Altered cholesterol metabolism in a hypercholesterolemia -resistant rabbit colony

A colony of rabbits has been developed at the University of Texas Medical School at Houston that is resistant to dietary-induced hypercholesterolemia. The liver of resistant rabbits had higher levels of ($\sp{125}$I) $\beta$-VLDL binding and 3-hydroxy-3-methylglutaryl (HMGCoA) reductase activity, but lower acyl coenzyme A:cholesterol acyltransferase (ACAT) activity than normal rabbits. Direct quantitation of intracellular cholesterol content of the liver revealed that the resistant rabbits had $<$10% of the intracellular free cholesterol present in normal rabbits. Fibroblasts isolated from normal and resistant rabbits exhibited differences in ($\sp{125}$I) LDL binding, HMGCoA reductase activity and ACAT activity that were similar to those found in the liver. No structural differences were found in the LDL receptor of normal and resistant fibroblasts that would account for the increased binding capacity of the resistant cells. The regulation of LDL receptor levels by exogenous oxygenated sterols was similar in normal and resistant fibroblasts. The regulation of LDL receptor binding capacity by LDL was attenuated in the resistant compared to normal fibroblasts, suggesting that the resistant fibroblasts have an alternate pathway for processing lipoprotein-derived cholesterol. Sterol-balance studies revealed that the cholesterol-fed resistant rabbits increased lithocholic acid excretion compared to the basal state, and had higher levels of deoxycholic acid excretion than cholesterol-fed normal rabbits. In addition, the specific activity and mRNA levels of cholesterol 7$\alpha$-hydroxylase (C7$\alpha$H) were higher in resistant rabbits than normal rabbits, suggesting that the increased bile acid excretion was due to an increase in bile acid synthesis. Increased clearance of cholesterol relieves the negative feedback inhibition cholesterol exerts on expression of the LDL receptor. The number of cell surface LDL receptors is then increased in resistant rabbits and allows rapid clearance of lipoproteins from the plasma compartment, thereby reducing plasma cholesterol levels. The low intracellular cholesterol level also relieves the negative feedback inhibition cholesterol exerts on HMGCoA reductase. Increased synthesis of cholesterol from acetate provides cells with cholesterol for bile acid synthesis and/or homeostasis. The activity of ACAT is then decreased due to the flux of cholesterol through the bile acid synthetic pathways. ^

Characterization of the cholesterol 7$\alpha$-hydroxylase promoter in hypercholesterolemia -resistant rabbits

Our laboratory has developed and partially characterized a strain of New Zealand white rabbits that are resistant to the hypercholesterolemia which typically occurs in normal rabbits when fed a cholesterol-enriched diet. This phenotype is most likely attributed to an increase in bile acid excretion by hypercholesterolemia-resistant (CRT) rabbits as a result of elevated enzyme activity of cholesterol 7$\alpha$-hydroxylase (C7$\alpha$H), the rate-limiting enzyme in bile acid synthesis. Northern analysis revealed that CRT rabbits, in comparison to normal rabbits, have a 7-fold greater steady-state C7$\alpha$H mRNA levels irrespective of dietary regimen. The C7$\alpha$H gene in both phenotypes was determined to be a single copy gene. The hypothesis was that the elevated C7$\alpha$H mRNA levels in CRT rabbits, in comparison to normal animals, was due to an increase in the transcription rate of the C7$\alpha$H gene as a result of a mutation in a cis-acting element and/or a trans-acting factor within the hepatocyte. To isolate the C7$\alpha$H gene from both normal and CRT rabbits, genomic libraries were prepared from both phenotypes into $\lambda$GEM12 vectors using conventional techniques. Three CRT and one normal phage clones that contained the C7$\alpha$H gene were identified by screening the library with a series of probes located within different exons of the C7$\alpha$H cDNA. Sequencing analysis confirmed that approximately 1100 bp of the C7$\alpha$H 5'-flanking region from both normal and CRT phenotypes was identical. The increase in C7$\alpha$H mRNA levels was not attributed to a cis-acting mutation within this region. Liver nuclear extracts were prepared from normal and CRT rabbits maintained either on a basal or 0.25% cholesterol-enriched diet and incubated with several radiolabeled DNA fragments from the C7$\alpha$H gene. A 37 basepair region, located between nucleotides $-$452 to $-$416 was identified that had altered binding patterns between normal and CRT rabbits as a function of diet. Two additional regions, $-$747 to $-$575 and $-$580 to $-$442, produced banding patterns which were identical, irrespective of phenotype or diet. In conclusion, these studies suggested that the increase in C7$\alpha$H mRNA in CRT rabbits was due to differences in binding of a cholesterol-responsive transcription factor to the C7$\alpha$H promoter. ^

Condensation in ducts

Phase changing flows are being considered for thermal management in space platforms. The resulting flow patterns are very complicated and extremely sensitive to gravity action. Concerning fluid flow in ducts, the available evidence indicates that although the pressure loss does not depend too much on the fluid flow pattern,the heat transfer (and resulting phase change) does. A simple exercise to illustrate this point is presented in this paper. It deals with condensing flow in straight circular cross-sectional ducts. Two extreme configurations are considered here, one corresponds to a stratified flow and the other to an annular flow. Both types of flow patterns have been extensively considered in the past and from this point of view almost nothing is new in the paper, but past results look conflictive and this could be due to the limitations and computational intricacies of the models used. Thus the problem has been reformulated from the onset and the results are presented as the evolution of the vapor quality (vapor to total mass flow rate) along the duct, in typical cases. The results presented here indicate that within the validity of the present models and the assumed ranges of mass flow rate, duct diameter, thermal conditions and fluid characteristics,the length of the ducts required to achieve complete condensation under zero gravity are an order of magnitude larger than in horizontal tubes under normal terrestrial conditions.

Static Pressure Impact on Aerodynamics of High Speed Railway Tunnels

A pressure wave is generated when a high speed train enters a tunnel. This wave travels along the tunnel back and forth, and is reflected at the irregularities of the tunnel duct (section changes, chimneys and tunnel ends). The pressure changes are associated to these waves can have an effect on passengers if the trains are not suitably sealed or pressurized. The intensity of the waves depends mainly on the train speed, and on the blockage ratio (train-section-to- tunnel-section area ratio). As the intensity of the waves is limited by regulations, and also by the effects on passengers and infrastructures, the sizing of the tunnel section area is largely influenced by the maximum train speed allowed in the tunnel. The aim of this study is to analyse the increase in cost in a tunnel due to the existence of this difference in ground level, and evaluate the increase of construction costs that this elevation might involve.

Diseño de laboratorio para la evaluación de silenciadores "in situ"

Se ha realizado el diseño de un sistema que permite realizar ensayos de silenciadores “in situ”. Para evaluar el comportamiento del sistema de ensayo de silenciadores se procede a caracterizar los paneles acústicos que son la base del sistema. De los paneles empleados en el sistema, se determinará por diferencia del nivel de presión sonora, el aislamiento a ruido aéreo de los mismos, partiendo del promedio de las medidas obtenidas en la sala emisora de la cámara reverberante con el material instalado y en la sala receptora. Se realizan distintas medidas de los niveles de presión sonora en sala emisora de la cámara reverberante y en la receptora así como del tiempo de reverberación con los paneles instalados en el hueco existente en el elemento de separación vertical de la cámara. Una vez ensayados los paneles, se ha procedido a medir los niveles de presión sonora que se obtienen antes y después de la interposición de distintos silenciadores en el sistema diseñado con el propósito de disponer de un laboratorio de medida de la atenuación de silenciadores. Para ello, se procede a efectuar el montaje, en la puerta sencilla de la cámara reverberante, de los componentes del sistema diseñado y se realizan mediciones de la atenuación que proporciona la colocación de un silenciador en el sistema y su posterior sustitución por un conducto. La medición de la atenuación del nivel de presión sonora que producen los distintos silenciadores se realiza por pérdidas por inserción, siguiendo las directrices de la Norma UNE-ISO 11820 y por el método de conductos forrados como cálculo teórico. ABSTRACT. A system has been designed for testing silencers “in situ”. In first place, to evaluate the behavior of the system in the test of silencers, it has been proceeded to customize the acoustic panels that are the basis of the system. The attenuation sound of the panels used in the system will be determined by the difference of sound pressure level. It will be made through the average of the measurements obtained in the source room of the reverberant chamber, the average of the measurements obtained in the receiving room, as well as the reverberation time, with the material installed in the hole of the walls. Once the panels are tested, and with the purpose of having a laboratory to measure the attenuation of silencers, the sound pressure levels has been measured before and after inserting the different silencers in the designed system. To obtain these measures, the components of the system designed have been installed in the hollow of the single door of the reverberant chamber and then the sound pressure level has been measured with a silencers first and after with a duct instead of the silencer. The measurement of the attenuation of the sound pressure level produced by different silencers has been made by insertion loss, following the 11820 UNE-ISO, as well as theoretical calculation has been made by the method of duct lined.

Analysis of air flow past and through the 2415-3S airfoil for an unmanned aerial vehicle with internal propulsion system

This paper deals with the prediction of velocity fields on the 2415-3S airfoil which will be used for an unmanned aerial vehicle with internal propulsion system and in this way analyze the air flow through an internal duct of the airfoil using computational fluid dynamics. The main objective is to evaluate the effect of the internal air flow past the airfoil and how this affects the aerodynamic performance by means of lift and drag forces. For this purpose, three different designs of the internal duct were studied; starting from the base 2415-3S airfoil developed in previous investigation, basing on the hypothesis of decreasing the flow separation produced when the propulsive airflow merges the external flow, and in this way obtaining the best configuration. For that purpose, an exhaustive study of the mesh sensitivity was performed. It was used a non-structured mesh since the computational domain is three-dimensional and complex. The selected mesh contains approximately 12.5 million elements. Both the computational domain and the numerical solution were made with commercial CAD and CFD software, respectively. Air, incompressible and steady was analyzed. The boundary conditions are in concordance with experimental setup in the AF 6109 wind tunnel. The k-e model is utilized to describe the turbulent flow process as followed in references. Results allowed obtaining velocity contours as well as lift and drag coefficients and also the location of separation and reattachment regions in some cases for zero degrees of angle of attack on the internal and external surfaces of the airfoil. Finally, the selection of the configuration with the best aerodynamic performance was made, selecting the option without curved baffles.

Numerical analysis of air flow past the 2415-3S airfoil for an unmanned aerial vehicle with internal propulsion system

This paper deals with the prediction of pressure and velocity fields on the 2415-3S airfoil which will be used for and unmanned aerial vehicle with internal propulsion system and in this way analyze the air flow through an internal duct of the airfoil using computational fluid dynamics. The main objective is to evaluate the effect of the internal air flow past the airfoil and how this affects the aerodynamic performance by means of lift and drag forces. For this purpose, three different designs of the internal duct were studied; starting from the base 2415-3S airfoil developed in previous investigation, basing on the hypothesis of decreasing the flow separation produced when the propulsive airflow merges the external flow, and in this way obtaining the best configuration. For that purpose, an exhaustive study of the mesh sensitivity was performed. It was used a non-structured mesh since the computational domain is tridimensional and complex. The selected mesh contains approximately 12.5 million elements. Both the computational domain and the numerical solution were made with commercial CAD and CFD software respectively. Air, incompressible and steady was analyzed. The boundary conditions are in concordance with experimental setup in the AF 6109 wind tunnel. The k-ε model is utilized to describe the turbulent flow process as followed in references. Results allowed obtaining pressure and velocity contours as well as lift and drag coefficients and also the location of separation and reattachment regions in some cases for zero degrees of angle of attack on the internal and external surfaces of the airfoil. Finally, the selection of the configuration with the best aerodynamic performance was made, selecting the option without curved baffles.

Isolation of a multispecific organic anion and cardiac glycoside transporter from rat brain

A novel multispecific organic anion transporting polypeptide (oatp2) has been isolated from rat brain. The cloned cDNA contains 3,640 bp. The coding region extends over 1,983 nucleotides, thus encoding a polypeptide of 661 amino acids. Oatp2 is homologous to other members of the oatp gene family of membrane transporters with 12 predicted transmembrane domains, five potential glycosylation, and six potential protein kinase C phosphorylation sites. In functional expression studies in Xenopus laevis oocytes, oatp2 mediated uptake of the bile acids taurocholate (Km ≈ 35 μM) and cholate (Km ≈ 46 μM), the estrogen conjugates 17β-estradiol-glucuronide (Km ≈ 3 μM) and estrone-3-sulfate (Km ≈ 11 μM), and the cardiac gylcosides ouabain (Km ≈ 470 μM) and digoxin (Km ≈ 0.24 μM). Although most of the tested compounds are common substrates of several oatp-related transporters, high-affinity uptake of digoxin is a unique feature of the newly cloned oatp2. On the basis of Northern blot analysis under high-stringency conditions, oatp2 is highly expressed in brain, liver, and kidney but not in heart, spleen, lung, skeletal muscle, and testes. These results provide further support for the overall significance of oatps as a new family of multispecific organic anion transporters. They indicate that oatp2 may play an especially important role in the brain accumulation and toxicity of digoxin and in the hepatobiliary and renal excretion of cardiac glycosides from the body.

Design of compounds that increase the absorption of polar molecules

Hydrophilic drugs are often poorly absorbed when administered orally. There has been considerable interest in the possibility of using absorption enhancers to promote absorption of polar molecules across membrane surfaces. The bile acids are one of the most widely investigated classes of absorption enhancers, but there is disagreement about what features of bile acid enhancers are responsible for their efficacy. We have designed a class of glycosylated bile acid derivatives to evaluate how increasing the hydrophilicity of the steroid nucleus affects the ability to transport polar molecules across membranes. Some of the glycosylated molecules are significantly more effective than taurocholate in promoting the intestinal absorption of a range of drugs, showing that hydrophobicity is not a critical parameter in transport efficacy, as previously suggested. Furthermore, the most effective glycosylated compound is also far less damaging to membranes than the best bile acid absorption promoters, presumably because it is more hydrophilic. The results reported here show that it is possible to decouple absorption-promoting activity from membrane damage, a finding that should spark interest in the design of new compounds to facilitate the delivery of polar drugs.

Transcriptional regulation of the rat Müllerian inhibiting substance type II receptor in rodent Leydig cells

Müllerian inhibiting substance (MIS) causes regression of the fetal Müllerian duct on binding a heteromeric complex of types I and II cell-surface receptors in the fetal urogenital ridge. The MIS type II receptor (MISRII), which provides specificity for MIS, is also expressed in the adult testis, ovary, and uterus. The rat MISRII promoter was cloned to study the molecular mechanisms underlying its temporal and cell-specific expression. The 1.6-kilobase (kb) promoter contained no recognizable TATA or CAAT box, but there was a consensus Sp1 site upstream of the transcription initiation site. Two binding sites for the orphan nuclear receptor steroidogenic factor-1 (SF-1) are occupied in vitro by using nuclear extracts from R2C cells, an MIS-responsive rat Leydig cell line that expresses endogenous MISRII, with differing affinities, indicating that the distal SF-1 site is bound more avidly than is the proximal SF-1 site. R2C cells transfected with MISRII promoter/luciferase reporter constructs show a 12-fold induction with the 1.6-kb fragment and deletion of sequences upstream of −282-bp lowered luciferase expression to one-third. Mutation of both SF-1 sites greatly inhibited luciferase expression, whereas mutation of either site alone resulted in continuing activation by endogenous SF-1, indicating redundancy. In vitro binding and transcriptional analyses suggest that a proximal potential Smad-responsive element and an uncharacterized element also contribute to activation of the MISRII gene. R2C cells and MISRII promoter regulation can now be used to uncover endogenous transcription factors responsible for receptor expression or repression.

Disruption of the murine gene encoding phosphatidylethanolamine N-methyltransferase

All nucleated cells make phosphatidylcholine via the CDP-choline pathway. Liver has an alternative pathway in which phosphatidylcholine is made by methylation of phosphatidylethanolamine catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). We investigated the function of PEMT and its role in animal physiology by targeted disruption of its gene, Pempt2. A targeting vector that interrupts exon 2 was constructed and introduced into mice yielding three genotypes: normal (+/+), heterozygotes (+/−), and homozygotes (−/−) for the disrupted PEMT gene. Only a trace of PE methylation activity remained in Pempt2(−/−) mice. Antibody to one form of the enzyme, PEMT2, indicated complete loss of this protein from Pempt2(−/−) mice and a decrease in Pempt2(+/−) mice, compared with Pempt2(+/+) mice. The levels of hepatic phosphatidylethanolamine and phosphatidylcholine were minimally affected. The active form of CTP:phosphocholine cytidylyltransferase, the regulated enzyme in the CDP-choline pathway, was increased 60% in the PEMT-deficient mice. Injection of [l-methyl-3H]methionine demonstrated that the in vivo PEMT activity was eliminated in the Pempt2(−/−) mice and markedly decreased in the Pempt2(+/−) mice. This experiment also demonstrated that the choline moiety derived from PEMT in the liver can be distributed via the plasma throughout the mouse where it is found as phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin. Mice homozygous for the disrupted Pempt2 gene displayed no abnormal phenotype, normal hepatocyte morphology, normal plasma lipid levels and no differences in bile composition. This is the first application of the “knockout mouse” technique to a gene for phospholipid biosynthesis.

The thiazide-sensitive Na–Cl cotransporter is an aldosterone-induced protein

Although the collecting duct is regarded as the primary site at which mineralocorticoids regulate renal sodium transport in the kidney, recent evidence points to the distal convoluted tubule as a possible site of mineralocorticoid action. To investigate whether mineralocorticoids regulate the expression of the thiazide-sensitive Na–Cl cotransporter (TSC), the chief apical sodium entry pathway of distal convoluted tubule cells, we prepared an affinity-purified, peptide-directed antibody to TSC. On immunoblots, the antibody recognized a prominent 165-kDa band in membrane fractions from the renal cortex but not from the renal medulla. Immunofluorescence immunocytochemistry showed TSC labeling only in distal convoluted tubule cells. Semiquantitative immunoblotting studies demonstrated a large increase in TSC expression in the renal cortex of rats on a low-NaCl diet (207 ± 21% of control diet). Immunofluorescence localization in tissue sections confirmed the strong increase in TSC expression. Treatment of rats for 10 days with a continuous subcutaneous infusion of aldosterone also increased TSC expression (380 ± 58% of controls). Furthermore, 7-day treatment of rats with an orally administered mineralocorticoid, fludrocortisone, increased TSC expression (656 ± 114% of controls). We conclude that the distal convoluted tubule is an important site of action of the mineralocorticoid aldosterone, which strongly up-regulates the expression of TSC.

Serial microanalysis of renal transcriptomes

Large-scale gene expression studies can now be routinely performed on macroamounts of cells, but it is unclear to which extent current methods are valuable for analyzing complex tissues. In the present study, we used the method of serial analysis of gene expression (SAGE) for quantitative mRNA profiling in the mouse kidney. We first performed SAGE at the whole-kidney level by sequencing 12,000 mRNA tags. Most abundant tags corresponded to transcripts widely distributed or enriched in the predominant kidney epithelial cells (proximal tubular cells), whereas transcripts specific for minor cell types were barely evidenced. To better explore such cells, we set up a SAGE adaptation for downsized extracts, enabling a 1,000-fold reduction of the amount of starting material. The potential of this approach was evaluated by studying gene expression in microdissected kidney tubules (50,000 cells). Specific gene expression profiles were obtained, and known markers (e.g., uromodulin in the thick ascending limb of Henle's loop and aquaporin-2 in the collecting duct) were found appropriately enriched. In addition, several enriched tags had no databank match, suggesting that they correspond to unknown or poorly characterized transcripts with specific tissue distribution. It is concluded that SAGE adaptation for downsized extracts makes possible large-scale quantitative gene expression measurements in small biological samples and will help to study the tissue expression and function of genes not evidenced with other high-throughput methods.

Actin Filaments and Microtubules are Involved in Different Membrane Traffic Pathways That Transport Sphingolipids to the Apical Surface of Polarized HepG2 Cells

In polarized HepG2 hepatoma cells, sphingolipids are transported to the apical, bile canalicular membrane by two different transport routes, as revealed with fluorescently tagged sphingolipid analogs. One route involves direct, transcytosis-independent transport of Golgi-derived glucosylceramide and sphingomyelin, whereas the other involves basolateral to apical transcytosis of both sphingolipids. We show that these distinct routes display a different sensitivity toward nocodazole and cytochalasin D, implying a specific transport dependence on either microtubules or actin filaments, respectively. Thus, nocodazole strongly inhibited the direct route, whereas sphingolipid transport by transcytosis was hardly affected. Moreover, nocodazole blocked “hyperpolarization,” i.e., the enlargement of the apical membrane surface, which is induced by treating cells with dibutyryl-cAMP. By contrast, the transcytotic route but not the direct route was inhibited by cytochalasin D. The actin-dependent step during transcytotic lipid transport probably occurs at an early endocytic event at the basolateral plasma membrane, because total lipid uptake and fluid phase endocytosis of horseradish peroxidase from this membrane were inhibited by cytochalasin D as well. In summary, the results show that the two sphingolipid transport pathways to the apical membrane must have a different requirement for cytoskeletal elements.

Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis

Mutations in the sister of P-glycoprotein (Spgp) or bile salt export pump (BSEP) are associated with Progressive Familial Intrahepatic Cholestasis (PFIC2). Spgp is predominantly expressed in the canalicular membranes of liver. Consistent with in vitro evidence demonstrating the involvement of Spgp in bile salt transport, PFIC2 patients secrete less than 1% of biliary bile salts compared with normal infants. The disease rapidly progresses to hepatic failure requiring liver transplantation before adolescence. In this study, we show that the knockout of spgp gene in mice results in intrahepatic cholestasis, but with significantly less severity than PFIC2 in humans. Some unexpected characteristics are observed. Notably, although the secretion of cholic acid in mutant mice is greatly reduced (6% of wild-type), total bile salt output in mutant mice is about 30% of wild-type. Also, secretion of an unexpectedly large amount of tetra-hydroxylated bile acids (not detected in wild-type) is observed. These results suggest that hydroxylation and an alternative canalicular transport mechanism for bile acids compensate for the absence of Spgp function and protect the mutant mice from severe cholestatic damage. In addition, the spgp−/− mice display a significant increase in the secretion of cholesterol and phospholipids into the bile. This latter observation in spgp−/− mice suggests that intrahepatic, rather than intracanalicular, bile salts are the major driving force for the biliary lipid secretion. The spgp−/− mice thus provide a unique model for gaining new insights into therapeutic intervention for intrahepatic cholestasis and understanding mechanisms associated with lipid homeostasis.