Sialic acid, electrophoretic mobility and transmembrane potentials of the Amphiuma red cell

Red cells from the giant salamander Amphiuma means are shown to contain sialic acid. The amount removed by the action of neuraminidase is equal to that released by acid hydrolysis, indicating that all of the sialic acid is present on the outer surface of the plasma membrane. These cells have a negative electrophoretic mobility and 100% enzymatic removal of sialic acid results in a 40% reduction in the mobility, suggesting that either a fraction of the sialic acid carboxyl groups are unavailable to the action of external electric fields, or other negatively charged groups contribute to the surface charge. A further reduction in mobility of normal and sialic acid-free cells is caused by an increased extracellular calcium concentration. The negative groups affected by calcium are most likely to be phosphate groups, since the isoelectric point of the cells is found to lie between the pK values for H₂PO₄⁻ groups and the carboxyl groups of sialic acid. Membrane potentials of single cells, from which 80% or more of the total sialic acid had been removed, were identical to those measured in normal cells, confirming that sialic acid plays little, if any, direct role in the maintenance of membrane potentials and ionic permeabilities.     

Changes in the glycoprotein composition of plasma membrane during the differentiation of friend erythroleukemia cells

Friend erythroleukemia cells display transient and permanent changes in the composition of their plasma membrane-bound glycoproteins during dimethyl sulfoxide-induced differentiation. The transient changes, as revealed by metabolic labeling with [¹⁴C]glucosamine, are most conspicuous around the time during which most cells become committed to terminal differentiation. Permanent changes are revealed by reductive tritiation after oxidation with NaIO₄ or galactose oxidase. In differentiated cells one glycoprotein fraction (M_r 150 000) could not be labeled by any of these methods, although it does contain neuraminic acid. We found no evidence in support of the hypothesis that the anomalous behavior of this fraction is caused by an increased degree of O-acetylated neuraminic acid in the plasma membrane of differentiated cells.     

Formation of α-keto acids from amino acids using immobilized bacteria and algae

Summary Both free and immobilized cells of the algae Chlorella vulgaris and Anacystis nidulans contain aminoacid oxidase (AAO) activity which is increased by illumination with red light. Both immobilized species are photosynthetically active. By co-immobilizing Chlorella with bacterial cells (Providencia sp. PCM 1298) containing high AAO activity an increased production of keto acid (up to tenfold) is observed due to improved oxygen supply.     

Characterization of two cDNAs that encode MAP kinase homologues in Arabidopsis thaliana and analysis of the possible role of auxin in activating such kinase activities in cultured cells

Two cDNA clones, cATMPK1 and CATMPK2, encoding MAP kinases (mitogen-activated protein kinases) have been cloned from Arabidopsis thaliana and their nucleotide sequences have been determined. Putative proteins encoded by ATMPK1 and ATMPK2 genes, designated ATMPK1 and ATMPK2, contain 370 and 376 amino acid residues, respectively, and are 88.7% identical at the amino acid sequence level. ATMPK1 and ATMPK2 exhibit significant similarity to rat ERK2 (49%) and Xenopus MAP kinase (50%). The amino acid residues corresponding to the sites of phosphorylation (Thr-Glu-Tyr) that are involved in the activation of MAP kinases are conserved in ATMPK1 and ATMPK2. Northern blot analysis indicates that the ATMPK1 and ATMPK2 mRNAs are significantly present in all the organs except seeds. Genomic Southern blot analysis suggests that there are a few additional genes that are related to ATMPK1 and ATMPK2 in the Arabidopsis genome. Purified Xenopus MAP kinase kinase (MAPK kinase) phosphorylates ATMPK1 and ATMPK2 proteins that have been expressed in Escherichia coli, activating these enzymes. A rapid and transient activation of 46-kDa protein kinase activity that phosphorylated myelin basic protein (MBP) was detected when auxinstarved tobacco BY-2 cells were treated with synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D). Protein kinase activities which phosphorylated the recombinant ATMPK2 protein also increased rapidly after auxin treatment in the auxin-starved BY-2 cells. These results suggest that auxin may function as an activator of plant MAP kinase homologues, as do various mitogens in animal systems.     

Transfer Agent of Immunity

Detection of the cells which contain antibody was accomplished by a method of immune adherence of human erythrocytes to a single cell, termed SCIA (single cell immune adherence) reaction. Peritoneal exudate cells were collected from mice immunized with flagella of either Salmonella enteritidis or S. tennessee. Serologically specific antibody was detectable in some of the peritoneal exudate cells of such mice. An immune ribonucleic acid (RNA) was extracted from the peritoneal exudate cells of mice immunized with salmonella flagella. When mice were injected intraperitoneally with this preparation, serologically specific antibody was found in some of their peritoneal exudate cells by the SCIA method. This preparation was inactivated by treatment with ribonuclease, but was resistant to proteinases, deoxyribonuclease and anti-flagella antibody, suggesting that this agent is of RNA nature and does not contain antigen or fragment thereof.     

Stomatococcus mucilaginosus produces a mannose-containing lipoglycan rather than lipoteichoic acid

Five strains of Stomatococcus mucilaginosus were investigated to determine whether the organism produces a lipoteichoic acid or a lipoglycan. Crude phenol extracts were purified by hydrophobic interaction chromatography and shown to contain lipoglycan. The major carbohydrate component present was mannose, indicating that the macroamphiphile is a lipomannan. The fatty acid composition of the lipoglycan was similar to that of stomatococcal whole cells. These data provide additional chemotaxonomic evidence supporting the suprageneric classification of the genus Stomatococcus within a group of actinomycete genera that also includes the genus Micrococcus.Abbreviations LTA Lipoteichoic acid - HIC Hydrophobic interaction chromatography - FAMEs Fatty acid methyl esters     

Fine Structure and Histochemistry of the Choroid Plexus of the Teleost Leuciscus rutilus

Analyses of the fine structure of the posterior choroid plexus in the teleost Leuciscus rutilus and the determination of the presence and function of the enzymes acid and alkaline phosphatases, ATPase and glucose-6-phosphatase confirm similarities between these epithelial cells and the saccus dorsalis and also with the epithelial cells of the choroid plexus found in mammals. The teleost plexus cells contain coated vesicles which are derived from the plasmalemma as well as from the Golgi complex. Moreover, they contain multivesicular bodies and Iysosomes. These organelles function in the absorption of substances from the cerebrospinal fluid and in the breakdown of these substances within the cells. The investigated enzymes play an important role in the secretion of electrolytes into the cerebrospinal fluid by active membrane transport.     

Sialic acid concentrations in plants are in the range of inadvertent contamination

The long held but challenged view that plants do not synthesize sialic acids was re-evaluated using two different procedures to isolate putative sialic acid containing material from plant tissues and cells. The extracts were reacted with 1,2-diamino-4,5-methylene dioxybenzene and the fluorescently labelled 2-keto sugar acids analysed by reversed phase and normal phase HPLC and by HPLC–electrospray tandem mass spectrometry. No N-glycolylneuraminic acid was found in the protein fraction from Arabidopsis thaliana MM2d cells. However, we did detect 3-deoxy-d-manno-octulosonic acid and trace amounts (3–18 pmol/g fresh weight) of a compound indistinguishable from N-acetylneuraminic acid by its retention time and its mass spectral fragmentation pattern. Thus, plant cells and tissues contain five orders of magnitude less sialic acid than mammalian tissues such as porcine liver. Similar or lower amounts of N-acetylneuraminic acid were detected in tobacco cells, mung bean sprouts, apple and banana. Yet even yeast and buffer blanks, when subjected to the same isolation procedures, apparently contained the equivalent of 5 pmol of sialic acid per gram of material. Thus, we conclude that it is not possible to demonstrate unequivocally that plants synthesize sialic acids because the amounts of these sugars detected in plant cells and tissues are so small that they may originate from extraneous contaminants.     

Synthesis of trans unsaturated fatty acids in Pseudomonas putida P8 by direct isomerization of the double bond of lipids

The phospholipids of Pseudomonas putida P8 contain monounsaturated fatty acids in the cis and trans configuration. Cells of this phenol-degrading bacterium change the proportions of these isomers in response to the addition or elimination of a membrane active compound such as 4-chlorophenol. This study undoubtedly reveals that the cis unsaturated fatty acids are directly converted into trans isomers without involvement of de novo synthesis of fatty acids. Oleic acid, which cannot be synthesized by this bacterium, was incorporated as a cis unsaturated fatty acid marker in the membrane lipids of growing cells. The conversion of this fatty acid into the corresponding trans isomer was demonstrated by gas chromatographic-mass spectrometric analysis and use of ¹⁴C-labeled oleic acid. Separation and isolation of the cellular membranes showed that the fatty acid isomerase is located in the cytoplasmic membrane of P. putida P8.Abbreviation 4-CP 4-chlorophenol     

The relationship of the charasome to chloride uptake in Chara corallina: physiological and histochemical investigations

A possible role of the charasome in terms of chloride transport into Chara corallina Klein ex. Willd., em. R.D.W. is examined. The branches of Chara contain the most charasome material and are shown to be very effective in acquiring Cl^- to support continued shoot growth. The early maturation of the branches, the rather large Cl^- fluxes into these cells, and their ability of translocate Cl^- to growing cells of the shoot indicate a special role of these branches in Cl^- accumulation. The structure of the charasome, with its extensive periplasmic space, appears especially suited as a site for H⁺–Cl^- cotransport (influx). We show, by histochemical assay, that the charasomes of mature cells contain ATPase activity; such activity is absent in growing charasomes of very young cells. ATPase activity is also associated with the plasmodesmata of C. corallina. Charasome ATPase activity and Cl^- uptake are both inhibited by p-chloromercuribenzenesulfonic acid (1 mM) or diethylstibestrol (40 M; 45 min). The anion transport inhibitor, 4,4-diisothiocyano-2,2-disulfonic acid stilbene (1 mM) had no effect on Cl^- transport and inhibited ATPase activity only when applied after chemical fixation of the cells. Results of an attempt to demonstrate the presence of Cl^- within the cytoplasmic tubules of the charasome, using a silver precipitation technique, proved difficult to interpret because of a reaction between the silver and a cellular substance produced in the light.Abbreviations CPW Chara pond water - DES diethylstilbestrol - DIDS 4,4-diisothiocyano-2,2-disulfonic acid stilbene - Mops 3-(N-morpholino)propanesulfonic acid - PCMBS p-chloromercuribenzenesulfonic acid     

Cultivation ofCandida lipolytica 4-1 on hydrocarbons

The length of the carbon chain of the hydrocarbon substrate affects markedly the fatty acid composition in the cell lipids of the yeastCandida lipolytica 4-1. During cell growth onn-hexadecane dissolved in deparaffinated gas oil, direct incorporation of palmitic acid into lipids takes place. During growth onn-dodecane, on the other hand, an immediate synthesis and incorporation into oleic acid is observed. The cells contain only little lauric acid (maximum 11%). During fermentation of then-alkanes dissolved in deparaffinated gas oil which contains a mixture of isoalkanes, alkylated aromatic and cyclic hydrocarbons, free fatty acids accumulate in the oil phase. The fatty acid composition in the oil differs markedly according to the growth stage of cells. At the beginning of the logarithmic phase of growth, the fatty acid composition in the oil phase reflects the acid composition in the cell lipids, toward the end of cell growth, the cooxidation products of the isoalkanes accumulate. The aqueous phase contains lower fatty acids and cooxidation products of isoalkanes and of alkylated aromatic and alicyclic hydrocarbons. Part III. Oxidation and Utilization of Individual Pure Hydrocarbons—seeFolia Microbiol. 14,334 (1969).     

Biochemical Effects of Fatty Acid and its Derivatives on l-Glutamic Acid Fermentation

Tween 60 (polyoxyethylene sorbitan monostearate) has been found to be the most effective derivative of fatty acid in accumulating l-glutamic acid in biotin-sufficient medium. The effect was exceedingly subject to the influence of the addition time of the ester, and this was observed also on the growth curve of Brev. lactofermentum. Changes of the growth curve caused by the varied addition time of the ester corresponded to those by the concentration of biotin in the medium that did not contain Tween 60. The patterns of fermentation course in the two corresponding conditions, such as biotin 3 μg/l and biotin 20 μg/l-Tween 60 mg/ml, agreed closely with each other. It seemed that identical cells were grown on the conditions. The only difference between the cells was observed as to the contents of intracellular biotin. Although l-glutamic acid was not accumulated by biotin-sufficient cells, cells with sufficient biotin and capable of accumulating l-glutamic acid were obtained in the presence of Tween 60, in which case the ester neither prevented the cells from taking up biotin nor controlled the level of intracellular biotin.     

Ultrastructure and histochemistry,of the stomach of Asplanchna sieboldi

Stomach cells of female Asplanchna sieboldi are specialized for absorption and intracellular digestion of nutrients. Evidence is presented to show that electron-opaque colloidal substances, present in the medium and within digestive vacuoles of the prey (Paramecium), are taken up by the stomach cells at the apical cell membrane and sequestered within food vacuoles which contain hydrolases working in both the acid and alkaline pH range. The stomach cells are also implicated in the absorption of molecules below the resolving power of the electron microscope. In rotifers possessing a complete digestive tract, this task is presumed to be handled by the intestine.     

Role of relA mutation in the survival of amino acid-starved Escherichia coli

Amino acid-starved cells of Escherichia coli relA ⁺, which contain a large number of glycogen particles, are able to survive in phosphate buffer for a longer time period than their relaxed counterparts. With regard to NH ₄ ⁺ starvation differences in the survival of both strains were not found. NH ₄ ⁺ starved cells of E. coli relA are able to synthesize glycogen but amino acid-starved cells of the relA strain are not. We suggest that the synthesis of glycogen triggered by guanosine tetraphosphate during amino acid starvation is responsible for the prolonged viability of the E. coli relA ⁺ strain.Abbreviations ppGpp guanosine tetraphosphate     

An improvement of auxin extraction procedure and its application to cultured plant cells

Summary The senescent cultured tobacco (Nicotiana tabacum L.) cells, XD6S, when extracted with customary procedure, contained much auxin. The use of new extraction procedure using dichloromethane which does not extract much indolepyruvic acid revealed that the cultured tobacco cells do not contain measurable auxin at any stage of culture.Abbreviations IAA indoleacetic acid - IPyA indolepyruvic acid - Trp tryptophan     

MASSIVE ACCUMULATION OF PHYTOENE INDUCED BY NORFLURAZON IN DUNALIELLA BARDAWIL (CHLOROPHYCEAE) PREVENTS RECOVERY FROM PHOTOINHIBITION1

The effects of nanomolar to micromolar concentrations of the herbicide norflurazon were studied in Dunaliella bardawil Ben-Amotz et Avron, a β-carotene-accumulating halotolerant alga. The large amount of β-carotene which Dunaliella bardawil can contain, around 8% of the algal dry weight, is reduced to 0.2% by treatment with 100 nm norflurazon. Simultaneously, phytoene is accumulated to a similar level of about 8%. The gradual increase in phytoene content, in response to increasing norflurazon concentrations, corresponds to the decrease in β-carotene, with no evident change in other isoprenoid intermediates. Carotene-rich Dunaliella bardawil is substantially resistant to high-intensity photoinhibition. This resistance is lost in cells grown to contain low β-carotene and in the norflurazon-treated phytoene-rich cells. These observations are in agreement with the hypothesis that the accumulated β-carotene in Dunaliella bardawil protects the cells against injury by excessive irradiation.     

Physical and Chemical Properties of Cilia Isolated from Tetrahymena pyriformis*

SYNOPSIS. Cilia devoid of their basal granules and isolated from Tetrahymena pyriformis strain W, by the method of Watson et al. (66) have been characterized in terms of physical, chemical and immunologic properties. The general chemical composition of cilia, based on mean values from these determinations is 66.3% protein, 24.3%“lipid,” 3.7 or 4.8% carbohydrate, and 0.4% nucleic acid. The amino acid composition of cilia includes hydroxyproline and is not significantly different from the amino acid composition of whole cells. Chloroformmethanol extracts of ciliary material contain free amino acids as well as neutral lipids and phospholipids. The lipid composition of cilia also is not significantly different from that of whole cells. Pentose, hexose and small but significant amounts of adenine nucleotide and ribonucleic acid are present. Cilia are a greater stimulus for the production of antibody than an equivalent amount of cellular antigen, and the ciliary antigen is strain specific.     

Responses of the African Armyworm and three species of borers to carbohydrates and phenolic substances: an electro- and behavioural physiological study

Electrophysiological recordings were made from the maxillary sensilla styloconica of fifth instars of the African Armyworm, Spodoptera exempta (Wlk.) (Lep.: Noctuidae), and the borers (Lep.: Pyralidae) Eldana saccharina Wlk. and Maruca testulalis (Geyer), and fifth and sixth instars of the borer Chilo partellus (Swinhoe). Stimuli were different concentrations of 13 carbohydrates and 11 phenolic substances. It appeared that a sucrose sensitive cell is present in the lateral styloconic sensilla of Spodoptera, Maruca and Chilo, and in both the lateral and medial sensilla of Eldana. The sucrose cell of fifth-instar Chilo is more sensitive than that of sixth instars. The additional responses of the medial cells to the phagostimulant sucrose are supposed to have contributed to the widespread infestation of sugarcane by Eldana. The medial sensilla of the Armyworm contain a cell type responding to both meso-inositol and D-ribose. Chilo appeared to be the only one of the three borer species able to perceive phenolic substances. It has a cell responsive to chlorogenic acid in its medial sensilla, which is a bit more sensitive in fifth- than in sixth-instar larvae. The presence of this cell may account for the positive correlation between total phenols content and resistance of sorghum cultivars to this borer. Spodoptera can perceive four phenolic acids. Its lateral sensilla contain a cell responsive to chlorogenic acid and its medial sensilla a cell responsive to 4-hydroxybenzoic acid, gallic acid as well as vanillic acid. Feeding experiments showed that the latter substances inhibit feeding in the Armyworm.So far, in the borers adequate stimuli could be determined for only tow or three of the four taste cells present in each sensillum styloconicum. This indicates that we are still unaware of chemicals that may be important to these caterpillars. On the other hand, for the sensilla styloconica of Spodoptera, electrophysiology suggests the presence of five or six taste cell types, whereas only four taste cells are known from morphological studies.     

Histochemistry of mucosubstances in Brunner's-gland cells and duodenal goblet cells of two New World monkey species (Saimiri sciureus andSaguinus fuscicollis)

Summary Duodenal goblet cells and Brunner's-gland cells obtained from two species of New World monkeys (Saimiri sciureus andSaguinus fuscicollis) were studied using conventional histochemical methods and by applying a panel of 17 labelled lectins. The secretions of both goblet and Brunner'-gland cells were found to contain neutral mucosubstances, while those of goblet cells also exhibit acid and sulphated carbohydrate components. Lectin binding studies allowed a more detailed analysis of the mucus glycoproteins. Marked differences between the two examined species were not detected.N-Acetyl-galactosamine, galactose, fucose andN-Acetyl-glucosamine were found to be the predominante sugar residues in Brunner's-glands glycoproteins, with mannose and glucose being only minor components.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Comparative studies of chromophore contents inside and outside the rhabdoms of arthropod compound eyes

Summary The intracellular distribution of visual pigments in the retinular cells of compound eyes of a butterfly (Papilio), a moth (Actias), a water scorpion (Ranatra), a dragonfly (Pantala), a crayfish (Procambarus), and a crab (Hemigrapsus) were investigated.The experiments measured the total surface area of rhabdom membrane in single compound eyes by light and electron microscopy, the densities of intramembrane particles in rhabdoms of compound eyes by freeze-fracture methods, and the quantities of chromophore molecules in compound eyes by high pressure liquid chromatography.Four species (moth, water scorpion, dragonfly, and crayfish) contain more than 75% of total chromophore probably as visual pigments in their rhabdoms, but the butterfly and the crab contain only 28.6% and 39.2%, respectively.The remainder of the chromophore molecules (Papilio 71.4%,Actias 5.7%,Ranatra 18.9%,Pantala 23.2%,Procambarus 24.3%, andHemigrapsus 60.8%) are supposed to exist mostly in the cytoplasm of the retinular cells. The localizing sites of such extrarhabdomeric chromophores are discussed in relation to the cytoplasmic membrane systems such as endoplasmic reticulum and lysosomal elements.In view of the results of the present experiment, it seems clear that the butterfly,Papilio, possesses a very large extra-rhabdomeric pool of chromophore that is available for rapid regeneration of visual pigment.Abbreviations HPLC high pressure liquid chromatography - ROR ratio of rhabdom to retinulae volume