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PubMed1992

Faster superoxide dismutase mutants designed by enhancing electrostatic guidance.

The enzyme Cu, Zn superoxide dismutase (SOD) protects against oxidative damage by dismuting the superoxide radical O2-. to molecular oxygen and hydrogen peroxide at the active-site Cu ion in a reaction that is rate-limited by diffusion and enhanced by electrostatic guidance. SOD has evolved to be one of the fastest enzymes known (V(max) approximately 2 x 10(9) M-1 s-1). The new crystal structures of human SOD show that amino-acid site chains that are implicated in electrostatic guidance (Glu 132, Glu 133 and Lys 136) form a hydrogen-bonding network. Here we show that site-specific mutants that increase local positive charge while maintaining this orienting network (Glu----Gln) have faster reaction rates and increased ionic-strength dependence, matching brownian dynamics simulations incorporating electrostatic terms. Increased positive charge alone is insufficient: one charge reversal (Glu----Lys) mutant is slower than the equivalent charge neutralization (Glu----Gln) mutant, showing that the newly introduced positive charge disrupts the orienting network. Thus, electrostatically facilitated diffusion rates can be increased by design, provided the detailed structural integrity of the active-site electrostatic network is maintained.

Amino Acid Sequence↗
PubMed1991

Role of protein D2 and lipopolysaccharide in diffusion of quinolones through the outer membrane of Pseudomonas aeruginosa.

Routes of quinolone permeation in Pseudomonas aeruginosa were investigated by using sparfloxacin as a prototype compound. [14C]sparfloxacin cell labeling was 13 to 28% lower in three protein D2-deficient mutants resistant to imipenem than in their imipenem-susceptible counterparts. In four impermeability-type quinolone-resistant strains isolated from pefloxacin-treated animals, we observed two- to fourfold-greater resistance to imipenem, reduced protein D2 expression in the outer membrane according to Western blotting (immunoblotting), and 25 to 29% decreased cell labeling with imipenem. In a protein D2-producing strain but not in its protein D2-deficient isogenic mutant, uptake of [14C]sparfloxacin was strongly inhibited by L-lysine and imipenem, which act as substrates for protein D2. Conversely, binding of [14C]imipenem in a porin D2-positive strain was reduced by sparfloxacin but not by the nonamphoteric quinolone nalidixic acid. Sparfloxacin, imipenem, and lysine possess a carboxyl group and a potentially protonated nitrogen separated from each other by 0.64 to 1.07 nm as calculated by computer. Hence, protein D2 may catalyze facilitated diffusion for sparfloxacin, as it does for imipenem. In addition, pefloxacin-selected isolates contained 41 to 113% more 3-deoxy-D-mannooctulosonic acid than their quinolone-susceptible counterparts, with MIC increases of 2- to 4-fold for WIN-57273 (n-octanol-phosphate buffer partition coefficient, 13.139), 4- to 8-fold for difloxacin (partition coefficient, 3.093) and sparfloxacin (partition coefficient, 0.431), and 8- to 16-fold for norfloxacin (partition coefficient, 0.059) and ciprofloxacin (partition coefficient, 0.056). Thus, we hypothetize that in quinolone-selected strains, increased amounts of lipopolysaccharide form a permeability barrier that acts preferentially against hydrophilic quinolones.

Anti-Infective Agents↗
PubMed1989

Active transport of ascorbic acid into lens epithelium of the rat.

The transport rates of radiolabeled ascorbic acid (AA) and dehydroascorbic acid, as well as 3-O-methyl-D-glucose and L-glucose from blood into aqueous humor, lens epithelium and lens 'cortex' compartments were studied in male Sprague-Dawley rats. In vivo pulse chase kinetic studies and modeling of transport from plasma and aqueous and on into idealized water compartments of lens epithelium and cortex allowed for the calculation of transport rate constants, Ki (min-1), in experiments utilizing L-glucose as a passive internal control. TLC chromatography was used to monitor intraocular labeled molecules deriving from labeled test molecules introduced via blood. Results indicate that AA enters aqueous humor at rates similar to L-glucose and likely via simple passive diffusion. In contrast, an active uptake of AA by lens epithelium was found with the calculated entry constant for ascorbate being more than 21 times faster than that of L-glucose. Concentrations in lens epithelium were found to be more than twice that of aqueous humor within only 7 min from the introduction of a [14C]AA bolus into blood. It was also found that very little AA continued on past the epithelium to the interior lens cortex compartment. Our data suggest no special uptake of AA by lens fiber cells. The non-metabolizable analog of D-glucose, 3-O-methyl-D-glucose, however, readily moves past the lens epithelium into fiber cells at much faster rates than the passive L-glucose marker and in a manner consistent with facilitated diffusion. The data suggest that even in a nocturnal species, such as a rat, which demonstrates relatively low circulating levels of ascorbic acid in plasma and aqueous humor, special mechanisms exist for moving ascorbic acid into intraocular tissues. More specifically, the lens epithelium actively takes up ascorbate for some, as yet unclear purpose while the interior fiber cells appear to have no special uptake mechanism for this molecule.

Animals↗
PubMed2001

Uptake of the neutral amino acids glutamine, leucine, and serine by Pneumocystis carinii.

Experiments to elucidate the mechanism by which Pneumocystis carinii transports glutamine, leucine, and serine were performed in this study. Uptake of all three radiolabeled amino acids exhibited first-order, saturation kinetics as extracellular substrate concentrations were increased, thus ruling out simple diffusion and indicating carrier-mediated transport. Kinetic analyses of amino acid uptake and the results of competitive inhibition experiments suggested that leucine, serine, and glutamine were taken up via a common transporter system. The uptake of serine was examined in greater detail to characterize the nature of the carrier. Serine uptake was not affected by N, N'-dicyclohexylcarbodiimide, carbonyl cyanide m-chlorophenyl hydrazone, ouabain, gramicidin, valinomycin, sodium azide, salicylhydroxamine acid (SHAM), iodoacetate, iodoacetate plus SHAM, KCN, and azide. Thus serine uptake did not require sodium or energy from ATP, an electrochemical proton gradient or a membrane potential across the cell surface (i.e., proton-motive force). Serine uptake was dependent on glucose in the extracellular compartment. In the presence of glucose, serine uptake was inhibited by chloramphenicol but not cycloheximide. The results from these experiments are most consistent with facilitated diffusion as the mechanism. After 30 min of incubation, most of the radioactivity was in the cellular soluble fraction. In most cases, incorporation into the extractable total lipids and the remaining particulate cellular components were detectable after this incubation period.

Biological Transport↗
PubMed1982

Comparison of lipid-mediated blood-brain-barrier penetrability in neonates and adults.

The permeability of compounds that penetrate brain capillaries by virtue of their lipoidal solubilities was studied in vivo after a single capillary transit by the intracarotid injection technique. Brain permeabilities of 14C-labeled test isotopes were measured relative to that of tritiated water, a highly diffusible reference substance, with correction for any test isotope remaining in the cerebral vasculature. The brain uptake indices of acetamide, antipyrine, benzyl alcohol, butanol, caffeine, cytosine, diphenyl hydantoin, ethanol, ethylene glycol, heroin, mannitol, methanol, phenobarbital, propylene glycol, thiourea, and urea were measured in ether-anesthetized newborn rabbits. A highly significant correlation (r = 0.86) between brain uptake indices and octanol-saline partition coefficients of these compounds was observed. An almost identical relationship was derived in the adult rat blood-brain barrier where brain uptakes and partition coefficients of some 48 compounds could be correlated (r = 0.86). The similarities in slope-intercept relationships indicate that newborn rabbit and adult rat brain endothelia are functionally similar with respect to lipid-mediated permeability [in contrast to previous studies that have established dramatic differences in selective permeabilities of metabolites transported by saturable, carrier-mediated ("facilitated diffusion") mechanisms]. Permeability-surface area products were also derived; these data confirmed no differences in permeability could be detected between newborn and adult blood-brain-barrier capillaries. A relationship between hydrogen bond number (an alternative indicator of hydrophobic properties( and brain uptake indices derived for the adult rat brain could not be confirmed in the case of the newborn rabbit.

Aging↗
PubMed1991

Utilization of Inorganic Carbon by Ulva lactuca.

Thalli discs of the marine macroalga Ulva lactuca were given inorganic carbon in the form of HCO(3) (-), and the progression of photosynthetic O(2) evolution was followed and compared with predicted O(2) evolution as based on calculated external formation of CO(2) (extracellular carbonic anhydrase was not present in this species) and its carboxylation (according to the K(m)(CO(2)) of ribulose-1,5-bisphosphate carboxylase/oxygenase), at two different pHs, assuming a photosynthetic quotient of 1. The K(m)(inorganic carbon) was some 2.5 times lower at pH 5.6 than at the natural seawater pH of 8.2, whereas V(max) was similar under the two conditions, indicating that the unnaturally low pH per se had no adverse effect on U. lactuca's photosynthetic performance. These results, therefore, could be evaluated with regard to differential CO(2) and HCO(3) (-) utilization. The photosynthetic performance observed at the lower pH largely followed that predicted, with a slight discrepancy probably reflecting a minor diffusion barrier to CO(2) uptake. At pH 8.2, however, dehydration rates were too slow to supply CO(2) for the measured photosynthetic response. Given the absence of external carbonic anhydrase activity, this finding supports the view that HCO(3) (-) transport provides higher than external concentrations of CO(2) at the ribulose-1,5-bisphosphate carboxylase/oxygenase site. Uptake of HCO(3) (-) by U. lactuca was further indicated by the effects of potential inhibitors at pH 8.2. The alleged band 3 membrane anion exchange protein inhibitor 4,4'-diisothiocyanostilbene-2,2'disulphonate reduced photosynthetic rates only when HCO(3) (-) (but not CO(2)) could be the extracellular inorganic carbon form taken up. A similar, but less drastic, HCO(3) (-)-competitive inhibition of photosynthesis was obtained with Kl and KNO(3). It is suggested that, under ambient conditions, HCO(3) (-) is transported into cells at defined sites either via facilitated diffusion or active uptake, and that such transport is the basis for elevated internal [CO(2)] at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation.

Journal Article↗
PubMed1997

Characteristics of Fps1-dependent and -independent glycerol transport in Saccharomyces cerevisiae.

Eadie-Hofstee plots of glycerol uptake in wild-type Saccharomyces cerevisiae W303-1A grown on glucose showed the presence of both saturable transport and simple diffusion, whereas an fps1delta mutant displayed only simple diffusion. Transformation of the fps1delta mutant with the glpF gene, which encodes glycerol transport in Escherichia coli, restored biphasic transport kinetics. Yeast extract-peptone-dextrose-grown wild-type cells had a higher passive diffusion constant than the fps1delta mutant, and ethanol enhanced the rate of proton diffusion to a greater extent in the wild type than in the fps1delta mutant. In addition, the lipid fraction of the fps1delta mutant contained a lower percentage of phospholipids and a higher percentage of glycolipids than that of the wild type. Fps1p, therefore, may be involved in the regulation of lipid metabolism in S. cerevisiae, affecting membrane permeability in addition to fulfilling its specific role in glycerol transport. Simultaneous uptake of glycerol and protons occurred in both glycerol- and ethanol-grown wild-type and fps1delta cells and resulted in the accumulation of glycerol at an inside-to-outside ratio of 12:1 to 15:1. Carbonyl cyanide m-chlorophenylhydrazone prevented glycerol accumulation in both strains and abolished transport in the fps1delta mutant grown on ethanol. Likewise, 2,4-dinitrophenol inhibited transport in glycerol-grown wild-type cells. These results indicate the presence of an Fps1p-dependent facilitated diffusion system in glucose-grown cells and an Fps1p-independent proton symport system in derepressed cells.

Aquaporins↗
PubMed2004

Further evidence for the existence of a specific process for the membrane transport of anandamide.

Indirect evidence for the existence of a specific protein-mediated process for the cellular uptake of endocannabinoids has been reported, but recent results suggested that such a process, at least for AEA [ N -arachidonoylethanolamine (anandamide)], is facilitated uniquely by its intracellular hydrolysis by FAAH (fatty acid amide hydrolase) [Glaser, Abumrad, Fatade, Kaczocha, Studholme and Deutsch (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 4269-4274]. In the present study, we show that FAAH alone cannot account for the facilitated diffusion of AEA across the cell membrane. In particular, (i) using a short incubation time (90 s) to avoid AEA hydrolysis by FAAH, AEA accumulation into rat basophilic leukaemia or C6 cells was saturable at low microM concentrations of substrate and non-saturable at higher concentrations; (ii) time-dependent and, at low microM concentrations of substrate, saturable AEA accumulation was observed also using mouse brain synaptosomes; (iii) using synaptosomes prepared from FAAH-deficient mice, saturable AEA accumulation was still observed, although with a lower efficacy; (iv) when 36 AEA and N -oleoylethanolamine analogues, most of which with phenyl rings in the polar head group region, were tested as inhibitors of AEA cellular uptake, strict structural and stereochemical requirements were needed to observe significant inhibition, and in no case the inhibition of FAAH overlapped with the inhibition of AEA uptake; and (v) AEA biosynthesis by cells and sensory neurons was followed by AEA release, and this latter process, which cannot be facilitated by FAAH, was still blocked by an inhibitor of AEA uptake. We suggest that at least one protein different from FAAH is required to facilitate AEA transport across the plasma membrane in a selective and bi-directional way.

Amidohydrolases↗
PubMed2001

Hepatic uptake of synthetic chlorogenic acid derivatives by the organic anion transport proteins.

Chlorogenic acid derivatives were recently identified as novel, potent, and specific inhibitors of the hepatic glucose 6-phosphate translocase. Inhibition of the glucose 6-phosphate translocase leads to a decrease in hepatic glucose production, rendering chlorogenic acid derivatives as potential novel therapeutics in patients with type 2 diabetes. The present study examines the hepatic uptake mechanism of the radiolabeled chlorogenic acid derivative S 1743 into freshly isolated rat hepatocytes. Initial uptake rates were Na(+)-independent and followed saturation kinetics with no superimposition of facilitated diffusion. Inhibition studies demonstrated that other chlorogenic acid derivatives inhibited uptake of the radiolabeled compound S 1743 into rat hepatocytes in the range of 1.1 to 11 microM, whereas the natural chlorogenic acid (up to 100 microM) had no effect at all. In addition, inhibition of S 1743 uptake into rat hepatocytes was found in the presence of sulfobromophthalein, sulfolithocholyltaurine, estrone-3-sulfate, cholyltaurine, verapamil, bumetanide, probenecide, phenol red, digoxin, and ouabain (in decreasing order) but not with N-methylnicotinamide, alpha-ketoglutarate, p-aminohippurate, geneticin sulfate, and 5-sulfosalicylate. The observed inhibition pattern suggested that members of the family of the organic anion transporting polypeptides (Oatps) could be involved in hepatic uptake of chlorogenic acid derivatives. Indeed, S 1743 uptake could be demonstrated in Oatp1- and Oatp2-expressing Xenopus laevis oocytes as well as in Oatp1-expressing Chinese hamster ovary cells. A comparison of the inhibition pattern obtained in hepatocytes compared with that obtained in Oatp1-expressing Chinese hamster ovary cells suggests that facilitated uptake by Oatp1 is a major contributor in total hepatic uptake of chlorogenic acid derivatives.

Animals↗
PubMed1994

Specificity of the retinol transporter of the rat small intestine brush border.

The uptake of vitamin A (all-trans-retinol) by the absorptive cell of the small intestine is the necessary first step in its utilization by the organism and appears to involve a specific carrier that operates by facilitated diffusion. We investigated the specificity of that process by determining the absorption of all-trans-, 13-cis-, and 9-cis-retinol, 3-dehydroretinol, and retinal (vitamin A aldehyde) by gut sheets from the small intestine of suckling rats. We found that radiolabeled all-trans-retinol and 3-dehydroretinol were absorbed at similar rates and that approximately 60% of the total absorption could be competed for by unlabeled all-trans-retinol. A similar level of inhibition could be achieved for all-trans-retinol absorption by treating the intestinal sheets with N-ethylmaleimide. The noncompetable, noninhibitable component of all-trans-retinol absorption corresponded to the total absorption rate for 13-cis- and 9-cis-retinol and retinal. Additionally, we found that the relative rates of transport of these retinoids were unrelated to their relative affinities for the abundant absorptive cell retinoid carrier protein, cellular retinol-binding protein, type II, and were not driven by esterification. This confirms that the absorption of retinol is facilitated by a transporter and establishes that it is specific for the all-trans alcohol forms of vitamin A.

Acyltransferases↗
PubMed2001

Helium inhalation enhances vasodilator effect of inhaled nitric oxide on pulmonary vessels in hypoxic dogs.

There are theoretical and experimental indications that the presence of He as a balance gas markedly increase the diffusion velocity of other gases contained in a gas mixture. We allowed dogs with pulmonary vasoconstriction induced by hypoxia to inhale a mixture of 5 parts per million (ppm) of nitric oxide (NO) and O(2) balanced with He (NO in He) instead of N(2) (NO in N(2)). The dilating effect of NO in He and NO in N(2) on the pulmonary artery was evaluated by determining conventional pulmonary hemodynamic parameters, mean pulmonary artery (PA) pressure (MPAP), and pulmonary vascular resistance indexed to body surface area (PVRI), pulmonary impedance (Z), and the recently developed hemodynamic index, time-corrected wave intensity (WI). The main findings in this study were as follows: 1) hypoxia increased MPAP, PVRI, Z at 0 Hz (Z(0)), Z at the first harmonics, characteristic impedance (Z(c)), the reflection coefficient (Gamma), and the first peak of WI; 2) NO in N(2) reduced Z(0) and Gamma; and 3) NO in He reduced the first peak of WI and reduced Z(0) and Gamma more than NO in N(2). The enhanced vasodilatory effect of NO in He might be associated with facilitated diffusion of NO diluted in the gas mixture with He. In conclusion, increased efficacy of NO in He offers the possibility to reduce the inhaled NO concentration.

Administration, Inhalation↗
PubMed1996

Theoretical predictions of end-capillary PO2 in muscles of athletic and nonathletic animals at VO2max.

Characterizing the resistances to O2 transport from the erythrocyte to the mitochondrion is important in understanding potential transport limitations. A steady-state model of this process was developed to predict the minimum (critical) end-capillary PO2 required to prevent hypoxia at maximal O2 consumption (VO2max) in a circular region of tissue surrounding the venular end of a capillary. Capillary density was used as a measure of O2 delivery, and mitochondrial density was used as a measure of O2 consumption. The effects of oxyhemoglobin dissociation kinetics and diffusion facilitation by hemoglobin in the erythrocytes and facilitation by myoglobin in the tissue were taken into account. Calculations made for selected skeletal muscles, diaphragm, and myocardium in three adaptive animal pairs (dog and goat, horse and cow, and pony and calf) yielded values of end-capillary PO2 that were consistent with measured values of mixed venous PO2 in maximally working animals. Values of end-capillary PO2 were found to be uncorrelated with values of VO2max in different muscles. No significant difference in end-capillary PO2 was found between similar muscles of athletic versus nonathletic animals. Predicted intracapillary O2 transport resistance ranged from 18 to 54% of the total transport resistance in the O2 pathway. Further investigation is required to explore the extent to which spatial and temporal heterogeneities in O2 delivery and consumption play a role in O2 transport.

Animals↗
PubMedDate not supplied

Mechanism of glucose transport across the human and rat placental barrier: a review.

Glucose is one of the most important substances transferred from the maternal blood to the fetal circulation in the placenta, and its transport across the cellular membranes is mediated by glucose transporters. Facilitated-diffusion glucose transporter GLUT1 is abundant in the placental barrier, as is the case in other blood-tissue barriers, where GLUT1 is present at the critical plasma membranes of the barrier cells. In the human placenta, the microvillous apical and the basal plasma membranes of the syncytiotrophoblast are rich in GLUT1, which molecule seems to be responsible for the transcellular transport of glucose across the placental barrier. In the rat placental labyrinth, two layers of syncytiotrophoblasts (termed syncytiotrophoblasts I and II from the maternal side) serve as a barrier. GLUT1 is abundant at the plasma membrane of syncytiotrophoblast I facing the maternal side, and the plasma membrane of syncytiotrophoblast II facing the fetal side. Numerous gap junctions, made of connexin 26, connect syncytiotrophoblasts I and II, comprising a channel for the transfer of glucose between them. GLUT1 in combination with the gap junction, therefore, seems to serve as the structural basis for the transport of glucose across the rat placental barrier.

Animals↗
PubMed1998

Hyperglycemia regulates the glucose-transport system of clonal choriocarcinoma cells in vitro. A potential molecular mechanism contributing to the adjunct effect of glucose in tumor therapy.

Glucose is taken up by tumor cells via sodium-independent facilitated diffusion along a concentration gradient. To examine the regulation of this process by substrate concentration, we investigated the effect of hyperglycemia on the glucose-transport system of choriocarcinoma-derived JAR and JEG-3 cells by culturing them for 24, 48 and 96 hr in medium containing either 5.5 (normoglycemia) or 25 (hyperglycemia) mM D-glucose, respectively. Immunocytochemically, choriocarcinoma cells expressed the high-affinity glucose transporter isoforms GLUT1 and GLUT3. Based on initial uptake measurements using 3-O-[14C]methyl-D-glucose, kinetic parameters were calculated as Km = 15 mM and Vmax = 95 fmol/sec per cell for JAR and Km = 9 mM and Vmax = 64 fmol/sec per cell for JEG-3 cells. In JAR cells cultured under hyperglycemic conditions, uptake rates were significantly increased at 15, 20 and 25 mM exogenous D-glucose concentrations as compared with normoglycemic conditions. This effect was due to an increase in Vmax, whereas Km remained unchanged. Using Northern blotting, GLUT1 mRNA levels were higher but GLUT3 transcripts were reduced upon hyperglycemia. Western blotting revealed elevated GLUT1 and GLUT3 expression under hyperglycemic conditions. Hyperglycemia did not significantly influence the glucose-transport system of JEG-3 cells. We conclude that sustained hyperglycemia stimulates the glucose-transport system of JAR, but not of JEG-3, choriocarcinoma cells in vitro due to changes in GLUT1 and GLUT3 expression levels. We speculate that this mechanism may contribute to the beneficial effects of induced hyperglycemia as an adjuvant in tumor therapy.

3-O-Methylglucose↗
PubMedDate not supplied

Methylmercury-cysteine uptake by rat erythrocytes: evidence for several transport systems.

The present study is a continuation of our experiments on methylmercury-cysteine (MeHg-cysteine) uptake by rat red blood cells (RBCs) at low temperature. Methylmercury-cysteine uptake by rat RBCs was conducted at 5 degrees C and 20 degrees C. The effects of Ca2+, colchicine, cytochalasin B, 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid (DIDS), N-ethylmaleimide (NEM), D-glucose, hexanol, L-homocysteine, ouabain, probenecid, sodium fluoride (NaF), vinblastine, and anisotonic changes on MeHg-cysteine uptake were examined. The results showed that MeHg-cysteine uptake at 5 degrees C could be described by Michaelis-Menten kinetics (v = VmaxS/(S+Km), where Km = 37.02 mM and Vmax = 320.84 mmol l-1 RBCs h-1), but MeHg-cysteine uptake at 20 degrees C could be described by Michaelis-Menten kinetics with a linear component (v = [VmaxS/(S + Km)] + KdS, where Km = 2.71 mM, Vmax = 250.72 mmol l-1 RBCs h-1, and Kd = 5.63 mM). Methylmercury-cysteine uptake was inhibited by colchicine, cytochalasin B, D-glucose, hexanol, NaF, NEM, ouabain, probenecid, vinblastine and 230 mosM hypotonicity but stimulated by Ca2+, DIDS, L-homocysteine and 460 mosM hypertonicity. The results in the present study suggest that MeHg-cysteine uptake by rat RBCs might be involved in the following three transport systems: an energy transport system sensitive to Ca2+, ouabain and NaF and subjected to Michaelis-Menten kinetics; an organic anion transport system sensitive to probenecid; and a facilitated diffusive transport for D-glucose sensitive to cytochalasin B. It is likely that most of the transport systems work at temperatures higher than 5 degrees C.

Animals↗
PubMedDate not supplied

Prodrug activation enzymes in cancer gene therapy.

Among the broad array of genes that have been evaluated for tumor therapy, those encoding prodrug activation enzymes are especially appealing as they directly complement ongoing clinical chemotherapeutic regimes. These enzymes can activate prodrugs that have low inherent toxicity using both bacterial and yeast enzymes, or enhance prodrug activation by mammalian enzymes. The general advantage of the former is the large therapeutic index that can be achieved, and of the latter, the non-immunogenicity (supporting longer periods of prodrug activation) and the fact that the prodrugs will continue to have some efficacy after transgene expression is extinguished. This review article describes 13 different prodrug activation schemes developed over the last 15 years, two of which - activation of ganciclovir by viral thymidine kinase and activation of 5-fluorocytosine to 5-fluorouracil - are currently being evaluated in clinical trials. Essentially all of these prodrug activation enzymes mediate toxicity through disruption of DNA replication, which occurs at differentially high rates in tumor cells compared with most normal cells. In cancer gene therapy, vectors target delivery of therapeutic genes to tumor cells, in contrast to the use of antibodies in antibody-directed prodrug therapy. Vector targeting is usually effected by direct injection into the tumor mass or surrounding tissues, but the efficiency of gene delivery is usually low. Thus it is important that the activated drug is able to act on non-transduced tumor cells. This bystander effect may require cell-to-cell contact or be mediated by facilitated diffusion or extracellular activation to target neighboring tumor cells. Effects at distant sites are believed to be mediated by the immune system, which can be mobilized to recognize tumor antigens by prodrug-activated gene therapy. Prodrug activation schemes can be combined with each other and with other treatments, such as radiation, in a synergistic manner. Use of prodrug wafers for intratumoral drug activation and selective permeabilization of the tumor vasculature to prodrugs and vectors should further increase the value of this new therapeutic modality.

Antineoplastic Agents↗
PubMed2000

Lysozyme microencapsulation within biodegradable PLGA microspheres: urea effect on protein release and stability.

Lysozyme was encapsulated within biodegradable poly(D, L-lactide-co-glycolide) microspheres by a double emulsion solvent evaporation method for studying its release mechanism associated with protein stability problems. When urea, a protein unfolding agent, was added into the incubation medium lysozyme release rate from the microspheres increased with the increase in urea concentration. The enhanced lysozyme release was attributed to the suppression of protein aggregation, to the facilitated diffusion of unfolded lysozyme by an efficient reptile motion of unfolded protein molecules through porous channels in microspheres, and to the largely decreased extent of nonspecific protein adsorption onto the enlarged surface area of degrading polymer microspheres in the presence of urea. Encapsulating lysozyme in an unfolded form within PLGA microspheres was attempted by using urea as an excipient. This new urea-based formulation exhibited a more sustained lysozyme release profile than the control formulation, and released lysozyme from the microspheres showed a much less amount of lysozyme dimer population while maintaining a correct conformation after refolding in the incubation medium. This study provides new insights for the formulation of protein encapsulated PLGA microspheres.

Animals↗
PubMed1983

Perturbation of alpha-aminoisobutyric acid transport in human placental membranes: direct effects by HgCl2, CH3HgCl, and CdCl2.

Mercuric chloride, methylmercuric chloride, and cadmium chloride directly affect the human placental syncytiotrophoblast microvillous membrane. These heavy metals alter the facilitated diffusion of alpha-aminoisobutyric acid (AIB) into vesicles of this membrane in microM concentrations. Mercuric chloride abolishes temporal kinetics of AIB transport, inducing an initial increase in AIB transport (27% at 100 microM) but subsequently lowering equilibrium values when compared to equilibrium time points in control. Methylmercuric chloride and cadmium chloride inhibited the initial rate of AIB transport (40% and 21%, respectively, at 200 microM), but did not affect the equilibrium value of AIB transported when compared to equilibrium levels in control. These effects were concentration dependent. Methylmercuric chloride was more potent in inhibiting AIB transport than cadmium chloride. Methylmercuric chloride and cadmium chloride effects on AIB transport were observed with minimal preincubation with placental vesicles. However, preincubation was necessary for mercuric chloride-induced perturbation of AIB transport. Cysteine protects against mercuric chloride- and methylmercuric chloride-induced effects on AIB transport but did not reverse these perturbations. Mercury- and cadmium-induced placental membrane toxicity result from interactions of these heavy metals with the placental plasma membranes.

Aminoisobutyric Acids↗