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.

In vitro assessment of bioavailability of selenium from a processed Japanese anchovy, Niboshi.

Niboshi is a commonly used foodstuff that is processed from Japanese anchovy (Engraulis japonicus) in Japanese cuisine. It was previously demonstrated that Niboshi and its water extract contained highly bioavailable selenium for selenium deficient mice. In this study, we assessed the selenium bioavailability from the extract of the Niboshi, using cultured cells. The activity of selenium-dependent glutathione peroxidase (GPx) of rat dorsal ganglion cells and human cervical carcinoma cells incubated with selenium from the Niboshi extract was over 2 times of that of the extract-free control cells and comparable to that of cells incubated with selenious acid of the same selenium concentration. These results suggest that selenium from the Niboshi extract was utilized for synthesis of the selenoprotein. Such in vitro selenium bioavailability was consistent with our previous results of in vivo assessment in mice.

Cardiac myoglobin participates in the metabolic pathway of selenium in rats.

As an essential micronutrient, selenium deficiency is a leading cause of cardiovascular diseases. The heart is continuously beating to deliver blood to the entire body, and this requires a high amount of energy. An adult heart normally obtains 50-70% of its adenosine 5'-triphosphate from fatty acid ß-oxidation. An increase in fatty acid oxidation activity induces the generation of larger amounts of by-products (reactive oxygen species, ROS) from mitochondrial oxidative phosphorylation. Selenium-dependent glutathione peroxidases play a critical role in the removal of these ROS, especially organic hydroperoxides, from the heart. The definitive transport and/or detailed metabolic pathways from the selenium-source compounds to the selenoproteins in the heart still remain unclear. We explored the selenium-binding proteins in a rat cardiac cell lysate using its reactive metabolic intermediate, selenotrisulfide (STS), and MALDI TOF-mass spectrometry. Several proteins with a free cysteine (Cys) thiol were found to be reactive with STS through a thiol-exchange reaction. The most distinctive Cys-containing protein in the cardiac cell lysate was identified as myoglobin (Mb) from a rat protein database search and tryptic fragmentation experiments. When separately examined in selenium adequate rats, selenium-binding to the cardiac Mb was verified using selenium-specific fluorometry. Cardiac Mb is thought to participate in the selenium metabolic pathway in the heart.

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.

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.

Elevated amyloid-ß plaque deposition in dietary selenium-deficient Tg2576 transgenic mice.

Selenium-containing proteins (e.g., glutathione peroxidases) are important antioxidants in neuronal defense against oxidative stress. In this study, the production of amyloid-ß (Aß) plaques in the brain of the Tg2576 transgenic mice was investigated under dietary selenium-deficient conditions. The 16-week-old mice were fed a selenium-deficient diet (0.004 µg-selenium g(-1)-diet) or a selenium-adequate diet (0.386 µg-selenium g(-1) diet) for 76 weeks. The selenium concentrations of the organs/tissues in the selenium-deficient diet-fed mice were significantly decreased in comparison to those in the selenium-adequate diet-fed mice; 1.7% of that in the selenium-adequate diet-fed mice in the liver and 43% of that in the selenium-adequate diet-fed mice in the brain. The Aß plaques formed in the brain were fluorescently stained with thioflavin T, and then the obtained images of the brain slices were qualitatively analyzed. The feeding of the selenium-deficient diet to the Tg2576 transgenic mice resulted in more than a two-fold increase in the total area of the Aß plaques in comparison to that of the selenium-adequate diet. The elevated Aß plaque deposition in the selenium-deficient mice can be explained as a consequence of decrease in the selenium concentration, which suggests that the selenium status is associated with the production and/or the clearance of the Aß peptide. The selenium-deficiency could possibly promote the onset and/or progression of Alzheimer's disease (AD) dementia, if the Aß peptides initiate a sequence of events that lead to AD dementia. Consequently, the results shown here suggest that AD has an important relation with the selenium status in vivo.

A thiol-mediated active membrane transport of selenium by erythroid anion exchanger 1 protein.

In this paper, we describe a thiol-mediated and energy-dependent membrane transport of selenium by erythroid anion exchanger 1 (AE1, also known as band 3 protein). The AE1 is the most abundant integral protein of red cell membranes and plays a critical role in the carbon dioxide transport system in which carbon dioxide is carried as bicarbonate in the plasma. This protein mediates the membrane transport of selenium, an essential antioxidant micronutrient, from red cells to the plasma in a manner that is distinct from the already known anion exchange mechanism. In this pathway, selenium bound to the cysteine 93 of the hemoglobin ß chain (Hb-Cysß93) is transported by the relay mechanism to the Cys317 of the amino-terminal cytoplasmic domain of the AE1 on the basis of the intrinsic interaction between the two proteins and is subsequently exported to the plasma via the Cys843 of the membrane-spanning domain. The selenium export did not occur in plain isotonic buffer solutions and required thiols, such as albumin, in the outer medium. Such a membrane transport mechanism would also participate in the export pathways of the nitric oxide vasodilator activity and other thiol-reactive substances bound to the Hb-Cysß93 from red cells to the plasma and/or peripherals.

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.

Absorption and retention characteristics of selenium in dorsal root ganglion neurons.

Selenium concentration in the brain tissue is far less variable than those in peripherals, such as the liver and kidneys, in rodents, when fed a selenium-deficient diet. This fact implies the importance of this element for maintaining the integrity of brain functions and the distinctive selenium metabolism and/or the regulatory mechanism in the brain. To obtain basic information concerning the homeostatically maintained selenium store in the brain, we investigated absorption and retention characteristics of selenium from selenious acid (SA) and seleno-l-methionine (SeMet) in rat dorsal root ganglion (DRG) neurons, in comparison to isolated rat hepatocytes and renal cells in vitro. When DRG neurons were cultured in an SA-free medium subsequent to an SA-supplemented one for 24 h, the DRG neurons maintained a higher selenium concentration than that before SA supplementation over a period of 96 h after removal of SA from the culture medium. The cellular glutathione peroxidase activity of the cells increased for 72 h after removal of SA from the culture medium. A similar retention characteristic of selenium was also observed for DRG neurons treated with SeMet-supplemented culture medium. Consequently, selenium from source compounds, in part, was thought to be retained in DRG neurons and then be utilized for the synthesis of selenium-containing proteins, which implied the presence of a neuron-specific selenium retention mechanism.

Synthesis and evaluation of novel chalcone derivatives with (99m)Tc/Re complexes as potential probes for detection of ß-amyloid plaques.

Four (99m)Tc-labeled chalcone derivatives and their corresponding rhenium analogues were tested as potential probes for imaging ß-amyloid plaques. The chalcones showed higher affinity for Aß(1-42) aggregates than did (99m)Tc complexes. In sections of brain tissue from an animal model of AD, the four Re chalcones intensely stained ß-amyloid plaques. In biodistribution experiments using normal mice, (99m)Tc-BAT-chalcone ([(99m)Tc]17) displayed high uptake in the brain (1.48% ID/g) at 2 min postinjection. The radioactivity washed out from the brain rapidly (0.17% ID/g at 60 min), a highly desirable feature for an imaging agent. [(99m)Tc]17 may be a potential probe for imaging ß-amyloid plaques in Alzheimer's brains.

Thiol-dependent membrane transport of selenium through an integral protein of the red blood cell membrane.

The molecular details of the selenium metabolism and transport in living systems are still not completely understood, despite their physiological importance. Specifically, little is known about the membrane transport of selenium from most of the selenium containing compounds. In the present study, we investigated the mechanism for the membrane transport of selenium from red blood cells (RBCs) to the blood plasma. When the selenium distribution in the RBC ghost membrane after treatment with selenious acid was analyzed, nearly 70% of the selenium in the membrane was found to bind to the anion exchanger 1 (AE1) protein, which suggested that the integral protein AE1 is responsible for the membrane transport of selenium. The thiol dependency of the selenium export from the RBC to the blood plasma was examined using membrane permeable thiol reagents, i.e., N-ethylmaleimide (NEM) and tetrathionate (TTN). Treatment of the RBC with NEM, a thiol-alkylating reagent, resulted in modification of the thiol groups in the amino-terminal cytoplasmic domain (N-CPD) of the AE1, but not those in the membrane domain. Such an NEM treatment provided a marked inhibition of the selenium export from the RBC to the blood plasma. In addition, the treatment with TTN, a thiol-oxidizing reagent that forms intermolecular disulfide bonds, appeared to oxidize thiol groups in both the N-CPD and the membrane domain of AE1, which resulted in complete inhibition of the selenium export even during the initial period in which the export had a maximum velocity when using the thiol reagent-free treatment. Such complete inhibition of the selenium export from the TTN-treated RBC appeared to be due to the oligomerized AE1 proteins resulting from the intermolecularly formed disulfide bonds. These inhibitory effects using NEM and TTN suggested that thiol groups in the integral protein AE1 play essential roles in the membrane transport of the selenium from the RBCs to the blood plasma.

Albumin-mediated selenium transfer by a selenotrisulfide relay mechanism.

In this study, we demonstrated a human serum albumin (HSA)-mediated selenium transfer; the selenium exported from red blood cells (RBCs) was bound to HSA through the selenotrisulfide and then transferred into the hepatocyte. After the treatment of the RBCs with selenite, the selenium efflux from the RBCs occurred in an HSA concentration-dependent manner. Pretreatment of HSA with iodoacetamide almost completely inhibited the selenium efflux from the RBC to the HSA solution. The selenium efflux experiment was carried out in an HSA solution (45 mg/mL), and subsequently the HSA solution was subjected to gel permeation chromatographic separation. The peak fraction of the selenium content was consistent with that of the HSA. The selenium bound to HSA in this solution was completely eliminated by a treatment with penicillamine (Pen), which resulted in the generation of penicillamine selenotrisulfide, PenSSeSPen. The selenium efflux from the RBCs was also occurred in a Pen solution, and PenSSeSPen was observed in the resulting Pen solution. The selenium exported from the RBC was thought to bind to the HSA via a selenotrisulfide linkage with its single free thiol. A model of the selenium-bound HSA was prepared by the reaction of the HSA with PenSSeSPen. The selenium from PenSSeSPen can bind to HSA by a thiol exchange between Pen and the free thiol of HSA, which produces the selenotrisulfide-containing HSA (HSA-SSeSPen). When HSA-SSeSPen was incubated with isolated rat hepatocytes, the selenium content in the hepatocytes increased along with its decrease in the incubation medium. To verify the results from the model experiments using HSA-SSeSPen, we conducted the HSA-mediated selenium transfer experiment from RBC treated with selenite to the hepatocytes. The selenotrisulfide-containing HSA was able to transport the selenium into the hepatocyte. Overall, the selenium transfer from the RBC to the hepatocytes involves a relay mechanism of thiol exchange that occurs between the selenotrisulfide and thiol compounds (selenotrisulfide relay mechanism: R-SSeS-R + HSA-SH --> HSA-SSeS-R + R'-SH --> R-SSeS-R').

Hemoglobin-mediated selenium export from red blood cells.

On the basis of the fact that selenium from selenite binds to hemoglobin (Hb), we investigated the missing process in the selenium export from red blood cells (RBCs), i.e., the transfer of selenium bound to Hb to RBC membrane proteins. To elucidate the molecular events of the Hb-associated selenium export from RBC, a Hb-Se complex was synthesized from thiol-exchange of Cys-beta93 in Hb with penicillamine-substituted glutathione selenotrisulfide, as a model of major metabolic intermediates, and then interactions between the Hb-Se complex and RBC inside-out vesicles (IOVs) were examined. Selenium bound to Hb was transferred to the IOV membrane on the basis of the intrinsic interactions between Hb and the cytoplasmic domains of band 3 protein (CDB3). The observed selenium transfer was inhibited by the pretreatments of IOVs with iodoacetamide and the alpha-chymotrypsin digestion, indicating that the Hb mediates the selenium transfer to the thiol groups of CDB3. In addition, it was found that deoxygenated Hb, with a high binding affinity for CDB3, more favorably transferred selenium to the IOV membranes than oxygenated Hb, with a low affinity. When selenium export from RBC to the plasma was examined by continuously introducing nitrogen gas, the selenium export rate was promoted with an increase in the rate of deoxygenated Hb. Overall, these data suggested that Hb could possibly play a role in the selenium export from RBC treated with selenite in an oxygen-linked fashion.

Synthesis and biological evaluation of (E)-3-styrylpyridine derivatives as amyloid imaging agents for Alzheimer's disease.

A new series of (E)-3-styrylpyridine derivatives as potential diagnostic imaging agents targeting amyloid plaques in Alzheimer's disease (AD) were synthesized and examined. When in vitro binding studies using AD brain homogenates were carried out with a series of styrylpyridine derivatives, (E)-2-Bromo-5-(4-dimethylaminostyryl)pyridine (7) with a dimethylamino group showed the highest binding affinity. Compound 7 intensely stained neuritic and diffused plaques and cerebrovascular amyloids on postmortem AD brain sections. (E)-2-Iodo-5-(4-dimethylaminostyryl)pyridine, the iodo derivative of compound 7, also stained senile plaques in human AD sections. The radioiodinated ligand [125I] was successfully prepared through an iododestannylation reaction from the corresponding tributyltin derivatives using hydrogen peroxide as the oxidant in high yields and with high radiochemical purity. A biodistribution study in normal mice after an intravenous injection of [125I] displayed high brain uptake and fast washout. Taken together, the data suggest that the new radio tracer, [125I], may be useful as a radioiodinated imaging agent for mapping A beta plaques in the brains of patients with AD.

Selenium binding to human hemoglobin via selenotrisulfide.

Selenotrisulfide (e.g., glutathione selenotrisulfide (GSSeSG)) is an important intermediate in the metabolism of selenite. However, its reactivity with biological substances such as peptides and proteins in the subsequent metabolism is still far from clearly understood, because of its chemical instability under physiological conditions. Penicillamine (Pen) is capable of generating a chemically stable and isolatable selenotrisulfide, PenSSeSPen. To explore the metabolic fate of selenite in red blood cells (RBC), we investigated the reaction of selenotrisulfide with human hemoglobin (Hb) using PenSSeSPen as a model. PenSSeSPen rapidly reacted with Hb under physiological conditions. From the analysis of selenium binding using the Langmuir type binding equation, the apparent binding number of selenium per Hb tetramer almost corresponded to the number of reactive thiol groups of Hb. The thiol group blockade of Hb by iodoacetamide treatment completely inhibited the reaction of PenSSeSPen with Hb. In addition, MALDI-TOF mass spectrometric analysis of the selenium-bound Hb revealed that PenSSe moiety binds to the beta subunits of Hb. Overall, the reaction of PenSSeSPen with Hb appears to involve the thiol exchange between Pen and the cysteine residues on the beta subunit of Hb.