Coincident isolation of a novel homoisoflavonoid from Resnova humifusa and Eucomis montana (Hyacinthoideae: Hyacinthaceae)

We report on the first phytochemical investigation of a member of the African genus Resnova (Hyacinthoideae: Hyacinthaceae). From the dichloromethane extract of the bulbs of both Resnova humifusa and Eucomis montana (Hyacinthoideae: Hyacinthaceae) a novel 3-benzyl-4-chromanone homoisoflavonoid, 5,6-dimethoxy-7-hydroxy-3-(4′-hydroxybenzyl)-4-chromanone, was isolated. A further 11 known homoisoflavonoids were also identified, the 12 in total presenting a clear biosynthetic sequence. Eight of the 12 compounds found were common to both species.     

Inhibition of thyroid secretion by sodium fluoride in vitro

NaF mimicked the activation by thyrotropin of iodide binding to proteins and of glucose C-I oxidation but not the accumulation of intracellular colloid droplets or the stimulation of secretion in dog thyroid slices in vitro. On the contrary, NaF inhibited the two latter thyrotropin effects. The inhibitory action of F⁻ was partially relieved by the addition of glucose to the medium; it was mimicked by sodium oxamate. These data suggest that NaF depresses the endocytosis of colloid and thyroid secretion by inhibiting aerobic glycolysis in the follicular cell. NaF inhibited the activation of colloid droplet accumulation and secretion by N⁶,O^2′-dibutyryl-adenosine 3′,5′-monophosphate (dibutyryl cyclic AMP) and the accumulation of cyclic AMP in thyrotropin-stimulated slices. This suggests an inhibition at the level of both cyclic AMP accumulation and cyclic AMP action. The inhibition by NaF and sodium oxamate of colloid droplet formation and thyroid secretion but not of glucose C-I oxidation in stimulated slices further confirms our conclusion that the latter effect is not merely a consequence of the activation by thyrotropin of colloid endocytosis.     

Tuning of functional heme reduction potentials in Shewanella fumarate reductases

The fumarate reductases from S. frigidimarina NCIMB400 and S. oneidensis MR-1 are soluble and monomeric enzymes located in the periplasm of these bacteria. These proteins display two redox active domains, one containing four c-type hemes and another containing FAD at the catalytic site. This arrangement of single-electron redox co-factors leading to multiple-electron active sites is widespread in respiratory enzymes. To investigate the properties that allow a chain of single-electron co-factors to sustain the activity of a multi-electron catalytic site, redox titrations followed by NMR and visible spectroscopies were applied to determine the microscopic thermodynamic parameters of the hemes. The results show that the redox behaviour of these fumarate reductases is similar and dominated by a strong interaction between hemes II and III. This interaction facilitates a sequential transfer of two electrons from the heme domain to FAD via heme IV.     

Integrin participates in the effect of thyroxine on plasma membrane in immature rat testis

Background

The secretory activity of Sertoli cells (SC) is dependent on ion channel functions and protein synthesis and is critical to ongoing spermatogenesis. The aim of this study was to investigate the mechanism of action associated with a non-metabolizable amino acid [¹⁴C]-MeAIB (α-(methyl-amino)isobutyric acid) accumulation stimulated by T₄ and the role of the integrin receptor in this event, and also to clarify whether the T₄ effect on MeAIB accumulation and on Ca²⁺ influx culminates in cell secretion.

Methods

We have studied the rapid and plasma membrane initiated effects of T₄ by using ⁴⁵Ca²⁺ uptake and [⁴⁵C]-MeAIB accumulation assays, respectively. Thymidine incorporation into DNA was used to monitor nuclear activity and quinacrine to analyze the secretory activity on SC.

Results

The stimulation of MeAIB accumulation by T₄ appears to be mediated by the integrin receptor in the plasma membrane since tetrac and RGD peptide were able to nullify the effect of this hormone. In addition, T₄ increases extracellular Ca²⁺ uptake and Ca²⁺ from intracellular stocks to enhance nuclear activity, but this genomic action seems not to influence SC secretion mediated by T₄. Also, the cytoskeleton and ClC-3 chloride channel contribute to the membrane-associated responses of SC.

Conclusions

T₄ integrin receptor activation ultimately determines the plasma membrane responses on amino acid transport in SC, but it is not involved in calcium influx, cell secretion or the nuclear effect of the hormone.

General significance

The integrin receptor activation by T₄ may take a role in plasma membrane processes involved in the male reproductive system.     

Phenolic analogs of the N,C-coupled naphthylisoquinoline alkaloid ancistrocladinium A, from Ancistrocladus cochinchinensis (Ancistrocladaceae), with improved antiprotozoal activities

The first N,8′-coupled naphthylisoquinoline alkaloids with free phenolic OH groups, 4′-O-demethylancistrocladinium A and 6,4′-O-didemethylancistrocladinium A, have been isolated from the leaves and bark of the Vietnamese liana Ancistrocladus cochinchinensis, along with its known, non-phenolic parent compound, ancistrocladinium A, and four C,C-coupled representatives. The structure elucidation was achieved by chemical, spectroscopic, and chiroptical methods. The mono-phenolic alkaloid showed excellent activities in particular against the pathogen causing Chagas’ disease, Trypanosoma cruzi.     

Anthocyanin 3-galactosides from Cornus alba 'Sibirica' with glucosidation of the B-ring

The three anthocyanins, delphinidin 3-O-beta-galactopyranoside-3',5'-di-O-beta-glucopyranoside (1), delphinidin 3-O-beta-galactopyranoside-3'-O-beta-glucopyranoside (2) and cyanidin 3-O-beta-galactopyranoside-3'-O-beta-glucopyranoside (3), and the 3-O-beta-galactopyranosides of delphinidin (4) and cyanidin (5) were isolated from the bluish white berries and compound umbel of Siberian dogwood, Cornus alba 'Sibirica'. The ornamental autumn leaves and the characteristic purplish red bark of this variety were found to contain only pigment 5.     

Slow Myosin ATP Turnover in the Super-Relaxed State in Tarantula Muscle

We measured the nucleotide turnover rate of myosin in tarantula leg muscle fibers by observing single turnovers of the fluorescent nucleotide analog 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-triphosphate, as monitored by the decrease in fluorescence when 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-triphosphate (mantATP) is replaced by ATP in a chase experiment. We find a multiexponential process with approximately two-thirds of the myosin showing a very slow nucleotide turnover time constant (∼ 30 min). This slow-turnover state is termed the super-relaxed state (SRX). If fibers are incubated in 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-diphosphate and chased with ADP, the SRX is not seen, indicating that trinucleotide-relaxed myosins are responsible for the SRX. Phosphorylation of the myosin regulatory light chain eliminates the fraction of myosin with a very long lifetime. The data imply that the very long-lived SRX in tarantula fibers is a highly novel adaptation for energy conservation in an animal that spends extremely long periods of time in a quiescent state employing a lie-in-wait hunting strategy. The presence of the SRX measured here correlates well with the binding of myosin heads to the core of the thick filament in a structure known as the “interacting-heads motif,” observed previously by electron microscopy. Both the structural array and the long-lived SRX require relaxed filaments or relaxed fibers, both are lost upon myosin phosphorylation, and both appear to be more stable in tarantula than in vertebrate skeletal or vertebrate cardiac preparations.     

Sterically hindered carotenoids with 3Z, 5Z configuration from the seeds of oriental bitter sweet, Celastrus orbiculatus

Sterically hindered cis-carotenoids 1 and 2 were isolated from seeds of the oriental bitter sweet, Celastrus orbiculatus. Their structures were determined to be (3′Z, 5′Z)-celaxanthin and (3′Z, 5′Z)-torulene, respectively, on the basis of spectroscopic data and iodine-catalyzed stereomutation. This is the first report on carotenoids with a 3Z, 5Z configuration.     

Evidence for major structural changes in subunit C of the vacuolar ATPase due to nucleotide binding

The ability of subunit C of eukaryotic V-ATPases to bind ADP and ATP is demonstrated by photoaffinity labeling and fluorescence correlation spectroscopy (FCS). Quantitation of the photoaffinity and the FCS data indicate that the ATP-analogues bind more weakly to subunit C than the ADP-analogues. Site-directed mutagenesis and N-terminal sequencing of subunit C from Arabidopsis (VHA-C) and yeast (Vma5p) have been used to map the C-terminal region of subunit C as the nucleotide-binding site. Tryptophan fluorescence quenching and decreased susceptibility to tryptic digestion of subunit C after binding of different nucleotides provides evidence for structural changes in this subunit caused by nucleotide-binding.     

Six naphthylisoquinoline alkaloids and a related benzopyranone from a Congolese Ancistrocladus species related to Ancistrocladus congolensis

From the roots of a recently discovered Ancistrocladus taxon, with close affinities to Ancistrocladus congolensis regarding molecular ITS sequence data, six naphthylisoquinoline alkaloids, 5'-O-demethylhamatine (2), 5'-O-demethylhamatinine (3), 6-O-demethylancistroealaine A (4), 6,5'-O,O-didemethylancistroealaine A (5), 5-epi-6-O-methylancistrobertsonine A (6), and 5-epi-4'-O-demethylancistrobertsonine C (7), have been isolated, along with a likewise benzopyranone carboxylic acid, 8. The structural elucidation succeeded by chemical, spectroscopic, and chiroptical methods. Their bioactivities were tested against protozoan parasites causing severe tropical diseases. Furthermore, eight known related alkaloids were identified.     

Synthesis and membrane behavior of a new class of unnatural phospholipid analogs useful as phospholipase A2 degradable liposomal drug carriers

A new and unnatural type of lipid analogs with the phosphocholine and phosphoglycerol head groups linked to the C-2 position of the glycerol moiety have been synthesized and the thermodynamic lipid membrane behavior has been investigated using differential scanning calorimetry. From the heat capacity measurements, it was observed that the pre-transition was abolished most likely due to the central position of the head groups providing better packing properties in the low temperature ordered gel phase. Activity measurements of secretory phospholipase A2 (PLA2) on unilamellar liposomal membranes revealed that the unnatural phospholipids are excellent substrates for PLA2 catalyzed hydrolysis. This was manifested as a minimum in the PLA2 lag time in the main phase transition temperature regime and a high degree of lipid hydrolysis over a broad temperature range. The obtained results provide new information about the interplay between the molecular structure of phospholipids and the lipid membrane packing constrains that govern the pre-transition. In addition, the PLA2 activity measurements are useful for obtaining deeper insight into the molecular details of the catalytic site of PLA2. The combined results also suggest new approaches to rationally design liposomal drug carries that can undergo a triggered activation in diseased tissue by overexpressed PLA2.     

Halogranum salarium sp. nov., a halophilic archaeon isolated from sea salt

Three halophilic archaea, strains B-1^T, B-3 and B-4, were isolated from evaporitic salt crystals from Namhae, Korea. Cells of the strains were Gram-stain-negative, motile and pleomorphic, and colonies were red-pigmented. The three isolates had identical 16S rRNA gene sequences and formed a tight phylogenetic clade with Halogranum rubrum RO2-11^T in the genus Halogranum, showing 99.5% sequence similarity. The next most closely related species were Halogranum amylolyticum and Halogranum gelatinilyticum (97.4 and 96.3% similarity to the respective type strains). The phylogeny based on the full-length RNA polymerase subunit B′ gene (rpoB′) was in agreement with the 16S rRNA gene sequence analysis, but allowed better discrimination. DNA-DNA hybridization between a representative strain (B-1^T) and the type strains of Hgn. rubrum, Hgn. amylolyticum and Hgn. gelatinilyticum revealed less than 40% relatedness. Polar lipid analysis showed that the three isolates contained phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and three glycolipids. Combined genotypic and phenotypic data supported the conclusion that strains B-1^T, B-3 and B-4 represent a novel species of the genus Halogranum, for which the name Halogranum salarium sp. nov. is proposed. The type strain is B-1^T (=KCTC 4066^T = DSM 23171^T).     

Differential activation of cAMP- and cGMP-dependent protein kinases by cyclic purine and pyrimidine nucleotides

The cyclic purine nucleotides cAMP and cGMP are well-characterized second messengers and activators of PKA and PKG, respectively. In contrast, the functions of the cyclic pyrimidine nucleotides cCMP and cUMP are poorly understood. cCMP induces relaxation of smooth muscle via PKGI, and phosphodiesterases differentially hydrolyze cNMPs. Here, we report that cNMPs differentially activate PKA isoforms and PKGIα. The combination of cCMP with cAMP reduced the EC₅₀ of cAMP for PKA. PKGIα exhibited higher specificity for the cognate cNMP than PKA. Our data support a role of cCMP and cUMP as second messengers.     

Distribution of reaction products in phospholipase A2 hydrolysis

We have monitored the composition of supported phospholipid bilayers during phospholipase A₂ hydrolysis using specular neutron reflection and ellipsometry. Porcine pancreatic PLA₂ shows a long lag phase of several hours during which the enzyme binds to the bilayer surface, but only 5 ± 3% of the lipids react before the onset of rapid hydrolysis. The amount of PLA₂, which resides in a 21 ± 1 Å thick layer at the water-bilayer interface, as well as its depth of penetration into the membrane, increase during the lag phase, the length of which is also proportional to the enzyme concentration. Hydrolysis of a single-chain deuterium labelled d₃₁-POPC reveals for the first time that there is a significant asymmetry in the distribution of the reaction products between the membrane and the aqueous environment. The lyso-lipid leaves the membrane while the number of PLA₂ molecules bound to the interface increases with increasing fatty acid content. These results constitute the first direct measurement of the membrane structure and composition, including the location and amount of the enzyme during hydrolysis. These are discussed in terms of a model of fatty-acid mediated activation of PLA₂.     

Two aurone glycosides from heartwood of Pterocarpus santalinus

Two new aurone glycosides, 6 hydroxy 5 methyl 3',4',5' trimethoxy aurone 4-O-alpha-L-rhamnopyranoside and 6,4' dihydroxy aurone 4-O-rutinoside have been isolated from the ethanolic extract of the wood of Pterocarpus santalinus. Their structures were determined on the basis of chemical and spectroscopic analysis (UV, IR, EIMS, (1)H and (13)C NMR).