Red clover necrotic mosaic virus replication proteins accumulate at the endoplasmic reticulum.
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Red clover necrotic mosaic virus (RCNMV) encodes N-terminally overlapping proteins of 27 and 88 kDa (p27 and p88) known to be required for replication. Green fluorescent protein (GFP) fusions were used to visualize the location of p27 and p88 within Nicotiana benthamiana cells. GFP:p27 fusions localized to the endoplasmic reticulum (ER), co-localized with ER-targeted yellow fluorescent protein and caused membrane restructuring and proliferation. Cellular fractionation of virus-inoculated N. benthamiana leaves confirmed the association of p27 with ER membranes. GFP:p88 fusions also localized to the ER and co-localized with GFP:p27. Both fusion proteins co-localize to the cortical and cytoplasmic ER and were associated with invaginations of the nuclear envelope. Independent accumulation in, and perturbation of, the ER suggests that p27 and p88 function together in the replication complex. This is the first report of a member of the Tombusviridae replicating in association with the ER. (+info)
Isoflavones and women's health.
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There is evidence that diets which contain high levels of phytoestrogenic isoflavanoids are associated with a low incidence of osteoporosis and menopausal vasomotor symptoms. Plant extracts such as red clover, which contain high levels of isoflavanoids, have been used to reduce menopausal symptoms and have been shown to reduce bone loss in healthy women. A placebo-controlled clinical trial [ISRCTN42940165] of red clover is reported in this issue of Breast Cancer Research and shows that these phytoestrogens do not cause any oestrogenic increase in breast density, which would indicate that they are unlikely to cause an increased risk of breast cancer. (+info)
Red-clover-derived isoflavones and mammographic breast density: a double-blind, randomized, placebo-controlled trial [ISRCTN42940165].
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INTRODUCTION: Isoflavones are hypothesized to protect against breast cancer, but it is not clear whether they act as oestrogens or anti-oestrogens in breast tissue. Our aim was to determine the effects of taking a red clover-derived isoflavone supplement daily for 1 year on mammographic breast density. Effects on oestradiol, follicle-stimulating hormone (FSH), luteinizing hormone (LH), lymphocyte tyrosine kinase activity and menopausal symptoms were also assessed. METHODS: A total of 205 women (age range 49-65 years) with Wolfe P2 or DY mammographic breast patterns were randomly assigned to receive either a red clover-derived isoflavone tablet (26 mg biochanin A, 16 mg formononetin, 1 mg genistein and 0.5 mg daidzein) or placebo. Change in mammographic breast density, serum oestradiol, FSH, LH, menopausal symptoms and lymphocyte tyrosine kinase activity from baseline to 12 months were assessed. RESULTS: A total of 177 women completed the trial. Mammographic breast density decreased in both groups but the difference between the treatment and placebo was not statistically significant. There was a significant interaction between treatment group and oestrogen receptor (ESR1) PvuII polymorphism for the change in estimated percentage breast density (mean +/- standard deviation): TT isoflavone 1.4 +/- 12.3% and TT placebo -9.6 +/- 14.2%; CT isoflavone -5.2 +/- 12.0% and CT placebo -2.8 +/- 10.3%; and CC isoflavone -3.4 +/- 9.7% and CC placebo -1.1 +/- 9.5%. There were no statistically significant treatment effects on oestradiol, FSH, or LH (assessed only in postmenopausal women), or on lymphocyte tyrosine kinase activity. Baseline levels of menopausal symptoms were low, and there were no statistically significant treatment effects on frequency of hot flushes or other menopausal symptoms. CONCLUSION: In contrast to studies showing that conventional hormone replacement therapies increase mammographic breast density, the isoflavone supplement did not increase mammographic breast density in this population of women. Furthermore, there were no effects on oestradiol, gonadotrophins, lymphocyte tyrosine kinase activity, or menopausal symptoms. (+info)
Disposition of flavonoids via enteric recycling: enzyme-transporter coupling affects metabolism of biochanin A and formononetin and excretion of their phase II conjugates.
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The purpose of this study was to continue our effort to determine how enzyme-transporter coupling affect disposition of flavonoids. The rat intestinal perfusion and Caco-2 cell models were used together with relevant microsomes. In perfusion model, isoflavone (i.e., formononetin and biochanin A) absorption and subsequent excretion of its metabolites were always site-dependent. Maximal amounts of intestinal and biliary conjugates excreted per 30 min were 31 and 51 nmol for formononetin, more than that for pure biochanin A (12 and 20 nmol). When a standardized red clover extract (biochanin A/formononetin = 10:7) was used, the results indicated that more metabolites of biochanin A than formononetin were found in the perfusate (36.9 versus 22.8 nmol) and bile (78 versus 51 nmol). In metabolism studies, rat intestinal and liver microsomes always glucuronidated biochanin A faster (p < 0.05) than formononetin, whereas intestinal microsomes glucuronidated both isoflavones faster (p < 0.05) than liver microsomes. However, rapid metabolism in the microsomes did not translate into more efficient excretion in either the rat perfusion model as shown previously or in the Caco-2 model. In the Caco-2 model, both isoflavones were rapidly absorbed, efficiently conjugated, and the conjugates excreted apically and basolaterally. More formononetin conjugates were excreted than biochanin A when used alone, but much more biochanin A conjugates were found when using the isoflavone mixture. In conclusion, efficiency of enzyme-transporter coupling controls the amounts of metabolites excreted by the intestine and liver and determines the relative contribution of enteric and enterohepatic recycling to the in vivo disposition of isoflavones. (+info)
Molecular cloning, functional expression in Escherichia coli and enzymatic characterisation of a cysteine protease from white clover (Trifolium repens).
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This paper presents the cloning and biochemical characterisation of the cysteine protease Tr-cp 14 from white clover (Trifolium repens). The predicted amino acid sequence of Tr-cp 14 is 71%, 74% and 74% identical to the cysteine proteases XCP1 and XCP2 from Arabidopsis thaliana, and p48h-17 from Zinnia elegans, respectively. These cysteine proteases have previously been shown to be involved in programmed cell death during tracheary element differentiation. The precursor polypeptide of Tr-cp 14 was expressed in Escherichia coli, purified from inclusion bodies and refolded. The precursor polypeptide could be processed to its active mature form autocatalytically at pH 5.0 and had a requirement for 20 mM l-cysteine for optimal activity. Mature Tr-cp 14 showed a preference for synthetic aminomethylcoumarin substrates with either Leu or Phe in the P2 position when tested with Arg in P1. A substrate with Arg in both the P1 and P2 position was not accepted as substrate. (+info)
In the prostatic epithelium, dietary isoflavones from red clover significantly increase estrogen receptor beta and E-cadherin expression but decrease transforming growth factor beta1.
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In mice fed a diet supplemented with red clover isoflavones the prostatic epithelium displays a significant increase in the production of estrogen receptor beta and the adhesion protein E-cadherin but a decrease in transforming growth factor beta1. These proteins are estrogenically-induced markers of proliferation, maintenance of histological architecture, preservation of cell phenotype and reduction of the potential for neoplastic and metastatic transformation. This study suggests that red clover isoflavones represent a non-toxic dietary treatment for prostatic hyperplasia and a reduction in the potential for neoplastic transformation. (+info)
Modest protective effects of isoflavones from a red clover-derived dietary supplement on cardiovascular disease risk factors in perimenopausal women, and evidence of an interaction with ApoE genotype in 49-65 year-old women.
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Data suggest that soy protein, a source of isoflavones, may have favorable effects on cardiovascular risk factors. Women (n = 205), ages 49-65 y, were randomized into this double blind, placebo-controlled trial of 43.5 mg red clover-derived isoflavones/d. A total of 177 women completed the trial. There were no differences between treatments for changes from baseline to 12 mo in total cholesterol, LDL cholesterol, triglycerides, HDL cholesterol, systolic and diastolic blood pressures, fibrinogen, and plasminogen activator inhibitor type 1 (PAI-1) (P >/= 0.1). Interactions between treatment and menopausal status were significant for changes in triglycerides and PAI-1 (P = 0.02 and P = 0.01), and changes were significant among perimenopausal women. In the isoflavone and placebo groups, changes in triglycerides were -0.2 +/- 0.6 and 0.4 +/- 0.6 mmol/L, P = 0.02, and changes in PAI-1 were -3.06 +/- 5.88 and 4.95 +/- 6.25 IU/L, P = 0.004, respectively. Interactions between apolipoprotein E (apoE) genotype and treatment tended to be significant for changes in total and LDL cholesterol (P = 0.06 and P = 0.05), and differences between treatments were significant in E2/E3 women. In the isoflavone and placebo groups, changes in total cholesterol were -0.61 +/- 0.79 and 0.18 +/- 0.79 mmol/L, P = 0.03, and changes in LDL cholesterol were -0.84 +/- 0.79 and -0.04 +/- 0.69 mmol/L, P = 0.02, respectively. Although there were potentially beneficial changes in triglycerides and PAI-1 among perimenopausal women consuming isoflavones, this study suggests that isoflavones alone are not responsible for the well-documented effects of soy protein on blood lipids. A larger study is required to confirm the effect modification by apoE genotype. (+info)
Clover proliferation phytoplasma (CPR) is designated as the reference strain for the CP phylogenetic group or subclade, on the basis of molecular analyses of genomic DNA, the 16S rRNA gene and the 16S-23S spacer region. Other strains related to CPR include alfalfa witches'-broom (AWB), brinjal little leaf (BLL), beet leafhopper-transmitted virescence (BLTV), Illinois elm yellows (ILEY), potato witches'-broom (PWB), potato yellows (PY), tomato big bud in California (TBBc) and phytoplasmas from Fragaria multicipita (FM). Phylogenetic analysis of the 16S rRNA gene sequences of BLL, CPR, FM and ILEY, together with sequences from 16 other phytoplasmas that belong to the ash yellows (AshY), jujube witches'-broom (JWB) and elm yellows (EY) groups that were available in GenBank, produced a tree on which these phytoplasmas clearly clustered as a discrete group. Three subgroups have been classified on the basis of sequence homology and the collective RFLP patterns of amplified 16S rRNA genes. AWB, BLTV, PWB and TBBc are assigned to taxonomic subgroup CP-A, FM belongs to subgroup CP-B and BLL and ILEY are assigned to subgroup CP-C. Genetic heterogeneity between different isolates of AWB, CPR and PWB has been observed from heteroduplex mobility assay analysis of amplified 16S rRNA genes and the 16S-23S spacer region. Two unique signature sequences that can be utilized to distinguish the CP group from others were present. On the basis of unique properties of the DNA from clover proliferation phytoplasma, the name 'Candidatus Phytoplasma trifolii' is proposed for the CP group. (+info)