Hikaru Suenaga

Comparison of the WST-8 colorimetric method and the CLSI broth microdilution method for susceptibility testing against drug-resistant bacteria.

The minimum inhibitory concentrations (MICs) obtained from the susceptibility testing of various bacteria to antibiotics were determined by a colorimetric microbial viability assay based on reduction of a tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8)} via 2-methyl-1,4-napthoquinone as an electron mediator and compared with those obtained by the broth microdilution methods approved by the Clinical and Laboratory Standard Institute (CLSI). Especially for drug-resistant bacteria, the CLSI method at an incubation time of 24h tended to give lower MICs. The extension of incubation time was necessary to obtain consistent MICs for drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococi (VRE) and multi-drug resistant Pseudomonas aeruginosa (MDRP) in the broth microdilution method. There was excellent agreement between the MICs determined after 24h using the WST-8 colorimetric method and those obtained after 48-96 h using the broth microdilution method. The results suggest that the WST-8 colorimetric assay is a useful method for rapid determination of consistent MICs for drug-resistant bacteria.

Screening of fluorescent boronic acids for sugar sensing which show a large fluorescence change

To search for such fluorescent boronic acids that are useful for sensitive detection of sugars in water, 8 aromatic boronic acids have been screened. Among them biphenyl-3-boronic acid and naphthalene-2-boronic acid could fully satisfy three prerequisites for sugar sensing: strong fluorescence intensity, large pH-dependent change in Imax, and shift of the pH-Imax profile to lower pH region in the presence of sugars.

Amine functionalized polythiophenes: synthesis and formation of chiral, ordered structures on DNA substrates

Abstract Highly regioregular, head-to-tail coupled, 3-(amino functionalized)-polythiophenes can be synthesized by CuO co-catalyzed Stille coupling. Salts of one example are water soluble, and become helically ordered upon addition of DNA.

Engineering a hybrid pseudomonad to acquire 3,4-dioxygenase activity for polychlorinated biphenyls

We constructed a hybrid strain that acquired 3,4-dioxygenase activity for polychlorinated biphenyls (PCBs). This strain, KF707-D34, possessed a chimeric biphenyl dioxygenase gene, of which a portion of bphA1 (coding for a large subunit of biphenyl dioxygenase) of Pseudomonas pseudoalcaligenes KF707 was replaced with that of a PCB-degrader, Burkholderia cepacia LB400 by homologous recombination. KF707-D34 retained the ability to degrade 4,4-dichlorobiphenyl via 2,3-dioxygenation in a fashion identical to that of KF707 and gained novel capability to degrade 2,5,4-trichlorobiphenyl and 2,5,2,5-tetrachlorobiphenyl via 3,4-dioxygenation in a fashion identical to that of LB400. Sequence analysis of bphA1 from KF707-D34 revealed that three nucleotides in the 3-terminal region of KF707 bphA1 were changed to correspond to those in LB400 bphA1. The resulting BphA1 protein in KF707-D34 was changed at position 376 from threonine (Thr) to asparagine (Asn). The results demonstrate that a minor alteration of the amino acid sequence in BphA1 improved the PCB degradation capability in biphenyl-utilizing bacteria.

Hydroxylation of various molecules including heterocyclic aromatics using recombinant Escherichia coli cells expressing modified biphenyl dioxygenase genes

Abstract Various molecules, in which heterocyclic aromatics are linked with phenyl or benzyl groups, were converted to their corresponding cis -dihydrodiols by recombinant Escherichia coli cells expressing modified biphenyl dioxygenase genes. Heterocyclic aromatic compounds with substituted phenyl or aliphatic moieties were also biotransformed to the hydroxylated products by the cells. Many of the converted products were novel compounds. These compounds are potentially useful as versatile starting materials for the chemical synthesis of pharmaceuticals and biologically active organic molecules.

Screening of boronic acids for strong inhibition of the hydrolytic activity of α-chymotrypsin and for sugar sensing associated with a large fluorescence change

The inhibitory effect of arylboronic acids which act as a transition state analogue for certain peptidases is efficiently intensified by added saccharides: this finding enables us to control the enzyme activity by a combination of arylboronic acids and polyols including sugars. In particular, the combined system of 3-biphenylboronic acid and D-glucose strongly inhibited the hydrolysis reaction mediated by a- chymotrypsin, the inhibitory effect ( Ki = 1.1 X mol dm3) being stronger than that of a specific inhibitor, chymostatin (Ki = 4.8 XlU7 mol d~n-~). Hence, saccharides act as a co-inhibitor in the boronic acid inhibition system. To search for such fluorescent boronic acids that are useful for sensitive detection of sugars in water, 8 aromatic boronic acids have been screened. Among them biphenyl-3-boronic acid and naphthalene-2-boronic acid could fully satisfy three prerequisites for sugar sensing: strong fluorescence intensity, large pH-dependent change in I-, and shift of the pH- Z,, profile to lower pH region in the presence of sugars.

Water-soluble [60]fullerene–cationic homooxacalix[3]arene complex which is applicable to the photocleavage of DNA

A new water-soluble, cationic homooxacalix[3]arene 1 was synthesized: this homooxacalix[3]arene solubilized [60]fullerene into water and the [60]fullerene–homooxacalix[3]arene complex acted as an efficient DNA photocleavage reagent.

Sugar-controlled association–dissociation equilibria between DNA and boronic acid-appended porphyrin

5,10,15,20-Tetrakis{1-[2-(dihydroxyboryl)benzyl]pyridin-1-ium-4-yl}porphyrin tetrabromide 1 has been synthesized to control the DNA-binding properties by physiologically-non-toxic saccharides. At pH 8.01 is dicationic and strongly bound to DNA whereas it becomes neutral through saccharide complexation with two boronic acid pendants and the complex is dissociated. Examination with absorption and CD spectroscopy established that poly(dGdC)·poly(dGdC) intercalates 1 whereas poly(dAdT)·poly(dAdT) binds 1 to the outside of the double strand. Since 1 in the presence of calf thymus DNA gave the CD spectrum similar to that in the presence of poly(dAdT)·poly(dAdT),1 should be bound to the AT region in an outside binding manner. When D-fructose was added, both the absorption and CD spectra changed simply from the 1·DNA complex to 1·D-fructose complex. This indicates that D-fructose can dissociate the 1·DNA complex in a one-step manner. A similar change is observed for D-glucose when the 1 concentration is low, but the dissociation occurred in a two-step manner when the concentration of 1 is high, suggesting the D-glucose-induced reorganization of 1 in DNA. This is a novel method for controlling the DNA-binding properties of porphyries by saccharides.

Sugars intensify the inhibitory effect of phenylboronic acid on the hydrolytic activity of α-chymotrypsin

The inhibitory effect of phenylboronic acid which acts as a transition state analogue for certain peptidases is efficiently intensified by added saccharides: this finding enables us to control the enzyme activity with sugars.

Strong inhibitory effect of sugar·biphenylylboronic acid complexes on the hydrolytic activity of α-chymotrypsin

Boronic acids act as transition-state analogues for certain peptidases. The inhibitory effect of 2-, 3- and 4-biphenylylboronic acids (2a, 2b and 2c) on the hydrolytic activity of α-chymotrypsin has been investigated. These inhibitors were employed to monitor the binding event [formation of covalent bond with either serine residue (195) or histidine residue (57)] occurring in the active site by a fluorescence method. It was shown that the decrease in the fluorescence intensity, which is induced by the formation of a covalent bond with the boronic acid moiety, is well correlated with the inhibitory effect estimated by kinetic measurements. The inhibitory effect appeared in the order 2a < 2c2b(Ki= 1.6 × 10–6 mol dm–3). Interestingly, the inhibitory effect was further intensified by added saccharides. In particular, the combined system of 2b and D-glucose strongly inhibited the enzyme reaction, the inhibitory effect(Ki= 1.1 × 10–7 mol dm–3)being stronger than that of a specific inhibitor, chymostatin(Ki= 4.8 × 10–7 mol dm–3). Hence, saccharides act as a ‘co-inhibitor’ in the boronic acid inhibition system. This is a novel and efficient inhibition system for α-chymotrypsin (and probably more generally for other peptidases).