Kidney Cell Line HEK293T Research Articles

Inhibition of veratridine-induced delayed inactivation of the voltage-sensitive sodium channel by synthetic analogs of crambescin B

Crambescin B carboxylic acid, a synthetic analog of crambescin B, was recently found to inhibit the voltage-sensitive sodium channels (VSSC) in a cell-based assay using neuroblastoma Neuro 2A cells. In the present study, whole-cell patch-clamp recordings were conducted with three heterologously expressed VSSC subtypes, Nav1.2, Nav1.6 and Nav1.7, in a human embryonic kidney cell line HEK293T to further characterize the inhibition of VSSC by crambescin B carboxylic acid. Contrary to the previous observation, crambescin B carboxylic acid did not inhibit peak current evoked by depolarization from the holding potential of −100mV to the test potential of −10mV in the absence or presence of veratridine (VTD). In the presence of VTD, however, crambescin B carboxylic acid diminished VTD-induced sustained and tail currents through the three VSSC subtypes in a dose-dependent manner, whereas TTX inhibited both the peak current and the VTD-induced sustained and tail currents through all subtypes of VSSC tested. We thus concluded that crambescin B carboxylic acid does not block VSSC in a similar manner to TTX but modulate the action of VTD, thereby causing an apparent block of VSSC in the cell-based assay.

Solid-Phase Synthesis and In Vitro Cytotoxicity of New Peptide Functionalized Cyclencarboxymethylen and L-DOPA Hybrids

The new small peptide functionalized cyclen and DOPA derivatives were synthesized: cyclen-HisHis, cyclen- AspHis, cyclen-GluHis, DOPA-HisHis were prepared. The solid-phase synthesis strategy was used for preparation of new compounds. Synthesized cyclen- and DOPA-oligopeptide hybrid conjugations were purified by HPLC and analyzed using MALDI-TOF MS spectrometer. The toxic effect was determined against mammalian cells human embryonic kidney cell line HEK293T ATCC®CLR-11268TM for each new compound. The inhibition effect of all tested cyclen– dipeptides on kidney cells was approximately about 30% after 24 hours. The minimal rate of toxicity against human liver cells showed all tested dipeptides with DOPA, their inhibition effect was maximal 10%. The acute inhibition effect of sample DOPA-GluHis-DOPA was 14% imme¬diately after adding. The antioxidant and anticancer activity studies will be next part of the ongoing project.

Antimycobacterial activity of novel hydrazide-hydrazone derivatives with 2H-chromene and coumarin scaffold

This study reports the synthesis of new 2H-chromene or coumarin based acylhydrazones, which were evaluated for their in vitro antimycobacterial activity against reference strain Mycobacterium tuberculosis H37Rv and compared to the first-line antituberculosis drugs, isoniazid (INH) and ethambutol (EMB). The most active compounds 7m (MIC 0.13μM), 7o (MIC 0.15μM) and 7k (MIC 0.17μM) demonstrated antimycobacterial activity at submicromolar concentration level and remarkably minimal associated cytotoxicity in the human embryonic kidney cell line HEK-293T. Structure-activity relationship for this class of compounds has been established.

Sprouty2 regulates proliferation and survival of multiple myeloma byinhibiting activation of the ERK1/2 pathway invitro and invivo.

Multiple myeloma (MM) is an incurable disease, and its pathogenesis remains unclear. MicroRNA (miR)-21 was detected at a high level in MM and plays a key role in the pathogenesis of MM. However, Sprouty2 (spry2), a downstream target of miR-21, has low expression, and its mechanism in MM is unknown. We investigated whether spry2 could exert an antimyeloma effect and further studied the potential pathogenesis and progression of MM. To address the functional consequences of spry2, we assessed the expression levels of spry2 in several myeloma cell lines and detected low expression levels in MMcells. Overexpression of spry2 suppressed growth and colony formation ability and decreased the phosphorylation of extracellular signal-regulated kinases 1 and 2. Spry2 also decreased secretion of vascular endothelial growth factor and partially enhanced the sensitivity of MMcells to an inhibitor of mitogen-activated protein kinases 1 and 2. Additionally, spry2 inhibited the tumorigenesis and angiogenesis of MMcells invivo. In summary, we report for the first time that spry2 can inhibit MMcell growth and survival with a concomitant reduction in phosphorylation of extracellular signal-regulated kinases 1 and 2 invitro and invivo.

Toxicological impact of JWH-018 and its phase I metabolite N-(3-hydroxypentyl) on human cell lines

The emergence and abuse of synthetic cannabinoids has been increasing as an alternative to cannabis, mainly among youth. As their appearance on the drug market has been recent, the pharmacological and toxicological profiles of these psychoactive substances are poorly understood. Current studies suggest that they have stronger effects compared to their natural alternatives and their metabolites retain affinity towards CB1 receptors in CNS. Since studies on its toxicological properties are scarce, the effects of the drug in human derived cell lines were investigated. The present study was designed to explore the toxicological impact of parent drug versus phase I metabolites of synthetic cannabinoids on human cells with and without CB1 receptor. The human cell line of neuroblastoma SH-SY5Y and human kidney cell line HEK-293T were exposed to JWH-018 and to its N-(3-hydroxypentyl) metabolite. Cell toxicity was evaluated using the MTT and LDH assay. Additionally, a dual staining methodology with fluorescent Annexin V-FITC and propidium iodide was performed to address the question of whether JWH-018 N-(3-hydroxypentyl) metabolite is inducing cell death through apoptosis or necrosis, in HEK293T and SH-SY5Y cell lines.The obtained results show that JWH-018 does not cause a statistically significant decrease in cell viability, in contrast to its N-(3-hydroxypentyl) metabolite, which at ≥25μM causes a significant decrease in cell viability. Both cell lines are affected by JWH-018 metabolite. Our results point to higher toxicity of JWH-018 metabolite when compared to its parent drug, suggesting a non-CB1 receptor mediated toxicological mechanism. Comparing the results from Annexin V/PI with MTT and LDH assays of SH-SY5Y and HEK293T in the presence of the synthetic cannabinoid metabolite, emerges the picture that cellular viability decreases and associated death is occurring through necrosis.

Cell surface expression level variation between two common Human Leukocyte Antigen alleles, HLA-A2 and HLA-B8, is dependent on the structure of the C terminal part of the alpha 2 and the alpha 3 domains.

Constitutive cell surface expression of Human Leukocyte Antigen (HLA) class I antigens vary extremely from tissue to tissue and individual antigens may differ widely in expression levels. Down-regulation of class I expression is a known immune evasive mechanism used by cancer cells and viruses. Moreover, recent observations suggest that even minor differences in expression levels may influence the course of viral infections and the frequency of complications to stem cell transplantation. We have shown that some human multipotent stem cells have high expression of HLA-A while HLA-B is only weakly expressed, and demonstrate here that this is also the case for the human embryonic kidney cell line HEK293T. Using quantitative flow cytometry and quantitative polymerase chain reaction we found expression levels of endogenous HLA-A3 (median 71,204 molecules per cell) 9.2-fold higher than the expression of-B7 (P = 0.002). Transfection experiments with full-length HLA-A2 and -B8 encoding plasmids confirmed this (54,031 molecules per cell vs. 2,466, respectively, P = 0.001) independently of transcript levels suggesting a post-transcriptional regulation. Using chimeric constructs we found that the cytoplasmic tail and the transmembrane region had no impact on the differential cell surface expression. In contrast, ~65% of the difference could be mapped to the six C-terminal amino acids of the alpha 2 domain and the alpha 3 domain (amino acids 176–284), i.e. amino acids not previously shown to be of importance for differential expression levels of HLA class I molecules. We suggest that the differential cell surface expression of two common HLA-A and–B alleles is regulated by a post-translational mechanism that may involve hitherto unrecognized molecules.

The NDR Kinase Cbk1 Downregulates the Transcriptional Repressor Nrg1 through the mRNA-Binding Protein Ssd1 in Candida albicans.

NDR (nuclear Dbf2-related) kinases are essential components for polarized morphogenesis, cytokinesis, cell proliferation, and apoptosis. The NDR kinase Cbk1 is required for the hyphal growth of Candida albicans; however, the molecular functions of Cbk1 in hyphal morphogenesis are largely unknown. Here, we report that Cbk1 downregulates the transcriptional repressor Nrg1 through the mRNA-binding protein Ssd1, which has nine Cbk1 phosphorylation consensus motifs. We found that deletion of SSD1 partially suppressed the defective hyphal growth of the C. albicans cbk1Δ/Δ mutant and that Ssd1 physically interacts with Cbk1. Cbk1 was required for Ssd1 localization to polarized growth sites. The phosphomimetic SSD1 allele (ssd1-9E) allowed the cbk1Δ/Δ mutant to form short hyphae, and the phosphodeficient SSD1 allele (ssd1-9A) resulted in shorter hyphae than did the wild-type SSD1 allele, indicating that Ssd1 phosphorylation by Cbk1 is important for hyphal morphogenesis. Furthermore, we show that the transcriptional repressor Nrg1 does not disappear during hyphal initiation in the cbk1Δ/Δ mutant but is completely absent in the cbk1Δ/Δ ssd1Δ/Δ double mutant. Deletion of SSD1 also increased Als3 expression and internalization of the cbk1Δ/Δ mutant in the human embryonic kidney cell line HEK293T. Collectively, our results suggest that one of the functions of Cbk1 in the hyphal morphogenesis of C. albicans is to downregulate Nrg1 through Ssd1.

Expression of recombinant alpha and beta tubulins from the yew Taxus cuspidata and analysis of the microtubule assembly in the presence of taxol.

Taxol was originally isolated from the yew Taxus brevifolia. Because taxol inhibits the depolymerization of microtubules, the presence of a self-resistance mechanism in Taxus spp. was hypothesized. The cloning of the cDNA for alpha and beta tubulins from Taxus cuspidata and those from the human embryonic kidney cell line HEK293T revealed that the (26)Asp, (359)Arg, and (361)Leu residues in the human beta tubulin, which are important for taxol binding, were replaced with Glu, Trp, and Met in the beta tubulin of T. cuspidata, respectively. The microtubule assembly of the recombinant alpha and beta tubulins was monitored turbidimetrically, and the results clearly demonstrated that the microtubule from T. cuspidata is less sensitive to taxol than that from HEK293T cells. The Taxus microtubule composed of the wild-type alpha tubulin and the beta tubulin with the E26D mutation restored the sensitivity to taxol. We thus postulated that the mutation identified in the beta tubulin of T. cuspidata plays a role in the self-resistance of this species against taxol.

Theophylline-7-acetic acid derivatives with amino acids as anti-tuberculosis agents

A series of amides were synthesized by condensation of theophylline-7-acetic acid and eight commercially available amino acid methyl ester hydrochlorides. Consecutive hydrolysis of six of the amido-esters resulted in the formation of corresponding amido-acids. The newly synthesized compounds were evaluated for their in vitro activity against Mycobacterium tuberculosis H37Rv. The activity varied depending on the amino acid fragments and in seven cases exerted excellent values with MICs 0.46–0.26μM. Assessment of the cytotoxicity revealed that the compounds were not cytotoxic against the human embryonal kidney cell line HEK-293T. The theophylline-7-acetamides containing amino acid moieties appear to be promising lead compounds for the development of antimycobacterial agents.

Polycationic Nanoparticles for siRNA Delivery: Comparing ARGET ATRP and UV-Initiated Formulations

In this work, we develop and evaluate polycationic nanoparticles for the delivery of small interfering RNA (siRNA). Delivery remains a major challenge for translating siRNA to the clinic, and overcoming the delivery challenge requires effective siRNA delivery vehicles that meet the demands of the specific delivery strategy. Cross-linked polycationic nanoparticle formulations were synthesized using ARGET ATRP or UV-initiated polymerization. The one-step, one-pot, surfactant-stabilized monomer-in-water synthesis technique may provide a simpler and faster alternative to complicated, multistep techniques and an alternative to methods that rely on toxic organic solvents. The polymer nanoparticles were synthesized using the cationic monomer 2-(diethylamino)ethyl methacrylate, the hydrophobic monomer tert-butyl methacrylate to tune pH responsiveness, the hydrophilic monomer poly(ethylene glycol) methyl ether methacrylate to improve biocompatibility, and cross-linking agent tetraethylene glycol dimethacrylate to enhance colloidal stability. Four formulations were evaluated for their suitability as siRNA delivery vehicles in vitro with the human embryonic kidney cell line HEK293T or the murine macrophage cell line RAW264.7. The polycationic nanoparticles demonstrated efficient and rapid loading of the anionic siRNA following complexation. Confocal microscopy as well as flow cytometry analysis of cells treated with polycationic nanoparticles loaded with fluorescently labeled siRNA demonstrated that the polycationic nanoparticles promoted cellular uptake of fluorescently labeled siRNA. Knockdown experiments using polycationic nanoparticles to deliver siRNA demonstrated evidence of knockdown, thus demonstrating potential as an alternative route to creating polycationic nanoparticles.

Protective effect of aronia melanocarpa fruit juice in a model of cisplatin-induced cytotoxicity in vitro

The aim of the present study was to investigate the protective potential of Aronia melanocarpa fruit juice in a model of cisplatin-induced cytotoxicity in the human embryonal kidney cell line HEK293T. The cellular viability was assessed using the MTT-dye reduction assay based on the reduction of the yellow tetrazolium dye MTT to a violet formazan product via the mitochondrial succinate dehydrogenase in viable cells. Cisplatin was applied in various concentrations either alone or after a 24-hour pretreatment of the cells with Aronia melanocarpa fruit juice at 0.1 and 0.05 mg/ml. The half maximal inhibitory concentrations (IC50 values) were derived from the concentration-response curves to cisplatin. Applied alone, the anticancer drug caused a prominent decrease of cellular viability with IC50 8.3 +/- 1.1 microM. The juice proved to significantly ameliorate the in vitro cytotoxicity of the platinum drug, in a concentration-dependent manner. The pretreatment of the cells with Aronia melanocarpa fruit juice resulted in a significant increase (p < 0.001) of IC50 for cisplatin to 25.1 +/- 2.7 microM (at 0.05 mg/ml) and 34.4 +/- 3.4 microM (at 0.1 mg/ml), respectively. The protective effect of Aronia melanocarpa fruit juice observed in this study is most probably due to its well appreciated antioxidant activity as oxidative stress plays a central role in the toxic effects of cisplatin.

VBP1 facilitates proteasome and autophagy‐mediated degradation of MutS homologue hMSH4

Ubiquitination is an important mechanism for the regulation of diverse cellular functions, including proteolysis and DNA repair. The human MutS family protein hMSH4 functions in meiotic recombinational DNA double-strand break (DSB) repair. It was previously observed that hMSH4 interacts with the von Hippel-Lindau binding protein 1 (VBP1), a partner of the VHL ubiquitin E3 ligase as well as a subunit of the prefoldin complex. In this study we address how ubiquitination regulates the homeostasis of hMSH4 in the human embryonic kidney cell line HEK293T. We demonstrate that VBP1 targets hMSH4 for degradation and identify a new VBP1 binding partner, p97, an AAA(+) ATPase involved in protein degradation and DNA damage response. VBP1, VHL, and p97 coexist in the hMSH4 immunocomplex and regulate the polyubiquitination of hMSH4. Furthermore, the results of this study demonstrate that VBP1 acts together with p97 to regulate hMSH4 degradation. Overall, this study has revealed a molecular mechanism by which VBP1 controls the levels of hMSH4 by ubiquitination in mitotic cells. Such a mechanism may be important for controlling the role of hMSH4 in regulating homologous recombination and nonhomologous DNA end joining-mediated DSB repair in human cells.

FOXO3 induces FOXO1-dependent autophagy by activating the AKT1 signaling pathway

Forkhead box O (FOXO) transcriptional protein family members, including FOXO1 and FOXO3, are involved in the modulation of autophagy. However, whether there is redundancy between FOXO1 and FOXO3 in the ability to induce autophagy remains unclear. In this study, we showed that FOXO3 induced a transcription-dependent autophagy, and FOXO1 was required for this process. Overexpression of wild-type FOXO3 (WT) or FOXO3 (3A), which harbors alanine mutations at residues Thr32, Ser253 and Ser315, but not transcription-inactive FOXO3 (∆DB3A), significantly induced autophagy in the human embryonic kidney cell line HEK293T and mouse embryonic fibroblast (MEF) cell lines. Interestingly, depletion of FOXO1 by siRNA attenuated FOXO3-induced autophagy. Our data also showed that FOXO3 overexpression did not increase the expression of FOXO1 at the protein level, although FOXO3 was capable of binding the promoter region of FOXO1 and inducing an increase in the transcription of FOXO1 mRNA. Furthermore, our results showed that FOXO3 promoted the translocation of FOXO1 from the nucleus to the cytoplasm, resulting in an increase in FOXO1-induced autophagy. Moreover, our results supported a mechanism whereby FOXO3 dramatically increased the expression of the class I PtdIns3K catalytic subunit PIK3CA, leading to an increase in AKT1 activity, which resulted in the phosphorylation and nuclear export of FOXO1. To the best of our knowledge, our data are the first to suggest that FOXO1 plays a central role in FOXO3-induced autophagy.

MYC Protein Inhibits Transcription of the MicroRNA Cluster MC-let-7a-1∼let-7d via Noncanonical E-box

The human microRNA cluster MC-let-7a-1∼let-7d, with three members let-7a-1, let-7f-1, and let-7d, is an important cluster of the let-7 family. These microRNAs play critical roles in regulating development and carcinogenesis. Therefore, precise control of MC-let-7a-1∼let-7d level is critical for cellular functions. In this study, we first showed that the expression of these three members was significantly reduced in human hepatocellular carcinoma HepG2 cells as compared with the immortalized human liver L02 cells. We demonstrated that the MC-let-7a-1∼let-7d cluster was encoded by a single polycistronic transcript driven by a 10-kb upstream promoter, with two MYC-binding sites. Importantly, MYC inhibited MC-let-7a-1∼let-7d promoter activity via binding to the noncanonical E-box 3 downstream of the transcription start sites, whereas it enhanced promoter activity by binding to the canonical E-box 2 upstream of the transcription start sites. We found that although the binding affinity of MYC to E-box 2 was stronger than E-box 3, the binding quantum of MYC to E-box 3 was significantly higher in cancerous HepG2 cells as compared with the noncancerous L02 cells. In addition, forced expression of let-7 could reverse the MYC-mediated cell proliferation. These findings suggested that in L02 cells with a low level of MYC, MYC binds mainly to E-box 2 to enhance MC-let-7a-1∼let-7d expression. However, in HepG2 cells with an elevated MYC, the extra MYC could bind to E-box 3 to suppress the transcription of MC-let-7a-1∼let-7d and thus enable HepG2 cells to maintain a high level of MYC and a low level of let-7 microRNAs simultaneously.

A loss‐of‐function variant of the antiviral molecule MAVS is associated with a subset of systemic lupus patients

Dysregulation of the antiviral immune response may contribute to autoimmune diseases. Here, we hypothesized that altered expression or function of MAVS, a key molecule downstream of the viral sensors RIG-I and MDA-5, may impair antiviral cell signalling and thereby influence the risk for systemic lupus erythematosus (SLE), the prototype autoimmune disease. We used molecular techniques to screen non-synonymous single nucleotide polymorphisms (SNPs) in the MAVS gene for functional significance in human cell lines and identified one critical loss-of-function variant (C79F, rs11905552). This SNP substantially reduced expression of type I interferon (IFN) and other proinflammatory mediators and was found almost exclusively in the African-American population. Importantly, in African-American SLE patients, the C79F allele was associated with low type I IFN production and absence of anti-RNA-binding protein autoantibodies. These serologic associations were not related to a distinct, functionally neutral, MAVS SNP Q198K. Hence, this is the first demonstration that an uncommon genetic variant in the MAVS gene has a functional impact upon the anti-viral IFN pathway in vivo in humans and is associated with a novel sub-phenotype in SLE. This study demonstrates the utility of functional data in selecting rare variants for genetic association studies, allowing for fewer comparisons requiring statistical correction and for alternate lines of evidence implicating the particular variant in disease.

Characterization of a novel Nav1.5 channel mutation, A551T, associated with Brugada syndrome

Brugada syndrome is a life-threatening, inherited arrhythmia disorder associated with autosomal dominant mutations in SCN5A, the gene encoding the human cardiac Na+ channel α subunit (Nav1.5). Here, we characterized the biophysical properties of a novel Brugada syndrome-associated Nav1.5 mutation, A551T, identified in a proband who was successfully resuscitated from an episode of ventricular fibrillation with sudden collapse. Whole-cell currents through wild-type (WT) Nav1.5 and mutant (A551T) channels were recorded and compared in the human embryonic kidney cell line HEK293T transfected with SCN5A cDNA and SCN1B cDNA, using the patch-clamp technique. Current density was decreased in the A551T mutant compared to the WT. In addition, the A551T mutation reduced Nav1.5 activity by promoting entry of the channel into fast inactivation from the closed state, thereby shifting the steady-state inactivation curve by -5 mV. Furthermore, when evaluated at -90 mV, the resting membrane potential, but not at the conventionally used -120 mV, both the percentage, and rate, of channel recovery from inactivation were reduced in the mutant. These results suggest that the DI-DII linker may be involved in the stability of inactivation gating process. This study supports the notion that a reduction in Nav1.5 channel function is involved in the pathogenesis of Brugada syndrome. The structural-functional study of the Nav1.5 channel advances our understanding of its pathophysiolgocial function.

Recombinant full-size human antibody to Ebola virus

A full-size human antibody to Ebola virus was constructed by joining genes encoding the constant domains of the heavy and light chains of human immunoglobulin with the corresponding DNA fragments encoding variable domains of the single-chain antibody 4D1 specific to Ebola virus, which was chosen from a combinatorial phage display library of single-strand human antibodies. Two expression plasmids. pCH1 and pCL1, containing the artificial genes encoding the light and heavy chains of human immunoglobulin, respectively, were constructed. Their cotransfection into the human embryonic kidney cell line HEK293T provided the production of a full-size recombinant human antibody. The affinity constant for the antibody was estimated by solid-phase enzyme-linked immunoassay to be 7.7 x 10(7) +/- 1.5 x 10(7) M(-1). Like the parent single-chain antibody 4DI, the resulting antibody bound the nucleoprotein of Ebola virus and did not interact with the proteins of Marburg virus.

Harnessing Functional Plasticity of Enzymes: A Fluorogenic Probe for Imaging 17β-HSD10 Dehydrogenase, an Enzyme Involved in Alzheimer's and Parkinson's Diseases

In this paper, we describe the development of a fluorogenic substrate for 17beta-hydroxysteroid-dehydrogenase type 10 (17beta-HSD10), which is a multifunctional metabolic enzyme fulfilling several metabolic roles (beta-oxidation of fatty acids, catabolism of isoleucine, and metabolism of steroids). In recent years, it has emerged as an important stress and pathological marker in neurons and glial cells (expression down-regulation in Parkinson's disease, up-regulation and association with beta-amyloid peptide in Alzheimer's disease). Through the iterative molecular design and chemical synthesis described herein, compound 1 was developed, which possesses all required properties for a selective optical reporter substrate: alcohol-ketone optical switching, the ability to function as a good enzyme substrate (expressed in kinetic parameters), cell permeability, and cell retention. Probe 1 provides a blue-to-green/yellow bright switch and enables non-invasive, real-time imaging of 17beta-HSD10 in live human cells. The selectivity of reporter 1 was established by the quantitative correlation of metabolic activity to protein expression in human kidney cell line HEK-293T.

Farnesoid X Receptor Agonist Reduces Serum Asymmetric Dimethylarginine Levels through Hepatic Dimethylarginine Dimethylaminohydrolase-1 Gene Regulation

The farnesoid X receptor (FXR, NR1H4) is a bile acid-responsive nuclear receptor that plays critical roles in the transcriptional regulation genes involved in cholesterol, bile acid, triglyceride, and carbohydrate metabolism. By microarray analysis of hepatic genes from female Zucker diabetic fatty (ZDF) rats treated with the FXR agonist GW4064, we have identified dimethylarginine dimethylaminohydrolase-1 (DDAH1) as an FXR target gene. DDAH1 is a key catabolic enzyme of asymmetric dimethylarginine (ADMA), a major endogenous nitric-oxide synthase inhibitor. Sequence analysis of the DDAH1 gene reveals the presence of an FXR response element (FXRE) located 90 kb downstream of the transcription initiation site and within the first intron. Functional analysis of the putative FXRE demonstrated GW4064 dose-dependent transcriptional activation from the element, and we have demonstrated that the FXRE sequence binds the FXR-RXR heterodimer. In vivo administration of GW4064 to female ZDF rats promoted a dose-dependent and >6-fold increase in hepatic DDAH1 gene expression. The level of serum ADMA was reduced concomitantly. These findings provide a mechanism by which FXR may increase endothelium-derived nitric oxide levels through modulation of serum ADMA levels via direct regulation of hepatic DDAH1 gene expression. Thus, beneficial clinical outcomes of FXR agonist therapy may include prevention of atherosclerosis and improvement of the metabolic syndrome.

Evidence That the Transmembrane Biogenesis of Aquaporin 1 Is Cotranslational in Intact Mammalian Cells

Most polytopic membrane proteins are believed to integrate into the membrane of the endoplasmic reticulum (ER) cotranslationally. However, recent studies with Xenopus oocytes and dog pancreatic microsomes have suggested that this is not the case for human aquaporin 1 (AQP1). These experiments indicate that membrane-spanning segments (MSSs) 2 and 4 of AQP1 do not integrate into the membrane cotranslationally so that this protein initially adopts a four MSS topology. A later maturation event involving a 180-degree rotation of MSS 3 from an N(lum)/C(cyt) to an N(cyt)/C(lum) orientation and the concomitant integration of MSSs 2 and 4 into the membrane results in the final six MSS topology. Here we examine the biogenesis of AQP1 in the human embryonic kidney cell line HEK-293T. To do this, we constructed an expression vector for a fusion protein consisting of the enhanced green fluorescent protein followed by an insertion site for AQP1 sequences and a C-terminal glycosylation tag. We then transiently transfected HEK-293T cells with this vector containing the AQP1 sequence truncated after each MSS. Glycosylation of the C-terminal tag was used to monitor its location relative to the ER lumen and consequently the membrane integration and orientation of successive MSSs. In contrast to previous studies our results indicate that AQP1 integrates into the ER membrane cotranslationally in intact HEK-293T cells.