We have identified a novel gene from Schizosaccharomyces pombe that we have named ecl1(+) (extender of the chronological lifespan). When ecl1(+) is provided on a high-copy number plasmid, it extends the viability of both the Deltasty1 MAP kinase mutant and the wild-type cells after entry into the stationary phase. ecl1(+) encodes an 80-amino acid polypeptide that had not been annotated in the current database. The ecl1(+)-mRNA increases transiently when the growth phase is changed from the log phase to the stationary phase. The Ecl1 protein is localized in the nucleus. Calorie restriction extends the chronological lifespan of wild-type and Deltaecl1 cells but not ecl1(+)-overproducing cells. The Deltapka1 mutant shows little, if any, additional extension of viability when Ecl1 is overproduced. The ste11(+) gene that is negatively controlled by Pka1 is up regulated when Ecl1 is overproduced. From these results we propose that the effect of Ecl1 overproduction may be mainly linked to and negatively affects the Pka1-dependent pathway.
Membrane type-1 matrix metalloproteinase (MT1-MMP) expressed on the tumor cell surface activates pro-MMP-2 and pro-MMP-13 to exacerbate the malignancy, suggesting its suitability as a target molecule for diagnosis by in vivo molecular imaging. Thus, we prepared radiolabeled anti-MT1-MMP monoclonal antibody (mAb) as a novel radiolabeled probe for detecting MT1-MMP in vivo and evaluated its usefulness in breast tumor-bearing rodents. Tc-anti-MT1-MMP mAb was prepared using HYNIC as a bifunctional chelating agent and immunoreactivity was evaluated by flow cytometry. MT1-MMP expression in breast carcinoma cells (rat: Walker-256 and MRMT-1, mouse: FM3A) was measured by Western blotting. In vivo biodistribution was examined for 48 h using tumor-implanted rodents followed by estimation of radiation absorbed by a standard quantitation platform Organ Level Internal Dose Assessment (OLINDA).
The Pseudomonas putida transcriptional
activator
XylS induces transcription from the Pm promoter in
the presence of several benzoic acid effectors, with m-toluic acid being the most effective and p-toluic
acid being much less effective. To alter the effector specificity
of XylS, we developed a dual selection system in Escherichia
coli, which consists of (i) an artificial operon of an ampicillin
resistance gene and tetR under Pm promoter control and (ii) a chloramphenicol resistance gene under tetR promoter control. This system enabled both positive
selection to concentrate XylS mutants recognizing a desired ligand
and negative selection to exclude undesired XylS mutants such as those
recognizing undesired ligands and those that are active without effectors.
Application of a random mutagenesis library of xylS to directed evolution that exploited this selection system yielded
two XylS mutants that recognize p-toluic acid more
effectively. Analysis of each missense mutation indicated three amino
acid residues (N7, T74, and I205) important for p-toluic acid recognition. Then, a codon-randomized xylS library at these three residues was similarly screened, resulting
in three XylS mutants with increased p-toluic acid-recognition
specificity. Analysis of each amino acid substitution revealed that
T74P attributes to both m-toluic acid sensitivity
loss and subtle p-toluic acid sensitivity acquisition,
and that N7R increases the overall ligand-sensitivity. Finally, the
combination of these two mutations generated a desirable XylS mutant,
which has a high p-toluic acid sensitivity and scarcely
responds to m-toluic acid. These results demonstrate
the effectiveness of the dual selection system in the directed evolution
of biosensors.
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