大島 信子

Four kinds of avian-derived H5N1 influenza virus, A/Vietnam/1194/2004 (Clade 1), A/Indonesia/5/2005 (Clade 2.1), A/Qinghai/1A/2005 (Clade 2.2), and A/Anhui/1/2005 (Clade 2.3), have been stocked in Japan for use as pre-pandemic vaccines. When a pandemic occurs, these viruses would be used as vaccines in the hope of inducing immunity against the pandemic virus. We analyzed the specificity of antibodies (Abs) produced by B lymphocytes present in the blood after immunization with these vaccines. Eighteen volunteers took part in this project. After libraries of Ab-encoding sequences were constructed using blood from subjects vaccinated with these viruses, a large number of clones that encoded Abs that bound to the virus particles used as vaccines were isolated. These clones were classified into two groups according to the hemagglutination inhibition (HI) activity of the encoded Abs. While two-thirds of the clones were HI positive, the encoded Abs exhibited only restricted strain specificity. On the other hand, half of the HI-negative clones encoded Abs that bound not only to the H5N1 virus but also to the H1N1 virus; with a few exceptions, these Abs appeared to be encoded by memory B cells present before vaccination. The HI-negative clones included those encoding broadly cross-reactive Abs, some of which were encoded by non-VH1-69 germline genes. However, although this work shows that various kinds of anti-H5N1 Abs are encoded by volunteers vaccinated with pre-pandemic vaccines, broad cross-reactivity was seen only in a minority of clones, raising concern regarding the utility of these H5N1 vaccine viruses for the prevention of H5N1 pandemics.

Neutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses.

Influenza viruses present a significant health challenge each year, as in the H3N2 epidemic of 2012-2013. Here we describe an antibody, F045-092, that possesses broadly neutralizing activity against the entire H3 subtype and accommodates the natural variation and additional glycosylation in all strains tested from 1963 to 2011. Crystal structures of F045-092 in complex with HAs from 1975 and 2011 H3N2 viruses reveal the structural basis for its neutralization breadth through insertion of its 23-residue HCDR3 into the receptor-binding site that involves striking receptor mimicry. F045-092 extends its recognition to divergent subtypes, including H1, H2 and H13, using the enhanced avidity of its IgG to overcome lower-affinity Fab binding, as observed with other antibodies that target the receptor-binding site. This unprecedented level of antibody cross-reactivity against the H3 subtype can potentially inform on development of a pan-H3 vaccine or small-molecule therapeutics.

Many people have a history of catching the flu several times during childhood but no additional flu in adulthood, even without vaccination. We analyzed the total repertoire of antibodies (Abs) against influenza A group 1 viruses induced in such a flu-resistant person after vaccination with 2009 H1N1 pandemic influenza virus. They were classified into two types, with no exceptions. The first type, the products of B cells newly induced through vaccination, binds near the sialic acid-binding pocket. The second type, the products of long-lived memory B cells established before vaccination, utilizes the 1-69 V-H gene, binds to the stem of HA, and neutralizes both H1N1 and H5N1 viruses with few exceptions. These observations indicate that the sialic acid-binding pocket and its surrounding region are immunogenically very potent and majority of the B cells whose growth is newly induced by vaccination produce Abs that recognize these regions. However, they play a role in protection against influenza virus infection for a short period since variant viruses that have acquired resistance to these Abs become dominant. On the other hand, although the stem of HA is immunogenically not potent, the second type of B cells eventually becomes dominant. Thus, a selection system should function in forming the repertoire of long-lived memory B cells and the stability of the epitope would greatly affect the fate of the memory cells. Acquisition of the ability to produce Abs that bind to the stable epitope could be a major factor of flu resistance.

Influenza A viruses are classified into 16 subtypes according to the serotypes of hemagglutinin (HA). It is generally thought that neutralizing antibodies (Abs) are not broadly cross-reactive among HA subtypes. We examined the repertoire of neutralizing Abs against influenza viruses in humans. B lymphocytes were collected from donors by apheresis, and Ab libraries were constructed by using phage-display technology. Anti-HA clones were isolated by screening with H3N2 viruses. Their binding activity was examined, and four kinds of Abs showing broad strain specificity were identified from one donor. Two of the Abs, F045-092 and F026-427, were extensively analyzed. They neutralized not only H3N2 but also H1N1, H2N2, and H5N1 viruses, although the activities were largely varied. Flow cytometry suggested that they have the ability to bind to HA and HA1 artificially expressed on the cell surface. They show hemagglutination inhibition activity and do not compete with C179, an Ab thought to bind to the stalk region. F045-092 competes with Abs that recognize sites A and B for binding to HA. Furthermore, the serine at residue 136 in site A could be a part of the epitope. Thus, it is likely that F045-092 and F026-427 bind to a conserved epitope in the head region formed by HA1. Interestingly, while the V(H)1-69 gene can encode MAbs against the HA stem that are group 1 specific, F045-092 and its relatives that recognize the head region also use V(H)1-69. The possible epitope recognized by these clones is discussed.

Through extensive isolation of neutralizing mAbs against H3N2 influenza viruses representing the in vivo repertoire in a human donor, we examined the relationships between antigenic drift of influenza virus and protective antibodies generated in an infected individual. The majority of mAbs isolated from a donor born in 1960 were divided into three major groups with distinct strain specificity: 1968-1973, 1977-1993 and 1997-2003. In the present study, we developed a new method that allowed us to comprehensively determine the location of epitopes recognized by many mAbs. Original haemagglutinins (HAs) of several strains and chimaeric variants, in which one of the seven sites (A, B1, B2, C1, C2, D or E) was replaced by some other strain-derived sequence, were artificially expressed on the cell surface. The binding activity of mAbs to the HAs was examined by flow cytometry. By using this method, we determined the location of epitopes recognized by 98 different mAbs. Clones that neutralize the 1968-1973 strains bind to site B2/D, A or A/B1. While sites C, E and B were recognized by clones that neutralized the 1977-1993 strains, the majority of these clones bind to site C. Clones that neutralize the 1997-2003 strains bind to site B, A/B1, A/B2 or E/C2.

We tried to reveal the strain specificity of neutralizing mAbs against H3N2 influenza viruses in individuals. A large number of B lymphocytes of a pediatrician were collected by apheresis and two Ab libraries were constructed at 2004 and 2007 by using the phage-display technology. The libraries were screened against 12 different H3 strains Of flu isolated between 1968 and 2004. Large numbers of clones that bound to the Ags were isolated and mAbs that specifically bound to H3 strain viruses were selected. Their binding activity to the 12 strains and neutralizing activity were studied by ELISA and focus reduction test, respectively. Furthermore, the binding activity to hemagglutinin (HA) was examined by Western blot. The majority of clones showing the neutralizing activity turned out to be anti-HA mAbs and could be divided into three major groups showing distinct strain specificity: 1968-1973, 1977-1993 and 1997-2003. (C) 2009 Elsevier Inc. All rights reserved.

HMN-176 ((E)-4-([2-N-[4-methoxybenzenesulfonyl]amino]stilbazole]1-oxide) is an active metabolite of HMN-214 ((E)-4-{2-[2-(N-acetyl-N-[4-methoxybenzenesulfonyl]amino)stilbazole]1- -oxide), which has a potent antitumor activity in mouse xenograft models. In this study, we show that HMN-176 circumvents multidrug resistance in a K2 human ovarian cancer subline selected for Adriamycin resistance (K2/ARS). Upon treatment of K2/ARS cells with 3 mum HMN-176, the GI(50) of Adriamycin for the cells decreased by similar to50%. To explore the molecular mechanism of this effect, we assessed the expression of the multidrug resistance gene (MDR1), which is constitutive in K2/ARS cells, at both the protein and the mRNA level. Western and reverse transcription-PCR analysis revealed that the expression of MDR1 was significantly suppressed by treatment with HMN-176. Furthermore, when administered p.o., HMN-214 suppressed the expression of MDR1 mRNA in a mouse xenograft model implanted with KB-A.1, an Adriamycin-resistant cell line. Luciferase reporter fusion gene analysis demonstrated that HMN-176 inhibited the Y-box-dependent promoter activity of the MDR1 gene in a dose-dependent manner. Moreover, we show by electrophoretic mobility shift assay that HMN-176 inhibits the binding of NF-Y, which is thought to be an essential factor for the basal expression of MDR1, to its target Y-box consensus sequence in the MDR-1 promoter. Inhibition of MDR-1 expression was achieved with pharmacological concentrations of HMN-176, suggesting that HMN-176 may act by two different mechanisms-cytotoxicity and MDR1 downregulation-simultaneously. The data presented strongly suggest that the antitumor mechanism of HMN-176 (or its prodrug HMN-214 in vivo) is quite different from those of known antitumor agents.

A human homologue (hDREF/KIAA0785) of Drosophila DREF, a transcriptional regulatory factor required for expression of genes involved in DNA replication and cell proliferation, was identified by BLAST search. Amino acid sequences corresponding to three regions highly conserved between two Drosophila species also proved to be very similar in the hDREF/KIAA0785 polypeptide. A consensus binding sequence (5'-TGTCG(C/T)GA(C/T)A) for hDREF/KIAA0785, determined by the CASTing method, overlapped with that for the Drosophila DREF (5'-TGTCGATA). We found hDREF/KIAA0785 binding sequences in the promoter regions of human genes related to cell proliferation. Analyses using a specific antibody revealed that an hDREF/KIAA0785 binds to the promoter region of the histone H1 gene. Co-transfection experiments with an hDREF/KIAA0785-expressing plasmid and a histone H1 promoter-directed luciferase reporter plasmid in HeLa cells revealed possible activation of the histone H1 promoter. Immunohistochemical analysis demonstrated that hDREF/KIAA0785 is localized in the nuclei. Although the expression level of the factor was found to be low in serum-deprived human normal fibroblasts, the amount was increased by adding serum to cultures and reached a maximum during S phase. RNA interference experiments targeting hDREF/KIAA0785 resulted in inhibition of S phase entry and reduction of histone H1 mRNA in HeLa cells. These results suggest that expression of hDREF/KIAA0785 may have a role in regulation of human genes related to cell proliferation.

A DNA replication-related element (DRE)-binding factor (DREF) has been revealed to be an important transcription factor for activating promoters of cell proliferation and differentiation related genes. The amino acid sequences of DREF are conserved in evolutionary separate Drosophila species, Drosophila inelanogaster (Dm) and Drosophila virilis (Dv) in three regions. In the present study, evidence was obtained that there are several highly conserved regions in the 51 flanking region between the DmDREF and DvDREF genes. Band mobility shift assays using oligonucleotides corresponding to these conserved regions revealed that specific trans-acting factors can bind to at least three regions - 554 to - 543 (5'-TTTGTTCTTGCG), - 81 to - 70 (5-GCCCACGTGGCT) and + 225 to + 234 (5'-GCAATCAGTG). Using a transient luciferase expression assay, we demonstrated that the region - 554 to - 543 functions as a negative regulatory element for DmDREF promoter activity. while the regions - 77 to - 70 (5'-ACGTGGCT) and + 225 to + 236 (5-GCAATCAGTGTT) function as positive regulatory elements. In previous studies, we observed that expression of the homeodomain protein Zerknullt (Zen) represses PCNA Gene transcription. by reducing the DNA binding activity of DREF. Here we show Zen downregulates DREF gene promoter activity through action on the region between +241 and +254 (5'-AGAATACTCAACA). In addition, the DmDREF promoter contains five DREs. Using a double stranded RNA-mediated interference method, we generated evidence that expression of DmDREF could be auto-regulated by DREF through the third DRE located at + 211 to + 218. In living flies we obtained results consistent with those obtained in vitro and in cultured cells. The study thus indicates that DmDREF is effectively regulated via highly conserved regions between the DmDREF and DvDREF promoters, suggesting the existence of common regulatory factors, and that DmDREF can be positively regulated by itself via the third DRE located in its most highly conserved region. (C) 2003 Elsevier Science B.V. All rights reserved.

Drosophila melanogaster DNA replication-related element (DRE) factor (dDREF) is a transcriptional regulatory factor required for the expression of genes carrying the 5'-TATCGATA DRE. dDREF has been reported to bind to a sequence in the chromatin boundary element, and thus, dDREF may play a part in regulating insulator activity. To generate further insights into dDREF function, we carried out a Saccharomyces cerevisiae two-hybrid screening with DREF polypeptide as bait and identified Mi-2 as a DREF-interacting protein. Biochemical analyses revealed that the C-terminal region of Drosophila Mi-2 (dMi-2) specifically binds to the DNA-binding domain of dDREF. Electrophoretic mobility shift assays showed that dMi-2 thereby inhibits the DNA-binding activity of dDREF. Ectopic expression of dDREF and dMi-2 in eye imaginal discs resulted in severe and mild rough-eye phenotypes, respectively, whereas flies simultaneously expressing both proteins exhibited almost-normal eye phenotypes. Half-dose reduction of the dMi-2 gene enhanced the DREF-induced rough-eye phenotype. Immunostaining of polytene chromosomes of salivary glands showed that dDREF and dMi-2 bind in mutually exclusive ways. These lines of evidence define a novel function of dMi-2 in the negative regulation of dDREF by its DNA-binding activity. Finally, we postulated that dDREF and dMi-2 may demonstrate reciprocal regulation of their functions.

The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory element DRE (5'-TATCGATA) in addition to E2F recognition sites. A specific DRE-binding factor, DREF, positively regulates DRE-containing genes. In addition, it has been reported that DREF can bind to a sequence in the hsp70 scs' chromatin boundary element that is also recognized by boundary element-associated factor, and thus DREF may participate in regulating insulator activity. To examine DREF function in vivo, we established transgenic flies in which ectopic expression of DREF was targeted to the eye imaginal discs. Adult flies expressing DREF exhibited a severe rough eye phenotype. Expression of DREF induced ectopic DNA synthesis in the cells behind the morphogenetic furrow, which are normally postmitotic, and abolished photoreceptor specifications of R1, R6, and R7. Furthermore, DREF expression caused apoptosis in the imaginal disc cells in the region where commitment to R1/R6 cells takes place, suggesting that failure of differentiation of R1/R6 photoreceptor cells might cause apoptosis. The DREF-induced rough eye phenotype was suppressed by a half-dose reduction of the E2F gene, one of the genes regulated by DREF, indicating that the DREF overexpression phenotype is useful to screen for modifiers of DREF activity. Among Polycomb/trithorax group genes, we found that a half-dose reduction of some of the trithorax group genes involved in determining chromatin structure or chromatin remodeling (brahma, moira, and osa) significantly suppressed and that reduction of Distal-less enhanced the DREF-induced rough eye phenotype. The results suggest a possibility that DREF activity might be regulated by protein complexes that play a role in modulating chromatin structure. Genetic crosses of transgenic flies expressing DREF to a collection of Drosophila deficiency stocks allowed us to identify several genomic regions, deletions of which caused enhancement or suppression of the DREF-induced rough eye phenotype. These deletions should be useful to identify novel targets of DREF and its positive or negative regulators.

A rapid and convenient new method for isolating the genes encoding cellular drug-binding proteins is described. This method, drug-western, is based on the use of the drug conjugated with a marker molecule as a probe for the screening of a cDNA library. Unlike the other methods, this method allows us to identify the genes for trace amounts of cellular drug-binding proteins without purification. We have used this approach to isolate human cDNA clones encoding binding proteins of HMN-154 ((E)-4-[2-[2-(p-methoxy-benzene-sulfonamide) phenyl]ethenyl] pyridine), a novel benzenesulfonamide anticancer compound (Katoh and Hidaka, 1997). The proteins encoded by two of the isolated clones are identical to NF-YB, B subunit of nuclear transcription factor NF-Y, and thymosin beta-10, respectively. Recombinants of both proteins bind specifically to HMN-154 in vitro. Comparison of amino acid sequences between these proteins shows the sequence similarity in a short amino acid stretch [K(X)AKXXK]. Deletion or mutation of this region causes the significant loss of binding of both proteins to HMN-154. Furthermore, HMN-154 inhibits DNA binding of NF-Y to the human major histocompatibility complex class II human leukocyte antigen DRA Y-box sequence in a dose-dependent manner. Interestingly, other binding proteins identified by this method also possess the same or a similar motif. These results clearly demonstrate that NF-YB and thymosin beta-10 are specific cellular binding proteins of HMN-154 and that this shared region is necessary for the binding to HMN-154. Hence, this new method is thought to be useful for the identification of drug-binding proteins.

The structure of a new niacin metabolite in cultured tobacco cells grown in a high concentration of niacin (1 mM) was confirmed to be 1-O-nicotinoyl-beta-D-glucopyranose. This glucoside is thought to be one of the detoxification forms of niacin in plants. Copyright (C) 1997 Elsevier Science Ltd.

Relative activities of pyridine nucleotides themselves to nicotinic acid as niacin nutriture were investigated using Lactobacillus plantarum ATCC 8014. This organism is generally used as a test organism for measuring the niacin contents in foods, because this organism lacks the de novo NAD biosynthetic pathway, therefore, nicotinic acid or nicotinamide is required for growth. Nicotinic acid mononucleotide and nicotinic acid adenine dinucleotide had little niacin activity, while nicotinamide mononucleotide, NAD(+), and NADP(+) had a little niacin activity and the relative activity of the respective compound to nicotinic acid as niacin nutriture was 1/14, 1/5, and 1/33.

A major metabolite of niacin in an extract from cultured tobacco cells when cultured with niacin at 1 mM was analyzed by various spectroscopic and chromatographic methods, and the structure was assigned as N-(beta-D-glucopyranosyl)nicotinic acid. It may be a detoxified form of excess niacin in cultured tobacco cells.

The relative activity of niacin-related compounds to nicotinic acid was tested as the niacin nutriture in Lactobacillus plantarum ATCC 8014, the strain for the measurement of niacin in foods. As the results, this microorganism could use nicotinuric acid as equimolar as nicotinic acid. The relative niacin activities of N'-methylnicotinamide, pyridine 3-aldehyde, and nicotinamide N-oxide to nicotinic acid were 1/20, 1/30 and 1/40 in molar basis, respectively. While pyridine 3-methanol, nicotinic acid N-oxide, 3-acetylpyridine, β-picoline, N^1-methylnicotinamide, N^1-methylnicotinic acid, 6-hydroxynicotinic acid, 6-aminonicotinamide, quinolinic acid, and cinchomeronic acid did not show niacin activity.