Non-hemagglutinating flaviviruses: molecular mechanisms for the emergence of new strains via adaptation to European ticks

Tick-borne encephalitis virus (TBEV) causes human epidemics across Eurasia. Clinical manifestations range from inapparent infections and fevers to fatal encephalitis but the factors that determine disease severity are currently undefined. TBEV is characteristically a hemagglutinating (HA) virus; the ability to agglutinate erythrocytes tentatively reflects virion receptor/fusion activity. However, for the past few years many atypical HA-deficient strains have been isolated from patients and also from the natural European host tick, Ixodes persulcatus. By analysing the sequences of HA-deficient strains we have identified 3 unique amino acid substitutions (D67G, E122G or D277A) in the envelope protein, each of which increases the net charge and hydrophobicity of the virion surface. Therefore, we genetically engineered virus mutants each containing one of these 3 substitutions; they all exhibited HA-deficiency. Unexpectedly, each genetically modified non-HA virus demonstrated increased TBEV reproduction in feeding Ixodes ricinus, not the recognised tick host for these strains. Moreover, virus transmission efficiency between infected and uninfected ticks co-feeding on mice was also intensified by each substitution. Retrospectively, the mutation D67G was identified in viruses isolated from patients with encephalitis. We propose that the emergence of atypical Siberian HA-deficient TBEV strains in Europe is linked to their molecular adaptation to local ticks. This process appears to be driven by the selection of single mutations that change the virion surface thus enhancing receptor/fusion function essential for TBEV entry into the unfamiliar tick species. As the consequence of this adaptive mutagenesis, some of these mutations also appear to enhance the ability of TBEV to cross the human blood-brain barrier, a likely explanation for fatal encephalitis. Future research will reveal if these emerging Siberian TBEV strains continue to disperse westwards across Europe by adaptation to the indigenous tick species and if they are associated with severe forms of TBE.

Lipids and the immune response: from molecular mechanisms to clinical applications

Purpose of review This review critically evaluates recent studies investigating the effects of fatty acids on immune and inflammatory responses in both healthy individuals and in patients with inflammatory diseases, with some reference to animal studies where relevant. It examines recent findings describing the cellular and molecular basis for the modulation of immune function by fatty acids. The newly emerging area of diet-genotype interactions will also be discussed, with specific reference to the anti-inflammatory effects of fish oil. Recent findings Fatty acids are participants in many intracellular signalling pathways. They act as ligands for nuclear receptors regulating a host of cell responses, they influence the stability of lipid rafts, and modulate eicosanoid metabolism in cells of the immune system. Recent findings suggest that some or all of these mechanisms may be involved in the modulation of immune function by fatty acids. Summary Human studies investigating the relationship between dietary fatty acids and some aspects of the immune response have been disappointingly inconsistent. This review presents the argument that most studies have not been adequately powered to take into account the influence of variation (genotypic or otherwise) on parameters of immune function. There is well-documented evidence that fatty acids modulate T lymphocyte activation, and recent findings describe a range of potential cellular and molecular mechanisms. However, there are still many questions remaining, particularly with respect to the roles of nuclear receptors, for which fatty acids act as ligands, and the modulation of eicosanoid synthesis, for which fatty acids act as precursors.

Flavonoids and cognition: The molecular mechanisms underlying their behavioural effects

Evidence Suggests that a group of phytochemicals known as flavonoids are highly effective in reversing age-related declines in neuro-cognitive performance through their ability to interact with the cellular and molecular architecture of the brain responsible for memory and by reducing neuronal loss due to neurodegenerative Processes. In particular, they may increase the number of, and strength of, connections between neurons, via their specific interactions with the ERK and Akt signalling pathways, leading to an increase in neurotrophins Such as BDNF. Concurrently, their effects on the peripheral and Cerebral vascular system may also lead to enhancements in cognitive performance through increased brain blood flow and an ability to initiate neurogenesis in the hippocampus. Finally, they have also been shown to reduce neuronal damage and losses induced by various neurotoxic species and neuroinflammation. Together, these processes act to maintain the number and quality of synaptic connections in the brain. a factor known to be essential for efficient LTP, synaptic plasticity and ultimately the efficient working of memory. (C) 2009 Elsevier Inc. All rights reserved.

Molecular mechanisms by which dietary isoflavones potentially prevent atherosclerosis

Dietary isoflavones are currently receiving much attention because of their potential role in preventing coronary artery disease and other chronic diseases. Accumulating evidence from cell culture and laboratory animal experiments indicates that isoflavones have the potential to prevent or delay atherogenesis. Suggested mechanisms of action include: a reduction in low-density lipoprotein (LDL) cholesterol and a potential reduction in the susceptibility of the LDL particle to oxidation; (2) an improvement in vascular reactivity; (3) an inhibition of pro-inflammatory cytokines, cell adhesion proteins and nitric oxide (NO) production; and (4) an inhibition of platelet aggregation. These mechanisms are consistent with the epidemiological evidence that a high consumption of isoflavone-rich soy products is associated with a reduced incidence of coronary artery disease. Biological effects of isoflavones are dependent on many factors, including dose consumed, duration of use, protein-binding affinity, and an individual's metabolism or intrinsic oestrogenic state. Further clinical studies are necessary to determine the potential health effects of isoflavones in specific population groups as we currently know little about age-related differences in exposure to these compounds and there are few guidelines on optimal dose for cardiovascular health benefits.

Identification of hepatic molecular mechanisms of action of alpha-tocopherol using global gene expression profile analysis in rats

The recent discovery that vitamin E (VE) regulates gene activity at the transcriptional level indicates that VE may exert part of its biological effects by mechanisms which may be independent of its well-recognised antioxidant function. The objective of this study was the identification of hepatic vitamin E-sensitive genes and examination of the effects of VE on their corresponding biological endpoints. Two groups of male rats were randomly assigned to either a VE-sufficient diet or to a control diet deficient in VE for 290 days. High-density oligonucleotide microarrays comprising over 7000 genes were used to assess the transcriptional response of the liver. Differential gene expression was monitored over a period of 9 months, at four different time-points, and rats were individually profiled. This experimental strategy identified several VE-sensitive genes, which were chronically altered by dietary VE. VE supplementation down-regulated scavenger receptor CD36, coagulation factor IX and 5-alpha-steroid reductase type 1 mRNA levels while hepatic gamma glutamyl-cysteinyl synthetase was significantly up-regulated. Measurement of the corresponding biological endpoints such as activated partial thromboplastin time, plasma dihydrotestosterone and hepatic glutathione substantiated the gene chip data which indicated that dietary VE plays an important role in a range of metabolic processes within the liver. (C) 2004 Elsevier B.V. All rights reserved.

Molecular mechanisms of oestrogen action on growth of human breast cancer cells in culture

Growth responses to oestrogen can be reproducibly obtained using a selection of oestrogen-receptor-containing human breast cancer cell lines, and molecular mechanisms have been shown to include modulation to growth factor/receptor/signalling pathways, cell-cycle proteins, apoptosis, differentiation, adhesion, motility and migration. Considerable progress has been made in understanding the molecular basis of oestrogen action on gene expression through the ligand-activated transcription factors human oestrogen receptor α (ERα) and ERβ and the resulting effects on global gene expression patterns, but the full profile of coordination of the alterations, which brings about changes in cell growth through genomic and non-genomic mechanisms remain to be fully elucidated. Oestrogen regulation of cell growth involves a complex cross-talk between oestrogen receptor and growth factor signalling pathways such that inhibition of one pathway may lead to stimulation of another, which may explain the remarkable ability of human breast cancer cells to escape from any mode of imposed growth inhibition be it oestrogen deprivation or administration of antioestrogen. Although studies on cell growth have focused to date on the effects of physiological oestrogens, many hundreds of environmental chemicals with oestrogenic properties have now been measured in the human breast. Whether or not the weight of evidence eventually establishes any causal link of complex mixtures of environmental oestrogenic chemicals with breast cancer, the presence of so many oestrogenic chemicals in the breast must influence resulting oestrogenic responses, and the impact of this additional oestrogenic burden needs to be taken into account in future studies on growth regulation of human breast cancer cells.

Molecular mechanisms of crosstalk between thyroid hormones and estrogens

Purpose of review Novel analyses of the relations between thyroid hormone receptor signaling and estrogen receptor—dependent mechanisms are timely for two sets of reasons. Clinically, both affect mood and foster neuronal growth and regeneration. Mechanistically, they overlap at the levels of DNA recognition elements, coactivators, and signal transduction systems. Crosstalk between thyroid hormone receptors and estrogen receptors is possibly important to integrate external signals to transcription within neurons. Recent findings It has been shown that reproductive functions, including behaviors, driven by estrogens can be antagonized by thyroid hormones, and it has been argued that such crosstalk is biologically adaptive to ensure optimal reproduction. Transcriptional facilitation during transient transfunction studies show that the interactions between thyroid receptor isoforms and estrogen receptor isoforms depend on cell type and promoter context. Overall, this pattern of interactions assures multiple and flexible means of transcriptional regulation. Surprisingly, in some brain areas, thyroid hormone actions can synergize with estrogenic effects, particularly when nongenomic modes of action are considered, such as kinase activation, which, as has been reported, affect later estrogen receptor—induced genomic events. Summary In summary, recent work with nerve cells has contributed to a paradigm shift in how the molecular and behavioral effects of hormones which act through nuclear receptors are viewed.

Associations between dairy consumption and body weight: a review of the evidence and underlying mechanisms

As the incidence of obesity is reaching 'epidemic' proportions, there is currently widespread interest in the impact of dietary components on body-weight and food intake regulation. The majority of data available from both epidemiological and intervention studies provide evidence of a negative but modest association between milk and dairy product consumption and BMI and other measures of adiposity, with indications that higher intakes result in increased weight loss and lean tissue maintenance during energy restriction. The purported physiological and molecular mechanisms underlying the impact of dairy constituents on adiposity are incompletely understood but may include effects on lipolysis, lipogeneis and fatty acid absorption. Furthermore, accumulating evidence indicates an impact of dairy constituents, in particular whey protein derivatives, on appetite regulation and food intake. The present review summarises available data and provides an insight into the likely contribution of dairy foods to strategies aimed at appetite regulation, weight loss or the prevention of weight gain.

SARS coronavirus unique domain: three-domain molecular architecture in solution and RNA binding

Nonstructural protein 3 of the severe acute respiratory syndrome (SARS) coronavirus includes a "SARS-unique domain" (SUD) consisting of three globular domains separated by short linker peptide segments. This work reports NMR structure determinations of the C-terminal domain (SUD-C) and a two-domain construct (SUD-MC) containing the middle domain (SUD-M) and the C-terminal domain, and NMR data on the conformational states of the N-terminal domain (SUD-N) and the SUD-NM two-domain construct. Both SUD-N and SUD-NM are monomeric and globular in solution; in SUD-NM, there is high mobility in the two-residue interdomain linking sequence, with no preferred relative orientation of the two domains. SUD-C adopts a frataxin like fold and has structural similarity to DNA-binding domains of DNA-modifying enzymes. The structures of both SUD-M (previously determined) and SUD-C (from the present study) are maintained in SUD-MC, where the two domains are flexibly linked. Gel-shift experiments showed that both SUD-C and SUD-MC bind to single-stranded RNA and recognize purine bases more strongly than pyrimidine bases, whereby SUD-MC binds to a more restricted set of purine-containing RNA sequences than SUD-M. NMR chemical shift perturbation experiments with observations of (15)N-labeled proteins further resulted in delineation of RNA binding sites (i.e., in SUD-M, a positively charged surface area with a pronounced cavity, and in SUD-C, several residues of an anti-parallel beta-sheet). Overall, the present data provide evidence for molecular mechanisms involving the concerted actions of SUD-M and SUD-C, which result in specific RNA binding that might be unique to the SUD and, thus, to the SARS coronavirus.

Investigating the mechanisms underlying neuronal death in ischemia using in vitro oxygen-glucose deprivation: potential involvement of protein SUMOylation

It is well established that brain ischemia can cause neuronal death via different signaling cascades. The relative importance and interrelationships between these pathways, however, remain poorly understood. Here is presented an overview of studies using oxygen-glucose deprivation of organotypic hippocampal slice cultures to investigate the molecular mechanisms involved in ischemia. The culturing techniques, setup of the oxygen-glucose deprivation model, and analytical tools are reviewed. The authors focus on SUMOylation, a posttranslational protein modification that has recently been implicated in ischemia from whole animal studies as an example of how these powerful tools can be applied and could be of interest to investigate the molecular pathways underlying ischemic cell death.

Detection and molecular characterisation of Pyrenopeziza brassicae isolates resistant to methyl benzimidazole carbamates

BACKGROUND Methyl benzimidazole carbamate (MBC) fungicides are used to control the oilseed rape pathogen Pyrenopeziza brassicae. Resistance to MBCs has been reported in P. brassicae, but the molecular mechanism(s) associated with reductions in sensitivity have not been verified in this species. Elucidation of the genetic changes responsible for resistance, hypothesised to be target-site mutations in β-tubulin, will enable resistance diagnostics and thereby inform resistance management strategies. RESULTS P. brassicae isolates were classified as sensitive, moderately resistant or resistant to MBCs. Crossing P. brassicae isolates of different MBC sensitivities indicated that resistance was conferred by a single gene. The MBC-target encoding gene β-tubulin was cloned and sequenced. Reduced MBC sensitivity of field isolates correlated with β-tubulin amino acid substitutions L240F and E198A. The highest level of MBC resistance was measured for isolates carrying E198A. Negative cross-resistance between MBCs and the fungicides diethofencarb and zoxamide was only measured in E198A isolates. PCR-RFLP was used to screen isolates for the presence of L240F and E198A. The substitutions E198G and F200Y were also detected in DNA samples from P. brassicae populations after cloning and sequencing of PCR products. The frequencies of L240F and E198A in different P. brassicae populations were quantified by pyrosequencing. There were no differences in the frequencies of these alleles between P. brassicae populations sampled from different locations or after fungicide treatment regimes. CONCLUSIONS The molecular mechanisms affecting sensitivity to MBCs in P. brassicae have been identified. Pyrosequencing assays are a powerful tool for quantifying fungicide-resistant alleles in pathogen populations.

The stimulation of mitogenic signaling pathways by N-POMC peptides

The N-terminal fragment of pro-opiomelancortin (POMC) has been shown previously to act as an adrenal mitogen. However, little is known about the molecular mechanisms by which mitogenesis is stimulated, although it has been shown that N-POMC1-28 Stimulates the ERK pathway in human H295R cells. We have investigated signaling stimulated by N-POMC1-28 and N-POMC1-49 in the mouse Y1 cell line and found that both peptides stimulate ERK phosphorylation with maximal stimulation being achieved within 5 min. Similar results were observed for both MEK and c-Raf phosphorylation, although N-POMC1-49 stimulated the phosphorylation of Akt more robustly than N-POMC1-28. We also investigated the expression of tyrosine kinase receptors in adrenal cells. PCR utilizing degenerate primers was performed on cDNA from both Y1 cells and rat adrenal tissue. Sequencing of 114 clones from each cDNA population revealed the expression of a number of receptors, several of which have not been described previously in the adrenal. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Use of global gene expression patterns in mechanistic studies of oestrogen action in MCF7 human breast cancer cells

Over the years, the MCF7 human breast cancer cell line has provided a model system for the study of cellular and molecular mechanisms in oestrogen regulation of cell proliferation and in progression to oestrogen and antioestrogen independent growth. Global gene expression profiling has shown that oestrogen action in MCF7 cells involves the coordinated regulation of hundreds of genes across a wide range of functional groupings and that more genes are down regulated than upregulated. Adaptation to long-term oestrogen deprivation, which results in loss of oestrogen-responsive growth, involves alterations to gene patterns not only at early time points (0-4 weeks) but continuing through to later times (20-55 weeks), and even involves alterations to patterns of oestrogen-regulated gene expression. Only 48% of the genes which were regulated >= 2-fold by oestradiol in oestrogen-responsive cells retained this responsiveness after long-term oestrogen deprivation but other genes developed de novo oestrogen regulation. Long-term exposure to fulvestrant, which resulted in loss of growth inhibition by the antioestrogen, resulted in some very large fold changes in gene expression up to 10,000-fold. Comparison of gene profiles produced by environmental chemicals with oestrogenic properties showed that each ligand gave its own unique expression profile which suggests that environmental oestrogens entering the human breast may give rise to a more complex web of interference in cell function than simply mimicking oestrogen action at inappropriate times. (C) 2009 Elsevier Ltd. All rights reserved.