Biosynthesis of terpenoids: 4-Diphosphocytidyl-2-C-methyl-d-erythritol kinase from tomato

The putative catalytic domain (residues 81–401) of a predicted tomato protein with similarity to 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase of Escherichia coli was expressed in a recombinant E. coli strain. The protein was purified to homogeneity and was shown to catalyze the phosphorylation of the position 2 hydroxy group of 4-diphosphocytidyl-2-C-methyl-d-erythritol at a rate of 33 μmol⋅mg−1⋅min−1. The structure of the reaction product, 4-diphosphocytidyl-2-C-methyl-d-erythritol 2-phosphate, was established by NMR spectroscopy. Divalent metal ions, preferably Mg2+, are required for activity. Neither the tomato enzyme nor the E. coli ortholog catalyzes the phosphorylation of isopentenyl monophosphate.

In Vitro Reconstitution of Electron Transport from Glucose-6-Phosphate and NADPH to Nitrite1

An NADPH-dependent NO2−-reducing system was reconstituted in vitro using ferredoxin (Fd) NADP+ oxidoreductase (FNR), Fd, and nitrite reductase (NiR) from the green alga Chlamydomonas reinhardtii. NO2− reduction was dependent on all protein components and was operated under either aerobic or anaerobic conditions. NO2− reduction by this in vitro pathway was inhibited up to 63% by 1 mm NADP+. NADP+ did not affect either methyl viologen-NiR or Fd-NiR activity, indicating that inhibition was mediated through FNR. When NADPH was replaced with a glucose-6-phosphate dehydrogenase (G6PDH)-dependent NADPH-generating system, rates of NO2− reduction reached approximately 10 times that of the NADPH-dependent system. G6PDH could be replaced by either 6-phosphogluconate dehydrogenase or isocitrate dehydrogenase, indicating that G6PDH functioned to: (a) regenerate NADPH to support NO2− reduction and (b) consume NADP+, releasing FNR from NADP+ inhibition. These results demonstrate the ability of FNR to facilitate the transfer of reducing power from NADPH to Fd in the direction opposite to that which occurs in photosynthesis. The rate of G6PDH-dependent NO2− reduction observed in vitro is capable of accounting for the observed rates of dark NO3− assimilation by C. reinhardtii.

Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase

Peppermint (Mentha × piperita L.) was independently transformed with a homologous sense version of the 1-deoxy-d-xylulose-5-phosphate reductoisomerase cDNA and with a homologous antisense version of the menthofuran synthase cDNA, both driven by the CaMV 35S promoter. Two groups of transgenic plants were regenerated in the reductoisomerase experiments, one of which remained normal in appearance and development; another was deficient in chlorophyll production and grew slowly. Transgenic plants of normal appearance and growth habit expressed the reductoisomerase transgene strongly and constitutively, as determined by RNA blot analysis and direct enzyme assay, and these plants accumulated substantially more essential oil (about 50% yield increase) without change in monoterpene composition compared with wild-type. Chlorophyll-deficient plants did not afford detectable reductoisomerase mRNA or enzyme activity and yielded less essential oil than did wild-type plants, indicating cosuppression of the reductoisomerase gene. Plants transformed with the antisense version of the menthofuran synthase cDNA were normal in appearance but produced less than half of this undesirable monoterpene oil component than did wild-type mint grown under unstressed or stressed conditions. These experiments demonstrate that essential oil quantity and quality can be regulated by metabolic engineering. Thus, alteration of the committed step of the mevalonate-independent pathway for supply of terpenoid precursors improves flux through the pathway that leads to increased monoterpene production, and antisense manipulation of a selected downstream monoterpene biosynthetic step leads to improved oil composition.

A combined kinetic and modeling study of the catalytic center subsites of human angiogenin.

Kinetic analysis and molecular modeling have been used to map the ribonucleolytic center of angiogenin (Ang). Pyrimidine nucleotides were found to interact very weakly with Ang, consistent with the inaccessible B1 pyrimidine binding site revealed by x-ray crystallography. Ang also lacks an effective phosphate binding site on the 5' side of B1. Although the B2 site that preferentially binds purines on the 3' side of B1 is also weak, its associated phosphate subsites make substantial contributions: both 3',5'-ADP and 5'-ADP have Ki values 6-fold lower than for 5'-AMP, and adding a 3'-phosphate to the substrate CpA increases Kcat/Km by 9-fold. Thus Ang has a functional P2 site on the 3' side of B2 and a site for a second phosphate on the 5' side of B2. Modeling of an Ang-d(ApTpApA) complex suggested that Arg-5 forms part of the P2 site and that a 2'-phosphate might bind more tightly than a 3'-phosphate. Both predictions were confirmed kinetically. The subsite map obtained by this combined approach indicated that 5'-diphosphoadenosine 2'-phosphate might be a more potent inhibitor than any of the nucleotides tested thus far. Indeed, its Ki value of 150 microM is 50-fold lower than that for the best nucleotide previously reported and 400-fold lower than the Km for the best dinucleotide substrate. This compound may serve as a suitable starting point for the eventual design of tight-binding inhibitors of Ang as antiangiogenic agents for human therapy.

A structural basis for a phosphoramide mustard-induced DNA interstrand cross-link at 5'-d(GAC).

Phosphoramide mustard-induced DNA interstrand cross-links were studied both in vitro and by computer simulation. The local determinants for the formation of phosphoramide mustard-induced DNA interstrand cross-links were defined by using different pairs of synthetic oligonucleotide duplexes, each of which contained a single potentially cross-linkable site. Phosphoramide mustard was found to cross-link dG to dG at a 5'-d(GAC)-3'. The structural basis for the formation of this 1,3 cross-link was studied by molecular dynamics and quantum chemistry. Molecular dynamics indicated that the geometrical proximity of the binding sites also favored a 1,3 dG-to-dG linkage over a 1,2 dG-to-dG linkage in a 5'-d(GCC)-3' sequence. While the enthalpies of 1,2 and 1,3 mustard cross-linked DNA were found to be very close, a 1,3 structure was more flexible and may therefore be in a considerably higher entropic state.

Temporally distinct accumulation of transcripts encoding enzymes of the prechorismate pathway in elicitor-treated, cultured tomato cells.

The accumulation of phenylalanine-derived phenolic compounds is a well-known element of a plant's defense in response to pathogen attack. Phenylalanine, as well as the other two aromatic amino acids, tyrosine and tryptophan, is synthesized by way of the shikimate pathway. The first seven steps of the shikimate pathway (the prechorismate pathway) are common for the biosynthesis of all three aromatic amino acids. We have studied transcript levels of six genes--i.e., two 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase genes, one shikimate kinase gene, one 5-enolpyruvylshikimate 3-phosphate synthase gene, and two chorismate synthase genes--corresponding to four steps of the prechorismate pathway, in cultured tomato cells exposed to fungal elicitors. The abundance of transcripts specific for some of these genes increased 10- to 20-fold within 6 h after elicitor treatment, as did the abundance of phenylalanine ammonialyase-specific transcripts and the synthesis of ethylene. Interestingly, transcript accumulation occurred more rapidly for shikimate kinase than for the enzymes preceding or following it in the prechorismate pathway. Neither the inhibition of ethylene biosynthesis by aminoethoxyvinylglycine nor inhibition of phenylalanine ammonia-lyase (EC 4.3.1.5) activity by 2-aminoindan-2-phosphonic acid affected the time course or extent of transcript accumulation. Thus, the increased demand for phenylalanine in the phenylpropanoid pathway required after elicitor treatment appears to be met by increased de novo synthesis of its biosynthetic enzymes.

Some account of a paper call'd the Telltale, begun in colledge Sept 1721

The small leather-bound volume holds two sections, a manuscript student periodical, and written tête-bêche, an exchange on smallpox inoculation followed by notes on the rules and activities of a Harvard College student club. The volume begins with thirteen numbered manuscript issues, written in one hand, of the Tell-Tale running from September 9, 1721 to November 1, 1721. Prefaced, "This paper was entitl'd the Telltale or Criticisms on the Conversation & Beheavour of Scholars to promote right reasoning & good manner," the work is modeled after literary periodicals of the time, including the "Spectator," and is considered the oldest student publication at Harvard. The periodical appears to have circulated in manuscript form. The content varies in format and includes letters between Telltale and correspondents, short essays, and advertisements. Topics discussed include conversation, detraction, and flattery. While not specifically about Harvard it does provide some information about the College including evidence of various student activities and organizations at Harvard in the 1720s. The entry explaining the rules of the Telltale Club is heavily faded and nearly illegible. The Telltale records multiple dreams, which are populated by various characters, such as “beautiful” Kate, two “learned Physicians” debating inoculation, “four Fellows” “pushing and shoving one another,” and a “person of a very Dark & swarthy complexion in a Slovenly Dress with 7 patches & 5 sparks on his Face.”

Mémoire de Denis Benjamin Viger, écuyer, et de Marie Amable Foretier, son épouse, appellans; contre Toussaint Pothier, écuyer, et autres, intimes, à la Cour provinciale d'appel, d'un jugement de la Cour du Banc du Roi de Montréal, pour les Causes civiles, du 20 Février, 1827.

"The case concerns the estate of Pierre Foretier, d. Dec. 3, 1815."--Toronto Public Library. A bibliography of Canadiana, no. 1471.

Cours d'agriculture.

Vols. 1 and 4, 3d éd.; v. 3, 4, éd.

Mémoires d'une contemporaine; ou, Souvenirs d'une femme sur les principaux personnages de la république, du consulat, de l'empire, etc. ...

"Lettres inédites de Napoléon Bonaparte, général en chef de l'armée d'Italie": v. 2, p. [347]-395.

Manuel de l'ingénieur des ponts et chaussées, rédigé conformément au programme annexé au Decret du 7 mars 1868 réglant l'admission des conducteurs des ponts et chaussées au grade d'ingénieur.

Text issued in 20 pts. Pt. 20, with title: Dictionnaire administratif des travaux publics is separately cataloged (HD4163.D3 1879)

Arf6-independent GPI-anchored protein-enriched early endosomal compartments fuse with sorting endosomes via a Rab5/phosphatidylinositol-3 '-kinase-dependent machinery

In the process of internalization of molecules from the extracellular milieu, a cell uses multiple endocytic pathways, consequently generating different endocytic vesicles. These primary endocytic vesicles are targeted to specific destinations inside the cell. Here, we show that GPI-anchored proteins are internalized by an Arf6-independent mechanism into GPI-anchored protein-enriched early endosomal compartments (GEECs). Internalized GPI-anchored proteins and the fluid phase are first visualized in GEECs that are acidic, primary endocytic structures, negative for early endosomal markers, Rab4, Rab5, and early endosome antigen (EEA)1. They subsequently acquire Rab5 and EEA1 before homotypic fusion with other GEECs, and heterotypic fusion with endosomes containing cargo from the clathrin-dependent endocytic pathway. Although, the formation of GEECs is unaffected by inhibition of Rab5 GTPase and phosphatidylinositol-3'-kinase (PI3K) activity, their fusion with sorting endosomes is dependent on both activities. Overexpression of Rab5 reverts PI3K inhibition of fusion, providing evidence that Rab5 effectors play important roles in heterotypic fusion between the dynamin-independent GEECs and clathrin- and dynamin-dependent sorting endosomes.

Synthesis, crystallography and biological activity of myo-inositol phosphates

The antioxidant property of myo-inositol hexakisphosphate is important in the prevention of hydroxyl radical formation which may allow it to act as a 'safe' carrier of iron within the cell. Here, the hypothesis that the recently discovered natural product, myo-inositol 1,2,3-trisphosphate represents the simplest structure to mimic phytate's antioxidant activity has been tested. The first synthesis of myo-inositol 1,2,3-trisphosphate has been completed, along with its X-ray structure determination and that of key synthetic intermediates. Iron binding studies of myo-inositol 1,2,3-trisphosphate demonstrated that phosphate groups with the equatorial-axial-equatorial conformation are required for complete inhibition of hydroxyl radical formation. myo-Inositol monophosphatase is a key enzyme in recycling myo-inositol from its monophosphates in the brain and its inhibition is implicated in lithium's antimanic properties. Current synthetic strategies require inositol compounds to be protected (often with more than one group), resolved, phosphorylated and deprotected to produce the desired optically active myo-inositol phosphates. Here, the synthesis of myo-inositol 3-phosphate has been achieved in only 4 steps from myo-inositol. The stereoselective addition of the chiral phosphorylating agent (2R,4S,5R)-2-chloro-3,4-dimethyl-5-phenyl-1,3,2-oxazaphospholidin-2-one to a protected inositol intermediate allowed separation of diastereoisomers and easy deprotection to myo-inositol 3-phosphate. This strategy also allows the possible introduction of labels of oxygen and sulphur to give a thiophosphate of known stereochemistry at phosphorus which would be useful for the analysis of the stereochemical course of phosphate hydrolysis catalysed by inositol monophosphatase.

The role of cytoskeletal proteins in the mechanism of insulin release

This thesis is concerned with the role of /3-cell cytoskeletal proteins in the mechanism of insulin release from islets of experimental animals, the Aston obese diabetic hyperglycaemic (ob/ob) mouse and their lean littermates and the cultural insulin secreting /?-cell lines, HIT-TT5 and RINm5F. Investigations were carried out into the glucose induced insulin response of the lean and obese mouse islets and HIT-TI5 cells and the D-glyceraldehyde response of RINm5F cells using a static incubation system. Colchicine was found to inhibit insulin release from both lean and obese mouse islets more significantly than cultured TTT-TI5 and RINm5F cells. (Colchicine pre-treatment also inhibited the second phase of insulin release from perifused lean mouse islets and HIT-TI5 cells). Cytocha-lasin B, used to investigate the role of the microfilamentous system in the mechanism of insulin release enhanced insulin release from both lean and obese mouse islets to a significantly greater degree than that from cultured HIT-TI5 and RINm5F cells. Pre-treatment of isolated lean and obese mouse islets and cultured /?-cells with a combination of colchicine and cytochalasin B significantly reduced the insulin response of the HIT-TI5 and RINm5F cells compared with the control values suggesting that intact microtubules are more important for the sustained release of insulin than the microfilamentous system. However, the response was not so clearly defined with the lean and obese mouse islets. Tubulin was separated from the extracts of lean mouse islets and the HIT-TI5 and RINm5F cells and actin was separated from all of the cell types including the obese mouse islets by SDS- polyacrylamide electrophoresis. A tubulin radioimmunoassay and a colchicine binding assay were developed to measure the tubulin content of lean and obese mouse islets, and the shift between the proportions of tubulin dimers and polymerized tubulin under stimulatory and non-stimulatory conditions. The assay methods developed were not prone to be accurate, sensitive and precise but gave some indication of the shift from unpolymerised to polymerised tubulin during glucose stimulated insulin release. These studies show that microtubules do play a fundamental role in the mechanism of insulin release from both islets and cultured HIT-TI5 and RINm5F cells.

APP controls the formation of PI(3,5)P2 vesicles through its binding of the PIKfyve complex

Phosphoinositides are signalling lipids that are crucial for major signalling events as well as established regulators of membrane trafficking. Control of endosomal sorting and endosomal homeostasis requires phosphatidylinositol-3-phosphate (PI(3)P) and phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2), the latter a lipid of low abundance but significant physiological relevance. PI(3,5)P2 is formed by phosphorylation of PI(3)P by the PIKfyve complex which is crucial for maintaining endosomal homeostasis. Interestingly, loss of PIKfyve function results in dramatic neurodegeneration. Despite the significance of PIKfyve, its regulation is still poorly understood. Here we show that the Amyloid Precursor Protein (APP), a central molecule in Alzheimer’s disease, associates with the PIKfyve complex (consisting of Vac14, PIKfyve and Fig4) and that the APP intracellular domain directly binds purified Vac14. We also show that the closely related APP paralogues, APLP1 and 2 associate with the PIKfyve complex. Whether APP family proteins can additionally form direct protein–protein interaction with PIKfyve or Fig4 remains to be explored. We show that APP binding to the PIKfyve complex drives formation of PI(3,5)P2 positive vesicles and that APP gene family members are required for supporting PIKfyve function. Interestingly, the PIKfyve complex is required for APP trafficking, suggesting a feedback loop in which APP, by binding to and stimulating PI(3,5)P2 vesicle formation may control its own trafficking. These data suggest that altered APP processing, as observed in Alzheimer’s disease, may disrupt PI(3,5)P2 metabolism, endosomal sorting and homeostasis with important implications for our understanding of the mechanism of neurodegeneration in Alzheimer’s disease.