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V H Lee

Publications and source records attributed to V H Lee.

At least 55 records · Page 3Linked to original sources
PubMed1997

Na(+)-dependent L-arginine transport in the pigmented rabbit conjunctiva.

The objective of this study was to characterize Na(+)-coupled L-arginine (L-Arg) transport in the pigmented rabbit conjunctiva. The excised pigmented rabbit conjunctiva was mounted in the modified Ussing chamber for measurement of short-circuit current (Isc), 3H-L-arginine (3H-L-Arg) flux, and 22Na flux. L-Arg when added to the mucosal side led to 0.32-2.65 microA cm-2 increases in the Isc at 37 degrees C, but not at 4 degrees C or in a Na(+)-free solution. L-Arg at 1 mM stimulated net Na+ absorption by 0.12 microEq cm-2 h-1. The evidence for carrier-mediated transport of L-Arg includes: (1) temperature dependence and saturability over 0.01-10 mM, (2) Na+ dependence and ouabain sensitivity, (3) 84 +/- 2% reduction in the apparent permeability coefficient (Papp) of 3H-L-Arg in the presence of excess unlabeled L-Arg (1 mM), and (4) 16-fold difference in L-Arg transport (at 0.1 mM) between the mucosal-to-serosal and the serosal-to-mucosal direction. Moreover, L-Arg transport was inhibited by basic amino acids, large neutral amino acids, and nitric oxide synthase inhibitors, but not by acidic and small neutral amino acids. Kinetic analysis revealed the possible existence of both high and low affinity processes for L-Arg transport. A half maximal concentration (Km) and maximal L-Arg flux (Jmax) values of the low and high affinity processes were 5.90 and 0.07 mM, and 1,248 and 111 pmol cm-2 min-1, respectively. Hill analysis of L-Arg transport at 0.1 mM in the presence of varying Na+ concentrations in the mucosal bathing fluid yielded a Hill coefficient of 0.93, suggesting a 1:1 coupling between Na+ and L-Arg. In conclusion, Na(+)-coupled transport process(es) for L-Arg in accordance with a 1:1 stoichiometry appear to be present on the mucosal side of the pigmented rabbit conjunctiva. The pattern of inhibition by basic and large neutral amino acids and Na+ dependency are suggestive of system B0,(+)-mediated L-Arg transport.

Amino Acids↗
PubMed1997

Size-dependent dextran transport across rat alveolar epithelial cell monolayers.

The transport of dextrans (approximately 4 to approximately 150 kDa) across an in vitro model of the alveolar epithelial barrier was studied to determine the effects of molecular size on pulmonary absorption of macromolecular drugs. Fluorescein isothiocyanate (FITC)-labeled dextrans (FDs) with average molecular weights (all in kDa) of 3.86 (FD4), 9 (FD10), 19.8 (FD20), 40.5 (FD40), 71.6 (FD70), and 156.9 (FD150) were utilized as model macromolecular drugs. Unidirectional fluxes of FDs at 37 and 4 degrees C were measured from the appearance rates of FD in the receiver fluid of open-circuited monolayers (>2000 omega-cm2) of rat alveolar epithelial cells. Apparent permeability coefficients (P(app)) were estimated from the observed flux and the corresponding concentration gradient of FD. Results showed that FD fluxes were the same in both apical-to-basolateral (AB) and opposite (BA) directions at each molecular weight studied. The P(app) was not significantly different at 0.5 and 1.0 mg/mL FD40 donor concentrations. The FD P(app) (x 10(-8)cm/s) decreased gradually from 1.35 for FD4 to 0.32 for FD40, indicating an apparent inverse relationship between P(app) and molecular weight of FD. By contrast, P(app) was about the same at 0.13 for both FD70 and FD150. When experimental temperature was lowered to 4 degrees C, P(app) decreased by approximately 40% for FDs of 4 through 40 kDa, whereas the decrease in P(app) was by approximately 80% for larger FDs of both 70 and 150 kDa. Moreover, these FDs were found to be relatively intact (approximately 90%) in either receiver fluid after 5-h flux experiments without detectable levels of metabolites in the respective donor fluid, suggesting that alveolar epithelial cells allow translocation of FDs intact across the barrier. Equivalent pore analysis, assuming restricted diffusion of FDs of 4-40 kDa via cylindrical, water-filled pores across the cell monolayer revealed a population of large equivalent pores with approximately 5.6 nm radius. These data suggest that smaller macromolecules (radius <5 nm) traverse the alveolar epithelial barrier via paracellular pathways, and that larger (i.e., radius > or = 6 nm) macromolecules likely cross the barrier via other pathways (e.g., pinocytosis).

Absorption↗
PubMed1997

Synthesis and purification of NB1-palmitoyl insulin.

A procedure for synthesizing NB1-palmitoyl insulin for incorporation into liposomes for targeting to hepatocytes was developed. The amino group of the first amino acid phenylalanine on the B chain (B1) of insulin was selected for conjugation with palmitic acid in anticipation that its binding to the insulin receptor would be preserved. Two other free amino groups present in insulin, the first amino acid glycine on the A chain (A1) and the 29th amino acid lysine on the B chain (B29), were first protected with a t-butoxycarbonyloxy (t-Boc) group to yield NA1, B29-di-(t-Boc) insulin. The identity of this di-(t-Boc) insulin was confirmed by amino acid analysis as well as by enzyme hydrolysis coupled with matrix-assisted laser-desorption time of flight mass spectrometry (MALDI-TOF MS). NA1,B29-Di-(t-Boc) insulin was then reacted with the N-hydroxysuccinimide ester of palmitic acid, followed by deblocking the t-Boc groups, to yield NB1-palmitoyl insulin, the structure of which was further confirmed by MALDI-TOF MS analysis. NB1-palmitoyl insulin was found to interact with the insulin receptor on fat cells, thereby catalyzing the conversion of [14C]glucose into lipids, at reduced efficiency (30-40%).

Animals↗
PubMed1997

Polar solute transport across the pigmented rabbit conjunctiva: size dependence and the influence of 8-bromo cyclic adenosine monophosphate.

PURPOSE: To characterize the conjunctival permeability to polar solutes ranging from 182 to 167,000 daltons in molecular weight (m.w.). METHODS: Solute transport across the excised pigmented rabbit conjunctiva with a baseline transepithelial electrical resistance (TEER) of 1,285 +/- 46 ohm.cm2 was evaluated in the modified Ussing chamber under open-circuit conditions. The model solutes were mannitol (m.w. 182), 6-carboxyfluorescein (m.w. 376), and fluorescein isothiocyanate-labeled dextrans (FD4, m.w. 4,400-FD150, m.w. 167,000). RESULTS: For a given solute, the apparent permeability coefficient (Papp) was independent of solute concentration and direction of transport. As expected, the Papp decreased with solute size, from 27.7 x 10(-8) cm/sec for mannitol to 0.31 x 10(-8) cm/sec for FD150. When the experimental temperature was lowered from 37 degrees C to 4 degrees C. Papp decreased by approximately 50% for FD4 through FD40 and by > 80% for both FD70 and FD150. Equivalent pore analysis, assuming restricted solute diffusion via cylindrical, water-filled pores across the isolated tissue, revealed a radius of 5.5 nm at a pore density of 1.9 x 10(8) pores per cm2. The addition of 1 mM 8-bromo cyclic adenosine monophosphate (8-BrcAMP), known to stimulate Cl- secretion and decrease TEER, to the mucosal side of the conjunctiva increased the transport of mannitol, FD4, and FD40 by 28%, while not affecting FD150 transport. CONCLUSIONS: Our findings suggest that polar solutes up to FD40 traverse the conjunctival epithelial barrier primarily by restricted diffusion through equivalent pores of 5.5 nm radius and that solute movement is affected by reduction of TEER. On the other hand, polar solutes of the FD70 or larger may cross the barrier primarily via non-diffusional pathways such as non-specific endocytosis.

Animals↗
PubMed1997

Kinetic evidence for Na(+)-glucose co-transport in the pigmented rabbit conjunctiva.

PURPOSE: To obtain kinetic evidence for the existence of a Na(+)-coupled glucose co-transport process on the mucosal (tear) side of the pigmented rabbit conjunctiva. METHODS: The excised pigmented rabbit conjunctiva was mounted in the modified Ussing chamber for measurement of 22Na and 3H-3-O-methyl-D-glucose (3-O-MG) fluxes. RESULTS: In the presence of 5 mM glucose, the conjunctival tissue showed net Na+ absorption in the mucosal-to-serosal direction at an approximate rate of 0.15 microEq/cm2/h. This net Na+ absorption was abolished by serosally added 0.5 mM ouabain, but not affected by mucosally added 0.1 mM or 1 mM amiloride. There was a 40-60% reduction in net Na+ absorption under the glucose-free condition or in the mucosal presence of 0.5 mM phlorizin. Moreover, serosally added ouabain and mucosally added phlorizin (both at 0.5 mM) significantly decreased the 3-O-MG permeability coefficient in the mucosal-to-serosal direction by about 70%, whereas mucosally instilled 0.1-1.0 mM amiloride was without any effect. Three-O-MG absorption in the mucosal-to-serosal direction appeared to be coupled with Na+ transport with a 1:1 stoichiometry. In addition, this process exhibited temperature dependency, saturability, and directionality. CONCLUSION: Our findings are consistent with Na(+)-glucose cotransport as being one of the mechanisms for mucosal Na+ entry into the epithelial cells of the pigmented rabbit conjunctiva.

3-O-Methylglucose↗
PubMed1997

Cidofovir transport in the pigmented rabbit conjunctiva.

PURPOSE: To characterize cidofovir (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine) transport in the pigmented rabbit conjunctiva and to evaluate the formulation influence on its transport. METHODS: The excised pigmented rabbit conjunctiva was mounted in the modified Ussing chamber. Cidofovir transport was initiated by applying 3H-cidofovir to the donor compartment and assayed by measuring the radioactivity accumulated in the receiver fluid over 180 min. RESULTS: Cidofovir flux in the mucosal-to-serosal direction increased proportionally with drug concentration over the 0.01 to 1 mM range. Cidofovir transport (0.01 mM) at 37 degrees C in the mucosal-to-serosal direction was not significantly different from that in the opposite direction or from that at 4 degrees C. Hypotonicity (80 mOsm/kg), 0.5% EDTA, and 0.0125% benzalkonium chloride increased the apparent permeability coefficient of cidofovir 3, 21, and 49 times, respectively. This was accompanied by a corresponding 43%, 86%, and 96% reduction in the transconjunctival electrical resistance over 180 min. The reduction in transepithelial electrical resistance elicited by hypotonicity was reversible. There was a good correlation between apparent permeability coefficient and the transconjunctival conductance, suggesting that cidofovir may undergo paracellular passive diffusion in the conjunctiva. CONCLUSION: Cidofovir transport in the rabbit conjunctiva may be via paracellular passive diffusion. Formulation changes may improve cidofovir absorption from the conjunctival route.

Animals↗
PubMed1996

Age-dependent expression of P-glycoprotein gp170 in Caco-2 cell monolayers.

PURPOSE: To determine whether the expression and activity of the P-glycoprotein (P-GP) drug efflux pump vary with the culture age of Caco-2 cell monolayers. METHODS: Caco-2 cell monolayers were grown for 3-27 days on tissue culture-treated Transwells. P-GP efflux function was determined by measuring transmonolayer fluxes of cyclosporin A (CsA) and verapamil, while P-GP expression level was evaluated by Western blot analysis using monoclonal antibody C219. RESULTS: The apparent permeability coefficient (Papp) of CsA (0.5 microM) in the basolateral-to-apical (B-->A) direction increased with culture age and was higher than the apical-to-basolateral (A-->B) direction at all times. Net secretory Papp significantly increased from day 17 onward compared to that observed during day 3 through 13. Verapamil (100 microM) significantly inhibited CsA transport in the B-->A direction from day 17 to 27, while elevating CsA transport in the A-->B direction from day 6 to 27. Interestingly, the Papp of verapamil (0.5 microM) in the B-->A direction was significantly higher than in the A-->B direction from day 6 to 27, rendering increases in net secretory Papp of verapamil with culture age. Western analysis revealed that P-GP expression level was in the order of 4 weeks approximately 1 week > 3 weeks > 2 weeks at equal loading of cell proteins. CONCLUSIONS: P-GP is continuously expressed throughout the culture period, but it may not be fully functional at an early age. Caco-2 cell monolayers of day 17 to 27 appear to be a good model to evaluate the functional role of P-GP in drug efflux.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Horseradish peroxidase transport across rat alveolar epithelial cell monolayers.

PURPOSE: To evaluate the transport characteristics of horseradish peroxidase (HRP, a nonspecific fluid-phase endocytosis marker) across an in vitro model of tight (> 2,000 ohm-cm2) rat alveolar epithelial cell monolayers grown on tissue culture-treated polycarbonate filters. METHODS: Unidirectional HRP fluxes were estimated from the appearance rate of HRP in the receiver fluid following instillation in the donor fluid as a function of donor [HRP] and temperature. Molecular species present in either bathing fluid were determined at the end of flux experiments using fluorescein isothiocyanate (FITC)-labeled HRP by gel permeation chromatography. Cell-associated HRP activity at the end of the transport experiment was determined, as were the rates of recycling and transcellular movement of HRP. An enzymatic assay was uses to quantify HRP activity in the bathing fluid and cells. RESULTS: Unidirectional HRP fluxes were symmetric and increased linearly with up to 50 microM donor [HRP]. The apparent permeability coefficient of HRP was reduced by 3.5 times upon lowering the temperature from 37 to 4 degrees C. About 50% of the FITC-labeled species present in either receiver fluid was intact HRP. Cell-associated HRP estimated from apical HRP incubation was about 4 times greater than that from basolateral incubation. Recycling into apical fluid of cell-associated HRP following apical incubation occurred rapidly with a half-time (T1/2) of approximately 5 min, reaching a plateau at approximately 67% of the initial cell-associated HRP, while transcellular movement of HRP (into basolateral fluid) took place with a T1/2 of approximately 20 min, attaining a steady-state at approximately 13% of the initial cell-associated HRP. Basolateral recycling of HRP was also rapid (T1/2 = approximately 5 min) reaching a steady-state at approximately 35% of the initial basolaterally-bound HRP. Transcellular movement of HRP following basolateral incubation was slower (T1/2 = approximately 70 min), leveling off at 50% of the initial cell-associated HRP. CONCLUSIONS: HRP appears to be transported relatively intact (approximately 50%) across rat alveolar epithelial barrier via nonspecific fluid-phase endocytosis. The transepithelial pinocytotic rate of alveolar epithelial cells is estimated to be about 25 nL/cm2/h.

Animals↗
PubMed1996

Basis for dosing time-dependent changes in the ocular and systemic absorption of topically applied timolol.

The objective of the present study was to determine the basis for dosing time-dependent changes in the ocular and systemic absorption of topically applied timolol in pigmented rabbits. The gamma scintigraphic technique was used to monitor the changes in precorneal solution retention following instillation. Changes in timolol concentration in the plasma over 120 min and in various anterior segment eye tissues at 30 min following the topical instillation of 25 microliters of 0.65% timolol maleate solutions at various dosing times were monitored using reversed phase HPLC. Corneal and conjunctival permeability at various dosing times was evaluated in the modified Ussing chamber. The results indicated that precorneal solution drainage was slowest at noon. Suppressing tear production by anesthesia led to an increase in ocular timolol absorption at 6 a.m. but not at other dosing times, in spite of the lowest corneal permeability then. There was no statistically significant dosing time influence on systemic timolol absorption following nasal or conjunctival dosing. In conclusion, possible diurnal changes in precorneal solution clearance may be the main factor underlying the diurnal changes in ocular as well as systemic timolol absorption in rabbits. In addition, diurnal changes in corneal permeability may also contribute to diurnal changes in ocular timolol absorption.

Absorption↗
PubMed1996

Cyclic AMP modulation of active ion transport in the pigmented rabbit conjunctiva.

This study was conducted to determine whether active ion transport in the pigmented rabbit conjunctiva was subjected to cyclic AMP modulation. 8-BrcAMP and compounds that may affect intracellular cAMP levels were tested for their effects on the conjunctival short-circuit current (Isc) following mucosal application. Conjunctival Isc was increased by 1 mM 8-BrcAMP, 0.001-20 microM forskolin, 0.01-2 mM theophylline, 2 microM 1-epinephrine, and 20 microM terbutaline in a Cl(-)-containing but not in a Cl(-)-free medium. The increase in Isc induced by 1 mM 8-BrcAMP was reversed by 1 mM N-phenylanthranilic acid, suggesting enhancement of Cl- secretion by cAMP. Similarly, the increase in Isc induced by 20 microM terbutaline was blunted by 1 microM timolol, suggesting a role for beta 2 adrenergic modulation of Cl- secretion. Collectively, these results are consistent with the notion of cAMP modulation of conjunctival active ion transport.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Localization of laminin proteins during early follicular development in pig and rabbit ovaries.

Laminin, a major component of the basal lamina, is known to be important in the differentiation of epithelial cells. The outer granulosa cell layer of ovarian follicles is attached to a basal lamina surrounding the follicle and it has been demonstrated that proteins of the basal lamina can alter the steroidogenic capacity and cytoskeletal composition of mature granulosa cells. The present studies were carried out to evaluate the developmental expression of laminin proteins in the basal lamina of ovarian follicles during early stages of granulosa cell differentiation. Ovaries from sexually immature pigs (0-20 weeks of age) and rabbits (0-12 weeks of age) were used for this study because formation of primordial follicles and initiation of follicular growth occur partially or completely postnatally. Specific antibodies were made against laminin beta 1/gamma 1 chains using protein purified by high resolution two-dimensional polyacrylmide gel electrophoresis. These antibodies were used in immunohistochemical localization studies to demonstrate that laminin is associated with the basal lamina surrounding egg clusters and their connections to the ovarian surface epithelium and ovarian rete during early ovarian development. In addition, laminin was observed to associate with a continuous matrix that surrounds forming primordial follicles as they are isolated from the egg clusters. Laminin is localized in the basal lamina of primordial, primary, secondary, and tertiary follicles of both pig and rabbit ovaries. Laminin proteins are not only expressed throughout early follicular development in pigs and rabbits, but are also found surrounding the germ cell compartments prior to initiation of meiosis and formation of primordial follicles. These results demonstrate that laminin proteins are deposited in the basal lamina well before granulosa cells undergo morphological differentiation. This pattern of expression suggests that laminin proteins alone do not control changes in granulosa cell morphology during early development but may be required to maintain cell lineage commitment.

Animals↗
PubMed1996

A primary culture model of rabbit conjunctival epithelial cells exhibiting tight barrier properties.

PURPOSE: The present study was conducted to develop and characterize a functional primary culture of pigmented rabbit conjunctival epithelial cells on permeable support exhibiting tight barrier properties. METHODS: Conjunctival epithelial cells were isolated by 0.2% protease treatment, cultured at 0.5-1.8 x 10(6) cells/cm2 onto collagen-treated Transwell filters, and were maintained either in the presence of 1% fetal bovine serum throughout or serum-free media from day 3 onwards. Transepithelial potential difference (PD) and transepithelial electrical resistance (TEER) were measured and equivalent short-circuit current (Ieq = PD/TEER) estimated. RESULTS: There appears to be a critical plating density of 1.5 x 10(6) cells/cm2 for functional development of tight epithelial cell cultures. The culture conditions as noted above did not affect either the time when peak bioelectric parameters were attained (days 8-10) or the magnitude of these parameters at a plating density of 1.5 x 10(6) cells/cm2. Specifically, cells grown in a serum-free media showed a peak TEER of 1.9 +/- 0.2 k omega.cm2, a PD of 14.2 +/- 1.6 V (apical side negative), and and Ieq of 8.0 +/- 0.4 microA/cm2 (mean +/- SEM, n = 45). Electron microscopy of serum-weaned cultures revealed a multilayered epithelium with numerous microvilli on the outermost layer of cells, while sporadic positive Periodic Acid Schiff (PAS) staining under light microscopy suggested the presence of mucin-secretory goblet cells. CONCLUSIONS: A functional, tight, epithelial barrier of the pigmented rabbit conjunctiva on a permeable support has been developed, which may be useful for mechanistic studies of ion and drug transport at the cellular level.

Animals↗
PubMed1996

Permeability characteristics of primary cultured rabbit conjunctival epithelial cells to low molecular weight drugs.

PURPOSE: To evaluate the permeability characteristics of primary cultured rabbit conjunctival epithelial cell (RCEC) layers to low molecular weight drugs of varying lipophilicity. METHODS: 3H-mannitol; hydrophilic sotalol and atenolol; moderately lipophilic metoprolol, timolol, propranolol; and highly lipophilic betaxolol were used as model compounds. RESULTS: The conjunctival apparent permeability coefficient (Papp) of mannitol (1 x 10(-7) cm/s) was 2.4 times lower than that of the most hydrophilic beta-blocker, sotalol (Papp = 2.4 x 10(-7) cm/s). Differences in the degree of tightness of the epithelial cell layers brought about a 30-fold difference in the transport of atenolol in favor of the leaky cell layers, while not affecting the transport of the lipophilic drug, propranolol. Within the log partition coefficient (PC) range of -0.62 (sotalol) and 3.44 (betaxolol), there was a hundred-fold difference in the Papp. A sigmoidal curve was used to depict the influence of lipophilicity on solute permeation across conjunctival epithelial cell layers. An effective half-maximal Papp was observed at a log PC value of 1.2. CONCLUSIONS: These findings on the lipophilicity effect on drug transport are generally similar to those reported for the isolated rabbit conjunctiva, suggesting the utility of cultured rabbit conjunctival epithelial cell layers as an in vitro model for evaluating drug transport.

Adrenergic beta-Antagonists↗
PubMed1996

Contribution of Na(+)-glucose cotransport to the short-circuit current in the pigmented rabbit conjunctiva.

The objective of this study was to determine whether Na(+)-glucose cotransport contributed to the residual short-circuit current (Isc) in the pigmented rabbit conjunctiva not accounted for by Cl- secretion. The Isc of the pigmented rabbit conjunctiva was measured following exposure of its mucosal or serosal side to varying concentrations of D-glucose or phloridzin in a glutathione-bicarbonated Ringer's solution (GBR). Addition of D-glucose to the mucosal side of the conjunctiva bathed in glucose-free GBR elevated Isc up to 26 +/- 6% in a dose-dependent manner (K0.5 = 1.2 mM), while mucosal (but not serosal) addition of phloridzin reduced the Isc (IC50 = 0.05 mM) of the conjunctiva bathed in regular GBR. In a mucosal Na(+)-free medium, neither 20 mM D-glucose nor 0.5 mM phloridzin was effective. In a mucosal glucose-containing medium, deletion of Na+ reduced Isc up to 27 +/- 4%. It was, however, restored to its initial value upon the addition of 17.5-141 mM Na+ to a medium containing 5 mM D-glucose. Hill plot analysis of Isc elevation at 141 mM Na+ in the presence of varying D-glucose concentrations in the mucosal bathing fluid yielded a Hill coefficient of 0.86. There is, therefore, evidence for the apical localization of phloridzin-sensitive Na(+)-coupled D-glucose transport that exhibits apparent 1:1 stoichiometry, being responsible for the maximal 26% increase in the Isc.

Animals↗
PubMed1996

Targeted drug delivery to the respiratory tract: solute permeability of air-interface cultured rabbit tracheal epithelial cell monolayers.

The permeability characteristics of air-interfaced rabbit tracheal epithelial cell monolayers to model macromolecules and low molecular weight solutes were evaluated. The model macromolecules were fluorescein isothiocyanate (FITC)-labeled dextrans of varying molecular sizes (4,000 to 70,000 daltons). The model low molecular weight solutes were beta-adrenergic compounds of similar sizes with widely different log octanol/pH 7.4 buffer partition coefficients (log P). FITC-dextrans were assayed spectrofluorimetrically and beta-adrenergic compounds were assayed by reverse phase HPLC with UV detection. The apparent permeability coefficients (Papp) for FITC alone to FITC-dextran 70,000 in the tracheal epithelial barrier were in the range of 11.2 to 0.3 x 10(8) cm/sec. A molecular cut-off at about 20,000 daltons, consistent with a single equivalent pore population of about 5 nm in radius, was found. A sigmoidal relationship best described the influence of drug lipophilicity on the Papp of beta-adrenergic compounds, where the log P at the half maximal Papp was 2.08. Thus, the air-interfaced rabbit tracheal epithelial cell culture model has been successfully applied to elucidate the permeability of tracheal epithelial barrier to model macromolecules and small solutes. It appears that the tracheal epithelial cell monolayers absorb drugs in a similar manner as do native excised tracheal and other epithelia.

Adrenergic beta-Agonists↗
PubMed1995

The trinucleotide repeat sequence d(GTC)15 adopts a hairpin conformation.

The structure of a single-stranded (ss) oligonucleotide containing (GTC)15 [ss(GTC)15] was examined. As a control, parallel studies were performed with ss(CTG)15, an oligonucleotide that forms a hairpin. Electrophoretic mobility, KMnO4 oxidation and P1 nuclease studies demonstrate that, similar to ss(CTG)15, ss(GTC)15 forms a hairpin containing base paired and/or stacked thymines in the stem. Electrophoretic mobility melting profiles performed in approximately 1 mM Na+ revealed that the melting temperature of ss(GTC)15 and ss(CTG)15 were 38 and 48 degrees C respectively. The loop regions of ss(GTC)15 and ss(CTG)15 were cleaved by single-strand-specific P1 nuclease at the T25-C29 and G26-C27 phosphodiester bonds respectively (where the loop apex of the DNAs is T28). Molecular dynamics simulations suggested that in ss(GTC)15 the loop was bent towards the major groove of the stem, apparently causing an increased exposure of the T25-C29 region to solvent. In ss(CTG)15 guanine--guanine stacking caused a separation of the G26 and C27 bases, resulting in exposure of the intervening phosphodiester to solvent. The results suggest that ss(GTC)15 and ss(CTG)15 form similar, but distinguishable, hairpin structures.

Base Sequence↗