We describe a method for simultaneous identification and quantitation of proteins within complex mixtures. The method consists of 18O-labeling, a simple stable isotope-coding that requires merely enzymatic digestion in 18O-water, in combination with a capillary-liquid chromatography electrospray ion-trap mass spectrometer. In a separate experiment using the same sample and a spike test, we demonstrate that the difference ration was calculated accurately using the 18O-labeling method even if the protein was part of a complex mixture. Our data also suggest that the accuracy of the quantitation can be improved by averaging the difference ratios of several peptides. In comparing our method with the isotope-coded affinity tag (ICAT) method, we show that the 18O-labeling method has the advantages of better recovery and fewer isotope effects. Therefore, the 18O-labeling method is a powerful tool for large-scale proteomics applications.
First-generation (FG) adenoviral vectors (AdVs) have been widely used not only for gene therapy but also for basic studies. Because vectors of this type lack the E1A gene that is essential for the expression of other viral genes, their expression levels in target cells have been considered low. However, we found that the viral pIX gene, located immediately downstream of the inserted expression unit of the transgene, was significantly coexpressed with the transgene in cells infected with FG AdV. Whereas CAG and SRalpha promoters activated the pIX promoter considerably through their enhancer effects, the EF1alpha promoter hardly did. Moreover, when the expression unit was inserted in the rightward orientation, not only the pIX protein but also a fusion protein consisting of the N-terminal part of transgene product and pIX were sometimes coexpressed with the transgene product through an aberrant splicing mechanism. In in vivo experiments, a LacZ-expressing AdV bearing the CAG promoter caused an elevation of alanine aminotransferase, but an AdV bearing the EF1alpha promoter produced no detectable levels. Whereas the FG AdV expressing human growth hormone under the control of the CAG promoter maintained a high hormone level for less than 1 month, the FG AdV under the control of the EF1alpha promoter maintained a high level for at least 6 months. These results suggest that pIX coexpression may be one of the main causes of AdV-induced immune responses, and that the EF1alpha promoter is probably valuable for the long-term expression of FG AdV. Thus, the in vivo utility of FG AdV should be reevaluated.
Aims/hypothesis: Metformin is widely used as a hypoglycaemic reagent for type 2 diabetes. While the reduction of hepatic gluconeogenesis is thought to be a key effect, the detailed molecular mechanism of action of metformin remains to be elucidated. To gain insight into this, we performed a global gene expression profiling study. Materials and methods: We performed DNA microarray analysis to study global gene expression in the livers of obese diabetic db/db mice 2 h after a single administration of metformin (400 mg/kg). Results: This analysis identified 14 genes that showed at least a 1.5-fold difference in expression following metformin treatment, including a reduction of glucose-6-phosphatase gene expression. The mRNA levels of glucose-6-phosphatase showed one of the best correlations with blood glucose levels among 12,000 genes. Enzymatic activity of glucose-6-phosphatase was also reduced in metformin-treated liver. Moreover, intensive analysis of the expression profile revealed that metformin effected significant alterations in gene expression across at least ten metabolic pathways, including those involved in glycolysis-gluconeogenesis, fatty acid metabolism and amino acid metabolism. Conclusions/interpretation: These results suggest that reduction of glucose-6-phosphatase activity, as well as suppression of mRNA expression levels of this gene, in liver is of prime importance for controlling blood glucose levels in vivo, at least at early time points after metformin treatment. Our results also suggest that metformin not only affects expression of specific genes, but also alters the expression level of multiple genes linked to the metabolic pathways involved in glucose and lipid metabolism in the liver.
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