Synthesis of naphthalene-fused imidazo[1,2-a]pyridinium salts showing green luminescence with high quantum yields and large Stokes shift

We have synthesized naphthalene-fused imidazo[1,2-a]pyridines by a novel electrophilic iodocyclization of 2-alkynyl-3-arylimidazo[1,2-a]pyridines. Notably, their quaternized salts exhibited green emission with unexpectedly high quantum yields and mega-large Stokes shift and were successfully used for mitochondrial imaging.


Interactive Analysis of UTX-114 Family With EGFR-tyk: Molecular Features of Acetyl Glycosylated Gefitinib.

Acetyl glucose adducts (UTX-114, -115, and -116) were prepared from gefitinib, and their characteristics (e.g., anticancer activity, structural property) were analyzed. Cytotoxicity and radiosensitizing properties of the UTX-114 family were examined using A431 cells. Supramolecular associations between the UTX-114 family compounds and the tyrosine kinase domain of epidermal growth factor receptor (EGFR-tyk) were also examined. The interactive analyses of the UTX-114 family compounds with EGFR-tyk were performed using docking simulation technique. The UTX-114 family showed a similar cytotoxicity as gefitinib, yielding IC values of 31.2 μM (gefitinib), 34.3 μM (UTX-114), 36.8 μM (UTX-115), and 39.4 μM (UTX-116). The EGFR-tyk inhibition ratios (IR) of UTX-114, -115, and -116 to gefitinib were 1.515, 0.983, and 0.551, respectively. The EGFR-tyk inhibitory activity of UTX-114 was higher than that of gefitinib. UTX-114 also showed the highest radiosensitizing activity among the tested compounds. UTX-114 expressed 1841 conformers (-8.989~15.718 kcal/mol) with the solvation free energy (dGW) of UTX-114 decreasing with increasing conformational energy, ranging between -354.955~ -260.815 kJ/mol. Interactive energies of gefitinib, UTX-114, -115, and -116 with EGFR-tyk were -123.640, -144.053, -120.830, and -124.658 kcal/mol, respectively. UTX-114 yielded the lowest interaction energy with EGFR-tyk among tested compounds. Given the association behavior between UTX-114 and EGFR-tyk, along with its other observed properties, UTX-114 appears to be a viable therapeutic possibility.


Blue Luminescent Boron Complexes Based on N,N-Type Imidazo[1,5-a]pyridine Ligand for Mitochondrial Imaging


Streptolysin S induces pronounced calcium-ion influx-dependent expression of immediate early genes encoding transcription factors

Anginosus group streptococci (AGS) are opportunistic human pathogens of the oral cavity. The β-hemolytic subgroup of Streptococcus anginosus subsp. anginosus secretes streptolysin S (SLS) and exhibits not only hemolytic activity but also cytotoxicity toward cultured human cell lines. However, the detailed mechanism of action of SLS and the cellular responses of host cells have not yet been fully clarified. To determine the pathogenic potential of SLS-producing β-hemolytic S. anginosus subsp. anginosus, the SLS-dependent response induced in the human oral squamous cell carcinoma HSC-2 cells was investigated to determine the pathogenic potential of SLS-producing β-hemolytic S. anginosus subsp. anginosus. This study revealed that the Ca influx and the expression of immediate early genes (IEGs) encoding transcription factors such as early growth responses (EGRs) and activator protein-1 (AP-1) were greatly increased in HSC-2 cells incubated with the culture supernatant of SLS-producing β-hemolytic S. anginosus subsp. anginosus. Moreover, this SLS-dependent increase in expression was significantly suppressed by Ca chelation, except for jun. These results suggest that SLS caused Ca influx into the cells following greatly enhanced expression of IEG-encoding transcription factors. The results of this study may help in understanding the pathogenicity of SLS-producing AGS.


A simple method to differentiate three classes of cholesterol-dependent cytolysins.

Cholesterol-dependent cytolysins (CDCs) are proteinaceous toxins widely distributed in gram-positive pathogenic bacteria. CDCs can be classified into three groups (I-III) based on the mode of receptor recognition. Group I CDCs recognize cholesterol as their receptor. Group II CDC specifically recognizes human CD59 as the primary receptor on the cell membrane. Only intermedilysin from Streptococcus intermedius has been reported as a group II CDC. Group III CDCs recognize both human CD59 and cholesterol as receptors. CD59 contains five disulfide bridges in its tertiary structure. Therefore, we treated human erythrocytes with dithiothreitol (DTT) to inactivate CD59 on membranes. Our data showed that DTT treatment caused a complete loss of recognition of intermedilysin and an anti-human CD59 monoclonal antibody. In contrast, this treatment did not affect the recognition of group I CDCs, judging from the fact that DTT-treated erythrocytes were lysed with the same efficiency as mock-treated human erythrocytes. The recognition of group III CDCs toward DTT-treated erythrocytes was partially reduced, and these results are likely due to the loss of human CD59 recognition. Therefore, the degree of human CD59 and cholesterol requirements of uncharacterized group III CDCs frequently found in Mitis group streptococci can be easily estimated by comparing the amounts of hemolysis between DTT-treated and mock-treated erythrocytes.


Human serum albumin stabilizes streptolysin S activity secreted in the extracellular milieu by streptolysin S-producing streptococci.

Anginosus group streptococci (AGS) are opportunistic pathogens of the human oral cavity; however, their pathogenicity has not been discussed in detail. Oral streptococci live in the gingival sulcus, from where they can easily translocate into the bloodstream due to periodontal diseases and dental treatment and cause hazardous effects on the host through their virulence factors. Streptolysin S (SLS), a pathogenic factor produced by β-hemolytic species/strains belonging to AGS, plays an important role in damaging host cells. Therefore, we investigated the SLS-dependent cytotoxicity of β-hemolytic Streptococcus anginosus subsp. anginosus (SAA), focusing on different growth conditions such as in the bloodstream. Consequently, SLS-dependent hemolytic activity/cytotoxicity in the culture supernatant of β-hemolytic SAA was stabilized by blood components, particularly human serum albumin (HSA). The present study suggests that the secreted SLS, not only from β-hemolytic SAA, but also from other SLS-producing streptococci, is stabilized by HSA. As HSA is the most abundant protein in human plasma, the results of this study provide new insights into the risk of SLS-producing streptococci which can translocate into the bloodstream.


Dual functions of discoidinolysin, a cholesterol-dependent cytolysin with N-terminal discoidin domain produced from strain Nm-76.

Some strains of exhibit β-hemolysis due to the β-hemolytic activity of cholesterol-dependent cytolysin (CDC). Recently, a gene encoding an atypical lectinolysin-related CDC was found in strain Nm-76. However, the product of this gene remains uncharacterized. We aimed to characterize this atypical CDC and its molecular functions and contribution to the pathogenicity of strain Nm-76. Phylogenetic analysis of the CDC gene was conducted based on the web-deposited information. The molecular characteristics of CDC were investigated using a gene-deletion mutant strain and recombinant proteins expressed in . The gene encoding CDC found in Nm-76 and its homolog are distributed among many strains. This CDC is phylogenetically different from other previously characterized CDCs, such as -derived human platelet aggregation factor (Sm-hPAF)/lectinolysin and mitilysin. Because this CDC possesses an additional N-terminal domain, including a discoidin motif, it was termed discoidinolysin (DLY). In addition to the preferential lysis of human cells, DLY displayed N-terminal domain-dependent facilitation of human erythrocyte aggregation and intercellular associations between human cells. DLY functions as a hemolysin/cytolysin and erythrocyte aggregation/intercellular association molecule. This dual-function DLY could be an additional virulence factor in .


Molecular Interaction Between Boron Tracedrug UTX-51 Derivatives and Bovine Serum Albumin: Application to an Analytical Model of AGEs Destruction by Thermal Neutron Irradiation

Boron tracedrugs possess global molecular tracking abilities and localized destructive power. We investigated the molecular properties of synthesized boron tracedrugs, including UTX-51, and their interactions with the advanced glycation end-product (AGE)-related protein bovine serum albumin (BSA). A conformational analysis of the compounds used in the present study was performed using CAChe (Fujitsu Inc., Tokyo, Japan) and the degree of stereo-hydrophobicity of the conformers obtained was verified using Mopac (Fujitsu Inc.). The interactive properties of global minimum conformers of the derivatives tested with BSA were assessed using Molegro Virtual Docker (CLC bio., Aarhus, Denmark). Among the compounds investigated, UTX-51 was confirmed to interact with BSA based on the formation of hydrogen bonds between BSA and UTX-51. UTX-51 is a promising boron tracedrug and can be used as the lead structure for developing a therapeutic agent for AGE-related diseases, including cancer.


Charge-independent protein adsorption characteristics of epitaxial graphene field-effect transistor on SiC substrate


Synthesis of D-π-A type benzothiazolepyridinium salt composite and its application as photo-degradation agent for amyloid fibrils

We have synthesized a cyan fluorescent benzothiazole-pyridinium salt composite based on D-p-A architecture. This salt was found to work as not only a two- and three-photon excitable fluorophore but also a degradation agent against amyloid fibrils under LED irradiation conditions.

Bioorganic & Medicinal Chemistry Letters


Construction of a Drug Release Evaluation System: Application of Mitochondrial Respiration to Monitor Drug Release

Efficient drug encapsulation and regulation of drug release are important factors for sustained drug release and application for release-controlled anti-cancer and anti-inflammatory drug delivery. In the present study, a direct evaluation system for drug-release from model carrier (e.g., alginate-gel beads) was examined using the mitochondrial oxygen consumption rate as an index. Alginate-gel beads were coated with the uncoupler SF6847 (SF beads) and used as a model microparticle-type drug. The real-time monitoring of SF6847 release from prepared alginate-gel beads was performed using the mitochondrial oxygen consumption rate. Release profiles of nonsteroidal anti-inflammatory drugs [NSAIDs, mefenamic acid (MEF) and diclofenac (DIC)] from alginate-gel beads as well as SF beads were investigated using the real time monitoring system. SF6847 release from alginate-gel beads was clearly detected using the rat liver mitochondrial oxygen consumption rate. The release features of MEF and DIC from alginate-gel beads were defined by the present trial monitoring system, and these NSAIDs exhibited different release profiles. The present drug monitoring system detected released drugs, and the release profile reflected the molecular properties of the test drugs. This system may be applied to the design and development of precise sustained drug release systems (e.g., anti-cancer and anti-inflammatory drugs).


Molecular characteristics of an adhesion molecule containing cholesterol-dependent cytolysin-motif produced by mitis group streptococci

Streptococcus pseudopneumoniae (SPpn) is a relatively new species closely related to S. pneumoniae (SPn) and S. mitis (SM) belonging to the Mitis group of the genus Streptococcus (MGS). Although genes encoding various pneumococcal virulence factors have been observed in the SPpn genome, the pathogenicity of SPpn against human, including the roles of virulence factor candidates, is still unclear. The present study focused on and characterized a candidate virulence factor previously reported in SPpn with deduced multiple functional domains, such as lipase domain, two lectin domains, and cholesterol-dependent cytolysin-related domain using various recombinant proteins. The gene was found not only in SPpn but also in the strains of SM and SPn. Moreover, the gene product was expressed in the gene-positive strains as secreted and cell-bound forms. The recombinant of gene product showed lipase activity and human cell-binding activity depending on the function of lectin domain(s), but no hemolytic activity. Thus, based on the distribution of the gene within the MGS and its molecular function, the gene product was named mitilectin (MLC) and its contribution to the potential pathogenicity of the MLC-producing strains was investigated. Consequently, the treatment with anti-MLC antibody and the mlc gene-knockout significantly reduced the human cell-binding activity of MLC-producing strains. Therefore, the multifunctional MLC was suggested to be important as an adhesion molecule in considering the potential pathogenicity of the MLC-producing strains belonging to MGS, such as SPpn and SM.


Complete Genome Sequence of Streptococcus mitis Strain Nm-65, Isolated from a Patient with Kawasaki Disease

Nm-65 is a human commensal streptococcal strain of the mitis group that was isolated from the tooth surface of a patient with Kawasaki disease. The complete genome sequence of Nm-65 was obtained by means of hybrid assembly, using two next-generation sequencing data sets. The final assembly size was 2,085,837 bp, with 2,039 coding sequences.


Synthesis and Optical Property of Quadrupolar Pyridinium Salt and Its Application as Bioimaging Agent


Two- and three-photon excitable quaternized imidazo[1,2-a]pyridines for mitochondrial imaging and potent cancer therapy agent


Effect of Isomerization of TX-2036 Derivatives on the Interaction With Tyrosine Kinase Domain of EGF Receptor

From the design and synthesis of enantiomers, we can expect to obtain two compounds with different pharmacokinetics and pharmacological activities at the same time, which is thought to lead to the development of efficient anticancer agents. Chiral-2-nitroimidazole TX-2036 derivatives exhibit stereo-configuration (R- and S-configuration)-dependent tyrosine kinase inhibitory activity, and the activity of the tyrosine kinase domain of EGF receptor (EGFR-tyk) is suppressed. In order to clarify the reason why the effects on EGFR-tyk activity differ depending on stereoisomers, we tried to analyze the interaction between each TX-2036 derivative and EGFR-tyk. The 2-nitroimidazole-based radiosensitizer TX-2036 series were synthesized and their molecular features were examined using protein kinase inhibition assay and molecular structural analysis. R-configured TXs (TX-2043, -2030, and -2036) exhibited more potent protein kinase inhibitory activity than S-configured TXs (TX-2044, - 2031, and -2037), and the IC value of TX-2036 was 1.8 μM. R-configured TXs interacted with Lys and Thr of EGFR-tyk. The combinations of amino acid residues targeted by the S-configured TXs were different from each other (Ile and Thr (TX-2044), Ser, Thr, and Thr (TX-2031), Gly, Cys, and Thr (TX-2037)). Preparing a series of isomers with different target sites was considered beneficial when the target was mutated.


Transcriptional regulator SpxA1a controls the resistance of the honey bee pathogen Melissococcus plutonius to the antimicrobial activity of royal jelly

Royal jelly (RJ), a brood food of honey bees, has strong antimicrobial activity. Melissococcus plutonius, the causative agent of European foulbrood of honey bees, exhibits resistance to this antimicrobial activity and infects larvae orally. Among three genetically distinct groups (CC3, CC12 and CC13) of M. plutonius, CC3 strains exhibit the strongest RJ resistance. In this study, to identify genes involved in RJ resistance, we generated an RJ-susceptible derivative from a highly RJ-resistant CC3 strain by UV mutagenesis. Genome sequence analysis of the derivative revealed the presence of a frameshift mutation in the putative regulator gene spxA1a. The deletion of spxA1a from a CC3 strain resulted in increased susceptibility to RJ and its antimicrobial component 10-hydroxy-2-decenoic acid. Moreover, the mutant became susceptible to low-pH and oxidative stress, which may be encountered in brood foods. Differentially expressed gene analysis using wild-type and spxA1a mutants revealed that 45 protein-coding genes were commonly upregulated in spxA1a-positive strains. Many upregulated genes were located in a prophage region, and some highly upregulated genes were annotated as universal/general stress proteins, oxidoreductase/reductase, chaperons and superoxide dismutase. These results suggest that SpxA1a is a key regulator to control the tolerance status of M. plutonius against stress in honey bee colonies.


Cytotoxic property of Streptococcus mitis strain producing two different types of cholesterol-dependent cytolysins


Change in membrane potential induced by streptolysin O, a pore-forming toxin: flow cytometric analysis using a voltage-sensitive fluorescent probe and rat thymic lymphocytes

Streptolysin O (SLO) is a bacterial pore-forming toxin that is employed to permeabilize cell membranes in some biological experiments. SLO forms various types of pores with different shapes, increasing membrane ion permeability and subsequently inducing changes in membrane potential. To characterize the pores formed by SLO, the changes in membrane potential induced by SLO in rat lymphocytes were considered using flow cytometry with a voltage-sensitive fluorescent probe, bis-(1,3-dibutylbarbituric acid)trimethine oxonol (Oxonol). SLO caused three types of membrane potential responses accessed with Oxonol. One type induces a great decrease in Oxonol fluorescence (large hyperpolarization) that may be elicited via the increase of Ca -dependent K permeability by SLO-induced influx of external Ca . A second type is an increase in Oxonol fluorescence (depolarization) that may be caused by a nonspecific increase in membrane cation permeability. The third type is a small decrease in Oxonol fluorescence (small hyperpolarization), probably via an increase in Cl permeability. That SLO transitionally changes membrane ion permeability may have implications in the pathology of pyogenic group streptococci infections in which SLO is thought to be one of the key virulence factors.


Fluorescent Imidazo[1,5-a]pyridinium Salt for a Potential Cancer Therapy Agent


Correlation Between Radiosensitizing Activity and the Stereo-structure of the TX-2036 Series of Molecules

The stereo-configuration (R-, S-configuration) of chiral-2-nitroimidazole derivatives alters their radiosensitizing activity. This study aimed at examining the molecular features of these enantiomers by molecular simulation techniques. A series of 2-nitroimidazole-based radiosensitizer TX-2036 molecules were synthesized, and their profiles were examined using molecular structural analysis such as conformation analysis, molecular orbital analysis, and electrostatic potential analysis. R-configured TXs (TX-2043, -2030, -2036) had a weaker radiosensitizing activity than S-configured TXs (TX-2044, -2031, -2037), and R-compounds had a small minus electrostatic potential (ESP) field in the cyclopentene-1,3-dione region. S-configured TX-2046 had weaker radiosensitizing activity than R-configured TX-2045, and TX-2046 had a small minus ESP field as well as R-configured TX-2043, -2030, - 2036. The cyclopentene-1,3-dione involved in the small minus ESP field affected the radiosensitizing activity of the TX-2036 series of molecules.


β-Hemolytic Streptococcus anginosus subsp. anginosus causes streptolysin S-dependent cytotoxicity to human cell culture lines in vitro

Streptococcus anginosus subsp. (SAA) is one of the opportunistic pathogens in humans that inhabits the oral cavity. The type strain of SAA, NCTC10713, showed clear β-hemolysis on blood agar plates, and the sole β-hemolytic factor revealed two streptolysin S (SLS) molecules. SLS is well known as the peptide hemolysin produced from the human pathogen and shows not only hemolytic activity on erythrocytes but also cytotoxic activity in cell culture lines and , such as in a mouse infection model. However, no cytotoxic activity of SLS produced from β-hemolytic SAA (β-SAA) has been reported so far. In this study, the SLS-dependent cytotoxicity of the β-SAA strains including the genetically modified strains was investigated . The SLS-producing β-SAA showed cytotoxicity in human cell culture lines under the co-cultivation condition and it was found that this cytotoxicity was caused by the SLS secreted into the extracellular milieu. The results from this study suggest that the SLS produced from β-SAA might indicate the cytotoxic potential similar to that of the SLS from and the SLS-producing β-SAA would be recognized as "a wolf in sheep's clothing" More attention will be paid to the pathogenicity of β-hemolytic Anginosus group streptococci.


Recognizability of heterologous co-chaperones with Streptococcus intermedius DnaK and Escherichia coli DnaK

Streptococcus intermedius DnaK complements the temperature-sensitive phenotype of an Escherichia coli dnaK null mutant only when co-chaperones DnaJ and GrpE are co-expressed. Therefore, whether S. intermedius DnaK and E. coli DnaK can recognize heterologous co-chaperones in vitro was examined. Addition of heterologous GrpE to DnaK and DnaJ partially stimulated adenosine triphosphatase (ATPase) activity, and almost completely stimulated luciferase refolding activity. Addition of heterologous DnaJ to GrpE and DnaK also stimulated ATPase activity; however, significant luciferase refolding activity was not observed. Moreover, E. coli DnaJ had a negative effect on the luciferase refolding activity of the S. intermedius DnaK chaperone system. In E. coli chaperone mutants, with the exception of E. coli DnaJ, stronger expression of the heterologous co-chaperones partially or almost completely complemented the temperature-sensitive-phenotype. These results indicate that all heterologous co-chaperones can at least partially recognize DnaK of a distantly related species. A region of the ATPase domain that is present in the DnaK of gram-negative bacteria is absent from the DnaK of gram-positive bacteria. This region is believed to be important for recognition of co-chaperones from gram-negative bacteria. However, insertion of this segment into S. intermedius DnaK failed to increase its ability to recognize E. coli co-chaperones, implying that this region is unnecessary or insufficient for the recognition of E. coli co-chaperones. Thus, our data suggest that a basic structural similarity is conserved among the components of the S. intermedius and E. coli DnaK chaperone systems, allowing weak associations between heterologous components.


Fluorescent N-Heteroarenes Having Large Stokes Shift and Water Solubility Suitable for Bioimaging


Structure-associated Functional Control of TX-1877 Series by Glyco-conjugation

Sugar molecules are often used as a tool to structurally modify chemical compounds. The features of synthesized sugar-conjugated TX-1877 derivatives were herein examined. The molecular stabilities (reactivity) and hydrophobicities of sugar (e.g., monosaccharide and tetra-O-acetylated monosaccharide)-conjugated TXs were analyzed using a molecular simulation (e.g. molecular mechanics (MM) and molecular orbital (MO) analysis). The hydrophobicities of TX-1877 derivatives were increased by tetra-O-acetylation, and TX-2244 exhibited the most potent radiosensitizing activity (enhancement ratio: ER=2.30). The conformations and hydrophobicities of chemical compounds may be controlled by adding monosaccharide- and tetra-O-acetyl-conjugated sugars to TX-1877.


Rapid screening method for detecting highly pathogenic Streptococcus intermedius strains carrying a mutation in the lacR gene.

Streptococcus intermedius is a member of the normal human commensal flora and secretes a human-specific cytolysin intermedilysin (ILY) as a major virulence factor. Expression of ily is repressed by LacR and loss-of-function mutations of LacR are observed in many ILY high-producing strains isolated from deep-seated abscesses, suggesting that high ILY production is necessary for increased virulence. However, because ILY exhibits no β-hemolysis on animal blood agar plates, differentiating ILY high- and low-producing strains using conventional laboratory methods is not possible. Interestingly, S. intermedius also produces glycosidases, including MsgA and NanA, which exhibit N-acetyl-β-d-glucosaminidase and neuraminidase activities, respectively. Moreover, MsgA expression, but not NanA, is negatively regulated by LacR. Here we measured the activities of MsgA, NanA and ILY in strains isolated from clinical specimens and dental plaque to determine the correlation between these glycosidase activities and ILY hemolytic activity. Hemolytic activity showed a strong positive correlation with MsgA and a weak negative correlation with NanA activities. Therefore, we calculated the ratio of MsgA and NanA activity (M/N ratio). This value showed a stronger positive correlation (r = 0.81) with ILY hemolytic activity and many strains with high M/N ratios (>2) were ILY-high producers with loss-of-function mutations in LacR.


A novel plasmid, pSAA0430-08, from Streptococcus anginosus subsp. anginosus strain 0430-08

Mobile genetic elements (MGEs) are the genetic material often involved in the interspecies and intraspecies genetic transduction in bacteria. However, little is known about MGEs in the Anginosus group of streptococci (AGS), one of the streptococcal groups found in the oral cavity of humans. We looked for the presence of MGEs in Streptococcus anginosus subsp. anginosus (SAA), a representative species belonging to AGS, and found a novel plasmid from SAA strain 0430-08. This plasmid was 7038bp and ~31% G/C content which we named pSAA0430-08, and examined its genetic structure and characteristics. Open reading frame (ORF) prediction revealed that pSAA0430-08 was composed of 10 ORFs including a putative plasmid replication protein (ORF1) and a putative toxin-antitoxin system (ORF9 and ORF10). Between ORF10 and ORF 1, four tandem repeats of 22bp each, generally termed as iteron, were also observed. Using variant plasmids of pSAA0430-08, we confirmed that both ORF1 and iteron were necessary for replication in host cells. Interestingly, the region from ORF4 to ORF7 showed homology with a genomic DNA segment of S. gordonii strains. Thus, this plasmid may travel between the different species in Streptococci, i.e., S. gordonii and S. anginosus.


Biphenyl increases the intracellular Ca2+ concentration in HL-60 cells


Effects of the Nonsteroidal Anti-inflammatory Drug Celecoxib on Mitochondrial Function

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to treat inflammation and pain. In the present study, we examined the effects of celecoxib, a cyclooxygenase-2 (COX-2)-selective NSAID, on rat liver mitochondrial function. Celecoxib dose-dependently induced mitochondria swelling, which was not suppressed by cyclosporine A (CsA). The oxygen consumption rate in mitochondria-suspended solution was facilitated by the addition of celecoxib, and its uncoupling activity was observed. Celecoxib also suppressed SF6847-induced uncoupling, and appeared to exert inhibitory effects on the electron transport chain. Celecoxib suppressed the state 3 oxygen consumption rate in the presence of ADP. Protein release from the mitochondrial matrix was detected following the addition of celecoxib, and aldehyde dehydrogenase 2 (ALDH2) and hydroxymethylglutaryl-CoA (HMG-CoA) synthase 2 (HMGCS2) bands were confirmed in a Western blot analysis. On the other hand, protein release of cytochrome C (CytC), which is an inducer of apoptosis, from the intermembrane space was not observed. Celecoxib enhanced the membrane permeability of human erythrocytes and synthesized liposomes dose-dependently. It then induced the membrane-involving mitochondrial swelling and suppressed mitochondrial function.


Positive- and Negative-Control Pathways by Blood Components for Intermedilysin Production in Streptococcus intermedius.

is an opportunistic bacterial pathogen secreting a human-specific cytolysin called intermedilysin (ILY) as a major pathogenic factor. This bacterium can degrade glycans into monosaccharides using two glycosidases, multisubstrate glycosidase A (MsgA) and neuraminidase (NanA). Here, we detected a stronger hemolytic activity mediated by ILY when PC574 was cultured in fetal bovine serum (FBS) than when it was grown in the standard culture medium. FBS-cultured cells also showed higher MsgA and NanA activity, although overproduction of ILY in FBS was undetectable in mutants -null and -null. Addition of purified MsgA and NanA to the FBS resulted in a release of 2.8 mM galactose and 4.3 mM -acetylneuraminic acid; these sugar concentrations were sufficient to upregulate the expression of ILY, MsgA, and NanA. Conversely, when strain PC574 was cultured in human plasma, no similar increase in hemolytic activity was observed. Moreover, addition of human plasma to the culture in FBS appeared to inhibit the stimulatory effect of FBS on ILY, MsgA, and NanA, although there were individual differences among the plasma samples. We confirmed that human plasma contains immunoglobulins that can neutralize ILY, MsgA, and NanA activities. In addition, human plasma had a neutralizing effect on cytotoxicity of toward HepG2 cells in FBS, and a higher concentration of human plasma was necessary to reduce the cytotoxicity of an ILY-high-producing strain than an ILY-low-producing strain. Overall, our data show that blood contains factors that stimulate and inhibit ILY expression and activity, which may affect pathogenicity of .


Evaluation of the effectiveness of the potent bis-quaternary ammonium compound, 4,4'-(α,ω-hexametylenedithio) bis (1-octylpyridinium bromide) (4DTBP-6,8) on Pseudomonas aeruginosa

Quaternary ammonium compounds (QACs), including benzalkonium chloride (BAC) and cetylpyridinium chloride (CPC) are cationic surfactants and have been used widely as general disinfectants in the medical field due to their strong antibacterial effects and low cytotoxicity to human cells. 4,4'-(α,ω-hexametylenedithio) bis (1-octylpyridinium bromide) (4DTBP-6,8) is one of the potent bis-QACs synthesized to improve the antimicrobial activities of mono-QACs such as BAC. This study aimed to assess the effectiveness of 4DTBP-6,8 against Pseudomonas aeruginosa, a prevalent hospital pathogen. The minimum inhibitory concentrations of 4DTBP-6,8, CPC and BAC against P. aeruginosa were measured. 4DTBP-6,8 exhibited strong antibacterial activity. We assessed the bactericidal effects of QACs against P. aeruginosa under certain conditions and their cytotoxicities in human epithelial cells using lactate dehydrogenase (LDH) release. 4DTBP-6,8 exerted excellent bactericidal effects against high concentrations of bacteria, biofilm cells and even in the presence of contaminated proteins. Cellular LDH was not released by the treatment with 4DTBP-6,8. 4DTBP-6,8 exhibited the strongest bactericidal activity against P. aeruginosa among the three QACs tested without any cytotoxicity. The potent bis-QAC, 4DTBP-6,8 has the potential to be an effective disinfectant in preventing hospital infections caused by P. aeruginosa.


Cytolytic Activity and Molecular Feature of Cardiotoxin and Cardiotoxin-like Basic Protein: The Electrostatic Potential Field Is an Important Factor for Cell Lytic Activity

Cardiotoxin (CT) is a well-known cell lytic protein and has been purified from cobra venom. Cardiotoxin-like basic protein (CLBP) has two amino acid insertions and does not exhibit cell lytic activity. The molecular features of these CT family proteins were examined in the present study using molecular modeling and molecular simulation techniques. Molecular models of CT and CLBP were constructed based on the X-ray data of Naja mossambica mossambica CT VII4 (Protein Data Bank ID: 1CDT). The structural features of these models were examined using molecular orbital and electrostatic potential parameters. The stereo-hydrophobicities and molecular torsions of CT and CLBP, which are indexes of structural features, were similar. Electrostatic potential fields (ESP) differed between CT and CLBP and this was considered one of the critical factors in molecular titer. The distribution of ESP fields may affect the cytolytic activity of the CT family.


Development of a Sortase A-mediated Peptide-labeled Liposome Applicable to Drug-delivery Systems

In order to develop an efficient drug-delivery system (DDS), a lipopeptide-loaded liposome that functions as a platform for the transpeptidase reaction mediated by sortase A (SrtA) was constructed and its stability, as well as cell-specific targeting were evaluated in the present study. Several lipopeptides possessing an acceptor peptide sequence (oligoglycine ≥ three residues) or donor peptide sequence (LPETG) for the SrtA-mediated reaction were chemically synthesized and then inserted into the liposome membrane composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol (DPPC-Chol-lipo) to obtain the lipopeptide-loaded liposomes. The transpeptidase reaction mediated by recombinant SrtA (His-ΔN59SrtA) was employed to modify the peptide moiety on the liposomal surface using a fluorescently-labeled substrate peptide corresponding to the species of each loaded lipopeptide. Furthermore, lung tumor-binding peptide (LTBP)-labeled liposomes, prepared by this transpeptidase reaction, were investigated for selective targeting to lung cancer cells in vitro. The His-ΔN59SrtA-mediated transpeptidation of fluorescently-labeled peptide on the lipopeptide-loaded DPPC-Chol-lipo was confirmed. The selective targeting of LTBP-labeled liposomes to the lung cancer cell line A549 was also observed in vitro. These results suggest that the labeling of acceptor or donor lipopeptide-loaded liposomes with the transpeptidase SrtA could be a useful method for developing a platform applicable to a cancer-targeting DDS.


5-O-Acyl plumbagins inhibit DNA polymerase activity and suppress the inflammatory response

We previously found that vitamin K3 (menadione, 2-methyl-1,4-naphthoquinone) inhibits the activity of human mitochondrial DNA polymerase (pol) γ. In this study, we focused on plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), and chemically synthesized novel plumbagins conjugated with C2:0 to C22:6 fatty acids (5-O-acyl plumbagins). These chemically modified plumbagins displayed enhanced mammalian pol inhibition, with plumbagin conjugated to docosahexaenoic acid (C22:6-acyl plumbagin) exhibiting the strongest inhibition of pol λ among the ten 5-O-acyl plumbagins synthesized. C22:6-acyl plumbagin selectively inhibited the activities of mammalian pol species, but did not influence the activities of other pols or DNA metabolic enzymes tested. The inhibition of pol λ, a DNA repair/recombination pol, by these compounds was significantly correlated with both their suppression of lipopolysaccharide (LPS) induced tumor necrosis factor-α (TNF-α) production by mouse RAW264.7 macrophages and the reduction of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in the mouse ear. These data indicate that 5-O-acyl plumbagins act as anti-inflammatory agents by inhibiting mammalian pol λ. These results further suggest that C22:6-acyl plumbagin is a promising anti-inflammatory candidate and that acylation could be an effective chemical modification to improve the anti-inflammatory activity of vitamin K3 derivatives, such as plumbagin.


Estrogen stimuli promote osteoblastic differentiation via the subtilisin-like proprotein convertase PACE4 in MC3T3-E1 cells.

Estrogenic compounds include endogenous estrogens such as estradiol as well as soybean isoflavones, such as daidzein and its metabolite equol, which are known phytoestrogens that prevent osteoporosis in postmenopausal women. Indeed, mineralization of MC3T3-E1 cells, a murine osteoblastic cell line, was significantly decreased in medium containing fetal bovine serum treated with charcoal-dextran to deplete endogenous estrogens, but estradiol and these soybean isoflavones dose-dependently restored the differentiation of MC3T3-E1 cells; equal was tenfold more effective than daidzein. These differentiation promoting effects were inhibited by the addition of fulvestrant, which is a selective downregulator of estrogen receptors. Analysis of the expression pattern of bone-related genes by reverse transcription PCR (RT-PCR)/quantitative real-time PCR (qRT-PCR), which focused on responsiveness to the estrogen stimuli, revealed that the transcription of PACE4, a subtilisin-like proprotein convertase, was tightly linked with the differentiation of MC3T3-E1 cells induced by estrogen stimuli. Moreover, treatment with RNAi of PACE4 in MC3T3-E1 cells resulted in a drastic decrease of mineralization in the presence of estrogen stimuli. These results strongly suggest that PACE4 participates in bone formation at least in osteoblast differentiation, and estrogen receptor-mediated stimuli induce osteoblast differentiation through the upregulation of PACE4 expression.


Molecular Analysis of Streptococcus anginosus-derived SagA Peptides

SagA1 and SagA2 molecules produced from beta-hemolytic Streptococcus anginosus subsp. anginosus are composed of a leader peptide and a propeptide, and their mature form has hemolytic activity as a well-known Streptococcal peptide toxin, streptolysin. The function of these SagA molecules is thought to be dependent on intra-molecular heterocycle formation. In this study, we examined the heterocycle-involved molecular features of SagA1, SagA2, and S. pyogenes SagA (SPySagA), focusing on their heterocycle formation. Molecular models of SagA1, SagA2, and SPySagA were constructed using a molecular modeling technique. Molecular dynamics and molecular mechanic analyses of the modeled SagA molecules were performed to obtain their energy profiles. Total energy of the modeled SagA1, SagA2, and SPySagA decreased with heterocycle formation, and the border between the leader peptide and propeptide was clearly observed after heterocycle formation. The flexibility of SagA molecules was changed by intramolecular heterocycle formation, and their function (e.g. hemolytic activity) seems to be regulated by structural transition with heterocycle formation.


Investigation on the Reaction Conditions of Staphylococcus aureus Sortase A for Creating Surface-modified Liposomes as a Drug-delivery System Tool

This study aimed to determine the preferred conditions for the transpeptidase reaction of sortase A from Staphylococcus aureus, for the purpose of creating functional liposomes useful for a drug-delivery system (DDS). His-tagged recombinant sortase A with 59 amino acids deleted from the N-terminus (His-ΔN59SrtA) was prepared using an Escherichia coli expression system. The pH dependency and sorting signal sequence dependency of the transpeptidase reaction of His-ΔN59SrtA were analyzed by monitoring the transfer of model donor-substrates (i.e. His-tagged mutant green fluorescent proteins with a C-terminal LPxTG sorting signal) to model acceptor-beads with a GGGGGC peptide. In addition, using preferred conditions, the sortase A reaction was used to modify liposome surface. The transpeptidase reaction of His-ΔN59SrtA was enhanced under weakly acidic conditions. Transfer efficiency, based on sorting signal recognition by His-ΔN59SrtA, was similar to or higher than that obtained using several substrates with amino acids other than Glu in the sorting signal position "x". Furthermore, liposomes containing GGGGGC peptide-linked dipalmitoylphosphatidylethanolamine were successfully modified using the preferred conditions for His-ΔN59SrtA determined in this study. Preferred conditions for the transpeptidase reaction of His-ΔN59SrtA, especially in a weakly acidic environment to enhance reaction, was established and successfully used to create functional liposomes applicable to DDS.


Identification and Characterization of a Novel Secreted Glycosidase with Multiple Glycosidase Activities in Streptococcus intermedius

Streptococcus intermedius is a known human pathogen and belongs to the anginosus group (S. anginosus, S. intermedius, and S. constellatus) of streptococci (AGS). We found a large open reading frame (6,708 bp) in the lac operon, and bioinformatic analysis suggested that this gene encodes a novel glycosidase that can exhibit β-d-galactosidase and N-acetyl-β-d-hexosaminidase activities. We, therefore, named this protein "multisubstrate glycosidase A" (MsgA). To test whether MsgA has these glycosidase activities, the msgA gene was disrupted in S. intermedius. The msgA-deficient mutant no longer showed cell- and supernatant-associated β-d-galactosidase, β-d-fucosidase, N-acetyl-β-d-glucosaminidase, and N-acetyl-β-d-galactosaminidase activities, and all phenotypes were complemented in trans with a recombinant plasmid carrying msgA. Purified MsgA had all four of these glycosidase activities and exhibited the lowest Km with 4-methylumbelliferyl-linked N-acetyl-β-d-glucosaminide and the highest kcat with 4-methylumbelliferyl-linked β-d-galactopyranoside. In addition, the purified LacZ domain of MsgA had β-d-galactosidase and β-d-fucosidase activities, and the GH20 domain exhibited both N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities. The β-d-galactosidase and β-d-fucosidase activities of MsgA are thermolabile, and the optimal temperature of the reaction was 40°C, whereas almost all enzymatic activities disappeared at 49°C. The optimal temperatures for the N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities were 58 and 55°C, respectively. The requirement of sialidase treatment to remove sialic acid residues of the glycan branch end for glycan degradation by MsgA on human α1-antitrypsin indicates that MsgA has exoglycosidase activities. MsgA and sialidase might have an important function in the production and utilization of monosaccharides from oligosaccharides, such as glycans for survival in a normal habitat and for pathogenicity of S. intermedius.


The diversity of receptor recognition in cholesterol-dependent cytolysins

Cholesterol-dependent cytolysins (CDCs) are bacterial pore-forming toxins secreted mainly by pathogenic Gram-positive bacteria. CDCs generally recognize and bind to membrane cholesterol to create pores and lyse target cells. However, in contrast to typical CDCs such as streptolysin O, several atypical CDCs have been reported. The first of these was intermedilysin, which is secreted by Streptococcus intermedius and has human cell-specificity, human CD59 (huCD59) being its receptor. In the study reported here, the diversity of receptor recognition among CDCs was investigated and multi-receptor recognition characteristics were identified within this toxin family. Streptococcus mitis-derived human platelet aggregation factor (Sm-hPAF) secreted by S. mitis strain Nm-65 isolated from a patient with Kawasaki disease was previously shown to hemolyze erythrocytes in a species-dependent manner, its maximum activity being in human cells. In the present study, it was found that Sm-hPAF recognizes both membrane cholesterol and huCD59 as receptors for triggering pore-formation. Moreover, vaginolysin (VLY) of Gardnerella vaginalis showed similar characteristics to Sm-hPAF regarding receptor recognition. On the basis of the results presented here, the mode of receptor recognition of CDCs can be categorized into the following three groups: (i) Group I, comprising typical CDCs with high affinity to cholesterol and no or very little affinity to huCD59; (ii) Group II, including atypical CDCs such as ILY, with no or very little affinity to cholesterol and high affinity to huCD59; and (iii) Group III, which contains atypical CDCs such as Sm-hPAF and VLY with affinity to both cholesterol and huCD59.


LacR mutations are frequently observed in Streptococcus intermedius and are responsible for increased intermedilysin production and virulence.

Streptococcus intermedius secretes a human-specific cytolysin, intermedilysin (ILY), which is considered to be the major virulence factor of this pathogen. We screened for a repressor of ily expression by using random gene disruption in a low-ILY-producing strain (PC574). Three independent high-ILY-producing colonies that had plasmid insertions within a gene that has high homology to lacR were isolated. Validation of these observations was achieved through disruption of lacR in strain PC574 with an erythromycin cassette, which also led to higher hemolytic activity, increased transcription of ily, and higher cytotoxicity against HepG2 cells, compared to the parental strain. The direct binding of LacR within the ily promoter region was shown by a biotinylated DNA probe pulldown assay, and the amount of ILY secreted into the culture supernatant by PC574 cells was increased by adding lactose or galactose to the medium as a carbon source. Furthermore, we examined lacR nucleotide sequences and the hemolytic activity of 50 strains isolated from clinical infections and 7 strains isolated from dental plaque. Of the 50 strains isolated from infections, 13 showed high ILY production, 11 of these 13 strains had one or more point mutations and/or an insertion mutation in LacR, and almost all mutations were associated with a marked decline in LacR function. These results strongly suggest that mutation in lacR is required for the overproduction of ILY, which is associated with an increase in pathogenicity of S. intermedius.


Profiles of ILY, VLY and Sm-hPAF interaction with human CD59

The molecular features of a new member of the bacterially -derived cytolysin family were examined. In particular, the interactive mechanisms of intermedilysin (ILY), vaginolysin (VLY), and Streptococcus mitis-derived human platelet aggregation factor (Sm-hPAF) with human CD59 (hCD59) were analyzed. Molecular models of VLY and Sm-hPAF were constructed based on X-ray data of ILY (protein data bank ID=1S3R), and their interactive profiles with hCD59 were examined using molecular simulation. Non-binding (NB) energy between ILY and hCD59 was three orders of magnitude higher than the energy between VLY and hCD59. NB energy between Sm-hPAF and hCD59 was similar to that between VLY and hCD59. A hydrogen bond (ILY Arg432-hCD59 Glu76) was observed between ILY and hCD59, and a stronger interaction was formed by flexible adjustment between them.


Construction of an improved drug delivery system tool with enhanced versatility in cell-targeting.

His-Z-CDC(ss)(IS) was revealed to be an improved DDS tool with enhanced versatility in cell targeting.


Novel Twin Streptolysin S-Like Peptides Encoded in the sag Operon Homologue of Beta-Hemolytic Streptococcus anginosus.

Streptococcus anginosus is a member of the anginosus group streptococci, which form part of the normal human oral flora. In contrast to the pyogenic group streptococci, our knowledge of the virulence factors of the anginosus group streptococci, including S. anginosus, is not sufficient to allow a clear understanding of the basis of their pathogenicity. Generally, hemolysins are thought to be important virulence factors in streptococcal infections. In the present study, a sag operon homologue was shown to be responsible for beta-hemolysis in S. anginosus strains by random gene knockout. Interestingly, contrary to pyogenic group streptococci, beta-hemolytic S. anginosus was shown to have two tandem sagA homologues, encoding streptolysin S (SLS)-like peptides, in the sag operon homologue. Gene deletion and complementation experiments revealed that both genes were functional, and these SLS-like peptides were essential for beta-hemolysis in beta-hemolytic S. anginosus. Furthermore, the amino acid sequence of these SLS-like peptides differed from that of the typical SLS of S. pyogenes, especially in their propeptide domain, and an amino acid residue indicated to be important for the cytolytic activity of SLS in S. pyogenes was deleted in both S. anginosus homologues. These data suggest that SLS-like peptides encoded by two sagA homologues in beta-hemolytic S. anginosus may be potential virulence factors with a different structure essential for hemolytic activity and/or the maturation process compared to the typical SLS present in pyogenic group streptococci.


Small heat shock protein AgsA: an effective stabilizer of enzyme activities.

A small heat shock protein, AgsA, possesses chaperone activity that can reduce the amount of heat-aggregated protein in vivo, and suppress the aggregation of chemical- and heat-denatured proteins in vitro. Therefore, we examined the ability of AgsA to stabilize the activity of several enzymes by using this chaperone activity. We observed that AgsA can stabilize the enzymatic activities of Renilla (Renilla reniformis) luciferase, firefly (Photinus pyralis) luciferase, and β-galactosidase, and showed comparable or greater stabilization of these enzymes than bovine serum albumin (BSA), a well-known stabilizer of enzyme activities. In particular, AgsA revealed better stabilization of Renilla luciferase and β-galactosidase than BSA under disulfide bond-reducing conditions with dithiothreitol. In addition, AgsA also increased the enzymatic performance of β-galactosidase and various restriction enzymes to a comparable or greater extent than BSA. These data indicate that AgsA may be useful as a general stabilizer of enzyme activities.

Journal of Bioscience and Bioengineering


Molecular Profiles of Cholesterol-dependent Cytolysin Family-derived 11mer Regions

Cholesterol-dependent cytolysins (CDCs) are secreted from various types of bacteria and are involved in various diseases (e.g. abscess formation). Traditional CDCs has a conserved 11mer region, which is a key structure in membrane recognition. Based on the X-ray data of traditional CDC perfringolysin O (PFO), molecular models of intermedilysin (ILY), pyolysin (PLO), vaginolysin (VLY), and Streptococcus mitis-derived human platelet aggregation factor (Sm-hPAF) were constructed. The 11mer regions of these models were extracted, and their molecular features were analyzed. The dipole moments of these 11mer regions were classified into four types, and their stereo-hydrophobicity (dGW) was different. It was thought that these results influenced the species specificity and membrane recognition of each cytolysin. Traditional CDCs, ILY, PLO, and VLY consisted of four domains (domains 1 to 4). Domain 0 existed on the N-terminal side in Sm-hPAF in addition to these four domains. The 11mer sequence of Sm-hPAF is the same as that of VLY, but Sm-hPAF has slightly different characteristics (e.g. species specificity, membrane recognition, cholesterol dependency) compared to VLY. Dynamic structure analysis of domain 0 might clarify these differences.


Investigation of a Bacterial Pore-forming Chimera Toxin for Application as a Novel Drug-delivery System Tool

Cholesterol-dependent cytolysins (CDCs) are pore-forming toxins from Gram-positive bacteria. The aim of this study was to investigate the potential of a CDC, intermedilysin, as a drug-delivery system (DDS) for clinical application. Intermedilysin was modified by the addition of a disulfide bridge to regulate pore formation, by swapping domain 4 to provide cholesterol-binding capacity, and by the introduction of a targeting domain. The resultant chimera protein, His-LTBP-CDC(ss)(IP), was investigated for its use as a DDS tool in vitro. His-LTBP-CDC(ss)(IP) exhibited a regulated pore-forming capacity under reducing conditions. This chimera protein was able to deliver a drug-carrier liposome specifically to the target cell, to be endocytosed into the cell with subsequent release of the components into the cytoplasm. A chimera protein derived from the bacterial pore-forming toxin intermedilysin (His-LTBP-CDC(ss)(IP)) forms the basis for a novel DDS tool.


Role of Streptococcus intermedius DnaK chaperone system in stress tolerance and pathogenicity

Streptococcus intermedius is a facultatively anaerobic, opportunistic pathogen that causes purulent infections and abscess formation. The DnaK chaperone system has been characterized in several pathogenic bacteria and seems to have important functions in stress resistance and pathogenicity. However, the role of DnaK in S. intermedius remains unclear. Therefore, we constructed a dnaK knockout mutant that exhibited slow growth, thermosensitivity, accumulation of GroEL in the cell, and reduced cytotoxicity to HepG2 cells. The level of secretion of a major pathogenic factor, intermedilysin, was not affected by dnaK mutation. We further examined the function and property of the S. intermedius DnaK chaperone system by using Escherichia coli ΔdnaK and ΔrpoH mutant strains. S. intermedius DnaK could not complement the thermosensitivity of E. coli ΔdnaK mutant. However, the intact S. intermedius DnaK chaperone system could complement the thermosensitivity and acid sensitivity of E. coli ΔdnaK mutant. The S. intermedius DnaK chaperone system could regulate the activity and stability of the heat shock transcription factor ς(32) in E. coli, although S. intermedius does not utilize ς(32) for heat shock transcription. The S. intermedius DnaK chaperone system was also able to efficiently eliminate the aggregated proteins from ΔrpoH mutant cells. Overall, our data showed that the S. intermedius DnaK chaperone system has important functions in quality control of cellular proteins but has less participation in the modulation of expression of pathogenic factors.


Characterization of recombinant Streptococcus mitis-derived human platelet aggregation factor

We previously purified Streptococcus mitis-derived human platelet aggregation factor (Sm-hPAF) from the culture supernatant of S. mitis strain Nm-65, isolated from the tooth surface of a patient with Kawasaki disease. Here we produced recombinant Sm-hPAF protein (rSm-hPAF) in Escherichia coli, to determine whether rSm-hPAF conserves its platelet aggregation activity. rSm-hPAF precursor (665 amino acids) shows up to 36-56% identity with the family of cholesterol-dependent cytolysins (CDCs), and rSm-hPAF displayed potent hemolytic activity toward mammalian erythrocytes, including human erythrocytes with platelet aggregation activity. The 162-amino acid amino-terminal domain of rSm-hPAF was found in no other CDCs except lectinolysin; this domain is homologous to a portion of pneumococcal fucolectin-related protein. Interestingly, suilysin (SLY) and pneumolysin (PLY) of CDCs also exhibit substantial human platelet aggregation activity, similar to rSm-hPAF, and the platelet aggregation by rSm-hPAF, SLY, and PLY was morphologically confirmed using light and electron microscopy.


Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells.

(Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca(2+)]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-B translocation in human hepatic HepG2 cells, ILY did not affect NF-B localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca(2+)]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells.)

Biochemical and Biophysical Research Communications


Role of catabolite control protein A in the regulation of intermedilysin production by Streptococcus intermedius.

Streptococcus intermedius is an opportunistic pathogen of humans that causes purulent infections, including brain and liver abscesses. This pathogen secretes a human-specific cytolysin, intermedilysin, which has been recognized as a major virulence factor. However, most of the expressional control mechanisms of ily are still unknown. To determine these mechanisms, we analyzed the nucleotide sequence of the ily promoter region. We found a highly homologous region to the catabolite-repressible element (cre) in the ily promoter region and observed a considerable decrease in the amount of secreted intermedilysin when cells were grown in a culture medium containing high concentrations of glucose/utilizable carbohydrates. Disruption of the ccpA gene, which encodes catabolite control protein A, did not induce catabolite repression of ily by glucose/utilizable carbohydrates. In cre mutants, catabolite repression of ily was partially restored, and purified catabolite control protein A bound to an oligonucleotide containing the cre consensus sequence in the ily promoter region. In addition, a prolonged lag phase and slower doubling time of the ccpA mutant cells were observed. Our data show that S. intermedius can modulate ily expression and growth rate through catabolite control protein A-mediated monitoring of the extracellular glucose/utilizable carbohydrate concentration.


Investigation of the chaperone function of the small heat shock protein AgsA.

A small heat shock protein AgsA was originally isolated from Salmonella enterica serovar Typhimurium. We previously demonstrated that AgsA was an effective chaperone that could reduce the amount of heat-aggregated proteins in an Escherichia coli rpoH mutant. AgsA appeared to promote survival at lethal temperatures by cooperating with other chaperones in vivo. To investigate the aggregation prevention mechanisms of AgsA, we constructed N- or C-terminal truncated mutants and compared their properties with wild type AgsA. AgsA showed significant overall homology to wheat sHsp16.9 allowing its three-dimensional structure to be predicted. Truncations of AgsA until the N-terminal 23rd and C-terminal 11th amino acid (AA) from both termini preserved its in vivo chaperone activity. Temperature-controlled gel filtration chromatography showed that purified AgsA could maintain large oligomeric complexes up to 50 degrees C. Destabilization of oligomeric complexes was observed for N-terminal 11- and 17-AA truncated AgsA; C-terminal 11-AA truncated AgsA could not form large oligomeric complexes. AgsA prevented the aggregation of denatured lysozyme, malate dehydrogenase (MDH) and citrate synthase (CS) but did not prevent the aggregation of insulin at 25 degrees C. N-terminal 17-AA truncated AgsA showed no chaperone activity towards MDH. C-terminal 11-AA truncated AgsA showed weak or no chaperone activity towards lysozyme, MDH and CS although it prevented the aggregation of insulin at 25 degrees C. When the same amount of AgsA and C-terminal 11-AA truncated AgsA were mixed (half of respective amount required for efficient chaperone activities), good chaperone activity for all substrates and temperatures was observed. Detectable intermolecular exchanges between AgsA oligomers at 25 degrees C were not observed using fluorescence resonance energy transfer analysis; however, significant exchanges between AgsA oligomers and C-terminal truncated AgsA were observed at 25 degrees C. Our data demonstrate that AgsA has several regions necessary for efficient chaperone activity: region(s) important for lysozyme chaperone activity are located outer surface of the oligomeric complex while those region(s) important for insulin are located inside the oligomeric complex and those for MDH are located within the N-terminal arm. In addition, the equilibrium between the oligomer and the dimer structures appears to be important for its efficient chaperone activity.


Anti-Annexin A2 IgM Antibody in Preterm Infants: Its Association with Chorioamnionitis

Intrauterine infection is associated with chorioamnionitis (CAM), which can lead to preterm delivery. We previously reported that the levels of IgM and the incidence of CAM were elevated in preterm infants with neonatal pulmonary emphysema. The pathogen and target of this IgM remain unclear. By using Western blot and amino acid sequences, we have determined one of the target proteins: annexin A2. Immunohistochemical analysis showed that annexin A2 was expressed at fetal chorion and amnion membranes. Among very low birth weight (VLBW) infants with hyper-IgM (> or = 30 mg/dL), 58.8% showed a high titer against annexin A2 (more than x 16), which accounted for about 20%-40% of the total IgM. Anti-annexin A2 IgM antibody inhibited plasmin generation. Furthermore, the median of anti-annexin A2 IgM titer from preterm infants who were delivered with high-grade (grade III) CAM was significantly higher than those from preterm infants without CAM (p = 0.011) and with low-grade CAM (grade I and II) (p = 0.010). Here, we indicate the fetal autoimmunoreactivity against the fetomaternal interface in preterm infants.


Expression and purification of recombinant human annexin A2 in Pichia pastoris and utility of expression product for detecting annexin A2 antibody

Annexin A2, a Ca2+-dependent phospholipid binding protein, is abundantly expressed in various human organs, which exists as either a membrane-associated, cytosolic or soluble form in serum. We constructed expression systems for recombinant human annexin A2 (rhA2) using Pichia pastoris. The systems are designed to secrete rhA2 as either the N- or C-terminally His6-tagged form to facilitate purification. Both types of rhA2 were overexpressed, but in the N-terminal-truncated form as revealed from the results of N-terminal amino acid sequencing and Western blotting. Therefore, further purification of N-terminally His6-tagged rhA2 was not feasible because of the removal of the N-terminal His6-tag sequence. C-terminally His6-tagged rhA2 was expressed as either a glycosylated or a nonglycosylated form, and the nonglycosylated form was purified using the combination of nickel-immobilized affinity, concanavalin A and cation exchanged column chromatographies. The solid-phase binding of rhA2 was examined by enzyme-linked immunosorbent assay (ELISA), which revealed the specific reactivity of rhA2 against an anti-annexin A2 monoclonal antibody. These results suggest that the expression system using P. pastoris is useful for the preparation of rhA2 that is applicable to the ELISA detection of the anti-annexin A2 antibody.

Journal of Bioscience and Bioengineering


Correlation between resistance of Pseudomonas aeruginosa to quaternary ammonium compounds and expression of outer membrane protein OprR

論文題目:緑膿菌の細胞膜タンパク質OprRの発現と第四アンモニウム塩に対する耐性化の相関 第四アンモニウム塩系消毒剤に耐性化した緑膿菌において顕著に発現量が増加する約25.6kDaの蛋白質を2次元電気泳動で分離し，そのN末端アミノ酸配列の解析によって，それが外膜蛋白質のOprRであることを明らかとした．またその耐性化株でOprRのノックアウト株を作成したところ，第四アンモニウム塩系消毒剤に対する感受性が野生株と同程度まで上昇することから，OprRがこの消毒薬耐性に重要な役割を果たしていることを示した．


Microbial Contamination in Home Laundry Operations in Japan

論文題目:日本における家庭洗濯の微生物汚染．家庭洗濯中の衛生レベルは生活様式の変化と共に変遷してきた．諸外国では洗濯中に微生物汚染や感染が報告されている．そこで日本における洗濯中の汚染について様々な着用後の繊維製品に付着している微生物種の同定，洗濯物間の微生物転移による感染の危険性の実態について明らかにした．さらに除菌洗剤と非除菌洗剤を用いて検討し，除菌洗剤の汚染や感染防止効果が高いことを明らかにした．


The Properties of Bacterial Adaptation to Mono-and Bis-Quaternary Ammonium Compounds

論文題目:モノ型およびビス型第四アンモニウム塩に対する細菌の適応の性質．モノ型(デシルピリジニウム塩)およびビス型第四アンモニウム塩(架橋構造がチオール，エステル，カルバモイルおよびアミド)に対する細菌の適応について検討した．最小発育阻止濃度(MIC)測定法により検討した結果，モノ型ではMIC値が上昇し，抵抗性化が見受けられたが，ビス型第四アンモニウム塩では全く耐性化が見られなかった．抵抗性菌の表面疎水性を解析した結果，モノ型の抵抗性化は細胞表面の疎水性が低下したためであるとした．


Characterization of Pseudomonas aeruginosa Resistant to a Quaternary Ammonium Compound

論文題目:第四アンモニウム塩化合物に対して薬剤耐性を示す緑膿菌の特徴 N-ドデシルピリジニウムアイオダイドに対する薬剤耐性化を最小濃度測定法である培地希釈法を用いて行うことに成功した．その結果，感受性菌よりも8倍高い耐性化細菌を得ることができた．電子顕微鏡で観察した結果，耐性化細胞は感受性細胞には見られないバイオフィルム様の凝集が観察された．さらに，耐性化した細胞の表面疎水性について測定した結果，感受性細胞よりも親水性化していることがわかった．


Gibberella 菌のDeoxynivalenol産生およびTRI遺伝子発現への光照射と稲わら水蒸気爆砕リグニンの影響

次世代光フォーラム2025 in 徳島

次世代光フォーラム2025 in 徳島


Blue Luminescent Boron Complexes Based on N,N-type Bidentate Imidazo[1,2-a]pyridine Ligands

次世代光フォーラム 2024 in 徳島 論文集

次世代光フォーラム 2024 in 徳島 論文集


Naphthalene-fused Imidazo[1,2-a]pyridinium Salts Showing Green Emission with High Quantum Yields and Large Stokes Shift

次世代光フォーラム 2024 in 徳島 論文集

次世代光フォーラム 2024 in 徳島 論文集


Fluorescent Imidazo[1,2-a]pyridinium Salt for Potent Cancer Therapeutic Agent

Proceedings of the LED General Forum 2021 Tokushima


Fluorescent N-Heteroarene as a DNA Photo-cleaving Agent under the LED Irradiation

LED総合フォーラム2019 in 徳島 論文集

LED総合フォーラム2019 in 徳島 論文集