TNF-α Downregulation Modifies Insulin Receptor Substrate 1 (IRS-1) in Metabolic Signaling of Diabetic Insulin-Resistant Hepatocytes.

One of the major mechanisms of hyperglycemia in type 2 diabetes is insulin resistance (IR) which can induce free fatty acids like palmitate. In hepatic cell, as an insulin target tissue, insulin resistance can be stimulated by inflammatory cytokine TNF-α. The interaction of intracellular TNF-α signal with the insulin signaling pathway is not well identified. Hence, we aimed to investigate the effect of TNF-α elimination on the diabetic model of palmitate-induced insulin-resistant hepatocytes (HepG2). The changes of phosphorylation rate in IRS-1 protein are determined to know the effect of TNF-α on this key protein of the insulin signaling pathway. HepG2 cells were treated with 0.5 Mm palmitate, and TNF-α gene knockdown was performed by shRNA-mediated technique. Western blot analysis was used to evaluate the phosphorylated activity of the insulin signaling pathway. Palmitate-induced IR could increase TNF-α protein expression 1.2-, 2.78-, and 2.25-fold compared to the control cells at times of 8 h, 16 h, and 24 h, respectively. TNF-α expression in downregulated cells transfected with shRNA-TNF-α is approximately 47.0% of normal cells and 49.0% in the case of scrambled cells. IRS-1 phosphorylation in TNF-α-downregulated and stimulated cells with 100 nM insulin, after treatment and in the absence of palmitate, was 45% and 29% higher than the normal cells. These data support the evidence that TNF-α downregulation strategy contributes to the improvement of IRS-1 phosphorylation after insulin stimulation and insulin response in HepG2 liver cells.

Development of Multiplex PCR Coupled DNA Chip Technology for Assessment of Endogenous and Exogenous Allergens in GM Soybean.

Allergenicity assessment of transgenic plants and foods is important for food safety, labeling regulations, and health protection. The aim of this study was to develop an effective multi-allergen diagnostic approach for transgenic soybean assessment. For this purpose, multiplex polymerase chain reaction (PCR) coupled with DNA chip technology was employed. The study was focused on the herbicide-resistant Roundup Ready soya (RRS) using a set of certified reference materials consisting of 0, 0.1%, 0.5%, and 10% RRS. Technically, the procedure included design of PCR primers and probes; genomic DNA extraction; development of uniplex and multiplex PCR systems; DNA analysis by agarose gel electrophoresis; microarray development, hybridization, and scanning. The use of the asymmetric multiplex PCR method is shown to be very efficient for DNA hybridization with biochip probes. We demonstrate that newly developed fourplex PCR methods coupled with DNA-biochips enable simultaneous identification of three major endogenous allergens, namely, Gly m Bd 28K, Gly m Bd 30K, and lectin, as well as exogenous 5-enolppyruvyl shikimate-phosphate synthase (epsps) expressed in herbicide-resistant roundup ready GMOs. The approach developed in this study can be used for accurate, cheap, and fast testing of food allergens.

MIG1 Glucose Repression in Metabolic Processes of Saccharomyces cerevisiae: Genetics to Metabolic Engineering.

BACKGROUND: Although Saccharomyces cerevisiae has several industrial applications, there are still fundamental problems associated with sequential use of carbon sources. As such, glucose repression effect can direct metabolism of yeast to preferably anaerobic conditions. This leads to higher ethanol production and less efficient production of recombinant products. The general glucose repression system is constituted by MIG1, TUP1 and SSN6 factors. The role of MIG1 is known in glucose repression but the evaluation of effects on aerobic/anaerobic metabolism by deletion of MIG1 and constructing an optimal strain brand remains unclear and an objective to be explored. METHODS: To find the impact of MIG1 in induction of glucose-repression, the Mig1 disruptant strain (ΔMIG1) was produced for comparing with its congenic wild-type strain (2805). The analysis approached for changes in the rate of glucose consumption, biomass yield, cell protein contents, ethanol and intermediate metabolites production. The MIG1 disruptant strain exhibited 25% glucose utilization, 12% biomass growth rate and 22% protein content over the wild type. The shift to respiratory pathway has been demonstrated by 122.86 and 40% increase of glycerol and pyruvate production, respectively as oxidative metabolites, while the reduction of fermentative metabolites such as acetate 35.48 and ethanol 24%. RESULTS: Results suggest that ΔMIG1 compared to the wild-type strain can significantly present less effects of glucose repression. CONCLUSION: The constructed strain has more efficient growth in aerobic cultivations and it can be a potential host for biotechnological recombinant yields and industrial interests.

New multiplex PCR methods for rapid screening of genetically modified organisms in foods.

We present novel multiplex PCR methods for rapid and reliable screening of genetically modified organisms (GMOs). New designed PCR primers targeting four frequently used GMO specific sequences permitted identification of new DNA markers, in particular 141 bp fragment of cauliflower mosaic virus (CaMV) 35S promoter, 224 bp fragment of Agrobacterium tumefaciens nopaline synthase (NOS) terminator, 256 bp fragment of 5-enolppyruvylshikimate-phosphate synthase (epsps) gene and 258 bp fragment of Cry1Ab delta-endotoxin (cry1Ab) gene for GMO screening. The certified reference materials containing Roundup Ready soybean (RRS) and maize MON 810 were applied for the development and optimization of uniplex and multiplex PCR systems. Evaluation of amplification products by agarose gel electrophoresis using negative and positive controls confirmed high specificity and sensitivity at 0.1% GMO for both RRS and MON 810. The fourplex PCR was developed and optimized that allows simultaneous detection of three common transgenic elements, such as: CaMV 35S promoter, NOS terminator, epsps gene together with soybean-specific lectin gene. The triplex PCR developed enables simultaneous identification of transgenic elements, such as: 35S promoter and cry1Ab gene together with maize zein gene. The analysis of different processed foods demonstrated that multiplex PCR methods developed in this study are useful for accurate and fast screening of GM food products.