Gold nanoparticles-based colorimetric assay for cathepsin B activity and the efficiency of its inhibitors.

Cathepsin B has been suggested to be a prognostic marker of melanoma, glioma, and a variety of cancers such as brain, breast, colon, esophageal, gastric, lung, ovarian, and thyroid cancers. Cathepsin B inhibitors have also been considered as anticancer drug candidates; hence, there has been a growing need for a probe which enables the selective and simple detection of cathepsin B and its inhibitors. For the purpose of selective assay, a cathepsin B-specific substrate, N,N'-diBoc-dityrosine-glycine-phenylalanine-3-(methylthio)propylamine (DBDY-Gly-Phe-MTPA) was synthesized in this study. Phe-MTPA, which was produced via cathepsin B-catalyzed hydrolysis of DBDY-Gly-Phe-MTPA, allowed aggregation of gold nanoparticles (AuNPs) leading to a color change from red to blue. When tested for cathepsins B, L, and S, this assay method exhibited AuNPs color change only in reaction to cathepsin B. The limits of detection for cathepsin B was 10 and 5 nM in the 1 and 2 h hydrolysis reactions, respectively. The efficiency of cathepsin B inhibitors such as leupeptin, antipain, and chymostatin was easily compared by the degree of color change. Moreover, IC50 values of leupeptin, antipain, and chymostatin were found to be 0.11, 0.48, and 1.78 µM, respectively, which were similar to the results of previous studies. Therefore the colorimetric assay of cathepsin B and cathepsin B inhibitors using DBDY-Gly-Phe-MTPA and AuNPs allowed not only the selective but also the simple assay of cathepsin B and its inhibitors, which was possible with the naked eye.

New strategy for selective and sensitive assay of cathepsin B using a dityrosine-based material.

The increasing number of reports for disease-related proteases has necessitated materials for the fast, sensitive, and specific assessment of protease activities. The purpose of this study was to synthesize and test a dityrosine-based substrate for the selective assay of a specific cysteine cathepsin. DBDY-Gly-INH)2 was synthesized from the conjugation of N,N'-diBoc-dityrosine (DBDY) with two molecules of glycine and isoniazid (INH) for this purpose. The fluorescence of DBDY (λex=284-320nm, λem=400-420nm) disappeared due to the quenching effect of INH. However, the protease-catalyzed hydrolysis resulted in the release of INH and recovered the fluorescence of DBDY. When reacted with 13 proteases, DBDY-Gly-INH)2 was hydrolyzed by the cysteine proteases only. Meeting the growing need to discriminate cysteine cathepsins (e.g., cathepsins B, L, and S found at high levels in various cancers), DBDY-Gly-INH)2 was tested as a substrate for cathepsins B, L, and S. Only cathepsin B catalyzed the hydrolysis reaction among the three cathepsins. The reaction rate followed the Michaelis-Menten kinetics, and the KM and kcat/KM values were 2.88µM and 3.87×10(3)M(-1)s(-1), respectively, which were comparable to those for the materials reported for the selective assay of cathepsin B. Considering the simple preparation of DBDY-(Gly-INH)2, DBDY-(Gly-INH)2 is believed to be valuable for the sensitive and selective assay of cathepsin B activity.

Fabrication of high quantum yield quantum dot/polymer films by enhancing dispersion of quantum dots using silica particles.

We have fabricated quantum dot (QD)/polymer films of high quantum yield by coating silica particles with quantum dots. When particles were dispersed in tetrahydrofuran, free QD suspension exhibited higher quantum yield than QD-coated silica particles. Scattering is a most likely reason for the drop in quantum yield for the QD-coated silica particles, as supported by results of silica particles with varying morphologies: for example, QD-coated hollow silica particles showed higher quantum yield than filled silica particles, as the hollowness gave rise to reduced scattering. In the QD/polymer films, however, QD-coated filled/hollow silica particles showed significant enhancement in quantum yield (i.e., up to 2.4 times higher than that of free QDs). Confocal microscopy revealed that the enhanced quantum yield likely results from improved dispersion of QD-coated silica particles. In addition, the quantum yield of QD-coated hollow silica particles in films was lower than that of filled particles because of lower structural stability. Introducing silica (either filled or hollow) particles prevents spectral redshift of emission peak when prepared in the form of film, as opposed to QD-only sample. Our findings point to the possibility that QD-coated filled/hollow silica particles exhibit superior stability, quantum efficiency, and color accuracy, which render them potentially useful for the next-generation light-emitting devices and photovoltaics.

Adhesion of nano-sized particles to the surface of bacteria: mechanistic study with the extended DLVO theory.

Due to the increasing production and application of nanoparticles, their release into the environment would be inevitable, which requires a better understanding of their fate in the environment. When considering their toxic behavior or biodegradation as their fate, their adhesion to the cell surface must be the first step to be thoroughly studied. In this study, nano-sized polymeric particles of urethane acrylate with various hydrophobicity and ionic properties were synthesized as model nanoparticles, and their adhesion to Pseudomonas putida strains was monitored. The higher hydrophobicity and positive charge density on the particle surface exhibited the larger adhesion to the bacteria, whereas negative charge density on the particle hindered their adhesion to the bacteria, albeit high hydrophobicity of particle. These observations were successfully explained with the extended Derjaguin-Landau-Verwey-Overbeek (DLVO) theory.

A dityrosine-based substrate for a protease assay: application for the selective assessment of papain and chymopapain activity.

N,N'-diBoc-dityrosine (DBDY), which was synthesized by the oxidative C-C coupling of 2 N-Boc-L-tyrosine molecules, was conjugated with two isoniazid (INH) molecules. Due to the quenching effect of INH, DBDY-(INH)(2) lacks the fluorescence of DBDY. As such, it was tested for use in the detection of proteases by measuring fluorescence recovery. In this study, serine proteases (chymotrypsin, trypsin, subtilisin, and proteinase K), metalloproteases (thermolysin and carboxypeptidase A, dispase, and collagenase), aspartic proteases (pepsin and aspergillopepsin) and cysteine proteases (papain and chymopapain) were chosen. Reported optimum assay conditions were chosen for each enzyme. Only papain and chymopapain catalyzed the hydrolysis of DBDY-(INH)(2) and led to fluorescence recovery, possibly due to their extensive binding sites and the INH-mediated inhibition of metalloproteases and aspartic proteases.

Transduction of striatum and cortex tissues by adeno-associated viral vectors produced by herpes simplex virus- and baculovirus-based methods.

Recombinant adeno-associated virus (AAV) vectors can be engineered to carry genetic material encoding therapeutic gene products that have demonstrated significant clinical promise. These viral vectors are typically produced in mammalian cells by the transient transfection of two or three plasmids encoding the AAV rep and cap genes, the adenovirus helper gene, and a gene of interest. Although this method can produce high-quality AAV vectors when used with multiple purification protocols, one critical limitation is the difficulty in scaling-up manufacturing, which poses a significant hurdle to the broad clinical utilization of AAV vectors. To address this challenge, recombinant herpes simplex virus type I (rHSV-1)- and recombinant baculovirus (rBac)-based methods have been established recently. These methods are more amenable to large-scale production of AAV vectors than methods using the transient transfection of mammalian cells. To investigate potential applications of AAV vectors produced by rHSV-1- or rBac-based platforms, the in vivo transduction of rHSV-1- or rBac-produced AAV serotype 2 (AAV2) vectors within the rat brain were examined by comparing them with vectors generated by the conventional transfection method. Injection of rHSV-1- or rBac-produced AAV vectors into rat striatum and cortex tissues revealed no differences in cellular tropism (i.e., predominantly neuronal targeting) or anteroposterior spread compared with AAV2 vectors produced by transient transfection. This report represents a step towards validating AAV vectors produced by the rHSV-1- and the rBac-based systems as promising tools, especially for delivering therapeutic molecules to the central nervous system.

Heparin-coated superparamagnetic nanoparticle-mediated adeno-associated virus delivery for enhancing cellular transduction.

Superparamagnetic iron oxide nanoparticles (SPIONs) have been exploited as an elegant vehicle to enhance gene delivery efficiencies in gene therapy applications. We developed a magnetically guided adeno-associated virus (AAV) delivery system for enhancing gene delivery to HEK293T and PC12 cell lines. Wild-type AAV2 and a novel AAV vector, AAVr3.45, which was directly evolved in a previous study to possess diverse cell tropisms, were used as gene carriers. Additionally, the affinity of each viral vector to heparin was employed as a moiety to immobilize virus onto heparin-coated SPIONs (HpNPs). Magnetically guided AAV delivery resulted fast and efficient cellular transduction. Importantly, a short exposure of virus to target cells under a magnetic field (<180min) yielded comparable transduction produced by the conventional gene-delivery protocol (i.e., 24h-incubation of virus with target cells prior to replacing with fresh medium). Additionally, magnetic guidance of AAV encoding nerve growth factor (NGF) produced sufficient functional NGF, leading to robust neurite elongation by PC12 as compared to direct NGF protein delivery or non-magnetic delivery. The successful establishment of a magnetically guided AAV delivery system, with the ability to efficiently and rapidly infect target cells, will provide a powerful platform for a variety of gene therapy applications.

Colorimetric detection of c-Kit mutations using electrostatic attraction induced aggregation of peptide nucleic acid modified gold nanoparticles.

We report a colorimetric detection of c-Kit mutations using selective aggregation of the peptide nucleic acid modified gold nanoparticles that is caused by electrostatic attraction.

Magnetically enhanced adeno-associated viral vector delivery for human neural stem cell infection.

Gene therapy technology is a powerful tool to elucidate the molecular cues that precisely regulate stem cell fates, but developing safe vehicles or mechanisms that are capable of delivering genes to stem cells with high efficiency remains a challenge. In this study, we developed a magnetically guided adeno-associated virus (AAV) delivery system for gene delivery to human neural stem cells (hNSCs). Magnetically guided AAV delivery resulted in rapid accumulation of vectors on target cells followed by forced penetration of the vectors across the plasma membrane, ultimately leading to fast and efficient cellular transduction. To combine AAV vectors with the magnetically guided delivery, AAV was genetically modified to display hexa-histidine (6xHis) on the physically exposed loop of the AAV2 capsid (6xHis AAV), which interacted with nickel ions chelated on NTA-biotin conjugated to streptavidin-coated superparamagnetic iron oxide nanoparticles (NiStNPs). NiStNP-mediated 6xHis AAV delivery under magnetic fields led to significantly enhanced cellular transduction in a non-permissive cell type (i.e., hNSCs). In addition, this delivery method reduced the viral exposure times required to induce a high level of transduction by as much as to 2-10 min of hNSC infection, thus demonstrating the great potential of magnetically guided AAV delivery for numerous gene therapy and stem cell applications.

Analysis of the adhesion of Pseudomonas putida NCIB 9816-4 to a silica gel as a model soil using extended DLVO theory.

The extended Derjaguin-Landau-Verwey-Overbeek (DLVO) theory was applied to explain the hydrophobic interaction-mediated adhesion of Pseudomonas putida NCIB 9816-4 to soil. Soil particles are heterogeneous, and it is difficult to define consistent physico-chemical properties such as a contact angle and zeta potential. Hence, a silica gel and a silanized (3-aminopropyltriethoxysilane-coated) silica gel, which showed greater hydrophobicity than the unmodified silica gel, were used as model soils. Gibbs energies for the cell adhesion to the silica gels were calculated with the physico-chemical properties of the microbes and the silica gels and then plotted as a function of the separation distance. The extended DLVO theory successfully explained that the adhesion of P. putida NCIB 9816-4 to the silica gel, a model soil, was primarily caused by hydrophobic interaction.

Influence of naphthalene biodegradation on the adhesion of Pseudomonas putida NCIB 9816-4 to a naphthalene-contaminated soil.

In an earlier study, Pseudomonas putida NCIB 9816-4, which was one of the most studied naphthalene-degrading bacteria, showed the preferred adhesion to the naphthalene-contaminated soil when it was in the exponential growth phase. The adhesion was found to take place through a hydrophobic interaction. We postulated that the surface hydrophobicity of P. putida NCIB 9816-4 in the exponential growth phase might be increased during the uptake of naphthalene, which caused the preferred adhesion to the naphthalene-contaminated soil. To verify this postulate, a plasmid-cured strain of P. putida NCIB 9816-4 was obtained in this study and compared with the wild-type for adhesion to the naphthalene-contaminated soil. Only the wild-type in the exponential growth phase showed increased adhesion to naphthalene-contaminated soil. The water contact angles of the two strains were measured in the presence and in the absence of naphthalene as indices of surface hydrophobicity. The water contact angle of the wild-type increased in the presence of naphthalene, whereas that of the cured strain did not change. We conclude that the uptake of naphthalene during naphthalene biodegradation in the exponential growth phase of P. putida NCIB 9816-4 made the cell surface more hydrophobic, resulting in increased adhesion to naphthalene-contaminated soil.

Manganese peroxidase-catalyzed oxidative degradation of vanillylacetone.

When 4-(4-hydroxy-3-methoxy-phenyl)-2-butanone (vanillylacetone) was tested for manganese peroxidase (MnP)-catalyzed oxidation, it was found to be degraded with the cleavage of an aromatic ring. Among numerous products of vanillylacetone oxidation, four major ones were purified by thin-layer chromatography and identified using mass spectroscopy (MS) and nuclear magnetic resonance (NMR) analysis. Three of them maintained the aromatic ring structure and were identified as 4-[6,2'-dihydroxy-5,3'-dimethoxy-5'-(3-oxo-butyl)-biphenyl]-butan-2-one, 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one, and 4-[6,2'-dihydroxy-5,3'-dimethoxy-5'-(3-oxo-butyl)-biphenyl]-3-buten-2-one. Even though the fourth product could not be purified to a single compound, data from infrared spectroscopy showed that it did not have a benzene ring. From MS and NMR analysis, 3-(3-oxo-butyl)-hexa-2,4-dienedioic acid-1-methyl ester was tentatively suggested as the dominant species. The reaction mechanism was suggested on the basis of the structural information of these products. To our knowledge, this paper is the first report on aromatic ring cleavage of the phenolic compound by MnP.

Oxidation of 17alpha-ethinylestradiol with Mn(III) and product identification.

With increasing concern about the contamination of aquatic environments by estrogenic pollutants, removal of synthetic estrogens such as 17alpha-ethinylestradiol (EE2) has been widely studied, especially with respect to the treatment methods. However, the degradation products have rarely been identified. The purpose of this study was to identify structurally the oxidation products of EE2. Mn(III) was used as an oxidizing agent. To obtain sufficient oxidation products for HPLC, LC-MS and NMR spectroscopy, a highly concentrated solution of EE2 (1mM) was prepared in a mixture of water and a water-miscible organic solvent. From HPLC of the reaction products, a single compound (I) was found to be predominant. From LC-MS, its molecular mass was found to be 294, and two hydrogens were believed to have been removed from EE2 (M.W. 296) to form a C=C . The structure of compound I (position of the double bond) was determined using 1H NMR, 13C NMR, H-H COSY, HSQC and HMBC. As minor products, isomeric dimers (M.W. 590) of EE2, as well as the products (M.W. 588) in which EE2 was coupled to compound I were also formed during the Mn(III)-mediated oxidation of EE2.

Absorption of a volatile organic compound by a jet loop reactor with circulation of a surfactant solution: performance evaluation.

Biofiltration shows high efficiency for the removal of industrial waste gases and reliable operational stability at low investment and operating cost, especially when the VOC concentration is low, such as 100 ppmv (micro LL(-1)) or less. However, it has been reported that the abrupt change in VOC concentrations leads to the failure of the biofilter. Hence, the pretreatment of waste gases is necessary to ensure the stable operation of the biofilter. The objective of this study is to develop a jet loop reactor (JLR) with circulation of a surfactant solution to lower the concentration of VOCs, especially hydrophobic VOCs. Toluene and Tween 81 were used as a model industrial waste gas and a surfactant, respectively. Among several non-ionic surfactants tested, Tween 81 showed the most rapid dissolution of toluene. When a JLR is replaced with fresh Tween 81 solution (0.3% w/v) every hour, it successfully absorbed for 48 h over 90% of the toluene in an inlet gas containing toluene at 1000 ppmv (microL L(-1)) or less. Therefore, JLR with circulation of a surfactant solution is believed to ensure the stable operation of the biofilter even with the unexpected increase in the VOC concentrations.

Adhesion of Pseudomonas putida NCIB 9816-4 to a naphthalene-contaminated soil.

The effect of a soil contaminant on the initial adhesion to the soil of a contaminant-degrading soil microorganism in the exponential phase was investigated using naphthalene as the soil contaminant and Pseudomonas putida strain NCIB 9816-4 as the naphthalene-degrading bacteria. P. putida strain DK-1, which is not capable of degrading naphthalene, was used as a control. P. putida NCIB 9816-4 in the exponential phase showed the more adhesion to the soil than that in the stationary phase. In contrast, P. putida DK-1 showed the increased adhesion to the soil when it was in the stationary phase. P. putida NCIB 9816-4 in the exponential phase showed the preferred adhesion to the naphthalene-contaminated soil, whereas the adhesion of P. putida DK-1 was not affected by naphthalene. From the data of surface hydrophobicities of the cells and the soil, the microbial adhesion, especially the initial adhesion to the naphthalene-contaminated soil, takes place through the hydrophobic interaction. We suspect that the surface hydrophobicity of P. putida NCIB 9816-4 in the exponential phase might be increased during the uptake of naphthalene, which caused the preferred adhesion to the naphthalene-contaminated soil.

Microscopic analysis of ester hydrolysis reaction catalyzed by Candida rugosa lipase.

The relationship between the kinetics of the lipase-catalyzed oil hydrolysis and the surface area distribution of oil droplets was investigated using ethyl decanoate and gum Arabic (GA) as a model oil and an emulsifier, respectively. Along an ethyl decanoate concentration gradient between 2 and 8 mM, the initial hydrolysis rate increased at 0.25% (w/v) GA but did not change at 1.0% (w/v) GA. At 0.25% GA, the surface area of droplets was narrowly distributed regardless of the ethyl decanoate concentration. However, at 1.0% GA and with ethyl decanoate concentrations higher than 2 mM, the fraction of relatively large droplets with a surface area larger than approximately 200 microm2, suddenly increased. The microscopy of ethyl decanoate emulsion during the hydrolysis reaction indicates that the large oil droplets were not hydrolyzed. At 20 mM ethyl decanoate where the hydrolysis rate remained the same between 0.25% and 1.0% GA, the surface area of droplets was narrowly distributed at 0.25% and 1.0% GA. Therefore, the constant hydrolysis rate observed in the emulsion of ethyl decanoate between 2 and 8 mM containing GA at 1.0%, is believed to be caused by the relatively large oil droplets with the interface quality differing from that of the small oil droplets.

Enhanced production of manganese peroxidase from immobilized Phanerochaete chrysosporium due to the increased autolysis of chlamydospore-like cells.

The autolysis of chlamydospore-like cells in Phanerochaete chrysosporium immobilized in polyurethane foam correlated with the production of manganese peroxidase (MnP). The maximum specific activity of MnP was 1055 U g dry mycelium(-1) in the immobilized culture, compared with 260 U g dry mycelium(-1) in the submerged culture. Scattered mycelial pellets were formed in the immobilized culture in which almost all of the chlamydospore-like cells were subject to autolysis. However, highly crowded pellets were formed in the free culture, in which only the chlamydospore-like cells in the exterior were subject to autolysis. We propose that the enhanced production of MnP in immobilized cultures of P. chrysosporium is due to increased autolysis of the chlamydospore-like cells.

Evaluation of cellulose-binding domain fused to a lipase for the lipase immobilization.

A cellulose-binding domain (CBD) fragment of a cellulase gene of Trichoderma hazianum was fused to a lipase gene of Bacillus stearothermophilus L1 to make a gene cluster for CBD-BSL lipase. The specific activity of CBD-BSL lipase for oil hydrolysis increased by 33% after being immobilized on Avicel (microcrystalline cellulose), whereas those of CBD-BSL lipase and BSL lipase decreased by 16% and 54%, respectively, after being immobilized on silica gel. Although the loss of activity of an enzyme immobilized by adsorption has been reported previously, the loss of activity of the CBD-BSL lipase immobilized on Avicel was less than 3% after 12 h due to the irreversible binding of CBD to Avicel.

Enzymatic esterification of beta-methylglucoside with acrylic/methacrylic acid in organic solvents.

The enzymatic esterifications of beta-methylglucoside with acrylic acid/methacrylic acid were carried out using Novozym 435. t-Butanol indicating the highest conversion value was determined as an optimal solvent. The molar ratio (beta-methylglucoside:acids) of 1:15 was most favorable to the esterification. The enzyme concentration of 5% (w/v), and the temperature (50 degrees C for beta-methylglucoside:acrylic acid, 45 degrees C for beta-methylglucoside:methacrylic acid) resulted in the highest final conversion. Beta-methylglucoside of 60gl(-1) was found to be most effective in terms of short reaction time as well as product concentrations. Under these conditions, the maximum conversions for the esterification of beta-methylglucoside with acrylic acid and beta-methylglucoside with methacrylic acid were 59.3% after 12h and 71.3% after 72h, respectively. The structural analysis of the products was performed by FT-IR spectroscopy and (1)H NMR.

Effect of additional carbon source on naphthalene biodegradation by Pseudomonas putida G7.

Addition of a carbon source as a nutrient into soil is believed to enhance in situ bioremediation by stimulating the growth of microorganisms that are indigenous to the subsurface and are capable of degrading contaminants. However, it may inhibit the biodegradation of organic contaminants and result in diauxic growth. The objective of this work is to study the effect of pyruvate as another carbon source on the biodegradation of polynuclear aromatic hydrocarbons (PAHs). In this study, naphthalene was used as a model PAH, ammonium sulfate as a nitrogen source, and oxygen as an electron acceptor. Pseudomonas putida G7 was used as a model naphthalene-degrading microorganism. From a chemostat culture, the growth kinetics of P. putida G7 on pyruvate was determined. At concentrations of naphthalene and pyruvate giving similar growth rates of P. putida G7, diauxic growth of P. putida G7 was not observed. It is suggested that pyruvate does not inhibit naphthalene biodegradation and can be used as an additional carbon source to stimulate the growth of P. putida G7 that can degrade polynuclear aromatic hydrocarbons.