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.     

Hodulcin: selective sweetness-reducing principle from Hovenia dulcis leaves

An aqueous extract of Hovenia dulcis leaves selectively reducedsweetness perception in humans. The taste-active principle,‘hodulcin’, was partially purified and comparedchromatographically with similarly prepared samples of the selective,sweetness-reducing compounds, gymnemic acids and ziziphins.Hodulcin appears to be a triterpene saponin glycoside, as arethe gymnemic acids and ziziphins. Nuclear magnetic resonance(NMR) spectra indicated an hodulcin aglycone structure differentfrom the gymnemic acids aglycone, and similar to, but not thesame as the ziziphins aglycone. Future comparative studies ofthe actions of hodulcin, gymnemic acids and ziziphins couldelucidate physiological mechanisms for the transduction andidentification of sweet stimuli and aid the development of newapproaches to the sweetening of foods.     

ON THE GUSTATORY EFFECTS OF MIRACULIN AND GYMNEMIC ACID IN THE MONKEY

The summated response from the chorda tympani proper nerve of9 monkeys was recorded during stimulation with solutions ofacetic and citric acids, sodium chloride, quinine sulfate, sucrose,glucose and fructose before and after application of extractsof Synsepalum dulcificum-miraculin- and Gymnema sylvestre-gymnemicacid-on the tongue. It was observed that (a) miraculin enhancedthe response to all acids used (b) miraculin had no significanteffect on the response of the other taste stimuli (c) its effectlasts for more than h and was not removed by rubbing of thetongue (d) gymnemic acid had no significant effect on the responseto any of the stimuli used if miraculin had not been appliedbeforehand (e) gymnemic acid applied after miraculin diminishedthe response to acid, then miraculin enhanced the response toacid again. It was concluded that these electrophysiologicalfindings in monkey parallel the psychophysical observationsin man with regard to the effect of miraculin and gymnemic acidon the response to acids, but that they differ with regard tothe effect of gymnemic acid on the response to sugars. ^* On leave from Dept. of Psychology, University of New Hampshire,U.S.A. ^** On leave from Dept. of Oral Physiology, Osaka University,Japan.     

Protein Synthesis Linked to Respiration and Phosphorylation in Intact Euglena Mitochondria

Highly purified condensed mitochondria obtained from bleachedmutant. W₁₀BSmL of Euglena gracilis Klebs var bacillaris Coriincorporate [³⁵S]methionine into protein when fortified withmalate, ADP, Mg²⁺, phosphate and a sucrose osmoticum. Twentyto twenty-five polypeptide bands were found to be labeled inorganello when the labeled protein was subjected to sodium dodecylsulfatepolyacrylamide gel electrophoresis. Methionine incorporation,but not respiration or oxidative phosphorylation, was blockedby chloramphenicol and other 70S ribosomal translation inhibitorsbut cycloheximide and ribonuclease were without effect. Inhibitorsof electron transport and uncouplers of oxidative phosphorylationwere excellent inhibitors of protein synthesis. Thus, thesemitochondrial preparations carry out protein synthesis in organellothat is linked to respiration and oxidative phosphorylation. ¹Present address: VA Hospital Outpatient Clinic, 17 Court St.,Boston, MA 02115, U.S.A. ²Present address: Laboratories de Microbiologia e Inmunologia,Universidad Catolica de Chile, Casilla 114-D, Santiago, Chile. ³Present address: Botany Department, University of Massachusetts,Amherst, MA 01003, U.S.A. (Received June 17, 1985; Accepted October 28, 1985)     

Molecular Cloning and Characterization of S-Adenosyl-L-Methionine:Scoulerine-9-O-Methyltransferase from Cultured Cells of Coptis japonica

S-Adenosyl-L-methionine : scoulerine-9-O-methyltransferase (SMT)catalyzes the transfer of the S-methyl group of S-adenosyl-L-methionineto the 9-hydroxyl group of scoulerine during the biosynthesisof berberine. We have isolated functionally active cDNA clones(pCJSMTs) from a cDNA library prepared from cultured cells ofCoptis japonica. The longest cDNA insert (pCJSMT1) had an openreading frame that encoded 351 amino acids, but the calculatedmolecular mass (38,364 Da) of the deduced product was slightlylower than the experimentally determined molecular mass of purifiedSMT. Rapid amplification of the 5' end of the cDNA indicatedthat the full-length cDNA of SMT consisted of 1,458 nucleotidesthat encoded 381 amino acids. When the full-length cDNA wasexpressed in E. coli, the molecular mass of the expressed SMTwas greater than that of native SMT in Coptis cells. This resultsuggests that SMT might be produced in a pre-mature form andprocessed post-translationally. SMT was also found to exhibitsequence homology to other O-methyltransferases from plantsand N-terminal region of the SMT polypeptide appeared to benecessary for enzymatic activity. ¹Present address: High Quality Life Research Laboratories, SumitomoMetal Industries, Ltd., 3-5 Hikaridai, Seika, Sourakugun, Kyoto,619-02 Japan ²Present address: Suntory Research Center, 1-1-1 Wakayamadai,Shimamoto, Mishima-gun, Osaka, 618 Japan ³Present address: Department of Cell Biology, The Scripps ResearchInstitute, La Jolla, CA 92037 U.S.A.     

Isolation and characterization of the 337 m{micro} UV-absorbing substance in red alga, Porphyra yezoensis UEDA

Using a variety of Sephadex gel filtrations, starch block zoneelectrophoresis, Avicel SF preparative TLC and DEAE cellulosecolumn chromatography, the characteristic 337 mµ UV-absorbingsubstance from marine red alga, Porphyra yezoensis UEDA wasisolated. The molecular weight of this substance was about 1,000,and its chemical composition was 43.11% C, 5.85% H, 7.23% N,34.73% O and 9.08% Na. In vivo it is located in the chloroplast.On irradiation at 378 mµ, it has a fluorescence actionspectrum peak at 470 mµ. Structural studies on this substanceare still underway, but it could be a kind of aminosugar judgingfrom NMR, IR spectra and other chemical properties. ¹This work was partially supported by the grant of the Ministryof Education in 1969 ²Present address: Department of Biochemistry and Biophysics,University of Hawaii, Honolulu, Hawaii 96822, U.S.A. (Received December 21, 1969; )     

Mechanisms of the acido- and thermophily of Cyanidium caldarium Geitler VI. Some physicochemical properties of acid and heat stable cytochrome c-553

An acid and heat stable cytochrome of c-type was purified froman acidophilic and thermophilic unicellular alga, Cyanidiumcaldarium. Physicochemical properties and acid and heat stabilitiesof the cytochrome were examined and compared with those of mesophilicalgal cytochromes. ¹ Based on a dissertation submitted to the Tokyo MetropolitanUniversity in partial fulfillment of the requirements for thedegree of Doctor of Science. (Received October 24, 1977; )     

Comparison of carbon dioxide fixation and the fine structure in various assimilatory tissues of Amaranthus retroflexus L

The pattern for primary products of CO₂-fixation and the chloroplaststructure of Amaranthus retrqflexus L., a species which incorporatescarbon dioxide into C₄ dicarboxylic acids as the primary productof photosynthesis, were compared in various chlorophyll containingtissues,i.e., foliage leaves, stems, cotyledons and pale-greencallus induced from stem pith. Despite some morphological differencesin these assimilatory tissues, malate and aspartate were identifiedas the major compounds labelled during a 10 sec fixation of¹⁴CO₂ in all tissues. Whereas, aspartate was the major componentin C₄-dicarboxylic acids formed in foliage leaves, malate predominatedas the primary product in stems, cotyledons and the pale-greencallus. The percentage of ¹⁴C-radioactivity incorporated intoPGA and sugar-P esters increased and ¹⁴C-sucrose was detectedin the prolonged fixation of ¹⁴CO₂ in the light, not only infoliage leaves, but also in stems and cotyledons. ¹ This work was supported by a Grant for Scientific ResearchNo. 58813, from the Ministry of Education, Japan. ² Present address: Institute of Applied Microbiology, Universityof Tokyo, Tokyo, Japan. ³ Present address: Department of Biochemistry, University ofGeorgia, Athens 30601. Georgia, U. S. A. (Received July 10, 1971; )     

Properties of S-adenosyl-L-methionine-magnesium-protoporphyrin IX methyltransferase from barley

S-Adenosyl-L-methionine-magnesium-protoporphyrin IX methyltransferase(EC 2.1.1.11 [EC] ) is present in greening barley seedlings associatedwith the particulate fraction. This enzyme was purified 20 foldusing protamine and ammonium sulfate precipitation. The enzymewas active over a wide pH range with highest activity at pH7.5. The Km values for Mg-protoporphyrin IX and S-adenosylmethioninewere 48 and 39 µM, respectively; S-adenosylethionine andS-adenosyihomocysteine were competitive inhibitors with respectto S-adenosylmethionine; hemin inhibition was non-competitivewith respect to Mg-protoporphyrin IX; thiol compounds exhibiteda stimulatory effect on enzyme activity. The properties of theenzyme are discussed and compared with the enzyme from otherorganisms. ¹ This research was supported in part by the Utah State AgriculturalExperiment Station. ² Present address: Department of Chemistry, Boston University,Boston, Massachusetts, U. S. A. ³ Present address: Department of Biochemistry and Microbiology,Faculty of Pharmacy, Comenius University, Bratislava, Czechoslovakia. (Received February 20, 1978; )     

Sodium Requirement of Monocotyledonous C4 Plants for Growth and Nitrate Reductase Activity

The effects of Na application on growth and nitrate reductaseactivity of seven C₄ plant species, Zea mays, Echinochloa crus-galli,Panicum miliaceum, Panicum coloratum, Panicum dichotomiflorum,Panicum maximum and Chloris gayana were studied. Except forZ. mays and P. miliaceum, Na application enhanced growth significantly,and concurrent increases in nitrate reductase activities weredetected in Panicum coloratum, Panicum dichotomiflorum, Panicummaximum and Chloris gayana. ¹Present address: International Research Institute, Ciba GeigyJapan Ltd., Takarazuka, Hyogo 665, Japan. ²Present address: Photobiology Lab., Research Institute forFood Science, Kyoto Univ., Uji, Kyoto 611, Japan. (Received May 2, 1988; Accepted August 22, 1988)     

Comparison of Some Catalytic Properties of the Purified and Membrane-Bound Reduced Nicotinamide Adenine Dinucleotide-Cytochrome o Reductase of Vitreoscilla

The NADH oxidase system of Vitreoscilla was localized in themembrane as shown by three assays of respiratory activity: NADHoxidation, oxygen consumption and NADH dehydrogenase activityusing p-iodonitrotetrazolium violet (INT) as electron acceptor.The purified metalloflavoprotein component of the NADH-cytochromeo reductase system catalyzes the aerobic reduction of impurecytochrome o but not of the pure cytochrome. The purified enzymeexhibited hyperbolic kinetics with NADH when impure cytochromeo was used as electron acceptor but sigmoid kinetics with TNTas electron acceptor. The kinetics of INT reduction became morehyperbolic when assayed in the presence of impure cytochromeo and the Hill coefficient decreased from 1.8 to 1.1. In contrast,both the NADH-INT reductase and NADH oxidase activities of membranevesicles showed hyperbolic kinetics with NADH. The NADH oxidaseactivity of membrane vesicles was subject to substrate inhibitionat NADH concentrations greater than 150 µM which was notobserved for the NADH-cytochrome o reductase activity of thepurfied enzyme under aerobic conditions. Respiratory inhibitorssuch as rotenone, cyanide, and salicylhydroxamic acid inhibitedthe oxidation of NADH by membrane vesicles with oxygen as electronacceptor but not with INT as electron acceptor. These comparativestudies of some catalytic properties of the purified and membranebound enzyme provided evidence that the NADH-cytochrome o oxidasesystem of Vitreoscilla consists of cytochrome o, the flavoproteinreductase, and at least one other component which may regulatethe catalytic properties of the reductase. ¹ This work was supported by National Science Foundation GrantPCM77-15915 and Public Health Service Grant GM20006. This paperwas submitted by V.G.P. in partial fulfillment of the requirementsfor the Ph.D. degree of Illinois Institute of Technology. ² Present address: Department of Biological Science, NorthwesternUniversity, Evanston, Illinois 60201, U.S.A. (Received July 28, 1981; Accepted November 9, 1981)     

COMPARATIVE STUDIES OF PHOTOCHEMICAL OXIDATION-REDUCTION REACTIONS IN LAMELLAR FRAGMENTS OF VARIOUS ALGAE AND SPINACH

The activity of various electron carriers, including DPIP, spinachplastocyanin, mammalian cytochrome c, and Anabaena cytochrome553, as donor in the reaction induced by the photochemical systemI was examined with lamellar fragments of various algae andspinach. Reduced DPIP was an effective electron donor irrespective ofthe organisms, when it was supplied at a high concentration(10^–3 M). Spinach plastocyanin was effective in the reactionswith the lamellae of green algae, Euglena, diatom Phaeodactyrumand red algae Porphyra yezoensis and Porphyra sp. Yamamoto II,whereas it was inactive in the lamellae of blue-green algae.Horse-heart cytochrome c and Anabaena cytochrome 553 were activein the reaction with the lamellae of bluegreen algae. The formercytochrome was also active in the reactions in Porphyridiumand Cyanidium. The cytochromes were less active in the reactionsin which spinach plastocyanin acted as effective electron donor. The data were interpreted as that the photochemical system Iin bluegreen algae differs from that of other photosyntheticorganisms with respect to the properties of the site of theelectron-input. ¹ Present address: Nomura Research Institute for Technologyand Economics, Kamakura, Kanagawa. ² Present address: Ocean Research Institute, University of Tokyo,Nakano, Tokyo.     

Trifluralin and root growth

Trifluralin inhibited root elongation and induced root tip swellingof Zea mays and Triticum aeslivum. Time-course experiments showedthese effects occurred within 6 hr of treatment. As the rootstreated with trifluralin enlarged, there was a concomitant increasein root growth inhibition. Bioassays were devised to quantitativelymeasure the radial enlargement of trifluralin-treated roots.Histological observations indicated that swollen root tissuewas growing in a non-polar manner. The root swelling effectof trifluralin was inhibited 70% by the reducing agent, 2,3-dimercaptopropanol.Trifluralin had no effect on SH content of root tip proteinof Z.mays. ¹ Published with the approval of the Director of die West VirginiaAgricultural Experiment Station as Scientific paper No. 1177. ² Present address: Department of Biology, Mercyhurst College,Erie, Pennsylvania 16501, U.S.A. (Received March 19, 1971; )     

Further studies on ribulose-l,5-diphosphate carboxylase from Rhodopseudomonas spheroides and Rhodospirillum rubrum

Some enzymic properties of RuDP carboxylase isolated from Rhodopseudomonasspheroides and Rhodospirillum rubrum, grown under autotrophic,semi-autotrophic and heterotrophic conditions were studied.Regardless of the growing condition, the nature of the enzymefrom each respective bacterium remained unchanged. The molecularweight of R. spheroides and R. rubrum RuDP carboxylase was estimatedto be approximately 2.4x10⁵ and 8.3x10⁴, respectively. Bothenzymes require Mg⁺⁺. Their kinetic properties were also examined. ¹Part XII. Structure and Function of Chloroplast Proteins ²Supported in part by research grants from the Ministry of Educationof Japan (No. 8719), USPHS (AM. 10792-03) and the Asahi Press(Tokyo) (Received March 18, 1970; )     

BIOTIN LIBERATION BY THE LICHEN ALGA COCCOMYXA SP. AND BY CHLORELLA PYRENOIDOSA

The unicellular green alga Coccomyxa, a component of the lichenPeltigera aphthosa, liberated about 7.2mµg biotin permg dry weight of cells into the culture medium during a growthperiod of 15–20 days. The corresponding figure for thefree-living alga Chlorella pyrenoidosa was 0.45mµg ofbiotin. Chromatographic analysis indicated that this was freebiotin and not a bound form of the vitamin. The biotin concentrationof rinsed Coccomyxa cells was 1.88mµg per mg dry weightof cells, of which less than 0.01mµg was extractable byhot water. Cells of Chlorella contained 0.16mµg of biotinper mg dry weight, of which 0.11mµg was extractable byhot water. The biotin content of Coccomyxa, which was about12 times that of Chlorella, is thus almost entirely in the boundform. The importance of biotin in the symbiotic interactionsbetween the alga and the fungus in Peltigera is discussed. ¹Present address: University Department of Agriculture, Oxford,England. ²Present address: Institute of Marine Resources, Universityof California, La Jolla, California, U.S.A.     

ASSESSMENT OF BIVALVE INTRACELLULAR DIGESTION BASED ON DIRECT MEASUREMENTS

Two methods of studying intracellular digestive state in bivalvemolluscs, (1) the subjective tubule grading technique and (2)an alternative method based on measurements of digestive tubuleand luminal widths, were compared using data from a starvation/feedingexperiment on the quahog, Mercenaria mercenaria (L.). Measurementsof digestive cell heights or lumen widths could not be directlycompared due to differences in tubule widths within and betweenindividuals (range = 56.6 to 77.4 µm; X = 63.0; SD 4.8(µm) which were unrelated to quahog length. Instead, themean proportion of the tubule width (MPTW) covered by digestivecell cytoplasm was used for comparisons between samples of animals.MPTW covaried directly with the subjectively established proportionof Type II tubules (r = 0.882). The inclusion of data from obliquesections of digestive tubules did not unduly affect statisticalresults . Of 4 sampling schemes compared, a systematic samplingscheme in which measurements were made on only one digestivegland tubule within an area containing a cluster of similartubule types, was shown to be best. ¹Contribution No. 105, Marine Science Institute, NortheasternUniversity, Nahant, MA., 01908 ²Current address: New England Aquarium, Central Wharf, Boston,MA, 02110 USA (Received 28 September 1981;     

Direct Evaluation of Effects of Fatty-Acid Unsaturation on the Thermal Properties of Photosynthetic Activities, as Studied by Mutation and Transformation of Synechocystis PCC6803

Glycerolipids of thylakoid membranes isolated from the cyanobacteriumSynechocystis PCC6803 contained high levels of dienoic and trienoicC₁₈ fatty acids, in addition to saturated C₁₆ and monoenoicC₁₈ fatty acids. A mutant (Fadl2) of this cyanobacterium wasdefective in the desaturation of C₁₈ fatty acids at the ¹² position,and its thylakoid membranes lacked trienoic acids and containeda very reduced level of dienoic acids. A derivative strain ofFadl2 (Fadl2/desA), which had been transformed with a gene fordesaturation at the ¹² position, fully recovered the abilityto desaturate the fatty acids in the glycerolipids of thylakoidmembranes. The thermal properties of the photosynthetic activitiesof the mutant and the transformant were compared with thoseof the wild-type strain. Despite great diversity in the extentof unsaturation of fatty acids between the wild-type, Fadl2,and Fad12/desA strains, no significant differences were foundeither in the temperature dependence of photosynthesis or inthe heat stability of photosynthetic, photosystem II and photosystemI activities. These results demonstrate that the trienoic fattyacids and, probably, the dienoic acids of the lipids in thethylakoid membrane do not affect the thermal properties of theabove-mentioned activities of photosynthesis. ³Permanent address: Institute of Plant Physiology, BiologicalResearch Center of Hungarian Academy of Sciences, H-6701 Szeged,P.O. Box 521, Hungary (Received August 9, 1990; Accepted December 7, 1990)     

Effect of malformin on root growth

Malformin induces curvatures, stimulates root hair and lateralroot formation, promotes radial expansion, inhibits elongation,wet and dry weight, cell division and cell wall synthesis inroots of Zea mays, but has no effect on protein synthesis. Thegrowth curves (elongation, wet and dry weight) of Z. mays rootstreated with malformin are cubic. Processes which are involvedin inhibition of elongation are considered the primary causeof root curvatures by malformin. ¹This research was supported by grant GB-7158 from the NationalScience Foundation and grant E-146-F from the American CancerSociety. Journal Paper No. 3536 of the Purdue Agricultural ExperimentStation. ²Present address: Volcani Institute of Agricultural Research,P.O.B. 6, Bet-Dagan, Israel. (Received December 24, 1969; )     

Separation and Partial Characterization of Membranes from Prochloron sp.

Two differently colored membrane preparations were separatedfrom the prochlorophyte, Prochloron sp., by mechanical disintegrationof the cells followed by sucrose density gradient centrifugation.An orange-colored preparation, containing zeaxanthin as themajor constituent pigment, seemed to comprise the cytoplasmicmembrane. The other green-colored membrane preparation, containingß-carotene and chlorophyll a and b as major pigmentconstituents, was identified as the thylakoid membrane. Thetwo types of membranes were compared as to their absorptionspectra and buoyant densities. ¹ This work is one of the results of the 8th International Expeditionon Prochloron organized by Dr. R. A. Lewin, University of Californiaat San Diego. ⁵ Present address: Solar Energy Research Group, The Algatron,The Institute of Physical and Chemical Research (RIKEN), Wako-shi,Saitama 351, Japan. ⁶ Present address: National Institute for Basic Biology, Okazaki444, Japan. (Received October 19, 1984; Accepted January 7, 1985)     

High Affinity of the 5'-Upstream Region of a Winged Bean Chymotrypsin Inhibitor Gene for Nuclear Matrix

The 5'-upstream region of a winged bean chymotrypsin inhibitorgene (WCI-3b) was found to have a high affinity for nuclearmatrix. The region, named WCI-3b MAR (matrix attachment region),is highly A+T-rich and contains multiple sites interacting withnuclear matrix. A MAR was also found in the corresponding regionof the WCI-x gene, another active gene of the WCI family. SeveralMAR-binding proteins were detected in the wheat nuclear matrix. ⁴Present address: Friedrich Miescher Institute, P.O. Box 2543,CH-4002 Basel, Switzerland. ⁵Present address: Research Institute for Biological Sciences(RIBS), Kayo-cho, Jyobo-gun, Okayama, 716–1241 Japan.