Showing papers on "Lipid biosynthesis published in 1996"

Sterol regulatory element binding protein binds to a cis element in the promoter of the farnesyl diphosphate synthase gene

Abstract: Sterol-regulated transcription of the gene for rat farnesyl diphosphate (FPP) synthase (geranyl-diphosphate:isopentenyl-diphosphate geranyltranstransferase, EC 2.5.1.10) is dependent in part on the binding of the ubiquitous transcription factor NF-Y to a 6-bp element within the proximal promoter. Current studies identify a second element in this promoter that is also required for sterol-regulated transcription in vivo. Mutation of three nucleotides (CAC) within this element blocks the 8-fold induction of FPP synthase promoter-reporter genes that normally occurs when the transfected cells are incubated in medium deprived of sterols. Gel mobility-shift assays demonstrate that the transcriptionally active 68-kDa fragment of the sterol regulatory element (SRE-1)-binding protein assays (SREBP-1) binds to an oligonucleotide containing the wild-type sequence but not to an oligonucleotide in which the CAC has been mutated. DNase 1 protection pattern (footprint) analysis indicates that SREBP-1 binds to nucleotides that include the CAC. Both the in vivo and in vitro assays are affected by mutagenesis of nucleotides adjacent to the CAC. Coexpression of SREBP with a wild-type FPP synthase promoter-reporter gene in CV-1 cells results in very high levels of reporter activity that is sterol-independent. In contrast, the reporter activity remained low when the promoter contained a mutation in the CAC trinucleotide. We conclude that sterol-regulated transcription of FPP synthase is controlled in part by the interaction of SREBP with a binding site that we have termed SRE-3. Identification of this element may prove useful in the identification of other genes that are both regulated by SREBP and involved in lipid biosynthesis.

Hepatotoxicity and -carcinogenicity of the fumonisins in rats. A review regarding mechanistic implications for establishing risk in humans.

Abstract: Cancer induction by the non-genotoxic mycotoxin, fumonisin B1, has been investigated by studying the mechanisms involved during cancer initiation and promotion in rat liver. Cancer initiation is effected through a toxic-proliferative response while the inhibitory effect on hepatocyte cell proliferation appears to be a key aspect determining cancer promotion. Dose-response effects of the fumonisins on the induction of early neoplastic lesions in both long- and short-term animal experiments have been established. The biphasic response of FB1 on hepatocyte proliferation will be discussed in relation to the known mechanisms of cancer induction by the genotoxic hepatocarcinogens. Recent investigations regarding the effect of the fumonisins on lipid biosynthesis and its inhibitory effect on hepatocyte growth stimulatory responses in vitro will be highlighted. Integration of our current knowledge regarding the carcinogenic potential of the fumonisins in setting a realistic and applicable risk assessment model for this non-genotoxic carcinogen will finally be addressed.

Hepatotoxicity and -Carcinogenicity of the Fumonisins in Rats

Abstract: Cancer induction by the non-genotoxic mycotoxin, fumonisin B1, has been investigated by studying the mechanisms involved during cancer initiation and promotion in rat liver Cancer initiation is effected through a toxic-proliferative response while the inhibitory effect on hepatocyte cell proliferation appears to be a key aspect determining cancer promotion Dose-response effects of the fumonisins on the induction of early neoplastic lesions in both long- and short-term animal experiments have been established The biphasic response of FB1 on hepatocyte proliferation will be discussed in relation to the known mechanisms of cancer induction by the genotoxic hepatocarcinogens Recent investigations regarding the effect of the fumonisins on lipid biosynthesis and its inhibitory effect on hepatocyte growth stimulatory responses in vitro will be highlighted Integration of our current knowledge regarding the carcinogenic potential of the fumonisins in setting a realistic and applicable risk assessment model for this non-genotoxic carcinogen will finally be addressed

Uses of biotechnology in modifying plant lipids

Abstract: This review discusses fatty acid modification of oilseeds with additional emphasis on production of oxygenated derivatives. In a relatively short period, less than a decade, our understanding of the enzymes involved in plant fatty acid synthesis has increased to the point where we understand how they might be used in oilseed modification. Further, through modern molecular biological techniques, the actual genes for many of these important enzymes have been cloned. Use of genetic transformation systems has allowed us to fundamentally alter the normal biosynthetic pathways in highly specific ways, in manners that would be either difficult or impossible using traditional breeding techniques. Alteration of plant lipid biosynthesis is not restricted to using genes from the plants themselves, but interspecies transfer is possible, either from completely unrelated plant species (often of no commercial value but possessing unusual biochemical properties) or from animals, fungi, and prokaryotic organisms. In this way “designer” plants possessing altered metabolism, tailored to the interests or needs of certain industries, nutritionists, and the consumer can be created.

Cardiac adaptations to low temperature in non-polar teleost fish

Abstract: Acclimation of teleost fish to low temperature results in a number of well-characterized adaptive responses in skeletal muscle. Our understanding of the response of cardiac muscle to such a challenge though is still poorly understood. It is considered of interest to learn more about the impact of chronic low temperature exposure on the cardiac response since the heart is required to support other tissues through the delivery of oxygen and metabolic fuels and this system may be a useful model in which to identify loci of cardiac plasticity. An overview of the available data reveals a number of fundamental responses. These include an increase in heart mass, an increase in maximal contraction frequency, an increase in complex lipid biosynthesis, and an increase in aerobic-based fatty acid metabolism. Two different strategies seem to emerge and lead to working hypotheses. As one example, rainbow trout develop larger hearts which may lead to enhanced stroke volume. The net accretion of protein is associated with either a more effective use of the available protein synthetic machinery and/or a decrease in protein degradation. The net accretion of lipid is associated with an increased rate of biosynthesis. Energy metabolism at low temperature appears to be much more dependent upon the oxidation of fatty acids. As another example, perch acclimated to low temperature show enhanced maximal rates of contraction which may be associated with increase in the volume of sarcoplasmic reticulum. The metabolic processes necessary to supply ATP appear quite adequate and require no further expansion as evidenced by constant mitochondrial volume density and activity of mitochondrial marker enzymes. The data however, in both cases are fragmentary and call for comprehensive studies on selected species. © 1996 Wiley-Liss, Inc.

Bacillus subtilis acyl carrier protein is encoded in a cluster of lipid biosynthesis genes.

Abstract: A cluster of Bacillus subtilis fatty acid synthetic genes was isolated by complementation of an Escherichia coli fabD mutant encoding a thermosensitive malonyl coenzyme A-acyl carrier protein transacylase. The B. subtilis genomic segment contains genes that encode three fatty acid synthetic proteins, malonyl coenzyme A-acyl carrier protein transacylase (fabD), 3-ketoacyl-acyl carrier protein reductase (fabG), and the N-terminal 14 amino acid residues of acyl carrier protein (acpP). Also present is a sequence that encodes a homolog of E. coli plsX, a gene that plays a poorly understood role in phospholipid synthesis. The B. subtilis plsX gene weakly complemented an E. coli plsX mutant. The order of genes in the cluster is plsX fabD fabG acpP, the same order found in E. coli, except that in E. coli the fabH gene lies between plsX and fabD. The absence of fabH in the B. subtilis cluster is consistent with the different fatty acid compositions of the two organisms. The amino acid sequence of B. subtilis acyl carrier protein was obtained by sequencing the purified protein, and the sequence obtained strongly resembled that of E. coli acyl carrier protein, except that most of the protein retained the initiating methionine residue. The B. subtilis fab cluster was mapped to the 135 to 145 degrees region of the chromosome.

Systematic sequencing of the 283 kb 210 degrees-232 degrees region of the Bacillus subtilis genome containing the skin element and many sporulation genes.

Abstract: As part of the Bacillus subtilis genome sequencing project, we have determined a 283 kb contiguous sequence from 210° to 232° of the B. subtilis genome. This region contains the 48 kb skin element which is excised during sporulation by a site-specific recombinase. In this region, 310 complete ORFs and one tRNA gene were identified: 66 ORFs have been sequenced and characterized previously by other workers, e.g. acc, ans, bfm, blt, bmr, comE, comG, dnaK, rpoD and sin operons; cwIA, gpr and lysA genes; many sporulation genes and operons, spoOA, spoIIA, spoIIM, spoIIP, spoIIIA, spoIIIC, spoIVB, spoIVCA, spoIVCB and spoVA, etc. The products of 84 ORFs were found to display significant similarity to proteins with known function in data banks, e.g., proteins involved in nucleotide metabolism, lipid biosynthesis, amino acid transport (ABC transporter), phosphate-specific transport, the glycine cleavage system, the two-component regulatory system, cell wall autolysis, ferric uptake and sporulation. However, the functions of more than half of the ORFs (52%, 160 ORFs) are still unknown. In the skin element containing 60 ORFs, 32 ORFs (53%) encode proteins which have significant homology to gene products of the B. subtilis temperate phage o105 and/or the defective phage PBSX.

The anhydrobiotic cyanobacterial cell

Abstract: The cyanobacterium Nostoc commune has been developed as the prokaryotic model for the anhydrobiotic cell and it provides the means to answer fundamental questions about desiccation tolerance. The anhydrobiotic cell is characterized by its singular lack of water — with contents as low as 0.02 g H2O g-1 dry weight. These levels are orders of magnitude lower than those found either in bacterial spores or in cells subjected to acute salt (osmotic) stress. Mechanisms that contribute to the desiccation tolerance of N. commune include the selective stabilization of anhydrous proteins, the secretion of water- and lipid-soluble UV-absorbing pigments, and the secretion of a complex glycan that immobilizes the cells, immobilizes water stress proteins and the UV-absorbing pigments, and which may confer the properties of a mechanical glass upon colonies. Rehydration of desiccated cells induces an instantaneous resumption of metabolic activities, including membrane transport and global lipid biosynthesis. These initial recoveries may not follow classical Arrhenius-based kinetics. The rehydrating cell exhibits a stringent, stepwise recovery of physiological capacities beginning with respiration, then photosynthesis and finally nitrogen fixation. Protein turnover, de novo protein synthesis and a rapid rise in the intracellular ATP pool accompany these recoveries. During the early stages of rehydration, the de novo transcription of one gene set (rpoC1C2) is achieved using an extant DNA-dependent RNA polymerase holoenzyme that remains stable in desiccated cells. These properties of desiccation-tolerant cyanobacleria, present in extant forms such as N. commune and Chroococcidiopsis spp., may have been utilized by the eoanhydrobiotes. However, it is the desiccation-tolerant cyanobacterium as a whole, and not some collection of disparate properties, that must be considered as the primary strategy for the achievement of desiccation tolerance.

Identification of Promoter and Stringent Regulation of Transcription of the fabH, fabD and fabG Genes Encoding Fatty Acid Biosynthetic Enzymes of Escherichia Coli

Abstract: In Escherichia coli, amino acid starvation results in the coordinate inhibition of a variety of metabolic activities, including fatty acid and phospholipid biosynthesis. By using primer extension analysis we identified the fabH promoter responsible for transcription of the fabH, fabD and fabG genes encoding fatty acid biosynthetic enzymes. The response of the fabH promoter to amino acid starvation was determined in vivo. Transcripts originating from the fabH promoter were quantified by employing a ribonuclease protection assay. The fabH promoter was subject to relA-dependent stringent control and was repressed approximately 4-fold upon amino acid starvation. The results suggest that inhibition of transcription initiation of lipid biosynthetic genes in starved cells contributes to the stringent control of lipid biosynthesis.

An introduction to the potential of 1H-, 31P- and 13C-NMR-spectroscopy.

Abstract: Multinuclear NMR spectroscopy ( 1 H, 13 C, 31 P) of biological samples reflects their metabolic composition. The analysis of high field and high resolution spectra of body fluids, cell and biopsy extracts requires no separation of a complex mixture into individual metabolites. Pattern recognition methods can be used to classify normal and pathological samples. Stable isotope enriched precursors are used to follow-up biochemical pathways and metabolic trafficking demonstrated for the lipid biosynthesis. Finally, volume selective in vivo spectroscopy or spectroscopic imaging depicts acute and time dependent metabolic events noninvasively in intact tissue within a few minutes.

Diacylglycerols Biosynthetic Intermediates and Lipid Second Messengers

Abstract: Diacylglycerols (DAGs), long-recognized intermediates of lipid biosynthesis (Bell and Coleman, 1980), are now established second messengers crucial to both short- and long-term regulation of cellular function (Bishop and Bell, 1989; Nishizuka, 1992). DAGs are formed by hydrolysis of membrane phospholipids through the action of phospholipases. These intracellular events are elicited in response to highly diverse agents impinging on the exterior surface of a cell, such as growth factors, hormones, and neurotransmitters. The location of DAG formation, the molecular species generated, and the time frame of DAG transients contribute to the versatility of this lipid-derived second messenger molecule. Extended elevations of DAGs, in particular derived from phosphatidylcholine, are important in the regulation of cell growth and transformation. Several functionally distinct intracellular pools of DAG exist, in particular at the plasma membrane and in the endoplasmic reticulum (ER). While DAG in the ER is a biosynthetic intermediate, plasma membrane DAGs are important for signaling. More recent studies indicate that DAG signals are also generated in intracellular compartments, such as the nucleus, where potential downstream effector molecules (protein kinase C or cytidylyltransferase) are also present. A functional distinction for DAGs on the basis of intracellular location is, therefore, no longer immediately apparent. Rather, metabolic channeling of DAGs is emerging as a critical determinant of their fate.

Cloning of a cDNA encoding the 21.2 kDa oleosin isoform from Arabidopsis thaliana and a study of its expression in a mutant defective in diacylglycerol acyltransferase activity

Abstract: A full-length cDNA clone (pA23) of 832 bp encoding an oleosin from Arabidopsis thaliana was isolated by differential screening of a silique-specific cDNA library with probes prepared from poly(A)+ RNA isolated from developing seeds of wild-type (WT) Arabidopsis and from mutant AS11 with a lesion affecting diacylglycerol acyltransferase (DGAT) activity during embryo development. The encoded protein has a calculated molecular mass of 21.2 kDa, and its amino acid sequence shows strong sequence homology and structural similarity to other known oleosins. Transcription of the oleosin gene during seed development was both reduced and delayed in AS11 compared to WT. However, the level of oleosin protein did not appear to be down-regulated during seed development, and at maturity, the overall level of oleosin protein was similar in both WT and AS11. These findings indicate that regulation of oleosin gene expression is part of a highly complex, and co-ordinated expression of storage lipid biosynthesis and related (oleosin) genes during oilseed development.

The apparent coupling between synthesis and posttranslational modification of Escherichia coli acyl carrier protein is due to inhibition of amino acid biosynthesis.

Abstract: Acyl carrier protein (ACP) is modified on serine 36 by the covalent posttranslational attachment of 4'-phosphopantetheine from coenzyme A (CoA), and this modification is required for lipid biosynthesis. Jackowski and Rock (J. Biol. Chem 258:15186-15191, 1983) reported that upon depletion of the CoA pool by starvation for a CoA precursor, no accumulation of the unmodified form of ACP (apo-ACP) was detected. We report that this lack of apo-ACP accumulation results from decreased translation of the acpP mRNAs because of the limitation of the synthesis of glutamate and other amino acids made directly from tricarboxylic acid cycle intermediates.

Method and compounds for inhibiting lipid biosynthesis of bacteria and plants

Abstract: This invention relates to InhA enzyme crystals and to methods of growing said crystals. This invention is further directed to the utilization of said crystals to determine the three dimensional structure of InhA enzyme utilizing heavy atom derivatives of said crystals, and to the identification and development of compounds which inhibit the biochemical activity of InhA enzyme in bacteria and plants.

Molecular Cloning of the Gene Encoding Stearoyl-Acyl Carrier Protein Desaturase in Brassica juncea

Abstract: Metabolic engineering of the pathways of lipid biosynthesis has generated transgenic oilseed crops with enhanced levels of specialty fatty acids of Industrial value. Stearic acid, a 18:0 saturated fatty acid, is one such important fatty acid. Stearoylacyl carrier protein (stearoyl-ACP) desaturase (EC 1.14.99.6) catalyzes the first desaturation step in seed oil biosynthesis and converts stearoyl-ACP to oleoyl-ACP. We have cloned the complete coding region of the gene for this enzyme in Brassica juncea. Based on the sequence information of the gene in B. napus, 27-mer forward and reverse primers were designed each of which incorporated a Sal I restriciton site at the end. The primers were used to fish out the desaturase gene from B. juncea genome by polymerase chain reaction (PCR). The PCR product conformed to the average size of the coding region of the gene in B. napus. The PCR product was cloned in the pGem-T vector. The cloning was reconfirmed by restriction enzyme analysis and by PCR of the recombinant plasmid. The potential use of this gene in molecular farming of designer oilseed brassicas is discussed.

Membrane Traffic and Compartmentalization within the Secretory Pathway

Abstract: A wide variety of processes necessary for cell growth and homeostasis are carried out within the secretory pathway of eukaryotic cells. These include biosynthesis and assembly of protein complexes, lipid biosynthesis and metabolism, and protein processing, degradation, and secretion. These functions occur within distinct membrane-bound compartments, or organelles, which communicate by membrane transport pathways. Movement of protein and lipid through these pathways involves the formation, targeting, and fusion of membrane-bound transport intermediates. Regulation of the organization and function of this membrane system is crucial for enabling cells to selectively transport and localize protein and lipid in response to changing cellular needs.

Metabolism of ricinoleate by Neurospora crassa

Abstract: Neurospora crassa is a potential expression system for evaluating fatty-acid-modifying genes from plants producing uncommon fatty acids. One such gene encodes the hydroxylase that converts oleate to ricinoleate, a fatty acid with important industrial uses. To develop this expression system, it is critical to evaluate the metabolism and physiological effects of the expected novel fatty acid(s). We therefore examined effects of ricinoleate on lipid biosynthesis and growth of N. crassa. Ricinoleate inhibited growth and reduced levels of phospholipids and of 2-hydroxy fatty acids in glycolipids, but led to increased lipid accumulation on a mass basis. To evaluate incorporation and metabolism of ricinoleate, we followed the fate of 14 μM–3 mM [1-14C]ricinoleate. The fate of the [14C]ricinoleate was concentration-dependent. At higher concentrations, ricinoleate was principally incorporated into triacylglycerols. At lower concentrations, ricinoleate was principally metabolized to other compounds. Thus, N. crassa transformants expressing the hydroxylase gene can be detected if the level of hydroxylase expression allows both growth and ricinoleate accumulation.