Studies on the Cell Wall of Chlorella V. Comparison of the Cell Wall Chemical Compositions in Strains of Chlorella ellipsoidea

Cell walls of four strains of Chlorella ellipsoidea (IAM C-27,C-87, C-102 and C-183) were compared as to their chemical compositions.Many differences were found: (1) The sugar composition of alkali-soluble cell walls differedin quantity as well as quality with glucuronic acid being foundonly in C-27 and C-87. (2) In alkali-insoluble cell walls glucosamine was found onlyin C-27. The other three strains contained mainly glucose. (3) The amino acid compositions of the alkali-insoluble cellwalls markedly differed among the four strains. The cell wallof C-102 contained more amino acids than carbohydrates, butC-27 and C-87 contained extremely little amino acid. In addition to the variation in cell wall composition, the opticalanisotropy findings also differed for these cell walls of Chlorellastrains which had been grouped as the same species. (Received August 16, 1983; Accepted December 27, 1983)     

Isolation and Characterization of Hardening-Induced Proteins in Chlorella vulgaris C-27: Identification of Late Embryogenesis Abundant Proteins

Hardening-induced soluble proteins of Chlorella vulgaris BeijerinkIAM C-27 (formerly Chlorella ellipsoidea Gerneck IAM C-27) wereisolated and purified by two-dimensional high-performance liquidchromatography (2D-HPLC) on an anion-exchange column, with subsequentreversed-phase chromatography. Some of the proteins were resolvedby SDS-PAGE, characterized by amino-terminal sequencing andidentified by searching for homologies in databases. Separationof the soluble proteins during the hardening of Chlorella bya combination of 2D-HPLC and SDS-PAGE revealed that at least31 proteins were induced or increased in abundance. Of particularinterest was the induction after 12 h of a 10-kDa protein withthe amino-terminal amino acid sequence AGNKPITEQISDAVGAAGQKVGand the induction after 6 h of a 14-kDa protein with the amino-terminalsequence ALGEESLGDKAKNAFEDAKDAVKDAAGNVKEAV. The amino-terminalsequences of these proteins indicated that they were homologousto late embryogenesis abundant (LEA) proteins. Furthermore,the level of a 22-kDa protein also increased after 12 h. Theamino-terminal sequence of this protein, AAPLVGGPAPDFTAAAVFD,indicated that it was homologous to thioredoxin peroxidase. (Received June 9, 1995; Accepted September 12, 1995)     

Growth Delay and Intracellular Changes in Chlorella ellipsoidea C-27 as a Result of Deuteration

The effects of deuterium (D) on Chlorella ellipsoidea C-27 wereinvestigated. Cells grown in a medium prepared with deuteriumoxide (D₂O) showed pronounced delays in cell growth and division;the length of a cell cycle in medium with 100 mol% D₂O was morethan 5 times longer than that in medium prepared in H₂O Thedelay caused by D₂O was not overcome by either indoleaceticacid or kinetin. The biological and ultrastractural characteristicsof deuterated .Chlorella (D-Chlorella) cells were examined.The responses of D-Chlorella to cell wall-digesting enzymesdid not differ from those of normal (H-Chlorella) cells. D-Chlorellacells were enlarged, and cellular components, such as proteins,nucleic acids, lipids and ATP, were present in larger quantitiesthan those in H-cells. The chloroplast of D-Chlorella was enlarged,but the levels of component photosynthetic pigments were significantlyreduced. By contrast, mitochondria of D-Chlorella were smallerthan those of H-cells. These changes in levels of cellular componentsand in the sizes of organelles seem to be unique to deuteration. (Received May 13, 1992; Accepted July 28, 1992)     

Differential Expression of an Auxin-Regulated Gene, parC, and a Novel Related Gene, C-7, from Tobacco Mesophyll Protoplasts in Response to External Stimuli and in Plant Tissues

As part of an extended search for auxin-regulated genes otherthan parA and parB in the cDNA library from cultured tobaccomesophyll protoplasts, we have isolated the cDNA for a genedesignated parC. This gene is expressed during the transitionfrom the G₀ to S phase of the cell cycle. The nucleotide sequenceof parC cDNA was similar to that of parA. Using the parC cDNAas a probe we have isolated cDNA for a gene designated C-7 byhybridization at reduced stringency. Even though C-7 is relatedto parC, as is parA, its mode of expression was, to our surprise,completely different from that of parC. The C-7 gene is predominantlyexpressed in mature leaves and in freshly prepared protoplasts,but its level of expression did not change in response to auxintreatment. Although parC and C-7 are related to one anotherand exhibit homology to the parA gene, the two former genesdemonstrated conspicuous differences in their responses to externalstimuli, which included auxin, as well as their tissue-specificexpression. These results provide us an interesting system forthe analysis of the differential expression of closely relatedgenes. The significance of our observations is discussed withreference to the other members of the family of parA genes. (Received May 8, 1992; Accepted June 17, 1992)     

Enzymatic Inactivation of Extracellular Carbonic Anhydrase and its Effect on K1/2 (CO2) for Photosynthesis in Chlorella ellipsoidea C-27

The ratio of the extracellular to the intracellular activityof carbonic anhydrase (CA) in cells of Chlorella ellipsoideaC-27, adapted to low levels of CO₂ for 24 h (low-CO₂ cells),was about one to one. Treatment of intact cells with PronaseP inactivated about one-half of the extracellular CA activitywithout affecting photosynthetic activity. The CA activity incell homogenates and in cell-wall ghosts liberated during celldivision was completely inactivated by the same treatment. Pretreatmentwith Glycosidase mix, Chitosanase and Macerozyme enhanced theinactivation of the CA activity in intact cells. These resultssuggest that extracellular CA is evenly distributed throughoutthe whole cell-wall region. The apparent K_1/2 for dissolved inorganic carbon (DIC) in low-CO₂cells doubled when extracellular CA was inactivated by treatmentwith Pronase P, but the K_1/2 obtained was still one-half ofthat in high-CO₂ cells. Photosynthetic ¹⁴CO₂-fixation in low-CO₂cells was enhanced by acetazolamide, whereas H¹⁴CO₃^–-fixationwas suppressed. The results suggest that CO₂ is a dominant substrateutilized by cells and that HCO₃^– is utilized after conversionto CO₂. The present results show that both intracellular andextracellular CA contribute to the increase in affinity forDIC during photosynthesis in low-CO₂ cells of Chlorella ellipsoideaC-27. (Received May 7, 1990; Accepted July 18, 1990)     

The oxidation of C-550 by exogenously added oxidants in the presence of dichlorophenyldimethylurea: The localization of the primary electron acceptor of Photosystem II in the thylakoid membrane

The oxidation of C-550 by exogenously added oxidants in spinachchloroplasts and digitonin-treated chloroplasts was studiedin the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea inan attempt to elucidate localization of the primary electronacceptor of Photosystem II in the thylakoid membrane. C-550was directly oxidized by various oxidants such as ferricyanide,N-methylphenazonium methosulfate (PMS) and quinones with redoxpotentials higher than that of C-550. Among the oxidants used,dibromothymoquinone was the most effective oxidant of C-550,followed by PMS. In spite of its high redox potential, ferricyanidewas rather a poor oxidant. The rates of C-550 oxidation by quinoneswere linearly proportional to the oxidant concentration, whereasthe rates tapered off with increasing concentrations of polaroxidants within the ranges of concentration used. These resultsindicate that C-550 is located inside the thylakoid membraneand is covered by a lipophilic shield. Addition of cations,especially divalent cations, significantly affected C-550 oxidationby ferricyanide or PMS but not by nonpolar oxidants. C-550 oxidationby ferricyanide was accelerated at low pH. Thus the accessibilityof C-550 to polar oxidants appears to be affected by electrostaticinteractions between oxidant ions and the negative charges ofthe thylakoid surface. When electrostatic interaction was minimized,ferricyanide oxidized C-550 as rapidly as several quinones did.This suggests that C-550 is located close to the surface ofthe membrane. The evidence indicates that the rates of C-550oxidation depend not only on the accessibility of C-550 to addedoxidants, but also on the reactivity between them. Reduction of cytochrome f by added reductants shows featuressimilar to those of C-550 oxidation by added oxidants, indicatingthat properties of the shield covering cytochrome f are similarto those of the shield covering C-550. (Received March 8, 1977; )     

Ethylene evolution in marine algae and a proteinaceous inhibitor of ethylene biosynthesis from red alga

Fronds of marine algae, especially green alga, Codium latum,and red alga, Porphyra tenera, evolved a quantity of ethylenewhen IAA was exogenously applied, while brown alga, Padina arborescens,evolved only a little. Propionic acid, when added together withIAA, noticeably enhanced IAA-induced ethylene evolution in P.tenera and P. arborescens. This evolution was also enhancedby added acrylic acid in P. arborescens but not in P. tenera.It was promoted by methionine, though only at a high concentration(0.1 M), in P. tenera but not in P. arborescens. The rate ofethylene evolution was highest at 12?C among the incubationtemperatures tested of 5, 12 and 15?C. The conversion of ¹⁴C-3-methionineto radioactive ethylene in P. tenera was remarkably inhibitedby a proteinaceous inhibitor from P. tenera. ¹Present address: Division of Environmental Biology, NationalInstitute for Environment, Yatabe, Ibaraki, Japan. (Received May 27, 1976; )     

Buckminsterfullerene (C-60 Carbon Allotrope) Inhibits Ethylene Evolution from 1-Aminocyclopropane-1-carboxylic acid (ACC)-treated Shoots of Pea (Pisum sativum), Broadbean (Vicia faba) and Flowers of Carnation (Dianthus caryophyllus)

When applied either in the form of a colloidal solution or inliposomes, buckyballs (C-60—buckminsterfullerene) markedlyreduced ethylene evolution from cut carnation (Dianthus caryophyllus)flowers, as well as pea (Pisum sativum) and broadbean (Viciafaba) foliage treated with ethylene precursor l-aminocyclopropane-l-carboxylicacid (ACC). The liposome preparation was approximately twiceas effective as colloidal solutions. Moreover, upon being incubatedin a closed atmosphere with ethylene, buckyballs induced a significantdepletion of ambient ethylene which was temperature and C-60—concentrationdependent. This mode of C-60 action is attributed to ethyleneadsorption stemming from the vast C-60 surface area, calculatedto be 1317 m² g_-1, and the affinity of its carbon atoms forthe component in the ethylene double bond.Copyright 1993, 1999Academic Press Dainthus caryophyllus, Pisum sativum, Vicia faba, adsorption, ethylene, fullerene     

Characteristics of the Photosynthetic Metabolism of Carbon in Deuterated Chlorella ellipsoidea C-27

The photosynthetic metabolism of carbon in fully deuteratedcells of Chlorella ellipsoidea C-27 (D-Chlorella), obtainedby culture in medium prepared with 100 mol% D₂O, was characterizedby examining the activities of several enzymes and the levelsof metabolic regulators in a comparison with those of ordinarycells (H-Chlorella). The cellular content of starch in D-Chlorellawas more than twice that in H-Chlorella, whereas those of sucroseand glucose were significantly lower in D-Chlorella. Deuterationof Chlorella caused marked alterations in the activities ofenzymes involved in starch metabolism. There was a significantdecrease in the activity of phosphorylase, a catabolic enzyme,and a significant increase in the activity of starch synthase,an anabolic enzyme. These alterations are probably responsiblefor the increase in the amount of starch in cells. By contrast,no marked changes were observed in the activities of enzymesand the levels of metabolic inhibitors that are involved inthe synthesis of sucrose. It seems likely, therefore, that thedecrease in the amount of sucrose in D-Chlorella was causedmainly by a deficiency in sources of carbon in the cytoplasm,as a consequence of an increase in levels of starch in chloroplasts. (Received May 13, 1992; Accepted December 1, 1992)     

Tritium labelling of amino sugars at C-2 by alkaline epimerization in tritiated water

N-Acetyl-D-[2-³H]glucosamine was synthesized from N-acetyl-D-mannosamineby alkaline 2-epimerization in pyridine containing ³H₂O andnickelous acetate. The reaction involves reversible formationof an enol intermediate and therefore also resulted in incorporationof tritium into N-acetylmannosamine. After completed reaction,the two N-acetylhexosamines were separated from other radioactiveproducts and Morgan-Elson chromogens by chromatography on acolumn of Sephadex G-10, which was eluted with 10% ethanol,and were then separated from each other by chromatography onSephadex G-15 in 0·27 M sodium borate (pH 7·8).The location of the incorporated tritium was established bytreatment of the N-acetylhexosamines with borate under the conditionsof the Morgan-Elson reaction, which converts the sugars to Kuhn'schromogen I with concomitant loss of the C-2 hydrogen. As expected,this treatment resulted in the formation of ³H₂O, indicatingthat the tritium was located at C-2. [2-³H]Glucosamine was preparedby acid hydrolysis of the labelled N-acetylglucosamine and wasconverted to [2-³H]glucosamine 6-phosphate by incubation withhexokinase and ATP. The sugar phosphate was used as a substratefor glucosamine 6-phosphate deaminase (isomerase, EC 5.3.1.10 [EC] )in a simple ³H₂O release assay. N-acetyl[2-³H]glucosamine N-acetyl[2-³H]mannosamine [2-³H]glucosamine glucosamine 6-phosphate deaminase [2-³H]mannosamine     

ERRATA

Page 678, line 3, for [4-¹⁴C] read [I-¹⁴C] Page 678, line 4, for [I-¹⁴C] read [4-¹⁴C] Page 679, line 17, for C-I of malate read C-4 of malate Page 679, line 18, for C-4 of malate read C-I of malate     

Enzymatic Conversion of Pheophorbide a to the Precursor of Pyropheophorbide a in Leaves of Chenopodium album

Soluble proteins extracted from leaves of Chenopodium albumcatalyzed the conversion of pheophorbide a to a precursor ofpyropheophorbide a, putatively identified as C-13²-carboxyl-pyropheophorbidea. The precursor was then decarboxylated non-enzymatically toyield pyropheophorbide a. Soluble proteins and pheophorbidea, as the substrate, were required for the formation of theprecursor, and boiled proteins were enzymatically inactive.The maximum rate of conversion of pheophorbide a to the precursoroccurred at pH 7.5. The K_m for pheophorbide a was 12.5 µMat pH 7.0. Both pheophorbide b and bacteriopheophorbide a couldserve as substrates, but protopheophorbide a could not. Formationof methanol was detected during the enzymatic reaction, an indicationthat the enzyme is an esterase. Among seven alcohol analogstested, only methanol inhibited the enzymatic activity uncompetitively,with a K₁ of 71.6 mM. Mass-spectrometric (MS) analysis of theprecursor yield a peak at m/z 579 that indicated the releaseof a methyl group from pheophorbide a. It appears thereforethat the enzyme catalyzes the demethylation of the carbomethoxygroup at C-13² of pheophorbide a by hydrolysis to yield methanoland the precursor, C-13²-carboxyl-pyropheophorbide a, whichis converted to pyropheophorbide a by spontaneous decarboxylation.We have tentatively designated the enzyme "pheophorbidase".The presence of the enzyme was dependent on plant species andit was expressed constitutively. ¹Present address: Faculty of Science, Shizuoka University, Ohya,Shizuoka, 422 Japan     

Studies on nucleic acids in plastids of the pigment mutant C-2A{acute} of Scenedesmus obliquus

Pigment mutant C-2A{acute} of Scenedesmus obliquus whose chlorophyllformation and chloroplast development are light dependent, wasstudied for the nucleic acid content of its plastids. The ribosomalRNA of plastids of the achlorophyllous or greened mutant C-2A{acute},did not show any difference from that of the wild type. Incorporationof [5-³H] uridine into mutant cells was partially inhibitedby rifampicin, indicating this part as being plastidial incorporation.Since there were no significant differences in the ribosomalRNA of plastids between the mutant and the wild type of Scenedesmus,the ribosomal system in the plastids of mutant C-2A' seemednot to be affected by the mutation. C_sCl gradient patterns ofScenedesmus mutant and wild-type DNA were almost identical withthose of Chlorella DNA. A peak at a buoyant density of 1.69g/cm³, the same as that of Chlorella chloroplast DNA, couldbe identified in Scenedesmus also as plastid DNA because itdisappeared after prolonged treatment with myxin and hybridizedwith rifampicin-sensitive pulse-labelled RNA. This peak waspresent to nearly the same degree in the mutant and the wildtype, indicating that a larger deficit of plastid DNA did notoccur in the mutant. Whether or not the mutation might be localizedin the plastid genome is discussed. (Received March 19, 1976; )     

Further Evidence For Cell Wall Deposition During Graft Formation

Cells of Sedum telephoides undergoing lethal cellular senescencein response to grafting with Solanum pennellii often possessplasmalemmal tubules (PT). The PT are unbranched, 27–31nm in diameter, and are bordered by a wall measuring 4.8–5.2nm in thickness. No regular substructure is discernible in thelumen of the PT. At 36 h after grafting PT extend into the cytoplasmof Sedum cells at the graft interface between Sedum and Solanum.By 10 days after grafting, however, PT are found embedded innewly deposited cell wall. These results indicate that cellwall deposition occurs during the early stages of graft development. grafting, plasmalemmal tubules, cell wall, Sedum telephoides, Solanum pennellii     

The interactions between nucleic acids and polyamines: I. High resolution carbon-13 and hydrogen-1 nuclear magnetic resonance studies of spermidine and 5'-AMP

In 0.5 M solution at pH 7.6, interaction of spermidine and 5'-AMP is demonstrated by downfield proton NMR shifts. Shifts of ribose and adenine protons support a model in which triprotonated spermidine engages the phosphate, anion with the C-3 diamine segment in a conformation to maximize interaction and the C-4 ammo segment extended to interact with adenine N-7 (base anti, 2'endo, g'g' and gg nucleoside conformation). Changes in carbon-13 chemical shifts for ribose C-5' (downfield), C-2' C-3', and C-4' (upfield) and for adenine C-6 and C-8 (upfield) support this model. In 0.006 M solution no significant changes in proton shifts and therefore no evidence for interaction was found. Spermidine and 5'-UMP (0.006 M) showed interaction at pH 10.5 (small upfield shifts in the proton nmr) interpreted as changing conformation by solvent interaction. In 0.006 M 3'-UMP at pH 10.5 small downfield proton shifts induced by spennidine are attributed to interactions with phosphate anion.     

Phased cell division in a natural population of Dinophysis sacculus and the in situ measurement of potential growth rate

A method for the in situ estimation of the potential growthrate of the dinoflagellate Dinophysis sacculus based on thepattern of cell division and the duration of the cell cyclephases according to the model of McDuff and Chisholm (Limnol.Oceanogr., 27, 783–788, 1982) is proposed. In order toobserve the cell cycle and measure the in situ growth rate ofD.sacculus, water samples were taken from Alfacs Bay in theEbro River Delta, Spain, in May and June 1994, and kept in thelaboratory under a light-dark cycle similar to that in the field.The pattern of cell division in the D.sacculus population wasestablished by intensive sampling over 24 h periods. The maximumfrequency of cells undergoing cytokinesis was observed at dawnand that of recently divided cells 2 h later. Based on the patternof cell division and the duration of the various cell cyclephases, a generation time between 2 and 5 days was estimated.Subsequently, in situ estimates of the growth rate for D.sacculuswere carried out in St Carles de la Ràpita harbour duringOctober 1995. A mean generation time of 7 days was estimated.     

HL 60 leukaemia cells chemically induced to differentiate retain some surface glycan features of undifferentiated cells not found in normal leukocytes

Human HL 60 myeloid leukaemia ells have the potential to differentiateinto either macrophage-like cells or granulocyte-like cellsunder the stimulus of chemical treatments. Using glycotechnologyprocedures, the glycosylation patterns of differentiated andundifferentiated HL 60 cells were analysed and compared withthose of normal human peripheral monocytes. Both in vitro differentiationsresult in significant morphologic and functional changes, butwe observed that the glycosylation patterns of undifferentiatedand differentiated HL 60 cells exhibit several common glycosidicfeatures that are absent in normal peripheral monocytes: thepresence of (i) bisecting ß-N-acetylglucosamine attachedat the C-4 position of the ß-mannose of polyantennarycomplex-type carbohydrate chains and (ii) complex-type carbohydratechains enriched with non-reducing terminal ß-N-acetylglucosamineresidues. Moreover, the three populations of HL 60 cells expresssmall amounts of biantemary complex-type structures (<6%),whereas normal peripheral monocytes expressed >20% of suchstructures. Thus, the cell glycosylation pattern could reflectthe pathological state of the HL 60 cells. differentiation glycosylation HL 60 cell monocytes     

Permeability to Water of the Fibre Cell Wall Material of Two Hardwoods

The permeability to water of the fibre cell wall of birch (Betulapubescens Ehrh.) and lime (Tilia x vulgaris Hayne) wood, wasmeasured in wood slices in which most of the void space wasfilled with paraffin wax or a polymerizing silicone elastomer.An osmotic technique was used with solutes of molecular weightgreat enough to prevent their molecules from penetrating thepores in the water-swollen cell wall. The solutes used weredextran and polyethylene glycol with molecular weights of 40000 and 6000 respectively. Values for permeability k x 10²¹, as defined by the Darcy equation,ranged between 3.6 m² for the tangential direction in birch,to 27 m² for the longitudinal direction in birch. These resultsare in good agreement with previously measured values but areat least ten times less than theoretical values. It is calculatedthat total emptying or filling of a wood cell through the wallmight be possible in as little as 5 min under a pressure differenceof 0.1 MPa if other flow pathways were blocked. Key words: Betula pubescens, Tilia x vulgaris, Water permeability, Fibre cell wall     

Monoclonal antibody LU-BCRU-G7 against a breast tumour-associated glycoprotein recognizes the disaccharide Gal{beta}1-3GlcNAc

The monoclonal antibody LU-BCRU-G7, that was generated by invitro immunization, shows clinical value as a prognostic markerin early stage breast carcinoma. It has now been characterizedwith regard to its binding epitope. Using a recently describedmethod based on the construction of N-substituted polyacrylamide(PAA) derivatives of carbohydrates (pseudopolysaccharides),the structure of the epitope for the monoclonal antibody LU-BCRU-G7has been determined. Competitive binding assays and inhibitoryenzyme-linked immunosorbent assays (ELISAs) using these pseudopolysaccharideshave shown the LU-BCRU-G7 epitope to be a disaccharide Galß1-3GlcNAc(Le^c; where Gal is D-galactose, Glc is D-glucose and GlcNAcis N-acetyl-D-glucosainine). Both galactose and N-acetyl glucosaminemoieties are essential for binding. Substitution on C-2 or C-3of the terminal galactose abolished binding, as did galactose-terminated oligosaccharides. The galactose moiety alone, asexpressed by the Galß-PAA conjugate, appeared to hea more important feature of the epitope than the GlcNAc-PAAconjugate, which failed to bind or inhibit the LU-BCRU-G7 antibody.In the N-acetyl glucosamine moiety, binding was decreased butnot eliminated by fucose substitution, as in Le^a, or changein configuration of C-4, as in Galß1-3GlcNAc. Omissionof the NAc group resulted in complete loss of activity. Thetetrasaccharide lacto-N-tetraose, although containing the terminalLe^c disaccharide, does not react with the antibody, suggestingconformational interference of the binding site. These findingsshow that the monoclonal antibody LU-BCRU-G7 recognizes a terminalisolactosamine fragment on a tumour-associated glycoprotein,which we have previously shown to be inversely related to survivalin breast cancer. breast cancer Galß1-3GlcNAc LU-BCRU-G7 monoclonal antibody pseudopolysaccharides