Characterization of candidate intermediates in the Black Box of the ecdysone biosynthetic pathway in Drosophila melanogaster: Evaluation of molting activities on ecdysteroid-defective larvae

Early steps of the biosynthetic pathway of the insect steroid hormone ecdysone remains the “Black Box” wherein the characteristic ecdysteroid skeleton is built. 7-Dehydrocholesterol (7dC) is the precursor of uncharacterized intermediates in the Black Box. The oxidation step at C-3 has been hypothesized during conversion from 7dC to 3-oxo-2,22,25-trideoxyecdysone, yet 3-dehydroecdysone is undetectable in some insect species. Therefore, we first confirmed that the oxidation at C-3 occurs in the fruitfly, Drosophila melanogaster using deuterium-labeled cholesterol. We next investigated the molting activities of candidate intermediates, including oxidative products of 7dC, by feeding-rescue experiments for Drosophila larvae in which an expression level of a biosynthetic enzyme was knocked down by the RNAi technique. We found that the administration of cholesta-4,7-dien-3-one (3-oxo-Δ^4,7C) could overcome the molting arrest of ecdysteroid-defective larvae in which the expression level of neverland was reduced. However, feeding 3-oxo-Δ^4,7C to larvae in which the expression levels of shroud and Cyp6t3 were reduced inhibited molting at the first instar stage, suggesting that this steroid could be converted into an ecdysteroid-antagonist in loss of function studies of these biosynthetic enzymes. Administration of the highly conjugated cholesta-4,6,8(14)-trien-3-one, oxidized from 3-oxo-Δ^4,7C, did not trigger molting of ecdysteroid-defective larvae. These results suggest that an oxidative product derived from 7dC is converted into ecdysteroids without the formation of this stable conjugated compound. We further found that the 14α-hydroxyl moiety of Δ⁴-steroids is required to overcome the molting arrest of larvae in loss of function studies of Neverland, Shroud, CYP6T3 or Spookier, suggesting that oxidation at C-14 is indispensable for conversion of these Δ⁴-steroids into ecdysteroids via 5β-reduction.     

Developmental arrest induced by juvenile hormone in larvae of Bombyx mori

Injection of the juvenile hormone analog (JHA) methoprene into day 3, fifthinstar larvae of Bombyx mori induced developmental arrest. Feeding activity declined, and the larvae remained as larvae for more than 2 weeks, after which they died. After JHA injection, the hemolymph ecdysteroid titer was low, and the prothoracic glands were almost inactive for 7 days. During this period, prothoracic glands were stimulated by prothoracicotropic hormone (PTTH) in vitro, indicating that JHA did not inhibit the competence of the glands to respond to PTTH. When brain-corpora cardiaca-corpora allata complexes were removed from intact fifth-instar larvae on day 4, the prothoracic glands became autonomously active and produced enough ecdysone for pupation. When PTTH injections were given to larvae previously injected with JHA (7 days before), the larvae recovered feeding activity, purged their guts, and pupated. Injections of 20-hydroxyecdysone into larvae that had been injected with JHA 7 days earlier induced larval molting. These results suggest that JHA affects both the brain and the prothoracic gland.     

Biotransformation of 20(S)-protopanaxadiol by Mucor spinosus

Biotransformation of 20(S)-protopanaxadiol (1) by the fungus Mucor spinosus AS 3.3450 yielded eight metabolites (2–9). On the basis of NMR and MS analyses, the metabolites were identified as 12-oxo-15α,27-dihydroxyl-20(S)-protopanaxadiol (2), 12-oxo-7β,11α,28-trihydroxyl-20(S)-protopanaxadiol (3), 12-oxo-7β,28-dihydroxyl-20(S)-protopanaxadiol (4), 12-oxo-15α,29-dihydroxyl-20(S)-protopanaxadiol (5), 12-oxo-7β,15α-dihydroxyl-20(S)-protopanaxadiol (6), 12-oxo-7β,11β-dihydroxyl-20(S)-protopanaxadiol (7), 12-oxo-15α-hydroxyl-20(S)-protopanaxadiol (8), and 12-oxo-7β-hydroxyl-20(S)-protopanaxadiol (9), respectively. Among them, 2–5, 7, and 8 are new compounds. These results indicated that M. spinosus could catalyze the specific C-12 dehydrogenation of 20(S)-protopanaxadiol, as well hydroxylation at different positions. These biocatalytic reactions may be difficult for chemical synthesis. The biotransformed products showed weak in vitro cytotoxic activities.     

Development of an in vitro assay for prothoracicotropic hormone of the gypsy moth,Lymantria dispar (L.) following studies on identification,titers and synthesis of ecdysteroids in last-instar females

Summary Hemolymph ecdysteroid titers and in vitro prothoracic gland ecdysteroid synthesis have been examined in last-instar larval (5th instar) females of Lymantria dispar. Ecdysteroids were quantified by radioimmunoassay and characterized by co-elution with known standards of ecdysteroids on reverse-phase high-performance liquid chromatography. Analysis of hemolymph yielded ecdysone and 20-OH-ecdysone in ratios of 1:1 (day 6, shortly after attainment of maximum weight) and 1:28 (day 10, molting peak). Analysis of in vitro culture media from glands challenged with extracts of brains or retrocerebral complexes, or left unchallenged, revealed only immunoreactive material co-eluting with a known standard of ecdysone. Time-course studies of in vitro prothoracic gland ecdysone secretion demonstrated a major peak on day 10, 1–2 days prior to pupal ecdysis, and a small elevation on days 5–6. On days 5 and 6, 2.29±0.41 and 2.65±0.72 ng ecdysone per gland, respectively, were secreted in 6-h cultures. On day 10, 25.69±4.36 ng was secreted in 6-h culture. The ability of prothoracic glands of various ages to respond to brain extracts containing prothoracicotropic hormone activity was tested by determining an activation ratio for each day of the instar. The activation ratio was determined over a 90-min period by dividing the amount of ecdysone secreted by one member of a pair of prothoracic glands in the presence of brain extract by that of its contralateral control gland in Grace's medium. Prior to the addition of brain extract, the activity of the glands was allowed to subside to basal level for 180 min in Grace's medium. The activition ratio was highest on days 3–7 and fell throughout the remainder of the instar as the inherent ability of the prothoracic gland to maintain high levels of ecdysteroid synthesis in vitro in the absence of prothoracicotropic hormone increased. A two-phase in vitro assay for prothoracicotropic hormone was established using activition ratios. This assay showed saturable doseresponse kinetics for prothoracic gland ecdysone secretion and specificity to extracts prepared from brain or retrocerebral complexes. A comparable assay for prothoracicotropic hormone purification, based on net synthesis and requiring half the number of prothoracic glands was also established.Abbreviations A _r activation ratio - HPLC high performance liquid chromatography - HPSEC high performance size-exclusion chromatography - PG prothoracic gland - PTTH prothoracicotropic hormone - RIA radioimmunoassay     

A Novel Type of Receptor cDNA from the Prothoracic Glands of the Silkworm,Bombyx mori

A cDNA encoding a novel heptahelical receptor from the prothoracic glands of the silkworm, Bombyx mori was cloned and sequenced during screening of a prothoracicotropic hormone (PTTH) receptor. Orthologs of this receptor are found not only in insects, but also in the vertebrates. In B. mori, ubiquitous expression of the mRNA was observed in the larva. Also, a higher expression level in the prothoracic glands was observed before molting and metamorphosis and was impaired after pupal molting. But, further analysis is required to confirm whether this receptor cDNA encodes the PTTH receptor.     

Australopithecus, Meganthropus and Ramapithecus

The South African members of Australopithecus form a single group, trending from earlier, more gracile or smaller forms, to later, more robust or larger forms, in accordance with the “Law of Cope”. This is supported by the evidence of the lower first deciduous molar: it is only slightly molarized in the earlier, smaller forms and astonishingly heavily molarized in the later, robust forms, such as that of Kromdraai. Hence, Robinson's view that two different genera are represented by the gracile and robust forms is not supported here. A. robustus is seen as a late offshoot of the Australopithecus stem. The resemblance of the Taung dm₁ to that of Sinanthropus, except for the more differentiated talonid of the Taung specimen, suggests that the separation of the Australopithecinae and Homininae must have taken place at an earlier stage than that represented by the oldest South African Australopithecinae. The lower jaw of Meganthropus of Java combines certain characteristics of A. africanus with those of A. robustus: Meganthropus might provisionally be called “Australopithecoid”. The geographically intermediate India has yielded a hominid, Ramapithecus punjabicus, but the author does not consider “Kenyapithecus” to be a hominid. “K. wickeri” is a pongid species of its own and “K. africanus” a Proconsul. Ramapithecus sensu stricto is known only from the Indian Siwaliks and the author suggests that the transition from Ramapithecus to a still unknown Australopithecus took place in the same region prior to their migration into Africa and Southeast Asia.     

Conversion of 3-oxo steroids into ecdysteroids triggers molting and expression of 20E-inducible genes in Drosophila melanogaster

Ecdysteroids, steroid hormones in insects, coordinate major developmental transitions. During postembryonic development, ecdysone is biosynthesized from dietary cholesterol in the prothoracic gland (PG). Despite extensive studies, the initial conversion process, the so-called "Black Box", has not been characterized. A cytochrome P450 enzyme, Spookier (Spok), is speculated as a rate limiting enzyme in the Black Box during larval-pupal transitions in Drosophila melanogaster. RNAi mediated knockdown of spok expression in the PG results in arrest of molting. Because the developmental arrest can be rescued by application of an appropriate intermediate, we examined potential activities of candidate intermediates in the RNAi-treated larvae. We found that two 3-oxo steroids, cholesta-4,7-diene-3,6-dione-14α-ol (Δ(4)-diketol) and 5β [H]cholesta-7-ene-3,6-dione-14α-ol (diketol), triggered molting of the RNAi-treated larvae. We also detected an enhancement of the amounts of ecdysteroids in the RNAi-treated larvae by feeding the Δ(4)-diketol or diketol, indicating that the dietary 3-oxo steroids were incorporated and converted into ecdysteroids in vivo. Furthermore, 20-hydroxyecdysone inducible genes were induced in the RNAi-treated larvae by feeding the Δ(4)-diketol or diketol. These results indicate that Δ(4)-diketol and diketol are components of the ecdysteroid biosynthetic pathway and lie downstream of a step catalyzed by Spok.     

Triterpenoids from Viburnum suspensum

Three triterpenoids, 3-oxo-11,13(18)-oleanadien-28-oic acid, 24-hydroxy-3-oxo-11,13(18)-oleanadien-28-oic acid, 6β-hydroxy-3-oxo-11,13(18)-oleanadien-28-oic acid have been isolated together with the previously known virgatic acid, vibsanin B and 3-hydroxyvibsanin E from the leaves of Viburnum suspensum. Their structures were determined by spectroscopic methods and by comparison of their NMR spectral data with those of the previously known 11,13(18)-oleanadien-3β-ol.     

Neuropeptides, second messengers and insect molting

Insect molting is elicited by a class of polyhydroxylated steroids, ecdysteroids, that originate in the prothoracic glands. Ecdysteroid synthesis in the prothoracic glands is controlled in large measure by a peptide hormone from the brain, prothoracicotropic hormone (PTTH), which exists in two forms and is released into the general circulation as a result of environmental and developmental cues. The means by which PTTH activates the prothoracic glands has been examined at the cellular level and the data reveal the involvement of cAMP, calcium, calmodulin, cAMP-dependent protein kinase and the ultimate phosphorylation of a 34 kDa protein tentatively identified as ribosomal protein S6.     

Effects of engrailed in the genital disc of Drosophila melanogaster

engrailed has been postulated to be the “selector gene” involved in the establishment of the anterior-posterior compartment border in several imaginal discs and in at least the first two abdominal segments of Drosophila melanogaster. Our study of the effects of different mutant engrailed genotypes on genital disc development provided the following major results: All three terminal primordia (female and male genitalia, and analia) were affected. Different heteroallelic combinations showed different expressivities, and the three terminal primordia were differently affected by the same mutant genotype. The engrailed genotypes deleted specific elements of the adult terminalia without causing associated pattern duplications. The reduced morphology of the male engrailed genital disc was analogous to the pattern deletions observed in the adult terminalia. That the engrailed phenotype is stable was demonstrated by culturing in vivo intact and fragmented engrailed genital discs. Cell death was found in a significant number of mature male en²/en³ genital discs. The results are discussed in terms of the segmental organization of the genital disc and in terms of the “selector gene” function postulated for the engrailed locus. The interpretation that each terminal primordium has an anterior and a posterior compartment is presented and it is assumed that in the genital disc engrailed transforms posterior cells into anterior cells that do not develop, thereby causing the deficiency pattern of the engrailed phenotype.     

PAF-antagonistic bicyclo[3.2.1]octanoid neolignans from leaves of Ocotea macrophylla Kunth. (Lauraceae)

Di-nor-benzofuran neolignan aldehydes, Δ⁷-3,4-methylenedioxy-3′-methoxy-8′,9′-dinor-4′,7-epoxy-8,3′-neolignan-7′-aldehyde (ocophyllal A) 1, Δ⁷-3,4,5,3′-tetramethoxy-8′,9′-dinor-4′,7-epoxy-8,3′-neolignan-7′-aldehyde (ocophyllal B) 2, and macrophyllin-type bicyclo[3.2.1]octanoid neolignans (7R, 8R, 3′S, 4′S, 5′R)-Δ⁸′-4′-hydroxy-5′-methoxy-3,4-methylenedioxy-2′,3′,4′,5′-tetrahydro-2′-oxo-7.3′,8.5′-neolignan (ocophyllol A) 3, (7R, 8R, 3′S, 4′S, 5′R)-Δ⁸′-4′-hydroxy-3,4,5′-trimethoxy-2′,3′,4′,5′-tetrahydro-2′-oxo-7.3′,8.5′-neolignan (ocophyllol B) 4, (7R, 8R, 3′S, 4′S, 5′R)-Δ⁸′-4′-hydroxy-3,4,5,5′-tetramethoxy-2′,3′,4′,5′-tetrahydro-2′-oxo-7.3′,8.5′-neolignan (ocophyllol C) 5, as well as 2′-epi-guianin 6 and (+)-licarin B 7, were isolated and characterized from leaves of Ocotea macrophylla (Lauraceae). The structures and configuration of these compounds were determined by extensive spectroscopic analyses. Inhibition of platelet activating factor (PAF)-induced aggregation of rabbit platelets were tested with neolignans 1–7. Although compound 6 was the most potent PAF-antagonist, compounds 3–5 showed some activity.     

S6 phosphorylation results from prothoracicotropic hormone stimulation of insect prothoracic glands: A role for S6 kinase

The insect prothoracic glands are the source of steroidal molting hormone precursors and the glands are stimulated by a brain neuropeptide, prothoracicotropic hormone (PTTH). Previous work from this laboratory revealed that PTTH acts via a cascade including Ca²⁺/calmodulin activation of adenylate cyclase, protein kinase A, and the subsequent phosphorylation of a 34 kDa protein (p34) hypothesized, but not proven, to be the 56 protein of the 40S ribosomal subunit. The jmmunosuppressive macrolide, rapamycin, is a potent inhibitor of cell proliferation, a signal transduction blocker, and also prevents ribosomal S6 phosphorylation in mammalian systems. We demonstrate here that rapamycin inhibited PTTH-stimulated ecdysteroidogenesis in vitro by the prothoracic glands of the tobacco hornworm, Manduca sexta, with half-maximal inhibition at a concentration of about 5 nM. At concentrations above 5 nM, there was a 75% inhibition of ecdysteroid biosynthesis. Similar results, were observed with the calcium ionophore (A23187), a known stimulator of ecdysteroidogenesis. Most importantly, the inhibition of ecdysteroid biosynthesis was accompanied by the specific inhibition of the phosphorylation of p34, indicating that p34 indeed is ribosomal protein S6. In vivo assays revealed that injection of rapamycin into day 6 fifth instar larvae resulted in a decreased hemolymph ecdysteroid titer and a dose-dependent delay in molting and metamor-phosis. When S6 kinase (S6K) activity was examined using rapamycin-treated prothoracic glands as the enzyme source and a synthetic peptide (S6-21) or a 40S ribosomal subunit fraction from Manduca tissues as substrate, the date revealed that rapamycin inhibited S6K activity. The composite data suggest that rapamycin inhibits a signal transduction element leading to p34 phosphorylation that is necessary for PTTH-stimulated ecdysteroidogenesis in this insect endocrine gland, and lend further support to the concept that p34 is S6. © 1994 Wiley-Liss, Inc.     

Neural cell protective compounds isolated from Phoenix hanceana var. formosana

A platform for screening drugs for their ability to protect neuronal cells against cytotoxicity was developed. Nerve growth factor (NGF) differentiates PC12 cells into nerves, and these differentiated PC12 cells enter apoptosis when challenged with 6-hydroxydopamine (6-OHDA). A screening spectrophotometer was used to assay cytotoxicity in these cells; pretreatment with test samples allowed identification of compounds that protected against this neuronal cytotoxicity. The 95% ethanol extract of Phoenix hanceana Naudin var. formosana Beccari. (PH) showed potential neuroprotective activity in these assays. The PH ethanol extract was further fractionated by sequential partitioning with n-hexane, ethyl acetate (EtOAc), n-butanol (n-BuOH), and water. Subsequent rounds of assaying resulted in the isolation of ten constituents, and their structures were characterized by various spectroscopic techniques and identified by comparison with previous data as: isoorientin (1), isovitexin (2), veronicastroside (3), luteolin-7-O-β-d-glucopyranoside (4), isoquercitrin (5), tricin-7-neohesperidoside (6), tricin-7-O-β-d-gluco-pyranoside (7), (+)-catechin (8), (−)-epicatechin (9), and orientin 7-O-β-d-glucopyranoside (10). Among these compounds, isovitexin (2), luteolin-7-O-β-d-glucopyranoside (4) and (+)-catechin (8) showed significant neuroprotective activity in cell viability (WST-8 reduction), anti-apoptosis (Annexin V-FITC/propidium iodide double-labeled flow cytometry), and cellular ROS scavenging assays (besides isovitexin (2)), as well as a decreased caspase-8 activity in 6-OHDA-induced PC12 cells. Hence, isovitexin (2), luteolin-7-O-β-d-glucopyranoside (4), and (+)-catechin (8) protected PC12 cells from 6-OHDA-induced apoptotic neurotoxicity.     

Antagonistic effect of enterocin CCM 4231 from Enterococcus faecium on “bryndza”, a traditional Slovak dairy product from sheep milk

“Bryndza” is a traditional Slovak dairy product (type of soft cheese) made from sheep cheese which was ripened for 14 days. Because its manufacture, transporting and/or storing represent conditions which facilitate contamination, the effect of enterocin CCM4231 in “bryndza” was investigated with the aim to reduce the contaminant agents. “Bryndza” was divided into equal portions (50 g). The experimental sample (ES) as well as the control sample one (C1) were inoculated with Listeria innocua Li1 strain. The other control samples C2 and C3 were without Li1 strain. C3 control was selected as a reference control. ES and C2 portions were treated with purified enterocin CCM4231 in a concentration of 6400 AU/ml. Before the experimental inoculation, “bryndza” was checked for the presence of contaminant agents. The experiment lasted 1 week and the samples were stored in the refrigerator at 4 °C. Sampling was performed on day 1, on day 4 and on day 7. The control samples C2 and C3 were checked only on day 1 and then after 1 week. The following contaminant agents were detected in “bryndza” before its experimental inoculation with L. innocua Li1 strain: Escherichia coli in the amount 10³ cfu/ml/g, Staphylococcus aureus (10² cfu/ml/g) and enterococci (10⁴ cfu/ml/g). In the control sample C2, the number of E. coli was reduced to 10² cfu/ml/g. Enterococci and staphylococci were totally eliminated there. Concerning C3 control, natural decrease of bacteria was found and/or their unchanged counts. The value of pH (5) was stable during the whole experiment. In the experimental sample inoculated with Li1 strain, its counts were decreased immediately after enterocin CCM4231 addition approximately by one order of magnitude. This inhibitory effect was also detectable on day 4 by the difference of one order of magnitude between ES and C1. On day 7, 10³ cfu/ml/g of Li1 strain were detected in both samples (ES, C1). The difference by one order of magnitude indicated, an inhibitory effect of enterocin CCM4231 in “bryndza”. However, bacteriocin activity was not determined by laboratory analyses.     

The Phylogenetics of Mycotoxin and Sclerotium Production in Aspergillus flavus and Aspergillus oryzae

Aspergillus flavus is a common filamentous fungus that produces aflatoxins and presents a major threat to agriculture and human health. Previous phylogenetic studies of A. flavus have shown that it consists of two subgroups, called groups I and II, and morphological studies indicated that it consists of two morphological groups based on sclerotium size, called “S” and “L.” The industrially important non-aflatoxin-producing fungus A. oryzae is nested within group I. Three different gene regions, including part of a gene involved in aflatoxin biosynthesis (omt12), were sequenced in 33 S and L strains of A. flavus collected from various regions around the world, along with three isolates of A. oryzae and two isolates of A. parasiticus that were used as outgroups. The production of B and G aflatoxins and cyclopiazonic acid was analyzed in the A. flavus isolates, and each isolate was identified as “S” or “L” based on sclerotium size. Phylogenetic analysis of all three genes confirmed the inference that group I and group II represent a deep divergence within A. flavus. Most group I strains produced B aflatoxins to some degree, and none produced G aflatoxins. Four of six group II strains produced both B and G aflatoxins. All group II isolates were of the “S” sclerotium phenotype, whereas group I strains consisted of both “S” and “L” isolates. Based on the omt12 gene region, phylogenetic structure in sclerotium phenotype and aflatoxin production was evident within group I. Some non-aflatoxin-producing isolates of group I had an omt12 allele that was identical to that found in isolates of A. oryzae.     

Expression in Escherichia coli and Simple Purification of Human Fhit Protein

The fragile histidine triad (Fhit) protein is a homodimeric protein with diadenosine 5′,5-P¹,P³-triphosphate (Ap₃A) asymmetrical hydrolase activity. We have cloned the human cDNA Fhit in the pPROEX-1 vector and expressed with high yield in Escherichia coli with the sequence Met-Gly-His₆-Asp-Tyr-Asp-Ile-Pro-Thr-Thr followed by a rTEV protease cleavage site, denoted as “H6TV,” fused to the N-terminus of Fhit. Expression of H6TV–Fhit in BL21(DE3) cells for 3 h at 37°C produced 30 mg of H6TV–Fhit from 1 L of cell culture (4 g of cells). The H6TV–Fhit protein was purified to homogeneity in a single step, with a yield of 80%, using nickel-nitrilotriacetate resin and imidazole buffer as eluting agent. Incubation of H6TV–Fhit with rTEV protease at 4°C for 24 h resulted in complete cleavage of the H6TV peptide. There were no unspecific cleavage products. The purified Fhit protein could be stored for 3 weeks at 4°C without loss of activity. The pure protein was stable at −20°C for at least 18 months when stored in buffer containing 25% glycerol. Purified Fhit was highly active, with a K_m value for Ap₃A of 0.9 μM and a k_cat(monomer) value of 7.2 ± 1.6 s⁻¹ (n = 5). The catalytic properties of unconjugated Fhit protein and the H6TV–Fhit fusion protein were essentially identical. This indicates that the 24-amino-acid peptide containing the six histidines fused to the N-terminus of Fhit does not interfere in forming the active homodimers or in the binding of Ap₃A.     

Dinucleoside 5′,5-P,P-Triphosphate Hydrolase from Yellow Lupin (Lupinus luteus) Seeds: Purification to Homogeneity and Hydrolysis of mRNA 5′-Cap Analogs

Three separable forms of diadenosine 5′,5-P¹,P³-triphosphate (Ap₃A)-degrading activity were revealed when proteins obtained from the meal of yellow lupin seeds by ammonium sulfate precipitation were chromatographed on a DEAE-Sephacel column. The major form, which eluted first at the lowest salt concentration (0.15MKCl), was free of any activity converting the reaction products, ADP and AMP. Its further purification by gel filtration on Sephadex G-200 and by affinity elution from an AMP-agarose column yielded homogeneous protein as demonstrated on SDS–polyacrylamide gel electrophorograms. The enzyme is a single polypeptide chain ofM_r41 kDa. Eleven guanosine-containing dinucleoside triphosphates, including analogs of the mRNA 5′-cap structure, have been tested as potential substrates of the lupin “Ap₃A hydrolase.” All have been hydrolyzed yielding mixtures of corresponding nucleoside mono- and diphosphates. Asymmetrical compounds gave four products; m⁷Gp₃G, et⁷Gp₃G, and bz⁷Gp₃G were hydrolyzed randomly, whereas m⁷Gp₃A, m⁷Gp₃C, and m⁷Gp₃U yielded at least 80% m⁷GMP plus corresponding NDP and 20% or less NMP plus m⁷GDP. Hydrolysis of the guanosine-containing hybrids, Ap₃G, Cp₃G, and Up₃G, yielded at least 85% GMP plus corresponding NDP. All dinucleotides containing the m⁷G- moiety were hydrolyzed 2- to 4.5-fold faster than Ap₃A. Thus a general name, “dinucleoside triphosphate hydrolase,” is more appropriate for the enzymatic activity described.     

Acylated flavonol diglucosides from Lotus polyphyllos

Three acylated flavonol diglucosides, kaempferol 3-O-β-(6″-O-E-p-coumaroylglucoside)-7-O-β-glucoside; quercetin 3-O-β-(6″-O-E-p-coumaroylglucoside)-7-O-β-glucoside; isorhamnetin 3-O-β-(6″-O-E-p-coumaroylglucoside)-7-O-β-glucoside were isolated from the whole plant aqueous alcohol extract of Lotus polyphyllos. The known 3,7-di-O-glucosides of the aglycones kaempferol, quercetin and isorhamnetin were also characterized. All structures were established on the basis of chemical and spectral evidence.