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Benzbromarone has been used for the treatment of gout for more than 30 years. Although it shows a high level of binding to plasma proteins (>99%), our knowledge of this binding is not sufficiently extensive to permit us to understand its pharmacokinetics and pharmacodynamics. To address this issue in more detail, we characterized the binding of benzbromarone to human serum albumin (HSA), the most abundant protein in plasma. Equilibrium dialysis and circular dichroism findings indicated that benzbromarone binds strongly to one primary as well as to multiple secondary sites on HSA and that the bromine atoms of benzbromarone play important roles in this high affinity binding. An X-ray crystallographic study revealed that benzbromarone molecules bind to hydrophobic pockets within subdomains IB, IIA, and IIIA. Inhibition experiments using site specific ligands (subdomain IB; fusidic acid, IIA; warfarin, IIIA; diazepam) indicated that the primary and secondary binding sites that benzbromarone binds to are within subdomains IIIA and IB/IIA, respectively. Lastly, a study of the effect of fatty acids on the benzbromarone-HSA interaction suggested that benzbromarone, when displaced from subdomain IIIA by sodium oleate, could transfer to subdomains IB or IIA. Thus, these data will permit more relevant assessments of the displacement interactions of benzbromarone especially in cases of co-administered drugs or endogenous compounds that also bind to subdomain IIIA. In addition, the findings presented herein will also be useful for designing drug combination therapy in which pharmacokinetic and pharmacodynamic performance need to be controlled.

We recently reported that aripiprazole binds strongly to human albumin. In continuing our investigations, we investigated the mechanism responsible for the binding and the related interactions of aripiprazole with α1-acid glycoprotein (AGP). The extrinsic Cotton effects for the binding of aripiprazole and its derivatives to AGP were generated, but the magnitudes of the induced circular dichroism intensities did not correlate with those for the binding affinities. It therefore appears that the binding mode of aripiprazole with AGP is somewhat complicated, compared with that of albumin. Isothermal titration calorimetry data obtained for the binding of aripiprazole with AGP were different from that for albumin systems in that the 3 driving reactions, entropy-driven, enthalpy-driven, and the entropy-enthalpy mixed type, were all found for the AGP system, but not albumin. Moreover, the weak binding mode of aripiprazole with the 2 proteins were supported by a molecular docking model analysis. The concentration of albumin in plasma is about 50 times higher than those of AGP, but AGP levels in plasma are increased by about 10 times under inflammatory disease. Therefore, the involvement of these 2 plasma proteins should be considered in more depth for understanding the pharmacokinetics of aripiprazole.

Nanosize plasma proteins could be used as a biomimetic drug delivery system (DDS) for cancer treatment when loaded with anticancer drugs based on the fact that plasma proteins can serve as a source of nutrients for cancer cells. This prompted us to investigate the potential of α1-acid glycoprotein (AGP) for this role because it is a nanosize plasma protein and binds a variety of anticancer agents. Pharmacokinetic analyses indicated that AGP is distributed more extensively in tumor tissue than human serum albumin, which was already established as a cancer DDS carrier. AGP is possibly being incorporated into tumor cells via endocytosis pathways. Moreover, a synthetic AGP-derived peptide which possesses a high ability to form an α-helix, as deduced from the primary structure of AGP, was also taken up by the tumor cells. AGP loaded with anticancer agents, such as paclitaxel or nitric oxide, efficiently induced tumor cell death. These results suggest that AGP has the potential to be a novel DDS carrier for anticancer agents.

Conjugated network polymer emitters are potential materials for use as solid detectors of chemicals. We demonstrated the synthesis of fluorescent network polymers through a facile route. Solid-state fluorescent network polymer emitters were synthesized through the Knoevenagel polycondensation of an arylaldehyde (tris(p-formylphenyl)amine) with an arylacetonitrile (phenylenediacetonitrile) in good yields. The molecular structure based on the electron-donor triphenylamine and electron-acceptor cyano-substituted phenylene-vinylene showed a highly efficient solid-state fluorescence. The synthesized model small molecule showed a well-defined solvatofluorochromism: the dielectric constants were esol of approximately 0–50 at λfl of 480–560 nm. The response to solvent chemicals was also shown for the network polymers. Surprisingly, the network polymer linked with para-phenylene-vinylene was only responsive to the solvents with a low dielectric constant (esol ca.

In this study, we report the synthesis of π-conjugated network polymers including unique fluorescent units via palladium-catalyzed direct (CH) arylation polycondensation of 1,2,4,5-tetrafluorobenzene with tetrabromoarenes. The obtained polymers, including tetraphenylethene (TPE) or pyrene (PYR) units, had microporous structures with the specific Brunauer–Emmett–Teller (BET) surface areas at 508 and 824 m2 g−1, respectively. These polymers possessed narrow pore distributions (

Mycoplasma bovis is a significant pathogen in bovine infections including mastitis, pneumonia, arthritis and otitis media, and is the cause of large economic losses in beef and dairy farms. During infection with M. bovis, recruited neutrophils are not sufficient to eradicate M. bovis from the infection site. The release of neutrophil extracellular traps (NETs) is one of the innate immune responses of neutrophils but the effect of M. bovis on NET formation by bovine neutrophils has not yet been clarified. The objective of our research was to examine the effect of M. bovis on NET formation and the killing activity of bovine neutrophils. We showed that NETs were not detected following stimulation of neutrophils by M. bovis alone or with Phorbol 12-myristate 13-acetat (PMA). Reactive oxygen species production is essential for NET formation but the levels in neutrophils stimulated with M. bovis at multiplicity of infections of 10, 100, and 1000 were similar to those of unstimulated cells. NET formation induced by PMA stimulated neutrophils disappeared following the addition of M. bovis but this phenomenon was not observed when ethylenediaminetetraacetic acid (EDTA) was added. M. bovis colony forming units were significantly decreased by the addition of EDTA in the presence of NETs. Our results suggested that M. bovis infection alone did not induce NETs and that M. bovis nucleases, as hypothesis-based, contributed to resistance against the killing activity of NETs.

Albumin is widely conserved from vertebrates to invertebrates, and nature of mammalian albumins permit them to bind various endogenous ligands and drugs in the blood. It is known that at least two major ligand binding sites are present on the albumin molecule, which are referred to as Site I and Site II. These binding sites are thought to be almost completely conserved among mammals, even though the degree of binding to these sites are different depending on the physical and chemical properties of drugs and differences in the microenvironment in the binding pockets. In addition, the binding sites for medium and long-chain fatty acids are also well conserved among mammals, and it is considered that there are at least seven binding sites, including Site I and Site II. These bindings properties of albumin in the blood are also widely known to be important for transporting drugs and fatty acids to various tissues. It can therefore be concluded that albumin is one of the most important serum proteins for various ligands, and information on human albumin can be very useful in predicting the ligand binding properties of the albumin of other vertebrates.

We have successfully synthesized π-conjugated microporous polymers (CMPs) via direct arylation of fluoroarenes with 2,4,6-tris(4-bromophenyl)-1,3,5-triazine. Both 1,2,4,5-tetrafluorobenzene and 1,3,5-trifluorobenzene were good monomers to give the networks, CMP1 and CMP2, respectively. Characterization of the insoluble materials was successfully achieved by high performance solid-state 13C and 19F CP/MAS NMR spectroscopy. The signals appeared in the aromatic region. No end group signals were observed in the 13C NMR spectrum of CMP1, but CMP2 showed the unreacted C–H signal of the trifluorobenzene unit. CMP2 showed higher Brunauer–Emmett–Teller (BET) surface area than CMP1. The number of branch controlled (or tailored) the porosity of the polymer networks. The polymers have microporocity with CO2 capture capacity. The CO2 capture capacity of CMP2 (16.75 cm3 g-1) was higher than that of CMP1 (11.49 cm3 g-1) at 1.0 bar.

Background The ultraviolet A (UVA) spectrum mainly includes the region associated with the phototoxicity of fluoroquinolone antimicrobial agents. This study investigated apoptosis induced with UVA light and enoxacin in HL-60 cells. Materials and methods HL-60 cells were irradiated by UVA (1.1 mW/cm2) for 20 min in the presence or absence of enoxacin. The induction of apoptosis was investigated by analysing cell morphology, flow cytometry of annexin V-positive cells, DNA ladder formation, and caspase-3 activation. Results Significant induction of apoptosis, DNA fragmentation, and caspase-3 activation were observed in cells treated with both UVA and enoxacin. UVA-induced apoptosis was significantly suppressed when NaN3, a singlet oxygen scavenger, was present. Conclusion Apoptosis was induced by the combination of UVA and enoxacin in HL-60 cells, and singlet oxygen appears to play an important role in photodynamically-induced apoptosis.

Lomefloxacin (LFX) is a widely used fluoroquinolone antimicrobial agent that plays an important role in the treatment of human and animal infections; however, it has been reported to cause phototoxicity. In this study, we investigated the induction of apoptosis due to ultraviolet A (UVA) light in the presence and absence of LFX in HL-60 human promyelocytic leukemia cells. HL-60 cells were exposed to UVA at an intensity of 1.1 mW/cm2 for 20 min in the presence and absence of LFX, and the induction of apoptosis was examined by analyzing cell morphology, DNA fragmentation, and caspase-3 activity. Cells treated with 100 μM LFX and UVA clearly showed membrane blebbing and cell shrinkage. The proportion of apoptotic cells was significantly higher in cells treated with both UVA and LFX than in those treated with UVA or LFX alone. In addition, DNA ladder formation and caspase-3 activation were observed in cells treated with both UVA and LFX. A significant reduction in the number of UVA-induced apoptotic cells and caspase-3 activation was observed when histidine was present, which suggested that photodynamically-generated singlet oxygen is an important mediator of apoptosis. These results indicate that the combination of UVA and LFX induces apoptosis in HL-60 cells.

Pancreatic cancer is one of the deadliest human cancers. In the current study, we investigated the possibility of a new treatment strategy using a combination of the new fluoroquinolone, enoxacin, and mild ultraviolet A (UVA) irradiation. Enoxacin with UVA irradiation increased the number of annexin V-positive (apoptotic) pancreatic cancer cells in time- and concentration-dependent manners, whereas alone neither had these effects. In addition, enoxacin with UVA irradiation induced cleavage of poly (ADP-ribose) polymerase in AsPC1 human pancreatic cancer cells. Moreover, the singlet oxygen scavengers, histidine and sodium azide, and the hydroxyl radical scavenger, mannitol, significantly suppressed apoptosis induced by enoxacin and UVA irradiation, respectively. These results suggest that UVA irradiation activates enoxacin, after which activated enoxacin induces apoptosis of AsPC1 cells through generation of reactive oxygen species. Therefore, the combination of enoxacin with mild UVA irradiation may be a useful method for treating pancreatic cancer.

Human α(1) -acid glycoprotein (AGP), a serum glycoprotein, is known to have anti-inflammatory activity. We recently reported that AGP was mainly incorporated into the liver in mice via a receptor-mediated pathway, although the mechanism for this was largely unknown. The objective of this study was to identify the specific cellular surface protein that recognizes the peptide moiety of AGP. Pharmacokinetic studies of (111) In-AGP and (111) In -recombinant glycan-deficient AGP (rAGP) in mice demonstrated that both AGPs are mainly distributed to the liver and kidney, but hepatic and renal uptake clearance of rAGP was higher than that for AGP. Hepatic uptake of rAGP was inhibited in the presence of 100-fold excess of unlabeled AGP, indicating that the hepatic uptake of rAGP shared a common route with that of AGP and that it recognized the peptide moiety of AGPs. In ligand blotting analyses using crude cellular membrane fraction of mice liver, a band corresponding to a 16 kDa protein was observed to bind to both AGPs. Interestingly, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry MALDI-TOF-MS and western blotting analyses indicated that this 16 kDa protein is the hemoglobin β-chain (HBB). It, therefore, appears that HBB is associated with the hepatic uptake of AGP via a direct interaction with its peptide moiety.

7-Hydroxystaurosporine (UCN-01) is a protein kinase inhibitor anticancer drug currently undergoing a phase II clinical trial. The low distribution volumes and systemic clearance of UCN-01 in human patients have been found to be caused in part by its extraordinarily high affinity binding to human alpha1-acid glycoprotein (hAGP). In the present study, we photolabeled hAGP with [3H]UCN-01 without further chemical modification. The photolabeling specificity of [3H]UCN-01 was confirmed by findings in which other hAGP binding ligands inhibited formation of covalent bonds between hAGP and [3H]UCN-01. The amino acid sequence of the photolabeled peptide was concluded to be SDVVYTDXK, corresponding to residues Ser-153 to Lys-161 of hAGP. No PTH derivatives were detected at the 8th cycle, which corresponded to the 160th Trp residue. This strongly implies that Trp-160 was photolabeled by [3H]UCN-01. Three recombinant hAGP mutants (W25A, W122A, and W160A) and wild-type recombinant hAGP were photolabeled by [3H]UCN-01. Only mutant W160A showed a marked decrease in the extent of photoincorporation. These results strongly suggest that Trp-160 plays a prominent role in the high affinity binding of [3H]UCN-01 to hAGP. A docking model of UCN-01 and hAGP around Trp-160 provided further details of the binding site topology.

Alpha(1)-acid glycoprotein (AGP) consists of 183 amino acid residues and 5 carbohydrate chains and binds to basic and neutral drugs as well as steroid hormones. We investigated the structural properties and ligand-binding capacity of AGP under mild acidic conditions and its interactions with liposomes prepared from neutral or anionic lipids and the neutral drug, progesterone. Interestingly, AGP had a unique structure at pH 4.5, at which the tertiary structure changed, whereas the secondary structure remained intact. Furthermore, the binding capacity of AGP for progesterone did not significantly change under these conditions. It was also observed that AGP was strongly bound to the anionic membrane at pH 4.5, forming an alpha-helix-rich structure from the original beta-sheet-rich structure, which significantly decreased the binding capacity of AGP for progesterone. The structural transitions as well as the membrane binding were suppressed by adding NaCl. These results indicate that AGP has a unique structure on the membrane surface under mild acidic conditions. The conformational change induces binding to the membrane aided by electrostatic interaction, and AGP subsequently takes on a predominantly alpha-helical conformation.

Human alpha1-acid glycoprotein (hAGP) is a plasma glycoprotein that functions as a major carrier of basic ligands. This is the first report of the recombinant hAGP (rhAGP). In this study, rhAGP was expressed in the methylotropic yeast Pichia pastoris (GS115) using the expression vector, pPIC9, and then purified by anionic exchange, hydrophobic interaction, and gel filtration chromatography. The molecular weight of rhAGP was much lower than that of hAGP, because of the difference in glycan chain content. Results of glycopeptidase F digestion suggest that the peptide moiety of rhAGP was the same as that of hAGP. The results of circular dichroism spectra measurement indicated that rhAGP predominantly formed a beta-sheet-rich structure that was the same as that of hAGP and typical of the lipocalin family. From the experiments using AGP-binding drugs (chlorpromazine, warfarin, and progesterone) and quinaldine red as a probe for the binding site, it was indicated that rhAGP also had the same ligand-binding capacity and binding site structure as hAGP. These findings strongly suggest that this recombinant hAGP (rhAGP) is very useful for the exploration of the ligand-binding site and biological function of hAGP.

Alpha(1)-acid glycoprotein (AGP) is a glycoprotein that consists of 183 amino acid residues and five carbohydrate chains and binds to neutral and basic drugs. We examined the structural properties and ligand-binding capacity of AGP in interactions with reverse micelles. Also, detailed information was obtained by comparing several different states of AGP. Interaction with reverse micelles induced a unique conformational transition (beta-sheet to alpha-helices) in AGP and decreased the binding capacity for the basic drug, chlorpromazine and the steroid hormone, progesterone to AGP. These structural conformations are very similar to those observed under conditions of acidity and high ionic strength (pH 2.0, 1.5 M NaCl). This structure seems to be an intermediate between the native state and the denatured state, possibly a molten globule. The present results suggest that when AGP interacts with the biomembrane, it undergoes a structural transition to a unique structure that differs from the native and denatured states and has a reduced ligand-binding capacity.

The goal of this study was to classify and identify the ligand binding sites on alpha(1)-acid glycoprotein (AGP) from 3 species, in order to understand species differences with respect to both ligand binding properties and ligand interaction on protein binding. These characteristics of human, dog and bovine AGP were examined using the basic ligands chlorpromazine and auramine O, the acidic ligand acenocoumarin, and the steroid hormone progesterone. Ultrafiltration and fluorescence techniques were used to characterize the nature of the interactions, and the data were analyzed according to the method of Kragh-Hansen. Using a model analysis of the interaction, the ligand binding site on human AGP consists of at least 3 partially overlapping subsites: a basic ligand binding site, an acidic ligand binding site and a steroid hormone binding site. Moreover, dog and bovine AGP each have a basic ligand binding site and a steroid hormone binding site, which significantly overlap and affect each other. However, dog and bovine AGPs do not contain an acidic ligand binding region. The results of the fluorescence experiments indicate that the hydrophobic nature of the ligand binding pockets on the 3 AGPs are similar, but that their microviscosities are markedly different.

Acyl-coenzyme A:cholesterol acyltransferases (ACATs), which are members of the membrane-bound O-acyltransferase family, catalyze the conversion of cholesterol to cholesteryl esters. Mammals have two isoenzymes: ACAT1 and ACAT2. Both enzymes are drug targets for treating human diseases. ACAT1 is present in various cell types. It contains nine transmembrane domains (TMDs), with the active site His460 located within TMD7, and the active site Asn421 located within the fourth large cytoplasmic loop. In human ACAT1, a single-nucleotide polymorphism exists for residue 526: the codon is either CAG for Gln, or CGG for Arg. Gln526/Arg526 is present within the C-terminal loop. Its biochemical significance is unknown. In addition, within the C-terminal half of ACAT1, numerous residues conserved with those of ACAT2 are present; the functions of these conserved residues are largely unknown. Here, we performed single-substitution mutagenesis experiments to investigate the roles of individual residues present in the C-terminal loop, including Gln526/Arg526, and the eight conserved Pro residues located near/in various TMDs. The results show that the enzyme activity of ACAT1 with Gln526 is less active than that of ACAT1 with Arg526 by 40%. In addition, several residues in the C-terminal loop are important for maintaining proper ACAT1 protein stability. Other results show that Pro347 plays an important role in modulating enzyme catalysis. Overall, our results imply that the CAG/CGG polymorphism can be utilized to perform ACAT1 activity/human disease susceptibility studies, and that Pro347 located near TMD5 plays an important role in modulating enzyme catalysis.

Background: In this study, we investigated the induction of apoptosis by ultrasound in the presence of the photochemically active chlorine, 4-formyloximeetylidene-3-hydroxyl-2-vinyl-deuterio-porphynyl (IX)-6-7-diaspartic acid (ATX-S10). Methods: HL-60 cells were exposed to ultrasound for up to 3 min in the presence and absence of ATX-S10, and the induction of apoptosis was examined by analyzing cell morphology, DNA fragmentation, and caspase-3 activity. Results: Cells treated with 80μM ATX-S10 and ultrasound clearly showed membrane blebbing and cell shrinkage, whereas significant morphologic changes were not observed in cells exposed to either ultrasound or ATX-S10 alone. Also, DNA ladder formation and caspase-3 activation were observed in cells treated with both ultrasound and ATX-S10 but not in cells treated with ultrasound or ATX-S10 alone. In addition, the combination of ATX-S10 and the same acoustical arrangement of ultrasound substantially enhanced nitroxide generation by the cells. Sonodynamically induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine. Conclusions: These results indicate that the combination of ultrasound and ATX-S10 induces apoptosis in HL-60 cells. The significant reduction in sonodynamically induced apoptosis, nitroxide generation, and caspase-3 activation by histidine suggests active species such as singlet oxygen are important in the sonodynamic induction of apoptosis. General significance: The results reported in this paper are experimental, but they significantly support the possibility of sonodynamic treatment for cancer using the induction of apoptosis.

In this study, we investigated the induction of apoptosis by ultrasound in the presence of the novel porphyrin derivative DCPH-P-Na(I). HL-60 cells were exposed to ultrasound for up to 3 min in the presence and absence of DCPH-P-Na(I), and the induction of apoptosis was examined by analyzing cell morphology, DNA fragmentation, and caspase-3 activity. Reactive oxygen species were measured by means of ESR and spin trapping technique. Cells treated with 8 μM DCPH-P-Na(I) and ultrasound clearly showed membrane blebbing and cell shrinkage, whereas significant morphologic changes were not observed in cells exposed to either ultrasound or DCPH-P-Na(I) alone. Also, DNA ladder formation and caspase-3 activation were observed in cells treated with both ultrasound and DCPH-P-Na(I) but not in cells treated with ultrasound or DCPH-P-Na(I) alone. In addition, the combination of DCPH-P-Na(I) and the same acoustical arrangement of ultrasound substantially enhanced nitroxide generation by the cells. Sonodynamically induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine. These results indicate that the combination of ultrasound and DCPH-P-Na(I) induced apoptosis in HL-60 cells. The significant reduction in sonodynamically induced apoptosis, nitroxide generation, and caspase-3 activation by histidine suggests active species such as singlet oxygen are important in the sonodynamic induction of apoptosis. These experimental results support the possibility of sonodynamic treatment for cancer using the induction of apoptosis.

Human α1-acid glycoprotein (hAGP) in serum functions as a carrier of basic drugs. In most individuals, hAGP exists as a mixture of two genetic variants, the F1*S and A variants, which bind drugs with different selectivities. We prepared a mutant of the A variant, C149R, and showed that its drug-binding properties were indistinguishable from those of the wild type. In this study, we determined the crystal structures of this mutant hAGP alone and complexed with disopyramide (DSP), amitriptyline (AMT), and the nonspecific drug chlorpromazine (CPZ). The crystal structures revealed that the drug-binding pocket on the A variant is located within an eight-stranded β-barrel, similar to that found in the F1*S variant and other lipocalin family proteins. However, the binding region of the A variant is narrower than that of the F1*S variant. In the crystal structures of complexes with DSP and AMT, the two aromatic rings of each drug interact with Phe-49 and Phe-112 at the bottom of the binding pocket. Although the structure of CPZ is similar to those of DSP and AMT, its fused aromatic ring system, which is extended in length by the addition of a chlorine atom, appears to dictate an alternative mode of binding, which explains its nonselective binding to the F1*S and A variant hAGPs. Modeling experiments based on the co-crystal structures suggest that, in complexes of DSP, AMT, or CPZ with the F1*S variant, Phe-114 sterically hinders interactions with DSP and AMT, but not CPZ. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Unglycosylated recombinant human alpha(1)-acid glycoprotein (hAGP) variants (rF1(*)S and rA) were prepared in an E. coli expression system using the Origami B strain and pET-3c vector. Thioredoxin was co-expressed to promote the appropriate folding of hAGP. SDS-PAGE under reducing conditions showed that rF1(*)S and rA migrate as single bands after purification. However, several bands derived from rA were observed under non-reducing conditions because of the high reactivity of a free cystein residue (C149). We therefore prepared a mutant of A variant (C149R-A), and confirmed that this mutant maintained homogeneity. Circular dichroism and intrinsic tryptophan fluorescence spectroscopic analyses indicated that rF1(*)S and C149R-A have almost the same conformational structures as F1(*)S and A purified from serum. Ligand binding experiments using propranolol as a F1(*)S ligand and disopyramide as an A specific ligand indicated that the capacity of rF1(*)S and C149R-A is equivalent to those ligands as well as F1(*)S and A from serum. These results suggest that the oligosaccharide moieties of hAGP have negligible effects on the structural and ligand binding properties of hAGP. Thus, rF1(*)S and C149R-A promise to be useful in studies on the drug binding sites of hAGP.

Summary: Human α 1 -acid glycoprotein (AGP), a serum glycoprotein, is thought to have anti-inflammatory effects by a mechanism that is not well understood. In this study, we investigated the pharmacokinetics of AGP in mice using enzymatically modified AGP (AGP with the sialic acids removed, asialo-AGP, and with both sialic acids and galactose removed, agalacto-AGP). It was observed that AGP was eliminated from the circulation slowly, and was mainly taken up by the liver. The elimination of labeled AGP, asialo-AGP and agalacto-AGP from the circulation was suppressed in the presence of excess unlabeled AGP, asialo-AGP and agalacto-AGP, respectively, suggesting the receptor-mediated uptake of these AGPs. Interestingly, the up-take of AGP by the liver competed with agalacto-AGP, but not with asialo-AGP, while agalacto-AGP competed with asialo-AGP. These results suggest that agalacto-AGP binds to at least two types of receptors, namely asialoglycoprotein receptor (ASGPR) and an as yet unidentified receptor that is shared with AGP, and that AGP is directly taken up by the liver through such a receptor and not via ASGPR. These findings help improve our understanding of the clearance mechanism of AGP.

The binding site of 7-hydroxystaurosporine (UCN-01) on alpha-acid glycoprotein (AGP) was studied by fluorescence and ultracentrifugation experiments. Three ligands, propranolol, warfarin and progesterone were employed as marker ligands and quinaldine red was employed as a fluorescent probe. The presence of UCN-01, pro- pranolol, warfarin and progesterone resulted in a significant quenching of the fluorescence of quinaldine red, when bound to AGP, depending upon the potency of the binding to AGP. The construction of Klotz plots indicated that the displacement effects of propranolol, warfarin and progesterone on UCN-01-AGP binding were competitive in nature. These data suggest that the binding site of UCN-01 on the AGP partly overlaps the binding site for basic drugs, acidic drugs, as well as steroid hormones.

The present study aims to investigate sonodynamically-induced apoptosis using the phthalocyanine, chloroaluminum phthalocyanine tetrasulfonate (AlPcTS). HL-60 cells were exposed to ultrasound for up to 3 min in the absence and presence of AlPcTS. Apoptosis was analyzed by cell morphology, DNA fragmentation, and caspase-3 activity. Electron spin resonance was used to measure reactive oxygen species. The number of apoptotic cells showing membrane blebbing and cell shrinkage after combined treatment (ultrasound and AlPcTS) was significantly higher than following other treatments, including ultrasound alone and AlPcTS alone. Furthermore, DNA ladder formation, caspase-3 activation and enhanced nitroxide generation were observed in cells treated with ultrasound and AlPcTS. Sonodynamically induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine. The significant reduction by histidine indicated that ultrasonically generated reactive oxygen species, such as singlet oxygen, is an important mediator of sonodynamically-induced apoptosis.

Human alpha(1)-acid glycoprotein (AGP), which is comprised of 183 amino acid residues and 5 carbohydrate chains, is a major plasma protein that binds to basic and neutral drugs as well as to steroid hormones. It has a beta-sheet-rich structure in aqueous solution. Our previous findings suggest that AGP forms an alpha-helix structure through an interaction with biomembranes. We report herein on a study of the mechanism of alpha-helix formation in AGP using various modified AGPs. The disulfide reduced AGP (R-AGP) was extensively unfolded, whereas asialylated AGP (A-AGP) maintained the native structure. Intriguingly, reduced and asialylated AGP (RA-AGP) increased the alpha-helix content as observed in the presence of biomembrane models, and showed a significant decrease in ligand binding capacity. This suggests that AGP has an innate tendency to form an alpha-helix structure, and disulfide bonds are a key factor in the conformational transition between the beta-sheet and alpha-helix structures. However, RA-AGP with all histidine residues chemically modified (HRA-AGP) was found to lose the intrinsic ability to form an alpha-helix structure. Furthermore, disulfide reduction of the H172A mutant expressed in Pichia pastoris also caused a similar loss of folding ability. The present results indicate that disulfide bonds and the C-terminal region, including H172 of AGP, play important roles in alpha-helix formation in the interaction of the protein with biomembranes.

Alpha1-acid glycoprotein (AGP) is a serum glycoprotein that mainly binds basic drugs. Previous reports have shown that AGP converts from a beta-sheet to an alpha-helix upon interaction with biomembranes. In the current studies, we found that alkanols, diols, and halogenols all induce this conformational change. Increased length and bulkiness of the hydrocarbon group and the presence of a halogen atom promoted this conversion, whereas the presence of a hydroxyl group inhibited it. Moreover, the effect was dependent on the hydrophobic and electrostatic properties of the alcohols. These results indicate that, in a membrane environment, hydrophobic and electrostatic factors cooperatively induce the transition of AGP from a beta-sheet to an alpha-helix.

The binding of flunitrazepam (FNZP) by human α1-acid glycoprotein (hAGP) and the relationships between the extent of drug binding and desialylation and the genetic variants of hAGP were examined. The photolabeling specificity of [3H]FNZP was confirmed by findings in which other hAGP-binding ligands inhibited the formation of covalent bonds between [3H]FNZP and hAGP. The photolabeling of asialo-hAGP suggested that sialic acid does not involve in the binding of [3H]FNZP. No difference in the labeling could be found between the F1 * S variants and A variant. Similarly, FNZP did not show a difference in binding affinity to the two genetic variants of hAGP. Sequence analysis of the photolabeled peptide indicated a sequence corresponding to Tyr91-Arg105 of hAGP.

To investigate the factors that contribute to the exceptionally high affinity binding of UCN-01 to human alpha1-acid glycoprotein (hAGP).Interactions between UCN-01, UCN-02, and staurosporine with native and chemically modified hAGPs were examined using ultracentrifugation and spectroscopic analysis.The binding affinity of staurosporine, as well as UCN-02, to hAGP was lower than that of UCN-01 by 20- and 100-fold respectively. The percentage of UCN-01 that binds to hAGP was low at acidic pH but increased with increasing pH, reaching a maximum at pH 7.4. The binding of UCN-01 to desialylated hAGP was comparable to that of hAGP. No significant difference was found for the binding of UCN-01 to F1*S and A variants of hAGP. Chemical modification of the His, Lys, Trp, and Tyr residues caused a decrease in percentage of bound UCN-01. Trp-modified hAGP showed the largest decrease in binding. Tryptophanyl fluorescence quenching results indicate that Trp residues play a prominent role in the binding of UCN-01 to hAGP.A substituent at position C-7 of UCN-01 appeared to influence the binding specificity of the drug, and Trp residues in hAGP play a prominent role in the high affinity binding of UCN-01 to hAGP.

Gemcitabine (GEM), a pyrimidine nucleoside analogue, is used in patients with pancreatic cancer as the standard chemotherapeutic agent. However, pancreatic cancer cells often show resistance to GEM in vivo and vitro. In this study, we explored the factors involved in the sensitivity of GEM in human pancreatic cancer cells, Miapaca-2 and AsPC-1, by a metabolomic analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). The sensitivity of Miapaca-2 (IC50: 66 nM) to GEM was higher than that of AxPC-1 (IC50: 16 nM), whereas there were little differences in the growth rates of these cells. This result indicates that the difference in the sensitivities between Miapaca-2 and AsPC-1 is not dependent on their growth rates. At 12 hr after adding GEM, intracellular level of GEM in Miapaca-2 was higher than that in AsPC-1, whereas levels of GEM monophosphate, diphosphate and triphosphate in Miapaca-2 were lower than that in AsPC-1. In addition, expression level of deoxycytidine kinase (dCK), which catalyzes the reaction from GEM to GEM monophosphate, in Miapaca-2 was lower than that in AsPC-1. Expression levels of human equilibrium transporter (hENT1), which is known to major transporter of GEM, were similar in both cells. These results suggest that dCK is rate-limiting enzyme in GEM phosphorylation and one of the factors involved in the sensitivity. In pyrimidine metabolism, GEM was observed to inhibit CTP synthase, which converts UTP to CTP, resulting increase in UTP level, decrease in CTP level and depletion of dCTP in both cells at 12 hr after adding GEM. The degree of increase in UTP and decrease in CTP was lower in Miapaca-2 than that of AsPC-1. Indeed, the ratio of UTP to CTP in Miapaca-2 was about 2.5, whereas that of AsPC-1 was about 11. In addition, the expression level of CTP synthase in Miapaca-2 was higher than that in AsPC-1. Ribonucleotide reductase, which catalyzes the conversion of CDP to dCDP, is known to be inhibited by GEM diphosphate and be involved in the sensitivity of pancreatic cancer cells to GEM. GEM decreased CDP in AsPC-1. This may be caused by inhibiting CTP synthase. Expression level of ribonucleotide reductase in Miapaca-2 was higher than that in AxPC-1. Therefore, ribonucleotide reductase also may be involved in the sensitivity to GEM. Furthermore, we previously found that GEM mainly inhibited CTP synthase in pyrimidine metabolism, resulting in depletion of dCTP. Our current study suggests that dCK, CTP synthase and ribonucleotide reductase are involved in the sensitivity of pancreatic cancer cells to GEM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 838. doi:1538-7445.AM2012-838