor antagonist, through rabbit skin and to compare the in vitro penetration with the in vivo absorption. BP penetrated across the skin slowly in the absence of enhancers in vitro. Isopropyl myristate and N-methyl-2-pyrrolidone enhanced the in vitro penetration, with a 3.6 times higher flux compared with that without enhancers. However, in the in vivo percutaneous absorption, the maximal penetration was obtained with the formulation added dlimonene, with a 3.0 times higher area under the concentration-time curve (AUC) than that for the formulation without enhancers. The plasma levels of BP determined, however, were extremely lower than the theoretical plasma steady-state concentrations predicted. The plasma levels of BP after application of these formulations were maintained in the range of 7-22 ng/ml for 30 h, of which concentrations were above the therapeutically effective concentration (1.5-4 ng/ml). Therefore, the transdermal systems will offer an efficient drug delivery system for the treatment of angina pectoris and tachycardia.