ABSTRACT
The interpretation of oligonucleotide array experiments depends on the quality of the target cRNA used. cRNA target quality is assessed by quantitative analysis of the representation of 5' and 3' sequences of control genes using commercially available Test arrays. The Test array provides an economically priced means of determining the quality of labeled target prior to analysis on whole genome expression arrays. This manuscript validates the use of a duplex RT-PCR assay as a faster (6 h) and less expensive (
Subject(s)
Humans , Oligonucleotide Array Sequence Analysis/methods , RNA, Complementary/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , BiotinylationABSTRACT
The present study compares two computer models of the first part of glucose catabolism in different organisms in search of evolutionarily conserved characteristics of the glycolysis cycle and proposes the main parameters that define the stable steady-state or oscillatory behavior of the glycolytic system. It is suggested that in both human pancreatic b-cells and Saccharomyces cerevisiae there are oscillations that, despite differences in wave form and period of oscillation, share the same robustness strategy: the oscillation is not controlled by only one but by at least two parameters that will have more or less control over the pathway flux depending on the initial state of the system as well as on extra-cellular conditions. This observation leads to two important interpretations: the first is that in both S. cerevisiae and human b-cells, despite differences in enzyme kinetics and mechanism of feedback control, evolution seems to have kept an oscillatory behavior coupled to the glucose concentration outside the cytoplasm, and the second is that the development of drugs to regulate metabolic dysfunctions in more complex systems may require further study, not only determining which enzyme is controlling the flux of the system but also under which conditions and how its control is maintained by the enzyme or transferred to other enzymes in the pathway as the drug starts acting.
Subject(s)
Humans , Glycolysis , Insulin-Secreting Cells/metabolism , Saccharomyces cerevisiae/metabolism , Computer Simulation , Enzyme Activation , Glucokinase/metabolism , Glucose/metabolism , Insulin-Secreting Cells/enzymology , Kinetics , Models, Biological , Oscillometry , Phosphofructokinases/metabolism , Saccharomyces cerevisiae/enzymologyABSTRACT
Calluses from stems and leaves of Mandevilla velutina were grown in culture for 30,45 and 60 days. Thin-layer chromatography of ethyl acetate extracts of calluses from stems of M. velutina revealed the presence of 6 compounds. Two of them had RF values identical to those of the anti-bradykinin (BK) compounds MV8609 and MV8610 previously isolated from the natural plant. The ethyl acetate extract (20 to 80 microng/ml) from stem callus culture antagonized in a concentration-dependent and reversible manner contractions caused by BK (0.1-100 nM) in the isolated rat uterus. The extracts obtained from calluses cultured for 30,45 and 60 days were about 79-, 63-and 31-fold more potent, respectively, in antagonizing BK than the crude extract obtained from the rhizome of the plant