Inhibitory and inductive effects of rifampin on the pharmacokinetics of bosentan in healthy subjects.

This study was conducted to investigate the effect of rifampin on the pharmacokinetics of bosentan. Healthy male subjects received bosentan 125 mg b.i.d. for 6.5 days in the presence or absence of rifampin 600 mg once a day. In vitro experiments were performed to investigate the effect of rifampin on the uptake of bosentan into Chinese hamster ovary cells expressing the human organic anion-transporting polypeptide (OATP)1B1, -1B3, and -2B1. Following the first concomitant administration, there was a fivefold increase in bosentan trough concentrations. At steady state, concomitant rifampin significantly decreased exposure to bosentan by 58%. Rifampin potently inhibited the uptake of bosentan into cells expressing human OATP1B1 and -1B3. Rifampin decreased the exposure to bosentan consistent with its known cytochrome P450 enzyme-inductive properties. The initial increase in bosentan concentrations can be explained by an inhibitory effect of rifampin on hepatic drug transporters.

Saccharomyces cerevisiae, a model to study sterol uptake and transport in eukaryotes.

The molecular mechanisms that govern intracellular transport of sterols in eukaryotic cells are only poorly understood. Saccharomyces cerevisiae is a facultative anaerobic organism that requires supplementation with unsaturated fatty acids and sterols to grow in the absence of oxygen, as the synthesis of these lipids requires molecular oxygen. The fact that yeast grows well under anaerobic conditions indicates that lipid uptake is rapid and efficient. To identify components in this lipid uptake and transport pathway, we screened the yeast mutant collection for genes that are essential under anaerobic conditions. Out of the approx. 4800 non-essential genes represented in the mutant collection, 37 were required for growth under anaerobic conditions. Uptake assays using radiolabelled cholesterol revealed that 16 of these genes are required for cholesterol uptake/transport and esterification. Further characterization of the precise role of these genes is likely to advance our understanding of this elusive pathway in yeast and may prove to be relevant to understand sterol homoeostasis in higher eukaryotic cells.

Incidence and management of Mirizzi syndrome during laparoscopic cholecystectomy.

BACKGROUND: Benign extrinsic obstruction of the hepatic duct, known as "Mirizzi syndrome" (MS), is an uncommon complication of longstanding cholelithiasis. Since laparoscopic cholecystectomy (LC) replaced the open approach, Mirizzi syndrome has regained the interest of biliary surgeons. METHODS: The Swiss Association for Laparoscopic and Thoracoscopic Surgery (SALTS) prospectively collected the data on 13,023 patients undergoing LC between 1995 and 1999. This database was investigated with special regard to patients with Mirizzi syndrome. RESULTS: There were 39 patients (14 men and 25 women; mean age, 61 years) with MS (incidence, 0.3%). Thirty-four patients had type 1 MS and five had type 2. A gallbladder carcinoma was found in four patients (incidence, 11%). In the type 1 group, 23 patients underwent cholecystectomy only, 10 patients had a bile duct exploration and T-tube insertion, and one patient had a Roux-en-Y reconstruction. In three patients with type 2, a hepaticojejunostomy was performed; two others underwent simple closure and drainage (via T-tube) of the biliary fistula. The conversion rate was 74% (24 of 34 patients) in the type 1 group and 100% (five of five patients) for type 2. The overall complication rate was 18%. There were no deaths. CONCLUSIONS: Although MS is rarely encountered during LC, it must be recognized intraoperatively. Conversion to an open approach is often needed, and prior to any surgical intervention, gallbladder cancer must be excluded.

Roles of phosphatidylethanolamine and of its several biosynthetic pathways in Saccharomyces cerevisiae.

Three different pathways lead to the synthesis of phosphatidylethanolamine (PtdEtn) in yeast, one of which is localized to the inner mitochondrial membrane. To study the contribution of each of these pathways, we constructed a series of deletion mutants in which different combinations of the pathways are blocked. Analysis of their growth phenotypes revealed that a minimal level of PtdEtn is essential for growth. On fermentable carbon sources such as glucose, endogenous ethanolaminephosphate provided by sphingolipid catabolism is sufficient to allow synthesis of the essential amount of PtdEtn through the cytidyldiphosphate (CDP)-ethanolamine pathway. On nonfermentable carbon sources, however, a higher level of PtdEtn is required for growth, and the amounts of PtdEtn produced through the CDP-ethanolamine pathway and by extramitochondrial phosphatidylserine decarboxylase 2 are not sufficient to maintain growth unless the action of the former pathway is enhanced by supplementing the growth medium with ethanolamine. Thus, in the absence of such supplementation, production of PtdEtn by mitochondrial phosphatidylserine decarboxylase 1 becomes essential. In psd1Delta strains or cho1Delta strains (defective in phosphatidylserine synthesis), which contain decreased amounts of PtdEtn, the growth rate on nonfermentable carbon sources correlates with the content of PtdEtn in mitochondria, suggesting that import of PtdEtn into this organelle becomes growth limiting. Although morphological and biochemical analysis revealed no obvious defects of PtdEtn-depleted mitochondria, the mutants exhibited an enhanced formation of respiration-deficient cells. Synthesis of glycosylphosphatidylinositol-anchored proteins is also impaired in PtdEtn-depleted cells, as demonstrated by delayed maturation of Gas1p. Carboxypeptidase Y and invertase, on the other hand, were processed with wild-type kinetics. Thus, PtdEtn depletion does not affect protein secretion in general, suggesting that high levels of nonbilayer-forming lipids such as PtdEtn are not essential for membrane vesicle fusion processes in vivo.

Depletion of acyl-coenzyme A-binding protein affects sphingolipid synthesis and causes vesicle accumulation and membrane defects in Saccharomyces cerevisiae.

Deletion of the yeast gene ACB1 encoding Acb1p, the yeast homologue of the acyl-CoA-binding protein (ACBP), resulted in a slower growing phenotype that adapted into a faster growing phenotype with a frequency >1:10(5). A conditional knockout strain (Y700pGAL1-ACB1) with the ACB1 gene under control of the GAL1 promoter exhibited an altered acyl-CoA profile with a threefold increase in the relative content of C18:0-CoA, without affecting total acyl-CoA level as previously reported for an adapted acb1Delta strain. Depletion of Acb1p did not affect the general phospholipid pattern, the rate of phospholipid synthesis, or the turnover of individual phospholipid classes, indicating that Acb1p is not required for general glycerolipid synthesis. In contrast, cells depleted for Acb1p showed a dramatically reduced content of C26:0 in total fatty acids and the sphingolipid synthesis was reduced by 50-70%. The reduced incorporation of [(3)H]myo-inositol into sphingolipids was due to a reduced incorporation into inositol-phosphoceramide and mannose-inositol-phosphoceramide only, a pattern that is characteristic for cells with aberrant endoplasmic reticulum to Golgi transport. The plasma membrane of the Acb1p-depleted strain contained increased levels of inositol-phosphoceramide and mannose-inositol-phosphoceramide and lysophospholipids. Acb1p-depleted cells accumulated 50- to 60-nm vesicles and autophagocytotic like bodies and showed strongly perturbed plasma membrane structures. The present results strongly suggest that Acb1p plays an important role in fatty acid elongation and membrane assembly and organization.

Molecular identification of virilizer, a gene required for the expression of the sex-determining gene Sex-lethal in Drosophila melanogaster.

Sex-lethal (Sxl) is a central switch gene in somatic sexual development of Drosophila melanogaster. Female-specific expression of Sxl relies on autoregulatory splicing of Sxl pre-mRNA by SXL protein. This process requires the function of virilizer (vir). Besides its role in Sxl splicing, vir is essential for male and female viability and is also required for the production of eggs capable of embryonic development. We have identified vir molecularly and found that it produces a single transcript of 6 kb that is ubiquitously expressed in male and female embryos throughout development. This transcript encodes a nuclear protein of 210 kD that cannot be assigned to a known protein family. VIR contains a putative transmembrane domain, a coiled-coil region and PEST sequences. We have characterized five different alleles of vir. Those alleles that affect both sexes are associated with large truncations of the protein, while alleles that affect only the female-specific functions are missense mutations that lie relatively close to each other, possibly defining a region important for the regulation of Sxl.

Elo1p-dependent carboxy-terminal elongation of C14:1Delta(9) to C16:1Delta(11) fatty acids in Saccharomyces cerevisiae.

Saccharomyces cerevisiae medium-chain acyl elongase (ELO1) mutants have previously been isolated in screens for fatty acid synthetase (FAS) mutants that fail to grow on myristic acid (C14:0)-supplemented media. Here we report that wild-type cells cultivated in myristoleic acid (C14:1Delta(9))-supplemented media synthesized a novel unsaturated fatty acid that was identified as C16:1Delta(11) fatty acid by gas chromatography-mass spectroscopy. Synthesis of C16:1Delta(11) was dependent on a functional ELO1 gene, indicating that Elo1p catalyzes carboxy-terminal elongation of unsaturated fatty acids (alpha-elongation). In wild-type cells, the C16:1Delta(11) elongation product accounted for approximately 12% of the total fatty acids. This increased to 18% in cells that lacked a functional acyl chain desaturase (ole1Delta mutants) and hence were fully dependent on uptake and elongation of C14:1. The observation that ole1Delta mutant cells grew almost like wild type on medium supplemented with C14:1 indicated that uptake and elongation of unsaturated fatty acids were efficient. Interestingly, wild-type cells supplemented with either C14:1 or C16:1 fatty acids displayed dramatic alterations in their phospholipid composition, suggesting that the availability of acyl chains is a dominant determinant of the phospholipid class composition of cellular membranes. In particular, the relative content of the two major phospholipid classes, phosphatidylethanolamine and phosphatidylcholine, was strongly dependent on the chain length of the supplemented fatty acid. Moreover, analysis of the acyl chain composition of individual phospholipid classes in cells supplemented with C14:1 revealed that the relative degree of acyl chain saturation characteristic for each phospholipid class appeared to be conserved, despite the gross alteration in the cellular acyl chain pool. Comparison of the distribution of fatty acids that were taken up and elongated (C16:1Delta(11)) to those that were endogenously synthesized by fatty acid synthetase and then desaturated by Ole1p (C16:1Delta(9)) in individual phospholipid classes finally suggested the presence of two different pools of diacylglycerol species. These results will be discussed in terms of biosynthesis of different phospholipid classes via either the de novo or the Kennedy pathway.

A novel cold-sensitive allele of the rate-limiting enzyme of fatty acid synthesis, acetyl coenzyme A carboxylase, affects the morphology of the yeast vacuole through acylation of Vac8p.

The yeast vacuole functions both as a degradative organelle and as a storage depot for small molecules and ions. Vacuoles are dynamic reticular structures that appear to alternately fuse and fragment as a function of growth stage and environment. Vac8p, an armadillo repeat-containing protein, has previously been shown to function both in vacuolar inheritance and in protein targeting from the cytoplasm to the vacuole. Both myristoylation and palmitoylation of Vac8p are required for its efficient localization to the vacuolar membrane (Y.-X. Wang, N. L. Catlett, and L. S. Weisman, J. Cell Biol. 140:1063-1074, 1998). We report that mutants with conditional defects in the rate-limiting enzyme of fatty acid synthesis, acetyl coenzyme A carboxylase (ACC1), display unusually multilobed vacuoles, similar to those observed in vac8 mutant cells. This vacuolar phenotype of acc1 mutant cells was shown biochemically to be accompanied by a reduced acylation of Vac8p which was alleviated by fatty acid supplementation. Consistent with the proposed defect of acc1 mutant cells in acylation of Vac8p, vacuolar membrane localization of Vac8p was impaired upon shifting acc1 mutant cells to nonpermissive condition. The function of Vac8p in protein targeting, on the other hand, was not affected under these conditions. These observations link fatty acid synthesis and availability to direct morphological alterations of an organellar membrane.

Brave little yeast, please guide us to thebes: sphingolipid function in S. cerevisiae.

Sphingolipids typically cover the exoplasmic leaflet of the plasma membrane of eukaryotic cells. They differ from the more abundant glycerophospholipids in that they contain ceramide instead of diacylglycerol as a hydrophobic anchor. Why did nature choose to invent this complex class of lipids, and why do eukaryotic cells follow elaborate remodelling pathways in order to generate dozens to hundreds of different molecular species of sphingolipid, depending on cell type? Yeast may, once again, serve as a model to dissect sphingolipid function at various levels. Almost the complete pathway for sphingolipid synthesis in yeast has been uncovered during the past two decades. More recently, key enzymes in sphingolipid degradation and signalling have been identified. Together with a wealth of genetic data obtained from the characterization of various suppressor mutants, this information now allows for an unprecedented analysis of sphingolipid function in this organism. This overview summarizes recent data on sphingolipid function in cell signalling, their role in the heat-stress response and Ca(2+) homeostasis, and addresses their function in transport of glycosylphosphatidylinositol-anchored proteins.

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane.

Nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) was employed to determine qualitative differences in the lipid molecular species composition of a comprehensive set of organellar membranes, isolated from a single culture of Saccharomyces cerevisiae cells. Remarkable differences in the acyl chain composition of biosynthetically related phospholipid classes were observed. Acyl chain saturation was lowest in phosphatidylcholine (15.4%) and phosphatidylethanolamine (PE; 16.2%), followed by phosphatidylserine (PS; 29.4%), and highest in phosphatidylinositol (53.1%). The lipid molecular species profiles of the various membranes were generally similar, with a deviation from a calculated average profile of approximately +/- 20%. Nevertheless, clear distinctions between the molecular species profiles of different membranes were observed, suggesting that lipid sorting mechanisms are operating at the level of individual molecular species to maintain the specific lipid composition of a given membrane. Most notably, the plasma membrane is enriched in saturated species of PS and PE. The nature of the sorting mechanism that determines the lipid composition of the plasma membrane was investigated further. The accumulation of monounsaturated species of PS at the expense of diunsaturated species in the plasma membrane of wild-type cells was reversed in elo3Delta mutant cells, which synthesize C24 fatty acid-substituted sphingolipids instead of the normal C26 fatty acid-substituted species. This observation suggests that acyl chain-based sorting and/or remodeling mechanisms are operating to maintain the specific lipid molecular species composition of the yeast plasma membrane.

The Saccharomyces cerevisiae hyperrecombination mutant hpr1Delta is synthetically lethal with two conditional alleles of the acetyl coenzyme A carboxylase gene and causes a defect in nuclear export of polyadenylated RNA.

In a screen for mutants that display synthetic lethal interaction with hpr1Delta, a hyperrecombination mutant of Saccharomyces cerevisiae, we have isolated a novel cold-sensitive allele of the acetyl coenzyme A (CoA) carboxylase gene, acc1(cs), encoding the rate-limiting enzyme of fatty acid synthesis. The synthetic lethal phenotype of the acc1(cs) hpr1Delta double mutant was only partially complemented by exogenous fatty acids. hpr1Delta was also synthetically lethal with a previously isolated, temperature-sensitive allele of ACC1, mtr7 (mRNA transport), indicating that the lethality of the acc1(cs) hpr1Delta double mutant was not allele specific. The basis for the interaction between conditional acc1 alleles and hpr1Delta was investigated in more detail. In the hpr1Delta mutant background, acetyl-CoA carboxylase enzyme activity was reduced about 15-fold and steady-state levels of biotinylated Acc1p and ACC1 mRNA were reduced 2-fold. The reduced Acc1p activity in hpr1Delta cells, however, did not result in an altered lipid or fatty acid composition of the mutant membranes but rendered cells hypersensitive to soraphen A, an inhibitor of Acc1p. Similar to mtr7, hpr1Delta and acc1(cs) mutant cells displayed a defect in nuclear export of polyadenylated RNA. Oversized transcripts were detected in hpr1Delta, and rRNA processing was disturbed, but pre-mRNA splicing appeared wild type. Surprisingly, the transport defect of hpr1Delta and acc1(cs) mutant cells was accompanied by an altered ring-shaped structure of the nucleolus. These observations suggest that the basis for the synthetic lethal interaction between hpr1Delta and acc1 may lie in a functional overlap of the two mutations in nuclear poly(A)+ RNA production and export that results in an altered structure of the nucleolus.

The yeast mic2 mutant is defective in the formation of mannosyl-diinositolphosphorylceramide.

The mic2 mutation dominantly blocks formation of mannosyl-diinositolphosphorylceramide, the most abundant sphingolipid of the yeast, Saccharomyces cerevisiae. Interestingly, lack of mannosyl-diinositolphosphorylceramide is not lethal but is compensated for by increased amounts of inositolphosphorylceramide and mannosyl-inositolphosphorylceramide in the plasma membrane and Golgi of the mutant. The level of negatively charged phospholipids in the plasma membrane of the mic2 strain is markedly reduced; the sterol composition is not altered. In spite of dramatic changes of its lipid composition the mutant grows like wild type on complex and minimal media, under osmotic stress conditions, at low pH, and in the presence of high ionic strength. While sensitivity to several drugs is not altered, the mic2 mutant strain becomes resistant to the polyene antibiotic nystatin.

Yeast acetyl-CoA carboxylase is associated with the cytoplasmic surface of the endoplasmic reticulum.

Acetyl-CoA carboxylase (ACC1) catalyzes the first and rate limiting step of de novo fatty acid synthesis. Defects in Acc1p were recently correlated with an altered structure/function of the nuclear envelope in yeast. The subcellular distribution of the enzyme was determined in wild-type and mutant cells by cell fractionation and confocal immunofluorescence microscopy. Even though fatty acid synthesis is generally considered to be a cytosolic reaction, we found that Acc1p cofractionated with nuclei and the ER (endoplasmic reticulum) marker BiP/Kar2p. Membrane-bound Acc1p was susceptible to proteinase K digestion and was solubilized by mild salt treatment indicating that it is loosely associated with the cytosolic surface of the nuclear ER membrane. Consistent with these observations, immunofluorescence analysis revealed that Acc1p was distributed in a gradient within the cytoplasm that had its highest concentration around the ER. Possible association of Acc1p with the nuclear pore complexes (NPCs) was investigated in strains that display NPC clustering. Results of these experiments suggest that Acc1p localization is independent of NPC distribution. We propose that association of Acc1p with the cytoplasmic surface of the ER membrane is physiologically relevant to "channel" the enzymatic product of Acc1p, malonyl-CoA, to a putative ER-localized fatty acid chain elongase complex.

A yeast acetyl coenzyme A carboxylase mutant links very-long-chain fatty acid synthesis to the structure and function of the nuclear membrane-pore complex.

The conditional mRNA transport mutant of Saccharomyces cerevisiae, acc1-7-1 (mtr7-1), displays a unique alteration of the nuclear envelope. Unlike nucleoporin mutants and other RNA transport mutants, the intermembrane space expands, protuberances extend from the inner membrane into the intermembrane space, and vesicles accumulate in the intermembrane space. MTR7 is the same gene as ACC1, encoding acetyl coenzyme A (CoA) carboxylase (Acc1p), the rate-limiting enzyme of de novo fatty acid synthesis. Genetic and biochemical analyses of fatty acid synthesis mutants and acc1-7-1 indicate that the continued synthesis of malonyl-CoA, the enzymatic product of acetyl-CoA carboxylase, is required for an essential pathway which is independent from de novo synthesis of fatty acids. We provide evidence that synthesis of very-long-chain fatty acids (C26 atoms) is inhibited in acc1-7-1, suggesting that very-long-chain fatty acid synthesis is required to maintain a functional nuclear envelope.

Phospholipids: synthesis, sorting, subcellular traffic - the yeast approach.

Most of the enzymes and genes required for lipid biosynthesis and degradation in the budding yeast Saccharomyces cerevisiae have now been identified and the global mechanisms that regulate their activity are being established. Synthesis of phospholipids is restricted to specific subcellular compartments, and the lipids migrate from their site of formation to their final destination. In addition to synthesis, remodelling and degradation of phospholipids controls the content of the lipid portion of cellular membranes, while highly specific phospholipases catalyse the release of lipid-based second messengers. In this review, we describe the current understanding of the organization and regulation of phospholipid metabolism in yeast, and discuss the mechanisms that have been proposed for intracellular lipid transport.

The gene virilizer is required for female-specific splicing controlled by Sxl, the master gene for sexual development in Drosophila.

The gene virilizer (vir) is needed for dosage compensation and sex determination in females and for an unknown vital function in both sexes. In genetic mosaics, XX somatic cells mutant for vir differentiate male structures. One allele, vir2f, is lethal for XX, but not for XY animals. This female-specific lethality can be rescued by constitutive expression of Sxl or by mutations in msl (male-specific lethal) genes. Rescued animals develop as strongly masculinized intersexes or pseudomales. They have male-specifically spliced mRNA of tra, and when rescued by msl, also of Sxl. Our data indicate that vir is a positive regulator of female-specific splicing of Sxl and of tra pre-mRNA.