As a potential disease-modifying treatment for AD, Alzhemed (tramiprosate) is a compound that binds to soluble amyloid-beta peptide (Abeta) and inhibits the formation of neurotoxic aggregates that lead to amyloid plaque deposition in the brain. The safety, tolerability, and pharmacodynamic effects of Alzhemed were assessed in a double-blind study in which 58 individuals with mild-to-moderate AD (MMSE 13-25) were randomized to receive placebo or Alzhemed 50, 100 or 150 mg BID for 3 months. At the end of the double-blind phase, 42 of these subjects entered a 36-month open-label (OL) phase in which they received Alzhemed 150 mg BID. Assessments included plasma and cerebrospinal fluid (CSF) Alzhemed concentrations, CSF levels of Abeta, as well as cognitive (Alzheimer's Disease Assessment Scale-cognitive subscale, Mini-Mental State Examination) and clinical performance (Clinical Dementia Rating scale, Sum-of-Boxes) measures. Alzhemed was safe and well tolerated, crossed the blood-brain barrier, and dose-dependently reduced CSF Abeta(42) levels after 3 months of treatment. Mild AD subjects (MMSE 19-25 at entry) displayed greater reduction of CSF Abeta(42) levels than moderate AD participants (MMSE 13-18 at entry). There was no effect of Alzhemed on the cognitive or clinical measures after 3 months of treatment. The OL follow-up suggested a stabilization of cognitive function especially in mild AD subjects over the 36-month study period. Alzhemed thus appears to be well tolerated with long-term exposure and reduces CSF Abeta(42) levels in mild-to-moderate AD subjects. These findings will be discussed in the context of two large-scale randomized, double-blind, placebo-controlled Phase III clinical trials that are currently being conducted to test the long-term safety and efficacy of Alzhemed.

We report a detailed analysis of the sterol composition of Trypanosoma cruzi epimastigotes grown in the absence or presence of two sterol analogs previously reported as inhibitors of delta 24(25) sterol methyltransferase (24(25)-SMT,E.C.2.1.1.43) in yeast and fungi, a cholestanol analog with a 6-membered aza ring as side chain (22,26-azasterol) and 24-(R,S),25-epiminolanosterol, as well as combinations of these compounds with the C14 demethylase inhibitor ketoconazole. Both sterol analogs produced a dose-dependent reduction in the incorporation of radioactivity from [methyl-14C]methionine with IC50 values of 640 nM and 70 nM for 22,26-azasterol and 24,25-(R,S)-epiminolanosterol, respectively, indicating a specific inhibition of 24(25)-SMT. Correspondingly, it was found that the sterols present in control cells (ergosterol, 24-ethylcholesta-5,7,22-trien-3 beta-ol and precursors) were almost completely replaced by zymosterol (cholesta-8,24-dien-3 beta-ol) or a mixture of zymosterol, cholesta-7,24-dien-3 beta-ol and cholesta-5,7,24-trien-3 beta-ol when the parasites were exposed to the minimal growth inhibitory concentrations of 22,26-azasterol and 24-(R,S),25-epiminolanosterol, respectively. At sub-optimal concentrations of the inhibitors a complete disappearance of the 24-ethyl sterols was observed and a concomitant increase in the proportion of 24-methyl sterols, particularly delta 24(24') sterols. This showed that in T. cruzi the second methenylation step (catalyzed by delta 24(24') sterol methyl transferase) was significantly more sensitive to these inhibitors than the first and that the sterol analogs were also powerful inhibitors of the delta 24(24') sterol reductase. In growth-arrested epimastigotes resulting from their treatment with low (1-3 microM) concentrations of either sterol analog combined with sub optimal (100-300 nM) levels of ketoconazole the main sterol was lanosterol with no evidence 24-methylenedihydrolanosterol, the main sterol found in cells treated with growth inhibitory concentrations of the azole alone. Taken together, these results indicated that 24-alkyl sterols are essential growth factors for T. cruzi and that the preferred substrate of the delta 24(25) sterol methyl transferase in this organism is zymosterol.

The volatiles emitted from cell cultures of myxobacterium Myxococcus xanthus were collected by use of a closed-loop stripping apparatus (CLSA) and analyzed by GC-MS. Two new natural products, (S)-9-methyldecan-3-ol ((S)-1) and 9-methyldecan-3-one (2), were identified and synthesized, together with other aliphatic ketones and alcohols, and terpenes. Biosynthesis of the two main components (S)-1 and 2 was examined in feeding experiments carried out with the wild-type strain DK1622 and two mutant strains JD300 and DK11017, which are impaired in the degradation pathway from leucine to isovaleryl-SCoA. Isovaleryl-SCoA is used as a starter, followed by chain elongation with two malonate units. Subsequent use of methyl malonate and decarboxylation leads to (S)-1 and 2. Furthermore, 3,3-dimethylacrylic acid (DMAA) can be used by the mutant strain to form isovaleryl-SCoA, which corroborates recent data on the detection of a novel variety of the mevalonate pathway giving rise to isovaleryl-SCoA from HMGCoA.

Several synthetic cannabinoids were found in 44 of 46 different kinds of herbal products that are currently distributed on the illegal drug market in Japan due to their expected narcotic effects. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) analyses indicated that most of the products contained two major synthetic cannabinoids: (1RS,3SR)-3-[2-hydroxy-4-(2-methylnonan-2-yl)phenyl]cyclohexan-1-ol, renamed cannabicyclohexanol with the agreement of Pfizer Inc., and/or 1-naphthalenyl(1-pentyl-1H-indol-3-yl)methanone, named JWH-018. Oleamide (cis-9,10-octadecenoamide), which is an endogenous cannabinoid, was also detected in 7 products. Additionally, two synthetic cannabinoids were identified as minor components in some products. One was (1RS,3SR)-3-[2-hydroxy-4-(2-methyloctan-2-yl)phenyl]cyclohexan-1-ol, which is named CP-47,497 and is a homolog of cannabicyclohexanol. The other was 1-naphthalenyl(1-butyl-1H-indol-3-yl)methanone, which is named JWH-073 and is a homolog of JWH-018. These compounds were reported as synthetic cannabinoids possessing pharmacological cannabimimetic activity. The concentrations of cannabicyclohexanol, JWH-018 and oleamide in the products ranged from 1.1 to 16.9mg/g, 2.0 to 35.9mg/g and 7.6 to 210.9mg/g, respectively, and showed considerable variation. In this study, details of the analysis and identification of these synthetic cannabinoids in herbal products being sold on the Japanese drug market are described.

The neurotransmitter dopamine plays important roles in motor control, learning, and motivation in mammals and probably other animals as well. The strong dopaminergic projection to striatal regions and more moderate dopaminergic projections to other regions of the telencephalon predominantly arise from midbrain dopaminergic neurons in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). Homologous dopaminergic cell groups in songbirds project anatomically in a manner that may allow dopamine to influence song learning or song production. The electrophysiological properties of SNc and VTA neurons have not previously been studied in birds. Here we used whole cell recordings in brain slices in combination with tyrosine-hydroxylase immunolabeling as a marker of dopaminergic neurons to determine electrophysiological and pharmacological properties of dopaminergic and nondopaminergic neurons in the zebra finch SNc and VTA. Our results show that zebra finch dopaminergic neurons possess physiological properties very similar to those of mammalian dopaminergic neurons, including broad action potentials, calcium- and apamin-sensitive membrane-potential oscillations underlying pacemaker firing, powerful spike-frequency adaptation, and autoinhibition via D2 dopamine receptors. Moreover, the zebra finch SNc and VTA also contain nondopaminergic neurons with similarities (fast-firing, inhibition by the mu-opioid receptor agonist [d-Ala(2), N-Me-Phe(4), Gly-ol(5)]-enkephalin (DAMGO)) and differences (strong h-current that contributes to spontaneous firing) compared with GABAergic neurons in the mammalian SNc and VTA. Our results provide insight into the intrinsic membrane properties that regulate the activity of dopaminergic neurons in songbirds and add to strong evidence for anatomical, physiological, and functional similarities between the dopaminergic systems of mammals and birds.

BACKGROUND

Despite recent successes with biological agents as therapy for autoimmune inflammatory diseases such as rheumatoid arthritis (RA), many patients fail to respond adequately to these treatments, making a continued search for new therapies extremely important. Recently, the prevailing hypothesis that reactive oxygen species (ROS) promote inflammation was challenged when polymorphisms in Ncf1, that decrease oxidative burst, were shown to increase disease severity in mouse and rat arthritis models. Based on these findings we developed a new therapy for arthritis using oxidative burst-inducing substances.

RESULTS

Treatment of rats with phytol (3,7,11,15-tetramethyl-2-hexadecene-1-ol) increased oxidative burst in vivo and thereby corrected the effect of the genetic polymorphism in arthritis-prone Ncf1(DA) rats. Importantly, phytol treatment also decreased the autoimmune response and ameliorated both the acute and chronic phases of arthritis. When compared to standard therapies for RA, anti-tumour necrosis factor-alpha and methotrexate, phytol showed equally good or better therapeutic properties. Finally, phytol mediated its effect within hours of administration and involved modulation of T cell activation, as injection prevented adoptive transfer of disease with arthritogenic T cells.

CONCLUSIONS

Treatment of arthritis with ROS-promoting substances such as phytol targets a newly discovered pathway leading to autoimmune inflammatory disease and introduces a novel class of therapeutics for treatment of RA and possibly other chronic inflammatory diseases.

Temporal lobe epilepsy is associated with loss of interneurons and inhibitory dysfunction in the dentate gyrus. While status epilepticus (SE) leads to changes in granule cell inhibition, whether dentate basket cells critical for regulating granule cell feedforward and feedback inhibition express tonic GABA currents (I(GABA)) and undergo changes in inhibition after SE is not known. We find that interneurons immunoreactive for parvalbumin in the hilar-subgranular region express GABAA receptor (GABA(A)R) δ-subunits, which are known to underlie tonic I(GABA). Dentate fast-spiking basket cells (FS-BCs) demonstrate baseline tonic I(GABA) blocked by GABA(A)R antagonists. In morphologically and physiologically identified FS-BCs, tonic I(GABA) is enhanced 1 wk after pilocarpine-induced SE, despite simultaneous reduction in spontaneous inhibitory postsynaptic current (sIPSC) frequency. Amplitude of tonic I(GABA) in control and post-SE FS-BCs is enhanced by 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), demonstrating the contribution of GABA(A)R δ-subunits. Whereas FS-BC resting membrane potential is unchanged after SE, perforated-patch recordings from FS-BCs show that the reversal potential for GABA currents (E(GABA)) is depolarized after SE. In model FS-BCs, increasing tonic GABA conductance decreased excitability when E(GABA) was shunting and increased excitability when E(GABA) was depolarizing. Although simulated focal afferent activation evoked seizurelike activity in model dentate networks with FS-BC tonic GABA conductance and shunting E(GABA), excitability of identical networks with depolarizing FS-BC E(GABA) showed lower activity levels. Thus, together, post-SE changes in tonic I(GABA) and E(GABA) maintain homeostasis of FS-BC activity and limit increases in dentate excitability. These findings have implications for normal FS-BC function and can inform studies examining comorbidities and therapeutics following SE.

There is a clear association between excessive exposure to estrogens and the development of cancer in several tissues including breast and endometrium. The risk factors for women developing these cancers are all associated with longer estrogen exposure, as may be facilitated by early menses, late menopause and long-term estrogen replacement therapy. Equilenin (1,3,5(10),6,8-estrapentaen-3-ol-17-one) or its 17-hydroxylated analogs make up 15% of the most widely prescribed estrogen replacement formulation, Premarin, and yet there is very little information on the human metabolism of these estrogens. In this study, we synthesized the catechol metabolite of equilenin, 4-hydroxyequilenin, and examined how aromatization of the B ring affects the formation and reactivity of the o-quinone (3,5-cyclohexadien-1,2-dione). 4-Hydroxyequilenin-o-quinone is much more redox-active and longer-lived than the endogenous catechol estrone-o-quinones, which suggests that the mechanism(s) of toxicity of the former could be quite different. Interestingly, the rate of reduction of the 4-hydroxyequilenin-o-quinone is increased at least 13-fold in the presence of NAD(P)H:quinone oxidoreductase (DT-diaphorase). Once NADH is consumed however, the catechol auto-oxidized rapidly to the o-quinone. NADH consumption was accompanied by dicumarol-sensitive oxygen uptake both with the purified enzyme and with cytosol from human melanoma cells with high levels of DT-diaphorase activity. P450 reductase and rat liver microsomes also catalyzed NADPH consumption and oxygen uptake. 4-Hydroxyestrone-o-quinone was also rapidly reduced by NAD(P)H; however, this o-quinone does not auto-oxidize and once the o-quinone is reduced the reaction terminates. Including oxidative enzymes in the incubation completes the redox couple and 4-hydroxyestrone-o-quinone behaves like 4-hydroxyequilenin-o-quinone. These data suggest that reduction of estrogen-o-quinones may not result in detoxification. Instead this could represent a cytotoxic mechanism involving consumption of reducing equivalents (NADH/NADPH) as well as formation of superoxide and other reactive oxygen species leading to oxidative stress. Finally, we have compared the cytotoxicity of 4-hydroxyequilenin with that of the estrone catechols in human melanoma cells. 4-Hydroxyequilenin is 5-fold more toxic in these cells compared with 4-hydroxyestrone (ED50 = 7.8 versus 38 microM, respectively) suggesting that formation of the longer-lived redox-active 4-hydroxyequilenin-o-quinone was responsible for the cytotoxic differences. These results substantiate the conclusion that the involvement of quinoids in catechol estrogen toxicity depends on a combination of the rate of formation of the o-quinone, the lifetime of the o-quinone, and the electrophilic/redox reactivity of the quinoids.

Ten healthy human subjects consumed 500 mL of Choladi green tea, containing 648 mumol of flavan-3-ols after which plasma and urine were collected over a 24 h period and analysed by HPLC-MS. Plasma contained a total of ten metabolites, in the form of O-methylated, sulphated and glucuronide conjugates of (epi)catechin and (epi)gallocatechin, with 29-126 nM peak plasma concentrations (C(max)) occurring 1.6-2.3 h after ingestion, indicative of absorption in the small intestine. Plasma also contained unmetabolised (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate with respective C(max) values of 55 and 25 nM. Urine excreted 0-24 h after consumption of green tea contained 15 metabolites of (epi)catechin and (epi)gallocatechin, but (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate were not detected. Overall flavan-3-ol metabolite excretion was equivalent to 8.1% of intake, however, urinary (epi)gallocatechin metabolites corresponded to 11.4% of (epi)gallocatechin ingestion while (epi)catechin metabolites were detected in amounts equivalent to 28.5% of (epi)catechin intake. These findings imply that (epi)catechins are highly bioavailable, being absorbed and excreted to a much greater extent than most other flavonoids. It is also evident that flavan-3-ol metabolites are rapidly turned over in the circulatory system and as a consequence C(max) values are not an accurate quantitative indicator of the extent to which absorption occurs.

Accumulation of iron at sites where neurons degenerate in Parkinson's disease (PD) and Alzheimer's disease (AD) is thought to have a major role in oxidative stress induced process of neurodegeneration. The novel non-toxic lipophilic brain- permeable iron chelators, VK-28 (5- [4- (2- hydroxyethyl) piperazine-1-ylmethyl]- quinoline- 8- ol) and its multi-functional derivative, M-30 (5-[N-methyl-N-propargylaminomethyl]-8-hydroxyquinoline), as well as the main polyphenol constituent of green tea (-)-epigallocatechin-3-gallate (EGCG), which possesses iron metal chelating, radical scavenging and neuroprotective properties, offer potential therapeutic benefits for these diseases. M-30 and EGCG decreased apoptosis of human SH-SY5Y neuroblastoma cells in a neurorescue, serum deprivation model, via multiple protection mechanisms including: reduction of the pro-apoptotic proteins, Bad and Bax, reduction of apoptosis-associated Ser139 phosphorylated H2A.X and inhibition of the cleavage and activation of caspase-3. M-30 and EGCG also promoted morphological changes, resulting in axonal growth-associated protein-43 (GAP-43) implicating neuronal differentiation. Both compounds significantly reduced the levels of cellular holo-amyloid precursor protein (APP) in SH-SY5Y cells. The ability of theses novel iron chelators and EGCG to regulate APP are in line with the presence of an iron-responsive element (IRE) in the 5'-untranslated region (5'UTR) of APP. Also, EGCG reduced the levels of toxic amyloid-beta peptides in CHO cells over-expressing the APP "Swedish" mutation. The diverse molecular mechanisms and cell signaling pathways participating in the neuroprotective/neurorescue and APP regulation/processing actions of M-30 and EGCG, make these multifunctional compounds potential neuroprotective drugs for the treatment of neurodegenerative diseases, such as PD, AD, Huntington's disease and amyotrophic lateral sclerosis.

Intraperitoneal injection of ethanol (1-2 g/kg) and chlordiazepoxide (2-16 mg/kg) suppressed susceptibility to audiogenically induced, clonic-tonic seizures and antagonized forelimb tremor in rats undergoing ethanol withdrawal, 30 min after treatment. However, a smaller dose of ethanol (0.5 g/kg) actually increased clonic seizure frequency, suggesting that ethanol exerts a biphasic proconvulsant/anticonvulsant action. Direct activation of gamma-aminobutyric acid (GABA) receptors by intracisternal administration of GABA (100-1000 micrograms), muscimol (0.3-1.0 micrograms) or 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) (0.3-3.0 micrograms) 5 to 10 min before testing also reduced susceptibility to audiogenic clonic-tonic seizures. In sharp contrast to these anticonvulsant actions, GABA, muscimol and THIP had no effect on withdrawal-induced forelimb tremors. Blockade of GABA uptake with 1-2,4-diaminobutyric acid (300 and 600 mg/kg i.p.) and inhibition of GABA transaminase with aminooxyacetic acid (12.5 and 25.0 mg/kg i.p.) both reduced susceptibility to seizures. However, anticonvulsant doses of these two drugs, unlike GABA, muscimol and THIP, also reduced forelimb tremor. Three other GABA transaminase inhibitors, gamma-vinyl GABA (450 and 900 mg/kg i.p.), gamma-acetylenic GABA (50-150 mg/kg i.p.) and ethanolamine-O-sulfate (250-750 mg/kg i.p.), were inactive against ethanol withdrawal audiogenic seizures and forelimb tremors. These results indicate that direct GABA receptor activation can selectively suppress one type of ethanol withdrawal response (i.e., audiogenic seizure susceptibility) while failing to influence another (forelimb tremors).

Apoptosis plays a crucial role in brain development by ensuring that only appropriately growing, migrating, and synapse-forming neurons and their associated glial cells survive. This process involves an intimate relationship between cell-cell interactions and developmental cues and is further impacted by environmental stress during neurogenesis and disease. Oligodendrocytes (OLs), the major myelin-forming cells in the central nervous system, largely form after this wave of neurogenesis but also show a selective vulnerability to cell death stimuli depending on their stage of development. This can affect not only embryonic and early postnatal brain formation but also the response to demyelinating pathologies. In the present review, we discuss the stage-specific sensitivity of OL lineage cells to damage-induced death and how this might impact myelin survival and regeneration during injury or disease.

O-linked oligosaccharide groups ranging from di- to hexasaccharide were beta-eliminated by mild alkaline treatment under reducting conditions from the peptidogalactomannan of Aspergillus fumigatus mycelial cell wall. The resulting reduced oligosaccharides, which were the minor components of the peptidogalactomannan fraction, were fractionated to homogeneity by successive gel filtration and high-performance liquid chromatography. Their primary structures were determined based on a combination of techniques including gas chromatography, ESI-QTOF-MS, 1H COSY and TOCSY, and 1H-13C HMQC NMR spectroscopy and methylation analysis, to be: alpha-Glcp-(1 ->> 6)-Man-ol, beta-Galf-(1 ->> 6)-alpha-Manp-(1 ->> 6)-Man-ol, beta-Galf-(1 ->> 5)-beta-Galf-(1 ->> 6)-alpha-Manp-(1 ->> 6)-Man-ol and beta-Galf-(1 ->> 5)-[beta-Galf-(1 ->> 5]3-beta-Galf-(1 ->> 6)-Man-ol. The beta-Galf containing oligosaccharides have not been previously described as fungal O-linked oligosaccharides. The peptidogalactomannan is antigenic and was recognized by human sera of patients with aspergillosis when probed by ELISA, but de-O-glycosylation rendered a 50% decrease in its reactivity. Furthermore, when tested in a hapten inhibition test, the isolated oligosaccharide alditols were able to block, on a dose-response basis, recognition between human sera and the intact peptidogalactomannan. The immunodominant epitopes were present in the tetra- and hexasaccharide, which contain a beta-Galf-(1 ->> 5)-beta-Galf terminal group. These results suggest that the O-glycosidically linked oligosaccharide chains, despite being the less abundant carbohydrate component of the A. fumigatus peptidogalactomannan, may account for a significant part of its antigenicity, other than the known activity associated with the galactomannan component.

Numerous synthetic agonists selectively stimulate beta3-adrenoceptors (ARs). The endogenous catecholamines, noradrenaline and adrenaline, however, stimulate all the beta-AR subtypes, and no selective physiological agonist for beta3-ARs has been described so far. The aim of this study was to investigate whether any naturally occurring amine can stimulate selectively beta3-ARs. Since activation of lipolysis is a well-known beta-adrenergic function, the efficacy and potency of various biogenic amines were compared with those of noradrenaline, isoprenaline, and beta3-AR agonists 4-(-{[2-hydroxy-(3-chlorophenyl)ethyl]-amino} propyl)phenoxyacetate (BRL 37,344) and (R,R)-5-(2-{[2-(3-chlorophenyl )-2-hydroxyethyl]-amino} propyl)-1,3-benzo-dioxole-2,2-dicarboxylate (CL 316,243) by testing their lipolytic action in white fat cells. Five mammalian species were studied: rat, hamster and dog, in which selective beta-AR agonists act as full lipolytic agents, and guinea-pigs and humans, in which beta3-AR agonists are less potent activators of lipolysis. Several biogenic amines were inefficient (e.g. dopamine, tyramine and beta-phenylethylamine) while others (synephrine, phenylethanolamine, epinine) were partially active in stimulating lipolysis in all species studied. Their actions were inhibited by all the beta-AR antagonists tested, including those selective for beta1- or beta2-ARs. Octopamine was the only amine fully stimulating lipolysis in rat, hamster and dog fat cells, while inefficient in guinea-pig or human fat cells, like the beta3-AR agonists. In rat white fat cells, beta-AR antagonists inhibited the lipolytic effect of octopamine with a relative order of potency very similar to that observed against CL 316,243. Competitive antagonism of octopamine effect resulted in the following apparent pA2 [-log(IC50), where IC50 is the antagonist concentration eliciting half-maximal inhibition] values: 7.77 (bupranolol), 6.48 [3-(2-ethyl-phenoxy)-1[(1 S)-1,2,3,4-tetrahydronaphth-1-ylaminol]-(2S)2-propanol oxalate, SR 59230A, a beta3-selective antagonist], 6.30[erythro-D,L-1(7-lethylindan-4-yloxy)-3-isopropylamino-+ ++butan-2-ol, ICI 118,551, a beta2-selective antagonist] and 4.71 [(+/-)-[2-(3-carbomyl-4-hydroxyphenoxy)-ethylamino]-3-[4-(1- methyl-4-trifluoromethyl-2-imidazolyl)-phenoxy]2-propanolmethane sulphonate, CGP 20712A, a beta1-selective antagonist]. Octopamine had other properties in common with beta3-AR agonists: stimulation of oxygen consumption in rat brown fat cells and very low affinity in displacing [3H]CGP 12,177 binding to [beta1- or beta2-ARs in dog and rat adipocyte membranes. In Chinese hamster ovary (CHO) cells expressing human beta3-ARs, octopamine inhibited [125I]ICYP binding with only twofold less affinity than noradrenaline while it exhibited an affinity around 200-fold lower than noradrenaline in CHO cells expressing human beta1- or beta2-ARs. These data suggest that, among the biogenic amines metabolically related to catecholamines, octopamine can be considered as the most selective for beta3-ARs.

Flavonoids have been investigated for possible inverse associations with various chronic degenerative diseases, but there are no epidemiologic data concerning a possible association between several of the main flavonoid categories and breast cancer risk. We have applied recently published data on the flavonoid content of several foods and beverages on dietary information collected in the context of a large case-control study of 820 women with breast cancer and 1548 control women, conducted in Greece. We found a strong, statistically significant inverse association of flavone intake with breast cancer. The odds ratio for an increment equal to one standard deviation of daily flavone intake (i.e. 0.5 mg day(-1)) was 0.87, with 95% confidence interval 0.77-0.97. The association persisted after controlling for fruit and vegetable consumption, or for other flavonoid intake. This inverse association is compatible with and may explain the reported inverse association of breast cancer with consumption of vegetables, particularly leafy vegetables. After controlling for dietary confounding, there was no association of breast cancer risk with flavanones, flavan-3-ols, flavonols, anthocyanidins or isoflavones.

Xylella fastidiosa is a pathogen that causes leaf scorch and related diseases in over 100 plant species, including Pierce's disease in grapevines (PD), phony peach disease (PP), plum leaf scald (PLS), and leaf scorch in almond (ALS), oak (OAK), and oleander (OLS). We used a high-resolution DNA sequence approach to investigate the evolutionary relationships, geographic variation, and divergence times among the X. fastidiosa isolates causing these diseases in North America. Using a large data set of 10 coding loci and 26 isolates, the phylogeny of X. fastidiosa defined three major clades. Two of these clades correspond to the recently identified X. fastidiosa subspecies piercei (PD and some ALS isolates) and X. fastidiosa subsp. multiplex (OAK, PP, PLS, and some ALS isolates). The third clade grouped all of the OLS isolates into a genetically distinct group, named X. fastidiosa subsp. sandyi. These well-differentiated clades indicate that, historically, X. fastidiosa has been a clonal organism. Based on their synonymous-site divergence ( approximately 3%), these three clades probably originated more than 15,000 years ago, long before the introduction of the nonnative plants that characterize most infections. The sister clades of X. fastidiosa subsp. sandyi and X. fastidiosa subsp. piercei have synonymous-site evolutionary rates 2.9 times faster than X. fastidiosa subsp. multiplex, possibly due to generation time differences. Within X. fastidiosa subsp. multiplex, a low level ( approximately 0.1%) of genetic differentiation indicates the recent divergence of ALS isolates from the PP, PLS, and OAK isolates due to host plant adaptation and/or allopatry. The low level of variation within the X. fastidiosa subsp. piercei and X. fastidiosa subsp. sandyi clades, despite their antiquity, suggests strong selection, possibly driven by host plant adaptation.

Under phosphate-limiting conditions, some bacteria replace their membrane phospholipids by lipids not containing any phosphorus. One of these phosphorus-free lipids is an ornithine-containing lipid (OL) that is widespread among eubacteria. In earlier work, we had identified a gene (olsA) required for OL biosynthesis that probably encodes an O-acyltransferase using acyl-acyl carrier protein (acyl-AcpP) as an acyl donor and that converts lyso-ornithine lipid into OL. We now report on a second gene (olsB) required for OL biosynthesis that is needed for the incorporation of radiolabelled ornithine into OL. Overexpression of OlsB in an olsA-deficient mutant of Sinorhizobium (Rhizobium) meliloti leads to the transient accumulation of lyso-ornithine lipid, the biosynthetic intermediate of OL biosynthesis. Overexpression of OlsB in Escherichia coli is sufficient to cause the in vivo formation of lyso-ornithine lipid in this organism and is the cause for a 3-hydroxyacyl-AcpP-dependent acyltransferase activity forming lyso-ornithine lipid from ornithine. These results demonstrate that OlsB is required for the first step of OL biosynthesis, in which ornithine is N-acylated with a 3-hydroxy-fatty acyl residue in order to obtain lyso-ornithine lipid. OL formation in a wild-type S. meliloti is increased upon growth under phosphate-limiting conditions. Expression of OlsB from a broad host range vector leads to the constitutive formation of relatively high amounts of OL (12-14% of total membrane lipids) independently of whether strains are grown in the presence of low or high concentrations of phosphate, suggesting that in S. meliloti the formation of OlsB is usually limiting for the amount of OL formed in this organism. Open reading frames homologous to OlsA and OlsB were identified in many eubacteria and although in S. meliloti the olsB and olsA gene are 14 kb apart, in numerous other bacteria they form an operon.

Tumor necrosis factor-alpha (TNF-alpha), a cytokine secreted by activated monocytes/macrophages and T lymphocytes, has been implicated in several disease states, including rheumatoid arthritis, inflammatory bowel disease, septic shock, and osteoporosis. Monocyte/macrophage production of TNF-alpha is dependent on the mitogen-activated protein kinase p38. RWJ 67657 (4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol -2-yl]-3-butyn-1-ol) inhibited the release of TNF-alpha by lipopolysaccharide (a monocyte stimulus)-treated human peripheral blood mononuclear cells with an IC(50) of 3 nM, as well as the release of TNF-alpha from peripheral blood mononuclear cells treated with the superantigen staphylococcal enterotoxin B (a T cell stimulus), with an IC(50) value of 13 nM. This compound was approximately 10-fold more potent than the literature standard p38 kinase inhibitor SB 203580 in all p38 dependent in vitro systems tested. RWJ 67657 inhibited the enzymatic activity of recombinant p38alpha and beta, but not gamma or delta, in vitro and had no significant activity against a variety of other enzymes. In contrast, SB 203580 significantly inhibited the tyrosine kinases p56 lck and c-src (IC(50) = 5 microM). RWJ 67657 did not inhibit T cell production of interleukin-2 or interferon-gamma and did not inhibit T cell proliferation in response to mitogens. RWJ 67657 inhibited TNF-alpha production in lipopolysaccharide-injected mice (87% inhibition at 50 mg/kg) and in rats (91% inhibition at 25 mg/kg) after oral administration. Based on these favorable biological properties, RWJ 67657 may have use as a treatment for inflammatory diseases.

OBJECTIVE

To investigate the association between physical performance measures and bone mineral density (BMD) in older women.

METHODS

Cross-sectional analysis.

METHODS

University research laboratory.

METHODS

Healthy postmenopausal women (N=116; mean age +/- standard deviation, 68.3+/-6.8y) in self-reported good health who were not taking medications known to affect bone, including hormone replacement therapy.

METHODS

Not applicable.

METHODS

Anthropometrics and BMD of the hip, spine, whole body, and forearm. Physical performance measures included normal and brisk 8-m gait speed, normal step length (NSL), brisk step length (BSL), timed 1-leg stance (OLS), timed sit-to-stand (STS), and grip strength.

RESULTS

NSL, BSL, normal gait speed, brisk gait speed, OLS, and grip strength correlated significantly with several skeletal sites ( r range, .19-.38; P <.05). In multiple regression models containing body mass index, hours of total activity, total calcium intake, and age of menarche, NSL, BSL, normal and brisk gait speeds, OLS, and grip strength were all significantly associated with BMD of various skeletal sites (adjusted R 2 range, .11-.24; P <.05). Analysis of covariance showed that subjects with longer step lengths and faster normal and brisk gait speeds had higher BMD at the whole body, hip, and spine (brisk speed only). Those with a longer OLS had greater femoral neck BMD, and those with a stronger grip strength had greater BMD in the whole body and forearm ( P <.05). STS was not related to any skeletal site.

CONCLUSIONS

Normal and brisk gait speed, NSL, BSL, OLS, and grip strength are all associated with BMD at the whole body, hip, spine, and forearm. Physical performance evaluation may help with osteoporosis prevention and treatment programs for postmenopausal women when bone density scores have not been obtained or are unavailable.

Recent research on the bioavailability of flavan-3-ols after ingestion of green tea by humans is reviewed. Glucuronide, sulfate, and methyl metabolites of (epi)catechin and (epi)gallocatechin glucuronide reach peak nanomolar per liter plasma concentrations 1.6-2.3 h after intake, indicating absorption in the small intestine. The concentrations then decline, and only trace amounts remain 8 h after ingestion. Urinary excretion of metabolites over a 24-h period after green tea consumption corresponded to 28.5% of the ingested (epi)catechin and 11.4% of (epi)gallocatechin, suggesting higher absorption than that of most other flavonoids. The fate of (-)-epicatechin-3-O-gallate, the main flavan-3-ol in green tea, is unclear because it appears unmetabolized in low concentrations in plasma but is not excreted in urine. Possible enterohepatic recirculation of flavan-3-ols is discussed along with the impact of dose and other food components on flavan-3-ol bioavailability. Approximately two-thirds of the ingested flavan-3-ols pass from the small to the large intestine where the action of the microbiota results in their conversion to C-6-C-5 phenylvalerolactones and phenylvaleric acids, which undergo side-chain shortening to produce C-6-C-1 phenolic and aromatic acids that enter the bloodstream and are excreted in urine in amounts equivalent to 36% of flavan-3-ol intake. Some of these colon-derived catabolites may have a role in vivo in the potential protective effects of tea consumption. Although black tea, which contains theaflavins and thearubigins, is widely consumed in the Western world, there is surprisingly little research on the absorption and metabolism of these compounds after ingestion and their potential impact on health.

Opioid receptor subtypes may have site-specific effects and play different roles in modulating serotonergic neurotransmission in the mammalian central nervous system. To test this hypothesis, we used in vivo microdialysis to measure changes in extracellular serotonin (5-hydroxytryptamine; 5-HT) in response to local infusion of mu-, delta-, and kappa-opioid receptor ligands into the dorsal raphe nucleus (DRN), median raphe nucleus (MRN), and nucleus accumbens (NAcc) of freely behaving rats. The mu-opioids [D-Ala(2)-N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), endomorphin-1, and endomorphin-2 were administered by reverse dialysis infusion into the DRN. In response, extracellular 5-HT was increased in the DRN, an effect that was blocked by the selective mu-receptor antagonist beta-funaltrexamine, but not by the delta-receptor antagonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI-174,864). Infusion of delta-receptor agonists, [D-Ala(2),D-Len(5)]enkephalin (DADLE), [D-Pen(2,5)]enkephalin (DPDPE), and deltophin-II into the DRN also increased extracellular 5-HT, an effect that was blocked by selective delta-receptor antagonists. In contrast to the DRN, local infusion of mu- and delta-opioids had no effect on 5-HT in the MRN or NAcc. These data indicate that mu- and delta-opioid ligands have a selective influence on serotonergic neurons in the DRN. Finally, the kappa-receptor agonist U-50,488 [trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide] produced similar decreases in 5-HT during local infusion into the DRN, MRN, and NAcc. These results provide evidence of differential regulation of 5-HT release by opioid receptor subtypes in the midbrain raphe and forebrain.

Neurosteroids are potent blockers of neuronal low-voltage activated (T-type) Ca(2+) channels and potentiators of GABA(A) ligand-gated channels, but their effects in peripheral pain pathways have not been studied previously. To investigate potential analgesic effects and the ion channels involved, we tested the ability of locally injected 5alpha-reduced neurosteroids to modulate peripheral thermal nociception to radiant heat in adult rats in vivo and to modulate GABA(A) and T-type Ca(2+) channels in vitro. The steroid anesthetic alphaxalone (ALPX), the endogenous neurosteroid allopregnanolone (3alpha5alphaP), and a related compound ((3alpha,5alpha,17beta)-3-hydroxyandrostane-17-carbonitrile, (ACN)), induced potent, dose-dependent, enantioselective anti-nociception in vivo and modulation of both T-type Ca(2+) currents and GABA(A)-mediated currents in vitro. Analgesic effects of ALPX were incompletely antagonized by co-injections of the GABA(A) receptor antagonist bicuculline. The neurosteroid analogue ((3alpha,5alpha)-3-hydroxy-13,24-cyclo-18,21-dinorchol-22-en-24-ol (CDNC24), a compound with GABAergic but not T-type activity, was not analgesic. However, (3beta,5alpha,17beta)-17-hydroxyestrane-3-carbonitrile (ECN)), which has effects on T-type channels but not on GABA(A) receptors, also induced potent enantioselective peripheral anti-nociception. ECN increased pain thresholds less than ALPX, 3alpha5alphaP and ACN. However, when an ineffective dose of CDNC24 was combined with ECN, anti-nociceptive activity was greatly enhanced, and this effect was bicuculline-sensitive. These results strongly suggest that GABA(A) channels do not contribute to baseline pain transmission, but they can enhance anti-nociception mediated by blockade of T-type Ca(2+) channels. In conclusion, we demonstrate that potent peripheral analgesia induced by 5alpha-reduced neurosteroid is mediated in part by effects on T-type Ca(2+) channels. Our results also reveal a role of GABA-gated ion channels in peripheral nociceptive signaling.

The fungus Botrytis cinerea is the causal agent of the economically important gray mold disease that affects more than 200 ornamental and agriculturally important plant species. B. cinerea is a necrotrophic plant pathogen that secretes nonspecific phytotoxins, including the sesquiterpene botrydial and the polyketide botcinic acid. The region surrounding the previously characterized BcBOT1 gene has now been identified as the botrydial biosynthetic gene cluster.Five genes including BcBOT1 and BcBOT2 were shown by quantitative reverse transcription-PCR to be co-regulated through the calcineurin signaling pathway. Inactivation of the BcBOT2 gene, encoding a putative sesquiterpene cyclase, abolished botrydial biosynthesis, which could be restored by in trans complementation.Inactivation of BcBOT2 also resulted in overproduction of botcinic acid that was observed to be strain-dependent. Recombinant BcBOT2 protein converted farnesyl diphosphate to the parent sesquiterpene of the botrydial biosynthetic pathway, the tricyclic alcohol presilphiperfolan-8beta-ol.

Engagement of toll-like receptors serve to link innate immune responses with adaptive immunity and can be exploited as powerful vaccine adjuvants for eliciting both primary and anamnestic immune responses. TLR7 agonists are highly immunostimulatory without inducing dominant proinflammatory cytokine responses. A structure-activity study was conducted on the TLR7-agonistic imidazoquinolines, starting with 1-(4-amino-2-((ethylamino)methyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol as a lead. Modifications of the secondary amine of the C2 ethylaminomethylene side chain are poorly tolerated. The 4-amino group must be retained for activity. Replacement of the imidazole ring of the scaffold with triazole or cyclic urea led to complete loss of activity. A systematic exploration of N(1)-benzyl-C2-alkyl substituents showed a very distinct relationship between alkyl length and TLR7-agonistic potency with the optimal compound bearing a C2-n-butyl group. Transposition of the N(1) and C2 substituents led to the identification of an extremely active TLR7-agonistic compound with an EC(50) value of 8.6 nM. The relative potencies in human TLR7-based primary reporter gene assays were paralleled by interferon-alpha induction activities in whole human blood models.