Secretory products from human adipocytes stimulate proinflammatory cytokine secretion from human endothelial cells

Hyperplasia and hypertrophy of fat cells can be found in obesity and increased adiposity is associated with endothelial dysfunction as an early event of atherosclerosis. However, it is unclear whether human adipocytes directly influence endothelial protein secretion. To study the crosstalk between fat and endothelial cells, human umbilical venous endothelial cells (HUVECs) were cultured in infranatants (Adipo) of primary differentiated human adipocytes. Interestingly, significantly increased secretion of 23 cytokines and chemokines from HUVECs was detected in four independent experiments after Adipo stimulation by protein array analysis detecting a total of 174 different proteins. Among those, time‐dependent Adipo‐induced upregulation of cytokine secretion in HUVECs was confirmed by ELISA for interleukin (IL)‐8, monokine induced by gamma interferon, macrophage inflammatory protein (MIP)‐1β, MIP‐3α, monocyte chemoattractant protein‐1, and IL‐6. Factors besides adiponectin, leptin, resistin, and tumor necrosis factor α appear to mediate these stimulatory effects. Our findings suggest that endothelial cell secretion is significantly influenced towards a proinflammatory pattern by adipocyte‐secreted factors. J. Cell. Biochem. 106: 729–737, 2009. © 2009 Wiley‐Liss, Inc.     

Prediction of helix-helix contacts and interacting helices in polytopic membrane proteins using neural networks

Despite rapidly increasing numbers of available 3D structures, membrane proteins still account for less than 1% of all structures in the Protein Data Bank. Recent high-resolution structures indicate a clearly broader structural diversity of membrane proteins than initially anticipated, motivating the development of reliable structure prediction methods specifically tailored for this class of molecules. One important prediction target capturing all major aspects of a protein's 3D structure is its contact map. Our analysis shows that computational methods trained to predict residue contacts in globular proteins perform poorly when applied to membrane proteins. We have recently published a method to identify interacting alpha-helices in membrane proteins based on the analysis of coevolving residues in predicted transmembrane regions. Here, we present a substantially improved algorithm for the same problem, which uses a newly developed neural network approach to predict helix-helix contacts. In addition to the input features commonly used for contact prediction of soluble proteins, such as windowed residue profiles and residue distance in the sequence, our network also incorporates features that apply to membrane proteins only, such as residue position within the transmembrane segment and its orientation toward the lipophilic environment. The obtained neural network can predict contacts between residues in transmembrane segments with nearly 26% accuracy. It is therefore the first published contact predictor developed specifically for membrane proteins performing with equal accuracy to state-of-the-art contact predictors available for soluble proteins. The predicted helix-helix contacts were employed in a second step to identify interacting helices. For our dataset consisting of 62 membrane proteins of solved structure, we gained an accuracy of 78.1%. Because the reliable prediction of helix interaction patterns is an important step in the classification and prediction of membrane protein folds, our method will be a helpful tool in compiling a structural census of membrane proteins.     

Analyzing plant defenses in nature

A broad range of chemical plant defenses against herbivores has been studied extensively under laboratory conditions. In many of these cases there is still little understanding of their relevance in nature. In natural systems, functional analyses of plant traits are often complicated by an extreme variability, which affects the interaction with higher trophic levels. Successful analyses require consideration of the numerous sources of variation that potentially affect the plant trait of interest. In our recent study on wild lima bean (Phaseolus lunatus L.) in South Mexico, we applied an integrative approach combining analyses for quantitative correlations of cyanogenic potential (HCNp; the maximum amount of cyanide that can be released from a given tissue) and herbivory in the field with subsequent feeding trials under controlled conditions. This approach allowed us to causally explain the consequences of quantitative variation of HCNp on herbivore-plant interactions in nature and highlights the importance of combining data obtained in natural systems with analyses under controlled conditions.Key words: natural systems, plant defensive traits, optimal defense hypothesis (ODH), cyanogenesis, lima bean, Phaseolus lunatus L., plant-herbivore interaction, plant-pathogen interaction, multiple defense syndromesAnalyzing plant defenses against herbivores in nature is often complicated by an extreme variability in multiple factors. Plant populations generally show high genetic variability resulting in substantial intraspecific variation of plant traits.¹ In addition to genotypic variability, phenotypic plasticity of plants is a source of variation.² At the level of individual plants, expression of defensive traits strongly depends on plant organ and ontogeny of plants or plant parts. Within an individual plant, it is quite common for reproductive structures and young leaves to be better chemically defended than older leaf tissues. To explain these within-plant variations of defenses, the optimal defense hypothesis (ODH) was formulated. Concerning the variability of chemical defenses of leaves, the ODH predicts that within the total foliage of a plant, young leaves make a larger contribution to plant fitness than old leaves as they have a higher potential photosynthetic value resulting from a longer expected life-time.^3–5 In addition, younger leaves are often more nutritious and thus more attractive to herbivores⁶ and should consequently be better defended.⁷ In this line, the basic assumption of the ODH is that three main factors—cost of defense, risk of attack and value of the respective plant organ—determine the investment in defensive secondary metabolites.^8,9 Thus, the higher the risk of a given plant tissue to be consumed by herbivores and the higher its value for plant fitness, the more energy should be allocated to its defense.^10,11 Beyond genotypic and ontogenetic variability of a given defense, potential co-variation with other defensive or nutritive traits expressed by the same plant individual can strongly affect its efficiency as defense against herbivores.^12,13 In addition to these endogenous sources of defensive variability, the expression of plant traits strongly depends on multiple external factors such as temperature or availability of plant nutrients, water or light (Fig. 1).¹⁴ At the same time, the outcome of herbivore-plant interactions is crucially determined by biotic interactions. Plant interactions with mutualistic microorganisms such as Rhizobia, mycorrhiza and above-ground fungal endophytes as well as tri-trophic interactions with predators and parasitoids of herbivores can all strongly impact plant fitness.¹⁵Open in a separate windowFigure 1Factors influencing variability of plant defenses. Plant defensive traits are affected by various endogenous and external factors. Endogenous factors comprise plant genotype and ontogeny of plants or plant parts. External factors can be categorized as abiotic or biotic. Important abiotic factors that can influence plant defenses are light exposure, temperature, soil salinity, as well as water and nutrient availability. Biotic factors that can have an effect on plant defense are interspecific interaction with Rhizobia (in the case of legumes), mycorrhizal and endophytic fungi, pathogens as well as the interaction with conspecifics or different plant species.Variability in herbivore-plant interactions can also be associated with herbivore variation. Different attackers of a particular plant species might be affected in different ways by toxins in food plants (Fig. 1). The efficiency of a specific defensive compound can also depend on the feeding mode, i.e., sucking or chewing, as well as on the degree of specialization of the herbivore to the respective plant.¹⁶ Defenses mediated by secondary plant compounds are generally believed not to affect specialist herbivores, because of their capacity to tolerate or to detoxify defensive compounds of their hosts by behavioral or physiological adaptations.^17–20 In this context, the specialist herbivore paradigm predicts that adapted herbivores are less affected by a given chemical defense than generalists,^21,22 although exceptions have been noted.^23–25While it is important to consider these numerous sources of variation affecting the outcome of herbivore-plant interactions when designing functional studies, a significant fraction of the variability in natural systems will always remain unidentified. Consequently, approaches combining field observations with experiments under controlled conditions provide a powerful tool to uncover functional interactions between plants and their multiple antagonists in nature.In a recent study, we analyzed the importance of wild lima bean''s cyanogenesis—i.e., the release of toxic hydrogen cyanide from preformed precursors in response to cell damage—as plant defense at a natural site in South Mexico.²⁵ Although cyanogenesis is generally considered an efficient direct defense against herbivores, in numerous studies plant cyanide production had little or no effect on herbivores.^26–28 One would like to think that most of these inconsistencies in cyanogenesis-based herbivore defense efficiency could be explained by one or more sources of variation mentioned above. Nevertheless, field studies analyzing the action of plant cyanogenesis on a quantitative basis have been scarce. In our study, a two-step approach was used to gain insight into the function of cyanogenesis in nature.²⁵ First, cyanide concentration and herbivore damage were quantified by measuring removed leaf area of individual leaves derived from different individual plants while considering microclimate conditions. Significant negative correlations between cyanogenesis and leaf damage were observed. Second, since existing correlations do not necessarily indicate causal associations, we conducted consecutive feeding experiments under controlled conditions. To consider natural variability of lima beans'' cyanogenesis observed in nature in our analysis, we prepared clones from field-grown plants with different but defined cyanogenic features. These clonal plants showed high constancy of cyanogenic traits compared to their respective mother plants and thus, could be used in comparative analyses. Every effort was made to duplicate natural conditions and so herbivore species selected for feeding trials represented those identified in the field as the most important plant consumers at the respective site (pers. observ.). Feeding trials supported our hypothesis that cyanogenesis has quantitative effects on herbivore behavior in nature and explained the negative correlation of lima bean''s cyanogenesis and herbivory observed in the field.Analytical approaches combining field observations with controlled experiments help to explain natural patterns and may represent a powerful methodological approach for functional analyses of herbivore-plant interactions.     

Monitoring,management, and mitigation of Karenia blooms in the eastern Gulf of Mexico

Annual blooms of the toxic dinoflagellate Karenia brevis in the eastern Gulf of Mexico represent one of the most predictable global harmful algal bloom (HAB) events, yet remain amongst the most difficult HABs to effectively monitor for human and environmental health. Monitoring of Karenia blooms is necessary for a variety of precautionary, management and predictive purposes. These include the protection of public health from exposure to aerosolized brevetoxins and the consumption of toxic shellfish, the protection and management of environmental resources, the prevention of bloom associated economic losses, and the evaluation of long term ecosystem trends and for potential future bloom forecasting and prediction purposes. The multipurpose nature of Karenia monitoring, the large areas over which blooms occur, the large range of Karenia cell concentrations (from 5 × 10³ cells L^?1 to >1 × 10⁶ cells L^?1) over which multiple bloom impacts are possible, and limitations in resources and knowledge of bloom ecology have complicated K. brevis monitoring, mitigation and management strategies. Historically, K. brevis blooms were informally and intermittently monitored on an event response basis in Florida, usually in the later bloom stages after impacts (e.g. fish kills, marine mammal mortalities, respiratory irritation) were noted and when resources were available. Monitoring of different K. brevis bloom stages remains the most practical method for predicting human health impacts and is currently accomplished by the state of Florida via direct microscopic counts of water samples from a state coordinated volunteer HAB monitoring program. K. brevis cell concentrations are mapped weekly and disseminated to stakeholders via e-mail, web and toll-free phone numbers and provided to Florida Department of Agriculture and Consumer Services (FDACS) for management of both recreational and commercial shellfish beds in Florida and to the National Oceanic and Atmospheric Administration (NOAA) for validation of the NOAA Gulf of Mexico HAB bulletin for provision to environmental managers. Many challenges remain for effective monitoring and management of Karenia blooms, however, including incorporating impact specific monitoring for the diverse array of potential human and environmental impacts associated with blooms, timely detection of offshore bloom initiation, sampling of the large geographic extent of blooms which often covers multiple state boundaries, and the involvement of multiple Karenia species other than K. brevis (several of which have yet to be isolated and described) with unknown toxin profiles. The implementation and integration of a diverse array of optical, molecular and hybrid Karenia detection technologies currently under development into appropriate regulatory and non-regulatory monitoring formats represents a further unique challenge.     

Short-term activation induces multifunctional dendritic cells that generate potent antitumor T-cell responses in vivo

Dendritic cell (DC) vaccines have emerged as a promising strategy to induce antitumoral cytotoxic T cells for the immunotherapy of cancer. The maturation state of DC is of critical importance for the success of vaccination, but the most effective mode of maturation is still a matter of debate. Whereas immature DC carry the risk of inducing tolerance, extensive stimulation of DC may lead to DC unresponsiveness and exhaustion. In this study, we investigated how short-term versus long-term DC activation with a Toll-like receptor 9 agonist influences DC phenotype and function. Murine DC were generated in the presence of the hematopoietic factor Flt3L (FL-DC) to obtain both myeloid and plasmacytoid DC subsets. Short activation of FL-DC for as little as 4 h induced fully functional DC that rapidly secreted IL-12p70 and IFN-α, expressed high levels of costimulatory and MHC molecules and efficiently presented antigen to CD4 and CD8 T cells. Furthermore, short-term activated FL-DC overcame immune suppression by regulatory T cells and acquired high migratory potential toward the chemokine CCL21 necessary for DC recruitment to lymph nodes. In addition, vaccination with short-term activated DC induced a strong cytotoxic T-cell response in vivo and led to the eradication of tumors. Thus, short-term activation of DC generates fully functional DC for tumor immunotherapy. These results may guide the design of new protocols for DC generation in order to develop more efficient DC-based tumor vaccines.     

Cell adaptation to aneuploidy by the environmental stress response dampens induction of the cytosolic unfolded-protein response

Aneuploid yeast cells are in a chronic state of proteotoxicity, yet do not constitutively induce the cytosolic unfolded protein response, or heat shock response (HSR) by heat shock factor 1 (Hsf1). Here, we demonstrate that an active environmental stress response (ESR), a hallmark of aneuploidy across different models, suppresses Hsf1 induction in models of single-chromosome gain. Furthermore, engineered activation of the ESR in the absence of stress was sufficient to suppress Hsf1 activation in euploid cells by subsequent heat shock while increasing thermotolerance and blocking formation of heat-induced protein aggregates. Suppression of the ESR in aneuploid cells resulted in longer cell doubling times and decreased viability in the presence of additional proteotoxicity. Last, we show that in euploids, Hsf1 induction by heat shock is curbed by the ESR. Strikingly, we found a similar relationship between the ESR and the HSR using an inducible model of aneuploidy. Our work explains a long-standing paradox in the field and provides new insights into conserved mechanisms of proteostasis with potential relevance to cancers associated with aneuploidy.     

Helicobacter pylori Infection in Pediatrics

This review summarizes the articles published on Helicobacter pylori infection in children between April 2008 and March 2009. Recent evidence highlights the decreasing prevalence trend of H. pylori infection and supports both intrafamilial and extrafamilial transmission. The association with various symptoms is still being debated. Interestingly, H. pylori infection seems inversely associated with allergic diseases. Monoclonal stool antigen tests are widely used and accurate for the diagnosis of H. pylori infection, but less accurate in young children. The new biprobe real-time PCR assay applied to stools showed a poor sensitivity in children. Using the urea hydrolysis rate next to the delta over baseline values, the ¹³C-urea breath test provides excellent results for all age children, even for young children. Treatment of H. pylori infection remains a challenge, considering suboptimal efficacy of current therapy. Among emerging alternatives, sequential treatment appears promising. The adjunction of probiotics to conventional regimens, although eliciting great interest, has shown limited therapeutic benefit.     

Control of c-myc mRNA stability by IGF2BP1-associated cytoplasmic RNPs

The RNA-binding protein IGF2BP1 (IGF-II mRNA binding protein 1) stabilizes the c-myc RNA by associating with the Coding Region instability Determinant (CRD). If and how other proteins cooperate with IGF2BP1 in promoting stabilization of the c-myc mRNA via the CRD remained elusive. Here, we identify various RNA-binding proteins that associate with IGF2BP1 in an RNA-dependent fashion. Four of these proteins (HNRNPU, SYNCRIP, YBX1, and DHX9) were essential to ensure stabilization of the c-myc mRNA via the CRD. These factors associate with IGF2BP1 in a CRD-dependent manner, co-distribute with IGF2BP1 in non-polysomal fractions comprising c-myc mRNA, and colocalize with IGF2BP1 in the cytoplasm. A selective shift of relative c-myc mRNA levels to the polysomal fraction is observed upon IGF2BP1 knockdown. These findings suggest that IGF2BP1 in complex with at least four proteins promotes CRD-mediated mRNA stabilization. Complex formation at the CRD presumably limits the transfer of c-myc mRNA to the polysomal fraction and subsequent translation-coupled decay.     

IGF2BP1 enhances HCV IRES-mediated translation initiation via the 3′UTR

The positive-strand RNA genome of the Hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) in the 5′untranslated region (5′UTR) and structured sequence elements within the 3′UTR, but no poly(A) tail. Employing a limited set of initiation factors, the HCV IRES coordinates the 5′cap-independent assembly of the 43S pre-initiation complex at an internal initiation codon located in the IRES sequence. We have established a Huh7 cell-derived in vitro translation system that shows a 3′UTR-dependent enhancement of 43S pre-initiation complex formation at the HCV IRES. Through the use of tobramycin (Tob)-aptamer affinity chromatography, we identified the Insulin-like growth factor-II mRNA-binding protein 1 (IGF2BP1) as a factor that interacts with both, the HCV 5′UTR and 3′UTR. We report that IGF2BP1 specifically enhances translation at the HCV IRES, but it does not affect 5′cap-dependent translation. RNA interference against IGF2BP1 in HCV replicon RNA-containing Huh7 cells reduces HCV IRES-mediated translation, whereas replication remains unaffected. Interestingly, we found that endogenous IGF2BP1 specifically co-immunoprecipitates with HCV replicon RNA, the ribosomal 40S subunit, and eIF3. Furthermore eIF3 comigrates with IGF2BP1 in 80S ribosomal complexes when a reporter mRNA bearing both the HCV 5′UTR and HCV 3′UTR is translated. Our data suggest that IGF2BP1, by binding to the HCV 5′UTR and/or HCV 3′UTR, recruits eIF3 and enhances HCV IRES-mediated translation.