This study evaluated the developmental gene and protein expression of proton-coupled oligopeptide transporters (POTs: PepT1, PepT2, PhT1 and PhT2) in different regions of rodent brain, and the age-dependent uptake of a POT substrate, glycylsarcosine, in brain slices. Slices were obtained from cerebral cortex, cerebellum and hypothalamus 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. Whereas 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.

Hsp90 requires cochaperone Cdc37 to load its clients to the Hsp90 superchaperone complex. The purpose of this study was to utilize split Renilla luciferase protein fragmentassisted complementation (SRL-PFAC) bioluminescence to study the full-length human Hsp90-Cdc37 complex and to identity critical residues and their contributions for Hsp90/ Cdc37 interaction in living cells. SRL-PFAC showed that fulllength human Hsp90/Cdc37 interaction restored dramatically high luciferase activity through Hsp90-Cdc37-assisted complementation of the N and C termini of luciferase (compared with the set of controls). Immunoprecipitation confirmed that the expressed fusion proteins (NRL-Hsp90 and Cdc37-CRL) preserved their ability to interact with each other and also with native Hsp90 or Cdc37. Molecular dynamic simulation revealed several critical residues in the two interaction patches (hydrophobic and polar) at the interface of Hsp90/Cdc37. Mutagenesis confirmed the critical residues for Hsp90-Cdc37 complex formation. SRL-PFAC bioluminescence evaluated the contributions of these critical residues in Hsp90/Cdc37 interaction. The results showed that mutations in Hsp90 (Q133A, F134A, and A121N) and mutations in Cdc37 (M164A, R167A, L205A, and Q208A) reduced the Hsp90/Cdc37 interaction by 70 -95% as measured by the resorted luciferase activity through Hsp90-Cdc37-assisted complementation. In comparison, mutations in Hsp90 (E47A and S113A) and a mutation in Cdc37 (A204E) decreased the Hsp90/Cdc37 interaction by 50%. In contrast, mutations of Hsp90 (R46A, S50A, C481A, and C598A) and mutations in Cdc37 (C54S, C57S, and C64S) did not change Hsp90/Cdc37 interactions. The data suggest that single amino acid mutation in the interface of Hsp90/Cdc37 is sufficient to disrupt its interaction, although Hsp90/Cdc37 interactions are through large regions of hydrophobic and polar interactions. These findings provides a rationale to develop inhibitors for disruption of the Hsp90/Cdc37 interaction.The 90-kDa heat shock protein (Hsp90) 4 is a ubiquitous and essential molecular chaperone with multiple functions in eukaryotic cells under both stressed and nonstressed conditions (1, 2). It plays a central role in post-translational folding and stability of over 100 signaling proteins, including steroid hormone receptors, the dioxin receptor, growth factor receptors, transcription factors, protein kinases, and enzymes (3). Many of these Hsp90 clients are crucial in tumorigenesis, and when these proteins are dysregulated, they contribute to the hallmark traits of cancer. It has also been reported that the expression of Hsp90 in cancer cells is 2-10-fold higher compared with their normal counterparts (4). Therefore, tumor cells show much more sensitivity when subjected to Hsp90 inhibition than nontransformed cells (5), which suggests the important function of Hsp90 in tumor progression.Hsp90 consists of the following three highly conserved domains: a 25-kDa N-terminal domain, a 35-kDa middle domain, and a 10-kDa C-terminal domain (6). A nucleotide bindin...

The proton-coupled oligopeptide transporter PEPT1 (SLC15A1) is abundantly expressed in the small intestine, but not colon, of mammals and found to mediate the uptake of di/tripeptides and peptide-like drugs from the intestinal lumen. However, species differences have been observed in both the expression (and localization) of PEPT1 and its substrate affinity. With this in mind, the objectives of this study were to develop a humanized PEPT1 mouse model (huPEPT1) and to characterize hPEPT1 expression and functional activity in the intestines. Thus, after generating huPEPT1 mice in animals previously nulled for mouse Pept1, phenotypic, PCR, and immunoblot analyses were performed, along with in situ single-pass intestinal perfusion and in vivo oral pharmacokinetic studies with a model dipeptide, glycylsarcosine (GlySar). Overall, the huPEPT1 mice had normal survival rates, fertility, litter size, gender distribution, and body weight. There was no obvious behavioral or pathological phenotype. The mRNA and protein profiles indicated that huPEPT1 mice had substantial PEPT1 expression in all regions of the small intestine (i.e., duodenum, jejunum, and ileum) along with low but measurable expression in both proximal and distal segments of the colon. In agreement with PEPT1 expression, the in situ permeability of GlySar in huPEPT1 mice was similar to but lower than wildtype animals in small intestine, and greater than wildtype mice in colon. However, a species difference existed in the in situ transport kinetics of jejunal PEPT1, in which the maximal flux and Michaelis constant of GlySar were reduced 7-fold and 2- to 4-fold, respectively, in huPEPT1 compared to wildtype mice. Still, the in vivo function of intestinal PEPT1 appeared fully restored (compared to Pept1 knockout mice) as indicated by the nearly identical pharmacokinetics and plasma concentration–time profiles following a 5.0 nmol/g oral dose of GlySar to huPEPT1 and wildtype mice. This study reports, for the first time, the development and characterization of mice humanized for PEPT1. This novel transgenic huPEPT1 mouse model should prove useful in examining the role, relevance, and regulation of PEPT1 in diet and disease, and in the drug discovery process.

Objectives-Relaxin induces the matrix metalloproteinase MMP-1 (collagenase-1) in TMJ fibrocartilaginous cells, and this response is potentiated by β-estradiol. We identified the MMP-1 promoter sites and transcription factors that are induced by relaxin with or without β-estradiol in fibrocartilaginous cells.Material & Methods-Early passage cells were transiently transfected with the pBLCAT2 plasmid containing specific segments of the human MMP-1 promoter regulating the chloramphenicol acyl transferase (CAT) gene and co-transfected with a plasmid containing the β-galactosidase gene. The cells were cultured in serum-free medium alone or medium containing 0.1 ng/ml relaxin, or 20 ng/ml β-estradiol or both hormones, and lysates assayed for CAT and β-galactosidase activity.Results-Cells transfected with the −1200/−42 or −139/−42 bp MMP-1 promoter-reporter constructs showed 1.5-fold and 3-fold induction of CAT by relaxin in the absence or presence of β-estradiol, respectively. Relaxin failed to induce CAT in the absence of the −137/−69 region of the MMP-1 promoter, which contains the AP-1-and PEA3-binding sites. Using wild type or mutated minimal AP-1 and PEA-3 promoters we found that both these promoter sites are essential for the induction of MMP-1 by relaxin. The mRNAs for transcription factors c-fos and c-jun, which together form the AP-1 heterodimer, and Ets-1 that modulates the PEA-3 site, were upregulated by relaxin or β-estradiol plus relaxin.Conclusion-These studies show that both the AP-1 and PEA-3 promoter sites are necessary for the induction of MMP-1 by relaxin in fibrocartilaginous cells.

Cefadroxil is a broad-spectrum β-lactam antibiotic that is widely used in the treatment of various infectious diseases. Currently, poor understanding of the drug’s pharmacokinetic profiles and disposition mechanism(s) prevents determining optimal dosage regimens and achieving ideal antibacterial responses in patients. In the present retrospective study, we developed a population pharmacokinetic model of cefadroxil in wildtype and Pept2 knockout mice using the NONMEM approach.Cefadroxil pharmacokinetics were best described by a two-compartment model, with both saturable and nonsaturable elimination processes to/from the central compartment. Through this modeling approach, pharmacokinetic parameters in wildtype and Pept2 knockout mice were well estimated, respectively, as: volume of central compartment V1 (3.43 vs. 4.23 mL), volume of peripheral compartment V2 (5.98 vs. 8.61 mL), inter-compartment clearance Q (0.599 vs. 0.586 mL/min), and linear elimination rate constant K10 (0.111 vs. 0.070 min−1). Moreover, the secretion kinetics (i.e., Vm1 = 17.6 nmoL/min and Km1 = 37.1 μM) and reabsorption kinetics (i.e., Vm2 = 15.0 nmoL/min and Km2 = 27.1 μM) of cefadroxil were quantified in kidney, for the first time, under in vivo conditions.Our model provides a unique tool to quantitatively predict the dose-dependent nonlinear disposition of cefadroxil, as well as the potential for transporter-mediated drug interactions.