Synthesis of 4-hydroxyisoleucine by the aldolase-transaminase coupling reaction and basic characterization of the aldolase from Arthrobacter simplex AKU 626

Arthrobacter simplex AKU 626 was found to synthesize 4-hydroxyisoleucine from acetaldehyde, alpha-ketobutyrate, and L-glutamate in the presence of Escherichia coli harboring the branched chain amino acid transaminase gene (ilvE) from E. coli K12 substrain MG1655. By using resting cells of A. simplex AKU 626 and E. coli BL21(DE3)/pET-15b-ilvE, 3.2 mM 4-hydroxyisoleucine was produced from 250 mM acetaldehyde, 75 mM alpha-ketobutyrate, and 100 mM L-glutamate with a molar yield to alpha-ketobutyrate of 4.3% in 50 mM Tris-HCl buffer (pH 7.5) containing 2 mM MnCl(2) x 4H(2)O at 28 degrees C for 2 h. An aldolase that catalyzes the aldol condensation of acetaldehyde and alpha-ketobutyrate was purified from A. simplex AKU 626. Mn(2+) and pyridoxal 5'-monophosphate were effective in stabilizing the enzyme. The native and subunit molecular masses of the purified aldolase were about 180 and 32 kDa respectively. The N-terminal amino acid sequence of the purified enzyme showed no significant homology to known aldolases.     

Two Sec13p homologs, AtSec13A and AtSec13B, redundantly contribute to the formation of COPII transport vesicles in Arabidopsis thaliana

COPII vesicles mediate protein transport from ER to Golgi. Sec13 makes up lattice structure with Sec31 to form COPII vesicles. We analyzed expression of two Arabidopsis thaliana Sec13 homologs, AtSec13A and AtSec13B. AtSec13A was expressed in most parts of seedlings, while AtSec13B was partially expressed. Interaction of AtSec13A or AtSec13B with Sec31 homolog was demonstrated by bimolecular fluorescence complementation (BiFC).     

Efficient and rapid purification of drug- and gene-carrying bio-nanocapsules, hepatitis B virus surface antigen L particles, from Saccharomyces cerevisiae

Bio-nanocapsules (BNCs) are hollow particles (approx. 50 nm diameter) consisting of hepatitis B virus surface antigen (HBsAg) large (L, pre-S1+pre-S2+S) proteins embedded in a unilamellar liposome, sharing the same transmembrane S region with an immunogen of hepatitis B vaccine (i.e., HBsAg small (S) protein particle). BNCs can incorporate drugs and genes into the hollow space and systemic administration of the BNCs can deliver the products to human liver via the human hepatocyte-specific receptor within the pre-S (pre-S1+pre-S2) region displayed on BNC's surface. Thus, BNCs are expected to offer efficient and safe non-viral nanocarriers to deliver human liver-specific genes and drugs. To date, BNCs have been purified from the crude extract of BNC-overexpressing yeast cells by fractionation with polyethylene glycol followed by one CsCl equilibrium and two sucrose density gradient ultracentrifugation steps. However, the process was inefficient in terms of yield and time, and was not suitable for mass production because of the ultracentrifugation step. Furthermore, trace contamination with yeast-derived proteinases degraded the pre-S region, which is indispensable for liver-targeting, during long-term storage. In this study, we developed a new purification method involving heat treatment and sulfated cellulofine column chromatography to facilitate rapid purification, completely remove proteinases, and enable mass production. In addition, the BNCs were functional for at least 14 months after lyophilization with 5% (w/v) sucrose as an excipient. This new process will significantly contribute to the development of forthcoming BNC-based nanomedicines as well as hepatitis B vaccines.     

Acute toxic impacts of three heavy metals (copper,zinc, and cadmium) on <Emphasis Type="Italic">Diaphanosoma brachyurum</Emphasis> (Cladocera: Sididae)

Diaphanosoma brachyurum (Cladocera: Sididae) is a common limnetic species in summer-temperate and tropical water bodies. Few studies have investigated the sensitivity of D. brachyurum to toxic chemicals despite this species often being dominant in natural lakes and ponds. We performed acute toxicity tests of three heavy metals, copper (Cu), zinc (Zn), and cadmium (Cd), to D. brachyurum. For D. brachyurum, the lethal concentration (LC)₅₀ values of Cu (24-h LC₅₀ = 16.4 μg/L, 48-h LC₅₀ = 10.4 μg/L) and Zn (24-h LC₅₀ = 253.4 μg/L, 48-h LC₅₀ = 174.1 μg/L) were lower than those for D. magna, one of the most used test organisms for toxic chemicals. On the other hand, for D. brachyurum the 24-h LC₅₀ of Cd (166.4 μg/L) was much greater than that for D. magna, and the 48-h LC₅₀ of Cd (69.8 μg/L) was comparable. Our results indicate that D. brachyurum may be more strongly influenced by Zn and Cu than is D. magna. It is likely that the summer plankton community in which Diaphanosoma species is dominant is more sensitive to heavy metals than a community in which Daphnia species are dominant.     

Random genetic drift and gamete frequency

An analytic expression of conditional expectation of transient gamete frequency, given that one of the two loci remains polymorphic, is obtained in terms of the diffusion process by calculating the moments of the distribution. Using this expression, a model where linkage disequilibrium is introduced by a single mutation is considered. The conditional expectation of the gamete frequency given that the locus with the mutant allele remains polymorphic is presented. The behavior is significantly different from the monotonic decrease observed in the deterministic model without random genetic drift.     

Mutual regulation of protein-tyrosine phosphatase 20 and protein-tyrosine kinase Tec activities by tyrosine phosphorylation and dephosphorylation

PTP20, also known as HSCF/protein-tyrosine phosphatase K1/fetal liver phosphatase 1/brain-derived phosphatase 1, is a cytosolic protein-tyrosine phosphatase with currently unknown biological relevance. We have identified that the nonreceptor protein-tyrosine kinase Tec-phosphorylated PTP20 on tyrosines and co-immunoprecipitated with the phosphatase in a phosphotyrosine-dependent manner. The interaction between the two proteins involved the Tec SH2 domain and the C-terminal tyrosine residues Tyr-281, Tyr-303, Tyr-354, and Tyr-381 of PTP20, which were also necessary for tyrosine phosphorylation/dephosphorylation. Association between endogenous PTP20 and Tec was also tyrosine phosphorylation-dependent in the immature B cell line Ramos. Finally, the Tyr-281 residue of PTP20 was shown to be critical for deactivating Tec in Ramos cells upon B cell receptor ligation as well as dephosphorylation and deactivation of Tec and PTP20 itself in transfected COS7 cells. Taken together, PTP20 appears to play a negative role in Tec-mediated signaling, and Tec-PTP20 interaction might represent a negative feedback mechanism.     

Analysis of the antigen binding site of anti-deoxycholate monoclonal antibody using a novel affinity labeling reagent, acyl adenylate

Large-scale analysis of protein-protein interaction sites is especially needed in the postgenomic era. The combination of affinity labeling with mass spectrometry is a potentially useful high-throughput screening method for this purpose. However, reagents in current use are not ideal as some cause damage to the target molecule and others have poor solubility in physiologic aqueous buffers. In this paper, we describe a novel affinity labeling reagent, acyl adenylate, which is highly soluble in aqueous solutions and reacts in a pH-dependent manner. The adenylate of deoxycholic acid reacts with amino groups on the side chain of a lysine residue and at the N-terminus of proteins/peptides. The reactivity and stability of this reagent were investigated, and it was confirmed that, after formation of a reversible ligand-protein complex under weakly acidic conditions, derivatization with acyl adenylate occurred at the target site under weakly alkaline condition. We further demonstrated the utility of this reagent for affinity labeling using a monoclonal antibody with high affinity for deoxycholic acid. Competitive ELISA indicated that deoxycholic acid was labeled around the antibody ligand binding site, thus enabling the structural elucidation of the ligand-protein interaction. In addition, LC/ESI-MS/MS analysis of the labeled peptide obtained by enzymatic digestion and affinity extraction allowed the identification of the structure surrounding the antigen binding site.     

A nonenzymatic modification of the amino-terminal domain of histone H3 by bile acid acyl adenylate

Although it has been proposed that the secondary bile acids, deoxycholic acid and lithocholic acid, increase the number of aberrant crypt foci in the colon and may act as colon tumor promoters, there is little evidence detailing their mechanism of action. Histones play an important role in controlling gene expression, and the posttranslational modification of histones plays a role in regulation of intracellular signal transduction. In particular, the amino-terminal tail domain of histone H3 is sensitive to several posttranslational modifications, and acetylation of this domain changes its electrostatic environment and results in the loss of native folding. Therefore, we studied the modification of epsilon-amino groups on human histone H3 by deoxycholyl adenylate, which is an active intermediate in deoxycholyl thioester biosynthesis. After incubation of recombinant human histone H3 with a smaller amount of acyl adenylate, followed by enzymatic digestion, the peptide fragment mixtures were analyzed by matrix-assisted laser desorption ionization mass spectrometry. These data showed the formation of only one adduct fragment, which corresponded to amino acids 3-8 with a deoxycholate adduct, suggesting that the epsilon-amino group of Lys(4) had the highest reactivity. This novel modification, formation of a bile acid adduct on the histone H3 amino-terminal tail domain through an active acyl adenylate, may relate to the carcinogenesis-promoting effects of secondary bile acids.     

Signals from intra-abdominal fat modulate insulin and leptin sensitivity through different mechanisms: neuronal involvement in food-intake regulation

Intra-abdominal fat accumulation is involved in development of the metabolic syndrome, which is associated with insulin and leptin resistance. We show here that ectopic expression of very low levels of uncoupling protein 1 (UCP1) in epididymal fat (Epi) reverses both insulin and leptin resistance. UCP1 expression in Epi improved glucose tolerance and decreased food intake in both diet-induced and genetically obese mouse models. In contrast, UCP1 expression in Epi of leptin-receptor mutant mice did not alter food intake, though it significantly decreased blood glucose and insulin levels. Thus, hypophagia induction requires a leptin signal, while the improved insulin sensitivity appears to be leptin independent. In wild-type mice, local-nerve dissection in the epididymis or pharmacological afferent blockade blunted the decrease in food intake, suggesting that afferent-nerve signals from intra-abdominal fat tissue regulate food intake by modulating hypothalamic leptin sensitivity. These novel signals are potential therapeutic targets for the metabolic syndrome.     

Identification of a novel bile acid in swans, tree ducks, and geese: 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid

By HPLC, a taurine-conjugated bile acid with a retention time different from that of taurocholate was found to be present in the bile of the black-necked swan, Cygnus melanocoryphus. The bile acid was isolated and its structure, established by (1)H and (13)C NMR and mass spectrometry, was that of the taurine N-acyl amidate of 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid. The compound was shown to have chromatographic and spectroscopic properties that were identical to those of the taurine conjugate of authentic 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid, previously synthesized by us from ursodeoxycholic acid. By HPLC, the taurine conjugate of 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid was found to be present in 6 of 6 species in the subfamily Dendrocygninae (tree ducks) and in 10 of 13 species in the subfamily Anserinae (swans and geese) but not in other subfamilies in the Anatidae family. It was also not present in species from the other two families of the order Anseriformes. 3alpha,7alpha,15alpha-Trihydroxy-5beta-cholan-24-oic acid is a new primary bile acid that is present in the biliary bile acids of swans, tree ducks, and geese and may be termed 15alpha-hydroxy-chenodeoxycholic acid.