ON THE GUSTATORY EFFECTS OF GYMNEMIC ACID AND MIRACULIN IN DOG, PIG AND RABBIT

The effects of gymnemic acid, 1 mg/ml, and miraculin, 1 mg/mlor in a tablet (Miralin^TM) on the taste response to sucrose,citric acid, NaCl and quinine have been studied in dog, pigand rabbit. In the dog gymnemic acid suppressed the responseto sucrose for about 5 min in two out of three animals. It alsoaffected the response to NaCl. Miraculin caused a slight short-lastingincrease in the response to acid. No effects of the taste modifierswere observed in the pig. In the rabbit gymnemic acid did notsuppress the response to sucrose while miraculin enhanced theresponse to citric acid in one out of three animals for a fewminutes.     

Extraction, purification and characterization of a sweetnessmodifying component from Ziziphus jujuba

An aqueous ethanol extract of Ziziphus jujuba leaves was fractionatedby solvent extraction. The active chloroform-ethanol fractionwas separated into two components - ZjE-A, which had surfaceactive properties, and ZjE-B, which did not. Bioassay by psychophysicaltests on humans revealed sweetness-modifying activity in ZjE-A,but not in ZjE-B. The combined chemistry and bioassay resultsindicated that the active component is either closely associatedwith, or is itself, an amphipathic molecule and supported thenotion of a role for surface active properties in the tastemodifyingaction of Z. jujuba. Thin layer chromatography (TLC) and otherdata indicated and/or suggested that the active component inZ. jujuba consists of a group of compounds (ziziphins), whichprobably are triterpene saponin glycosides, as had been indicatedpreviously for the taste-modifying component in Gymnema sylvestre(gymnemic acids). However, the ziziphins differed from the gymnemicacids in TLC mobility and in coloration by a Liebermann-Burchardreagent. Therefore, the ziziphins are not the same compoundsas the gymnemic acids. ZjE-A is a potent, purified component,consisting of 60–80% ziziphins, which should be usefulfor future physiological and chemical studies. ^*A preliminary report of this research was presented at theChemical Senses and Intake Society Annual Meeting, Natick, MA,April, 1977. An earlier version of this paper is included inKennedy,L.M., Ph. D.Thesis, Harvard University, 1979 ^**Now at the Worcester Foundation for Experimental Biology,Shrewsbury, MA 01545, USA, Reprint requests should be sent tothis address     

EFFECT OF MANGANESE IONS ON THE IAA-INDUCED ELONGATION OF AVENA COLEOPTILE SECTIONS INHIBITED BY SOME ORGANIC ACIDS

1.Organic acids, such as citric, -ketoglutaric, succinic, fumaricand L-malic acids, inhibit the IAA-induced growth of Avena coleoptilesections. But pyruvic acid has no effect on the growth. 2.High concentrations of MnCl₂ (for example 10^–3 m) alleviatethe inhibition due to L-malic, -ketoglutaric, succinic and fumaricacids, but not that due to D-malic, tartaric and malonic acids. 3.A mechanism of the alleviating effect of Mn⁺⁺ on the inhibitiondue to the organic acids is discussed with the reference tothe activating effect of Mn⁺⁺ on "malic" enzyme. ¹Contribution No. 6 from the Botanical Gardens. Faculty of Science,University of Tokyo, Koishikawa, Tokyo.     

Qualitative psychophysical studies on the gustatory effects of the sweet tasting proteins thaumatin and monellin

The gustatory effects of the sweet tasting proteins thaumatinand monellin were studied aftei application to small areas onthe anterior third of the tongue or to single fungiform papillae.The sweet sensation caused by thaumatin and monellin developedmore slowly, but reached a higher intensity and had a longerduration than that given by sucrose. Also, the response evokedby these sweet tasting proteins was more pronounced at the lateraledges, whereas that evoked by sucrose was stronger at the tipof the tongue. The taste modifier, miraculin, had no noticeableeffect on the sweet taste elicited by thaumatin, monellin andsucrose. Gymnemic acid abolished the sweet taste of all threecompounds. Experiments with time intervals of less than one minute betweenstimuli showed strong crossadaptation between thaumatin andmonellin, between the two proteins and sucrose, and betweenthe two proteins and miraculin-induced sweet taste of citricacid. While the differences in response to the sweet tasting proteinsand sucrose may be taken as evidence in favor of the existenceof more than one kind of sweet receptor, the cross-adaptationnoted between the various substances tested, would seem to indicatethat, at some point, they engage a common neural mechanism. ¹On leave from Dept. of Prosthetics, Faculty of Odontology,Karolinska Institutet. Present address: Dept. of Histology,Karolinska Institutet, S-104 01 STOCKHOLM, Sweden.     

EFFECTS OF PHOTOPERIOD AND GIBBERELLIN ON THE GERMINATION OF SEEDS OF BEGONIA EVANSIANA ANDR.

1. Seed germination of Begonia Evansiana ANDR. was investigatedat 29?C.
2. The germination was induced under long-day conditions,the criticaldaylength being about 8 hours. Exposure to at least2 or 3 cyclesof long days was necessary for germination. Theseeds couldgerminate under otherwise non-inductive photoperiods,when thedark period was interrupted with a short period ofillumination.Thus the photoperiodic behaviour of Begonia seedsin germinationis similar to that of typical long-day plantsin flowering.
3. The application of gibberellin brought aboutno germinationin complete darkness, but markedly reduced thecritical daylengthfor germination, even 1-minute photoperiodsbeing inductive.The germination under continuous light wasalso favoured bygibberellin application. The action of gibberellinin germinationof Begonia seeds may be to intensify the lightaction or tosubstitute for a part of it.

¹Present address: Dept. of Botany, Hokkaido University, Sapporo. (Received October 19, 1959; )     

Effect of temperature on the ethylene-synthesizing systems in apple, tomato and Penicillium digitatum

Arrhenius plots of ethylene-synthesizing systems of apple andtomato showed discontinuities while the plot for Penicilliumdigitatum was linear. Triton X-100 markedly lowered the activationenergies of the apple and tomato systems without altering thatof the fungal system. The data presented suggest that the lipidmicro-environment of the ethylene-synthesizing enzyme systemsin higher plants and P. digitatum are different, and that cellmembrane-cell wall complex may be the site of ethylene-synthesizingenzymic systems in higher plants. ¹ On leave from the M. S. University of Baroda, India. ² On leave from the Agricultural Research Organization, TheVolcani Center, Israel. (Received February 7, 1977; )     

CHANGES IN HYDROGENASE ACTIVITY DURING THE SYNCHRONOUS GROWTH OF SCENEDESMUS OBLIQUUS D3

1. A fairly good synchronization of Scenedesmus cells was obtainedby transferring the cells grown in a medium containing a lowconcentration of iron into a medium containing relatively highconcentration of iron.
2. During the synchronous culture in themineral medium, a goodparallelism between the average cellvolume and hydrogenaseactivity was observed.
3. Effect of glucoseon the development of the hydrogenase activitywas variabledepending on the stage of algal growth.
4. Iron is essentialfor the development of the hydrogenase activityand glucosesupplementary.

¹On leave from Laboratory of Applied Botany, Faculty of Agriculture,Kyoto University, Kyoto.     

On the sense of taste in two Malagasy Primates (Microcebus murinus and Eulemur mongoz)

The relationship between phylogeny and taste is of growing interest.In this study we present recordings from the chorda tympaniproper (CT) nerve of two lemuriforme primates, the lesser mouselemur (Microcebus murinus) and the mongoose lemur (Eulemur mongoz),to an array of taste stimuli which included the sweeteners acesulfame-K,alitame, aspartame, D-glucose, dulcin, monellin, neohesperidindihydrochalcone (NHDHC), saccharin, sodium superaspartame, stevioside,sucralose (TGS), sucrose, suosan, thaumatin and xylitol, aswell as the non–sweet stimuli NaC1, citric acid, tanninand quinine hydrochloride. In M.murinus the effects of the tastemodifiers gymnemic acid and miraculin on the CT response wererecorded. Conditioned taste aversion (CTA) experiments in M.murinusand two-bottle preference (TBP) tests in E.mongoz were alsoconducted. We found that all of the above tastants except thaumatinelicited a CT response in both species. The CTA technique showedthat M.murinus generalized from sucrose to monellin but notto thaumatin. The intake of aspartame, ranging in concentrationfrom 0.1 to 30 mM was measured in E.mongoz with TBP tests. Atno concentration did we see a preference, but there was a significantrejection of 10 and 30 mM aspartame (P     

Purification and properties of a nitrite reductase from Porphyra yezoensis Ueda

Nitrite reductase was extracted from the red alga Porphyra yezoensisUeda and purified through precipitation with ammonium sulfate,column chromatographies, and polyacrylamide gel disk electrophoresis.The enzyme preparation thus obtained showed a single band ondisk electrophoresis. The absorption spectrum had three maxima at 385 nm (Soret band),580 nm (-band), and 278 nm; the ratio of absorbance of the Soretband to the -band was 4.3. The molecular weight and the numberof amino acid residues were estimated to be 63,000 and 601,respectively. The enzyme activity was optimal at around pH 7.5, and its activitywas heat labile as indicated by reduction of activity by about70% when heated at 37°C for 10 min. The enzyme used ferredoxin and methyl viologen, but not NADP⁺or NAD⁺, as the electron carriers. Moreover, reduced forms ofthe latter two showed no effect on its activity. Km values ofthis enzyme for NO₂^–, Fd, and MV were 8.1 x 10^–4M, 4.3 x 10^–8 M, and 3.7 x 10^–4 M, respectively.Almost half of its activity was lost when potassium cyanidewas added at a concentration as low as 10^–5 M, and theKi value was 1.8 x 10^–5 M. Thus, the nitrite reductaseof Porphyra must be systematically grouped in EC 1.7.7.1 [EC] . Itresembled closely that of Chlorella, except for the amountsof some amino acids. ¹ Present address: Department of Biological Sciences, Universityof Tsukuba, Sakura-Mura, Ibaraki, 300-31 Japan. ² Present address: Department of Fisheries, College of Agricultureand Veterinary Medicine, Nihon University, Shimouma, Setagaya-ku,Tokyo, 154 Japan. (Received June 10, 1975; )     

Compositions and Positional Distributions of Fatty Acids in Phospholipids from Leaves of Chilling-Sensitive and Chilling-Resistant Plants

The compositions and positional distributions of fatty acidsin the major leaf phospholipids of phosphatidylglycerol, phosphatidylcholineand phosphatidylethanolamine were analyzed by gas-liquid chromatographyand enzymic hydrolysis, and chilling-sensitive and chilling-resistantplants were comparcd with respect to the relative contents ofpalmitic and trans-³-hexadecenoic acids in the separated phospholipids.A distinct difference between these plants was found in thefatty acid compositions of phosphatidylglycerol, in which thesum of palmitic and trans-³-hexadecenoic acids ranged from 60to 78% of the total fatty acids in 8 species of chilling-sensitiveplants, and from 50 to 57% in 11 species of chilling-resistantplants. The only exception among the chilling sensitive plantsin this respect was the tomato, in which the sum of palmiticand trans-³-hexadecenic acids in phosphatidylglycerol amountedto 54%. The fatty acid compositions and the positional distributionsof fatty acids in phosphatidylglycerol suggest that the occurrenceof high proportions of dipalmitoyl and 1-palmitoyl-2-(trans-³-hexadecenoyl)species in this lipid is correlated with the susceptibilityto chilling of the leaves of higher plants. In the compositionsand positional distributions of fatty acids in phosphatidylcholineand phosphatidylethanolamine, no difference was found betweenthe chilling-sensitive and chilling-resistant plants. ¹ Present address: Department of Biology, Faculty of Science,Universityof Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan. (Received May 21, 1982; Accepted June 25, 1982)     

Assimilation and metabolism of glucose by Dunaliella tertiolecta I. Uptake by whole cells and metabolism by cell free systems

Dunaliella tertiolecta, a green euryhaline flagellate, is unableto use glucose as a substitute for photosynthetically fixedCO₂ to maintain growth. Glucose, acetate, pyruvate, succinate,sucrose, glycerol, alanine and -ketoglutarate do not stimulateendogenous respiration in this alga. By incubating whole cellswith these compounds labelled with ¹⁴C, it was shown that onlyacetate, pyruvate and glycerol penetrated the cell at rateswhich might affect growth. These rates were still only of theorder of 10 m/(moles/hr/mg protein. Only acetate and pyruvatewere metabolized to CO₂ at appreciable rates, 20 and 80% ofthe total assimilated, respectively. Cell free preparations of D. tertiolecta metabolized glucoserapidly, up to 2 µmoles/hr/mg protein, with over 75% ofthe ¹⁴C-label being recovered as triose phosphate. Both thehexose monophosphate shunt and the Embden-Meyerhof pathway wereactive. When specifically labelled glucose was supplied, CO₂from the C-6 carbon was released more rapidly than from theC-6 position, in both whole cells and in the cell free extract. It is concluded that the failure of D. tertiolecta to use glucoseis due to membrane impermeability, not lack of hexokinase. Apossible basis for this impermeability is discussed in the lightof the metabolic sequence which seems to be active in this alga. ¹Colombo Plan Fellow, 1968–69. Present address: NaturalProducts Research Institute, Seoul National University, Seoul,Korea. ²Present address: Biology Dept. Queen's University, Kingston,Ont., Canada. (Received August 13, 1970; )     

A biphasic model for action of the gymnemic acids and ziziphins on taste receptor cell membranes

A biphasic membrane penetration process for the action of thegymnemic acids and ziziphins on taste receptor cells is proposed.According to this model, the penetrating modifier moleculesinteract first, with the receptor cell plasma membrane surfaceand second, with the lipid interior of the membrane. The initialinteraction is postulated to involve a selective effect on processesfunctional in the transduction and quality specification ofa sweet stimulus and the second interaction to involve a generaldisruption of membrane function and nonselective effect on tasteperception. This biphasic model can account for available chemical,physiological and psyebophysical data, and it makes explicitpredictions which can be tested in future experiments. If themodel holds, it has important implications for the design andinterpretation of psychophysical, neurophysiological and chemicalexperiments that use the gymnemic acids and ziziphins as toolsto study taste perception. Moreover if the model holds, it placesat least one process involved in the specification of a sweetstimulus at the surface of the receptor cell membrane. ^*An earlier version of this paper is included in Kennedy.L.M.,Ph.D. Thesis, Harvard University, 1979 ^**Now at Worcester Foundation for Experimental Biology, Shrewsbury,MA 01545, USA. Reprint requests should be sent to this address.     

THE REPRODUCTIVE BIOLOGY OF THE AMPULLARIID SNAIL POMACEA URCEUS (MULLER)

Investigations of the reproductive biology, life cycle and populationdynamics have been undertaken to assess the potential of Pomaceaurceus as a culture species in Trinidad. The species is dioeciousand evidence from microscopic gonad analyses, together withmaturity indices and size frequency distributions in the population,all indicate that the reproductive cycle is annual. Adults spawnat the end of the rainy season months. Hatching and early developmentof the young occur in the dry season (January to May) whileadult females aestivate. The total developmental period variedbetween 22 to 30 days. Mean fecundity is 54 eggs/female witha range of 21 to 93 for egg masses deposited in the field andthose produced under laboratory conditions. ^*Present address: Zoology Dept., University of Aberdeen, TillydroneAve., Aberdeen AB9 2TN. Scotland. (Received 13 January 1988; accepted 11 April 1988)     

ORIGIN OF AMINO ACIDS CONSTITUTING CELLULAR PROTEIN IN GERMINATING CONIDIOSPORES OF ASPERGILLUS NIGER

Formation of pool amino acids in germinating spores of Aspergillusniger strain 1617 was investigated. The pool amino acids comprisedmainly glutamic acid and alanine. Small amounts of pyruvateand -ketoglutarate were found to increase almost in parallelwith the course of increase in the amount of free amino acidsup to the stage of onset of active protein synthesis. Asparticglutamictransaminase activity was exhibited even in dormant spores andit developed in response to the increase in cellular protein.Alanine-glutamic transaminase activity, on the other hand, waslacking in dormant spores and appeared at the stage of accumulationof amino acids preceding protein synthesis. It was revealed from the experiments with ³⁵S-labeled sporesthat the dormant spores of this fungus contain two unidentifiedsulfur substances, and the sulfur of these substances is incorporatedinto the sulfur amino acids of the protein synthesized in germinatingspores. ¹Present address: Institute of Applied Microbiology, Universityof Tokyo, Tokyo (Received September 11, 1959; )     

Purification and characterization of {delta}-aminolevulinic acid dehydratase from Chlorella regularis

-Aminolevulinic acid dehydratase (-aminolevulinic acid hydrolyaseEC 4.2.1.24 [EC] ) which catalyzes the formation ofporphobilinogenfrom two molecules of -aminolevulinic acid (ALA) was purifiedfrom Chlorella regularis 737-fold by acetone and ammonium sulfatefractionations, DEAE-cellulose column chromatography, and SephadexG-200 gel filtration. The enzyme had an optimum pH of 8.5 inTris-HCl buffer and required either Mg²⁺ or Mn²⁺ for its maximumactivity. The Km values for Mg²⁺, Mn²⁺ and ALA were 15 µM,10µM, and 0.5 mM, respectively. The enzyme was not activatedby thiol compounds, but was inhibited by p-chloromercuribenzoate.The molecular weight estimated by gel filtration was 316,000and the isoelectric point was 5.25. (Received October 18, 1978; )     

Effect of abscisic acid and auxin on ribonuclease during ageing of bean endocarp tissue sections

The activity of RNase increases rapidly upon cutting sectionsof bean (Phascolus vulgaris L. var. Kentucky Wonder) endocarp,peaks within 4 to 8 hr and then declines. This rapid developmentof RNase activity is inhibited by cycloheximide. Auxin (naphthaleneaceticacid, NAA) accelerates the rate of decline of RNase. Abscisicacid (ABA) enhances the level of RNase between 4 and 24 hr,associated with a decline in RNA, and this effect of ABA isobscured in the presence of auxin. ¹ This work was supported by National Science Foundation Grant(GB-8316) to J. A. Sacher. ² On leave from Laboratorio di Radiobiochimica ed EcofisiologiaVegetale, C. N. R., Roma, (Italy), with a Fellowship supportedby North Atlantic Threaty Organization. ³ Present address: Instituto di Botanica, Universita di Ban,Bari, 70126, Italy. (Received April 1, 1971; )     

Incorporation of some possible radioactive intermediates into chlorogenic acid in sliced sweet potato tissue

t-Cinnamic acid-2-¹⁴C, p-coumaric acid-2-¹⁴C and caffeic acid-2-¹⁴Cwere administered to discs of sweet potato roots and incorporationof each radioactive compound into chlorogenic acid was compared.The data suggest that chlorogenic acid is synthesized througheither or both of two major pathways, phenylalanine t-cinnamate t-cinnamoyl derivative p-coumaroyl derivative chlorogenicacid and phenylalanine t-cinnamate p-coumarate p-coumaroylderivative chlorogenic acid. ¹Part 75 of the phytopathological chemistry of sweet potatowith black rot and injury. ²Present address : Department of Biology, Tokyo MetropolitanUniversity, Setagaya-ku, Tokyo. (Received December 23, 1968; )     

ON THE NATURE OF MOSS OXALIC ACID OXIDASE

Moss oxalic acid oxidase freed from catalase by boiling is stronglyinhibited by the "metal-complexing" compounds such as thiocyanate,azide, diethyldithiocarbamate, and hydrosulfite. Inactivatedby dialysis against thiocyanate or azide, the enzyme can bereactivated to a considerable extent by the addition of ferricsalt, cytochrome-c or hemoglobin, not by other metal ions, suchas Cu²⁺, Zn²⁺ , Mn²⁺, and Fe²⁺. Nitrate, chlorate, monoiodoacetate,and iodide also act as strong inhibitors towards moss oxalicacid oxidase. Some enzyme fractions which were obtained by the sodium sulfateprecipitation method were stimulated by Fe³⁺, but not by cytochrome-cor by other metallic ions. This stimulation was inhibited bythiocyanate, azide and monoiodoacetate. ¹ Present address: Biological Institute, University of Toyama,Toyama     

Partial purification of a mannosyltransferase involved in the O-mannosylation of glycoproteins from Saccharomyces cerevisiae

The mannosyltransferase that catalyses the transfer of mannosefrom dolichyl-phosphate-mannose (Dol-P-Man) to the hydroxylgroup of serine/threonine residues in the acceptor peptide (Tyr-Asn-Pro-Thr-Ser-Val)was partially purified 150-fold from the microsomal membranefraction of Saccharomyces cerevisiae. The membrane-bound enzymewas solubilized with 0.5% Triton X-100 at a protein:detergentratio of 2: 1, and was then purified by ionexchange chromatographyon DEAE-cellulose, followed by hydroxyapatite column chromatography.The partially purified enzyme had a pH optimum of 7.2 and requiredMg²⁺ at an optimum concentration of 10 mM for activity. Theapparent mol. wt of the enzyme, as estimated by gel filtrationon Sephacryl S-300, was 125 kDa. The activity of the partiallypurified enzyme was greatly stimulated by phosphatidylcholine(PC), while other naturally occurring phosphoglycerides hadno significant effect.The extent of activation of mannosyltransferaseactivity was greatly affected by the number of carbons and thedegree of saturation/unsaturation of the fatty acid substituents,as well as by their position on the glycerol moiety of the PCmolecule. Maximum stimulation of the mannosyltransferase activitywas induced by a PC derivative in which both sn-1 and sn-2 positionson the glycerol moiety were occupied by C_12:0 fatty acids. Ingeneral, mannosyltransferase was found to exhibit greater specificityfor the L--PC derivatives in which the sn-2 position of theglycerol contained a saturated fatty acid. The mannosyltransferaseshowed a greatly reduced K_m value (five times lower) for thehexapeptide substrate in the presence of PC than in its absence,indicating that the stimulation of mannosyltransferase activitywas at least partially due to the increased affinity for theacceptor peptide. Upon ß-elimination of the radiolabelledmannosyl-peptide formed during the incubation of Dol-P-[¹⁴C]Manand unlabelled acceptor peptide with the partially purifiedenzyme, total radioactivity was released as mannose, confirmingthat a single mannose unit was linked to the serine/threonineresidues via an O-glycosidic bond. mannosyltransferase purification Saccharomyces cerevisiae ¹Present address: Department of Biosciences and Biotechnology,University of Roorkee,Roorkee 247 667, India ²Present address: Department of Biochemistry and Molecular Biology,University of Arkansas for Medical Sciences, Little Rock, AR72205, USA