Genetic translation

The proteolytic activities of the ADAM (a disintegrin and metalloproteinase),
ADAMTS (a disintegrin and metalloproteinases with thrombospondin motifs) and
MMP (matrix metalloproteinase) protein families play important roles in normal and
multiple pathological conditions. These metalloproteases have potential implications
in the degradation of the extracellular matrix and in the processing of bioactive
molecules. Under pathological conditions these proteases are involved in many
diverse processes from tumor cell migration to cartilage destruction in rheumatoid
arthritis. In the present study, the gene expression levels of six ADAMs, eight MMPs,
and four ADAMTSs were analyzed by Real Time PCR. RNA was isolated from
multiple normal fibroblast and metastatic melanoma cell lines, as well as the isogenic
normal tissue and tumor samples. This method allowed for detected changes in mRNA expressiOn of the individual metalloproteainase genes to be compared
between normal and metastatic states, and also between tissue and cultured cells.
Substantial differences have been observed in the level of ADAM and MMP mRNA
expression between tissue and cell lines. In general, the level of expression is several
folds higher in cultured cells compared to the isogenic tissue they are derived from.
Protein microarrays were utilized in order to evaluate the correlations between MMP
and TIMP mRNA copy numbers and protein abundance in cell culture. In several
cases, distinct differences were observed regarding the localization of the proteins
examined. In order to determine if the metalloprotease genes that were elevated at the
level of RNA expression produce functional proteins, the foundations of an in situ
FRET assay have been established. This will greatly aid in a better understanding of
the behavior of metallopeptidases in a cellular context.

The Mexican axolotl, Ambystoma mexicanum, possesses a naturally-occurring
lethal mutation, designated gene "c", for cardiac non-function. Hearts form but fail to
beat, lack organized myofibrils, and are deficient in tropomyosin. Treatment with a noncoding
RNA MIR (Myofibril-Inducing RNA) rescues this defect in organ culture.
Rescued mutant hearts have restored tropomyosin, form organized myofibrils, and beat
vigorously. Studies to elucidate the mechanism of MIR heart rescue are underway.
Current evidence suggests that MIR acts by binding with at least two proteins. The yeast
three-hybrid system is being used to screen an axolotl eDNA library for these two
proteins and other possible MIR-binding candidates. This is a method utilizing two
hybrid proteins and a hybrid RNA. An interaction between these three components will
activate the expression of reporter genes, whose activity is assayed through phenotypical
and biochemical methods. In this study, the protocol for yeast three-hybrid technology is
being established for analyzing the MIR in the Mexican axolotl, cardiac mutant animal
model.

We have studies oxidative damage of RNA, a major type of cellular macromolecules. RNA is a primary target of reactive oxygen species (ROS). Under oxidative stress, most nucleic acid damages in Escherichia coli (E.coli) are present in RNA as shown by high levels of 8-oxo-G, an oxidized form of guanine. Increased RNA oxidation is closely correlated to cell death under oxidative stress. Surprisingly, neither RNA structure nor association with proteins protects RNA from oxidation... Our results demonstrate a major role for RNA degradation in controlling oxidized RNA. We have identified activities that may work in specific pathways for selectively degrading damaged RNA. These activities may play pivotal rold in controlling oxidized RNA and protecting cells under oxidative stress.