Peptide analogs of the mud crab pumping pheromone: structure-function studies

In the mud crab, Rhithropanopeus harrisii (Gould), larval releaseis highly synchronous and is controlled by peptide pheromonesreleased from the hatching eggs. The pheromones and syntheticpeptide analogs, containing neutral amino acids at the arnino-terminusand a basic amino acid at the carboxy-terminus, evoke larvalrelease or pumping behavior in the female. Here we describestructure–function relationships between behavioral responsesand peptide analogs of the pumping pheromone. Compounds testedincluded 11 peptides containing 1–7 neutral amino acidresidues and a carboxy-terminal arginine residue, and modifiedarginine compounds. Complex feeding-stimulant mixtures and groupsof peptides were tested to provide information about the numberof different types of receptors. Pumping behavior was describedin terms of response threshold, maximum percentage responseand effective concentration range. All carboxy-terminal argininepeptides tested evoked pumping responses. Response thresholdsranged from 10^-15 M for the most potent peptide Ile-Gly-Argto 10^-8 M for the least potent Tyr-Arg. Effective concentrationranges for the peptides varied from one to four orders of magnitudeand the responses normally declined with further increase inconcentration. Maximum percentage response was largely independentof response threshold and ranged from 30–60%, comparableto assay responses of larval hatch water. Responses within sizegroups of peptides were more similar than between groups. Studieswith modified arginines showed that hydrophobic structures similarin size and shape to small peptides evoked responses. Stericallyhindered hydrophobic moieties were ineffective. Tests with mixturesof active compounds support the hypothesis that there is onetype of pumping peptide receptor.     

Behavioral responses of the brittle star Ophiura ophiura to amino acids, acetylcholine and related low-molecular-weight compounds

Feeding behavior of the brittle star Ophiura ophiura includesorienting posture, orienting movements, arm ‘walking’,changing the direction of ‘walking’ arm coilingand ingestion. All sequential behavior patterns were releasedor enhanced by single low-molecular-weight compounds. Stimuliwhich released ‘walking’ behavior at high concentrations(10^–4 M) in all the test animals are listed in decreasingorder of sensitivity: sarcosine, glycine, urea, L-valine, L-leucine,L-methionine, L-homocysteine, L-norvaline, L-norleucine, L-threonine,L-serine, S-methyl-L-cysteine, L-proline. Threshold values forsingle amino acids were as much as 100 times different in differentindividuals and ranged from 3 x 10^–9 to 3 x 10^–7M for the most effective stimulus, sarcosine, and from 10^–6to 10^–4 M for proline. Above 10^–5 M, only L-prolineregularly released a second behavior pattern, the arm coilingresponse, which temporarily inhibited the ‘walking’behavior. Behavioral thresholds for the ‘walking’behavior for L(+)-lactate and L-alanine were higher than thosefor the orienting movements. Thyoglycolic acid and ß-alaninereleased tube feet walking, which is not part of the feedingbehavior. Structure—activity comparisons were studied at estimated10^–5 M concentrations. Gycline, sarcosine, L-valine, L-norvaline,L-leucine, L-isoleucine, DL-norleucine and DL-homocysteine releasedarm ‘walking’ behavior in more than 75% of all thetests. With the exceptions of S-methyl-and S-ethyl-cysteine,and glycine methylester, derivatives of amino acids were noteffective behavioral stimuli in Ophiura ophiura. L-Isomers ofvaline and leucine regularly stimulated the ‘walking’behavior while their D-isomers were effective in some testsand ineffective in others. Acetylcholine iodide, acetyl-ß-methylcholine chloride and choline phosphate chloride regularly released‘walking’ behavior at concentrations above 10^–5M.     

The Genetic Basis of Preference for Sweet Substances Among Inbred Strains of Mice: Preference Ratio Phenotypes and the Alleles of the Sac and dpa Loci

Five inbred strains (129/J, BALB/cByJ, C3HeB/FeJ, C57BI/6J andDBA/2J) were examined with two-bottle (48 h) preference ratiotesting across concentrations of sodium saccharin (3 x 10^–4M, 10^–3 M, 3 x 10^–3 M and 10^–2 M), d-phenylalanine(10^–3 M, 10^–2 M and 10^–1 M), and l-glutamine(10^–2 M, 3 x 10^–2 M, 10^–1 M and 3 x 10^–1M). Three consistent groupings of strains were observed acrosssubstances and concentrations:

1. C57BI/6J (preference at low andhigh concentrations);
2. BALB/cByJ and C3HeB/FeJ (preferenceat high concentrations);
3. 129/J and DBA/2J (preference at highconcentration for sodiumsaccharin and indifference to d-phenylalanineand l-glutamine).

If a single locus (presumably dpa or Sac) determines these phenotypes,there are likely to be three alleles. If two independent loci(presumably dpa and Sac) determine these phenotypes, an allelicassignment of Sac^b/dpa^+s for the C57BI/6J strain, Sac^b/dpa^–sfor the BALB/cByJ and C3HeB/FeJ strains, and either Sac^d/dpa^+sor Sac^d/dpa^–s for the 129/J and DBA/2J strains is suggested.Chem. Senses 20: 291–298, 1995.     

Effect of Spermidine and Methylglyoxal-bis (Guanyl-hydrazone) (MGBG) on In Vitro Pollen Germination and Tube Growth in Catharanthus roseus

Incorporation of polyamine-spermidine into the nutrient mediumat 10^–6 and 10^–5 M concentrations stimulates pollen-tubegrowth in vitro in Catharanthus roseus L. G. Don. MGBG, an inhibitorof spermidine biosynthesis, at 0.5 x 10^–3 and 1 x 10^–3M concentrations reduced the percentage of germination as wellas tube growth and at a concentration of 1.5 x 10^–3 Mgermination was totally inhibited. Pollen grains incubated inthe medium containing 1.5 x 10^–3 M MGBG, when transferredto a fresh medium with 10^–5 M spermidine, resulted in80% germination recovery, along with considerable tube growth.Experiments with actinomycin-D indicate that stimulation ofpollen-tube growth by spermidine may involve de novo synthesisof protein. Catharanthus roseus, pollen germination, tube growth, spermidine, MGBG, inhibition, actinomycin-D     

Effect of abscisic acid on differentiation and ribulose diphosphate carboxylase of chloroplasts

Daucus carota tissues were grown on Murashige-Skoog medium (MS)at different concentrations with abscisic acid (ABA). Sevenbands of chloroplast fractions were obtained on a sucrose gradient.At 10^–5M, ABA highly increased chlorophyll and proteinnitrogen content of medium density chloroplasts. With increasingage of the tissues, the most active chloroplasts according totheir ¹⁴CO₂ fixation were found in smaller numbers. When treatedwith 10^–5 M ABA, 34 day-old tissues cultivated in vitroshowed the chloroplast pattern of 110 day-old tissues. The effectof ABA—given to the tissues during a short pretreatmentor continuously present in the culture medium—on the ribulosediphosphate carboxylase activity was analysed. It was foundthat ABA at 10^–5 M strongly inhibited ¹⁴CO₂ fixation. (Received December 20, 1977; )     

The role of amino acids in the chemosensory swarming and feeding of marine copepods

Behavioural observations were made on two copepods, Eurytemoraherdmani and Acartia hudsonica, presented with 18 dissolvedL-amino acids the concentrations of which ranged from 10^–8M to 10^–2 M. The onset and duration of the swarming behaviourwere determined by the structure and concentration of a restrictednumber of molecules, which differed depending on the copepodspecies. Dicarboxylic amino acids were the most stimulatoryagent for E. herdmani, whereas A. hudsonica responded preferentiallyto aliphatic amino acids. Both the feeding and swarming of A.hudsonica could be induced by the same kind of molecules. Thethreshold sensitivity of the copepods appeared to be compatiblewith the natural concentrations of the dissolved free aminoacids measured at sea. These findings are discussed with referenceto the chemically mediated interactions between phytoplanktonand zooplankton.     

Regulation of Free Calcium Concentration in the Pitchers of the Carnivorous Plant Sarracenia purpurea: A Model for Calcium in the Higher Plant Apoplast?

The pitcher of the carnivorous plant Sarracenia purpurea L.contains an entrapped body of liquid within which its prey isdigested. Free calcium in the pitcher is derived from eitherthe pitcher walls or from prey falling into the pitcher; inthe absence of exogenous (prey-derived) calcium it will dependon the active and passive calcium regulatory properties of thepitcher walls and may to some extent therefore mimic calciumin the apoplast of plant cells. Using a calcium-specific electrode,the free calcium concentration of the pitchers of Sarraceniaplants was investigated and the effect of adding a variety ofconcentrations of calcium in water determined. The mean pitcherfree calcium concentration in vivo was 2.3 x 10^–5 M±2.5x 10^–5 M; when pitchers were washed and filled with watercontaining lower calcium concentrations, the concentration inthe pitcher water rose to 1–5 x 10^–5 M. When highercalcium concentrations (up to 1 x 10^–4 M) were added,the pitcher calcium concentration declined to 1–7 x 10^–5M. Concentrations of calcium above 1 x 10^–4 M were alsoreduced, but to a lesser extent. Metabolic inhibition of activeion transport, while inhibiting pitcher acidification, did notinhibit regulation of pitcher free calcium, suggested that itoccurs as a result of calcium exchange sites in the pitcherwalls. The data are discussed in relation to the physiologyof Sarracenia pitchers and to the usefulness of the pitcheras a model for free calcium in the higher plant apoplast. Sarracenia purpurea L., carnivorous plant, pitcher, free calcium, plant apoplast     

Behavioral and Electrophysiological Studies on Chemoreception in Aplysia

Using the typical mouth opening response (MOR) as the indexof chemore-ception, several amino acid constituents of naturalfood for Aplysia were effective in eliciting MOR in concentrationsas low is 10^–6 10^–7 M (glutamic acid) and 10^–510^–6 M (aspartic acid). It was suggested that these substancesmay serve as food attractants for this animal. Combined resultsof behavioral and electrophysiological experiments indicatedthat the most sensitive food receptors of Aplysia are predominantlylocated in the anterior tentacular groove area of the oral veil.These receptors appeared to strongly project to the caudal darkcell clusters of the cerebral ganglion. Extracellular recordingof single afferent units following oral veil stimulation withvarious chemicals showed that the chemoreceptors in the anteriortentacular groove area were 100 to 1000 times more sensitiveto food substances than other stimuli tested. It was suggestedthat these receptors are relatively "specific" to food attractants.There was a correlation between behavioral and electrophysiologicalthreshold data for the individual compounds. The electrophysiologicalthresholds however were 5–15 times higher than the behavioralthresholds.     

Identification of Antheridic Acid as an Antheridiogen in Anemia rotundifolia and Anemia flexuosa

Antheridic acid was identified by retention time and full massspectra from GCMS analysis as an antheridiogen in Anemia rotundifoliaand A. flexuosa. In the dark spore germination assay, antheridicacid was active down to 10^–10 and 5 ? 10^–12g.ml^–1in A. rotundifolia and A.flexuosa, respectively. In the antheridiumformation assay, antheridic acid was active down to 10^–10g.ml^–1 in both A. rotundifolia and A.flexuosa (Received April 14, 1987; Accepted July 8, 1987)     

Nitrogen regeneration by the subtropical marine copepod Eucalanus pileatus

Ammonium release rates by individual marine copepods (Eucalanuspileatus) were examined kinetically over successive 10-min intervalsafter the animals were exposed to three concentrations of thediatom Thalassiosira fluviatilis as food. Food concentrationsspanned those expected in the natural environment. "Well-fed"(3 mm³ T. fluviatilis l^–1) copepods released ammoniumsignificantly (p<0.05) faster than those fed zero or lowconcentrations of food, but differences were not dramatic. Meanexcretion rates (± SE) for animals removed from foodfor 18 –22 h [30 ± 6 nmol NH₄(mg ash free dry weight)^–1h ^–1] were 60% of those for "well-fed" animals [49 ±8 nmol NH₄ (mg ash free dry weight)^–1 h^–1]. Continuedhigh release rates after extended periods without food suggestthat E. pileatus must feed frequently in nature to maintainan adequate nitrogen balance. Depending on food concentration,animals released ammonium at rates ranging from 40 to > 100%of their N ingestion rates over the previous 18–22 h.Long-term (4–6 h) kinetic excretion experiments with "well-fed"animals indicated that, on average, release rates decreasedwith time after food removal, but patterns varied among individualcopepods. In addition to ammonium release, o-phthalaldehydereactive amino acid nitrogen was occasionally released by E.pileatus in large "spurt events" lasting from 20 to 60 min. ¹GLERL Contribution No. 283     

Action of Benzoic Acid and its o-, m- and p-Hydroxy-Derivatives on the Dark- and Light-Induced Leaflet Movements in Cassia fasciculata Michx.

Benzoic acid and its o-, m- and p-hydroxy derivatives appliedon excised leaves of Cassia fasciculata modified the dark-induced(scotonasty) and light-induced (photonasty) leaflet movements.Benzoic acid inhibited the scotonastic closure in a dose-dependentmanner from 10^–4M to 10^–3M and promoted the photonasticopening at optimum concentration of 5.10^–4M. These effectswere dependent upon the position of hydroxyl group on the benzoicring, the o-derivative inducing a stronger effect than the m-and p-derivatives. Experiments showed that treatment with o-hydroxybenzoicacid had not to exceed 30–60 min and that the maximumeffect was obtained at pH 5.5. (Received September 16, 1986; Accepted June 22, 1987)     

MORPHOGENESIS IN SPIEODELA OLIGORRHIZA: EFFECTS OF PYRIMIDINE BASE ANALOGUES ON INITIATION, ELONGATION AND DIFFERENTIATION OF FRONDS

The effect of four pyrimidine base analogues and their antidoteson S. oligorrhiza was studied. FUdR stopped cell division at concentrations of 4 10^–7M and higher. This effect could be nullified by the additionof 4 10^–6 M thymidine. Neither uridine nor uracil hadan antidotal effect on FUdR. FU (8 10^–6 M or higher concentration) affected celldivision, frond elongation and differentiation, and could notbe counteracted by either thymidine or uracil. TU (8 10^–4 M) rather specifically inhibited differentiationof frond tissues, while not preventing cell division or theinitiation of new generations. Uracil and uridine at about equimolarconcentrations completely counteracted the TU effect. AzU (10^–3 M) suppressed cell division, frond elongationand frond differentiation. When thymidine (10^–3 M) wasadded simultaneously with AzU only cell elongation and differentiationof fronds were inhibited, but cell division proceeded. 10^–3M uracil (but not uridine) counteracted all effects of AzU. ¹ Based on a portion of the senior author's Ph.D. Thesis.     

Effects of indoleacetic, p-chlorophenoxyisobutyric and 2,4,6-trichlorophenoxyacetic acids on three phases of rooting in Azukia cuttings

In adventitious root formation of disbudded epicotyl cuttingstaken from light-grown, 5-day-old Azukia angularis seedlings,indoleacetic acid (IAA), 1 x 10^–4 M, applied during thefirst day showed no effect, but enhanced the effect of IAA,1 x 10^–4 M, applied during the second day. Treatment duringthe second day promoted rooting by about 70%, and a combinationof treatments for the first and second days promoted rootingsome 200%. p-Chlorophenoxyisobutyric acid (PCIB), 3 x 10^–4M, and2,4,6-trichlorophenoxyacetic acid (2,4,6-T), 2 x 10^–44M, applied the first day also enhanced the effect of IAA, 2x 10^–4 M, applied the second day. When applied the second day, PCIB, 2 x 10^–4M, increasedthe number of root primordia or clusters of small cells, butnot die number of protruded roots. Formation of the cell clusterwas inhibited by 2,4,6-T, 3 x 10^–4M, applied the secondday. Rooting processes in Azukia cuttings seem to include at leastthree phases: the first phase is induced not only by IAA butalso by PCIB or 2,4,6-T, the second phase is induced by IAAor PCIB and the diird phase depends specifically on IAA. (Received October 28, 1970; )     

Strain differences among mice in taste psychophysics of sucrose octaacetate

SWR/J inbred mice consistently avoided a 10^–4 M sucroseoctaacetate (SOA) solution in unconditioned two-bottle preferencetests whereas mice of all other inbred strains tested did not(confirming a previous report that used SWR mice of a differentsubline). In a conditioned taste aversion procedure SWR/J miceavoided SOA at concentrations from 10^–3 M to 10^–7M but not at 10^–8 M. Various other inbred strains firstfailed to avoid SOA at concentrations from 10^–3 M to 10^–5M. The major strain difference between SWR and other inbredmice was robust across rearing regimes and when tested withother psychophysical procedures. In single-bottle, free-lickingtests SWR/J mice differed from C57L/J mice in response to SOAfollowing extremely brief exposure to the SOA.The SOA detectionthreshold differences indicated by these psychophysical proceduresare also consistent with differences reported from electrophysiologicalrecordings from glossopharyngeal and chorda tympani nerves inmice of several of the same strains.     

Non-cyclic photophosphorylation by spinach grana treated with 0.8 M Tris buffer

1. Photophosphorylation was measured with spinach grana sampleswashed by 0.8 M Tris buffer at pH 8.0, which no longer catalyzedthe ferricyanide and NADP HILL reactions with water as the electrondonor. The photophosphorylation with the reaction mixture containing2 10^–4 M 2,6-dichlorophenol indophenol (DCPI) plus above2 10^–3 M ascorbate as the electron donor system insteadof water under anaerobic conditions was, in the most part, dependenton the addition of both PPNR (a nonheme iron protein requisitefor photosynthetic pyridine nucleotide reduction ; spinach ferredoxin)and NADP as the electron acceptor system. However, when ascorbateconcentration only was lowered to 2 10^–4 M, the entirephotophosphorylation proceeded, even in the absence of the electronacceptor system. 2. When the NADP added in the reaction mixture had been reducedby glucose-6-phosphate and glucose-6-phosphate dehydrogenasebefore illumination, the photophosphorylation with 2 10^–4M DCPI plus 6.7 10^–3 M ascorbate decreased to aboutthe same rate as that obtained without NADP. 3. The time course for photophosphorylation in the presenceof NADP was consistent with the time course for the photoreductionof NADP: On the complete reduction of NADP, the photophosphorylationstopped. 4. In the presence of 6.7 10^–3 M dichloropheny 1.1,1-dimethylureaor 3 10^–4 M o-phenanthroline, non-cyclic photophosphorylationwith 2 10^–4 M DCPI plus 6.7 10^–3 M ascorbateas the electron donor system decreased to about half that ofthe control, and the remaining activities were hardly affectedeven at higher concentrations of both inhibitors. The P/2e^–ratios of non-cyclic photophosphorylation in the absence andpresence of ophenanthroline were 0.74 and 0.48, respectively. ¹Present address: Department of Biology, University of California,San Diego, La Jolla, California 92037, U. S. A.     

Morphological and physiological characterization of IAA-less-sensitive and IAA-sensitive phases in rooting of Azukia cuttings

In disbudded epicotyl cuttings taken from light grown 5-dayold Azukia angularis Phaseolus angularis) seedlings, all adventitiousrootlets appeared on the second day of incubation. No root primordiawere observed within the first 24 hr and no increase in thenumber of roots occurred after 48 hr. Puromycin (5.5?10^–5M), p-fluorophenylalanine (1?10^–3M),2-thiouracil (2.3?10^–4M) and 2,6-diaminopurine (2?10^–5M)inhibited rooting when applied to cuttings on the second day,but showed no inhibition when applied on the first day. Unlike these inhibitors, pyrithiamine (7.2?10^–5M) inhibitedrooting when it was applied to cuttings on the first day. A rooting promoting effect was observed with actinomycin D (2.4?10^–6M),2,4-dinitrophenol (3?10^–5M) and p-fluorophenylalanine(1?10^–4M) applied to the cuttings on the first day, whereasindoleacetic acid (1.7?10^–4M) showed its promoting effectmost effectively on the second day. ¹Contribution No. 17 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Tokyo, Japan. (Received June 4, 1969; )     

Involvement of cellulose synthesis in actions of gibberellin and kinetin on cell expansion. Gibberellin-coumarin and kinetin-coumarin interactions on stem elongation

In azuki bean (Azukia angularis = Vignia angularis) epicotylsections, 5 ? 10^–4 M coumarin inhibited the incorporationof radioactivity from [U^–14C]glucose into the cellulosefraction by 35% in the absence of indole-3-acetic acid (IAA)and by 40% in the presence of 1 ? 10^–4 M IAA. There wasno inhibitory effect on the incorporation of radioactivity intothe other fractions. Coumarin at 5 ? 10^–4 M reversed thepromoting effect of 1 ? 10^–5 M gibberellin A₃ (GA) andthe inhibitory effect of 1 ? 10^–5 M kinetin on IAA-inducedelongation of sections with no significant effects on IAA-inducedelongation. Neither GA nor kinetin had any appreciable effectson cellulose synthesis. No inhibition of cellulose syntheiswas observed with 1 ? 10^–3 M colchichine, which has beenreported to have effects similar to those of coumarin on GA-or kinetin-affected stem elongation. Coumarin at 5 ? 10^–4M was ineffectual in breaking up wall microtubules, while adisrupting effect on wall microtubules was clearly demonstratedwith 3 ? 10^–4M colchicine. From these results, the possible involvement of cellulose synthesisin cell expansion controlled by GA or kinetin was suggested. (Received August 3, 1973; )     

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; )     

Endogenous factors affecting the curved growth of seminal roots of Zea mays L. seedlings grown in liquid culture

Seminal roots of Zea mays L. show curved growth, i.e. waving,meandering and spiral growth, when water cultured. Root curvaturewas accelerated by exogenously applied indole-3-acetic acidat 10^–9 M and gibberellic acid at 10^–6M; this curvaturedisappeared when 10^–7 M p-chlorophenoxyisobutyric acidwas added. Roots curved more when the tops of seedlings wereexposed to light than when the tops of seedlings were covered.These results suggest that auxin may induce root curvature. (Received February 29, 1980; )