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.

Molecular characterisation of the gut microflora of healthy and inflammatory bowel disease cats using fluorescence in situ hybridisation with special reference to Desulfovibrio spp

Inflammatory bowel disease (IBD) is a common cause of chronic large bowel diarrhoea in cats. Although the aetiology of IBD is unknown, an immune-mediated response to a luminal antigen is thought to be involved. As knowledge concerning the colonic microflora of cats is limited and requires further investigation, the purpose of this study was to determine the presence of specific bacterial groups in normal and IBD cats, and the potential role they play in the health of the host. Total bacterial populations, Bacteroides spp., Bifidobacterium spp., Clostridium histolyticum subgp., Lactobacillus-Enterococcus subgp. and Desulfovibrio spp. were enumerated in 34 healthy cats and 11 IBD cats using fluorescence in situ hybridisation. The study is one of the first to show the presence of Desulfovibrio in cats. Total bacteria, Bifidobacterium spp. and Bacteroides spp. counts were all significantly higher in healthy cats when compared with IBD cats, whereas Desulfovibrio spp. (producers of toxic sulphides) numbers were found to be significantly higher in colitic cats. The information obtained from this study suggests that modulation of bacterial flora by increasing bifidobacteria and decreasing Desulfovibrio spp. may be beneficial to cats with IBD. Dietary intervention may be an important aspect of their treatment.

Effects of glucose, propionate and splanchnic hormones on neuropeptide mRNA concentrations in the ovine hypothalamus

The capacity for glucose, propionate or hormones of splanchnic origin to influence appetite by directly regulating the expression of neuropeptides in the feeding centres of the hypothalamus of the ruminant is not described. Therefore, our objective was to measure the direct effect of metabolites (glucose and propionate) or hormones [insulin, cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1) and polypeptide YY (PYY)] on hypothalamic mRNA concentrations for neuropeptide Y (NPY), agouti-related peptide (AgRP) and proopiomelanocortin (POMC) following in vitro incubation. Hypothalamic tissue from 4- to 5-month-old lambs was obtained at slaughter and immediately incubated in culture media for 2 h at 36 °C. Treatments included a control Dulbecco’s modified Eagle medium (DMEM) containing 1 mm glucose or DMEM with the following additions: 10 mm glucose, 1 mm propionate, 1 nm insulin, 120 pm GLP-1, 100 pm PYY, 80 pm CCK or 10 mm glucose plus 1 nm insulin. The abundance of mRNA for NPY, AgRP and POMC was measured using quantitative reverse transcriptase PCR. Fisher’s protected LSD test was used to compare changes in relative mRNA concentrations for the hypothalamus incubated in the control media vs. the rest of the treatments. The media containing glucose plus insulin increased POMC mRNA concentration (p < 0.05), but did not affect NPY or AgRP mRNA concentration. There were no effects observed for the other treatments (p > 0.20). Results of the present study are consistent with the concept that effects of propionate on feed intake in ruminants is not mediated through direct effects on the hypothalamus, and that insulin is required for an effect of glucose on hypothalamic POMC expression.

Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor

Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with beta-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK(1)R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK(1)R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca(2+) signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK(1)R. SP induced association of beta-arrestin1 and PP2A with noninternalized NK(1)R. beta-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK(1)R with PP2A, indicating that beta-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping beta-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK(1)R with PP2A. Resensitization of NK(1)R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK(1)R and mediates resensitization. PP2A interaction with NK(1)R requires beta-arrestin1. ECE-1 promotes this process by releasing beta-arrestin1 from NK(1)R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs.

Impact of variation in structure of condensed tannins from sainfoin (Onobrychis viciifolia) on in vitro ruminal methan production and fermentation characteristics

Our study investigated the effects of condensed tannins (CT) on rumen in vitro methane (CH4) production and fermentation characteristics by incubating lucerne in buffered rumen fluid in combination with different CT extracts at 0 (control), 40, 80 and 120 g CT/kg of substrate DM. Condensed tannins were extracted from four sainfoin accessions: Rees ‘A’, CPI63763, Cotswold Common and CPI63767. Gas production (GP) was measured using a fully automated GP apparatus with CH4 measured at distinct time points. Condensed tannins differed substantially in terms of polymer size and varied from 13 (Rees ‘A’) to 73 (CPI63767) mean degree of polymerization, but had relatively similar characteristics in terms of CT content, procyanidin: prodelphinidin (PC: PD) and cis:trans ratios. Compared to control, addition of CT from CPI63767 and CPI63763 at 80 and 120 g CT/kg of substrate DM reduced CH4 by 43% and 65%, and by 23% and 57%, respectively, after 24-h incubation. Similarly, CT from Rees ‘A’ and Cotswold Common reduced CH4 by 26% and 46%, and by 28% and 46% respectively. Addition of increasing level of CT linearly reduced the maximum rates of GP and CH4 production, and the estimated in vitro organic matter digestibility. There was a negative linear and quadratic (p < 0.01) relation between CT concentration and total volatile fatty acid (VFA) production. Inclusion of 80 and 120 g CT/kg of substrate DM reduced (p < 0.001) branched-chain VFA production and acetate: propionate ratio and was lowest for CPI63767. A decrease in proteolytic activity as indirectly shown by a change in VFA composition favouring a shift towards propionate and reduction in branched-chain VFA production varied with type of CT and was highest for CPI63767. In conclusion, these results suggest that tannin polymer size is an important factor affecting in vitro CH4 production which may be linked to the CT interaction with dietary substrate or microbial cells.

Efficient animal-serum free 3D cultivation method for adult human neural crest-derived stem cell therapeutics

Due to their broad differentiation potential and their persistence into adulthood, human neural crest-derived stem cells (NCSCs) harbour great potential for autologous cellular therapies, which include the treatment of neurodegenerative diseases and replacement of complex tissues containing various cell types, as in the case of musculoskeletal injuries. The use of serum-free approaches often results in insufficient proliferation of stem cells and foetal calf serum implicates the use of xenogenic medium components. Thus, there is much need for alternative cultivation strategies. In this study we describe for the first time a novel, human blood plasma based semi-solid medium for cultivation of human NCSCs. We cultivated human neural crest-derived inferior turbinate stem cells (ITSCs) within a blood plasma matrix, where they revealed higher proliferation rates compared to a standard serum-free approach. Three-dimensionality of the matrix was investigated using helium ion microscopy. ITSCs grew within the matrix as revealed by laser scanning microscopy. Genetic stability and maintenance of stemness characteristics were assured in 3D cultivated ITSCs, as demonstrated by unchanged expression profile and the capability for self-renewal. ITSCs pre-cultivated in the 3D matrix differentiated efficiently into ectodermal and mesodermal cell types, particularly including osteogenic cell types. Furthermore, ITSCs cultivated as described here could be easily infected with lentiviruses directly in substrate for potential tracing or gene therapeutic approaches. Taken together, the use of human blood plasma as an additive for a completely defined medium points towards a personalisable and autologous cultivation of human neural crest-derived stem cells under clinical grade conditions.

Targeting the cell cycle machinery for the treatment of cardiovascular disease

Cardiovascular disease represents a major clinical problem affecting a significant proportion of the world's population and remains the main cause of death in the UK. The majority of therapies currently available for the treatment of cardiovascular disease do not cure the problem but merely treat the symptoms. Furthermore, many cardioactive drugs have serious side effects and have narrow therapeutic windows that can limit their usefulness in the clinic. Thus, the development of more selective and highly effective therapeutic strategies that could cure specific cardiovascular diseases would be of enormous benefit both to the patient and to those countries where healthcare systems are responsible for an increasing number of patients. In this review, we discuss the evidence that suggests that targeting the cell cycle machinery in cardiovascular cells provides a novel strategy for the treatment of certain cardiovascular diseases. Those cell cycle molecules that are important for regulating terminal differentiation of cardiac myocytes and whether they can be targeted to reinitiate cell division and myocardial repair will be discussed as will the molecules that control vascular smooth muscle cell (VSMC) and endothelial cell proliferation in disorders such as atherosclerosis and restenosis. The main approaches currently used to target the cell cycle machinery in cardiovascular disease have employed gene therapy techniques. We will overview the different methods and routes of gene delivery to the cardiovascular system and describe possible future drug therapies for these disorders. Although the majority of the published data comes from animal studies, there are several instances where potential therapies have moved into the clinical setting with promising results.

Effects of cis-9,trans-11 and trans-1 0,cis-12 conjugated linoleic acid on immune cell function in healthy humans

Background: Animal studies have suggested that conjugated linoleic acid (CLA), a natural component of ruminant meat and dairy products, may confer beneficial effects on health. However, little information on the effects of CLA on immune function is available, especially in humans. Furthermore, the effects of individual isomers of CLA have not been adequately investigated. Objective: This study investigated the effects of supplementing the diet with 3 doses of highly enriched cis-9,trans-11 CLA (0.59, 1.19, and 2.38 g/d) or trans-10,cis-12 CLA (0.63, 1.26, and 2.52 g/d) on immune outcomes in healthy humans. Design: The study had a randomized, double-blind, crossover design. Healthy men consumed 1, 2, and 4 capsules sequentially that contained 80% of either cis-9,trans-11 CLA or trans-10,cis-12 CLA for consecutive 8-wk periods. This regimen was followed by a 6-wk washout and a crossover to the other isomer. Results: Both CLA isomers decreased mitogen-induced T lymphocyte activation in a dose-dependent manner. There was a significant negative correlation between mitogen-induced T lymphocyte activation and the proportions of both cis-9,trans-11 CLA and trans-10,cis-12 CLA in peripheral blood mononuclear cell lipids. However, CLA did not affect lymphocyte subpopulations or serum concentrations of C-reactive protein and did not have any consistent effects on ex vivo cytokine production. Conclusion: CLA supplementation results in a dose-dependent reduction in the mitogen-induced activation of T lymphocytes. The effects of cis-9,trans-l I CLA and trans-10,cis-12 CLA were similar, and there was a negative correlation between mitogen-induced T lymphocyte activation and the cis-9,trans-11 CLA and trans-10,cis-12 CLA contents of mononuclear cells.

Permanent changes in growth and physiology of Bacillus subtilis ToC46 after loss of a recombinant plasmid

The physiology and growth of plasmid-bearing Bacillus subtilis carrying plasmid pPFF1, the non-transformed host, and cells after loss of the plasmid (so-called plasmid-cured cells) were investigated. It was found that, following plasmid loss, cells exhibited phenotypic characteristics different from those of the non-transformed host strains. Compared to plasmid-bearing cells and non-transformed host cells, an approximate 25% increase in the maximum specific growth rate and a more rapid increase in total RNA per unit cell mass were observed in plasmid-cured cells. The total enthalpy associated with irreversible denaturation events was determined in whole cells by differential scanning calorimetry. This showed higher enthalpies for plasmid-cured cells compared with the non-transformed host, which suggests increased ribosome numbers. The result from cellular DNA hybridisation suggests that there was no direct evidence of plasmid integration into the host chromosome. (C) 2004 Elsevier Inc. All rights reserved.

Architecture for neuronal cell control of a mobile robot

It is usually expected that the intelligent controlling mechanism of a robot is a computer system. Research is however now ongoing in which biological neural networks are being cultured and trained to act as the brain of an interactive real world robot - thereby either completely replacing or operating in a cooperative fashion with a computer system. Studying such neural systems can give a distinct insight into biological neural structures and therefore such research has immediate medical implications. In particular, the use of rodent primary dissociated cultured neuronal networks for the control of mobile `animals' (artificial animals, a contraction of animal and materials) is a novel approach to discovering the computational capabilities of networks of biological neurones. A dissociated culture of this nature requires appropriate embodiment in some form, to enable appropriate development in a controlled environment within which appropriate stimuli may be received via sensory data but ultimate influence over motor actions retained. The principal aims of the present research are to assess the computational and learning capacity of dissociated cultured neuronal networks with a view to advancing network level processing of artificial neural networks. This will be approached by the creation of an artificial hybrid system (animal) involving closed loop control of a mobile robot by a dissociated culture of rat neurons. This 'closed loop' interaction with the environment through both sensing and effecting will enable investigation of its learning capacity This paper details the components of the overall animat closed loop system and reports on the evaluation of the results from the experiments being carried out with regard to robot behaviour.

Spatial and temporal effects of free-air CO2 enrichment (POPFACE) on leaf growth, cell expansion and cell production in a closed canopy of poplar

Leaf expansion in the fast-growing tree,Populus × euramericana was stimulated by elevated [CO2] in a closed-canopy forest plantation, exposed using a free air CO2 enrichment technique enabling long-term experimentation in field conditions. The effects of elevated [CO2] over time were characterized and related to the leaf plastochron index (LPI), and showed that leaf expansion was stimulated at very early (LPI, 0–3) and late (LPI, 6–8) stages in development. Early and late effects of elevated [CO2] were largely the result of increased cell expansion and increased cell production, respectively. Spatial effects of elevated [CO2] were also marked and increased final leaf size resulted from an effect on leaf area, but not leaf length, demonstrating changed leaf shape in response to [CO2]. Leaves exhibited a basipetal gradient of leaf development, investigated by defining seven interveinal areas, with growth ceasing first at the leaf tip. Interestingly, and in contrast to other reports, no spatial differences in epidermal cell size were apparent across the lamina, whereas a clear basipetal gradient in cell production rate was found. These data suggest that the rate and timing of cell production was more important in determining leaf shape, given the constant cell size across the leaf lamina. The effect of elevated [CO2] imposed on this developmental gradient suggested that leaf cell production continued longer in elevated [CO2] and that basal increases in cell production rate were also more important than altered cell expansion for increased final leaf size and altered leaf shape in elevated [CO2].

Development of an ex vivo human skin model for intradermal vaccination : tissue viability and Langerhans cell behaviour

The presence of resident Langerhans cells (LCs) in the epidermis makes the skin an attractive target for DNA vaccination. However, reliable animal models for cutaneous vaccination studies are limited. We demonstrate an ex vivo human skin model for cutaneous DNA vaccination which can potentially bridge the gap between pre-clinical in vivo animal models and clinical studies. Cutaneous transgene expression was utilised to demonstrate epidermal tissue viability in culture. LC response to the culture environment was monitored by immunohistochemistry. Full-thickness and split-thickness skin remained genetically viable in culture for at least 72 h in both phosphate-buffered saline (PBS) and full organ culture medium (OCM). The epidermis of explants cultured in OCM remained morphologically intact throughout the culture duration. LCs in full-thickness skin exhibited a delayed response (reduction in cell number and increase in cell size) to the culture conditions compared with split-thickness skin, whose response was immediate. In conclusion, excised human skin can be cultured for a minimum of 72 h for analysis of gene expression and immune cell activation. However, the use of split-thickness skin for vaccine formulation studies may not be appropriate because of the nature of the activation. Full-thickness skin explants are a more suitable model to assess cutaneous vaccination ex vivo.

Influence of substrate on corneal epithelial cell viability within ocular surface models

Corneal tissue engineering has improved dramatically over recent years. It is now possible to apply these technological advancements to the development of superior in vitro ocular surface models to reduce animal testing. We aim to show the effect different substrates can have on the viability of expanded corneal epithelial cells and that those which more accurately mimic the stromal surface provide the most protection against toxic assault. Compressed collagen gel as a substrate for the expansion of a human epithelial cell line was compared against two well-known substrates for modeling the ocular surface (polycarbonate membrane and conventional collagen gel). Cells were expanded over 10 days at which point cell stratification, cell number and expression of junctional proteins were assessed by electron microscopy, immunohistochemistry and RT-PCR. The effect of increasing concentrations of sodium lauryl sulphate on epithelial cell viability was quantified by MTT assay. Results showed improvement in terms of stratification, cell number and tight junction expression in human epithelial cells expanded upon either the polycarbonate membrane or compressed collagen gel when compared to a the use of a conventional collagen gel. However, cell viability was significantly higher in cells expanded upon the compressed collagen gel. We conclude that the more naturalistic composition and mechanical properties of compressed collagen gels produces a more robust corneal model.

Cell walls of developing wheat starchy endosperm: comparison of composition and RNA-Seq transcriptome

The transcriptome of the developing starchy endosperm of hexaploid wheat (Triticum aestivum) was determined using RNA-Seq isolated at five stages during grain fill. This resource represents an excellent way to identify candidate genes responsible for the starchy endosperm cell wall, which is dominated by arabinoxylan (AX), accounting for 70% of the cell wall polysaccharides, with 20% (1,3; 1,4)-beta-D-glucan, 7% glucomannan, and 4% cellulose. A complete inventory of transcripts of 124 glycosyltransferase (GT) and 72 glycosylhydrolase (GH) genes associated with cell walls is presented. The most highly expressed GT transcript (excluding those known to be involved in starch synthesis) was a GT47 family transcript similar to Arabidopsis (Arabidopsis thaliana) IRX10 involved in xylan extension, and the second most abundant was a GT61. Profiles for GT43 IRX9 and IRX14 putative orthologs were consistent with roles in AX synthesis. Low abundances were found for transcripts from genes in the acyl-coA transferase BAHD family, for which a role in AX feruloylation has been postulated. The relative expression of these was much greater in whole grain compared with starchy endosperm, correlating with the levels of bound ferulate. Transcripts associated with callose (GSL), cellulose (CESA), pectin (GAUT), and glucomannan (CSLA) synthesis were also abundant in starchy endosperm, while the corresponding cell wall polysaccharides were confirmed as low abundance (glucomannan and callose) or undetectable (pectin) in these samples. Abundant transcripts from GH families associated with the hydrolysis of these polysaccharides were also present, suggesting that they may be rapidly turned over. Abundant transcripts in the GT31 family may be responsible for the addition of Gal residues to arabinogalactan peptide.

Interaction between attaching and effacing Escherichia coli serotypes O157:H7 and O26:K60 in cell culture

Ruminants harbour both O157:H7 and non-O157 Attaching Effacing Escherichia coli (AEEC) strains but to date only nonO157 AEEC have been shown to induce attaching effacing lesions in naturally infected animals. However, O157 may induce lesions in deliberate oral inoculation studies and persistence is considered dependent upon the bacterially encoded locus for enterocyte effacement. In concurrent infections in ruminants it is unclear whether non-O157 AEEC contribute either positively or negatively to the persistence of E. coli O157:H7. To investigate this, and prior to animal studies, E. coli O157:H7 NCTC 12900, a non-toxigenic strain that persists in conventionally reared sheep, and non-toxigenic AEEC O26:K60 isolates of sheep origin were tested for adherence to Hep-2 tissue culture alone and in competition one with another. Applied together, both strains adhered in similar numbers but lower than when either was applied separately. Pre-incubation of tissue culture with either one strain reduced significantly (P < 0.05) the extent of adherence of the strain that was applied second. It was particularly noticeable that AEEC O26 when applied first reduced adherence and inhibited microcolony formation, as demonstrated by confocal microscopy, of E. coli 01 57:H7. The possibility that prior colonisation of a ruminant by non-O157 AEEC such as O26 may antagonise O157 colonisation and persistence in ruminants is discussed. (C) 2004 Elsevier B.V. All rights reserved.