Advanced glycation end products regulate extracellular matrix protein and protease expression by human glomerular mesangial cells

Advanced glycation end products (AGEs) may play a role in the pathogenesis of diabetic nephropathy, by modulating extracellular matrix turnover. AGEs are known to activate specific membrane receptors, including the receptor for AGE (RAGE). In the present study, we analyzed the various receptors for AGEs expressed by human mesangial cells and we studied the effects of glycated albumin and of carboxymethyl lysine on matrix protein and remodelling enzyme synthesis. Membrane RAGE expression was confirmed by FACS analysis. Microarray methods, RT-PCR, and Northern blot analysis were used to detect and confirm specific gene induction. Zymographic analysis and ELISA were used to measure the induction of tPA and PAI-1. We show herein that cultured human mesangial cells express AGE receptor type 1, type 2 and type 3 and RAGE. AGEs (200 microg/ml) induced at least a 2-fold increase in mRNA for 10 genes involved in ECM remodelling, including tPA, PAI-1 and TIMP-3. The increase in tPA synthesis was confirmed by fibrin zymography. The stimulation of PAI-1 synthesis was confirmed by ELISA. AGEs increased PAI-1 mRNA through a signalling pathway involving reactive oxygen species, the MAP kinases ERK-1/ERK-2 and the nuclear transcription factor NF-kappaB, but not AP-1. Carboxymethyl lysine (CML, 5 microM), which is a RAGE ligand, also stimulated PAI-1 synthesis by mesangial cells. In addition, a blocking anti-RAGE antibody partially inhibited the AGE-stimulated gene expression and decreased the PAI-1 accumulation induced by AGEs and by CML. Inhibition of AGE receptors or neutralization of the protease inhibitors TIMP-3 and PAI-1 could represent an important new therapeutic strategy for diabetic nephropathy.

Risk scoring for setting priorities in a monitoring of antimicrobial resistance in meat and meat products

Meat and meat products can be contaminated with different species of bacteria resistant to various antimicrobials. The human health risk of a type of meat or meat product carry by emerging antimicrobial resistance depends on (i) the prevalence of contamination with resistant bacteria, (ii) the human health consequences of an infection with a specific bacterium resistant to a specific antimicrobial and (iii) the consumption volume of a specific product. The objective of this study was to compare the risk for consumers arising from their exposure to antibiotic resistant bacteria from meat of four different types (chicken, pork, beef and veal), distributed in four different product categories (fresh meat, frozen meat, dried raw meat products and heat-treated meat products). A semi-quantitative risk assessment model, evaluating each food chain step, was built in order to get an estimated score for the prevalence of Campylobacter spp., Enterococcus spp. and Escherichia coli in each product category. To assess human health impact, nine combinations of bacterial species and antimicrobial agents were considered based on a published risk profile. The combination of the prevalence at retail, the human health impact and the amount of meat or product consumed, provided the relative proportion of total risk attributed to each category of product, resulting in a high, medium or low human health risk. According to the results of the model, chicken (mostly fresh and frozen meat) contributed 6.7% of the overall risk in the highest category and pork (mostly fresh meat and dried raw meat products) contributed 4.0%. The contribution of beef and veal was of 0.4% and 0.1% respectively. The results were tested and discussed for single parameter changes of the model. This risk assessment was a useful tool for targeting antimicrobial resistance monitoring to those meat product categories where the expected risk for public health was greater.

Governance Structure Choices in Supply Chain Management Evidence from Spanish and Chinese Pork Chain Cases

The author participated in the 6 th EU Framework Project ―Q-pork Chains (FP6-036245-2)‖ from 2007 to 2009. With understanding of work reports from China and other countries, it is found that compared with other countries, China has great problems in pork quality and safety. By comparing the pork chain management between China and Spain, It is found that the difference in governance structure is one of the main differences in pork chain management between Spain and China. In China, spot-market relationship still dominates governance structure of pork chain, especially between the numerous house-hold pig holders and the great number of small slaughters. While in Spain, chain agents commonly apply cooperatives or integrations to cooperate. It also has been proven by recent studies, that in quality management at the chain level that supply chain integration has a direct effect on quality management practices (Han, 2010). Therefore, the author started to investigate the governance structure choices in supply chain management. And it has been set as the first research objective, which is to explain the governance structure choices process and the influencing factors in supply chain management, analyzing the pork chains cases in Spain and in China. During the further investigation, the author noticed the international trade of pork between Spain and China is not smooth since the signature of bi-lateral agreement on pork trade in 2007. Thus, another objective of the research is to find and solve the problems exist in the international pork chain between Spain and China. For the first objective, to explain the governance structure choices in supply chain management, the thesis conducts research in three main sections. 10 First of all, the thesis gives a literature overview in chapter two on Supply Chain Management (SCM), agri-food chain management and pork chain management. It concludes that SCM is a systems approach to view the supply chains as a whole, and to manage the total flow of goods inventory from the supplier to the ultimate customer. It includes the bi-directional flow of products (materials and services) and information, and the associated managerial and operational activities. And it also is a customer focus to create unique and individual source of customer value with an appropriate use of resources, leading to customer satisfaction and building competitive chain advantages. Agri-food chain management and pork chain management are applications of SCM in agri-food sector and pork sector respectively. Then, the research gives a comparative study in chapter three in the pork chain and pork chain management between Spain and China. Many differences are found, while the main difference is governance structure in pork chain management. Furthermore, the author gives an empirical study on governance structure choice in chapter five. It is concluded that governance structure of supply chain consists of a collection of rules/institutions/constraints structuring the transactions between the various stakeholders. Based on the overview on literatures closely related with governance structure, such as transaction cost economics, transaction value analysis and resource-based view theories, seven hypotheses are proposed, which are: Hypothesis 1: Transaction cost has positive relationship with governance structure choice Hypothesis 2: Uncertainty has positive relationship with transaction cost; higher uncertainty exerts high transaction cost Hypothesis 3: The relationship between asset specificity and transaction cost is positive Hypothesis 4: Collaboration advantages and governance structure choice have positive relationship11 Hypothesis 5: Willingness to collaborate has positive relationship with collaboration advantages Hypothesis 6: Capability to collaborate has positive relationship with collaboration advantages Hypothesis 7: Uncertainty has negative effect on collaboration advantages It is noted that as transaction cost value is negative, the transaction cost mentioned in the hypotheses is its absolute value. To test the seven hypotheses, Structural Equation Model (SEM) is applied and data collected from 350 pork slaughtering and processing companies in Jiangsu, Shandong and Henan Provinces in China is used. Based on the empirical SEM model and its results, the seven hypotheses are proved. The author generates several conclusions accordingly. It is found that the governance structure choice of the chain not only depends on transaction cost, it also depends on collaboration advantages. Exchange partners establish more stable and more intense relationship to reduce transaction cost and to maximize collaboration advantages. ―Collaboration advantages‖ in this thesis is defined as the joint value achieved through transaction (mutual activities) of agents in supply chains. This value forms as improvements, mainly in mutual logistics systems, cash response, information exchange, technological improvements and innovative improvements and quality management improvements, etc. Governance structure choice is jointly decided by transaction cost and collaboration advantages. Chain agents take different governance structures to coordinate in order to decrease their transaction cost and to increase their collaboration advantages. In China´s pork chain case, spot market relationship dominates the governance structure among the numerous backyard pig farmer and small family slaughterhouse 12 as they are connected by acquaintance relationship and the transaction cost in turn is low. Their relationship is reliable as they know each other in the neighborhood; as a result, spot market relationship is suitable for their exchange. However, the transaction between large-scale slaughtering and processing industries and small-scale pig producers is becoming difficult. The information hold back behavior and hold-up behavior of small-scale pig producers increase transaction cost between them and large-scale slaughtering and processing industries. Thus, through the more intense and stable relationship between processing industries and pig producers, processing industries reduce the transaction cost and improve the collaboration advantages with their chain partners, in which quality and safety collaboration advantages be increased, meaning that processing industries are able to provide consumers products with better quality and higher safety. It is also drawn that transaction cost is influenced mainly by uncertainty and asset specificity, which is in line with new institutional economics theories developed by Williamson O. E. In China´s pork chain case, behavioral uncertainty is created by the hold-up behaviors of great numbers of small pig producers, while big slaughtering and processing industries having strong asset specificity. On the other hand, ―collaboration advantages‖ is influenced by chain agents´ willingness to collaborate and chain agents´ capabilities to cooperate. With the fast growth of big scale slaughtering and processing industries, they are more willing to know and make effort to cooperate with their chain members, and they are more capable to create joint value together with other chain agents. Therefore, they are now the main chain agents who drive more intense and stable governance structure in China‘s pork chain. For the other objective, to find and solve the problems in the international pork chain between Spain and China, the research gives an analysis in chapter four on the 13 international pork chain. This study gives explanations why the international trade of pork between Spain and China is not sufficient from the chain perspective. It is found that the first obstacle is the high quality and safety requirement set by Chinese government. It makes the Spanish companies difficult to get authorities to export. Other aspects, such as Spanish pork is not competitive in price compared with other countries such as Denmark, United States, Canada, etc., Chinese consumers do not have sufficient information on Spanish pork products, are also important reasons that Spain does not export great quantity of pork products to China. It is concluded that China´s government has too much concern on the quality and safety requirements to Spanish pork products, which makes trade difficult to complete. The two countries need to establish a more stable and intense trade relationship. They also should make the information exchange sufficient and efficient and try to break trade barriers. Spanish companies should consider proper price strategies to win the Chinese pork market

Isolation of recombinant antibody fragments (scFv) by phage display technology for detection of almond allergens in food products

Tree nut allergies are considered an important health issue in developed countries. To comply with the regulations on food labeling, reliable allergen detection methods are required. In this work we isolated almond-specific recombinant antibody fragments (scFv) from a commercial phage display library bypassing the use of live animals, hence being consistent with the latest policies on animal welfare. To this end an iterative selection procedure employing the Tomlinson I phage display library and a crude almond protein extract was carried out. Two different almond-specific scFv (named PD1F6 and PD2C9) were isolated after two rounds of biopanning, and an indirect phage ELISA was implemented to detect the presence of almond protein in foodstuffs. The isolated scFvs demonstrated to be highly specific and allowed detection of 40 ng mL?1 and 100 ng mL?1 of raw and roasted almond protein, respectively. The practical detection limit of the assay in almond spiked food products was 0.1 mg g?1 (110e120 ppm). The developed indirect phage ELISA was validated by analysis of 92 commercial food products, showing good correlation with the results obtained by a previously developed real-time PCR method for the detection of almond in foodstuffs. The selected phage clones can be affinity maturated to improve their sensitivity and genetically engineered to be employed in different assay formats.

Epitopes close to the apolipoprotein B low density lipoprotein receptor-binding site are modified by advanced glycation end products

Advanced glycation end products (AGEs) are thought to contribute to the abnormal lipoprotein profiles and increased risk of cardiovascular disease of patients with diabetes and renal failure, in part by preventing apolipoprotein B (apoB)-mediated cellular uptake of low density lipoproteins (LDL) by LDL receptors (LDLr). It has been proposed that AGE modification at one site in apoB, almost 1,800 residues from the putative apoB LDLr-binding domain, may be sufficient to induce an apoB conformational change that prevents binding to the LDLr. To further explore this hypothesis, we used 29 anti-human apoB mAbs to identify other potential sites on apoB that may be modified by in vitro advanced glycation of LDL. Glycation of LDL caused a time-dependent decrease in its ability to bind to the LDLr and in the immunoreactivity of six distinct apoB epitopes, including two that flank the apoB LDLr-binding domain. ApoB appears to be modified at multiple sites by these criteria, as the loss of glycation-sensitive epitopes was detected on both native glycated LDL and denatured, delipidated glycated apoB. Moreover, residues directly within the putative apoB LDLr-binding site are not apparently modified in glycated LDL. We propose that the inability of LDL modified by AGEs to bind to the LDLr is caused by modification of residues adjacent to the putative LDLr-binding site that were undetected by previous immunochemical studies. AGE modification either eliminates the direct participation of the residues in LDLr binding or indirectly alters the conformation of the apoB LDLr-binding site.

The orphan nuclear receptor LXRα is positively and negatively regulated by distinct products of mevalonate metabolism

LXRα is an orphan member of the nuclear hormone receptor superfamily that displays constitutive transcriptional activity. We reasoned that this activity may result from the production of an endogenous activator that is a component of intermediary metabolism. The use of metabolic inhibitors revealed that mevalonic acid biosynthesis is required for LXRα activity. Mevalonic acid is a common metabolite used by virtually all eukaryotic cells. It serves as a precursor to a large number of important molecules including farnesyl pyrophosphate, geranylgeranyl pyrophosphate, cholesterol, and oxysterols. Inhibition of LXRα could be reversed by addition of mevalonic acid and certain oxysterols but not by other products of mevalonic acid metabolism. Surprisingly, the constitutive activity of LXRα was inhibited by geranylgeraniol, a metabolite of mevalonic acid. These findings suggest that LXRα may represent a central component of a signaling pathway that is both positively and negatively regulated by multiple products of mevalonate metabolism.

Heterogeneity of primer extension products in asymmetric PCR is due both to cleavage by a structure-specific exo/endonuclease activity of DNA polymerases and to premature stops.

In PCR, DNA polymerases from thermophilic bacteria catalyze the extension of primers annealed to templates as well as the structure-specific cleavage of the products of primer extension. Here we show that cleavage by Thermus aquaticus and Thermus thermophilus DNA polymerases can be precise and substantial: it occurs at the base of the stem-loop structure assumed by the single strand products of primer extension using as template a common genetic element, the promoter-operator of the Escherichia coli lactose operon, and may involve up to 30% of the products. The cleavage is independent of primer, template, and triphosphates, is dependent on substrate length and temperature, requires free ends and Mg2+, and is absent in DNA polymerases lacking the 5'-->3' exonuclease, such as the Stoffel fragment and the T7 DNA polymerase. Heterogeneity of the extension products results also from premature detachment of the enzyme approaching the 5' end of the template.

Formation of chimeric DNA primer extension products by template switching onto an annealed downstream oligonucleotide.

Oligodeoxynucleotide sequences are described that anneal to a template downstream of a priming site. During polymerase-catalyzed extension of the primer, the extending primer shifts from the original template to a segment of the annealed oligonucleotide that acts as an alternative template. The resulting chimeric extended primer has one segment that is complementary to the template and a second segment that is complementary to the oligonucleotide. The influence of the sequence elements of the oligonucleotide and the reaction conditions on template switching have been explored. The sequence requirements for template switching are compared to those for transposon excision.

An introduction to the chemistry of plant products by Paul Haas and T. G. Hill.

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An Introduction to the chemistry of plant products / by Paul Haas, ... and TG. Hill, ... Vol. 2 : Metabolic processes.

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