Affinity Improvement of a Cancer-Targeted Antibody through Alanine-Induced Adjustment of Antigen-Antibody Interface

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Rapid evolution of an introduced insect Ophraella communa LeSage in new environments: temporal changes and geographical differences in photoperiodic response

The accidentally introduced beetle Ophraella communa was first found in 1996 in Japan and has rapidly expanded its distribution range to include regions with substantially different climates. During this range expansion and subsequent establishment in new habitats, the life history traits of O. communa have changed depending on new habitat environmental conditions. In this study, we investigated the photoperiodic response of O. communa controlling its reproductive diapause, an important trait that adjusts the life cycle to local climate and phenology of host‐plants. We examined temporal changes and geographical differences in this trait. In the Tsukuba population of O. communa, the diapause incidence under conditions of 12 h light : 12 h dark (LD 12:12), 13:11 and 14:10 did not consistently increase or decrease during 2005–2012, although it fluctuated yearly. The diapause incidence in this period, however, was lower than that in 1999 reported previously and the critical day‐length inducing diapause was shortened by >1 h from 1999 to 2005. These results suggest that the photoperiodic response of O. communa shifted during this period. To examine geographical differences in this trait, we compared the Hirosaki, Morioka, Tsukuba and Koshi lines. The diapause incidence at LD 13:11 was significantly different between the O. communa lines: 86.5% in the Hirosaki line (collected in 2010) , 81.4% in Morioka (collected in 2010), 45.0% in Tsukuba (collected in 2011) and 7.1% in Koshi (collected in 2009), and was positively correlated with the latitude of the origin. These results suggest that this trait shifted, responding to the environmental conditions in the colonized regions.     

The molecular interaction of a protein in highly concentrated solution investigated by Raman spectroscopy

We used Raman spectroscopy to investigate the structure and interactions of lysozyme molecules in solution over a wide range of concentrations (2.5–300 mg ml^?1). No changes in the amide‐I band were observed as the concentration was increased, but the width of the Trp band at 1555 cm^?1 and the ratios of the intensities of the Tyr bands at 856 and 837 cm^?1, the Trp bands at 870 and 877 cm^?1, and the bands at 2940 (CH stretching) and 3420 cm^?1 (OH stretching) changed as the concentration was changed. These results reveal that although the distance between lysozyme molecules changed by more than an order of magnitude over the tested concentration range, the secondary structure of the protein did not change. The changes in the molecular interactions occurred in a stepwise process as the order of magnitude of the distance between molecules changed. These results suggest that Raman bands can be used as markers to investigate the behavior of high‐concentration solutions of proteins and that the use of Raman spectroscopy will lead to progress in our understanding not only of the basic science of protein behavior under concentrated (i.e., crowded) conditions but also of practical processes involving proteins, such as in the field of biopharmaceuticals. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 237–246, 2015.     

The Atomic Structure of the HIV-1 gp41 Transmembrane Domain and Its Connection to the Immunogenic Membrane-proximal External Region

The membrane-proximal external region (MPER) C-terminal segment and the transmembrane domain (TMD) of gp41 are involved in HIV-1 envelope glycoprotein-mediated fusion and modulation of immune responses during viral infection. However, the atomic structure of this functional region remains unsolved. Here, based on the high resolution NMR data obtained for peptides spanning the C-terminal segment of MPER and the TMD, we report two main findings: (i) the conformational variability of the TMD helix at a membrane-buried position; and (ii) the existence of an uninterrupted α-helix spanning MPER and the N-terminal region of the TMD. Thus, our structural data provide evidence for the bipartite organization of TMD predicted by previous molecular dynamics simulations and functional studies, but they do not support the breaking of the helix at Lys-683, as was suggested by some models to mark the initiation of the TMD anchor. Antibody binding energetics examined with isothermal titration calorimetry and humoral responses elicited in rabbits by peptide-based vaccines further support the relevance of a continuous MPER-TMD helix for immune recognition. We conclude that the transmembrane anchor of HIV-1 envelope is composed of two distinct subdomains: 1) an immunogenic helix at the N terminus also involved in promoting membrane fusion; and 2) an immunosuppressive helix at the C terminus, which might also contribute to the late stages of the fusion process. The unprecedented high resolution structural data reported here may guide future vaccine and inhibitor developments.     

Stable nuclear transformation of the diatom Chaetoceros sp.

A nuclear transformation system for the centric diatom Chaetoceros sp. has been established using two plasmids pTpfcp/nat and pTpNR/green fluorescent protein (GFP) that had been used for Thalassiosira pseudonana transformation. These contain the nourseothricin resistance gene (nat) with the fucoxanthin chlorophyll a/c binding protein (fcp) promoter/terminator from T. pseudonana and the enhanced green fluorescent protein gene (egfp), with the nitrate reductase (NR) promoter/terminator from T. pseudonana, respectively. Transformants were recovered in the presence of the antibiotic nourseothricin. One to four copies of both nat and egfp genes were integrated into genomic DNA of the transformants. Transformation efficiency was 1.5–6.0 transformants per 10⁸ cells. This work is the first report of stable genetic transformation of Chaetoceros, which is important as not only a constituent member of marine ecosystem but also feed for aquaculture.     

Regulation of food intake by melanin-concentrating hormone in goldfish

Melanin-concentrating hormone (MCH), originally discovered in the teleost pituitary, is a hypothalamic neuropeptide involved in the regulation of body color in fish. Although MCH is also present in the mammalian brain, it has no evident function in providing pigmentation. Instead, this peptide is now recognized to be one of the key neuropeptides that act as appetite enhancers in mammals such as rodents and primates. Although there has been little information about the central action of MCH on appetite in fish, recent studies have indicated that, in goldfish, MCH acts as an anorexigenic neuropeptide, modulating the α-melanocyte-stimulating hormone signaling pathway through neuronal interaction. These observations indicate that there may be major differences in the mode of action of MCH between fish and mammals. This paper reviews what is currently known about the regulation of food intake by MCH in fish, especially the goldfish.     

Syntheses, structures and antimicrobial activities of various metal complexes of hinokitiol

Metal-oxygen bonding complexes (M = Mg^II, Mn^II, Ni^II, Mo^VI, W^VI, Pd^II, Sb^III, Bi^III, Fe^III, Ti^IV, K^I, Ba^II, Zr^IV and Hf^IV) with a hinokitiol (Hhino; 2-hydroxy-4-isopropylcyclohepta-2,4,6-trienone or β-thujaplicin) ligand, which has two unequivalent oxygen donor atoms, were synthesized and characterized by elemental analysis, TG/DTA, FT-IR and solution (¹H and ¹³C) NMR spectroscopy. Single-crystal X-ray structure analysis revealed various molecular structures for the complexes, which were classified into several families of family, i.e. type A [M^II(hino)₂(L)]₂ (M = Mg^II, Mn^II, Ni^II; L = EtOH or MeOH), with a dimeric structure consisting of one bridging hino⁻ anion, one chelating hino⁻ anion and one alcohol or water molecule, type B, with the octahedral, cis-dioxo, bis-chelate complexes cis-[M^VIO₂(hino)₂] (M = Mo^VI, W^VI), type C, with square planar complex [M^II(hino)₂] (M = Pd^II), type D, with tris-chelate, 7-coordinate complexes with one inert electron pair [M^III(hino)₃] (M = Sb^III, Bi^III), type D′, with the bis-chelate, pseudo-6-coordinate complexes with one inert electron pair [M^III(hino)₂X] (M = Sb^III, X = Br), type E, with tris-chelate, 6-coordinate complexes with Δ and Λ isomers [M^III(hino)₃] (M = Fe^III), type E′ of bis-chelate, 6-coordinate complex [M^IV(hino)₂X₂] (M = Ti^IV, X = Cl), type F, with water-soluble alkali metal salts [M^I(hino)] (M = K^I), and type H, with tetrakis-chelate, 8-coordinate complexes [M^IV(hino)₄](M = Zr^IV, Hf^IV). These structural features were compared with those of metal complexes with a related ligand, tropolone (Htrop). The antimicrobial activities of these complexes, evaluated in terms of minimum inhibitory concentration (MIC; μg mL⁻¹) in two systems, were compared to elucidate the relationship between structure and antimicrobial activity.     

Crystal structure of the C-terminal domain of Mu phage central spike and functions of bound calcium ion

Bacteriophage Mu, which has a contractile tail, is one of the most famous genus of Myoviridae. It has a wide host range and is thought to contribute to horizontal gene transfer. The Myoviridae infection process is initiated by adhesion to the host surface. The phage then penetrates the host cell membrane using its tail to inject its genetic material into the host. In this penetration process, Myoviridae phages are proposed to puncture the membrane of the host cell using a central spike located beneath its baseplate. The central spike of the Mu phage is thought to be composed of gene 45 product (gp45), which has a significant sequence homology with the central spike of P2 phage (gpV). We determined the crystal structure of shortened Mu gp45Δ1-91 (Arg92–Gln197) at 1.5 Å resolution and showed that Mu gp45 is a needlelike structure that punctures the membrane. The apex of Mu gp45 and that of P2 gpV contained iron, chloride, and calcium ions. Although the C-terminal domain of Mu gp45 was sufficient for binding to the E. coli membrane, a mutant D188A, in which the Asp amino acid residue that coordinates the calcium ion was replaced by Ala, did not exhibit a propensity to bind to the membrane. Therefore, we concluded that calcium ion played an important role in interaction with the host cell membrane.     

Investigation of Proteomic Profiles of Lamina of Ecklonia kurome (Laminariales): Homology-Based Cross-Species Protein Identification and Analysis of the Post-translational Processing of Vanadium-Dependent Bromoperoxidases Using MALDI-TOF/TOF

Proteomic profiles of the lamina of Ecklonia kurome Okamura, one of the Japanese dominant laminarialean kelps, were investigated by two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF. Due to the absence of E. kurome DNA or protein databases, homology-based cross-species protein identification was performed using a combination of three database-searching algorithms, Mascot peptide mass fingerprinting, Mascot MS/MS ion search, and mass spectrometry-based BLAST. Proteins were extracted from the lamina by an ethanol/phenol method and subjected to 2-DE (pI 4–7, 10 % polyacrylamide gel). More than 700 spots were detected in the 2-DE gel with CBB, and 93 spots (24 proteins) were successfully identified by MALDI-TOF/TOF and the cross-species database searching. The identified proteins mainly consisted of cytoplasmic carbohydrate metabolic enzymes, chloroplast proteins involved in photosynthesis, and haloperoxidases. Interestingly, vanadium-dependent bromoperoxidases (vBPO), which is thought to be involved in halogen uptake, synthesis of halogenated products, and detoxification of reactive oxygen species, were separated into at least 23 different spots. By comparing mass spectra, amino acid sequences predicted from tandem mass spectra and haloperoxidase activities of the vBPOs, we found that (1) at least two types of vBPOs were expressed in the lamina of E. kurome and (2) two pro-vBPOs might be activated by specific cleavage at N- and C-terminal regions.