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Membrane transporter PhT2 (SLC15A3), which belongs to the proton-coupled oligopeptide transporter family, mediates the transport of di/tripeptides and histidine utilizing an inwardly directed proton gradient and negative membrane potential. The aim of this study was to elucidate the molecular expression of PhT2 in macrophages and mouse tissues and to explore the regulation of PhT2 by lipopolysaccharide (LPS). The results showed relatively high expression of PhT2 in J774A.1 and THP-1 macrophage cells, mouse spleen, and lung. Using an LPS-induced inflammatory cell model, we found that hPhT2 mRNA expression was up-regulated in THP-1 cells and that the up-regulation was suppressed by pyrrolidine dithiocarbamate, a specific inhibitor of NF-κB. Similar results were observed in mouse spleen during LPS-induced acute inflammation. Using dual-labeling immunofluorescence and confocal laser scanning microscopy, we confirmed that mPhT2 was colocalizing with lysosome-associated membrane protein 1 in transfected HEK293 cells. These results suggested that PhT2, a lysosomal membrane transporter, was up-regulated by LPS via the NF-κB signaling pathway.

This study evaluated the developmental gene and protein expression of proton-coupled oligopeptide transporters (POTs: peptide transporter, PepT1 and PepT2; peptide-histidine transporter, PhT1 and PhT2) in different regions of rodent brain, and the age-dependent uptake of a POT substrate, glycylsarcosine (GlySar), in brain slices. Slices were obtained from cerebral cortex, cerebellum and hippocampus of wildtype and PepT2 null mice, and from rats at different ages. Gene and protein expression were determined by real-time PCR and immunoblot analyses. Brain slice uptakes of radiolabeled glycylsarcosine were determined in the absence and presence of excess unlabeled glycylsarcosine or l-histidine, the latter being an inhibitor of PhT1/2 but not PepT1/2. As PepT2 and PhT1 transcripts were abundantly expressed in all three regions of mouse brain, little to no expression was observed for PepT1 and PhT2. PhT1 protein was present in brain regions of adult but not neonatal mice and expression levels increased with age in rats. Glycylsarcosine uptake, inhibition and transporter dominance did not show regional brain or species differences. However, there were clear age-related differences in functional activity, with PepT2 dominating in neonatal mice and rats, and PhT1 dominating in adult rodents. These developmental changes may markedly impact the neural activity of both endogenous and exogenous (drug) peptides/mimetics. Developmental gene and protein expression of peptide transporters was evaluated in various regions of rodent brain, along with age-dependent uptake of dipeptide. We found marked changes in protein expression and functional activity of PhT1 and PepT2, the former predominating in adult and the latter in neonate. These developmental changes may markedly impact the neural activity of endogenous and exogenous peptides/mimetics., (© 2014 International Society for Neurochemistry.)

Objective: To explore the risk factors for Type 1 cardio-renal syndrome (CRS1) after ST-segment elevation myocardial infarction (STEMI)., Methods: A total of 378 patients with STEMI were divided into two groups: a CRS1 group (n=98) and a non-CRS1 group (n=280). Clinical characteristics in the 2 groups were compared, and independent risk factors for CRS1 after STEMI were analyzed, and the effect of emergency percutaneous coronary intervention (PCI) on CRS1 in patients after STEMI were assessed., Results: In the 378 STEMI patients, CRS1 was found in 98 patients (25.9%). Between the 2 groups, there was significant difference in 12 parameters, including age, history of diabetes, admission mean arterial pressure, admission systolic blood pressure, admission heart rate, Killip classification, left ventricular ejection fraction, baseline serum creatinine, baseline evaluated glomerular filtration rate (eGFR), emergency PCI, β-blockers and angiotensin converting enzyme inhibitor/angiotensin, receptor antagonist (ACEI/ARB) application (all P<0.05). Multivariate logistic regression showed that age, history of diabetes, admission systolic blood pressure, Killip classification, reduced left ventricular ejection fraction, reduced eGFR, emergency PCI nonundergo and ACEI/ARB non-use were independent risk factors for CRS1 after STEMI. In the 256 patients undergoing emergency PCI, 50 patients (19.5%) had CRS1. The door-ball time and the amount of contrast agent in the CRS1 group were significantly higher than those in the non- CRS1 group (both P<0.05), but there was no significant difference in the blood flow in the "culprit vessel" after the PCI (P>0.05)., Conclusion: CRS1 is a common complication of STEMI, which is associated with many factors. Immediate revascularization can reduce the incidence of CRS1 in patients with ST-segment elevation myocardial infarction.

Mass-selected IR plus UV/VUV spectroscopy and mass spectrometry have been coupled into a powerful technique to investigate chemical, physical, structural, and electronic properties of radicals, molecules, and clusters. Advantages of the use of vacuum ultraviolet (VUV) radiation to create ions for mass spectrometry are its application to nearly all compounds with ionization potentials below the energy of a single VUV photon, its circumventing the requirement of UV chromophore group, its inability to ionize background gases, and its greatly reduced fragmenting capabilities. In this review, mass-selected IR plus VUV (118 nm) spectroscopy is introduced first in a general manner. Selected application examples of this spectroscopy are presented, which include the detections and structural analysis of radicals, molecules, and molecular clusters in a supersonic jet., (© 2013 Wiley Periodicals, Inc.)

As a typical Raman reporter molecule, 6-Mercaptonicotinic acid (C6H5NO2S, MNA) has three atoms (i.e., N, O, and S), which could be potential to chemically interact with gold nano-surface. In the present report, the adsorption behavior of MNA on self-assembled gold nano-substrates is explored by means of surface-enhanced Raman scattering and theoretical calculations. The results reveal that all the enhanced bands are assigned to in-plane vibrations. The ring triangle breathing coupling with CS stretching band at 1108cm(-)(1) is enhanced a lot and redshifts to 1088cm(-)(1) in the SERS spectra. Furthermore, most of the bands related to N atom are apparently enhanced and shift in the SERS spectra. Based on the surface selection rule, it infers that MNA is vertically chemisorbed on self-assembled gold substrates through the S and the N atoms. The spectroscopic results are further interpreted by the density functional theory (DFT) calculations., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The primary objective of this study was to determine the in vivo absorption properties of valacyclovir, including the potential for saturable proton-coupled oligopeptide transporter 1 (PepT1)-mediated intestinal uptake, after escalating oral doses of prodrug within the clinical dose range. A secondary aim was to characterize the role of PepT1 on the tissue distribution of its active metabolite, acyclovir. [³H]Valacyclovir was administered to wild-type (WT) and PepT1 knockout (KO) mice by oral gavage at doses of 10, 25, 50, and 100 nmol/g. Serial blood samples were collected over 180 minutes, and tissue distribution studies were performed 20 minutes after a 25-nmol/g oral dose of valacyclovir. We found that the C(max) and area under the curve (AUC)₀₋₁₈₀ of acyclovir were 4- to 6-fold and 2- to 3-fold lower, respectively, in KO mice for all four oral doses of valacyclovir. The time to peak concentration of acyclovir was 3- to 10-fold longer in KO compared with WT mice. There was dose proportionality in the C(max) and AUC₀₋₁₈₀ of acyclovir in WT and KO mice over the valacyclovir oral dose range of 10-100 nmol/g (i.e., linear absorption kinetics). No differences were observed in the peripheral tissue distribution of acyclovir once these tissues were adjusted for differences in perfusing drug concentrations in the systemic circulation. In contrast, some differences were observed between genotypes in the concentrations of acyclovir in the distal intestine. Collectively, the findings demonstrate a critical role of intestinal PepT1 in improving the rate and extent of oral absorption for valacyclovir. Moreover, this study provides definitive evidence for the rational development of a PepT1-targeted prodrug strategy.

Site-selective ionization of ethanol dimer and the subsequent fragmentation were studied by synchrotron vacuum ultraviolet (VUV) photoionization mass spectrometry. With photoionization efficiency spectra measurements and theoretical calculations, the detailed mechanisms of the ionization-dissociation processes of ethanol dimer under VUV irradiation were explored. In 9.49-10.89 eV photon energy range, it was found that the ejection of the highest occupied molecular orbital (HOMO) electron from hydrogen bond donor induces a rapid barrierless proton-transfer process followed by two competitive dissociation channels, generating (C2H5OH)[middle dot]H(+) and CH2O[middle dot](C2H5OH)H(+), respectively. The latter comes from a carbon-carbon bond cleavage in the donor. While the photon energy is 10.9-11.58 eV, the electron of HOMO-1 of the hydrogen bond acceptor, is removed. Besides the dissociation channel to produce C2H5OH and C2H5OH(+), a new channel to generate (C2H5OH)[middle dot]CH2OH(+) is opened, where the cleavage of the carbon-carbon bond occurs in the acceptor. When the photon energy increases to 11.58 eV, the electron from HOMO-2 is ejected.

Recently, unethical manufacturers illegally adulterated foods to increase the nitrogen content of protein through the use of melamine. Although surface-enhanced Raman scattering (SERS) methods have been widely used to detect melamine, the details of the interaction mechanism between a melamine molecule and silver colloids are still unclear. In this paper, we present the adsorption behavior of melamine on the surface of silver nanoparticles, which we explored by using SERS and density functional theory calculation methods. The calculated results demonstrate that the melamine molecule interacts with the silver surface mainly via the heterocyclic nitrogen. When comparing the predicted spectra of the complexes of melamine-Ag(+) and melamine-Ag(0), we found that the spectrum of the former agrees better with the experimental spectra than that of the latter. In the structure of the melamine-Ag(+) complex, we found that the melamine molecule "stands" on the surface of nanoparticles, with a small tilt angle. The concentration-dependent SERS spectra reveal that the melamine molecule stands on the silver surface with a small tilt angle in high concentration, and the tilt angle becomes larger when the concentrations are diluted.

The aim of this study was to determine the expression and function of proton-coupled oligopeptide transporters (POTs) in spleen and macrophages and their contribution to innate immune response induced by bacterial peptidomimetics γ-iE-DAP and MDP. Quantitative real-time PCR (qRT-PCR) and Western blot results revealed the mRNA and protein expression of PepT2, PhT1, and PhT2, but not PepT1, in the spleen of mice and humans. In comparison to lymphocytes of the spleen, macrophages had higher transcript levels of PepT2 and PhT2. The cellular uptake of Ala-Lys-AMCA in mouse splenic macrophages was pH-dependent with maximum uptake at pH 6.0, and the kinetic parameters were K(m) = 75.5 ± 14.3 μM and V(max) = 25.4 ± 2.1 pmol/min per mg protein. The uptake of Ala-Lys-AMCA by mouse splenic macrophages was not inhibited by histidine but was significantly inhibited by glycyl-sarcosine (GlySar) and carnosine (P < 0.01), and by bacterial peptidomimetics γ-iE-DAP and MDP, ligands of nucleotide-binding oligomerization domain (NOD)-containing proteins. Carnosine and GlySar, but not histidine, attenuated the inflammatory response induced by γ-iE-DAP and MDP in mouse splenic macrophages. Functional expression of POTs was also demonstrated in THP-1 cells, and dipeptides reduced the immune response induced by γ-iE-DAP. In conclusion, our findings are novel by providing important information on the molecular and functional expression of POTs in the spleen. Moreover, it appears that the PepT2-mediated uptake of γ-iE-DAP and MDP in macrophages further contributes to the innate immune response.

Benzoic acid (BA) and phthalic acid (PTA) are the simplest aromatic carboxylic acids, and they can be regarded as typical model compounds in investigating the interaction of aromatic carboxylic acids with metal surfaces by use of SERS spectroscopy. In this work, we have investigated the structure and adsorption behavior of benzoic acid and phthalic acid on the gold surface with combination of SERS and DFT calculation methods. The experimental results show that both BA and PTA may be adsorbed on the Au surface with a bidentate bridging structure, namely, the carboxylate group(s) being bound to gold via two oxygen atoms in the carboxylate group(s). Comparison of the observed SERS and predicted spectra of the complexes of these two substances with Au atoms indicates that BA is favorable to adsorb on the gold surface with a vertical orientation rather than a flat one, and PTA could "stand up" on the Au surface as a slight tilt with a two-legged geometry, i.e. all four oxygen atoms in two carboxylate groups interact on the metal surface. Apart from that, we compare the discrepancy of SERS spectra between those two molecules, which could be taken as a potential analysis technique in food safety field., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Pancreatic cancer is characterized by early metastasis and high mortality. In this study, the role of miR-143 in invasion and metastasis was investigated in pancreatic cancer cells. miR-143 expression was established by an adenovirus-carried miR-143 expression cassette. mRNA and protein levels of gene expression were examined by RT-PCR and Western blot assay, respectively. Rho GTPases activity was measured by the pull down assay. The role of miR-143 in migration and invasion of Panc-1 cells was tested in vitro. The antimetastatic effect of miR-143 was tested in a liver metastasis model, while its antitumor growth effect was tested in a xenograft Panc-1 tumor model. Results demonstrated that ARHGEF1 (GEF1), ARHGEF2 (GEF2), and K-RAS genes are the targets of miR-143. miR-143 expression significantly decreased mRNA and protein levels of GEF1, GEF2, and K-RAS genes; lowered the constitutive activities of RhoA, Rac1, and Cdc42 GTPases; decreased the protein levels of MMP-2 and MMP-9; but significantly increased the protein level of E-cadherin. miR-143 expression also significantly inhibited the migration and invasion of Panc-1 cells in vitro, liver metastasis, and xenograft tumor growth in vivo. Our study suggested that miR-143 plays a central role in the invasion and metastasis of pancreatic cancer and miR-143 is a potential target for pancreatic cancer therapy.

In present study, photoionization and dissociation of acetic acid dimers have been studied with the synchrotron vacuum ultraviolet photoionization mass spectrometry and theoretical calculations. Besides the intense signal corresponding to protonated cluster ions (CH(3)COOH)(n)·H(+), the feature related to the fragment ions (CH(3)COOH)H(+)·COO (105 amu) via β-carbon-carbon bond cleavage is observed. By scanning photoionization efficiency spectra, appearance energies of the fragments (CH(3)COOH)·H(+) and (CH(3)COOH)H(+)·COO are obtained. With the aid of theoretical calculations, seven fragmentation channels of acetic acid dimer cations were discussed, where five cation isomers of acetic acid dimer are involved. While four of them are found to generate the protonated species, only one of them can dissociate into a C-C bond cleavage product (CH(3)COOH)H(+)·COO. After surmounting the methyl hydrogen-transfer barrier 10.84 ± 0.05 eV, the opening of dissociative channel to produce ions (CH(3)COOH)(+) becomes the most competitive path. When photon energy increases to 12.4 eV, we also found dimer cations can be fragmented and generate new cations (CH(3)COOH)·CH(3)CO(+). Kinetics, thermodynamics, and entropy factors for these competitive dissociation pathways are discussed. The present report provides a clear picture of the photoionization and dissociation processes of the acetic acid dimer in the range of the photon energy 9-15 eV.

The purpose of this study was to determine whether glycylsarcosine (a model dipeptide) and oseltamivir (an antiviral prodrug) exhibited a species-dependent uptake in yeast Pichia pastoris expressing the rat, mouse, and human homologs of PEPT1. Experiments were performed with [(3)H]glycylsarcosine (GlySar) in yeast P. pastoris expressing human, mouse, and rat peptide transporter 1 (PEPT1), in which uptake was examined as a function of time, concentration, potential inhibitors, and the dose-response inhibition of GlySar by oseltamivir. Studies with [(14)C]oseltamivir were also performed under identical experimental conditions. We found that GlySar exhibited saturable uptake in all three species, with K(m) values for human (0.86 mM) > mouse (0.30 mM) > rat (0.16 mM). GlySar uptake in the yeast transformants was specific for peptides (glycylproline) and peptide-like drugs (cefadroxil, cephradine, and valacyclovir), but was unaffected by glycine, l-histidine, cefazolin, cephalothin, cephapirin, acyclovir, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, tetraethylammonium, and elacridar. Although oseltamivir caused a dose-dependent inhibition of GlySar uptake [IC(50) values for human (27.4 mM) > rat (18.3 mM) > mouse (10.7 mM)], the clinical relevance of this interaction would be very low in humans. Of importance, oseltamivir was not a substrate for the intestinal PEPT1 transporter in yeast expressing the three mammalian species tested. Instead, the prodrug exhibited nonspecific binding to the yeast vector and PEPT1 transformants. Finally, the mouse appeared to be a better animal model than the rat for exploring the intestinal absorption and pharmacokinetics of peptides and peptide-like drugs in human.

Endogenous retrotransposons have caused extensive genomic variation within mammalian species, but the functional implications of such mobilization are mostly unknown. We mapped thousands of endogenous retrovirus (ERV) germline integrants in highly divergent, previously unsequenced mouse lineages, facilitating a comparison of gene expression in the presence or absence of local insertions. Polymorphic ERVs occur relatively infrequently in gene introns and are particularly depleted from genes involved in embryogenesis or that are highly expressed in embryonic stem cells. Their genomic distribution implies ongoing negative selection due to deleterious effects on gene expression and function. A polymorphic, intronic ERV at Slc15a2 triggers up to 49-fold increases in premature transcriptional termination and up to 39-fold reductions in full-length transcripts in adult mouse tissues, thereby disrupting protein expression and functional activity. Prematurely truncated transcripts also occur at Polr1a, Spon1, and up to ∼5% of other genes when intronic ERV polymorphisms are present. Analysis of expression quantitative trait loci (eQTLs) in recombinant BxD mouse strains demonstrated very strong genetic associations between the polymorphic ERV in cis and disrupted transcript levels. Premature polyadenylation is triggered at genomic distances up to >12.5 kb upstream of the ERV, both in cis and between alleles. The parent of origin of the ERV is associated with variable expression of nonterminated transcripts and differential DNA methylation at its 5'-long terminal repeat. This study defines an unexpectedly strong functional impact of ERVs in disrupting gene transcription at a distance and demonstrates that ongoing retrotransposition can contribute significantly to natural phenotypic diversity.

Purpose: To determine if fasting would affect the intestinal expression and in vivo functional activity of PEPT1 as determined after oral dosing of the dipeptide glycylsarcosine (GlySar)., Methods: Systemic exposure and tissue distribution studies were performed in wild-type and Pept1 knockout mice, under fed and fasted conditions, following both intravenous and oral doses of [(14)C]GlySar at 5 nmol/g body weight. Intestinal PEPT1 expression was evaluated by real-time PCR and immunoblot analyses., Results: We found that expression of PEPT1 protein in the small intestine was increased ~2-fold in wild-type mice during fasted as compared to fed conditions. In agreement, systemic exposure and peak plasma concentrations of orally administered GlySar were 40 and 65% greater, respectively, in wild-type mice during fasted vs. fed state. No significant differences were observed between fed and fasted animals during PEPT1 ablation. Tissue distribution of GlySar was unchanged after oral dosing for all four treatment groups., Conclusions: As little as 16 h of fasting can cause significant upregulation of PEPT1 protein expression in the small intestine, which then translates into a significant increase in in vivo oral absorption of GlySar.

This study evaluated the in vivo absorption and disposition of glycylsarcosine (GlySar), after escalating oral doses, in wild-type and peptide transporter 1 (Pept1) knockout mice. [(3)H]GlySar was administered to mice at doses of 1, 10, 100, 1000, and 5000 nmol/g b.wt. Serial blood samples were obtained over 480 min, the plasma was harvested, and the area under the plasma concentration-time curve (AUC) was determined. It was observed that the GlySar AUC was 60, 45, and 30% lower in knockout than wild-type mice when evaluated over 2, 4, and 8 h, respectively (p < 0.01). Plasma levels of GlySar reached a plateau at 90 min in knockout mice and then rose to a second plateau at 240 min. In wild-type mice, the plasma levels rose continuously to reach a single plateau at 90 min. When partial AUC (0-120 min) was used as an indicator for rate of absorption, there was a 60% reduction in GlySar absorption rate in knockout mice compared with wild-type animals. Tissue distribution studies were also performed after 10 nmol/g oral doses of [(3)H]GlySar. When sampled 1 h after dosing, GlySar tissue concentrations were significantly lower in knockout versus wild-type mice and, with the exception of intestines, reflected differences in the systemic exposure of dipeptide between these two genotypes. Overall, PEPT1 ablation in mice resulted in significant reductions, in vivo, in the rate and extent of GlySar absorption. The AUC of GlySar was proportional to dose in both genotypes over 1 to 100 nmol/g, with minor decrements at the two highest doses.

In this report, we present a novel approach to detect clenbuterol based on competitive surface-enhanced Raman scattering (SERS) immunoassay. Herein, a SERS nanoprobe that relies on gold nanoparticle (GNP) is labeled by 4,4'-dipyridyl (DP) and clenbuterol antibody, respectively. The detection of clenbuterol is carried out by competitive binding between free clenbuterol and clenbuterol-BSA fastened on the substrate with their antibody labeled on SERS nanoprobes. The present method allows us to detect clenbuterol over a much wider concentration range (0.1-100 pg mL(-1)) with a lower limit of detection (ca. 0.1 pg mL(-1)) than the conventional methods. Furthermore, by the use of this new competitive SERS immunoassay, the clenbuterol-BSA (antigen) is chosen to fasten on the substrate instead of the clenbuterol antibody, which could reduce the cost of the assay. Results demonstrate that the proposed method has the wide potential applications in food safety and agonist control., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The aim of this paper was to determine the clinical significance of the MHC class I chain-related molecule A(MICA) and NKG2D receptor on NK cells in pancreatic cancer. We compared MICA expression in malignant (n = 103), inflammatory (n = 28), and normal (n = 17) pancreatic tissues using immunohistochemistry and assessed serum levels of soluble MICA (sMICA) and NKG2D expression on NK cells in patients with pancreatic cancer (n = 103), in patients with chronic pancreatitis (n = 28), and in healthy volunteers (n = 43). Expression of MICA was detected in 89.3% of pancreatic cancer tissues, whereas fewer were expressed in inflammatory and normal pancreatic tissues. The levels of sMICA were frequently elevated in patients with advanced pancreatic cancer. The elevation of sMICA was associated with down-regulated NKG2D expression and impaired activity of NK cells. The successful tumor resection significantly decreased serum levels of sMICA and increased the NKG2D expression; there was an inverse correlation between change in sMICA levels and that in NKG2D expression. MICA expression, preoperative sMICA levels and NKG2D intensity were found to be independent prognostic factors in resected pancreatic cancer. This study supports the clinical significance of release of MICA for the malignant progression of pancreatic cancer. The successful tumor resection for pancreatic cancer may have a beneficial effect on NKG2D-mediated antitumor immunity. Our results also suggest sMICA and NKG2D expression on NK cells may be useful to identify risk patients at time point of diagnosis.

2-Phenylethylamine (PEA) is the simplest aromatic amine neurotransmitter, as well as one of the most important. In this work, the conformational equilibrium and hydrogen bonding in liquid PEA were studied by means of Raman spectroscopy and theoretical calculations (DFT/MP2). By changing the orientation of the ethyl and the NH(2) group, nine possible conformers of PEA were found, including four degenerate conformers. Comparison of the experimental Raman spectra of liquid PEA and the calculated Raman spectra of the five typical conformers in selected regions (550-800 and 1250-1500 cm(-1)) revealed that the five conformers can coexist in conformational equilibrium in the liquid. The NH(2) stretching mode of the liquid is red-shifted by ca. 30 cm(-1) relative to that of an isolated PEA molecule (measured previously), implying that intermolecular N-H···N hydrogen bonds play an important role in liquid PEA. The relative intensity of the Raman band at 762 cm(-1) was found to increase with increasing temperature, indicating that the anti conformer might be favorable in liquid PEA at room temperature. The blue shift of the band for the bonded N-H stretch with increasing temperature also provides evidence of the existence of intermolecular N-H···N hydrogen bonds.

First of all the surface enhanced Raman spectroscopy (SERS) and normal Raman spectra of caffeine aqueous solution were obtained at different pH values. In order to obtain the detailed vibrational assignments of the Raman spectroscopy, the geometry of caffeine molecule was optimized by density functional theory (DFT) calculation. By comparing the SERS of caffeine with its normal spectra at different pH values; it is concluded that pH value can dramatically affect the SERS of caffeine, but barely affect the normal Raman spectrum of caffeine aqueous solution. It can essentially affect the reorientation of caffeine molecule to the Ag colloid surface, but cannot impact the vibration of functional groups and chemical bonds in caffeine molecule., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The purpose of this study was to determine the relative importance of peptide transporter 1 (PEPT1) in the uptake of peptides/mimetics from mouse small intestine, using glycylsarcosine (GlySar). After isolating jejunal tissue from wild-type and Pept1 null mice, 2 cm intestinal segments were everted and mounted on glass rods for tissue uptake studies. [(14)C]GlySar (4 μM) was studied as a function of time, temperature, sodium and pH, concentration, and potential inhibitors. Compared with wild-type animals, Pept1 null mice exhibited a 78% reduction in GlySar uptake at pH 6.0 at 37°C. GlySar uptake showed pH dependence, with peak values between pH 6.0 and 6.5 in wild-type animals, whereas no such tendency was observed in Pept1 null mice. GlySar exhibited Michaelis-Menten uptake kinetics and a minor nonsaturable component in wild-type animals. In contrast, GlySar uptake occurred only by a nonsaturable process in Pept1 null mice. GlySar uptake was significantly inhibited by dipeptides, aminocephalosporins, angiotensin-converting enzyme inhibitors, and the antiviral prodrug valacyclovir; these inhibitors had little, if any, effect on the uptake of GlySar in Pept1 null mice. The findings demonstrate that PEPT1 plays a critical role in the uptake of GlySar in jejunum and suggest that PEPT1 is the major transporter responsible for the intestinal absorption of small peptides., (Copyright © 2010 Wiley-Liss, Inc.)

The purpose of this study was to define the cerebrospinal fluid (CSF) clearance kinetics, choroid plexus uptake, and parenchymal penetration of PEPT2 substrates in different regions of the brain after intracerebroventricular administration. To accomplish these objectives, we performed biodistribution studies using [(14)C]glycylsarcosine (GlySar) and [(3)H]cefadroxil, along with quantitative autoradiography of [(14)C]GlySar, in wild-type and Pept2 null mice. We found that PEPT2 deletion markedly reduced the uptake of GlySar and cefadroxil in choroid plexuses at 60 mins by 94% and 82% (P<0.001), respectively, and lowered their CSF clearances by about fourfold. Autoradiography showed that GlySar concentrations in the lateral, third, and fourth ventricle choroid plexuses were higher in wild-type as compared with Pept2 null mice (P<0.01). Uptake of GlySar by the ependymal-subependymal layer and septal region was higher in wild-type than in null mice, but the half-distance of penetration into parenchyma was significantly less in wild-type mice. The latter is probably because of the clearance of GlySar from interstitial fluid by brain cells expressing PEPT2, which stops further penetration. These studies show that PEPT2 knockout can significantly modify the spatial distribution of GlySar and cefadroxil (and presumably other peptides/mimetics and peptide-like drugs) in brain.

The purpose of this study was to evaluate the role, relevance, and regional dependence of peptide transporter (PEPT) 1 expression and function in mouse intestines using the model dipeptide glycylsarcosine (GlySar). After isolating specific intestinal segments, in situ single-pass perfusions were performed in wild-type and Pept1 knockout mice. The permeability of [(3)H]GlySar was measured as a function of perfusate pH, dipeptide concentration, potential inhibitors, and intestinal segment, along with PEPT1 mRNA and protein. We found the permeability of GlySar to be saturable (K(m) = 5.7 mM), pH-dependent (maximal value at pH 5.5), and specific for PEPT1; other peptide transporters, such as PHT1 and PHT2, were not involved, as judged by the lack of GlySar inhibition by excess concentrations of histidine. GlySar permeabilities were comparable in the duodenum and jejunum of wild-type mice but were much larger than that in ileum (approximately 2-fold). A PEPT1-mediated permeability was not observed for GlySar in the colon of wild-type mice (<10% residual uptake compared to proximal small intestine). Moreover, GlySar permeabilities were very low and not different in the duodenum, jejunum, ileum, and colon of Pept1 knockout mice. Functional activity of intestinal PEPT1 was confirmed by real-time polymerase chain reaction and immunoblot analyses. Our findings suggest that a loss of PEPT1 activity (e.g., due to polymorphisms, disease, or drug interactions) should have a major effect in reducing the intestinal absorption of di-/tripeptides, peptidomimetics, and peptide-like drugs.

Neuropeptide inactivation is generally thought to occur via peptidase-mediated degradation. However, a recent study found increased analgesia after L-kyotorphin (L-Tyr-L-Arg; L-KTP) administration in mice lacking an oligopeptide transporter, PEPT2. The current study examines the role of PEPT2 in L-KTP uptake by astrocytes and compares it to astrocytic L-KTP degradation. L-[(3)H]KTP uptake was measured in primary cultures of neonatal astrocytes from rats and from Pept2(+/+) and Pept2(-/-) mice. Uptake was further characterized using potential inhibitors. L-[(3)H]KTP degradation was examined in primary astrocyte cultures from Pept2(-/-) mice by following the formation of L-[(3)H]tyrosine. The uptake of L-[(3)H]KTP in both rat and Pept2(+/+) mouse neonatal astrocytes was inhibited by known PEPT2 inhibitors. L-[(3)H]KTP uptake was also reduced in Pept2(-/-) astrocytes as compared to those from Pept2(+/+) mice. Kinetic analysis indicated the presence of a high affinity (K(m) approximately 50 microM) transporter for L-[(3)H]KTP, identified as Pept2, and a low affinity transporter (K(m) approximately 3-4 mM), inhibited by amastatin, bestatin and tyrosine. Astrocytes also degraded L-KTP through a low affinity peptidase (K(m) approximately 2 mM). Astrocytic clearance of L-KTP occurs via both peptidase activity and transport. These processes occur at similar rates and may be linked. This supports the contention that oligopeptide transport may have an impact on the extracellular clearance (and potentially activity) of certain neuropeptides., (Copyright 2010 Elsevier B.V. All rights reserved.)

We report the first observation of infrared (IR) and mass spectra of neutral, aliphatic amino acid clusters: the example presented herein is for (valine)(n), n = 2-5. The clusters are generated in a supersonic expansion and their IR spectra are recorded in various fragment and (valine)(n-1)H(+) (n = 2-5) mass channels. The ions are created by single photon ionization with a VUV laser at 118 nm (10.5 eV/photon) following IR absorption in the single photon energy range 2500-4000 cm(-1). Mass channels obviously associated with valine clusters lose intensity and mass channels associated with valine monomers gain intensity under IR irradiation. No free OH modes are identified in any of these spectra suggesting that for (valine)(n), n = 1-5, all the OH groups are hydrogen bonded. The infrared transition for the hydrogen bonded OH moiety appears as a very broad, shifted feature ca. 3000 cm(-1) (approximately 2800 to approximately 3200 cm(-1)). Free and perturbed NH(2) modes can also be identified in the cluster spectra. CH modes ca. 3000 cm(-1) can be identified and appear to be coupled to the shifted and broadened OH modes of the clusters. Fragmentation pathways for three (valine)(2) isomers under 118 nm ionization are proposed and discussed.

L-Kyotorphin (L-KTP), an endogenous analgesic neuropeptide, is a substrate for aminopeptidases and a proton-coupled oligopeptide transporter, PEPT2. This study examined the CSF efflux, antinociceptive response, and hydrolysis kinetics in brain of L-KTP and its synthetic diastereomer D-kyotorphin (D-KTP) in wild-type and Pept2 null mice. CSF clearance of L-KTP was slower in Pept2 null mice than in wild-type animals, and this difference was reflected in greater L-KTP-induced analgesia in Pept2 null mice. Moreover, dose-response analyses showed that the ED50 of L-KTP in Pept2-deficient animals was one-fifth of the value observed in Pept2-competent animals (4 vs. 21 nmol for null vs. wild-type mice, respectively). In contrast, the ED50 of D-KTP was very similar between the two genotypes (9-10 nmol). Likewise, there was little difference between genotypes in slope factor or baseline effects of L-KTP and D-KTP. The enhanced antinociceptive response to L-KTP in Pept2 null mice could not be explained by differences in neuropeptide degradation as Vmax and Km values did not differ between genotypes. Our results demonstrate that PEPT2 can significantly impact the analgesic response to an endogenous neuropeptide by altering CSF (and presumably brain interstitial fluid) concentrations and that it may influence the disposition and response to exogenous peptide/mimetic substrates.

Carnosine (beta-alanyl-l-histidine), an endogenous dipeptide substrate of the proton-coupled oligopeptide transporter PEPT2, plays an important role in many physiological processes. This study examined the effect of PEPT2 on the disposition of endogenous and exogenous carnosine in wild-type and Pept2 null mice. After exogenous dosing of [(3)H]carnosine (1 nmol/g iv bolus), a marked increase was observed in its systemic clearance in Pept2 null mice (0.50 vs. 0.29 ml/min), resulting in a decreased systemic exposure of dipeptide (area under the curve = 43.7 vs. 73.0 microM). Carnosine uptake was substantially reduced in the kidney of Pept2 null mice, and renal clearance increased 18-fold in this genotype (206 vs. 11.5 microl/min). Fractional reabsorption of carnosine in Pept2 null mice was only one-fifth that in wild-type animals (0.20 vs. 0.94). PEPT2 also had a substantial impact in brain where the cerebrospinal fluid (CSF)-to-plasma concentration ratio of carnosine was eightfold greater in Pept2 null mice (0.70 vs. 0.08). With respect to endogenous carnosine levels, significant reductions were observed in Pept2 null compared with wild-type mice for choroid plexus (0.026 vs. 0.20 mmol/kg), olfactory bulb (1.12 vs. 1.79 mmol/kg), and spleen (0.019 vs. 0.029 mmol/kg). In contrast, carnosine levels in the skeletal muscle of Pept2 null mice were significantly increased (1.70 vs. 1.14 mmol/kg), and no differences were observed between genotypes for endogenous carnosine levels in plasma and CSF. These results demonstrate that PEPT2 significantly modulates the disposition of exogenous carnosine. However, endogenous carnosine levels may be under homeostatic control to maintain systemic and central concentrations under physiological in vivo conditions.

Aniline-methanol mixed clusters are ionized by single photon vacuum ultraviolet (VUV, 118 nm) radiation with which absorption to an excited intermediate S(1) state is not required. Aniline ion (An(+)), a series of (An)(n)(+)-(CH(3)OH)(m) (n = 1, 2) cluster ions, and their hydrogenated cluster ions, (An)(n)(+)-(CH(3)OH)(m)H (n = 1, 2) are observed by mass spectrometry. Infrared (IR) absorption spectra of aniline-methanol cluster cations and neutrals are measured through IR and VUV (118 nm) "ion dip" spectroscopy in the range 2500-4000 cm(-1). The observed mid-IR spectrum of the An(+)-CH(3)OH has two sharp absorption bands, at 3438 and 3668 cm(-1), which are assigned to the free NH stretch vibration of the aniline cation and the free OH stretch vibration of methanol, respectively. Calculations demonstrated that a change in the charges on the nitrogen atom of the amine group upon ionization of the neutral to the cluster cation alters the role of aniline from hydrogen acceptor to hydrogen donor in its interaction with methanol. Theoretical and experimental results suggest that a hydrogen bond forms between one of the H atoms of the aniline amine group and the lone pair of electrons of the methanol oxygen atom in the aniline-methanol cluster cation. Measured IR spectra and theoretical results for neutral clusters suggest that the H atom of the methanol OH moiety is bonded to the aniline amine group lone pair electrons for the neutral ground state aniline-methanol cluster.

Purpose: The aim of this study was to investigate the transport properties of carnosine in kidney using SKPT cell cultures as a model of proximal tubular transport, and to isolate the functional activities of renal apical and basolateral transporters in this process., Methods: The membrane transport kinetics of 10 microM [3H]carnosine was studied in SKPT cells as a function of time, pH, potential inhibitors and substrate concentration. A cellular compartment model was constructed in which the influx, efflux and transepithelial clearances of carnosine were determined. Peptide transporter expression was probed by RT-PCR., Results: Carnosine uptake was 15-fold greater from the apical than basolateral surface of SKPT cells. However, the apical-to-basolateral transepithelial transport of carnosine was severely rate-limited by its cellular efflux across the basolateral membrane. The high-affinity, proton-dependence, concentration-dependence and inhibitor specificity of carnosine supports the contention that PEPT2 is responsible for its apical uptake. In contrast, the basolateral transporter is saturable, inhibited by PEPT2 substrates but non-concentrative, thereby, suggesting a facilitative carrier., Conclusions: Carnosine is expected to have a substantial cellular accumulation in kidney but minimal tubular reabsorption in blood because of its high influx clearance across apical membranes by PEPT2 and very low efflux clearance across basolateral membranes.

The aliphatic amino acids glycine, valine, leucine, and isoleucine are thermally placed into the gas phase and expanded into a vacuum system for access by time of flight mass spectroscopy and infrared (IR) spectroscopy in the energy range of 2500-4000 cm(-1) (CH, NH, OH, and stretching vibrations). The isolated neutral amino acids are ionized by a single photon of 10.5 eV energy (118 nm), which exceeds by less than 2 eV their reported ionization thresholds. As has been reported for many hydrogen bonded acid-base systems (e.g., water, ammonia, alcohol, acid clusters, and acid molecules), the amino acids undergo a structural rearrangement in the ion state (e.g., in simplest form, a proton transfer) that imparts sufficient excess vibrational energy to the ion to completely fragment it. No parent ions are observed. If the neutral ground state amino acids are exposed to IR radiation prior to ionization, an IR spectrum of the individual isomers for each amino acid can be determined by observation of the ion intensity of the different fragment mass channels. Both the IR spectrum and fragmentation patterns for individual isomers can be qualitatively identified and related to a particular isomer in each instance. Thus, each fragment ion detected presents an IR spectrum of its particular parent amino acid isomer. In some instances, the absorption of IR radiation by the neutral amino acid parent isomer increases a particular fragmentation mass channel intensity, while other fragmentation mass channel intensities decrease. This phenomenon can be rationalized by considering that with added energy in the molecule, the fragmentation channel populations can be modulated by the added vibrational energy in the rearranged ions. This observation also suggests that the IR absorption does not induce isomerization in the ground electronic state of these amino acids. These data are consistent with theoretical predictions for isolated amino acid secondary structures and can be related to previous IR spectra of amino acid conformers.

Background: To assess the outcome of patients with stable angina pectoris treated with percutaneous coronary intervention versus medically treated patients., Methods: Eighty patients with stable angina pectoris and coronary stenosis as confirmed in coronary angiography were treated with (n = 31) or without (n = 49) percutaneous coronary intervention in our department. All patients received optimal medical therapy and were followed up for a period of 24 months., Results: Baseline clinical characteristics, including risk factors of coronary heart disease and coronary lesion type did not differ between the two groups (all p > 0.05). There was no significant difference in major adverse cardiac events (22.4% vs. 22.6%) during the 24 month follow-up between the two groups (p > 0.05)., Conclusions: Percutaneous coronary intervention did not provide extra benefit in this group of patients with stable angina pectoris receiving standard medical treatment in terms of 24 months major adverse outcomes.

The proton-coupled oligopeptide transporter PEPT2 (or SLC15A2) is the major protein involved in the reclamation of peptide-bound amino acids and peptide-like drugs in kidney. PEPT2 is also important in effluxing peptides and peptidomimetics from CSF at the choroid plexus, thereby limiting their exposure in brain. In this study, we report a neuroprotective role for PEPT2 in modulating the toxicity of a heme precursor, 5-aminolevulinic acid (5-ALA). Our findings demonstrate that in PEPT2-deficient mice, 5-ALA administration results in reduced survivability, a worsening of neuromuscular dysfunction, and CSF concentrations of substrate that are 8-30 times higher than that in wild-type control animals. The ability of PEPT2 to limit 5-ALA exposure in CSF suggests that it may also have relevance as a secondary genetic modifier of conditions (such as acute hepatic porphyrias and lead poisoning) in which 5-ALA metabolism is altered and in which 5-ALA toxicity is important.

The aim of this study was to examine the role of PEPT2, a proton-coupled oligopeptide transporter of the SLC15 family, on the disposition of the antibiotic cefadroxil in the body, particularly the kidney and brain. Pharmacokinetic, tissue distribution, and renal clearance studies were performed in wild-type and PEPT2 null mice after intravenous bolus administration of [(3)H]cefadroxil at 1, 12.5, 50, and 100 nmol/g body weight. Studies were also performed in the absence and presence of probenecid and quinine. Cefadroxil disposition kinetics was clearly nonlinear over the dose range studied (1-100 nmol/g), which was attributed to both saturable renal tubular secretion and reabsorption of the antibiotic. After an intravenous bolus dose of 1 nmol/g cefadroxil, PEPT2 null mice exhibited a 3-fold greater total clearance and 3-fold lower systemic concentrations of drug compared with wild-type animals. Renal clearance studies further demonstrated that the renal reabsorption of cefadroxil was almost completely abolished in PEPT2 null versus wild-type mice (3% versus 70%, p < 0.001). Of the 70% of cefadroxil reabsorbed in wild-type mice, PEPT2 accounted for 95% and PEPT1 accounted for 5% of reabsorbed substrate. Tissue distribution studies indicated that PEPT2 had a dramatic effect on cefadroxil tissue exposure, especially in brain where the cerebrospinal fluid (CSF)-to-blood concentration ratio of cefadroxil was 6-fold greater in PEPT2 null mice compared with wild-type animals. These findings demonstrate that renal PEPT2 is almost entirely responsible for the reabsorption of cefadroxil in kidney and that choroid plexus PEPT2 limits the exposure of cefadroxil (and perhaps other aminocephalosporins) in CSF.

5-aminolevulinic acid (ALA) and carnosine have important physiological and pathophysiological roles in the CNS. Both are substrates for the proton-coupled oligopeptide transporter PEPT2. The purpose of the current study was to determine the importance of PEPT2 in the uptake of ALA and carnosine in rat and mouse (PEPT2+/+ and PEPT2-/-) cultured neonatal astrocytes. Although neonatal astrocytes are known to express PEPT2, its quantitative importance in the transport of these compounds is not known. [14C]ALA uptake in neonatal rat astrocytes was inhibited by dipeptides, an alpha-amino containing cephalosporin (which is a PEPT2 substrate) but was not affected by a non-amino containing cephalosporin (which is not a PEPT2 substrate). Uptake was pH sensitive as expected from a proton-coupled transporter and was saturable (Vmax=715+/-29 pmol/mg/min, Km=606+/-14 microM). [3H]Carnosine uptake in neonatal rat astrocytes was inhibited by dipeptides but not by histidine (a substrate for the peptide/histidine transporters PHT1 and PHT2) and also showed saturable transport (Vmax=447+/-23 pmol/mg/min, Km=43+/-5.5 microM). Neonatal astrocytes from PEPT2-/- mice had a 62% reduction in [14C]ALA uptake and a 92% reduction in [3H]carnosine uptake compared to PEPT2+/+ mice. These results demonstrate that PEPT2 is the primary transporter responsible for the astrocytic uptake of ALA and carnosine.

The aims of the current study were (1) to quantify the role of PEPT2 in the uptake of glycylsarcosine (GlySar) in cultured neonatal astrocytes and (2) to examine GlySar transport and PEPT2 expression in two glioma cell lines. The uptake of [(14)C]GlySar was measured in astrocytes cultured from neonatal mouse (PEPT2(+/+) and PEPT2(-/-)) and rat, as well as rat C6 and F98 glioma cells. PEPT2 expression was examined by reverse transcription-polymerase chain reaction (RT-PCR). Neonatal astrocytes from PEPT2(-/-) mice had a 94% reduction in [(14)C]GlySar uptake compared to wild type mice and there was no saturable transport. In PEPT2(+/+) mice, [(14)C]GlySar uptake was saturable (V(max) 58 +/- 12 pmol/mg/min, K(m) 107 +/- 46 microM, K(d) 0.043 +/- 0.004 microl/mg/min). In neonatal rat astrocytes, kinetic analysis also suggested that [(14)C]GlySar uptake was via a single transporter. The inhibitor profile and pH dependence of that transport process was consistent with PEPT2. In C6 and F98 glioma cells, [(14)C]GlySar uptake was markedly reduced ( approximately 96-98%) compared to that in neonatal astrocytes and this was reflected by an absence of PEPT2 mRNA expression. These results indicate that PEPT2 is the sole transporter involved in the uptake of GlySar into neonatal cultured astrocytes. However, PEPT2 mRNA appears to be absent from two glioma cell lines.

Cefadroxil is a cephalosporin antibiotic used in the treatment of infection. However, cerebrospinal fluid (CSF) concentrations of cefadroxil and other aminocephalosporins are not adequate for the treatment of bacterial meningitis. To evaluate the relevance of PEPT2 in affecting the exposure of aminocephalosporins in brain, we investigated the transport properties of cefadroxil at the blood-CSF interface using primary-cultured epithelial cells and isolated whole tissues of choroid plexus. Our results indicated that cefadroxil was preferentially taken up from the apical as opposed to basal side of the monolayer (5-fold), and its apical uptake was stimulated by an inwardly directed proton gradient. The concentration-dependent apical uptake of cefadroxil was characterized by a high-affinity/low-capacity transport system (Km = 39.0 +/- 22.7 microM; Vmax = 22.9 +/- 6.6 pmol/mg/min) and a nonsaturable component (Kd = 0.15 +/- 0.01 microl/mg/min); in contrast, only a nonsaturable component was found for the basal uptake of cefadroxil (Kd = 0.14 +/- 0.01 microl/mg/min). The apical-to-basal transepithelial transport of 2 microM cefadroxil was greater than its basal-to-apical transport, but no differences were observed in directionality when 5 mM concentrations of cefadroxil were studied. Moreover, the cellular efflux of cefadroxil was not saturable in either direction (i.e., to apical or basal side). Finally, no differences were observed in the choroid plexus tissue efflux of 2 microM cefadroxil from wild-type and PEPT2 null mice. These findings demonstrate that PEPT2 has an important role in limiting the exposure of cefadroxil in CSF. Located at the apical membrane of choroid plexus epithelium, PEPT2 acts in a unidirectional (as opposed to bidirectional) manner in transporting cefadroxil from CSF into the cell.

The strategic localization of peptide transporter 2 (PEPT2), a proton-coupled oligopeptide transporter, to the apical membrane of epithelial cells in the kidney and choroid plexus suggests that it plays an important role in the disposition of peptides/mimetics in the body. Therefore, the in vivo significance of PEPT2 was investigated in wild-type and PEPT2 null mice following an i.v. bolus dose (0.05 micromol/g body weight) of [14C]glycylsarcosine (GlySar). In PEPT2 null mice, the clearance (total and renal) of GlySar was markedly increased (2-fold), resulting in concomitantly lower systemic concentrations. In addition, renal reabsorption was almost abolished, and GlySar was eliminated by glomerular filtration. Of the 46% of GlySar reabsorbed in wild-type mice, PEPT2 accounted for 86% and PEPT1 accounted for 14% of reabsorbed substrate. Analysis of GlySar uptake in kidney sections revealed that PEPT2 was primarily localized in the outer medullary region. Wild-type mice also had greater choroid plexus concentrations of GlySar and a 5-fold greater choroid plexus/cerebrospinal fluid (CSF) ratio as compared with null mice at 60 min. Null mice exhibited a greater CSF/blood ratio at 60 min (0.9 versus 0.2) and area under the curve (AUC)(CSF)/AUC(blood) ratio over 60 min (0.45 versus 0.12), indicating that PEPT2 significantly reduces the exposure of GlySar in CSF. Our in vivo results demonstrate that PEPT2 is the predominant peptide transporter in kidney and that it acts as an efflux transporter in choroid plexus. Thus, PEPT2 may have profound effects on the sensitivity and/or toxicity of peptides and peptide-like drugs.

Purpose: The purpose of this research was to determine the polarity and directionality of the PEPT2-mediated uptake and transepithelial transport of the neuropeptide glycyl-L-glutamine (GlyGln) in choroid plexus., Methods: The transport kinetics of [3H]GlyGln was studied in neonatal rat choroid plexus epithelial cells in primary culture grown on laminin-coated Transwell filter inserts. Using a bicarbonate artificial cerebrospinal fluid (CSF) buffer (pH 7.4) at 37 degrees C, GlyGln studies were performed as a function of time, substrate concentration, and the presence of potential inhibitors (at 1 mM)., Results: GlyGln (2 microM) accumulation was about three to four times greater when introduced from the apical (CSF-facing) as opposed to the basal (blood-facing) side of the cell monolayer, and transepithelial transport was about two times greater in the apical-to-basal direction. The apical uptake of radiolabeled GlyGln (2 microM) was inhibited significantly by dipeptides (i.e., unlabeled GlyGln and cysteinylglycine) and some neuropeptides (i.e., carnosine, N-acetylaspartylglutamate, kyotorphin), but was unaffected by amino acids (i.e., glycine, glutamine) as well as by [D-Arg2]-kyotorphin and glutathione. The concentration-dependent apical uptake of GlyGln (2-1000 microM) was characterized by a high-affinity process (i.e., Vmax of 72 pmol/mg/min; Km of 136 microM), consistent with the properties of PEPT2. The intracellular hydrolysis of GlyGln was extensive, however, with only 40% of the dipeptide remaining intact after 1 h., Conclusions: The results demonstrate that PEPT2 plays an important role in regulating the apical uptake of GlyGln at the blood-CSF interface. Once inside the cell, GlyGln is rapidly degraded to its constitutive amino acids for further processing.

Purpose: To determine the importance of PEPT2 in the uptake of glycylsarcosine (GlySar) and 5-aminolevulinic acid (5-ALA) in mouse choroid plexus whole tissue., Methods: Uptake studies were performed in bicarbonate artificial cerebrospinal fluid buffer using choroid plexuses isolated from PEPT2+/+ and PEPT2-/- mice. [14C]GlySar and [14C]5-ALA were studied as a function of temperature, concentration, potential inhibitors, and low sodium conditions., Results: PEPT2-/- mice exhibited a 90% reduction in GlySar uptake (p < 0.001) and a 92% reduction in 5-ALA uptake (p < 0.001) as compared to wild type animals. At 4 degrees C (vs. 37 degrees C), GlySar uptake was reduced by 95% in PEPT2+/+ mice; no difference was observed in null animals. Unlabeled GlySar inhibited the uptake of [14C]GlySar in PEPT2+/+ mice (p < 0.01); self-inhibition did not occur in PEPT2-/- mice. GlySar demonstrated saturable uptake in PEPT2+/+ mice (Vmax = 16.4 pmol mg(-1) min(-1), Km = 70 microM, Kd = 0.014 microl mg(-1) min(-1)), however, uptake was linear in PEPT2-/- mice (Kd = 0.023 microl mg(-1) min(-1)). Low sodium buffer (1 mM) resulted in 75% and 59% reductions, respectively, in GlySar (p < 0.001) and 5-ALA (p < 0.01) uptake in PEPT2+/+ mice; no differences were observed in PEPT2-/- mice. Overall, about 90-95% of the choroid plexus uptake of GlySar and 5-ALA was mediated by PEPT2, with about 5-10% of the residual uptake occurring by nonspecific mechanisms., Conclusions: The results demonstrate that PEPT2 is the only transporter responsible for the choroid plexus uptake of GlySar and 5-ALA. They also suggest a role for PEPT2 in the clearance of dipeptides and endogenous peptidomimetics from cerebrospinal fluid.

Purpose: To investigate the mechanism of electroacupuncture (EA) plus intra-carotid drug injection for treating cerebral infarction., Methods: Rheoencephalogram was recorded with a RG-2B type of bridge rheoencephalograph and findings were compared before and after the treatment., Results: After the treatment, the prolonged rising time was shortened, and the decreased amplitude obviously elevated., Conclusion: The therapy can dilate cerebral blood vessels, increase the cerebral blood flow, and improve the elasticity of cerebral blood vessels, leading to sufficient blood and oxygen supply in the ischemic brain tissues and to restoration of their functions.

The choroid plexus uptake of [(3)H]cefadroxil was studied in peptide transporter 2 (PEPT2) wild-type and null mice as a function of temperature, transport inhibitors, pH, and saturability. At normal pH (7.4) and temperature (37 degrees C), the uptake of 1 microM cefadroxil was reduced by 83% in PEPT2(-/-) mice as compared with PEPT2(+/+) mice (p < 0.001). A further reduction was achieved in null animals by reducing the temperature to 4 degrees C, or by adding saturating concentrations of unlabeled cefadroxil or p-aminohippurate (p < 0.05). Glycylsarcosine coadministration could inhibit the uptake of cefadroxil in PEPT2(+/+) mice (p < 0.01) but not PEPT2(-/-) mice. Although a proton-stimulated uptake of cefadroxil was demonstrated in PEPT2(+/+) mice (pH 6.5 versus pH 7.4; p < 0.01), no pH dependence was observed in PEPT2(-/-) mice. Kinetic parameters for cefadroxil (without p-aminohippurate) in wild-type mice were: V(max) = 5.4 pmol/mg/min, K(m) = 34 microM, and K(d) = 0.0069 microl/mg/min; in the presence of p-aminohippurate, the parameters were: V(max) = 4.1 pmol/mg/min, K(m) = 27 microM, and K(d) = 0.0064 microl/mg/min. In null animals, the kinetic parameters of cefadroxil (without p-aminohippurate) were: V(max) = 2.7 pmol/mg/min, K(m) = 110 microM, and K(d) = 0.0084 microl/mg/min; in the presence of p-aminohippurate, only a K(d) = 0.010 microl/mg/min was observed. Based on kinetic and inhibitor analyses, it was determined that (under linear conditions), 80 to 85% of cefadroxil's uptake in choroid plexus is mediated by PEPT2, 10 to 15% by organic anion transporter(s), and 5% by nonspecific mechanisms. These findings demonstrate that PEPT2 is the primary transporter responsible for cefadroxil uptake in the choroid plexus. Moreover, the data suggest a role for PEPT2 in the clearance of peptidomimetics from cerebrospinal fluid.