Borealin-Derived Peptides as Survivin-Targeting Cancer Imaging and Therapeutic Agents.

Survivin is overexpressed in most cancer cells but is rarely expressed in normal adult tissues. It is associated with poor prognosis and resistance to radiation therapy and chemotherapy. In this study, we designed and synthesized borealin-derived small peptides (Bor peptides) to function as survivin-targeting agents for the diagnosis and treatment of cancers. These peptides exhibited binding affinities for recombinant human survivin (Kd = 49.6-193 nM), with Bor65-75 showing the highest affinity (Kd = 49.6 nM). Fluorescence images of fluorescein isothiocyanate-labeled Bor65-75 showed its co-localization with survivin expression in the human pancreatic cancer cell line, MIA PaCa-2. In the WST-1 assay, cell penetrable nona-d-arginine-conjugated Bor65-75 (r9-Bor65-75) inhibited the growth of MIA PaCa-2 cells and MDA-MB-231 cells (89 and 88% inhibition at 10 µM, respectively), whereas it had almost no effect on the human mammary epithelial cell line, MCF-10A, that inherently does not have high survivin expression. Flow cytometry with annexin V and propidium iodide staining revealed that r9-Bor65-75 induced apoptosis in MIA PaCa-2 cells in a dose-dependent manner. An increase in cleaved poly ADP-ribose polymerase protein expression was observed in MIA PaCa-2 cells exposed to r9-Bor65-75 by western blotting, suggesting that r9-Bor65-75 inhibits cell proliferation by inducing apoptosis. In vivo, r9-Bor65-75 significantly suppressed tumor growth in MIA PaCa-2 xenograft mice, without any marked weight loss. Hence, Bor peptides are promising candidates for the development of cancer imaging and anticancer agents targeting survivin.

Synthesis and Characterization of Hydroxyethylamino- and Pyridyl-Substituted 2-Vinyl Chromone Derivatives for Detection of Cerebral Abnormal Prion Protein Deposits.

Prion diseases are fatal neurodegenerative diseases characterized by the deposition of abnormal prion protein aggregates (PrPSc) in the brain. In this study, we developed hydroxyethylamino-substituted styrylchromone (SC) and 2-(2-(pyridin-3-yl)vinyl)-4H-chromen-4-one (VPC) derivatives for single-photon emission computed tomography (SPECT) imaging of PrPSc deposits in the brain. The binding affinity of these compounds was evaluated using recombinant mouse prion protein (rMoPrP) aggregates, which resulted in the inhibition constant (Ki) value of 61.5 and 88.0 nM for hydroxyethyl derivative, (E)-2-(4-((2-hydroxyethyl)amino)styryl)-6-iodo-4H-chromen-4-one (SC-NHEtOH) and (E)-2-(4-((2-hydroxyethyl)(methyl)amino)styryl)-6-iodo-4H-chromen-4-one (SC-NMeEtOH), respectively. However, none of the VPC derivatives showed binding affinity for the rMoPrP aggregates. Fluorescent imaging demonstrated that the accumulation pattern of SC-NHEtOH matched with the presence of PrPSc in the brain slices from mouse-adapted bovine spongiform encephalopathy-infected mice. A biodistribution study of normal mice indicated low initial brain uptake of [125I]SC-NHEtOH (0.88% injected dose/g (% ID/g) at 2 min) despite favorable washout from the brain (0.26% ID/g, at 180 min) was displayed. [125I]SC-NHEtOH exhibited binding affinities to both artificial prion aggregates as well as prion deposits in the brain. However, significant improvement in the binding affinity for PrPSc and blood-brain barrier permeability is necessary for the development of successful in vivo imaging probes for the detection of cerebral PrPSc in the brain.

Peptidyl-prolyl cis-trans isomerase A participates in the selenium transport into the rat brain.

Selenium, an essential micronutrient, plays vital roles in the brain. Selenoprotein P (SELENOP), a major plasma selenoprotein, is thought to transport selenium to the brain. However, Selenop-knockout mice fed a diet containing an adequate amount of selenium shows no objective neurological dysfunction which is observed in the selenium-deficient diet-fed Selenop-knockout mice. This fact indicated that selenium from low-mass selenium-source compounds can be transported by SELENOP-independent alternative pathways to the brain. In this study, to obtain the basic information about the SELENOP-independent transport pathways, we performed ex vivo experiments in which the rat brain cell membrane fraction was analyzed to find selenium-binding and/or -interactive proteins using its reactive metabolic intermediate, selenotrisulfide (STS), and MALDI TOF-mass spectrometry. Several membrane proteins with the cysteine (C) thiol were found to be reactive with STS through the thiol-exchange reaction. One of the C-containing proteins in the brain cell membrane fraction was identified as peptidyl-prolyl cis-trans isomerase (PPIase) A from tryptic fragmentation experiments and database search. Among the 4 C residues in rat PPIase A, 21st C was proved to react with STS by assessment using C mutated recombinant proteins. PPIase A is ubiquitously expressed and also associates with a variety of biologically important events such as immunomodulation, intracellular signaling, transcriptional regulation and protein trafficking. Consequently, PPIase A was thought to participate in the selenium transport into the rat brain.

Discovery of inner centromere protein-derived small peptides for cancer imaging and treatment targeting survivin.

Survivin belongs to the inhibitor of apoptosis protein family, which is consistently overexpressed in most cancer cells but rarely expressed in normal adult tissues. Therefore, the detection and inhibition of survivin are regarded as attractive strategies for cancer-specific treatment. In this study, we designed and synthesized 7-19 residues of inner centromere protein (INCENP)-derived small peptides (INC peptides) as novel survivin-targeting agents. The INC peptides showed binding affinity for the human survivin protein (Kd  = 91.4-255 nmol L-1 ); INC16-22 , which contains residues 16-22 of INCENP, showed the highest affinity (91.4 nmol L-1 ). Confocal fluorescence imaging showed consistent colocalization of FITC-INC16-22 and survivin in cell lines. Nona-arginine-linked INC16-22 (r9-INC16-22 ) rendered INC16-22 cells penetrable and strongly inhibited cell growth of MIA PaCa-2 cells (52% inhibition at 1.0 µmol L-1 ) and MDA-MB-231 cells (60% inhibition at 10 µmol L-1 ) as determined by MTT assays. The exposure of MIA PaCa-2 cells to 40 µmol L-1 r9-INC16-22 apparently reduced the intracellular protein expression levels of survivin. However, cleaved caspase-3 was significantly increased in cells treated with r9-INC16-22 , even at 10 µmol L-1 , compared to untreated cells. Flow cytometry revealed that r9-INC16-22 strongly induced apoptosis in MIA PaCa-2 cells. These results indicate that the cytotoxic effects of r9-INC16-22 could be mediated mainly through the disruption of survivin-dependent antiapoptotic functions and partly because of the direct degradation of the survivin protein. Our findings suggest that INC peptides can act as useful scaffolds for novel cancer imaging and anticancer agents.

Synthesis and evaluation of radioactive/fluorescent peptide probes for imaging of legumain activity.

Legumain or asparaginyl endopeptidase is an enzyme overexpressed in some cancers and involved in cancer migration, invasion, and metastasis. We have developed radioiodine- ([125I]I-LCP) or fluorescein-labeled peptides (FL-LCP) with a cell-permeable d-Arg nonamer fused to an anionic d-Glu nonamer via a legumain-cleavable linker, to function as peptide probes that measure and monitor legumain activity. Non-cleavable probes of FL-NCP and [125I]I-NCP were similarly prepared and evaluated as negative control probes by altering their non-cleavable sequence. Model peptides with the legumain-cleavable or non-cleavable sequence (LCP and NCP, respectively) reacted with recombinant human legumain, and only LCP was digested by this enzyme. [125I]I-LCP uptake in legumain-positive HCT116 cells was significantly higher than that of [125I]I-NCP (11.2 ±â€¯0.44% vs 1.75 ±â€¯0.06% dose/mg). The accumulation of FL-LCP in the HCT116 cells was rather low (4.75 ±â€¯0.29% dose/mg protein), but not significantly different from the levels of FL-NCP. It is possible that low concentrations of [125I]I-LCP (40 pM) can be effectively internalized after legumain cleavage. On the other hand, the cellular uptake of much higher concentrations of the FL-LCP derivative (1 mM) may be restricted by high concentrations of polyanions. The in vivo biodistribution studies in tumor-bearing mice demonstrated that the tumor uptake of [125I]I-LCP was 1.34% injected dose per gram (% ID/g) at 30 min. The tumor/blood and tumor/muscle ratios at 30 min were 0.63 and 1.77, respectively, indicating that the [125I]I-LCP accumulation in tumors was inadequate for in vivo imaging. Although further structural modifications are necessary to improve pharmacokinetic properties, [125I]I-LCP has been demonstrated to be an effective scaffold for the development of nuclear medicine imaging probes to monitor legumain activity in living subjects.

Synthesis and characterization of radioiodinated 3-phenethyl-2-indolinone derivatives for SPECT imaging of survivin in tumors.

Survivin, overexpressed in most cancers, is associated with poor prognosis and resistance to radiation therapy and chemotherapy. Herein, we report the synthesis of three 3-phenethyl-2-indolinone derivatives and their application as in vivo imaging agents for survivin. Of these, 3-(2-(benzo[d][1,3]dioxol-5-yl)-2-oxoethyl)-3-hydroxy-5- iodoindolin-2-one (IPI-1) showed the highest binding affinity (Kd = 68.3 nM) to recombinant human survivin, as determined by quartz crystal microbalance (QCM). In vitro studies demonstrated that the [125I]IPI-1 binding in survivin-positive MDA-MB-231 cells was significantly higher than that in survivin-negative MCF-10A cells. In addition, uptake of [125I]IPI-1 by MDA-MB-231 cells decreased in a dose-dependent manner in the presence of the high-affinity survivin ligand S12; this is indicative of specific binding of [125I]IPI-1 to cellular survivin protein in vitro. Biodistribution studies in MDA-MB-231 tumor-bearing mice demonstrated the moderate uptake of [125I]IPI-1 in the tumor tissue (1.37% ID/g) at 30 min that decreased to 0.32% ID/g at 180 min. Co-injection of S12 (2.5 mg/kg) slightly reduced tumor uptake and the tumor/muscle ratio of [125I]IPI-1. Although further structural modifications are necessary to improve pharmacokinetic properties, our results indicate that PI derivatives may be useful as tumor-imaging probes targeting survivin.

Synthesis and characterization of 11 C-labeled benzyl amidine derivatives as PET radioligands for GluN2B subunit of the NMDA receptors.

GluN2B-containing NMDA receptors (NMDARs) play fundamental roles in learning and memory, although they are also associated with various brain disorders. In this study, we synthesized and evaluated three 11 C-labeled N-benzyl amidine derivatives 2-[11 C]methoxybenzyl) cinnamamidine ([11 C]CBA), N-(2-[11 C]methoxybenzyl)-2-naphthamidine ([11 C]NBA), and N-(2-[11 C]methoxybenzyl)quinoline-3-carboxamidine ([11 C]QBA) as PET radioligands for these receptors. The 11 C-benzyl amidines were synthesized via conventional methylation of corresponding des-methyl precursors with [11 C]CH3 I. In vitro binding characteristics were examined in brain sagittal sections using various GluN2B modulators and off-target ligands. Further, in vivo brain distribution studies were performed in normal mice. The 11 C-labeled benzyl amidines showed high-specific binding to the GluN2B subunit at in vitro. In particular, the quinoline derivative [11 C]QBA had the best binding properties in terms of high-brain localization to GluN2B-rich regions and specificity to the GluN2B subunit. Conversely, these 11 C-radioligands showed the brain distributions were inconsistent with GluN2B expression in biodistribution experiments. The majority of the radiolabeled compounds were identified as metabolized forms of which amido derivatives seemed to be the major species. Although these 11 C-ligands had high-specific binding to the GluN2B subunit, significant improvement in metabolic stability is necessary for successful positron emission tomography (PET) imaging of the GluN2B subunit of NMDARs.

Development of radioiodinated acridine derivatives for in vivo imaging of prion deposits in the brain.

Prion diseases are caused by deposition of abnormal prion protein aggregates (PrPSc) in the central nervous system. This study aimed to develop in vivo imaging probes that can detect cerebral PrPSc deposits. We synthesized several quinacrine-based acridine (AC) derivatives with 2,9-substitution and radioiodinated them. The AC derivatives were evaluated as prion-imaging probes using recombinant mouse prion protein (rMoPrP) aggregates and brain sections of mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice. The distribution of these compounds in mice was also evaluated. The 2-methoxy derivative [125I]2 exhibited the highest binding affinity for rMoPrP aggregates with an equilibrium dissociation constant (Kd) value of 43.4nM. Fluorescence imaging with 2 showed clear signals at the thioflavin T (ThT)-positive amyloid deposits in the mBSE-infected mouse brain. Although a discrepancy was observed between the in vitro binding of AC derivatives to the aggregates and in vivo distribution of these compounds in the brain and we failed to identify prospective prion-imaging probes in this study, the AC derivatives may be considered a useful scaffold for the development of in vivo imaging probes. Further chemical modification of these AC derivatives may discover clinically applicable prion imaging probes.

Characterization of Selenium Species in the Shijimi Clam.

Selenium is an essential trace element for humans and animals. Fish and shellfish are known to be rich in selenium and suppose to be an effective selenium source. In this study, we characterized the selenium species in the Shijimi clam (Corbicula japonica), which is a typical clam eaten in Japan. The Shijimi clam contains a relatively high concentration of selenium (3.5 µg-selenium/g-dry Shijimi). Approximately 30% of the total selenium in the Shijimi clam meat was extractable with water, while selenium in the Shijimi clam was hardly extracted with ethanol, chloroform and hexane. Based on an ultrafiltration study, the molecular mass of the major selenium species in the Shijimi water-extract was estimated to be less than 5000. Because amphoteric selenium species were contained in the Shijimi water-extract, which was indicated by ion-exchange chromatographic separation, an ion-pair reagent was utilized to extract the ionic selenium species into an organic solvent. A matrix assisted laser desorption ionization (MALDI) time of flight (TOF)-mass spectrometric analysis revealed the selenium isotopic pattern involving one selenium atom in a molecule with the 80Se molecular ion peak at m/z 534. This selenium species was mainly found in the visceral part of the Shijimi clam by imaging mass spectrometry.

Synthesis and evaluation of a radioiodinated 4,6-diaryl-3-cyano-2-pyridinone derivative as a survivin targeting SPECT probe for tumor imaging.

Survivin is overexpressed in most of the cancerous tissues but not in terminally differentiated normal tissues, making it an attractive target for diagnosis and therapy of various types of cancers. In this study, we aimed to develop 4,6-diaryl-3-cyano-2-pyridinone (DCP) derivatives, as novel cancer imaging probes that target survivin. Chloro and iodo analogs of DCP (CDCP and IDCP, respectively) were successfully synthesized by using a previously unreported carbon monoxide-free procedure. IDCP exhibited a slightly higher binding affinity for recombinant human survivin (Kd=34 nM) than that of CDCP (Kd=44 nM). Fluorescence staining indicated that both CDCP and IDCP showed high signals in MDA-MB-231 cells with high levels of survivin expression. Significantly low fluorescent signals were observed in MCF-10A cells, which showed low levels of survivin expression. [(125)I]IDCP was synthesized for the application of IDCP to single photon emission computed tomography (SPECT) imaging. Quantitative in vitro binding of [(125)I]IDCP in cell cultures showed results consistent to those observed after fluorescent staining. In vivo biodistribution studies in tumor-bearing mice demonstrated that the tumor uptake of [(125)I]IDCP increased gradually with time and was 0.65% injected dose per gram (% ID/g) at 180 min. The maximum tumor/blood and tumor/muscle ratio at 60 min were 0.87 and 2.27, respectively, indicating inadequate [(125)I]IDCP accumulation in tumors necessary for in vivo imaging. Although further structural modifications are necessary to improve pharmacokinetic properties of IDCP, this study demonstrates the feasibility of using the DCP backbone as a scaffold for the development of survivin-targeting tumor imaging probes.

Evaluation for Peritoneal Injury at an Early Stage Using Dual Macromolecular Markers.

Long-term peritoneal dialysis (PD) frequently produces morphological and functional changes of the peritoneum, making continuation of PD difficult. Therefore, it is necessary to evaluate peritoneal injury at an early stage and develop appropriate therapies. The aims of the present study were to evaluate peritoneal injury at an early stage and assess a drug for prevention of peritoneal injury using our previously developed novel evaluation method. Peritoneal injury was induced in model animals by intraperitoneal injection of methylglyoxal (MGO) for 1 to 5 consecutive days or chlorhexidine digluconate (CG) for 1 to 14 consecutive days. Tetramethylrhodamine-dextran (RD)-10 and fluorescein isothiocyanate-dextran (FD)-2000 were then injected into the peritoneal cavity and recovered after 120 min to evaluate peritoneal injury. The ratio of the concentration of RD-10 to FD-2000 (RD-10/FD-2000 ratio) significantly decreased in animals that had been treated with MGO or CG for 1 d. Moreover, the RD-10/FD-2000 ratio significantly increased in CG- and thalidomide-treated animals. The RD-10/FD-2000 ratio can be used to evaluate peritoneal injury at an early stage and assess the drug efficacy of thalidomide for prevention of peritoneal injury. This study will contribute to the development of therapeutic treatments for peritoneal injury.

A Comprehensive Analysis of Selenium-Binding Proteins in the Brain Using Its Reactive Metabolite.

The intracellular metabolism of selenium in the brain currently remains unknown, although the antioxidant activity of this element is widely acknowledged to be important in maintaining brain functions. In this study, a comprehensive method for identifying the selenium-binding proteins using PenSSeSPen as a model of the selenium metabolite, selenotrisulfide (RSSeSR, STS), was applied to a complex cell lysate generated from the rat brain. Most of the selenium from L-penicillamine selenotrisulfide (PenSSeSPen) was captured by the cytosolic protein thiols in the form of STS through the thiol-exchange reaction (R-SH+PenSSeSPen→R-SSeSPen+PenSH). The cytosolic protein species, which reacted with the PenSSeSPen mainly had a molecular mass of less than 20 kDa. A thiol-containing protein at m/z 15155 in the brain cell lysate was identified as the cystatin-12 precursor (CST12) from a rat protein database search and a tryptic fragmentation experiment. CST12 belongs to the cysteine proteinase inhibitors of the cystatin superfamily that are of interest in mechanisms regulating the protein turnover and polypeptide production in the central nervous system and other tissues. Consequently, CST12 is suggested to be one of the cytosolic proteins responsible for the selenium metabolism in the brain.

An effective method for profiling the selenium-binding proteins using its reactive metabolic intermediate.

Currently, the intracellular reduction and/or transport of selenium still remain unknown. Certain reduced forms of selenium species are thought to be reactive with various endogenous molecules, particularly thiol-containing proteins. In this study, a profiling method for identifying the selenium-binding proteins using L-penicillamine selenotrisulfide (PenSSeSPen) as a model of the selenium metabolic intermediate was applied to the cell lysate generated from the rat liver. Several proteins with cysteine thiol were found to be reactive with PenSSeSPen through the thiol-exchange reaction by MALDI TOF-MS analysis. The most distinctive cysteine-containing protein at m/z 14,313 in the liver cell lysate was identified as the liver fatty acid-binding protein based on a rat protein database search and a tryptic fragmentation experiment. This methodology could be used for determining the selenium-binding proteins and/or selenium-interactive species and provide a better understanding of the selenium metabolism and utilization in biological systems.

Development of alkoxy styrylchromone derivatives for imaging of cerebral amyloid-ß plaques with SPECT.

We report here the development of radioiodinated styrylchromone derivatives with alkoxy groups as single photon emission computed tomography (SPECT) imaging probes for cerebral amyloid-ß (Aß) plaques. Among the derivatives, the methoxy derivative 14 and the dimethoxy derivative 15 displayed relatively high affinity for the Aß(1-42) aggregates with K(i) values of 22 and 46 nM, respectively. Fluorescent imaging demonstrated that 14 and 15 clearly labeled thioflavin-S positive Aß plaques in the brain sections of Tg2576 transgenic mice. In the in vivo studies, [(125)I]14 and [(125)I]15 showed high initial brain uptake expressed as the percentage of the injected dose per gram (2.25% and 2.49% ID/g at 2 min, respectively) with favorable clearance (0.12% and 0.20% ID/g at 180 min, respectively) from the brain tissue of normal mice. Furthermore, in vitro autoradiography confirmed that [(125)I]15 binds thioflavin-S positive regions in Tg2576 mouse brain sections. The derivative 15 may be a potential scaffold for the development of in vivo imaging probes targeting Aß plaques in the brain. In particular, further structural modifications are required to improve the compounds binding affinity for Aß.

Development of PET and SPECT probes for glutamate receptors.

L-glutamate and its receptors (GluRs) play a key role in excitatory neurotransmission within the mammalian central nervous system (CNS). Impaired regulation of GluRs has also been implicated in various neurological disorders. GluRs are classified into two major groups: ionotropic GluRs (iGluRs), which are ligand-gated ion channels, and metabotropic GluRs (mGluRs), which are coupled to heterotrimeric guanosine nucleotide binding proteins (G-proteins). Positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging of GluRs could provide a novel view of CNS function and of a range of brain disorders, potentially leading to the development of new drug therapies. Although no satisfactory imaging agents have yet been developed for iGluRs, several PET ligands for mGluRs have been successfully employed in clinical studies. This paper reviews current progress towards the development of PET and SPECT probes for GluRs.

Enhancement of binding affinity for amyloid aggregates by multivalent interactions of 99mTc-hydroxamamide complexes.

Deposition of amyloid aggregates has been regarded as an early stage of amyloidosis progression. An imaging probe that can image amyloid aggregates enables the early diagnosis of amyloidosis and contributes to the development of new medical therapies. High binding affinity for amyloid aggregates is essential to develop a useful molecular imaging probe. This article describes a new strategy to enhance the binding affinity of imaging agents targeting amyloid aggregates. We designed and synthesized novel (99m)Tc-hydroxamamide ((99m)Tc-Ham) complexes with a bivalent amyloid ligand and evaluated their binding affinity for amyloid aggregates by using ß-amyloid peptide (Aß(1-42)) aggregates as a model. In vitro inhibition assay indicated that bivalent (99m)Tc-Ham complexes had much higher binding affinity for amyloid aggregates than monovalent complexes. In vitro autoradiography using Tg2576 mice showed the specific binding of bivalent (99m)Tc-Ham complexes to Aß plaques in the mouse brain, as reflected in the results of the inhibition assay. The preliminary results suggest that a new molecular design based on bivalent (99m)Tc-Ham complexes may be reasonable to develop an imaging probe targeting amyloid aggregates.

Synthesis and evaluation of ethyleneoxylated and allyloxylated chalcone derivatives for imaging of amyloid ß plaques by SPECT.

We report radioiodinated chalcone derivatives as new SPECT imaging probes for amyloid ß (Aß) plaques. The monoethyleneoxy derivative 2 and allyloxy derivative 8 showed a high affinity for Aß(1-42) aggregates with Ki values of 24 and 4.5 nM, respectively. Fluorescent imaging demonstrated that 2 and 8 clearly stained thioflavin-S positive Aß plaques in the brain sections of Tg2576 transgenic mice. In vitro autoradiography revealed that [(125)I]2 displayed no clear accumulation toward Aß plaques in the brain sections of Tg2576 mice, whereas the accumulation pattern of [(125)I]8 matched with the presence of Aß plaques both in the brain sections of Tg2576 mice and an AD patient. In biodistribution studies using normal mice, [(125)I]2 showed preferable in vivo pharmacokinetics (4.82%ID/g at 2 min and 0.45%ID/g at 60 min), while [(125)I]8 showed only a modest brain uptake (1.62%ID/g at 2 min) with slow clearance (0.56%ID/g at 60 min). [(125)I]8 showed prospective binding properties for Aß plaques, although further structural modifications are needed to improve the blood brain barrier permeability and washout from brain.

Novel diagnostic method of peritoneal injury using dual macromolecular markers.

Long-term peritoneal dialysis (PD) frequently produces morphological and functional changes of the peritoneum, which makes continuation of PD difficult. Moreover, the progression of peritoneal injury causes complications and poor prognosis. Since therapeutic treatments for peritoneal injury during PD have yet to be established, it is important to diagnose peritoneal injury as early as possible. The aim of this study was to develop a method of monitoring peritoneal function to diagnose peritoneal injury. Model rats of peritoneal injury were prepared by intraperitoneal injection of methylglyoxal (MGO) for five consecutive days. Then, marker substances of various molecular weights (phenolsulfonphthalein, fluorescein isothiocyanate-dextran (FD)-10, FD-40, FD-70, FD-2000 or tetramethylrhodamine-dextran (RD)-10) were injected into the peritoneal cavity. At 120 min after injection, the remaining amounts of all marker substances were significantly decreased in the MGO-treated rats compared with those in the vehicle-treated rats. Molecular weight dependence of the peritoneal permeability was observed. A substance with a molecular weight of approximately 10000 was found to be suitable to diagnose peritoneal injury. Moreover, coadministration of RD-10 with FD-2000 enabled us to monitor enhanced peritoneal permeability and the transfer of water simultaneously, without the recovery of whole PD fluid, even in the case of different ultrafiltration volumes. We demonstrated the usefulness of administering substances to evaluate peritoneal permeability and the transfer of water simultaneously to diagnose peritoneal injury. This study should be valuable for safe and effective PD.

Peptide-Based Turn-On Fluorescent Probes for Highly Specific Detection of Survivin Protein in the Cancer Cells.

Survivin is highly expressed in most human cancers, making it a promising target for cancer diagnosis and treatment. In this study, we developed peptide probes consisting of Bor65-75, a high-affinity survivin-binding peptide, and a survivin protein segment using peptide linkers as survivin-sensitive fluorescent probes (SSFPs). All conjugates were attached to 5(6)-carboxyfluorescein (FAM) at the C-terminal as a fluorophore and to 4((4(dimethylamino)phenyl)azo)benzoic acid (DABCYL) at the N-terminal as a quencher. Fluorescence (or Förster) resonance energy transfer (FRET) quenching via intramolecular binding of Bor65-75 with survivin protein segment could be diminished by the approach of survivin to SSFPs, which dissociate Bor65-75 from SSPF and increased the distance between FAM and DABCYL. A binding assay using recombinant human survivin protein (rSurvivin) demonstrated moderate to high affinity of SSFPs for survivin (dissociation constants (K d) = 121-1740 nM). Although the SSFPs (0.5 µM) had almost no fluorescence under baseline conditions, a dose-dependent increase in fluorescence intensity was observed in the presence of rSurvivin (0.1-2.0 µM). In particular, the proline-rich SSFP (SSFP5) showed the highest (2.7-fold) fluorescence induction at 2.0 µM survivin compared to the signals in the absence of survivin. Confocal fluorescence imaging demonstrated that SSFP5 exhibited clear fluorescence signals in survivin-positive MDA-MB-231 cells, whereas no marked fluorescence signals were observed in survivin-negative MCF-10A cells. Collectively, these results suggest that SSFPs can be used as survivin-specific FRET imaging probes.

Characterization of selenium species in extract from Niboshi (a processed Japanese anchovy).

Fish are selenium rich foodstuffs and a major selenium source for the Japanese population. Niboshi is processed from Japanese anchovy (Engraulis japonicus) and commonly used to prepare soup stock for Japanese dishes. In this study, we characterized selenium species in the Niboshi extract by ultrafiltration, ion-exchange chromatography and mass spectrometry. Selenium species in the Niboshi were more extractable by polar solvents (water and ethanol) than an apolar one (hexane) along with amino acids and proteinous species. Selenium in the water-extract from the Niboshi was mostly ascribed to organoselenium compounds with a molecular mass less than 5 kDa. Although selenoamino acids and selenoproteins and their fragments were involved in the extract, a large portion of the selenium species appeared to be low-molecular-mass organoselenium compounds other than selenoamino acids and their derivatives. Ion-exchange chromatographic separations revealed that most of the selenium species in the extract possess anionic and/or amphoteric characteristics. One of these selenium species from the Niboshi extract was detected at m/z 577 for 80Se by mass spectrometry subsequent to ion-pair extraction.