Biosynthesis of vitamin B12

Radioactivity from [1-¹⁴C]riboflavin was incorporated into the 5,6-dimethylbenzimidazole moiety of Vitamin B₁₂ in the aerobes Bacillus megaterium, Nocardia rugosa and Streptomyces sp. as well as in the aerotolerant anaerobe Propionibacterium freudenreichii, but not in the anaerobe Eubacterium limosum.As recently published for E. limosum, also in the anaerobe Clostridium barkeri radioactivity from [1-¹⁴C]glycine and [2-¹⁴C]glycine was found in the 5,6-dimethylbenzimidazole moiety, but not in the corrin moiety. The addition of l-[methyl-¹⁴C]methionine to C. barkeri led to the labeling of the corrin moiety and the 5,6-dimethylbenzimidazole moiety, showing that the seven extra methyl groups in the corrin ring as well as the two methyl groups of the base part originate from this precursor.In Clostridium thermoaceticum, forming the vitamin B₁₂ analog 5-methoxybenzimidazolylcobamide, [1-¹⁴C]glycine and [2-¹⁴C]glycine were also incorporated into the 5-methoxybenzimidazole moiety, but not into the corrin ring.In E. limosum l-[U-¹⁴C]glutamate led to the labeling of the corrin ring of vitamin B₁₂, but not of its base moiety.There results together with data from the literature indicate that a common biosynthetic pathway might exist for the corrinoid biosynthesis in aerobic microorganisms, and in those aerotolerant anaerobes like the Propionibacteria, which form the 5,6-dimethylbenzimidazole moiety of vitamin B₁₂ only under aerobic conditions. They also show that this pathway differs from the pathway found in anaerobic bacteria.     

1H-NMR spectroscopic characterization of dansyl glyco-asparagines derived from hen egg white clycoproteins

Dansyl glyco-asparagines were prepared from a partially fractionated mixture of hen egg white glycoproteins. Reverse-phase high performance liquid chromatography (HPLC) on a silica-based octadecyl column yielded ten such derivatives in a virtually pure state. The detailed structures of the glyco-asparagines were identified by 500-MHz¹H nuclear magnetic resonance (NMR) spectroscopy. Two of them were found to be of the oligomannosideN-type, four were of the intersected-hybridN-type and another four were of the intersected multi-antennaryN-type. In monogalactosylated, intersected structures the galactose residue was proved by¹H-NMR to be attached in (1–4)-linkage to the GlcNAc1-4Man1–3 branch.Dansyl glyco-asparagines turned out to be suitable derivatives for¹H-NMR spectroscopic analysis. The combination of HPLC and high-resolution NMR spectroscopy of such derivatives proved to be a powerful technique in studying the (micro-)heterogeneity of sugar chains in glycoproteins.Abbreviations dns dansyl (5-dimethylaminonaphthalene-1-sulfonyl) - ODS octadecyl-silica - WEFT water-eliminating Fourier transform - DSS sodium 4,4-dimethyl-4-silapentane-1-sulfonate - OVA ovalbumin - OVM ovomucoid - OVT ovotransferrin     

Synthesis of mono- and disaccharide amino-acid derivatives for use in solid phase peptide synthesis

N-Fluorenylmethyloxycarbonyl-protected serine and threonine derivatives, carryingO-glycosidically - or -linked peracetylated -d-Galp-(1–3)-d-GalNAcp carbohydrate chains, were prepared. These derivatives are intended for use in solid phase glycopeptide synthesis. Suitably protected mono- and disaccharide thioglycosides were used as carbohydrate intermediates. These were activated by treatment with bromine to give the glycosyl bromides, which were then used in silver triflate-promoted glycosidations ofN-fluorenylmethyloxycarbonyl amino-acid phenacyl esters. Removal of the phenacyl esters with zinc gave the target free acids.     

Interaction between new neoglycoproteins and thed-Man/l-fuc receptor of rabbit alveolar macrophages

New types of neoglycoproteins, -caseins coupled with ovalbumin-derived asparagine oligosaccharides (AO), aspartate aminotransferase-phosphopyridoxylated AO complex (AAT-PG), and streptavidin-biotinylated AO complex (SA-BAO), were tested for their inhibitory effect on binding of bovine serum albumin derivatized with thiomannoside, Man-AI-BSA [Lee YC, Stowell CP, Krantz MJ (1976) Biochemistry 15:3956–63] by rabbit alveolar macrophages. The -casein derivatives and the AAT-PG complex increased binding affinity as the number of oligosaccharide chains attached was increased. Their inhibitory potencies were closely related to those of the Man-Al-BSA derivatives [Hoppe CA, Lee YC (1983) J Biol Chem 258:14193–99] on the basis of terminal mannose density. The SA-BAO complex containing three AO chains gave stronger inhibitory potency than the -casein derivative with three AO residues, suggesting that proper orientation of the oligosaccharides on the protein can affect the receptor-ligand interaction.Abbreviations BSA bovine serum albumin - Man₄₃-BSA BSA derivative containing on average 43 residues of Man linked through an amidino-linkage [7] - AO asparagine oligosaccharide (Man₅-GlcNAc₂-Asn) from ovalbumin - AAT aspartate aminotransferase - PG phosphopyridoxylated AO - SA streptavidin - BAO biotinylated AO     

Species and Tissue Distribution of Proteins Binding 16α,17α-Cycloalkanoprogesterone Derivatives

The binding of [³H]progesterone and [³H]16,17-cycloalkanoprogesterones to proteins from rat, rabbit, and human uteri and other organs was studied. We found that 16,17-cycloalkanoprogesterone derivatives display affinities for the uterine progesterone receptors comparable with that of the natural hormone and no substantial species differences in the affinity. Rabbit uterus was found to have no proteins distinct from the progesterone receptor that specifically bind [³H]16,17-cycloalkanoprogesterones. At the same time, in the human uterus, we found another protein that binds some of these progesterone derivatives; it turned out to be similar to the protein from rat uterus. A similar protein with the same selectivity and affinity for steroids was also found in rat and human kidneys. Blood serum, liver, lung, and a number of other tissues were found to contain a protein of the third type that binds the same 16,17-cycloalkanoprogesterones and exhibits submicromolar K _d values for these steroids and a very low affinity for progesterone. We speculated that the introduction of a bulky substituent adjacently to the 17-side chain of progesterone could result in a change in the general biodynamics of the derivative including its transport, uptake, and accumulation in tissues, which may determine the selectivity of its effect.     

Synthesis of methyl 2-acetamido-2-deoxy-3-O-(β-d-galactopyranosyl)-α-d-galactopyranoside from a corresponding thioglycoside derivative

The title disaccharide glycoside was synthesized by halide ion-promoted glycosidation, using methanol and the disaccharide bromide derived from methyl 2-azido-3-O-(2,3,4,6-tetra-O-benzoyl--d-galactopyranosyl)-4,6-O-benzylidene-2-deoxy-1-thio--d-galactopyranoside. This derivative in turn was prepared by silver triflate-promoted condensation of monosaccharide derivatives.     

The Synthesis of Phosphates of Long-Chain ω-Hydroxyalkyl Esters of 11-Deoxyprostaglandin E1

Di(p-methylbenzyl) phosphates of -hydroxyalkyl esters of 11-deoxyprostaglandin E₁ were synthesized from disubstituted 1,10-decane and 1,22-docosane derivatives for studying the permeability of bilayer membranes.     

Lysine Dendrimers and Their Starburst Polymer Derivatives: Possible Application for DNA Compaction and in vitro Delivery of Genetic Constructs

We attempted to find some compounds for the effective delivery of gene constructs into cells and obtained two trispherical dendrimers on the basis of lysine, (Lys)₈-(,-Lys)₄-(,-Lys)₂-(,-Lys)-Ala-NH₂ (D1) and ((Lys)₈-(,-Lys)₄-(,-Lys)₂-,-Lys)-Ala-[Lys(Plm)]₂-Ala-NH₂ (D2), as well as the starburst polymeric derivatives of D1, (pVIm) ₈ -D1 and (pLys) _n -D1, containing poly(N-vinylimidazole) and polylysine chains single-point bound to the dendrimer amino groups. The conditions of dendrimer–plasmid DNA complex formation were studied. The intracellular localization of these complexes and the expression of gene constructs delivered with their help were analyzed in transfection experiments on the HeLa cell cultures of human epithelial carcinoma and on mouse C2C12 myoblasts. It was found that the chemical structure of dendrimer D1 and its derivatives significantly affected the structure and properties of complex.     

Synthesis ofN-Acetylglucosamine derivatives as probes for specificity of chicken hepatic lectin

Syntheses of the following compounds are described: 6-(Trifluoroacetylamino)hexyl 2-acetamido-2,6-dideoxy--d-glucopyranoside and 2-acetamido-2-deoxy--d-xylopyranoside, two allyl 2-acetamido-2-deoxy--d-glucopyranosiduronic acid derivatives, and several allyl 2-acylamido-2-deoxy--d-glucopyranosides having different acyl groups. These and other compounds were used as inhibitors in the binding assay for the chicken hepatic lectin specific forN-acetylglucosamine. We found that: 1) The inhibitory potency ofN-acylglucosamine derivatives decreased progressively with increase in the size of acyl group, 2) absence of either 3-or 4-OH group ofN-acetylglucosamine lowered the binding affinity more than 100-fold, and 3) the presence of a negatively charged group (carboxylic acid) at the C-6 position did not lower the affinity. The first two items are similar to the mammalian hepatic galactose/N-acetylgalactosamine lectins, but the last item is in a strong contrast to the mammalian lectins.Abbreviations XyLNAc N-acetyl-d-xylosamine - BSA bovine serum albumin - NeuAc N-acetylneuraminic acid - GlcNAc34-BSA amidino-type neoglycoprotein [6] containing on the average 34N-acetylglucosaminyl residues per BSA molecule     

Introgression of diverse genes for resistance to rusts into an improved wheat variety, Kalyansona

Breeding for resistance to the three rusts of wheat usually requires incorporation of genetically independent factors conferring resistance to each rust. Linked resistance genes in some alien translocation stocks permit concurrent transference of resistance for more than one rust. Alien derived resistances, however, are often reported to be associated with reduced yield and other undesirable characters. In our experience, backcross breeding when given a limited number of backcrosses (3–6) and with suitable selection procedures has resulted in lines giving yields higher or comparable to the recurrent wheat parent Kalyansona and resistance to one, two or all three rusts without any adverse effects. Some of the rust resistant derivatives also show resistance to Neovossia indica (Karnal bunt). The derivatives thus developed when used as parents in a breeding programme have produced several improved cultivars with high yields, superior grains and diversity for resistance to rust pathogens. One of the cultivars, named Vaishali (DL784-3), has been officially released for cultivation in the country.     

Effect of a corrinoid on <Emphasis Type="Italic">Methanosarcina barkeri</Emphasis> DNA synthesis

Methanosarcina barkeri is capable of synthesizing large amounts of corrinoids, compounds of the vitamin B₁₂ group, although not cobalamin. In the present work, exogenous cobalamin was demonstrated to upregulate DNA synthesis in M. barkeri cell suspensions incubated under air. The effect is similar to the one in Propionibacterium freudenreichii cells, though less pronounced. The growth of the archaeon under anaerobic conditions was shown to be suppressed by cobalamin and 5,6-dimethylbenzimidazole. The data obtained suggest the presence of a corrinoid-dependent ribonucleotide reductase in the archaeal cells which provides for deoxyribose precursors for DNA biosynthesis independently of the presence of molecular oxygen in the medium. Growth suppression under anoxic conditions by cobalamin and 5,6-dimethylbenzimidazole may be due to a decrease in the concentration of factor III, a polyfunctional corrinoid dominating in M. barkeri cells.     

Effect of α(2–6)-linked sialic acid and α(1–3)-linked fucose on the interaction ofN-linked glycopeptides and related oligosaccharides with immobilizedPhaseolus vulgaris leukoagglutinating lectin (L-PHA)

The effects of branching and substitution of branches by sialic acid and fucose on the interaction ofN-linked glycopeptides and related oligosaccharides with immobilizedPhaseolus vulgaris leukoagglutinating lectin (L-PHA) were examined. Asialo bi-, tri-and tetra-antennary glycans were all retarded but to different extents on a long column of L-PHA-agarose. Asialo tri- and tetra-antennary glycans containing the pentasaccharide unit Gal1-4GlcNAc1-2[Gal1-4GlcNAc1-6]Man were strongly retarded, whereas asialo bi- and tri-antennary glycans lacking the Gal1-4GlcNAc1-6 branch were only weakly retarded. In all instances the interaction with the lectin was completely abolished when either (2–6)-linkedN-acetylneuraminic acid or (1–3)-linked fucose was present at the galactose orN-acetylglucosamine residue of the Gal1-4GlcNAc1-6Man1-6 branch, respectively. The same substitutions on the Gal1-4GlcNAc1-6Man1-6 branch decreased but did not abolish the affinity of the lectin for the glycans. The presence of NeuAc2-6 and Fuc1-3 on the other two branches did not interfere with the binding of the glycans to L-PHA. Furthermore, it appeared that the presence of the Man1-4GlcNAc unit is requried for interaction with the lectin. In order to obtain reliable information on the relative occurrence of tri- and tetra-antennary glycopeptides, this study shows that it is essential to desialylate and to defucosylate the glycans prior to application to L-PHA-agarose.Abbreviations L-PHA leukoagglutinating phytohemagglutinin - CMP-NeuAc cytidine-5-monophospho-N-acetylneuraminic acid - GP glycopeptide - OS oligosaccharide - HPLC high-performance liquid chromatography - FNR fraction not retarded - FR fraction retarded suffixes MS, BS and TS indicate mono-, bi- and trisialyl derivatives respectively; suffix MF indicates monofucosyl derivatives.structures of the substratesOS2, OS3, OS3, OS4, GP2, GP3, GP4, GP4-MF, OS2(3) andOS2(-) are presented in Fig. 2     

Morphology of the eye and visual acuities in the settlement-intervals of some Coral Reef Fishes (Labridae, Scaridae)

The morphology of the retina and photoreceptors of settlement-interval larvae and early juveniles and some adults of 12 species of Caribbean labrids and scarids were examined using histological techniques. The retinal structure is described in these species and life intervals. Larvae have a pure cone retina and unorganized mosaic and organization into a square mosaic pattern occurs during metamorphosis. Early post-settlement juveniles have an organized mosaic with structures that may enable them to detect polarized and UV light. Visual acuities were calculated for all species and life intervals and acuities ranged between 86.6–29.4min of arc in the settlement-intervals and 16–1.8min of arc in the adults. The visual abilities of the settlement-interval fishes and the possibility of the use of vision during settlement are discussed.     

Dissolution of ferric phosphate by alfalfa (Medicago sativa L.) root exudates

Alfalfa (Medicago sativa L.) was grown in hydroponic culture to investigate adaptation to Fe-deficiency. Root exudates released into the nutrient solution from Fe-deficient plants were trapped and condensed on an amberlite XAD-4 resin column. The diethyl ether fraction of these exudates dissolved ferric phosphate remarkably. The dissolving capability was about 62 times higher than that of root exudates obtained from Fe-sufficient plants in complete nutrient solution. The Fe-dissolving compound was separated and identified. It was a new natural compound with molecular formula C₁₄H₁₀O₅ and was identified as 2-(3,5-dihydroxyphenyl)-5,6-dihydroxybenzofuran by means of mass spectrometry and ¹H-nuclear magnetic resonance. This new compound worked as a phytoalexin and inhibited completely the fungal growth of Fusarium oxysporum f. sp. phaseoli.     

New Photosensitizers of Bacteriochlorin Series for Photodynamic Cancer Therapy

New derivatives of bacteriochlorophyll a bearing an extra glutarimide exocycle were synthesized, and their reactivity was studied. Acetyl group in 3-acetyl-2,7,12,18-tetramethyl-8-ethyl-13,15-dicarboxy-17-carboxyethyl-7,8,17,18-tetrahydroporphyrin (bacteriochlorin p) was chemically modified into -hydroxyethyl and vinyl groups. A simple method of preparation of vinylbacteriopurpurin esters under the catalysis by p-toluenesulfonic acid was proposed. The resulting compounds exhibit a high adsorption in the visible and near IR areas of electronic spectra, a reasonable stability, and amphiphilic properties and, therefore, may be regarded as promising photosensitizers for the photodynamic cancer therapy.     

Chemotaxonomic features of iridoids occurring inVerbenoxylum reitzii (Verbenaceae)

The familyVerbenaceae sensu stricto is chemically characterized by the accumulation of 10-hydroxy or 8-methyl-8-hydroxy derivatives of 4-carboxylated iridoids compounds. The former were supposed to be characteristic of some genera of tribeLantaneae, once were never found in another tribe. This paper reports the occurrence of theviridoside (10-hydroxy-iridoid) in leaves ofVerbenoxylum reitzii (tribeCitharexyleae) and its taxonomic implication.     

Monoterpene und Systematik der GattungArtemisia (Asteraceae—Anthemideae)

Leaf monoterpenes from 166 provenances representing 71 species of the genusArtemisia were identified by gas-liquid (using 3 columns with different polarity) and thin layer co-chromatography with authentic samples. Cineol and thujane and camphane derivatives together with small quantities of pinanes, monocyclic and acyclic monoterpenes apparently characterize sect.Artemisia and sect.Abrotanum (pro parte), and probably also the North American sect.Tridentatae. The subg.Dracunculus and the ser.Laciniatae andLatifoliae of sect.Abrotanum are clearly distinguished by only small amounts of pinanes, monocyclic and acyclic monoterpenes together with a nearly complete reduction of all major components, i.e. cineol and thujane and camphane derivatives. Different trends towards -thujone or myrcene accumulation appear to be of some systematic relevance within the ser.Frigidae of sect.Absinthium.

     

Flavonoids ofBignoniaceae from the “cerrado” and their possible taxonomic significance

Flavones and glycosides of flavones and flavonols were obtained from leaves of five species ofBignoniaceae from the cerrado, a savanna ecosystem of Central and Southeast Brazil. A predominance of flavonol glycosides was observed in all samples investigated. Flavones were often found in species of tribeTecomeae and rarely inBignonieae. The species of the former yielded derivatives of 6-hydroxyluteolin. No 6-oxygenated compounds were found in species ofBignonieae. The results point to the flavonoid chemistry evolution ofBignoniaceae following a path of simplification. More complex chemical profiles characterize the more woodyTecomeae rather than the more advancedBignonieae.     

Ecogeography of the grass genusHelictotrichon (Poaceae: Aveneae) in the Mediterranean and adjacent regions

The geographical distribution of the grass genusHelictotrichon in the Mediterranean Region is analysed and mapped for 33 taxa. Based on only a single life form (perennial herb), a variety of edaphically, climatically and altitudinally differently adapted species complexes has evolved in the area. Most of these complexes show west-east disjunctions and contain geographically, sometimes even edaphically vicarious taxa with complementary distribution. A transition from mesomorphic to xeromorphic habit occurred independently in different species groups and led to the establishment of the modern Mediterranean taxa which are in part highly polyploid derivatives of more mesophilic diploids. The significance of polyploidy, patterns of parapatric and sympatric distribution, biogeographical borders, and centres of species diversity are discussed in context with the history of the Mediterranean vegetation. New combinations are:Helictotrichon setaceum subsp.petzense, H. pratense subsp.lusitanicum, H. praetutianum.     

An envelope-shaped film culture vessel for plant cell suspension cultures and metabolite production without agitation

An envelope-shaped film culture vessel (named Culture Bag) made of fluorocarbon polymer film, which is much more permeable to oxygen, nitrogen and carbon dioxide than other films, was found to be suitable to grow plant cells in liquid medium without agitation. Proliferous BY-2 tobacco cells showed almost the same growth in a Culture Bag of 12.5 m-thick film as that in a shake flask; the growth was lower in a Culture Bag of a thicker film. Lithospermum erythrorhizon cells produced almost the same amount of red naphthoquinone pigments (shikonin derivatives) in a Culture Bag of 12.5 m-thick film as those in a shake flask although the productivity was suppressed as the film thickness increased. L. erythrorhizon cells in a Culture Bag produced much less abnormal stress metabolites (orange-colored benzoquinone derivatives) than those in a shake flask, suggesting that culturing cells in the Culture Bag was less stressful due to its stationary liquid environment.