A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl β-d-lactoside derivatives

4-Methylumbelliferyl 6-O-benzyl--d-lactoside (6Bn-MU-Lac) and some related compounds were synthesizedvia different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, theK _m value is 0.232 mM at pH 5. Abbreviations: CGase, ceramide glycanase; Gal, galactose; Glc, Glucose; Lac, lactose; MU, 4-methylumbelliferone; MU-Lac, 4-methylumbelliferyl -d-lactoside; bBn-Lac, 6-O-benzyl-lactose; 6Bn-MU-Lac, 4-methylumbelliferyl 6-Obenzyl--d-lactoside; 46Bd-MU-Lac, 4-methylumbelliferyl 4,6-O-benzylidene--d-lactoside; MU-Cel, 4-methylumbellifery -d-cellobioside; 46Bd-MU-Cel, 4-methylumbelliferyl 4,6-O-benzylidene--d-cellobioside; TLC, thin layer chromatography;¹H-NMR, proton nuclear magnetic resonance; GSL, glycosphingolipids; CSA, 10-camphorsulfonic acid. See Scheme 1 for chemical structures.     

Adventitious shoot regeneration from cultured petal explants of carnation

Adventitious shoot regeneration was compared among leaf, stem and petal explants of carnation (Dianthus caryophyllus L.) cv. Scania on MS medium containing different concentrations of 6-benzyladenine (BA) and -naphthaleneacetic acid (NAA). High frequency regeneration was obtained only from petal explants on the media containing 5 to 10 M BA with or without 5 M NAA. Among the cytokinins tested, N-2-chloro-4-pyridyl-N-phenylurea and N-1,2,3-thiadiazol-5-yl-N-N-phenylurea were more effective than BA, kinetin, N⁶-2-isopentenyl adenine and zeatin on regeneration from petal explants. Although, high frequency shoot regeneration was obtained from all petal explants harvested from various developmental stages of buds, a significant decrease in regeneration capacity was observed in the explants obtained from fully-opened flowers. High frequency shoot regeneration was also obtained from the petal explants of cvs. Coral. Lena, Nora and White Sim, and an interspecific cultivar Eolo using the method developed in this study.Abbreviations NAA -naphthaleneacetic acid - BA 6-benzyladenine - GA₃ gibberellic acid - 2iP N⁶-2-isopentenyl adenine - KT-30 N-2-chloro-4-pyridyl-N-phenylurea (also called 4PU) - TDZ N-1,2,3-thiadiazol-5-yl-N-phenylurea (also called thidiazuron)     

Facile synthesis of 2′-substituted lactoses

Isopropylidenation of lactose with 2,2-dimethoxypropane in the presence ofp-toluenesulfonic acid gave two products, which were identified by¹H- and¹³C-NMR as 2,35,63,4-tri-O-isopropylidenelactose dimethyl acetal (1) and its 6-O-(2-methoxy)-isopropyl derivative (2). These products were used for the synthesis of 2-O-methyllactose (7), 2,6-di-O-methyllactose (9) and 2-O-benzyllactose (13).     

In vitro biosynthesis of the C-glycosidic bond in aloin

Biosynthetic studies with cell-free extracts from Aloe arborescens Mill. demonstrate the transfer of the glucose moiety from UDP-glucose to aloe emodin anthrone, forming the C-glycosidic linkage in the anthracene derivative aloin. The pH-dependence and the specificity of UDP-glucose and aloe emodin anthrone for the biosynthesis of the C-glycosidic bond in aloin are shown.Abbreviations ADP-Glc adenosine-5-diphosphate glucose - AEA aloe emodin anthrone (1,8-dihydroxy-3-(hydroxymethyl)-9(10 H)-anthracenone) - CoASAc acetyl coenzyme A - GDP-Glc guanosine-5-diphosphate glucose - Glc glucose - Glc-1-P glucose-1-phosphate - HPLC high performance liquid chromatography - TLC thin layer chromatography - UDP-Gal uridine-5-diphosphate galactose - UDP-Glc uridine-5-diphosphate glucose     

A novel PH-CT-COSY methodology for measuring JPH coupling constants in unlabeled nucleic acids. Application to HIV-2 TAR RNA

A quantitative analysis of J_PH scalar couplings in nucleic acids is difficult due to small couplings to phosphorus, the extreme overlap of the sugar protons and the fast relaxation of the spins involved in the magnetization transfer. Here we present a new methodology that relies on heteronuclear Constant Time Correlation Spectroscopy (CT-COSY). The three vicinal ³J_PH3, ³J_PH5 and ³J_PH5 scalar couplings can be obtained by monitoring the intensity decay of the P_i-H3_{i – 1} peak as a function of the constant time T in a 2D correlation map. The advantage of the new method resides in the possibility of measuring the two ³J_PH5 and ³J_PH5 scalar couplings even in the presence of overlapped H5/H5 resonances, since the quantitative information is extracted from the intensity decay of the P-H3 peak. Moreover, the relaxation of the H3 proton is considerably slower than that of the H5/H5 geminal protons and the commonly populated conformations of the phosphate backbone are associated with large ³J_PH3 couplings and relatively small ³J_{PH5 / H5}. These two facts lead to optimal signal-to-noise ratio for the P-H3 correlation compared to the P-H5/H5 correlation.The heteronuclear CT-COSY experiment is suitable for oligonucleotides in the 10–15 kDa molecular mass range and has been applied to the 30mer HIV-2 TAR RNA. The methodology presented here can be used to measure P-H dipolar couplings (D_PH) as well. We will present qualitative results for the measurement of P-H_base and P-H2 dipolar couplings in the HIV-2 TAR RNA and will discuss the reasons that so far precluded the quantification of the D_PHs for the 30mer RNA.     

Two-dimensional 1H NMR study of two cyclobutane type photodimers of thymidylyl-(3′→5′)-thymidine

2D NMR spectroscopy and J coupling constant analysis are applied to resolve the structure of two photoproducts of thymidylyl-(35)-thymidine. These products are cyclobutane type thymine dimers possessing the cis-syn (the predominant one) and trans-syn geometry. The cis-syn is formed in an ANTI-ANTI conformation about the N-glycosyl linkages and resembles the normal base-stacked configuration. The glycosidic conformation in solution of the 5 terminal fragment differs from the crystal in which the less common SYN conformation is observed. In this isomer only the sugar pucker of the 3 terminal fragment is changed substantially with respect to the dinucleotide. The trans-syn isomer is formed in a SYN-ANTI glycosidic conformation. In this isomer the sugar puckers of both deoxyribose rings are affected and a preference for a pure 2-endo conformation is observed.Abbreviations dTpdT 2-deoxythymidylyl-(35)-2-deoxythymidine - dTp[]dT cyclobutane type photodimers of dTpdT - dTp- and dTp[]- their 5' terminal fragments (fragment A) - -pdT and-[]pdT their 3 terminal fragments (fragment B) - RP-HPLC reversed-phase high-performance liquid chromatography - COSY two-dimensional correlated spectroscopy - 2D NOE two-dimensional nuclear Overhauser spectroscopy     

Localization of 5′-nucleotidase in bovine brain myelin fraction and myelin subfractions

Purified myelin from fresh calf brain white matter was subfractionated in a discontinuous sucrose gradient; significant recovery of protein and 2,3-cyclic nucleotide 3-phosphohydrolase (CNP) and 5-nucleotidase (5N) activities occurred in all three obtained subfractions, the highest recovery being in the light subfraction; highest 5N and CNP specific activities were in medium myelin. Purified myelin was also subfractionated in a continuous sucrose gradient, with a similar localization of protein; CNP activity and 5N activity maxima suggest that myelin may be a predominant locus of 5N in bovine brain white matter. Freezing of brain white matter caused an increase in protein and in CNP and 5N total activity recoveries in denser myelin subfractions. Cytochemistry showed the reaction product of 5N in the whole myelin fraction to be associated with the innermost, outermost and medial compact myelin layers. Effects of non-ionic detergent (Lubrol WX) on 5N activity were studied, and the results also suggest the intrinsic nature of 5N as an ectoenzyme in myelin membranes. Lubrol WX was viewed as an advisable detergent for the stimulation of myelin 5N activity, but not for the solubilization of this enzyme.     

Theβ-subunit of human chorionic gonadotropin containsN-glycosidic trisialo tri- and tri′-antennary carbohydrate chains

TheN-linked carbohydrate chains of the-subunit of highly purified urinary human chorionic gonadotropin have been re-investigated. The oligosaccharides were released enzymatically by peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F, and fractionated by a combination of FPLC and HPLC. As a result of the application of improved fractionation methods, apart from the earlier reported carbohydrate chains, also small amounts of trisialo tri- and tri-antennary oligosaccharides were found. The primary structures of the latter carbohydrate chains have been determined by 500-MHz¹H-NMR spectroscopy to beAbbreviations hCG human chorionic gonadotropin - hCG- -subunit - hCG- -subunit - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F (E.C. 3.5.1.52) - endo-F endo--N-acetylglucosaminidase-F (E.C. 3.2.1.96) - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - CBB coomassie brilliant blue R 250 - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - Man mannose - Gal galactose - Fuc fucose     

Evidence of β-carotene 7,8(7′,8′) oxygenase (β-cyclocitral,crocetindial generating) in Microcystis

A -carotene oxygenase is described which occurs in the Cyanobacterium Microcystis. It cleaves -carotene and zeaxanthin specifically at the positions 7,8 and 7,8, while echinenone and myxoxanthophyll are not affected. The oxidative cleavage of -carotene leads to the formation of -cyclocitral and crocetindial and that of zeaxanthin to hydroxy--cyclocitral and crocetindial in nearly stoichiometric amounts. Oxidant is dioxygen as has been demonstrated by high incroporation (86%) of ¹⁸O₂ into -cyclocitral. -Carotene oxygenase is membrane bound, sensitive to sulfhydryl reagents, antioxidants and chelating agents. Iron seems to be an essential part of the enzyme activity. Cofactors necessary for the reaction could not be detected.Abbreviations TLC thin layer-chromatography - PIPES piperazine-N,N-bis-(2-ethanesulfonate) Na - TES 2{[tris-(hydroxymethyl)-methyl]-amino} ethanesulfonic acid Dedicated to Professor G. Drews on occasion of his 60th birthday     

Cyclic AMP regulation of glucose transport in germinating Pilobolus longipes spores

Pilobolus longipes spores were activated by either glucose or 6-deoxyglucose. Glucose-induced spore activation was previously shown to follow an increase in intracellular cyclic AMP. Concurrent with glucose-induced spore activation, were shifts in 6-deoxyglucose transport kinetics towards higher V _max and K _m values. Cyclic AMP derivatives also caused spore activation and similar changes in the kinetic parameters of 6-deoxyglucose transport. The time course of activation was paralleled by changes in transport activity. Inhibition of phosphodiesterase alone did not cause activation or induce changes in transport activity, but in combination with sub-optimal levels of either 6-deoxyglucose or cAMP derivatives, it amplified the germination signals to produce large increases in both spore activation and 6-deoxyglucose transport activity. These results support the conclusion that glucose transport in germinating spores is regulated by cAMP.Abbreviations IBMX 3-isobutyl-1-methylxanthine; monobutyryl cyclic AMP - N⁶ monobutyryladenosine 3:5-cyclic monophosphate - 8-bromo cyclic AMP 8-bromoadenosine 3:5-cyclic monophosphate     

In vitro degradation of guanosine 3′,5′-bis(diphosphate) [ppGpp] by the spoT gene product [ppGppase] from auxotrophic strains of Escherichia coli: Effects of various antibiotics and drugs

The enzyme specifically hydrolyzing guanosine 3,5-bis(diphosphate) [ppGpp] has been isolated from the ribosomal fraction of Escherichia coli; it released pyrophosphate from the 3-position of ppGpp. The effects of various drugs and antibiotics known to interfere with protein and/or RNA synthesis were investigated in the ppGpp degrading reaction. It was determined that tetracycline, chlorotetracycline, and thiostrepton strongly inhibited the reaction, whereas levallorphan gave a moderate inhibition. Only the tetracycline-mediated inhibition could be reversed by manganese ions. Oxytetracycline, rifampicin, fusidic acid, kirromycin, streptomycin, puromycin, chloramphenicol, and morphine did not inhibit the decay reaction.Abbreviations ppGpp guanosine 3,5-bis(diphosphate)     

Carotenoid pigments in a red strain of Rhizobium from Lotononis bainesii Baker

The carotenoid pigments of a Rhizobium strain isolated from Lotononis bainesii were found to be diglucosyl-4,4-diapocarotene-4,4-dioate and glucosyl-4,4-diapocarotene-4-oate-4-oic acid.5th publication in the series Carotenoids of Rhizobia [4th publication: Helv. chim. Acta 62: 2551–2557 (1979)]     

Structural analysis of Tpn1, a transposable element isolated from Japanese morning glory bearing variegated flowers

The 6.4 kb transposable element Tpn1 belonging to the En/Spm family was found within one of the DFR (dihydroflavonol-4-reductase) genes for anthocyanin biosynthesis in a line of Japanese morning glory (Pharbitis nil) bearing variegated flowers. Sequencing of the Tpn1 element revealed that it is 6412 by long and carries 28-bp perfect terminal inverted repeats. Its subterminal repetitive regions, believed to be the cis-acting sequences for transposition, show striking structural features. Twenty-two copies of the 10-bp sequence motif GACAACGGTT can be found as direct or inverted repeats within 650 by of the 5 end of the element, and 33 copies of the sequence motif lie within 800 by of the 3 terminus. All these 22 copies of the sequence motif near the 5 terminus and 30 copies in the 3 terminal region are arranged as inverted repeats and 3–8 by AT-rich sequences are detected between these inverted repeats. In addition, four copies of 122-bp tandem repeats and six copies of 104-bp tandem repeats are present in the 5 and 3 subterminal repetitive regions, respectively. No large open reading frame characteristic of autonomous elements of the En/Spm family can be detected within the element. The results are discussed with respect to heritable changes in flower variegation in this line of Japanese morning glory.     

Eubacteria have 3 growth modes keyed to nutrient flow

Aerobic growth of Escherichia coli and Paracoccus denitrificans has been studied in chemostat, fed batch, and recycling fermentor modes under carbon and energy limitation. Two abrupt drops or discontinuities in molar growth yield, Y, have been found that occur over relatively short ranges in the value of specific growth rate.Before the first discontinuity, Y is constant and maximal. After the first discontinuity, at a doubling time of 33 h, Y becomes constant again and independent of until the second discontinuity appears at a doubling time of about 50 h, corresponding to a of about 0.014. At this point, Y drops to a lower value that is constant at doubling times longer than 100 h, corresponding to a of about 0.007.The second discontinuity is associated in Paracoccus with elevated levels of guanosine tetraphosphate (ppGpp) that impose stringent regulation as has been found previously with Bacillus and Escherichia species. It is thus likely that the stringent response generally occurs in bacteria in vivo at a doubling time of about 50 h. The cause of the first discontinuity is unknown. All experiments indicate that Pirt-type calculations relating , Y, and maintenance energy demand are no longer valid. In chemostat experiments, the intercept of the relationship between specific substrate utilization and specific growth rate is defined as maintenance. However, this intercept most probably is caused by stringent regulation at low dilution rates. Three regions of bacterial growth rates are defined by this study, corresponding to doubling times of 0.5 to 15 h, 33 to 50 h, and >100 h. Some growth behavior in each region is unique to that region.Abbreviations ppGpp guanosine 5 diP 3 diP - pppGpp guanosine 5 triP 3 diP - SPR substrate provision rate (mol/l h)     

The catalysis of nucleotide polymerization by compounds of divalent lead

Summary Soluble lead salts and a number of lead-containing minerals catalyze the formation of oligonucleotides from nucleoside 5-phosphorimidazolides. The effectiveness of lead compounds correlates strongly with their solubility. Under optimal conditions we were able to obtain 18% of pentamer and higher oligomers from ImpA. Reactions involving ImpU gave smaller yields.Abbreviations A adenosine - U uridine - Im imidazole - MeIm 1-methyl-imidazole - EDTA ethylenediaminetetraacetic acid - pA adenosine 5-phosphate - pU uridine 5-phosphate - Ap adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - AppA P₁,P₂-diadenosine 5-diphosphate - pNp (N = A,U) nucleotide 2(3), 5-diphosphate - ImpA adenosine 5-phosphoreimidazolide - ImpU uridine 5-phosphorimidazolide - A ²pA adenylyl-[25]-adenosine - A ³pA adenylyl-[35]-adenosine - pA ²pA 5-phospho-adenylyl-[25]-adenosine - pA ³pA 5-phospho-adenylyl-[35]-adenosine - pUpU 5-phospho-uridylyl-uridine - pApU 5-phospho-adenylyl-uridine - pUpA 5-phospho-uridylyladenine - (pA)n (n, 2,3,4,) oligoadenylates with 5 terminal phosphate - ImpApA 5-phosphorimidazolide of adenylyl adenosine - (pA) ₅₊ pentamer and higher oligoadenylates with 5 terminal phosphate - (Ap)nA (n = 2,3,4) oligoadenylates without terminal phosphates In the following we do not specify the nature of the internucleotide linkageIn the following we do not specify the nature of the internucleotide linkage     

Observations on the reduction of non-activated carboxylates by Clostridium formicoaceticum with carbon monoxide or formate and the influence of various viologens

Crude extracts or supernatants of broken cells of Clostridium formicoaceticum reduce unbranched, branched, saturated and unsaturated carboxylates at the expense of carbon monoxide to the corresponding alcohols. The presence of viologens with redox potentials varying from E ₀=-295 to-650 mV decreased the rate of propionate reduction. The more the propionate reduction was diminished the more formate was formed from carbon monoxide. The lowest propionate reduction and highest formate formation was observed with methylviologen. The carbon-carbon double bond of E-2-methyl-butenoate was only hydrogenated when a viologen was present. Formate as electron donor led only in the presence of viologens to the formation of propanol from propionate. The reduction of propionate at the expense of a reduced viologen can be followed in cuvettes. With respect to propionate Michaelis Menten behavior was observed. Experiments are described which lead to the assumption that the carboxylates are reduced in a non-activated form. That would be new type of biological reduction.Non-standard abbreviations glc Gas liquid chromatography - HPLC high performance liquid chromatography - RP reverse phase; Mediators (the figures in parenthesis of the mediators are redox potentials E ₀ in mV) - CAV²⁺ carbamoylmethylviologen, 1,1-carbamoyl-4,4-dipyridinium dication (E ₀=-296 mV) - BV²⁺ benzylviologen, 1,1-dibenzyl-4,4-dipyridinium dication (E ₀=-360 mV) - MV methylviologen, 1,1-dimethyl-4,4-dipyridinium-dication (E ₀=-444 mV) - DMDQ²⁺ dimethyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-ethylendication (E ₀=-514 mV) - TMV²⁺ tetramethylviologen, 1,1,4,4-tetramethyl-4,4-dipyridinium dication (E ₀=-550 mV) - PDQ²⁺ propyldiquat, 2,2-dipyridino-1,1-propenyl dication (E ₀=-550 mV) - DMPDQ²⁺ dimethylpropyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-propenyl dication (E ₀=-656 mV) - PN productivity number=mmol product (obtained by the uptake of one pair of electrons) x (biocatalyst (dry weight) kg)^-1×h^-1     

Temperature-sensitive coupling and uncoupling of ATPase-mediated,nonradiative energy dissipation: Similarities between chloroplasts and leaves

The effects of temperature on the dark relaxation kinetics of nonradiative energy dissipation in photosystem II were compared in lettuce (Lactuca sativa L.) chloroplasts and leaves of Aegialitis annulata R. Br. After high levels of violaxanthin de-epoxidation in the light, Aegialitis leaves showed a marked delay in the dark relaxation of nonradiative dissipation, measured as non-photochemical quenching (NPQ) of photosystem II chlorophyll a fluorescence. Aegialitis leaves also maintained a moderately high adenylate energy charge at low temperatures during and after high-light exposure, presumably because of their limited carbon-fixation capacity. Similarly, dark-sustained NPQ could be induced in lettuce chloroplasts after de-epoxidizing violaxanthin and light-activating the ATP synthase. The duration and extent of dark-sustained NPQ were strongly enhanced by low temperatures in both chloroplasts and leaves. Further, the NPQ sustained at low temperatures was rapidly reversed upon warming. In lettuce chloroplasts, low temperatures sharply decreased the ATP-hydrolysis rate while increasing the duration and extent of the resultant trans-thylakoid proton gradient that elicits the NPQ. This was consistent with a higher degree of energy-coupling, presumably due to reduced proton diffusion through the thylakoid membrane at the lower temperatures. The chloroplast adenylate pool was in equilibrium with the adenylate kinase and therefore both ATP and ADP contributed to reverse coupling. The low-temperature-enhanced NPQ quenched the yields of the dark level (F_o) and the maximal (F_m) fluorescence proportionally in both chloroplasts and leaves. The extent of NPQ in the dark was inversely related to the efficiency of photosystem II, and very similar linear relationships were obtained over a wide temperature range in both chloroplasts and leaves. Likewise, the dark-sustained absorbance changes, caused by violaxanthin de-epoxidation (A_508nm) and energy-dependent light scattering (A_536nm) were strikingly similar in chloroplasts and leaves. Therefore, we conclude that the dark-sustained, low-temperature-stimulated NPQ in chloroplasts and leaves is apparently directly dependent on lumen acidification and chloroplastic ATP hydrolysis. In leaves, the ATP required for sustained NPQ is evidently provided by oxidative phosphorylation in the mitochondria. The functional significance of this quenching process and implications for measurements of photo-protection versus photodamage in leaves are discussed.Abbreviations and Symbols A antheraxanthin - Chl chlorophyll - DPS de-epoxidation state of the xanthophyll cycle, ([Z+A]/[V+A+Z]) - F, F steady-state fluorescence in the absence, presence of thylakoid energization - F_o, F_o dark fluorescence level in the absence, presence of thylakoid energization - F_m, F_m maximal fluorescence in absence, presence of thylakoid energization - NPQ nonphotochemical quenching (F_m/F_m)–1 - V violaxanthin - Z zeaxanthin - NRD nonradiative dissipation - PFD photon flux density - [2ATP+ADP] - pH trans-thylakoid proton gradient - S pH-dependent light scattering - _PSII (F_m–F)/F_m, photon yield of PSII photochemistry at the actual reduction state in the light or dark - [ATP+ADP+AMP] We thank Connie Shih for skillful assistance in growing plants and for conducting HPLC analyses. Support from an NSF/USDA/DOE postdoctoral training grant to A.G. is gratefully acknowledged. A.G. also wishes to thank Prof. Govindjee for valuable discussions. C.I.W.-D.P.B. Publication No. 1197.     

Specificity of recA441-mediated (tif-1) mutational events.

Summary To investigate the impact of SOS induction on the distribution of spontaneous mutation, 111 recA441-mediated mutations were characterized at the DNA sequence level in the lacI gene of Escherichia coli. A 2.6-fold enhancement in lacI ^– mutation frequency was observed after induction of the SOS system in the absence of mutagenic treatment, and specific classes of mutational events were induced. G : C C : G, G : C T : A and A : T T : A transversion events were specifically enhanced after SOS induction. A preferential 5-Y-Purine-3 neighbouring base specificity for these transversion events is reported here (normalised for mutation of the purine residue). In addition, a preference for transversion events at 5-C/GTGG-3 sequences is also observed. Fifty events were recovered at the lacI frameshift hotspot site and were equally represented by 4 bp addition and deletion events. This 1:1 ratio deviates significantly from the 4:1 distribution characteristic of spontaneous frameshift mutation in the RecA⁺ background and is a consequence of the fourfold induction of the (–)4 event. This abberrant distribution was confirmed by oligomeric probing of 474 independent recA441-mediated spontaneous lacI ^– mutations.     

Involvement of cyclic adenosine-3′, 5′-monophosphate in chloronema differentiation in protonema cultures of Funaria hygrometrica

The role of purine and pyrimidine ribosides, nucleotides and substituted xanthines in the differentiation of chloronema filaments in suspension cultures of protonema of the moss Funaria hygrometrica Hedw. has been examined. Cyclic adenosine-3,5-monophosphate (cAMP) and mono-and dibutyryl cAMP evoked the maximum response in wild-type protonema. ADP and ATP also enhanced chloronema differentiation but were less active than cAMP; pyrimidine derivatives were completely inactive. Inhibitors of cyclic-nucleotide phosphodiesterase aminophylline, theophylline and ICI 58, 301 (3-acetamido-6-methyl-8-n-propyl-s-triazolo-(4,3a)-pyrazine)-mimicked the effect of cAMP. A leaky, chloronema-repressed mutant was isolated and in this mutant cAMP was much more active than cyclic guanosine monophosphate and ADP in enhancing chloronema differentiation. These results strongly indicate that cAMP is involved in chloronema differentiation in Funaria, and a hypothesis on growth regulation in protonema cell cultures is proposed.Abbreviations cAMP, cyclic AMP cyclic adenosine-3, 5-monophosphate - cCMP, cGMP, cIMP cyclic cytosine-, guanosine-and inosine-3, 5-monophosphates, respectively - IAA indole-3-acetic acid - ICI 58,301 3-acetamido-6-methyl-8-n-propyl-s-triazolo-(4,3a)-pyrazine     

The effect of ionic stress on photosynthesis in Dunaliella tertiolecta

A comparison of the effects of ionic stress and an uncoupler on long-term fluorescence transients (the Kautsky effect) in the green alga Dunaliella tertiolecta indicated that the large quenching induced by ionic stress was caused by a pH gradient across the thylakoid membrane. This possiblity was given support by the increase in the slow phase of 3-(3,4-dichlorophenyl)-1,1-dimethylurea-induced fluorescence relaxation in algae subjected to ionic stress. Low-temperature fluorescence emission spectra indicated that salt stress enhanced photosystem-I emission in the dark, and a comparison of simultaneous emissions at 695 and 720 nm at room temperature indicated a further increase in photosystem-I emission during the fluorescence transients. Taken together with the decrease in the fast phase of 3-(3,4-dichlorophenyl)-1,1-dimethylurea-induced fluorescence relaxation in stressed algae, our results indicate that ionic stress stimulates cyclic electron flow, and that non-cyclic flow is inhibited. The effect of sucrose-induced osmotic stress was similar to, but less marked than, the effects of NaCl and KCl; the effect of decreasing the external salinity was small.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FCCP carbonylcyanide p-trifluoromethoxyphenylhydrazone - PSI, II photosystem I, II