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David E Smith

Publications and source records attributed to David E Smith.

33 records · Page 2Linked to original sources
PubMed2004

Free energy, energy, and entropy of swelling in Cs-, Na-, and Sr-montmorillonite clays.

A Monte Carlo method for grand canonical and grand isoshear ensemble simulations has been used to characterize the free energy, energy, and entropy of clay mineral swelling. The Monte Carlo approach was found to be more efficient at simulating water content fluctuations in the highly constrained clay environment than a previously developed molecular dynamics method. Swelling thermodynamics calculated for Cs-, Na-, and Sr-montmorillonite clays indicate a strong dependence of swelling on the interlayer ion identity, in agreement with various experimental measurements. The Sr clay swells most readily, and both the Na and Sr clays prefer expanded states (two-layer hydrate or greater) when in contact with bulk water. In contrast, swelling is inhibited in the Cs clay. Differences in swelling behavior are traced directly to the tendency of the different ions to hydrate. The swelling free energies are decomposed into their energetic and entropic components, revealing an overall energetic driving force for the swelling phenomena. Entropic effects provide a smaller, mediating role in the swelling processes. The results provide a unique molecular perspective on experimentally well-characterized swelling phenomena.

Journal Article↗
PubMed2004

Disposition of WR-1065 in the liver of tumor-bearing rats following regional vs systemic administration of amifostine.

PURPOSE: Amifostine is a prodrug in which selectivity is largely determined by the preferential formation and uptake of its cytoprotective metabolite, WR-1065, in normal tissues as a result of differences in membrane-bound alkaline phosphatase activity. It was hypothesized that amifostine may be a good candidate for regional drug delivery to the liver because of its large hepatic extraction and total body clearance. METHODS: Rat livers were implanted with Walker-256 tumors. The tumor-bearing rats received 15 min infusions of amifostine (200 mg/kg) via the portal vein or the femoral vein. WR-1065 concentrations in the blood, liver and tumor were measured at various times. RESULTS: The WR-1065 tumor portal dosing AUC15-60 was 40% of systemic dosing, and tumor concentrations following portal dosing were one-fifth of that following systemic dosing. The portal dosing WR-1065 liver AUC15-60 was 60% higher than the values for systemic dosing. The liver/tumor concentration ratios of WR-1065 following portal dosing were up to 8-fold higher than the ratio following systemic administration. Unfortunately, systemic exposure to WR-1065 was greater following portal vs systemic amifostine. CONCLUSIONS: Amifostine may provide increased liver protection and decreased tumor protection from radio- or chemotherapy when administered by the portal vein. However, portal dosing also increases systemic exposure to WR-1065, which is associated with hypotension.

Amifostine↗
PubMed2004

Role of PEPT2 in the choroid plexus uptake of glycylsarcosine and 5-aminolevulinic acid: studies in wild-type and null mice.

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.

Aminolevulinic Acid↗
PubMed2004

Carnosine uptake in rat choroid plexus primary cell cultures and choroid plexus whole tissue from PEPT2 null mice.

PEPT2 is functionally active and localized to the apical membrane of rat choroid plexus epithelial cells. However, little is known about the transport mechanisms of endogenous neuropeptides in choroid plexus, and the role of PEPT2 in this process. In the present study, we examined the uptake kinetics of carnosine in rat choroid plexus primary cell cultures and choroid plexus whole tissue from wild-type (PEPT2(+/+)) and null (PEPT2(-/-)) mice. Our results indicate that carnosine is preferentially taken up from the apical as opposed to basolateral membrane of cell monolayers, and that basolateral efflux in limited. Transepithelial flux of carnosine was not distinguishable from that of paracellular diffusion. The apical uptake of carnosine was characterized by a high affinity (K(m) = 34 microM), low capacity (V(max) = 73 pmol/mg protein/min) process, consistent with that of PEPT2. The non-saturable component was small (K(d) = 0.063 microL/mg protein/min) and, under linear conditions, was only 3% of the total uptake. Studies in transgenic mice clearly demonstrated that PEPT2 was responsible for over 90% of carnosine's uptake in choroid plexus whole tissue. These findings elucidate the unique role of PEPT2 in regulating neuropeptide homeostasis at the blood-cerebrospinal fluid interface.

Amino Acids↗
PubMed2003

Mechanisms of cefadroxil uptake in the choroid plexus: studies in wild-type and PEPT2 knockout mice.

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.

Animals↗
PubMed2003

Identification and characterization of mechanistically distinct inducers of gamma-globin transcription.

Inhibition of HbS polymerization is a major target for therapeutic approaches in sickle cell anemia. Toward this goal, initial efforts at pharmacological elevation of fetal hemoglobin (HbF) has shown therapeutic efficacy. In order to identify well-tolerated, novel agents that induce HbF in patients, we developed a high-throughput screening approach based on induction of gamma-globin gene expression in erythroid cells. We measured gamma-globin transcription in K562 cells transfected with either gamma promoter elements fused with the locus control region hypersensitivity site 2 and luciferase reporter gene (HS2 gamma) or a beta-yeast artificial chromosome in which the luciferase reporter gene was recombined into the gamma-globin coding sequences (gamma YAC). Corresponding pharmacological increases in HbF protein were confirmed in both K562 cells and in human primary erythroid progenitor cells. Approximately 186,000 defined chemicals and fungal extracts were evaluated for their ability to increase gamma gene transcription in either HS2 gamma or gamma YAC models. Eleven distinct classes of compounds were identified, the majority of which were active within 24-48 hr. The short chain hydroxamate-containing class generally exhibited delayed maximal activity, which continued to increase transcription up to 120 hr. The cyclic tetrapeptide OSI-2040 and the hydroxamates were shown to have histone deacetylase inhibitory activity. In primary hematopoietic progenitor cell cultures, OSI-2040 increased HbF by 4.5-fold at a concentration of only 40 nM, comparable to the effects of hydroxyurea at 100 microM. This screening methodology successfully identifies active compounds for further mechanistic and preclinical evaluation as potential therapeutic agents for sickle cell anemia.

Anemia, Sickle Cell↗
PubMed2003

Preliminary investigation into the expression of proton-coupled oligopeptide transporters in neural retina and retinal pigment epithelium (RPE): lack of functional activity in RPE plasma membranes.

PURPOSE: To determine the expression and functional activity of proton-coupled oligopeptide transporters (POT) in retinal pigment epithelial (RPE) cells. METHODS: RT-PCR was used to probe the presence of POT mRNA in freshly isolated bovine RPE (BRPE) and human RPE (HRPE) cells, a human RPE cell line (ARPE-19), and human and bovine neural retina. [14C]GlySar uptake was used to characterize POT activity in cultured ARPE-19 cells and freshly isolated BRPE cell sheet suspensions. RESULTS: PHT1 mRNA was expressed in BRPE, HRPE, ARPE-19, and bovine and human neural retina. In contrast, PEPT2 and PHT2 were expressed only in bovine and human retina, and PEPT1 could not be detected. GlySar exhibited a linear uptake over 6 h at pH values of 6.0 and 7.4, with greater uptake at pH 7.4 (p < 0.01). GlySar uptake did not exhibit saturability (5-2000 microM) and was unchanged when studied in the presence of 1 mM L-histidine. In contrast, GlySar uptake was significantly decreased when studied at 4 degrees C or in the presence of endocytic inhibitors at 37 degrees C (p < 0.01). Studies in BRPE cell sheet suspensions validated the results obtained in ARPE-19 cells and strongly suggested the absence of POT on the apical and basolateral membranes of RPE. CONCLUSIONS: PHT1 mRNA is present in native bovine and human RPE and a human RPE cell line. However, the data argue against PHT1 being expressed on plasma membranes of RPE. Overall, GlySar appears to be taken up by RPE cells via a low-affinity, endocytic process.

Animals↗
PubMed2003

Effects of organic anion, organic cation, and dipeptide transport inhibitors on cefdinir in the isolated perfused rat kidney.

Cefdinir (Omnicef; Abbott Laboratories) is a cephalosporin antibiotic primarily eliminated by the kidney. Nonlinear renal elimination of cefdinir has been previously reported. Cefdinir renal transport mechanisms were studied in the erythrocyte-free isolated perfused rat kidney. Studies were performed with drug-free perfusate and perfusate containing cefdinir alone to establish the baseline physiology and investigate cefdinir renal elimination characteristics. To investigate cefdinir renal transport mechanisms, inhibition studies were conducted by coperfusing cefdinir with inhibitors of the renal organic anion (probenecid), organic cation (tetraethylammonium), or dipeptide (glycylsarcosine) transport system. Cefdinir concentrations in biological samples were determined using reversed-phase high-performance liquid chromatography. Differences between treatments and controls were evaluated using analysis of variance and Dunnett's test. The excretion ratio (ER; the renal clearance corrected for the fraction unbound and glomerular filtration rate) for cefdinir was 5.94, a value indicating net renal tubular secretion. Anionic, cationic, and dipeptide transport inhibitors all significantly affected the cefdinir ER. With probenecid, the ER was reduced to 0.59, clearly demonstrating a significant reabsorptive component to cefdinir renal disposition. This finding was confirmed by glycylsarcosine studies, in which the ER was elevated to 7.95, indicating that reabsorption was mediated, at least in part, by the dipeptide transporter system. The effects of the organic cation tetraethylammonium, in which the ER was elevated to 7.53, were likely secondary in nature. The anionic secretory pathway was found to be the predominant mechanism for cefdinir renal excretion.

Animals↗
PubMed2002

Targeted disruption of the PEPT2 gene markedly reduces dipeptide uptake in choroid plexus.

The presence of multiple oligopeptide transporters in brain has generated considerable interest as to their physiological role in neuropeptide homeostasis, pharmacologic importance, and potential as a target for drug delivery through the blood-brain and blood-cerebrospinal fluid barriers. To understand further the purpose of specific peptide transporters in brain, we have generated PEPT2-deficient mice by targeted gene disruption. Homozygous PepT2 null mice lacked expression of PEPT2 mRNA and protein in choroid plexus and kidney, tissues in which PepT2 is normally expressed, whereas heterozygous mice displayed PepT2 expression levels that were intermediate between those of wild-type and homozygous null animals. Mutant PepT2 null mice were found to be viable, grew to normal size and weight, and were without obvious kidney or brain abnormalities. Notwithstanding the lack of apparent biological effects, the proton-stimulated uptake of 1.9 microm glycylsarcosine (a model, hydrolysis-resistant dipeptide) in isolated choroid plexus was essentially ablated (i.e. residual activity of 10.9 and 3.9% at 5 and 30 min, respectively). These novel findings provide strong evidence that, under the experimental conditions of this study, PEPT2 is the primary member of the peptide transporter family responsible for dipeptide uptake in choroid plexus tissue.

Animals↗
PubMed2002

Arrhythmogenic right ventricular cardiomyopathy/dysplasia: a case report of identical twins with heart failure.

Arrhythmogenic right ventricular cardiomyopathy/dysplasia is characterized by the progressive replacement of myocardium by fatty or fibrofatty tissue. Presenting symptoms are generally related to ventricular arrhythmias, including sudden cardiac death. Heart failure due to right ventricular and sometimes left ventricular dysfunction is uncommon in the early stages of the disease, but is known to occur in advanced cases. This case report describes identical adolescent twins with presenting symptoms related predominantly to right heart failure.

Adult↗
PubMed2002

Regional pharmacokinetics of amifostine in anesthetized dogs: role of the liver, gastrointestinal tract, lungs, and kidneys.

Amifostine is a prodrug in which selectivity is largely determined by the preferential formation and uptake of its cytoprotective metabolite, WR-1065, in normal tissues as a result of differences in membrane-bound alkaline phosphatase activity. In this study, we characterized the sites and extent of organ-specific activation by the liver, gastrointestinal tract, lungs, and kidneys after systemic administrations of amifostine. A total of 10 dogs were infused via the cephalic vein using sequential dose rates of drug at 0.125, 0.500, and 1.00 micro mol/min/kg. Infusion of each dose rate lasted 2 h, at which time steady-state plasma concentrations were obtained (i.e., portal vein, carotid artery, hepatic vein, pulmonary artery, and renal vein). The hepatic arterial, portal venous, and renal arterial blood flows, and cardiac output, were measured. The hepatic and splanchnic extraction of amifostine remained high at 90%, whereas gastrointestinal extraction decreased from 43 to 12 to 15% with increasing dose. Pulmonary extraction of amifostine was low at 7%, whereas renal extraction was intermediate at 57%. Because blood flow measurements were relatively constant during the drug infusions, clearance parameters paralleled that of organ extraction. As a result, saturability was observed in the gastrointestinal blood clearance (i.e., from 9.8 to 2.8-3.3 ml/min/kg) and total body plasma clearance of amifostine (i.e., from 52.6 to about 37.3 ml/min/kg), as the doses increased. Due to the drug's high activation in liver, these findings suggest that amifostine may offer good protection of this organ against the toxicities of chemotherapy and radiation.

Amifostine↗
PubMed2002

Role of PEPT2 in peptide/mimetic trafficking at the blood-cerebrospinal fluid barrier: studies in rat choroid plexus epithelial cells in primary culture.

Recent studies have established the functional and molecular presence of a high-affinity peptide transporter, PEPT2, in whole tissue rat choroid plexus. However, the precise membrane location and directionality of PEPT2-mediated transport is uncertain at present. In this study, we examined the transport kinetics of a model dipeptide, glycylsarcosine (GlySar), along with the protein expression of PEPT2 using primary cell cultures of choroidal epithelium from neonatal rats. GlySar accumulation and transepithelial transport were 3 to 4 times higher when introduced from the apical as opposed to the basal side of the monolayers. GlySar apical uptake was also stimulated by an inwardly directed proton gradient. The uptake of GlySar was inhibited by di/tripeptides, carnosine, and alpha-amino cephalosporins but was unaffected by amino acids, cephalosporins lacking an alpha-amino group, and organic anions and cations. The Michaelis constant (K(m)) of GlySar was 59.6 microM for apical uptake and 1.4 mM for basal uptake; this is consistent with the high-affinity properties of PEPT2 at the apical membrane. Immunoblot analyses and immunofluorescent confocal microscopy demonstrated the presence of PEPT2, but not PEPT1, in rat choroid plexus epithelial cells. Moreover, PEPT2 was present in the apical and subapical regions of the cell but was absent in the basolateral membrane. These findings demonstrate, for the first time, that PEPT2 protein is present at the apical membrane of choroidal epithelial cells and that it is functionally active at this membrane surface. The results suggest that PEPT2 may have a role in the efflux of peptides and/or mimetics from cerebrospinal fluid to the blood.

Animals↗
PubMedDate not supplied

Immunolocalization of the proton-coupled oligopeptide transporter PEPT2 in developing rat brain.

This study examined the tissue distribution, cellular localization, and developmental expression of the PEPT2 protein in rat brain. Immunoblot and immunocytochemistry analyses were performed with specific rat PEPT1 and PEPT2 antisera developed in our laboratory. Rats were examined from fetus (gestation for 17 days) to adult (day 75). On immunoblot analysis, the PEPT2 protein was detected in cerebral cortex, olfactory bulb, basal ganglia, cerebellum, and hindbrain sections of adult brain, with the strongest signals in cerebral cortex. No PEPT1 protein was found in brain. Expression levels of the PEPT2 protein in cerebral cortex were maximal in the fetus and declined rapidly with advancing age. Adult protein levels were approximately 14% of that observed in fetus. In immunofluorescence experiments, the strongest PEPT2 signals were observed in epithelial cells of the choroid plexus for both adult and neonate brains. The PEPT2 protein was exclusively expressed on the apical membrane (CSF-facing) of choroid plexus epithelia. In double labeling experiments, PEPT2 immunoreactivity in adult brain colocalized with NeuN, a neuronal marker, but not with GFAP, an astrocyte marker. In contrast, in neonatal brain, PEPT2 immunoreactivity colocalized with both GFAP and NeuN. These findings demonstrate that the PEPT2 protein is found throughout the brain. The apical expression of PEPT2 in choroid plexus suggests that it is involved in the export of neuropeptides, peptide fragments, and peptide-like drugs from cerebrospinal fluid. PEPT2 may also play a role in the regulation of neuropeptide concentrations in extracellular fluid, especially during early development.

Aging↗
PubMedDate not supplied

Effect of diabetes mellitus and insulin on the regulation of the PepT 1 symporter in rat jejunum.

This investigation focused on studying the effects of insulin-dependent diabetes mellitus and insulin treatment on absorption of glycylsarcosine (Gly-Sar) across the Sprague-Dawley rat jejunum, using in situ perfusion in a physiologic acidic microenvironment at pH 6.0. Rats were divided into five groups: normal controls in group I, normal colchicine-treated rats in group II, normal cytochalasin-treated rats in group III, streptozotocin-induced diabetic rats in group IV, and insulin-treated diabetic rats in group V. Histologic studies of the five different groups showed morphologic changes upon induction of diabetes and treatments with colchicine and cytochalasin and several variations in post-1 month diabetic rats treated with insulin. The rate of uptake of Gly-Sar was significantly reduced in the diabetic state. The comparison of colchicine-treated and cytochalasin-treated rats to the diabetic group suggests that an intact cytoskeleton and tight junctions may play a role in jejunal dipeptide absorption. In the diabetic and insulin-treated group, the dipeptide influx rate was significantly increased compared to that of the nontreated controls. The regulation of the PepT 1 symporter was further assessed by immunostaining and Western blot analyses in the normal, diabetic, and diabetic and insulin-treated groups. Our results showed that a downregulation of PepT 1 in the diabetics seemed to be due in part to the low systemic insulin levels, and not necessarily to hyperglycemia. In addition, the results suggest a probable role of systemic insulin binding at the vascular site of the jejunal epithelium, and the role that this hormone may be playing in the regulation and probably cellular trafficking of PepT1.

Animals↗