Quality indicators for the management of urinary incontinence in vulnerable community-dwelling elders.
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Aged↗
Biomedical subjects
R L Smith
Publications and source records attributed to R L Smith.
In situ stimulation of groundwater denitrification with formate to remediate nitrate contamination.
In situ stimulation of denitrification has been proposed as a mechanism to remediate groundwater nitrate contamination. In this study, sodium formate was added to a sand and gravel aquifer on Cape Cod, MA, to test whether formate could serve as a potential electron donor for subsurface denitrification. During 16- and 10-day trials, groundwater from an anoxic nitrate-containing zone (0.5-1.5 mM) was continuously withdrawn, amended with formate and bromide, and pumped back into the aquifer. Concentrations of groundwater constituents were monitored in multilevel samplers after up to 15 m of transport by natural gradient flow. Nitrate and formate concentrations were decreased 80-100% and 60-70%, respectively, with time and subsequent travel distance, while nitrite concentrations inversely increased. The field experiment breakthrough curves were simulated with a two-dimensional site-specific model that included transport, denitrification, and microbial growth. Initial values for model parameters were obtained from laboratory incubations with aquifer core material and then refined to fit field breakthrough curves. The model and the lab results indicated that formate-enhanced nitrite reduction was nearly 4-fold slower than nitrate reduction, but in the lab, nitrite was completely consumed with sufficient exposure time. Results of this study suggest that a long-term injection of formate is necessary to test the remediation potential of this approach for nitrate contamination and that adaptation to nitrite accumulation will be a key determinative factor.
Biodegradation, Environmental↗
Long-lasting rebound cue effects following single doses of nicotine and amphetamine: implications for understanding tolerance.
RATIONALE: Previous drug-discrimination studies have, with the exception of nicotine (NIC), demonstrated tolerance to the cue effects of a broad range of drugs of abuse. Barrett et al. have shown that tolerance to a drug's cue properties reflects drug-induced rebound shifts in the discrimination baseline and not a weakened or less salient cue. OBJECTIVES: The objective of the present study was to use a discrimination task sensitive to bidirectional cue changes to characterize the interoceptive cues associated with both the primary and rebound cues produced by nicotine in an attempt to understand why a recent study by Shoaib et al. failed to observe tolerance to the nicotine cue. METHODS: Since dopamine (DA) has been implicated in mediating the NIC cue, rats were trained to discriminate between 0.25 mg/kg amphetamine (AMPH), an indirect DA agonist, and 0.033 mg/kg haloperidol (HAL), a DA antagonist at the D2 receptor site. Training doses were chosen so that rats responded about equally on both levers when tested on saline (SAL) following acquisition. This procedure provided a behavioral baseline to assess NIC-related changes along a presumed continuum of DA-mediated cues. Following acquisition of the discrimination: (i) NIC substitution tests were conducted, (ii) rats were tested for lever choice at intervals from 2 h to 48 h following treatment with single doses of 0.25 mg/kg and 0.50 mg/kg NIC, and (iii) rats were challenged with test doses of NIC during a period of NIC rebound. RESULTS: (i) NIC substituted for AMPH in a dose- dependent manner. (ii) At short intervals after treatment with 0.25 mg/kg and 0.50 mg/kg NIC, rats responded primarily on the AMPH lever followed by a shift to predominant responding on the HAL lever 16-24 h post-treatment, before returning to predrug levels. (iii) No evidence was observed for acute tolerance to NIC. CONCLUSIONS: The robust and long-lasting rebound cues associated with training level doses of NIC suggest that maximal tolerance would likely develop to the NIC cue during the acquisition phase of the conventional NIC-saline discrimination study.
Amphetamine↗
Predicting evaporation rates and times for spills of chemical mixtures.
Spreadsheet and short-cut methods have been developed for predicting evaporation rates and evaporation times for spills and constrained baths of chemical mixtures. Steady-state and time-varying predictions of evaporation rates can be made for six-component mixtures, including liquid-phase non-idealities as expressed through the UNIFAC method for activity coefficients. A group-contribution method is also used to estimate vapor-phase diffusion coefficients, which makes the method completely predictive. The predictions are estimates that require professional judgement in their application. One application that the evaporation time calculations suggest is a method for labeling chemical containers that allows one to quickly assess the time for complete evaporation of spills of both pure components and mixtures. The labeling would take the form of an evaporation time that depends on the local environment. For instance, evaporation time depends on indoor or outdoor conditions and the amount of each chemical among other parameters. This labeling would provide rapid information and an opportunity to premeditate a response before a spill occurs.
Hazardous Substances↗
The effect of the choline head group on phospholipid hydration.
The hydration behavior of three naturally occurring sphingomyelins (SM) has been studied. Adsorption isotherms for these SM, singly and in combination with other lipids, were obtained and the isotherms analyzed by the application of BET theory. The results are compared with those found previously by us for the phosphatidylcholines (PC). We find that, depending on the SM studied, both "weak" and "strong" water adsorption can be observed and thus, the presence of choline in the phospholipid head group, does not guarantee "strong" water adsorption. When the head group is removed from SM, the resulting ceramides are found to be very weak, water adsorbers. Cholesterol, when present in a mixture with SM, has a rather dramatic effect on the hydration of the SM. A mixed system of PC and SM exhibits water adsorption characteristics very similar to those exhibited by PC itself. We speculate that these hydration results may well play a role in the cell signaling action of SM and, moreover, may be closely associated with the hypothesized formation of lipid "rafts."
Adsorption↗
Central nervous control of gill filament muscles in channel catfish.
The gills of fish are innervated by cranial nerves IX and X. There have been a number of studies on the characteristics of sensory activity carried by these nerves but remarkably little is known about motor control of the gills. Efferent, motor activity to the first gill arch was recorded from the glossopharyngeal nerve in spontaneously breathing channel catfish, Ictalurus punctatus. This study addressed two objectives. The first objective was to characterize efferent branchial nerve activity in spontaneously breathing fish. Nerve recordings show bursts of activity firing in synchrony with ventilation. These bursts occurred once during either abduction or adduction of the operculum with each breath. The observed patterns of neural activity indicate that it represents motor control of gill filament abductor and adductor muscles. The data show that rhythmic output from a central pattern generator controls filament musculature during the ventilatory cycle. The second objective was to use this efferent branchial nerve activity as an index of ventilation (fictive ventilation) in fish before and after paralysis to determine if feedback from phasic mechanoreceptors affects ventilatory timing. Breath-to-breath intervals measured before and after paralysis with gallamine were not significantly different, demonstrating that rhythmic feedback from phasic mechanoreceptors in the gills and/or ventilatory musculature is not involved in the breath-to-breath timing of the normal ventilatory cycle. During the course of these experiments many fish exhibited coughing. Coughs were characterized by a distinctive pattern of nerve activity that was not altered by paralysis. Overall, the data indicate that phasic mechanoreceptor feedback during normal breathing has no effect on the pattern of central motor control of gill filament muscles.
Animals↗
In vitro reaction to orthopaedic biomaterials by macrophages and lymphocytes isolated from patients undergoing revision surgery.
Periprosthetic tissues observed at sites of loose total joint implants exhibit abundant macrophages, lymphocytes, fibroblasts and particulate debris. Macrophages phagocytose orthopaedic debris and release proinflammatory cytokines, chemokines, matrix metalloproteinases and other substances. In addition, other cell types present in tissues harvested from the bone-implant interface are thought to influence periprosthetic bone resorption. The present study examined the effects of polymethylmethacrylate (PMMA), cobalt chrome molybdenum alloy (CoCr), and titanium-alloy particle challenge on macrophages co-cultured with lymphocytes in vitro. Potential synergistic effects of lymphocytes on macrophage activation were determined by measuring interleukin-6 and tumor necrosis factor-alpha release following exposure to orthopaedic biomaterial particles. Exposure of macrophages or macrophages co-cultured with lymphocytes to all three types of particles resulted in increased release of interleukin-6 and tumor necrosis factor-alpha at 48 h, when compared to macrophages or macrophages co-cultured with lymphocytes, respectively, cultured in the absence of particles. Lymphocytes isolated from periprosthetic tissues secreted increased basal levels of cytokines relative to peripheral blood lymphocytes. Higher doses of PMMA and titanium-alloy particles stimulated increased levels of cytokine release in the macrophage and macrophage/lymphocyte groups. In contrast, a higher dose of CoCr particles (0.075% v/v) was not as effective as the 0.015% v/v dose, indicating probable CoCr toxicity. The macrophage/lymphocyte co-culture did not show synergism between the two types of cells with respect to cytokine release. T-cells at the bone-implant interface may alter the biological response to particulate debris.
Biocompatible Materials↗
Interleukin-10 inhibits polymethylmethacrylate particle induced interleukin-6 and tumor necrosis factor-alpha release by human monocyte/macrophages in vitro.
Periprosthetic membranes commonly observed at sites of total joint implant loosening exhibit abundant macrophages and particulate debris. Macrophages phagocytose orthopedic debris and release the pro-inflammatory mediators interleukin-1, interleukin-6, tumor necrosis factor-alpha, and prostaglandin E2. Populations of activated lymphocytes are often seen in periprosthetic membranes. These lymphocytes may modulate the monocyte/macrophage response to particulate debris and influence aseptic loosening. In addition, other immunologic agents, such as interleukin-10, are present in tissues harvested from the bone-implant interface of failed total joint arthroplasties. The present study examined the effects of interleukin-10 on polymethylmethacrylate (PMMA) particle challenged human monocyte/macrophages in vitro. Human monocyte/macrophages isolated from buffy coats of five healthy individuals were exposed to 1-10 microm PMMA particles. Interleukin-10 was added to the monocyte/macrophages with and without the addition of PMMA particles. Interleukin-10-induced alterations in monocyte/macrophage metabolism were determined measuring interleukin-6 and tumor necrosis factor-alpha release by the cells following exposure to PMMA particles. Exposure of the monocyte/macrophages to PMMA particles resulted in a dose-dependent release of interleukin-6 and tumor necrosis factor-alpha at 48 h. Interleukin-10 reduced the levels of interleukin-6 and tumor necrosis factor-alpha release by macrophages in response to PMMA particles in a dose-dependent manner. At 48 h, particle-induced interleukin-6 release was inhibited by 60 and 90% with 1.0 and 10.0 ng/ml treatments of interleukin-10, respectively. At 48 h, particle-induced tumor necrosis factor-alpha release was inhibited by 58 and 88% with 1.0 and 10.0 ng/ml treatments of interleukin-10, respectively. Interleukin-10 challenge alone did not significantly alter basal interleukin-6 or tumor necrosis factor-alpha release relative to control cultures. The data presented in this study demonstrate that the anti-inflammatory cytokine, interleukin-10, inhibits monocyte/macrophage release of the pro-inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha in response to PMMA particle challenge in vitro.
Biocompatible Materials↗
Lack of effect of treatment with penciclovir or acyclovir on the establishment of latent HSV-1 in primary sensory neurons in culture.
Recent studies suggest reductions in establishment of herpes simplex virus, type 1 (HSV-1) latency using the nucleoside analog penciclovir compared with acyclovir in the murine model. These observations raise the possibility that the new analogs may have novel activities that directly interfere with the establishment of the latent infection, suggesting a mechanism other than simply blocking the productive infection. To determine if penciclovir has a direct action on the establishment of latency, we compared the effects of penciclovir versus acyclovir in an in vitro model of HSV-1 latency in rat dorsal root ganglia neurons in culture. In neurons in culture, both penciclovir and acyclovir were highly effective in blocking the productive infection. However, neither penciclovir nor acyclovir blocked establishment of latency as demonstrated by similar percentages of neurons expressing the latency-associated transcript (LAT). Following removal of the respective nucleoside analog, latency was maintained until reactivation was induced by nerve growth factor deprivation. Similar virus titers were recovered after induction of reactivation of latent infections, which were established in the presence of either penciclovir or acyclovir. These results indicate that neither penciclovir nor acyclovir treatment directly prevents the establishment of latent HSV-1 infections in primary sensory neurons in culture.
Acyclovir↗
An assessment of the release of inorganic cyanide from the fragrance materials benzyl cyanide, geranyl nitrile and citronellyl nitrile applied dermally to the rat.
Organonitriles are widely used as components of fragrances that are incorporated into consumer products, many of which are for human topical use. Some organontriles are readily broken down metabolically to potentially toxic inorganic cyanide. Studies were therefore undertaken to assess whether this occurs with three representative fragrance nitriles, namely, benzyl cyanide, geranyl nitrile and citronellyl nitrile when applied dermally to the rat. The nitriles (benzyl cyanide, 150 mg/kg; geranyl and citronellyl nitriles, 400 mg/kg) were applied to the shaved backs of rats and maintained under occlusion for 24 h. Urine samples were collected for 0-24 h, 24-48 h and 48-72 h from the time of first application. These samples were analysed for thiocyanate, a biomarker for cyanide formation in vivo, as described previously (Potter, J., Smith, R.L., Api, A.M., 2000. Urinary thiocyanate levels as a biomarker for the generation of inorganic cyanide from benzyl cyanide in the rat. Food and Chemical Toxicology 39, 141-146). In the case of benzyl cyanide, there was a marked increase in urinary thiocyanate levels attributable to the release of cyanide in vivo. The amount of thiocyanate recovered was equivalent to 37% of the dose for males and 32% for females. For geranyl nitrile there was no significant increase in urinary thiocyanate excretion and there was only a marginal increase in the case of citronellyl nitrile that was equivalent to 0.40% of the applied dose for males and 0.29% for females.
Acetonitriles↗
Urinary thiocyanate levels as a biomarker for the generation of inorganic cyanide from benzyl cyanide in the rat.
A colorimetric procedure was developed and validated for the determination of thiocyanate in rat urine over the concentration range of 7-7000 microg/ml. It was applied to the determination of thiocyanate following its oral administration to male and female rats. The mean percentage urinary recoveries of sodium thiocyanate given by oral gavage at 10 and 100 mg/kg were 60 and 39%, respectively, for male rats and 89 and 73% for females over a period of 3 days. Most of the elimination occurred in the 0-48-h period post-dosing but significant amounts were still being excreted in the 48-72-h period. It was concluded from these results that the recoveries of urinary thiocyanate were such that this anion was suitable for use as a biomarker for the release of cyanide from organonitriles such as benzyl cyanide. Benzyl cyanide (150 mg/kg) administered orally to rats led to markedly increased urinary thiocyanate levels; for male rats this was equivalent to 54% of the dose and for females this was 65% over a period of 3 days. When adjusted for incomplete recoveries of the marker, thiocyanate, these values equated to 61 and 89%, respectively. It was concluded that this validated assay could be used to assess cyanide release from topically applied fragrance organonitriles (Potter, J., Smith, R.L., Api, A.M., 2000. An assessment of the release of inorganic cyanide from the fragrance materials, benzyl cyanide, geranyl nitrile and citronellyl nitrile applied dermally to the rat. Food and Chemical Toxicology 39, 147-151).
Acetonitriles↗
Fibroblast expression of C-C chemokines in response to orthopaedic biomaterial particle challenge in vitro.
C-C chemokines are soluble mediators that occur in a periprosthetic granuloma and influence recruitment, localization and activation of inflammatory cells. This study tested effects of titanium and polymethylmethacrylate (PMMA) particles on expression of selected C-C chemokines in cultured human fibroblasts. The C-C chemokines analyzed included monocyte chemoattractant protein-1. 2 (MCP-1. 2), monocyte inflammatory protein-1 alpha (MIP-1 alpha), and regulated on activation, normal T-cell expressed and secreted protein (RANTES). Interleukin-1 beta (IL-1 beta) served as a known stimulator of chemokine release while interleukin-6 (IL-6) expression served as a marker for fibroblast activation. Protein and mRNA signal levels were determined by ELISA and RT-PCR, respectively. The results demonstrated that exposure of fibroblasts to titanium and PMMA particles resulted in increased release of MCP-1 in a dose- and time-dependent manner. After 24 h, titanium particles maximally upregulated MCP-1 release 7-fold while PMMA particles increased MCP-1 levels 2-fold, when compared to unchallenged fibroblasts. MCP-2, MIP-1 alpha and RANTES levels remained unchanged following exposure of fibroblasts to titanium or PMMA particles at any concentration or time point tested. However, IL-1 beta stimulated release of MCP-1, MCP-2, and RANTES, but not MIP-1 alpha from the fibroblasts. IL-1 beta, not particles, exhibited the most prominent effect on MCP-1 mRNA levels. Increased release of MCP-1 from fibroblasts exposed to titanium and PMMA particles coincided with increased release of IL-6. This study suggests that release of chemoattractant factors from fibroblasts localized in periprosthetic membranes enhances the chronic inflammatory process leading to bone resorption and implant loosening.
Bone Cements↗
An evaluation of the clinical performance of newly qualified nurses: a competency based assessment.
The clinical performance of newly qualified staff nurses, or their 'fitness for purpose' has become a central professional and corporate issue and highlighted as a central causes for the reforms recommended by the Peach report (Peach 1999). However the ability to gauge the performance of newly qualified nurses remains a largely subjective exercise relying upon anecdotal evidence or general statements of newly qualified nurses' feelings of inadequacy on qualification. This article seeks to address this issue by reporting the findings of project that sought to compare the expectations of senior nurses regarding the level of competence of newly qualified nurses with that of the actual level of competency as assessed by the preceptors after 8 weeks in post. Using a specifically designed instrument to assess clinical competency a comparison has been made between 139 senior nurses' expectations of the competency of newly qualified nurses and the actual competence as assessed by preceptors of 36 newly qualified nurses after 8 weeks in post. The findings indicate that the senior nurses have clear subjective expectations of the competence level of newly qualified nurses. However these expectations are consistently lower than the actual level of competency demonstrated by the newly qualified nurses as assessed by their preceptors. This research demonstrates that through the use of a validated tool newly qualified nurses consistently perform at a higher level of competency than that expected by senior nurses. The ability to estimate competency levels by objective means should be developed. Further research is needed involving a larger sample of Trusts and Schools of Nursing to replicate the results of this study and to compare methods of the assessment of performance in terms of competency in practice on qualification. It is through developments based upon research such as this that a systematic evaluation of the contribution of Schools of Nursing to the competence of newly qualified nurses can be addressed.
Clinical Competence↗
Pharmacokinetics and absolute bioavailability of sitafloxacin, a new fluoroquinolone antibiotic, in healthy male and female Caucasian subjects.
1. The aim was to compare the pharmacokinetics of sitafloxacin from a capsule formulation (dose of 500 mg sitafloxacin) and an intravenous (i.v.) formulation infused over 1 h (dose of 400 mg sitafloxacin) in healthy male and female subjects and to estimate the absolute bioavailability of sitafloxacin from the capsule formulation. 2. Following oral administration, sitafloxacin was rapidly absorbed, with a mean maximum concentration in plasma of 4.65 microgml(-1) occuring at median tmax = 1.25 h giving a mean AUC(0-infinity) = 28.1 microg h ml(-1). For the i.v. administration, a mean Cmax = 5.53 microm(-1) occurred at the end of the 1-h infusion with a mean AUC(0-infinity) = 25.4 microg h ml(-1). The mean terminal elimination half-life was 7.0 h (oral) and 6.6 h (i.v.). For the oral and i.v. formulations, the mean total plasma clearance was 296 and 263 mlmin(-1), respectively and the mean volume of distribution was 180 and 150 litres, respectively. 3. Within 48 h post-dose, approximately 61% (range 22-86%) of the administered dose was excreted unchanged in urine following capsule administration, compared with approximately 75% (range 42-101%) following the i.v. formulation. For both formulations, the renal clearance of sitafloxacin (means of 181 and 198 ml min(-1) for the capsule and i.v. doses, respectively) implies active tubular secretion of the drug. 4. The absolute bioavailability of sitafloxacin from the capsule formulation was high at 89%, with a 95% CI of 84-94%. The intersubject variability (CV%) in the sitafloxacin AUC(0-infinity) for the capsule was low at 18.6%. 5. Gender differences in the pharmacokinetics of sitafloxacin were small and would not warrant dose adjustment. 6. The findings show that the capsule formulation offers good oral bioavailability and merits further clinical evaluation of sitafloxacin as an orally effective fluoroquinolone antibacterial.
Administration, Oral↗
Inducible cyclic AMP early repressor produces reactivation of latent herpes simplex virus type 1 in neurons in vitro.
Herpes simplex virus type 1 (HSV-1) establishes a latent infection in neurons of the peripheral nervous system. During latent HSV-1 infection, viral gene expression is limited to latency-associated transcripts (LAT). HSV-1 remains latent until an unknown mechanism induces reactivation. The ability of the latent virus to periodically reactivate and be shed is essential to the transmission of disease. In vivo, the stimuli that induce reactivation of latent HSV-1 include stress, fever, and UV damage to the skin at the site of initial infection. In vitro, in primary neurons harboring latent HSV-1, nerve growth factor (NGF) deprivation or forskolin treatment induces reactivation. However, the mechanism involved in the induction of reactivation remains poorly understood. An in vitro neuronal model of HSV-1 latency was used to investigate potential mechanisms involved in the induction of reactivation of latent HSV-1. In situ hybridization analysis of neuronal cultures harboring latent HSV-1 showed a marked, rapid decrease in the percentage of LAT-positive neurons following induction of reactivation by NGF deprivation or forskolin treatment. Western blot analysis showed a corresponding increase in expression of the cellular transcription factor inducible cyclic AMP early repressor (ICER) during reactivation. In transient-transfection assays, ICER downregulated LAT promoter activity. Expression of ICER from a recombinant adenoviral vector induced reactivation and decreased the percentage of LAT-positive neurons in neuronal cultures harboring latent HSV-1. These results indicate that ICER represses LAT expression and induces reactivation of latent HSV-1.
Animals↗
ClC-2 chloride channels contribute to HTC cell volume homeostasis.
Membrane Cl(-) channels play an important role in cell volume homeostasis and regulation of volume-sensitive cell transport and metabolism. Heterologous expression of ClC-2 channel cDNA leads to the appearance of swelling-activated Cl(-) currents, consistent with a role in cell volume regulation. Since channel properties in heterologous models are potentially modified by cellular background, we evaluated whether endogenous ClC-2 proteins are functionally important in cell volume regulation. As shown by whole cell patch clamp techniques in rat HTC hepatoma cells, cell volume increases stimulated inwardly rectifying Cl(-) currents when non-ClC-2 currents were blocked by DIDS (100 microM). A cDNA closely homologous with rat brain ClC-2 was isolated from HTC cells; identical sequence was demonstrated for ClC-2 cDNAs in primary rat hepatocytes and cholangiocytes. ClC-2 mRNA and membrane protein expression was demonstrated by in situ hybridization, immunocytochemistry, and Western blot. Intracellular delivery of antibodies to an essential regulatory domain of ClC-2 decreased ClC-2-dependent currents expressed in HEK-293 cells. In HTC cells, the same antibodies prevented activation of endogenous Cl(-) currents by cell volume increases or exposure to the purinergic receptor agonist ATP and delayed HTC cell volume recovery from swelling. These studies provide further evidence that mammalian ClC-2 channel proteins are functional and suggest that in HTC cells they contribute to physiological changes in membrane Cl(-) permeability and cell volume homeostasis.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Ontogeny of CLCN3 chloride channel gene expression in human pulmonary epithelium.
Human fetal bronchopulmonary epithelia secrete liquid, and this chloride (Cl)-dependent process is important for normal lung growth. At the time of birth there is a maturational transition from a secretory to an absorptive phenotype. The pathways for Cl exit from the apical membrane which are required for fetal lung liquid secretion are unknown but are thought to be independent of the cystic fibrosis transmembrane conductance regulator. We determined the ontogeny of expression of the CLCN family of voltage-dependent Cl channel genes (CLCN2 through 6, K(a) and K(b)) in the human lung to identify potential pathways for pulmonary liquid secretion. Only CLCN3 and CLCN6 messenger RNA were detected by Northern analysis of fetal whole lung tissue. Ribonuclease protection assays confirmed the expression of CLCN3 and also revealed expression of CLCN2. The ontogeny of expression of these two channels was similar, peaking in midgestation and declining postnatally. In situ hybridization localized the CLCN2 and CLCN3 messages to airway and distal pulmonary epithelia and to pulmonary blood vessels. We conclude that CLCN3 is expressed in human airway epithelia and expression is developmentally regulated. The contribution of these channels to pulmonary epithelial liquid transport and lung development remains to be determined.
Blotting, Northern↗
Spatial expression patterns of peptide transporters in the human and rat gastrointestinal tracts, Caco-2 in vitro cell culture model, and multiple human tissues.
This study sought to identify the spatial patterns of expression of peptide transporter 1 (PepT1), peptide transporter 3 (PTR3), peptide/histidine transporter 1 (PHT1), and the human peptide transporter 1 (HPT-1) mRNA in complementary DNA (cDNA) libraries of the human and rat gastrointestinal tracts (GIT), Caco-2 in vitro cell culture model, and in a human multiple tissue panel. Human PTR3 and PHT1 are putative peptide transporters recently discovered. Using sequence-specific primers designed to amplify regions of PepT1, PTR3, PHT1, and HPT-1, we were able to identify the expression of mRNA for each of these transporters in human cDNA panels (Clontech, Palo Alto, CA), the rat GIT, and in Caco-2 cDNA libraries by the polymerase chain reaction (PCR) and Southern Blot analysis. These studies suggest that in the human GIT, PepT1 appears to be localized predominantly in the duodenum, with decreasing expression in the jejunum and ileum. In contrast, PTR3 and HPT-1 were widely expressed in the human GIT, with predominant expression in the different regions of the colon. PHT1 appeared to be expressed in low levels throughout the human GI tract. Interestingly, the mRNAs for all 4 peptide transporters were expressed in Caco-2 cells throughout 30 days of culture. PepT1, PTR3, PHT1, and HPT-1 were also widely expressed in the rat GIT. Human tissue cDNA panel screening suggests that PTR3 and PHT1 are more uniformly expressed, whereas PepT1 and HPT-1 demonstrated site-specific expression. These results suggest that PepT1, PTR3, PHT1, and HPT-1 all may act to facilitate the diffusion of peptides and peptide-based pharmaceuticals in the GIT. PTR3, PHT1, and HPT-1 expressions in Caco-2 cell monolayers strongly suggest that their function needs to be further elucidated and their contribution to peptide transport not ignored. Taken together, these results demonstrate the potential for molecular biological characterization in localizing active transporter systems that can potentially be targeted for enhancing the absorption of peptide-based pharmaceuticals.
Animals↗