A synthetic globotriaosylceramide analogue inhibits HIV-1 infection <Emphasis Type="Italic">in vitro</Emphasis> by two mechanisms

Previously, it was shown that the cell-membrane-expressed glycosphingolipid, globotriaosylceramide (Gb₃/P^k/CD77), protects against HIV-1 infection and may be a newly described natural resistance factor against HIV infection. We have now investigated the potential of a novel, water soluble, non-toxic and completely synthetic analogue of Gb₃/P^k (FSL-Gb₃) to inhibit HIV-1 infection in vitro. A uniquely designed analogue, FSL-Gb₃, of the natural Gb₃/P^k molecule was synthesized. HIV-1_IIIB (X4 virus) and HIV-1_Ba-L (R5 virus) infection of PHA/interleukin-2-activated, peripheral blood mononuclear cells (PBMCs) and Jurkat T cells in vitro was assessed, as well as infection of U87.CD4.CCR5 by various clinical R5 tropic viruses after treatment with FSL-Gb₃. We monitored Gb₃, CD4 and CXCR4 expression by fluorescent antibody cell sorting and viral replication by p24 ^gag ELISA. Total cellular Gb₃ was examined by glycosphingolipid extraction and thin layer chromatography. In vivo toxicity was monitored in mice by histological assessment of vital organs and lymphoid tissue. FSL-Gb₃ blocked X4 and R5 of both lab and clinical viral strains in activated PBMCs or the U87.CD4.CCR5 cell line with a 50% inhibitory concentration (IC₅₀) of approximately 200–250 μM. FACS and TLC overlay showed that FSL-Gb₃ can insert itself into cellular plasma membranes and that cellular membrane-absorbed FSL-Gb₃ is able to inhibit subsequent HIV-1 infection. There was no effect of FSL-Gb₃ on cell surface levels of CD4 or CXCR4. Thus, FSL-Gb₃ can inhibit HIV-1 by two mechanisms: direct inhibition of virus and inhibition of viral entry. Infusion of FSL-Gb₃ into laboratory mice at doses well in excess of theoretical therapeutic doses was tolerated with no untoward reactions. Our results demonstrate the potential utility of using a completely synthetic, water soluble globotriaosylceramide analogue, FSL-Gb₃, having low toxicity, for possible future use as a novel therapeutic approach for the systemic treatment of HIV/AIDS.     

Effect of mannosylated derivatives on HIV-1 infection of macrophages and lymphocytes

We previously demonstrated that gpl20/160 (Env) of HIV-1 interactin a carbohydrate-specific manner with mannosyI/N-acetylglucosaminylderivatives and that HTV-1_LAI infection of monocytic U937 andlymphoid CEM cells was inhibited by CD4-free Concanavalin A-reactiveglyco-peptides from U937 cells. We report here that the naturalglycoproteins bovine fetuin and asialofetuin, human oroso-mucoidand a-fetoprotein, and mannan, which all specifically interactwith Env, inhibited infection of primary mac-rophages by macrophage-tropicHIV-1 strains, whereas dextran had no such effect This activitywas conserved if fetuin, asialofetuin, or orosomucoid were heat-treated,which rules out the role of their three-dimensional structure.Orosomucoid and mannan partially inhibited Env binding to macrophagesbut not to U937 or CEM cells. This indicates that Env does notbind in the same manner to primary macrophages and to immortalizedCD4⁺ cells, and that orosomucoid and mannan act at CD4-independentstages of virus binding to macrophages. Mannan also inhibitedEnv binding to surface glycopeptides obtained after trypsintreatment of macrophages. Furthermore, orosomucoid and fetuininteracted with, and they inhibited the binding of a V3 loopB clade consensus peptide to several macrophage membrane proteins,including two 36 and 42 kDa proteins. These data indicate thatthese glycoproteins interfere with post-binding events duringHIV-1 infection of primary macrophages. In contrast, the compoundsdid not affect infection of U937 or CEM cells by T-cell tropicHIV-1_LAI nor infection of peripheral blood lymphocytes by HIV-1_LAIor HIV-1_BA-L. Thus, carbohydrate-specific inhibition of HIVinfection depends on the cell type more than on the viral strain,and differences in the glycan structure of cell-type-specificcofactors may be important for HIV entry into cells. HIV macrophage lymphocyte inhibition glycoprotein     

Modulation of K+ currents in monocytes by VCAM-1 and E-selectin on activated human endothelium

Resting membrane potential (RMP) and whole cell currents wererecorded in human THP-1 monocytes adherent to polystyrene, unstimulated human umbilical vein endothelial cells (HUVECs),lipopolysaccharide (LPS)-treated HUVECs, immobilizedE-selectin, or vascular cell adhesion molecule 1 (VCAM-1)using the patch-clamp technique. RMP after 5 h on polystyrene was24.3 ± 1.7 mV (n = 42) with delayed rectifier K⁺(I_dr) andCl currents(I_Cl) presentin >75% of the cells. Inwardly rectifying K⁺ currents(I_ir) werepresent in only 14% of THP-1 cells. Adherence to unstimulated HUVECsor E-selectin for 5 h had no effect on I_ir orI_Cl but decreasedI_dr. Five hoursafter adherence to LPS-treated HUVECs, outward currents were unchanged,but I_ir waspresent in 81% of THP-1 cells. A twofold increase inI_ir and ahyperpolarization (41.3 ± 3.7 mV,n = 16) were abolished by pretreatmentof THP-1 cells with cycloheximide, a protein synthesis inhibitor, orherbimycin A, a tyrosine kinase inhibitor, or by pretreatment of theLPS-treated HUVECs with anti-VCAM-1. Only a brief (15-min) interactionbetween THP-1 cells and LPS-treated HUVECs was required toinduce I_ir expression 5 h later. THP-1 cells adherent to VCAM-1 exhibited similarconductances to cells adherent to LPS-treated HUVECs. Thus engagementof specific integrins results in selective modulation of differentK⁺ conductances.

     

Oligosaccharide profiles of HIV-2 external envelope glycoprotein: dependence on host cells and virus isolates

The glycosylation pattern of the external envelope glycoproteinof human immunodeficiency virus type 2 (HIV-2) was studied independence on host cells and virus isolates. Strains HIV-2_ALT,HIV-2_ROD and HIV-2_D194, differing in their biological propertiesand in the amino acid sequences of their env genes, were propagatedin MOLT4, HUT78 and U937 cells, in human peripheral blood lymphocytesand monocytes/macrophages in the presence of [6-³] glucosamine.Radiolabelled viral glycoproteins were isolated from the cell-freesupernatants and digested with trypsin. Glycans were sequentiallyliberated by endo-ß-N-acetylglucosaminidase H andpeptide-N⁴-(N-acetyl-ß-glucosaminyl) asparagine amidaseF, and fractionated according to charge and size. Comparisonof the oligosaccharide profiles revealed that the envelope glycoproteinsof different virus isolates, propagated in the same host cells,yielded very similar glycan patterns, whereas cultivation ofan isolate in different host cells resulted in markedly divergentoligosaccharide maps. Variations concerned the proportion ofhigh-mannose-, hybrid- and complex-type substituents, as wellas the state of charge and structural parameters of the complex-typespecies. As a characteristic feature, complex-type glycans ofmacrophage-derived viral glycoprotein were almost exclusivelysubstituted by lactosamine repeats. Hence, glycosylation ofthe HIV-2 external envelope glycoprotein seems to be primarilygoverned by host cell-specific factors rather than by the aminoacid sequence of the corresponding polypeptide backbone. envelope glycoprotein glycosylation human immunodeficiency virus type 2     

Impaired trafficking of choline transporter-like protein-1 at plasma membrane and inhibition of choline transport in THP-1 monocyte-derived macrophages

The present study investigates choline transport processes and regulation of choline transporter-like protein-1 (CTL1) in human THP-1 monocytic cells and phorbol myristate 13-acetate (PMA)-differentiated macrophages. Choline uptake is saturable and therefore protein-mediated in both cell types, but its transport characteristics change soon after treatments with PMA. The maximal rate of choline uptake intrinsic to monocytic cells is greatly diminished in differentiated macrophages as demonstrated by alterations in V_max values from 1,973 ± 118 to 380 ± 18 nmol·mg^–1·min^–1, when the binding affinity did not change significantly (K_m values 56 ± 8 and 53 ± 6 µM, respectively). Treatments with hemicholinim-3 effectively inhibit most of the choline uptake, establishing that a choline-specific transport protein rather than a general transporter is responsible for the observed kinetic parameters. mRNA screening for the expression of various transporters reveals that CTL1 is the most plausible candidate that possesses the described kinetic and inhibitory properties. Fluorescence-activated cell sorting analyses at various times after PMA treatments further demonstrate that the disappearance of CTL1 protein from the cell surface follows the same trend as the reduction in choline uptake. Importantly, the loss of functional CTL1 from the cell surface occurs without significant changes in total CTL1 protein or its mRNA level indicating that an impaired CTL1 trafficking is the key contributing factor to the reduced choline uptake, subsequent to the PMA-induced THP-1 differentiation to macrophages. protein trafficking     

Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose

A significant percentage of eukaryotic proteins contain posttranslationalmodifications, including glycosylation, which are required forbiological function. However, the understanding of the structure–functionrelationships of N-glycans has lagged significantly due to themicroheterogeneity of glycosylation in mammalian produced proteins.Recently we reported on the cellular engineering of yeast toreplicate human N-glycosylation for the production of glycoproteins.Here we report the engineering of an artificial glycosylationpathway in Pichia pastoris blocked in dolichol oligosaccharideassembly. The PpALG3 gene encoding Dol-P-Man:Man₅GlcNAc₂-PP-Dolmannosyltransferase was deleted in a strain that was previouslyengineered to produce hybrid GlcNAcMan₅GlcNAc₂ human N-glycans.Employing this approach, combined with the use of combinatorialgenetic libraries, we engineered P. pastoris strains that synthesizecomplex GlcNAc₂Man₃GlcNAc₂ N-glycans with striking homogeneity.Furthermore, through expression of a Golgi-localized fusionprotein comprising UDP-glucose 4-epimerase and ß-1,4-galactosyltransferase activities we demonstrate that this structure isa substrate for highly efficient in vivo galactose addition.Taken together, these data demonstrate that the artificial invivo glycoengineering of yeast represents a major advance inthe production of glycoproteins and will emerge as a practicaltool to systematically elucidate the structure–functionrelationship of N-glycans. ¹ These authors contributed equally to this work. ² To whom correspondence should be addressed; e-mail: swildt{at}glycofi.com     

The Influence of NO-3 and NH+4 Nutrition on the Gas Exchange Characteristics of the Roots of Wheat (Triticum aestivum) and Maize (Zea mays) Plants

Respiratory oxygen consumption by roots was 1·4- and1·6-fold larger in NH⁺₄-fed than in NO^-₃-fed wheat (Triticumaestivum L.) and maize (Zea mays L.) plants respectively. Higherroot oxygen consumption in NH⁺₄-fed plants than in NO^-₃-fedplants was associated with higher total nitrogen contents inNH⁺4-fed plants. Root oxygen consumption was, however, not correlatedwith growth rates or shoot:root ratios. Carbon dioxide releasewas 1·4- and 1·2-fold larger in NO⁺3-fed thanin NH⁺₄-fed wheat and maize plants respectively. Differencesin oxygen and carbon dioxide gas exchange rates resulted inthe gas exchange quotients of NH^-₄-fed plants (wheat, 0·5;maize, 0·6) being greatly reduced compared with thoseof NO^-₃-fed plants (wheat, 1·0; maize, 1·1). Measuredrates of HCO^-₃ assimilation by PEPc in roots were considerablylarger in 4 mM NH⁺₄-fed than in 4 NO^-₃ plants (wheat, 2·6-fold;maize, 8·3-fold). These differences were, however, insufficientto account for the observed differences in root carbon dioxideflux and it is probable that HCO^-₃ uptake is also importantin determining carbon dioxide fluxes. Thus reduced root extension in NH⁺₄-fed compared with NO^-₃-fedwheat plants could not be ascribed to differences in carbondioxide losses from roots.Copyright 1993, 1999 Academic Press Triticum aestivum, wheat, Zea mays, maize assimilation, ammonium assimilation, root respiration     

Nitrate Effects on Growth of the First Four Main Stem Leaves of a Range of Temperate Cereals and Pasture Grasses

The effects of different applied NO₃^– concentrations onextension growth and final length and area of leaves 1–4of five cereals and six pasture grasses of temperate originwere examined. Increased applied NO₃^– in the range 0.1–0.5.0mol m^–3 caused decreased duration of growth but increasedgrowth rate and final length of leaves 2–4 of the cerealsAvena saliva, Hordeum vulgare, Secale cereale, x Triticosecaleand Triticum aestivum. For all cereals, increased NO₃^–resulted in increased area of leaves 1–4. Pasture grasseswere supplied either 0.5 or 50 mol m^–3 NO₃^–. Increasedapplied NO₃^– (0.5–5.0 mol m^–3) resulted indecreased duration of growth and increased growth rate and finalarea of leaves 1–4 of Bromus wiltdenowii, leaves 2–4ofFestuca arundinaceae and leaves 3 and 4 of Lolium muitiflorum.In addition, length of leaves 3 and 4 of B. witidenowii increasedwith increased NO₃^–. Increased NO₃^– resulted inincreased area of leaves 2–4 of Dactylis gtomerata andLolium perenne and leaves 3 and 4 of Phalaris aquaiica but hadno effect on extension growth of all three species. Avena sativa L, oat, Hordeum vulgare L, barley, Secale cereale L, rye, x Triticosecale Wittm, triticale, Triticum aestivum L, wheat, Bromus willdenowii Kunth, prairie grass, Dactylis gtomerata L, cocksfoot, Festuca arundinaceae Shreb, tall fescue, Lolium multijlorum Lam, Italian ryegrass, Lolium perenne L, perennial ryegrass, Phalaris aquatica L, nitrate, leaf extension, leaf expansion     

Some Experimental and Theoretical Observations Concerning Mass Flow in the Vascular Bundles of Heracleum

The theory and practice of applying the thermodynamics of irreversibleprocesses to mass-flow theories is presented. Onsager coefficientswere measured on cut and uncut phloem and cut xylem strandsof Heracleum muntegazzimum. In 0.3 N sucrose + 1 mN KC1 theyare as follows. In phloem, L_EE = 5 ? 10–⁴ mho cm^–1,L_pE = 9 ? 10^–6 cm³ s^–1 cm^–2 volt^–1 cm,and L_PP = 0.16 cm³ s^–1 cm^–2 (J cm^–3)^–1cm. In uncut phloem strands L_EE is about 1 ? 10^–3 mhocm^–1. In xylem in 2 x 10^–3 N KCI, L_pp = 50 to 225,L_PE = 2 ? 10^–4, and L^EE = 4 ? 10^–3. The measurementsare tentative since the blockage of the sieve plates is an interferingfactor, but if they are valid they lead to the conclusion thatneither a pressure-flow nor an electro-kinetic mechanism envisaginga ‘long distance’ current pathway can be the majormotive ‘force’ for transport in mature phloem. Measurementsof biopotentials along conducting but laterally detached phloembundles of Heracleum suggest, nevertheless, that there may bea small electro-osmotic component of at least 0.1 mV cm^–1endogenous in the phloem.     

Nitrate Effects on Growth of the First Four Main Stem Leaves of a Range of Temperate Cereals and Pasture Grasses

The effects of different applied NO₃^– concentrations onextension growth and final length and area of leaves 1–4of five cereals and six pasture grasses of temperate originwere examined. Increased applied NO₃^– in the range 0.1–50mol m^–3; caused decreased duration of growth but increasedgrowth rate and final length of leaves 2–4 of the cerealsAvena saliva, Hordeum vulgare, Secale cereale x Triticosecaleand Triticum aestivum. For all cereals, increased NO₃^–resulted in increased area of leaves 1-4. Pasture grasses weresupplied either 0.5 or 50 mol m^–3; NO₃^–. Increasedapplied NO₃^– (0.5–50 mol m^–3) resulted indecreased duration of growth and increased growth rate and finalarea of leaves 1–4 of Bromus willdenowii leaves 2–4of Festuca arundinaceae and leaves 3 and 4 of Lolium multiflorum.In addition, length of leaves 3 and 4 of B. willdenowii increasedwith increased NO₃. Increased NO₃ resulted in increased areaof leaves 2–4 of Daciylis glomerata and Lolium perenneand leaves 3 and 4 of Phalaris aquatica but had no effect onextension growth of all three species. Avena saliva L., oat, Hordeum vulgare L., barley, Secale cereaie L., rye, x Triticosecale Wittm, triticale, Triticum aestivum L., wheat, Bromus willdenowii Kunth, prairie grass, Dactylis glomerata L., cocksfoot, Festuca arundinaceae Shreb, tall fescue, Lolium multiflorum Lam, Italian ryegrass, Lolium perenne L, perennial ryegrass, Phalaris aquatica L, nitrate,, leaf extension, leaf expansion     

Interactions of NH4+ and L-glutamate with NO3- transport processes of non-mycorrhizal Fagus sylvatica roots

The processes of NO₃^– uptake and transport and the effectsof NH₄⁺ or L-glutamate on these processes were investigatedwith excised non-mycorrhizal beech (Fagus sylvatica L.) roots.NO₃^– net uptake followed uniphasic Michaelis-Menten kineticsin a concentration range of 10µM to 1 mM with an apparentK_m of 9.2 µM and a V_max of 366 nmol g^–1 FW h^–1.NH₄⁺, when present in excess to NO₃^–, or 10 mM L-glutamateinhibited the net uptake of NO₃^– Apparently, part of NO₃^–taken up was loaded into the xylem. Relative xylem loading ofNO₃^– ranged from 3.21.6 to 6.45.1% of NO₃^– netuptake. It was not affected by treatment with NH₄⁺ or L-glutamate.¹⁶N/¹³N double labelling experiments showed that NO₃^–efflux from roots increased with increasing influx of NO₃^–and, therefore, declined if influx was reduced by NH₄⁺ or L-glutamateexposure. From these results it is concluded that NO₃^–net uptake by non-mycorrhizal beech roots is reduced by NH₄⁺or L-glutamate at the level of influx and not at the level ofefflux. Key words: Nitrate transport, net uptake, influx, efflux, ammonium, Fagus, Fagaceae     

Photosynthesis in Panicum milioides, a Species with Reduced Photorespiration

The capacity for C₄ photosynthesis in Panicum milioides, a specieshaving reduced levels of photorespiration, was investigatedby examining the activity of certain key enzymes of the C₄ pathwayand by pulse-chase experiments with ¹⁴CO₂. The ATP$P₁ dependentactivity of pyruvate,P₁ dikinase in the species was extremelylow (0.14–0.18 µmol mg chlorophyll^–1 min^–1).Low activity of the enzyme was also found in Panicum decipiensand Panicum hians (related species with reduced photorespiration)and in Panicum laxum (a C₃ species). The antibody to pyruvate,P₁dikinase caused about 70% inhibition of the ATP$P₁ dependentactivity of the enzyme in P. milioides. The activity of NAD-malicenzyme and NADP-malic enzyme in ^P. ^milioides was equally low(approximately 0.1–0.2 µmol mg chlorophyll^–1min^–1) and similar to the activity in P. decipiens, P.hians and P. laxum. Photosynthetic pulse-chase experiments underatmospheric conditions showed a typical C₃-like pattern of carbonassimilation including the labelling of glycine and serine asexpected during photorespiration. During the pulse with ¹⁴CO₂only about 1% of the labelled products appeared in malate and2–3% in aspartate. During a chase in atmospheric levelsof CO₂ for up to 6 min there was a slight increase in labellingin the C₄ acids. The amount of label in carbon 4 of aspartatedid not change during the chase, indicating little or no turnoverof the C₄ acid via decarboxylation. The results indicate thatunder atmospheric conditions P. milioides assimilates carbondirectly through the C₃ pathway. Photorespiration as indicatedby the CO₂ compensation point may be repressed in the speciesby a more efficient recycling of photorespired CO₂. (Received June 8, 1982; Accepted July 22, 1982)     

The Effect of Salinity Changes Upon the Physiology of Eulittoral Green Macroalgae from Antarctica and Southern Chile: II INTRACELLULAR INORGANIC IONS AND ORGANIC COMPOUNDS

The effects of hypo- and hypersaline treatments ranging from7–68% on the intracellular inorganic ion and organic soluteconcentrations were determined in the eulittoral green macroalgaeUlothrix implexa, Ulothrix subflaccida, Enteromorpha bulbosa,Acrosiphonia arcta, and Ulva rigida from Antarctica and SouthernChile. The main inorganic cations were K⁺, Na⁺, and Mg²⁺ inall species. The major osmolyte in E. bulbosa, A. arcta, andU. rigida was K⁺ at increasing salinities. In both Ulothrixspecies, however, K⁺ levels declined during hypersaline stressand Na⁺ concentrations rose significantly. The main inorganicanions were Cl^-, SO²_4-, and PO³_4- in all algae, while E. bulbosaand U. rigida also contained NO⁺₃. A. arcta showed an extremelyhigh SO^2-₄ content. The organic solutes proline, sucrose, andß-dimethylsulphoniopropionate (DMSP) played an importantrole in osmotic acclimation. The occurrence of three organicosmolytes suggests an additional function of these solutes ascryoprotectants in the cold-water macroalgae investigated.     

Maitotoxin and P2Z/P2X7 purinergic receptor stimulation activate a common cytolytic pore

The effects ofmaitotoxin (MTX) on plasmalemma permeability are similar to thosecaused by stimulation of P2Z/P2X₇ionotropic receptors, suggesting that1) MTX directly activatesP2Z/P2X₇ receptors or2) MTX andP2Z/P2X₇ receptor stimulationactivate a common cytolytic pore. To distinguish between these twopossibilities, the effect of MTX was examined in1) THP-1 monocytic cells before andafter treatment with lipopolysaccharide and interferon-, a maneuverknown to upregulate P2Z/P2X₇receptor, 2) wild-type HEK cells andHEK cells stably expressing theP2Z/P2X₇ receptor, and3) BW5147.3 lymphoma cells, a cellline that expresses functional P2Z/P2X₇ channels that are poorlylinked to pore formation. In control THP-1 monocytes, addition of MTXproduced a biphasic increase in the cytosolic freeCa²⁺ concentration([Ca²⁺]_i);the initial increase reflects MTX-inducedCa²⁺ influx, whereas the secondphase correlates in time with the appearance of large pores and theuptake of ethidium. MTX produced comparable increases in[Ca²⁺]_iand ethidium uptake in THP-1 monocytes overexpressing theP2Z/P2X₇ receptor. In bothwild-type HEK and HEK cells stably expressing theP2Z/P2X₇ receptor, MTX-inducedincreases in[Ca²⁺]_iand ethidium uptake were virtually identical. The response of BW5147.3cells to concentrations of MTX that produced large increases in[Ca²⁺]_ihad no effect on ethidium uptake. In both THP-1 and HEK cells, MTX- andBz-ATP-induced pores activate with similar kinetics and exhibit similarsize exclusion. Last, MTX-induced pore formation, but not channelactivation, is greatly attenuated by reducing the temperature to22°C, a characteristic shared by theP2Z/P2X₇-induced pore. Together,the results demonstrate that, although MTX activates channels that aredistinct from those activated byP2Z/P2X₇ receptor stimulation, thecytolytic/oncotic pores activated by MTX- and Bz-ATP are indistinguishable.

     

Selective inhibition of antimycin A-insensitive respiration in Ustilago maydis and Ceratocystis ulmi

Sporidia of Ustilago maydis and conidia of Ceratocystis ulmipossess an antimycin A and azide-tolerant electron transportpathway which apparently diverts electrons to O₂ from some pointon the substrate side of the antimycin A block. The alternatepathway (induced by 0.5 µg/ml antimycin A or 5x10^–4M sodium azide) supports a respiratory rate 1.5–2 timesthat of the normal system, but has a terminal oxidase with alower than normal affinity for O₂. A similarly high respiratoryrate in U. maydis is supported by the normal pathway when uncoupledby 4 µg/ml of 4,5-dichloro-2-trifluoromethylbenzimidazole,but a high affinity for O₂ in this case indicates that the normalterminal oxidase is utilized. Respiration by the normal pathway in both fungi is only slightlyor moderately inhibited by 1.5x10^–3 M benzohydroxamicacid (BHAM) and 5x10^–4 M 8-hydroxyquinoline. The alternatepathway in U. maydis, however, is inhibited as much as 84 and92% respectively by these two compounds, while alternate respirationin C. ulmi can be inhibited as much as 86 and 76% respectively.BHAM, 8-hydroxyquinoline, 2-pyridinethiol-1-oxide, a,a'-dipyridyl,carboxin, and diphenylamine inhibit alternate respiration ata site on the alternate pathway which is not part of the normalelectron transport system. Antimycin A and azide-insensitiverespiration found in U. maydis and C. ulmi closely resemblesinhibitor insensitivity noted in several fungi and some higherplants. Such an alternate respiratory pathway may be an earlystep in the evolution of oxidative phosphorylation. (Received June 27, 1972; )     

Nitrogen utilization by plant species from acid heathland soils: II. Growth and shoot/root partitioning of NO3- assimilation at constant low pH and varying NO3-/NH4+ ratio

The growth of four heathland species, two grasses (D. flexuosa,M. caerulea) and two dwarf shrubs (C. vulgaris, E. tetralix),was tested in solution culture at pH 4.0 with 2 mol m^–3N, varying the N0₃^–/NH₄⁺ ratio up to 40% nitrate. In addition,measurements of NRA, plant chemical composition, and biomassallocation were carried out on a complete N0₃^–/NH₄⁺ replacementseries up to 100% nitrate. With the exception of M. caerulea, the partial replacement ofNH₄⁺ by NO₃^– tended to enhance the plant's growth ratewhen compared to NH₄⁺ only. In contrast to the other species,D. flexuosa showed a very flexible response in biomass allocation:a gradual increase in the root weight ratio (RWR) with NO₃^–increasing from 0 to 100%. In the presence of NH₄⁺, grassesreduced nitrate in the shoot only; roots did not become involvedin the reduction of nitrate until zero ambient NH₄⁺. The dwarfshrubs, being species that assimilate N exclusively in theirroots, displayed an enhanced root NRA in the presence of nitrate;in contrast to the steady increase with increasing NO₃^–in Calluna roots, enzyme activity in Erica roots followed arather irregular pattern. Free nitrate accumulated in the tissuesof grasses only, and particularly in D. flexuosa. The relative uptake ratio for NO₃^– [(proportion of nitratein N uptake)/(proportion of nitrate in N supply)] was lowestin M. caerulea and highest in D. flexuosa. Whereas M. caeruleaand the dwarf shrubs always absorbed ammonium highly preferentially(relative uptake ratio for NO₃^– <0.20), D. flexuosashowed a strong preference for NO₃^– at low external nitrate(the relative uptake ratio for N0₃^– reaching a value of2.0 at 10% NO₃^–). The ecological significance of thisprominent high preference for NO₃^– at low NO₃^–/NH₄⁺ratio by D. flexuosa and its consequences for soil acidificationare briefly discussed. Key words: Ammonium, heathland lants, N0₃^–/NH₄⁺ ratio, nitrate, nitrate reductase activity, soil acidification, specific absorption rate     

Growth and development of Neocalanus flemingeri/plumchrus in the northern Gulf of Alaska: validation of the artificial-cohort method in cold waters

In situ growth and development of Neocalanus flemingeri/plumchrusstage C1–C4 copepodites were estimated by both the artificial-cohortand the single-stage incubation methods in March, April andMay of 2001–2005 at 5–6°C. Results from thesetwo methods were comparable and consistent. In the field, C1–C4stage durations ranged from 7 to >100 days, dependent ontemperature and chlorophyll a (Chl a) concentration. Averagestage durations were 12.4–14.1 days, yielding an averageof 56 days to reach C5, but under optimal conditions stage durationswere closer to 10 days, shortening the time to reach C5 (fromC1) to 46 days. Generally, growth rates decreased with increasingstage, ranging from 0.28 day^–1 to close to zero but weretypically between 0.20 and 0.05 day^–1, averaging 0.110± 0.006 day^–1 (mean ± SE) for single-stageand 0.107 ± 0.005 day^–1 (mean ± SE) forartificial-cohort methods. Growth was well described by equationsof Michaelis–Menten form, with maximum growth rates (G_max)of 0.17–0.18 day^–1 and half saturation Chl a concentrations(K_chl) of 0.45–0.46 mg m^–3 for combined C1–3,while G_max dropped to 0.08–0.09 day^–1 but K_chl remainedat 0.38–0.93 mg m^–3 for C4. In this study, in situgrowth of N. flemingeri/plumchrus was frequently food limitedto some degree, particularly during March. A comparison withglobal models of copepod growth rates suggests that these modelsstill require considerable refinement. We suggest that the artificial-cohortmethod is the most practical approach to generating the multispeciesdata required to address these deficiencies.     

Clustering of very late antigen-4 integrins modulates K(+) currents to alter Ca(2+)-mediated monocyte function

Endothelialcell vascular cell adhesion molecule-1 (VCAM-1) activates adherentmonocytes by clustering their very late antigen-4 (VLA-4) receptors,resulting in the modulation of the inwardly rectifying(I_ir) and delayed rectifying(I_dr) K⁺ currents, hyperpolarizationof the cells, and enhanced Ca²⁺ influx (Colden-Stanfield Mand Gallin EK. Am J Physiol Cell Physiol 275:C267-C277, 1998; Colden-Stanfield M and Scanlon M. Am JPhysiol Cell Physiol 279: C488-C494, 2000). The present studywas undertaken to test the hypothesis that monoclonal antibodies(MAbs) against VLA-4 (MAbVLA-4) mimic VCAM-1 to cluster VLA-4integrins, which play a key role in signaling an increase in thesecretion of the proinflammatory cytokine interleukin-8 (IL-8). Wholecell ionic currents and IL-8 secretion from THP-1 monocytes that wereincubated on polystyrene, VCAM-1-immobilized MAbVLA-4 or anisotype-matched MAb against CD45 (MAbCD45) were measured. Clustering ofVLA-4 integrins with a cross-linked MAbVLA-4, but not a monovalentMAbVLA-4, modulated the K⁺ currents in an identical mannerto incubation of cells on VCAM-1. Similarly, cross-linked MAbVLA-4 orVCAM-1 augmented Ca²⁺-mediated IL-8 secretion from THP-1monocytes and was completely abolished by exposure to CsCl, anI_ir blocker. Thus VLA-4 integrin clustering bycross-linked MAbVLA-4 mimics VCAM-1/VLA-4 interactions sufficiently tobe associated with events leading to monocyte differentiation, enhancedCa²⁺-mediated macrophage function, and possiblyatherosclerotic plaque formation.

     

Ammonium release by zooplankton in suspensions of heat-killed algae and an evaluation of the flow-cell method

The maximum excretion rate of NH₄ (39 nmol mg dry wt^–1h^–1) was directly measured for Daphnia pulex by measuringNH₄ accumulation in bottles containing D. pulex and dense, satiatingsuspensions of heat-killed algae. Ammonium release rates inthe algal suspensions were compared to those of individual animalsremoved from the suspension and placed in flow cells. Ammoniumrelease rate, R (nmol mg dry wt^–1 h^–1). in the flowcell decreased very rapidly with time, t (min), after removalaccording to the relation R = 26 + 25e^–0.16t. Ammoniumexcretion obtained by the flow cell method after extrapolationto time zero was not significantly different from that obtainedin the bottles. The considerable experiment-to-experiment variationin NH₄ excretion was in large part correlated (r² = 0.73) withthe feeding rate on the algae.     

Utilization Modes of Inorganic Carbon for Photosynthesis in Various Species of Chlorella

Rates of CO₂ and HCC₃^– fixation in cells of various Chlorellaspecies in suspension were compared from the amounts of ¹⁴Cfixed during the 5 s after the injection of a solution containingonly ¹⁴CO₂ or H¹⁴CO₃^–. Results indicated that irrespectiveof the CO₂ concentration during growth, Chlorella vulgaris 11h and C. miniata mainly utilized CO₂, whereas C. vulgaris C-3,C. sp. K. and C. ellipsoidea took up HCO₃^– in additionto CO₂. Cells of C. pyrenoidosa that had been grown with 1.5%CO₂ (high-CO₂ cells) mainly utilized CO₂, whereas those grownwith air (low-CO₂ cells) utilized HCO₃^– in addition toCO₂. Cells that utilized HCO₃^– had carbonic anhydrase(CA) on their surfaces. The effects of Diamox and CA on the rates of CO₂ and HCO₃^–fixation are in accord with the inference that HCO₃^– wasutilized after conversion to CO₂ via the CA located on the cellsurface. CA was found in both the soluble and insoluble fractions;the CA on the cell surface was insoluble. Independent of the modes of utilization, the apparent K_m (NaHCO₃)for photosynthesis was much lower in low-CO₂ cells than in high-CO₂ones. The fact that the CA in the soluble fraction in C. vulgarisC-3 was closely correlated with the K_m(NaHCO₃) indicates thatsoluble CA lowers the K_m. ¹ Dedicated to the late Professor Joji Ashida, one of the foundersand first president of the Japanese Society of Plant Physiologists. ⁴ On leave from Research and Production Laboratory of Algology,Bulgarian Academy of Sciences, Sofia. (Received September 14, 1982; Accepted March 1, 1983)