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The accumulation of β-amyloid (Aβ) aggregates in the brain occurs early in the progression of Alzheimer's disease (AD), and non-fibrillar soluble Aβ oligomers are particularly neurotoxic. During binding to Aβ fibrils, curcumin, which can exist in an equilibrium state between its keto and enol tautomers, exists predominantly in the enol form, and binding activity of the keto form to Aβ fibrils is much weaker. Here we described the strong binding activity the keto form of curcumin derivative Shiga-Y51 shows for Aβ oligomers and its scant affinity for Aβ fibrils. Furthermore, with imaging mass spectrometry we revealed the blood-brain barrier permeability of Shiga-Y51 and its accumulation in the cerebral cortex and the hippocampus, where Aβ oligomers were mainly localized, in a mouse model of AD. The keto form of curcumin derivatives like Shiga-Y51 could be promising seed compounds to develop imaging probes and therapeutic agents targeting Aβ oligomers in the brain., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: It remains unclear how the head and stem diameters for the radial head prosthesis could affect mechanical properties of the lateral collateral ligament measured by strain changes during elbow and forearm motions., Methods: Eight cadaveric specimens were secured to the device, which allows elbow flexion-extension and forearm pro-supination. Using six different implant combinations comprising 2 sizes for the head (long- and short-axis of the native head) and 3 sizes for the stem (press-fit, -1 mm, and -2 mm downsizing), prostheses were attached via the posterior approach. A differential variable reluctance transducer placed on the central portion of the radial collateral ligament were used for strain measurement with elbow flexion at 0°, 30°, 60°, and 90°. At each position, the strain patterns with the forearm in the neutral and 45° pro-supination positions were also assessed., Findings: Specimens implanted with long-axis head component showed greater increases in the ligament strain during elbow flexion than intact specimens or those implanted with short-axis head. Compared to press-fit stem, implants with downsizing to -1 mm approximated strain patterns during pro-supination with elbow extension to intact condition., Interpretation: Morphologic variation of the head and stem components in radial head prostheses led to altered strain patterns in the lateral collateral ligament during elbow and forearm motions. A short-axis head component can be used to prevent excessive strain changes after the prosthesis application. Downsizing of the stem component might be an option for approximating the biomechanics at the radiocapitellar joint during forearm rotation to the intact elbow., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Human CYP3A4 catalyzes the 10,11-epoxidation of carbamazepine (CBZ). However, the epoxide is less stable in terms of potential energy than hydroxides of the six-membered aromatic ring. To clarify the reason why CYP3A4 produces such an energetically unfavorable compound, the mechanism of epoxidation of CBZ by CYP3A4 was investigated by theoretical calculations. The reaction consisted of two elementary processes in which two C-O bonds were generated stepwise. The rate-determining step was the first one and the activation energy was 21.3kcal/mol at the DFT (B3LYP/6-31G( * *)) level. The activation energy level of the first step of the 10,11-epoxidation was lower than that of the hydroxylation of the aromatic ring. For this reason, 10,11-epoxidation is more probable than hydroxylation of the aromatic ring, and only 10,11-epoxide is formed.

The choroid plexus produces cerebrospinal fluid, providing a specialized environment for the CNS. We previously demonstrated that choroid plexus ependymal cells can enhance nerve regeneration in vivo and promote neurite outgrowth in vitro. To understand the molecular mechanisms of choroid plexus functions, we isolated genes predominantly expressed in the mouse choroid plexus using suppression subtractive hybridization. Out of the 49 complementary DNA (cDNA) fragments isolated in two types of screening, 43 matched known sequences in the database and six were novel. In one type of screening where choroid plexus cDNAs were subtracted with cerebral cortex cDNAs, transthyretin and phosphodiesterase I alpha were predominant. This is consistent with previous reports and supports the authenticity of our approach. In the other type of screening, cDNAs derived from the choroid plexus of neonatal (postnatal day 5) mice were subtracted with cDNAs from the choroid plexus of adult mice. RNA blot and/or in situ hybridization confirmed abundant expression, in the mouse choroid plexus, of the mRNA encoding gelsolin, phospholipid transfer protein, ATP-binding cassette transporter A8 (ABCA8), androgen-inducible aldehyde reductase, and Na(+)/sulfate cotransporter SUT-1. Also, one novel gene (FS88) was found to be expressed in the choroid plexus from neonatal mice. Our data suggest that the choroid plexus cells produce molecules involved in processes such as prevention of fibrillization of amyloid beta-protein (transthyretin and gelsolin), lipid metabolism (phospholipid transfer protein and ABCA8), and detoxification (androgen-inducible aldehyde reductase).

Synaptosomal-associated protein 25 has been regarded as one of the target-associated soluble N-ethylmaleimide-sensitive fusion attachment protein receptors essential for exocytosis of vesicles in synapses. We have previously reported that cleavage of syntaxin, which is another target-associated soluble N-ethylmaleimide-sensitive fusion attachment protein receptor, with botulinum neurotoxin C1 resulted in inhibition of neurite extension and morphological changes including growth cone collapse and large vacuole formation. As an attempt to explore the mechanism of growth cone extension, we examined the ultrastructural localization of synaptosomal-associated protein 25 in growth cones with or without treatment of botulinum neurotoxin A, which cleaves synaptosomal-associated protein 25. In dorsal root ganglion neurons, light microscopy demonstrated synaptosomal-associated protein 25 immunoreactivity throughout the neurons, including the cell bodies, neurites and growth cones. Using electron microscopy, gold signals immunoreactive for synaptosomal-associated protein 25 were identified diffusely in the cytoplasm of the growth cones. In contrast, in PC-12 cells, a large number of gold signals were localized on the plasma membranes. High levels of signal were also found in the cytoplasm in the central region of the growth cones. We also confirmed that botulinum neurotoxin A treatment reduced neurite extension by about 50%. However, both in dorsal root ganglion neurons and in PC-12 cells we found no differences in the ultrastructure nor in the localization of synaptosomal-associated protein 25 between growth cones with and without toxin treatment. These results indicate that cleavage of synaptosomal-associated protein 25 inhibits growth cone extension in a manner different than that of syntaxin cleavage. The results of this study suggest the possibility that synaptosomal-associated protein 25 is involved in growth cone extension through a process independent of vesicle fusion.

The contribution of induced cytochrome P450 (P450) isozymes (CMLa; CYP2B, CMLb; CYP2A and CMLc; CYP3A) and related enzymes to trimethadione (TMO) metabolism in phenobarbital-treated rhesus monkey were investigated. The animals received a single dose of TMO (4 mg kg-1) and plasma samples were withdrawn before this administration and again at 0.08, 0.25, 0.5, 1 and 2 h later. Phenobarbital-treatment (20 mg kg-1 day-1 for 3 days; i.p.) significantly increased the plasma dimethadione (DMO)/TMO ratios at 0.08, 0.5, 1 and 2 h one's appropriate controls. Phenobarbital treatment also increased the P450 content (1.7-fold) and activity of aniline p-hydroxylase (1.3-fold), p-nitroanisole O-demethylase (1.8-fold) and benzphetamine N-demethylase (2.3-fold). The content of CMLa, CMLb and CMLc were increased about 12.8, 2.3 and 2.7-fold by phenobarbital pretreatment, respectively. The activity of TMO N-demethylation was inhibited by anti-P450 CMLa and anti-P450 CMLb. However, the anti-P450 CMLc antibody had no effect on this activity in liver microsomes. The results of both in vivo and in vitro studies of the effects of phenobarbital treatment on TMO metabolism indicate that these effects may be attributed to the induction of CMLa. These findings suggest that plasma DMO/TMO ratio in a single blood sampling after TMO administration is very useful for determination the degree of hepatic induction in clinical study.

Chemiluminescence was observed in a rat heart mitochondrial suspension containing NADH, FeC1(3) and doxorubicin (Adriamycin) (DXR). There was good correlation between the total intensity of chemiluminescence and the total amount of thiobarbituric acid reactive substances (TBARS) produced during DXR redox cycling. Thus, the chemiluminescence was shown to be associated with lipid peroxidation. The chemiluminescence was quenched by superoxide dismutase (SOD), suggesting that superoxide anion radicals contributed to its production. Upon addition of 1,4-diazabicyclo[2,2,2]-octane (DABCO), a singlet oxygen emission enhancer, to the mitochondrial suspension emitting the chemiluminescence, the chemiluminescence intensity increased transiently, indicating the involvement of singlet oxygen. Furthermore, spectral analysis of the chemiluminescence showed it to be due to singlet oxygen and excited carbonyls.

The role of cytochrome P450 (P450) in lipid peroxidation induced by NADPH or peroxide was investigated in a reconstituted system. When cumene hydroperoxide, t-butyl hydroperoxide and hydrogen peroxide were used as initiators, the rates of malondialdehyde (MDA) formation were much higher in a reconstituted system containing P450 1A1 than those observed in a reconstituted system containing P450 1A2. In contrast to peroxide-induced lipid peroxidation, P450 1A2 catalysed NADPH-induced lipid peroxidation more effectively than did P450 1A1 regardless of the presence of ADP-Fe(NO3)3. Carbon monoxide inhibited NADPH-induced formation of MDA in a reconstituted system containing P450 1A2, but not P450 1A1. In addition, superoxide dismutase (SOD) was an effective inhibitor in a NADPH-induced lipid peroxidation system catalysed by P450 1A2 but not by P450 1A1. These results suggest that a peroxide-induced reaction might proceed readily with P450 1A1, whereas P450 1A2 mainly functions in NADPH-induced lipid peroxidation via generation of an active oxygen species. It is furthermore indicated that the difference in the effect of SOD in NADPH-induced lipid peroxidation depends on the P450 used.

Lipid peroxidation is known to be a mechanism for Adriamycin-induced toxicity. In the present study, two methods which detect fluorescent substances and high molecular weight protein aggregates in peroxidized membranes were applied to Adriamycin-induced lipid peroxidation in liver microsomes. A rat liver microsomal suspension containing an NADPH-generating system was incubated with Adriamycin. Thiobarbituric acid reactive substances (TBA-RS), formed during this incubation, were transferred from the microsomes to the medium. Fluorescent substances determined by the fluorescence emitted from both the microsomes themselves and the chloroform/methanol extracts of the microsomes, were found to be formed during this incubation. High molecular weight protein aggregates determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were also formed. Fluorescent substances and high molecular weight protein aggregates were found in microsomal membranes themselves and increased time dependently. These substances retained in membranes can be of great use to delineate the site of Adriamycin-induced lipid peroxidation in vitro and in vivo and to determine how this lipid peroxidation affects the membrane.

Cytochrome P-450, designated as P-450-MK1, which is cross-reactive with antibodies to rat P-450-male, was purified to an electrophoretical homogeneity from liver microsomes of the untreated male crab-eating monkey. The molecular weight of P-450-MK1 was estimated to be 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The oxidized form of P-450-MK1 showed a peak at 418 nm, indicating that this cytochrome is in a low spin state. The carbon monoxide-bound reduced form showed a peak at 451 nm. The first 22 amino acid residues of the NH2-terminal sequence of P-450-MK1 was fairly homologous to those of P-450-male (75% identity, not including unidentified amino acid residues). Unlike the P-450-male, P-450-MK1 did not exhibit catalytic activities for testosterone 2 alpha- and 16 alpha-hydroxylations and catalyzed testosterone 6 beta-hydroxylation. It is, therefore, suggested that although the spectral and immunochemical properties and the N-terminal amino acid sequence of P-450-MK1 were similar to those of P-450-male, the physiological functions of P-450-MK1 may be somewhat different from those of P-450-male. Comparison of the physico-chemical properties of P-450-MK1 with those of P-450-D1 and P-450-HM2, which are cross-reactive with anti-P-450-male antibodies, purified from liver microsomes of dogs and humans, respectively, are also discussed.

Acetone stimulated NADPH-dependent aniline hydroxylation catalysed by cytochrome P-450 purified from phenobarbital-treated rats. No activation by acetone occurred in a reconstituted system containing cytochrome P-448 purified from 3-methylcholanthrene-treated rats. Cumene hydroperoxide-supported aniline hydroxylation catalysed by cytochrome P-450 was not increased by the addition of acetone at the concentration which stimulated NADPH-dependent aniline hydroxylation in the reconstituted system. The NADPH-dependent cytochrome P-450 reduction was stimulated by acetone in the presence or absence of aniline. On the other hand, acetone did not exhibit any stimulatory effect on NADPH-dependent reduction of cytochrome P-448. The stimulatory effect of acetone on aniline hydroxylation was decreased with increasing pH of the reaction media. Furthermore, in the system containing cytochrome P-450, the ratio of product to hydrogen peroxide formation was virtually unchanged by the addition of acetone.

The activities of drug oxidation in a reconstituted system which contains two forms of cytochrome P-450 and a limiting amount of NADPH-cytochrome P-450 reductase were determined. Cytochrome P-450 (termed MC P-4481 and MC P-4482) purified from liver microsomes of 3-methyl-cholanthrene-treated rats was active in both 2- and 4-hydroxylation of biphenyl but cytochrome P-450 (termed PB P-450) purified from liver microsomes of phenobarbital-treated rats was active in 4-hydroxylation of biphenyl only. PB P-450, MC P-4481 and MC P-4482 were most active toward benzphetamine N-demethylation, aniline hydroxylation and 7-ethoxycoumarin O-deethylation, respectively. PB P-450 inhibited the activity of biphenyl 2-hydroxylation supported by MC P-4481 or MC P-4482. On the contrary, no inhibition of PB P-450 supported benzphetamine N-demethylation was observed when MC P-4481 or MC P-4482 was added to the system containing PB P-450 and limited amount of the reductase. The apparent Km of PB P-450 for the reductase obtained from double reciprocal plot of the reductase concentration and the activity of biphenyl hydroxylase or benzphetamine N-demethylation was lower than that of MC P-4481 or MC P-4482. These and other results suggest that there is a certain hierarchy among the cytochrome P-450 species for receiving electrons from reductase.

The purpose of this study was to clarify the pharmacological and physiological significance of P-450 HFLa. Thus, correlations between cytochrome P-450 (P-450 HFLa) level and different monooxygenase activities were investigated in liver homogenates from human fetuses. Poor correlation was seen between P-450 HFLa level and the activity of benzphetamine N-demethylation or aniline hydroxylation. In contrast, the content of P-450 HFLa was highly correlated with the activity of benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation or testosterone 6 beta-hydroxylation. In microsomes from human adult livers, a moderate relationship was also observed between testosterone 6 beta-hydroxylation and P-450 HFLa level. Furthermore, antibodies to P-450 HFLa inhibited testosterone 6 beta-hydroxylase activity in fetal and adult livers to similar extents. We conclude that P-450 HFLa is a form of cytochrome P-450 which catalyzes testosterone 6 beta-hydroxylation and limited drug oxidations in human fetal and adult livers.

Effect of successive administration of Aztreonam on microsomal monooxygenase system was investigated in male and female Sprague-Dawley rats. The activities of benzphetamine N-demethylase, aminopyrine N-demethylase, p-nitroanisole O-demethylase and aniline hydroxylase in liver microsomes from male rats were decreased dose-dependently by Aztreonam. On the contrary, the activities in liver microsomes from female rats were slightly increased rather than decreased by the administration of Aztreonam. In addition, Aztreonam was found to decrease the specific content of microsomal cytochrome P-450 in male rats but not in female rats. The decreases in the activities observed in male rats were accompanied by a parallel decrease in the specific content of cytochrome P-450. Furthermore, the results of quantitation of P-450 (M-1), one of the male specific forms of cytochrome P-450, indicated that the administration of Aztreonam resulted in a dose-dependent decrease in the content of P-450 (M-1) in liver microsomes from male rats.

The effect of cyanide on 7-ethoxycoumarin O-deethylation by two cytochrome P-450 isozymes obtained from 3-methylcholanthrene treated rat liver microsomes was investigated. 7-Ethoxycoumarin O-deethylation was stimulated by the addition of cyanide to a reconstituted monooxygenase system consisting of NADPH, dilauroyl 3-L-phosphatidylcholine, NADPH-cytochrome P-450 reductase and MC P-448(2) (low spin form of cytochrome). In contrast, a weak inhibitory effect of cyanide on 7-ethoxycoumarin O-deethylation was observed when MC P-448(1) (high spin form of cytochrome) was used in the reconstituted system. Cyanide did not influence the apparent Km for 7-ethoxycoumarin when either form of cytochrome P-450 was used in the reconstituted system and did not stimulate the cumene hydroperoxide dependent O-deethylation by MC P-448(2). The stimulatory effect of cyanide on O-deethylation by MC P-448(2) was decreased with increasing the concentration of the reductase added to the reconstituted system. On the other hand, the effect of cyanide on O-deethylation by MC P-448(1) was virtually independent on the amount of the reductase added.

The effects of administration of buserelin, a synthetic agonist of lutenizing hormone-releasing hormone (LH-RH), on the content of P-450-male, a male specific form of cytochrome P-450, and the activities of drug and testosterone hydroxylases were examined in liver microsomes of male rats. Administration of buserelin resulted in a decrease in the serum level of testosterone in a dose-dependent manner. Similar decreases were seen in the activities of aminopyrine N-demethylase, 7-propoxycoumarin O-depropylase and testosterone 2 alpha- and 16 alpha-hydroxylases, but not in the activity of aniline p-hydroxylase. Thus, correlations were observed between the serum level of testosterone and the activities of drug and testosterone hydroxylases except for aniline p-hydroxylase. These results indicate that the drug and testosterone hydroxylases are affected sensitively and sequentially by the changes in the testosterone levels in the body.

A form of cytochrome P-450, P-450-D3, cross reactive with antibodies to rat P-450d was purified from liver microsomes of polychlorinated biphenyl (PCB)-treated female Beagle dogs to an electrophoretic homogeneity. Judging from the result of sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of P-450-D3 was estimated to be 54,000. The oxidized form of P-450-D3 showed a peak at 416 nm indicating that the cytochrome is mostly in a low spin state. The carbon monoxide bound reduced form of P-450-D3 showed a peak at 448 nm. In a reconstituted system, P-450-D3 catalyzed drug oxidations including benzphetamine and aminopyrine N-demethylations, 7-ethoxycoumarin and p-propoxyaniline O-dealkylations, and aniline and benzo(a)pyrene hydroxylations. The rate of aniline hydroxylation catalyzed by P-450-D3 was similar to that catalyzed by P-450c which is a low spin form of cytochrome P-450 purified from liver microsomes of PCB-treated rats, whereas the catalytic activities of P-450-D3 for 7-ethoxycoumarin O-deethylation and benzo(a)pyrene hydroxylation were considerably lower than those of P-450c. The amino terminal portion of P-450-D3 was found to be highly similar to those of P-450d, human P3-450 and P3-450 when four amino acid deletions were tentatively inserted between fifth and sixth amino acids from the N-terminal, but not that of P-450c which is a low spin form of cytochrome P-448 purified from rat liver microsomes. These results indicate that Beagle dogs possess a low spin form of cytochrome P-450 with spectral properties similar to P-450c but with catalytic and structural properties similar to P-450d.