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V H Lee

Publications and source records attributed to V H Lee.

At least 19 recordsLinked to original sources
PubMed1998

Molecular identification of a role for tyrosine 167 in the function of the human intestinal proton- coupled dipeptide transporter (hPepT1).

hPepT1 is a proton-coupled peptide transporter that mediates the absorption of di- and tripeptides. Here we show that tyrosine 167 (Y167) in transmembrane domain 5 (TMD5) of this 12-transmembrane spanning protein contributes to its transport function. We identified this particular amino acid by a computer model of the arrangement of the TMDs of hPepT1 and investigated its role by site-directed mutagenesis and dipeptide uptake studies. [3H]Gly-sar uptake in cells transiently transfected with Y167A-hPepT1 was abolished completely, even though the level of Y167A-hPepT1 expression by Western blot analysis and cell surface expression by immunofluorescence microscopy was similar to those of the wild type. Therefore, mutation affected transport function, but apparently not the steady-state protein level or trafficking of the transporter to the plasma membrane. Moreover, mutation of Y167 into phenylalanine, serine, or histidine all abolished gly-sar uptake in transfected HEK 293 cells. Taken together, these findings suggest that Y167 plays an essential role in hPepT1 function, perhaps due to the unique chemistry of its phenolic side chain.

Blotting, Western↗
PubMed1998

Modulation of chloride secretion across the pigmented rabbit conjunctiva.

The purpose of the present study was to investigate whether active Cl- secretion in the pigmented rabbit conjunctiva was subject to cAMP, Ca2+ and protein kinase C (PKC) modulation. The excised pigmented rabbit conjunctivas were mounted in the modified Ussing-type chambers for measurement of unidirectional 36Cl fluxes under the open-circuit condition and of the short-circuit current (Isc), potential difference, and transconjunctival electrical resistance. The results indicate that Cl- secretion across the conjunctiva was abolished by mucosal application of 1 mM N-phenylanthranilic acid and was reduced by 40% by serosal application of 10 microM bumetanide. Net Cl- flux was stimulated by 133% by 1 mM 8-Br cAMP, 107% by 10 microM A23187, and 87% by 1 microM phorbol 12-myristate-13-acetate (PMA), suggesting that cAMP, Ca2+, and PKC all modulated active Cl- secretion, respectively. There existed a linear correlation between measured changes in net Cl- flux and observed changes in Isc (r2=0.99). The serial treatment of the conjunctiva with (a) 1 mM 8-Br cAMP and 10 microM A23187 and (b) 10 microM A23187 and 1 microM PMA resulted in sequence-independent, additive stimulation of Isc. In the case of 1 mM 8-Br cAMP and 1 microM PMA, additive stimulation of Isc was observed only when 1 mM 8-Br cAMP was added prior to 1 microM PMA. These results suggest that a given pharmacological agent may affect more than one channel type and that there might be a possible connection among the channels at the signal transduction level. In summary, Cl- appears to enter the pigmented rabbit conjunctiva from the serosal fluid via Na+-(K+)-2Cl- cotransport process and exit to the mucosal fluid via channels, resulting in active Cl- secretion. Active Cl- secretion in the pigmented rabbit conjunctiva appears to be modulated by cAMP, Ca2+, and PKC.

Animals↗
PubMed1998

Structure, function, and molecular modeling approaches to the study of the intestinal dipeptide transporter PepT1.

The proton-coupled intestinal dipeptide transporter, PepT1, has 707 amino acids, 12 putative transmembrane domains (TMD), and is of importance in the transport of nutritional di- and tripeptides and structurally related drugs, such as penicillins and cephalosporins. By using a combination of molecular modeling and site-directed mutagenesis, we have identified several key amino acid residues that effect catalytic transport properties of PepT1. Our molecular model of the transporter was examined by dividing it into four sections, parallel to the membrane, starting from the extracellular side. The molecular model revealed a putative transport channel and the approximate locations of several aromatic and charged amino acid residues that were selected as targets for mutagenesis. Wild type or mutagenized human PepT1 cDNA was transfected into human embryonic kidney (HEK293) cells, and the uptake of tritiated glycylsarcosine [3H]-(Gly-Sar) was measured. Michaelis-Menton analysis of the wild-type and mutated transporters revealed the following results for site-directed mutagenesis. Mutation of Tyr-12 or Arg-282 into alanine has only a very modest effect on Gly-Sar uptake. By contrast, mutation of Trp-294 or Glu-595 into alanine reduced Gly-Sar uptake by 80 and 95%, respectively, and mutation of Tyr-167 reduced Gly-Sar uptake to the level of mock-transfected cells. In addition, preliminary data from fluorescence microscopy following the expression of N-terminal-GFP-labeled PepT1Y167A in HEK cells indicates that the Y167A mutation was properly inserted into the plasma membrane but has a greatly reduced Vmax.

Amino Acid Sequence↗
PubMed1998

Ocular absorption of Pz-peptide and its effect on the ocular and systemic pharmacokinetics of topically applied drugs in the rabbit.

PURPOSE: To determine the corneal and conjunctival penetration of 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (Pz-peptide) and to evaluate its effect on the corneal and conjunctival penetration of hydrophilic solutes as well as on the ocular and systemic absorption of topically applied atenolol and propranolol in the rabbit. The hydrophilic solutes were mannitol, fluorescein, FITC-dextran 4,000, and FITC-dextran 10,000. METHODS: Drug penetration across the rabbit cornea and conjunctiva was evaluated using the modified Ussing chamber. Ocular and systemic absorption of topically applied atenolol and propranolol was evaluated by analyzing the drug concentration in various anterior segment tissues at 45 min and in the blood over 240 min, respectively, following topical instillation of 25 microl of 20 mM atenolol or propranolol solution to the rabbit eye. RESULTS: The conjunctiva was 29 times more permeable than the cornea to 3 mM Pz-peptide. Conjunctival Pz-peptide transport was 1.7 times more extensive in the mucosal-to-serosal than in the opposite direction, whereas corneal Pz-peptide transport showed no directionality. The apparent permeability coefficient of Pz-peptide across the cornea and the conjunctiva increased over the 1-5 mM range, suggesting that Pz-peptide enhanced its own transport across both epithelial tissues. The cornea appeared to be more sensitive than the conjunctiva to the penetration enhancement effect of Pz-peptide. Thus, whereas Pz-peptide elevated the corneal transport of mannitol, fluorescein, and FD4 by 50%, 57%, and 106%, respectively, it did not affect the conjunctival transport of mannitol and fluorescein, while enhancing FD4 transport by only 46%. Moreover, while Pz-peptide enhanced the ocular absorption of topically applied hydrophilic atenolol, it did not affect the ocular absorption of lipophilic propranolol. Interestingly, Pz-peptide did not affect the systemic absorption of either beta adrenergic antagonist. CONCLUSIONS: Pz-peptide appears to facilitate its own penetration across the cornea and the conjunctiva. Pz-peptide appears to increase the ocular absorption of topically applied hydrophilic but not lipophilic drugs, while not affecting the systemic absorption of either type of drugs.

Actins↗
PubMed1998

Dipeptide uptake and transport characteristics in rabbit tracheal epithelial cell layers cultured at an air interface.

PURPOSE: To determine the functional presence ofa H+/peptide cotransport process in rabbit tracheal epithelial cell layers cultured at an air-interface and its contribution to transepithelial dipeptide transport. METHODS: Rabbit tracheocytes were isolated, plated on Transwells, and cultured at an air-interface. After 5 or 6 days in culture, uptake and transepithelial transport of carnosine were examined. RESULTS: Carnosine uptake by tracheocytes was pH-dependent and was saturable with a Michaelis-Menten constant of 170 microM. Moreover, carnosine uptake was inhibited 94% by Gly-L-Phe, 28% by beta-Ala-Gly, but not at all by Gly-D-Phe or by the amino acids beta-Ala and L-His. Unexpectedly. transepithelial carnosine transport at pH 7.4 (i.e., in the absence of a transepithelial pH gradient) was similar in both the apical-to-basolateral (ab) and basolateral-to-apical (ba) directions. Lowering the apical fluid pH to 6.5 reduced ab transport 1.6 times without affecting ba transport, consistent with predominantly paracellular diffusion of carnosine under an electrochemical potential gradient. CONCLUSIONS: The kinetic behavior of carnosine uptake into cultured tracheal epithelial cell layers is characteristic of a H+-coupled dipeptide transport process known to exist in the small intestine and the kidney. Such a process does not appear to be rate-limiting in the transport of carnosine across the tracheal epithelial barrier.

Animals↗
PubMed1998

Spatio-temporal pattern for expression of galectin-3 in the murine utero-placental complex: evidence for differential regulation.

In mice, immunoreactive galectin-3 protein has previously been localized in uterine epithelial cells adjacent to implanting blastocysts as well as in the decidualized endometrium of implantation sites, uterine natural killer cells, and several types of placental trophoblast cells. Because galectin-3 is a soluble extracellular molecule, protein localization by immunohistochemical methods does not demonstrate its cellular origin. Therefore, the present study was undertaken to determine precisely which cell types in the utero-placental complex express galectin-3 mRNA. In situ hybridization results demonstrated that galectin-3 mRNA was expressed throughout the utero-placental complex in all cell types previously shown to contain immunoreactive protein, including uterine epithelium, decidualized endometrium, uterine natural killer cells, and placental trophoblasts. These results indicate that galectin-3 protein is not synthesized in a restricted cell type and translocated through the extracellular spaces to other tissue compartments. Furthermore, Northern blot analysis of total RNA prepared from separated fetal and maternal components of utero-placental complexes demonstrated different patterns of expression for galectin-3 mRNA in the uterus and placenta. Relative levels of galectin-3 mRNA peak at midgestation in the implantation site and during the second half of gestation remain elevated in the placenta but decline in the uterus. Separate mechanisms for regulating expression of galectin-3 on opposite sides of the feto-maternal interface are indicated.

Animals↗
PubMed1998

Nucleoside transport mechanisms in the pigmented rabbit conjunctiva.

PURPOSE: To elucidate the mechanisms of nucleoside transport in the pigmented rabbit conjunctiva using [3H]uridine as the substrate. METHODS: Excised pigmented rabbit conjunctiva was mounted in a modified Ussing chamber for measurement of short-circuit current (Isc) and [3H]uridine transport. RESULTS: [3H]Uridine transport in the mucosal-to-serosal direction at 10 microM exhibited directionality, temperature dependency, and phlorizin sensitivity. Uridine transport appeared to be mediated via saturable Na(+)-dependent and nitrobenzylthioinosine-insensitive Na(+)-independent processes. The corresponding Michaelis-Menten constants (K(m)) were 1.9 microM and 200 microM, and the maximal uridine fluxes (Jmax) were 29.3 and 46.7 pmol/cm2 per minute. When added to the mucosal side containing 141 mM Na+, uridine increased the Isc in a dose-dependent manner from 0.005 mM to 1.0 mM at 37 degrees C. The K(m) value was 7.6 microM, and the maximal increase in Isc was 0.71 microA/cm2. Hill analysis of uridine transport at 10 microM in the presence of varying Na+ concentrations in the mucosal bathing fluid yielded a Hill coefficient of 1.1, suggesting a 1:1 coupling between Na+ and uridine. Na(+)-dependent uridine transport was inhibited by 10 microM adenosine, guanosine, and inosine, but not by thymidine, suggesting that the transport process may be mainly selective for purine nucleosides. Moreover, 2'-deoxyuridine, 5-iodo-2'-deoxyuridine, and 5-(2-bromovinyl)-2'-deoxyuridine were potent inhibitors of Na(+)-dependent uridine transport. CONCLUSIONS: Na(+)-dependent and Na(+)-independent nucleoside transport processes appeared to be localized on the mucosal aspect of the pigmented rabbit conjunctiva. One or more Na(+)-coupled uridine cotransport processes exhibited a 1:1 stoichiometry and an apparent preference toward purine nucleosides.

Animals↗
PubMed1998

Carrier-mediated transport of NG-nitro-L-arginine, a nitric oxide synthase inhibitor, in the pigmented rabbit conjunctiva.

In this study, the transport mechanism of NG-nitro-L-arginine (L-NA), a nitric oxide synthase inhibitor that may be useful for alleviating intraocular inflammation, was characterized in the pigmented rabbit conjunctiva. L-NA, when applied to the mucosal side of the conjunctiva, led to dose-dependent increases in the short-circuit current (Isc) at 37 degrees C but not at 4 degrees C or under the Na+-free condition. Serosally added 1 mM L-NA did not elicit any change in the Isc. Mucosally added 1 mM L-NA elicited a net absorptive Na+ flux of 0.09 microEq/(cm2.hr), comparable with the Isc change. L-NA transport at 0.1 mM in the mucosal-to-serosal (ms) direction was 22 times greater than that in the serosal-to-mucosal direction. There was a good correlation between the ms flux of L-NA and the Isc changes elicited by L-NA under the same experimental conditions. L-NA transport was saturable, with a Km of 0.35 mM and a maximal flux of 290 pmol/(cm2.min). Hill analysis of L-NA flux observed at 0.1 mM L-NA in response to varying Na+ concentrations in the mucosal bathing fluid yielded a Hill coefficient of 0.98, suggesting a 1:1 coupling between Na+ and L-NA. Moreover, ms 3H-L-NA transport was inhibited by basic amino acids (L-Arg and L-Lys) and a neutral amino acid (L-Leu), but not by an acidic amino acid (L-Glu) and the D-stereoisomer of L-NA. In the case of L-Arg, inhibition was competitive with a Ki of 0.034 mM. Taken together, the above findings are consistent with the involvement of the L-Arg transport system B0,+ in the conjunctival transport of L-NA.

Animals↗
PubMed1998

Existence of a p-glycoprotein drug efflux pump in cultured rabbit conjunctival epithelial cells.

PURPOSE: To determine whether a p-glycoprotein (P-gp) drug efflux pump exists in cultured rabbit conjunctival epithelial cells (RCEs) to restrict the absorption of cyclosporin A (CSA) and other lipophilic drugs such as verapamil and dexamethasone. METHODS: The anti-P-gp monoclonal antibody (mAb) C219 was used in western blot analysis to reveal the presence of P-gp in freshly isolated and cultured RCEs. Bidirectional transport of tritiated CSA, verapamil, and dexamethasone (0.5 or 5.0 microM) across cultured RCEs was evaluated in the absence and presence of P-gp inhibitors and an external mAb to P-gp (4E3). RESULTS: Western blot analysis of lysates of freshly isolated and cultured RCEs with C219 mAb revealed a 170-kDa membrane protein band. At 0.5 microM CSA, the basal-to-apical (ba) apparent permeability coefficient (P(app) that is, efflux) was 9.3 times higher than that in the apical-to-basal direction (that is, influx). At 5 microM, this ratio was halved. Net CSA secretion was blocked completely at 4 degrees C. Verapamil (100 microM), progesterone (100 microM) and 4E3 mAb (5 microg/ml) increased CSA influx three times, while reducing efflux by 50% to 70%. Verapamil and progesterone inhibited CSA efflux in a concentration-dependent manner. In all cases, net secretory CSA flux was markedly reduced. The P(app) for verapamil (0.5 microM) and dexamethasone (0.5 microM) in the ba direction was 3.4 and 1.6 times, respectively, which was higher than that in the opposite direction. The 4E3 mAb reduced net verapamil secretion by 65%. CONCLUSIONS: There may exist a P-gp-mediated drug efflux pump on the apical aspect of the rabbit conjunctiva to restrict the absorption of cyclosporin A and other lipophilic drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Carrier-mediated transport of monocarboxylate drugs in the pigmented rabbit conjunctiva.

PURPOSE: To determine whether an Na+-dependent monocarboxylate transport process exists on the mucosal side of the pigmented rabbit conjunctiva and to evaluate how it may contribute to the absorption of ophthalmic monocarboxylate drugs. METHODS: L-lactate was used as a model substrate. The excised pigmented rabbit conjunctiva was mounted in a modified Ussing chamber for the measurement of short-circuit current (Isc) and 14C-L.-lactate transport. RESULTS: When added to the mucosal side at 37 degrees C and at pH 7.4, applications of as much as 40 mM L- and D-lactate increased Isc in a saturable manner. By contrast, no change in Isc was observed at 4 degrees C or under the mucosal Na+-free condition. 14C-L-lactate transport in the mucosal-to-serosal (m-s) direction at 0.01 mM revealed directionality, temperature dependency, Na+ dependency, and ouabain sensitivity, but not pH dependency. L-lactate transport in the m-s direction consisted of a saturable Na+-dependent process by the transcellular pathway and a nonsaturable process by the paracellular pathway. For the saturable process, the apparent Michaelis-Menten constant was 1.9 mM, the maximum flux was 8.9 nanomoles/cm2 per hour, and the apparent Na+ :L-lactate coupling ratio was 2:1. 14C-L-lactate transport in the m-s direction was significantly inhibited (46% to 83%) by the mucosal presence of various monocarboxylate compounds, but not by dicarboxylate compounds, zwitterionic compound, D-glucose, amino acids, and peptidomimetic antibiotics. Monocarboxylate nonsteroidal anti-inflammatory drugs and the antibacterial fluoroquinolones inhibited 14C-L-lactate transport by 40% to 85%, whereas prostaglandins and cromolyn had no effect. CONCLUSIONS: An Na+-dependent monocarboxylate transport process that may be used by non-steroidal anti-inflammatory and fluoroquinolone antibacterial drugs for transport appears to be present on the mucosal side of the pigmented rabbit conjunctiva. A possible physiologic role for the Na+-dependent monocarboxylate transport process may be to salvage tear lactate.

Animals↗
PubMed1998

Proton-driven dipeptide uptake in primary cultured rabbit conjunctival epithelial cells.

PURPOSE: To characterize proton-driven carrier-mediated dipeptide uptake in primary cultured conjunctival epithelial cells of the pigmented rabbit using beta-alanyl-L-histidine (L-carnosine) as a model dipeptide substrate. METHODS: Uptake of tritiated L-carnosine was monitored using conjunctival epithelial cells on days 6 through 8 in culture on a filter support. The structural features of dileucine stereoisomers and cephalexin contributing to interaction with the dipeptide transporter were evaluated by computer modeling and inhibition of tritiated L-carnosine uptake. RESULTS: Uptake of L-carnosine by primary cultured conjunctival epithelial cells in the presence of an inwardly directed proton gradient showed directional asymmetry (favoring apical uptake by a factor of five), temperature dependence, and saturability correlated with substrate concentration, with a Michaelis-Menten constant (Km) of 0.3 +/- 0.03 mM and a maximum uptake rate (Vmax) of 22.0 +/- 1.0 picomoles per milligram protein per minute. L-Carnosine uptake was optimal at pH 6.0 and was reduced by 60% and 35%, respectively, by 50 microM p-trifluoromethoxyphenylhydrazone (a proton ionophore) and by acid preloading with 50 mM NH4Cl. The constituent amino acids did not inhibit L-carnosine uptake. L-Carnosine uptake was inhibited, however, from 50% to 80% by other dipeptides and structurally similar drugs such as bestatin, beta-lactam antibiotics, and angiotensin-converting enzyme inhibitors. The LL, LD, or DL forms of the dipeptide Leu-Leu inhibited tritiated L-carnosine uptake by approximately 60%, 40%, and 70%, respectively. By contrast, the DD form did not inhibit uptake. Results from computer modeling suggest that an appropriate dipeptide N-terminal to C-terminal distance and a favorable orientation of the side chains may be important for substrate interaction with the conjunctival dipeptide transporter. CONCLUSIONS: Uptake of the dipeptide L-carnosine in primary cultured pigmented rabbit conjunctival epithelial cells is probably mediated by a proton-driven dipeptide transporter. This transporter may be used for optimizing the uptake of structurally similar peptidomimetic drugs.

Ammonium Chloride↗
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↗