Purification, cloning, and expression of an alpha/beta-galactoside alpha-2,3-sialyltransferase from a luminous marine bacterium, Photobacterium phosphoreum

A novel sialyltransferase, alpha/beta-galactoside alpha-2,3-sialyltransferase, was purified from the cell lysate of a luminous marine bacterium, Photobacterium phosphoreum JT-ISH-467, isolated from the Japanese common squid (Todarodes pacificus). The gene encoding the enzyme was cloned from the genomic library of the bacterium using probes derived from the NH(2)-terminal and internal amino acid sequences. An open reading frame of 409 amino acids was identified, and the sequence had 32% identity with that of beta-galactoside alpha-2,6-sialyltrasferase in Photobacterium damselae JT0160. DNA fragments that encoded the full-length protein and a protein that lacked the sequence between the 2nd and 24th residues at the NH(2) terminus were amplified by polymerase chain reactions and cloned into an expression vector. The full-length and truncated proteins were expressed in Escherichia coli, producing active enzymes of 0.25 and 305 milliunits, respectively, per milliliter of the medium in the lysate of E. coli. The truncated enzyme was much more soluble without detergent than the full-length enzyme. The enzyme catalyzed the transfer of N-acetylneuraminic acid from CMP-N-acetylneuraminic acid to disaccharides, such as lactose and N-acetyllactosamine, with low apparent K(m) and to monosaccharides, such as alpha-methyl-galactopyranoside and beta-methyl-galactopyranoside, with much lower apparent K(m). Thus, this sialyltransferase is unique and should be very useful for achieving high productivity in E. coli with a wide substrate range.     

Retinal Attachment Instability Is Diversified among Mammalian Melanopsins

Melanopsins play a key role in non-visual photoreception in mammals. Their close phylogenetic relationship to the photopigments in invertebrate visual cells suggests they have evolved to acquire molecular characteristics that are more suited for their non-visual functions. Here we set out to identify such characteristics by comparing the molecular properties of mammalian melanopsin to those of invertebrate melanopsin and visual pigment. Our data show that the Schiff base linking the chromophore retinal to the protein is more susceptive to spontaneous cleavage in mammalian melanopsins. We also find this stability is highly diversified between mammalian species, being particularly unstable for human melanopsin. Through mutagenesis analyses, we find that this diversified stability is mainly due to parallel amino acid substitutions in extracellular regions. We propose that the different stability of the retinal attachment in melanopsins may contribute to functional tuning of non-visual photoreception in mammals.     

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.     

Effects of a bout of traditional and original sumo training on neutrophil immune function in amateur university sumo wrestlers

In order to examine in detail the influence on the neutrophil immune function in sumo wrestlers of performing traditional and original training we examined changes in the neutrophil immune function in 17 male amateur university sumo wrestlers (aged 20.2 ± 1.5 years), before (‘Pre’) and after the training (‘Post’) for 2.5 h under fasting conditions. Assays included blood leukocyte and neutrophil counts, serum concentration of immunoglobulins, complements, myogenic enzymes and neutrophil oxidative burst activity (OBA) and phagocytic activity (PA). Myogenic enzymes, neutrophil counts, the ratio of neutrophil counts:leukocyte counts significantly increased and immunoglobulins and complements decreased in Post compared with Pre. There was a positive correlation between the change of neutrophil counts before and after the training and the change of creatine kinase (r = 0.667, p < 0.01). The Post OBA significantly increased and PA significantly decreased compared with Pre. It was concluded that sumo training causes muscular damage and an increase in the neutrophil count as a response. In this time, although OBA increased, PA decreased after training. Compensation between PA and reactive oxygen species production may exist to maintain the overall integrity of the neutrophil immune function. Copyright © 2008 John Wiley & Sons, Ltd.     

TGFβ induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

Background and aims: Transforming growth factor-beta (TGFβ) is known to potently inhibit cell growth. Loss of responsiveness to TGFβ inhibition on cell growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGFβ and HB-EGF signal transduction via ADAM activation.Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGFβ. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGFβ was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGFβ was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown.Result: TGFβ-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGFβ induced shedding of proHB-EGF allowing HB-EGF-CTF to translocate to the nucleus. ADAM inhibitors blocked this nuclear translocation. TGFβ enhanced gastric cancer cell growth and ADAM inhibitors suppressed this effect. EGFR phosphorylation, HB-EGF-CTF nuclear translocation, and cell growth were suppressed in ADAM17 knockdown cells.Conclusion: HB-EGF-CTF nuclear translocation and EGFR transactivation from proHB-EGF shedding mediated by ADAM17 activated by TGFβ might be an important pathway of gastric cancer cell proliferation by TGFβ.     

Effects of Mutations of Rad50, Rad51, Rad52, and Related Genes on Illegitimate Recombination in Saccharomyces Cerevisiae

To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rad51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.     

Binding mode of ecdysone agonists to the receptor: comparative modeling and docking studies

Three-dimensional structure models of the ligand-binding domain of the ecdysone receptor of Heliothis virescens were built by the homology modeling technique from the crystal structures of nuclear receptors. Two models were created based both on known ligand-binding domain structures of the receptors with the highest sequence identity to the ecdysone receptor, and on those of steroid hormone receptors. The latter model, which was found to have better stereochemical quality and be in good agreement with the binding of the steroidal framework of the endogenous agonist 20-hydroxyecdysone, was used for docking studies. The docking of 20-hydroxyecdysone to the receptor model revealed that the ligand molecule can interact with the receptor in a similar manner to other steroid hormone-receptor complexes. The docking of a dibenzoylhydrazine agonist, chromafenozide, was performed based on the correspondences between the molecule and 20-dydroxyecdysone expected by molecular comparison. The interactions of the ligands with the receptor in the complexes modeled were investigated and found to be consistent with known structure-activity relationships.     

Soluble branched beta-(1,4)glucans from Acetobacter species show strong activities to induce interleukin-12 in vitro and inhibit T-helper 2 cellular response with immunoglobulin E production in vivo

An extracellular polysaccharide, AC-1, produced by Acetobacter polysaccharogenes is composed of beta-(1,4)glucan with branches of glucosyl residues. We found that AC-1 showed a strong activity to induce production of interleukin-12 P40 and tumor necrosis factor-alpha by macrophage cell lines in vitro. Cellulase treatment completely abolished the activity of AC-1 to induce tumor necrosis factor-alpha production by macrophages, whereas treatment of AC-1 with polymyxin B or proteinase did not affect the activity. Results of experiments using toll-like receptor (TLR) 4-deficient mice and TLR4-transfected human cell line indicated that TLR4 is involved in pattern recognition of AC-1. In vivo administration of AC-1 significantly reduced the serum levels of ovalbumin (OVA)-specific IgE and interleukin-4 production by T cells in response to OVA in mice immunized with OVA. AC-1, a soluble branched beta-(1,4)glucan may be useful in prevention and treatment of allergic disorders With IgE production.