Δ9-tetrahydrocannabivarin suppresses in vitro epileptiform and in vivo seizure activity in adult rat

Purpose: We assessed the anticonvulsant potential of the phytocannabinoid Δ9-tetrahydrocannabivarin (Δ9-THCV) by investigating its effects in an in vitro piriform cortex (PC) brain slice model of epileptiform activity, on cannabinoid CB1 receptor radioligand-binding assays and in a generalized seizure model in rats. Methods: Δ9-THCV was applied before (10 μmΔ9-THCV) or during (10–50 μmΔ9-THCV) epileptiform activity induced by Mg2+-free extracellular media in adult rat PC slices and measured using multielectrode array (MEA) extracellular electrophysiologic techniques. The actions of Δ9-THCV on CB1 receptors were examined using [3H]SR141716A competition binding and [35S]GTPS assays in rat cortical membranes. Effects of Δ9-THCV (0.025–2.5 mg/kg) on pentylenetetrazole (PTZ)–induced seizures in adult rats were also assessed. Results: After induction of stable spontaneous epileptiform activity, acute Δ9-THCV application (≥20 μm) significantly reduced burst complex incidence and the amplitude and frequency of paroxysmal depolarizing shifts (PDSs). Furthermore, slices pretreated with 10 μmΔ9-THCV prior to induction of epileptiform activity exhibited significantly reduced burst complex incidence and PDS peak amplitude. In radioligand-binding experiments, Δ9-THCV acted as a CB1 receptor ligand, displacing 0.5 nm [3H]SR141716A with a Ki∼290 nm, but exerted no agonist stimulation of [35S]GTPS binding. In PTZ-induced seizures in vivo, 0.25 mg/kg Δ9-THCV significantly reduced seizure incidence. Discussion: These data demonstrate that Δ9-THCV exerts antiepileptiform and anticonvulsant properties, actions that are consistent with a CB1 receptor–mediated mechanism and suggest possible therapeutic application in the treatment of pathophysiologic hyperexcitability states.

Antifungal activity of Bacillus subtilis filtrate to control Fusarium oxysporum f.sp. lentis, the causal organism of lentil vascular wilt

In dual cultures, the supernatant filtrate of the biological control agent Bacillus subtilis was evaluated against (Fusarium oxysporum f.sp. lentis) the causal organism of lentil vascular wilt. The antagonistic activity was evaluated as percent reduction of fungal growth (certainly due, in part, to the antifungal metabolites produced by the antagonistic bacterium). The in-vitro experiments showed that B. subtilis filtrate, whether solid or liquid media, had a strong inhibiting activity on the spore germination and mycelial growth of F. oxysporum f. sp. lentis. In a glasshouse experiment, soil was drenched with B. subtilis filtrate at 30 ml/kg (vol/wt) around seedlings of a susceptible lentil line (ILL 4605). In this treatment there was only 31% mortality compared with 100% kill of plants in the control treatment (P≤0.05).

Nitrogen fertilizer and seed rate effects on Hagberg failing number of hybrid wheats and their parents are associated with alpha-amylase activity, grain cavity size and dormancy

Field experiments were carried out to assess the effects of nitrogen fertilization and seed rate on the Hagberg falling number (HFN) of commercial wheat hybrids and their parents. Applying nitrogen (200 kg N ha(-1)) increased HFN in two successive years. The HFN of the hybrid Hyno Esta was lower than either of its parents (Estica and Audace), particularly when nitrogen was not applied. Treatment effects on HFN were negatively associated with a-amylase activity. Phadebas grain blotting suggested two populations of grains with different types of a-amylase activity: Estica appeared to have a high proportion of grains with low levels of late maturity endosperm a-amylase activity (LMEA); Audace had a few grains showing high levels of germination amylase; and the hybrid, Hyno Esta, combined the sources from both parents to show heterosis for a-amylase activity. Applying nitrogen reduced both apparent LMEA and germination amylase. The effects on LMEA were associated with the size and disruption of the grain cavity, which was greater in Hyno Esta and Estica and in zero-nitrogen treatments. External grain morphology failed to explain much of the variation in LMEA and cavity size, but there was a close negative correlation between cavity size and protein content. Applying nitrogen increased post-harvest dormancy of the grain. Dormancy was greatest in Estica and least in Audace. It is proposed that effects of seed rate, genotype and nitrogen fertilizer on HFN are mediated through factors affecting the size and disruption of the grain cavity and therefore LMEA, and through factors affecting dormancy and therefore germination amylase. (c) 2004 Society of Chemical Industry.

In vitro biological activity of tannins from Acacia and other tree fruits: Correlations with colorimetric and gravimetric phenolic assays

This Study was designed to investigate impact of tannins on in vitro ruminal fermentation parameters as well as relationships between concentration and in vitro biological activity of tannins present in tree fruits. Dry and mature fruits of known phenolic content harvested from Acacia nilotica, A. erubescens, A. erioloba, A. sieberiana, Piliostigima thonningii and Dichrostachys cinerea tree species were fermented with rumen fluid in vitro with or without polyethylene glycol (PEG). Correlation between in vitro biological activity and phenolic concentration was determined. Polyethylene glycol inclusion increased Cumulative gas production from all fruit substrates. The largest Increase (225%) after 48 h incubation was observed in D. cinerea fruits while the least (12.7%) increase was observed in A. erubescens fruits. Organic matter degradability (48 h) was increased by PEG inclusion for all tree species except A. erubescens and P. thonningii. For D. cinerea fruits, colorimetric assays were poorly correlated to Increases In gas production due to PEG treatment. Ytterbium precipitable phenolics (YbPh) were also poorly correlated with response to PEG for A. erioloba and P. thonningii fruits. However, YbPh were strongly and positively correlated to the increase In Cumulative gas production due to PEG for A. erubescens and A. nilotica. Folin-Ciocalteau assayed phenolics (SPh) were not correlated to response to PEG in P. thonningii and A. sieberiana. It was Concluded that the PEG effect oil in vitro fermentation was closely related to some measures of phenolic concentration but the relationships varied with tree species.

Antifungal activity of bacterium symbiotically associated with Steinernema abbasi towards Fusarium oxysporum

Fusarium oxysporum f.sp. lycopersici (Fol) is the causal agent of the Fusarium wilt disease of tomato. Soil fumigant (mainly methyl bromide) applications are in use for its control. With the increasing environmental awareness, biological control methods are under investigation for their effectiveness, including the use of antagonists. Pseudomonas oryzihabitans (=Flavimonas oryzihabitans), a symbiont of the entomopathogenic nematode Steinernema abbasi was investigated as an antagonism of a Fol isolate in two laboratory and two glasshouse experiments. Bacteria and cell-free filtrate antifungal activity were tested both in dual cultures and in broth culture. In pot experiments, suspensions of bacteria in five concentrations (106, 105, 104, 103 and 102 cells/ml) were tested for their ability to control the pathogen at 25±3°C. In all tests the bacterium significantly inhibited the growth of Fol mycelium in vitro. Similar results were obtained when the bacterium was also tested against Fusarium oxysporum f.sp. radicis lycopersici and against Rhizoctonia solani. Moreover, when it was introduced into the soil, it was able to suppress the Fusarium wilt of tomato.

Muscle hypertrophy driven by myostatin blockade does not require stem/precursor-cell activity

Myostatin, a member of the TGF-beta family, has been identified as a powerful inhibitor of muscle growth. Absence or blockade of myostatin induces massive skeletal muscle hypertrophy that is widely attributed to proliferation of the population of muscle fiber-associated satellite cells that have been identified as the principle source of new muscle tissue during growth and regeneration. Postnatal blockade of myostatin has been proposed as a basis for therapeutic strategies to combat muscle loss in genetic and acquired myopathies. But this approach, according to the accepted mechanism, would raise the threat of premature exhaustion of the pool of satellite cells and eventual failure of muscle regeneration. Here, we show that hypertrophy in the absence of myostatin involves little or no input from satellite cells. Hypertrophic fibers contain no more myonuclei or satellite cells and myostatin had no significant effect on satellite cell proliferation in vitro, while expression of myostatin receptors dropped to the limits of detectability in postnatal satellite cells. Moreover, hypertrophy of dystrophic muscle arising from myostatin blockade was achieved without any apparent enhancement of contribution of myonuclei from satellite cells. These findings contradict the accepted model of myostatin-based control of size of postnatal muscle and reorient fundamental investigations away from the mechanisms that control satellite cell proliferation and toward those that increase myonuclear domain, by modulating synthesis and turnover of structural muscle fiber proteins. It predicts too that any benefits of myostatin blockade in chronic myopathies are unlikely to impose any extra stress on the satellite cells.

Sterol content analysis suggests altered eburicol 14 alpha-demethylase (CYP51) activity in isolates of Mycosphaerella graminicola adapted to azole fungicides

The recent decline in the effectiveness of some azole fungicides in controlling the wheat pathogen Mycosphaerella graminicola has been associated with mutations in the CYP51 gene encoding the azole target, the eburicol 14 alpha-demethylase (CYP51), an essential enzyme of the ergosterol biosynthesis pathway. In this study, analysis of the sterol content of M. graminicola isolates carrying different variants of the CYP51 gene has revealed quantitative differences in sterol intermediates, particularly the CYP51 substrate eburicol. Together with CYP51 gene expression studies, these data suggest that mutations in the CYP51 gene impact on the activity of the CYP51 protein.

Oestrogenic activity of benzyl salicylate, benzyl benzoate and butylphenylmethylpropional (Lilial) in MCF7 human breast cancer cells in vitro

Benzyl salicylate, benzyl benzoate and butylphenylmethylpropional (Lilial) are added to bodycare cosmetics used around the human breast. We report here that all three compounds possess oestrogenic activity in assays using the oestrogen-responsive MCF7 human breast cancer cell line. At 3 000 000-fold molar excess, they were able to partially displace [H-3]oestradiol from recombinant human oestrogen receptors ER alpha and ER beta, and from cytosolic ER of MCF7 cells. At concentrations in the range of 5 x 10(-5) to 5 x 10(-4) M, they were able to increase the expression of a stably integrated oestrogen-responsive reporter gene (ERE-CAT) and of the endogenous oestrogen-responsive pS2 gene in MCF7 cells, albeit to a lesser extent than with 10(-8) M 17 beta-oestradiol. They increased the proliferation of oestrogen-dependent MCF7 cells over 7 days, which could be inhibited by the antioestrogen fulvestrant, suggesting an ER-mediated mechanism. Although the extent of stimulation of proliferation over 7 days was lower with these compounds than with 10(-8) M 17 beta-oestradiol, given a longer time period of 35 days the extent of proliferation with 10(-4) M benzyl salicylate, benzyl benzoate or butylphenylmethylpropional increased to the same magnitude as observed with 10(-8) M 17 beta-oestradiol over 14 days. This demonstrates that benzyl salicylate, benzyl benzoate and butylphenylmethylpropional are further chemical components of cosmetic products which give oestrogenic responses in a human breast cancer cell line in culture. Further research is now needed to investigate whether oestrogenic responses are detectable using in vivo models and the extent to which these compounds might be absorbed through human skin and might enter human breast tissues. Copyright (C) 2009 John Wiley & Sons, Ltd.

PECAM-1 expression and activity negatively regulate multiple platelet signaling pathways

Platelet endothelial cell adhesion molecule-1 (PECAM-1) inhibits platelet response to collagen and may also inhibit two other major platelet agonists ADP and thrombin although this has been less well explored. We hypothesized that the combined effect of inhibiting these three platelet activating pathways may act to significantly inhibit thrombus formation. We demonstrate a negative relationship between PECAM-1 surface expression and platelet response to cross-linked collagen related peptide (CRP-XL) and ADP, and an inhibitory effect of PECAM-1 clustering on platelet response to CRP-XL, ADP and thrombin. This combined inhibition of multiple signaling pathways results in a marked reduction in thrombus formation. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Oestrogenic and androgenic activity of triclosan in breast cancer cells

As a consequence of its widespread use as an antimicrobial agent in consumer goods, triclosan has become distributed ubiquitously across the ecosystem, and recent reports that it can cause endocrine disruption in aquatic species has increased concern. It is reported here that triclosan possesses intrinsic oestrogenic and androgenic activity in a range of assays in vitro which could provide some explanation for the endocrine disrupting properties described in aquatic populations. In terms of oestrogenic activity, triclosan displaced [H-3]oestradiol from oestrogen receptors (ER) of MCF7 human breast cancer cells and from recombinant human ER alpha/ER beta. Triclosan at 10(-5) M completely inhibited the induction of the oestrogen-responsive ERE-CAT reporter gene in MCF7 cells by 10(-10) M 17 beta-oestradiol and the stimulation of growth of MCF7 human breast cancer cells by 10(-10) M 17 beta-oestradiol. On its own, 1 mu M triclosan increased the growth of MCF7 cells over 21 days. Triclosan also had androgenic activity. It displaced [H-3]testosterone from binding to the ligand binding domain of the rat androgen receptor (AR). Triclosan was able to inhibit the induction of the androgen-responsive LTR-CAT reporter gene in S115 mouse mammary tumour cells by 10(-9) M testosterone and in T47D human breast cancer cells by 10(-8) M testosterone at concentrations of 10(-7) M and 10(-6) M, respectively. Triclosan at 2 x 10(-5) M antagonized the stimulation of the growth of S115+A mouse mammary tumour cells by 10(-9) M testosterone. The finding that triclosan has oestrogenic and androgenic activity warrants further investigation in relation to both endocrine disruption of aquatic wildlife and any possible impact on human health. Copyright (C) 2007 John Wiley & Sons, Ltd.

Antioxidant activity and protective effects of green and dark coffee components against human low density lipoprotein oxidation

The in vitro antioxidant activity and the protective effect against human low density lipoprotein oxidation of coffees prepared using different degrees of roasting was evaluated. Coffees with the highest amount of brown pigments (dark coffee) showed the highest peroxyl radical scavenging activity. These coffees also protected human low-density lipoprotein (LDL) against oxidation, although green coffee extracts showed more protection. In a different experiment, coffee extracts were incubated with human plasma prior to isolation of LDL particles. This showed, for the first time, that incubation of plasma with dark, but not green coffee extracts protected the LDL against oxidation by copper or by the thermolabile azo compound AAPH. Antioxidants in the dark coffee extracts must therefore have become associated with the LDL particles. Brown compounds, especially those derived from the Maillard reaction, are the compounds most likely to be responsible for this activity.

Effect of sulphation on the oestrogen agonist activity of the phytoestrogens genistein and daidzein in MCF-7 human breast cancer cells

The phytoestrogens genistein, daidzein and the daidzein metabolite equol have been shown previously to possess oestrogen agonist activity. However, following consumption of soya diets, they are found in the body not only as aglycones but also as metabolites conjugated at their 4'- and 7-hydroxyl groups with sulphate. This paper describes the effects of monosulphation on the oestrogen agonist properties of these three phytoestrogens in MCF-7 human breast cancer cells in terms of their relative ability to compete with [H-3]oestradiol for binding to oestrogen receptor (ER), to induce a stably transfected oestrogen-responsive reporter gene (ERE-CAT) and to stimulate cell growth. In no case did sulphation abolish activity. The 4'-sulphadon of genistein reduced oestrogen agonist activity to a small extent in whole-cell assays but increased the relative binding affinity to ER. The 7-sulphation of genistein, and also of equol, reduced oestrogen agonist activity substantially in all assays. By contrast, the position of monosulphation of daidzein acted in an opposing manner on oestrogen agonist activity. Sulphation at the 4'-position of daidzein resulted in a modest reduction in oestrogen agonist activity but sulphation of daidzein at the 7-position resulted in an increase in oestrogen agonist activity. Molecular modelling and docking studies suggested that the inverse effects of sulphation could be explained by the binding of daidzein into the ligand-binding domain of the ER in the opposite orientation compared with genistein and equol. This is the first report of sulphation enhancing activity of an isoflavone and inverse effects of sulphation between individual phytoestrogens.