Isolation of portulaca oleracea (regla) mucilage and identification of its structure

Portulaca oleracea leaves were found to contain 0.42% of a mucilage mixture. The mucilage was fractionated into an acidic and a neutral fraction. The acidic fraction consists of galacturonic acid residues joined by α-(1→4)-linkages; 60% of these residues are present as the calcium salt, and esterified galacturonic acid residues are absent. The neutral fraction is composed of 41% of arabinose and 43% of galactose residues, besides traces of rhamnose residues.     

Water-soluble polysaccharides of Opuntia ficus-indica cv “Burbank's Spineless”

Carbohydrate-containing polymers have been extracted with water from the fleshy, lobed stems of Opuntia ficus-indica cv “Burbank's Spineless”. By ion exchange chromatography, the material was separated into one neutral and two acidic fractions. Each fraction was separated in two by gel filtration. The neutral fractions consisted of two glucans and a glycoprotein, containing arabinose and galactose. All four acidic fractions contained galacturonic acid, arabinose, rhamnose, galactose and xylose in different proportions. The cell wall structure of O. ficus-indica is discussed.     

Variations in endogenous gibberellins in developing bean seeds I. Occurrence of neutral and acidic substances

Activities of separated and chromatographed substances in the nonacidic, acidic ethyl acetate and acidic butanol fractions from bean seeds, Phaseolus vulgaris L., cv. Bountiful and Kentucky Wonder, were measured in the Progress No. 9 dwarf pea bioassay grown under red light. Activity in the nonacidic fraction was shown to be attributable only to neutral substances and was free of acidic gibberellin-like substances. As the seed matures, neutral substances and one of the acidic butanol-soluble substances (B-I) increase in activity. The acidic ethyl acetate substances and butanol-soluble substance (B-II) initially increase and then almost disappear.     

Variations in Endogenous Gibberellins in Developing Bean Seeds II. Changes Induced in Acidic and Neutral Fractions by GA(1)

Immature (8-mm), medium mature (11-mm), and mature green (16- and 17-mm) bean seeds (Phaseolus vulgaris L. cv. Kentucky Wonder and Bountiful) were incubated in gibberellin A₁ solutions for 24 hours at 20°. Extracts from the seeds were separated into nonacidic, acidic ethyl acetate, and acidic butanol fractions. These were chromatographed. The eluates of the chromatograms were tested on Progress No. 9 dwarf peas grown under red light. The level of neutral gibberellin-like substances remained unchanged in immature seed, but they increased markedly in mature green seeds. Coincident with increased levels of the neutral substances, there were significant decreases in acidic ethyl acetate-soluble gibberellin-like substances, including applied GA₁, and in 1 acidic butanol-soluble gibberellin-like substance. Seed incubation in GA₁ brought about increased activity of substance B-II in immature and medium mature seeds. The level of butanol-soluble gibberellin-like substance B-I in seeds of any size was not affected by incubation in GA₁. Considering the marked increases in activity induced in the neutral fraction and the decreases in activity of certain eluates from the chromatograms of the acidic fractions, it was concluded that the neutral fraction may serve as a reserve form of gibberellins in the dry seed. The acidic ethyl acetate substances and substance B-II may be required for normal development of the bean seed.     

Structural studies of the acidic oligosaccharide units from bovine glycophorin

The O-glycosidically-linked carbohydrate units of glycophorin from bovine erythrocyte membrane were released by alkaline borohydride treatment. These oligosaccharides were separated into the neutral fractions and the acidic fractions by ion-exchange chromatography followed by gel filtration. The two acidic fractions (fractions 10 and 13) which have the smallest molecular weight in acidic oligosaccharides, were further purified by gel filtration on Bio-Gel P-4 column. Two acidic oligosaccharides (fractions 10-I and 10-II), heptasaccharides, were separated by gel filtration on a Bio-Gel P-4 column from fraction 10. These structures were determined by methylation analyses, nitrous acid deamination after hydrazinolysis and Smith degradation after desialylation. In addition, the structures were also analyzed by direct-probe mass spectrometry of the permethylated derivatives before and after desialylation. These studies indicated that one of them (fraction 10-I) was NeuNGcα(2→3)Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)GlcNAcβ(1→3)Galβ(1→3) GalNAcol and another heptasaccharide (fraction 10-II) was Galβ(1→4)GlcNAcβ(1→3)Galβ(1→3) [NeuNGcα(2→3)Galβ(1→4)GlcNAcβ(1→6)]GalNAcol. Athough another acidic fraction (fraction 13) was obtained as a single peak on a Bio-Gel P-4 column, it appeared to be the mixture of a heptasaccharide, NeuNGcα(2→3)Galβ(1→4)GlcNAcβ(1→3 or 6)[Galβ(1→4)GlcNAcβ(1→6 or 3)]Galβ(1→3)GalNAcol and an oligosaccharide similar to fraction 10-II, by analysis of two products obtained by Smith degradation after desialylation.     

Arabinogalactans in a purified allergen preparation from pollen of timothy (Phleum pratense)

The purified allergen preparation representing a certain fraction of an aqueous timothy pollen extractcontained ca. 20% carbohydrate, mainly as arabinose (7%) and galactose (13%). The protein content was 63%. Fractionation on DEAE-Sephadex and Sephadex G-100 gave one neutral and two acidic fractions, all containing protein, arabinose and galactose. The structure of the carbohydrate moiety was investigated by methylation analysis, periodate oxidation and enzyme incubation. The acidic fraction contained (1→6)-linked galactose residues, some being substituted on O-3 with arabinose. The neutral fraction consisted of a more extensively branched arabinogalactan with longer side chains of (1→3)- and (1→5)-linked arabinose. The arabinose was present mainly as α-l-arabinofuranosyl residues. Alkaline degradation and subsequent fractionation indicated the presence of a covalent linkage between hydroxyproline and arabinose. Periodate oxidation or incubation with α-l-arabinofuranosidase did not affect the allergenic activity of the extract.     

Lipid-linked saccharides formed during pullulan biosynthesis in Aureobasidium pullulans

Radioactive glycolipids were extracted from cells of Aureobasidium pullulanspulsed with d-[¹⁴C]glucose. Labelled, alkali-stable lipids were resolved into one neutral and two acidic fractions. The neutral fraction was stable to mild hydrolysis with acid, whereas the acidic fractions could be hydrolysed, yielding d-glucose and a series of oligosaccharides having mobilities corresponding to those of isomaltose, panose, and isopanose. Amyloglucosidase (EC 3.2.1.3) catalysed the hydrolysis of 60% of the liberated radioactive oligosaccharides to d-glucose, indicating the presence of (1→4)-α- and (1 → 6)-α-d-glucosidic bonds. Since these lipid-linked saccharides are produced during pullulan biosynthesis in A. pullulans, it is proposed that they are intermediates in the biosynthetic pathway of that extracellular polysaccharide. A mechanism incorporating these glycolipids into a possible scheme of polysaccharide assembly is presented.     

Evidence for the presence of N2-(1,3-dicarboxypropyl)-L-amino acids in crown-gall tumors induced by Agrobacterium tumefaciens strains 181 and EU6

Using crown-gall tumors induced by Agrobacterium tumefaciens strains 181 and EU6 several unusual compounds have been isolated in a single fraction. Physicochemical analysis of the compounds in this fraction showed that they are N²-(1,3-dicarboxypropyl)-L-amino acids derived from neutral and acidic amino acids. Asparagine and glutamine derivatives (asparaginopine and glutaminopine) are the main components. Synthesis of asparaginopine from asparagine, α-ketoglutarate and NADPH, and degradation of asparaginopine to asparagine have also been demonstrated using enzyme prepared from Pinto bean strain 181 tumor.     

Structural investigations of the extracellular polysaccharides elaborated by Beijerinckia mobilis

The extracellular mucilage from Beijerinckia mobilis, a member of the Azotobacteriaceae, after removal of contaminating protein, was separated into a neutral polysaccharide (N-2, 10%); a neutral, dialysable fraction (N-1, 5%), consisting of glucose and oligosaccharides containing glucose, arabinose, and rhamnose; and an acidic polysaccharide (85%). N-2 (mol. wt, 1900) was highly branched and comprised glucopyranose, mannopyranose, and arabinofuranose residues (1:1:1). The various linkages were determined. The acid fraction was a polymer of high molecular weight composed of L-guluronic acid (65%), D-glucose (15%), and D-glycero-D-mannoheptose (20%), together with acetic and pyruvic acids. From the results of methylation, periodate oxidation, and partial hydrolysis, a branched molecule with a backbone of guluronic acid and heptose, and side chains of glucose and guluronic acid is proposed. Pyruvic acid was found to be acetal-linked to 2?5% of the heptose residues. The similarities between this polysaccharide and that from the related species Azotobacter indicum are discussed.     

Reaction between Nitrite and Low Salt-soluble Diffusible Fraction of Meat. Some Compounds Influencing Nitrite Depletion and Producing Unidentified-N Compounds

A low salt-soluble, diffusible fraction of meat was separated into basic, neutral and acidic fractions. Investigations were then conducted on the reaction of each fraction with nitrite and on the recovery of the added nitrite.

The basic fraction shared the lowest ability to decompose nitrite and had 88% recovery of added nitrite-N. Hypoxanthine was one of the main components that affected the recovery in the basic fraction. In the neutral fraction, 42% of nitrite was decomposed, and the recovery of the nitrite was 107%. In the acidic fraction, more than 80% of nitrite was decomposed and 30% was converted to unidentified-N compounds. Among endogenous acidic substances tested, cysteic acid showed the highest ability to decompose nitrite, accompanying the production of unidentified-N compounds.     

Epicuticular wax of Pinus radiata needles

Epicuticular wax isolated from the cotyledons and primary needles of 10-week-old Pinus radiata seedlings is similar in composition and contains 86% neutral compounds, viz. alkyl esters (25%, C₂₄–C₆₄), nonacosan-10-ol (52%), heptacosane-5,10-diol (2%), nonacosane-4,10-diol, nonacosane-5,10-diol, and nonacosane-10,13-diol (total 12%) and estolides, MW ca 800 (2%), MW ca 1100 (6%), and MW ca 1500 (1%). The acidic fraction (14%) contains n-acids (78%, C₁₂–C₃₂) and diterpene acids (22%, mainly abieta-8,11,13-trien-18-oic, with lesser amounts of pimara-8(14),15-dien-18-oic, isopimara-7,15-dien-18-oic and hydroxylated aromatic, diene and mono-ene acids). Wax isolated from primary needles of 1-yr-old seedlings had a similar neutral fraction composition, but the acidic fraction contained predominantly the diterpene acid mixture, with only trace amounts of n-acids. The wax from 1-yr-old secondary, needles from P. radiata forest trees aged 5 yr and 40 yr contained an acid fraction (12% 5 yr, 17% 40 yr trees) comprising the diterpene acid mixture, with trace amounts of n-acids together with ω-hydroxy acids (C₁₂, C₁₄ and C₁₆). The neutral fraction from both young and old trees had a similar composition containing alkyl esters (7%, C₂₄–C₆₆), estolides (90%, MW 566-ca 1500), nonacosan-10-ol (2%) and the heptacosane and nonacosane diols (1%). During growth and maturation of P. radiata, the nonacosan-10-ol content of the needle wax decreases while the proportion of estolides and diterpene acids increases, the latter probably being located around the stomatal pore.     

Expolygalacturonase from Suspension Cultures of Marchantia polymorpha: Its Presence and Involvement in Pectic Polysaccharide Degradation

Polygalacturonase was isolated from cell suspension cultures of a thalloid liverwort, Marchantia polymorpha. The enzyme in the `buffer-soluble' protein fraction was dialyzed at pH 5.2 and further purified 91-fold by a combination of chromatographic techniques including CM-Sephadex, Sephacryl S-200, DEAE-Sephadex, and Sephadex G-200. The purified enzyme had an optimum activity in the pH range at 3.6 to 3.8 and molecular weight of 76,000 daltons, and its activity was not stimulated by cations. The enzyme was identified as an exohydrolase from viscometric data and chromatographic analysis of the reaction products.

The polysaccharides extracted from the Marchantia cell walls with 2% (w/v) Na hexametaphosphate solution were separated into two fractions, neutral polysaccharides (fraction P-N) and acidic polysaccharides (fraction P-A) by a DEAE-Sephadex column. The fraction P-N was not susceptible to the purified exopolygalacturonase, whereas fraction P-A was partially degraded. This resulted in hydrolysis of 19.5% of the glycosyl linkages of fraction P-A with the release of galacturonic acids. The specific activity of exopolygalacturonase increased during the growth cycle.

     

Metabolism of DL-Tryptophan-3-14C by the Fruitlets of Citrus aurantifolia

Developing lime fruit [Citrus aurantifolia (Christm.) Swingle] was supplied with dl-tryptophan-3-¹⁴C in a special medium. An incubation period of six hours was sufficient for the radioactivity to reach an equilibrium between the fruit tissue and the incubation medium. Analyses of the fruit tissue and the medium for acidic and neutral metabolites of tryptophan indicated the existence of indolic products. The auxin indole-3-acetic acid (IAA) was identified among the products by dry column chromatography and biological assay. Other acidic metabolites included indolepyruvic acid and an unidentified material. Neutral metabolites included indolealdehyde, indoleacetaldehyde, and two unidentified compounds. Biological activity in the Avena curvature test was obtained from extracted compounds which corresponded to IAA and indolepyruvic acid in the acidic fraction and indoleacetaldehyde in the neutral fraction. Radioactive tryptophan was also found in both the acidic and the neutral fractions due to its amphoteric nature. The experiment demonstrated the conversion of tryptophan to its indolic metabolites, including indole-3-acetic acid, in this Citrus tissue.     

Nanoarchaeota,Their Sulfolobales Host,and Nanoarchaeota Virus Distribution across Yellowstone National Park Hot Springs

Nanoarchaeota are obligate symbionts with reduced genomes first described from marine thermal vent environments. Here, both community metagenomics and single-cell analysis revealed the presence of Nanoarchaeota in high-temperature (∼90°C), acidic (pH ≈ 2.5 to 3.0) hot springs in Yellowstone National Park (YNP) (United States). Single-cell genome analysis of two cells resulted in two nearly identical genomes, with an estimated full length of 650 kbp. Genome comparison showed that these two cells are more closely related to the recently proposed Nanobsidianus stetteri from a more neutral YNP hot spring than to the marine Nanoarchaeum equitans. Single-cell and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) analysis of environmental hot spring samples identified the host of the YNP Nanoarchaeota as a Sulfolobales species known to inhabit the hot springs. Furthermore, we demonstrate that Nanoarchaeota are widespread in acidic to near neutral hot springs in YNP. An integrated viral sequence was also found within one Nanoarchaeota single-cell genome and further analysis of the purified viral fraction from environmental samples indicates that this is likely a virus replicating within the YNP Nanoarchaeota.     

HISTOCHEMICAL STAINING AND CHARACTERIZATION OF GLYCOPROTEINS IN ACID-SECRETING CELLS OF FROG STOMACH

Glycoproteins were histochemically localized in oxyntic cells of the frog stomach by staining with periodic acid-silver methenamine. Reduction of silver was most intense on (a) the outer aspect of the apical plasmalemma, (b) within the tubular smooth membrane system characteristic of oxyntic cells, and (c) within cisternae and vesicles of the Golgi complex. Other membrane components such as those from the mitochondria, nucleus, junctional complex, lateral and basal cell membranes showed little or no stainability. Gastric mucosal homogenates were fractionated by centrifugation for further morphological and chemical analysis. The staining reaction of the microsomal fraction (40,000 g x 60 min) was similar to that of the tubular membranous components of intact oxyntic cells. Carbohydrate analyses showed that all cell fractions are extremely low in acidic sugars, uronic and sialic acids, while neutral sugars and hexosamines are relatively abundant. The microsomal fraction contains the largest proportion of carbohydrates, ca. 9% of the fat-free dry weight. Another distinguishing feature is that glucosamine is the only detectable hexosamine in the microsomal fraction. These histochemical and chemical data indicate that neutral glycoproteins are associated with membranous components which have been implicated in the process of HCl secretion by oxyntic cells. The staining pattern within the cells supports the hypothesis of interrelationships between the Golgi membranes, tubular smooth membranes, and apical surface membrane.     

The acidic polysaccharide from Palmaria decipiens (Palmariales,Rhodophyta)

Palmaria decipiens, one of the most abundant red seaweeds of the chilean Antarctic, was collected in King George Island. The hot water extract (26% yield) showed by acid hydrolysis to contain xylose, galactose and traces of glucose. Fractionation with cetrimide gave a soluble neutral xylan and an insoluble fraction. The insoluble fraction afforded an acidic polysaccharide that contained 4.8% of uronic acids, 2.8% of sulfate and 18.9% of protein. Polyacrylamide gel electrophoresis showed that it was homogeneous. The GLC and HPLC analysis of the total acidic hydrolysis products showed that the acidic polysaccharide was composed of the neutral sugars galactose and xylose in the molar ratio 8.2:1.0 and of galacturonic and glucuronic acid in the ratio 1.5:1.0. The second-derivative FT-IR spectrum showed the characteristic amide I, II and III bands of proteins. Alkaline cleavage with 0.1 M NaOH indicated the presence of a glycoprotein with O-glycosidic linkage.Results found in this work suggest that the acidic polysaccharide extracted from Palmaria decipiens is an acidic xylogalactan-protein complex.     

Water-soluble glucans from true cardamom (Elettaria cardamomum White at Maton) seeds

Water-soluble polysaccharides from seeds of true cardamom (Elettaria cardamomum White at Maton, family Zingiberaceae) have been studied. The study has shown the presence of neutral and acidic components in these polysaccharides. Three polysaccharides (380, 166, and 27 kDa) have been isolated from the neutral fraction. According to the structural analysis data, they represent α-glucans with different degrees of branching (7.1–46.1%); α-(1→4)-D-glucopyranose residues of their backbone chains are substituted at the C6 position with single α-D-glucopyranose residues. Polysaccharides with such structures have a wide range of biological activity. The presence of branched α-glucans in E. cardamomum seeds has been demonstrated.     

Subcellular Localization of Proteases in Developing Leaves of Oats (Avena sativa L.)

The distribution and subcellular localization of the two major proteases present in oat (Avena sativa L. cv Victory) leaves was investigated. Both the acidic protease, active at pH 4.5, and the neutral protease, active at pH 7.5, are soluble enzymes; a few percent of the enzyme activity was ionically bound or loosely associated with organellar structures sedimenting at 1000g. On the average, 16% of the acidic protease could be washed out of the intercellular space of the leaf. Since isolated protoplasts contained correspondingly lower activities as compared to crude leaf extracts, part of the acidic activity is associated with cell walls. No neutral protease activity was recovered in intercellular washing fluid. Of the activities present in protoplasts, the acidic protease was localized in the vacuole, whereas the neutral protease was not. The localization of the acidic protease in vacuoles did not change during leaf development up to an advanced stage of senescence, when more than 50% of the leaf protein had been degraded. These observations indicate that protein degradation during leaf senescence is not due to a redistribution of acidic protease activity from the vacuole to the cytoplasm.     

Mucilage from a fresh-water red alga of the genus Batrachospermum

The gelatinous polysaccharides of a Batrachospermum species have been extracted from the alga. The major polysaccharide is acidic and has been separated from neutral polysaccharides by chromatography on DEAE-cellulose. The constituent sugars of the acidic polysaccharide include d- and l-galactose, d-mannose, d-xylose, l-rhamnose, d-glucuronic acid, and two O-methyl sugars, which have been characterized as 3-O-methyl-l-rhamnose (l-acofriose and 3-O-methyl-d-galactose. Partial acid hydrolysis of this polysaccharide has given a complex mixture of neutral and acidic oligosaccharides. The two preponderant acidic oligosaccharides contained galactose and glucuronic acid in 1:1 ratio, suggesting the presence of a repeating sequence of these two residues as a major structural feature of the polysaccharide.     

Pectic polysaccharides in carrot cells growing in suspension culture

Pectic polysaccharides in the cell wall of suspension-cultured carrot cells (Daucus carota L.) were fractionated into high- and low-molecular-weight components by molecular-sieve chromatography with a Sepharose 4B column. During the phase of cell-wall expansion, the relative content of low-molecular-weight polymers rapidly increased. Electrophoretic analyses of these fractions showed that the high-molecular-weight components were largely composed of neutral and weakly acidic polymers while the low-molecular-weight fraction contained, in addition to neutral polymers, strongly acidic polyuronides in which the content of neutral sugars was very small. The accumulation of a large amount of the strongly acidic polyuronides occurred in a late stage of cell-wall growth, concomitant with a marked decrease in the high-molecular-weight components.Abbreviation MW molecular weight