The small molecule CBR-5884 inhibits the Candida albicans phosphatidylserine synthase.

Systemic infections by Candida spp. are associated with high mortality rates, partly due to limitations in current antifungals, highlighting the need for novel drugs and drug targets. The fungal phosphatidylserine synthase, Cho1, from Candida albicans is a logical antifungal drug target due to its importance in virulence, absence in the host, and conservation among fungal pathogens. Inhibitors of Cho1 could serve as lead compounds for drug development, so we developed a target-based screen for inhibitors of purified Cho1. This enzyme condenses serine and cytidyldiphosphate-diacylglycerol (CDP-DAG) into phosphatidylserine (PS) and releases cytidylmonophosphate (CMP). Accordingly, we developed an in vitro nucleotidase-coupled malachite-green-based high throughput assay for purified C. albicans Cho1 that monitors CMP production as a proxy for PS synthesis. Over 7,300 molecules curated from repurposing chemical libraries were interrogated in primary and dose-responsivity assays using this platform. The screen had a promising average Z' score of ~0.8, and seven compounds were identified that inhibit Cho1. Three of these, ebselen, LOC14, and CBR-5884, exhibited antifungal effects against C. albicans cells, with fungicidal inhibition by ebselen and fungistatic inhibition by LOC14 and CBR-5884. Only CBR-5884 showed evidence of disrupting in vivo Cho1 function by inducing phenotypes consistent with the cho1∆∆ mutant, including a reduction of cellular PS levels. Kinetics curves and computational docking indicate that CBR-5884 competes with serine for binding to Cho1 with a Ki of 1,550 ± 245.6 nM. Thus, this compound has the potential for development into an antifungal compound. IMPORTANCE: Fungal phosphatidylserine synthase (Cho1) is a logical antifungal target due to its crucial role in the virulence and viability of various fungal pathogens, and since it is absent in humans, drugs targeted at Cho1 are less likely to cause toxicity in patients. Using fungal Cho1 as a model, there have been two unsuccessful attempts to discover inhibitors for Cho1 homologs in whole-cell screens prior to this study. The compounds identified in these attempts do not act directly on the protein, resulting in the absence of known Cho1 inhibitors. The significance of our research is that we developed a high-throughput target-based assay and identified the first Cho1 inhibitor, CBR-5884, which acts both on the purified protein and its function in the cell. This molecule acts as a competitive inhibitor with a Ki value of 1,550 ± 245.6 nM and, thus, has the potential for development into a new class of antifungals targeting PS synthase.

Clinical variety and prognosis of intracranial arachnoid cysts in children.

Arachnoid cysts (AC) occur in different intracranial locations. Management and prognosis depend on the clinical presentation and treatment guidelines do not exist. With this study, we want to demonstrate the clinical variety of arachnoid cysts in children and place a focus on outcome factors in operated cases. This retrospective study of a consecutive single unit series of children, who underwent AC surgery between January 2010 and September 2019, provides demographic, clinical, imaging data, and information about surgical treatment and outcome. Overall, 63 patients (71.4 male) underwent surgery. Mean age was 50 months (0-191). Mean follow-up was 40 months (0-121). Eighty-one percent of patients presented with symptoms/signs of raised ICP. Focal neurological deficits were present in 15.9%, headache in 11.1% of children. Galassi cysts represented the predominant type (30.2%), followed by suprasellar (14.3%), quadrigeminal (12.7%), retrocerebellar, CPA and midline (each 11.1%), and hemispheric cysts (7.9%). Endoscopic and microsurgical fenestrations were performed in 27% and 58.7%, stent or shunt insertion in 6.3%/57.9% of the cases. In 33.3% of the cases one and in 12.7%, a second reintervention became necessary. Reoperation rate was significantly higher in children < 1 year (p = 0.003). Cyst volume decreased in 85.7%. Seventy percent of the patients were symptom free, 5% suffered from headache, and 22% from developmental disorders. All focal neurological symptoms resolved. Complication rate and outcome are depending on age and cyst location. Recurrence and revision rates are significantly higher in young infants (p = 0.003). Midline cysts with CCA are associated with developmental disorders.

Genetically Compromising Phospholipid Metabolism Limits Candida albicans' Virulence.

Perturbing ergosterol synthesis has been previously shown to reduce the virulence of Candida albicans. We tested the hypothesis that further altering cell membrane composition by limiting phospholipid synthesis or remodeling will have the same effect. To model partial inhibition, C. albicans strains independently harboring heterozygous deletion of four genes that encode for enzymes that mediate phospholipid synthesis or modification were generated. Quantitative PCR determined that heterozygous deletion routinely caused a nearly 50% reduction in the respective gene's transcript abundance. Compensatory increased transcript abundance was only found with the deletion of LRO1, a homolog of phospholipid diacylglycerol acyltransferases. Virulence of the mutants was assayed in a Caenorhabditis elegans host model. Even modestly reduced expression of LRO1, phosphatidylserine synthase (CHO1), and lysophospholipid acyltransferase (LPT1) significantly reduced virulence by 23-38%. Reintroducing a second functional allele, respectively, to all three mutants restored virulence. Heterozygous deletion of SLC1, a homolog of 1-acylglycerol-3-phosphate O-acyltransferases, did not significantly reduce virulence. Electrospray ionization tandem mass spectrometry analysis of phospholipid composition followed by principal component analysis identified comprehensive changes in the LRO1 and CHO1 deletion heterozygotes. Strikingly (p < 0.001), univariate comparisons found that both deletion heterozygotes had 20% more phosphatidylinositol, 75% less lysophosphatidylcholine, and 35% less lysophosphatidylethanolamine compared to wild type. Heterozygous deletion of LPT1 also significantly increased phosphatidylinositol abundance. No growth phenotype, including filamentation, was affected by any mutation. Together, these data predict that even partial pharmacological inhibition of Lro1p, Cho1p, and Lpt1p will limit C. albicans virulence through altering phospholipid composition.

Semester-long inquiry-based molecular biology laboratory: Transcriptional regulation in yeast.

A single semester molecular biology laboratory has been developed in which students design and execute a project examining transcriptional regulation in Saccharomyces cerevisiae. Three weeks of planning are allocated to developing a hypothesis through literature searches and use of bioinformatics. Common experimental plans address a cell process and how three genes that encode for proteins involved in that process are transcriptionally regulated in response to changing environmental conditions. Planning includes designing oligonucleotides to amplify the putative promoters of the three genes of interest. After the PCR, each product is cloned proximal to ß-galactosidase in a yeast reporter plasmid. Techniques used include agarose electrophoresis, extraction of DNA from agarose, plasmid purification from bacteria, restriction digestion, ligation, and bacterial transformation. This promoter/reporter plasmid is then transformed into yeast. Transformed yeast are cultured in conditions prescribed in the experimental design, lysed and ß-galactosidase activity is measured. The course provides an independent research experience in a group setting. Notebooks are maintained on-line with regular feedback. Projects culminate with the presentation of a poster worth 60% of the grade. Over the last three years, about 65% of students met expectations for experimental design, data acquisition, and analysis. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(2):145-151, 2017.

Four Acyltransferases Uniquely Contribute to Phospholipid Heterogeneity in Saccharomyces cerevisiae.

Diverse acyl-CoA species and acyltransferase isoenzymes are components of a complex system that synthesizes glycerophospholipids and triacylglycerols. Saccharomyces cerevisiae has four main acyl-CoA species, two main glycerol-3-phosphate 1-O-acyltransferases (Gat1p, Gat2p), and two main 1-acylglycerol-3-phosphate O-acyltransferases (Lpt1p, Slc1p). The in vivo contribution of these isoenzymes to phospholipid heterogeneity was determined using haploids with compound mutations: gat1Δlpt1Δ, gat2Δlpt1Δ, gat1Δslc1Δ, and gat2Δslc1Δ. All mutations mildly reduced [3H]palmitic acid incorporation into phospholipids relative to triacylglycerol. Electrospray ionization tandem mass spectrometry identified few differences from wild type in gat1Δlpt1Δ, dramatic differences in gat2Δslc1Δ, and intermediate changes in gat2Δlpt1Δ and gat1Δslc1Δ. Yeast expressing Gat1p and Lpt1p had phospholipids enriched with acyl chains that were unsaturated, 18 carbons long, and paired for length. These alterations prevented growth at 18.5°C and in 10% ethanol. Therefore, Gat2p and Slc1p dictate phospholipid acyl chain composition in rich media at 30°C. Slc1p selectively pairs acyl chains of different lengths.

Energy Storage in Yeast: Regulation and Competition with Ethanol Production.

Mechanisms that may regulate the storage of energy as triacylglycerol in Saccharomyces cerevisiae were examined. First, the kinetics of Dga1p, which mediates the majority of diacylglycerol esterification, the lone committed step in triacylglycerol synthesis, was measured in vitro. With an apparent K m of 17.0 µM, Dga1p has higher affinity for oleoyl-CoA than the only S. cerevisiae acyltransferase previously kinetically characterized, Lpt1p. Lpt1p is a 1-acylglycerol-3-phosphate O-acyltransferase that produces phosphatidate, a precursor to diacylglycerol. Therefore, limiting triacylglycerol synthesis to situations of elevated acyl-CoA concentration is unlikely. However, Dga1p's apparent V max of 5.8 nmol/min/mg was 20 times lower than Lpt1p's. This supports Dga1p being rate limiting for TAG synthesis. Dga1p activity was not activated or inhibited when seven different molecules (e.g., ATP) which reflect cellular energy status were provided at physiological concentrations. Thus, allosteric regulation was not found. Coordination between triacylglycerol and glycogen synthesis was also tested. Yeast genetically deficient in triacylglycerol synthesis did not store more energy in glycogen and vice versa. Lastly, we tested whether genetically limiting energy storage in triacylglycerol, glycogen, steryl esters, or combinations of these will increase ethanol production efficiency. In nutrient-rich media containing 5 % glucose, solely limiting glycogen synthesis had the greatest affect, increasing ethanol production efficiency by 12 %. Since limiting glycogen synthesis only had a modest effect on growth in media containing 10 % ethanol, such genetic manipulation may improve commercial ethanol production.

Saccharomyces cerevisiae lysophospholipid acyltransferase, Lpt1, requires Asp146 and Glu297 for catalysis.

The esterification of lysophospholipids contributes to phospholipid synthesis, remodeling, and scavenging. Acyl-CoA-dependent lysophospholipid acyltransferase activity with broad substrate use is mediated by Saccharomyces cerevisiae Lpt1p. We sought to identify Lpt1p active site amino acids besides the histidine conserved among homologs and repeatedly found to be required for catalysis. In vitro Lpt1p assays with amino acid modifying agents implicated aspartate, glutamate, and lysine as active site residues. Threonine and tyrosine were not ruled out. Aligning the primary structures of functionally characterized LPT1 homologs from fungi, plants, and animals identified 11 conserved aspartate, glutamate, lysine, threonine, and tyrosine residues. Site-directed mutagenesis of the respective codons showed that changing D146 and E297 abolished activity without abolishing protein expression. The mechanism of Lpt1p was further analyzed using monounsaturated acyl-CoA species with different double bond positions. Delta 6 species showed the highest catalytic efficiency. We propose that D146 and E297 act in conjunction with H382 as nucleophiles that attack the hydroxyl group in lysophospholipids in a general acid/base mechanism. This sequential mechanism provides a precedent for other members of the membrane bound O-acyltransferase family. Also, Lpt1p optimally orients acyl-CoA substrates with 7.5 Å between a double bond and the thioester bond.

Characterization of a lysophospholipid acyltransferase involved in membrane remodeling in Candida albicans.

Phospholipid remodeling involves phospholipase activity to remove acyl chains and acyltransferases to replace acyl chains. We here describe the characterization of a lysophospholipid acyltransferase in the opportunistic fungal pathogen, Candida albicans. Expression of this gene, C.a. LPT1, complemented the lysophospholipid acyltransferase defect in Saccharomyces cerevisiae strains lacking the homologous LPT1 gene. In vitro, lysophospholipid acyltransferase activity in these strains showed acyl-CoA substrate specificity, as measured by apparent Vmax/Km ratios, to be linolenoyl-CoA>oleoyl-CoA>linoleoyl-CoA>stearoyl-CoA. To address the physiological importance of C.a. LPT1, homozygous deletion strains were generated. Lysophospholipid acyltransferase activity with amine containing lysophospholipids was dramatically reduced while lysophosphatidylinositol and lysophosphatidic acid esterification was not significantly lowered. However, C.a. LPT1 over-expression yielded an increased amount of lysophosphatidic acyltransferase activity, suggesting a role in de novo phospholipid synthesis. LPT1 deletion strains showed slightly slowed growth in standard liquid media but no phenotype in media containing three antifungals that target sterols. To assess the role of C.a. Lpt1 in phospholipid remodeling, an in vivo, pulse-chase assay utilizing polysorbitan palmitate and mass spectrometry was developed. Cellular phospholipid composition became atypical with the provision of palmitate and gradually returned to the typical distribution when palmitate was removed. Deletion of C.a. LPT1 showed a modest yet significant effect on remodeling under these conditions.

Sterol and diacylglycerol acyltransferase deficiency triggers fatty acid-mediated cell death.

Deletion of the acyltransferases responsible for triglyceride and steryl ester synthesis in Saccharomyces cerevisiae serves as a genetic model of diseases where lipid overload is a component. The yeast mutants lack detectable neutral lipids and cytoplasmic lipid droplets and are strikingly sensitive to unsaturated fatty acids. Expression of human diacylglycerol acyltransferase 2 in the yeast mutants was sufficient to reverse these phenotypes. Similar to mammalian cells, fatty acid-mediated death in yeast is apoptotic and presaged by transcriptional induction of stress-response pathways, elevated oxidative stress, and activation of the unfolded protein response. To identify pathways that protect cells from lipid excess, we performed genetic interaction and transcriptional profiling screens with the yeast acyltransferase mutants. We thus identified diacylglycerol kinase-mediated phosphatidic acid biosynthesis and production of phosphatidylcholine via methylation of phosphatidylethanolamine as modifiers of lipotoxicity. Accordingly, the combined ablation of phospholipid and triglyceride biosynthesis increased sensitivity to saturated fatty acids. Similarly, normal sphingolipid biosynthesis and vesicular transport were required for optimal growth upon denudation of triglyceride biosynthesis and also mediated resistance to exogenous fatty acids. In metazoans, many of these processes are implicated in insulin secretion thus linking lipotoxicity with early aspects of pancreatic beta-cell dysfunction, diabetes, and the metabolic syndrome.

Characterization of human lysophospholipid acyltransferase 3.

Esterifying lysophospholipids may serve a variety of functions, including phospholipid remodeling and limiting the abundance of bioactive lipids. Recently, a yeast enzyme, Lpt1p, that esterifies an array of lysophospholipids was identified. Described here is the characterization of a human homolog of LPT1 that we have called lysophosphatidylcholine acyltransferase 3 (LPCAT3). Expression of LPCAT3 in Sf9 insect cells conferred robust esterification of lysophosphatidylcholine in vitro. Kinetic analysis found apparent cooperativity with a saturated acyl-CoA having the lowest K0.5 (5 microM), a monounsaturated acyl-CoA having the highest apparent Vmax (759 nmol/min/mg), and two polyunsaturated acyl-CoAs showing intermediate values. Lysophosphatidylethanolamine and lysophosphatidylserine were also utilized as substrates. Electrospray ionization mass spectrometric analysis of phospholipids in Sf9 cells expressing LPCAT3 showed a relative increase in phosphatidylcholine containing saturated acyl chains and a decrease in phosphatidylcholine containing unsaturated acyl chains. Targeted reduction of LPCAT3 expression in HEK293 cells had essentially an opposite effect, resulting in decreased abundance of saturated phospholipid species and more unsaturated species. Reduced LPCAT3 expression resulted in more apoptosis and distinctly fewer lamellipodia, suggesting a necessary role for lysophospholipid esterification in maintaining cellular function and structure.

Identification of a novel lysophospholipid acyltransferase in Saccharomyces cerevisiae.

The incorporation of unsaturated acyl chains into phospholipids during de novo synthesis is primarily mediated by the 1-acyl-sn-glycerol-3-phosphate acyltransferase reaction. In Saccharomyces cerevisiae, Slc1 has been shown to mediate this reaction, but distinct activity remains after its removal from the genome. To identify the enzyme that mediates the remaining activity, we performed synthetic genetic array analysis using a slc1Delta strain. One of the genes identified by the screen, LPT1, was found to encode for an acyltransferase that uses a variety of lysophospholipid species, including 1-acyl-sn-glycerol-3-phosphate. Deletion of LPT1 had a minimal effect on 1-acyl-sn-glycerol-3-phosphate acyltransferase activity, but overexpression increased activity 7-fold. Deletion of LPT1 abrogated the esterification of other lysophospholipids, and overexpression increased lysophosphatidylcholine acyltransferase activity 7-fold. The majority of this activity co-purified with microsomes. To test the putative role for this enzyme in selectively incorporating unsaturated acyl chains into phospholipids in vitro, substrate concentration series experiments were performed with the four acyl-CoA species commonly found in yeast. Although the saturated palmitoyl-CoA and stearoyl-CoA showed a lower apparent Km, the monounsaturated palmitoleoyl-CoA and oleoyl-CoA showed a higher apparent Vmax. Arachidonyl-CoA, although not abundant in yeast, also had a high apparent Vmax. Pulse-labeling of lpt1Delta strains showed a 30% reduction in [3H]oleate incorporation into phosphatidylcholine only. Therefore, Lpt1p, a member of the membrane-bound o-acyltransferase gene family, seems to work in conjunction with Slc1 to mediate the incorporation of unsaturated acyl chains into the sn-2 position of phospholipids.

Identification of two novel human acyl-CoA wax alcohol acyltransferases: members of the diacylglycerol acyltransferase 2 (DGAT2) gene superfamily.

The esterification of alcohols such as sterols, diacylglycerols, and monoacylglycerols with fatty acids represents the formation of both storage and cytoprotective molecules. Conversely, the overproduction of these molecules is associated with several disease pathologies, including atherosclerosis and obesity. The human acyl-CoA:diacylglycerol acyltransferase (DGAT) 2 gene superfamily comprises seven members, four of which have been previously implicated in the synthesis of di- or triacylglycerol. The remaining 3 members comprise an X-linked locus and have not been characterized. We describe here the expression of DGAT2 and the three X-linked genes in Saccharomyces cerevisiae strains virtually devoid of neutral lipids. All four gene products mediate the synthesis of triacylglycerol; however, two of the X-linked genes act as acyl-CoA wax alcohol acyltransferases (AWAT 1 and 2) that predominantly esterify long chain (wax) alcohols with acyl-CoA-derived fatty acids to produce wax esters. AWAT1 and AWAT2 have very distinct substrate preferences in terms of alcohol chain length and fatty acyl saturation. The enzymes are expressed in many human tissues but predominate in skin. In situ hybridizations demonstrate a differentiation-specific expression pattern within the human sebaceous gland for the two AWAT genes, consistent with a significant role in the composition of sebum.

Acyl-coenzymeA (CoA):cholesterol acyltransferase inhibition in rat and human aortic smooth muscle cells is nontoxic and retards foam cell formation.

OBJECTIVE: Studies in vitro and in vivo of macrophage foam cells have shown evidence of cytotoxicity after acyl-CoA:cholesterol acyltransferase (ACAT) inhibition. Foam cells of smooth muscle origin are also found in human and animal atherosclerotic lesions. METHODS AND RESULTS: To study whether cytotoxicity from ACAT inhibition is independent of cell type, we first established a protocol to conveniently induce aortic smooth muscle foam cell formation using cholesterol-cyclodextrin complexes (CCC). Rat aortic smooth muscle cells (ASMCs) treated for 48 hours with CCC (20 microg/mL) became foam cells by morphological (oil-red-O staining) and biochemical (approximately 1200% and approximately 180% increase in cellular esterified and free cholesterol, respectively) criteria. ACAT activity increased 500% (P<0.01 versus untreated). Similar results were obtained in human ASMC, but ACAT activity increased to an even greater extent (3200%; P<0.01 versus untreated). Western blots indicated that CCC treatment increased human (to 380+/-20% of untreated, P<0.001), but not rat, ACAT protein expression. ACAT inhibition by Fujirebio compound F1394 suppressed CCC-induced foam cell formation in rat and human ASMC, but, notably, did not induce significant cytotoxicity. CONCLUSIONS: ASMC might be more resistant to FC-induced adverse effects than are macrophages.

Overexpression of human diacylglycerol acyltransferase 1, acyl-coa:cholesterol acyltransferase 1, or acyl-CoA:cholesterol acyltransferase 2 stimulates secretion of apolipoprotein B-containing lipoproteins in McA-RH7777 cells.

The relative importance of each core lipid in the assembly and secretion of very low density lipoproteins (VLDL) has been of interest over the past decade. The isolation of genes encoding diacylglycerol acyltransferase (DGAT) and acyl-CoA:cholesterol acyltransferases (ACAT1 and ACAT2) provided the opportunity to investigate the effects of isolated increases in triglycerides (TG) or cholesteryl esters (CE) on apolipoprotein B (apoB) lipoprotein biogenesis. Overexpression of human DGAT1 in rat hepatoma McA-RH7777 cells resulted in increased synthesis, cellular accumulation, and secretion of TG. These effects were associated with decreased intracellular degradation and increased secretion of newly synthesized apoB as VLDL. Similarly, overexpression of human ACAT1 or ACAT2 in McA-RH7777 cells resulted in increased synthesis, cellular accumulation, and secretion of CE. This led to decreased intracellular degradation and increased secretion of VLDL apoB. Overexpression of ACAT2 had a significantly greater impact upon assembly and secretion of VLDL from liver cells than did overexpression of ACAT1. The addition of oleic acid (OA) to media resulted in a further increase in VLDL secretion from cells expressing DGAT1, ACAT1, or ACAT2. VLDL secreted from DGAT1-expressing cells incubated in OA had a higher TG:CE ratio than VLDL secreted from ACAT1- and ACAT2-expressing cells treated with OA. These studies indicate that increasing DGAT1, ACAT1, or ACAT2 expression in McA-RH7777 cells stimulates the assembly and secretion of VLDL from liver cells and that the core composition of the secreted VLDL reflects the enzymatic activity that is elevated.

Sterol and fatty acid regulatory pathways in a Giardia lamblia-derived promoter: evidence for SREBP as an ancient transcription factor.

The sterol regulatory element binding-proteins (SREBPs) are transcription factors that regulate the genes of lipid metabolism. Cholesterol and unsaturated fatty acids regulate SREBPs. Giardia lamblia (GL) is an intestinal parasite and one of the earliest derived members within the eukaryotic lineage. GLs exist as trophozoites and cysts. Growth in cholesterol depletion induces transcription of cyst-wall protein (CWP) genes that are upregulated during encystation. The hypothesis was investigated that SREBP-like pathways have a role in cwp gene transcription. Chinese hamster ovary cells were transfected with a cwp-2 promoter reporter construct. Incubation with cholesterol or oleate reduced cwp-2 mediated gene transcription to about half of the control. Incubation in sterol-depleted media, or in the presence of either an inhibitor of intracellular cholesterol movement or inhibitor of cholesterol synthesis, increased gene expression up to 3-fold. Overexpression of SREBPs increased reporter gene activity 2.5-fold. In the absence of functional SREBPs, cwp-2 was not regulated by cholesterol. Footprint analysis of cwp-2 reveals three novel binding sites for mammalian SREBPs with no homologies in other species or humans. The data show that SREBP binds to and can modulate transcription of a regulatory element from an ancient eukaryote and suggest the existence of an SREBP homolog in GL.

Posttranscriptional control of the expression and function of diacylglycerol acyltransferase-1 in mouse adipocytes.

Acyl-CoA:diacylglycerol acyltransferase-1 (DGAT1) catalyzes the final step of triglyceride synthesis in mammalian cells. Data obtained from DGAT1-knockout mice have indicated that this enzyme plays an important role in energy homeostasis. We investigated the regulation of the expression and function of DGAT1 in mouse 3T3-L1 cell as a model for mammalian adipocytes. We demonstrated that the DGAT1 protein level increased by approximately 90-fold following differentiation of 3T3-L1 into mature adipocytes, a change that was accompanied by approximately 7-fold increase in DGAT1 mRNA. On the other hand, forced overexpression of DGAT1 mRNA by >20-fold via a recombinant adenovirus only resulted in approximately 2-fold increase in DGAT1 protein in mature adipocytes and little increase in preadipocytes. These results indicated that gene expression of DGAT1 in adipocytes is subjected to rigorous posttranscriptional regulation, which is modulated significantly by the differentiation status of 3T3-L1 cells. Protein stability is not a significant factor in the control of DGAT1 expression. The steady-state levels of DGAT1 were unaffected by blockage of proteolytic pathways by ALLN. However, translational control was suggested by sequence analysis of the 5'-untranslated region of human DGAT1 (hDGAT1) mRNA. We found that the level of DGAT1 activity was predominantly a function of the steady-state level of DGAT1 protein. No significant functional changes were observed when the conserved tyrosine phosphorylation site in hDGAT1 was mutated by a single base pair substitution. Despite only a approximately 2-fold increase in DGAT1 protein caused by recombinant viral transduction, a proportionate increase in cellular triglyceride synthesis resulted without affecting the triglyceride lipolysis rate, leading to >2-fold increase in intracellular triglyceride accumulation. No change in adipocyte morphology or in the expression levels of lipoprotein lipase, proxisomal proliferation-activating receptor-gamma, and aP2 was evident secondary to DGAT1 overexpression at different stages in 3T3-L1 differentiation. These data suggest that dysregulation of DGAT1 may play a role in the development of obesity, and manipulation of the steady-state level of DGAT1 protein may offer a potential means to treat or prevent obesity.

FAIR and dynamic susceptibility contrast-enhanced perfusion imaging in healthy subjects and stroke patients.

PURPOSE: To compare dynamic susceptibility contrast-enhanced magnetic resonance imaging (DSC-MRI) and the flow-sensitive alternating inversion recovery (FAIR) technique for measuring brain perfusion. MATERIALS AND METHODS: We investigated 12 patients with acute stroke, and 10 healthy volunteers with FAIR and DSC maps of regional cerebral blood volume (rCBV), mean transit time (MTT), and regional cerebral blood flow (rCBF). RESULTS: In volunteers good gray/white-matter contrast was observed in FAIR, rCBF, and rCBV maps. Regions with high signal intensities in FAIR matched well with high values of rCBV and rCBF. In ischemic stroke patients a high correlation (r = 0.78) of the ipsi- to contralateral signal intensity ratios in FAIR and rCBF was observed in areas with perfusion abnormalities. In contrast, FAIR and rCBV (r = 0.50), and FAIR and MTT (r = -0.22) correlated only modestly. Furthermore, FAIR and rCBF demonstrated similar sizes of perfusion abnormality. CONCLUSION: This study demonstrates for the first time that FAIR and rCBF depict similar relations of perfusion in ischemic stroke patients and healthy subjects.

The DGA1 gene determines a second triglyceride synthetic pathway in yeast.

Diacylglycerol esterification provides an excellent target for the pharmacological reduction of triglyceride accumulation in several human disease states. We have used Saccharomyces cerevisiae as a model system to study this critical component of triglyceride synthesis. Recent studies of an oleaginous fungus, Mortierella ramanniana, identified a new family of enzymes with in vitro acyl-CoA:diacylglycerol acyltransferase activity. We show here that DGA1, the sole member of this gene family in yeast, has a physiological role in triglyceride synthesis. Metabolic labeling of DGA1 deletion strains with triglyceride precursors detected significant reductions in triglyceride synthesis. Triglyceride synthesis was virtually abolished in four different growth conditions when DGA1 was deleted in concert with LRO1, an enzyme that esterifies diacylglycerol from a phospholipid acyl donor. The relative contributions of the two enzymes depended on growth conditions. The residual synthesis was lost when ARE2, encoding an acyl-CoA:sterol acyltransferase, was deleted. In vitro microsomal assays verified that DGA1 and ARE2 mediate acyl-CoA:diacylglycerol acyltransferase reactions. Three enzymes can thus account for diacylglycerol esterification in yeast. Yeast strains deficient in both diacylglycerol and sterol esterification showed only a slight growth defect indicating that neutral lipid synthesis is dispensable under common laboratory conditions.