ABSTRACT
Phenotypic variation is the raw material of adaptive Darwinian evolution. The phenotypic variation found in organismal development is biased towards certain phenotypes, but the molecular mechanisms behind such biases are still poorly understood. Gene regulatory networks have been proposed as one cause of constrained phenotypic variation. However, most pertinent evidence is theoretical rather than experimental. Here, we study evolutionary biases in two synthetic gene regulatory circuits expressed in Escherichia coli that produce a gene expression stripe-a pivotal pattern in embryonic development. The two parental circuits produce the same phenotype, but create it through different regulatory mechanisms. We show that mutations cause distinct novel phenotypes in the two networks and use a combination of experimental measurements, mathematical modelling and DNA sequencing to understand why mutations bring forth only some but not other novel gene expression phenotypes. Our results reveal that the regulatory mechanisms of networks restrict the possible phenotypic variation upon mutation. Consequently, seemingly equivalent networks can indeed be distinct in how they constrain the outcome of further evolution.
Subject(s)
Biological Evolution , Escherichia coli/genetics , Gene Regulatory Networks , Models, Genetic , Phenotype , Synthetic Biology/methods , Arabinose/metabolism , Arabinose/pharmacology , Cloning, Molecular , Culture Media/chemistry , Culture Media/pharmacology , Escherichia coli/drug effects , Escherichia coli/metabolism , Gene Expression Regulation , Genetic Variation , Genotype , Mutation , Selection, GeneticABSTRACT
This paper focuses on the synthesis, characterization and biological activity of new N,N'-methylene modified cyclohexyl ethylenediamine-N,N'-diacetate (edda)-type ligands and their Pt(IV) complexes. Both the ligands and complexes were characterized by infrared, UV-vis, ESI-MS, 1D ((1)H, (13)C, (195)Pt) and 2D (COSY, HSQC, HMBC) NMR spectroscopy and elemental analysis. The possible correlation between the reduction potentials and the cytotoxicity of the complexes was examined. The potential antitumoral activity of all compounds was tested in vitro on human melanoma A375, human glioblastoma U251, human prostate cancer PC3, human colon cancer HCT116, mouse melanoma B16 and mouse colon cancer CT26CL25 cells, as well as primary fibroblasts and keratinocytes. The results obtained revealed strong antitumor potential of the newly synthesized drugs with preserved efficacy against cisplatin resistant lines and less toxicity towards nonmalignant counterparts. The mechanism found to be responsible for the observed tumoricidal action of each synthesized compound was induction of apoptosis generally accompanied with caspase activation. Taken together, the effective response to the treatment of a wide range of different cell lines, including cisplatin resistant subclones, as well as induction of apoptosis, as the mechanism suggested to be the most desirable way of eliminating malignant cells, represents a great advantage of this novel group of drugs in comparison to other members in this metallo-drug family.
Subject(s)
Antineoplastic Agents/chemical synthesis , Coordination Complexes/chemical synthesis , Cyclohexanes/chemical synthesis , Ethylenediamines/chemical synthesis , Organoplatinum Compounds/chemical synthesis , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Coordination Complexes/chemistry , Coordination Complexes/pharmacology , Cyclohexanes/chemistry , Cyclohexanes/pharmacology , Edetic Acid/chemistry , Ethylenediamines/chemistry , Ethylenediamines/pharmacology , Flow Cytometry , Humans , Magnetic Resonance Spectroscopy , Mice , Molecular Structure , Organoplatinum Compounds/chemistry , Organoplatinum Compounds/pharmacology , Spectrometry, Mass, Electrospray Ionization , Spectroscopy, Fourier Transform InfraredABSTRACT
Natural monocrystalline pyrite as a new indicator electrode for the potentiometric titration of weak acids in acetonitrile, propionitrile and benzonitrile was studied. The investigated electrode showed a linear dynamic response for p-toluenesulfonic acid concentrations in the range from 0.1 to 0.001 M, with a Nernstian slope of 74 mV per decade. Sodium methylate, potassium hydroxide and tetrabutylammonium hydroxide (TBAH) proved to be very suitable titrating agent for this titration. The response time was less than (11 s) and the lifetime of the electrode is long. The advantages of the electrode are log-term stability, fast response, and reproducibility, while the sensor is easy to prepare and of low cost.