Is heritability of alopecia areata sex-specific? A nationwide population-based cohort study.

Previous studies have demonstrated the heritability of alopecia areata (AA). However, whether the heritability of AA is sex-specific has not been examined. A nationwide population-based retrospective cohort study was performed using the Taiwan Maternal and Child Health Database from 2004 to 2017. We examined the heritability of AA in offspring of parents with and without AA, and determined whether the transmission of AA from parents to the next generation may occur in opposite directions depending on sex. We found that the risk ratio (RR) for heritability of AA between parents with and without AA was approximately two-fold. In addition, for fathers with AA, the risk of AA in offspring tended to be higher in girls than in boys (RR: 2.97; 95% confidence interval: 0.94, 9.31). Therefore, the present study confirms the heritability of AA, and further studies examining the sex-specific heritability of AA with a larger sample are warranted.

Abundantly expressed hepatic genes and their differential expression in liver of prelaying and laying geese.

Geese have a short egg-laying period and a low egg production rate. To induce and maintain egg laying, genes related to generating hepatic lipid for yolk deposition should be adequately expressed. Liver mRNA from 6 laying geese was extracted and used for construction of a full-length enriched cDNA library. About 2,400 clones containing gene sequences were determined and National Center for Biotechnology Information Gallus gallus Gene Index databases were used to compare and analyze these sequences. Ten highly expressed genes were selected to determine the differential expression between laying and prelay goose liver. Tissue distribution data showed that very low density apolipoprotein II, liver type fatty acid binding protein, vitellogenin I, and vitellogenin II transcripts were specifically expressed in the liver of laying geese. Ovoinhibitor, preproalbumin, alpha-2-hs-glycoprotein, and vitamin D binding protein mRNA were highly expressed in the liver and to a lesser extent in other tissues. Ovotransferrin mRNA was expressed in liver, ovary, oviduct, shell gland, brain, and adipose tissues. The concentration of transthyretin mRNA was high in the liver and brain. The mRNA concentrations of liver type fatty acid binding protein, alpha-2-hs-glycoprotein, and transthyretin in the livers of laying and prelay geese were not different. The concentrations of hepatic ovotransferrin, ovoinhibitor, preproalbumin, very low density apolipoprotein II, vitellogenin I, vitellogenin II, and vitamin D binding protein mRNA were higher in the liver of laying geese than in prelay geese, suggesting that these genes may be involved in laying function or lipid metabolism related to egg formation.

Psychiatric comorbidity of internet addiction in college students: an interview study.

OBJECTIVE: This study was aimed to evaluate the association between Internet addiction and depressive disorder, social phobia and adult attention-deficit/hyperactivity disorder (ADHD) in a sample of Taiwanese college students; and examine gender differences in the psychiatric comorbidity of Internet addiction in this student population. METHODS: Two hundred sixteen college students (132 males, 84 females) were recruited. Internet addiction, major depressive disorder, dysthymic disorder, social phobia, and adult ADHD of all participants were diagnosed based on psychiatric diagnostic interview. RESULTS: This study revealed that adult ADHD and depressive disorders were associated with Internet addiction among college students. However, depressive disorders were associated with Internet addiction in the males but not the females. CONCLUSION: With these results, it seems reasonable to suggest that effective evaluation of, and treatment for, adult ADHD and depressive disorders is required for college students with Internet addiction.

The differential expression of hepatic genes between prelaying and laying geese.

Suppression subtractive hybridization was used to detect differential expression of genes in the livers of laying and prelaying geese. Liver tissues from prelaying and laying geese were dissected for mRNA extraction. The cDNA, reverse transcribed from liver mRNA of prelaying geese, was subtracted from the cDNA generated from the laying geese (forward subtraction). Five hundred seventy-six clones with possible differentially expressed gene fragments were observed by forward subtraction hybridization. After differential screening using the reverse and forward subtraction cDNA, 164 clones were subjected to gene sequence determination and further analysis. Using Northern analysis, 5 known and 8 unknown genes were shown to be highly expressed in the livers of laying geese compared with prelaying geese. Vitellogenin I, apoVLDL-II, ethanolamine kinase, G-protein gamma-5 subunit, and leucyl-tRNA synthase were highly expressed in the livers of laying geese compared with that from the prelaying geese (P<0.05). The expression of these known genes suggests that their function in the liver of laying geese is primarily involved in lipid and lipoprotein metabolism. Several of these differentially expressed genes were found to be responsive to estrogen stimulation, confirming the involvement of these genes in the egg-laying function of the goose.

Measuring Force-Induced Dissociation Kinetics of Protein Complexes Using Single-Molecule Atomic Force Microscopy.

Proteins respond to mechanical force by undergoing conformational changes and altering the kinetics of their interactions. However, the biophysical relationship between mechanical force and the lifetime of protein complexes is not completely understood. In this chapter, we provide a step-by-step tutorial on characterizing the force-dependent regulation of protein interactions using in vitro and in vivo single-molecule force clamp measurements with an atomic force microscope (AFM). While we focus on the force-induced dissociation of E-cadherins, a critical cell-cell adhesion protein, the approaches described here can be readily adapted to study other protein complexes. We begin this chapter by providing a brief overview of theoretical models that describe force-dependent kinetics of biomolecular interactions. Next, we present step-by-step methods for measuring the response of single receptor-ligand bonds to tensile force in vitro. Finally, we describe methods for quantifying the mechanical response of single protein complexes on the surface of living cells. We describe general protocols for conducting such measurements, including sample preparation, AFM force clamp measurements, and data analysis. We also highlight critical limitations in current technologies and discuss solutions to these challenges.

The adaptive decision-making, risky decision, and decision-making style of Internet gaming disorder.

BACKGROUND: Persistent gaming, despite acknowledgment of its negative consequences, is a major criterion for individuals with Internet gaming disorder (IGD). This study evaluated the adaptive decision-making, risky decision, and decision-making style of individuals with IGD. METHODS: We recruited 87 individuals with IGD and 87 without IGD (matched controls). All participants underwent an interview based on the Diagnostic and Statistical Manual of Mental Disorders (5th Edition) diagnostic criteria for IGD and completed an adaptive decision-making task; the Preference for Intuition and Deliberation Scale, Chen Internet Addiction Scale, and Barratt Impulsivity Scale were also assessed on the basis of the information from the diagnostic interviews. RESULTS: The results demonstrated that the participants in both groups tend to make more risky choices in advantage trials where their expected value (EV) was more favorable than those of the riskless choice. The tendency to make a risky choice in advantage trials was stronger among IGD group than that among controls. Participants of both groups made more risky choices in the loss domain, a risky option to loss more versus sure loss option, than they did in the gain domain, a risky option to gain more versus sure gain. Furthermore, the participants with IGD made more risky choices in the gain domain than did the controls. Participants with IGD showed higher and lower preferences for intuitive and deliberative decision-making styles, respectively, than controls and their preferences for intuition and deliberation were positively and negatively associated with IGD severity, respectively. CONCLUSIONS: These results suggested that individuals with IGD have elevated EV sensitivity for decision-making. However, they demonstrated risky preferences in the gain domain and preferred an intuitive rather than deliberative decision-making style. This might explain why they continue Internet gaming despite negative consequences. Thus, therapists should focus more on decision-making styles and promote deliberative thinking processes to mitigate the long-term negative consequences of IGD.

The expression of pituitary gland genes in laying geese.

The purpose of this study was to detect differential expression of genes in the pituitary gland in laying geese by suppression subtractive hybridization (SSH). Pituitary glands from prelaying and laying geese were dissected for mRNA extraction. The cDNA from pituitary glands of prelaying geese was subtracted from the cDNA from the pituitary glands of laying geese (forward subtraction); the reverse subtraction was also performed. We screened 384 clones with possible differentially expressed gene fragments by differential screening. Sixty-five clones from the differential screening results were subjected to gene sequencing and further analysis. We found that at least 19 genes were highly expressed in the pituitary glands of laying geese compared with prelaying geese. Among these, 6 genes (including 4 novel genes) were confirmed by virtual Northern analysis. We found that prolactin and visinin-like protein were highly expressed in the pituitary glands of laying geese compared with prelaying geese (P < 0.05). Further investigation is needed to demonstrate specific functions of the novel genes discovered in the current study.

Regulation of low-density lipoprotein receptors and assessment of their functional role in Burkitt's lymphoma cells.

The status of low-density lipoprotein receptors (LDLR) in Daudi Burkitt's lymphoma (BL) cells was examined using a flow cytometric assay employing the fluorescent ligand DiI-LDL, and a radioligand-binding assay using [125I]LDL. The binding is concentration-and time-dependent; and is specific, as judged by its competitive displacement in the presence of unlabeled LDL, and inhibition by heparin, EGTA, and 4 degrees C incubation. The regulation of the receptor and its functional role were then explored. Our results suggest the following: (a) In sharp contrast to normal peripheral blood lymphocytes, the LDLR levels in BL cells are basally elevated when cultured in fetal bovine serum (FBS) medium. (b) In accord with normal peripheral blood lymphocytes, incubation in lipoprotein-deficient serum (LPDS) medium further up-regulates the level of the receptor in BL cells, and co-incubation with LDL or 25-hydroxycholesterol down-regulates the receptor level. The magnitude of the up-regulation is significantly smaller than in normal peripheral blood lymphocytes. (c) Northern blots using a plasmid-DNA probe for LDLR mRNA point to a similar pattern for message regulation as is observed in direct binding studies. (d) Although the LDLR level is constitutively high in BL cells, availability of LDL, unlike transferrin, is not a growth requirement since incubation of cells in LPDS medium does not prevent proliferation of these cells. (e) In contrast to anti-transferrin receptor antibody which results in apoptosis upon binding, anti-LDLR antibody does not inhibit growth or induce apoptosis. Our results suggest LDLR is expressed at a significantly higher level in BL cells than in normal peripheral blood lymphocytes. Although up-regulation and down-regulation of LDLR are observed, this applies only to a small population of LDLR. The bulk receptor population is significantly resistant to down-regulation. Furthermore, notable differences in the functional role of the LDLR are found relative to the transferrin receptor which is also up-regulated in the BL cells.

Cloning and expression of the genes associated with lipid metabolism in Tsaiya ducks.

Sterol regulatory element binding protein 1 (SREBP1) drives the expression of several lipogenic genes, whereas SREBP2 dictates the expression of every gene involved in cholesterolgenesis in mammals. In the current study, we cloned the cDNA fragments for SREBP1, SREBP2, fatty acid synthase (FAS), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), and very low density apolipoprotein-II (apoVLDL-II), the genes associated with lipid metabolism. Fifteen ducks immediately before the first egg was laid (18 wk old) and 15 ducks from the same population at an egg production rate of 80% were killed. Total RNA was extracted from liver and used to amplify the targeted genes by reverse transcription-PCR and screening of a cDNA library. The sequence data showed that Tsaiya duck SREBP1, SREBP2, FAS, and HMG-CoA reductase were highly homologous to that of chicken. Tsaiya duck SREBP1 mRNA was expressed in adipose tissue, cardiac muscle, skeletal muscle, liver, and ovary. The SREBP2 mRNA concentration was highest in liver and ovary. Concentrations of FAS and HMG-CoA reductase mRNA were high in liver and lower in other tissues. The apoVLDL-II mRNA was specifically expressed in the liver. The differences between mRNA concentrations of SREBP1, SREBP2, and FAS in the livers of laying and prelay ducks were not significant. However, the concentrations of hepatic HMG-CoA reductase and apoVLDL-II mRNA were higher in the laying ducks than in prelay ducks. Therefore, laying may affect particular aspects of lipid metabolism, especially biochemical pathways that involved apoVLDL-II and HMG-CoA reductase.

Estrogen and diphosphonate treatment provide long-term protection against osteopenia in ovariectomized rats.

The goal of this study is to determine whether the previously observed, short-term protective effect of estrogen and diphosphonate compounds against osteopenia in ovariectomized (OVX) rats can be maintained for an entire year. Sham-operated control and OVX rats were treated intermittently with vehicle alone, estrogen, or the diphosphonate compounds etidronate disodium (EHDP) and risedronate (NE-58095) for 360 days after surgery. Their proximal tibiae and first lumbar vertebrae were processed undecalcified for quantitative bone histomorphometry. Both skeletal sites in vehicle-treated OVX rats were characterized by decreased cancellous bone volume and increases in most cellular and fluorochrome-based indices of bone formation and resorption. Treatment of OVX rats with estrogen or diphosphonate compounds depressed bone turnover and provided nearly complete protection against cancellous bone loss. Long-term EHDP treatment induced a moderate mineralization defect, as indicated by increased absolute osteoid volume and a high proportion of osteoid surfaces devoid of adjacent osteoblasts. In contrast, NE-58095 had minimal effects on bone mineralization. These findings indicate that diphosphonate compounds and estrogen provide long-term protection against tibial and vertebral osteopenia in OVX rats. They further indicate that diphosphonate compounds merit consideration as an alternative to estrogen for the prevention of postmenopausal bone loss.

Parathyroid hormone is more effective than estrogen or bisphosphonates for restoration of lost bone mass in ovariectomized rats.

The study was designed to compare the therapeutic efficacy of estrogen, the bisphosphonate risedronate (NE-58095), and PTH for restoration of lost bone mass in osteopenic, ovariectomized (OVX) rats. In addition, the skeletal effects of these single treatments were compared to those of concurrent treatments with PTH + estrogen or PTH + NE-58095. OVX rats were untreated for the first 4 weeks postovariectomy to allow for the development of moderate tibial osteopenia. These animals were then subjected to the various treatments for periods of 5, 10, and 15 weeks. Their proximal tibiae were processed undecalcified for quantitative bone histomorphometry. Treatment of osteopenic OVX rats with estrogen or NE-58095 alone depressed bone turnover and prevented additional cancellous bone loss from occurring during the treatment period. However, these therapeutic agents failed to restore lost bone in OVX rats to control levels. In contrast, OVX rats treated with PTH alone exhibited a marked stimulation of bone formation which resulted in augmentation of cancellous bone mass to a level 2-fold greater than that of vehicle-treated control rats. Concurrent treatment of OVX rats with PTH + estrogen as well as PTH + NE-58095 also effectively reversed cancellous osteopenia in OVX rats, but did not appear to be more beneficial to the estrogen-deplete skeleton than treatment with PTH alone. The results indicate that PTH is a powerful stimulator of bone formation and completely restores lost cancellous bone in osteopenic OVX rats. Furthermore, the bone anabolic effects of PTH are much more pronounced than those of estrogen or bisphosphonates. These findings in an animal model of estrogen depletion provide support for PTH as a potentially effective treatment for oophorectomized and postmenopausal women with established osteoporosis.

Skeletal effects of calcitonin in ovariectomized rats.

Although calcitonin (CT) has been shown to be effective for the prevention of bone loss in early postmenopausal women, the skeletal effects of the hormone specifically during the early stages of estrogen deficiency have not been characterized histomorphometrically to date. The current study involves use of the ovariectomized (OVX) rat as an animal model for early postmenopausal bone loss to perform such a histomorphometric analysis. One group of OVX rats was injected sc with salmon CT on alternate days for a 6-week period. Additional groups of OVX and sham-operated control rats were treated with vehicle alone. In comparison to control rats, the proximal tibia of vehicle-treated OVX rats were characterized by a 3-fold decrease in cancellous bone volume and significant increases in osteoblast surface (+200%), osteoclast surface (+143%), mineralizing surface (+111%), mineral apposition rate (+36%), bone formation rate (+181%), and longitudinal bone growth (+38%). In contrast, treatment of OVX rats with CT normalized tibial cancellous bone volume and significantly decreased all of the above cellular- and fluorochrome-based indices of bone turnover to near control levels. The results indicate that CT treatment depresses bone turnover and prevents the development of osteopenia in OVX rats. These findings are consistent with the bone protective effect of CT in early postmenopausal women and further support the OVX rat as an animal model for the preclinical evaluation of prophylactic treatments for postmenopausal bone loss.

Insulin increases intracellular magnesium transport in human platelets.

Evidence suggests that magnesium (Mg) deficiency may play a key role in cardiovascular disease. In particular, Mg deficiency may lead to a potentiation of platelet aggregation. However, the factor(s) regulating intracellular-free Mg concentration ([Mg2+]i) in platelets is not known. We studied the effects of insulin on the changes of [Mg2+]i in human platelets. Preincubation of hirudinized platelet-rich plasma with insulin had a dose- and time-dependent effect on the increase of [Mg2+]i measured in Mag-fura-2-loaded cells with a fluorescence spectrophotometer. The maximal effect was achieved by incubation with 200 microU/mL insulin for 30 min. [Mg2+]i increased from the basal value of 266 +/- 23 mumol to 355 +/- 46 (SD, P < 0.001). In the presence of an antiinsulin receptor monoclonal antibody the effect of insulin was abolished suggesting that the Mg transport mechanism was an insulin-receptor mediated process. Furthermore, the insulin-stimulated Mg transport was inhibited by the addition of chelating agent ethylenediaminete-traacetate while the receptor binding was not altered. These findings suggest that insulin can translocate Mg from the extracellular space. Insulin alone had no effect on the changes of intracellular calcium (Ca) concentration using Ca-Fura-2 as a probe. In addition, glucose (5 mg/mL) was not effective in altering either the Mg or Ca concentration. Insulin (100 microU/mL) decreased thrombin-induced platelet aggregation (washed platelets resuspended in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-Tyrode buffer). Similarly, the production of the proaggregatory eicosanoids thromboxane B2 was inhibited by insulin from 16 +/- 1 ng/10(8) platelets to 13 +/- 2 (P < 0.05). The results suggest that insulin, through the interactions with its receptors may be a key factor regulating, Mg transport in human platelets.

Anabolic effects of parathyroid hormone on cortical bone in ovariectomized rats.

The purpose of this study was to determine whether PTH has anabolic effects on cortical bone in ovariectomized (OVX) rats similar to those previously observed in cancellous bone. Groups of OVX rats were untreated for the first 4 weeks postovariectomy followed by treatment for 5-, 10-, or 15-week periods with vehicle, estrogen, the bisphosphonate rise-dronate (NE-58095), PTH, PTH+estrogen or PTH+NE-58095. A group of sham-operated control rats was treated with vehicle alone. Cross sections of the tibial diaphysis immediately proximal to the tibiofibular junction were subjected to quantitative bone histomorphometry. In comparison to vehicle-treated control rats, the following structural changes were detected in cortical bone of OVX rats after 15 weeks of vehicle treatment: increased cortical bone tissue area, increased cortical bone area, and a trend for increased bone marrow area. Indices of periosteal bone formation were increased in vehicle-treated OVX rats at 5 weeks, but declined to control levels at the later time points. In addition, vehicle-treated OVX rats exhibited a nonsignificant trend for increased endocortical bone formation relative to vehicle-treated control rats throughout most of the study. Estrogen, but not NE-58095, prevented most of the changes in cortical bone structure associated with ovariectomy. In comparison to vehicle treatment of OVX rats, estrogen decreased both periosteal and endocortical bone formation whereas NE-58095 decreased endocortical, but not periosteal, bone formation. Treatment of OVX rats with PTH alone increased cortical bone area and width as well as decreased bone marrow area compared to vehicle-treated OVX rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow cytometric evaluation of LDL receptors using DiI-LDL uptake and its application to B and T lymphocytic cell lines.

Low density lipoprotein receptors (LDL-R) on Daudi Burkitt's lymphoma (BL) cells were assessed using fluorescent DiI (3,3'-dioctadecylindocarbocyanine iodide)-LDL and flow cytometric analyses. Receptor-specific binding of DiI-LDL is followed by internalization of the bound complex and lysosomal hydrolysis of the ligand. Increase in the fluorescence intensity per cell is hence used as a measure of DiI-LDL uptake and, implicitly, as an indication of LDL-R presence. Our results show that uptake was observed in > 98% of the Daudi cells, and the level of uptake was significant and clearly distinguishable from autofluorescence, suggesting that: (a) this assay is comparable to the iodinated LDL uptake assay, although the ED50 values for the ligands are different; (b) this assay is comparable to the flow-cytometric detection of LDL-R using a commercial antibody directed against the receptor itself, and superior to a similar assay based on an antibody directed against membrane-bound LDL; (c) LDL uptake could be monitored along with transferrin uptake, suggesting that multiple endocytic receptor activities can be concurrently studied; (d) DiI-LDL uptake can be examined along with fluorescein-conjugated anti-CD10, -CD19, and -CD71, with little cross-interference, offering the added advantage that endocytic uptake and phenotyping can be simultaneously monitored; (e) the expression of LDL-R is intrinsically elevated in diverse cell lines such as Daudi, Raji, Ramos, Jurkat, and WIL2-NS, but not in normal lymphocytes. Our results therefore indicate that flow cytometric analysis of DiI-LDL uptake has potentially useful applications in the detection and study of endocytic receptor LDL-R in B and T lymphocytic cell lines.

Effect of extracellular magnesium on platelet activation and intracellular calcium mobilization.

A dose-dependent effect of magnesium on the inhibition of platelet aggregation and release of ATP from dense granules was observed in human platelets (in whole blood, platelet-rich plasma, or washed platelets) against various aggregation agents (ADP, U46619, collagen, or thrombin). The synthesis and release of the proaggregatory cyclooxygenase (CO) and lipoxygenase (LO) products, thromboxane A2 (TXA2) and 12-hydroxyeicosatetraenoic acid (12-HETE), respectively, in platelets were also inhibited by Mg in a dose-dependent manner (IC50 4 to 6 mmol/L). These Mg-mediated activities were further enhanced when platelets were preincubated with insulin (100 microU/mL). The effect of extracellular Mg on the change of intracellular calcium concentration ([Ca2+]i) was assessed using Fura-2/AM loaded cells in the presence or absence of extracellular Ca. Thrombin-stimulated influx of Ca ions decreased from 194 +/- 30 nmol/L to 156 +/- 21 nmol/L in the presence of 5 mmol/L Mg and to 111 +/- 16 nmol/L in 10 mmol/L Mg. However, the intracellular Ca release (as determined in the presence of 5 mmol/L EGTA) was not affected by Mg. The intracellular Ca-dependent protein kinase C and myosin light chain kinase activities on the phosphorylation of endogenous p47 and p20 proteins studied after 2 min of thrombin addition decreased only 10 to 25% in the presence of 5 to 10 mmol/L Mg. Similar results were obtained when EGTA was added prior to the initiation of protein phosphorylation. We conclude that Mg can dose dependently inhibit a wide variety of agonists on platelet aggregation. Furthermore, insulin can potentiate the inhibitory effects of Mg on platelet activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Highly functionalized Bicyclo[2.2.2]octenone-fused [60]fullerenes from masked o-benzoquinones and C(60).

[reaction: see text] The Diels-Alder reactions of masked o-benzoquinones (MOBs) with [60]fullerene, affording novel and highly functionalized bicyclo[2.2. 2]octenone-fused [60]fullerene derivatives, are described.

Release of different fractions of epidermal growth factor from human platelets in vitro: preferential release of 140 kDa fraction.

Platelet-rich plasma in acidic-citrate-dextrose anticoagulant was kept for 5 days in an oxygen-permeable bag at 22 degrees C in an incubator/rotator. Platelet count remained stable throughout the experiment. On days 0, 3 and 5, aliquots were removed; platelets were isolated by centrifugation at 22 degrees C, 1500 g for 20 min, reconstituted to the original volume with PBS buffer, and the contents of alpha-granules were released by repeated freezing and thawing. Epidermal growth factor (EGF) and beta-thromboglobulin (beta-TG) in the platelet-poor plasma and platelet lysates were determined by radioimmunoassays. Results indicated that in platelet-free plasma, both total EGF and beta-TG increased 3-5-fold after 5 days; this amount represented 10-20% of the factors stored in the platelets. Correspondingly, the EGF and beta-TG contents of the platelet lysates exhibited accompanying decreases. HPLC fractionation showed that the main EGF fraction which progressively decreased in the lysates and increased in plasma had a molecular mass of 140 kDa. The contents of the 67 kDa and 6 kDa fractions did not change substantially. We conclude that under these conditions, the 140 kDa fraction was released preferentially. In view of these and previous experiments, it seems likely that different organs contribute to plasma EGF fractions.

Intestinal absorption studies on peptide mimetic alpha-methyldopa prodrugs.

Two dipeptide mimetic prodrugs, 1 and 2, and two tripeptide mimetic prodrugs, 3 and 4, of L-alpha-methyldopa were evaluated for intestinal absorption by in-situ single pass rat jejunal perfusion studies and by in-vitro uptake experiments in brush-border membrane vesicles (BBMVs) prepared from rat intestine. In the perfusion studies, compound 1 demonstrated a 3.5-fold increase in permeability (Pm* = 2.27) as compared with that of alpha-methyldopa (Pm* = 0.65), indicating that this prodrug was better absorbed in the intestine than its parent drug. Other prodrugs showed no significant improvement in intestinal permeability. The results correlated with the results of BBMV uptake studies. In the presence of an inward proton gradient, compound 1 showed Michaelis-Menton saturable kinetics of BBMV uptake with a low value of K(m) (0.06 +/- 0.13 mM) and a high value of Vmax/K(m)(36.38 nmol (mg protein)-1/30s mM-1) at a low concentration range and a linear uptake at high concentrations with Kd = 0.14 +/- 0.02 mM. Compounds 2 and 3 were mainly taken up in BBMVs via passive diffusion. Compound 4 was taken up in BBMVs basically via the carrier-mediated transport system, while the rate of uptake was much lower than that of compound 1. The uptake of compounds 1 and 4 was significantly inhibited by dipeptides L-Gly-L-Pro and L-Gly-L-Phe, and cephradine, a beta-lactam known to be transported via the dipeptide carrier system, indicating that both compounds were taken up in BBMVs via the H(+)-coupled dipeptide-mediated transport system. In contrast to the complicated uptake profile of alpha-methyldopa, the higher rate of BBMV uptake with less variation demonstrated on compound 1 suggested that the attached nonessential amino acid moiety, D-phenylglycine, is a feasible delivery tool in carrying the parent drug through the intestine.