Creation of an artificial oscillating gene expression. system is one of the most challenging issues in synthetic biology. Here, we constructed a simple system to manipulate gene expression molarity calculator patterns to be circadian, reflecting the intrinsic cellular clock, by fusing a core clock protein, BMAL1 or CLOCK,, with a zinc finger type DNA binding domain. Circadian rhythmic gene expression was induced only when the target gene contained zinc finger binding sequences. To our knowledge, this simple approach is the first to Manipulate gene expression patterns into circadian rhythms and would be applicable to various endogenous genes.
It been known for nearly a half century that human tumors, including those derived from the nervous system such as glioblastomas, medulloblastoma, and neuroblastomas are much more sensitive than normal tissues to L-methionine (L-Met)starvation.
Inhibitors,Modulators,Libraries More recently, systemic L-Met depletion by administration of Pseudomonas putida methionine-gamma-lyase Inhibitors,Modulators,Libraries (MGL) could effectively inhibit human tumors xenografted in mice. However, bacterial-derived MGLs are unstable in serum (t(1/2) = 1.9 +/- 0.2 h) and highly immunogenic in primates. Since the human genome does not Inhibitors,Modulators,Libraries encode a human MGL enzyme, we created de novo a methionine degrading enzyme by : reengineering the structurally homologous pyridoxal phosphate-dependent human enzyme cystathionine-gamma-lyase (hCGL). hCGL degrades L-cystathionine but displays no promiscuous activity toward L-Met. Rational design and scanning saturation mutagenesis led to the generation of a variant containing three amino acid substitutions (hCGL-NLV) that degraded L-Met with a k(cat)/K-M of 5.
6 x 10(2) Inhibitors,Modulators,Libraries M-1 s(-1) and displayed a serum deactivation t(1/2) = 78 +/- 5 h (non-PEGylated). In vitro, the cytotoxicity of hCGL-NLV toward 14 neuroblastoma cell lines was essentially indistinguishable from that of the P. putida MGL. Intravenous Dacomitinib administration of PEGylated hCGL-NLV in mice reduced serum L-Met from 123 mu M to <5 mu M for over 30 h. Importantly, treatment of neuroblastoma mouse xenografts with PEGylated hCGL-NLV resulted in near complete cessation of tumor growth. Since the mode of action of hCGL-NLV does not require breaching the blood-brain barrier, this enzyme may have potential application for sensitive tumors that arise from or metastasize to the central nervous system.
G protein-coupled receptor kinase 2 (GRK2) is a well-established further info therapeutic target for the treatment of heart,failure. Herein we identify the Selective: serotonin reuptake inhibitor.(SSRI) paroxetine as a selective inhibitor of GRK2; activity both in Vitro and in living cells. In the crystal structure of the GRK2.paroxetine-G beta gamma complex, paroxetine binds in the active site of GRK2 and stabilizes the kinase domain in a novel conformation in which a unique regulatory loop forms part of the ligand binding site.