Glycosylation pattern and processing of envelope gene products encoded by glycosylation mutants of Friend spleen focus-forming virus

The protein encoded by the envelope gene of Friend spleen focus-formingvirus is responsible for the acute leukaemogenicity of thisvirus. In order to correlate glycosylation and intracellularprocessing of this protein with viral pathogenicity, envelopegene products of pathogenic and apathogenic glycosylation mutantswere expressed in Rat-1 cells and metabolically labelled with[6-³H] glucosamine. Following immunoprecipitation, primary andsecondary gene products (gp55, gp65) were separated by preparativepolyacrylamide gel electrophoresis. Oligosaccharides were releasedfrom tryptic glycopeptides by treatment with endo-ß-N-acetylglucosaminidaseH (gp55), peptide-N⁴-(N-acetyl-ß-glucosaminyl)asparagineamidase F (gp65) or by reductive ß-elimination. Resultingglycans were characterized by cochromatography with authenticoligosaccharide standards using different HPLC systems and digestionwith exoglycosidases. The results revealed that the primaryenvelope gene products of pathogenic glycosylation mutants were,in part, further processed in Rat-1 cells similar to wild-typeglycoprotein, resulting in polypeptides carrying complex-typeN-glycans as well as partially sialylated O-linked oligosaccharides.In contrast, corresponding glycoproteins encoded by apathogenicmutants were found to remain at the level of the primary translationproduct exclusively comprising high-mannose-type N-glycans.Hence, intracellular maturation of the envelope gene productsin this model cell line seems to correlate with the in vivopathogenicityof the glycosylation mutants studied. carbohydrate structure glycoprotein murine leukaemia virus oligosaccharide processing SFFV     

O-Linked fucose in glycoproteins from Chinese hamster ovary cells

We report our discovery that many glycoproteins synthesizedby Chinese hamster ovary (CHO) cells contain fucose in O-glycosidiclinkage to polypeptide. To enrich for the possible presenceof O-linked fucose, we studied the lectin-resistant mutant ofCHO cells known as Lec1. Lec1 cells lack N-acetylglucosaminyltransferaseI and are therefore unable to synthesize complex-type N-linkedoligosaccharides. Lec1 cells were metabolically radiolabelledwith [6-³H]fucose and total glycoproteins were isolated. Glycopeptideswere prepared by proteolysis and fractionated by chromatographyon a column of concanavalin A (Con A)— Sepharose. Thesets of fractionated glycopeptides were treated with mild base/borohydrideto effect the ß-elimination reaction and release potentialO-linked fucosyl residues. The ß-elimination produced[³H]fucitol quantitatively from [³H]fucose-labelled glycopeptidesnot bound by Con A-Sepharose, whereas none was generated bytreatment of glycopeptides bound by the lectin. The total [³H]fucose-labelledglycoproteins from Lec1 cells were separated by SDS—PAGEand detected by fluorography. Treatment of selected bands ofdetectable glycoproteins with mild base/borohydride quantitativelygenerated [³H]fucitol. Pretreatment of the glycoproteins withN-glycanase prior to the SDS—PAGE method of analysis causedan enrichment in the percentage of radioactivity recovered as[³H]fucitol. Trypsin treatment of [³H]fucose-labelled intactCHO cells released glycopeptides that contained O-linked fucose,indicating that it is present in surface glycoproteins. Thesefindings demonstrate that many glycoproteins from CHO cellscontain O-linked fucosyl residues and raise new questions aboutits biosynthesis and possible function. fucose glycoproteins monosaccharide O-linked     

A new approach to mapping co-localization of multiple glycosyl transferases in functional Golgi preparations

We have developed a new method to co-localize multiple glycosyltransferases in different Golgi compartments. The approach relieson the proven ability of intact, sealed rat liver Golgi preparationsto concentrate exogenous labeled sugar nucleotides into thelumen where they glycosylate either endogenous or artificialacceptors. The premise is that if two glycosyl transferasesare co-localized within the same compartment, they will competefor the limited amount of transported donor. If the donor isconsumed in glycosylating a permeable artificial glycoside withina Golgi compartment, it will be unavailable to glycosylate endogenousproducts within that same compartment. The greater the degreeof transferase co-localization, the greater the potential decreasein glycosylation of endogenous acceptors. We provide an exampleconsistent with these predictions. Adding 1 µM UDP[³H]Galto Golgi preparations followed by a chase with a cocktail ofunlabeled sugar nucleotides labels mostly endogenous N-linkedoligosaccharides containing both ß1,3- and ß1,4[³H]Galresidues with and without sialic acid. Addition of increasingamounts of 4-methylumbelliferyl-ß-xyloside (XylßMU)produces [³H]Gal1ß,4XylßMU and leads toa reciprocal decrease in labeling of a restricted set of theendogenous acceptors. This decrease is preferential for [³H]Galß1     

Metabolism and Translocation of Gibberellins in the Seedlings of Pharbitis nil (II). Photoperiodic Effects on Metabolism and Translocation of Gibberellins Applied to Cotyledons

We investigated the metabolism and translocation of two gibberellins(GAs), [³H]GA₂₀ and [³H]GA₁, which were applied at low concentrationto the cotyledons of Pharbitis nil (cv. Violet). Seedlings weregrown under three different photoperiodic conditions: continuouslight (CL-CL), continous light followed by short day conditions(CL-DT) and long day conditions followed by short day conditions(DT-DT). Translocation of the applied [³H]GAs from cotyledonsto hypocotyls was promoted by DT for all GAs examined. Whilethe conversion of the translocated [³H]GA₁ to [³H]GA₈ and itsconjugates was rapid in hypocotyl, the conversion of translocated[³H]GA₂₀ to [³H]GA₂₉ was slow. Radioactivity in epicotyls wasdetected much more rapidly on application of [³H]GA₂₀ than of[³H]GA₁, [³H]GA₈ and [³H]GA₂₉ and their conjugates. The conversionof [³H]GA₂₀ to [³H]GA₁ in the epicotyl was more rapid underCL-CL conditions. This result in consistent with the higherlevel of endogenous GA₁ existing in epicotyls under CL-DT thanDT-DT conditions. However, when [³H]GA₁ was applied to the cotyledon,only small amounts of [³H]GA₈ and its conjugates were detectedin the epicotyl regardless of the photoperiodic conditions.This result may suggest that the translocation and metabolismof [³H]GA₂₀ from cotyledons to epicotyl was faster under CL-CLthan DT-DT conditions and may correlate with the increased epicotylelongation of GA₂₀ treated plants under CL-DT than DT-DT conditions. (Received June 28, 1995; Accepted November 2, 1995)     

Characterization of the synthesis and release of catecholamine in the rat carotid body in vitro

The aim of this work was to determinecontents and turnover rates for dopamine (DA) andnorepinephrine (NE) and to identify the catecholamine (CA) releasedduring stimulation of the rat carotid body (CB). Turnover rates and therelease of CA were measured in an in vitro preparation using acombination of HPLC and radioisotopic methods. Mean rat CB levels of DAand NE were 209 and 45 pmol/mg tissue, respectively. With[³H]tyrosine asprecursor, rat CB synthesized[³H]CA in a time- andconcentration-dependent manner; calculated turnover times for DA and NEwere 5.77 and 11.4 h, respectively. Hypoxia and dibutyryl adenosine3',5'-cyclic monophosphate significantly increased[³H]CA synthesis. Innormoxia, rat CB released[³H]DA and[³H]NE in a ratio of5:1, comparable to that of the endogenous tissue CA. Hypoxia and highK⁺ preferentially released[³H]DA, nicotinepreferentially released[³H]NE, and acidicstimuli released both amines in proportion to tissue content. Releaseof [³H]CA induced byhypoxia and high K⁺ was nearlyfully dependent on extracellularCa²⁺, whereas basal normoxicrelease was not altered by removal of Ca²⁺ from the incubating solution.We conclude that the rat CB is an organ with higher levels of DA thanNE that preferentially releases DA or NE in a stimulus-specific manner.

     

Abscisic acid and the accumulation, biological activity and metabolism of four derivatives of [3H]gibberellin A1 in barley aleurone layers

Tritiated GA₁ and four of its synthetic derivatives were studiedin relation to their biological activity, uptake and metabolismby barley aleurone layers. Incubation was done in the presenceand absence of ABA. Tentative identification of some of themetabolites was made by TLC and GLC radiocounting of the metaboliteand its acid hydrolyzed derivative. Only GA₁ promoted -amylase synthesis. Uptake ranged from 20to 42%, varying with the derivative. ABA enhanced uptake of[³H]GA₁ and [³H]pseudoGA₁ and inhibited uptake of [³H]ketoGA₁the Wagner-Meerwein rearrangement product of [³H]GA₁ Uptakeof [³H]GA₁ methyl ester ([³H]GA₁-Me) and [³H]dihydroGA₁ wasunaffected by ABA. [³H]GA₁ was converted to an amphoteric GA₁ derivative ([³H]amphoGA₁)and [³H]GA₁-glycosyl ester. GA₁-Me was metabolized to four products,all of them GA₁ derivatives, including an apparent amphotericGA₁ derivative. DihydroGA₁ was quite stable; only one metabolitewas produced in sufficient yield to analyze. This product didnot cochromatograph with either of the expected acid hydrolyzedepimers of [³H]dihydroGA₁. [³H]ketoGA₁ was readily metabolizedto one product, probably the glycoside. [³H]pseudoGA₁ remainedessentially unmetabolized. Metabolism of all compounds testedwas not dramatically affected by ABA. Surprisingly, no metabolitesfrom hydroxylation at the 2-position were found. ¹ Present address: Monsanto Agricultural Co., 800 N. LindberghBlvd., St. Louis, MO 63166, U.S.A. (Received January 31, 1977; )     

The hormonal control of protein N-glycosylation in the developing rabbit mammary gland and its effect upon transferrin synthesis and secretion

Pregnant rabbit mammary gland explants cultured with insulin, prolactin and cortisol, synthesise and secrete transferrin radiolabelled with [³H]leucine or [³H]mannose. Omission of prolactin from the culture medium inhibited the incorporation of [³H]leucine into casein but not transferrin. Total transferrin secreted under these conditions was approx. 75% of the control (+ prolactin) value measured by rocket immunoelectrophoresis. Little incorporation of [³H]mannose into transferrin was seen in the absence of prolactin suggesting a lack of glycosylation of the protein. Dual label experiments with [³H]mannose and [¹⁴C]leucine confirmed this. The decreased incorporation of [³H]mannose into dolichol linked intermediates suggests a general effect on protein N-glycosylation in the absence of prolactin. Thus, while the synthesis of the polypeptide backbone of transferrin does not require prolactin its glycosylation does.     

Relationship between [125I]RTI-55-labeled cocaine binding sites and the serotonin transporter in rat placenta

We investigated the characteristics of cocainelike binding sitesin rat placenta using[¹²⁵I]RTI-55.[³H]paroxetine bindingand immunocytochemical staining for serotonin [5-hydroxytryptamine (5-HT)] and for the 5-HT transporterwere also used to obtain evidence for rat placental 5-HT uptake.[¹²⁵I]RTI-55saturation analyses with membranes from normal gestational day 20 placentas yielded curvilinear Scatchard plots that were resolved intohigh- and low-affinity components (mean dissociation constants of 0.29 and 7.9 nM, respectively). Drug competition studies with variousmonoamine uptake inhibitors gave rise to complex multiphasicdisplacement curves, although the results obtained with the selective5-HT uptake inhibitor citalopram suggest that the 5-HT transporter isan important component of placental high-affinity[¹²⁵I]RTI-55 binding.The presence of a rat placental 5-HT uptake system was additionallysupported by the[³H]paroxetine bindingexperiments and by the presence throughout the placenta ofimmunoreactivity for 5-HT and the 5-HT transporter. Immunostaining withboth antibodies was most intense in the junctional zone, whereas thedensity of[¹²⁵I]RTI-55 bindingsites was greater in the placental labyrinth. This discrepancy may bedue to the fact that[¹²⁵I]RTI-55 appearsto be labeling additional cellular components besides the 5-HTtransporter. The presence of cocaine- and antidepressant-sensitive 5-HTtransporters in the placenta has important implications for thepossible effects of these compounds on pregnancy and fetal development.

     

Effects of overexpression of {beta}1, 4-galactosyltransferase on glycoprotein biosynthesis in F9 embryonal carcinoma cells

ß1,4-Galactosyltransferase (GalTase) plays a centralrole in the biosynthesis of N-acetyllactosamine-containing oligo-saccharides.However, despite this seemingly important function, little isknown about how changes in the levels of GalTase affect oligosaccharidebiosynthesis. We have examined the effects of overexpressingGalTase on the glycosylation of endogenous glycoproteins inF9 mouse embryonal carcinoma cells. Cells transfected with eitherthe short form of the GalTase cDNA (encoding a protein of 386amino acids) or the long form of the GalTase cDNA (encodinga protein of 399 amino acids) had a 3-fold increase in totalGalTase activity, relative to control F9 cells. Analysis ofpronase-digested glycopeptides obtained from control and transfectedcells after metabolic labelling with [6-³H]galactose revealedno significant qualitative or quantitative differences, as assessedby Bio-Gel P-6 gel filtration chromatography and Tomato lectinaffinity chroma-tography. Furthermore, SDS-PAGE analysis ofimmuno-precipitated [³H]galactose-labelled lysosomal-associatedmembrane protein-1 (LAMP-1) glycoprotein showed no differencein amounts or mobility. Pronase digestion and subsequent analysisof the gel-fractionated LAMP-1 glycoproteins also indicatedno differences between the various cell lines. The inabilityof elevated GalTase activity to affect glycosylation was notdue to limiting levels of GalTase substrates, since an excessof substrates was detectable in lysed cells using either endogenousor exogenous GalTase and UDP-[³H]galactose. Finally, the subcellulardistribution of GalTase, as assessed by sucrose gradient fractionation,was similar between all cell types, thus suggesting that GalTasewas appropriately compartmentalized in the transfected cells.More importantly, GalTase specific activities in the Golgi membranesof the transfected cells were 3–4 times greater than incontrol cells. These results show that selectively increasingGalTase activity does not alter glycoprotein biosynthesis inF9 cells. F9 cells galactosyltransferase glycoprotein biosynthesis     

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     

Incomplete glycosylation of ASN 563 in mouse immunoglobulin M

Mouse immunoglobulin IgM was prepared from MOPC 104E ascites fluid and [3H]-mannose labeled tumor cells. The purified protein was used to prepare Fc fragments which were cleaved by cyanogen bromide. Gel filtration allows complete separation of the C-terminal glycosylation site. Amino acid and carbohydrate analyses show that Asn 563 of murine IgM is glycosylated only about 44% of the time.     

Pharmacological characterization of swelling-induced D-[3H]aspartate release from primary astrocyte cultures

During stroke orhead trauma, extracellular K⁺concentration increases, which can cause astrocytes to swell. In vitro,such swelling causes astrocytes to release excitatory amino acids, which may contribute to excitotoxicity in vivo. Several putative swelling-activated channels have been identified through which suchanionic organic cellular osmolytes can be released. In the presentstudy, we sought to identify the swelling-activated channel(s) responsible forD-[³H]aspartaterelease from primary cultured astrocytes exposed to either KCl orhypotonic medium. KCl-inducedD-[³H]aspartaterelease was inhibited by the anion channel inhibitors 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), dideoxyforskolin, L-644711, ATP, ITP, 3'-azido-3'-deoxythymidine, DIDS, andtamoxifen but not by cAMP. The cell swelling caused by raised KCl wasnot inhibited by extracellular ATP or tamoxifen as measured by an electrical impedance method, which suggests that these anion channel inhibitors directly blocked the channel responsible for efflux. Extracellular nucleotides and DIDS, however, had no or only partial effects onD-[³H]aspartaterelease from cells swollen by hypotonic medium, but such release wasinhibited by NPPB, dideoxyforskolin, and tamoxifen. Of theswelling-activated channels so far identified, our data suggest that avolume-sensitive outwardly rectifying channel is responsible forD-[³H]aspartaterelease from primary cultured astrocytes during raised extracellularK⁺ and possibly during hypotonicmedium-induced release.

     

Inhibition of cell differentiation and cell cohesion by tunicamycin in Dictyostelium discoideum

The effects of tunicamycin on protein glycosylation and cell differentiation were examined during early development of Dictyostelium discoideum. Tunicamycin inhibited cell growth reversibly in liquid medium. At a concentration of 3 μg/ml, tunicamycin completely inhibited morphogenesis and cell differentiation in developing cells. These cells remained as a smooth lawn and failed to undergo chemotactic migration. The expression of EDTA-resistant contact sites was also inhibited. The inhibition by tunicamycin was reversible if cells were washed free of the drug within the first 10 hr of incubation. After 12 hr of development, cells were protected from the drug by the sheath. When cells were treated with tunicamycin during the first 10 hr of development, incorporation of [³H]mannose and [³H] fucose was inhibited by approximately 75% within 45 min while no significant inhibition of [³H]leucine incorporation was observed during the initial 3 hr of drug treatment. The inhibition of protein glycosylation was further evidenced by the reduction in number of glycoproteins “stained” with ¹²⁵I-labelled con A. A number of developmentally regulated high-molecular-weight glycoproteins, including the contact site A glycoprotein (gp80), were undetectable when cells were labelled with [³H]fucose in the presence of tunicamycin. It is therefore evident that glycoproteins with N-glycosidically linked carbohydrate moieties may play a crucial role in intercellular cohesiveness and early development of D. discoideum.     

Characterization of ganglioside associated with the thyrotrophin receptor

The receptor protein for thyrotrophin (thyroid-stimulating hormone;TSH) is associated with a glycosphingolipid moiety. The proteinbelongs to the family of receptors that couple to guanine nucleotidebinding proteins; the glycosphingolipid contains sialic acidand belongs to the family of gangliosides. This report definesthe structure of the receptor ganglioside in the Fisher ratthyroid cell line (FRTL-5). Receptor protein was purified byTSH affinity chromatography from FRTL-5 cells, biosyntheticallylabelled with [³H]galactose and [³H]glucosamine, and resolvedby SDS-PAGE. A single radiolabelled band of M_r     

Kinetics of Integration of Xyloglucan into the Walls of Suspension-Cultured Rose Cells

To study the kinetics of synthesis, wall-binding and degradationof xyloglucan, we incubated suspension-cultured rose cells for0–5–24 h in L-[1-³H]arabinose. >95% of the [³H]arabinosewas taken up within 2 h. UDP-Pentoses were maximally labelledwithin 0–5 h and had lost most of their ³H by 2 h afterthe addition of [³H]arabinose. Therefore, the 24 h experimentresembled a pulse-chase rgime. The [³H]xyloglucan formed wasfractionated into four cellular pools [detergent-extractable(interpreted as cytoplasmic), and guanidinium thiocyanate-,06 M NaOH- and 60 M NaOH-extractable (interpreted as progressivelymore firmly wall-bound)]; soluble extracellular xyloglucan wascollected as a fifth pool. All five pools of xyloglucan hadstarted accumulating ³H at their respective maximal rates by     

Effects of ABA on Synthesis of Cell-Wall Polysaccharides in Segments of Etiolated Squash Hypocotyl I. Changes in Incorporation of Glucose and myo-Inositol into Cell-Wall Components

Externally supplied [³H]myo-inositol and [¹⁴C]glucose were incorporatedin cell-wall fractions of segments of etiolated squash hypocotyl.The extent of incorporation of [¹⁴C]glucose into cell-wall fractionswas very much greater than that of [³H]myo-inositol. Radioactivityfrom [¹⁴C]-glucose was effectively incorporated into hemicelluloseB and cellulose fractions and was incorporated uniformly intohexose, pentose and uronic acid residues, but radioactivityfrom [³H]myo-inositol was incorporated predominantly into uronicacid and pentose residues in the pectin and hemicellulose Bfractions. Exogenously applied ABA significantly suppressed the elongationof segments of squash hypocotyl and the incorporation of radioactivityfrom [^l4C]glucose and [³H]myo-inositol into the segments. Furthermore,ABA significantly inhibited the distribution of incorporatedradioactivity from [¹⁴C]glucose into the cellulose fraction,but did not affect distribution into the pectic fraction. Bycontrast, ABA only slightly inhibited the distribution of theincorporated radioactivity from [³H]myo-inositol into the pecticfraction. These results suggest that most of the cell-wall polysaccharidesin segments of squash hypocotyl are synthesized via the UDP-sugarpathway, and that ABA significantly inhibits the synthesis ofcellulose but not the synthesis of pectic polysaccharides whenABA suppresses the elongation of the segments. (Received March 25, 1988; Accepted November 15, 1988)     

Role of microtubules in the regulation of metabolism in isolated cerebral microvessels

We used¹³C-labeled substrates and nuclearmagnetic resonance spectroscopy to examine carbohydrate metabolism invascular smooth muscle of freshly isolated pig cerebral microvessels(PCMV). PCMV utilized[2-¹³C]glucose mainlyfor glycolysis, producing[2-¹³C]lactate.Simultaneously, PCMV utilized the glycolytic intermediate [1-¹³C]fructose1,6-bisphosphate (FBP) mainly for gluconeogenesis, producing[1-¹³C]glucose withonly minor[3-¹³C]lactateproduction. The dissimilarity in metabolism of[2-¹³C]FBP derivedfrom [2-¹³C]glucosebreakdown and metabolism of exogenous[1-¹³C]FBPdemonstrates that carbohydrate metabolism is compartmented in PCMV.Because glycolytic enzymes interact with microtubules, we disruptedmicrotubules with vinblastine. Vinblastine treatment significantlydecreased[2-¹³C]lactate peakintensity (87.8 ± 3.7% of control). The microtubule-stabilizing agent taxol also reduced[2-¹³C]lactate peakintensity (90.0 ± 2.4% of control). Treatment with both agentsfurther decreased[2-¹³C]lactateproduction (73.3 ± 4.0% of control). Neither vinblastine, taxol,or the combined drugs affected[1-¹³C]glucose peakintensity (gluconeogenesis) or disrupted the compartmentation ofcarbohydrate metabolism. The similar effects of taxol and vinblastine, drugs that have opposite effects on microtubule assembly, suggest thatthey produce their effects on glycolytic rate by competing withglycolytic enzymes for binding, not by affecting the overall assemblystate of the microtubule network. Glycolysis, but not gluconeogenesis,may be regulated in part by glycolytic enzyme-microtubule interactions.

     

Continuous binding of the PAF molecule to its receptor is necessary for the long-term aggregation of platelets

Human and rabbit platelets fully aggregated byplatelet-activating factor (PAF) underwent slow disaggregation but wererapidly disaggregated by the PAF receptor antagonists WEB-2086,Y-24180, SM-12502, and CV-3988. Whereas the1-alkyl-2-[³H]acetyl-sn-glycero-3-phosphocholine([³H]acetyl-PAF)specifically bound to platelet receptors underwent slow and spontaneousdissociation, it dissociated promptly from its receptor when WEB-2086was added, in parallel with platelet disaggregation and disappearanceof P-selectin on the cell surface. Extracellular[³H]acetyl-PAF wasrapidly deacetylated by normal rabbit platelets; some of the[³H]acetyl-PAF wasbound to the cells and a very small amount of [³H]acetate wasdetected in the cells. In contrast, when1-[³H]alkyl-2-acetyl-sn-glycero-3-phosphocholinewas added to the platelets, the radioactivity was rapidly incorporatedinto the 1-alkyl-2-acyl-sn-glycero-3-phosphocholinefraction. These results indicate that1) continuous binding of PAF to itsreceptor is necessary for prolonged platelet aggregation, which may bemediated through an unknown signaling system for a long-term cellresponse rather than a transient signaling system, and2) most of the[³H]acetyl-PAF boundto platelets is metabolized extracellularly by ecto-type PAFacetylhydrolase, with the lyso-PAF generated being incorporated rapidlyinto the cells and converted to1-alkyl-2-acyl-sn-glycero-3-phosphocholine.

     

The Effect of Growth in D2O on the Metabolism of Winter Rye

The metabolism of winter rye seedlings (Secale cereale, L. ev.Winter) cultured in 99.8 per cent D₂O was investigated. Comparedwith water-grown seedlings, the protein content was much lowerin the D₂O-cultured seedlings and the pattern of incorporationof [³H]leucine and [³H]phenylalanine into protein was substantiallydifferent. Seedlings cultured in D₂O incorporated [³H]thymidineinto DNA, but did not take up [³H]uridine. The results suggestthat some of the toxic effects of D₂O culture on higher plantscan be attributed to a partial block of protein synthesis.     

Carbon-specific phytoplankton growth rates: a comparison of methods

Measurements of biomass and growth rate of two axenic algalcultures were carried out using three different methodologicalapproaches: the specific ¹⁴C-labelling of chlorophyll a, [³H]adenineincorporation into DNA and net organic carbon assimilation.Time-course experiments revealed that the specific activitiesof chlorophyll a were significantly higher than the specificactivity of total algal carbon in six of seven experiments.When the specific activity of chlorophyll a is used to calculatethe carbon biomass and growth rate, the carbon biomass of thealgae will thus be underestimated and the specific growth ratewill be too high. Determination of growth rates from incorporationof [³H]adenine gave lower values than those obtained from netorganic carbon assimilation and from ¹⁴C incorporation intochlorophyll a. Problems with adenine saturation are suggested.When [³H]adenine is used to measure growth rates in dense algalcultures, additions of >1 µM [³H]adenine are oftenrequired to maximally label the extracellular and intracellularadenine pools and hence DNA.