A proposal for a convenient notation for P-chiral nucleotide analogues. Part 3. Compounds with one nucleoside residue and nonnucleosidic derivatives

Recently, we have proposed a new D_P/L_P stereochemical notation for P-chiral dinucleoside monophosphate analogues that permits simple correlation between spatial arrangement of the substituents and the configuration at the phosphorus center. As an extension of this work, we present here applications of the D_P/L_P notation to derivatives containing only one nucleoside unit (e.g., alkyl nucleoside phosphodiesters, nucleoside phosphomonoesters, cyclic phosphate derivatives, nucleoside di-, and triphosphates) and to nonnucleosidic phosphorus compounds.     

A proposal for a convenient notation for P-chiral nucleotide analogues. Part 2. Dinucleoside monophosphate analogues

A configuration of ligands around a phosphorus atom in P-chiral dinucleoside monophosphate analogues can be described using D_P/L_P stereochemical notation, which allows immediate correlation between the notation of configuration and the actual spatial arrangement of the phosphorus ligands. The area of applications of this new stereochemical nomenclature covers dinucleoside units bridged by virtually any type of tri-and tetra-coordinated phosphorus moieties, that is, phosphorothioates, phosphoramidates, phosphoramidites, boranephosphates, methanephosphonates, H-phosphonates, and many others.     

Compounds stimulating cytosolic phospholipase A2 activity with a combinational action mode

The screening of small synthetic compound libraries is a useful means of identifying molecules that modulate various cellular responses. We screened more than 10,000 different small compounds and identified three synthetic compounds that stimulate arachidonic acid (AA) release in a combinational manner in neutrophil-like differentiated HL60 cells. These three compounds were designated as AARIC-1, -2, and -3, representing AA release inducing compounds-1, -2, and -3. Although AA release was not induced by any single one of these compounds, it was dramatically stimulated by the three compounds in combination. Moreover, the effect of combined treatment by these compounds on AA release was completely abolished by MAFP and AACOCF(3), specific cytosolic phospholipase A(2) inhibitors. Furthermore, we found that AARIC-3 stimulates cytosolic calcium influx, while AARIC-1 induces ERK activation. Taken together, we demonstrate a useful approach to the study of complicated and nonlinear intracellular signaling networks using small synthetic compounds in combination.     

Oriented solids as a colloid suspension in nematic liquid crystal – New tool for IR-spectroscopic and structural elucidation of inorganic compounds and glasses

Possibilities of the linear-polarized infrared (IR-LD) spectroscopy of oriented colloid suspensions in nematic liquid crystals, for structural and local structural elucidation for first time are demonstrated of inorganic compounds and glasses. The advantages of the method for tellurite and borate glasses are shown. The IR-band assignment of the typical local structural units in the glasses are proposed by a comparison with the IR-characteristics of appropriate crystalline analogues as α-TeO₂, V₂O₅, MoO₃ · H₂O and its high temperature form. The IR-spectroscopic characteristics of BO₃, BO₄ and boroxol ring are elucidated, using crystalline β-BaB₂O₄, SrB₄O₇, H₃BO₃ and B₂O₃ as model systems, where the structural moieties have been refined by single crystal X-ray diffraction.     

Generation of a homology model for the human cytochrome P450, CYP24A1, and the testing of putative substrate binding residues by site-directed mutagenesis and enzyme activity studies

A systematic analysis of conserved H-bonding patterns and tertiary structural motifs from 13 crystal structures was used to create a homology model for the human multicatalytic cytochrome P450, CYP24A1, involved in catabolism of 1alpha,25-dihydroxyvitamin D3. The substrate was docked in the active site and used to identify potential substrate contact residues in the B' helix, B'/C loop, F-helix and the beta-5 hairpin. Seven CYP24A1 mutants were created and studied by mammalian cell transfection and CYP24A1 activity assay. Mutants showed reduced metabolic rates and altered metabolite patterns compared to wild-type. We conclude that: Ile-131 positions substrate via A-ring and cis-triene contacts; Trp-134 and Gly-499 are determinants of substrate access; Leu-148 contacts the substrate side-chain; Met-246 is important in mediating regioselectivity. Our findings validate the new model of CYP24A1, which can now be used to predict structural modifications for rational vitamin D drug design.     

Cluster-root production and organic anion exudation in a group of old-world lupins and a new-world lupin

We examined the capacity of several Old-World lupin species (Lupinus luteus L., L. hispanicus Boiss. et Reuter and L. angustifolius L.) and one species of a New-World lupin (L. mutabilis Sweet) to form cluster roots under a range of conditions in solution culture. The effect of the synthetic auxin, IBA (indole-3-butyric acid), on cluster-root development in L. luteus and L. albus L. provided with an adequate phosphorus (P) supply was also investigated. In addition, the effect of a high nitrate-N (NO₃-N) supply on the efflux of citrate and malate from roots of L. angustifolius was examined to determine if specific regions of the root system exuded these organic anions. When P-deficient, L. hispanicus, L. luteus and L. mutabilis formed cluster roots that secreted organic anions. Citrate was generally the dominant organic anion exuded, although succinate was also exuded in large quantities from L. luteus. Citrate efflux by L. hispanicus and L. luteus was at least comparable to that reported for P-deficient L. albus[up to 1.092 nmol g^–1 fresh weight (FW) s^–1], but was over an order of magnitude lower in L. mutabilis (0.036 nmol g^–1 FW s^–1). Citrate and malate were not detected in significant amounts from either the lateral roots or the root tips of any species grown under P-sufficient or -deficient conditions. Citrate efflux from cluster roots of L. luteus showed a diurnal pattern, similar to that reported for L. albus, with maximum efflux during the day, and declining to a minimum before dawn. IBA added to the nutrient solution induced cluster-root formation on both L. albus and L. luteus at concentrations of P that would normally suppress the production of these roots. However, the IBA-induced cluster roots did not exude significant amounts of citrate. Although L. angustifolius did not produce cluster roots when P-deficient, it produced cluster-like root structures that exuded citrate (0.053 nmol g^–1 FW s^–1) when grown at a high nitrate-N (NO₃-N) supply. L. angustifolius did not exude significant citrate or malate from lateral roots or root tips when grown at either high or low NO₃-N supply. Our findings for L. hispanicus and L. luteus are the first reports of cluster-root formation in response to P deficiency for these Old-World species, and for L. mutabilis, it is the first report of cluster roots for a New-World lupin species. These reports indicate that evolutionary and biogeographical aspects of cluster-root formation in the genus Lupinus need to be revised. Furthermore, investigation is warranted to determine the capacity of species of the large group of New-World lupins to form cluster roots in soils of their native habitats.     

New substance P receptor antagonists. Conception, synthesis and biological evaluation of spirolactam derivatives and their tripeptide Cbz-Pro-Leu-Trp-OBzl(CF3)2 and lactam pseudopeptide derivatives

New chemical entities were designed from the C-terminal sequence of GR71251, an NK₁ pseudopeptide antagonist. Three new NK₁ antagonists were identified with high affinity and selectivity for NK₁ receptors.     

A simple and rapid harvesting method for microalgae by in situ magnetic separation

A simple and rapid harvesting method by in situ magnetic separation with naked Fe₃O₄ nanoparticles has been developed for the microalgal recovery of Botryococcus braunii and Chlorella ellipsoidea. After adding the magnetic particles to the microalgal culture broth, the microalgal cells were adsorbed and then separated by an external magnetic field. The maximal recovery efficiency reached more than 98% for both microalgae at a stirring speed of 120 r/min within 1 min, and the maximal adsorption capacity of these Fe₃O₄ nanoparticles reached 55.9 mg-dry biomass/mg-particles for B. braunii and 5.83 mg-dry biomass/mg-particles for C. ellipsoidea. Appropriate pH value and high nanoparticle dose were favorable to the microalgae recovery, and the adsorption mechanism between the naked Fe₃O₄ nanoparticles and the microalgal cells was mainly due to the electrostatic attraction. The developed in situ magnetic separation technology provides a great potential for saving time and energy associated with improving microalgal harvesting.     

A sugar ester and an iridoid glycoside from Scrophularia ningpoensis

From cytotoxic extracts of the roots of Scrophularia ningpoensis Hemsl. (Scrophulariaceae) a new sugar ester, ningposide D (3-O-acetyl-2-O-p-methoxycinnamoyl-alpha(beta)-L-rhamnopyranose) (1) and a new iridoid glycoside, scrophuloside B4 (6-O-(2'-O-acetyl-3'-O-cinnamoyl-4'-O-p-methoxycinnamoyl-alpha-L-rhamnopyranosyl) catalpol) (2) along with known compounds: oleanonic acid (3), ursolonic acid (4), cinnamic acid (5), 3-hydroxy-4-methoxy benzoic acid (6), 5-(hydroxymethyl)-2-furfural (7) and beta-sitosterol (8) were isolated. The structures of the new compounds were elucidated by spectral data (1, 2D NMR, EI, HRESI-MS and MS/MS). Oleanonic acid (3) and ursolonic acid (4) were found to be cytotoxic against a series of human cancer cell lines with IC50=4.6, 15.5 microM on MCF7; 4.2, 14.5 microM on K562; 14.8, 44.4 microM on Bowes; 24.9, 43.6 microM on T24S; 61.3, 151.5 microM on A549, respectively. Beta-sitosterol (8) inhibited Bowes cells growth at IC50=36.5 microM. Scrophuloside B4 (2) showed activity on K562 and Bowes cells at IC50=44.6, 90.2 microM, respectively.     

The essentiality of His-47 and the N-terminal region for the binding of 8-anilinonaphthalene-1-sulfonate with Taiwan cobra phospholipase A2

The binding of the apolar fluorescent dye 8-anilinonaphthalene-1-sulfonate (ANS) toNaja naja atra phospholipase A₂ (PLA₂) as well as the enhancement of ANS fluorescence of the PLA₂-ANS complex decreased with increasing pH in a pH range from 3 to 9. These pH-dependent curves can be well interpreted as the perturbation of an ionizable group with pK value of 5.8, which was assigned as His-47 in the active site of PLA₂. The ionizable group with pK 5.8 was no longer observed after methylation of His-47, supporting the idea that thepH dependence of ANS binding arose from an electrostatic interaction between His-47 and the bound ANS. Removal of the N-terminal octapeptide of PLA₂ caused a precipitous drop in the capability of PLA₂ for binding with ANS and enhancing ANS fluorescence, reflecting that the integrity of the N-terminal region was essential for maintaining the hydrophobic character of the ANS-binding site. However, the nonpolarity of the ANS-binding site in the N-terminus-removed derivative was still partially retained at lowpH, but was completely lost at highpH. Evidently, the N-terminal region plays a more crucial role in ANS binding at highpH than at lowpH. These results indicate that hydrophobic interaction as well as electrostatic interaction are involved in the binding of ANS to PLA₂, and that the relative contributions of both interactions in ANS fluorescence enhancement may be different under differentpH.     

Charge equilibria and pK(a) of malvidin-3-glucoside by electrophoresis

Paper electrophoresis has been used over the pH range 1.2 to 10.4 to measure apparent pK(a) values for malvidin-3-O-glucoside of pK(a(1)) 1.76+/-0.07, pK(a(2)) 5.36+/-0.04, and pK(a(3)) 8.39+/-0.07. Using solvent partitioning between buffered aqueous solutions and n-octanol, several micro-pK(a) constants for malvidin-3-O-glucoside were also identified, highlighting the complex nature of malvidin-3-glucoside equilibria. As a nonspectrophotometric procedure, the charge-dependent electrophoretic mobility method provided independent information on the net charge and color of anthocyanin species at wine pH (ca. 3.6). At this pH, the color of malvidin-3-glucoside in red wines is consistent only with the uncharged quinonoidal base as a major colored component of the equilibria.     

A validated liquid chromatography-mass spectrometry method for the determination of leukotrienes B4 and B5 produced by stimulated human polymorphonuclear leukocytes

A validated high-performance liquid chromatography (HPLC)-mass spectrometry method has been developed for the simultaneous assay of leukotrienes (LTs) B4 and B5, derived from omega-6 arachidonic acid and omega-3 polyunsaturated fatty acids (PUFA), respectively, produced by human polymorphonuclear leukocytes (PMNLs) stimulated with calcium ionophore A23187. The HPLC separation of PMNL ether extracts was performed on a reversed-phase column using a gradient elution program of 15 mM ammonium acetate and MeOH. Detection was performed by electrospray ionization-single quadripole mass spectrometry using single ion reaction monitoring in the negative mode at m/z 333.3 [M-H](-) and m/z 335.2 for prostaglandin B2/LTB5 and LTB4, respectively. The calibration curves for LTB4 and LTB5 were linear over the ranges 165-990 and 0.825-13.2 ng/ml, respectively. The lower limit of quantification for LTB5 was 0.66 ng/ml. The mean absolute recoveries for LTB4 and LTB5 were 81+/-4.8% and 82+/-5.9%, respectively. The method is precise with mean interday CVs for LTB4 and LTB5 within 7.1-10.7, and 3.8-9.4%, respectively, and accurate (range of interday deviations for LTB4 and LTB5 were -7.8 to 1, and -5 to 9% , respectively). The method has been validated and is being applied to the simultaneous quantification of the leukotrienes B4 and B5 in stimulated PMNLs in a clinical protocol studying the influence of a diet enriched in omega-3 PUFA on various surrogate markers of inflammation in young cystic fibrosis patients.     

Synthesis and preliminary evaluation of [H]PSB-0413, a selective antagonist radioligand for platelet P2Y12 receptors

The selective antagonist radioligand [³H]2-propylthioadenosine-5′-adenylic acid (1,1-dichloro-1-phosphonomethyl-1-phosphonyl) anhydride ([³H]PSB-0413) was prepared by catalytic hydrogenation of its propargyl precursor with a high specific radioactivity of 74 Ci/mmol. In preliminary saturation binding studies, [³H]PSB-0413 showed high affinity for platelet P2Y₁₂ receptors with a K_D value of 4.57 nM. Human platelets had a high density of P2Y₁₂ receptors exhibiting a B_max value of 7.66 pmol/mg of protein.     

Development of a one-step enzyme immunoassay for the quantitation of gibberellin A3 in barley seeds

The development of a sensitive and specific enzyme immunoassay for GA₃ is reported. This method was based on the use of peroxidase labelled GA₃ and immobilized antibodies. In order to obtain a rapid immunoassay, several steps of purification were analyzed to show their necessity. Barley seed extracts were assayed at different steps of purification to exhibit the effect of extract components on the assay. It was demonstrated that HPLC had to be performed when a selective quantitation of GA₃ was required. This assay allowed GA₃ to be measured with reproducibility as its unmethylated form and the quantitation of GA₃ in barley seeds with this enzyme immunoassay was correlated to a GC-MS method.Abbreviations GA₃ gibberellin A₃ - EIA enzyme immunoassay - DMF dimethylformamide - TEA tri(n)ethylamine - BSA bovine serum albumin - OVA ovalbumine - ECF ethylchloroformate - PB phosphate buffer     

Site-specific discrimination by cyanovirin-N for alpha-linked trisaccharides comprising the three arms of Man(8) and Man(9)

Cyanovirin-N (CVN) is a novel cyanobacterial protein that selectively binds with nanomolar affinities the mammalian oligosaccharides Man(8) and Man(9). Consequently, CVN potently blocks HIV entry through highly avid carbohydrate-mediated interactions with the HIV-envelope glycoprotein gp120, and is under preclinical investigation as an anti-HIV microbicide. CVN contains two non-overlapping carbohydrate-binding sites that bind the disaccharide Manalpha(1-2)Manalpha (which represents the terminal disaccharide of all three arms of Man(9)) with low to sub-micromolar affinities. The solution structure of a 1:2 CVN:Manalpha(1-2)Manalpha complex revealed that CVN recognizes the stacked conformation of Manalpha(1-2)Manalpha through a deep hydrophilic-binding pocket on one side of the protein (site 2) and a semi-circular cleft on the other (site 1). With the prominent exception of the C1 hydroxyl group of the reducing mannopyranose ring, the bound disaccharide is positioned so that each hydroxyl group is involved in a direct or water-mediated hydrogen bond to the polar or charged side-chains comprising the binding pocket. Thus, to determine whether the next-most reducing mannopyranose ring will augment CVN affinity and selectivity, we have characterized by NMR and ITC the binding of CVN to three synthetic trisaccharides representing the full-length D1, D2 and D3 arms of mammalian oligomannosides. Our findings demonstrate that site 1 is able to discriminate between the three related trisaccharides methyl Manalpha(1-2)Manalpha(1-2)Man, methyl Manalpha(1-2)Manalpha(1-3)Man and methyl Manalpha(1-2)Manalpha(1-6)Man with remarkable selectivity, and binds these trisaccharides with K(A) values ranging from 8.1x10(3)M(-1) to 6.6x10(6)M(-1). Site 2 is less selective in that it binds all three trisaccharides with similar K(A) values ranging from 1.7 to 3.7(+/-0.3)x10(5)M(-1), but overall binds these trimannosides with higher affinities than site 1. The diversity of pathogenic organisms that display alpha(1-2)-linked mannosides on their cell surfaces suggests a broad defensive role for CVN in its cyanobacterial source.     

Probing the sweet determinants of brazzein: wild-type brazzein and a tasteless variant, brazzein-ins(R18a-I18b), exhibit different pH-dependent NMR chemical shifts

Brazzein is a small, intensely sweet protein. As a probe of the functional properties of its solvent-exposed loop, two residues (Arg-Ile) were inserted between Leu18 and Ala19 of brazzein. Psychophysical testing demonstrated that this mutant is totally tasteless. NMR chemical shift mapping of differences between this mutant and brazzein indicated that residues affected by the insertion are localized to the mutated loop, the region of the single alpha-helix, and around the Cys16-Cys37 disulfide bond. Residues unaffected by this mutation included those near the C-terminus and in the loop connecting the alpha-helix and the second beta-strand. In particular, several residues of brazzein previously shown to be essential for its sweetness (His31, Arg33, Glu41, Arg43, Asp50, and Tyr54) exhibited negligible chemical shift changes. Moreover, the pH dependence of the chemical shifts of His31, Glu41, Asp50, and Tyr54 were unaltered by the insertion. The insertion led to large chemical shift and pKa perturbation of Glu36, a residue shown previously to be important for brazzein's sweetness. These results serve to refine the known sweetness determinants of brazzein and lend further support to the idea that the protein interacts with a sweet-taste receptor through a multi-site interaction mechanism, as has been postulated for brazzein and other sweet proteins (monellin and thaumatin).     

Purification of a post-synaptic neurotoxic phospholipase A2 from Naja naja venom and its inhibition by a glycoprotein from Withania somnifera

A post-synaptic neurotoxic phospholipase A(2) (PLA(2)) has been purified from Indian cobra Naja naja venom. It was associated with a peptide in the venom. The association was disrupted using 8 M urea. It is denoted to be a basic protein by its behavior on both ion exchange chromatography and electrophoresis. It is toxic to mice, LD(50) 1.9 mg/kg body weight (ip). It is proved to be post-synaptic PLA(2) by chymographic experiment using frog nerve-muscle preparation. A glycoprotein, (WSG) was isolated from a folk medicinal plant Withania somnifera. The WSG inhibited the phospholipase A(2) activity of NN-XIa-PLA(2,) isolated from the cobra venom, completely at a mole-to-mole ratio of 1:2 (NN-XIa-PLA(2): WSG) but failed to neutralize the toxicity of the molecule. However, it reduced the toxicity as well as prolonged the death time of the experimental mice approximately 10 times when compared to venom alone. The WSG also inhibited several other PLA(2) isoforms from the venom to varying extent. The interaction of the WSG with the PLA(2) is confirmed by fluorescence quenching and gel-permeation chromatography. Chemical modification of the active histidine residue of PLA(2) using p-brophenacyl bromide resulted in the loss of both catalytic activity as well as neurotoxicity of the molecule. These findings suggest that the venom PLA(2) has multiple sites on it; perhaps some of them are overlapping. Application of the plant extract on snakebite wound confirms the medicinal value associated with the plant.     

Effect of the leukotriene A4 hydrolase aminopeptidase augmentor 4-methoxydiphenylmethane in a pre-clinical model of pulmonary emphysema

The leukotriene A(4) hydrolase enzyme is a dual functioning enzyme with the following two catalytic activities: an epoxide hydrolase function that transforms the lipid metabolite leukotriene A(4) to leukotriene B(4) and an aminopeptidase function that hydrolyzes short peptides. To date, all drug discovery efforts have focused on the epoxide hydrolase activity of the enzyme, because of extensive biological characterization of the pro-inflammatory properties of its metabolite, leukotriene B(4). Herein, we have designed a small molecule, 4-methoxydiphenylmethane, as a pharmacological agent that is bioavailable and augments the aminopeptidase activity of the leukotriene A(4) hydrolase enzyme. Pre-clinical evaluation of our drug showed protection against intranasal elastase-induced pulmonary emphysema in murine models.     

A ‘phase-shift’ fusion system for the regulation of foreign gene expression by lambda represser in Gram-negative bacteria

A ‘phase-shift’ translation fusion vector was constructed in which mutually compatible restriction sites BamHI, BelI and BglII are positioned in such a manner that the cut point is in a different reading frame, immediately following the ATG start codon and ribosome-binding site of the λ cro gene. The λ cro gene is expressed from promoter p_R and controlled by a thermosensitive (cI857) λ repressor. The usefulness of the expression vector was demonstrated using a gal gene lacking the ATG start codon and fusing this to the p_R promoter and ATG start codon of the λ cro gene, resulting in cI857-regulated expression of galactokinase. The vector is of general use for foreign gene expression in Escherichia coli when the target gene has a compatible cohesive end (5′-GATC-3′) at the N terminus (provided, for example, by a BamHI linker). The A λ cI857-p_R-cro-galK cassette was cloned into pJRD215, a wide-host-range plasmid and transferred by conjugation to a variety of Gram-negative bacteria. In all cases, thermosensitive regulation of galactokinase could be demonstrated, though the levels of induction varied considerably. These results show that the powerful λ p_R promoter and the efficient A repressor can be used to regulate expression of foreign genes in Gram-negative organisms other than E. coli.