Lipid Composition of Pea and Bean Leaves during Chloroplast Development

The changes in composition of the complex lipids were followed during the greening of dark-grown pea (Pisum sativum) and bean (Phaseolus vulgaris) seedlings. No significant changes in glycerolipid concentrations in the leaves were observed during the early stages of greening (0-8 hour for peas and 0-12 hour for beans). On further greening, there was an increase in the proportion of galactolipids and a decrease in the phospholipids. The fatty acid composition of the galactolipids remained constant during 24 hours of greening, but there was a slight increase in α-linolenic acid at 72 hours in the bean. The percentage of α-linolenic acid in the phospholipids and in sulfolipid showed a marked increase between 24 and 72 hours in the bean. Trans-δ³-hexadecenoic acid was the major fatty acid of phosphatidyl glycerol in bean leaves at 72 hours, but it was barely detectable at 24 hours. The lipid composition of greening leaves is discussed in relation to the fine structure and photochemical activity of the developing plastids.     

Synthesis and Secretion of Hydroxyproline-containing Macromolecules in Carrots: II. In vivo Conversion of Peptidyl Proline to Peptidyl Hydroxyproline

The chelator α,α′-dipyridyl prevents the formation of protein-bound hydroxyproline without affecting protein synthesis as measured by the incorporation of ¹⁴C-proline. The inhibitory effect can be overcome by exogenous ferrous ions. Neither α,α′-dipyridyl nor Fe²⁺ interferes in any other known way with the synthesis and secretion of the hydroxyproline-rich proteins normally found in the cell wall. This reversal by Fe²⁺ of the inhibition of proline hydroxylation by α,α′-dipyridyl is used for a study in vivo of the hydroxylation reaction. This reaction has a temperature coefficient of 2.2, suggesting that it is an enzymatic process. Reversal by Fe²⁺ of the α,α′-dipyridyl inhibition can occur after protein synthesis has been arrested with cycloheximide, indicating that peptidyl proline is the substrate of the hydroxylation reaction. Hydroxylation occurs in the cytoplasm, and not in the cell wall, and only for a limited time after the incorporation of proline into the polypeptide chain. This suggests a spatial separation of the enzyme and the substrate.     

Involvement of the Terminal Oxygenase β Subunit in the Biphenyl Dioxygenase Reactivity Pattern toward Chlorobiphenyls

Biphenyl dioxygenase (BPH dox) oxidizes biphenyl on adjacent carbons to generate 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356 and in Pseudomonas sp. strain LB400. The enzyme comprises a two-subunit (α and β) iron sulfur protein (ISP_BPH), a ferredoxin (FER_BPH), and a ferredoxin reductase (RED_BPH). B-356 BPH dox preferentially catalyzes the oxidation of the double-meta-substituted congener 3,3′-dichlorobiphenyl over the double-para-substituted congener 4,4′-dichlorobiphenyl or the double-ortho-substituted congener 2,2′-dichlorobiphenyl. LB400 BPH dox shows a preference for 2,2′-dichlorobiphenyl, and in addition, unlike B-356 BPH dox, it can catalyze the oxidation of selected chlorobiphenyls such as 2,2′,5,5′-tetrachlorobiphenyl on adjacent meta-para carbons. In this work, we examine the reactivity pattern of BPH dox toward various chlorobiphenyls and its capacity to catalyze the meta-para dioxygenation of chimeric enzymes obtained by exchanging the ISP_BPH α or β subunit of strain B-356 for the corresponding subunit of strain LB400. These hybrid enzymes were purified by an affinity chromatography system as His-tagged proteins. Both types, the chimera with the α subunit of ISP_BPH of strain LB400 and the β subunit of ISP_BPH of strain B-356 (the α_LB400β_B-356 chimera) and the α_B-356β_LB400 chimera, were functional. Results with purified enzyme preparations showed for the first time that the ISP_BPH β subunit influences BPH dox’s reactivity pattern toward chlorobiphenyls. Thus, if the α subunit were the sole determinant of the enzyme reactivity pattern, the α_B-356β_LB400 chimera should have behaved like B-356 ISP_BPH; instead, its reactivity pattern toward the substrates tested was similar to that of LB400 ISP_BPH. On the other hand, the α_LB400β_B-356 chimera showed features of both B-356 and LB400 ISP_BPH where the enzyme was able to metabolize 2,2′- and 3,3′-dichlorobiphenyl and where it was able to catalyze the meta-para oxygenation of 2,2′,5,5′-tetrachlorobiphenyl.     

Differences in conformational dynamics of [Pt3(HPTAB)]6+–DNA adducts with various cross-linking modes

We present here molecular dynamics simulations and DNA conformational dynamics for a series of trinuclear platinum [Pt₃(HPTAB)]⁶⁺-DNA adducts [HPTAB = N,N,N′,N′,N′′,N′′-hexakis (2-pyridyl-methyl)-1,3,5-tris(aminomethyl) benzene], including three types of bifunctional crosslinks and four types of trifunctional crosslinks. Our simulation results reveal that binding of the trinuclear platinum compound to a DNA duplex induces the duplex unwinding in the vicinity of the platination sites, and causes the DNA to bend toward the major groove. As a consequence, this produces a DNA molecule whose minor groove is more widened and shallow compared to that of an undamaged bare-DNA molecule. Notably, for trifunctional crosslinks, we have observed extensive DNA conformational distortions, which is rarely seen for normal platinum–DNA adducts. Our findings, in this study, thus provide further support for the idea that platinum compounds with trifunctional intra-strand or long-range-inter-strand cross-linking modes can generate larger DNA conformational distortions than other types of cross-linking modes.     

The Two Genes Encoding Starch-Branching Enzymes IIa and IIb Are Differentially Expressed in Barley

The sbeIIa and sbeIIb genes, encoding starch-branching enzyme (SBE) IIa and SBEIIb in barley (Hordeum vulgare L.), have been isolated. The 5′ portions of the two genes are strongly divergent, primarily due to the 2064-nucleotide-long intron 2 in sbeIIb. The sequence of this intron shows that it contains a retro-transposon-like element. Expression of sbeIIb but not sbeIIa was found to be endosperm specific. The temporal expression patterns for sbeIIa and sbeIIb were similar and peaked around 12 d after pollination. DNA gel-blot analysis demonstrated that sbeIIa and sbeIIb are both single-copy genes in the barley genome. By fluorescence in situ hybridization, the sbeIIa and sbeIIb genes were mapped to chromosomes 2 and 5, respectively. The cDNA clones for SBEIIa and SBEIIb were isolated and sequenced. The amino acid sequences of SBEIIa and SBEIIb were almost 80% identical. The major structural difference between the two enzymes was the presence of a 94-amino acid N-terminal extension in the SBEIIb precursor. The (β/α)₈-barrel topology of the α-amylase superfamily and the catalytic residues implicated in branching enzymes are conserved in both barley enzymes.     

Effects of prostaglandins e(2) and f(2alpha) on the electrofusion of pea mesophyll protoplasts

The effects of prostaglandins E₂ and F_2α on the electrofusion of pea (Pisum sativum cv Ran 1) mesophyll protoplasts were examined. Prostaglandins E₂ and F_2α influenced electrofusion by lowering the threshold voltage necessary for fusion of dielectrophoretically arranged pairs of protoplasts. The direct current voltage threshold decreased with increasing Ca²⁺ concentration up to 0.1 millimolar CaCl₂ and the effects of prostaglandins E₂ and F_2α were more pronounced when CaCl₂ was present in the medium. Treatment with calcium channel blocker methoxy verapamil did not change the prostaglandin effects, while the addition of ethyleneglycol-bis (β-aminoethyl either)-N,N,N′,N′-tetraacetic acid, which binds free Ca²⁺, increased the threshold voltage. Influence of prostaglandins E₂ and F_2α and Ca²⁺ on the membrane fluidity was investigated by analysis of pyrene fluorescence spectra. The values of the ratio between the maximum fluorescence emission intensities of the excimer and the monomer forms (I_ex/I_mon) indicated that prostaglandins and Ca²⁺ decrease the membrane fluidity. It is proposed that electrically evoked displacement of plasmalemma components takes part in the fusion process (U Zimmermann 1982 Biochim Biophys Acta 694: 227-277). We suggest that prostaglandins E₂ and F_2α facilitate the electrofusion of pea mesophyll protoplasts by changing the fluidity of plasmalemma.     

Chromosomal Location of Genes Encoding Barley (1-->3, 1-->4)-beta-Glucan 4-Glucanohydrolases

Preparations of DNA from wheat (Triticum aestivum, cv Chinese Spring), barley (Hordeum vulgare, cv Betzes) and six euplasmic wheat-barley addition lines were digested to completion with restriction endonucleases and the products probed by Southern blot analysis using a cDNA-encoding barley (1→3, 1→4)-β-glucanase isoenzyme II. It is shown that one of the barley (1→3, 1→4)-β-glucanase genes is located on chromosome 1.     

Purification and Characterization of the Soluble F(1)-ATPase of Oat Root Mitochondria

The properties of the soluble moiety (F₁) of the mitochondrial H⁺-ATPase from oat roots were examined and compared to those of the native mitochondrial membrane-bound enzyme. The chloroform soluble preparation was purified by Sephadex G-200 and DEAE-cellulose chromatography. The purified F₁ preparation contained major polypeptides corresponding to α, β, γ, δ, and ε of apparent molecular mass 58, 55, 35, 22, and 14 kilodaltons, respectively. The purified F₁-ATPase, like the native enzyme, was inhibited by azide (I₅₀ = 10 micromolar), nitrate (I₅₀ = 7-10 millimolar), 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (I₅₀ = 1-3 micromolar), and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (I₅₀ = 3 micromolar). F₁-ATPase activity was stimulated by bicarbonate but not by chloride. In both the native and the F₁-form of the ATPase, ATP was hydrolyzed in preference to GTP. The results indicate that these properties of the native membrane-bound mitochondrial ATPase have been conserved in the purified F₁. In contrast to the membrane-bound enzyme, the F₁-ATPase was not inhibited by oligomycin or by N,N′-dicyclohexylcarbodiimide. The mitochondrial F₁-ATPase from oat roots is analogous to other known F₁F₀-ATPases.     

Proteins in intercellular washing fluid from noninoculated and rust-affected leaves of wheat and barley

Proteins in intercellular washing fluid (IWF) from wheat (Triticum aestivum) and barley (Hordeum vulgare) leaves were separated by two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue (CBB) or silver. Intracellular protein from the cut ends of leaves accounted for only a small proportion of total protein in IWF from wheat leaves. When these were heavily infected with the stem rust fungus (Puccinia graminis f. sp. tritici) and grown at 19°C, four infection-related CBB-stainable proteins were detected in IWF.

To compare IWF proteins from wheat and barley leaves infected with the same pathogen, conditions were established that permitted luxuriant growth of stem rust of wheat in barley (exposure to chloroform before inoculation and maintenance at 25°C thereafter). Under these conditions, at least 10 infection-related silver-stainable proteins were detected in IWF from infected wheat in addition to the more than 50 that were of host origin. The electrophoretic properties of 8 of the infection-related proteins were the same as those of 8 infection-related proteins in IWF from barley.

IWF from wheat and barley grown under these conditions was analyzed for Concanavalin A-binding glycoproteins immobilized on nitrocellulose membrane replicas made from gels. Of the many infection-related glycoproteins that were detected in IWF from stem rust-affected wheat, approximately 20 occupied the same positions as those from stem rust-affected barley. The glycoprotein pattern of IWF prepared from wheat leaves grown at 19°C and infected with the leaf rust fungus (P. recondita f. sp. tritici) was markedly different to that of IWF from the same host infected with the stem rust fungus. We conclude that IWF from rust-affected cereal leaves may be a useful source of surface or extracellular proteins from the parasitic mycelium.

     

Characterization of an Acidic Chitinase from Seeds of Black Soybean (Glycine max (L) Merr Tainan No. 3)

Using 4-methylumbelliferyl-β-D-N,N′,N″-triacetylchitotrioside (4-MU-GlcNAc₃) as a substrate, an acidic chitinase was purified from seeds of black soybean (Glycine max Tainan no. 3) by ammonium sulfate fractionation and three successive steps of column chromatography. The purified chitinase was a monomeric enzyme with molecular mass of 20.1 kDa and isoelectric point of 4.34. The enzyme catalyzed the hydrolysis of synthetic substrates p-nitrophenyl N-acetyl chitooligosaccharides with chain length from 3 to 5 (GlcNAc_n, n = 3-5), and pNp-GlcNAc₄ was the most degradable substrate. Using pNp-GlcNAc₄ as a substrate, the optimal pH for the enzyme reaction was 4.0; kinetic parameters K _m and k_cat were 245 µM and 10.31 min⁻¹, respectively. This enzyme also showed activity toward CM-chitin-RBV, a polymer form of chitin, and N-acetyl chitooligosaccharides, an oligomer form of chitin. The smallest oligomer substrate was an N-acetylglucosamine tetramer. These results suggested that this enzyme was an endo-splitting chitinase with short substrate cleavage activity and useful for biotechnological applications, in particular for the production of N-acetyl chitooligosaccharides.     

Polyamine metabolism and its relation to response of the aleurone layers of barley seeds to gibberellic Acid

Polyamine metabolism and its relation to the induction of α-amylase formation in the aleurone layers of barley seeds (Hordeum vulgare cv Himalaya) in response to gibberellic acid (GA₃) has been investigated. A high-performance liquid chromatographic system has been employed for qualitative and quantitative analyses of putrescine (Put), cadaverine (Cad), spermidine (Spd), spermine (Spm), and agmatine (Agm).

Active polyamine metabolism occurs in the aleurone cells of deembryonate barley half seeds during imbibition. The aleurone layers isolated from fully imbibed half seeds contain about 880 nanomoles of Put, 920 nanomoles of Spd, and 610 nanomoles of Spm as free form per gram tissue dry weight while the levels of Cad and Agm are relatively low. The polyamine levels do not change significantly in the aleurone layers in response to added GA₃ (1.5 micromolar) during the 8-hour lag period of the growth substance-induced formation of α-amylase. Also, the polyamine levels are not altered by the presence of abscisic acid (3 micromolar) which inhibits the enzyme induction by GA₃. Kinetic studies show that both applied [U-¹⁴C]ornithine and [U-¹⁴C]arginine are primarily incorporated into Put during 2 hours of incubation, but the incorporation is not significantly affected by added GA₃. Additionally, added GA₃ does not affect the uptake and turnover of [1,4-¹⁴C]Put, nor does it affect the conversion of Put → Spd or Spd → Spm. Treatment of the aleurone layers with GA₃ for 2 hours results in no significant changes in the total activities or the specific activities of ornithine decarboxylase and arginine decarboxylase.

Experiments with polyamine synthesis inhibitors demonstrate that the level of Spd in the aleurone layers could be substantially reduced by the presence of methylglyoxal-bis(guanylhydrazone) (MGBG) during imbibition. MGBG treatment does not affect in vivo incorporation of [8-¹⁴C] adenosine into ATP. The lower the level of Spd the less α-amylase formation is induced by added GA₃. The reduction of GA₃-induced α-amylase formation by MGBG treatment can be either completely or partially overcome by added Spd, depending upon the concentration of MGBG used in the imbibition medium. The results indicate that the early action of GA₃, with respect to induction of α-amylase formation in barley aleurone layers, appears to be not on polyamine metabolism. However, polyamines, particularly Spd, may be involved in regulation of the growth substance-dependent enzyme induction.

     

Interactions of lipoidal materials and a pyridazinone inhibitor of chloroplast development

Formation of chloroplast pigments was inhibited, and free fatty acids accumulated in mustard (Brassica juncea [L.] Coss.) cotyledons and in barley (Hordeum vulgare L.) first leaves developed after treatment with 4-chloro-5- (dimethylamino)-2- (α, α, α-trifluoro-m-tolyl) -3 (2H) -pyridazinone. The inhibitor reduced the amount of fatty acids found in polar lipids (galactolipids) of barley chloroplasts and increased the amount in nonpolar lipids while having little effect on total content of bound fatty acids. The inhibition of chlorophyll formation was circumvented by D-α-tocopherol acetate, phytol, farnesol, and squalene, and by unsaturated fatty acids and their methyl esters. The protective action can be explained partially by an interaction external to the plant whereby 4-chloro-5- (dimethylamino) -2- (α, α, α-trifluoro-m-tolyl) -3 (2H) -pyridazinone partitioned out of the aqueous phase and into the lipid phase, thus limiting availability of the inhibitor to plants. However, the amount of inhibitor reaching the cotyledons of tocopherol-protected mustard seedlngs was still in excess of the amount necessary to cause white foliage, but it failed to produce the effect. Tocopherol treatment did not prevent the 4-chloro-5- (dimethylamino) -2- (α, α, α-trifluoro-m-tolyl) -3 (2H) -pyridazinone-induced buildup of fatty acids in mustard cotyledons but did partially circumvent the effect in barley leaves. The amount of linolenic acid relative to linoleic acid was reduced in barley leaves and chloroplasts by 4-chloro-5- (dimethylamino) -2- (α, α, α-trifluoro-m-tolyl) -3 (2H) -pyridazinone action and this effect was circumvented by tocopherol.     

Cytohistochemical techniques for calcium localization and their application to diseased plants

Lesion delimitation and resistance of old bean (Phaselous vulgaris L., cv. Red Kidney) plants to Rhizoctonia solani Kühn have been suggested to result from increased calcium pectate formation in walls. Ultrastructural histochemistry was used to determine the site of calcium in tissues adjacent to lesions and in older bean hypocotyls. Hypocotyl lesion tissue and uninoculated control tissue were treated with ammonium oxalate or potassium pyroantimonate during fixation. Treatment with potassium pyroantimonate, but not with oxalate, resulted in granular deposits in cell walls of healthy and lesion tissue. Granules also occurred on the plasma membrane of cells adjacent to lesions and in organelles of damaged cells, but wall granule density was not increased. Cell walls from healthy 24-day-old plants had a greater granule density than those for 8-day-old plants. Wall granules were removed from thin sections with ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid. Energy dispersive analysis of x-rays also suggested that potassium pyroantimonate localized calcium. Chemical analyses showed that some calcium was retained in tissues after fixation. The results suggest that there are different mechanisms for lesion delimitation and age-induced resistance.     

Estimation of residual N effect of faba bean and pea on two succeeding cereals using15N methodology

A field experiment was conducted using¹⁵N methodology to study the effect of cultivation of faba bean (Vicia faba L.), pea (Pisum sativum L.) and barley (Hordeum vulgare L.) on the N status of soil and their residual N effect on two succeeding cereals (sorghum (Sorghum vulgare) followed by barley). Faba bean, pea and barley took up 29.6, 34.5 and 53.0 kg N ha^–1 from the soil, but returned to soil through roots only 11.3, 10.8 and 5.7 kg N ha^–1, respectively. Hence, removal of faba bean, pea and barley straw resulted in a N-balance of about –18, –24, and –47 kg ha^–1 respectively. A soil nitrogen conserving effect was observed following the cultivation of faba bean and pea compared to barley which was of the order of 23 and 18 kg N ha^–1, respectively. Cultivation of legumes resulted in a significantly higher A_N value of the soil compared to barley. However, the A_N of the soil following fallow was significantly higher than following legumes, implying that the cultivation of the legumes had depleted the soil less than barley but had not added to the soil N compared to the fallow. The beneficial effect of legume cropping also was reflected in the N yield and dry matter production of the succeeding crops. Cultivation of legumes led to a greater exploitation of soil N by the succeeding crops. Hence, appreciable yield increases observed in the succeeding crops following legumes compared to cereal were due to a N-conserving effect, carry-over of N from the legume residue and to greater uptake of soil N by the succeeding crops when previously cropped to legumes.     

Characterization of the alpha-Amylases Synthesized by Aleurone Layers of Himalaya Barley in Response to Gibberellic Acid

The gibberellic acid (GA₃)-induced α-amylases from the aleurone layers of Himalaya barley (Hordeum vulgare L. cv Himalaya) have been purified by cycloheptaamylose-Sepharose affinity chromatography and fractionated by DEAE-cellulose chromatography. Four fractions (α-amylases 1-4) were obtained which fell into two groups (A and B) on the basis of a number of characteristics. Major differences in serological characteristics and in proteolytic fingerprints were found between group A (α-amylases 1 and 2) and group B (α-amylases 3 and 4). Also, the lag time for appearance of group B enzyme activity was longer than for group A, and the appearance of group B required higher GA₃ levels than group A. The components of each group behaved similarly, although differences in proteolytic fingerprints were detected.

These results together with those from other studies indicate that GA₃ differentially controls the expression of two α-amylase genes or groups of genes giving rise to two groups of α-amylases with many different properties.

     

The Catabolism of (+/-)-Abscisic Acid by Excised Leaves of Hordeum vulgare L. cv Dyan and Its Modification by Chemical and Environmental Factors

Excised light-grown leaves and etiolated leaves of Hordeum vulgare L. cv Dyan catabolized applied (±)-[2-¹⁴C]abscisic acid ([±]-[2-¹⁴C]ABA) to phaseic acid (PA), dihydrophaseic acid (DPA), and 2′-hydroxymethyl ABA (2′-HMABA). Identification of these catabolites was made by microchemical methods and by combined capillary gas chromatographymass spectrometry (GC-MS) following high dose feeds of nonlabeled substrate to leaves. Circular dichroism analysis revealed that 2′-HMABA was derived from the (−) enantiomer of ABA. By selecting tissue samples in which endogenous catabolites were undetectable by gas chromatography, it was possible to identify unequivocally ABA catabolites by GC-MS without the need to employ deuteriated substrate to distinguish the (±)-ABA catabolites from the same endogenous compounds. Refeeding studies were used to confirm the catabolic route. The methyl ester of (±)-[2¹⁴C]-ABA was hydrolyzed efficiently by light-grown leaves of H. vulgare. Leaf age played a significant role in (±)-ABA catabolism, with younger leaves being less able than their older counterparts to catabolize this compound. The catabolism of (±)-ABA was inhibited markedly in water-stressed Hordeum leaves which was characterized by a decreased incorporation of label into 2′-HMABA, DPA, and conjugates. The specific, mixed function oxidase inhibitor, ancymidol, did not inhibit, dramatically, (±)-ABA catabolism in light-grown leaves of Hordeum whereas the 80s ribosome, translational inhibitor, cycloheximide, inhibited this process markedly. The 70s ribosome translational inhibitors, lincomycin and chloramphenicol, were less effective than cycloheximide in inhibiting (±)-ABA catabolism, implying that cytoplasmic protein synthesis is necessary for the catabolism of (±)-ABA in Hordeum leaves whereas chloroplast protein synthesis plays only a minor role. This further suggests that the enzymes involved in (±)-ABA catabolism in this plant are cytoplasmically synthesized and are `turned-over' rapidly, although the enzyme responsible for glycosylating (±)-ABA itself appeared to be stable.     

In Vitro Synthesis and Processing of Wheat alpha-Amylase : TRANSLATION OF GIBBERELLIC ACID-INDUCED WHEAT ALEURONE LAYER RNA BY WHEAT GERM AND XENOPUS LAEVIS OOCYTE SYSTEMS

Wheat (Triticum aestivum) RNA was used to program synthesis of the α-amylase protein by Xenopus laevis oocytes. A 41,500-dalton protein was made which was identified as α-amylase by immunoprecipitation with rabbit anti-α-amylase antiserum raised against the purified wheat protein and by its co-migration with authentic α-amylase on sodium dodecyl sulfate polyacrylamide gels. Synthesis of α-amylase was dependent upon injection of RNA extracted from gibberellic acid-induced aleurone layers from wheat. The amount of α-amylase produced was proportional to the amount of RNA injected and reached a plateau within 4 hours after injection. When the same RNA was translated in a wheat germ cell-free translation system, a 43,000-dalton protein was produced. Addition of dog pancreas microsomal membranes to the wheat germ translation system resulted in processing of the α-amylase protein to a form which co-migrated with authentic α-amylase purified from malted wheat and with the protein synthesized in oocytes.     

Adenosine 5′-monophosphate ameliorates D-galactosamine/lipopolysaccharide-induced liver injury through an adenosine receptor-independent mechanism in mice

D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced lethality and acute liver failure is dependent on endogenously produced inflammatory cytokines. Adenosine has been proven to be a central role in the regulation of inflammatory response. It is not entirely clear that which adenosine action is actually crucial to limiting inflammatory tissue destruction. Here we showed that GalN/LPS challenge elevated hepatic adenosine and induced lethality in adenosine receptor-deficient mice with equal efficiency as wild-type mice. In GalN/LPS-treated mice, pretreatment with adenosine 5′-monophosphate (5′-AMP) significantly elevated hepatic adenosine level and reduced mortality through decreasing cytokine and chemokine production. In RAW264.7 cells, 5′-AMP treatment inhibited the production of inflammatory cytokines, which is not mediated through adenosine receptors. 5′-AMP failed to attenuate LPS-induced nuclear factor-κB (NF-κB) p65 nuclear translocation, but reduced LPS-induced recruitment of NF-κB p65 to inflammatory gene promoters and decreased LPS-induced enrichment of H3K4 dimethylation at the tumor necrosis factor-α (TNF-α) promoter, which was involved in 5′-AMP-induced elevation of cellular adenosine and a decline of methylation potential. In vitro biochemical analysis revealed that adenosine directly attenuated recruitment of NF-κB to the TNF-α and interleukin-6 promoters. Our findings demonstrate that 5′-AMP-inhibiting inflammatory response is not mediated by adenosine receptors and it may represent a potential protective agent for amelioration of LPS-induced liver injury.     

Phosphatidylcholine biosynthesis in castor bean endosperm : purification and properties of cytidine 5'-triphosphate:choline-phosphate cytidylyltransferase

Cytidine 5′-triphosphate:choline-phosphate cytidylyltransferase (EC 2.7.7.15) has been purified to near homogeneity (3350-fold) from castor bean (Ricinus communis L. var Hale) endosperm. The steps of purification included a differential solubilization of this enzyme with n-octyl β-d-glucopyranoside (OGP) and column chromatography on sequential DEAE-sepharose, sepharose-6B, and second DEAE-sepharose columns. The uses of appropriate concentrations of the detergent, OGP, in each step were crucial to obtain the highly purified enzyme. The purified enzyme gave a single protein band on nondenaturing polyacrylamide gel electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed one major protein band of 40 kilodaltons. Gel filtration chromatography indicated that native cytidylyltransferase was approximately 155 kilodaltons, suggesting that it exists naturally as a tetramer. The purified enzyme used methylethanolamine-phosphate as a substrate but not ethanolamine-phosphate and dimethylethanolamine-phosphate. ATP and other nucleotides tested showed little effect on the purified enzyme. The purified enzyme activity was stimulated by both phospholipids extracted from castor bean endosperm and phosphatidylcholineoleate vesicles.