Sterol distribution in intracellular organelles isolated from tobacco leaves

All membrane-containing fractions isolated from tobacco leaves contained free sterols, sterol glycosides, and sterol esters. The three sterol forms increased, on a dry weight basis, with a decrease in particle size. The supernatant fraction contained only trace amounts of sterol. The major sterols in all cellular fractions, in the order of decreasing amounts, were: stigmasterol, β-sitosterol, campesterol, and cholesterol. The 500g pellet contained the largest percentage of free sterol, while the 46,000g pellet contained the largest percentage of esterified sterol. The individual sterol composition of the free sterol and sterol glycoside fraction was very similar; however, the composition of the sterol ester fraction varied widely among intracellular fraction. The intracellular distribution pattern of cholesterol-¹⁴C added to the isolation medium provided evidence that the intracellular sterol distribution pattern is not an artifact. These results support the suggestion that sterols in plant cells may have a physiological function associated with membranes.     

Reduced VLDL clearance in Apoe−/−Npc1−/− mice is associated with increased Pcsk9 and Idol expression and decreased hepatic LDL-receptor levels

Niemann-Pick type C1 (NPC1) promotes the transport of LDL receptor (LDL-R)-derived cholesterol from late endosomes/lysosomes to other cellular compartments. NPC1-deficient cells showed impaired regulation of liver_X receptor (LXR) and sterol regulatory element-binding protein (SREBP) target genes. We observed that Apoe^−/−Npc1^−/− mice displayed a marked increase in total plasma cholesterol mainly due to increased VLDL, reflecting decreased clearance. Although nuclear SREBP-2 and Ldlr mRNA levels were increased in Apoe^−/−Npc1^−/− liver, LDL-R protein levels were decreased in association with marked induction of proprotein convertase subtilisin/kexin type 9 (Pcsk9) and inducible degrader of the LDL-R (Idol), both known to promote proteolytic degradation of LDL-R. While Pcsk9 is known to be an SREBP-2 target, marked upregulation of IDOL in Apoe^−/−Npc1^−/− liver was unexpected. However, several other LXR target genes also increased in Apoe^−/−Npc1^−/− liver, suggesting increased synthesis of endogenous LXR ligands secondary to activation of sterol biosynthesis. In conclusion, we demonstrate that NPC1 deficiency has a major impact on VLDL metabolism in Apoe^−/− mice through modulation of hepatic LDL-R protein levels. In contrast to modest induction of hepatic IDOL with synthetic LXR ligands, a striking upregulation of IDOL in Apoe^−/−Npc1^−/− mice could indicate a role of endogenous LXR ligands in regulation of hepatic IDOL.     

Effects of Abnormal-Sterol Accumulation on Ustilago maydis Plasma Membrane H+-ATPase Stoichiometry and Polypeptide Pattern

Accumulation of 14α-methylated sterols or Δ⁸-sterols in Ustilago maydis affected three aspects of the plasma membrane H⁺-ATPase. Proton transport was reduced in Δ⁸-sterol-accumulating samples, due to an altered H⁺/ATP stoichiometry. ATP hydrolytic activity was increased, but no direct correlation with the extent or type of abnormal sterol accumulated could be drawn. Finally, Western blot analysis with antibodies against yeast PMA1 revealed a second lighter band (99-kDa band) in all samples from abnormal-sterol-accumulating sporidia. The conclusions are that the 99-kDa band and a reduced stoichiometry are directly linked to the presence of abnormal sterols, while changes in hydrolytic activity are linked only indirectly.     

A reappraisal of sterol biosynthesis and metabolism in aphids

Aphids of Schizaphis graminum (Rondani) (biotype C) reared on its host-plant, Sorghum bicolor (L.) Moench, sequestered campesterol, stigmasterol and sitosterol. Aphids reared for 72 hr on holidic diets supplemented with [4-¹⁴C]-sitosterol contained both [¹⁴C]-sitosterol and [¹⁴C]-cholesterol, indicating that these aphids are capable of dealkylation at C-24. When aphids were reared on artificial diets containing [2-¹⁴C]-mevalonic acid, no detectable amounts of radioactively labelled desmethyl sterols, nor metabolic intermediates in sterol synthesis (i.e. squalene, 2,3-oxidosqualene, 4,4-dimethyl and 4-monomethyl sterols) were found to accumulate in their tissues. The relevance of these findings to previous research suggesting the ability of aphids, via their symbiotes, to synthesize sterols is discussed.     

Amo 1618 effects on incorporation of 14C-MVA and 14C-acetate into sterols in Nicotiana and Digitalis seedlings and cell-free preparations from Nicotiana

Incorporation of radioactivity from acetate-[¹⁴C] and MVA-[¹⁴C] into sterols and sterol precursors in tobacco was inhibited by Amo 1618; differing patterns of accumulation were obtained with the two precursors, suggesting more than one point of inhibition. This was borne out with cell-free preparations with which it was demonstrated that both HMG-CoA reductase and squalene-2,3-epoxide cyclase were inhibited, the latter more strongly than the former. GLC analysis of gross sterol and hydrocarbon fractions confirmed previous indications that incorporation of radioactivity into individual sterols was inhibited by Amo 1618. Finally, incorporation of MVA-[¹⁴C] into sterols and sterol precursors of Digitalis was significantly altered by the retardant, thus expanding the generality of the relationship between sterol (particularly 4-desmethylsterol) biosynthesis inhibition and retardant effect.     

Cell-free transfer of sterols from dictyosome-like structures to plasma membrane vesicles of guinea pig testes

Summary Transfer of radiolabeled lipids from dictyosome-like structures (DLS) from testis tubules of the guinea pig as donor to unlabeled plasma membrane from testis tubules immobilized on nitrocellulose as acceptor was studied in a completely cell-free system. As a general label for lipids of the donor DLS, isolated testis tubules were incubated with [¹⁴C]acetate. Time- and temperature-dependent transfer of [¹⁴C]acetate labeled constituents was observed in the cellfree system. However, despite the fact that phospholipids and other constituents were highly labeled in the donor fraction, primarily radioactive sterols were transferred to the plasma membrane acceptor vesicles. Transfer at 37°C represented 0.4 to 0.7% of the total radiolabeled cholesterol at 37°C but little or no transfer occurred at 4°C. The sterols transferred exhibited Chromatographic mobilities corresponding to those of cholesterol and lanosterol. Similar results were obtained with [¹⁴C]mevalonic acid. In subsequent experiments, cholesterol transfer from DLS to plasma membrane was demonstrated by incubation of DLS with [³H]squalene which was converted into sterol or with [¹⁴C]cholesterol. Transfer of sterols required ATP, but not cytosol, and was both time- and temperature-dependent. DLS were more effective than either endoplasmic reticulum or plasma membrane as the donor fraction. The results from the cell-free analysis suggest a possible functional role of the DLS in sterol biogenesis and transfer to the plasma membrane during spermatid development.Abbreviations DLS dictyosome-like structure(s) - PBS phosphatebuffered saline - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - BSA bovine serum albumin     

A Mutation in a Purported Regulatory Gene Affects Control of Sterol Uptake in Saccharomyces cerevisiae

Aerobically growing wild-type strains of Saccharomyces cerevisiae are unable to take exogenously supplied sterols from media. This aerobic sterol exclusion is vitiated under anaerobic conditions, in heme-deficient strains, and under some conditions of impaired sterol synthesis. Mutants which can take up sterols aerobically in heme-competent cells have been selected. One of these mutations, designated upc2-1, gives a pleiotropic phenotype in characteristics as diverse as aerobic accumulation of sterols, total lipid storage, sensitivity to metabolic inhibitors, response to altered sterol structures, and cation requirements. During experiments designed to ascertain the effects of various cations on yeast with sterol alterations, it was observed that upc2-1 was hypersensitive to Ca²⁺. Using resistance to Ca²⁺ as a screening vehicle, we cloned UPC2 and showed that it is YDR213W, an open reading frame on chromosome IV. This belongs to a fungal regulatory family containing the Zn(II)2Cys6 binuclear cluster DNA binding domain. The single guanine-to-adenine transition in upc2-1 gives a predicted amino acid change from glycine to aspartic acid. The regulatory defect explains the semidominance and pleiotropic effects of upc2-1.     

Anion-Sensitive, H-Pumping ATPase of Oat Roots : Direct Effects of Cl, NO(3), and a Disulfonic Stilbene

To understand the mechanism and molecular properties of the tonoplast-type H⁺-translocating ATPase, we have studied the effect of Cl⁻, NO₃⁻, and 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) on the activity of the electrogenic H⁺-ATPase associated with low-density microsomal vesicles from oat roots (Avena sativa cv Lang). The H⁺-pumping ATPase generates a membrane potential (Δψ) and a pH gradient (ΔpH) that make up two interconvertible components of the proton electrochemical gradient (μh⁺). A permeant anion (e.g. Cl⁻), unlike an impermeant anion (e.g. iminodiacetate), dissipated the membrane potential ([¹⁴C]thiocyanate distribution) and stimulated formation of a pH gradient ([¹⁴C]methylamine distribution). However, Cl⁻-stimulated ATPase activity was about 75% caused by a direct stimulation of the ATPase by Cl⁻ independent of the proton electrochemical gradient. Unlike the plasma membrane H⁺-ATPase, the Cl⁻-stimulated ATPase was inhibited by NO₃⁻ (a permeant anion) and by DIDS. In the absence of Cl⁻, NO₃⁻ decreased membrane potential formation and did not stimulate pH gradient formation. The inhibition by NO₃⁻ of Cl⁻-stimulated pH gradient formation and Cl⁻-stimulated ATPase activity was noncompetitive. In the absence of Cl⁻, DIDS inhibited the basal Mg,ATPase activity and membrane potential formation. DIDS also inhibited the Cl⁻-stimulated ATPase activity and pH gradient formation. Direct inhibition of the electrogenic H⁺-ATPase by NO₃⁻ or DIDS suggest that the vanadate-insensitive H⁺-pumping ATPase has anion-sensitive site(s) that regulate the catalytic and vectorial activity. Whether the anion-sensitive H⁺-ATPase has channels that conduct anions is yet to be established.     

Deletion of Ubiquitin Fold Modifier Protein Ufm1 Processing Peptidase Ufsp in L. donovani Abolishes Ufm1 Processing and Alters Pathogenesis

Previously, we showed Leishmania donovani Ufm1 has a Gly residue conserved at the C-terminal region with a unique 17 amino acid residue extension that must be processed prior to conjugation to target proteins. In this report, we describe for the first time the isolation and characterization of the Leishmania Ufm1-specific protease Ufsp. Biochemical analysis of L. donovani Ufsp showed that this protein possesses the Ufm1 processing activity using sensitive FRET based activity probes. The Ufm1 cleavage activity was absent in a mutant Ufsp in which the active site cysteine is altered to a serine. To examine the effects of abolition of Ufm1 processing activity, we generated a L. donovani null mutant of Ufsp (LdUfsp^−/−). Ufm1 processing activity was abolished in LdUfsp^−/− mutant, and the processing defect was reversed by re-expression of wild type but not the cys>ser mutant in the LdUfsp^−/− parasites. Further LdUfsp^−/− mutants showed reduced survival as amastigotes in infected human macrophages but not as promastigotes. This growth defect in the amastigotes was reversed by re-expression of wild type but not the cys>ser mutant in the Ufsp^−/− indicating the essential nature of this protease for Leishmania pathogenesis. Further, mouse infection experiments showed deletion of Ufsp results in reduced virulence of the parasites. Additionally, Ufsp activity was inhibited by an anti-leishmanial drug Amphotericin B. These studies provide an opportunity to test LdUfsp^−/− parasites as drug and vaccine targets.     

Changes in sterol biosynthesis accompanying cessation of glial cell growth in serum-free medium

C-6 glioma cells, grown in medium supplemented with 5% delipidated foetal calf serum, were induced to enter a quiescent state by removing serum from the medium. Within 24h there was a 75–80% decline in the rate of incorporation of [¹⁴C]acetate or ³H₂O into digitonin-precipitable sterols. Experiments with [³H]mevalonolactone as a labelled sterol precursor suggested that the decline in sterol synthesis was regulated primarily at a point in the pathway before the formation of mevalonate. The specific activities of 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA reductase decreased sharply in conjunction with the decline in sterol synthesis in the serum-free cultures; however, the activity of acetoacetyl-CoA thiolase was altered only slightly. The magnitude of the initial decline in reductase activity was not affected when 50-mm-NaF was included in the preincubation and assay buffers to prevent activation of physiologically inactive enzyme. However, after 6h of serum deprivation the decline in 3-hydroxy-3-methylglutaryl-CoA reductase activity was due to a decrease in the amount of latent activity. The sterol concentration in C-6 cells was unchanged after 24h in serum-free medium, although a 20% decrease in the sterol/fatty acid molar ratio occurred as a result of a small increase in the fatty-acid concentration. Incorporation of ³H₂O into fatty acids was inhibited in the serum-deprived glial cells; however, this inhibition developed more slowly and was not as pronounced as the diminution in sterol synthesis. The results suggest that in C-6 glia, which resemble the glial stem cells of the developing brain, the decreased demand for membrane sterols in the quiescent state results in a decline in sterol synthesis, mediated primarily through co-ordinate changes in the activities of 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA reductase.     

Synthesis of Sterols In Herpetomonas Samuelpessoai: Influence of Growth Conditions*

Ergosterol was the only sterol detected in Herpetomonas samuelpessoai grown in a defined, lipid-free medium. When cultivated in a complex medium, this flagellate was found to contain 6 additional sterols. As measured by incorporation of L-[Me-¹⁴C]methionine, in the absence of acetate, the sterol synthesis was greater at 28 C than at 37 C; in the presence of acetate, however, this synthesis was greater at 37 C. When [2-¹⁴C]acetate was used as the sterol precursor, the synthesis level at 37 C exceeded that at 28 C.     

Leishmania Subtilisin Is a Maturase for the Trypanothione Reductase System and Contributes to Disease Pathology

Proteases are a ubiquitous group of enzymes that play key roles in the life cycle of parasites, in the host-parasite relationship, and in the pathogenesis of parasitic diseases. Furthermore, proteases are druggable targets for the development of new anti-parasitic therapy. The subtilisin protease (SUB; Clan SB, family S8) of Leishmania donovani was cloned and found to possess a unique catalytic triad. This gene was then deleted by gene knock-out, which resulted in reduced ability by the parasite to undergo promastigote to amastigote differentiation in vitro. Electron microscopy of SUB knock-out amastigotes revealed abnormal membrane structures, retained flagella, and increased binucleation. SUB-deficient Leishmania displayed reduced virulence in both hamster and murine infection models. Histology of spleens from SUB knock-out-infected hamsters revealed the absence of psammoma body calcifications indicative of the granulomatous lesions that occur during Leishmania infection. To delineate the specific role of SUB in parasite physiology, two-dimensional gel electrophoresis was carried out on SUB^−/− versus wild-type parasites. SUB knock-out parasites showed altered regulation of the terminal peroxidases of the trypanothione reductase system. Leishmania and other trypanosomatids lack glutathione reductase, and therefore rely on the novel trypanothione reductase system to detoxify reactive oxygen intermediates and to maintain redox homeostasis. The predominant tryparedoxin peroxidases were decreased in SUB^−/− parasites, and higher molecular weight isoforms were present, indicating altered processing. In addition, knock-out parasites showed increased sensitivity to hydroperoxide. These data suggest that subtilisin is the maturase for tryparedoxin peroxidases and is necessary for full virulence.     

Role of the Trypanosoma brucei HEN1 Family Methyltransferase in Small Interfering RNA Modification

Parasitic protozoa of the flagellate order Kinetoplastida represent one of the deepest branches of the eukaryotic tree. Among this group of organisms, the mechanism of RNA interference (RNAi) has been investigated in Trypanosoma brucei and to a lesser degree in Leishmania (Viannia) spp. The pathway is triggered by long double-stranded RNA (dsRNA) and in T. brucei requires a set of five core genes, including a single Argonaute (AGO) protein, T. brucei AGO1 (TbAGO1). The five genes are conserved in Leishmania (Viannia) spp. but are absent in other major kinetoplastid species, such as Trypanosoma cruzi and Leishmania major. In T. brucei small interfering RNAs (siRNAs) are methylated at the 3′ end, whereas Leishmania (Viannia) sp. siRNAs are not. Here we report that T. brucei HEN1, an ortholog of the metazoan HEN1 2′-O-methyltransferases, is required for methylation of siRNAs. Loss of TbHEN1 causes a reduction in the length of siRNAs. The shorter siRNAs in hen1^−/− parasites are single stranded and associated with TbAGO1, and a subset carry a nontemplated uridine at the 3′ end. These findings support a model wherein TbHEN1 methylates siRNA 3′ ends after they are loaded into TbAGO1 and this methylation protects siRNAs from uridylation and 3′ trimming. Moreover, expression of TbHEN1 in Leishmania (Viannia) panamensis did not result in siRNA 3′ end methylation, further emphasizing mechanistic differences in the trypanosome and Leishmania RNAi mechanisms.     

Degradation of Host Sphingomyelin Is Essential for Leishmania Virulence

In eukaryotes, sphingolipids (SLs) are important membrane components and powerful signaling molecules. In Leishmania, the major group of SLs is inositol phosphorylceramide (IPC), which is common in yeast and Trypanosomatids but absent in mammals. In contrast, sphingomyelin is not synthesized by Leishmania but is abundant in mammals. In the promastigote stage in vitro, Leishmania use SL metabolism as a major pathway to produce ethanolamine (EtN), a metabolite essential for survival and differentiation from non-virulent procyclics to highly virulent metacyclics. To further probe SL metabolism, we identified a gene encoding a putative neutral sphingomyelinase (SMase) and/or IPC hydrolase (IPCase), designated ISCL (Inositol phosphoSphingolipid phospholipase C-Like). Despite the lack of sphingomyelin synthesis, L. major promastigotes exhibited a potent SMase activity which was abolished upon deletion of ISCL, and increased following over-expression by episomal complementation. ISCL-dependent activity with sphingomyelin was about 20 fold greater than that seen with IPC. Null mutants of ISCL (iscl⁻) showed modest accumulation of IPC, but grew and differentiated normally in vitro. Interestingly, iscl⁻ mutants did not induce lesion pathology in the susceptible BALB/c mice, yet persisted indefinitely at low levels at the site of infection. Notably, the acute virulence of iscl⁻ was completely restored by the expression of ISCL or heterologous mammalian or fungal SMases, but not by fungal proteins exhibiting only IPCase activity. Together, these findings strongly suggest that degradation of host-derived sphingomyelin plays a pivotal role in the proliferation of Leishmania in mammalian hosts and the manifestation of acute disease pathology.     

Induction and Characterization of Penicillium caseicolum Mutants Resistant to Ergosterol Biosynthesis Inhibitors

The isolation of Penicillium caseicolum mutants resistant to different fungicides which inhibit ergosterol biosynthesis is reported. Mutational frequencies for resistance were high (3 × 10⁻³ to 3 × 10⁻⁵). The levels of resistance toward the inhibitors of sterol C-14 demethylation were always low (<10), whereas high values were obtained with mutants resistant to inhibitors of sterol Δ14 reduction or Δ8→Δ7 isomerization, or both. Generally, there was a positive cross-resistance between fungicides showing the same biochemical mode of action but not between compounds of two different groups. Mycelial growth rate and sporulation were tested; several mutants were not affected for these characteristics. We conclude that resistance to ergosterol biosynthesis inhibitors may be used as a good marker for genetic studies through protoplast fusion.     

Effects of Irradiance during Growth on Adaptive Photosynthetic Characteristics of Velvetleaf and Cotton

We grew velvetleaf (Abutilon theophrasti Medic.) and cotton (Gossypium hirsutum L. var. Stoneville 213) at three irradiances and determined the photosynthetic responses of single leaves to a range of six irradiances from 90 to 2000 μeinsteins m⁻²sec⁻¹. In air containing 21% O₂, velvetleaf and cotton grown at 750 μeinsteins m⁻²sec⁻¹ had maximum photosynthetic rates of 18.4 and 21.9 mg of CO₂ dm⁻²hr⁻¹, respectively. Maximum rates for leaves grown at 320 and 90 μeinsteins m⁻²sec⁻¹ were 15.3 and 10.3 mg of CO₂ dm⁻²hr⁻¹ in velvetleaf and 12 and 6.7 mg of CO₂ dm⁻²hr⁻¹ in cotton, respectively. In 1 O₂, maximum photosynthetic rates were 1.5 to 2.3 times the rates in air containing 21% O₂, and plants grown at medium and high irradiance did not differ in rate. In both species, stomatal conductance was not significantly affected by growth irradiance. The differences in maximum photosynthetic rates were associated with differences in mesophyll conductance. Mesophyll conductance increased with growth irradiance and correlated positively with mesophyll thickness or volume per unit leaf area, chlorophyll content per unit area, and photosynthetic unit density per unit area. Thus, quantitative changes in the photosynthetic apparatus help account for photosynthetic adaptation to irradiance in both species. Net assimilation rates calculated for whole plants by mathematical growth analysis were closely correlated with single-leaf photosynthetic rates.     

CXCL14 Deficiency in Mice Attenuates Obesity and Inhibits Feeding Behavior in a Novel Environment

Background

CXCL14 is a chemoattractant for macrophages and immature dendritic cells. We recently reported that CXCL14-deficient (CXCL14 ^−/−) female mice in the mixed background are protected from obesity-induced hyperglycemia and insulin resistance. The decreased macrophage infiltration into visceral adipose tissues and the increased insulin sensitivity of skeletal muscle contributed to these phenotypes.

Methodology/Principal Findings

In this study, we performed a comprehensive study for the body weight control of CXCL14 ^−/− mice in the C57BL/6 background. We show that both male and female CXCL14 ^−/− mice have a 7–11% lower body weight compared to CXCL14 ^+/− and CXCL14 ^+/+ mice in adulthood. This is mainly caused by decreased food intake, and not by increased energy expenditure or locomotor activity. Reduced body weight resulting from the CXCL14 deficiency was more pronounced in double mutant CXCL14^−/− ob/ob and CXCL14 ^−/−A^y mice. In the case of CXCL14 ^−/−A^y mice, oxygen consumption was increased compared to CXCL14 ^+/−A^y mice, in addition to the reduced food intake. In CXCL14 ^−/− mice, fasting-induced up-regulation of Npy and Agrp mRNAs in the hypothalamus was blunted. As intracerebroventricular injection of recombinant CXCL14 did not change the food intake of CXCL14 ^−/− mice, CXCL14 could indirectly regulate appetite. Intriguingly, the food intake of CXCL14 ^−/− mice was significantly repressed when mice were transferred to a novel environment.

Conclusions/Significance

We demonstrated that CXCL14 is involved in the body weight control leading to the fully obese phenotype in leptin-deficient or A^y mutant mice. In addition, we obtained evidence indicating that CXCL14 may play an important role in central nervous system regulation of feeding behavior.