Mutational activation of niche-specific genes provides insight into regulatory networks and bacterial function in a complex environment

The genome of the plant-colonizing bacterium Pseudomonas fluorescens SBW25 harbors a subset of genes that are expressed specifically on plant surfaces. The function of these genes is central to the ecological success of SBW25, but their study poses significant challenges because no phenotype is discernable in vitro. Here, we describe a genetic strategy with general utility that combines suppressor analysis with IVET (SPyVET) and provides a means of identifying regulators of niche-specific genes. Central to this strategy are strains carrying operon fusions between plant environment-induced loci (EIL) and promoterless 'dapB. These strains are prototrophic in the plant environment but auxotrophic on laboratory minimal medium. Regulatory elements were identified by transposon mutagenesis and selection for prototrophs on minimal medium. Approximately 106 mutants were screened for each of 27 strains carrying 'dapB fusions to plant EIL and the insertion point for the transposon determined in approximately 2,000 putative regulator mutants. Regulators were functionally characterized and used to provide insight into EIL phenotypes. For one strain carrying a fusion to the cellulose-encoding wss operon, five different regulators were identified including a diguanylate cyclase, the flagella activator, FleQ, and alginate activator, AmrZ (AlgZ). Further rounds of suppressor analysis, possible by virtue of the SPyVET strategy, revealed an additional two regulators including the activator AlgR, and allowed the regulatory connections to be determined.

Effect of hyaluronidase on ocular motility and eyelid function in sub-Tenon's anaesthesia: randomised controlled trial

Purpose: To assess the effect of hyaluronidase on eye and eyelid movements when used as an adjunct in sub-Tenon's anaesthesia. Methods: A total of 60 patients who had sub-Tenon's anaesthesia prior to phacoemulsification surgery were divided into two equal groups in a double-masked randomised controlled fashion. Of these, Group A had 4 ml lignocaine 2%, while Group B had 4ml lignocaine 2% with the addition of sodium hyaluronidase 75 IU/ml. Ocular motility, levator, and orbicularis oculi function were measured in all patients at 5 and 8 min. Levator function was scored from 0 (no function) to 3 (complete function) while orbicularis function was scored from 0 to 2. The score for ocular motility was the sum in four positions of gaze, each position scoring from 0 to 2. Results were compared using a nonparametric test. Results Group B achieved significantly better ocular and lid akinesia than Group A both at 5 and 8 min with P < 0.01. The median scores for levator function at 5 and 8 min were 2 for Group A and 0 for Group B. For orbicularis function, the median scores at both time intervals were 2 for Group A and 1 for Group B. For ocular motility, the median score for Group A at 5 min was 3 and at 8 min was 2.5; for Group B at 5 min was 0.5 and at 8 min was 0. Conclusions: The addition of hyaluronidase in sub-Tenon's anaesthesia has a significant effect in improving ocular and lid (levator and orbicularis) akinesia.

Sulfation of genistein alters its antioxidant properties and its effect on platelet aggregation and monocyte and endothelial function

Soy isoflavones have been extensively studied because of their possible benefits to human health. Genistein, the major isoflavone aglycone, has received most attention; however, it undergoes extensive metabolism (e.g. conjugation with sulfuric acid) in the gut and liver, which may affect its biological proper-ties. This study investigated the antioxidant activity and free radical-scavenging properties of genistein, genistein-4'-sulfate and genistein-4'-7-disulfate as well as their effect on platelet aggregation and monocyte and endothelial function. Electron spin resonance spectroscopy (ESR) and spin trapping data and other standard antioxidant assays indicated that genistein is a relatively weak antioxidant compared to quercetin and that its sulfated metabolites are even less effective. Furthermore, genistein-4'-sulfate was less potent than genistem, and genistein-4'-7-disulfate even less potent, at inhibiting collagen-induced platelet aggregation, nitric oxide (NO) production by macrophages, and secretion by primary human endothelial cells of monocyte chemoattractant protein 1 (MCP-1), intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1). The current data suggest that sulfation of genistein, with the associated loss of hydroxyl groups, decreases its antioxidant activity and its effect on platelet aggregation, inflammation, cell adhesion and chemotaxis. (C) 2004 Elsevier B.V All rights reserved.

Effect of genistein and daidzein on platelet aggregation and monocyte and endothelial function

There has been much recent interest in the cardiovascular benefits of dietary isoflavones. The aim of the present in vitro studies was to investigate potential anti-thrombogenic and anti-atherogenic effects of the isoflavones genistein and daidzein in platelets, macrophages and endothelial cells. Pre-treatment with either isoflavone inhibited collagen-induced platelet aggregation in a dose-dependent manner. In a macrophage cell line (RAW 264-7) activated with interferon gamma plus lipopolysaccharide, both isoflavones were found to inhibit NO production and tumour necrosis factor alpha (TNF-alpha) secretion dose-dependently, but they did not affect mRNA levels for inducible nitric oxide synthase and cyclo-oxygenase-2. Both isoflavones also dose-dependently decreased monocyte chemoattractant protein-1 secretion induced by TNF-alpha in human umbilical vein endothelial cells. Compared with daidzein, genistein exerted greater inhibitory effects for all parameters studied. The present data contributes to our knowledge on the molecular mechanisms by which isoflavones may protect against coronary artery disease. Further studies are required to determine whether the effects of isoflavones observed in the current in vitro studies are relevant to the aetiology of coronary artery disease in vivo.

Calculation of converged rovibrational energies and partition function for methane using vibrational-rotational configuration interaction

The rovibration partition function of CH4 was calculated in the temperature range of 100-1000 K using well-converged energy levels that were calculated by vibrational-rotational configuration interaction using the Watson Hamiltonian for total angular momenta J=0-50 and the MULTIMODE computer program. The configuration state functions are products of ground-state occupied and virtual modals obtained using the vibrational self-consistent field method. The Gilbert and Jordan potential energy surface was used for the calculations. The resulting partition function was used to test the harmonic oscillator approximation and the separable-rotation approximation. The harmonic oscillator, rigid-rotator approximation is in error by a factor of 2.3 at 300 K, but we also propose a separable-rotation approximation that is accurate within 2% from 100 to 1000 K. (C) 2004 American Institute of Physics.

Fatty acids and immune function: new insights into mechanisms

Fatty acids are known to play diverse roles in immune cells. They are important as a source of energy, as structural components of cell membranes, as signaling molecules and as precursors for the synthesis of eicosanoids and similar mediators. Recent research has suggested that the localization and organisation of fatty acids into distinct cellular pools has a direct influence on the behaviour of a number of proteins involved in immune cell activation, including those associated with T cell responses, antigen presentation and fatty acid-derived inflammatory mediator production. This article reviews these studies and places them in the context of existing literature in the field. These studies indicate the existence of several novel mechanisms by which altered fatty acid availability can modulate immune responses and impact upon clinical outcomes

Modulation of the fecal microflora profile and immune function by a novel trans-galactooligosaccharide mixture (B-GOS) in healthy elderly volunteers

Background: Aging is associated with reduced numbers of beneficial colonic bifidobacteria and impaired immunity. Galactooligosaccharides (GOSs) stimulate the growth of bifidobacteria in younger adults, but little is known about their effects in the elderly and their immunomodulatory capacity. Objective: We assessed the effect of a prebiotic GOS mixture (B-GOS) on immune function and fecal microflora composition in healthy elderly subjects. Design: In a double-blind, placebo-controlled, crossover study, 44 elderly subjects were randomly assigned to receive either a placebo or the B-GOS treatment (5.5 g/d). Subjects consumed the treatments for 10 wk, and then went through a 4-wk washout period, before switching to the other treatment for the final 10 wk. Blood and fecal samples were collected at the beginning, middle (5 wk), and end of the test period. Predominant bacterial groups were quantified, and phagocytosis, natural killer (NK) cell activity, cytokine production, plasma cholesterol, and HDL cholesterol were measured. Results: B-GOS significantly increased the numbers of beneficial bacteria, especially bifidobacteria, at the expense of less beneficial groups compared with the baseline and placebo. Significant increases in phagocytosis, NK cell activity, and the production of antiinflammatory cytokine interleukin-10 (IL-10) and significant reduction in the production of proinflammatory cytokines (IL-6, IL-1 beta , and tumor necrosis factor-alpha) were also observed. B-GOS exerted no effects on total cholesterol or HDL-cholesterol production, however. Conclusions: B-GOS administration to healthy elderly persons resulted in positive effects on both the microflora composition and the immune response. Therefore, B-GOS may be a useful dietary candidate for the enhancement of gastrointestinal health and immune function in elderly persons. Am J Clin Nutr 2008; 88: 1438-46.

Sulfation of genistein alters its antioxidant properties and its effect on platelet aggregation and monocyte and endothelial function

Soy isoflavones have been extensively studied because of their possible benefits to human health. Genistein, the major isoflavone aglycone, has received most attention; however, it undergoes extensive metabolism (e.g. conjugation with sulfuric acid) in the gut and liver, which may affect its biological proper-ties. This study investigated the antioxidant activity and free radical-scavenging properties of genistein, genistein-4'-sulfate and genistein-4'-7-disulfate as well as their effect on platelet aggregation and monocyte and endothelial function. Electron spin resonance spectroscopy (ESR) and spin trapping data and other standard antioxidant assays indicated that genistein is a relatively weak antioxidant compared to quercetin and that its sulfated metabolites are even less effective. Furthermore, genistein-4'-sulfate was less potent than genistem, and genistein-4'-7-disulfate even less potent, at inhibiting collagen-induced platelet aggregation, nitric oxide (NO) production by macrophages, and secretion by primary human endothelial cells of monocyte chemoattractant protein 1 (MCP-1), intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1). The current data suggest that sulfation of genistein, with the associated loss of hydroxyl groups, decreases its antioxidant activity and its effect on platelet aggregation, inflammation, cell adhesion and chemotaxis. (C) 2004 Elsevier B.V All rights reserved.

Green tea (Camellia sinensis) catechins and vascular function

The health benefits of green tea (Camellia sinensis) catechins are becoming increasingly recognised. Amongst the proposed benefits are the maintenance of endothelial function and vascular homeostasis and an associated reduction in atherogenesis and CVD risk. The mounting evidence for the influential effect of green tea catechins on vascular function from epidemiological, human intervention and animal studies is subject to review together with exploration of the potential mechanistic pathways involved. Epigallocatechin-3-gallate, one of the most abundant and widely studied catechin found in green tea, will be prominent in the present review. Since there is a substantial inconsistency in the published data with regards to the impact of green tea catechins on vascular function, evaluation and interpretation of the inter- and intra-study variability is included. In conclusion, a positive effect of green tea catechins on vascular function is becoming apparent. Further studies in animal and cell models using physiological concentrations of catechins and their metabolites are warranted in order to gain some insight into the physiology and molecular basis of the observed beneficial effects.

Flavonoids and cognitive function: a review of human randomized controlled trial studies and recommendations for future studies

Evidence in support of the neuroprotective effects of flavonoids has increased significantly in recent years, although to date much of this evidence has emerged from animal rather than human studies. Nonetheless, with a view to making recommendations for future good practice, we review 15 existing human dietary intervention studies that have examined the effects of particular types of flavonoid on cognitive performance. The studies employed a total of 55 different cognitive tests covering a broad range of cognitive domains. Most studies incorporated at least one measure of executive function/working memory, with nine reporting significant improvements in performance as a function of flavonoid supplementation compared to a control group. However, some domains were overlooked completely (e.g. implicit memory, prospective memory), and for the most part there was little consistency in terms of the particular cognitive tests used making across study comparisons difficult. Furthermore, there was some confusion concerning what aspects of cognitive function particular tests were actually measuring. Overall, while initial results are encouraging, future studies need to pay careful attention when selecting cognitive measures, especially in terms of ensuring that tasks are actually sensitive enough to detect treatment effects.

Isoflavones and endothelial function

Dietary isoflavones are thought to be cardioprotective due to their structural similarity to oestrogen. Oestrogen is believed to have beneficial effects on endothelial function and may be one of the mechanisms by which premenopausal women are protected against CVD. Decreased NO production and endothelial NO synthase activity, and increased endothelin-1 concentrations, impaired lipoprotein metabolism and increased circulating inflammatory factors result from oestrogen deficiency. Oestrogen acts by binding to oestrogen receptors alpha and beta. Isoflavones have been shown to bind with greater affinity to the latter. Oestrogen replacement therapy is no longer thought to be a safe treatment for prevention of CVD; isoflavones are a possible alternative. Limited evidence from human intervention studies suggests that isoflavones may improve endothelial function, but the available data are not conclusive. Animal studies provide stronger support for a role of isoflavones in the vasculature, with increased vasodilation and endothelial NO synthase activity demonstrated. Cellular mechanisms underlying the effects of isoflavones on endothelial cell function are not yet clear. Possible oestrogen receptor-mediated pathways include modulation of gene transcription, and also non-genomic oestrogen receptor-mediated signalling pathways. Putative non-oestrogenic pathways include inhibition of reactive oxygen species production and up regulation of the protein kinase A pathway (increasing NO bioavailability). Further research is needed to unravel effects of isoflavones on intracellular regulation of the endothelial function. Moreover, there is an urgent need for adequately powered, robustly designed human intervention studies in order to clarify the present equivocal findings.

Effect of genistein and daidzein on platelet aggregation and monocyte and endothelial function

There has been much recent interest in the cardiovascular benefits of dietary isoflavones. The aim of the present in vitro studies was to investigate potential anti-thrombogenic and anti-atherogenic effects of the isoflavones genistein and daidzein in platelets, macrophages and endothelial cells. Pre-treatment with either isoflavone inhibited collagen-induced platelet aggregation in a dose-dependent manner. In a macrophage cell line (RAW 264-7) activated with interferon gamma plus lipopolysaccharide, both isoflavones were found to inhibit NO production and tumour necrosis factor alpha (TNF-alpha) secretion dose-dependently, but they did not affect mRNA levels for inducible nitric oxide synthase and cyclo-oxygenase-2. Both isoflavones also dose-dependently decreased monocyte chemoattractant protein-1 secretion induced by TNF-alpha in human umbilical vein endothelial cells. Compared with daidzein, genistein exerted greater inhibitory effects for all parameters studied. The present data contributes to our knowledge on the molecular mechanisms by which isoflavones may protect against coronary artery disease. Further studies are required to determine whether the effects of isoflavones observed in the current in vitro studies are relevant to the aetiology of coronary artery disease in vivo.