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4-Aryloxy boron dipyrromethene (BODIPY) modification of the surface of silica nanoparticles (NPs) improved permeability through the membrane of HaCaT skin cells and swine skin tissue. The 35 nm BODIPY-modified NPs penetrated tape-stripped skin and reached the dermis within 1 h. Since these NPs can encapsulate a variety of molecules including macromolecules, they are expected to serve as effective carriers for the delivery of drugs, genes, and other compounds through skin and into cells.

We developed a simple method to prepare PEG-based soft nanoparticles that encapsulate dual imaging probes. Because the probes could be encapsulated by either chemical or physical means, a variety of probe molecules were encapsulated within the nanoparticles simultaneously. The nanoparticles were administrated to mice and the pharmacokinetics of the nanoparticles was analyzed by means of MRI, fluorescence spectroscopy, and transmission electron microscopy. The soft nanoparticles were excreted by the mice rapidly through the urine without collapse of the nanoparticles and without leaking of the probe molecules, and no accumulation of the nanoparticles in the body was observed. The pharmacokinetics of the nanoparticles was not changed by the encapsulated molecules and acute toxicity to mice was negligible. It was expect that these PEG-based soft nanoparticles will be applicable for use as a safe diagnostic agent.

Recently, nanoparticles have garnered considerable attention, and the demand for a rapid and simple method for their analysis has increased accordingly. The bimodal pores (few μm- and few tens nm-sized pores) of monolithic columns were thought to be suitable for the separation of nanoparticles and small molecules; however, the residual silanol groups on the column surface resulted in the strong adsorption of liposomes and hindered their analysis. To overcome this problem, we modified the surface of the silica monolith via a two-step process and developed three silica monolithic columns coated with three different polymers: glycidyl methacrylate (GMA), 2-hydroxyethyl methacrylate (HEMA), and N-vinylpyrrolidone (VP). These were used for the analysis of 100-nm liposomal nanoparticles. Since 15% polymer coating prevented the nanoparticle adsorption, liposomes (AmBisome®) and pegylated liposomes (DOXIL®) were eluted rapidly (within 1min) using these columns, without using organic solvents in the mobile phase. Molecular leaching from the liposomes, as well as protein adsorption to the liposomes (corona formation) could be evaluated using the polymer-coated columns, thus illustrating their utility in the rapid and simple analysis of 100-nm liposomal nanoparticles.

Although local protein activity in the cells is one of the hot topics in life science study, it still presents many difficulties with regard to measuring or changing its activity. Here, we prepared 20 nm-sized protein (caspase)-encapsulated photodegradable nanoparticles and used them to control the amount of caspase released within HeLa cells. Encapsulated caspase was released into the cytosol following irradiation. The increased amount of released caspase led to enhanced cell membrane permeability. These results indicate that the amount of protein released within cells can be regulated by the amount of protein-encapsulated nanoparticles; therefore, this technique would be useful for detailed study of protein function within cells or the control of cellular functions.

Nanoparticles have been used in diverse areas, and even broader applications are expected in the future. Since surface modification can influence the configuration and toxicity of nanoparticles, a rapid screening method is important to ensure nanoparticle quality.We examined the effect of the nanoparticle surface morphology on the HPLC elution profile using two types of 100-nm liposomal nanoparticles (AmBisome(Ⓡ) and DOXIL(Ⓡ)).These 100-nm-sized nanoparticles eluted before the holdup time (about 4 min), even when a column packed with particles with a relatively large pore size (30 nm) was used. The elution time of the nanoparticles increased with pegylation of the nanoparticles and protein adsorption to the nanoparticles; however, the nanoparticles still eluted before the holdup time.The results of this study indicate that HPLC is a suitable tool for rapid evaluation of the surface of liposomal nanoparticles.

Drug-containing nanoparticles, the foundation of nanomedicine, provide promise for the safe and effective delivery of drugs to their targets. In this study, we developed a simple method to determine the relative quantities of nanoparticle-encapsulated drugs by HPLC using a commercially available monolithic silica column. Amphotericin B- and irinotecan-containing nanoparticles produced nearly simultaneous elution peaks (~7 min), suggesting that elution was largely driven by hydrodynamic effects and was relatively unaffected by differences in the encapsulated drug. A good correlation was observed between the intensity of the nanoparticle peak and the relative quantity of encapsulated drug. We used our method to characterize the effects of drug quantity and nanoparticle size on drug encapsulation rates within the nanoparticles. Encapsulation increased with increasing quantities of the drug in the preparation solution. This effect was greater for irinotecan than for amphotericin B. Although absolute encapsulation also increased with increasing nanoparticle size, encapsulation efficiency decreased. Thus, the monolith column is suitable for evaluating nanomedicine quality and may be used to evaluate many kinds of nanomaterials. Graphical Abstract Evaluation method of quantity of drug encapsulated within nanoparticles was developed. The method can be applicable for a rapid quality assurance of nanomedicine.

Nanoparticles, spherical particles with diameters less than 100 nm, are promising theranostic devices for noninvasive diagnosis and therapy. In this study, nanoparticles composed of polyethylene glycol and silica were prepared, and their migration behavior was examined using capillary electrophoresis. The effects of the sodium dodecyl sulfate concentration in the electrolyte, the nanoparticle size, and the encapsulated molecule on the migration were examined. The addition of sodium dodecyl sulfate into the electrolyte had a significant effect on the electrophoretic mobility of polyethylene glycol nanoparticles, but a small effect on that of silica nanoparticles. As for the size effect, the mobility became a little faster for smaller nanoparticle sizes for both polyethylene glycol and silica nanoparticles. The encapsulated molecule affected the mobility of the nanoparticles through interactions between the encapsulated molecules and sodium dodecyl sulfate. We propose that the large effect of sodium dodecyl sulfate on the migration of the polyethylene glycol nanoparticles was due to the large spaces within the nanoparticles. These results indicate that nanoparticle migration is mainly determined by the nanoparticle components.

A simple method based on the vortex mixing of precursor chemicals in surfactant-free aqueous conditions has been developed for preparing nanoparticles that encapsulate functional molecules within a four-armed star polymer. Through investigation into the effects of altering such variables as the vortex intensity, chemical composition, temperature etc., it was found that the size of these nanoparticles could be controlled within a range of 20 to 200 nm. Furthermore, a variety of functional molecules could be encapsulated without changing the size of the nanoparticles, suggesting that this method has the potential to be used for a wide range of applications, such as medicine, industrial production and cosmetics.

The spatiotemporal control of molecular function is important but there are currently few techniques for noninvasively controlling various types of molecules in live cells. Herein we developed nanoparticles with a boron dipyrromethene structure, which are responsive to blue-green visible light. Fluorophores (fluorescein, rhodamine B, and Nile blue A) encapsulated within the nanoparticles were released by irradiation for 3 min with visible light. Nanoparticles were internalised by HeLa cells without the aid of a cell-penetrating peptide, serving as vehicles for the delivery of cargo molecules to the cytoplasm. The release and activation of encapsulated molecules by visible light irradiation demonstrate a novel method for the spatiotemporal control of molecular function that can be used to activate molecules inside the skin that cannot be reached by UV light, which has limited tissue penetration.

Chemical derivatization is often used to improve the separation efficiency and to enhance the detectability of the target compounds in high-performance liquid chromatography and capillary electrophoresis. The derivatization reagents having a benzofurazan (2,1,3-benzoxadiazole) structure are one of the most often used reagent for this purpose. In this paper, the recent advances in the development and the application of benzofurazan derivatization reagents are reviewed. Copyright © 2013 John Wiley & Sons, Ltd.

To effectively and safely use molecules, it is important to be able to control the timing and site of molecule activation. We developed a spatiotemporal method to control molecular function by using silica-based photodegradable nanoparticles that can be prepared under mild conditions. The function of various molecules, such as rhodamine B, Nile blue A, propidium iodide (PI), and rhodamine 110, bis-(N-CBZ-l-arginine amide), dihydrochloride (BZAR), was restricted by wrapping in the network structure of the nanoparticle gel. The encapsulated molecule was released from the gel by the light stimulus and its function was restored. Hence, this technique is applicable to the functional control of various molecules. The PI-encapsulated nanoparticles were internalized by the cells after being conjugated with the cell membrane permeability peptide, octaarginine, and were localized to the cytoplasm. Short-term irradiation (20 s) induced PI release from the nanoparticles and the rapid movement (less than 2 min) of the released PI to the nucleus. These nanoparticles are thus useful tools for the spatiotemporal control of various molecular functions because they permit the quick and transient release of encapsulated molecules after short-term irradiation and can be prepared under mild conditions.

A high-performance liquid chromatography method using a commercially available silica monolithic column for the simultaneous analysis of nanoparticles and small molecules was developed. The method uses the micrometer-sized flow-through pores and nanometer-sized mesopores of the monolithic column for separation: first, size separation of nanoparticles was performed by the micrometer-sized pores using the hydrodynamic mode, and then small molecules were separated by the nanometer-sized pores using the normal-phase mode. The method was used to evaluate and compare three existing methods for purifying nanoparticles and to analyse nanoparticle stability. The bimodal structure of the monolithic column is promising for the simultaneous separation of nanoparticles and small molecules.

This article summarizes detailed facts obtained from the questionnaire conducted in 2010 at about 14 National Universities on the topic of "Research programs and advanced educational programs for undergraduate students". The contents of the questionnaire included: (1) Research programs based on the coalition of university and hospital and/or community pharmacy, other Graduate Schools, such as School of Medicine etc., and the University Hospital, (2) Educational systems for the achievement of research programs and their research outcomes, (3) Research programs based on pharmacist practices, (4) Ongoing advanced educational programs for undergraduate students, taking advantage of the coalition with Graduate School, School of Medicine (and Dentistry), and University Hospital. Some of the advanced educational programs outlined in this questionnaire will be carried out by our group in the coming years and the educational benefits together with associated problems shall as well be clarified. This approach will be informative for the development of the leader-oriented pharmacist programs for the college of Pharmacy.

Peroxisomal biogenesis disorders represent a group of genetically heterogeneous conditions that have in common failure of proper peroxisomal assembly. Clinically, they are characterized by a spectrum of dysmorphia, neurological, liver, and other organ involvement. To date, mutations in 13 PEX genes encoding peroxins have been identified in patients with peroxisomal biogenesis disorders. Mutations in PEX13, which encodes peroxisomal membrane protein PEX13, are among the least common causes of peroxisomal biogenesis disorders with only three mutations reported so far. Here, we report on two infants whose clinical and biochemical profile was consistent with classical Zellweger syndrome and whose complementation analysis assigned them both to group H of peroxisomal biogenesis disorders. We show that they harbor two novel mutations in PEX13. One patient had a genomic rearrangement resulting in a 147 kb deletion that spans the whole of PEX13, while the other had an out-of-frame deletion of 14 bp. This represents the first report of a PEX13 deletion and suggests that further work is needed to examine the frequency of PEX13 mutations among Arab patients with peroxisomal biogenesis disorders.

Benzofurazan derivatization reagents, 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminopentylamino)-2,1,3-benzoxadiazole (DAABD-AP) and 4-[2-(N,N-dimethylamino) ethylaminosulfonyl]-7-(2-aminobutylamino)-2,1,3-benzoxadiazole (DAABD-AB), for short-chain carboxylic acids in liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) were synthesized. These reagents reacted with short chain carboxylic acids in the presence of the condensation reagents at 60 degrees C for 60 min. The generated derivatives were separated on the reversed-phase column and detected by ESI-MS/MS with the detection limits of 0.1-0.12 pmol on column. Upon collision-induced dissociation, a single and intense product ion at m/z 151 was observed. These results indicated that DAABD-AP and DAABD-AB are suitable as the derivatization reagents in LC/ESI-MS/MS analysis.

Benzofurazan derivatization reagent, 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-N-methylhydrazino-2,1,3-benzoxadiazole (DAABD-MHz), for aldehydes in liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS), was synthesized. DAABD-MHz reacted with aliphatic aldehydes under mild conditions. The generated derivatives were separated on a reversed-phase column and detected by ESI-MS/MS with detection limits of 30-60 fmol on-column. Upon collision-induced dissociation, a single and intense fragment ion at m/z 151 was observed. These results suggested that DAABD-MHz was suitable as a derivatization reagent in LC/ESI-MS/MS.

Kynurenine (KYN), a tryptophan metabolite, is a precursor of kynurenic acid, which is an antagonist of N -methyl- d -aspartate receptor. In this study, an enantiomeric separation of d,l -KYN derivatized with the benzofurazan fluorescence reagent 4- N,N -dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) (DBD- d,l -KYN) was first investigated by using a high-performance liquid chromatography (HPLC) with several chiral columns. As a consequence, DBD- d,l -KYN was enantiomerically separated on a cellulose-type chiral column (CHIRALCEL OJ-RH) with a mobile phase of H 2 O/CH 3 CN/MeOH (40/50/10) containing 0.1% acetic acid. Under this condition, the separation factor and resolution were 1.48 and 1.28, respectively. Next, a column-switching HPLC consisting of both octadecylsilica and chiral columns was developed and used to determine both d - and l -KYN enantiomers in 10 μL of rat plasma following the intraperitoneal administration of d , l -KYN to rats (10 mg kg −1 ). The result revealed that the concentration of l -KYN was higher than that of d -KYN, suggesting that d -KYN was eliminated faster than l -KYN.

Kynurenine (KYN), a tryptophan metabolite, is a crucial compound for modulating neurotransmission because it can be metabolized in vivo into both quinolinic acid and kynurenic acid, which are the agonist and antagonist, respectively, of N -methyl- d -aspartate receptor. For the highly sensitive detection of KYN by high-performance liquid chromatography (HPLC), a fluorescence derivatization of KYN with a benzofurazan-type fluorogenic reagent, 4- N , N -dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was investigated in the present study. KYN was derivatized with DBD-F (DBD-KYN) at 60 °C for 30 min, and separated on an octadecylsilica column with a gradient elution of the mobile phase, which consists of 0.1% formic acid in acetonitrile/methanol/water. DBD-KYN was detected fluorimetrically at 553 nm with an excitation wavelength of 431 nm. The limits of detection and quantification were approximately 0.30 pmol [signal-to-noise ratio (S/N) 3] and 1.0 pmol (S/N, 10) on column, respectively. Plasma KYN levels were successfully determined using 10 μL of rat plasma with satisfactory precision and accuracy. Intra- and inter-day precisions and accuracies were 1.7–6.8%, and −10 to 9.6%, respectively. KYN levels in plasma of male Sprague–Dawley rats (7 weeks old) were approximately 2.4 ± 0.32 μmol L −1 ( n = 4). The proposed HPLC method was applied to determine KYN levels in the plasma of ketamine-treated rats—the animal model of schizophrenia.

We describe a new approach applicable to the determination of organic acids that serve as diagnostic markers for several inherited metabolic disorders. We utilized liquid chromatography/tandem mass spectrometry for analysis of organic acid derivatives of a recently described benzofurazan reagent. The derivatization step was necessary to obtain organic acid derivatives suitable for analysis by reversed-phase liquid chromatography with high ionization efficiency for mass spectrometry in the positive-ion mode. In this work, a group of related dicarboxylic acid markers containing five or six carbon atoms were analyzed and validation was performed for glutaric and 3-hydroxyglutaric acids, the specific markers for glutaric acidemia type 1. Derivatization was achieved by reacting untreated urine with the derivatization reagent under mild conditions. The reaction mixture was analyzed on a C18 ultra-performance liquid chromatography (UPLC) column (50x2.1 mm, 1.7 microm) and detected in the multiple reaction monitoring mode in 5 min. Calibration curves were linear up to at least 1000 microM with detection limits for glutaric and 3-hydroxyglutaric acids of 0.025 and 0.02 microM, respectively (signal-to-noise ratio of 3). Intra-day (n=11) and inter-day (n=6) coefficients of variation were better than 11.2%. The assay was successfully applied to control (n=134) and glutaric acidemia type 1 (n=55) urine samples.

Estrogen receptor (ER) is a member of the nuclear receptor superfamily, and functions as a ligand-dependent transcription factor with roles in cell growth and differentiation. In addition to endogenous estrogen, 17beta-estradiol (E(2)) and artificial antagonists, many suspected environmental estrogenic chemicals are reported to bind to ER, with various affinities and transcriptional responses. ER is also an allosteric protein and shows a positive cooperative interaction with E(2). Cooperativity affects inter-subunit interaction, and while ligand-bound ER interacts with coactivators, antagonist-bound ER does not. We therefore hypothesized that ligand-binding characteristics influence coactivator recruitment to the ER dimer, and thereby affect transcriptional activity. We investigated the interaction between ER and human Steroid Receptor Coactivator-1 (SRC-1), in the presence of compounds exhibiting various Hill coefficients. In the case of both ER subtypes (ERalpha and ERbeta), the Hill coefficients of the compounds tested correlated with the affinity of the ER-ligand complex to SRC-1, with the exception of ERbeta-4-n-nonylphenol and ER-antagonist complexes. This is the first report to investigate the relationship between Hill coefficients of ligand binding and coactivator interaction with the ER-ligand complex. We also examined the proteolytic digestion of ER using trypsin, in the presence and absence of compounds with various Hill coefficients, to investigate ligand-dependent conformational changes in ER. We used not only agonists and antagonists but also compounds of weak biological activity (partial agonists). Our results shed light on the subtle modulation of transcriptional activation by chemical agents.

The applicability of benzofurazan derivatization regents to carboxylic acids analysis in LC/ESI-MS/MS (high-performance liquid chromatography/electrospray ionization tandem mass spectrometry) was examined. The product ion spectra of DAABD-AE {4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole}, DAABD-PZ {4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-N-piperazino-2,1,3-benzoxadiazole}, DAABD-PiCZ {4-[4-carbazoylpiperidin-1-yl]-7-[2-(N,N-dimethylamino)ethylaminosulfonyl]-2,1,3-benzoxadiazole}, DAABD-ProCZ {4-[2-carbazoylpyrrolidin-1-yl]-7-[2-(N,N-dimethylamino) ethylaminosulfonyl]-2,1,3-benzoxadiazole} and DAABD-Apy {4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole}, and their acetylated compounds were obtained. An intense fragment ion at m/z 151 corresponding to (dimethylamino)ethylaminosulfonyl moiety was observed in each spectra, suggesting that these reagents were suitable for ESI-MS/MS analysis. DAABD-AE, DAABD-APy and DAABD-PZ were applied to the analysis of octanoic acid and it was found that DAABD-AE and DAABD-APy gave high signal intensity suitable for LC/ESI-MS/MS. Copyright © 2007 John Wiley & Sons, Ltd.

A rapid and mild purification method for nanoparticles using the commercially available monolithic silica disk, MonoSpin(®), was developed. The nanoparticles were purified from a dispersed solution by filtration with the aid of centrifugation at 2290×g for 2min. The purification conditions were rapid, mild, and simple compared with those of the conventional purification methods such as ultracentrifugation, dialysis, size exclusion chromatography, and ultrafiltration. The method was shown to be applicable for the purification of various nanoparticles, regardless of their size (from 21 to 100nm), composition material (silica, polyethylene glycol, and pegylated liposome), and encapsulated molecule (rhodamine 110 and doxorubicin). It was shown that this method is applicable to the purification of a wide range of nanoparticles in many different fields.

New bi- and tricyclic deoxycytidine derivatives (dChpd, dCmpp, dCtpp, dCppp) were synthesized as analogues of a fluorescent nucleoside, dChpp, previously reported. The carbamoyl group of dChpd and the 5-position of the cytosine ring are bridged via an ethylene linker so that the modified group forms a nonplanar structure with the cytosine ring. The fluorescent study of dChpd indicated that the coplanar structure between the carbamoyl group and the cytosine ring is of importance. N-Methylation of the carbamoyl group (dCmpp) weakened the intensity of the fluorescence of dChpp, and the derivative (dCtpp), which had a thiocarbamoyl group, lost its fluorescent property. Moreover, addition of a pyrrolo-ring (dCppp) to dChpp enhanced the intensity of fluorescence, and an emission light was observed with a marked Stokes shift of 120 nm.

Kynurenic acid (KYNA), an endogenous antagonist of ionotropic glutamate and α7 nicotinic receptors, was fluorometrically determined by column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. The HPLC system consists of two octadecyl silica (ODS) columns, both of which are connected with an anion-exchange column (trapping column). Following sample injection onto the HPLC column, KYNA was separated on the first ODS column with a mobile phase of H 2 O/acetonitrile (95/5) containing 0.1% acetic acid. The peak fraction of KYNA was trapped on the anion-exchange column by changing the position of a six-port valve and then introduced into the second ODS column. Subsequently, KYNA was detected fluorometrically as a fluorescence complex formed with zinc ion which was pumped constantly. Instrumental limit of detection was approximately 0.16 nM, which corresponded to 8.0 fmol (per 50 μl injection, signal to noise ratio 3), and the limit of quantification was 0.53 nM (signal to noise ratio 10). Intra- and inter-day relative standard deviations were 1.1–3.9% ( n = 3) and 3.0–5.3% ( n = 3), respectively. The peak of KYNA in rat plasma was clearly detected by the proposed column-switching HPLC system after a facile pretreatment procedure. Intra- and inter-day relative mean errors were −1.6–1.4% ( n = 3) and −2.4 to −0.4% ( n = 3), respectively, with a satisfactory precision (within 5.0%). A calibration curve for the determination of KYNA showed a good linearity ( r 2 > 0.999) in the range of 25–200 nM. The KYNA concentrations in the plasma of male Sprague-Dawley rats (8-week-old) were 44 ± 5.5 nM (mean ± S.E., n = 5). In ketamine-treated rats, which are animal models of schizophrenia, the plasma KYNA concentrations were significantly increased compared with those in the control rats ( p

The derivatization of the reduced-form thiols with SBD-F (7-fluoro-2,1,3-benzoxadiazole-4-sulfonate) and ABD-F (4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole) was studied. The yields of the derivatives of the reduced-form thiols (cysteine, homocysteine, reduced-form glutathione) with SBD-F at 60 degrees C for 45 min in the borate buffer (pH 9.3) were significantly decreased in the presence of the oxidized-form thiols (cystine, homocystine, oxidized-form glutathione) because of the thiol exchange reaction between the reduced-form and the oxidized-form thiols. The use of ABD-F at low temperature enabled the suppression of these thiol exchange reactions, and the recommended conditions were below 5 degrees C for 90 min in borate buffer (pH 9.3). These results suggest that ABD-F is a preferred derivatization reagent for the accurate determination of the reduced-form thiols in samples containing the oxidized-form thiols. In addition, it was also suggested that the derivatization of the reduced-form thiols should also be performed at low temperature when derivatization reagents such as o-phthalaldehyde (OPA) and monobromobimane (BrB) are used.

It has been reported that the repeated administration of a sub-anesthetic dose of an N-methyl-D-aspartate receptor antagonist, ketamine, can produce an animal model of schizophrenia. Since no information is available on the alterations of the amino acid levels in ketamine-treated rats, we investigated the amino acid composition in the plasma and cerebrospinal fluid of rats that were repeatedly administered with ketamine for 5 consecutive days (30 mg/kg/day). The plasma and cerebrospinal fluid amino acid compositions in the fifth week after cessation of repeated ketamine administration were determined by highperformance liquid chromatography with fluorescence detection using a pre-column fluorescence reagent, i.e. 4-fluoro-7nitro-2,1,3-benzoxadiazole. Among the amino acids investigated in the present study, the level of plasma glutamic acid increased significantly (p < 0.05), while that of the cerebrospinal fluid glutamic acid decreased significantly in the ketamine-treated rats as compared with these levels in control rats injected with saline (p < 0.05, n = 7). These alterations in the glutamic acid level in the plasma and cerebrospinal fluid resemble those in schizophrenic patients, suggesting that ketamine-treated rats may be a useful model for performing research on the pathophysiology of schizophrenia.

Hydrophilic fluorescent derivatization reagents, sensors and probes are of great value because of their use in accurate analyses under water-rich condition. In this study, 13 7-substituted-2,1,3-benzoxadiazole-4-sulfonates (called SBD derivatives) as representatives of ionic benzofurazans were synthesized and their absorption and fluorescence spectra were obtained in solvents of varying polarity. The fluorescence quantum yields ( Φ f ) of the investigated compounds were significantly affected by the substituent at the 4-position. Solvent effects on the Φ f values were also observed. It was found that the combination of the PM3 and PM3-CAS/CI (CI = 6) methods afforded a good relationship between the experimental and calculated S 1 ← S 0 transition energies of the SBD derivatives.

We previously reported that therapeutic drug effects in the silkworm infection model are largely influenced by midgut permeability. In this report, we describe the effects of drug molecular mass and hydrophobicity on transport through the silkworm larva midgut membrane. Hydrophilic compounds with a molecular mass of greater than 400Da did not permeate the silkworm larva midgut, and the hydrophobicity of similar-sized compounds had positive effects on the transport rate. Furthermore, we compared transport rates through the midgut membrane between cefcapene sodium (CFPN-Na) and cefcapene pivoxil (CFPN-PI), which is a CFPN-Na prodrug. The in vitro transport rate of CFPN-PI was three times faster than that of CFPN-Na. Moreover, when CFPN-PI and CFPN-Na were injected into the living silkworm larva midgut, CFPN-PI appeared rapidly in the haemolymph, whereas CFPN-Na did not. The 50% effective dose (ED50) of CFPN-PI administered via the midgut was one-sixth that of CFPN-Na. These findings suggest that the general features of the non-specific transport route are similar between silkworm larvae and mammals.

The derivatization regents for carboxylic acids, DAABD-AE (4-[2-(N,N-dimethylamino)ethylaminosulfonyl]7-(2-aminoethylamino)-2,1,3-benzoxadiazole), MePZBD-AE ([4-(4-N-methyl)piperazinosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole) and APZBD-NHMe ([4-(4-N-aminoethyl)piperazinosulfonyl]-7-methylamino-2,1,3-benzoxadiazole) were developed for electrospray ionization-mass spectrometry (ESI-MS). The derivatization reaction with fatty acids was completed at 60 degrees C within 30 min. The derivatives of fatty acids were separated on a reversed-phase column and detected with ESI-MS. The detection limits attained for fatty acids were femtomol range and the calibration curves were linear over the range from 0.1 to 100 pmol (r2 > 0.992) for DAABD-AE and MePZBD-AE. DAABD-NHMe was applied to the analysis of fatty acids in rat plasma samples.

Background Asymmetric NG,NG-dimethylarginine (ADMA) is an endogenous inhibitor of endothelial nitric oxide (NO) synthase and its plasma concentration is elevated in patients with cardiovascular risk factors, including hyperlipidemia, hypertension, diabetes, and hyperhomocysteinemia. Obstructive sleep apnea syndrome (OSAS) has been attracting attention as a risk factor for cardiovascular disorders because it often accompanies hypertension, obesity, glucose impairment, and dyslipidemia, all of which are factors in metabolic syndrome and risk factors for cardiovascular disease. Methods and Results In the present study, flow-mediated vasodilatation (FMD) of the brachial artery and plasma concentrations of ADMA were measured before and after nasal continuous positive airway pressure (nCPAP) therapy, which abrogates apnea, in 10 male patients aged 36-69 years old, who were given a diagnosis of OSAS by polysomnography. The percent FMD (%FMD) improved significantly from 3.3±0.3% to 5.8±0.4% (p

The injection of bacteria ( Staphylococcus aureus , Stenotrophomonas maltophilia ) or true fungi ( Candida albicans , Candida tropicalis ) that are pathogenic to humans into the silkworm hemolymph leads to death of the larvae within 2 days. Antibiotics used for clinical purposes have therapeutic effects on silkworms infected with these pathogens. The 50% effective doses obtained by injection into the silkworm hemolymph are consistent with those reported for mice. Injection of vancomycin and kanamycin into the silkworm hemolymph was effective, but oral administration was not. Chloramphenicol, which is effective by oral administration, appeared in the silkworm hemolymph soon after injection into the midgut, whereas vancomycin did not. Isolated midgut membranes were impermeable to vancomycin. Thus, the ineffectiveness of oral administration of vancomycin to silkworms is due to a lack of intestinal absorption.

A fully automated amino acid analyzer using NBD-F (4- fluoro-7-nitro-2,1,3-benzoxadiazole) as a fluorescent derivatization reagent was developed. The whole analytical process was fully automated from derivatization, injection to HPLC separation and quantitation. The derivatization reaction conditions were re-evaluated and optimized. Amino acids were derivatized by NBD-F for 40 min at room temperature in the borate buffer (pH 9.5). The derivatives were separated within 100 min and fluorometrically detected at 540 nm with excitation at 470 nm. The detection limits for amino acids were in the range of 2.8-20 fmol. The calibration curves were linear over the range of 20 fmol to 20 pmol on column with the correlation coefficients of 0.999. The coefficients of variation were less than 5% at 3 pmol injection for all amino acids. Amino acids in rat plasma were determined by the proposed HPLC method.

In this study, we developed a rapid, simple and homogeneous human recombinant estrogen receptor-beta (hrER-beta) binding assay method using fluorescence polarization (FP) by applying a fluorescent ligand, fluorescein-labeled estradiol (F-E2). A Scatchard plot and a Hill plot analysis of the saturation binding assay using F-E2 and hrER-beta were studied. F-E2 showed a high affinity for hrER-beta, the dissociation constant was 5.53 nM, indicating that F-E2 is applicable to the hrER-beta binding assay. Competitive binding assays using F-E2, in which the FP values decreased upon the addition of compounds (competitors) were carried out to evaluate the binding characteristics of compounds with and without biological activities to hrER-beta. Twenty-one compounds, such as hormones, pharmaceuticals, industrial chemicals and phytoestrogens, were examined. The obtained sigmoidal inhibition curves were transformed into pseudo-Hill plots and the concentrations at 50% inhibition (IC50) and Hill coefficients, the degree of cooperativity in ER-ligand binding, were obtained from the regression equations. Antagonists exhibited larger Hill coefficients than agonists, showing the correlation between the Hill coefficients and the estrogenic/antiestrogenic activities.

Many fluorescent reagents with a benzofurazan (2,1,3-benzoxadiazole) skeleton have been developed and widely used in bio-analyses. In this study, we try to elucidate the fluorescence on-off switching mechanism of three fluorogenic reagents and their derivatives. Ten 4,7-disubstituted benzofurazans were used for this purpose and the measurements of their fluorescence, phosphorescence, photolysis, and time-resolved thermal lensing signal in acetonitrile were obtained in order to understand the relaxation processes of these compounds. These results indicate that the competition of fluorescence with a fast intersystem crossing or fast photoreaction plays a key role in the fluorescence on-off switching. Semi-empirical molecular orbital calculations show that the existence of the triplet n pi* state is responsible for the fast intersystem crossing while the proximity of the reactive second single pi pi* state to the first singlet pi pi* state contributes to the fast photoreaction in the excited states.

The derivatization procedure with a suitable fluorescence or chemiluminescence reagent is performed for the purpose of increasing the detection sensitivity and selectivity, in high-performance liquid chromatography (HPLC) and/or capillary electrophoresis (CE). In this article, recent derivatization methods and their applications to biosamples are described. In HPLC, femto mol order of mass detection limits are obtained by derivatization. Regarding the fluorescence reagents, the use of water-soluble reagents has been effective to avoid an undesired adsorption in the process of determination of peptides. In CE, the advantages of having extremely low mass detection limits (ranging from atto to yocto mol level) and requiring only a very short analysis time (less than a few minutes) are made possible by using laser-induced fluorescence or near infra-red detections.

7-Fluoro-2,1,3-benzoselenadiazole-4-sulfonate (SBSeD-F) and 7-fluoro-2,1,3-benzothiadiazole-4-sulfonate (SBThD-F) were synthesized and proposed as the water-soluble fluorescent derivatization reagents for thiols bearing anew type of fluorophores (benzoselenadiazole and benzothiadiazole). The maximum excitation wavelengths of the derivatives of cysteine with SBSeD-F (340 nm) and SBThD-F (315 nm) were shorter than that with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F) (365 nm), respectively, while the maximum emission wavelength with SBSeD-F (542 nm) was longer than those with SBThD-F (517 nm) and SBD-F (514 nm). The time required for the quantitative derivatization of cysteine with SBSeD-F or SBThD-F was less than 24 h at pH 10.0 and 60 °C, while the derivatization was achieved within 1 h with SBD-F. The appropriate derivatization condition was settled with 2 mM SBSeD-F or SBThD-F as 24 h reaction at 60 °C in 0.1 M borate buffer (pH 10.0) in the presence of 0.5 mM EDTA.

Nine 5-substituted benzofurazans including the non-substituted benzofurazan were synthesized, and measurements of the fluorescence, photolysis, transient absorption, and time-resolved thermal lensing signal were performed in order to understand the relaxation processes of these compounds. The results indicated that the main relaxation process was a photoreaction from an excited singlet state and the rate of the primary bond cleavage in the excited state tended to increase in the compound in which the S2 ← S0 absorption band is located close to the S1 ← S0 absorption band. These results suggest that the reactive state might be the S2 state, and that the interaction between the S1 and S2 states promotes the photoreaction. The separation of the S1 state from the S2 state decreases the rate of photoreaction, resulting in an increase in the Φf values. The Φf values of the 5-substituted benzofurazans in cyclohexane and acetonitrile were compared with their ΔE(S1, S2) (energy gap between the S1 and S2 states) values calculated by using a combination of AM1(EXCITED) and AM1-CAS/CI(CI = 6) for geometric optimization and calculation of the energy levels, respectively. The ΔE(S1, S2) values correlated well with the Φf values in each solvent, thus enabling us to predict the fluorescence properties of the 5-substituted benzofurazans based on their chemical structures.

A rapid, simple and nonhazardous assay method for endcrine disruptors was developed using an estrogen receptor (ER) and fluorescence polarization (FP). Among the fluorescent compounds, the 17alpha-fluorescein-labeled estradiol derivative was selected as the most suitable ligand for the ER binding assay, since it showed the highest affinity to ER. In the Scatchard plot analysis, its convex curve exhibited a positive cooperative binding, indicating the induction of a conformational change of the ER with the binding of the ligand to form a dimer and to increase the affinity for the additional ligand. On the basis of the Hill plot analysis, its dissociation constant and Hill coefficient were 10.4 nM and 1.63, respectively. A competitive binding assay with an unlabeled 17beta-estradiol (E2) yielded an IC50 value of 2.82 nM and a Hill coefficient of 1.67, thus providing a Ki value of 0.65 nM. In the same manner, the Hill coefficients for estrone, estriol, diethylstilbestrol, and tamoxifen were determined to be 0.99, 1.17, 1.59, and 2.44, respectively.

We studied the effects of spacer length on the fluorescence quantum yields (Φ) of photoinduced electron transfer (PET) reagents, using nitrobenzoxadiazole (NBD) derivatives that have the –NMe2 moiety and NBD–NH– fluorophore as electron donor (D) and electron acceptor (A), respectively. The Φ values were reduced as the spacer length became shorter (n ≤ 4; n is the number of methylene units of the spacer) and the fluorescence recovered by suppression of the PET process. It is necessary for the useful PET reagents to link D and A with a short spacer to obtain a difference in the Φ values between fluorescent ‘off-state’ and ‘on-state’. Copyright © 2002 John Wiley & Sons, Ltd.

A hydrophilic fluorescent derivatization reagent for fatty acids, 4-N-(4-N-aminoethyl)piperazino-7-nitro-2,1,3-benzoxadiazole (NBD-PZ-NH2), was designed and synthesized. NBD-PZ-NH2 possesses not only a fluorophore and a reacting group but also a positive charge group and, thus, was hydrophilic and suitable for application to capillary electrophoresis. NBD-PZ-NH2 reacted with fatty acids in the presence of triphenylphosphine (TPP) and 2,2′-dipyridyl disulfide (DPDS) at room temperature within 10 min. The derivatives were strongly fluoresced and were positively charged at pH below 3. The derivatives of C4-C20 fatty acids were separated within 10 min in 50% acetonitrile in water containing 30 mM ammonium acetate and 1.0 M acetic acid by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. The detection limits attained were 6.5 nM (signal-to-noise ratio of 3). It is proposed that NBD-PZ-NH2 is a prominent derivatization reagent for fatty acids which is suitable for CE-LIF application. Copyright © 2002 John Wiley & Sons, Ltd.

Nanoparticles, spherical particles with diameters less than 100 nm, are promising theranostic devices for noninvasive diagnosis and therapy. In this study, nanoparticles composed of polyethylene glycol and silica were prepared, and their migration behavior was examined using capillary electrophoresis. The effects of the sodium dodecyl sulfate concentration in the electrolyte, the nanoparticle size, and the encapsulated molecule on the migration were examined. The addition of sodium dodecyl sulfate into the electrolyte had a significant effect on the electrophoretic mobility of polyethylene glycol nanoparticles, but a small effect on that of silica nanoparticles. As for the size effect, the mobility became a little faster for smaller nanoparticle sizes for both polyethylene glycol and silica nanoparticles. The encapsulated molecule affected the mobility of the nanoparticles through interactions between the encapsulated molecules and sodium dodecyl sulfate. We propose that the large effect of sodium dodecyl sulfate on the migration of the polyethylene glycol nanoparticles was due to the large spaces within the nanoparticles. These results indicate that nanoparticle migration is mainly determined by the nanoparticle components.

We have developed a general method for controlling molecular functions using a photodegradable hydrogel; gels containing molecules made from such materials are capable of release and activation by light stimulus. As the elimination of molecular leaching from the gel before irradiation was a barrier to the precise control of molecular function, we optimized the monomer used in gel preparation during this study. The addition of N,N'-methylenebis(acrylamide) (MBAA) inhibited molecular leaching from the gel; the MBAA concentration is a critical factor in controlling the leaching of encapsulated molecules. We succeeded in preparing a gel that halved the leaching of small encapsulated molecules, while the leaching of large molecules, such as albumin (66 kDa) and ferritin (450 kDa), was at negligible levels, or disappeared. The on/off ratios (released amount/leached amount) of albumin and ferritin were 8 and 17, respectively.

We used a coarse-grained (CG) molecular dynamics model with potentials convertible to actual units to simulate the polymerization of a gel of a tetra-armed poly(ethylene glycol) derivative (MW ≈ 6000) under aqueous conditions and analysed its three-dimensional network structure. The radius of gyration of individual pre-polymers after gelation was slightly increased compared with that of the single pre-polymer before gelation, and its distribution was broad, attributable to inter- and intra-molecular bonds. The largest pores in the unit cell were about 3.5–3.9 nm. The existence of large pores seems to explain the protein encapsulation capability of and protein leakage from the gel indicating that the CG simulation, which maintains information about potentials in actual units, is an effective tool for investigating gel properties that are difficult to measure in real experiments.

A fully automated analyzer for methylated L-arginine metabolites [N,N-dimethyl-L-arginine (ADMA), N-methylarginine (NMMA) and N,N'-dimethyl-L-arginine (SDMA)] by high-performance liquid chromatography with post-column fluorescence derivatization was developed. This system consists of an on-line extraction, a separation on a reversed phase ion-pair chromatograph, a post-column derivatization by o-phthaladehyde (OPA) and thiol reaction, and fluorescence detection. NMMA, ADMA and SDMA were separated in 40 min with isocratic elution by a combination of octanoate and cyclohexane carboxylate as ion-pair reagents. The eluate was monitored at 450 nm with excitation at 337 nm. The calibration curves for NMMA, ADMA and SDMA showed linearity over the range from 0.05 micromol l(-1) (0.5 pmol on column) to 5.0 micromol l(-1) (50 pmol on column). This method does not require any time-consuming pre-treatment and requires only 10 microl of plasma sample for assay.

A modified Edman procedure using phenylisothiocyanate (PITC) and boron trifluoride (BF3)–methanol for amino acid sequencing and d / l configuration determination was developed. The utilization of BF3–methanol solution resulted in the direct generation of phenylthiohydantoin (PTH)-amino acids from phenylthiocarbamoyl (PTC)-peptide excluding the extraction step after cyclization/cleavage reaction. BF3–methanol as a cyclization/cleavage/conversion reagent in the Edman sequencing procedure afforded a great suppression of the racemization of liberated PTH-amino acids avoiding the extraction step.

D-amino acid-containing peptides with biological activities have been isolated from invertebrates and amphibians, and partial racemization of amino acid residues in mammalian peptides associated with aging and diseases have been discussed. Here, we review the amino acid configuration determination methods in these peptides and recent progress of simultaneous determination method for sequence and configuration of amino acid residues. The applicability of C-terminus sequence analysis and mass spectrometry to configuration determination of amino acids is also discussed.

Fluorogenic and fluorescent labeling reagents having a benzofurazan (2,1,3-benzoxadiazole) skeleton such as 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F), 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F), ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H), 4-N,N-dimethylaminosulfonyl-7-hydrazino-2,1,3-benzoxadiazole (DBD-H), 4-nitro-7-N-piperazino-2,1,3-benzoxadiazole (NBD-PZ), 4-N,N-dimethylaminosulfonyl-7-N-piperazino-2,1,3-benzoxadiazole (DBD-PZ), 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl) and 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) are reviewed in terms of synthetic method, reactivity, fluorescence characteristics, sensitivity and application to analytes.

A modified Edman sequencing method using 7- N , N -dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) for amino acid sequence and d / l -configuration determination has been developed. DBD-thiazolinone (TZ)-amino acids generated by Edman sequencing method were hydrolyzed and then oxidized to DBD-carbamoyl (CA)-amino acids, instead of the conversion reaction to thiohydantoin (TH)-amino acid in the conventional Edman method. The oxidative desulfuration step operated manually with formation of DBD-CA-amino acids was difficult to control because of the production of by-products by the side-chain oxidation of amino acid and inapplicability to the conventional automated protein sequencer. Therefore, post-column desulfuration method in which the reaction was carried out after the separation of analyte on a column was designed. DBD-thiocarbamoyl (TC)-amino acids generated by the modified sequencing method were separated on the reversed phase or Pirkle type chiral stationary phase column and then oxidized to DBD-CA-amino acids with hydrogen peroxide as a post-column reagent and detected fluorimetrically. Using the method, the determination of the amino acid sequence and d / l -configuration of a d -amino acid containing peptide was achieved and gave us the recognition to the utility of the method.