Full 1H NMR assignment of a 24-nucleotide RNA hairpin: Application of the 1H 3D-NOE/2QC experiment

The subject RNA models the binding site for the coat protein of the R17 virus, as well as the ribosome recognition sequence for the R17 replicase gene. With an RNA of this size, overlaps among the sugar protons complicate assignments of the ¹H NMR spectrum. The cross peaks that overlap significantly in 2D-NOE spectra can frequently be resolved by introducing a third, in our approach the double-quantum, frequency axis. In particular the planes in a 3D-NOE/2QC spectrum perpendicular to the 2Q axis are extremely useful, showing a highly informative repeating NOE-2Q pattern. In this experiment substantial J-coupling confers special advantages. This always occurs for geminal pairs (H5/H5 for RNA plus H2/H2 for DNA), as well as for H5/H6, for H3/H4 in sugars with substantial populations of the N-pucker, for H1/H2 for S-puckered sugars, and usually for H2/H3. For the 24-mer RNA hairpin the additional information from the 3D-NOE/2QC spectrum allowed assignment of all of the non-exchangeable protons, eliminating the need for stable-isotope labeling.     

Cyanine dye structural and voltage-induced variations in photo-voltages of bilayer membranes

Summary Flash illumination alters the voltage across bilayer lipid membranes in the presence of certain cyanine dyes. The waveforms of the photo-voltage vary systematically with dye structure and imposed transmembrane voltage. Experimental results are reported for 27 positively charged cyanine dyes, primarily oxazole derivatives, using lecithin/oxidized cholesterol bilayer membranes and 10-mm sodium chloride solutions. Several dyes do not induce any photo-voltages. Examples are 3,3 diethyl 9 ethyl 2,2 oxacarbocyanine iodide, 3,3 diethyl 2 oxa 2 thiacyanine iodide, and 3,3 dimethyl 2,2 indocarbocyanine iodide. Several dyes, when added to one side of the membranes, induce monophasic waveforms. Examples are 3,3 dimethyl 2,2 oxacarbocyanine chloride, and 3,4,3,4 tetramethyl 2,2 oxazalinocarbocyanine iodide. Other dyes induce a photo-voltage only if transmembrane voltages are imposed. These waveforms are biphasic with some dyes (3,3 diethyl 2,2 oxacarbocyanine iodide, for example) and monophasic with other dyes (3,3 dibutyl 2,2 oxacarbocyanine iodide, for example).The photo-voltage waveforms are explained by models that consider the movement of charged dye molecules within the membrane, following optical excitation. The dye movements are probably induced through charge rearrangements in the dye associated with long-lived triplet states, isomerization, or through excimer formation. These results provide information on the location and orientation of the dye molecules within bilayer membranes. The variations which occur in the waveforms with applied voltage indicate that these membranes are fluid in the direction perpendicular to the membrane plane.     

A novel PH-CT-COSY methodology for measuring JPH coupling constants in unlabeled nucleic acids. Application to HIV-2 TAR RNA

A quantitative analysis of J_PH scalar couplings in nucleic acids is difficult due to small couplings to phosphorus, the extreme overlap of the sugar protons and the fast relaxation of the spins involved in the magnetization transfer. Here we present a new methodology that relies on heteronuclear Constant Time Correlation Spectroscopy (CT-COSY). The three vicinal ³J_PH3, ³J_PH5 and ³J_PH5 scalar couplings can be obtained by monitoring the intensity decay of the P_i-H3_{i – 1} peak as a function of the constant time T in a 2D correlation map. The advantage of the new method resides in the possibility of measuring the two ³J_PH5 and ³J_PH5 scalar couplings even in the presence of overlapped H5/H5 resonances, since the quantitative information is extracted from the intensity decay of the P-H3 peak. Moreover, the relaxation of the H3 proton is considerably slower than that of the H5/H5 geminal protons and the commonly populated conformations of the phosphate backbone are associated with large ³J_PH3 couplings and relatively small ³J_{PH5 / H5}. These two facts lead to optimal signal-to-noise ratio for the P-H3 correlation compared to the P-H5/H5 correlation.The heteronuclear CT-COSY experiment is suitable for oligonucleotides in the 10–15 kDa molecular mass range and has been applied to the 30mer HIV-2 TAR RNA. The methodology presented here can be used to measure P-H dipolar couplings (D_PH) as well. We will present qualitative results for the measurement of P-H_base and P-H2 dipolar couplings in the HIV-2 TAR RNA and will discuss the reasons that so far precluded the quantification of the D_PHs for the 30mer RNA.     

Carotenoid pigments of Sorangium compositum (Myxobacterales) including two new carotenoid glucoside esters and two new carotenoid rhamnosides

Summary The carotenoid pigments of the myxobacterium Sorangium compositum were analyzed by chromatographical and chemical techniques and by visible, infra red, and mass spectroscopy. Besides -carotene, neurosporene, torulene, lycopene, and 1,2-dihydro-1-hydroxy--carotene, four new carotenoid glycosides were found. These pigments were identified as 1,2-dihydro-1-hydroxy-torulene glucoside ester (I), 1,2-dihydro-3,1-dihydroxy-torulene glucoside ester (III), 1,2-dihydro-1-hydroxy-torulene rhamnoside (II), and 1,2-dihydro-3,1-dihydroxytorulene rhamnoside (IV).Fifth communication on the carotenoids of myxobacteria. Fourth communication see Arch. Mikrobiol. 76, 364–380 (1971).     

The catalysis of nucleotide polymerization by compounds of divalent lead

Summary Soluble lead salts and a number of lead-containing minerals catalyze the formation of oligonucleotides from nucleoside 5-phosphorimidazolides. The effectiveness of lead compounds correlates strongly with their solubility. Under optimal conditions we were able to obtain 18% of pentamer and higher oligomers from ImpA. Reactions involving ImpU gave smaller yields.Abbreviations A adenosine - U uridine - Im imidazole - MeIm 1-methyl-imidazole - EDTA ethylenediaminetetraacetic acid - pA adenosine 5-phosphate - pU uridine 5-phosphate - Ap adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - AppA P₁,P₂-diadenosine 5-diphosphate - pNp (N = A,U) nucleotide 2(3), 5-diphosphate - ImpA adenosine 5-phosphoreimidazolide - ImpU uridine 5-phosphorimidazolide - A ²pA adenylyl-[25]-adenosine - A ³pA adenylyl-[35]-adenosine - pA ²pA 5-phospho-adenylyl-[25]-adenosine - pA ³pA 5-phospho-adenylyl-[35]-adenosine - pUpU 5-phospho-uridylyl-uridine - pApU 5-phospho-adenylyl-uridine - pUpA 5-phospho-uridylyladenine - (pA)n (n, 2,3,4,) oligoadenylates with 5 terminal phosphate - ImpApA 5-phosphorimidazolide of adenylyl adenosine - (pA) ₅₊ pentamer and higher oligoadenylates with 5 terminal phosphate - (Ap)nA (n = 2,3,4) oligoadenylates without terminal phosphates In the following we do not specify the nature of the internucleotide linkageIn the following we do not specify the nature of the internucleotide linkage     

Degradation of RNA in Escherichia coli. A hypothesis

Summary A hypothesis to explain RNA degradation in Escherichia coli is proposed. In this hypothesis all classes of RNA are potentially degradable unless they are protected. The proposed mechanism for mRNA degradation requires a combination of endonuclease(s) and exonuclease(s) which degrades RNA in the 3 to 5 direction. Ribosomes attached to the newly synthesized 5 end of an mRNA molecule protect it from being attacked endonucleolytically; a delay in attachment of ribosomes to this end exposes it to endonucleolytic cleavage, followed by exonucleolytic digestion from the newly exposed 3 end to the 5 end. This mechanism is consistent with an overall 5 to 3 direction of degradation for mRNA. Exoribonucleases that degrade polyribonucleotides from the 5 end to 3 end are not required. The 3 end of the messenger is protected by its association with DNA. In order to enable the mRNA to remain anchored to the DNA while serving as an efficient template for protein synthesis, a special region near the 3 end of the mRNA is envisaged. This hypothetical region would not be translated.     

Sugar conformation of a stereospecific 2′-R or 2′-S deuterium-labeled DNA decamer studied with proton-proton J coupling constants

The sugar conformation of a DNA decamer was studied with proton-proton ³J coupling constants. Two samples, one comprising stereospecifically labeled 2-R-²H for all residues and the other 2-S-²H, were prepared by the method of Kawashima et al. [J. Org. Chem. (1995) 60, 6980–6986; Nucleosides Nucleotides (1995) 14, 333–336], the deuterium labeling being highly stereospecific 99% for all 2-²H, 98% for 2-²H of A, C, and T, and 93% for 2-²H of G). The ³J values of all H1-H2 and H1-H2 pairs, and several H2-H3 and H2-H3 pairs were determined by line fitting of 1D spectra with 0.1–0.2 Hz precision. The observed J coupling constants were explained by the rigid sugar conformation model, and the sugar conformations were found to be between C3-exo and C2-endo with _m values of 26° to 44°, except for the second and 3 terminal residues C2 and C10. For the C2 and C10 residues, the lower fraction of S-type conformation was estimated from J_H1H2 and J_H1H2 values. For C10, the N–S two-site jump model or Gaussian distribution of the torsion angle model could explain the observed J values, and 68% S-type conformation or C1-exo conformation with 27° distribution was obtained, respectively. The differences between these two motional models are discussed based on a simple simulation of J-coupling constants.     

Geometry of the dry-state oligomerization of 2′,3′-cyclic phosphates

Summary Evaporation of a solution of thymidine plus either theexo or theendo diastereomer of uridine cyclic 2,3-O, O-phosphorothioate (U > p(S) in 1,2-diaminoethane hydrochloride buffer gave the 2,5 and 3,5 isomers of (P-thio) uridylylthymidine (Up(S)dT) in a ratio of 1:2 with a combined yield of about 20%. These isomers were re-converted to U > p(S) and dT by a reaction that is known to proceed by an in-line mechanism. Both the 2,5 and 3,5 isomers gave as product the same diastereomer of U > p(S) that had been used originally in their formation. These dry-state prebiotic reactions (Verlander, Lohrmann, and Orgel 1973) are thus shown to be stereospecific, and both the 2,5 and 3,5 internucleotide bonds are formed by an in-line mechanism.Abbreviations DAE 1,2-diaminoethane - HPLC high pressure liquid chromatography - RNase bovine pancreatic ribonuclease A, EC 3.1.4.22 - TEAB triethylammonium bicarbonate - tris tris(hydroxymethyl)aminomethane - UMP(S) uridine monophosphorothioate - U > p uridine cyclic 2,3-phosphate - U > p(S) uridine cyclic 2,3-O, O-phosphorothioate - Up(S)dT (P-thio)uridylylthymidine - U2p(R_p-S)5dT (P-thio)uridylylthymidine with theR configuration at phosphorous, and a 2,5 internucleotide linkage     

Prebiotic Formation of ADP and ATP from AMP,Calcium Phosphates and Cyanate in Aqueous Solution

Adenosine-5-triphosphate was synthesized by the phosphorylation of adenosine-5-diphosphate in aqueous solution containing cyanate as a condensing reagent and insoluble calcium phosphate produced from phosphate and calcium chloride. In a similar manner, adenosine-5-diphosphate was synthesized from adenosine-5-monophosphate. When the experiment was carried out in the conditions of 4 °C and pH 5.75, the formation of adenosine-5-diphosphate and adenosine-5-triphosphate from adenosine-5-monophosphate was observed in the yields of 19 and 7%, respectively. The other nucleoside-5-triphosphates were also produced from their respective diphosphates.     

The formation of peptides from the 2′(3′)-glycyl ester of a nucleotide

Summary We have synthesized 2(3)-O-(glycyl)-adenosine-5-(O-methylphos-phate), an analogue of the 3-terminus of aminoacylated tRNA. A 0.4M solution of this compound maintained at pH 8.2, yields 5.5% of diglycine and 11.5% of diketopiperazine, in addition to the hydrolysis products glycine and adenosine-5-(O-methylphosphate). Under the same conditions, glycine ethyl ester reacts much more slowly, but ultimately gives similar yields of diglycine and diketopiperazine.The aminolysis of 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) by free glycine is relatively inefficient, but serine reacts 20 times more rapidly and yields up to 50% of N-glycylserine. The prebiotic significance of these reactions is discussed.Abbreviations MepA adenosine-5-(O-methylphosphate) - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - MepA-bis-gly 2,3-O-(bis-glycyl)-adenosine-5-(O-methylphosphate) - DKP diketopiperazine - gly Et glycine ethyl ester - gly-ser N-glycylserine - O-gly-ser O-glycylserine - O-(gly)-gly-ser O-(glycyl)-glycylserine - Boc-gly N-tert-butyloxycarbonylglycine - MepA-Boc-gly 2(3)-O-(Boc-glycyl)-adenosine-5-(O-methylphosphate) - MepA-bis-Boc-gly 2,3-O-(bis-Boc-glycyl)-adenosine-5(O-methylphosphate) - (gly)₂ diglycine - (gly)₃ triglycine     

The investigation of the hcn derivative diiminosuccinonitrile as a prebiotic condensing agent. The formation of phosphate esters

Diiminosuccinonitrile (DISN) is formed readily by the Fe³⁺ oxidation of diaminomaleonitrile, a tetramer of HCN. DISN effects the phosphorylation of uridine in 13% yield to a mixture of the isomers of UMP when the reaction is performed in dimethylformamide solution. A 4% yield of the UMP isomers is obtained in neutral aqueous solution using 2 times the DISN concentration and 7 times the phosphate concentration used in DMF. DISN did not effect the conversion of adenosine to AMP or 5-AMP to 5-ADP in aqueous solution. The cyclization of 3-AMP and 3-UMP to the corresponding 2,3-cyclic phosphates proceeds in yields as high as 40–50% at 60°C in pH 6 aqueous solutions in the presence of divalent metal ions. Lower yields of the cyclic phosphate are observed when 2-AMP is the starting material. Substitution of acetate buffer for imidazole buffer results in a decrease in the yield of cyclic phosphate, the extent of which depends on the metal ion used in the reaction. No 3,5-cyclic AMP was detected as a reaction product with either 5-AMP or 3-AMP as the starting material except for a 2.4% yield from 3-AMP in the presence of Zn²⁺. BrCN effects the conversion of 3-AMP to the 2,3-cyclic AMP in 37–65% yield depending on the divalent cation used as catalyst. A mechanism has been proposed for these cyclization reactions and their potential significance to the prebiotic synthesis of ribonucleic acid derivatives is discussed.Chemical Evolution 41. For previous paper see Ferris, J.P., Hagan, W.J., Jr., Alwis, K. W., and McCrea, J.: 1982,J. Mol. Evol. 18, 304–309.Presented at the 7th International Conference on The Origins of Life, Mainz, F.R.G., 1983.     

Adenosine-5′-phosphosulfate (APS) as sulfate donor for assimilatory sulfate reduction in Rhodospirillum rubrum

Crude extracts of Rhodospirillum rubrum catalyzed the formation of acid-volatile radioactivity from (³⁵S) sulfate, (³⁵S) adenosine-5-phosphosulfate, and (³⁵S) 3-phosphoadenosine-5-phosphosulfate. An enzyme fraction similar to APS-sulfotransferases from plant sources was purified 228-fold from Rhodospirillum rubrum. It is suggested here that this enzyme is specific for adenosine-5-phosphosulfate, because the purified enzyme fraction metabolized adenosine-5-phosphosulfate, however, only at a rate of 1/10 of that with adenosine-5-phosphosulfate. Further, the reaction with 3-phosphoadenosine-5-phosphosulfate was inhibited with 3-phosphoadenosine-5-phosphate whereas this nucleotide had no effect on the reaction with adenosine-5-phosphosulfate. For this activity with adenosine-5-phosphosulfate the name APS-sulfotransferase is suggested. This APS-sulfotransferase needs thiols for activity; good rates were obtained with either dithioerythritol or reduced glutathione; other thiols like cysteine, 2-3-dimercaptopropanol or mercaptoethanol are less effective. The electron donor methylviologen did not catalyze this reaction. The pH-optimum was about 9.0; the apparent K _m for adenosine-5-phosphosulfate was determined to be 0.05 mM with this so far purified enzyme fraction. Enzyme activity was increased with K₂SO₄ and Na₂SO₄ and was inhibited by 5-AMP. These properties are similar to assimilatory APS-sulfotransferases from spinach and Chlorella.Abbreviations APS adenosine-5-phosphosulfate - PAPS 3-phosphoadenosine-5-phosphosulfate - 5-AMP adenosine-5-monophosphate - 3-AMP adenosine-3-monophosphate - 3-5-ADP 3-phosphoadenosine-5-phosphate (PAP) - DTE dithiorythritol - GSH reduced glutathione - BAL 2-3-dimercaptopropanol     

Effects of midazolam on the contraction and relaxation of segments of thoracic aorta stripped of endothelium and stimulated by adrenaline – experimental study in rabbits

To evaluate the effects of midazolam on the angiokinesis of segments of rabbits' thoracic aorta stripped of endothelium and stimulated by adrenaline.Two groups of aortic rings removed from albinic rabbits anesthetized with thiopental were used (Group I – 6 animals; Group II – 12 animals), stripped of endothelium, studied in an organ chamber, perfused by Krebs-Henseleit solution. The groups were stimulated by adrenaline, recording the maximum contraction and dT/dt at 12, 36, 60 and 120. When the plateau phase was reached, the vessel was washed with perfusion solution, recording relaxation at 2, 4 and 6. When the base values were reached, Group I underwent a new adrenergic stimulus; and Group II was stimulated with midazolam and then with adrenaline, and the same values were recorded. T test was applied as a statistical analysis when two variables were studied. When studying more than two variables the Anova test was used, supplemented by the Tuckey test.Group I did not show any significant difference between the two stimuli. Group II – the midazolam significantly reduced the maximum contraction induced by adrenaline (83.01 ± 4.11%) (p < 0.01). The dT/dt was reduced at 12 (57.06 ± 8.47%), and also at 36 (70.59 ± 5.26%). There was no significance at 60 and 120 (p < 0.01).The relaxation increased significantly at all measurements – at 2-adrenaline 39.31 ± 9.60%; adrenaline/midazolam: 44.06 ± 9.62% (p < 0.05). At 4-adrenaline: 53.08 ± 8.3%; adrenaline/midazolam: 61.68 ± 8.50% (p < 0.01). At 6-adrenaline: 76.26 ± 5.45%; adrenaline/midazolam: 84.20 ± 7.96% (p < 0.01).Midazolam significantly reduced the maximum contraction obtained by the adrenergic stimulus as well as the dT/dt in the initial phases of contraction. The relaxation speed also increased.     

Occurrence of pppApp-synthesizing activity in actinomycetes and isolation of purine nucleotide pyrophosphotransferase

The occurrence of adenosine 5-triphosphate-3-diphosphate-synthesizing activity was detected in five strains of actinomycetes; Streptomyces morookaensis, Streptomyces aspergilloides, Streptomyces hachijoensis, Actinomyces violascens and Streptoverticillium septatum, out of 825 strains of actinomycetes, bacteria, fungi and imperfecti. Purine nucleotide pyrophosphotransferase were extracellularly excreted associating with the cell growth, and were purified partially or to apparent homogeniety from the culture filtrate. The enzymes are a monomeric protein with a molecular weight of 18000–26000 and synthesize adenosine, guanosine and inosine 5-phosphate (mono, di or tri)-3-diphosphate such as pApp, ppApp, pppApp, pGpp, ppGpp, pppGpp and pppIpp by transferring a pyrophosphoryl group from the 5-position of ATP, dATP and pppApp to the 3-position of purine nucleotides in the presence of a divalent cation and in alkaline state.Abbreviations pppApp adenosine 5-triphosphate 3-diphosphate - ppApp adenosine 5-diphosphate 3-diphosphate - pApp adenosine 5-monophosphate 3-diphosphate - pppGpp guanosine 5-triphosphate 3-diphosphate     

Oligomerization of deoxynucleoside-bisphosphate dimers: Template and linkage specificity

Evidence is presented that a poly(U) template selectively favors the oligomerization of the activated, 3–5 pyrophosphate-linked dimer pdAppdAp, in comparison with the 3–3 and 5–5 linked dimers. In the absence of poly(U), the 5–5linked dimer is the most reactive, and chains are formed which are more than 60 monomer units in length.Nucleic Acid-Like Structures V. For the previous paper in this series see Visscher and Schwartz (1988).     

Integration of biochemical functions of different cells of RAT gastric mucosa for hydrochloric acid secretion

Summary The regulation patterns of gastric acid secretion in rats were investigated. Pentagastrin and histamine stimulate gastric acid secretion, but the inhibitors of DNA-dependent synthesis of RNA and of proteins prevent only the pentagastrin action. It has been found that pentagastrin induces histidine decarboxylase in gastric mucosa, ensuring local accumulation of histamine. The latter activates adenylate cyclase and results in 3,5-AMP accumulation in gastric tissues. The administration of pentagastrin, histamine or 3,5-AMP enhances the activity of gastric carbonic anhydrase, the enzyme which takes part in HCI formation. The data suggest that these three compounds act sequentially (pentagastrin histamine 3,5-AMP) and the effect of the last one could be mediated through 3,5-AMP dependent protein kinase. The experiments in vitro demonstrated that gastric carbonic anhydrase can be separated into two isoenzymes and the phosphorylation of one of them by the 3,5-AMP dependent protein kinase sharply increases its activity. The findings raise the possibility that histamine and 3,5-AMP, mediating gastrin action, form together with enzymes (histidine decarboxylase, adenylate cyclase, protein kinase, carbonic anhydrase) a cascade of amplifiers.Autoradiographic studies have shown that [³H]-pentagastrin is not bound by oxyntic cells but adheres preferentially to histamine-producing-like endocrine cells and to the chief cells, while³H-histamine adheres preferentially to oxyntic and to chief cells. Electron microscopy indicates that only pentagastrin (but not histamine) initiates in-like endocrine cells ultrastructural changes characteristic for induction. Pentagastrin, histamine and 3,5-AMP administration produces in oxyntic cells ultrastructural changes typical for the secretion processes.These results lead to assumption that pentagastrin (gastrin) induces histidine decarboxylase in-like endocrine cells of gastric glands. Histamine which is secreted enhances adenylate cyclase activity in the neighbouring oxyntic cells where 3,5-AMP dependent protein kinase activates carbonic anhydrase by means of phosphorylation. These different cells form, probably, a multicellular functional unit for gastric acid secretion.An invited article.     

The composition of Erythrosins, Fluorescein, Phloxine and Rose Bengal: a study using thin-layer chromatography and solvent extraction

Synopsis Commercial samples of Erythrosin B (CI 45430), Erythrosin Y (CI 45425), Fluorescein (CI 45350), Phloxine (CI 45410) and Rose Bengal (CI 45440) have been analysed by thin-layer chromatography. The Erythrosins were found to be mixtures consisting in the main of 4-iodofluorescein, 4,5-di-iodofluorescein, 2,4,5-triiodofluorescein and 2,4,5,7-tetraiodofluorescein, in some instances together with 2,4,5-tri-iodo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetraiodo-4,5,6,7-tetrachlorofluorescein. Samples of Fluorescein were mixtures of the nominal dye usually with traces of several unidentified, fluorescent components. Those of Phloxine consisted mainly of mixtures of 4-bromo-4,5,6,7-tetrachlorofluorescein, 4,5-dibromo-4,5,6,7-tetrachlorofluorescein, 2,4,5-tribromo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetrabromo-4,5,6,7-tetrachlorofluorescein, often with 4,5,6,7-tetrachlorofluorescein Samples of Rose Bengal were mixtures of 4-iodo-4,5,6,7-tetrachlorofluorescein, 4,5-di-iodo-4,5,6,7-tetrachlorofluorescein, 2,4,5-tri-iodo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetraiodo-4,5,6,7-tetrachlorofluorescein together with some unidentified components.Most of the commercial dye samples gave an insoluble residue when extracted with methanol. This residue was usually inorganic carbonate or halide. Some possible practical consequences of the various impurities are discussed.     

Genetic and biochemical studies on flavonoid 3′-hydroxylation in flowers of Petunia hybrida

Summary In flower extracts of defined genotypes of Petunia hybrida, an enzyme activity was demonstrated which catalyses the hydroxylation of naringenin and dihydrokaempferol in the 3-position. Similar to the flavonoid 3-hydroxylases of other plants, the enzyme activity was found to be localized in the microsomal fraction and the reaction required NADPH as cofactor. A strict correlation was found between 3-hydroxylase activity and the gene Ht1, which is known to be involved in the hydroxylation of flavonoids in the 3-position in Petunia. Thus, the introduction of the 3-hydroxyl group is clearly achieved by hydroxylation of C₁₅-intermediates, and the concomitant occurrence of the 3,4-hydroxylated flavonoids quercetin and cyanidin (paeonidin) in the presence of the functional allele Ht1 is due to the action of one specific hydroxylase catalysing the hydroxylation of common precursors for both flavonols and anthocyanins.     

Regioselective alkylation of benzylβ-d-lactoside and its derivatives by stannylation

The preparation of benzyl 2,3,6,2,6-penta-O-benzyl--d-lactoside, which is a key intermediate for chemical synthesis of oligosaccharide components of glycosphingolipids, was achieved by an improved method. The 3-O-p-methoxybenzyl and 3-O-methyl derivatives were prepared from benzyl 2,3,6,2,6-penta-O-benzyl--d-lactoside through stannylation. By using benzyl -d-lactoside as starting material, benzyl 3-O-methyl-, 3-O-benzyl- and 3-O-p-methoxybenzyl--d-lactoside were regioselectively synthesized using the same procedure.     

A practical approach to crosslinking

The various aspects of chemical crosslinking are addressed. Crosslinker reactivity, specificity, spacer arm length and solubility characteristics are detailed. Considerations for choosing one of these crosslinkers for a particular application are given as well as reaction conditions and practical tips for use of each category of crosslinkers.Abbreviations ABH azidobenzoyl hydrazide - ANB- NOS N-5-azido-2-nitrobenzoyloxysuccinimide - ASIB 1-(p-azidosalicylamido)-4-(iodoacetamido)butane - ASBA 4-(p-azidosalicylamido)butylamine - APDP N-[4-(p-azidosalicylamido) butyl]-3(2-pyridyldithio)propionamide - APG p-azidophenyl glyoxal monohydrate - BASED bis-[-(4-azidosalicylamido)ethyl] disulfide - BMH bismaleimidohexane - BS³ bis(sulfosuccinimidyl) suberate - BSOCOES bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone - DCC N,N-dicyclohexylcarbodiimide - DFDNB 1,5-difluoro-2,4-dinitrobenzene - DMA dimethyl adipimidate·2HCl - DMP dimethyl pimelimidate·2HCl - DMS dimethyl suberimidate·2HCl - DPDPB 1,4-di-(3,2-pyridyldithio)propionamido butane - DMF dimethylformamide - DMSO dimethylsulfoxide - DSG disuccinimidyl glutarate - DSP dithiobis(succinimidylpropionate) - DSS disuccinimidyl suberate - DST disuccinimidyl tartarate - DTSSP 3,3-dithiobis (sulfosuccinimidylpropionate) - DTBP dimethyl 3,3-dithiobispropionimidate·2HCl - EDC or EDAC 1-ethyl-3-(3-dimethylaminopropyl)carbodimide hydrochloride - EDTA ethylenediaminetetraacetic acid disodium salt, dihydrate - EGS ethylene glycolbis(succinimidylsuccinate) - GMBS N--maleimidobutyryloxysuccinimide ester - HSAB N-hydroxysuccinimidyl-4-azidobenzoate - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - MBS m-maleimidobenzoyl-N-hydroxysuccinimide ester - MES 4-morpholineethanesulfonic acid - NHS N-hydroxysuccinimide - NHS-ASA N-hydroxysuccinimidyl-4-azidosalicylic acid - PMFS phenylmethylsulfonyl fluoride - PNP-DTP p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate - SAED sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide) ethyl-1,3-dithiopropionate - SADP N-succinimdyl (4-azidophenyl)1,3-dithiopropionate - SAND sulfosuccinimidyl 2-(m-azido-o-nitrobenzamido)-ethyl-1,3-dithiopropionate - SANPAH N-succinimidyl-6(4-azido-2-nitrophenyl-amino)hexanoate - SASD sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3-dithiopropionate - SATA N-succinimidyl-S-acetylthioacetate - SDBP N-hydroxysuccinimidyl-2,3-dibromopropionate - SIAB N-succinimidyl(4-iodoacetyl)aminobenzoate - SMCC succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate - SMPB succinimidyl 4-(p-maleimidophenyl) butyrate - SMPT 4-succinimidyloxycarbonyl--methyl--(2-pyridyldithio)-toluene - sulfo-BSOCOES bis[2-sulfosuccinimidooxycarbonyloxy) ethyl]sulfone - sulfo-DST disulfosuccinimidyl tartarate - sulfo-EGS ethylene glycolbis(sulfosuccinimidylsuccinate) - sulfo-GMBS N--maleimidobutyryloxysulfosuccinimide ester - sulfo-MBS m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester - sulfo-SADP sulfosuccinimidyl(4-azidophenyldithio)propionate - sulfo-SAMCA sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate - sulfo-SANPAH sulfosuccinimidyl 6-(4-azido-2-nitrophenylamino)hexanoate - sulfo-SIAB sulfosuccinimidyl(4-iodoacetyl)aminobenzoate - sulfo-SMPB sulfo-succinimidyl 4-(p-maleimidophenyl)butyrate - sulfo-SMCC sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate - SPDP N-succinimidyl 3-(2-pyridyldithio)propionate