Regional and segmental flexibility of antibodies in interaction with antigens of different size

The interaction of antibodies (Abs) with protein antigens (Ags) of different size, such as hen egg white lysozyme, ovalbumin, and bovine serum albumin, was examined using analytical ultracentrifugation, electrospray ionization time-of-flight mass spectrometry, and surface plasmon resonance in order to estimate regional and segmental Ab flexibility. When both Abs and Ags were free in solution, sedimentation equilibrium and surface plasmon resonance analyses showed the formation of an Ag(2)Ab(1) complexes regardless of Ag size, suggesting that the Fab arms were able to move to avoid interference between Ags bound to Ab combining sites. The Ag(2)Ab(1) complex, as well as the Ag(1)Ab(1) complex, was observed by MS. However, when Abs were immobilized on the surface of a sensor chip through the Fc region, the stoichiometry of the Ag-Ab complex was dependent on the Ag size; Ag(2)Ab(1) forming with hen egg white lysozyme and Ag(1)Ab(1) with ovalbumin and bovine serum albumin. These results indicated that immobilization of the Fc region reduces the dynamic range of the Fab arms and results in interference from the first Ag bound to either combining site, which in turn prevents the binding of the second Ag to the other combining site. Our results allow us to propose that the Fab arms of B-cell receptors whose Fc regions are immobilized on cell surface have a reduced dynamic range.     

Structure toxicity relationships of synthetic azaspiracid-1 and analogs in mice

Synthetic azaspiracid-1 (AZA-1, 1), 6-, 10-, 13-, 14-, 16-, 17-, 19-, 20-epi-azaspiracid-1 (C₁–C₂₀-epi-AZA-1, 2), and twelve truncated azaspiracid-1 analogs (3–14) were synthesized and tested for their toxicity effects in mice. Of these compounds only AZA-1 (1) and its diastereomer C₁–C₂₀-epi-AZA-1 (2) exhibited significant toxicity in mice with the latter compound (2) being one-fourth as toxic as the former (1). The lack of toxicity exhibited by the severely truncated analogs (3–14) implies that the entire or at least a major part of the structure of AZA-1 (1) is required for biological activity.     

Enrichment of phosphorylated proteins from cell lysate using a novel phosphate-affinity chromatography at physiological pH

While phosphoproteins have attracted great interest toward the post-genome research (e.g. clinical diagnosis and drug design), there have been few procedures for the specific enrichment of native phosphoproteins from cells or tissues. Here, we describe a simple and efficient protocol to enrich phosphoproteins comprehensively from a complex mixture containing solubilized cellular proteins. This method is based on immobilized metal affinity chromatography using a phosphate-binding tag molecule (i.e. a dinuclear zinc(II) complex) attached on a highly cross-linked agarose. The binding, washing, and elution processes were all conducted without a detergent or a reducing agent at pH 7.5 and room temperature. An additive, 1.0 M CH3COONa, was necessary in the binding and washing buffers (0.10 M Tris-CH3COOH, pH 7.5) to prevent the nonphosphorylated protein from binding. The absorbed phosphoproteins were eluted using a mixed buffer solution (pH 7.5) consisting of 0.10 M Tris-CH3COOH, 10 mM NaH2PO4-NaOH, and 1.0 M NaCl. In this study, we demonstrate a typical example of phosphate-affinity chromatography using an epidermal growth factor-stimulated A431 cell lysate. The total time for the column chromatography (1 mL gel scale) was less than 1 h. The strong enrichment of the phosphoproteins into the elution fraction was evaluated using SDS-PAGE followed by Western blotting analysis.     

Effects of fasting and re-feeding on the expression of Dec1, Per1, and other clock-related genes

To elucidate the food-entrainable oscillatory mechanism of peripheral clock systems, we examined the effect of fasting on circadian expression of clock genes including Dec1 and Dec2 in mice. Withholding of food for 2 days had these effects: the expression level of Dec1 mRNA decreased in all tissues examined, although Per1 mRNA level markedly increased; Per2 expression was reduced in the liver and heart only 42-46 h after the start of fasting; and expression profiles of Dec2 and Bmal1 were altered only in the heart and in the liver, respectively, whereas Rev-erbalpha mRNA levels did not change significantly. Re-feeding after 36-h starvation erased, at least in part, the effect of fasting on Dec1, Dec2, Per1, Per2, and Bmal1 within several hours, and restriction feeding shifted the phase of expression profiles of all examined clock genes including Dec1 and Dec2. These findings indicate that short-term fasting and re-feeding modulate the circadian rhythms of clock genes to different extents in peripheral tissues, and suggest that the expression of Dec1, Per1, and some other clock genes was closely linked with the metabolic activity of these tissues.     

Attenuation of MPTP-induced neurotoxicity and locomotor dysfunction in Nucling-deficient mice via suppression of the apoptosome pathway

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity is one of the experimental models most commonly used to study the pathogenesis of Parkinson's disease (PD). Although the biochemical mechanisms underlying the cell death induced by MPTP remain to be clarified, it has been found that the mitochondrial apoptotic signaling pathway plays an important role in the neurotoxicity of MPTP. Nucling is a novel type of apoptosis-associated molecule, essential for cytochrome c, apoptosis protease activating factor 1 (Apaf-1), pro-caspase-9 apoptosome induction and caspase-9 activation following pro-apoptotic stress. Here we found that Nucling-deficient mice treated with MPTP did not exhibit locomotor dysfunction in an open-field test. The substantia nigra dopaminergic neurons of Nucling-deficient mice were resistant to the damaging effects of the neurotoxin MPTP. Up-regulated expression of apoptosome was attenuated in Nucling-deficient mice treated with MPTP. These results indicate an important role for Nucling in MPTP-induced neuronal degeneration and suggest that the suppression of Nucling would be of therapeutic benefit for the treatment of neurodegeneration in PD.     

Controlling systems of nucleic acid sensing-TLRs restrict homeostatic inflammation

Nucleic acid sensing-TLRs recognize pathogen-derived nucleic acids (NAs) and induce anti-microbial immune responses. NA-sensing TLRs are reported to have a risk of mistakenly responding to self-derived NAs and causing a variety of autoimmune diseases. To warrant innate immune responses without detrimental inflammation, NA-sensing TLRs need to be tightly controlled in multiple aspects including expression and downstream signaling pathways. Along this line, TLR7 vs 9 balance has emerged as a new mechanism that controls activation of RNA-sensor TLR7. In this review, we discuss mechanisms controlling NA-sensing TLRs and autoimmune diseases as consequences of the dysregulated control mechanisms.     

Phylogenetic and biochemical characterization of the oil-producing yeast Lipomyces starkeyi

Lipomyces starkeyi is an oleaginous yeast, and has been classified in four distinct groups, i.e., sensu stricto and custers α, β, and γ. Recently, L. starkeyi clusters α, β, and γ were recognized independent species, Lipomyces mesembrius, Lipomyces doorenjongii, and Lipomyces kockii, respectively. In this study, we investigated phylogenetic relationships within L. starkeyi, including 18 Japanese wild strains, and its related species, based on internal transcribed spacer sequences and evaluated biochemical characters which reflected the phylogenetic tree. Phylogenetic analysis showed that most of Japanese wild strains formed one clade and this clade is more closely related to L. starkeyi s.s. clade including one Japanese wild strain than other clades. Only three Japanese wild strains were genetically distinct from L. starkeyi. Lipomyces mesembrius and L. doorenjongii shared one clade, while L. kockii was genetically distinct from the other three species. Strains in L. starkeyi s.s. clade converted six sugars, d-glucose, d-xylose, l-arabinose, d-galactose, d-mannose, and d-cellobiose to produce high total lipid yields. The Japanese wild strains in subclades B, C, and D converted d-glucose, d-galactose, and d-mannose to produce high total lipid yields. Lipomyces mesembrius was divided into two subclades. Lipomyces mesembrius CBS 7737 converted d-xylose, l-arabinose, d-galactose, and d-cellobiose, while the other L. mesembrius strains did not. Lipomyces doorenjongii converted all the sugars except d-cellobiose. In comparison to L. starkeyi, L. mesembrius, and L. doorenjongii, L. kockii produced higher total lipid yields from d-glucose, d-galactose, and d-mannose. The type of sugar converted depended on the subclade classification elucidated in this study.     

Identification of NIPSNAP1 as a nocistatin-interacting protein involving pain transmission

4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) is a molecule of physiologically unknown function, although it is predominantly expressed in the brain, spinal cord, liver, and kidney. We identified NIPSNAP1 as a protein that interacts with the neuropeptide nocistatin (NST) from synaptosomal membranes of mouse spinal cord using high-performance affinity latex beads. NST, which is produced from the same precursor protein as an opioid-like neuropeptide nociceptin/orphanin FQ (N/OFQ), has opposite effects on pain transmission evoked by N/OFQ. The calculated full-length pre-protein of NIPSNAP1 was 33 kDa, whereas the N-terminal truncated form of NIPSNAP1 (29 kDa) was ubiquitously expressed in the neuronal tissues, especially in synaptic membrane and mitochondria of brain. The 29-kDa NIPSNAP1 was distributed on the cell surface, and NST interacted with the 29-kDa but not the 33-kDa NIPSNAP1. Although intrathecal injection of N/OFQ induced tactile allodynia in both wild-type and NIPSNAP1-deficient mice, the inhibition of N/OFQ-evoked tactile allodynia by NST seen in wild-type mice was completely lacking in the deficient mice. These results suggest that NIPSNAP1 is an interacting molecule of NST and plays a crucial role in pain transmission.     

Identification and functions of amino acid residues in PotB and PotC involved in spermidine uptake activity

Amino acid residues on PotB and PotC involved in spermidine uptake were identified by random and site-directed mutagenesis. It was found that Trp(8), Tyr(43), Trp(100), Leu(110), and Tyr(261) in PotB and Trp(46), Asp(108), Glu(169), Ser(196), Asp(198), and Asp(199) in PotC were strongly involved in spermidine uptake and that Tyr(160), Glu(172), and Leu(274) in PotB and Tyr(19), Tyr(88), Tyr(148), Glu(160), Leu(195), and Tyr(211) in PotC were moderately involved in spermidine uptake. Among 11 amino acid residues that were strongly involved in spermidine uptake, Trp(8) in PotB was important for insertion of PotB and PotC into membranes. Tyr(43), Trp(100), and Leu(110) in PotB and Trp(46), Asp(108), Ser(196), and Asp(198) in PotC were found to be involved in the interaction with PotD. Leu(110) and Tyr(261) in PotB and Asp(108), Asp(198), and Asp(199) in PotC were involved in the recognition of spermidine, and Trp(100) and Tyr(261) in PotB and Asp(108), Glu(169), and Asp(198) in PotC were involved in ATPase activity of PotA. Accordingly, Trp(100) in PotB was involved in both PotD recognition and ATPase activity, Leu(110) in PotB was involved in both PotD and spermidine recognition, and Tyr(261) in PotB was involved in both spermidine recognition and ATPase activity. Asp(108) and Asp(198) in PotC were involved in PotD and spermidine recognition as well as ATPase activity. These results suggest that spermidine passage from PotD to the cytoplasm is coupled to the ATPase activity of PotA through a structural change of PotA by its ATPase activity.