An investigation of phenolic glycoside and condensed tannin homeostasis in Populus by salicyl alcohol feeding to cell cultures and by transgenic manipulation of the sucrose transporter, PTSUT4, in planta

Secondary metabolites play an important role in plant protection against biotic and abiotic stress. In Populus, phenolic glycosides (PGs) and condensed tannins (CTs) are two such groups of compounds derived from the common phenylpropanoid pathway. The basal levels and the inducibility of PGs and CTs depend on genetic as well as environmental factors, such as soil nitrogen (N) level. Carbohydrate allocation, transport and sink strength also affect PG and CT levels. A negative correlation between the levels of PGs and CTs was observed in several studies. However, the molecular mechanism underlying such relation is not known. We used a cell culture system to understand negative correlation of PGs and CTs. Under normal culture conditions, neither salicin nor higher-order PGs accumulated in cell cultures. Several factors, such as hormones, light, organelles and precursors were discussed in the context of aspen suspension cells’ inability to synthesize PGs. Salicin and its isomer, isosalicin, were detected in cell cultures fed with salicyl alcohol, salicylaldehyde and helicin. At higher levels (5 mM) of salicyl alcohol feeding, accumulation of salicins led to reduced CT production in the cells. Based on metabolic and gene expression data, the CT reduction in salicin-accumulating cells is partly a result of regulatory changes at the transcriptional level affecting carbon partitioning between growth processes, and phenylpropanoid CT biosynthesis. Based on molecular studies, the glycosyltransferases, GT1-2 and GT1-246, may function in glycosylation of simple phenolics, such as salicyl alcohol in cell cultures. The uptake of such glycosides into vacuole may be mediated to some extent by tonoplast localized multidrug-resistance associated protein transporters, PtMRP1 and PtMRP6. In Populus, sucrose is the common transported carbohydrate and its transport is possibly regulated by sucrose transporters (SUTs). SUTs are also capable of transporting simple PGs, such as salicin. Therefore, we characterized the SUT gene family in Populus and investigated, by transgenic analysis, the possible role of the most abundantly expressed member, PtSUT4, in PG-CT homeostasis using plants grown under varying nitrogen regimes. PtSUT4 transgenic plants were phenotypically similar to the wildtype plants except that the leaf area-to-stem volume ratio was higher for transgenic plants. In SUT4 transgenics, levels of non-structural carbohydrates, such as sucrose and starch, were altered in mature leaves. The levels of PGs and CTs were lower in green tissues of transgenic plants under N-replete, but were higher under N-depleted conditions, compared to the levels in wildtype plants. Based on our results, SUT4 partly regulates N-level dependent PG-CT homeostasis by differential carbohydrate allocation.

CE provides evidence of the stereoselective hydroxylation of norketamine in equines

CE with multiple isomer sulfated-CD as selector was used for the simultaneous analysis of the stereoisomers of ketamine, norketamine, 5,6-dehydronorketamine and hydroxylated metabolites of norketamine in liquid/liquid extracts of (i) in vitro incubations with ketamine or norketamine and equine liver microsomes and (ii) plasma and urine of ponies receiving a target-controlled infusion of ketamine under isoflurane anesthesia. Hydroxynorketamine metabolites with the hydroxy group at the cyclohexanone ring could be shown to be formed stereoselectively both in vitro and in vivo. Due to the lack of standard compounds, urinary extracts were fractionated by HPLC followed by characterization of the collected fractions with CE and LC-MS(n) with 0.7 mmu mass discrimination. Comparison of LC-MS(n) data obtained with the fractions, an in vitro microsomal sample, and both pony urine and hydrolyzed pony urine led to the identification of four hydroxylated norketamine metabolites with hydroxylation at the cyclohexanone ring, two with hydroxylation at the aromatic ring and four hydroxylated metabolites of ketamine. Due to the lower detection sensitivity, only the four hydroxynorketamine metabolites with hydroxylation at the cyclohexanone ring were observed by CE. The data suggest that demethylation of ketamine followed by hydroxylation of norketamine at the cyclohexanone ring is the major metabolic pathway in equine species and that the ketamine metabolism is highly stereoselective.

Studies on the catalytic hydrogenation of Baylis Hillman derivatives of substituted isoxazolecarbaldehydes

Results of the catalytic hydrogenation of Baylis-Hillman adducts obtained from substituted 3-, 4- and 5-isoxazolecarbox-aldehydes and their corresponding acetates in the presence of Raney-Ni and Pd-C are presented. The hydrogenation of Baylis-Hillman adducts of substituted 5-isoxazolecarbaldehydes and 3-isoxazolecarbaldehydes in the presence of Raney-Ni furnishes diastereoselectively syn enaminones over anti and in the presence of boric acid as an additive further enhancement of diastereoselectivity in favor of syn isomer is observed. The Pd-C-promoted hydrogenation of these substrates is also diastereoselective in favor of syn isomer but occurs without the hydrogenolysis of isoxazole-ring. The presence of boric acid as additive in this hydrogenation exhibits no pronounced effect on diastereoselectivity. The Raney-Ni-mediated hydrogenation of Baylis-Hillman adducts of substituted 4-isoxazolecarbaldehydes yield pyridone derivatives and Pd-C-promoted hydrogenation of the same substrate is diastereoselective to afford the anti isomer of the resulting products. The enaminones derived from Baylis-Hillman adducts of 3- and 5-isoxazolecarbaldehydes serve as versatile precursors for '-hydroxy-1, 3-diketones, which undergo acid-catalyzed ring-closure reaction to afford the furanone derivatives in excellent yields

Watson–Crick and Sugar-Edge Base Pairing of Cytosine in the Gas Phase: UV and Infrared Spectra of Cytosine·2-Pyridone

While keto-amino cytosine is the dominant species in aqueous solution, spectroscopic studies in molecular beams and in noble gas matrices show that other cytosine tautomers prevail in apolar environments. Each of these offers two or three H-bonding sites (Watson–Crick, wobble, sugar-edge). The mass- and isomer-specific S1 ← S0 vibronic spectra of cytosine·2-pyridone (Cyt·2PY) and 1-methylcytosine·2PY are measured using UV laser resonant two-photon ionization (R2PI), UV/UV depletion, and IR depletion spectroscopy. The UV spectra of the Watson–Crick and sugar-edge isomers of Cyt·2PY are separated using UV/UV spectral hole-burning. Five different isomers of Cyt·2PY are observed in a supersonic beam. We show that the Watson–Crick and sugar-edge dimers of keto-amino cytosine with 2PY are the most abundant in the beam, although keto-amino-cytosine is only the third most abundant tautomer in the gas phase. We identify the different isomers by combining three different diagnostic tools: (1) methylation of the cytosine N1–H group prevents formation of both the sugar-edge and wobble isomers and gives the Watson–Crick isomer exclusively. (2) The calculated ground state binding and dissociation energies, relative gas-phase abundances, excitation and the ionization energies are in agreement with the assignment of the dominant Cyt·2PY isomers to the Watson–Crick and sugar-edge complexes of keto-amino cytosine. (3) The comparison of calculated ground state vibrational frequencies to the experimental IR spectra in the carbonyl stretch and NH/OH/CH stretch ranges strengthen this identification.

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds.

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl](1/2) at 3.4-5M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys(36)-Cys(49) and two disulfide bonds formed by two pair of consecutive cysteines, Cys(22)-Cys(23) and Cys(56)-Cys(57), a unique disulfide structure of polypeptide that has not been documented previously.

Switching on the fluorescence of 2-aminopurine by site-selective microhydration

​2-Aminopurine (​2AP) is a fluorescent isomer of ​adenine and has a fluorescence lifetime of ~11 ns in water. It is widely used in biochemical settings as a site-specific fluorescent probe of DNA and RNA structure and base-flipping and -folding. These assays assume that ​2AP is intrinsically strongly fluorescent. Here, we show this not to be the case, observing that gas-phase, jet-cooled ​2-aminopurine and ​9-methyl-2-aminopurine have very short fluorescence lifetimes (156 ps and 210 ps, respectively); they are, to all intents and purposes, non-fluorescent. We find that the lifetime of ​2-aminopurine increases dramatically when it is part of a hydrate cluster, 2AP·(H2O)n, where n = 1–3. Not only does it depend on the presence of water molecules, it also depends on the specific hydrogen-bonding site to which they attach and on the number of H2O molecules at that site. We selectively microhydrate ​2-aminopurine at its sugar-edge, cis-amino or trans-amino sites and see that its fluorescence lifetime increases by 4, 50 and 95 times (to 14.5 ns), respectively.

One-pot heterogeneous synthesis of Δ(3)-tetrahydrocannabinol analogues and xanthenes showing differential binding to CB(1) and CB(2) receptors

Δ(9)-tetrahydrocannabinol (Δ(9)-THC) is the major psychoactive cannabinoid in hemp (Cannabis sativa L.) and responsible for many of the pharmacological effects mediated via cannabinoid receptors. Despite being the major cannabinoid scaffold in nature, Δ(9)-THC double bond isomers remain poorly studied. The chemical scaffold of tetrahydrocannabinol can be assembled from the condensation of distinctly substituted phenols and monoterpenes. Here we explored a microwave-assisted one pot heterogeneous synthesis of Δ(3)-THC from orcinol (1a) and pulegone (2). Four Δ(3)-THC analogues and corresponding Δ(4a)-tetrahydroxanthenes (Δ(4a)-THXs) were synthesized regioselectively and showed differential binding affinities for CB1 and CB2 cannabinoid receptors. Here we report for the first time the CB1 receptor binding of Δ(3)-THC, revealing a more potent receptor binding affinity for the (S)-(-) isomer (hCB1Ki = 5 nM) compared to the (R)-(+) isomer (hCB1Ki = 29 nM). Like Δ(9)-THC, also Δ(3)-THC analogues are partial agonists at CB receptors as indicated by [(35)S]GTPγS binding assays. Interestingly, the THC structural isomers Δ(4a)-THXs showed selective binding and partial agonism at CB2 receptors, revealing a simple non-natural natural product-derived scaffold for novel CB2 ligands.

Distamycin-NA: A DNA Analog with an Aromatic Heterocyclic Polyamide Backbone. Part 1. Synthesis and structural analysis of monomers and dimers containing the nucleobase uracil

The synthesis of the monomeric building block 13 and its constitutional isomer 12 of a new type of DNA analog, distamycin-NA, is presented (Schemes 1 and 2). This building block consists of a uracil base attached to a thiophene core unit via a biaryl-like axis. Next to the biaryl-like axis on the thiophene chromophore, a carboxy and an amino substituent are located allowing for oligomerization via peptide coupling. The proof of constitution and the conformational preferences about the biaryl-like axis were established by means of X-ray analyses of the corresponding nitro derivatives 10 and 11. Thus, the uracil bases are propeller-twisted relative to the thiophene core, and bidentate H-bonds occur between two uracil bases in the crystals. The two amino-acid building blocks 12 and 13 were coupled to give the dimers 15 and 16 using dicyclohexylcarbodiimide (DCC) in THF/LiCl and DMF, respectively. While the dimer 15 showed no atropisomerism on the NMR time scale at room temperature, its isomer 16 occurred as distinct diastereoisomers due to the hindered rotation around its biaryl-like axis. Variable-temperature 1H-NMR experiments allowed to determine a rotational barrier of 19 ± 1 kcal/mol in 16. The experimental data were complemented by AM1 calculations.

Modeling the Histidine–Phenylalanine Interaction: The NH···π Hydrogen Bond of Imidazole·Benzene

NH···π hydrogen bonds occur frequently between the amino acid side groups in proteins and peptides. Data-mining studies of protein crystals find that ~80% of the T-shaped histidine···aromatic contacts are CH···π, and only ~20% are NH···π interactions. We investigated the infrared (IR) and ultraviolet (UV) spectra of the supersonic-jet-cooled imidazole·benzene (Im·Bz) complex as a model for the NH···π interaction between histidine and phenylalanine. Ground- and excited-state dispersion-corrected density functional calculations and correlated methods (SCS-MP2 and SCS-CC2) predict that Im·Bz has a Cs-symmetric T-shaped minimum-energy structure with an NH···π hydrogen bond to the Bz ring; the NH bond is tilted 12° away from the Bz C₆ axis. IR depletion spectra support the T-shaped geometry: The NH stretch vibrational fundamental is red shifted by −73 cm⁻¹ relative to that of bare imidazole at 3518 cm⁻¹, indicating a moderately strong NH···π interaction. While the Sₒ(A1g) → S₁(B₂u) origin of benzene at 38 086 cm⁻¹ is forbidden in the gas phase, Im·Bz exhibits a moderately intense Sₒ → S₁ origin, which appears via the D₆h → Cs symmetry lowering of Bz by its interaction with imidazole. The NH···π ground-state hydrogen bond is strong, De=22.7 kJ/mol (1899 cm⁻¹). The combination of gas-phase UV and IR spectra confirms the theoretical predictions that the optimum Im·Bz geometry is T shaped and NH···π hydrogen bonded. We find no experimental evidence for a CH···π hydrogen-bonded ground-state isomer of Im·Bz. The optimum NH···π geometry of the Im·Bz complex is very different from the majority of the histidine·aromatic contact geometries found in protein database analyses, implying that the CH···π contacts observed in these searches do not arise from favorable binding interactions but merely from protein side-chain folding and crystal-packing constraints. The UV and IR spectra of the imidazole·(benzene)₂ cluster are observed via fragmentation into the Im·Bz+ mass channel. The spectra of Im·Bz and Im·Bz₂ are cleanly separable by IR hole burning. The UV spectrum of Im·Bz₂ exhibits two 000 bands corresponding to the Sₒ → S₁ excitations of the two inequivalent benzenes, which are symmetrically shifted by −86/+88 cm⁻¹ relative to the 000 band of benzene.

Plant Adenosine 5′-phosphosulfate Reductase Is a Novel Iron-Sulfur Protein

Adenosine 5′-phosphosulfate reductase (APR) catalyzes the two-electron reduction of adenosine 5′-phosphosulfate to sulfite and AMP, which represents the key step of sulfate assimilation in higher plants. Recombinant APRs from both Lemna minorand Arabidopsis thaliana were overexpressed inEscherichia coli and isolated as yellow-brown proteins. UV-visible spectra of these recombinant proteins indicated the presence of iron-sulfur centers, whereas flavin was absent. This result was confirmed by quantitative analysis of iron and acid-labile sulfide, suggesting a 4Fe-4S cluster as the cofactor. EPR spectroscopy of freshly purified enzyme showed, however, only a minor signal at g = 2.01. Therefore, Mössbauer spectra of 57Fe-enriched APR were obtained at 4.2 K in magnetic fields of up to 7 tesla, which were assigned to a diamagnetic 4Fe-4S2+ cluster. This cluster was unusual because only three of the iron sites exhibited the same Mössbauer parameters. The fourth iron site gave, because of the bistability of the fit, a significantly smaller isomer shift or larger quadrupole splitting than the other three sites. Thus, plant assimilatory APR represents a novel type of adenosine 5′-phosphosulfate reductase with a 4Fe-4S center as the sole cofactor, which is clearly different from the dissimilatory adenosine 5′-phosphosulfate reductases found in sulfate reducing bacteria.

THE ESTROGENIC PROPERTIES OF O,P'-DDT

o,p'-DDT is a major component of the pesticide DDT (dichlorodiphenyltrichloro ethane, technical grade). Although possessing little insecticidal ability, the o,p'- isomer has two major biological activities which affect mammalian reproductive systems: it is estrogenic, and it induces hepatic mixed function oxidase enzymes. The focus of this work is the characterization of the estrogenic properties of o,p'-DDT in rodents.^ Initial studies examined the ability of o,p'-DDT to bind to and interact with elements of the estrogen receptor system. In an in vitro assay, DDT was shown to compete with 17(beta)-estradiol (E(,2)) for binding to cytoplasmic estrogen receptors (R(,c)) from normal and neoplastic tissues in two rodent species. The following phenomena were studied by measuring receptor levels from uteri (whole uteri and/or uterine cell types) taken from immature ovariectomized rats given one acute injection of o,p'-DDT or E(,2): the translocation of the R(,c) to the nucleus, nuclear receptor (R(,n)) retention patterns, and the subsequent reappearance of R(,c) in the cytoplasm.^ The magnitude and temporal patterns of the biological responses of uteri from similar immature rats were compared following o,p'-DDT and E(,2) exposure. The responses examined included increased "Induced Protein" synthesis (in vitro); and uterine wet weight, DNA synthesis and mitosis (in vivo).^ From dose-response data, correlations were made between R(,n) levels and levels of subsequent biological responses. The aim was to lend support to the premise that biological responses to o,p'-DDT exposure occur as a result of its interaction with the classical estrogen receptor system. Correlation coefficients of 0.95 to 0.98 were obtained between R(,n) levels and levels of responses examined, strongly supporting this hypothesis.^ Finally, o,p'-DDT was shown to be as effective as E(,2) in supporting the growth of a transplantable estrogen-responsive mammary tumor in adult rats (although it was unable to support the growth of a transplantable estrogen-dependent renal tumor in hamsters). While the positive result cannot be directly extrapolated to human or animal exposure to environmental estrogens, it suggests that hyperplastic responses of estrogen sensitive tissues should be considered as a possible toxicity of o,p'-DDT, related compounds having estrogenic properties, and other environmental estrogens. ^

A four-year record of UK'37- and TEX86-derived sea surface temperature estimates from sinking particles in the filamentous upwelling region off Cape Blanc, Mauritania

Lipid biomarker records from sinking particles collected by sediment traps are excellent tools to study the seasonality of biomarker production as well as processes of particle formation and settling, ultimately leading to the preservation of the biomarkers in sediments. Here we present records of the biomarker indices UK'37 based on alkenones and TEX86 based on isoprenoid glycerol dialkyl glycerol tetraethers (GDGTs), both used for the reconstruction of sea surface temperatures (SST). These records were obtained from sinking particles collected using a sediment trap moored in the filamentous upwelling zone off Cape Blanc, Mauritania, at approximately 1300 water depth during a four-year time interval between 2003 and 2007. Mass and lipid fluxes are highest during peak upwelling periods between October and June. The alkenone and GDGT records both display pronounced seasonal variability. Sinking velocities calculated from the time lag between measured SST maxima and minima and corresponding index maxima and minima in the trap samples are higher for particles containing alkenones (14-59 m/d) than for GDGTs (9-17 m/d). It is suggested that GDGTs are predominantly exported from shallow waters by incorporation in opal-rich particles. SST estimates based on the UK'37 index faithfully record observed fluctuations in SST during the study period. Temperature estimates based on TEX86 show smaller seasonal amplitudes, which can be explained with either predominant production of GDGTs during the warm season, or a contribution of GDGTs exported from deep waters carrying GDGTs in a distribution that translates to a high TEX86 signal.