C31-C34 methylated squalenes from a Bolivian strain of Botryococcus braunii

Three new triterpenes, synthesized by a Bolivian strain of the green microalga Botryococcus braunii, were isolated and their chemical structures determined by 1D and 2D NMR, and mass spectrometry. These compounds are tri-, di-, and mono-methylsqualenes, co-occurring with the previously identified tetramethylsqualene and some C(30)-C(32) botryococcenes. In this strain, methylated squalenes constitute up to 24% of the total hydrocarbons and 4.5% of the dry biomass. The results of a pulse-chase experiment with L-[Me-(13)C] methionine provide evidence for the origin of these compounds via methylation of squalene at positions 3, 7, 18 and 22.     

Prenylated xanthone glucosides from Ural's lichen Umbilicaria proboscidea

Two new compounds isolated from an extract of a Central Asian lichen [Umbilicaria proboscidea (L.) Schrader=Syn.: Gyrophora proboscidea (L.) Ach.] are glucosides with mono- and di-prenylated xanthones as the aglycones and a saccharide moiety from two glucoses linked at C-7. The structures were elucidated on the basis of extensive spectroscopic analysis (1D and 2D NMR, MS, IR and UV) and by hydrolysis.     

Peptide transport in yeast: utilization of leucine- and lysine-containing peptides by Saccharomyces cerevisiae.

A variety of leucine-containing di- and tripeptides and two lysine-containing dipeptides supported the growth of strain Z1-2D, a leucine, lysine auxotroph of Saccharomyces cerevisiae. However, (Lys)2, (Lys)3, (Lys)4, and (Lys)5 as well as Gly-Leu-Gly, three tetra- and one pentapeptide containing leucine were not utilized by the mutant. Cellular peptidases released leucine or lysine from all of these non-growth-supporting peptides, suggesting that the failure of strain Z1-2D to utilize these compounds reflects their failure to enter the yeast. Competition studies employing phenylalanine or non-leucine-containing peptides showed that the uptake of peptides into S. cerevisiae Z1-2D is distinct from that of amino acids and that di- and oligopeptides may share a common transport system. The failure of strain Z1-2D to utilize any peptide larger than (Leu)3 may indicate a transport size limit. Such a size limit would influence the construction of models that explain the action of yeast mating factors.     

Selective phosphinate transition-state analogue inhibitors of the protease of human immunodeficiency virus

The phosphinic acid isosteres of di-, tetra- and hexapeptides containing a hydrophobic amino acid side chains at the P1-P'1 positions are powerful inhibitors of Human Immunodeficiency Virus protease. Ki's ranged from 0.4 nM to 26 microM at pH 6.5 and were lower at pH 4.5. The compounds showed no activity against trypsin, weak activity against renin at pH 6.5, moderate activity against pepsin at pH 2.0 (Ki values in the microM range) and substantial activity against cathepsin D at pH 3.5 (Ki values from 9 to 300 nM).     

Glycoside esters from lichens of central Asia

Ten compounds isolated from the extract of the central Asian lichens comprised new glycosides and glycoside esters having 18R-hydroxy-dihydroalloprotolichesterinic, 18S-hydroxy-dihydroprotolichesterinic and 18S-hydroxy-neodihydroprotolichesterinic acids, as the aglycones and a saccharide moiety linked at C-18 and also at C-21 made by glucose, xylose or rhamnose. The structures were elucidated using extensive spectroscopic analysis (1D and 2D NMR, MS, IR, UV and ORD) and by biochemical methods.     

Purification of micromolar quantities of nucleotide analogs by reverse-phase high-performance liquid chromatography using a volatile buffer at neutral pH

The separation of 5'-adenosine di- and triphosphates from inorganic pyrophosphate or imidodiphosphate can be accomplished with reverse-phase HPLC by using a solvent system buffered by triethylammonium bicarbonate (pH 6.7). This buffer was used because it was neutral, readily volatile at 20 degrees C, and formed ion pairs with phosphate compounds to allow their separation by reverse-phase chromatography. Micromolar amounts of radioactive or fluorescent nucleotide analogs have been purified using C-18 columns or a polystyrene divinylbenzene column (PRP-1, Hamilton) with the solvent system described. The method is particularly advantageous in preparing salt-free acid-, base-, or thermally-labile nucleotide analogs. It is possible with this method to remove 32Pi (173 mumol) from ATP (50 mumol, 30 mg) in one run using a C-18 analytical column demonstrating that this approach can be useful for selected semipreparative purifications.     

Acyl chain-length asymmetry alters the interfacial elastic interactions of phosphatidylcholines.

Phosphatidylcholines (PCs) with stearoyl (18:0) sn-1 chains and variable-length, saturated sn-2 acyl chains were synthesized and investigated using a Langmuir-type film balance. Surface pressure was monitored as a function of lipid molecular area at various constant temperatures between 10 degrees C and 30 degrees C. Over this temperature range, 18:0-10:0 PC displayed only liquid-expanded behavior. In contrast, di-14:0 PC displayed liquid-expanded behavior at 24 degrees C and 30 degrees C, but two-dimensional phase transitions were evident at 20 degrees C, 15 degrees C, and 10 degrees C. The average molecular area of 18:0-10:0 PC was larger than that of liquid-expanded di-14:0 PC at equivalent surface pressures, and the shapes of their liquid expanded isotherms were somewhat dissimilar. Analysis of the elastic moduli of area compressibility (Cs(-1)) as a function of molecular area revealed shallower slopes in the semilog plots of 18:0-10:0 PC compared to di-14:0 PC. At membrane-like surface pressures (e.g., 30 mN/m), 18:0-10:0 PC was 20-25% more elastic (in an in-plane sense) than di-14:0 PC. Other PCs with varying degrees of chain-length asymmetry (18:0-8:0 PC, 18:0-12:0 PC, 18:0-14:0 PC, 18:0-16:0 PC) were also investigated to determine whether the higher in-plane elasticity of fluid-phase 18:0-10:0 PC is a common feature of PCs with asymmetrical chain lengths. Two-dimensional phase transitions in 18:0-14:0 PC and 18:0-16:0 PC prevented meaningful comparison with other fluid-phase PCs at 30 mN/m. However, the Cs(-1) values for fluid-phase 18:0-8:0 PC and 18:0-12:0 PC were similar to that of 18:0-10:0 PC (85-90 mN/m). These values showed chain-length asymmetrical PCs to have 20-25% greater in-plane elasticity than fluid-phase PCs with mono- or diunsaturated acyl chains.     

Glycosidic compounds of murolic, protoconstipatic and allo-murolic acids from lichens of Central Asia

Eleven compounds isolated from the extract of the Central Asian lichens comprised eight new glycosides having murolic, protoconstipatic and allo-murolic acids, as the aglycones and a saccharide moiety linked at C-18 made up of one or two sugars (glucose and apiose or rhamnose or xylose or arabinose). The structures were elucidated by using extensive spectroscopic analysis (1D and 2D NMR, MS, IR, UV and CD) and chemical methods.     

Phase diagrams of pseudo-binary phospholipid systems. I. Influence of the chain length differences on the miscibility properties of cephaline/cephaline/water systems

The miscibility properties of homologous cephalines (PEs) were studied by means of differential scanning calorimetry (DSC). The phase diagrams of 5 pseudo-binary cephaline/cephaline/water systems (50% water) are discussed. In the high temperature L alpha-phase, all the homologous cephalines of fatty acid chain length from C = 12 to C = 18 were completely miscible. On the other hand in the low temperature L beta-phase, a miscibility gap occurred in dependence on the differences of the acyl chain lengths. Further, a distinct succession of the phase diagram types was observed according to increasing chain length differences of the PEs: complete miscibility (systems di-(C12:O)-PE/di-(C14:O)-PE/H2O; di-(C14:O)-PE/di-C(16:O)-PE/H2O)----peritectic mixing behaviour (systems di-(C12:O)-PE/di-(C16:O)-PE/H2O; di-(C14:O)-PE/di-(C18:O)-PE/H2O)----eutectic mixing behaviour (system di-(C12:O)-PE/di-(C18:O)-PE/H2O). The change in the type of phase diagram from azeotropic to semi-azeotropic and from semi-azeotropic to eutectic is interpreted by means of the Landau theory.     

Comparative physical studies of phosphatidylsulfocholine and phosphatidylcholine. Calorimetry,fluorescence polarization and electron paramagnetic resonance spectroscopy

Transition temperatures of phosphatidylsulfocholines (PSCs; di-14 : 0-, di-16 : 0-, di-18 : 0- and di-18 : 1-) were compared with those of the corresponding phosphatidylcholines (PCs) using the techniques of differential scanning calorimetry, fluorescent polarization with diphenylhexatriene (DPH) and cis- and trans- parinaric acids as probes, and electron paramagnetic resonance (EPR) with 5-doxyl stearic acid as probe. Liposomal dispersions of the sulfonium analogs showed the typical multibilayer structure by electron microscopy (EM) and were in general very similar in physical behaviour to those of the corresponding PCs. However, the fully hydrated saturated PSCs consistently showed sharp main transitions 2–4°C above those of the corresponding PCs, by all three techniques used; the unsaturated PSC (di-18 : 1) had a transition 2–3°C below that of di-18 : 1-PC and only the di-14 : 0-PSC and di-18 : 1-PSC showed a well-defined pretransition. Fluorescence polarization studies with cis- and trans-parinaric acids showed that the PSC bilayers were less ordered than the corresponding PC bilayers in both the gel and liquid crystalline states.These results provide a rationale for the observed ability of the sulfonium analogs to substitute for PC in some natural membranes.     

The influence of the lipid composition on the degree of lipid-peroxidation of liposomes

Liposomes containing dilinoleoyl lecithin (di-18(2)PC) and diarachidonyl lecithin (di-20(4)PC) were peroxidized with Fe++/ascorbate. Lipidperoxidation was measured as generated malondialdehyde, the disappearance of polyunsaturated fatty acids, conjugated diene formation and chemiluminescense. It was found that di-20(4)PC was oxidized rapidly, while in the same time period hardly any oxidation of di-18(2)PC took place. Incorporation of different lipid classes into the di-20(4)PC liposomes influenced the peroxidation for each lipid in a characteristic way. Phosphatidylethanolamine stimulated the formation of fluorescent chromolipids. The possible meaning of these reactions in human pathology is discussed.     

Synthesis and biological studies of 5-aminolevulinic acid-containing dendrimers for photodynamic therapy.

Using a convergent growth approach, a series of novel 5-aminolevulinic acid (ALA)-containing dendrimers have been synthesized. In these molecules, ALA residues are attached to the periphery by ester linkages, with amide bonds connecting the dendrons. Three first-generation dendrimers, bearing either 6 or 9 ALA residues, were synthesized by attachment of a tris(Boc-protected ALA)-containing wedge (1) to a di- or tripodent aromatic, or tripodent aliphatic core. Two second generation 18-ALA-containing dendrimers were also synthesized using a 3,3'-iminodipropionic acid spacer unit between wedge 1 and the aromatic core. These compounds differed only in the distance between the core and the linker unit. The Boc-protected dendrimers were deprotected using trifluoroacetic acid and isolated as their TFA salts. The potential of these ALA ester dendrimers as macromolecular prodrugs for photodynamic therapy has been demonstrated in the tumorigenic keratinocyte PAM 212 cell line.     

Human placental cytoplasmic 5'-nucleotidase. Kinetic properties and inhibition

The kinetic properties of highly purified human placental cytoplasmic 5'-nucleotidase were investigated. Initial velocity studies gave Michaelis constants for AMP, IMP, and CMP of 18, 30, and 2.2 microM, respectively. The enzyme shows the following relative Vmax values: CMP greater than UMP greater than dUMP greater than GMP greater than AMP greater than dCMP greater than IMP. The activity was magnesium-dependent, and this cation binds sequentially with a Km of 14 microM for AMP and an apparent Km of 6 mM for magnesium. A large variety of purine, pyrimidine, and pyridine compounds exert an inhibitory effect on enzyme activity. IMP, GMP, and NADH produce almost 100% inhibition at 1.0 mM. Nucleoside di- and triphosphates are potent inhibitors. ATP and ADP are competitive inhibitors with respect to AMP and IMP as substrates with Ki values of 100 and 15 microM, respectively. Inorganic phosphate is a noncompetitive inhibitor with Ki values of 19 and 43 mM. Nucleosides and other compounds studied produce only a modest decrease of enzyme activity at 1 mM. Our findings suggest that the enzyme is regulated under physiological conditions by the concentrations of magnesium, nucleoside 5'-monophosphates, and nucleoside di- and triphosphates. The nucleotide pool concentration regulates the enzyme possibly by a mechanism of heterogeneous metabolic pool inhibition. These properties of human placental cytoplasmic 5'-nucleotidase may be related to the control of nucleotide degradation in vivo.     

Peroxidase-catalyzed cross linking of proteins

Incubation of casein and water-soluble soybean protein, separately or together, at 10 mg/ml with horseradish peroxidase (2.4 or 24 M) and H₂O₂ (1.8 or 18 mM) at pH 9.0 (0.2 M borate buffer) for 24 hr at 37°C in air led to formation of higher molecular weight compounds as determined by sodium dodecyl sulfate polyacrylamide disc gel electrophoresis. Incubation under the same conditions with peroxidase alone (in air) gave a smaller amount of higher molecular weight compounds. Incubation of lysozyme separately or with water-soluble soybean protein did not produce detectable amounts of higher molecular weight compounds. These results are discussed in terms of previously observed di- and tertyrosine isolated from peroxidase/H₂O₂-treated and naturally occurring proteins following acid hydrolysis. Transglutaminase, lipoxygenase, polyphenol oxidase, and lysyl oxidase are examples of other enzymes that can cross link proteins.     

Two new guaiane-sesquiterpenes from the cultured lichen mycobiont of Diorygma pruinosum

Lichens and the mycobionts derived from lichen-source are believed to be a valuable source of bioactive compounds. Diorygma pruinosum is a native lichen in Vietnam. A mycobiont of Diorygma pruinosum was separated, then cultivated. Phytochemical data on this lichen and its cultured mycobionts are scarce. The present study described the isolation and structural elucidation of two new guaiane-type sesquiterpenes, namely pruinosone (1) and hydroxypruinosone (2). Their absolute chemical structures were elucidated by extensive 1D and 2D NMR analysis, high-resolution mass spectroscopy, electronic circular dichroism (ECD), and comparisons in the literatures. Compounds 1 and 2 were evaluated for alpha-glucosidase inhibition and antimicrobial activity.     

On the specificity of vitamin D compounds binding to chick pig intestinal 1,25-dihydroxyvitamin D3 receptor

The binding activity of four vitamin D metabolites and/or analogs for the intestinal 1,25-dihydroxyvitamin D3 receptor was evaluated after incubation at 25 degrees C for 1 h or at 0-4 degrees C for 18 h. The incubation conditions, which had no effect on the binding of 1,25-dihydroxyvitamin D3, had a dramatic effect on the binding of 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3 and a small but reproducible effect on 24,25-dihydroxyvitamin D3 binding to receptor. Affinities 10- to 20-fold higher were obtained for 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3, and affinities 3-fold higher were obtained for 24,25-dihydroxyvitamin D3 at the 0-4 degrees C/18-h incubation. A comparison of intestinal receptor from chick and pig with nine vitamin D compounds showed no major differences between the two species. The relative affinity of the vitamin D analogs to compete with tritiated 1,25-dihydroxyvitamin D3 for the receptor in pig nuclear extract, expressed as ratios of the molar concentration required for 50% binding of the tritiated 1,25-dihydroxyvitamin D3 compared to nonradioactive 1,25-dihydroxyvitamin D3, are as follows: 1,25-dihydroxyvitamin D3 (1) = 1,25-dihydroxyvitamin D2 = 24-homo-1,25-dihydroxyvitamin D3 greater than 1,24,25-trihydroxyvitamin D3 (4) greater than 25-hydroxyvitamin D3 (21) = 10-oxo-19-nor-25-hydroxyvitamin D3 = 1 alpha-hydroxyvitamin D3 (37) greater than 24,25-dihydroxyvitamin D2 (257) much much greater than vitamin D3 (greater than 10(6)).     

The effect of fuel composition on the mutagenicity of diesel engine exhaust

The effect of fuel composition on the mutagenicity of diesel engine emission was investigated. To this end, a fuel matrix comprising fuels with different contents of aromatic and naphthenic compounds was used. Extracts of the organic phase of raw exhausts obtained with different fuels were tested for mutagenicity in bacterial reversion assays. The results obtained demonstrate that the mutagenicity of diesel exhaust is largely dependent on the aromatic content of the fuel. In fact, mutagenicity was greatly reduced when the aromatic content of the fuel was lowered by hydrogen treatment. Conversely, mutagenicity was enhanced when the fuel was enriched with fractions of di- or triaromatic compounds. The addition of di- and trinaphthenic compounds only produced borderline mutagenicity. No clear relationship was observed between sulfur content of the fuel and mutagenicity of the exhaust. Assays in bacterial strains with different sensitivity to nitroaromatic compounds suggest a low contribution of the highly mutagenic dinitropyrenes to the responses observed, and a relatively greater contribution of 1-nitropyrene or other nitroaromatics processed by the same bacterial nitroreductase.     

New diterpene furanoids from the Antarctic lichen Huea sp

In the course of ongoing research on protein tyrosine phosphatase 1B (PTP1B) inhibitory compounds from Antarctic lichens, four new diterpene furanoids, hueafuranoids A–D (1–4) have been isolated from the MeOH extract of Antarctic lichen Huea sp. by various chromatographic methods. The structures of these compounds were elucidated by analysis of NMR and MS data, and comparing their spectral data with those in the literature. Compound 1 showed inhibitory activity against therapeutically targeted protein, PTP1B with an IC₅₀ value of 13.9 μM. The kinetic analysis of PTP1B inhibition by hueafuranoid A (1) suggested that the diterpene furanoids encountered in this study inhibited PTP1B activity in a non-competitive manner.     

New compatible solutes related to Di-myo-inositol-phosphate in members of the order Thermotogales.

The accumulation of intracellular organic solutes was examined in six species of the order Thermotogales by nuclear magnetic resonance spectroscopy. The newly discovered compounds di-2-O-beta-mannosyl-di-myo-inositol-1,1'(3,3')-phosphate and di-myo-inositol-1,3'-phosphate were identified in Thermotoga maritima and Thermotoga neapolitana. In the latter species, at the optimum temperature and salinity the organic solute pool was composed of di-myo-inositol-1,1'(3,3')-phosphate, beta-glutamate, and alpha-glutamate in addition to di-myo-inositol-1,3'-phosphate and di-2-O-beta-mannosyl-di-myo-inositol-1,1'(3,3')-phosphate. The concentrations of the last two solutes increased dramatically at supraoptimal growth temperatures, whereas beta-glutamate increased mainly in response to a salinity stress. Nevertheless, di-myo-inositol-1,1'(3,3')-phosphate was the major compatible solute at salinities above the optimum for growth. The amino acids alpha-glutamate and proline were identified under optimum growth conditions in Thermosipho africanus, and beta-mannosylglycerate, trehalose, and glycine betaine were detected in Petrotoga miotherma. Organic solutes were not detected, under optimum growth conditions, in Thermotoga thermarum and Fervidobacterium islandicum, which have a low salt requirement or none.     

Transplantation of lichen thalli: a case study on Cetraria islandica for conservation and pharmaceutical purposes

Changes in biomass growth and chemical composition of transplanted Cetraria islandica lichen thalli were investigated in a natural stand in the Bory Tucholskie region (northern Poland) over 3 yr. Transplants consisted of either 3.85 or 7.88 g fragments, 12.62 g clumps, or control plots where all vegetation and lichens were removed. The initial mass of C. islandica significantly influenced the percentage cover of investigated lichens at the study plots. At the last set of study measurements the highest cover, biomass accumulation (4×) and mean final mass (31.99 g m⁻²) of C. islandica were recorded at the study plots at which 7.77 g fragments were transplanted. Analysis showed that the content of the determined chemical compounds in each sample were similar. The potential influence of other species growing at the study area on the occurrence of C. islandica was also examined through monitoring species diversity in the cleared forest floor plots. Altogether 18 species of plants and lichens were recorded in study plots, and among most abundant species Dicranum polysetum and Pleurozium schreberi were observed. The mean percentage cover for C. islandica was 14.61 %.