Properties of the Biotin Transport System in Bifidobacterium breve N4

Binding of biotin to resting cells of Bifidobacterium breve N4, which grew in a biotin-deficient medium, was independent of pH from 1 to 9 and of temperature below 50 C. It was not inhibited by metabolic inhibitors including sulfhydryl reagents, but it was inhibited by treatment with 80% ethanol or 5% trichloroacetic acid. It was also competitively inhibited by biotin-sulfone, but not by tetrahydrothiophene nor dethiobiotin. The binding constant was calculated to be 3.3 × 10⁸m^?1. The amount of biotin unextractable with hot water, representing part of the transported biotin, increased gradually for 20 min, this increase was inhibited by NaF, hydroxylamine and low temperature. ¹⁴C-biotin on the cells was displaced by cold biotin and biotin-sulfone; the displacement was not inhibited by metabolic inhibitors, but it was dependent on temperature. A few minutes after binding, the biotin was released to the medium. The release was dependent on pH and temperature, was affected by energy sources and was inhibited by metabolic inhibitors, e.g. NaF, p-chloromercuribenzoic acid and hydroxylamine. It could be stopped at any time by cooling to 0 C or by adding NaF, and the amount of accumulated biotin did not increase under those conditions. These results suggest that the binding sites on the cell surface decreased in number or in their binding affinity for biotin through an energy-dependent process.     

Immunohistological classification of ionocytes in the external gills of larval Japanese black salamander,Hynobius nigrescens Stejneger

In this cytological and immunohistological study, we clarified the localization of the membrane transporters Na⁺, K⁺‐ATPase (NKA), vacuolar‐type H⁺‐ATPase (VHA), and epithelial sodium channel (ENaC) and distinguished ionocyte subtypes in the gill of the Japanese salamander (Hynobius nigrescens). In larvae (IY stages 43–65), NKA immunoreactivity was observed on the basolateral plasma membrane in more than 60% cells and less than 20% cells in the primary filaments and secondary lamellae of the external gills, respectively. VHA immunoreactivity was observed on the apical membrane of some epithelial cells in the secondary lamellae of the external gills. High ENaCα immunoreactivity was widely observed on the apical cell membrane of a population of squamous cells, presumably pavement cells (PVCs), and mitochondria‐rich cells (MRCs), in the primary filaments and secondary lamellae of the external gills. Using double immunofluorescence microscopy, epithelial cell types involved in ionic regulation were characterized and divided into three ionocyte types: NKA‐, NKA‐ and ENaC‐, and VHA‐positive cells. VHA‐immunoreactive cells as well as NKA‐positive cells were observed during IY stages 43–65 of the salamander larvae. During late stages of metamorphosis, NKA, VHA, and ENaCα immunoreactivities in the external gills decreased and finally disappeared during the completion of metamorphosis (IY stage 68). PVCs and MRCs in the external gills are probably involved in acid–base balance regulation and osmoregulation in urodele amphibian larvae. The results are discussed in relation to the ionocytes previously reported in fish gills and the frog skin epithelium. J. Morphol., 2011. © 2011Wiley‐Liss, Inc.     

Seasonal changes in proteomic profiles of Japanese kelp: <Emphasis Type="Italic">Saccharina japonica</Emphasis> (Laminariales,Phaeophyceae)

The seasonal variation in protein expression in the sporophyte of Saccharina japonica (Areschoug) Lane, Mays, Druehl and Saunders was investigated. High-quality proteins that are available for protein profiling were extracted by the ethanol/phenol extraction method, and 564 protein spots in total were detected. Proteins were identified through database search by combining Mascot and MS BLAST for 100 spots, and significant difference of expression level between the samples collected in winter and in summer was observed in the case of 95 spots. Within 67 spots upregulated in the samples collected in summer, vanadium-dependent bromoperoxidase (vBPO) were identified for 21spots. It is thought that the elevation of expression level of vBPO in summer depend on the activation of the functions: (1) elimination of active oxygen species and protection of the algal body from oxygen injury, (2) prevention of the growth inhibition due to the adherence of attached organisms, in the season.     

Identification and determination of urinary acetylpolyamines in cancer patients by electrospray ionization and time-of-flight mass spectrometry

A method for the quantification of acetylpolyamines, N¹,N¹²-diacetylspermine (DiAcSpm), monoacetylspermidine (AcSpd), and N¹,N⁸-diacetylspermidine (DiAcSpd), identifying each compound simultaneously, was developed with the goal of evaluating these acetylpolyamines as potential biomarkers of cancer. The method consists of prepurification of acetylpolyamines in urine with commercially available cartridges and derivatization with heptafluorobutyric (HFB) anhydride. HFB derivatives of acetylpolyamines were determined simultaneously using ¹⁵N-labeled acetylpolyamines as internal standards by electrospray ionization and time-of-flight mass spectrometry (ESI-TOF MS). After the method was validated, the urinary acetylpolyamines of 38 cancer patients were quantified with this method. A comparison of the concentrations of DiAcSpm with those measured by a colloidal gold aggregation method demonstrated a correlation coefficient of 0.996, showing that the two methods were equally satisfactory. Analysis of the correlation between DiAcSpd or AcSpd and DiAcSpm, performed for the first time, indicated the usefulness of DiAcSpm as a urinary biomarker of cancer. During the course of this work, two simple methods for the preparation of α,ω-diacetylpolyamines were developed, and a possibility to separate and determine the concentrations of the two isomers, N¹-acetylspermidine and N⁸-acetylspermidine in AcSpd, was shown by tandem mass spectrometry (MS/MS).     

Molecular identification of monomeric aspartate racemase from Bifidobacterium bifidum.

Bifidobacterium bifidum is a useful probiotic agent exhibiting health-promoting properties and contains d-aspartate as an essential component of the cross-linker moiety in the peptidoglycan. To help understand D-aspartate biosynthesis in B. bifidum NBRC 14252, aspartate racemase, which catalyzes the racemization of D- and L-aspartate, was purified to homogeneity and characterized. The enzyme was a monomer with a molecular mass of 27 kDa. This is the first report showing the presence of a monomeric aspartate racemase. Its enzymologic properties, such as its lack of cofactor requirement and susceptibility to thiol-modifying reagents in catalysis, were similar to those of the dimeric aspartate racemase from Streptococcus thermophilus. The monomeric enzyme, however, showed a novel characteristic, namely, that its thermal stability significantly increased in the presence of aspartate, especially the D-enantiomer. The gene encoding the monomeric aspartate racemase was cloned and overexpressed in Escherichia coli cells. The nucleotide sequence of the aspartate racemase gene encoded a peptide containing 241 amino acids with a calculated molecular mass of 26 784 Da. The recombinant enzyme was purified to homogeneity and its properties were almost the same as those of the B. bifidum enzyme.     

First enzymological characterization of selenocysteine β-lyase from a lactic acid bacterium,Leuconostoc mesenteroides

We succeeded in expressing selenocysteine β-lyase (SCL) from a lactic acid bacterium, Leuconostoc mesenteroides LK-151 (Lm-SCL), in the soluble fractions of Escherichia coli Rosetta (DE3) using a novel expression vector of pET21malb constructed by ourselves that has both maltose binding protein (MBP)- and 6?×?His-tag. Lm-SCL acted on l-selenocysteine, l-cysteine, and l-cysteine sulfinic acid but showed a high preference for l-selenocysteine. The k_cat and k_cat/K_m values of Lm-SCL were determined to be 108 (min^?1) and 42.0 (min^?1?mM^?1), respectively, and this was enough catalytic efficiency to suggest that Lm-SCL might also be involved in supplying elemental selenium from l-selenocysteine to selenoproteins like other SCLs. The optimum temperature and optimum pH of Lm-SCL were determined to be 37 °C and pH 6.5, respectively. Lm-SCL was stable at 37–45 °C and pH 6.5–7.5. Lm-SCL was completely inhibited by the addition of hydroxylamine, semicarbazide, and iodoacetic acid. The enzyme activity of Lm-SCL was decreased in the presence of various metal ions, especially Cu²⁺. The quaternary structure of Lm-SCL is a homodimer with a subunit molecular mass of 47.5 kDa. The similarity of the primary structure of Lm-SCL to other SCLs from Citrobacter freundii, Escherichia coli, humans, or mouse was calculated to be 47.0, 48.0, 12.5, or 24.0%, respectively. Unlike Ec-SCL, our mutational and molecular docking simulation studies revealed that C362 of Lm-SCL might also catalyze the deselenation of l-selenocysteine in addition to the desulfuration of l-cysteine.