Leakage of Endotoxins through the Endotoxin Retentive Filter: An in vitro Study.

INTRODUCTION: Ultrapurification of dialysis fluid has enabled highly efficient dialysis treatments. Online hemodiafiltration is one such treatment that uses a purified dialysis fluid as a supplemental fluid. In this method, an endotoxin retentive filter (ETRF) is used in the final step of dialysis fluid purification, with the aim of preventing leakage of endotoxins. Sodium hypochlorite and peracetic acid are used as disinfecting agents for the dialysis fluid pipes containing the ETRF; however, the effects of these agents on ETRF membrane pores have not been fully clarified. METHODS: Water permeability (flux) and endotoxin permeability were assessed in 3 types of ETRFs made with different membrane materials: polyester polymer alloy (PEPA), polyether sulfone (PES), and polysulfone (PS). High-concentration sodium hypochlorite and 2 types of peracetic acid were used as disinfecting agents, and the changes in flux and the endotoxin sieving coefficient (SC) were measured. RESULTS: After repeated use of high concentrations of sodium hypochlorite and peracetic acid, the PEPA and PES ETRFs did not permit passage of endotoxins, regardless of their flux. However, in the PS ETRF, the flux and endotoxin SC increased with the number of cleaning cycles. No differences were observed according to the concentration of peracetic acid disinfecting agents. CONCLUSION: PEPA and PES ETRFs completely prevent endotoxin leakage and can be disinfected at concentrations higher than the conventionally recommended concentration without affecting pore expansion. Even new PS ETRFs have low levels of endotoxin leakage, which increase after disinfection cycles using sodium hypochlorite and peracetic acid.

Discovery of a Novel Diterpene in Brown Propolis from the State of Parana, Brazil.

Propolis is a resinous substance collected by honeybees from certain plant sources. The components of propolis depend on the vegetation of the area in which apiculture is practiced. In Brazil, there are several types of propolis including 'green,' 'red' and 'brown'. Brazilian brown propolis from the state of Parana characteristically includes diterpenes, and we discovered a novel clerodane diterpene, rel-(5S,6S,8R,9R,10S,18R,19S)-18,19-epoxy-2-oxocleroda-3,12(E),14- triene-6,18,19-triol 18,19-diacetate 6-benzoate (3) and five known diterpenes (1, 2, 4, 5 and 6). The chemical structure of the novel diterpene 3 was determined using 1D- and 2D-NMR spectroscopic analyses. Furthermore, the activities of the isolated diterpenes on growth inhibition of several human cancer cell lines (LNCaP, MCF-7, DLD-1 and A549) were evaluated in vitro; diterpene 3 exhibited a potent inhibition of cell growth, and its activity was approximately 15 times higher than that of the other diterpenes.

New cassane-type diterpenoids of Caesalpinia echinata (Leguminosae) exhibiting NF-κB inhibitory activities.

Seven new cassane-type diterpenoids, echinalides A-G (1-7), were isolated from the stem of Caesalpinia echinata LAM. (Leguminosae). The structures were established on the basis of their chemical properties and spectroscopic evidence, including two dimensional (2D)-NMR analysis. These compounds were assessed for inhibitory activity against nuclear factor κB (NF-κB). Echinalides C and D, in particular, significantly inhibited NF-κB-responsive reporter gene expression at 5.0 µM, an effect almost equivalent to that of parthenolide, a known potent inhibitor of NF-κB.

Culcitiolides A-D, four new eremophilane-type sesquiterpene derivatives from Senecio culcitioides.

Four new eremophilane-type sesquiterpenes, culcitiolides A-D, were isolated from the stem of Senecio culcitioides Sch. Bip (Asteraceae). Their structures were established by detailed 2D NMR spectroscopic and single-crystal X-ray experiments. These compounds were assessed for inhibitory activity against nuclear factor kappaB (NF-kappaB). Culcitiolides C and D at 20 microM showed 97 and 100% inhibition of NF-kappaB activity, respectively.

Enzymatic (13)C labeling and multidimensional NMR analysis of miltiradiene synthesized by bifunctional diterpene cyclase in Selaginella moellendorffii.

Diterpenes show diverse chemical structures and various physiological roles. The diversity of diterpene is primarily established by diterpene cyclases that catalyze a cyclization reaction to form the carbon skeleton of cyclic diterpene. Diterpene cyclases are divided into two types, monofunctional and bifunctional cyclases. Bifunctional diterpene cyclases (BDTCs) are involved in hormone and defense compound biosyntheses in bryophytes and gymnosperms, respectively. The BDTCs catalyze the successive two-step type-B (protonation-initiated cyclization) and type-A (ionization-initiated cyclization) reactions of geranylgeranyl diphosphate (GGDP). We found that the genome of a lycophyte, Selaginella moellendorffii, contains six BDTC genes with the majority being uncharacterized. The cDNA from S. moellendorffii encoding a BDTC-like enzyme, miltiradiene synthase (SmMDS), was cloned. The recombinant SmMDS converted GGDP to a diterpene hydrocarbon product with a molecular mass of 272 Da. Mutation in the type-B active motif of SmMDS abolished the cyclase activity, whereas (+)-copalyl diphosphate, the reaction intermediate from the conversion of GGDP to the hydrocarbon product, rescued the cyclase activity of the mutant to form a diterpene hydrocarbon. Another mutant lacking type-A activity accumulated copalyl diphosphate as the reaction intermediate. When the diterpene hydrocarbon was enzymatically synthesized from [U-(13)C(6)]mevalonate, all carbons were labeled with (13)C stable isotope (>99%). The fully (13)C-labeled product was subjected to (13)C-(13)C COSY NMR spectroscopic analyses. The direct carbon-carbon connectivities observed in the multidimensional NMR spectra demonstrated that the hydrocarbon product by SmMDS is miltiradiene, a putative biosynthetic precursor of tanshinone identified from the Chinese medicinal herb Salvia miltiorrhiza. Hence, SmMDS functions as a bifunctional miltiradiene synthase in S. moellendorffii. In this study, we demonstrate that one-dimensional and multidimensional (13)C NMR analyses of completely (13)C-labeled compound are powerful methods for biosynthetic studies.

Using the pER8:GUS reporter system to screen for phytoestrogens from Caesalpinia sappan.

Arabidopsis thaliana pER8:GUS, a low-cost, highly efficient, and convenient transgenic plant system, was used to assay the estrogen-like activity of 30 traditional Chinese medicines. The MeOH extract of Caesalpinia sappan exhibited significant bioactivity in this assay, and subsequent bioactivity-guided fractionation of the extract led to the isolation of one new compound, (S)-3,7-dihydroxychroman-4-one (1), and 10 known compounds. Both the plant pER8:GUS and in vitro estrogen response element reporter assays were used to evaluate the estrogenic activity of the isolated compounds, and these two systems produced comparable results. Compounds 6, 8, and 11 showed significant estrogenic activity comparable to genistein. These active compounds were determined to be nontoxic new sources of phytoestrogens. In addition, compounds 2 and 3 inhibited ERE transcription induced by 17ß-estradiol. A docking model revealed that compounds 6, 8, and 11 showed high affinity to the estrogen receptor. The pER8:GUS reporter system was demonstrated to be a useful and effective technique in phytoestrogen discovery.

Active core structure of terfestatin A, a new specific inhibitor of auxin signaling.

The auxins, plant hormones, regulate many aspects of the growth and development of plants. Terfestatin A (TrfA), a novel auxin signaling inhibitor, was identified in a screen of Streptomyces sp. F40 extracts for inhibition of the expression of an auxin-inducible gene. However, the mode of action of TrfA has not been elucidated. To identify the active core structure, 25 derivatives of TrfA were synthesized and their inhibitory activities against auxin-inducible gene expression were evaluated. The structure-activity relationships revealed the essential active core structure of TrfA, 3-butoxy-4-methylbiphenyl-2,6-diol, which will lead to the design of biotin-tagged active TrfA.

ent-Kaurane-type diterpenoids from a cell suspension culture of the liverwort Jungermannia subulata.

A cell suspension culture of the liverwort Jungermannia subulata was established from callus tissue induced by culturing spores. From the suspension culture, three new ent-kaurane-type diterpenoids were isolated together with three previously reported ent-kaurane-type diterpenoids. Five ent-kaurane-type diterpenoids were isolated from the intact J. subulata, two of which were new compounds. Their structures were determined by spectroscopic methods, chemical reaction, and X-ray crystallographic analysis.

Inhibitory activity of plant stilbene oligomers against DNA topoisomerase II.

The inhibitory activity of 40 stilbene oligomers isolated from six plant species against topoisomerase II was evaluated, of which nine compounds showed a potent inhibitory effect, stronger than daunorubicin, a topoisomerase II inhibitor, used as an anti-cancer drug. The specificity of active stilbene oligomers on topoisomerase II was assessed by their effect on DNA restriction enzyme. In particular, specific inhibitory activity was observed in alpha-viniferin 13-O-beta-glucopryranoside (2) and hemsleyanol C (13).

Three new agarofuran sesquiterpenes reissantins F-H from Reissantia buchananii.

Three new agarofuran sesquiterpenes, reissantins F-H, were isolated from a methanol extract of Reissantia buchananii. Their structures as well as their relative stereochemistry were established on the basis of modern mass and spectroscopic methods. Compounds were assayed for cytotoxicity against HepG2 and Hep3B human liver cancer cell lines.

The transgenic Arabidopsis plant system, pER8-GFP, as a powerful tool in searching for natural product estrogen-agonists/antagonists.

The transgenic Arabidopsis plant system, pER8-GFP, may be used as a powerful tool in searching for natural estrogen-agonists/antagonists. Among selected plant extracts and natural products, the method was able to distinguish active extracts (e.g., Glycine max and Pueraria lobata) and pure compounds (e.g., 17beta-estradiol (1), genistein (10), and daidzein (11)) and also to distinguish effects of structural changes on activity. Thus, this rapid sensitive system was proven to be suitable for the discovery of natural products with estrogenic activity.

Gaudichanolides A and B, clerodane diterpenes from Baccharis gaudichaudiana.

Two new clerodane diterpenoids, gaudichanolides A (1) and B (2), were isolated from the dried twigs of Baccharis gaudichaudiana, together with 7-oxo-16,19-dihydroxy-3,4-dehydroclerodan-15,20-diacid dilactone, spathulenol, 4,10-aromadendranediol, kobusone, trans-cosanyl ferulate, and defuscin. The structures and relative stereochemistry of 1 and 2 were elucidated by detailed 2D NMR spectroscopic experiments, and the absolute stereochemistry of 1 was determined by X-ray crystallographic analysis.

Nine new cytotoxic monotetrahydrofuranic Annonaceous acetogenins from Annona montana.

Nine monotetrahydrofuranic Annonaceous acetogenins, montalicins A-E (1-5), cis-annoreticuin (6), montalicins F (7), I (8), and J (9), along with eight known acetogenins 10-17, were isolated from the seeds of Annona montana by a high performance liquid chromatographic (HPLC) method. The structures of all new isolates were elucidated and characterized by spectroscopic and chemical methods. These monotetrahydrofuranic Annonaceous acetogenins showed selectively potent cytotoxicity against two human cancer cell lines, 1A9 and Hep G2.

Antitumor agents. 233. Lantalucratins A-F, new cytotoxic naphthoquinones from Lantana involucrata.

In a continuing study to isolate novel antitumor agents from rainforest plants, three new isopropenylfurano-beta-naphthoquinones, designated lantalucratins A (1), B (2), and C (3), and three new isoprenyl-alpha-naphthoquinones, designated lantalucratins D (4), E (5), and F (6), were isolated from Lantana involucrata. Their structures were determined on the basis of NMR and X-ray crystallographic analyses. Compounds 1 and 2 showed cytotoxic activities against various human tumor cell lines, including drug-resistant variants, with IC50 values of 1.0-4.9 microM.

Antitumor agents. 228. five new agarofurans, Reissantins A-E, and cytotoxic principles from Reissantia buchananii.

Twenty-one compounds, including five new agarofuran sesquiterpenes, reissantins A-E (1-5), were isolated from Reissantia buchananii by means of bioassay-directed fractionation and their structures identified from spectral data. Reissantins A-C are the first reported simple agarofuran sesquiterpenes to contain a 5-carboxy-N-methyl-2-pyridone (CNMP) substituent, which has previously been found only in macroring agarofuran pyridine alkaloids. The major terpenoid components, celastrol (6) and its methyl ester derivative, pristimerin (7), were significantly active against nine cancer cell lines, including A549, MCF-7, HCT-8, KB, KB-VIN, U-87-MG, PC-3, 1A9, and PTX10 cell lines, with ED(50) values ranging from 0.076 to 0.34 microg/mL.

Antitumor agents. 221.1 buceracidins A and B, two new flavanones from Bucida buceras.

As part of a study on antitumor agents from rainforest plants, two new flavanones, buceracidin A (1), 5,2'-dihydroxy-3-methoxy-6,7-(2' ',2' '-dimethylchromene)flavanone, and buceracidin B (2), 5,2'-dihydroxy-6,7-(2' ',2' '-dimethylchromene)flavanone, were isolated together with four known prenylated flavanones, minimiflorin (3), 3-hydroxyminimiflorin (4), 3-methoxyminimiflorin (5), and mundulinol (6), from Bucida buceras. The structures of buceracidins A and B were elucidated from detailed 2D NMR analyses. Among the six flavanones, 3, 5, and 6 were marginally cytotoxic in a human tumor cell line panel.

Antitumor agents. Part 212. Bucidarasins A-C, three new cytotoxic clerodane diterpenes from Bucida buceras.

As part of a study on antitumor agents from rainforest plants, four new clerodane diterpenes, bucidarasins A--D (1-4), were isolated from Bucida buceras. Their structures were elucidated from detailed 2D NMR analyses. Compounds 1-3 showed potent cytotoxicity against human tumor cell lines with IC(50) values of 0.5-1.9 microM. The potency was retained in drug resistant lines.