Signal ratios generated by wild-type probe reacted with PCR products from the control construct were all close to unity (i.e., there was no cross-reaction), as were those from wild-type PCR products reacted with the control sequence probe. End-point dilution of the control construct confirmed the spectrophotometric estimate of 10 11 RNA copies/L. Assay of the final construct for DNA copies showed that this contained ϳ10 5 DNA copies/mL (ϳ10 Ϫ6 of the RNA copies). It was intended that the RNA construct be added to the final assay at a concentrations of 100 RNA copies/reaction. At this dilution, the DNA contaminants would be diluted to Ͻ0.001 copies/reaction; thus it was decided not to risk the integrity of the RNA construct by the use of more vigorous DNase treatment.Quantitative molecular assays, particularly quantitative PCR, have become an important tool in medical research and routine clinical investigation, particularly for the assay of microbial genomes in clinical samples (9 ) and for the quantification of gene expression in mammalian cells (10 ). In such assay systems, accurate quantification depends on standardization and control of all steps in the protocol. In the case of quantitative PCR, this includes controls for nucleic acid extraction efficiency, amplification efficiency, and nonspecific inhibition of the reaction (11 ). Additionally, there is a need for accurately quantified internal standards with which to compare unknowns to obtain absolute nucleic acid copy numbers in the sample.The method described here allows molecular constructs for use as internal controls in quantitative molecular assays to be synthesized in a simple two-step PCR protocol. The simplicity of the procedure allows controls to be synthesized in laboratories that do not have facilities available for molecular biology procedures beyond PCR, such as diagnostic laboratories that use molecular detection methods. The method has been applied to the control of a quantitative PCR for MV RNA but could also be applied to the generation of DNA targets. Controls may be synthesized that are appropriate to any source of nucleic acid (e.g., mRNA in the quantification of microbial or mammalian gene expression) that is being detected or quantified by PCR or any other molecular method. Analysis of antioxidant molecules is potentially important to understanding the role of oxidative stress (1-6 ) in disease, as oxidative damage is accompanied or preceded by their depletion.Reduced glutathione (GSH) is ubiquitous and abundant; it can be oxidized to its disulfide form (GSSG) in response to an oxidative perturbation. Usually, however, this species is rapidly reduced by the action of glutathione reductase (7 ). If GSSG accumulates within the cell, it can create protein-glutathione adducts via thiol-disulfide exchange reactions. Thus, in addition to the ratio of GSH to GSSG, the content of glutathionylated proteins (GSSPs) can indicate oxidative stress. The analysis of GSSPs has potential advantages over measurements of GSH and GSSG because GSSPs ar...
The proto-oncogene c-myb, whose expression is usually limited to cells of the hematopoietic lineages, can be expressed in fibroblasts if placed under the control of a constitutive promoter, such as the simian virus SV40 early promoter. 3T3 cells carrying a constitutively expressed human c-myb were found to grow in 1% serum or in a serum-free, platelet-derived growth factor-supplemented medium, whereas the parent cell line, BALB/c 3T3, needed insulinlike growth factor 1 (IGF-1) in addition to platelet-derived growth factor for growth. myb-carrying cells, however, could not grow in platelet-poor plasma. In fibroblasts, therefore, a constitutively expressed c-myb can abrogate the requirement for platelet-poor plasma or IGF-1. When 3T3 cells constitutively expressed both c-myc and c-myb, they could grow in serum-free medium without added growth factors. The ability of c-myb to abrogate in fibroblasts the IGF-1 requirement seems to be due to its ability to induce overexpression of IGF-1, as indicated by an increase in steady-state levels of IGF-1 mRNA. These results have some important implications; for instance, they suggest a commonality of pathways for entry into S phase in different cell types and the possibility of a myb-like or myb-equivalent gene product of critical importance for entry of fibroblasts into S phase.
An innovative approach to identify new conformational antigens of Aβ 1–42 recognized by IgG autoantibodies as biomarkers of state and stage in Alzheimer’s disease (AD) patients is described. In particular, through the use of bioinformatics modeling, conformational similarities between several Aβ 1–42 forms and other amyloid-like proteins with F1 capsular antigen (Caf1) of Yersinia pestis were first found. pVIII M13 phage display libraries were then screened against YPF19, anti-Caf1 monoclonal antibody, and IgGs of AD patients, in alternate biopanning cycles of a so-called “double binding” selection. From the selected phage clones, one, termed 12III1, was found to be able to prevent in vitro Aβ 1–42 -induced cytotoxicity in SH-SY5Y cells, as well as to promote disaggregation of preformed fibrils, to a greater extent with respect to wild-type phage (pC89). IgG levels detected by 12III1 provided a significant level of discrimination between diseased and nondemented subjects, as well as a good correlation with the state progression of the disease. These results give significant impact in AD state and stage diagnosis, paving the way for the development not only for an innovative blood diagnostic assay for AD precise diagnosis, progressive clinical assessment, and screening but also for new effective treatments.
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