Lien Nguyen - Academia.edu (original) (raw)

Papers by Lien Nguyen

Myotonic dystrophy is a debilitating genetic disorder which currently does not have a therapeutic... more Myotonic dystrophy is a debilitating genetic disorder which currently does not have a therapeutic treatment. It is understood that CTG expansions lead to formation of stable poly(CUG) mRNA which mislocalize splicing factors such as MBNL1 and lead to missplicing in the cell. One therapeutic strategy is to target such mutant mRNA with small molecules to prevent the sequestration of splicing factors which will prevent the multiple missplicing events in the cell and reverse the symptoms of the disorder. In the following thesis I describe my work in identifying small molecule inhibitors of the RNA-protein complex through the optimization of gel shift assays for characterization of rationally-designed compounds and the development of a fluorescence anisotropy assay for a high-throughput screening of the NCI Diversity Set III compound library. From such studies I was able to identify several lead compounds that are successful at inhibiting the pathological nuclear aggregation.

Proceedings of The National Academy of Sciences, 1998

The regulation of dietary cholesterol absorption was examined in C57BL/6 and transgenic mice with... more The regulation of dietary cholesterol absorption was examined in C57BL/6 and transgenic mice with liver overexpression of the scavenger receptor BI (SR-BI Tg). In C57BL/6 animals, feeding 0.02 to 1% (wt/wt) dietary cholesterol resulted in a dose-dependent decrease in the percentage of dietary cholesterol absorbed. A plot of total daily mass of dietary cholesterol absorbed versus the percentage by weight of cholesterol in the diet yielded a curve suggesting a saturable process with a Km of 0.4% (wt/wt) and a Vmax of 0.65 mg cholesterol/g body weight per day. Dietary cholesterol suppressed hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity, stimulated cholesterol 7alpha-hydroxylase activity, and enhanced fecal excretion of bile acids, but none of these changes correlated with the percentage of dietary cholesterol absorption. Dietary cholesterol also caused an increase in biliary cholesterol concentration, and in this case the concentration of biliary cholesterol was strongly and inversely correlated with the percentage dietary cholesterol absorption (r = -0.63, P &amp;amp;amp;lt; 0.0001). Biliary cholesterol concentration was also directly correlated with daily cholesterol intake, dietary cholesterol mass absorption, and liver cholesterol ester content. Transgene-induced overexpression of SR-BI resulted in a stimulation of excretion of cholesterol into the bile and suppressed percentage dietary cholesterol absorption. Furthermore, biliary cholesterol levels in SR-BI Tg mice were strongly and inversely correlated with the percentage of dietary cholesterol absorbed (r = -0.99, P &amp;amp;amp;lt; 0.0008). In summary, these results suggest that the excretion of cholesterol into the bile plays an important role in regulating the percentage absorption of dietary cholesterol.

We investigated the effects of lovastatin, cholestyramine, and dietary sterol restriction on chol... more We investigated the effects of lovastatin, cholestyramine, and dietary sterol restriction on cholesterol synthesis and low density lipoprotein receptor function in freshly isolated mononuclear leukocytes from two unrelated sitosterolemic families. Total plasma sterol concentrations were elevated in the two homozygous sitosterolemic subjects (343 and 301 vs. 185 mg/dl in controls) and contained increased amounts of plant sterols and 5a-saturated stanols (20% and 8% vs. less than 1% in con-

Hepatology, 1995

We eg~mined the effects of feeding deoxycholic acid (1% and 0.4% of diet), alone and in combinati... more We eg~mined the effects of feeding deoxycholic acid (1% and 0.4% of diet), alone and in combination with ursodeoxycholic acid, on serum and biliary bile acid concentrations, hepatic morphology, and the activities and steady-state messenger RNA (mRNA) levels of HMG-CoA reductase and cholesterol 7a-hydroxylase in the rat. Feeding 1% deoxycholic acid increased serum bile acid concentrations (cholestasis), produced portal triad in-flamrn~tion, bile duct proliferation, and severe hepatocyte necrosis with nuclear pleomorphism. Hepatic clamage was prevented when ursodeoxycholic acid (1%) was combined with the deoxycholic acid (1%), or when deoxycholic acid intake was reduced to 0.4%. HMG-CoA reductase and cholesterol 7a-hydroxylase activities were markedly inhibited (-56% and -55%, respectively) with either 1% or 0.4% deoxycholic acid. Ursodeoxycholic acid alone produced an insignificant decline in HMG-CoA reductase and cholesterol 7a-hydroxylase activities, and when combined with 1% deoxycholic acid did not lessen the inhibitory effect of the latter. Steadystate mRNA levels increased 20-fold for HMG-CoA reductase and 53-fold for cholesterol 7a-hydroxylase in rats fed 1% deoxycholic acid. In contrast, 0.4% deoxycholic acid decreased HMG-CoA reductase mRNA levels 76%, and cholesterol 7a-hydroxylase mRNA levels 82%. Ursodeoxycholic acid alone did not affect HMG-CoA reductase or cholesterol 7a-hydroxylase steady-state mRNA levels. Steady-state mRNA levels and activities of sterol 27-hydroxylase, a key enzyme in the alternative acidic pathway of bile acid synthesis, did not change with either high or low doses of deoxycholic acid. In conclusion, 1% deoxycholic acid induced hepatocyte destruction and regeneration associated with increased mRNA levels for Abbreviations: mRNA, messenger RNA; SDS, sodium dodecyl sulfate. From the 1Department of Medicine and the Sammy Davis Jr.

Journal of Clinical Investigation, 1999

We investigated the effects of cholesterol, cholestyramine, and taurocholate feeding on steady st... more We investigated the effects of cholesterol, cholestyramine, and taurocholate feeding on steady state specific activities and mRNA levels of hepatic 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase and cholesterol 7a-hydroxylase in the rat. Interruption of the enterohepatic circulation of bile acids (cholestyramine feeding) increased total HMG-CoA reductase activity 5-fold. Cholesterol and taurocholate administration suppressed total microsomal HMG-CoA reductase activities 87 % and 65%, respectively. HMG-CoA reductase mRNA levels increased 3-fold with cholestyramine, did not decrease significantly with cholesterol feeding, but were markedly decreased after taurocholate treatment. Cholesterol 7a-hydroxylase activity increased 4-fold with cholestyramine and 29% during cholesterol feeding, but decreased 64% with taurocholate. Cholesterol 7ahydroxylase mRNA levels rose 150% and 50% with cholestyramine and cholesterol feeding, respectively, but decreased 73% with taurocholate. The administration of cholesterol together with taurocholate prevented the decline in cholesterol 7ahydroxylase mRNA levels, but inhibition of enzyme activity persisted ( -76%). Hepatic microsomal cholesterol concentrations increased 2-fold with cholesterol feeding but did not change with taurocholate or cholestyramine treatment.

Metabolism-clinical and Experimental, 1999

The regulation of the classic and alternative bile acid synthetic pathways by key hepatic enzyme ... more The regulation of the classic and alternative bile acid synthetic pathways by key hepatic enzyme activities (microsomal cholesterol 7oL-hydroxylase and mitochondrial sterol 27-hydroxylase, respectively) was examined in bile acid depletion and replacement and cholesterol-feeding experiments with rats, guinea pigs, and rabbits. The bile acid pool was depleted by creating a bile fistula (BF) and collecting bile for 2 to 5 days, and it was replaced by intraduodenal infusion of the major biliary bile acids (taurocholic acid [TCA], glycochenodeoxycholic acid [GCDCA], and glycocholic acid [GCA] in the rat, guinea pig, and rabbit, respectively) at rates equivalent to the measured hepatic flux of the bile acids. To study the effects of cholesterol, the animals were fed for 7 days on a basal diet with and without 2% cholesterol. Cholesterol 7~-hydroxylase and sterol 27-hydroxylase activities, measured by isotope incorporation assays, were related to bile acid output and composition and hepatic cholesterol concentrations. Intraduodenal infusion of bile acids increased the output of the tested bile acids, but did not significantly change hepatic cholesterol concentrations and had no effect on sterol 27-hydroxylase activity. Neither bile acid depletion nor replacement affected sterol 27-hydroxylase activity when three different substrates (cholesterol, 51~-cholestane-3~,7~-diol, and 51~-cholestane-3~,7~,12~-triol) were tested. In contrast, feeding 2% cholesterol increased hepatic cholesterol concentrations in rats, guinea pigs, and rabbits threefold, twofold, and eightfold, respectively, and increased hepatic mitochondrial stero127-hydroxylase activity (conversion of cholesterol to 27-hydroxycholesterol) in all three animal models. The stimulation and feedback inhibition of cholesterol 7~-hydroxylase activity by bile acid depletion and replacement were observed in all three animal models, whereas the effect of cholesterol feeding was species-dependent (cholesterol 7~-hydroxylase activity increased in the rat, did not change in the guinea pig, and was inhibited in the rabbit). Thus, in contrast to sterol 27-hydroxylase, which was upregulated by cholesterol but not affected by bile acid depletion and replacement in all three animal models, cholesterol 7~-hydroxylase activity was controlled consistently and inversely by the hepatic flux of bile acids, but was species-dependent in its response to a 1-week feeding with 2% cholesterol.

Metabolism-clinical and Experimental, 1988

The hypothesis that abnormal low density lipoprotein (LDL) sterol content and composition in sito... more The hypothesis that abnormal low density lipoprotein (LDL) sterol content and composition in sitosterolemia with xanthomatosis affects LDL uptake and/or degradation was tested. Monocytes and lymphocytes from three patients and 12 age- and sex-matched controls were incubated at 37 degrees C in lipid-free medium with 125I-labeled LDL prepared from sitosterolemic patients (LDLs) and controls (LDLn) in the presence or absence of excess unlabeled lipoproteins. Normal monocytes and lymphocytes took up and degraded LDLs 13% to 30% less than LDLn (P less than .05). Sitosterolemic monocytes and lymphocytes degraded LDLn 13% and LDLs 67% more actively than control cells (P less than .05). Sitosterolemic monocytes contained three times more sterols and stanols than controls (P less than .01), of which 12% were plant sterols and 2% were 5 alpha-saturated stanols. In one patient, stimulating bile acid synthesis by ileal bypass surgery reduced plasma and monocyte sterol and stanol concentrations about 60%, and was associated with a 40% to 50% increase in LDLn and LDLs receptor-mediated degradation. The decreased uptake and degradation of LDLs relative to LDLn by normal cells suggest that abnormal plant sterols in LDLs may reduce its affinity for the native LDL receptor. Increased receptor-mediated uptake and degradation of LDLs by sitosterolemic cells in the presence of high cellular sterol content may result from failure of the sitosterolemic cells to down-regulate LDL receptor synthesis. Ileal bypass surgery increased cellular LDL receptor activity, reduced plasma and cellular sterol concentrations, and may diminish the risk of premature atherosclerosis in sitosterolemia.

Sitosterolemia is a recessively inherited disorder characterized by abnormally increased plasma a... more Sitosterolemia is a recessively inherited disorder characterized by abnormally increased plasma and tissue plant sterol concentrations. Patients have markedly reduced whole body cholesterol biosynthesis associated with suppressed hepatic, ileal, and mononuclear leukocyte 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the ratecontrolling enzyme in cholesterol biosynthetic pathway, coupled with significantly increased low density lipoprotein (LDL) receptor expression. To investigate the mechanism of downregulated cholesterol biosynthesis, we assayed several other key enzymes in the cholesterol biosynthetic pathway including acetoacetyl-CoA thiolase, HMG-CoA synthase, squalene synthase, and 7-dehydrocholesterol ⌬ 7 -reductase activities in liver and freshly isolated mononuclear leukocytes from four sitosterolemic patients and 19 controls. Hepatic acetoacetyl-CoA thiolase, HMG-CoA synthase, reductase, and squalene synthase activities were significantly decreased ( P Ͻ 0.05) Ϫ 39%, Ϫ 54%, Ϫ 76%, and Ϫ 57%, respectively, and 7-dehydrocholesterol ⌬ 7 -reductase activity tended to be lower ( Ϫ 35%) in the sitosterolemic compared with control subjects. The reduced HMG-CoA synthase, reductase, and squalene synthase activities were also found in mononuclear leukocytes from a sitosterolemic patient. Thus, reduced cholesterol synthesis is caused not only by decreased HMG-CoA reductase but also by the coordinate down-regulation of entire pathway of cholesterol biosynthesis.

Faseb Journal, 2001

Apolipoprotein E (apo E) deficiency (or its abnormalities in humans) is associated with a series ... more Apolipoprotein E (apo E) deficiency (or its abnormalities in humans) is associated with a series of pathological conditions including dyslipidemia, atherosclerosis, Alzheimer's disease, and shorter life span. The purpose of this study was to characterize these conditions in apo E-deficient C57BL/6J mice and relate them to human disorders. Deletion of apo E gene in mice is associated with changes in lipoprotein metabolism [plasma total cholesterol (TC) (>؉400%), HDL cholesterol (؊80%), HDL/TC, and HDL/LDL ratios (؊93% and ؊96%, respectively), esterification rate in apo B-depleted plasma (؉100%), plasma triglyceride (؉200%), hepatic HMG-CoA reductase activity (؊50%), hepatic cholesterol content (؉30%)], decreased plasma homocyst(e)ine and glucose levels, and severe atherosclerosis and cutaneous xanthomatosis. Hepatic and lipoprotein lipase activities, hepatic LDL receptor function, and organ antioxidant capacity remain unchanged. Several histological/immunohistological stainings failed to detect potential markers for neurodegenerative disease in the brain of 37-wk-old male apo E-KO mice. Apo E-KO mice may have normal growth and development, but advanced atherosclerosis and xanthomatosis may indirectly reduce their life span. Apo E plays a crucial role in regulation of lipid metabolism and atherogenesis without affecting lipase activities, endogenous antioxidant capacity, or appearance of neurodegenerative markers in 37-wk-old male mice.-Moghadasian, M. H., McManus, B. M., Nguyen, L. B., Shefer, S., Nadji, M., Godin, D. V., Green, T. J., Hill, J., Yang, Y., Scudamore, C. H., Frohlich, J. J. Pathophysiology of apolipoprotein E deficiency in mice: relevance to apo E-related disorders in humans. FASEB J. 15, 2623-2630 (2001) 2623 0892-6638/01/0015-2623 © FASEB

Proceedings of The National Academy of Sciences, 2000

The present study examined the role of apolipoprotein E (apoE) in the regulation of dietary chole... more The present study examined the role of apolipoprotein E (apoE) in the regulation of dietary cholesterol absorption and biliary cholesterol excretion. Increasing dietary cholesterol from 0.02% to 0.5% in C57BL͞6 wild-type mice decreased the percentage of dietary cholesterol that is absorbed by 25%, and this decrease was associated with a 2-fold increase in gallbladder biliary cholesterol concentration. In contrast, increasing dietary cholesterol from 0.02% to 0.5% in C57BL͞6 apoE knockout mice produced no significant suppression of the percentage dietary cholesterol absorption and increased gallbladder biliary cholesterol concentration only 16%. Whereas in wild-type mice, the increase in dietary cholesterol increased the hepatic excretion of biliary cholesterol 4-fold, there was only a 2-fold increase in apoE knockout mice. On both the low-and the high-cholesterol diets, whole liver and isolated hepatocyte cholesterol content was higher in the apoE knockout mice. These results suggest that, in response to dietary cholesterol, apoE may play a critical role in decreasing the percentage absorption of dietary cholesterol and increasing biliary cholesterol excretion. These observations suggest a mechanism whereby the absence of apoE contributes to the propensity for tissue cholesterol deposition and accelerated atherogenesis in apoE knockout mice.

Metabolism-clinical and Experimental, 1990

We investigated the mechanism for reduced cholesterol biosynthesis in sitosterolemia with xanthom... more We investigated the mechanism for reduced cholesterol biosynthesis in sitosterolemia with xanthomatosis. The conversion of acetate to cholesterol and total and active hydroxymethylglutaryl (HMGI coenzyme A (CoA) reductase activities, enzyme protein mass, and catalytic efficiency were related to low-density lipoprotein (LDL) receptor function in freshly isolated mononuclear leukocytes collected at 9 AM after a 12-hour fast from two affected sisters and 12 control subjects. Active HMG-CoA reductase activity was determined in mononuclear leukocyte microsomes prepared and assayed in the presence of sodium fluoride, while total HMG-CoA reductase activity was determined in the absence of the phosphatase inhibitor. Enzyme protein was assayed using rabbit polyclonal anti-rat liver microsomal HMG-CoA reductase serum. The rates at which [?Z]acetate was transformed to cholesterol by sitosterolemic mononuclear leukocytes were decreased 29% and 41%. respectively, compared with the mean value for monunuclear leukocytes from 12 control subjects. Similarly, total HMG-CoA reductase activities were 71% and 66% lower in sitosterolemic mononuclear leukocyte microsomes and were associated with 62% and 65% less enzyme protein than the mean for the control microsomal preparations. This marked decrease in HMG-CoA reductase protein mass in sitosterolemic microsomes was partially compensated for by an increase in the proportion of active enzyme. Sitosterolemic plasma and mononuclear leukocyte cholesterol concentrations were not significantly different from control values, although total sterol levels were increased about 20% because of abundant plant sterols. In contrast, receptor-mediated LDL degradation by sitosterolemic mononuclear leukocytes was increased 50% over control. Our findings demonstrate reduced cholesterol formation in sitosterolemic mononuclear leukocytes that results from a deficiency of HMG-CoA reductase, the rate-controlling

We investigated the effect of ileal bile acid transport on the regulation of classic and alternat... more We investigated the effect of ileal bile acid transport on the regulation of classic and alternative bile acid synthesis in cholesterol-fed rats and rabbits. Bile acid pool sizes, fecal bile acid outputs (synthesis rates), and the activities of cholesterol 7 ␣ -hydroxylase (classic bile acid synthesis) and cholesterol 27-hydroxylase (alternative bile acid synthesis) were related to ileal bile acid transporter expression (ileal apical sodium-dependent bile acid transporter, ASBT). Plasma cholesterol levels rose 2.1-times in rats (98 ؎ 19 mg/dl) and 31-times (986 ؎ 188 mg/dl) in rabbits. The bile acid pool size remained constant (55 Ϯ 17 mg vs. 61 ؎ 18 mg) in rats but doubled (254 ؎ 46 to 533 ؎ 53 mg) in rabbits. ASBT protein expression did not change in rats but rose 31% ( P Ͻ 0.05) in rabbits. Fecal bile acid outputs that reflected bile acid synthesis increased 2-and 2.4-times ( P Ͻ 0.05) in cholesterol-fed rats and rabbits, respectively. Cholesterol 7 ␣ -hydroxylase activity rose 33% (24 ؎ 2.4 vs. 18 ؎ 1.6 pmol/mg/min, P Ͻ 0.01) and mRNA levels increased 50% ( P Ͻ 0.01) in rats but decreased 68% and 79%, respectively, in cholesterol-fed rabbits. Cholesterol 27-hydroxylase activity remained unchanged in rats but rose 62% ( P Ͻ 0.05) in rabbits. Classic bile acid synthesis (cholesterol 7 ␣hydroxylase) was inhibited in rabbits because an enlarged bile acid pool developed from enhanced ileal bile acid transport. In contrast, in rats, cholesterol 7 ␣ -hydroxylase was stimulated but the bile acid pool did not enlarge because ASBT did not change.

Journal of Neuroscience Research, 2000

Previous reports have suggested that elevated levels of phenylalanine inhibit cholesterol synthes... more Previous reports have suggested that elevated levels of phenylalanine inhibit cholesterol synthesis. The goals of this study were to investigate if perturbations in cholesterol synthesis exist in the PAH(enu2) genetic mouse model for phenylketonuria (PKU), and if so, initiate studies determining if they might underlie the white matter pathology that exists in PKU forebrain. Gross sections and electron microscopy showed that select tracts were hypomyelinated in adult PKU mouse forebrain but not hindbrain. The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the rate controlling enzyme in the cholesterol biosynthetic pathway, was examined in isolated microsomes from forebrain, hindbrain, and liver to assess if perturbations in cholesterol biosynthesis were occurring. HMGR activity was normal in unaffected PKU hindbrain and was increased 2-4-fold in PKU liver compared to control. HMGR activity in the forebrain, however, was decreased by 30%. Because normal numbers of MBP-expressing glia (oligodendrocytes) were present, but the number of glia expressing HMGR was reduced by 40% in the hypomyelinated tracts, the decreased HMGR activity seemed to result from a down-regulation of HMGR expression in affected oligodendrocytes. Exposure of an oligodendrocyte-like glioma cell line to physiologically relevant elevated levels of Phe resulted in a 30% decrease in cholesterol synthesis, a 28% decrease in microsomal HMGR activity, and a 28% decrease in HMGR protein levels. Measurement of HMGR activity after addition of exogenous Phe to control brain microsomes revealed that Phe is a noncompetitive inhibitor of HMGR; physiologically relevant elevated levels of exogenous Phe inhibited HMGR activity by 30%. Taken together, these data suggest that HMGR is moderately inhibited in the PKU mouse. Unlike other cell types in the body, a subset of oligodendrocytes in the forebrain seems to be unable to overcome this inhibition. We speculate that this may be the cause of the observed pathology in PKU brain.

Ophthalmology, 2002

To evaluate the safety and efficiency of the Artisan iris-supported phakic intraocular lens (Opht... more To evaluate the safety and efficiency of the Artisan iris-supported phakic intraocular lens (Ophtec BV, Groningen, Netherlands) for the correction of high myopia. Nonrandomized, prospective, multicenter trial conducted under a United States Food and Drug Administration (FDA) Investigational Device Exemption. One hundred fifty-five eyes of 155 patients with myopia from -5.5 to -22.5 diopters (D) underwent implantation of an Artisan lens, as part of a phase I, II, or III FDA trial. Eyes were examined at 1 day (154 eyes), 2 weeks (142 eyes), 2 months (130 eyes), and 6 months (84 eyes). Intraocular pressure and presence of flare and cell on slit-lamp biomicroscopy were recorded at each visit. Achieved correction, stability of spherical equivalent refraction, change in astigmatism, postoperative uncorrected vision, change in best spectacle-corrected acuity, and change in endothelial cell count. Mean spherical equivalent manifest refraction stabilized on postoperative day 1. Mean difference between attempted and achieved correction at 2 months was -0.32 +/- 0.95 D (mean +/- standard deviation; range, -4.42 to +2.2 D). At 6 months, 85% of eyes saw 20/40 or better uncorrected, and 90% of eyes were within 1 D of the attempted correction. Refractive astigmatism increased in 4.8% of eyes and decreased in 17% of eyes at 6 months. At 2 months, six eyes (4.8%) lost two or more lines of best spectacle-corrected visual acuity; by 6 months, no eyes lost two or more lines of best-corrected visual acuity. Endothelial cell count was unchanged at 6 months compared with the preoperative count. Nonprogressive lens opacities developed in four eyes as a result of surgical trauma. Chronic inflammation was not detected in any eye by slit-lamp biomicroscopy, nor did any eye develop angle closure or glaucoma. Short-term results suggest that the Artisan lens is an accurate and safe method for the correction of high myopia. Surgical skill is important in avoiding lens opacities. Longer-term data are needed to assess the impact of the lens on the endothelium, the crystalline lens, and the iris.

To determine the efficacy of systemic acyclovir in decreasing complications and improving the out... more To determine the efficacy of systemic acyclovir in decreasing complications and improving the outcome of penetrating keratoplasty for herpes simplex virus (HSV) keratitis. Retrospective study of 53 primary penetrating keratoplasties for HSV keratitis at an eye hospital from January 1, 1989, through December 31, 1996. Medical records were analyzed for history of HSV keratitis, preoperative neovascularization, and disease activity. Postoperative use of acyclovir, recurrence of HSV keratitis, rejection, uveitis or edema, and graft failure were evaluated. Twenty-four patients (mean +/- SD follow-up, 44.7 +/- 32.6 months) received no acyclovir and were compared with 20 patients, (mean +/- SD follow-up, 28.8 +/- 16.7 months), who received 400 mg acyclovir twice a day for at least 1 year. No patient in the acyclovir group had a recurrence of dendritic keratitis in the first year compared with 5 (21%) of the patients who did not receive acyclovir (P = .03). No patient had graft failure in the acyclovir group compared with 4 (17%) in the group without acyclovir after 1 year of follow-up (P = .06). Postoperative systemic acyclovir therapy after penetrating keratoplasty for HSV keratitis is associated with a reduced rate of recurrent HSV dendritic keratitis and possible graft failure at 1 year of follow-up.