A novel method for repetitive peptide synthesis in solution without isolation of intermediates.

A novel method was developed for the large-scale manufacture of peptides in solution, called DioRaSSP-Diosynth Rapid Solution Synthesis of Peptides. This method combines the advantages of the homogeneous character of classical solution-phase synthesis with the universal character and the amenability to automation inherent to the solid-phase approach. The process consists of repetitive cycles of coupling and deprotection in a permanent organic phase and is further characterized by the fact that intermediates are not isolated. Couplings are mediated by water-soluble carbodiimide. Several types of function may be applied for temporary amino protection depending on the sequence of the actual peptide, including Z, Fmoc, Msc and Nsc. Formate is the preferred hydrogen donor during hydrogenolysis of the Z function, while 1,8-diazabicyclo[5.4.0]undec-7-ene is used to deprotect Fmoc, Msc and Nsc. Morpholine is added during the deprotection of Msc and Nsc to scavenge the arising alkenes. Processes according to this highly efficient synthesis method are easy to scale up and yield products of reproducible high purity, which is guaranteed by a new quenching method for residual activated compounds, applying an anion-forming amine such as a beta-alanine ester. This ester should display a lability similar to that of the temporary amino-protecting function, allowing simultaneous deprotection of the growing peptide and the quenched compound. The DioRaSSP approach assures the completely quantitative removal of deprotected quenched compounds before the coupling step of the next cycle of the synthesis by basic aqueous (that is active) extraction, while the growing peptide remains anchored in the organic phase due to the presence of hydrophobic protecting functions.     

Immunomodulating properties of bidistilled modified water

The effect of bidistilled modified water obtained by treating bidistilled water with static electric field in the cathode compartment of a diaphragm electrolyzer on the production of tumor necrosis factor and activity of natural killer cells of mice was studied. It was found that bidistilled modified water added to cultured murine macrophages increases the tumor necrosis factor production 2.5-3-fold. A similar degree of stimulation of tumor necrosis factor production was observed after peroral administration of bidistilled modified water for 10-25 days. The activity of natural killer cells under these conditions increased by 150%. The stimulation of these immunity system components responsible for the resistance of the organism to tumors led to a suppression of tumor growth in tumor-bearing mice receiving bidistilled modified water.     

Microsatellite markers for population studies of Phytophthora megakarya (Pythiaceae), a cacao pathogen in Africa

? Premise of the study: Phytophthora megakarya is the agent of black pod disease of cacao and is the main pathogen of this crop in Africa. Population genetic studies are required to investigate how this pathogen emerged. To this end, we developed 12 novel polymorphic microsatellite markers for P. megakarya. ? Methods and Results: Microsatellite sequences were obtained by pyrosequencing of multiplex-enriched libraries. Candidate loci with di- or trinucleotide motifs were selected, and primer pairs were tested with nine P. megakarya isolates. The 12 most polymorphic and unambiguous loci were selected to develop three multiplex PCR pools. The total number of alleles varied from two to nine, depending on loci, and higher than expected heterozygosity was observed. ? Conclusions: These markers were used for population genetic studies of P. megakarya in Cameroon and for comparison with reference strains from West Africa. This is the first time that microsatellite markers have been developed for P. megakarya.     

TGFβ signaling and cardiovascular diseases

Transforming growth factor β (TGFβ) family members are involved in a wide range of diverse functions and play key roles in embryogenesis, development and tissue homeostasis. Perturbation of TGFβ signaling may lead to vascular and other diseases. In vitro studies have provided evidence that TGFβ family members have a wide range of diverse effects on vascular cells, which are highly dependent on cellular context. Consistent with these observations genetic studies in mice and humans showed that TGFβ family members have ambiguous effects on the function of the cardiovascular system. In this review we discuss the recent advances on TGFβ signaling in (cardio)vascular diseases, and describe the value of TGFβ signaling as both a disease marker and therapeutic target for (cardio)vascular diseases.     

Quantitative proteomic analysis of yeast DNA replication proteins

Chromatin is dynamically regulated, and proteomic analysis of its composition can provide important information about chromatin functional components. Many DNA replication proteins for example bind chromatin at specific times during the cell cycle. Proteomic investigation can also be used to characterize changes in chromatin composition in response to perturbations such as DNA damage, while useful information is obtained by testing the effects on chromatin composition of mutations in chromosome stability pathways. We have successfully used the method of stable isotope labeling by amino acids in cell culture (SILAC) for quantitative proteomic analysis of normal and pathological changes to yeast chromatin. Here we describe this proteomic method for analyzing changes to Saccharomyces cerevisiae chromatin, illustrating the procedure with an analysis of the changes that occur in chromatin composition as cells progress from a G1 phase block (induced by alpha factor) into S phase (in the presence of DNA replication inhibitor hydroxyurea).     

Characterization of novel diesel-degrading strains <Emphasis Type="Italic">Acinetobacter haemolyticus</Emphasis> MJ01 and <Emphasis Type="Italic">Acinetobacter johnsonii</Emphasis> MJ4 isolated from oil-contaminated soil

The diesel-degrading strains, designated as MJ01 and MJ4, were isolated from oil-contaminated soil in Daejeon (South Korea) and were taxonomically characterized using a polyphasic approach and their diesel oil degradation abilities were analyzed. The isolates MJ01 and MJ4 were identified as Acinetobacter haemolyticus and Acinetobacter johnsonii, respectively, based on their 16S rDNA gene sequences, DNA–DNA relatedness, fatty acid profiles and various physiological characteristics. Strains MJ01 and MJ4 were able to use diesel oil as the sole carbon and energy source. Both strains could degrade over 90% of diesel oil with an initial concentration of 20,000 mg/l after incubation for 7 days, the most significant degradation occurred during the first 3 days. To our knowledge, this is the first report on diesel oil-degrading microorganisms among bacterial strains belonging to A. haemolyticus and A. johnsonii.