Seasonal variations in polyhydroxysteroids and related glycosides from digestive tissues of the starfish Patiria (=Asterina) pectinifera

Seasonal variations in the concentrations of individual polyhydroxysteroids and related low molecular weight glycosides in pyloric caeca and stomach of the starfish Patiria (=Asterina) pectinifera collected at one location near Vladivostok have been studied. HPLC analysis on the fractions containing these substances showed a fairly constant composition of steroids in digestive tissues of P. pectinifera in spite of small seasonal variations in the relative concentrations of individual compounds.     

Steroidal monoglycosides from the Far Eastern starfish Hippasteria kurilensis and hypothetic pathways of polyhydroxysteroid biosynthesis in starfish

Five new steroidal monoglycosides, kurilensosides E (1), F (2), G (3), H (4) and 15-O-sulfate of echinasteroside C (5) were isolated along with the previously known echinasteroside C (6) from the alcoholic extract of the Far Eastern starfish Hippasteria kurilensis collected near Kuril Islands. Compounds 1-3 were determined to contain unusual polyhydroxysteroidal aglycons lacking 6-hydroxy group. Aglycon moiety of kurilensoside H (4) was shown to be the first case of marine polar steroids containing 4,5-epoxy functionality. Hypothetic pathways of the biosynthesis of polyhydroxysteroids and related glycosides in starfish and the existence of the late C-6 oxidation pathway in H. kurilensis are discussed.     

Accumulation of multiacetylated forms of histones by trichostatin A and its developmental consequences in early starfish embryos

Summary External application of 10 rig/ml (R)-trichostatin A (TSA), a potent and specific inhibitor of mammalian histone deacetylase, to the embryo of the starfish Asterina pectinifera inhibited development during the early gastrula stage before formation of mesenchyme cells. The TSA-sensitive period was limited to the mid-blastula stage before hatching. The pulse-chase experiment clearly demonstrated that TSA induced an accumulation of acetylated histone species in blastulae through inhibition of historic deacetylation. Similar blockage of development at the early gastrula stage was observed with n-butyrate, which has been known as a weak inhibitor of historic deacetylase. These results suggest an intimate role for historic acetylation-deacetylation equilibria in starfish development. Correspondence to: S. Ikegami     

Metabolite profiling of polar steroid constituents in the Far Eastern starfish Aphelasterias japonica using LC–ESI MS/MS

Numerous polar steroids are characteristic metabolites of starfish which determine physiological activities of their extracts. The Far Eastern starfish Aphelasterias japonica is a rich source of different steroid glycosides and polyhydroxysteroids. For detailed analysis of complicated mixture of steroids from this species, isolated by solid-phase extraction, a liquid chromatography–electrospray tandem mass spectrometry (LC–ESI MS/MS) approach was selected and applied. The characteristic fragmentations in ESI MS/MS spectra of steroid glycosides allowed determining types of aglycones, presence of sulfate groups, sugar sequences as well as branching. In addition, main structural features of polyhydroxylated polar steroids including position of hydroxylation and level of sulfation were also established. Totally, 68 metabolites, comprising of 33 asterosaponins, 28 sulfated polyhydroxysteroid mono- and biosides and 7 sulfated polyhydroxysteroid compounds were found by this method. In addition to 15 previously isolated compounds from A. japonica, many new steroid glycosides including asterosaponins with unusual carbohydrate chains were discovered and characterized by their ESI product ion mass spectra. Some details of biosynthesis of polyhydroxylated steroids and their conjugated forms in the species studied such as a role of sulfation and order of introduction of hydroxyl group in A. japonica were proposed.     

Biosynthesis of polar steroids from the Far Eastern starfish Patiria (=Asterina) pectinifera. Cholesterol and cholesterol sulfate are converted into polyhydroxylated sterols and monoglycoside asterosaponin P1 in feeding experiments

For the first time, it is experimentally established that the dietary cholesterol and cholesterol sulfate are biosynthetic precursors of polyhydroxysteroids and related low molecular weight glycosides in starfishes. These deuterium labeled precursors were converted into partly deuterated 5α-cholestane-3β,6α,7α,8,15α,16β,26-heptaol, 5α-cholestane-3β,4β,6α,7α,8,15β,16β,26-octaol, and steroid monoside asterosaponin P₁ in result of feeding experiments on the Far Eastern starfish Patiria (=Asterina) pectinifera. The incorporations of deuterium were established by MS and NMR spectroscopy. Scheme of the first stages of biosynthesis of polar steroids in these animals was suggested on the basis of inclusion of three from six deuterium atoms and determination of their positions in biosynthetic products, when [2,2,3,4,4,6-²H₆]cholesterol 3-sulfate was used as precursor. It was also shown that labeled cholesterol is transformed into Δ⁷-cholesterol (lathosterol) in digestive organs and gonads of the starfish.     

Chemical Constituents and Bioactivities of Starfishes: An Update

Starfishes produce various structurally unique secondary metabolites with diverse biological activities. This review is an update summary of the new compounds and their bioactivities from starfish (the Asteroidea Class) with 71 references covering from January 2007 to December 2018. During this period, 216 new compounds were obtained from 36 species. The chemical constituents are mostly steroids, steroidal glycosides, and gangliosides. These components have been found to possess various bioactivities, including anticancer, anti‐inflammation, etc.     

Steroid Compounds from the Far Eastern Starfish Diplopteraster multipes

Sodium salt of (20R)-3,4-dihydroxycholest-5-ene-21-yl sulfate and disodium salts of (20R)-4-hydroxycholest-5-ene-3,21-diyl disulfate, (20R)-24-methylcholest-5,24(28)-diene-3,21-diyl disulfate, (20R)-24-methyl-5-cholest-24(28)-ene-3,21-diyl disulfate, (20R)-cholest-5-ene-3,21-diyl disulfate, (20R)-5-cholestane-3,21-diyl disulfate, and (20R)-3-hydroxycholest-5-ene-2,21-diyl disulfate were isolated from the far eastern starfish Diplopteraster multipes and characterized. These compounds differ structurally from sulfated polyhydroxysteroids in other starfish species. At the same time, they are typical secondary metabolites of Ophiuroidea and have some structural features characteristic of the ophiuroid-isolated steroids, namely the 3-hydroxy (or 3-sulfoxy) and 21-sulfoxy groups. These data support the opinion of some taxonomists that starfishes and ophiuroids are phylogeneteically related classes and are closer to each other than to other classes of the Echinodermata phylum.     

Six New Polyhydroxysteroidal Glycosides,Anthenosides S1 – S6, from the Starfish Anthenea sibogae

Six new polyhydroxysteroidal glycosides, anthenosides S1  –  S6 ( 1  –  6 ), along with a mixture of two previously known related glycosides, 7 and 8 , were isolated from the methanolic extract of the starfish Anthenea sibogae. The structures of 1  –  6 were established by NMR and HR‐ESI‐MS techniques as well as by chemical transformations. All new compounds have a 5α‐cholest‐8(14)‐ene‐3α,6β,7β,16α‐tetrahydroxysteroidal nucleus and differ from majority of starfish glycosides in positions of carbohydrate moieties at C(7) and C(16) ( 1  –  4 , 6 ) or only at C(16) ( 5 ). The 4‐O‐methyl‐β‐d ‐glucopyranose residue ( 2 ) and Δ²⁴‐cholestane side chain ( 3 ) have not been found earlier in the starfish steroidal glycosides. The mixture of 7 and 8 slightly inhibited the proliferation of human breast cancer T‐47D cells and decreased the colony size in the colony formation assay.     

Cariniferosides A–F and other steroidal biglycosides from the starfish Asteropsis carinifera

Six new steroidal biglycosides, cariniferosides A–F (1–6), were isolated along with six previously known glycosides, 7–12, from the alcoholic extract of the starfish Asteropsis carinifera. The structures of 1–6 were determined by extensive NMR and ESIMS techniques. The isolated compounds did not show any apparent cytotoxicity in cancer cell lines HCT-116, RPMI-7951, and T-47D, but sulfated compounds 6, 11, and 12 demonstrated a significant inhibition of RPMI-7951 and T-47D cell colony formation in a soft agar clonogenic assay.     

Enzymatic properties of sialidase from the ovary of the starfish, Asterina pectinifera

A sialidase [EC 3.2.1 18] was isolated and highly purified from the ovary of the starfish, Asterina pectinifera, and its enzymatic properties were compared with those of human placental sialidase. The final preparation gave one broad protein band corresponding to sialidase activity on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 360 000 by HPLC on Sigma Chrome GFC-1300 and Sephadex G-150 column chromatography, and 55 000 by SDS-PAGE, suggesting the presence of a hexamer in the native protein. The optimum pH was between 3.0 and 4.0, and the enzyme liberated sialyl residues from the following compounds: α(2-3) and α(2-6) sialyllactose, colominic acid, fetuin, transferrin, gangliosides GM₃, GD_1a and GD_1b. The enzyme was strongly inhibited by 4-aminophenyl and methyl thio-glycosides of sialic acid, but not by those glycosides of 5-amino sialic acid or sialic acid methyl ester. The enzyme was also highly inhibited by sulfated glucan and glycosaminoglycans. The substrate specificity and the effects of inhibitors on starfish sialidase were very similar to those of human placental sialidase.     

The distribution of free sterols, polyhydroxysteroids and steroid glycosides in various body components of the starfish Patiria (=Asterina) pectinifera

The distribution of free sterols, polyhydroxysteroids and steroid glycosides in different body components of the Far-eastern starfish Patiria (=Asterina) pectinifera has been studied. It was shown that free sterol fractions from aboral and oral body walls, gonads, stomach and pyloric ceca contained Delta(7) sterols with a preponderance of 5alpha-cholest-7-en-3beta-ol. All these body components had also toxic steroid oligoglycosides. However, polyhydroxysteroids and related low molecular weight steroid glycosides were found in stomach and pyloric ceca only. In pyloric ceca, the sulfated monoside 'asterosaponin' P(1) was identified as a main polar steroid, whereas 6-sodium sulfate of cholestane-3beta,4beta,6alpha,7alpha,8,15beta,16beta,26-octaol predominated in the stomach. Probable biological functions of polar steroids and free sterols in this starfish were discussed. It was suggested that some polyhydroxysteroids and related monoglycosides play the same biological role as bile alcohols and bile acids do in vertebrates.     

Antibacterial activity and phytochemical evidence for the plant origin of Turkish propolis from different regions

Honeybees collect propolis from practically any abundant plant source in the neighborhood of the hive, be it populus, eucalyptus, pine, sugarcane, cashew nut or orange trees. We have described that the origin plants of Turkish propolis are Populus sp., Eucalyptus sp. and Castanea sativa. In our previous study, propolis samples from Middle Anatolia displayed the typical pattern of “poplar” propolis: they contained pinobanksin, caffeic and ferulic acids and their esters. The propolis samples examined in this study were shown not to contain polar phenolics. The main components of Eucalyptus propolis were aromatic acids, mainly cinnamic acid and its esters, that are usually found in Eucalyptus species resins. The second distinct sample originated from West Anatolia. Although it contained low amounts of phenolic substances and aromatic acids, its main components were sugars and glycosides. The study revealed that there was no significant difference between propolis samples in antibacterial activity, however the yeasts were shown to be more sensitive to eucalyptus-propolis. Gram negative bacteria were susceptible to none of the samples tested.     

Sea Cucumbers Triterpene Glycosides, the Recent Progress in Structural Elucidation and Chemotaxonomy

Triterpene glycosides are characteristic metabolites of sea cucumbers (Holothurioidea, Echinodermata). Majority of the glycosides belong to holostane type (lanostane derivatives with 18(20)-lactone). Carbohydrate chains of these glycosides contain xylose, glucose, quinovose, 3-O-methylglucose and 3-O-methyl sylose. During the last 5 years, main investigations were focused on holothurians belonging to the order Dendrochirotida collected in the North Pacific, North Atlantic, Antarctic and in subtropical waters. The glycosides of holothurians belonging to the order Aspidochirotida have also been studied. The most uncommon structural features of carbohydrate chains of new glycosides were: (1) the presence of quinovose as fifth terminal monosaccharide unit and the presence of two quinovose residues; (2) the presence of glucose instead of common xylose as fifth terminal monosaccharide unit; (3) trisaccharide carbohydrate chain; (4) the presence of two 3-O-methylxylose terminal monosaccharide units; (5) the presence of sulfate group at C-3 of quinovose residue. New glycosides without lactone or with 18(16)-lactone and having shortened side chains have also been isolated. The presence of 17α and 12α-hydroxyls, which are characteristic for glycosides from holothurians belonging to the family Holothuriidae (Aspidochirotida) in glycosides of dendrochirotids confirms parallel and relatively independent character of evolution of glycosides. All three families belonging to the order Aspidochirotida: Holothuriidae, Stichopodidae and Synallactidae have similar and parallel trends in evolution of the glycosides carbohydrate chains, namely from non-sulfated hexaosides to sulfated tetraosides. Sets of aglycones in glycosides from holothurians belonging to the genus Cucumaria (Cucumariidae, Dendrochirotida) are specific for each species. The carbohydrate chains are similar in all representatives of the genus Cucumaria.     

Evaluation of antiprotozoal and antimycobacterial activities of the resin glycosides and the other metabolites of Scrophularia cryptophila

Resin glycosides are secondary metabolites exclusive to the convolvulaceous plants. In this study, crypthophilic acids A–C (1–3), the first resin glycosides occurring in another family (Scrophulariaceae), and the other constituents of Scrophularia cryptophila were examined for in vitro antiprotozoal and antimycobacterial potentials. Except for crypthophilic acid B (2), all tested compounds exhibited growth-inhibitory effect against Trypanosoma brucei rhodesiense, with l-tryptophan (6) and buddlejasaponin III (7) being the most potent ones (IC₅₀'s 4.1 and 9.7 μg/ml). In contrast, the activity towards Trypanosoma cruzi was poor, and only crypthophilic acid C (3), 6 and 7 were trypanocidal at concentrations above 40 μg/ml. With the exception of 2 and 6, all compounds were active against Leishmania donovani. Harpagide (4) and 3 emerged as the best leishmanicidal agents (IC₅₀'s 2.0 and 5.8 μg/ml). Only compounds 3, 6 and 7 showed antimalarial activity against Plasmodium falciparum with IC₅₀ values of 4.2, 16.6 and 22.4 μg/ml. Overall the best and broadest spectrum activity was presented by compounds 3 and 7, as they inhibited all four parasitic protozoa. None of the isolates had significant activity against Mycobacterium tuberculosis (MICs >100 μg/ml) or were toxic towards mammalian (L6) cells. This is the first report of antiprotozoal activity for natural resin glycosides, as well as for harpagide (4), acetylharpagide (5), tryptophan (6) and buddlejasaponin III (7).     

cDNA cloning and sequencing of phospholipase A2 from the pyloric ceca of the starfish Asterina pectinifera

Three cDNA from the pyloric ceca of the starfish Asterina pectinifera, (namely, cDNA 1, 2, and 3), encoding phospholipase A₂ (PLA₂), were isolated and sequenced. These cDNAs were composed of 415 bp with an open reading frame of 414 bp at nucleotide positions 1–414, which encodes 138 amino acids including N-terminal Met derived from the PCR primer. The amino acid sequence deduced from the cDNA 1 was completely consistent with the sequence determined with the starfish PLA₂ protein, while those deduced from cDNA 2 and cDNA 3 differed at one and twelve amino acid residual positions, respectively, from the sequence of the PLA₂ protein, suggesting the presence of multiple forms in the starfish PLA₂. All of the sequences deduced from cDNA 1, 2, and 3 required two amino acid deletions in pancreatic loop region, and sixteen insertions and three deletions in β-wing region when aligned with the sequence of mammalian pancreatic PLA₂. In phylogenetic tree, the starfish PLA₂ should be classified into an independent group, but hardly to the established groups IA and IB. The characteristic structure in the pancreatic loop and β-wing regions may account for the specific properties of the starfish PLA₂, e.g. the higher activity and characteristic substrate specificity compared with commercially available PLA₂ from porcine pancreas.     

Effects of different leaf traits on growth rates of insect herbivores on willows

We examined relative effects of traits of leaf quality of ten willow species (Salix: Salicaceae) on growth rates of five species of insect herbivores found in interior Alaska (a willow sawfly, Nematus calais; the tiger swallowtail butterfly, Papilio canadensis; and three species of chrysomelid beetles, Gonioctena occidentalis, Calligrapha verrucosa, and Chrysomela falsa). Leaf traits examined were water content, toughness, total nitrogen contnet, pubescence, and presence or absence of phenolic glycosides. Of ten Salix species, four species contain phenolic glycosides in their leaves. We examined relative effects of water content, toughness, and nitrogen content of the Salix leaves on larval growth rates at three different levels, i.e., on a single host species, between different host species, and between herbivore species. The within-host analyses showed that effects of water content, toughness and/or nitrogen content on herbivore growth rates were generally significant in early-season herbivores but not in late-season herbivores. For each herbivore species, differences in growth rates between hosts were not explained by differences in water content, toughness, or nitrogen content. The between-herbivore analysis showed that the interspecific difference in larval growth rates were related to difference in water and nitrogen content of the hosts. Pubescence of Salix leaves had little effects on herbivore growth rates. Presence of phenolic glycosides had a positive effects on growth rates of a specialist, N. calais, but no effect on the other specialist, Ch. falsa. Presence of phenolic glycosides had, in general, negative effects on growth rates of nonspecialists, G. occidentalis, C. verrucosa, and P. canadensis.     

Tracking down the "head blob": comparative analysis of wingless expression in the developing insect procephalon reveals progressive reduction of embryonic visual system patterning in higher insects

The evolution of larval head morphology in holometabolous insects is characterized by reduction of antennal appendages and the visual system components. Little insight has been gained into molecular developmental changes underlying this morphological diversification. Here we compare the expression of the segment polarity gene wingless (wg) in the pregnathal head of fruit fly, flour beetle and grasshopper embryos. We provide evidence that wg activity contributes to segment border formation, and, subsequently, the separation of the visual system and protocerebrum anlagen in the anterior procephalon. In directly developing insects like grasshopper, seven expression domains are formed during this process. The activation of four of these, which correspond to polar expression pairs in the optic lobe anlagen and the protocerebral ectoderm, has shifted to postembryonic stages in flour beetle and Drosophila. The remaining three domains map to the protocerebral neuroectoderm, and form by disintegration of a large precursor domain in flour beetle and grasshopper. In Drosophila, the precursor domain remains intact, constituting the previously described “head blob”. These data document major changes in the expression of an early patterning gene correlated with the dramatic evolution of embryonic visual system development in the Holometabola.     

Preparation of cluster glycosides ofN-acetylgalactosamine that have subnanomolar binding constants towards the mammalian hepatic Gal/GalNAc-specific receptor

Preparation of di-and tri-valent cluster glycosides containingN-acetyl-d-galactosamine (GalNAc) is described. Oligopeptides that contain a protected amino group and two or three free carboxyl groups are activated by methyl chloroformate and then coupled to 6-aminohexyl 2-acetamido-2-deoxy--d-galactopyranoside. The concentrations of the divalent GalNAc glycosides needed to produce 50% inhibition of the binding of asialoorosomucoid to the isolated, purified rat liver receptor specific for galactose and GalNAc and to the receptor on the hepatocyte surface were of the order of 10^–8 M and 10^–9 M, respectively. The binding affinity of the trivalent glycoside was 10-to 20-fold stronger than the divalent glycosides towards both the soluble receptor and intact hepatocyte.Abbreviations Z benzyloxycarbonyl - EDAC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride - AH 6-aminohexyl - ASOR aslaloorosomucoid - DMF N-dimethylformamide - DMSO dimethylsulfoxide - Lac lactosyl     

Hydroxylation of steroids by Fusarium oxysporum, Exophiala jeanselmei and Ceratocystis paradoxa

The potential of Fusarium oxysporum var. cubense UAMH 9013 to perform steroid biotransformations was reinvestigated using single phase and pulse feed conditions. The following natural steroids served as substrates: dehydroepiandrosterone (1), pregnenolone (2), testosterone (3), progesterone (4), cortisone (5), prednisone (6), estrone (7) and sarsasapogenin (8). The results showed the possible presence of C-7 and C-15 hydroxylase enzymes. This hypothesis was explored using three synthetic androstanes: androstane-3,17-dione (9), androsta-4,6-diene-3,17-dione (10) and 3α,5α-cycloandrost-6-en-17-one (11). These fermentations of non-natural steroids showed that C-7 hydroxylation was as a result of that position being allylic. The evidence also pointed towards the presence of a C-15 hydroxylase enzyme.The eleven steroids were also fed to Exophialajeanselmei var. lecanii-corni UAMH 8783. The results showed that the fungus appears to have very active 5α and 14α-hydroxylase enzymes, and is also capable of carrying out allylic oxidations.Ceratocystis paradoxa UAMH 8784 was grown in the presence of the above-mentioned steroids. The results showed that monooxygenases which effect allylic hydroxylation and Baeyer–Villiger rearrangement were active. However, redox reactions predominated.     

The Synthesis and Cytotoxic Activity of D-Ribofuranosides and 2-Deoxy-D-Ribofuranosides of Substituted Bis(indolyl)furan, Bis(indolyl)pyrrole, and Indolo[2,3-a]carbazole Derivatives

1-(2,3,5-Tri-O-acetyl)--D-ribofuranosyl indole, the key compound in the synthesis of glycosides with the bis(indole) aglycone, was obtained for the first time by the indoline–indole method. There were synthesized 3-(1-methylindol-3-yl)-4-(1-glycosylindol-3-yl)furan(or pyrrole)-2,5-diones containing the residue of -D-ribofuranose or 2-deoxy--D-ribofuranose and analogous glycosides of indolo[2,3-a]furano(or pyrrolo)[3,4-]carbazol-5,7-diones, which are structurally relative to the antitumor antibiotic rebeccamycin. Their cytotoxicities toward a number of human tumor cell lines were studied in vitro, and the carbazole N-glycosides were shown to be more active than the bis(indole) glycosides. At the same time, the ribofuranosides were found to be less active than the corresponding ribopyranosides synthesized previously.