Physiological changes of a green alga (Micractinium sp.) involved in an early-stage of association with Tetrahymena thermophila during 5-year microcosm culture

Endosymbioses between phototrophic algae and heterotrophic organisms are an important symbiotic association in that this association connects photo- and heterotrophic metabolism, and therefore, affects energy/matter pathways and cycling in the ecosystem. However, little is known about the early processes of evolution of an endosymbiotic association between previously non-associated organisms. In previous studies, we analyzed an early process of the evolution of an endosymbiotic association between an alga and a ciliate by using a long-term culture of an experimental model ecosystem (CET microcosm) composed of a green alga (Micractinium sp.), a bacterium (Escherichia coli), and a ciliate (Tetrahymena thermophila). The results revealed that an algal type, isolated from 5-year cultures of the microcosm, prolonged the longevity of the ancestral and derived clones of T. thermophila in the absence of bacteria, suggesting that a cooperative algal phenotype that benefited the ciliate had evolved in the microcosm. Here, we investigated the physiological changes of the derived Micractinium clones that benefited Tetrahymena, focusing on the release of carbohydrates by and abundance of photopigments in the ancestral and 2 derived algal clones (SC10-2 and SC9-1) isolated from inside Tetrahymena cells. Analyses using HPLC revealed that the algal isolates released glycerol and sucrose at higher concentrations per cell and also contained higher levels of photopigments per cell at pH 7.2, in comparison with the ancestral strain. These phenotypic characters were considered responsible for the increased longevity of Tetrahymena cells, and thus supported the cooperator alga hypothesis.     

Allergenic glaucans from dermatophytes. II. Enzymic degradation

The action of pyruvic acid on glycerol leads principally to two isomeric, bicyclic lactones 1 and 2; this reaction is compared with that employing methyl and ethyl esters of pyruvic acid. The action of pyruvaldehyde or 2,3-butanedione on glycerol leads to bicyclic heterocycles having a secondary (5) or tertiary (6) hemiacetal group, respectively. These compounds, and likewise the derived alkoxy, acetoxy, and chloro analogs, are subject to the anomeric effect, the endo isomers being thermodynamically more stable than the exo isomers.     

Glyceroglycolipids,a novel class of platelet-activating factor antagonists from Kalimeris indica

The bioassay-guided purification of chloroform extracts of Kalimeris indica (Linn) Schultz-Bip led to the isolation of three diacylglycerols: 1-O-(9Z,12Z,15Z-octadecatrienoyl)-2-O-hexadecanoylglycerol (1), 1-O-(9Z,12Z,15Z-octadecatrienoyl)-2-O-hexadecanoyl-3-O-α-(6-sulfoquinovopyranosyl)glycerol (2), 1-O-(9Z,12Z,15Z-octadecatrienoyl)-2-O-hexadecanoyl-3-O-[α-d-galactopyranosyl(1 → 6)-O-β-d-galactopyranosyl]glycerol (3). Their structures were established on the basis of spectral and chemical methods. The glyceroglycolipids 2 and 3 exhibited significant PAF receptor binding inhibitory activities. Our studies have identified diacylglycolipids as a novel class of potent PAF antagonist.     

Synthesis of 2-(6-aminohexanamido)ethyl 1-thio-β-d-galactopyranoside and 1-thio-β-d-glucopyranoside,and related compounds

2-(6-Aminohexanamido)ethyl 1-thio-β-d-galactopyranoside (5) and 1-thio-β-d-glucopyranoside (9) were prepared by the following scheme: 2,3,4,6-tetra-O-acetyl-1-thio-β-d-aldopyranoses, generated from 2-S-(2,3,4,6-tetra-O-acetyl-β-d-aldopyranosyl)-2-thiopseudourea hydrobromides, were aminoethylated with ethylenimine, followed by N-acylation of the products with 6-(trifluoroacetamido)hexanoic acid (1), and O-deacylation. These reactions could be carried out consecutively without isolation of intermediates, and the products obtained after gel chromatography were de(trifluoroacetyl)ated to obtain the final products. The chain lengths of the aglycons were further extended by repeating the acylation and the de(trifluoroacetyl)ation. An analog containing glycerol in lieu of a sugar was prepared by a similar reaction-scheme.     

Two new antioxidant flavones from the twigs of Eriosema robustum (Fabaceae)

Phytochemical study of the ethanol extract of the twigs of Eriosema robustum, a Cameroonian medicinal plant resulted to the isolation of two new flavones, 2′,3′,5′,5,7-pentahydroxy-3,4′-dimethoxyflavone (1) and 2′,3,5′,5,7-pentahydroxy-4′-methoxyflavone (2), along with five known compounds: 6-prenylpinocembrin (3), 1-O-heptatriacontanoyl glycerol (4), β-sitosterol (5), stigmasterol (6) and 3-O-β-d-glucopyranoside of sitosterol (7). The structure of the isolated compounds were elucidated on the basis of their NMR, UV and MS data, and by comparison with those reported in the literature. The ethanol crude extract, fractions and some isolated compounds (1–4) were evaluated for their radical scavenging capacity using 2,2-diphenyl-1-picryhydrazyl (DPPH). The crude extract, fraction II, the new compounds namely robusflavones A (1) and B (2) exhibited significant antioxidant activity.     

(2S)-Prenylflavanones and taraxerane triterpenoids from Mallotus mollissimus

Three prenylflavanones, (2S)-5,7-dihydroxy-4′-methoxy-8-(3″,3″-dimethylallyl)flavanone (3), (2S)-5,4′-dihydroxy-7-methoxy-6-(3″,3″-dimethylallyl)flavanone (6), 8-prenylnaringenin (11), and a new epimeric pair (2″S/2″R)-(2S)-5,7-dihydroxy-4′-methoxy-6-(2″-hydroxy-3″-methylbut-3″-enyl)flavanones (4a/4b) were isolated together with taraxerone, taraxerol, epitaraxerol, β-sitosterol, oleanolic acid, 1-O-docosanoyl glycerol, apigenin, and apigenin 7-O-β-D-glucopyranoside from the MeOH extract of the leaves of Mallotus mollissimus. The structures of the isolated compounds were determined on the basis of 1D/2D NMR and HR-MS spectroscopic data; the 2S configuration of the prenylflavanones 3, 4, and 6 was deduced from CD spectroscopic data. The presence of three taraxerane triterpenoids reinforces the inclusion of M. mollissimus (syn. Croton mollissimus) in Mallotus genus. Among species of Mallotus the occurrence of the (2S)-prenylflavanones 3, 4, and 6 is confined to M. mollissimus.     

Anti-microfouling properties of compounds isolated from several Mediterranean Dictyota spp.

Brown algae of the genus Dictyota are widespread around the world and are common along the coasts of the Mediterranean Sea. These marine organisms keep their surface relatively free from biofouling and are known for their ability to produce a wide array of bioactive compounds, mostly diterpenes, whose ecological functions are not clearly defined. In this study, an evaluation of the chemodiversity of the Dictyota genus was conducted on three samples, harvested on both NW and SW Mediterranean coasts (France and Algeria, respectively). Ten compounds were purified from the organic extracts of these samples; their chemical structures were elucidated by 1D and 2D NMR spectroscopy and were compared with literature data. Among them, three new diterpenes [one dolabellane (1), one xenicane (2), and one prenylated guaiane (3)] were characterized together with five previously described compounds [3,4-epoxy-14-oxo-7,18-dolabelladiene (4), acetoxycrenulide (5), dictyol E (6), 10,18-dihydroxydolabella-2,7-diene (7), and 10-acetoxy-18-hydroxydolabella-2,7-diene (8)]. In addition, the occurrence of two known glycerol derivatives [1-Ο-octadecenoylglycerol (9) and sn-3-Ο-(geranylgeranyl)glycerol (10)] was also determined. Some of the isolated compounds (4–6 and 8–10) were screened for their potential to prevent the adhesion of three bacterial strains isolated from marine biofilms in comparison with four commercial antifoulants (TBTO, Zineb, ZnPT, and CuPT): those bearing a glycerol moiety (compounds 9 and 10) exhibited the strongest anti-adhesion effects, whatever the strain, and with a moderate toxicity. Thus, these chemical structures should be further explored for both their putative involvement in keeping the algal surface free of biofouling and the development of effective and environmentally benign antifoulants.     

Monoglycerol ester,galloylglucoside and phenolic derivatives from Gymnosporia senegalensis leaves

Phytochemical investigation of the leaves of Gymnosporia senegalensis (Lam.) Loes. led to the isolation and characterization of eighteen compounds: one new polyunsaturated fatty acid-derived monoglyceride, (2S)-1-O-(4′Z,7′Z,10′Z-octadecatrienoyl) glycerol (1); four galloylglucoside derivatives (2–5), including one new named (2R)-methyl [(6′-O-galloyl)-β-D-glucopyranosyloxy]phenylacetate (2), eleven phenolics (6–16), and two sterols (17–18). The structures of the isolates were determined by means of spectroscopic and spectrometric data, as well as by comparison with literature data. Galloylglucosides, especially cyanogenic constituents (3, 4) are described here for the first time in the genus. Antibacterial and cytotoxic activities of isolated compounds were investigated. None of the tested compounds showed growth inhibition against Staphylococcus aureus NBRC 13276, Bacillus subtilis NBRC 3134 and Escherichia coli NBRC 3972 at 50 μM, while 1, 7, 9, 17, and 18 at 50 μM showed relatively weak to moderate decrease of viability against colon (DLD1), breast (MCF7) and gastric (MKN45) cancer cells. Furthermore, the chemotaxonomic significance of the isolated compounds was discussed.     

Novel androgen receptor full antagonists: Design,synthesis, and a docking study of glycerol and aminoglycerol derivatives that contain p-carborane cages

Based on the co-crystal structure of bicalutamide with a T877A-mutated androgen receptor (AR), glycerol and aminoglycerol derivatives were designed and synthesized as a novel type of carborane-containing AR modulators. The (R)-isomer of 6c, whose chirality is derived from the glycerol group, showed 20 times more potent cell inhibitory activity against LNCaP cell lines expressing T877A-mutated AR than the corresponding (S)-isomer. Docking studies of both isomers with AR suggested that (R)-6c is in closer spatial proximity to helix-12 of the AR than (S)-6c, which is the most important common motif in the secondary structure of AR for the expression of antagonistic activity.     

Enzymatic synthesis of 1,3-dihydroxyphenylacetoyl-sn-glycerol: Optimization by response surface methodology and evaluation of its antioxidant and antibacterial activities

In this study, the enzymatic synthesis of phenylacetoyl glycerol ester was carried out as a response to the increasing consumer demand for natural compounds. 1,3-dihydroxyphenylacetoyl-sn-Glycerol (1,3-di-HPA-Gly), labeled as “natural” compound with interesting biological properties, has been successfully synthesized for the first time in good yield by a direct esterification of glycerol (Gly) with p-hydroxyphenylacetic acid (p-HPA) using immobilized Candida antarctica lipase as a biocatalyst. Spectroscopic analyses of purified esters showed that the glycerol was mono- or di-esterified on the primary hydroxyl group. These compounds were evaluated for their antioxidant activity using two different tests. The glycerol di-esters (1,3-di-HPA-Gly) showed a higher antiradical capacity than that of the butyl hydroxytoluene. Furthermore, compared to the p-HPA, synthesized ester (1,3-di-HPA-Gly) exhibited the most antibacterial effect mainly against Gram + bacteria. Among synthesized esters the 1,3-di-HPA-Gly was most effective as antioxidant and antibacterial compound. These findings could be the basis for a further exploitation of the new compound, 1,3-di-HPA-Gly, as antioxidant and antibacterial active ingredient in the cosmetic and pharmaceutical fields.     

Synthesis and absolute structures of Mycoplasma pneumoniae β-glyceroglycolipid antigens

Just recently, a pair of β-glycolipids was isolated from the cell membrane of Mycoplasma pneumoniae as a mixture of the two compounds. They are the major immunodeterminants of this pathogenic Mycoplasma and indicate high medicinal potential. They have a β-(1→6)-linked disaccharide structure close to each other; one has β-d-galactopyranoside (β-Gal-type 1) at the non-reducing terminal, and another has β-d-glucopyranoside (β-Glc-type 2). In the present study, the first stereoselective synthesis was conducted for each of the two β-glycolipids 1 and 2. ¹H NMR and TLC-immunostaining studies of the synthetic compounds enable us to establish the absolute structures having the β-(1→6)-linked disaccharides at the glycerol sn-3 position.     

Automated determination of monosaccharides using p-hydroxybenzoic acid hydrazide.

An automated procedure has been described based on the manual method of Hagen and Hagen [(1962) Canad. J. Biochem.40, 1129] which will rapidly and reproducibly measure glycerol concentrations. The method was developed primarily for the determination of glycerol in adipose tissue and various incubation media. The glycerol in solution is estimated by its partial conversion to dihydroxyacetone by glycerol dehydrogenase from Aerobacter aerogenes. The range of glycerol concentrations able to be measured is more extensive than with other automated methods.     

Chemical constituents from Ligularia purdomii (Turrill) Chittenden

The first phytochemical investigation on the roots of Ligularia purdomii led to the isolation and identification of 18 compounds, including two eremophilane sesquiterpenoids (1 and 2), three benzofuran derivatives (3–5), a triterpenoid (6), two steroids (7 and 8), nine phenolic components (9–17), and a monofatty glyceride (18). The structural elucidation of the isolated compounds was performed by spectroscopic data and comparison with the literature. Compounds (−)-syringaresinol (11), scopoletin (13), 3,5-dimethoxy-4-hydroxy-benzaldehyde (14), and glycerol monolinoleate (18) have not been recorded in Ligularia genus previously. The chemotaxonomic significance of these isolated compounds has been summarized.     

Physical-Chemical Basis of the Protection of Slowly Frozen Human Erythrocytes by Glycerol

One theory of freezing damage suggests that slowly cooled cells are killed by being exposed to increasing concentrations of electrolytes as the suspending medium freezes. A corollary to this view is that protective additives such as glycerol protect cells by acting colligatively to reduce the electrolyte concentration at any subzero temperature. Recently published phase-diagram data for the ternary system glycerol-NaCl-water by M. L. Shepard et al. (Cryobiology, 13:9-23, 1976), in combination with the data on human red cell survival vs. subzero temperature presented here and in the companion study of Souzu and Mazur (Biophys. J., 23:89-100), permit a precise test of this theory. Appropriate liquidus phase-diagram information for the solutions used in the red cell freezing experiments was obtained by interpolation of the liquidus data of Shepard and his co-workers. The results of phase-diagram analysis of red cell survival indicate that the correlation between the temperature that yields 50% hemolysis (LT₅₀) and the electrolyte concentration attained at that temperature in various concentrations of glycerol is poor. With increasing concentrations of glycerol, the cells were killed at progressively lower concentrations of NaCl. For example, the LT₅₀ for cells frozen in the absence of glycerol corresponds to a NaCl concentration of 12 weight percent (2.4 molal), while for cells frozen in 1.75 M glycerol in buffered saline the LT₅₀ corresponds to 3.0 weight percent NaCl (1.3 molal). The data, in combination with other findings, lead to two conclusions: (a) The protection from glycerol is due to its colligative ability to reduce the concentration of sodium chloride in the external medium, but (b) the protection is less than that expected from colligative effects; apparently glycerol itself can also be a source of damage, probably because it renders the red cells susceptible to osmotic shock during thawing.     

Vernoguinamide: A new ceramide and other compounds from the root of Vernonia guineensis Benth. and their chemophenetic significance

The chemical investigation of the roots of Vernonia guineensis Benth. (Asteraceae) resulted in the isolation of a new ceramide, named vernoguinamide (1), together with fifteen known compounds, including three anthraquinones, physion (2), erythroglaucin (3) and emodin (4), three triterpenoids, hop-17(21)-en-3β-yl acetate (5), lupeol (6) and betulinic acid (7), six steroids, vernoguinoside A (8), vernoguinoside (9), β-sitosterol 3-O-β-D-glucoside (10), stigmasterol 3-O-β-D-glucoside (11), stigmasterol (12) and β-sitosterol (13) and three fatty acid derivatives, tetracosanoic acid (14), tricosanic acid (15) and arachidic acid glycerol ester (16). The structure of the new compound as well as those of the known compounds were established by spectrometric analysis including HRESI-MS, 1D and 2D-NMR and by comparison with the previously reported data. Among these compounds, the anthraquinones 2–4 and the triterpene 5 were isolated for the first time from Vernonia genus and compounds 6, 7 and 14–16 were extracted for the first time from the species. The isolated compounds were tested for their antibacterial activity and 3, 8 and 9 were the most active compounds against the tested bacteria. Furthermore, the chemophenetic relationships of the isolated compounds and their significance were also discussed.     

A new biologically active triterpenoid saponin from the aerial parts of Neanotis wightiana

Phytochemical investigation of the aerial parts of Neanotis wightiana for the first time has led to the isolation of one new triterpenoid saponin, neanoside A (1), along with seven known compounds, oleanolic acid (2), ursolic acid (3), β-sitosterol (4) and its glucoside (5), stigmasterol (6) and its glucoside (7) and hexacosanoic acid (8). The structures of these compounds were elucidated by means of spectroscopic (NMR, MS and other) and chemical techniques as well as comparison with literature data. The structure of 1 was elucidated as 3-O-α-l-rhamnopyranosyl-(1 → 2)-β-d-xylopyranosyl (1 → 3)-β-d-glucopyranosyl bayogenin. The in vitro biochemical analysis of compound 1 against the activity of human serum liposomal enzymes, SGOT (serum glutamate oxaloacetate transaminase), SGPT (serum glutamate pyruvate transaminase) and ALP (alkaline phosphatase) and glycerol kinase showed significant reduction of their activity.     

Synthesis of an α-kojibiosyl substituted glycerol teichoic acid hexamer

In this paper the synthesis of an Enterococcus Faecalis teichoic acid (TA) hexamer is presented. The key kojibiosyl-glycerol phosphoramidite building block was obtained by condensation of thioglucose donors, provided with various protecting groups at the C2 hydroxyl function with an orthogonally protected glycerol acceptor. After selective deprotection, the resulting 1,2-cis-linked pseudodisaccharide acceptor was coupled to an α-directing thioglucose donor, giving the corresponding pseudotrisaccharide, which is then transformed to a phosphoramidite synthon. The kojibiosyl-glycerol phosphoramidite in combination with a glycerolphosphoramidite, an aminohexylphosphoramidite and dibenzylglycerol were coupled to a fully protected glycerol TA hexamer, using chemistry that can be amended for future automated synthesis. Global deprotection afforded the target hexamer kojibiosyl-glycerol containing TA (1).     

Phylogeny,morphology, and physiology of Micractinium strains isolated from shallow ephemeral freshwater in Antarctica

Cryotolerant eukaryotic microalgae were isolated from meltwater streams on Ardley Island and King George Island in Antarctica, and their morphological, molecular, and physiological characteristics were investigated. Owing to their simple morphology, distinctive characters were not observed with neither light microscopy nor transmission electron microscopy. However, molecular phylogenetic inferences drawn from the concatenated small subunit rRNA and internal transcribed spacer sequence data indicated that these microalgal strains belonged to the genus Micractinium. All the Micractinium strains showed cryotolerant properties, while their optimum growth temperature was around 20°C. Similar to other cryotolerant organisms, these Antarctic microalgae also contained a higher ratio of polyunsaturated fatty acids to saturated fatty acids. In this study, new Antarctic Micractinium spp. were discovered and added to the culture collection. These cryotolerant strains may serve as a promising source of nutritionally important linoleic (C_18:2 ω6) and α‐linolenic (C_18:3 ω3) acids.     

Large scale isolation and storage of Neurospora plasma membranes.

Functionally inverted plasma membrane vesieles isolated from the eukaryotic microorganism Neurospora crassa generate and maintain a transmembrane electrical potential via ATP hydrolysis catalyzed by a plasma membrane ATPase (G. A. Scarborough, 1976, Proc. Nat. Acad. Sci. USA73, 1485–1488). In order to facilitate investigation of the molecular mechanism of the electrogenic ATPase, and other transport systems, we have developed a method for the large scale isolation and storage of Neurospora plasma membranes in a stable form. Large quantities of open plasma membrane sheets (ghosts) are isolated by a scaled-up modification of the original method (G. A. Scarborough, 1975, J. Biol. Chem.250, 1106–1111) and stored at ?26°C in 60% glycerol ($\text{v}\text{v}$). As needed, the ghosts are washed free of glycerol and then converted to closed vesicles by a modification of the original method. With this technique, plasma membrane vesicles with normal electrogenic pump activity can be prepared daily in approximately 2.5 h.     

Radiosynthesis and evaluation of a novel monoacylglycerol lipase radiotracer: 1,1,1,3,3,3-hexafluoropropan-2-yl-3-(1-benzyl-1H-pyrazol-3-yl)azetidine-1-[11C]carboxylate

Monoacylglycerol lipase (MAGL) is a major serine hydrolase that hydrolyses 2-arachidonoylglycerol (2-AG) into arachidonic acid (AA) and glycerol in the brain. Because 2-AG and AA are endogenous biologically active ligands in the brain, the inhibition of MAGL is an attractive therapeutic target for neurodegenerative diseases. In this study, to visualize MAGL via positron emission tomography (PET), we report a new carbon-11-labeled radiotracer, namely 1,1,1,3,3,3-hexafluoropropan-2-yl-3-(1-benzyl-1H-pyrazol-3-yl)azetidine-1-[¹¹C]carboxylate ([¹¹C]6). Compound 6 exhibited high in vitro binding affinity (IC₅₀ = 0.41 nM) to MAGL in the brain with a suitable lipophilicity (cLogD = 3.29). [¹¹C]6 was synthesized by reacting 1,1,1,3,3,3-hexafluoropropanol (7) with [¹¹C]phosgene ([¹¹C]COCl₂), followed by a reaction with 3-(1-benzyl-1H-pyrazol-3-yl)azetidine hydrochloride (8), which resulted in a 15.0 ± 6.8% radiochemical yield (decay-corrected, n = 7) based on [¹¹C]CO₂ and a 45 min synthesis time from the end of bombardment. A biodistribution study in mice showed high uptake of radioactivity in MAGL-rich organs, including the lungs, heart, and kidneys. More than 90% of the total radioactivity was irreversibly bound in the brain homogenate of rats 5 min and 30 min after the radiotracer injection. PET summation images of rat brains showed high radioactivity in all brain regions. Pretreatment with 6 or MAGL-selective inhibitor JW642 significantly reduced the uptake of radioactivity in the brain. [¹¹C]6 is a promising PET tracer which offers in vivo specific binding and selectivity for MAGL in rodent brains.