Synthesis of Trilaurin by Developing Pisa Seeds (Actinodaphne hookeri)

The developing seeds of Actinodaphne hookeri were investigated to delineate their ability to synthesize large amounts of trilaurin. Until 88 days after flowering the embryos contained 71% neutral lipids (NL) and 29% phospholipids (PL) and both these components contained C-16:0, C-18:0, C-18:2, and C-18:3 as the major fatty acids (FA). At 102 days after flowering the seeds began to accumulate triacylglycerols (TAG) and to synthesize lauric acid (C-12:0). By 165 days after flowering, when the seeds were mature, they contained about 99% NL and 1% FL. At this stage the TAG contained exclusively C-12:0, while the PL consisted of long-chain fatty acids (LCFA) only. Leaf lipids in contrast did not contain any C-12:0. Experiments on [1-C-14]acetate incorporation into developing seed slices showed that at 88 days after flowering only 4% of the label was in TAG, 1% in diacylglycerols (DAG), and 87% in FL. One hundred two days after flowering seeds incorporated only 2% of the label into TAG, 30% into DAG, and 64% into FL. In contrast at 114 days after flowering 71% of the label was incorporated into TAG, 25% into DAG, and only 2% into FL. Analysis of labeled FA revealed that up to 102 days after flowering it was incorporated only into LCFA, whereas at 114 days after flowering it was incorporated exclusively into C-12:0. Furthermore, 67% of the label in PL at 114 days after flowering was found to be dilaurylglycerophosphate. Analysis of the label in DAG at this stage showed that it was essentially in dilaurin species. These observations indicate the induction of enzymes of Kennedy pathway for the specific synthesis of trilaurin at about 114 days after flowering, Homogenates of seeds (114 days after flowering) incubated with labeled FA in the presence of glycerol-3-phosphate and coenzymes A and ATP incorporated 84% of C-12:0 and 61% of C-14:0, but not C-16:0, C-18:2, and C-18:3, into TAG. In contrast the LCFA were incorporated preferentially into FL. It is concluded that, between 102 and 114 days after flowering, a switch occurs in A. hookeri for the synthesis of C-12:0 and trilaurin which is tissue specific. Since the seed synthesizes exclusively C-12:0 at 114 days after flowering onwards and incorporates specifically into TAG, this system appears to be ideal for identifying the enzymes responsible for medium-chain fatty acid as well as trilaurin synthesis and for exploiting them for genetic engineering. (C) 1994 Academic Press, Inc.

SecB binds only to a late native-like intermediate in the folding pathway of barstar and not to the unfolded state

SecB is a cytosolic, tetrameric chaperone of Escherichia coli which maintains precursor proteins in a translocation competent state. We have investigated the effect of SecB on the refolding kinetics of the small protein barstar in I M guanidine hydrochloride at pH 7.0 and 25 degrees C using fluorescence spectroscopy. We show that SecB does not bind either the native or the unfolded states of barstar but binds to a late near-native intermediate along the folding pathway. For barstar, polypeptide collapse and formation of a hydrophobic surface are required for binding to SecB. SecB does not change the apparent rate constant of barstar refolding. The kinetic data for SecB binding to barstar are not consistent with simple kinetic partitioning models.

The effect of pH on the unfolding pathway and stability of ribosome-inactivating protein abrin-II

The effect of pH on the unfolding pathway acid the stability of the toxic protein abrin-II have been studied by increasing denaturant concentrations of guanidine hydrochloride and by monitoring the change in 8,1-anilino naphthalene sulfonic acid (ANS) fluorescence upon binding to the hydrophobic sites of the protein. Intrinsic protein fluorescence, far and near UV-circular dichroism (CD) spectroscopy and ANS binding studies reveal that the unfolding of abrin-II occurs through two intermediates at pH 7.2 and one intermediate at pH 4.5. At pH 7.2, the two subunits A and B of abrin-II unfold sequentially. The native protein is more stable at pH 4.5 than at pH 7.2. However, the stability of the abrin-II A-subunit is not affected by a change in pH. These observations may assist in an understanding of the physiologically relevant transmembrane translocation of the toxin.

Localisation of Plasmodium falciparum uroporphyrinogen III decarboxylase of the heme-biosynthetic pathway in the apicoplast and characterisation of its catalytic properties

Uroporphyrinogen decarboxylase (UROD) is a key enzyme in the heme-biosynthetic pathway and in Plasmodium falciparum it occupies a strategic position in the proposed hybrid pathway for heme biosynthesis involving shuttling of intermediates between different subcellular compartments in the parasite. In the present study, we demonstrate that an N-terminally truncated recombinant P. falciparum UROD (r(Δ)PfUROD) over-expressed and purified from Escherichia coli cells, as well as the native enzyme from the parasite were catalytically less efficient compared with the host enzyme, although they were similar in other enzyme parameters. Molecular modeling of PfUROD based on the known crystal structure of the human enzyme indicated that the protein manifests a distorted triose phosphate isomerase (TIM) barrel fold which is conserved in all the known structures of UROD. The parasite enzyme shares all the conserved or invariant amino acid residues at the active and substrate binding sites, but is rich in lysine residues compared with the host enzyme. Mutation of specific lysine residues corresponding to residues at the dimer interface in human UROD enhanced the catalytic efficiency of the enzyme and dimer stability indicating that the lysine rich nature and weak dimer interface of the wild-type PfUROD could be responsible for its low catalytic efficiency. PfUROD was localised to the apicoplast, indicating the requirement of additional mechanisms for transport of the product coproporphyrinogen to other subcellular sites for its further conversion and ultimate heme formation.

Molecular convergence of hexosamine biosynthetic pathway and ER stress leading to insulin resistance in L6 skeletal muscle cells

Augmentation of hexosamine biosynthetic pathway (HBP) and endoplasmic reticulum (ER) stress were independently related to be the underlying causes of insulin resistance. We hypothesized that there might be a molecular convergence of activated HBP and ER stress pathways leading to insulin resistance. Augmentation of HBP in L6 skeletal muscle cells either by pharmacological (glucosamine) or physiological (high-glucose) means, resulted in increased protein expression of ER chaperones (viz., Grp78, Calreticulin, and Calnexin), UDP-GlcNAc levels and impaired insulin-stimulated glucose uptake. Cells silenced for O-glycosyl transferase (OGT) showed improved insulin-stimulated glucose uptake (P < 0.05) but without any effect on ER chaperone upregulation. While cells treated with either glucosamine or high-glucose exhibited increased JNK activity, silencing of OGT resulted in inhibition of JNK and normalization of glucose uptake. Our study for the first time, demonstrates a molecular convergence of O-glycosylation processes and ER stress signals at the cross-road of insulin resistance in skeletal muscle.

The mechanism of intestinal absorption of phosphatidylcholine in rats

1. The mechanism of absorption of phosphatidylcholine was studied in rats by injecting into the intestine phosphatidylcholine specifically labelled either in the fatty acid or in the glycerol moiety or with 32P, when considerable amounts of 1-acyl-lysophosphatidylcholine were found in the intestinal lumen. 2-([14C]Acyl)phosphatidylcholine gave markedly more radioactive unesterified fatty acids in the lumen, compared with the 1-([14C]acyl) derivative. Some of the radioactivity from either the fatty acid or the glycerol moiety of the injected phosphatidylcholine appeared in the mucosal triacylglycerols. 2. Injection of 32P-labelled phosphatidylcholine or 32P-labelled lysophosphatidylcholine led to the appearance of radioactive glycerylphosphorylcholine, glycerophosphate and Pi in the mucosa. 3. Rat mucosa was found to contain a highly active glycerylphosphorylcholine diesterase. 4. It was concluded that the dietary phosphatidylcholine is hydrolysed in the intestinal lumen by the pancreatic phospholipase A to 1-acylglycerylphosphorylcholine, which on entering the mucosal cell is partly reacylated to phosphatidylcholine, and the rest is further hydrolysed to glycerylphosphorylcholine, glycerophosphate, glycerol and Pi. The fatty acids and glycerophosphate are then reassembled to give triacylglycerols via the Kennedy (1961) pathway.

Identification of key DNA elements involved in promoter recognition by Mxr1p, a master regulator of methanol utilization pathway in Pichia pastoris

Mxr1p (methanol expression regulator 1) functions as a key regulator of methanol metabolism in the methylotrophic yeast Pichia pastoris. In this study, a recombinant Mxr1p protein containing the N-terminal zinc finger DNA binding domain was overexpressed and purified from E coli cells and its ability to bind to promoter sequences of AOXI encoding alcohol oxidase was examined. In the AOXI promoter, Mxr1p binds at six different regions. Deletions encompassing these regions result in a significant decrease in AOXI promoter activity in vivo. Based on the analysis of AOXI promoter sequences, a consensus sequence for Mxr1p binding consisting of a core 5' CYCC 3' motif was identified. When the core CYCC sequence is mutated to CYCA, CYCT or CYCM (M = 5-methylcytosine), Mxr1p binding is abolished. Though Mxr1p is the homologue of Saccharomyces cerevisiae Adr1p transcription factor, it does not bind to Adr1p binding site of S. cerevisiae alcohol dehydrogenase promoter (ADH2UAS1). However, two point mutations convert ADH2UAS1 into an Mxr1p binding site. The identification of key DNA elements involved in promoter recognition by Mxr1p is an important step in understanding its function as a master regulator of the methanol utilization pathway in P. pastoris.

Structural Insights into the acyl-intermediates of plasmodium falciparum fatty acid synthesis pathway; the mechanism of expansion of acyl carrier protein core

Acyl carrier protein (ACP) plays a central role in fatty acid biosynthesis. However, the molecular machinery that mediates its function is not yet fully understood. Therefore, structural studies were carried out on the acyl-ACP intermediates of Plasmodium falciparum using NMR as a spectroscopic probe. Chemical shift perturbation studies put forth a new picture of the interaction of ACP molecule with the acyl chain, namely, the hydrophobic core can protect up to 12 carbon units, and additional carbons protrude out from the top of the hydrophobic cavity. The latter hypothesis stems from chemical shift changes observed in C-alpha and C-beta of Ser-37 in tetradecanoyl-ACP. C-13, N-15-Double-filtered nuclear Overhauser effect (NOE) spectroscopy experiments further substantiate the concept; in octanoyl (C-8)- and dodecanoyl (C-12)-ACP, a long range NOE is observed within the phosphopantetheine arm, suggesting an arch-like conformation. This NOE is nearly invisible in tetradecanoyl (C-14)-ACP, indicating a change in conformation of the prosthetic group. Furthermore, the present study provides insights into the molecular mechanism of ACP expansion, as revealed from a unique side chain-to-backbone hydrogen bond between two fairly conserved residues, Ile-55 HN and Glu-48 O. The backbone amide of Ile-55 HN reports a pK(a) value for the carboxylate, similar to 1.9 pH units higher than model compound value, suggesting strong electrostatic repulsion between helix II and helix III. Charge-charge repulsion between the helices in combination with thrust from inside due to acyl chain would energetically favor the separation of the two helices. Helix III has fewer structural restraints and, hence, undergoes major conformational change without altering the overall-fold of P. falciparum ACP.

Backbone chemical shift assignments of the acyl-acyl carrier protein intermediates of the fatty acid biosynthesis pathway of Plasmodium falciparum

We report the backbone chemical shift assignments of the acyl-acyl carrier protein (ACP) intermediates of the fatty acid biosynthesis pathway of Plasmodium falciparum. The acyl-ACP intermediates butyryl (C4), -octanoyl (C8), -decanoyl (C10), -dodecanoyl (C12) and -tetradecanoyl (C14)-ACPs display marked changes in backbone HN, Cα and Cβ chemical shifts as a result of acyl chain insertion into the hydrophobic core. Chemical shift changes cast light on the mechanism of expansion of the acyl carrier protein core.

Structural Insights into the Acyl Intermediates of the Plasmodium falciparum Fatty Acid Synthesis Pathway THE MECHANISM OF EXPANSION OF THE ACYL CARRIER PROTEIN CORE

Acyl carrier protein (ACP) plays a central role in fatty acid biosynthesis. However, the molecular machinery that mediates its function is not yet fully understood. Therefore, structural studies were carried out on the acyl-ACP intermediates of Plasmodium falciparum using NMR as a spectroscopic probe. Chemical shift perturbation studies put forth a new picture of the interaction of ACP molecule with the acyl chain, namely, the hydrophobic core can protect up to 12 carbon units, and additional carbons protrude out from the top of the hydrophobic cavity. The latter hypothesis stems from chemical shift changes observed in C-alpha and C-beta of Ser-37 in tetradecanoyl-ACP. C-13, N-15-Double-filtered nuclear Overhauser effect (NOE) spectroscopy experiments further substantiate the concept; in octanoyl (C-8)- and dodecanoyl (C-12)-ACP, a long range NOE is observed within the phosphopantetheine arm, suggesting an arch-like conformation. This NOE is nearly invisible in tetradecanoyl (C-14)-ACP, indicating a change in conformation of the prosthetic group. Furthermore, the present study provides insights into the molecular mechanism of ACP expansion, as revealed from a unique side chain-to-backbone hydrogen bond between two fairly conserved residues, Ile-55 HN and Glu-48 O. The backbone amide of Ile-55 HN reports a pK(a) value for the carboxylate, similar to 1.9 pH units higher than model compound value, suggesting strong electrostatic repulsion between helix II and helix III. Charge-charge repulsion between the helices in combination with thrust from inside due to acyl chain would energetically favor the separation of the two helices. Helix III has fewer structural restraints and, hence, undergoes major conformational change without altering the overall-fold of P. falciparum ACP.

Reactions of tetrahalogeno-1,2-benzoquinones. Part III. Reaction of tetrachloro-1,2-benzoquinone with tetralones and naphthols: Pathway to the condensates

With a view to understanding the mechanism of the formation of 6-methoxy-2,2-(tetrachloro--phenylenedioxy)-naphthalen-1 (2H)-one (IIIa) in the reaction of 6-methoxy-1-tetralone (Ia) with tetrachloro-1,2-benzoquinone (II), the reaction of (II) with various tetralones and naphthols has been studied. Reaction with either α-tetralone or α-naphthol gives 2,2-(tetrachloro-o-phenylenedioxy)naphthalen-1 (2H)-one (IIIb), whereas reaction with either β-tetralone or β-naphthol gives a mixture of (IIIb) and ,1-(tetrachloro-o-phenylenedioxy)-naphthalen-2 (1H)-one (IX), with the former predominating. Further, reactions of (II) with 7-methoxy-3,4-dihydrophenanthren- 1 (2H)-one and m-methoxyphenol gave respectively 7-methoxy- ,2-(tetrachloro-o- phenylenedioxy)phenanthren-1 (2H)-one (VII) and 3-methoxy-6,6-(tetrachloro-o- phenylenedioxy)cyclohexa-2,4-dien-1-one (VIII). Structures of all these compounds have been proved on the basis of i.r. and n.m.r. data. The pathway to the formation of the condensates (III) is discussed.

Japanese Encephalitis Virus Utilizes the Canonical Pathway To Activate NF-kappa B but It Utilizes the Type I Interferon Pathway To Induce Major Histocompatibility Complex Class I Expression in Mouse Embryonic Fibroblasts

Flaviviruses have been shown to induce cell surface expression of major histocompatibility complex class I (MHC-I) through the activation of NF-kappa B. Using IKK1(-/-), IKK2(-/-), NEMO-/-, and IKK1-/- IKK2-/- double mutant as well as p50(-/-) RelA(-/-) cRel(-/-) triple mutant mouse embryonic fibroblasts infected with Japanese encephalitis virus (JEV), we show that this flavivirus utilizes the canonical pathway to activate NF-kappa B in an IKK2- and NEMO-, but not IKK1-, dependent manner. NF-kappa B DNA binding activity induced upon virus infection was shown to be composed of RelA: p50 dimers in these fibroblasts. Type I interferon (IFN) production was significantly decreased but not completely abolished upon virus infection in cells defective in NF-kappa B activation. In contrast, induction of classical MHC-I (class 1a) genes and their cell surface expression remained unaffected in these NF-kappa B-defective cells. However, MHC-I induction was impaired in IFNAR(-/-) cells that lack the alpha/beta IFN receptor, indicating a dominant role of type I IFNs but not NF-kappa B for the induction of MHC-I molecules by Japanese encephalitis virus. Our further analysis revealed that the residual type I IFN signaling in NF-kappa B-deficient cells is sufficient to drive MHC-I gene expression upon virus infection in mouse embryonic fibroblasts. However, NF-kappa B could indirectly regulate MHC-I expression, since JEV-induced type I IFN expression was found to be critically dependent on it.

Essential role of PI3-kinase pathway in p53-mediated transcription: Implications in cancer chemotherapy

The PI3-kinase pathway is the target of inactivation in achieving better cancer chemotherapy. Here, we report that p53-mediated transcription is inhibited by pharmacological inhibitors and a dominant-negative mutant of PI3-kinase, and this inhibition was relieved by a constitutively active mutant of PI3-kinase. Akt/PKB and mTOR, the downstream effectors of PI3-kinase, were also found to be essential. LY294002 (PI3-kinase inhibitor) pre-treatment altered the post-translational modifications and the sub-cellular localization of p53. Although LY294002 increased the chemosensitivity of cells to low concentrations of adriamycin (adriamycin-low), it protected the cells from cytotoxicity induced by high concentrations of adriamycin (adriamycin-high) in a p53-dependent manner. Further, we found that LY294002 completely abolished the activation of p53 target genes (particularly pro-apoptotic) under adriamycin-high conditions, whereas it only marginally repressed the p53 target genes under adriamycin-low conditions; in fact, it further activated the transcription of NOXA, HRK, APAF1 and CASP5 genes. Thus, the differential effect of PI3-kinase on p53 functions seems to be responsible for the differential regulation of DNA damage-induced cytotoxicity and cell death by PI3-kinase. Our finding becomes relevant in the light of ongoing combination chemotherapy trials with the PI3-kinase pathway inhibitors and underscores the importance of p53 status in the careful formulation of combination chemotherapies. Oncogene (2010) 29, 3605-3618; doi: 10.1038/onc.2010.123; published online 26 April 2010