FIXED CARBON PARTITIONING IN THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA)

The main products of carbon fixation in the red algae are sulfated cell-wall polysaccharides, floridean starch, and low molecular weight (LMW) carbohydrates, mainly floridoside. In the red microalga Porphyridium sp., sulfated polysaccharide—cell bound and soluble—comprises up to 70% of the algal biomass. The purpose of this study was to elucidate the partitioning of fixed carbon in Porphyridium sp. toward the different products of carbon fixation. Using pulse-chase technique with [¹⁴C]bicarbonate, we followed ¹⁴C flow into the major compounds, namely, cell-wall polysaccharide, floridoside, starch, and protein, under various environmental conditions (i.e. carbon dioxide enrichment and nitrate starvation). ¹³C-NMR and gas chromatography analysis showed the main LMW product in Porphyridium sp. to be floridoside. After the short [¹⁴C]bicarbonate pulse (20 min), 42%–53% of total ¹⁴C uptake was initially found in floridoside. The appearance of ¹⁴C in the soluble polysaccharide was evident immediately at the end of the 20-min [¹⁴C]bicarbonate pulse. The specific radioactivity in the floridoside fraction declined by 80% after the 48-h chase, this decline being accompanied by increased labeling of starch and the soluble polysaccharide. In cells exposed to high CO₂ concentration, larger amounts of ¹⁴C (about twice as much) were channeled into starch and soluble polysaccharide than in cells under low CO₂ concentration. The most significant increase (1500%) in labeling during chase was found in the soluble polysaccharide of the nitrate-deprived cultures. It therefore seems likely that the large amounts of carbon incorporated by Porphyridium sp. cells into floridoside were subsequently used for the synthesis of macromolecular components. The data thus support the premise that floridoside serves as a dynamic carbon pool, which channels the fixed carbon toward polysaccharides and other end products according to the ambient conditions.     

THE PRODUCTION OF EXTRACELLULAR POLYSACCHARIDE BY THE UNICELLULAR RED ALGA PORPHYRIDIUM AERUGINEUM1,2

The unicellular red alga Porphyridium aerugineum was shown to be encapsulated by an amorphous, water-soluble, polyanionic polysaccharide of high molecular weight. The encapsulating polysaccharide is qualitatively identical with polysaccharide found dissolved in large quantity in the culture medium. The kinetics of extracellular polysaccharide production as a function of cell age was studied. Rates of production (on a per cell basis) of both encapsulating and dissolved polysaccharides are greatest in stationary phase light-grown cultures. Dissolved polysaccharide was quantitatively isolated by precipitation with cetyl pyridinium chloride, conversion to the calcium salt, and reprecipitation with ethanol. The procedure yields a spectrally pure product, which is composed of glucose, galactose, xylose, and 2 undetermined, sugar components, and has a sulfate content of 7.6% by weight. Electron microscopy of Porphyridium revealed that Golgi vesicles transport, polymerized polysaccharides to and through the cell membrane. Similar vesicles were observed in the multicellular Pseudogloiophloea, indicating that the Golgi complex plays a crucial role in the production of extracellular polysaccharides by the red algae. H¹⁴CO₃^- pulse-label experiments resulted in labeled extracellular polysaccharide in which all the constituent components contained ¹⁴C. Rates of excretion of polysaccharide were found, to follow a cyclic pattern, correlated generally with the division cycle, of the cell.     

CELL-WALL FORMATION DURING THE CELL CYCLE OF PORPHYRIDIUM SP. (RHODOPHYTA)

The cell wall of the red microalgae Porphyridium sp. (UTEX 637) comprises a complex amorphous polysaccharide (6–7 × 10⁶ Da). The polysaccharide is made up of xylose, glucose, and galactose as the main sugars, as well as some minor sugars, protein, sulfate, and glucuronic acid, the latter two conferring a negative charge on the polysaccharide. In this study, we used synchronized cultures as one of the ways of unraveling the mechanism of biosynthesis of this complex polysaccharide by following cell-wall formation during the cell cycle. Synchronization of Porphyridium sp. was achieved with an alternating light:dark regime of 12:12 h LD and dilution of the culture at the end of the cycle. Under these conditions, cell duplication occurred between the 12th and 14th hours of the cycle. The following order of building toward formation of the final polysaccharide appeared to take place: Intermediate polysaccharides with molecular masses ranging from 0.5 × 10⁶ to 2 × 10⁶ Da appeared in succession during hours 2–6 of the cycle, and the full-sized polysaccharide was detected by the 8th hour. At the beginning of the cycle, xylose was the predominant sugar. Sulfur peaked at hours 2–4; glucose, galactose, and glucuronic acid at hours 8–12; and the minor sugars at hours 12–14. Upon incubation of low molecular mass polymer (0.5 × 10⁶ Da) collected from the 4th hour with cellular crude extract from cells of the 6th hour of the cycle, two intermediates were formed (0.8 × 10⁶ Da and 2 × 10⁶ Da). We suggest that the 0.5 × 10⁶ Da polymer intermediate, which is composed mainly of xylose, is the first polymer secreted into the medium, where it is further polymerized enzymatically to produce the 2 × 10⁶ Da polymer via an intermediate 0.8 × 10⁶ Da polymer. Later, the full-size polysaccharide is produced.     

DEGRADATION OF THE CELL-WALL POLYSACCHARIDE OF PORPHYRIDIUM SP. (RHODOPHYTA) BY MEANS OF ENZYMATIC ACTIVITY OF ITS PREDATOR,GYMNODINIUM SP. (PYRROPHYTA)1

The dinoflagellate Gymnodinium sp., which preys specifically on cells of the red microalga Porphyridium sp., possesses enzymes that degrade exocellular polysaccharides of the Porphyridium sp. A crude extract of Gymnodinium sp. was applied to this polysaccharide, and the degradation products were characterized by charge and size separations. Charge separation revealed the presence of a fraction that was not found in the native polysaccharide. This fraction, which was eluted from an anion-exchange resin with water alone, was composed mostly of glucose and xylose (in a 1:1 weight ratio). Size separation of the degradation products revealed three fractions; the molecular weight of the main one was 5 × 10⁶ daltons, whereas that of the native polysaccharide was 7 × 10⁶ daltons. The carbohydrate composition of these fractions was determined. Although the main product of degradation had a relatively high molecular weight, its viscosity was significantly reduced relative to the native polysaccharide. Additional enzymatic degradation is required for further exploration of the structure of the exocellular polymer of Porphyridium sp.     

A GLYCOPROTEIN NONCOVALENTLY ASSOCIATED WITH CELL‐WALL POLYSACCHARIDE OF THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA)1

The cells of the red microalga Porphyridium sp. (UTEX 637) are encapsulated in a cell wall of a negatively charged mucilaginous polysaccharide complex composed of 10 different sugars, sulfate, and proteins. In this work, we studied the proteins associated with the cell‐wall polysaccharide. A number of noncovalently associated proteins were resolved by SDS‐PAGE, but no covalently bound proteins were detected. The most prominent protein detected was a 66‐kDa glycoprotein consisting of a polypeptide of approximately 58 kDa and a glycan moiety of approximately 8 kDa containing N‐linked terminal mannose. In size‐exclusion chromatography, the 66‐kDa protein was coeluted with the polysaccharide and could be separated from the polysaccharide only after denaturation of the protein, indicating that the 66‐kDa protein was tightly bound to the polysaccharide. Western blot analysis revealed that the 66‐kDa protein was specific to Porphyridium sp. and P. cruentum, because it was not detected in the other species of red microalgae examined. Indirect immunofluorescence assay confirmed the location of the protein in the algal cell wall. The sequence of cDNA clone encoding the 66‐kDa glycoprotein, detected in our in‐house expressed sequence tag database of Porphyridium sp., revealed that this is a novel protein with no similarity to any protein in the public domain databases and our in‐house expressed sequence tag database of the red microalga Rhodella reticulata. The 66‐kDa protein bound polysaccharides from red algae but not from those of other origins tested. Possible roles of the 66‐kDa protein in the biosynthesis of the polysaccharide are discussed.     

Control of starch and exocellular polysaccharides biosynthesis by gibberellic acid with cells of sweet potato cultured in vitro

The regulation of starch synthesis and exocellular polysaccharide synthesis by GA₃ was studied with cells of sweet potato grown as suspension in glycerol medium. In the presence of GA₃, and under normal cell growth, starch formation was inhibited. The incorporation activity (starch synthesis) from ADP-[¹⁴C] glucose or UDP-[¹⁴C] glucose with GA₃ treated cells was reduced. On the other hand, the synthesis of exocellular polysaccharides composed of glucose, galactose, mannose and arabinose etc., was stimulated and a clear increase of the Man/Ara ratio was observed in the presence of GA₃. These results may indicate that GA₃ affects the regulation of starch synthesis and exocellular polysaccharide synthesis.     

Extracellular polysaccharide production in outdoor mass cultures of Porphyridium sp. in flat plate glass reactors

This work concerns an attempt to develop large scalecultivation of Porphyridium sp. outdoors. Theimpact on cell growth and production of solublesulphated polysaccharides of light-path length (LP)was studied in flat plate glass reactors outdoors. TheLP of the plate reactors ranged from 1.3–30 cm,corresponding to culture volumes of 3–72 L. The sidewalls of all reactors were covered, ensuring similarilluminated surfaces for all reactors. Maximal daytemperature was maintained at 26 ±1 ^°C.Growth conditions of pH (7.5), stirring (withcompressed air) and mineral nutrients, were optimal.Maximal volumetric concentration of the soluble sulfated polysaccharide (1.32 g L^-1) was obtained in winter with the smallest light-pathreactor (1.3 cm ) at a cell density of 1.37 ×10¹¹cells L^-1. Under these conditions, theviscosity of the culture medium was also highest,being inversely proportional to the culture'slight-path. Highest areal concentration of solublepolysaccharides (60 g m^-2) and areal cell density(3.01 × 10¹²m^-2) was recorded in the 20 cmLP reactor, progressively lower values being obtainedas the light path became shorter. A similar patternwas obtained for the areal productivity ofpolysaccharides, the highest being 4.15 g m^-2day^-1 (considering the total illuminated reactorsurface), produced in the 20-cm LP reactor.The main sugar composition (i.e. xylose, galactose andglucose) of the sulfated polysaccharides was similarin all reactors. As viscosity increased with timeduring culture growth, there was a substantial declinein bacterial population. Cultivation throughout mostof the year provided good evidence that a light pathlength of 20 cm in flat plate reactors under theseconditions is optimal for maximal areal solublepolysaccharide production of Porphyridium sp.     

Antioxidant activity of the polysaccharide of the red microalga <Emphasis Type="Italic">Porphyridium</Emphasis> sp

The cells of the red microalga Porphyridium UTEX 637 are encapsulated within a sulfated polysaccharide whose external part (i.e., the soluble fraction) dissolves into the medium. It is thought that the main function of the polysaccharide is to protect the algal cells from the extreme environmental conditions, such as drought and high light, prevailing in their native sea-sand habitat. In this study, we evaluated the antioxidant properties of the water-soluble polysaccharide of Porphyridium sp. by determining the ability of a polysaccharide solution to inhibit: (1) autooxidation of linoleic acid, as determined by the standard thiobarbituric acid (TBA) and ferrous oxidation (FOX) assays; and (2) oxidative damage to 3T3 cells as determined by the dichlorofluorescein (DCFH) assay. In all three assays, the polysaccharide inhibited oxidative damage in a dose-dependent manner. Antioxidant activity was also exhibited by fractions of the polysaccharide obtained by sonication followed by separation on a reverse-phase HPLC with a C₈ semi-preparative column. It is suggested that the antioxidant activity of the sulfated polysaccharide protects the alga against reactive oxygen species produced under high solar irradiation, possibly by scavenging the free radicals produced in the cell under stress conditions and transporting them from the cell to the medium.     

NDP-sugars, floridoside and floridean starch levels in relation to activities of UDP-glucose pyrophosphorylase and UDP-glucose-4-epimerase in Solieria chorda l is (Rhodophyceae) under experimental conditions

Under limited nutrient availability (i.e. unenriched sea‐water) and under 75 mol photons m^–2 s^–1 irradiance 12:12 LD, thalli of Solieria chordalis J. Agardh accumulated floridean starch and floridoside. When they were transferred into nutrient‐enriched seawater (150 umol L^?1 NO3¹‐ and 7 umol L^?1 P0₄³i at 35 umol photons m^?2 s^?1 in irradiance 12:12 LD, starch and floridoside levels decreased. The main nucleotide diphosphate (NDP) sugars (i.e. UDP‐glucose, UDP‐galactose and ADP‐glucose) and the activities of UDP‐glucose pyrophosphorylase [Enzyme Code (EC) 2.7.7.9] and UDP‐glucose‐4‐epimerase (EC 5.1.3.2) were measured under these controlled culture conditions. Both UDP‐glucose and UDP‐galactose in the thal l i increased under conditions known to favor the accumulation of floridean starch and floridoside, whereas they decreased under conditions leading to floridean starch and floridoside breakdown. On the other hand, ADP‐glucose level only varied slightly. Although UDP‐glucose pyrophosphorylase activity rose under conditions of floridean starch synthesis, little variation was observed in UDP‐glucose‐4‐epimerase activity. These results suggest a possible enzymatic regulation of the NDP‐sugar and carbohydrate pool in which UDP‐glucose pyrophosphorylase would play a major role.     

LOW MOLECULAR WEIGHT CARBOHYDRATES OF THE BANGIOPHYCIDAE (RHODOPHYTA)1

In the order Porphyridiales there are three clades based on molecular evidence. These show parallels with the low molecular weight carbohydrate (LMWCs) in different genera. Clade Porphyridiales 1 includes Dixoniella, Glaucosphaera, Rhodella, and one undescribed genus (3987) that all contain mannitol. Clade Porphyridiales 2 comprises taxa of the Stylonematales Rhodosorus and Stylonema species and contains digeneaside and sorbitol, whereas Chroodactylon has only sorbitol. In clade Porphyridiales 3 Flintiella, Porphyridium, and the undescribed genus (3797) all possess only floridoside. In the Erythropeltidales Rhodochaete contains floridoside and digeneaside, Erythrotrichia species contain only floridoside, Sahlingia subintegra has floridoside and traces of D‐floridoside, and Smithora has L‐isofloridoside plus floridoside. In the Compsopogonales Boldia and Compsopogon have only floridoside. Within these genera as presently circumscribed, the LMWCs appear to be a reliable character to supplement the usual cytological characters.     

Antiviral effect of red microalgal polysaccharides on Herpes simplex and Varicella zoster viruses

The cell-wall sulphated polysaccharide of the red microalga Porphyridium sp. has impressive antiviral activity against Herpessimplex viruses types 1 and 2 (HSV 1, 2) and Varicella zoster virus(VZV). Treatment of cells with 1 g mL^-1 polysaccharideresulted in 50% inhibition of HSV-infection as measured by the plaqueassay. Inhibition of the production of new virus particles was also shownwhen pre-infected cell cultures were treated with the polysaccharide. Inaddition, there was indirect evidence for a strong interaction between thepolysaccharide and HSV and a weak interaction with the cell surface.Depending on the concentration, the polysaccharide completely inhibitedor slowed down the development of the cytopathic effect in HSV or VZVpreinfected cells, but did not show any cytotoxic effects on Vero cells evenwhen a concentration as high as 250 g mL^-1 was used. Itseems therefore that the polysaccharide is able to inhibit viral infection bypreventing adsorption of virus into the host cells and/or by inhibiting theproduction of new viral particles inside the host cells. Thus, this alga seems tobe a good candidate for the development of an antiviral drug.     

In vitro biosynthesis of 1,4-β-galactan attached to rhamnogalacturonan I

 The biosynthesis of galactan was investigated using microsomal membranes isolated from suspension-cultured cells of potato (Solanum tuberosum L. var. AZY). Incubation of the microsomal membranes in the presence of UDP-[¹⁴C]galactose resulted in a radioactive product insoluble in 70% methanol. The product released only [¹⁴C]galactose upon acid hydrolysis. Treatment of the product with Aspergillus niger endo-1,4-β-galactanase released 65–70% of the radioactivity to a 70%-methanol-soluble fraction. To a minor extent, [¹⁴C]galactose was also incorporated into proteins, however these galactoproteins were not a substrate for Aspergillus niger endo-1,4-β-galactanase. Thus, the majority of the ¹⁴C-labelled product was 1,4-β-galactan. Compounds released by the endo-1,4-β-galactanase treatment were mainly [¹⁴C]galactose and [¹⁴C]galactobiose, indicating that the synthesized 1,4-β-galactan was longer than a trimer. In vitro synthesis of 1,4-β-galactan was most active with 6-d-old cells, which are in the middle of the linear growth phase. The optimal synthesis occurred at pH 6.0 in the presence of 7.5 mM Mn²⁺. Aspergillus aculeatus rhamnogalacturonase A digested at least 50% of the labelled product to smaller fragments of approx. 14 kDa, suggesting that the synthesized [¹⁴C]galactan was attached to the endogenous rhamnogalacturonan I. When rhamnogalacturonase A digests of the labelled product were subsequently treated with endo-1,4-β-galactanase, radioactivity was not only found as [¹⁴C]galactose or [¹⁴C]galactobiose but also as larger fragments. The larger fragments were likely the [¹⁴C]galactose or [¹⁴C]galactobiose still attached to the rhamnogalacturonan backbone since treatment with β-galactosidase together with endo-1,4-β-galactanase digested all radioactivity to the fraction eluting as [¹⁴C]galactose. The data indicate that the majority of the [¹⁴C]galactan was attached directly to the rhamnose residues in rhamnogalacturonan I. Thus, isolated microsomal membranes contain enzyme activities to both initiate and elongate 1,4-β-galactan sidechains in the endogenous pectic rhamnogalacturonan I. Received: 24 June 1999 / Accepted: 30 August 1999     

CARBON SOURCE DEPENDENCE OF THE RATIO OF δ‐AMINOLEVULINIC ACID BIOSYNTHESIS VIA THE C5 AND SHEMIN PATHWAYS IN EUGLENA GRACILIS (EUGLENOPHYCEAE)1

The ratio of two biosynthetic pathways was estimated, the C5 and Shemin pathways, to δ‐aminolevulinic acid (ALA, a biosynthetic intermediate of tetrapyrrole) from the ¹³C‐enrichment ratios (¹³C‐ER) at the carbon atoms of chl a (after conversion to methyl pheophorbide a) biosynthesized by Euglena gracilis G. A. Klebs when l ‐[3‐¹³C]alanine was used as a carbon source. On the basis of these estimations, we confirmed that ALA was efficiently biosynthesized via both the C5 and Shemin pathways in the plastids of E. gracilis, and we determined that the ratio of ALA biosynthesis via the Shemin pathway was increased in the ratio of 14%–67%, compared with that in our previous d ‐[1‐¹³C]glucose feeding experiment ( Iida et al. 2002 ). This carbon source dependence of the contributions of the two biosynthetic pathways might be related to activation of gluconeogenesis by the amino acid substrate. The methoxy carbon of the methoxycarbonyl group at C‐13² of chl a was labeled with the ¹³C‐carbon of l ‐[methyl‐¹³C]methionine derived from l ‐[3‐¹³C]alanine via [2‐¹³C]acetyl coenzyme A (CoA), through the atypical tricarboxylic acid (TCA) cycle, gluconeogenesis, and l‐ [3‐¹³C]serine. The phytyl moiety of chl a was also labeled on C‐P2, C‐P3¹, C‐P4, C‐P6, C‐P7¹, C‐P8, C‐P10, C‐P11¹, C‐P12, C‐P14, C‐P15¹, and C‐P16 from ¹³C‐isoprene (2‐[1,2‐methyl,3‐¹³C₃]methyl‐1,3‐butadiene) generated from l ‐[3‐¹³C]alanine via [2‐¹³C]acetyl CoA.     

Isolation of a Bacterium Capable of Degrading Peanut Hull Lignin

Thirty-seven bacterial strains capable of degrading peanut hull lignin were isolated by using four types of lignin preparations and hot-water-extracted peanut hulls. One of the isolates, tentatively identified as Arthrobacter sp., was capable of utilizing all four lignin preparations as well as extracted peanut hulls as a sole source of carbon. The bacterium was also capable of degrading specifically labeled [¹⁴C]lignin-labeled lignocellulose and [¹⁴C]cellulose-labeled lignocellulose from the cordgrass Spartina alterniflora and could also degrade [¹⁴C]Kraft lignin from slash pine. After 10 days of incubation with [¹⁴C]cellulose-labeled lignocellulose or [¹⁴C]lignin-labeled lignocellulose from S. alterniflora, the bacterium mineralized 6.5% of the polysaccharide component and 2.9% of the lignin component.     

Incorporation of D-[14C]galactose into organelle glycoprotein in castor bean endosperm

Excised casto bean (Ricinus communis L.) endosperm tissue supplied with [¹⁴C]galactose incorporates radioactivity into particulate cell components. Fractionation of homogenates established that ¹⁴C-labeled trichloroacetic acid-insoluble material was located primarily in the microsomal and glyoxysomal fractions. The capacity of the tissue to incorporate [¹⁴C]galactose into organelle glycoprotein varied during seedling development, increasing during the first 3 days of germination and subsequently declining. The kinetics of incorporation into the major organelle fractions of 2-day old endosperm tissue showed that the endoplasmic reticulum was immediately labeled whereas a lag period preceded the labeling of glyoxysomes. Sub-fractionation of the isolated organelles established that the greatest proportion of the [¹⁴C]-galactose labeled glycoprotein was located in the membrane, although a significant incorporation into the matrix protein was also observed.The results indicate that the addition of the carbohydrate moiety to the polypeptide cores occurs in the endoplasmic reticulum during or immediately after their synthesis on membrane-bound ribosomes.Abbreviations ER endoplasmic reticulum - SDS sodium dodecyl sulphate - TCA trichloroacetic acid     

Lipid synthesis in the laticifers of Euphorbia lathyris L. seedlings

Various solutions of labeled precursors were absorbed by the cotyledons of etiolated Euphorbia lathyris L. seedlings. Incorporation of ¹⁴C into triterpenes from [2-¹⁴C]mevalonic acid, [1-¹⁴C]acetate, [3-¹⁴C]pyruvate, [U-¹⁴C]glyoxylate, [U-¹⁴C]glycerol, [U-¹⁴C]serine, [U-¹⁴C]xylose, [U-¹⁴C]glucose, and [U-¹⁴C]sucrose was obtained. The [¹⁴] triterpenes synthesized from [¹⁴C] sugars were mainly of latex origin. [¹⁴C]mevalonic acid was only involved in terpenoid synthesis outside the laticifers. Exogenously supplied glyoxylate, serine, and glycerol were hardly involved in lipid synthesis at all. The ¹⁴C-distribution over the various triterpenols was consistent with the mass distribution of these constituents in gas liquid chromatography when [¹⁴C]sugars, [¹⁴C]acetate, and [¹⁴C]pyruvate were used. These precursors were supplied to the seedlings in the presence of increasing amounts of unlabeled substrates. The amount of substrate directly involved in lipid synthesis as well as the absolute triterpenol yield was calculated from the obtained [¹⁴C]triterpenols. The highest yield was obtained in the sucrose incorporated seedlings, being 25% of the daily increase of latex triterpenes in growing seedlings.     

Lipid synthesis by oligodendrocytes from adult pig brain maintained in long-term culture

Oligodendrocytes were isolated from adult pig brain and cultivated for 18–24 days. [¹⁴C]acetate, [³H]galactose or [³⁵S]sulfate were added to the medium for an additional 24 h. Lipids were extracted and separated by high-performance thin-layer chromatography. The labeled lipids were studied by fluorography and scintillation counting. [¹⁴C]acetate was incorporated in decreasing order into neutral lipids, phosphatidylcholine, ethanolamine phosphatides, galactocerebrosides, phosphatidylinositol, phosphatidylserine, sulfatides and sphingomyelin. From the [¹⁴C]acetate incorporated into ethanolamine and choline phosphatides, 71.6 and 14.8%, respectively, were found in plasmalogens. Among neutral lipids, [¹⁴C]acetate labeled not only cholesterol but also large amounts of triglycerides. No cholesterol esters were synthesized. [³H]galactose primarily labeled galactocerebrosides, sulfatides, and monogalactosyl diglyceride. [³⁵S]sulfate incorporation was restricted to sulfatides. Together with our previous results concerning proteins, these data show that: (1) oligodendrocytes remain highly differentiated in long-term cultures; (2) they are able to synthesize the major components of myelin; (3) they synthesize surprisingly high amounts of triglycerides and of monogalactosyl diglyceride, a marker for myelination.     

Protoplast isolation and culture from carob (Ceratonia siliqua) hypocotyls: ability of regenerated protoplasts to produce mannose-containing polysaccharides

The aim of this study was to isolate protoplasts from carob (Ceratonia siliqua L.) embryonic tissues with the ability to regenerate cell walls, divide and synthesize galactomannan, a valuable polysaccharide for industry. Protoplasts isolated from carob hypocotyl hooks regenerated cell walls within 24 h. The first divisions of the regenerated cells were observed after 2 days of culture. The highest percentage that successfully divided was achieved when the seedlings were grown under diffuse light, the hypocotyl hooks were plasmolysed for 1 h before incubation in the protoplast isolation solution and the protoplasts were cultured under diffuse light. After 9 days of culture, cell clusters, consisting of eight cells, had been produced, which underwent further mitotic divisions and which were expected to lead to callus formation. Polysaccharide and oligosaccharide synthesis during protoplast regeneration was studied by radiolabelling with exogenous d ‐[U‐¹⁴C]glucose, d ‐[U‐¹⁴C]mannose or d ‐[2‐³H]mannose, which gave rise to uniform, moderately specific and highly specific labelling, respectively. As revealed by the radioactivity distribution in cell wall monosaccharides, the regenerants deposited new wall polymers that differed markedly from those being synthesized by the hypocotyls from which the protoplasts had been isolated. The regenerants deposited large amounts of callose and smaller amounts of galactose‐, arabinose‐ and mannose‐containing polymers. The latter included glucuronomannan, as demonstrated by a new method involving partial acid hydrolysis followed by β‐glucuronidase (EC 3.2.1.31) digestion. The regenerating protoplasts also released soluble extracellular carbohydrates: polysaccharides which appeared to be mainly acidic arabinogalactans, and oligosaccharides which were mainly neutral and contained glucose, galactose and mannose. We conclude that regenerating carob protoplasts are a useful system for studying carbohydrate secretion, including mannose‐rich poly‐ and oligosaccharides.     

Protein-bound oligosaccharides of Semliki Forest virus

Semliki Forest virus was grown in BHK cells and labeled in vivo with radio-active monosaccharides. promnase digenst of the virus chromatographer on Bio-Gel P 6 revealed glycopeptides of A-type and B-type. (For the nomenclature see Johnson J. and Clamp J.R. (1971) Biochem. J. 123, 739–745) The former was labeled with [³H]fucose, [³H]galactose, [³H]mannose and [¹⁴C]glucosamine, the latter only with [³H]mannose and [¹⁴C]glucosamine. The three envelope glycoproteins E₁, E₂ and E₃ were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to pronase digestion. The glycoproteins E₁ and E₃ revealed glycopeptides of A-type. E₂ revealed glycopeptides of B-type. E₂ yielded additionally a glycopeptide (M_r3100) which was heavily labeled from [³H]galactose, but only marginally from [¹⁴C]glucosamine, [³H]fucose and [³H]mannose. Wether this glycopeptide belongs to the A-type or not remains uncertain. The apparent molecular weights of the A-type units measured by gel filtration were 3400 in E₁ and 4000 in E₃; the B-type unit of E₂ had an apparent molecular weight of 2000. Combined with the findings of our earlier chemical analysis these data suggast that E₁ and E₃ contain on the average one A-type unit; E₂ probably contains one 3100 dalton unit plus one or two B-type units.     

The temperature-mediated metabolism of 1-acyl-lysophosphatidyl glycerol in cerulenin-treated cultures of Bacillus megaterium.

When [U-¹⁴C]palmitate was added to a culture of $\text{B. megaterium}$ that had been grown at 35°, transferred to 20° and treated with cerulenin, label was initially incorporated into lysophosphatidyl glycerol. The labeled lyso derivative, in turn, was converted to phosphatidyl glycerol, apparently by esterification of the 2-position with endogenous acyl groups. Labeled lysophosphatidyl glycerol synthesis at 20° was observed only when a culture was treated with cerulenin prior to the addition of [U-¹⁴C]palmitate. When [U-¹⁴C]palmitate was added before cerulenin, labeled lysophosphatidyl glycerol formation was not detected. When chloramphenicol was added with cerulenin at the time of culture transfer from 35° to 20°, the synthesis of lysophosphatidyl glycerol was unaffected but the rate of its esterification to phosphatidyl glycerol was significantly retarded. Transfer of such a culture back to 35° resulted in a marked acceleration in the rate of conversion of lysophosphatidyl glycerol to phosphatidyl glycerol.