Investigations on the mode of action of gephyronic acid, an inhibitor of eukaryotic protein translation from myxobacteria.

The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate.

Hyafurones, hyapyrrolines, and hyapyrones: polyketides from Hyalangium minutum.

Seven new polyketides, for which the trivial names hyafurones A1-B (1-3), hyapyrrolines A (4) and B (5), and hyapyrones A (6) and B (7) are proposed, were isolated from the fermentation broth of the myxobacteria Hyalangium minutum, strains NOCB-2(T) and Hym-3. Their structures were elucidated from NMR and HRESIMS data, and their geometric configuration was assigned based on NOE and vicinal (1)H coupling data. Both hyafurone B (3) and hyapyrone B (7) inhibited growth of the Gram-positive bacterium Nocardia flava, while 7 showed antifungal activity against Mucor hiemalis.

Improved production of medium-chain-length polyhydroxyalkanoates in glucose-based fed-batch cultivations of metabolically engineered Pseudomonas putida strains.

One of the major challenges in metabolic engineering for enhanced synthesis of value-added chemicals is to design and develop new strains that can be translated into well-controlled fermentation processes using bioreactors. The aim of this study was to assess the influence of various fed-batch strategies in the performance of metabolically engineered Pseudomonas putida strains, Δgcd and Δgcd-pgl, for improving production of medium-chain-length polyhydroxyalkanoates (mcl-PHAs) using glucose as the only carbon source. First we developed a fed-batch process that comprised an initial phase of biomass accumulation based on an exponential feeding carbon-limited strategy. For the mcl-PHA accumulation stage, three induction techniques were tested under nitrogen limitation. The substrate-pulse feeding was more efficient than the constant-feeding approach to promote the accumulation of the desirable product. Nonetheless, the most efficient approach for maximum PHA synthesis was the application of a dissolved-oxygen-stat feeding strategy (DO-stat), where P. putida Δgcd mutant strain showed a final PHA content and specific PHA productivity of 67% and 0.83 g·l(-1)·h(-1), respectively. To our knowledge, this mcl-PHA titer is the highest value that has been ever reported using glucose as the sole carbon and energy source. Our results also highlighted the effect of different fed-batch strategies upon the extent of realization of the intended metabolic modification of the mutant strains.

Sulfangolids, macrolide sulfate esters from Sorangium cellulosum.

Sulfangolids are the first sulfate ester containing secondary metabolites from myxobacteria. The metabolites 1-4 and the structurally related kulkenon (5) were isolated from different strains of the species Sorangium cellulosum. In the course of isolation all metabolites proved to be rather sensitive due to their conjugated double bond systems and the strong acidic nature of the sulfate ester in sulfangolids. The relative configuration of sulfangolid C (3) was assigned by extensive 1D and 2D NMR analysis and molecular modelling. In addition, the biosynthesis of 3 was studied by feeding experiments.

Simple defined autoinduction medium for high-level recombinant protein production using T7-based Escherichia coli expression systems.

Protein production under the control of lac operon regulatory elements using autoinduction is based on diauxic growth of Escherichia coli on lactose after consumption of more preferred carbon substrates. A novel simple and cost-effective defined autoinduction medium using a mixture of glucose, glycerol, and lactose as carbon substrate and NH(4)(+) as sole nitrogen source without any supplementation of amino acids and vitamins was developed for T7-based E. coli expression systems. This medium was successfully employed in 96-well microtiter plates, test tubes, shake flasks, and 15-L bioreactor cultivations for production of different types of proteins achieving an average yield of 500 mg L(-1) product. Cell-specific protein concentrations and solubility were similar as during conventional isopropyl ß-D-1-thiogalactopyranoside induction using Luria-Bertani broth. However, the final yield of target proteins was about four times higher, as a higher final biomass was achieved using this novel defined autoinduction broth.

Roimatacene: an antibiotic against Gram-negative bacteria isolated from Cystobacter ferrugineus Cb G35 (Myxobacteria).

Roimatacene (1) was isolated from the myxobacterium Cystobacter ferrugineus strain Cb G35 in a bioactivity-guided process, by following the antimicrobial activity against Escherichia coli. Since 1 was extremely sensitive to oxygen, a protective isolation and handling protocol was developed, by utilizing the free radical scavenger 4-ethoxyphenol. The structure of 1 was determined by HRMS, 1D and 2D NMR spectroscopy and chemical derivatization to acetonides and Mosher esters to finally establish the absolute configuration. Methionine and acetate were identified as building blocks in the biosynthesis of 1 by feeding experiments with differently (13)C-labeled precursors. The antimicrobial activity of 1 was determined in a broad screening revealing 1 to inhibit several Gram-negative bacteria.

Patients' perception of preoperative information by interactive computer program-exemplified by cholecystectomy.

Patients' perception of preoperative information by interactive computer program informing about cholecystectomy was assessed. Patients were asked to qualify the computer program after hospital discharge by an anonymous questionnaire. Two hundred and fifty-seven of 278 consecutive patients completed the questionnaire resulting in a response rate of 92%. Sixty-three percent were very satisfied and 37% were satisfied, whereas none were dissatisfied or undecided. Patients' satisfaction was not associated with sex, age, surgeon or prior knowledge of computers. Most of the patients considered the clarity of text and illustrations and the volume of information as good or excellent and found the information well adapted to their prior knowledge. Ninety-eight percent stated that they would like to be informed again by an interactive computer program in case of another surgical intervention. Thus, interactive computer programs seem to be a valuable adjunct to written and oral information in the preoperative informed consent procedure.

Comparison of a production process in a membrane-aerated stirred tank and up to 1000-L airlift bioreactors using BHK-21 cells and chemically defined protein-free medium.

The applicability of a protein-free medium for the production of recombinant human interleukin-2 with baby hamster kidney cells in airlift bioreactors was investigated. For this purpose, a BHK-21 cell line, adapted to grow and produce in protein-free SMIF7 medium without forming spheroids in membrane-aerated bubble-free bioreactors, was used as the producer cell line. First, cultivation of the cells was established at a 20-L scale using an internal loop airlift bioreactor system. During the culturing process the medium formulation was optimized according to the specific requirements associated with cultivation of mammalian cells under protein-free conditions in a bubble-aerated system. The effects of the addition of an antifoam agent on growth, viability, productivity, metabolic rates, and release of lactate dehydrogenase were investigated. Although it was possible to establish cultivation and production at a 20-L scale without the use of antifoaming substances, the addition of 0.002% silicon-oil-based antifoaming reagent improved the cultivation system by completely preventing foam formation. This reduced the release of lactate dehydrogenase activity to the level found in bubble-free aerated stirred tank membrane bioreactors and led to a reduction in generation doubling times by about 5 h (17%). Using the optimized medium formulation, cells were cultivated at a 1000-L scale, resulting in a culture performance comparable to the 20-L airlift bioreactor. For comparison, cultivations with protein-containing SMIF7 medium were carried out at 20- and 1000-L scales. The application of protein supplements did not lead to a significant improvement in the cultivation conditions. The results were also compared with experiments performed in a bubble-free aerated stirred tank membrane bioreactor to evaluate the influence of bubbles on the investigated culture parameters. The data implied a higher metabolic activity of the cells in airlift bioreactors with a 150% higher glucose consumption rate. The results of this study clearly demonstrate the applicability of a protein-free chemically defined medium for the production of recombinant proteins with BHK cells in airlift bioreactors.

Treatment of a case of primary retroperitoneal mucinous cystadenocarcinoma: is adjuvant hysterectomy and bilateral salpingo-oophorectomy justified?

OBJECTIVE: We present a case of primary retroperitoneal mucinous cystadenocarcinoma in a 38-year-old woman. STUDY DESIGN: The tumor was resected with a segment of adjacent descending colon. Five years after the operation, the patient is well, without evidence of recurring disease, based on clinical investigation and modern imaging techniques. RESULTS: In the light of the literature, it appears most likely that this rare tumor is caused by coelomic metaplasia. On the basis of the histopathologic findings in our case and the reports from the literature, we recommend radical tumor excision en bloc with all infiltrated adjacent structures. CONCLUSION: Added removal of unaffected uterus and adnexes makes young women infertile and climacteric and is not yet validated by long-term results.