Taste of methyl-{alpha}-D-mannopyranoside: Effects of cross adaptation and Gymnema sylvestre

Methyl--D-mannopyranoside is a glycoside with a bitter-sweettaste. Adaptation to sucrose reduces the sweetness and adaptationto quinine sulphate reduces the bitterness of methyl--Dmannopyranoside.Application of Gymnema sylvestre reduces the sweetness of methyl--D-mannopyranosidewithout reducing its bitterness. These results, predicted byprevious studies, contradict a recent hypothesis and reportby Birch and Mylvaganam. ¹Supported by NIH Grant 2-RO1-NS07873-9     

Endocrine, Gonadal and Behavioral Responses of Male Crucian Carp to the Hormonal Pheromone 17{alpha},20{beta}-dihydroxy-4-pregnen-3-one

The olfactory-mediated responses to the sex hormone 17,20ß-dihydroxy-4-pregnen-3-one(17,20ß-P) were studied in spermiated and regressedmale crucian carp (Carassius carassius L). The position andspontaneous locomotor activity of single male crucian carp werecontinuously recorded in an artificial stream. 17,20ß-P(final concentration 10^–11 M) was supplied to one halfand its ethanol carrier to the other half of the test area.Milt volume and gonadotropin (GtH-II) concentration in the plasmawere also measured. The smell of 17, 20ß-P significantlyincreased both the GtH-II concentration in the plasma and thevolume of strippable milt in spermiated crucian carp. Behaviorally,the side of the test area scented with 17,20ß-P wassignificantly avoided by spermiated males. None of the describedeffects of 17,20ß-P on spermiated males were observedfor the regressed crucian carp. In view of the lack of responsefrom regressed crucian carp we suggest that the observed avoidancebehavior of 17,20ß-P by spermiated males is a relevantreaction for spawning male crucian carp. The results are wellin accordance with responses obtained in the closely relatedgoldfish and gives strong support that the wild male cruciancarp use the 17,20ß-P signal from the females to preparefor the coming spawning.     

Structure-activity relationships in sweeteners. I. Nitroanilines, sulphamates, oximes, isocoumarins and dipeptides

Besides the hydrophilic AH-B moiety in sweeteners, the morehydrophobic ‘third binding sites’ play an essentialrole in inducing sweetness. The distances between these molecularfunctions seem to be very critical, but exact data are lacking.To describe stereochemical requirements more precisely, newconceptual parameters were introduced, namely , and (minimum,optimum and maximum distances between these third binding sitesand the atoms A, H and B of the AH-B moieties respectively,especially appropriate for homologous series) and the S value(shortest distance between the position of an atom and the planeformed by the atoms A, H and B of the AH-B moiety). The dimensionsof the relevant side chains of five homologous series of sweeteners– sulphamates, oximes, nitroanilines, isocoumarins anddipeptides — were determined. We calculated that the positionsof the , and parameters with regard to the AH-B moieties arelocated around two main axes forming 95? angles with the H-Baxis in the AH-B moieties for sulphamates and isocoumarins and125? angles for oximes, nitroanilines and dipeptides. The positionsof are, for all potent sweeteners, situated at 70—85%of the maximum distance of viewed from the centre of the Aatom, while for , this value was found to be 15% for oximes,25% for nitroanilines, 40% for sulphamates, 50—70% fordipeptides and 65% for isocoumarins. The results indicate thereare at least three — but a maximum of four — differentreceptor sites. They have very narrow site clefts with maximumheights of -0.6 nm.     

Structure-activity relationships in sweeteners. II. Saccharins, acesulfames, chlorosugars, tryptophans and ureas

The previously introduced conceptual parameters (, , and S)to describe the stereochemical requirements for organic compoundsto taste sweet, were now applied to another series of sweetenersand to some well-known potent substances. With the help of anadapted STERIMOL computer program, the positions of relevant,hydrophobic side chains were determined in ureas, saccharins,tryptophans, chlorosugars and acesulfame derivatives in relationto their AH-B moieties. The results obtained were compared withprevious findings for five other homologous series of sweeteners.There is evidence to suggest that 6-substituted acesulfame derivativesand saccharin employ the same receptor site. in 5-substitutedacesulfame derivatives coincides with that of sulphamates calculatedearlier. in 6-chloro-D-tryptophan was found to be at equaldistances from H and B, a position which was earlier also observedfor the methyl ester group in aspartame. In the dulcin seriesof the urea derivatives, two AH-B moieties can be distinguished:the HN-CO group gives rise to , and 's which fit in the earliercalculated nitroaniline receptor site, while for the OC-NH₂group they are located near those found for isocoumarins. Thechlorine atoms in 1' '₁6'-dichlorosucrose are located aboveand below the plane of the pyranose ring at almost the samepositions with respect to the OH groups at positions 3 and 4(in fact, two equal 's), which are supposed to form the AH-Bmoiety. The high relative sweetness values of 1',6'-dichlorosucroseand 1',4,6'-trichlorogalactosucrose are most probably due tothe fact that both sweeteners can interact with the receptorsite in two ways (as such and upside-down). It is remarkablethat the average positions belonging to sweeteners with similarAH-B moieties are located very close to each other.     

Regulation of sexual agglutinability in Saccharomyces cerevisiae of {alpha} and {alpha} types by sex-specific factors produced by their respective opposite mating types

The sexual agglutinability of haploid cells of heterothallicSaccharomyces cerevisiae was repressed when they were culturedin the absence of easily fermentable sugars, such as glucoseand mannose. The repression was reversed by the action of hormone-likesubstances of the opposite mating types. The substance producedby mating type cells was identical to subtsance-I which isknown to induce sexual agglutinability of inducible matingtype cells. The mating type cells produce a new hormone-likesubstance which induces or enhances sexual agglutinability of mating type cells. A crude fraction of the mating type-specific substance ( substance-I)was obtained by passing the culture filtrate of mating typecells through Amberlite CG-50 (H⁺ form), followed by elutionwith 1.5 M ammonia. ² On leave from Osaka City University. (Received December 25, 1975; )     

Developmental Responses to Drought and Abscisic Acid in Sunflower Roots: I. ROOT GROWTH, APICAL ANATOMY, OSMOTIC ADJUSTMENT

Aeroponically grown sunflower seedlings (Helianthus annuus L.cv. Russian Giant) were droughted or treated with abscisic acid(ABA) for 7 d. Drought stress prompted a three-phase growthresponse in sunflower roots: an initial phase of increased rootelongation was followed by a period of almost complete inhibitionbetween about 6 h and 72 h; this was followed, in turn, by aphase of partial recovery in the rate of root elongation. Droughtdecreased the size of the apical meristem as cells in the proximalregion of the meristem vacuolated and elongated. Root-to-shootbiomass ratios (R:S) increased initially but declined after72 h. Drought stress decreased water potential () and osmoticpotential ( and increased turgor pressure _p in the apical 30mm of the roots. These initial changes were transitory, lastingabout 3 h. Thereafter, and began to rise; _p fell back to controllevels. In the later stages of treatment, fell as the stressgrew more severe, but fp was maintained by osmotic adjustment.Desiccation for 1 h increased turgor pressures in excised 30mm apical segments. The transitory increase in root elongationwas contemporary with the initial rise in _p in the root apices,while the periods of greatest inhibition and partial recoveryin root elongation were contemporary with the periods of declineand partial recovery in the length of the apical meristem respectively.The inhibition of root elongation and the anatomical changesin the root apices were not determined by loss of turgor orlack of photosynthate, but rather appeared to be an active responseby the meristem to a drop in external . Treatment with ABA triggeredmany of the same changes as drought stress: ABA promoted a three-phasegrowth response, increased R:S, triggered the same initial changesin , , and _p, increased _p in excised 3.0 mm apical segments,and induced the same pattern of anatomical changes in the rootapices as drought stress. It is proposed that ABA mediates drought-inducedchanges in the primary development of sunflower roots. Key words: Abscisic acid, apical meristem, drought, osmotic adjustmen     

Isolation of Subunits of Coupling Factor 1 from Maize and Spinach Chloroplasts and Properties of Combinations of Subunits with ATPase Activity

Subunits (, ß, ) and mixtures of subunits ( ß, , ß , ß ) were isolated without denaturationfrom a chloroform extract of chloroplast coupling factor 1 (CF₁)from maize (Zea mays var. Ushiku 5-4) and from spinach by fastprotein liquid chromatography (FPLC), on an anion-exchange columnof Mono-Q in the presence of n-octylglucoside (OG) and on achromatofocusing column of Mono-P. The ß -subunitcomplex (CF¹ ß ) was the minimum unit required forATPase activity, as was confirmed by the reconstituted complexof ß and subunits. An subunit isolated from maizeinhibited the ATPase activity of CF₁ ß from bothmaize and spinach. CF₁ ß was found to contain anOG-dependent Mg²⁺-ATPase. The ATPase activity of CF₁ ß required divalent cations, such as Mg²⁺ or Mn²⁺, for its expressionin the presence of OG; its optimum pH was 8.0 and it was markedlyinhibited by NaN₃. The enzyme hydrolyzed ATP in prefernece toGTP but not CTP, UTP, ADP, AMP or pNPP. Lineweaver-Burk plotsof its activity were curvilinear in the range of 0.6–0.7mM ATP.Mg²⁺. ¹Present address: Department of Biology, School of Education,Waseda University, Shinjuku-ku, Tokyo, 160 Japan. (Received February 15, 1989; Accepted April 20, 1989)     

Alternative Methods of Analysing Water Potential Isotherms: Some Cautions and Clarifications: I. THE IMPACT OF NON-IDEALITY AND OF SOME EXPERIMENTAL ERRORS

In recent years alternative ways have been proposed to transformmeasurements of leaf water potential, , and relative water content,R^*, in order to derive values of osmotic pressure at full turgidityin leaves and shoots, ^o(when 0). Two types of transformationsare usually considered: 1/ versus R^* and versus 1/R^*, and linearregression is used to fit the data in the region where turgoris thought to be zero. It appears that when ^o is estimated bylinear extrapolation of 1/Psi; versus R^* then apoplastic watermight not influence the accuracy of ^o but when the versus \/R^*transformation is used apoplastic water causes an underestimateof ^o. We examine the accuracy of the estimate of ^o obtainedfrom the two transformations when there are random errors in, systematic errors in , and when the osmotic solutions arenon-ideal. The 1/ versus R^* transformation generally producesthe best estimate of ⁰ by linear extrapolation.     

G-protein {beta}{gamma} Subunit Genes Expressed in Olfactory Receptor Neurons

The expression of genes encoding G-protein ß subunitswas investigated in isolated olfactory receptor neurons fromchannel catfish. DNA sequencing of PCR products showed thatthe ß1, ß2, 2 and 3 genes were expressedin the neurons. Western blotting showed that at least threeof these subunit proteins were expressed. This first analysisof the expression of ß genes in olfactory receptorneurons suggests that these subunits may be involved in a varietyof transduction events in these cells. Chem. Senses 22: 587–592,1997.     

Binding and metabolism of the urinous odorant 5{alpha}-androstan-3-one in sheep olfactory mucosa

The existence of specific receptors for the urinous smellingsteroid 5-androstan-3-one has been investigated in sheep olfactorymucosa using ligand-binding methods. [16,17-³H]5-Androstan-3-one(52.5 Ci/mmol) was synthesized from the precursor 5-androst-16-cn-3-one,which was prepared by two novel methods. A specific bindingcomponent, absent from control tissues was identified. The affinityconstant was 9.6 ± 6.1 x 10⁸ M^-1 (mean ± SD) andcompetition experiments with other compounds indicate high specificity.Binding activity was localized in a high M_r tissue fractionand was distinct from 3-ketosteroid dehydrogenase and otherenzymatic activity.     

Adaptation of the Euryhaline Charophyte Lamprothamnium papulosum to Brackish and Freshwater: Turgor Pressure and Vacuolar Solute Concentrations During Steady-State Culture and After Hypo-osmotic Treatment

The euryhaline charophyte Lamprothamnium papulosum (Wallr.)J. Gr. was adapted to media with decreasing salinities rangingfrom 550 to 0 mosmol kg^–1. Vegetative plants grown inmedia with osmotic pressures (⁰) in the range of 550 to 130mosmol kg^–1 maintained a constant turgor pressure () at309 + 7 mosmol kg^–1. The ions K+, Na+ and Cl–, werethe predominant solutes in the vacuole. Changes in their concentrationsaccount for the variation in internal osmotic pressure (¹) with,⁰. The divalent ions Mg²⁺, Ca²⁺ and were also present in significant amounts, but their concentrationsdid not alter with changes in, ⁰. In cells subjected to hypo-osmotic shock the regulation of was incomplete. The turgor pressure increased from 302 to 383mosmol kg^–1. The first rapid response to the sudden decreasein ⁰ was a loss of K⁺ and Cl^–. In contrast to the decreasein ionic concentrations an accumulation of sucrose occurredwhich could account for the increase of . The increase in sucroseconcentration started 24 to 48 h after the downshock and reachedits highest value after 3 to 4 weeks. The sucrose concentrationin the vacuole was up to 320 mol m^–3. During this timethe ionic content continued to decrease but did not counterbalancethe sucrose concentration sufficiently to regain the original. High sucrose levels accompanied by an enhanced were also observedduring the period of fructification (sexual reproduction: formationof antheridia and oogonia) in Lamprothamnium kept under conditionsof constant salinity. It is concluded that high sucrose content and elevated arecharacteristic of sexual reproduction in this charophyte. Lamprothamniumis able to tolerate different during various developmentalstages (e.g. vegetative and reproductive phases). Key words: Lamprothamnium papulosum, sucrose, turgor pressure     

The Incorporation of C14-labelled Substrates into the Amino-acids of Groundnut Plants (Arachis hypogaea)

The amino-acid metabolism of groundnut plants has been studiedwith special reference to -methyleneglutamic acid (-MGA) andy-methyleneglutamine (-MG), constituents not found in the greatmajority of plant species. The sequence in which C¹⁴ from radioactive C¹⁴-carbon dioxideenters the amino-acids of leaves was determined. The patternof labelling was very similar to that found for leaves of otherspecies. The metabolic relationships existing between photosynthesisand amino-acid synthesis therefore do not seem to be affectedby the large quantities of -MGA and -MG present in the leaves.-MGA and -MG only gained traces of radioactivity. Experiments designed to study the incorporation of C¹⁴ fromuniformly labelled C¹⁴-alanine into the amino-acids of roots,immature leaves and cotyledons of seedlings and young plantsindicated that the main site of synthesis of -MGA was the cotyledons. Various C^I4-labelled substrates were fed to germinating seedsand, after a period of growth, the specific activities of theamino-acids of seedlings receiving different treatments weredetermined. Comparison of the specific activities enabled certaindeductions to be made concerning the probable biosynthetic pathwaysleading to -MGA and -MG. The results were consistent with theintact incorporation of pyruvate molecules, or another related3-carbon-atom containing compound, into -MGA.     

Odor thresholds of some derivatives of strawberry aldehyde

The olfactory threshold concentrations of 19 derivatives ofethyl ß-methyl-ß-phenyl-,ß-epoxypropionate(strawberry aldehyde) were determined in water solution usinguntrained panellists. In spite of their similar carbon skeletons,the compounds differed considerably in their odor thresholdvalues. The lowest and highest values were found to be 0.5 p.p.m.(0.5 parts of the odorant per 10⁶ parts of water) for ß-(bromophenyl)-,ß-epoxypropionateand 720 p.p.m. for n-octyl-,ß-epoxy propionate, respectively.     

Characterization of Spring Wheat Genotypes Differing in Drought-Induced Abscisic Acid Accumulation: II. LEAF WATER RELATIONS

Aspects of the water relations of spring wheat (Triticum aestivumL.) are described for cultivars Highbury (low ABA) and TW269/9(high ABA), and low and high ABA accumulating F⁶selections derivedfrom a cross between them. In a pot experiment, pressure-volume (P-V) curves were constructedfor main stem leaf four (MSL4) of well-watered plants of Highburyand TW269/9. Estimates of solute potential (²) from these curveswere similar for the two cultivars, but varied with the timeof sampling and the time allowed for hydration in dim light. In a field experiment with four low and four high ABA F⁶lines,P-V curves for flag leaves from both droughted and irrigatedplants gave at both zero turgor (^p) and zero water potential(¹) which differed with degree of stress, sampling time andgenotype. ¹was strongly dependent on the initial_L of the leafand was reduced on average by c. 0.4 MPa per MPa decline ininitial L.5, was lower (more negative) by c. 0.1-MPa in theafternoon than in the morning. Overall, was also 0.1 MPa lowerin low ABA lines than in high ABA lines. In another field experiment, flag leaves of five low and fivehigh ABA F⁶lines were sampled over a 4 week period from droughtedplots and _L and 5, measured (the latter by osmometry with expressedsap). For these leaves 5, at zero p or zero _L was consistentlylower by 0.3–0.5 MPa than estimates of 5, from the P-Vcurves with flag leaves. However, data for the low ABA lineswere again lower (by c. 0.1 MPa) than those for high ABA lines. The consequences of these differences in 1 are discussed inrelation to the stimulation of ABA accumulation in low and highABA selections. Key words: Water potential, Solute potential, P-V curves, Wheat (Triticum aestivum), Drought stress     

Nucleoside Triphosphate(NTP)-Binding Proteins and Endogenous ADP-ribosyl Transferase in Neurospora crassa

Nucleoside triphosphate(NTP)-binding proteins were detectedin the crude extract of mycelia of Neurospora crassa, whichwas treated with 1% Lubrol PX and fractionated by gel filtration.Protein fractions showing the capacity to bind [³⁵S]ATPS or[³⁵S]GTPS were designated as AGN1 to 6. The binding of [³⁵S]ATPSor [³⁵S]GTPS was prevented in the presence of 0.1 mM ATP orGTP except that in fractions AGN1 and 2, the presence of GTPstimulated the binding of [³⁵S] ATPS to ATP(NTP)-binding proteins.ATP or GTP was 1 to 2 orders of magnitude more effective thanCTP or UTP in preventing the binding of [³⁵S]GTPS in AGN1, 2and 5. Among these fractions AGN1, 2, 5 and 6 showed activityto hydrolyze 1 nM [–³²P]ATP or [–³²P]GTP. NTP-bindingproteins bound with [³⁵S]ATPS or [³⁵S]GTPS had lower apparentmolecular weights than the same proteins without bound nucleotide.Proteins bound with [³⁵S]ATPS or [³⁵S]GTPS and those [³²P]ADP-ribosylatedby endogenous ADP-ribosyl transferase in each fraction wereanalyzed by SDS-PAGE. About 20 species of ATP or ATP-GTP-bindingproteins were detected, several of which were ADP-ribosylated.The binding of [³⁵S]ATPS or [³⁵S]GTPS to NTP-binding proteinswas confirmed by the comparison of non-boiled and boiled samplesimmediately before loading to SDS-PAGE. ATP, GTP, CTP or UTPat the concentration of 0.1 mM effectively removed [³³S]ATPSor [³⁵S]GTPS bound to NTP-binding proteins. (Received December 10, 1990; Accepted April 18, 1991)     

{delta}15N and {delta}13C Measurements of Antarctic Peninsula Fauna: Trophic Relationships and Assimilation of Benthic Seaweeds

Measurements of ¹³C, ¹⁵N, and C/N for a variety of Antarcticpeninsula fauna and flora were used to quantify the importanceof benthic brown algae to resident organisms and determine foodweb relationships among this diverse littoral fauna. ¹³C valuesranged from–16.8 for benthic algal herbivores (limpets)to –29.8 for the krill, Euphausia superba; the averagepooled value for brown macroalgae, including their attachedfilamentous diatoms, was–20.6. There was no correlationbetween biomass ¹³C or ¹⁵N with C/N content, and consequentlyboth ¹³C and ¹⁵N values were useful in evaluating trophic relationships.¹⁵N values of the fauna ranged from 3.1 to 12.5, with lowestvalues recorded in suspension feeders (e.g., bryozoans) andhighest values in Adelie penguins (12.5) collected in 1989.The comparatively lower ¹⁵N value for a Chinstrap penguin (6.9)collected in 1997 is attributed to the different dietary foodsources consumed by these species as reflected in their respective¹³C values. Significant amounts of benthic macroalgal carbonis incorporated into the tissues of invertebrates and fishesthat occupy up to four trophic levels. For many benthic andepibenthic species, including various crustaceans and molluscs,assimilation of benthic algal carbon through detrital pathwaysranges from 30 to 100%. Consequently, the trophic importanceof benthic brown algae may well extend to many pelagic organismsthat are key prey species for birds, fishes, and marine mammals.These data support the hypothesis that benthic seaweeeds, togetherwith their associated epiphytic diatoms, provide an importantcarbon source that is readily incorporated into Antarctic peninsulafood webs.     

{delta}15N signatures of marine mesozooplankton and seston size fractions in Kiel Fjord, Baltic Sea

The ¹⁵N of marine mesozooplankton species was measured on fouroccasions. Significant differences were found between copepodsand meroplanktonic larvae, yet not between holoplanktonic species.On average, mesozooplankton was enriched by 3.4 ± 0.9relative to selected seston size fractions. Despite suggestingsmall differences (0.5 to 1) in the ¹⁵N of different phytoplanktontaxa on one occasion, the size fractionation procedure generallyproved inadequate in separating major taxonomic groups composingseston. This circumstance, and phase-shifts in the transmissionof rapid changes (>2) in seston ¹⁵N to mesozooplankton complicatethe calculation of mesozooplankton trophic levels.     

An NTP-Binding Protein That Cross with a Ras-Specific Antibody in the Myceia of Neurospora crassa

A Ras-related NTP-binding protein was partially purified froma membrane fraction derived from the mycelia of Neurospora crassa.[-³²P]ATP and [-³²P]GTP were incubated with mem brane and solublefractions which were then irradiated with UV light to inducecrosslinking of tightly bound nucleotides. After SDS-polyacrylamidegel electrophoresis, blotting onto a nitrocellulose filter andautoradiography it was apparent that most of the proteins thatbound [-³²P]-GTP also bound [-³²P]ATP. Pretreatment of the membranefraction with Ras-specific antibody effectively blocked thebinding of [-³²P]ATP and [-³²P]GTP to several ATP-GTP-bindingproteins. The band of a protein with a molecular weight of 26kDa on the SDS-polyacrylamide gel cross-reacted strongly withthe Ras-specific antibody. The protein was extracted from thegel and further purified by repeated gel electrophoresis. Thepurified protein bound [-³²P]ATP, [-³²P]-GTP, [-³²P]CTP and[-³²P]UTP at 1.6x10^– M and was autophosphorylated in thepresence of [-³²P]ATP and [-³²P]GTP at 1.7x10 M. Pretreatmentof the protein with Ras-specific antibody partially blockedthe autophosphorylation in the presence of these nucleotides.The binding of [-³²P]ATP to the NTP-binding protein was blockedby addition of ATP at 10^–4–10^–3 M. ATP ata concentration of 10^–4 M prevented the binding of [-³²P]to a greater extent than did GTP at the same concentration.Binding of [-³²P]CTP and [-³²P]UTP to the protein was also observed. (Received October 7, 1991; Accepted July 14, 1992)     

Phosphorylation and subcellular location of {alpha}-L-fucosidase in Iymphoid cells from patients with I-cell disease and pseudo-Hurler polydystrophy

Mannose 6-phosphate is a recognition marker used by many newlymade acid hydrolases for their transport to lyso-somes. Previously,we investigated the incorporation of ³²Pi into -L-fucosidaseof lymphoid cell lines from a healthy individual (control) andan I-cell disease patient [DiCioccio and Miller, Glycobiology,1, 595–604 (1991)]. Phosphoserine was found in immunoprecipitable-L-fucosidase of both control and I-cell lymphoid cells, butmannose 6-phosphate was identified only in enzyme of controlcells. Extension of this investigation to lymphoid culturesof a pseudo-Hurler polydystrophy patient also identified onlyphosphoserine in -L-fucosidase. Using [³H] mannose instead of³²Pi, the precise identification of mannose 6-phosphate in -L-fucosidaseof control cells, and its absence in -L-fucosidase of I-celland pseudo-Hurler cells, was established. The stoichiometryof phosphorylation of -L-fucosidase in I-cell, pseudo-Hurlerand control lymphoid cells was 3, 4 and 10 mol Pi/mol enzyme,respectively. -L-Fucosidase was located in lysosomes isolatedfrom control, I-cell and pseudo-Hurler lymphoid cells by subcelluarfractionation on Percoll density gradients. Both I-cell andpseudo-Hurler lymphoid cells displayed normal intralysosomalactivity of -L-fucosidase despite lack of the mannose 6-phosphatemarker. Thus, I-cell and pseudo-Hurler lymphoid cells must possessa mannose 6-phosphate-independent mechanism for directing -L-fucosidaseto lysosomes. Phosphorylation of -L-fucosidase in pseudo-Hurlerand I-cell lymphoid cultures was found almost exclusively inintracellular and not in extracellular enzyme, suggesting thatphosphoserine may participate in the localization of -L-fucosidasein lysosomes of these cells. -L-fucosidase I-cell disease lysosome phosphorylation pseudo-Hurler polydystrophy     

3D Confocal Laser Scanning Microscopy for the Analysis of Chlorophyll Fluorescence Parameters of Chloroplasts in Intact Leaf Tissues

We analyzed the chlorophyll fluorescence parameters in a 3Dcellular arrangement in vivo by using a modified Nipkow disk-typeconfocal laser scanning microscope (CLSM). We first definedthe 3D values of _PSII (photochemical yield of PSII) and NPQ(non-photochemical quenching) in mesophyll, epidermal and guardcell chloroplasts from the leaf surface to several tens of micronsin depth. We also used this CLSM method to analyze the relationshipsbetween actinic light intensity and the chlorophyll fluorescenceparameters for Boston fern and broad bean leaf specimens. Asthe actinic light intensity increased, the mean _PSII valuesdecreased and the NPQ values increased in all chloroplasts ofBoston fern and broad bean leaf. These values differed withcell type and species. The Boston fern chloroplasts had lower_PSII values than the broad bean chloroplasts, and vice versafor the NPQ values. The _PSII values of Boston fern chloroplastsdecreased in the order mesophyll, epidermal and guard cell chloroplasts.The NPQ values decreased in the order guard cell, mesophylland epidermal chloroplasts, except at 12 µmol m^–2s^–1 actinic light, when the mesophyll value was slightlylower than that of the epidermis. The trend in the _PSII andNPQ values of broad bean mesophyll and guard cell chloroplastswas opposite to that of Boston fern chloroplasts. As 3D CLSMcan provide the _PSII and NPQ values of each chloroplast in a3D cellular arrangement, this method has potential for investigatingdifferences in the functions of chloroplasts in vivo.