Bibenzyls and dihydroisocoumarins from white salsify (Tragopogon porrifolius subsp. porrifolius)

A phytochemical investigation of three accessions of Tragopogon porrifolius L. subsp. porrifolius (Asteraceae, Lactuceae) yielded three new bibenzyl derivatives, 5,4'-dihydroxy-3-alpha-l-rhamnopyranosyl-(1-->3)-beta-d-xylopyranosyloxybibenzyl, 2-carboxyl-3,4'-dihydroxy-5-beta-d-xylopyranosyloxybibenzyl, tragopogonic acid (2'carboxyl-3',5',4'-trihydroxyphenylethanone) and three dihydroisocoumarin derivatives, including the new natural product 6-O-methylscorzocreticoside I. One of the isolated bibenzyl derivatives is considered to be a precursor to the biosynthesis of dihydroisocoumarins. Structures of new compounds were established by HR mass spectrometry, extensive 1D and 2D NMR spectroscopy, and CD spectroscopy. Moreover, radical scavenging activities of the polyphenolic compounds were measured using the 2,2-diphenyl-1-picrylhydrazyl assay; two of the bibenzyls showed moderate and two of the dihydroisocoumarins showed weak radical scavenging activities. The chemosystematic impact of bibenzyls and dihydroisocoumarins is discussed briefly.     

Rubralactone, rubralides A, B and C, and rubramin produced by Penicillium rubrum

Rubralactone (1), rubralides A, B and C (2-4), rubramin (5), and 2-formyl-3,5-dihydroxy-4-methylbenzoic acid (6), were isolated from Penicillium rubrum, and their structures established by spectroscopic methods including 2D NMR. The effects on plant growth of 1-6 were examined using the lettuce seedling bioassay. Compound 1 promoted root growth. Compounds 2, 3 and 5 inhibited the growth of lettuce seedlings, but 4 and 6 did not have any inhibitory effect on their growth.     

Abnormal metabolites produced by Daucus carota roots stored under conditions of stress

The formation of dihydroisocoumarins (I and II), the chromones (III and IV) and scopoletin (V) is induced in carrot roots by storage in the presence of low concentrations of ethylene and by inoculation with various fungi. There has been concern over possible toxic effects of dihydroisocoumarins and some preliminary toxicological data on (I), (II) and (IV) are included.     

Structure-requirements of isocoumarins, phthalides, and stilbenes from Hydrangeae Dulcis Folium for inhibitory activity on histamine release from rat peritoneal mast cells.

We examined the structure-activity relationships of isocoumarins, phthalides and stilbenes isolated from Hydrangeae Dulcis Folium and related compounds for the inhibition of histamine release in rat peritoneal mast cells. The activities of isocoumarins such as thunberginols A and B were more potent than those of dihydroisocoumarins such as hydrangenol and thunberginol G. The double bond at the 3-position seemed to be essential to potentiate the activity. The hydroxyl groups at the 8-, 3'- and 4'-positions of isocoumarin were essential for the activity, while the hydroxyl group at the 6-position was scarcely needed. Since the activities of benzylidenephthalides such as thunberginol F were more potent than those of hydramacrophyllols A and B, the presence of a double bond at the 3-position was needed to increase the activity. Moreover, the hydroxyl group at the 8-position was essential for the activity. On the time course study, thunberginols A, B and F completely inhibited histamine release by pretreatment at 100 microM for 1 to 15 min, whereas DSCG inhibited histamine release only following 1-min pretreatment at 1000 microM. These results suggested that the mechanisms of the inhibitory effect of thunberginols are different from that of DSCG.     

Dihydroisocoumarin from Xyris pterygoblephara active against dermatophyte fungi

The ethanol extract from Xyris pteygoblephara aerial parts was evaluated against five microorganism strains, by the microdilution and agar diffusion methods. Extract fractionation led to the isolation of three compounds, whose structures were assigned by spectrometric data (1D and 2D NMR, IR, MS and UV) as (3R,4R)-(-)-6-methoxy-3,4-dihydro-3-n-pentil-4-acethoxy-1H-2-benzopyran-1-one (1), moronic acid and quercetin. The absolute configuration of 1 was defined by circular dichroism spectroscopy and comparison with data reported for other dihydroisocoumarins. Assay of 1 (100 microg/disc) by the agar diffusion method against clinical isolates of the dermatophytes Epidermophyton floccosum (inhibition zone, mm+/-s.d.: 4.5+/-0.8), Trichophyton mentagrophytes (4.8+/-0.4) and Trichophyton rubrum (10.2+/-0.8) revealed similar inhibition zones to the positive control amphotericin B (32 microg/disc; 5.0+/-0.2; 5.0+/-0.6 and 8.8+/-1.2, respectively). The result corroborates the ethnomedical use of Xyris species to treat dermatitis.     

Effect of fibroblast growth factors 1, 2, 4, 5, 6, 7, 8, 9, and 10 on avian chondrocyte proliferation.

It has been demonstrated that fibroblast growth factor receptors are key regulators of endochondral bone growth. However, it has not been determined what fibroblast growth factor ligand(s) (FGFs) are important in this process. This study sought to determine whether FGFs 1, 2, 4, 5, 6, 7, 8, 9, and 10 were capable of stimulating avian chondrocyte proliferation in vitro. We have found that FGFs 2, 4, and 9 strongly stimulate avian chondrocyte proliferation while FGFs 6 and 8 stimulate proliferation to a lesser extent. RT-PCR indicates that FGF-2 and FGF-4 are expressed in the postnatal avian epiphyseal growth plate (EGP) while FGF-8 and FGF-9 are not. Thus, FGF-2 and FGF-4 stimulate chondrocyte proliferation and are both present in the EGP. This suggests that FGF-2 and FGF-4 may be important ligands, in vivo, for the regulation of endochondral bone growth. These observations coupled with our observation that multiple avian FGF receptors (Cek1, Cek2, Cek3, and FREK) are expressed in proliferative chondrocytes highlights the complexity of FGF signaling pathways in postnatal endochondral bone growth.     

Deletion of ribosomal S6 kinases does not attenuate pathological, physiological, or insulin-like growth factor 1 receptor-phosphoinositide 3-kinase-induced cardiac hypertrophy

Ribosomal S6 kinases (S6Ks) have been depicted as critical effectors downstream of growth factor pathways, which play an important role in the regulation of protein synthesis by phosphorylating the ribosomal protein, S6. The goal of this study was to determine whether S6Ks regulate heart size, are critical for the induction of cardiac hypertrophy in response to a pathological or physiological stimulus, and whether S6Ks are critical downstream effectors of the insulin-like growth factor 1 (IGF1)-phosphoinositide 3-kinase (PI3K) pathway. For this purpose, we generated and characterized cardiac-specific S6K1 and S6K2 transgenic mice and subjected S6K1(-/-), S6K2(-/-), and S6K1(-/-) S6K2(-/-) mice to a pathological stress (aortic banding) or a physiological stress (exercise training). To determine the genetic relationship between S6Ks and the IGF1-PI3K pathway, S6K transgenic and knockout mice were crossed with cardiac-specific transgenic mice overexpressing the IGF1 receptor (IGF1R) or PI3K mutants. Here we show that overexpression of S6K1 induced a modest degree of hypertrophy, whereas overexpression of S6K2 resulted in no obvious cardiac phenotype. Unexpectedly, deletion of S6K1 and S6K2 had no impact on the development of pathological, physiological, or IGF1R-PI3K-induced cardiac hypertrophy. These studies suggest that S6Ks alone are not essential for the development of cardiac hypertrophy.     

Rubralactone,Rubralides A,B and C,and Rubramin Produced by Penicillium rubrum

Rubralactone (1), rubralides A, B and C (2–4), rubramin (5), and 2-formyl-3,5-dihydroxy-4-methylbenzoic acid (6), were isolated from Penicillium rubrum, and their structures established by spectroscopic methods including 2D NMR. The effects on plant growth of 1–6 were examined using the lettuce seedling bioassay. Compound 1 promoted root growth. Compounds 2, 3 and 5 inhibited the growth of lettuce seedlings, but 4 and 6 did not have any inhibitory effect on their growth.     

MEK, ERK, and p90RSK are present on mitotic tubulin in Swiss 3T3 cells: a role for the MAP kinase pathway in regulating mitotic exit

The mitogen-activated protein (MAP) kinase pathway has been implicated in cell cycle control for some time. Several reports have suggested a role for this pathway in growth factor stimulation of DNA synthesis, while other reports have proposed a role in the transition of cells through mitosis. Here, we have examined the potential involvement of the extracellular signal-related kinase (ERK)1/2 MAP kinases, their upstream regulators, and downstream effectors in the regulation of mitosis. Inhibition of MAP kinase/ERK kinase (MEK) activity reduced the serum-stimulated DNA synthesis and proliferation of Swiss 3T3 cells. To study the potential mechanisms of this effect, we examined the subcellular localization of members of the MAP kinase pathway including regulators (MEK1/2), substrates (90-kDa ribosomal S6 kinases (RSKs): RSK1, RSK2 and RSK3), and ERK itself. We show that there is enrichment of ERK, MEK, and the RSK enzymes on both the spindle and midbody tubulin of dividing cells. Inhibition of MEK1/2 activity in cells released from mitotic arrest results in an inability of cells to complete mitosis. This failure to exit mitosis correlated with altered cyclin-dependent kinase (cdk) activities. Thus, the MAP kinase pathway may act to coordinate passage through mitosis in Swiss 3T3 fibroblasts by regulation of cdk activity.     

yucca6, a dominant mutation in Arabidopsis, affects auxin accumulation and auxin-related phenotypes

Auxin plays critical roles in many aspects of plant growth and development. Although a number of auxin biosynthetic pathways have been identified, their overlapping nature has prevented a clear elucidation of auxin biosynthesis. Recently, Arabidopsis (Arabidopsis thaliana) mutants with supernormal auxin phenotypes have been reported. These mutants exhibit hyperactivation of genes belonging to the YUCCA family, encoding putative flavin monooxygenase enzymes that result in increased endogenous auxin levels. Here, we report the discovery of fertile dominant Arabidopsis hypertall1-1D and hypertall1-2D (yucca6-1D, -2D) mutants that exhibit typical auxin overproduction phenotypic alterations, such as epinastic cotyledons, increased apical dominance, and curled leaves. However, unlike other auxin overproduction mutants, yucca6 plants do not display short or hairy root phenotypes and lack morphological changes under dark conditions. In addition, yucca6-1D and yucca6-2D have extremely tall (>1 m) inflorescences with extreme apical dominance and twisted cauline leaves. Microarray analyses revealed that expression of several indole-3-acetic acid-inducible genes, including Aux/IAA, SMALL AUXIN-UP RNA, and GH3, is severalfold higher in yucca6 mutants than in the wild type. Tryptophan (Trp) analog feeding experiments and catalytic activity assays with recombinant YUCCA6 indicate that YUCCA6 is involved in a Trp-dependent auxin biosynthesis pathway. YUCCA6:GREEN FLUORESCENT PROTEIN fusion protein indicates YUCCA6 protein exhibits a nonplastidial subcellular localization in an unidentified intracellular compartment. Taken together, our results identify YUCCA6 as a functional member of the YUCCA family with unique roles in growth and development.     

Stereoselectivity and stereospecificity of the alpha,beta-dihydroxyacid dehydratase from Salmonella typhimurium.

1. In addition to the known 2R,3R- and 2R, 3S-2,3-dihydroxy-3-methylpentanoic acids (DHI), the 1S,3S- and sS,DR-isomers were prepared. 2S-2,3-Dihydroxy-3-methylbutanoic acid (DHV) was also prepared in addition to the known 2R-isomer. 2. The six dihydroxy acids were examined for their ability to promote the growth of isoleucine-valine (ilv)-requiring strains of Salmonella typhimurium and to serve as substrates for the alpha,beta-dihydroxyacid dehydratase of the same organism. 3. Only 2R,3R-2,3-dihydroxy-3-methylpentanoic and 2R-2,3-dihydroxy-3-methylbutanoic acids supported growth of the ilv strains of S. typhimurium. 4. alpha,beta-Dihydroxyacid dehydratase utilized the three isomers with the 2R-configuration as substrates but not those with the 2S-configuration. 5. In an additional growth study that utilized the 3R- and 3S-isomers of 3-methyl-2-oxopentanoic acid, the alpha-keto acid analogue of isoleucine, only the 3S-isomer supported growth. 6. It is concluded that the mechanism of action of the dehydratase is stereospecific in that the proton that is attached to C-3 of the substrate occupies the same steriochemical position as the departing hydroxyl group (Fig. 6).     

IL-6 is an accessory signal in the alternative CD2-mediated pathway of T cell activation

Recent studies have demonstrated that IL-1 and IL-6 are synergistic accessory signals for activation of T cells. In this study, highly purified human T cells were cultured with either a stimulating pair of anti-CD2 mAb or with immobilized anti-CD3 mAb. Monocytes, a cellfree monocyte culture supernatant or IL-1 were required for anti-CD2-stimulated T cell proliferation, and they each strongly enhanced anti-CD3-induced T cell growth. IL-6 was synergistic with IL-1 as a helper factor for T cell growth after activation via CD2, but we could not demonstrate any effect of IL-6 in the CD3 pathway. The mechanism of the synergistic helper activity of IL-1 and IL-6 on T cell activation in the CD2 pathway was further examined. IL-1 (but not IL-6) was required for induction of IL-2 production. Both IL-1 and IL-6 enhanced IL-2R (p55) expression and the proliferative response to IL-2. T cell proliferation after stimulation with anti-CD2 and IL-1 or IL-1/IL-6 proceeded through an autocrine IL-2-dependent pathway. Moreover we found that, in the absence of IL-1, IL-6 still supported a transient and limited proliferation of anti-CD2- (but not of anti-CD3-) stimulated T cells, which apparently was independent of the autocrine growth factors IL-2 or IL-4. Our data suggest that IL-6 is important as an accessory signal for T cell growth in the CD2 pathway of T cell activation.     

Synthesis and cytotoxic activity evaluation of indolo-, pyrrolo-, and benzofuro-quinolin-2(1H)-ones and 6-anilinoindoloquinoline derivatives

Certain indolo-, pyrrolo-, and benzofuro-quinolin-2(1H)-ones 4a,b, 6, 8, 16a-c and 6-anilinoindoloquinoline derivatives 10a,b, 11a,b, 12a,b have been synthesized and evaluated in vitro against a 3-cell lines panel consisting of MCF7 (Breast), NCI-H460 (Lung), and SF-268 (CNS). Those active compounds 4a,b, 6, 8, 10a,b, 11a,b, 12a,b were then evaluated in the full panel of 60 human tumor cell lines derived from nine cancer cell types. The results have shown that cytotoxicity decreases in the order of 6-anilinoindoloquinolines>indoloquinolin-2(1H)-ones>pyrroloquinolin-2(1H)-ones>benzofuroquinolin-2(1H)-ones. Among them, 1-[3-(11H-indolo[3,2-c]quinolin-6ylamino)phenyl]ethanone oxime hydrochloride (11a) and its 2-chloro derivative (11b) were most active, with mean GI(50) values of 1.70 and 1.35 microM, respectively. Both compounds 11a,b were also found to inhibit the growth of SNB-75 (CNS cancer cell) with a GI(50) value of less than 0.01 microM, and, therefore, were selected for further evaluation for in vivo antitumor activity.     

Effects of abscisic Acid on growth, RNA metabolism, and respiration in germinating bean axes

The effect of abscisic acid on growth, respiration, the ATP pool, and rate and amount of RNA synthesis in aseptically cultured axes of Phaseolus vulgaris during the first 24 hours of germination has been measured in experiments where the duration of abscisic acid application and its concentration have been varied. At concentrations from 10(-7) to 10(-4)m, abscisic acid inhibits synthesis of RNA with maximal inhibition (80%) at 10(-5)m. RNA synthesis is inhibited by abscisic acid at all times examined (12, 18, and 24 hours), but the extent of inhibition is maximal at 18 hours. In 18-hour axes RNA synthesis is inhibited 42%, ATP pool size is reduced 3%, and O(2) consumption is decreased by 6% after 75 minutes of abscisic acid treatment. Inhibition of RNA synthesis is complete by 2 hours of treatment with abscisic acid, and recovery to near control levels occurs by the 3rd hour after removal from abscisic acid.     

Pfaffosides,nortriterpenoid saponins,from Pfaffia paniculata

Three new nortriterpene saponins having inhibitory effects on the growth of cultured tumor cells, named pfaffosides D, E and F, have been isolated from Pfaffia paniculata. Their structures have been established as 3β-O-[β-d-xylopyranosyl-(1 → 2)-β-d-(6-O-n-butyl) glucuronopyranosyl]-pfaffic acid-(28 → 1)-β-d-glucopyranosyl ester, 3β-O-[β-d-xylopyranosyl-(1 → 2)-β-d(6-O-methyl)glucuronopyranosyl]-pfaffic acid-(28 → 1)-β-d glucopyranosyl ester and 3β-O[β-d-glucuronopyranosyl]-pfaffic acid respectively, based on their chemical and spectroscopic properties     

Genetic analysis of Raf1, Mdm2, c-Myc, Cdc25a and Cdc25b proto-oncogenes in 2',3'-dideoxycytidine- and 1,3-butadiene-induced lymphomas in B6C3F1 mice

We have previously identified activation of ras proto-oncogenes and inactivation of tumor suppressor genes including p53, p16(INK4a) and p15(INK4b) in 2',3'-dideoxycytidine (ddC)- and/or 1,3-butadiene (BD)-induced lymphomas derived from B6C3F1 (C57BL/6xC3H/He) mice, indicating that alterations of ras signaling pathway, p53 and pRb growth control pathways are important in the development of these chemically induced lymphomas. However, there is still a subset of tumors that displayed no changes in these genes. Thus, we investigated whether the Raf1, Mdm2, c-Myc, Cdc25a and Cdc25b proto-oncogenes, which are implicated in the ras or p53 or pRb pathways, are alternative oncogenic target genes. Analyses of gross genomic alterations by Southern blots failed to reveal rearrangement or amplification in any of the tumors examined. Frequent point mutations on the substrate binding domain of the Raf1 gene has been reported in 1-ethyl-1-nitrosourea (ENU)-induced murine lymphomas and lung tumors, along with a conspicuous lack of ras mutations [U. Naumann, I. Eisenmann-Tappe, U.R. Rapp, The role of raf kinases in development and growth of tumors, Recent Results Cancer Res., 143 (1997) 237-244]. To investigate whether Raf1 mutation is involved in our set of tumor especially those without ras mutations, the PCR-based single-strand conformation analyses (SSCA) and direct DNA sequencing were employed. No mutations but four genetic polymorphisms between C57BL/6 and C3H/He were found, with two of them reported as point mutations previously (op. cit.). The polymorphisms were utilized for allelic loss study of Raf1 locus. Losses of heterozygosity were found in six of 31 BD-induced lymphomas. These results indicate that genetic alterations of c-Myc, Cdc25, Raf1 and Mdm2 proto-oncogenes may not be involved in the development of ddC- and BD-induced lymphomas and the inactivation of tumor suppressor gene(s) located close to Raf1 gene might be important in the development of a subset of BD-induced lymphomas.