Evidence from knockout mice against physiologically significant aquaporin 8-facilitated ammonia transport

Aquaporin (AQP)8-facilitated transport of NH₃ has been suggested recently by increased NH₃ permeability in Xenopus oocytes and yeast expressing human or rat AQP8. We tested the proposed roles of AQP8-facilitated NH₃ transport in mammalian physiology by comparative phenotype studies in wild-type vs. AQP8-null mice. AQP8-facilitated NH₃ transport was confirmed in mammalian cell cultures expressing rat or mouse AQP8, in which the fluorescence of a pH-sensing yellow fluorescent protein was measured in response to ammonia (NH₃/NH₄⁺) gradients. Relative AQP8 single-channel NH₃-to-water permeability was 0.03. AQP8-facilitated NH₃ and water permeability in a native tissue was confirmed in membrane vesicles isolated from testes of wild-type vs. AQP8-null mice, in which BCECF was used as an intravesicular pH indicator. A series of in vivo studies were done in mice, including 1) serum ammonia measurements before and after ammonia infusion, 2) renal ammonia clearance, 3) colonic ammonia absorption, and 4) liver ammonia accumulation and renal ammonia excretion after acute and chronic ammonia loading. Except for a small reduction in hepatic ammonia accumulation and increase in ammonia excretion in AQP8-null mice loaded with large amounts of ammonia, there were no significant differences in wild-type vs. AQP8-null mice. Our results support the conclusion that AQP8 can facilitate NH₃ transport but provide evidence against physiologically significant AQP8-facilitated NH₃ transport in mice. water transport; transgenic mouse; liver     

Chemical Factors Controlling Floral Bud Formation of Torenia Stem Segments Cultured in Vitro I. Effects of Mineral Nutrients and Sugars

Internodal segments of Torenia fournieri Lind. were culturedon various media to investigate chemical factors influencingin vitro flowering. The elimination or dilution of ammoniumnitrate from Murashige and Skoog's medium increased the formationof adventitious buds which subsequently differentiated floralbuds. The dilution of mineral salts in Murashige and Skoog'smedium enhanced adventitious bud formation, but did not influencethe ratio of cultures with floral buds to those with adventitiousbuds. Among various media tested, in vitro floral bud formationand development of Torenia was best on a medium having 1/5 ofthe mineral salts and no NH₄NO₃. Eighty-seven percent of thecultures produced floral buds on this medium. Using this medium,the effects of various sugars were also examined. Increasingthe concentration of sucrose in the medium (up to 60 g/liter)increased the rate of cultures with floral buds, and stimulatedthe development of floral buds led to anthesis. (Received January 17, 1981; Accepted February 21, 1981)     

Activity of Gibberellins A1 to A0 in the Avena First-leaf Bioassay, and Location after Chromatography

Activity curves are determined for gibberellins A₁ to A₀ bythe Avena first-leaf bioassay method. Gibberellins A¹, A₄ andA₅ can be detected at 10^-11 or 10^-10 g/ml and give optimum activityof approximately 230 per cent elongation (water controls = 100per cent). Gibberellins A²A³, and A⁹ can be detected at 10^-3g/mland give optimum activity of approximately 200 per cent. GibberellinsA₆ and A₇ can be detected at 10^-5g/ml; GA₇ gives optimum activityof around 190 per cent. All the gibberellins except GA₈ canbe detected by this bioassay method after chromatography inn-butanol: 1.5 N ammonia (3: 1) and benzene: acetic acid: water(4: 2: 1) when applied to the paper at concentrations from O.Ito µg. The sensitivity of the method is compared withthat of other gibberellin bioassay methods.     

Effects of sulfite and pH on abscisic acid-dependent transpiration and on stomatal opening

In rice, alday, wheat and tobacco (Nicotiana tabacum L. Samsunand Samsun NN) plants which contained large amounts of ABA,the transpiration rate decreased rapidly with 2 ppm SO₂ fumigationand reached 20 to 65% of the initial level after 5- to 30-minexposure depending on their ABA contents. In the cases of broadbean and tobacco (N. glutinosa L.) with low ABA contents, therate slightly increased for 20 and 40 min, respectively, afterthe start of the fumigation and then decreased gradually. Thetranspiration rates of corn and sorghum, in spite of their extremelylow ABA contents, pronouncedly decreased with SO₂ fumigationand reached 65 and 50%, respectively, of the initial levelsafter 40-min exposure. Foliar application of 0.04 N HC1 to N.tabacum L. Samsun NN leaves remarkably depressed the transpirationrate, while the application of 0.04 M Na₂SO₃ decreased the rateonly to the same level as water treatment. Foliar applicationof either HCl or Na₂SO₃ to N. glutinosa L. leaves exerted littlechange in the transpiration rate. When 10^–4M ABA was appliedto broad bean leaves prior to HCl and Na₂SO₃ treatment, theirtranspiration rate was decreased by HCl, but not by Na₂SO₃ application.In sonicated epidermal strips peeled from broad bean leaves,Na₂SO₃ produced a slight increase in the stomatal aperture sizein the absence of ABA, but showed no effect in the presenceof ABA. The aperture size was identical in the pH range of 3.0to 7.0 in the incubation medium. In the presence of ABA in themedium, the aperture size was small in the acidic region ofpH with a minimal value at pH 4.0. ABA decreased the aperturesize at concentrations above 10^–9 M at pH 4.0 and 10^–6M at pH 7.0 in the medium. [2_–14C] ABA uptake by epidermalstrips was large in the acidic region, especially at pH 4.0. (Received February 28, 1980; )     

Studies on the nutrition of rice cell culture I. A simple, defined medium for rapid growth in suspension culture

We investigated the nutrient requirements of rice in liquidculture and developed a revised medium of mineral salts, sucrose,thiamine and 2,4-dichlorophenoxyacetic acid. The following majornutrients at the indicated concentrations were beneficial: NO₃-N(40 mM), NH₄-N (5.0 mM), P (2.0 mM) and K (40 mM). Cobalt, iodine,pyridoxine, nicotinic acid and m-inositol were not essentialand were excluded from the revised R-2 medium. Growth was betterwith ammonium and nitrate together than with nitrate as thesole nitrogen source. Cell growth in the R-2 medium was superiorto that obtained in B5, Heller, Murashige-Skoog and White media. (Received March 31, 1973; )     

Ammonia Induces Starch Degradation in Chlorella Cells

When ammonia was added to cells of Chlorella which had fixed¹⁴CO₂ photo synthetically, ¹⁴C which had been incorporated intostarch was greatly decreased. A similar effect was observedwhen potassium nitrate and sodium nitrite were added. The ammonia-induceddecrease in ¹⁴C-starch was observed in all species of Chlorellatested. With cells of C. vulgaris 11h, most of the radioactivityin starch was recovered in sucrose, indicating that ammoniainduces the conversion of starch into sucrose. The percent of¹⁴C recovered in sucrose differed from species to species andpractically no recovery in sucrose was observed in C. pyrenoidosa.In most species tested, the enhancing effects of blue lightand ammonia on O₂ uptake as well as the ammonia effect on starchdegradation were greater in cells which had been starved inphosphate medium in the dark than in non-starved cells. In contrast,the enhancing effect of ammonia on dark CO₂ fixation was muchgreater in non-starved cells. C. pyrenoidosa was unique in thatblue light did not show any effect on its O₂ uptake. (Received August 15, 1984; Accepted November 16, 1984)     

Alkalization of the Medium by Unicellular Green Algae during Uptake Dissolved Inorganic Carbon

When Chlorella vulgaris 11h, Chlorella vulgaris C-l, Chlamydomonasreinhardtii, Chlamydomonas moewusii, Scenedesmus obliquus, orDunaliella tertiolecta were illuminated in with 0.5 mM NaHCO₃,the pH of the medium increased in a few minutes from 6 to about9 or 10. The alkalization, which was accompanied by O₂ evolution,was dependent on light, external dissolved inorganic carbon(DIC) as HCO^-₃, and algae grown or adapted to a low, air-levelCO₂ in order to develop a DIC concentrating mechanism. Therewas little pH increase by algae without a DIC concentratingprocess from growth on 3% CO₂ in air. Photosynthetic O₂ evolutionwithout alkalization occurred using either internal DIC or externalCO₂ at acidic pH. The PH increase stopped between pH 9 to 10,but the alkalization would restart upon re-acidification betweenpH 6 and 8. Alkalization was suppressed by the carbonic anhydraseinhibitors, acetazolamide, ethoxyzolamide or carbon oxysulfide.The pH increase appeared to be the consequence of the externalconversion of HCO^–₃ into CO₂ plus OH^– during photosynthesisby cells with a high affinity for CO₂ uptake. Cells grown onhigh CO₂ to suppress the DIC pump, when given low levels ofHCO^–₃ in the light, acidified the medium from pH 10 to7. Air adapted Scenedesmus cells with a HCO^–₃ pump, aswell as a CO₂ pump, alkalized the medium very rapidly in thelight to a pH of over 10, as well as slower in the dark or inthe light with DCMU or without external DIC and O₂ evolution.Alkalization of the medium during photosynthetic DIC uptakeby algae has been considered to be part of the global carboncycle for converting H₂CO₃ to HCO^–₃ and for the formationof carbonate salts by calcareous algae from the alkaline conversionof bicarbonate to carbonate. These processes seem to be a consequenceof the algal CO₂ concentrating process. ¹Present address: Department of Biology, Faculty of Science,Niigata University, Niigata, 950-21 Japan.     

The Effect of Inorganic Nutrients on the Hormonal Requirements of Normal, Habituated, and Crown-gall Tissue 4

The addition of Braun and Wood's inorganic supplements (845mg l^–1 KCl, 1800 mgl^–1 NaNO₃, 300 mg l^–1 NaH₂PO₄.2H₂O,790 mg l^–1 (NH₄)₂SO₄) to White's medium caused markedincreases in the growth of normal tissues of Helianthus annuus,Nicotiana rustica, Daucus carota, and Vinca rosea and crown-galltumour tissues of H. annuus. However, no evidence was obtainedwhich suggested that the presence of these extra salts markedlyinfluenced the essential requirements of normal callus for auxinsand kinetin. In contrast their presence significantly influencedthe hormonal requirements of certain habituated cultures ofH. annuus and V. rosea. These habituated cultures had specificauxin requirements on White's medium while either an auxin orkinetin was sufficient on high-salts medium. These results arediscussed in relation to previous reports which suggested thatthe biosyntheses of auxins and other growth factors in normaland crown-gall cultures are specifically activated by certaininorganic ions.     

The Permeability of Ammonia, Methylamine and Ethylamine in the Charophyte Chara corallina (C. australis)

Ritchie, R. J. 1987. The permeability of ammonia, methylamineand ethylamine in the charophyte Chara corallina (C. australis).—J.exp. Bot. 38: 67–76 The permeabilities of the amines, ammonia (NH₃), methylamine(CH₃NH₂) and ethylamine (CH₃CH₂NH₂) in the giant-celled charophyteChara corallina (C. australis) R.Br. have been measured andcompared. The permeabilities were corrected for uptake fluxesof the amine cations. Based on net uptake rates, the permeabilityof ammonia was 6?4?0?93 µm s^–1 (n = 38). The permeabilitiesof methylamine and ethylamine were measured in net and exchangeflux experiments. The permeabilities of methylamine were notsignificantly different in net and exchange experiments, norto that of ammonia (P_methylamine = 6?0?0?49 µm s^–1(n = 44)). In net flux experiments the apparent permeabilityof ethylamine was slightly greater than that of ammonia andmethylamine (P_{ethylamine, net} = 8?4?1?2 µm s^–1 (n= 40)) but the permeability of ethylamine based on exchangeflux data was significantly higher (P_{ethylamine, exchange} =14?1?2 µm s^–1 (n = 20)). Methylamine can be validlyused as an ammonium analogue in permeability studies in Chara. The plasmalemma of Chara has acid and alkaline bands; littlediffusion of uncharged amines would occur across the acid bands.The actual permeability of amines across the alkaline bandsis probably about twice the values quoted above on a whole cellbasis i.e. the permeability of ammonia across the permeablepart of the plasmalemma is probably about 12 µm s^–1. Key words: Chara, permeability, ammonia, methylamine     

Nitrate Stimulation of Mobilization of Seed Reserves in Temperate Cereals: Importance of Water Uptake

Relationships between nitrate (NO^-₃) supply, uptake and assimilation,water uptake and the rate of mobilization of seed reserves wereexamined for the five main temperate cereals prior to emergencefrom the substrate. For all species, 21 d after sowing (DAS),residual seed dry weight (d.wt) decreased while shoot plus rootd.wt increased (15–30%) with increased applied NO^-₃concentrationfrom 0 to 5–20 mM . Nitrogen (N) uptake and assimilationwere as great with addition of 5 mM ammonium (NH⁺₄) or 5 mMNO^-₃but NH⁺₄did not affect the rate of mobilization of seedreserves. Chloride (Cl^-) was similar to NO^-₃in its effect onmobilization of seed reserves of barley (Hordeum vulgare L.).Increased rate of mobilization of seed reserves with additionalNO^-₃or Cl^-was associated with increases in shoot, root and residualseed anion content, total seedling water and residual seed watercontent (% water) 21 DAS. Addition of NH⁺₄did not affect totalseedling water or residual seed water content. For barley suppliedwith different concentrations of NO^-₃or mannitol, the rate ofmobilization of seed reserves was positively correlated (r >0.95)with total seedling water and residual seed water content. Therate of mobilization of seed reserves of barley was greaterfor high N content seed than for low N content seed. Seed watercontent was greater for high N seed than for low N seed, 2 DAS.Additional NO^-₃did not affect total seedling water or residualseed water content until 10–14 DAS. The effects of seedN and NO^-₃on mobilization of seed reserves were detected 10and 14 DAS, respectively. It is proposed that the increasedrate of mobilization of seed reserves of temperate cereals withadditional NO^-₃is due to increased water uptake by the seedlingwhile the seed N effect is due to increased water uptake bythe seed directly. Avena sativa L.; oat; Hordeum vulgare L.; barley; Secale cereale L.; rye; xTriticosecale Wittm.; triticale; Triticum aestivum L.; wheat; nitrate; seed; germination; seed reserve mobilization     

Regulation of water permeability in rabbit conjunctival epithelium by anisotonic conditions

Effects of unilateral exposure to anisotonic conditions on diffusional water permeability of the isolated rabbit conjunctiva were determined. A segment of the bulbar-palpebral conjunctiva was mounted between Ussing-type hemichambers under short-circuit conditions. Unidirectional water fluxes (J_dw) were measured in either direction by adding ³H₂O to one hemichamber and sampling from the other. Electrical parameters were measured simultaneously. J_dw were determined under control isosmotic conditions and after introduction of either hyper- or hypotonic solutions against the tear or stromal sides of the preparations. In each of these four separate conditions, the anisotonic medium produced an 20–30% reduction in J_dw across the tissue, with the exception that to obtain such reduction with increased tonicity from the stromal side (medium osmolality increased by adding sucrose), conditions presumptively inhibiting regulatory volume increase mechanisms (e.g., pretreatment with amiloride and bumetanide) were also required. All reductions in J_dw elicited by the various anisotonic conditions were reversible on restoration of control tonicity. In experiments in which preparations were pretreated with the protein cross-linking agent glutaraldehyde, anisotonicity-elicited reductions in J_dw were not observed. Such reductions were also not observed in the presence of HgCl₂, implying the involvement of aquaporins. However, it is possible that the mercurial may be toxic to the epithelium, preventing the tonicity response. Nevertheless, from concomitant changes in transepithelial electrical resistance, as well as [¹⁴C]mannitol fluxes, [¹⁴C]butanol fluxes, and Arrhenius plots, arguments are presented that the above effects are best explained as a cell-regulated reduction in membrane water permeability that occurs at the level of water-transporting channels. Presumably both apical and basolateral membranes can downregulate their water permeabilities as part of a protective mechanism to help maintain cell volume. unidirectional water fluxes; net water fluxes; Ussing chamber; short-circuit current; electrolyte transport; cell volume regulation; paracellular mannitol permeability     

Relationship between Coleoptile Elongation and Alcoholic Fermentation in Rice Exposed to Anoxia. I. Importance of Treatment Conditions and Different Tissues

The relationship between coleoptile elongation and alcoholicfermentation of rice under anoxia is examined using seeds either:(a) N₂ flushed during submergence, (b) incubated in stagnantdeoxygenated agar at 0·1% w/v to simulate the stagnantconditions of waterlogged soil, or (c) incubated in waterloggedsoil. Coleoptile elongation growth was greater for N₂ flushing> stagnant agar > soil; seed survival was also greatestin this order over 1-5 d. Ethanol concentrations in coleoptiles and intact seeds (cv.IR42) were approximately 300 and 100 mol m^-3 respectively whenseeds were grown 3 d in stagnant agar, however 92% of the ethanolin seeds diffused into the external medium when solutions weremixed for 5-10 s. Coleoptile growth under anoxia was relatedto rates of ethanol synthesis (R_E) in different treatments;there was greater coleoptile growth and R_E for seeds in N₂ flushedsolutions than in stagnant deoxygenated agar. Coleoptile growthof individual seeds was also related to the R_E of each seedat 2-3 d after anoxia (r² = 0·46). Analysis of different tissues was important in evaluating growthand metabolism of coleoptiles. Although the coleoptile onlyaccounted for 0·7% of seed dry weight at 3 d after anoxia,it contained 21% of the ethanol produced by rice seeds. Therewere also three-fold higher rates of R_E on a fresh weight basisin expanding tissues in the base of the coleoptile relativeto the elongated tissues at the apex. Results are discussedin terms of the importance of environmental conditions usedto impose anoxia, quantification of R_E in specific tissues andthe possibility that under stagnant conditions high ethanolconcentrations in tissues may limit R_E and coleoptile growth.Copyright1994, 1999 Academic Press Anoxia, ethanol, alcoholic fermentation, Oryza sativa L., rice, submergence     

Inhibition of Ethylene Production by Fatty Acids in Fruit and Vegetable Tissues

Fatty acids of chain length from C₄ to C₁₂ inhibited ethyleneproduction in wounded albedo tissue of Hassaku (Citrus hassakuHort. ex Tanaka) fruit. Of the fatty acids tested, caprylicacid (C₈) and capric acid (C₁₀) were the most effective. Lauricacid (C₁₂) was less effective, and caproic acid (C₆) and butyricacid (C₄) were the least effective. Caprylic acid at 5 mM markedlyinhibited ethylene production in not only wounded albedo tissueof citrus fruit but also apple (Malus sylvestris Mill.) cortex,tomato (Lycopersicon esculentum Mill.) pericarp, cucumber (Cucumissativus L.) cortex, banana (Musa AAA group Cavendish subgroup)pulp, broccoli (Brassica oleracea L.) floret, spinach (Spinaciaoleracea L.) leaf, lettuce (Lactuca sativa L.) leaf and mungbean (Vigna radiata [L.] Wilczek) hypocotyl. Caprylic acid inhibitedethylene production at the step of conversion of l-aminocyclopropane-l-carboxylicacid to ethylene. The inhibition could be partially relievedby transferring the tissue to caprylic acid-free medium. (Received June 15, 1982; Accepted August 13, 1982)     

Germination of Talinum triangulare L. Seeds as Affected by Various Chemical and Physical Treatments

Three-month ‘old’ and ‘fresh’ seedsof Talinum triangulare were subjected to various treatmentsto induce early and rapid germination. Scarification and activated carbon were the most effective treatmentsin improving total germination in fresh seeds, while the 3 and5 per cent thiourea treatments were most effective in improvingtotal germination in old seeds. Activated carbon, scarificationand 5 per cent thiourea treatments enhanced early germinationin both old and fresh seeds. Cumulative percentage germinationwas very high in fresh seeds after scarification or after treatmentwith activated carbon and 5 per cent thiourea, and lowest inseeds treated with 3 per cent thiourea and hot water. In oldseeds, highest cumulative percentage germination was obtainedwith 3 and 5 per cent thiourea treatments and scarification.Generally, higher germination was obtained with fresh seedsthan with old seeds. Partial seed-coat removal and treatment with 5 per cent thiouriaresulted in a higher rate of and cumulative percentage germinationcompared with seeds with the coat partially removed but nottreated with thiourea. Constantly high temperature (34 °C) increased both rateand total germination compared with seeds planted at room temperature(20–23 °C). Treatments that did not induce germinationwere 1 per cent thiourea, H₂SO₄, cold water soaking at roomtemperature, 6 per cent hydrogen peroxide and soil planting.These treatments effected less than 3 per cent germination. Talinum triangulare L, seed scarification, activated charcoal, thiourea, germination     

Shell-Boring Mechanism of the Gastropod, Purpura (Thais) lapillus: a Physiological Demonstration of the Role of Carbonic Anhydrase in the Dissolution of CaCO3

Measurement of carbonic anhydrase (CA) activity in homogenatesof accessory boring organs of the muricid gastropod,Purpuralapillus, by Meldrum and Roughton's manometric method showedthat CA is always present in boring as well as in inactive ABOs,but in variable amounts. Tests by the same method on whole ABOsin an isotonic solution were negative, proving that CA remainsintracellularly. Experiments in vivo on inhibition and activation demonstratedclearly that CA is responsible for demineralization of the valvesof lamellibranchs by Purpura:(a) at low concentrations of Diamox,partial inhibition of the enzymeoccurred, that is, the numberof complete holes decreased or disappeared and etchings increased;and at higher concentrations full inhibition took place. Inhibitionis reversed when snails are replaced in normal sea water; (b)action of pure CO₂ or mixtures of CO₂ and O₂ accelerated boring:in the optimal mixture, three times more boreholes were producedby snails than in the controls, and in about half the time.Under these conditions the reaction catalyzed by CA goes tothe right with hydration of CO₂ and is accompanied by releaseof H⁺ ions; in the presence of an increased concentration ofCO₂, the reaction is intensified and results in an additionalrelease of H⁺ ions. Consequently, destruction of CaCO₃ by theABO of Purpura in sea water enriched with CO₂ is accelerated. To identify the nature of the exchanged ion, we tested the effectof NaCl and KC1 on the boring mechanism in vivo by increasingthe amount of these salts in the seawater of snails with prey.In this case augmentation of the boring activity was noticed.These results suggest that the boring activity is accompaniedby complex ionic exchanges.     

Flower-inducing Activity of Vitamin K in Lemna paucicostata

Vitamins K₁ K₃ and K₅ induced flowering in Lemna paucicostata151, a short-day plant, cultured in 1/10 strength M medium (1/10M medium) under continuous light, and their activity was greatlyintensified by simultaneous application of benzyladenine. Themost active of these was vitamin K₅ L. paucicostata 6746 ismore sensitive to vitamin K₅ than strain 151, but the effectof vitamin K₅ on strain 6746 was not intensified by benzyladenine.The flower-inducing activity of vitamin K₅ was intensified bythe addition of benzoic acid in both strains and by the additionof copper or ferricyanide in Strain 6746, when these chemicalswere added at such low concentrations that they would scarcelyinduce flowering. In strain 6746, vitamin K₅ added to 1/10 M had little effecton flowering under a subcritical photoperiod, while it clearlyinduced flowering under continuous light. In this strain, vitaminK₅ added to full strength M medium, in which this plant wasmore sensitive to short photoperiods than in 1/10 M medium,did not induce flowering even under continuous light, and wasrather inhibitory under short photoperiods. (Received August 14, 1984; Accepted October 16, 1984)     

Regulation of C4 Photosynthesis: Inactivation of Pyruvate,Pi Dikinase in Leaf and Chloroplast Extracts in Relation to Dark/Light Regulation in Vivo

Active pyruvate, P₁ dikinase in leaf or chloroplast extractsisolated from illuminated leaves was inactivated by incubatingwith ADP. With chloroplast extracts neither ATP nor AMP alonewas effective. Half the maximum rate of inactivation was observedwith about 55 µM ADP. The following evidence supportedthe view that ADP-mediated inactivation had a co-requirementfor low concentrations of ATP [Buchanan (1980) Ann. Rev. PlantPhysiol. 31: 341], adding hexokinase and glucose prevented inactivationby ADP [Feldhaus et al. (1975) Eur. J. Biochem. 57: 197], whenGDP and UDP were added in place of ADP they mediated rapid inactivationonly when ATP was also provided; GTP was not effective. ATPwas apparently optimally effective at about 1 µM or less.The rate of inactivation was approximately proportional to thesquare of extract concentration suggesting dependancy on a factorin the extracts in addition to active enzyme. The involvementof one or more heat labile protein factors was confirmed bytrypsin treatment of extracts. Pyruvate, P₁ dikinase inactivatedby treatment with ADP was reactivated by incubating with P₁,a property common to the inactive enzyme extracted from darkenedleaves. Thiol/disulphide interconversion was apparently notcritical in the regulation of pyruvate, P₁ dikinase. ³Present address: Department of Agricultural Chemistry, Facultyof Agriculture, Nagoya University, Chikusa, Nagoya 464, Japan. (Received September 22, 1980; Accepted December 6, 1980)     

Calcification in the Green Alga Halimeda: II. THE EXCHANGE OF CA2+ AND THE OCCURRENCE OF AGE GRADIENTS IN CALCIFICATION AND PHOTOSYNTHESIS

In Halimeda cylindracea and H. tuna segments, the concentrationof CaCO₃, MgCO₃, protein, and chlorophyll, as well as segmentvolume and wet and dry weight, increase with ‘age’i.e. from the apex of a branch downwards. Photosynthetic andcalcification rates decrease with age as does the degree oflight stimulation of calcification. Studies of the exchange of ⁴⁵Ca between the Halimeda thallusand the sea water under various conditions showed that mostof the Ca exchange is between the cell walls, the aragonitecrystals, and the intercellular space. The cell wall has twodistinguishable phases with half-times (t_0?5) of 200 and 35min while the CaCO₃ has a rapidly exchanging phase with a t_0?5of approximately 6 min. The t_0?5 of the exchange of Ca betweenthe intercellular space and the external medium is estimatedat about 6 min, on the basis of uptake studies. If the integrityof the barrier between the intercellular space and the externalsea water, created by the adpressed peripheral utricles is destroyedthe t_0?5 is smaller (<<3 min). These kinetic studies as well as comparative measurements ofcalcification rates by both isotopic and chemical methods showthat the ⁴⁵Ca method for measuring calcification rates overestimatesthe calcification rate, due to binding of ⁴⁵Ca in the cell wallsand retention of ⁴⁵Ca in the intercellular space. The ¹⁴C methodgives more accurate results and has the further advantage ofallowing simultaneous measurement of the photosynthetic andcalcification rate on the same segment.     

The regulation of ammonia uptake in Chara australis

The regulation of ammonia uptake was investigated in internodalcells of the freshwater alga Chara australis. Ammonia uptakewas estimated by monitoring (i) its depletion from the bathingsolution, (ii) the uptake of radiolabelled methylamine, an analogueof ammonia, and (iii) depletion of ammonia in the unstirredlayer with the microelectrode ion-flux estimation technique(MIFE). Distribution of methylamine (¹⁴CH₃NH₃⁺) between thevacuole and cytoplasm was estimated with efflux analysis. Whencells were bathed continuously in solutions containing ammoniaor methylamine, the uptake rates of both amines decreased over12 to 48 h despite the continuing existence of a large electrochemicalgradient favouring influx of the NH⁺₄ and CH₃NH⁺₄ cations. Treatmentwith 1.0 to 10.0 mM MSX, an inhibitor of glutamine synthetase,caused the internal ammonia concentration to rise and reducedthe subsequent uptake of ammonia and methylamine by up to 70%within 2 h. These results suggest that the permease facilitatingNH⁺₄/CH₃NH⁺₄ influx is under feedback or kinetic regulationfrom either internal ammonia or an intermediate of nitrogenassimilation. Treatment with metabolic inhibitors (CCCP, azide and DCMU) andsome weak acids (DMO and butyric acid) for 30 to 60 min inhibitedmethylamine uptake, but the changes in the electrical potentialdifference across the plasma membrane could not account forthe magnitude of inhibition. The rate of cytopiasmic streaming,which is an indicator of the cellular ATP concentration in Chara,was inhibited by many of these treatments. However, under certainconditions of external pH and concentration, butyric acid couldreversibly inhibit ammonia and methylamine uptake without affectingcytoplasmic streaming, demonstrating that a decrease in cytoplasmicATP concentration was not responsible for the inhibition. Theeffect of butyric acid was rapid, causing a 60% inhibition ofuptake in 15 min. We conclude that weak acids can inhibit theNH⁺₄/CH₃NH⁺₄ permease by acidifying the cytoplasm and suggestthat this may also explain the effects of the metabolic inhibitorson ammonia and methylamine uptake. Key words: Ammonia, methylamine, uptake, regulation, Chara     

Homonojirimycin and N-methyl-homonojirimycin inhibit N-Iinked oligosaccharide processing

Homonojirimycin (HNJ) and N-methylhomonojirimycin (MHNJ) weretested as inhibitors of the purified glycopro-tein processingenzymes, glucosidase I and glucosidase II. MHNJ was a reasonablygood inhibitor of glucosidase I (K₁ = 1 x 10^–6 M) andwas about three times as effective on this enzyme as was HNJ.On the other hand, HNJ inhibited glucosidase II with a K₁ ofabout 1 x 10^x6 M, whereas MHNJ was three times less effective(K₁ = 3 x 10^–5 M). However, the butyl derivative of HNJhad very low activity toward these two processing glucosidases.HNJ and its methyl derivative were also tested in vivo usinginfluenza virus-infected MDCK cells, and measuring the inhibitionof N-linked oligosaccharide processing of the viral envelopeglycoproteins. With 100 µg/ml of MHNJ in the medium, essentiallyall of the N-linked oligosaccharide chains of the virus wereof the "high-mannose" type with the major structure being characterizedas Glc₃Man₉(GlcNAc)₂. Similar results were obtained with HNJalthough this compound was less effective in vivo as well asin vitro. These results are in keeping with these inhibitorsbeing effective at the glucosidase I step. Both inhibitors werealso tested in MDCK cell cultures to determine whether theyaffected the in vivo synthesis of proteins, or of lipid-linkedsaccharides. In contrast to deoxynojirimycin, which has beenreported to inhibit the formation of lipid-linked saccharides,no effects were seen on either the incorporation of mannoseinto lipid-linked saccharides or the incorporation of leucineinto protein. glucosidase lipid influenza virus oligosaccharide