松下 文雄

Bitiscetin-1 (aka bitiscetin) and bitiscetin-2 are C-type lectin-like proteins purified from the venom of Bitis arietans (puff adder). They bind to von Willebrand factor (VWF) and-at least bitiscetin-1-induce platelet agglutination via enhancement of VWF binding to platelet glycoprotein Ib (GPIb). Bitiscetin-1 and -2 bind the VWF A1 and A3 domains, respectively. The A3 domain includes the major site of VWF for binding collagen, explaining why bitiscetin-2 blocks VWF-to-collagen binding. In the present study, sequences for a novel bitiscetin protein-bitiscetin-3-were identified in cDNA constructed from the B. arietans venom gland. The deduced amino acid sequences of bitiscetin-3 subunits α and β share 79 and 80% identity with those of bitiscetin-1, respectively. Expression vectors for bitiscetin-3α and -3β were co-transfected to 293T cells, producing the heterodimer protein recombinant bitiscetin-3 (rBit-3). Functionally, purified rBit-3 (1) induced platelet agglutination involving VWF and GPIb, (2) did not compete with bitiscetin-1 for binding to VWF, (3) blocked VWF-to-collagen binding, and (4) lost its platelet agglutination inducing ability in the presence of an anti-VWF monoclonal antibody that blocked VWF-to-collagen binding. These combined results suggest that bitiscetin-3 binds to the A3 domain, as does bitiscetin-2. Except for a small N-terminal fragment of a single subunit-which differs from that of both bitiscetin-3 subunits-the sequences of bitiscetin-2 have never been determined. Therefore, by identifying and analyzing bitiscetin-3, the present study is the first to present the full-length α- and β-subunit sequences and recombinant expression of a bitiscetin-family toxin that blocks the binding of VWF to collagen.

With the recent establishment of robust reverse genetics systems for rotavirus, rotavirus is being developed as a vector to express foreign genes. However, insertion of larger sequences such as those encoding multiple foreign genes into the rotavirus genome has been challenging because the virus segments are small. In this paper, we attempted to insert multiple foreign genes into a single gene segment of rotavirus to determine whether it can efficiently express multiple exogenous genes from its genome. At first, we engineered a truncated NSP1 segment platform lacking most of the NSP1 open reading frame and including a self-cleaving 2A sequence (2A), which made it possible to generate a recombinant rotavirus stably expressing NanoLuc (Nluc) luciferase as a model foreign gene. Based on this approach, we then demonstrated the generation of a replication-competent recombinant rotavirus expressing three reporter genes (Nluc, EGFP, and mCherry) by separating them with self-cleaving 2As, indicating the capacity of rotaviruses as to the insertion of multiple foreign genes. Importantly, the inserted multiple foreign genes remained genetically stable during serial passages in cell culture, indicating the potential of rotaviruses as attractive expression vectors. The strategy described here will serve as a model for the generation of rotavirus-based vectors designed for the expression and/or delivery of multiple foreign genes.

Von Willebrand factor (VWF) is one of the plasma protein carrying ABO(H) blood group antigens, but the combining process of these antigens is not clear. In the present study, we examined whether plasma glycosyltransferase affects the blood group antigens on VWF. VWF expressing H-antigen (H-VWF) from blood group O and bovine serum albumin conjugated with H-antigen (H-BSA) were incubated with recombinant α1-3-N-acetylgalactosaminyltransferase (rA-transferase) and A-plasma with or without an additional UDP-GalNAc. Transformed antigens were detected by western blotting and ELISA, using an anti-A antibody. Both H-VWF and H-BSA acquired the A-antigen after incubation with rA-transferase and UDP-GalNAc. Incubation with A-plasma very weakly converted the H-antigen on BSA and VWF to A-antigen only in the presence of supplemented UDP-GalNAc. This conversion was enhanced on desialylation of H-VWF. These results indicate that sugar chains of plasma VWF can be modified by the external glycosyltransferase, but that plasma glycosyltransferase has no effect on the blood group antigens of VWF due to its low activity and the lack of donor sugars. Further, sialic acid residues of VWF may exert a protective effect against post-translational glycosylation. Our results clearly exclude the possibility that blood group antigens of VWF are constructed extracellularly in plasma.

Background Botrocetin-2 (Bot2) is a botrocetin-like protein composed of and subunits that have been cloned from the snake Bothrops jararaca. Bot2 binds specifically to von Willebrand factor (VWF), and the complex induces glycoprotein (GP) Ib-dependent platelet agglutination. Objectives To exploit Bot2's VWF-binding capacity in order to attempt to create a mutant Bot2 that binds to VWF but inhibits platelet agglutination. Methods and Results Several point mutations were introduced into Bot2 cDNA, and the recombinant protein (recombinant Bot2 [rBot2]) was purified on an anti-botrocetin column. The mutant rBot2 with either Ala at Asp70 in the subunit (Asp70Ala), or Arg115Ala and Lys117Ala, showed reduced platelet agglutination-inducing activity. rBot2 with Asp70Ala showed little binding activity towards immobilized VWF on an ELISA plate, whereas rBot2 with Arg115Ala/Lys117Ala showed reduced binding activity towards GPIb (glycocalicin) after forming a complex with VWF. rBot2 point-mutated to oppositely charged Glu at both Arg115 and Lys117 showed normal binding activity towards VWF but no platelet-agglutinating activity. Furthermore, this doubly mutated protein inhibited ristocetin-induced or high shear stress-induced platelet aggregation, and restrained thrombus formation under flow conditions. Conclusions Asp70 in the subunit of botrocetin is important for VWF binding, and Arg115 and Lys117 in the subunit are essential for interaction with GPIb. Doubly mutated rBot2, with Arg115Glu and Lys117Glu, repels GPIb and might have potential as an antithrombotic reagent that specifically blocks VWF function. This is the first report on an artificial botrocetin that can inhibit the VWF-GPIb interaction.

Background: Three members of the Myt/NZF family of transcription factors are involved in many processes of vertebrate development. Several studies have reported that Myt1/NZF-2 has a regulatory function in the development of cultured oligodendrocyte progenitors or in neuronal differentiation during Xenopus primary neurogenesis. However, little is known about the proper function of Myt/NZF family proteins during mammalian nervous system development. To assess the possible function of Myt/NZF transcription factors in mammalian neuronal differentiation, we determined the comparative spatial and temporal expression patterns of all three types of Myt/NZF family genes in the embryonic mouse nervous system using quantitative reverse transcriptase polymerase chain reaction and in situ hybridization. Results: All three Myt/NZF family genes were extensively expressed in developing mouse nervous tissues, and their expression was transient. NZF-1 was expressed later in post-mitotic neurons. NZF-2 was initially expressed in neuronal cells a little earlier than NZF-3. NZF-3 was initially expressed in neuronal cells, just after proliferation was complete. Conclusion: These expression patterns suggest that the expression of NZF family genes is spatially and temporally regulated, and each Myt/NZF family gene may have a regulatory function in a specific phase during neuronal differentiation. Developmental Dynamics 243:588-600, 2014. (c) 2013 Wiley Periodicals, Inc.

Botrocetin is a heterodimer snake venom protein that induces von Willebrand factor (VWF)- and platelet glycoprotein Ib (GPIb)-dependent platelet agglutination in vitro. We have cloned cDNAs for a botrocetin-2 from a cDNA library of the venom gland of Bothrops jararaca having a high similarity with botrocetin subunits. Recombinant botrocetin-2, expressed in 293T cells, showed cofactor activity comparable to natural botrocetin. In a single subunit expression experiment, a dimer of the beta subunit was obtained, and it showed reduced, but apparent, platelet agglutination activity. Ala scanning mutagenesis showed that substitutions at Asp62, Asp70, Arg115, or Lys117 in the beta subunit reduced platelet agglutination activity. The 3D homology modeling of botrocetin-2 complexed with the VWF Al domain and GPIb alpha indicated that Asp62, Arg115, and Lys117 of the beta subunit are located near Arg218 and Asp222 of GPIb alpha, respectively, and that Asp beta 70 is in proximity to Gln1391 of the Al domain. Our results indicate that these charged amino acid residues in the beta subunit have a preferential role in the activity of botrocetin-2. Since it has been time-consuming and difficult to obtain homogeneous botrocetin from natural venom, recombinant botrocetin-2 has potential benefits for clinical and basic investigations into hemostasis and thrombosis as a standard reagent.

During mammalian central nervous system development, neural stem cells differentiate and then mature into various types of neurons. Myelin transcription factor (Myt)/neural zinc finger (NZF) family proteins were first identified as myelin proteolipid protein promoter binding factors and were shown to be involved in oligodendrocyte development. In this study, we found that Myt/NZF family molecules were expressed during neuronal differentiation in vivo and in vitro. Transient over-expression of Myt/NZF family genes could convert undifferentiated P19 cells into neurons without induction by retinoic acid (RA), and the ability of these genes to induce neuronal differentiation was comparable to that of Neurog1 and Neurod1. Additionally, we found that St18 (or NZF-3) was induced by several bHLH transcription factors. When NZF-3 and Neurog1 were co-expressed in P19 cells, the rate of neuronal differentiation was significantly increased. These data suggest not only that NZF-3 works downstream of Neurog1 but also that it plays a crucial role together with Neurog1 in neuronal differentiation. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

NZF-2 (MyT1) is a member of C2HC-type zinc finger transcription factors. A novel form of mouse NZF-2 has been isolated. This novel form, NZF-2b, has an additional C2HC-type zinc finger motif. The expression levels of NZF-2b are by far the more predominant than those of the already known form of NZF-2. In embryonic mouse nervous system, the expression of NZF-2b starts as early as at 9.5 days post-coitum (dpc) in newly differentiated neurons in the central nervous system (CNS) and the peripheral nervous system (PNS). (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Vomeroglandin, a subform of mouse CRP-ductin, is a protein strongly expressed in the glands of mouse vomeronasal system. Both the proteins contain several of scavenger receptor cysteine-rich and CUB domains and one ZP domain. This domain arrangement is similar to those of rat Ebnerin, human DMBT1, and rabbit hensin, In situ hybridization analysis shows strong expression of vomeroglandin mRNA in the glands of vomeronasal system. Immunological analyses detect both membrane-bound and secreted forms of vomeroglandin, The secreted protein seems to be localized in the lumen of the vomeronasal organ, playing a certain role in the pheromone perception. (C) 2000 Academic Press.

We report the molecular cloning and characterization of two secretory proteins specifically expressed in vomeronasal and posterior glands of the nasal septum, the ducts of which open into the lumen of the vomeronasal organ. These two proteins are members of the lipocalin superfamily, consisting of hydrophobic ligand carriers. We immunohistochemically localized one of the proteins in the mucus covering the vomeronasal sensory epithelium, where the primary reception of pheromone takes place. The immunoreactivity on the vomeronasal sensory epithelium was evident in the neonatal and post-pubertal periods, when the close contact between animals plays critical roles in suckling and sexual behaviors, respectively. These results suggest that small lipophilic molecules stimulate the accessory olfactory system to regulate the reproductive behavior of mice.

Alcohol dehydrogenase (ADH), a bacterial membrane-bound protein containing pyrroloquinoline quinone (PQQ) and heme c was held by adsorption on electrodes of gold silver, glassy carbon, or pyrolytic graphite. All the electrodes with adsorbed ADH produced anodic currents which oxidized ethanol, in which the adsorbed ADH catalyzed the electrolysis of ethanol. The electrocatalysis behavior could be described by a theoretical equation for bioelectrocatalysis at an enzyme-coated electrode, and was characterized by two quantities, the Michaelis constant K(m), and maximum current density I(max)/A. Using electroreflectance measurements with an ADH-coated gold electrode it was revealed that electron transfer occurred between heme c of the adsorbed ADH and the electrode. On the basis of these results, the reaction mechanism of the bioelectrocatalysis is discussed and oriented adsorption of ADH is proposed with the heme c moiety being in close contact with the electrode and with the PQQ moiety, the site reacting with the substrate, facing toward the solution.

D-Gluconate dehydrogenase, a flavohemoprotein, and alcohol dehydrogenase, a quinohemoprotein, are absorbed strongly on carbon and metal electrodes. All the electrodes with the adsorbed enzymes produce anodic currents due to the electro-enzymatic oxidation of the substrates, where the adsorbed enzymes donate electrons directly to the electrodes; neither mediators nor promoters are necessary.

n-Alkanethiols (CH3(CH2)(n)SH, n=3,5,11,13,15,17) have been adsorbed at mercury electrodes prom ethanolic solutions. A. c. voltammetry has been carried out of the adsorbed alkanethiol on mercury electrodes in aqueous solutions containing 1 M KCl at 25 degrees C. Cyclic d.c. voltammetry of several metal ions also has been performed at these electrodes. The results show that long-chain alkanethiols n >= 11 can form densely packed monolayers with fully extended alkyl chains in the potentia,1 range between 0 and -0.7 volt vs. Ag/AgCl. Metal ions such as Pb2+ are unable to penetrate into the monolayer. In contrast, short-chain alkanethiols with n = 3 and 5 form less ordered and loosely packed adsorption layers, which are permeable to the metal ions. A pH dependent a.c. voltammetric peak is observed at a mercury electrode with adsorbed alkanethiol(n=5) in buffer solutions, which is attributable to the redox reaction of the adsorbed thiol: [CH3(CH2)(5)S](ad)Hg + H+ + e(-) reversible arrow [CH3(CH2)SH](ad)Hg.

An enzyme-modified electrode is constructed by immobilizing an oxidoreductase behind a dialysis membrane on a carbon paste electrode containing a redox compound. The redox compound functions as an electron transfer mediator between the electrode and the immobilized enzyme, thus the electrode can oxidize or reduce the substrate electro-enzymatically. Many oxidoreductases, especially dehydrogenases which use artificial electron acceptors are the enzymes suitable for constructing the enzyme-modified electrodes. They can be used as mediated amperometric biosensors which are insensitive to oxygen tension in test solutions. Examples of the mediated enzyme electrodes are given and characteristics of the current response of the electrodes to the substrates are described. Use of the mediated enzyme electrodes with high sensitivity are also described. Enzyme-modified electrodes based on oxidoreductases from bacterial membranes can oxidize the substrates electro-enzymatically in the absence of mediators. Such electrodes can be used as mediatorless enzyme electrodes.

Fructose dehydrogenase (EC 1.1.99.11) from bacterial membranes was immobilized on a carbon paste electrode by covering the enzyme layer with a dialysis membrane. The fructose dehydrogenase-modified carbon paste electrode showed a current response to D-fructose without the addition of any external electron transfer mediators. The current response was independent of the oxygen concentration in the solution. Steady-state currents were obtained when measured at fixed electrode potentials. The dependence of the steady-state current on the potential, the pH of the solution and the temperature was studied. On the basis of this investigation, it was shown that the fructose dehydrogenase-modified carbon paste electrode could be used as an unmediated amperometric fructose sensor. D-Fructose in fruits was measured using the present electrode. A method of eliminating the effect of L-ascorbic acid is also described.

D-Fructose dehydrogenase was immobilized behind a dialysis membrane on a carbon paste electrode containing p-benzoquinone. The electrode showed a current response to D-fructose due to the p-benzoquinone-mediated bioelectrocatalytic oxidation of D-fructose. The dependence of the current response on the potential applied to the electrode, pH of the solution, and temperature was studied. On this basis, measurements of D-fructose were done at 0.5 V vs. Ag/AgCl, pH 4.5 and 25°C using the fructose dehydrogenase-modified carbon paste electrode. D-Fructose in fruits was measured by this electrode method, in which the interference from L-ascorbic acid was taken into account. A method of elimination of the interference, which uses ascorbate oxidase, is also described. © 1990 by the Japan Society for Bioscience, Biotechnology, and Agrochemistry.