Activation of enzymes for nonaqueous biocatalysis by denaturing concentrations of urea.
(65/1563)Urea is one of the most commonly used denaturants of proteins. However, herein we report that enzymes lyophilized from denaturing concentrations of aqueous urea exhibited much higher activity in organic solvents than their native counterparts. Thus, instead of causing deactivation, urea effected unexpected activation of enzymes suspended in organic media. Activation of subtilisin Carlsberg (SC) in the organic solvents (hexane, tetrahydrofuran, and acetone) increased with increasing urea concentrations up to 8 M. Active-site titration results and activity assays indicated the presence of partially unfolded but catalytically active SC in 8 M urea; however, the urea-modified enzyme retained high enantioselectivity and was ca. 80 times more active than the native enzyme in anhydrous hexane. Likewise, the activity of horseradish peroxidase (HRP) lyophilized from 8 M urea was ca. 56 times and 350 times higher in 97% acetone and water-saturated hexane, respectively, than the activity of HRP lyophilized from aqueous buffer. Compared with the native enzyme, the partially unfolded enzyme may have a more pliant and less rigid conformation in organic solvents, thus enabling it to retain higher catalytic activity. However, no substantial activation was observed for alpha-chymotrypsin lyophilized from urea solutions in which the enzyme retained some activity, illustrating that the activation effect is not completely general. (+info)
Statistical analysis on toxicity of a nitrofuran derivative, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide.
(66/1563)A food additive, furylfuramide or AF-2, which had been used in Japan since 1965 and structurally is composed of 5-nitro-2-furyl radical and acrylamide, was re-examined mainly on chronic toxicity by statistically reviewing published data. The conclusions are as follows: 1) The maximum safety dosage which shows no demonstrable change in rats must be corrected at least to 1/170 of the value which has been accepted by the Ministry of Health and Welfare of Japan (MHW). 2) The minimum effective dose to bacterial growth in food can not be lowered below the standard usage level with MHW determined, because the inactivation factor in food, decreasing effectivity to 1/20, must be taken into consideration. 3) In view of these two facts, AF-2 is found to be unacceptable as a food additive. 4) Great importance must also be attached to the possibility of mutagenicity and carcinogenicity of AF-2, pointed out recently. Both neurotoxicity and dermatitis observed in tofu (soybean curd) makers are also memtioned. (+info)
Aplysiallene, a new bromoallene as an Na, K-ATPase inhibitor from the sea hare, Aplysia kurodai.
(67/1563)A new bromoallene metabolite, named aplysiallene, was isolated from the Japanese sea hare, Aplysia kurodai, as an Na, K-ATPase inhibitor. Its structure was elucidated by spectroscopic methods. The known metabolites, laurinterol and debromolaurinterol, isolated from this animal were also evaluated for their Na, K-ATPase inhibitory activity. (+info)
In vitro metabolism of the COX-2 inhibitor DFU, including a novel glutathione adduct rearomatization.
(68/1563)The metabolic profile of DFU [5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone], a potent and selective COX-2 inhibitor, was characterized using in vitro microsomal and hepatocyte incubations. A single product, corresponding to p-hydroxylation, p-OH-DFU [(5,5-dimethyl-3-(3-fluoro-4-hydroxyphenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-fur anone)], was produced in rat microsomal incubations of DFU. In contrast, three metabolites were produced in incubations using suspensions of freshly isolated rat hepatocytes. Microsomal production of the p-O-glucuronide metabolite of DFU from synthetic p-OH-DFU was shown to have chromatographic and mass spectrometric properties identical to the earliest eluting hepatocyte metabolite (M1). The molecular weights of the other two hepatocyte metabolites were readily obtained using capillary high-performance liquid chromatography continuous-flow liquid secondary ion mass spectrometry (HPLC/CF-LSIMS); however, the elemental composition of these metabolites was not. Unlike typical metabolic products, which produce readily identified increments in molecular weight, metabolites M2 and M3 produced molecular ions in positive- and negative-ion CF-LSIMS that were consistent with oxidation of DFU (+16 Da), followed by addition of glutathione (+306 Da) and subsequent loss of 20 and 18 Da, respectively. Capillary HPLC/high-resolution CF-LSIMS was used to generate accurate mass data for M2 and M3 that provided evidence that the losses of 20 and 18 Da, respectively, corresponded to a rearomatization through loss of HF or H(2)O. Isolation and NMR characterization provided the definitive structural proof for these metabolites. Overall, the metabolism of DFU in rat hepatocytes is proposed to proceed through an epoxide intermediate, which then either rearranges to the p-OH-DFU and is conjugated with glucuronic acid, or is trapped with glutathione, followed by rearomatization with loss of HF (M2) or H(2)O (M3). (+info)
Human intelectin is a novel soluble lectin that recognizes galactofuranose in carbohydrate chains of bacterial cell wall.
(69/1563)Galactofuranosyl residues are present in various microorganisms but not in mammals. In this study, we identified a human lectin binding to galactofuranosyl residues and named this protein human intelectin (hIntL). The mature hIntL was a secretory glycoprotein consisting of 295 amino acids and N-linked oligosaccharides, and its basic structural unit was a 120-kDa homotrimer in which 40-kDa polypeptides were bridged by disulfide bonds. The hIntL gene was split into 8 exons on chromosome 1q21.3, and hIntL mRNA was expressed in the heart, small intestine, colon, and thymus. hIntL showed high levels of homology with mouse intelectin, Xenopus laevis cortical granule lectin/oocyte lectin, lamprey serum lectin, and ascidian galactose-specific lectin. These homologues commonly contained no carbohydrate recognition domain, which is a characteristic of C-type lectins, although some of them have been reported as Ca(2+)-dependent lectins. Recombinant hIntL revealed affinities to d-pentoses and a d-galactofuranosyl residue in the presence of Ca(2+), and recognized the bacterial arabinogalactan of Nocardia containing d-galactofuranosyl residues. These results suggested that hIntL is a new type lectin recognizing galactofuranose, and that hIntL plays a role in the recognition of bacteria-specific components in the host. (+info)
Protective effect of bilobalide against nitric oxide-induced neurotoxicity in PC12 cells.
(70/1563)AIM: To examine the effects of bilobalide on nitric oxide-induced neurotoxicity in pheochromocytoma-derived PC12 cells (PC12 cells). METHODS: PC12 cell survival was monitored by LDH release and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. Superoxide dismutases (SOD) and catalase (CAT) activities were measured based on their abilities to inhibit the oxidation of epinephrine by the xanthine-xanthine oxidase system or to decompose H2O2 respectively. The content of malondialdehyde (MDA) was measured by a fluorometric assay to indicate the lipid peroxidation. RESULTS: 3-Morpholinosydnonimine (SIN-1, 50-300 mumol.L-1) induced PC12 cell damage. After the cells had been pretreated with 10 mumol.L-1 bilobalide for 24 h, the cell viability was increased to 91% +/- 30% from 52% +/- 14% in SIN-1 alone group. Moreover, the activities of SOD and CAT were increased after cells were treated with bilobalide. CONCLUSION: The NO-induced neurotoxicity can be protected by bilobalide in PC12 cells. The bilobalide-induced increase in SOD and CAT activities may serve as one of the mechanisms underlying the neuroprotective effect of bilobalide. (+info)
Comparison of sestamibi, tetrofosmin, and Q12 retention in porcine myocardium.
(71/1563)Although there are several 99mTc perfusion tracers introduced for clinical use, there are no data available directly comparing these tracers with microsphere-determined flow. The aim of this study was to compare the myocardial retention of sestamibi, tetrofosmin, and Q12 in a porcine model. METHODS: We used a pig model with (n = 6) or without (n = 3) coronary occlusion. Each pig received a simultaneous injection of sestamibi and either tetrofosmin (group 1, n = 5) or Q12 (group 2, n = 4) labeled with either 99mTc or 95mTc (physical half-life, 61 d; photon energy, 204 keV) during pharmacologic vasodilation. Absolute myocardial retention of each tracer was calculated from the myocardial tracer activity and arterial input function. RESULTS: The plot of all three tracers versus flow achieved a plateau at a higher flow range. However, sestamibi showed a higher mean retention than either tetrofosmin (group 1, 0.27 +/- 0.11 vs. 0.16 +/- 0.06 mL/g/min, respectively; P < 0.01) or Q12 (group 2, 0.32 +/- 0.13 vs. 0.09 +/- 0.03 mL/g/min, respectively; P < 0.01). Furthermore, when a linear regression analysis was performed to assess the relationship between retention and microsphere-determined flow, sestamibi showed a greater increment in retention than did tetrofosmin or Q12. CONCLUSION: Although all of the tracers showed a nonlinear increase in retention as flow increased, sestamibi may display more favorable characteristics as a flow tracer in the porcine heart. (+info)
Use of perfluorochemical liquid allows earlier detection of gene expression and use of less vector in normal lung and enhances gene expression in acutely injured lung.
(72/1563)One of the obstacles to successful lung gene transfer is effective delivery of vector to lung, particularly injured or diseased lung. We have previously demonstrated that intratracheal instillation of perfluorochemical (PFC) liquids along with instillation of recombinant adenovirus and adeno-associated virus vectors, or with cationic liposome vectors, increased total lung gene expression and enhanced distribution of gene expression throughout the lung. To further explore the potential benefits of PFC liquid use, we evaluated the effect of PFC liquid instillation on several other aspects of adenovirus-mediated gene expression in lung. Use of PFC liquid resulted in earlier detection of gene expression and allowed the use of less vector to achieve expression comparable to that observed with the use of higher amounts of vector alone. Using PFC liquid also enhanced gene expression in a rodent model of acute lung injury. PFC liquid did cause a transient inflammation when instilled into normal lungs but did not cause any additional inflammation when instilled alone or with adenovirus vector into acutely injured lungs. Thus, PFC liquid may be a useful adjunct for clinical lung gene transfer, particularly for injured or diseased lungs. (+info)