Comparative study on the action of tocopherols and tocotrienols as antioxidant: chemical and physical effects

alpha-Tocopherol is known as the most abundant and active form of vitamin E homologues in vivo, but recently the role of other forms of vitamin E has received renewed attention. The antioxidant properties were compared for alpha-, beta-, gamma- and delta-tocopherols and tocotrienols. The following results were obtained: (1). the corresponding tocopherols and tocotrienols exerted the same reactivities toward radicals and the same antioxidant activities against lipid peroxidation in solution and liposomal membranes; (2). tocopherols gave more significant physical effect than tocotrienols on the increase in rigidity at the membrane interior; (3). tocopherols and tocotrienols showed similar mobilities within the membranes, but tocotrienols were more readily transferred between the membranes and incorporated into the membranes than tocopherols; (4). alpha-tocopherol and alpha-tocotrienol, but not the other forms, reduced Cu(II) to give Cu(I) together with alpha-tocopheryl and alpha-tocotrienyl quinones, respectively and exerted prooxidant effect in the oxidation of methyl linoleate in SDS micelles.     

Capillary electrophoresis-electrospray ionization mass spectrometry for rapid and sensitive N-glycan analysis of glycoproteins as 9-fluorenylmethyl derivatives

It is well known that most protein therapeutics such as monoclonal antibody pharmaceuticals and other biopharmaceuticals including cancer biomarkers are glycoproteins, and thus the development of high-throughput and sensitive analytical methods for glycans is essential in terms of their determination and quality control. We previously reported a novel alternative labeling method for glycans involving 9-fluorenylmethyl chloroformate (Fmoc-Cl) instead of the conventional reductive amination procedure. The derivatives were analyzed by high-performance liquid chromatography (HPLC) (Kamoda S, Nakano M, Ishikawa R, Suzuki S, Kakehi K. 2005. Rapid and sensitive screening of N-glycans as 9-fluorenylmethyl derivatives by high-performance liquid chromatography: A method which can recover free oligosaccharides after analysis. J Proteome Res. 4:146-152). This method was rapid and simple; however, it was time-consuming in terms of analysis by HPLC and did not provide so much information such as the detailed structures and mass numbers of glycans. Here we have developed a high-throughput and highly sensitive method. It comprises three steps, i.e., release of glycans, derivatization with Fmoc, and capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI MS) analysis. We analyzed several glycoproteins such as fetuin, alpha1 acid glycoprotein, IgG, and transferrin in order to validate this method. We were able to analyze the above glycoproteins with the three-step procedure within only 5 h, which provided detailed N-glycan patterns. Moreover, the MS/MS analysis allowed identification of the N-glycan structures. As novel applications, the method was employed for the analysis of N-glycans derived from monoclonal antibody pharmaceuticals and also from alpha-fetoprotein; the latter is known as one of the tumor markers of hepatocellular carcinomas. We were able to easily and rapidly determine the detailed structures of the N-glycans. The present method is very useful for the analysis of large numbers of samples such as a routine analysis.     

The role of α-tocopherol in motor hypofunction with aging in α-tocopherol transfer protein knockout mice as assessed by oxidative stress biomarkers

It has been hypothesized that oxidative stress plays a key role in aging. In order to elucidate the role of the antioxidant network — including α-tocopherol (αT) and αT transfer protein — in aging in vivo, α-tocopherol transfer protein knockout (αTTP^?/?) mice were fed a vitamin-E-depleted diet, and wild-type (WT) mice were fed a diet containing 0.002 wt.% αT from the age of 3 months to 1 1/2 years. The lipid oxidation markers total hydroxyoctadecadienoic acid (tHODE) and 8-iso-prostaglandin F₂α, and antioxidant levels in the blood, liver and brain were measured at 3, 6, 12 and 18 months. tHODE levels in the plasma of αTTP^?/? mice were elevated at 6 months compared to 3 months, and were significantly higher those in WT mice, although they decreased thereafter. On the other hand, tHODE levels in the liver and brain were constantly higher in αTTP^?/? mice than in WT mice. Motor activities decreased with aging in both mouse types; however, those in the αTTP^?/? mice were lower than those in the WT mice. It is intriguing to note that motor activities were significantly correlated with the stereoisomer ratio (Z,E/E,E) of HODE, which is a measure of antioxidant capacity in vivo, in the plasma, in the liver and even in the brain, but not with other factors such as antioxidant levels.In summary, using the biomarker tHODE and its stereoisomer ratio, we demonstrated that αT depletion was associated with a decrease in motor function, and that this may be primarily attributable to a decrease in the total antioxidant capacity in vivo.     

Affinity-tagged phosphorylation assay by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (ATPA-MALDI): application to calcium/calmodulin-dependent protein kinase

A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based kinase assay using a peptide substrate tagged with a biotinyl group has been developed. The peptide moiety was designed to serve as an efficient substrate for calcium/calmodulin-dependent protein kinase II, based on the in vivo phosphorylation site of phosrestin I, a Drosophila homolog of arrestin. In the assay, the quantitative relationship was determined from the ratio of the peak areas between the two peaks respectively representing the unphosphorylated and the phosphorylated substrate. Attempts to assay phosphorylated peptides directly from the reaction mixture, gave inaccurate results because of the high noise level caused by the presence of salts and detergents. In contrast, after purifying the substrate peptides with the biotin affinity tag using streptavidin-coated magnetic beads, peak areas accurately represented the ratio between the unphosphorylated and phosphorylated peptide. By changing the substrate peptide to a peptide sequence that serves as a kinase substrate, it is expected that an efficient non-radioactive protein kinase assay using MALDI-TOF MS can be developed for any type of protein kinase. We call this technique "Affinity-Tagged Phosphorylation Assay by MALDI-TOF MS (ATPA-MALDI)." ATPA-MALDI should serve as a quick and efficient non-radioactive protein kinase assay by MALDI-TOF MS.     

Molecular characterization of four β-tubulin genes from dinitroaniline susceptible and resistant biotypes of Eleusine indica

Dinitroaniline herbicides are antimicrotubule drugs that bind to tubulins and inhibit polymerization. As a result of repeated application of dinitroaniline herbicides, resistant biotypes of goosegrass (Eleusine indica) developed in previously susceptible wild-type populations. We have previously reported that -tubulin missense mutations correlate with dinitroaniline response phenotypes (Drp) (Plant Cell 10: 297–308, 1998). In order to ascertain associations of other tubulins with dinitroaniline resistance, four -tubulin cDNA classes (designated TUB1, TUB2, TUB3, and TUB4) were isolated from dinitroaniline-susceptible and -resistant biotypes. Sequence analysis of the four -tubulin cDNA classes identified no missense mutations. Identified nucleotide substitutions did not result in amino acid replacements. These results suggest that the molecular basis of dinitroaniline resistance in goosegrass differs from those of colchicine/dinitroaniline cross-resistant Chlamydomonas reinhardtii and benzimidazole-resistant fungi and yeast. Expression of the four -tubulins was highest in inflorescences. This is in contrast to -tubulin TUA1 that is expressed predominantly in roots. Collectively, these results imply that -tubulin genes are not associated with dinitroaniline resistance in goosegrass. Phylogenetic analysis of the four -tubulins, together with three -tubulins, suggests that the resistant biotype developed independently in multiple locations rather than spreading from one location.     

Development of an ultrasensitive immunoassay using affinity maturated antibodies for the measurement of rodent insulin

The measurement of plasma insulin is important for clinical diagnosis of diabetes and for preclinical research of metabolic diseases, especially in rodent models used in drug discovery research for type 2 diabetes. Fasting immunoreactive insulin (F-IRI) concentrations are used to calculate the homeostasis model assessment ratio (HOMA-R), an index of insulin sensitivity. However, even the most sensitive commercially available enzyme-linked immunosorbent assay (ELISA) kits cannot measure the very low F-IRI concentrations in normal rats and mice. Therefore, we sought to develop a new rodent insulin ELISA with greater sensitivity for low F-IRI concentrations. Despite repeated efforts, high-affinity antibodies could not be generated by immunizing mice with mouse insulin (self-antigen). Therefore, we generated two weak monoclonal antibodies (13G4 and 26B2) that were affinity maturated and used to develop a highly sensitive ELISA. The measurement range of the sandwich ELISA with the affinity maturated antibodies (13G4m1 and 26B2m1) was 1.5 to 30,000 pg/ml, and its detection limit was at least 10 times lower than those of commercially available kits. In conclusion, we describe the development of a new ultrasensitive ELISA suitable for measuring very low plasma insulin concentrations in rodents. This ELISA might be very useful in drug discovery research in diabetes.     

Examination of the dose distribution of volumetric modulated arc radiotherapy using a high-definition multi-leaf collimator for breast cancer patients with irradiated regional lymph nodes

BackgroundA high-definition multi-leaf collimator (HD-MLC) with 5- and 10-mm fine MLCs is useful for radiotherapy. However, it is difficult to irradiate the mammary gland and supraclavicular region using a HD-MLC because of the narrow field of volumetric modulated arc radiotherapy (VMAT). Therefore, we aimed to evaluate the dose distribution of the VMAT dose using a HD-MLC in 15 patients with left breast cancer undergoing postoperative irradiation of breast and regional lymph nodes, including the internal mammary node.Materials and methodsThe following four plans were generated: three-arc VMAT using HD-MLC (HD-VMAT), two tangential arcs and one-arc VMAT using HD-MLC (tHD-VMAT), three-dimensional conformal radiotherapy (3DCRT) using HD-MLC, and two-arc VMAT using the Millennium 120-leaf MLC (M-VMAT). We assessed the doses to the target volume and organs at risk.ResultsThe target dose distributions were higher for HD-VMAT than 3DCRT. There were no significant differences in the heart mean dose (D_mean) or lung volume receiving 20 Gy (V20 Gy) between HD-VMAT and 3DCRT. The heart D_mean and lung V20 Gy of tHD-VMAT were higher than those of HD-VMAT, and the heart D_mean of M-VMAT was higher than that of HD-VMAT. However, the target doses of tHD-VMAT, M-VMAT, and HD-VMAT were equivalent.ConclusionsIn cases of the mammary gland and regional lymph node irradiation, including the internal mammary node in patients with left breast cancer, HD-VMAT was not inferior to M-VMAT and provided a better dose distribution to the target volume and organs at risk compared with 3DCRT and tHD-VMAT.     

Presence of aquaporin and V-ATPase on the contractile vacuole of Amoeba proteus

Background information. The results of water permeability measurements suggest the presence of an AQP (aquaporin) in the membrane of the CV (contractile vacuole) in Amoeba proteus [Nishihara, Shimmen and Sonobe ( 2004 ) Cell Struct. Funct. 29 , 85–90]. Results. In the present study, we cloned an AQP gene from A. proteus [ApAQP (A. proteus AQP)] that encodes a 295‐amino‐acid protein. The protein has six putative TMs (transmembrane domains) and two NPA (Asn‐Pro‐Ala) motifs, which are conserved among various AQPs and are thought to be involved in the formation of water channels that span the lipid bilayer. Using Xenopus oocytes, we have demonstrated that the ApAQP protein product can function as a water channel. Immunofluorescence microscopy with anti‐ApAQP antibody revealed that ApAQP is detected on the CV membrane and on the vesicles around the CV. The presence of V‐ATPase (vacuolar H⁺‐ATPase) on the vesicle membrane around the CV was also detected. Conclusions. Our data on ApAQP allow us to provide the first informed explanation of the high water permeability of the CV membrane in amoeba. Moreover, the results suggest that vesicles possessing V‐ATPase are involved in generating an osmotic gradient. Based on our findings, we propose a new hypothesis for the mechanism of CV function.     

Development of an amperometric flow analysis sensor for specific detection of D-psicose

The aim of this study was to develop a simple, rapid and highly sensitive sensor for measuring the rare sugar d-psicose. The proposed system adopts amperometric flow analysis and two consecutive enzyme reactions consisting of a reactor packed with d-tagatose 3-epimerase (DTE)-immobilized beads, which converts d-psicose to d-fructose, and a carbon-paste electrode containing d-fructose dehydrogenase (DFDH). In order to fabricate a robust sensor system, various experimental parameters were optimized including the buffer composition, flow rate for the two enzyme reactions and the size of micro-flow cell. The developed sensor responded linearly to d-psicose concentration in the range from 0.08 to 50mM (R(2)=0.988). The signal/noise ratio was 3.0 for the 0.08 mM d-psicose solution, and the relative standard deviations were 1.7 (n=20) and 2.6% (n=20) for the 10 and 20mM d-psicose solutions, respectively. One round of assay was completed within 8 min. Our results suggest that the sensor can be used not only for the detection of d-psicose in food samples but also for monitoring d-psicose within the environment. Moreover, the sensor system can be applied to the detection of many other rare sugars by using the same measurement principle.