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Prenatal morphine exposure throughout pregnancy can induce a series of neurobehavioral and neurochemical disturbances by affecting central nervous system development. This study was designed to investigate the effects of an enriched environment on behavioral deficits and changes in hippocampal brain-derived neurotrophic factor (BDNF) levels induced by prenatal morphine in rats. On pregnancy days 11-18, female Wistar rats were randomly injected twice daily with saline or morphine. Offspring were weaned on postnatal day (PND) 21. They were subjected to a standard rearing environment or an enriched environment on PNDs 22-50. On PNDs 51-57, the behavioral responses including anxiety and depression-like behaviors, and passive avoidance memory as well as hippocampal BDNF levels were investigated. The light/dark (L/D) box and elevated plus maze (EPM) were used for the study of anxiety, forced swimming test (FST) was used to assess depression-like behavior and passive avoidance task was used to evaluate learning and memory. Prenatal morphine exposure caused a reduction in time spent in the EPM open arms and a reduction in time spent in the lit side of the L/D box. It also decreased step-through latency and increased time spent in the dark side of passive avoidance task. Prenatal morphine exposure also reduced immobility time and increased swimming time in FST. Postnatal rearing in an enriched environment counteracted with behavioral deficits in the EPM and passive avoidance task, but not in the L/D box. This suggests that exposure to an enriched environment during adolescence period alters anxiety profile in a task-specific manner. Prenatal morphine exposure reduced hippocampal BDNF levels, but enriched environment significantly increased BDNF levels in both saline- and morphine-exposed groups. Our results demonstrate that exposure to an enriched environment alleviates behavioral deficits induced by prenatal morphine exposure and up-regulates the decreased levels of BDNF. BDNF may contribute to the beneficial effects of an enriched environment on prenatal morphine-exposed to rats.

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We investigate a graphene quantum pump, adiabatically driven by two thin potential barriers vibrating around their equilibrium positions. For the highly doped leads, the pumped current per mode diverges at the Dirac point due to the more efficient contribution of the evanescent modes in the pumping process. The pumped current shows an oscillatory behavior with an increasing amplitude as a function of the carrier concentration. This effect is in contrast to the decreasing oscillatory behavior of a similar normal pump (i.e. a pump based on a two-dimensional electron gas). The graphene pump driven by two vibrating thin barriers operates more efficiently than the graphene pump driven by two oscillating thin barriers.

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BACKGROUND: Possible regulation of riboflavin (RF) intestinal uptake process by dietary substrate level is not known and was examined in this study. METHODS: RF uptake was examined using intestinal brush border membrane vesicles (BBMV). RESULTS: Oversupplementation with RF caused significant decrease (P < 0.05; down-regulation) in the uptake of 3 mumol/L RF compared with control. This effect was not due to differences in the relative purity of the intestinal BBMV preparations and appeared to be specific for RF. This down-regulation was mediated through a significant (P < 0.01) decrease in the Vmax of the RF uptake process with no significant change in the apparent Michaelis constant (Km). In contrast, RF deficiency caused a significant (P < 0.01) enhancement (up-regulation) in the uptake of 3 mumol/L RF compared with pair-fed control. Again the enhancement was not due to differences in the relative purity of the BBMV preparations and appeared to be specific for RF. This up-regulation was mediated via a significant (P < 0.01) increase in the Vmax of the RF uptake process with no significant change in the apparent Km. CONCLUSIONS: These findings show that the RF intestinal uptake process is regulated by the level of the vitamin in the diet (and/or body stores) and that the regulation is mediated via changes in the number (and/or activity) of the RF uptake carriers with no change in their affinity.

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The mechanism of riboflavin (RF) uptake by intestinal basolateral membrane vesicles (BLMV) was examined in this study. BLMV were isolated by an established Percoll-gradient methodology from rabbit small intestine. Uptake of riboflavin was mainly the result of transport of the substrate into an osmotically active intravesicular space with less binding to membrane surfaces. Uptake of RF with time was similar in the presence of a Na+ and a K+ gradient (out > in) and was not significantly influenced by changes in incubation buffer pH. The initial rate of uptake of riboflavin as a function of concentration was saturable in both jejunal and ileal BLMV and occurred with apparent Km values of 5.0 microM and 4.4 microM and Vmax values of 91.6 and 60.8 pmol/mg protein per 5 s, respectively. Unlabeled riboflavin and the structural analogues lumiflavin, isoriboflavin and 8-aminoriboflavin all caused significant inhibition (but to different degrees) in the uptake of [3H]riboflavin. On the other hand, 8-hydroxyriboflavin, lumichrome, lumazine and D-ribose failed to inhibit [3H]riboflavin uptake. Trans-stimulation of [3H]riboflavin efflux from preloaded BLMV by unlabeled riboflavin or lumiflavin was also observed. Altering transmembrane electrical potential by anion substitution and valinomycin-induced K+ diffusion did not affect the riboflavin uptake process. These results demonstrate the existence of a specialized carrier-mediated mechanism for riboflavin uptake by intestinal BLMV. Furthermore, the system appears to transport the vitamin by a process which is Na(+)- and pH-independent, and electroneutral in nature.

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Uptake of luminal riboflavin (RF) into the absorptive cells of rabbit small intestine was examined using purified brush border membrane vesicle (BBMV) preparations. These preparations were used in order to eliminate the interference of intracellular metabolism that occurs to the RF molecule during absorption. Uptake of RF by BBMV was found to be mainly (> 76%) the result of transport of the vitamin into the intracellular space with less binding to membrane surfaces. All 3H radioactivity that appeared in the intravesicular space after incubation with [3H]RF was found to be in the form of intact RF. Uptake of RF with time was independent of the presence or absence of a Na+ or a K+ gradient (out > in) and occurred without transaccumulation of the substrate in the intravesicular space. Furthermore, changing the incubation buffer pH showed minimal effect on RF uptake. When examined as a function of concentration, the initial rate of RF uptake was found to be saturable both in jejunal and ileal BBMV with an apparent Km of 7.24 +/- 1.06 and 8.88 +/- 0.90 microM and Vmax of 24.31 +/- 1.48 and 34.24 +/- 1.55 pmol/mg protein/5 sec, respectively. Unlabeled RF and the related compounds lumiflavin, 8-aminoriboflavin, isoriboflavin, and lumichrome all inhibited (but to different degrees) the uptake of physiologic concentration of [3H]RF. On the other hand, 8-hydroxyriboflavin, lumazine, and D-ribose all failed to inhibit [3H]RF uptake. Similarly, the membrane transport inhibitors DIDS, SITS, and furosemide all failed to inhibit [3H]RF uptake. The uptake of RF was found to be insensitive to changes in the transmembrane electrical potential, as shown by studies with anion substitution and valinomycin K(+)-induced negative or positive intravesicular potential methodologies. These results indicate that RF uptake by rabbit intestinal BBMV occurs via a carrier-mediated system that is Na+ independent in nature and transports the substrate by an electroneutral process. The role of this system in the overall absorption process of RF is discussed.

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We examined the possible existence of histidine residue(s) in the folate transporter of rabbit intestine. This was done with use of the histidine-specific reagent diethyl pyrocarbonate (DEPC) and purified intestinal brush-border-membrane vesicles. DEPC caused significant concentration- and time-dependent inhibition of folic acid transport. The inhibition was only seen when transport was examined in vesicles incubated in buffer at pH 5.2 and not in those incubated in buffer at pH 7.4. The addition of unlabelled folic acid to vesicle suspension before treatment with DEPC (2.5 mM) led to a significant (P < 0.01) protection (84%) against the inhibition of folic acid transport. Treating vesicles pretreated with DEPC (2.5 mM) with reducing reagents (dithiothreitol, 2-mercaptoethanol and 2,3-dimercaptopropanol, all at a final concentration of 10 mM) did not reverse the inhibitory effect of DEPC on folic acid transport. On the other hand, treating the DEPC-pretreated vesicles with hydroxylamine (140 mM) led to a significant reversal (P < 0.01) (54%) of the inhibition of folic acid transport. The inhibitory effect of DEPC on carrier-mediated folic acid transport was found to be mediated through a decrease in the Vmax. (i.e. a decrease in the number and/or activity) of the carriers and an increase in the apparent Km (i.e. a decrease in their affinity), classifying the effect as a mixed-type inhibition. These results demonstrate the existence of critical histidine residue(s) in the intestinal brush-border-membrane folate transporter which is essential for its interaction with, and transport of, the vitamin. These findings also suggest that the histidine residue(s) is located at (or near) the substrate-binding site.

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This study examined the mechanism of biotin uptake by liver mitochondria. Mitochondria were isolated from rat liver by an established procedure and demonstrated normal respiratory control ratios. Uptake of biotin with time at incubation buffer pH 6.1 was rapid and linear and occurred with a distinct "overshoot" phenomenon that peaked at approximately 1 min of incubation. At incubation buffer pH 7.4, however, uptake of biotin with time was significantly slower and no overshoot was observed. Gradual lowering of incubation buffer pH from 7.9 to 6.1 caused a similar pattern of increase in uptake of low (0.024 microM) and high (8 and 30 microM) concentrations of biotin. At incubation buffer pH 6.1 and 7.4, uptake of biotin as a function of concentration (0.012-30 microM) was linear and occurred at rates of 3.62 and 1.90 pmol.mg protein-1.5 s-1, respectively. Addition to the incubation medium of high concentrations of unlabeled biotin, its related compounds (biocytin, desthiobiotin, biotin methyl ester, thioctic acid, and thioctic amide), or substrates of known mitochondrial transporters (succinate, pyruvate, glutamate, malate, and phosphate) failed to cause any significant inhibition in uptake of [3H]biotin by mitochondria incubated in buffer pH 6.1 and 7.4. Furthermore, preloading mitochondria with unlabeled biotin, biocytin, malate, or aspartate failed to cause any significant stimulation in biotin uptake. At incubation buffer pH 6.1, treatment of mitochondria with the protonophore FCCP caused significant inhibition in pH-dependent overshoot of biotin uptake. However, treatment of mitochondria with the potassium ionophore valinomycin caused significant stimulation in the pH-dependent overshoot of biotin uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

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Possible involvement of histidine residues and sulfhydryl groups in the function of the intestinal brush-border membrane (BBM) transporter of biotin was investigated. This was done by examining the effects of pretreatment of BBM vesicle (BBMV) isolated from rabbit intestine with the histidine-specific reagent diethyl pyrocarbonate (DEPC) and the sulfhydryl group-specific reagents p-chloromercuribenzenesulfonic acid (p-CMBS) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) on carrier-mediated biotin transport. Pretreatment of BBMV with DEPC caused significant inhibition in the initial rate of biotin transport without affecting the substrate uptake at equilibrium. Addition of biotin plus Na+ to vesicle suspensions prior to treatment with DEPC provided significant protection to biotin transport. Treatment of DEPC-pretreated vesicles with the reducing agents dithiothreitol and 2,3-dimercaptopropanol failed to reverse the inhibitory effect of DEPC on biotin transport. The inhibitory effect of DEPC was found to be mediated through a marked decrease in the number of the functional biotin transport carriers with no change in their affinity, as indicated by the severe inhibition in the Vmax but not the apparent Km of the biotin transport process, respectively. Pretreatment of BBMV with p-CMBS and NBD-Cl also caused significant inhibition in the initial rate of biotin transport without affecting the substrate uptake at equilibrium. Addition of biotin plus Na+ to vesicle suspensions prior to treatment with p-CMBS (or NBD-Cl) failed to protect biotin transport from inhibition. On the other hand, treatment of vesicles pretreated with p-CMBS (or NBD-Cl) with the reducing agents dithiothreitol and mercaptoethanol caused significant reversal in the inhibition of biotin transport. The inhibitory effects of p-CMBS (and NBD-Cl) on biotin transport was also found to be mediated through inhibition in the Vmax, but not the apparent Km, of biotin transport process. These results indicate the involvement of histidine residues and sulfhydryl groups in the normal function of the biotin transport system of rabbit intestinal BBM. Furthermore, the results also suggest that the histidine residues are probably located at (or near) the substrate-binding site while the sulfhydryl groups are located at a site other than the substrate binding region.

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The characteristics of biotin transport into human liver were examined using purified liver basolateral membrane vesicle (BLMV) preparations. Biotin uptake by BLMVs was mostly due to transport of the vitamin into the inside of vesicles. In the presence of an Na+ gradient (out greater than in), biotin transport with time was significantly higher than that in the presence of a K+ gradient and showed transient accumulation (overshoot). High concentrations of unlabeled biotin and related compounds caused significant cis inhibition in biotin transport in the presence of an Na+ (but not a K+) gradient. Transport of biotin as a function of concentration in the presence of an Na+ gradient included a saturable component, while it was lower and linear in the presence of a K+ gradient. Apparent Km and Vmax of the saturable Na+ gradient-dependent component were 1.22 mumol/L and 4.76 pmol.mg protein-1 x 10 s-1, respectively. Induction of a negative or positive intravascular potential using valinomycin-K diffusion methodology did not affect biotin transport into BLMVs. Also, neither the anion-exchange inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid nor 4-acetamido-4-isothiocyanostilbene-2,2'-disulfonic acid caused significant inhibition in biotin transport. These results indicate that biotin transport into human liver occurs via a specialized, carrier-mediated transport system. This system is Na(+)-gradient dependent and transports the vitamin via an electroneutral process.

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