Structural analysis of the O-specific polysaccharide isolated from Plesiomonas shigelloides O51 lipopolysaccharide

Plesiomonasshigelloides strain CNCTC 110/92 (O51) was identified as a new example of plesiomonads synthesising lipopolysaccharides (LPSs) that show preference for a non-aqueous surrounding during phenol/water extraction. Chemical analyses combined with ¹H and ¹³C NMR spectroscopy, MALDI-TOF and ESI mass spectrometry showed that the repeating units of the O-specific polysaccharides isolated from phenol and water phase LPSs of P. shigelloides O51 have the same structure: →4)-β-d-GlcpNAc3NRA-(1→4)-α-l-FucpAm3OAc-(1→3)-α-d-QuipNAc-(1→, containing the rare sugar constituent 2,3-diamino-2,3-dideoxyglucuronic acid (GlcpNAc3NRA), and substituents such as d-3-hydroxybutyric acid (R) and acetamidino group (Am). The HR-MAS NMR spectra obtained for the isolated LPSs and directly on bacteria indicated that the O-acetylation pattern was consistent throughout the entire preparation. The ¹H chemical shift values of the structure reporter groups identified in the isolated O-antigens matched those present in bacteria. We have found that the O-antigens recovered from the phenol phase showed a higher degree of polymerisation than those isolated from the water phase.     

Two pentacyclic triterpenes from Rubia cordifolia

Rubicoumaric acid and rubifolic acid isolated from Rubia cordifolia have been shown to be 30-hydroxy-3β-p-hydroxycoumaryloxy-urs-12-ene-28-oic acid and 3β,30-dihydroxy-urs-12-ene-28-oic acid(30-hydroxyursolic acid) respectively on the basis of ¹H NMR, ¹³C NMR and mass spectral and chemical evidence.     

Characterization and kinetics of isoenzymes of pyruvate kinase from developing castor bean endosperm

Isozymes of pyruvate kinase (PK) have been isolated from developing castor bean endosperm. One isozyme, PK_c, is localized in the cytosol, and the other, PK_p, is in the plastid. Both isozymes need monovalent and divalent cations for activity, requirements which can be filled by K⁺ and Mg²⁺. Both isozymes are inhibited by citrate, pyruvate, and ATP. PK_c has a much broader pH profile than PK_p and is also more stable. Both have the same K_m (0.05 millimolar) for PEP, but PK_p has a 10-fold higher K_m (0.3 millimolar) for ADP than PK_c (0.03 millimolar). PK_c also has a higher affinity for alternate nucleotide substrates than PK_p. The two isozymes have different kinetic mechanisms. Both have an ordered sequential mechanism and bind phosphoenolpyruvate before ADP. However, the plastid isozyme releases ATP first, whereas pyruvate is the first product released from the cytosolic enzyme. The properties of the two isozymes are similar to those of their counterparts in green tissue.     

Hydroxycinnamic acid esters of isocitric acid: Accumulation and enzymatic synthesis in Amaranthus cruentus

New hydroxycinnamic acid esters have been isolated from the cotyledons of Amaranthus cruentus. (E)-Caffeoylisocitric acid was identified as the major constituent and p-coumaroyl-and feruloylisocitric acids as minor ones on the basis of ¹H and ¹³C NMR spectroscopy. FAB mass spectrometry, high-performance liquid and thin-layer chromatography. The structure of caffeoylisocitric acid was confirmed by chromatographic comparison with synthetic material. The accumulation of the new hydroxycinnamoylisocitric acids and their enzymatic synthesis via the hydroxycinnamoyl-CoA thioesters as acyl donors are described. The caffeoyl-CoA-dependent acyltransferase showed a rapid transient increase in activity reaching ca 5 pkat per cotyledon pair (i.e. ca 390 pkat per mg protein) at day six of seedling development.     

Nucleotide alterations in the bacteriophage T4 glutamine transfer RNA that affect ochre suppressor activity

We have determined the nucleotide sequences of the glutamine transfer RNAs that are coded by wild-type and psu₂⁺ ochre-suppressor strains of bacteriophage T4. The two transfer RNAs have the same sequence except for their anticodons, where NUG in the wild-type species is mutated to NUA in the psu₂⁺ species (N is a modified residue of U). This mutation is believed to confer suppressor activity on the psu₂⁺ glutamine tRNA. Three mutants derived from psu₂⁺ by loss of suppressor activity have been characterized with respect to their sequence alterations. Each mutant specifies a transfer RNA differing from the psu₂⁺ species by a nucleotide substitution that occupies a base-paired region in the cloverleaf arrangement of the molecule. The mutants synthesize a reduced amount of tRNA that is defective in nucleotide modifications and processing at the 5′ and 3′ termini.     

Inhibition of a Discrete Bacterial DNA Polymerase by 6-(<Emphasis Type="Italic">p</Emphasis>-Hydroxyphenylazo)-uracil and 6-(<Emphasis Type="Italic">p</Emphasis>-Hydroxyphenylazo-)-isocytosine

6-(p-HYDROXYPHENYLAZO)-URACIL (HPUra) specifically inhibits the semi-conservative replication of DNA in Gram-positive bacteria^1–3. We have reported that HPUra inhibits ATP-dependent polymerization of deoxyribonucleotides in vitro in toluene-treated B. subtilis⁴. Further studies of the effect of HPUra and its amino analogue, HPIsocytosine (6-(p-hydroxyphenylazo)-2-amino, 4-keto pyrimidine), in toluene-treated B. subtilis have provided considerable information on the mechanism of arylazopyrimidine action. First, HPUra and HPIsocytosine do not inhibit DNA synthesis unless they first are reduced to their colourless, hydrazo forms (refs. 4 and 5 and Mackenzie, Wright and Brown, unpublished results). Second, the inhibitory action of reduced HPUra and that of reduced HPIsocytosine are completely antagonized, respectively, by dGTP and dATP⁵. Third, drug-resistant mutants have been isolated which catalyse drug-resistant DNA synthesis following their permeabilization with toluene. These observations suggest that reduced HPUra and HPIsocytosine inhibit DNA replication by interfering competitively with the enzymatic polymerization of specific purine deoxyribonucleotides. We examined, therefore, cell free preparations of ^{B. subtilis} in an effort to identify a discrete DNA polymerase as the site of drug action. We report here experiments with crude and partially fractionated extracts of DNA polymerase I-deficient mutants which indicate the existence of at least one drug sensitive polymerase. Bazill and Gross⁶ have independently isolated chromatographically discrete HPUra-sensitive polymerases from extracts of B. subtilis.     

Structural and immunochemical studies of neutral exopolysaccharide produced by Lactobacillus johnsonii 142

This paper describes the structure of neutral exopolysaccharide (EPS) produced by Lactobacillus johnsonii 142, strain of the lactic acid bacteria isolated from the intestine of mice with experimentally induced inflammatory bowel disease (IBD). Sugar and methylation analyses along with ¹H and ¹³C NMR spectroscopy, including two-dimensional ¹H,¹H COSY, TOCSY, NOESY, and ¹H,¹³C HSQC experiments revealed that the repeating unit of the EPS is a pentasaccharide:→3)-α-d-Galp-(1→3)-β-d-Glcp-(1→5)-β-d-Galf-(1→3)-α-d-Galp-(1→3)-α-d-Galp-(1→The rabbit antiserum raised against whole cells of L. johnsonii 142 reacted with homologous EPS, and cross-reacted with exopolysaccharide from Lactobacillus animalis/murinus 148 isolated also from mice with IBD, but not reacted with EPS of L. johnsonii 151 from healthy mice.     

Interaction of γ-conglutin from Lupinus albus with model phospholipid membranes: Investigations on structure,thermal stability and oligomerization status

Interaction with model phospholipid membranes of lupin seed γ-conglutin, a glycaemia-lowering protein from Lupinus albus seeds, has been studied by means of Fourier-Transform infrared spectroscopy at p²H 7.0 and at p²H 4.5. The protein maintains the same secondary structure both at p²H 7.0 and at p²H 4.5, but at p²H 7.0 a higher ¹H/²H exchange was observed, indicating a greater solvent accessibility. The difference in T_m and T_D1/2 of the protein at the abovementioned p²H's has been calculated around 20 °C. Infrared measurements have been then performed in the presence of DMPG and DOPA at p²H 4.5. DMPG showed a little destabilizing effect while DOPA exerted a great stabilizing effect, increasing the T_m of γ-conglutin at p²H 4.5 of more than 20 °C. Since γ-conglutin at p²H 4.5 is in the monomeric form, the interaction with DOPA likely promotes the oligomerization even at p²H 4.5. Interaction between DMPG or DOPA and γ-conglutin has been confirmed by turbidity experiments with DMPC:DMPG or DOPC:DOPA SUVs. Turbidity data also showed high-affinity binding of γ-conglutin to anionic SUVs made up with DOPA. The molecular features outlined in this study are relevant to address the applicative exploitation and to delineate a deeper comprehension of the natural functional role of γ-conglutin.     

The biosynthesis of ubiquinone in isolated rat heart cells

Beating heart cells were isolated from the adult rat and the biosynthesis of ubiquinone was studied. These cells were able to incorporate p-hydroxy[U-¹⁴C]benzoate into ubiquinone and some unidentified compounds, presumably intermediates in the biosynthesis of ubiquinone. The unidentified compounds were labile to alkali and were also labeled by [5-³H]-mevalonate and [methyl-³H]methionine, but not by p-hydroxy[carboxy-¹⁴C]benzoate. They appear to be chromatographically different from 5-demethoxy ubiquinone and 5-desmethyl ubiquinone. Addition of unlabeled mevalonate stimulated the incorporation of p-hydroxy [U-¹⁴C]benzoate into ubiquinone and the other compounds. The addition of dimethylsulfoxide to the isolated cells or the isolation medium caused inhibition of ubiquinone biosynthesis. Adriamycin was not inhibitory to the biosynthesis of ubiquinone in the cells. The advantages of these cells are the rapidity and ease in studying the biosynthesis of ubiquinone from various precursors and its regulation.     

Carotane sesquiterpenes from Ferula lancerottensis

Six new carotane sesquiterpenes, the p-hydroxybenzoate of epoxyjaeschkeanadiol the p-methoxybenzoate and p-hydroxybenzoate of lancerodiol, lancerodiol, the p-hydroxybenzoate of lancerotriol and the p-methoxybenzoate of linkitriol, and the already known p-hydroxybenzoate of jaeschkeanadiol have been isolated from Ferula lancerottensis.     

Blockade by Polyphloretin Phosphate of the Prostaglandin F<Subscript>2α</Subscript>, Action on Isolated Human Bronchi

POLYPHLORETIN phosphate (PPP), a polymer with an inhibitory effect on some enzyme systems, such as alkaline phosphatase and hyaluronidase, has been synthesized by Diczfalusy et al.¹ by phosphorylating phloretin with phosphorus oxychloride. PPP has a molecular weight of about 15,000 and is readily soluble in water at pH 7. It has been tried in the treatment of oedema^2,3 and in vitro it inhibits the aggregation of red blood corpuscles⁴ and selectively antagonizes the effect of prostaglandin E₂ (PGE₂) on the intraocular pressure of the rabbit eye⁵. In the light of this finding we have examined whether PPP (supplied by Dr B. Högberg) could also modify the action of prostaglandins, when tested on some other preparation— in this case isolated human bronchi. During this study an investigation of the inhibition by PPP of the PGE₂ and PGF_2α action on isolated bird colon, rabbit jejunum and uterus has been reported⁶. It has been shown that PGE₁ and usually also PGE₂ have a bronchodilating effect in guinea-pig^7,8 and man⁹, while PGF_2α constricts bronchial smooth muscle in vivo and invitro^8,10,11. PGF_2α and PGE₂ have been isolated from human lungs^12,13 and both seem to be released in the anaphylactic reaction of isolated guinea-pig lung.     

Disaccharide 1-phosphate polymers of some representatives of the Bacillus subtilis group

Disaccharide 1-phosphate polymers as well as teichoic acids of various structures have been found in the cell walls of the representatives of the Bacillus subtilis group, namely Bacillus subtilis subsp. spizizenii VKM B-720 and VKM B-916, B. subtilis VKM B-517, and Bacillus vallismortis VKM B-2653^T. Disaccharide 1-phosphate polymers are composed of repeating units of the following structure: -P-4)-β-D-GlcpNAc-(1→6)-α-D-Galp-(1-, the N-acetylglucosamine residues are partially acetylated at positions O3 and O6 (VKM B-720 and VKM B-916); -P-4)-β-D-Glcp-(1→6)-α-D-GlcpNAc-(1-, the glucopyranose residues are partially acetylated at positions O2 or O3 (VKM B-517); -P-6)-α-D-GlcpNH ₃ ⁺ /α-D-GlcpNAc-(1→2)-α-D-Glcp-(1-, the N-acetylglucosamine residues are partially deacetylated (VKM B-2653^T). The structures of the two last disaccharide 1-phosphate polymers have not been reported so far for Gram-positive bacteria. The teichoic acids in the studied strains are O-D-alanyl-1,5-poly(ribitol phosphates) substituted with β-D-glucopyranose (VKM B-517, VKM B-720, VKM B-916) or 2-acetamido-2-deoxy-β-D-glucopyranose (VKM B-2653^T). The structures of the phosphate-containing polymers have been studied by chemical methods and by NMR spectroscopy.     

Aryl sulfatase of unusual specificity from the liver of marine mollusk Littorina kurila

An aryl sulfatase of unusual specificity has been isolated from the liver of marine mollusk Littorina kurila. It hydrolyzes p-nitrophenyl sulfate, does not affect the natural fucoidan, and catalyzes splitting off the sulfate group in position C4 of xylose residues within the carbohydrate chains of holostane triterpene glycosides from sea cucumbers. The properties of the enzyme were studied at pH 5.4. The protein is homogeneous according to electrophoresis and has M 45 ± 1 kDa. The semiinactivation time of the enzyme at 60°C is 20 min, and its K _m value for the hydrolysis of p-nitrophenyl sulfate is 8.7 ± 1 mM. It was shown that natural sulfated polyhydroxysteroids inhibit activity of the sulfatase; their I ₅₀ values depend on their structures and are within the range from 10^?3 to 10^?5 M.     

9-Fluorenylmethyl chloroformate (Fmoc-Cl) as a useful reagent for the synthesis of pentafluorophenyl, 2,4,5-trichlorophenyl, pentachlorophenyl, p-nitrophenyl, o-nitrophenyl and succinimidyl esters of Nα-urethane protected amino acids

9-Fluorenylmethyl chloroformate has been demonstrated to be useful reagent for the synthesis of several commonly used activeesters of Fmoc-/Boc-/Z-amino acids. These include pentafluorophenyl, 2,4,5-trichlorophenyl, pentachlorophenyl, p-nitrophenyl, o-nitrophenyl, and succinimidyl esters. The method is simple, rapid and efficient. All the compounds made have been isolated as crystaline solids in good yield and optical purity. They were fully characterized by IR, and ¹H NMR.     

Isolation, Purification, and Identification of 2-(p-Hydroxyphenoxy)-5, 7-Dihydroxychromone: A Fungal-Induced Phytoalexin from Cassia obtusifolia

A single phytoalexin was isolated and purified from 12- to 14-day-old leaves of Cassia obtusifolia L. inoculated with Alternaria cassiae Jurair & Khan. The structure was elucidated by ¹H- and ¹³C-nuclear magnetic resonance and mass spectrometry as 2-(p-hydroxyphenoxy)-5,7-dihydroxychromone. The compound was shown to be derived in part from phenylalanine. Radial growth of A. cassiae was inhibited 50% by the compound at 0.3 millimolar. This moderate toxicity is compensated for by the relatively high levels (3 millimolar) accumulated. Phenoxychromones have been previously reported only as constitutive secondary metabolites in Artemisia capillaris Thunb., in which their function is unknown.     

A general method for the high-yield isolation of mesophyll protoplasts from deciduous tree species

High yields (1.2–8.0 × 10⁶ g fresh wt.^?1) of viable leaf mesophyll protoplasts have been isolated from a range of mature deciduous woody-plant species (Betula pendula, Alnus glutinosa, Salix caprea var. Kuroyanagi, S. alba var. Tristis, Populus Tacatricho (= P. tacamahacca × trichocarpa 32), Ulmus glabra var. camperdown), and juvenile glasshouse-grown material (B. pendula, B. pubescens, A. glutinosa). Protoplasts are only released if chopped leaf tissue is thoroughly washed prior to digestive enzyme addition. The nature of the washing requirement has been investigated and it has been demonstrated that water soluble compounds are released from chopped leaves which modify their cell-wall structure rendering them resistant to enzymic digestion. When analyzed by paper chromatography the leachate from B. pendula leaves was found to contain the hydroxycinnamic acids p-coumaric acid (PCA) and o-coumaric acid (OCA). Pre-incubation of B. pendula tissue (which is normally susceptible to enzymic digestion) in authentic samples of PCA and OCA prior to enzymic incubation, completely suppressed protoplast yields. The relevance of hydroxycinnamic acids to protoplast isolation and plant tissue culture is discussed.     

Dioscin and methylprotodioscin isolated from the root of Asparagus cochinchinensis suppressed the gene expression and production of airway MUC5AC mucin induced by phorbol ester and growth factor

BackgroundThe root of Asparagus cochinchinensis (Lour.) Merr. has been utilized as mucoregulators and expectorants for controlling the airway inflammatory diseases in folk medicine.Hypothesis/purposeWe investigated whether dioscin and methylprotodioscin isolated from the root of Asparagus cochinchinensis (Lour.) Merr. suppress the gene expression and production of airway MUC5AC mucin induced by phorbol ester and growth factor.Study designConfluent NCI-H292 cells were pretreated with dioscin or methylprotodioscin for 30 min and then stimulated with EGF or PMA for 24 h. The MUC5AC mucin gene expression was measured by RT-PCR. Production of MUC5AC mucin protein was measured by ELISA.Results(1) Dioscin and methylprotodioscin suppressed the expression of MUC5AC mucin gene induced by EGF or PMA; (2) dioscin suppressed the production of MUC5AC mucin induced by either EGF at 10⁻⁵ M (p < 0.05) and 10⁻⁶ M (p < 0.05) or PMA at 10⁻⁴ M (p < 0.05), 10⁻⁵ M (p < 0.05) and 10⁻⁶ M (p < 0.05); (3) methylprotodioscin also suppressed the production of MUC5AC mucin induced by either EGF at 10⁻⁴ M (p < 0.05) or PMA at 10⁻⁴ M (p < 0.05).ConclusionThese results suggest that dioscin and methylprotodioscin isolated from the root of Asparagus cochinchinensis suppress the gene expression and production of MUC5AC mucin, by directly acting on airway epithelial cells, and the results are consistent with the traditional use of Asparagus cochinchinensis as remedy for diverse inflammatory pulmonary diseases.     

Proteases from the fungus Metarhizium anisopliae toxic for Galleria mellonella larvae

The high molecular fraction of the extract from Metarhizium anisopliae grown on wheat bran contains proteolytic enzymes which are toxic for Galleria mellonella larvae. The complex of proteases was fractionated using precipitation with ammonium sulfate, gel filtration, and electrofocusing. Two components have been found: one with the optimum of activity on hemoglobin at pH 6.5, and the second with the optimum around pH 9. The prevailing protease acting at pH 6.5 was inhibited by phenylmethylsulfonyl fluoride and the inhibition was followed by decrease of toxicity. The molecular weights of the enzymes are 35 × 10³ and 71 × 10³.     

An acetylated galactomannoglucan from the stems of Dendrobium nobile Lindl

A water-soluble polysaccharide DNP-W2 composed of glucose, mannose, and galactose in the molar ratio of 6.1:2.9:2.0 had been isolated from the stems of Dendrobium nobile. Its molecular weight was 1.8 × 10⁴ Da determined by HPGPC. Structural features of DNP-W2 were investigated by a combination of chemical and instrumental analysis, including FTIR, GC, GC-MS, periodate oxidation-Smith degradation, methylation analysis, partial acid hydrolysis, and NMR spectroscopy. The results showed that DNP-W2 is a 2-O-acetylgalactomannoglucan and has a backbone consisting of (1→4)-linked β-d-Glcp, (1→6)-linked β-d-Glcp, and (1→4)-linked β-d-Manp, with branches at O-6 of (1→4)-linked β-d-Glcp and β-d-Manp. The branches are composed of α-d-Galp. The acetyl groups are substituted at O-2 of (1→4)-linked Manp. Preliminary tests in vitro reveals that DNP-W2 can stimulate ConA- and LPS-induced T and B lymphocyte proliferation.