Freedom and constraint in engineered noncolinear polyketide assembly lines.

Many pharmacologically important natural products are assembled by modular type I polyketide synthases (PKS), which typically act in a unidirectional fashion. The synthases producing the unusual nitro-substituted polyketides neoaureothin (nor, also called spectinabilin) and aureothin (aur) are exceptional, as they employ individual modules iteratively. Here, we investigate the plasticity of the nor PKS and the factors governing the number of elongations catalyzed by the noncanonical module. Surprisingly, we observe that the nor PKS can mediate an additional chain elongation to yield the higher homolog homoneoaureothin. Furthermore, we design several truncated variants of the nor PKS to use them in the context of artificial assembly lines for aureothin and homoaureothin. The resulting polypropionate derivatives provide valuable insights into chain length control and reveal structure-activity relationships relating to the size of the polypropionate backbones. Overall, we show that iterative modules are remarkably adaptable while downstream modules are gatekeepers that select for correct polyketide chain length.

Bioelectromagnetic field effects on cancer cells and mice tumors.

We present possibilities and trends of ELF bioelectromagnetic effects in the mT amplitude range on cancer cells and on mice bearing tumors. In contrast to invasive electrochemotherapy and electrogenetherapy, using mostly needle electrodes and single high-amplitude electropulses for treatment, extremely low-frequency (ELF) pulsating electromagnetic fields (PEMF) and sinusoidal electromagnetic fields (SEMF) induce tumor cell apoptosis, inhibit angiogenesis, impede proliferation of neoplastic cells, and cause necrosis non invasively, whereas human lymphocytes are negligibly affected. Our successful results in killing cancer cells-analyzed by trypan blue staining or by flow cytometry-and of the inhibition of MX-1 tumors in mice by 15-20 mT, 50 Hz treatment in a solenoid coil also in the presence of bleomycin are presented in comparison to similar experimental results from the literature. In conclusion, the synergistic combinations of PEMF or SEMF with hyperthermia (41.5°C) and/or cancerostatic agents presented in the tables for cells and mice offer a basis for further development of an adjuvant treatment for patients suffering from malignant tumors and metastases pending the near-term development of suitable solenoids of 45-60 cm in diameter, producing >20 mT in their cores.

Electroporation and alternating current cause membrane permeation of photodynamic cytotoxins yielding necrosis and apoptosis of cancer cells.

In order to increase the permeability of cell membranes for low doses of cytostatic drugs, two bioelectrochemical methods have been compared: (a) electric pore formation in the plasma membranes by single electric impulses (electroporation), and (b) reordering of membrane structure by alternating currents (capacitively coupled). These treatments were applied to human leukemic K-562 cells and human lymphoma U-937 cells, yielding apoptotic and necrotic effects, determined by flow cytometry. Additional cell death occurs after exposure to light irradiation at wavelengths lambda > 600 nm, of cells which were electroporated and had incorporated actinomycin-C or daunomycin (daunorubicin). It is observed that drug uptake after an exponentially decaying electroporation pulse of the initial field strength Eo=1.4 kV/cm and pulse time constants in the time range 0.5-3 ms is faster than during PEMF-treatment, i.e., application of an alternating current of 16 kHz, voltage U<100 V, I=55 mA, and exposure time 20 min. However, at the low a.c. voltage of this treatment, more apoptotic and necrotic cells are produced as compared to the electroporation treatment with one exponentially decaying voltage pulse. Thus, additional photodynamic action appears to be more effective than solely drugs and electroporation as applied in clinical electrochemotherapy, and more effective than the noninvasive pulsed electromagnetic fields (PEMFs), for cancer cells in general and animals bearing tumors in particular.

Non-colinear polyketide biosynthesis in the aureothin and neoaureothin pathways: an evolutionary perspective.

Aureothin and neoaureothin (spectinabilin) represent rare nitroaryl-substituted polyketide metabolites from Streptomyces thioluteus and Streptomyces orinoci, respectively, which only differ in the lengths of the polyene backbones. Cloning and sequencing of the 39 kb neoaureothin (nor) biosynthesis gene cluster and its comparison with the aureothin (aur) pathway genes revealed that both polyketide synthase (PKS) assembly lines are remarkably similar. In both cases the module architecture breaks with the principle of colinearity, as individual PKS modules are used in an iterative fashion. Parsimony and neighbour-joining phylogenetic studies provided insights into the evolutionary process that led to the programming of these unusual type I PKS systems and to prediction of which modules act iteratively. The iterative function of the first module in the neoaureothin pathway, NorA, was confirmed by a successful cross-complementation.

ELF fields and photooxidation yielding lethal effects on cancer cells.

The lethal effect on human cancer cells was studied under three types of treatment: A) an ELF pulsed sinusoidal of 50 Hz electromagnetic field (PEMF) with amplitudes between 10 and 55 mT; B) the field and a cytostatic agent (actinomycin-C); and C) the field, the cytostatic agent, which has a photodynamic effect, and exposure to a halogen lamp. The results show a decreasing vitality of human K-562 and U-937 cancer cells in suspension with each additional treatment. Combination with other parameters as hyperthermia and/or hyperacidity could yield high killing rates by this noninvasive method.