Inertial forces affect fluid front displacement dynamics in a pore-throat network model.

The seemingly regular and continuous motion of fluid displacement fronts in porous media at the macroscopic scale is propelled by numerous (largely invisible) pore-scale abrupt interfacial jumps and pressure bursts. Fluid fronts in porous media are characterized by sharp phase discontinuities and by rapid pore-scale dynamics that underlie their motion; both attributes challenge standard continuum theories of these flow processes. Moreover, details of pore-scale dynamics affect front morphology and subsequent phase entrapment behind a front and thereby shape key macroscopic transport properties of the unsaturated zone.

Interfacial jumps and pressure bursts during fluid displacement in interacting irregular capillaries.

The macroscopically regular motion of fluid displacement fronts in porous media often results from numerous pore scale interfacial jumps and associated pressure fluctuations. Such rapid pore scale dynamics defy postulated slow viscous energy dissipation and may shape phase entrapment and subsequent macroscopic transport properties. Certain displacement characteristics are predictable from percolation theory; however, insights into rapid interfacial dynamics require mechanistic models for hydraulically interacting pores such as found along fluid displacement fronts.

Discovery of high-affinity ligands of sigma1 receptor, ERG2, and emopamil binding protein by pharmacophore modeling and virtual screening.

ERG2, emopamil binding protein (EBP), and sigma-1 receptor (sigma(1)) are enzymes of sterol metabolism and an enzyme-related protein, respectively, that share high affinity for various structurally diverse compounds. To discover novel high-affinity ligands, pharmacophore models were built with Catalyst based upon a series of 23 structurally diverse chemicals exhibiting K(i) values from 10 pM to 100 microM for all three proteins. In virtual screening experiments, we retrieved drugs that were previously reported to bind to one or several of these proteins and also tested 11 new hits experimentally, of which three, among them raloxifene, had affinities for sigma(1) or EBP of <60 nM.

Cloning of an emopamil-binding protein (EBP)-like protein that lacks sterol delta8-delta7 isomerase activity.

EBP (emopamil-binding protein) is a high-affinity binding protein for [3H]emopamil and belongs to the family of so-called sigma receptors. Mutations that disrupt EBP's 3beta-hydroxysteroid sterol delta8-delta7 isomerase activity (EC 5.3.

Isolation of differentially expressed genes in NPM-ALK-positive anaplastic large cell lymphoma.

In this study, we used subtractive suppression hybridization to compare gene expression between an ALK-positive anaplastic large cell lymphoma (ALCL)-derived cell line and a clinical case of ALK-negative ALCL. Construction and screening of a subtracted library resulted in the cloning of 29 cDNAs which were differentially expressed. Most of these clones corresponded to novel genes with unknown function (EST) or to genes implicated in the differentiation, activation or signalling of T cells such as Ran/TC4, interleukin 1-receptor, thymosin beta4, thymosin beta10, moesin and cytohesin-1.

7-Dehydrocholesterol-dependent proteolysis of HMG-CoA reductase suppresses sterol biosynthesis in a mouse model of Smith-Lemli-Opitz/RSH syndrome.

Smith-Lemli-Opitz/RSH syndrome (SLOS), a relatively common birth-defect mental-retardation syndrome, is caused by mutations in DHCR7, whose product catalyzes an obligate step in cholesterol biosynthesis, the conversion of 7-dehydrocholesterol to cholesterol. A null mutation in the murine Dhcr7 causes an identical biochemical defect to that seen in SLOS, including markedly reduced tissue cholesterol and total sterol levels, and 30- to 40-fold elevated concentrations of 7-dehydrocholesterol. Prenatal lethality was not noted, but newborn homozygotes breathed with difficulty, did not suckle, and died soon after birth with immature lungs, enlarged bladders, and, frequently, cleft palates.

Regulation of melastatin, a TRP-related protein, through interaction with a cytoplasmic isoform.

The TRP (transient receptor potential) superfamily includes a group of subfamilies of channel-like proteins mediating a multitude of physiological signaling processes. The TRP-melastatin (TRPM) subfamily includes the putative tumor suppressor melastatin (MLSN) and is a poorly characterized group of TRP-related proteins. Here, we describe the identification and characterization of an additional TRPM protein TRPM4.

Expression of the purported sigma(1) (sigma(1)) receptor in the mammalian brain and its possible relevance in deficits induced by antagonism of the NMDA receptor complex as revealed using an antisense strategy.

Sigma (sigma) receptors have generated a great deal of interest on the basis of their possible role in psychosis, neuroprotection and various other behaviors including learning processes. The existence of at least two classes of sigma receptor binding sites (sigma(1) and sigma(2)) is now well established. The recent cloning of the mouse, guinea pig and human sigma(1) receptors has allowed the study of the discrete distribution of the sigma(1) receptor mRNA in rodent and human brain tissues using in situ hybridization.

Mutational spectrum in the Delta7-sterol reductase gene and genotype-phenotype correlation in 84 patients with Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS), an autosomal recessive malformation syndrome, ranges in clinical severity from mild dysmorphism and moderate mental retardation to severe congenital malformation and intrauterine lethality. Mutations in the gene for Delta7-sterol reductase (DHCR7), which catalyzes the final step in cholesterol biosynthesis in the endoplasmic reticulum (ER), cause SLOS. We have determined, in 84 patients with clinically and biochemically characterized SLOS (detection rate 96%), the mutational spectrum in the DHCR7 gene.

Genetic defects in postsqualene cholesterol biosynthesis.

In humans and mice, four different genetic defects in the nine biosynthetic steps from lanosterol to cholesterol have been identified. They impair the activity of a putative C3-sterol dehydrogenase (Nshdl, X-linked dominant bare patches/striated mutation in mice), the sterol delta 8-delta 7 isomerase/EBP (Ebp, X-linked dominant tattered mutation in mice; chondrodysplasia punctata (CDPX2) in humans), the delta 24-sterol reductase (autosomal recessive desmosterolosis) and the delta 7-sterol reductase (DHCR7 gene, autosomal recessive Smith-Lemli-Opitz syndrome in humans). These inborn errors in postsqualene cholesterol metabolism result in dysmorphogenetic syndromes of variable severity.

Mutations in the gene encoding 3 beta-hydroxysteroid-delta 8, delta 7-isomerase cause X-linked dominant Conradi-Hünermann syndrome.

X-linked dominant Conradi-Hünermann syndrome (CDPX2; MIM 302960) is one of a group of disorders with aberrant punctate calcification in cartilage, or chondrodysplasia punctata (CDP). This is most prominent around the vertebral column, pelvis and long bones in CPDX2. Additionally, CDPX2 patients may have asymmetric rhizomesomelia, sectorial cataracts, patchy alopecia, ichthyosis and atrophoderma.

Molecular genetics of the Smith-Lemli-Opitz syndrome and postsqualene sterol metabolism.

The Smith-Lemli-Opitz syndrome is a disorder of morphogenesis resulting from an enzymatic defect in the last step of cholesterol metabolism (reduction of 7-dehydrocholesterol). Analysis of the defective gene and identification of mutations therein have paved the way for the study of the molecular genetics of the disorder which is caused by numerous different mutations. Future efforts should identify a postulated intracellular signalling activity of sterol intermediates, isolate proteins that govern the sterol traffic between intracellular compartments, structurally characterize the enzyme delta 7-sterol reductase defective in the Smith-Lemli-Opitz syndrome and investigate the pathomechanism of sterol depletion-induced dysmorphogenesis.

Histidine77, glutamic acid81, glutamic acid123, threonine126, asparagine194, and tryptophan197 of the human emopamil binding protein are required for in vivo sterol delta 8-delta 7 isomerization.

The human emopamil binding protein (hEBP) exhibits sterol Delta8-Delta7 isomerase activity (EC 5.3.3.

Pharmacological analysis of sterol delta8-delta7 isomerase proteins with [3H]ifenprodil.

Sterol Delta8-Delta7 isomerases (SIs) catalyze the shift of the double bond from C8-9 to C7-8 in the B-ring of sterols. Surprisingly, the isoenzymes in fungi (ERG2p) and vertebrates [emopamil binding protein (EBP)] are structurally completely unrelated, whereas the sigma1 receptor, a mammalian protein of unknown function, bears significant similarity with the yeast ERG2p. Here, we compare the drug binding properties of SIs and related proteins with [3H]ifenprodil as a common high affinity radioligand (Kd = 1.

Mutations in the Delta7-sterol reductase gene in patients with the Smith-Lemli-Opitz syndrome.

The Smith-Lemli-Opitz syndrome (SLOS) is an inborn disorder of sterol metabolism with characteristic congenital malformations and dysmorphias. All patients suffer from mental retardation. Here we identify the SLOS gene as a Delta7-sterol reductase (DHCR7, EC 1.

Molecular cloning and expression of the human delta7-sterol reductase.

Inhibitors of the last steps of cholesterol biosynthesis such as AY9944 and BM15766 severely impair brain development. Their molecular target is the Delta7-sterol reductase (EC 1.3.

High affinity of sigma 1-binding sites for sterol isomerization inhibitors: evidence for a pharmacological relationship with the yeast sterol C8-C7 isomerase.

1. The sigma-drug binding site of guinea-pig liver is carried by a protein which shares significant amino acid sequence similarities with the yeast sterol C8-C7 isomerase (ERG2 protein). Pharmacologically-but not structurally-the sigma 1-site is also related to the emopamil binding protein, the mammalian sterol C8-C7 isomerase.

Yeast sterol C8-C7 isomerase: identification and characterization of a high-affinity binding site for enzyme inhibitors.

The yeast gene ERG2 encodes a sterol C8-C7 isomerase and is essential for ergosterol synthesis and cell proliferation. Its striking homology with the so-called sigma1 receptor of guinea pig brain, a polyvalent steroid and drug binding protein, suggested that the yeast sterol C8-C7 isomerase (ERG2) carries a similar high affinity drug binding domain. Indeed the sigma ligands [3H]haloperidol (Kd = 0.

Identification of PK-A phosphorylation sites in the carboxyl terminus of L-type calcium channel alpha 1 subunits.

Full length L-type calcium channel alpha 1 subunits are rapidly phosphorylated by protein kinase A (PK-A) in vitro and in vivo at sites located in their long carboxyl terminal tails. In skeletal muscle, heart, and brain the majority of biochemically isolated alpha 1 subunits lacks these phosphorylation sites due to posttranslational proteolytic processing. Truncation may therefore modify the regulation of channel activity by PK-A.

Purification, molecular cloning, and expression of the mammalian sigma1-binding site.

Sigma-ligands comprise several chemically unrelated drugs such as haloperidol, pentazocine, and ditolylguanidine, which bind to a family of low molecular mass proteins in the endoplasmic reticulum. These so-called sigma-receptors are believed to mediate various pharmacological effects of sigma-ligands by as yet unknown mechanisms. Based on their opposite enantioselectivity for benzomorphans and different molecular masses, two subtypes are differentiated.

Phenylalkylamine Ca2+ antagonist binding protein. Molecular cloning, tissue distribution, and heterologous expression.

We recently characterized (Moebius, F. F., Burrows, G.

Purification and amino-terminal sequencing of the high affinity phenylalkylamine Ca2+ antagonist binding protein from guinea pig liver endoplasmic reticulum.

A high affinity phenylalkylamine Ca2+ antagonist binding polypeptide (Moebius, F. F., Burrows, G.

Identification of a 27-kDa high affinity phenylalkylamine-binding polypeptide as the sigma 1 binding site by photoaffinity labeling and ligand-directed antibodies.

The verapamil-like arylazide (-)-[3H]azidopamil specifically photoaffinity labeled two low molecular mass polypeptides, with apparent molecular masses of 22 and 27 kDa, in the endoplasmic reticulum of guinea pig liver, kidney, adrenal gland, and lung. It was recently shown that the 22-kDa polypeptide binds the anti-ischemic phenylalkylamine (-)-[3H]emopamil and other anti-ischemic drugs with high affinity. We now provide evidence that the photolabeling of the 27-kDa polypeptide is blocked by nanomolar concentrations of sigma ligands [order of potency, haloperidol > pentazocine > 1,3-ditolylguanidine > dextromethorphan > (+)-SKF10,047].