Effect of α-MSH upon cyclic AMP levels induced by the glutamatergic agonists NMDA, quisqualic acid, and kainic acid

This study was carried out to investigate possible interactions between some glutamatergic agonists and the peptide α-MSH upon the cyclic AMP levels. We used an in vitro tissue slice preparation incubated in the presence of different glutamatergic agonists such as N-methyl- -aspartic acid (NMDA), quisqualic acid (QUIS), kainic acid (KA), and the peptide α-MSH together with each agonist. Slices containing caudate putamen and accumbens were chosen according to neurochemical data indicating that the striatum contains a moderate amount of MSH binding sites and also receives glutamatergic innervation. Exposure of these slices to either MSH or to the agonists NMDA or QUIS resulted in an increase in the cAMP levels in relation to controls. Nevertheless, incubation with KA resulted in no changes in the nucleotide levels. The combination of MSH/NMDA induced a reduction of cAMP levels in relation to those obtained with NMDA alone. The combinations of QUIS/MSH or KA/MSH also induced variations in the values of nucleotide in relation to the those obtained with the peptide alone or with the corresponding agonist; these changes were related to the dose of agonist used in each case. The results obtained in these experiments suggest the existence of some interaction between the peptide and the agonist used.     

Uncovering mechanisms of rubber biosynthesis in Taraxacum koksaghyz – role of cis‐prenyltransferase‐like 1 protein

The Russian dandelion Taraxacum koksaghyz synthesizes considerable amounts of high‐molecular‐weight rubber in its roots. The characterization of factors that participate in natural rubber biosynthesis is fundamental for the establishment of T. koksaghyz as a rubber crop. The cis‐1,4‐isoprene polymers are stored in rubber particles. Located at the particle surface, the rubber transferase complex, member of the cis‐prenyltransferase (cisPT) enzyme family, catalyzes the elongation of the rubber chains. An active rubber transferase heteromer requires a cisPT subunit (CPT) as well as a CPT‐like subunit (CPTL), of which T. koksaghyz has two homologous forms: TkCPTL1 and TkCPTL2, which potentially associate with the rubber transferase complex. Knockdown of TkCPTL1, which is predominantly expressed in latex, led to abolished poly(cis‐1,4‐isoprene) synthesis but unaffected dolichol content, whereas levels of triterpenes and inulin were elevated in roots. Analyses of latex from these TkCPTL1‐RNAi plants revealed particles that were similar to native rubber particles regarding their particle size, phospholipid composition, and presence of small rubber particle proteins (SRPPs). We found that the particles encapsulated triterpenes in a phospholipid shell stabilized by SRPPs. Conversely, downregulating the low‐expressed TkCPTL2 showed no altered phenotype, suggesting its protein function is redundant in T. koksaghyz. MS‐based comparison of latex proteomes from TkCPTL1‐RNAi plants and T. koksaghyz wild‐types discovered putative factors that convert metabolites in biosynthetic pathways connected to isoprenoids or that synthesize components of the rubber particle shell.     

A novel leptin antagonist peptide inhibits breast cancer growth in vitro and in vivo

The role of the obesity cytokine leptin in breast cancer progression has raised interest in interfering with leptin's actions as a valuable therapeutic strategy. Leptin interacts with its receptor through three different binding sites: I–III. Site I is crucial for the formation of an active leptin–leptin receptor complex and in its subsequent activation. Amino acids 39‐42 (Leu‐Asp‐Phe‐Ile‐ LDFI) were shown to contribute to leptin binding site I and their mutations in alanine resulted in muteins acting as typical antagonists. We synthesized a small peptide based on the wild‐type sequence of leptin binding site I (LDFI) and evaluated its efficacy in antagonizing leptin actions in breast cancer using in vitro and in vivo experimental models. The peptide LDFI abolished the leptin‐induced anchorage‐dependent and ‐independent growth as well as the migration of ERα‐positive (MCF‐7) and ‐negative (SKBR3) breast cancer cells. These results were well correlated with a reduction in the phosphorylation levels of leptin downstream effectors, as JAK2/STAT3/AKT/MAPK. Importantly, the peptide LDFI reversed the leptin‐mediated up‐regulation of its gene expression, as an additional mechanism able to enhance the peptide antagonistic activity. The described effects were specific for leptin signalling, since the developed peptide was not able to antagonize the other growth factors' actions on signalling activation, proliferation and migration. Finally, we showed that the LDFI pegylated peptide markedly reduced breast tumour growth in xenograft models. The unmodified peptide LDFI acting as a full leptin antagonist could become an attractive option for breast cancer treatment, especially in obese women.     

Parameter identification of a dynamic model of CHO cell cultures: an experimental case study

In this paper, we address the problem of parameter identification in dynamic models of animal cultures, and we propose a step-by-step procedure, which gradually considers more detailed models. This procedure allows subsets of parameters to be estimated at each step, which can be used in the initialization of the next identification step. Finally, the full parameter set can be re-estimated starting from the results of the last step. The efficiency of the procedure is illustrated with a simulation case study and with the identification of a dynamic model from experimental data collected in CHO cell culture.     

Spread of a Distinct Stx2-Encoding Phage Prototype among Escherichia coli O104:H4 Strains from Outbreaks in Germany, Norway, and Georgia

Shiga toxin 2 (Stx2)-producing Escherichia coli (STEC) O104:H4 caused one of the world's largest outbreaks of hemorrhagic colitis and hemolytic uremic syndrome in Germany in 2011. These strains have evolved from enteroaggregative E. coli (EAEC) by the acquisition of the Stx2 genes and have been designated enteroaggregative hemorrhagic E. coli. Nucleotide sequencing has shown that the Stx2 gene is carried by prophages integrated into the chromosome of STEC O104:H4. We studied the properties of Stx2-encoding bacteriophages which are responsible for the emergence of this new type of E. coli pathogen. For this, we analyzed Stx bacteriophages from STEC O104:H4 strains from Germany (in 2001 and 2011), Norway (2006), and the Republic of Georgia (2009). Viable Stx2-encoding bacteriophages could be isolated from all STEC strains except for the Norwegian strain. The Stx2 phages formed lysogens on E. coli K-12 by integration into the wrbA locus, resulting in Stx2 production. The nucleotide sequence of the Stx2 phage P13374 of a German STEC O104:H4 outbreak was determined. From the bioinformatic analyses of the prophage sequence of 60,894 bp, 79 open reading frames were inferred. Interestingly, the Stx2 phages from the German 2001 and 2011 outbreak strains were found to be identical and closely related to the Stx2 phages from the Georgian 2009 isolates. Major proteins of the virion particles were analyzed by mass spectrometry. Stx2 production in STEC O104:H4 strains was inducible by mitomycin C and was compared to Stx2 production of E. coli K-12 lysogens.     

Genome Sequence of Radiation-Resistant Modestobacter marinus Strain BC501, a Representative Actinobacterium That Thrives on Calcareous Stone Surfaces

Here we report the full genome sequence of Modestobacter marinus strain BC501, an actinobacterial isolate that thrives on stone surfaces. The generated chromosome is circular, with a length of 5.57 Mb and a G+C content of 74.13%, containing 5,445 protein-coding genes, 48 tRNAs, and 3 ribosomal operons.     

Influence of the metal center and linker on the intracellular distribution and biological activity of organometal–peptide conjugates

Organometallic complexes conjugated to cell-penetrating peptides (CPPs) are promising systems for diagnostic imaging and therapeutic applications in human medicine. Recently, we reported on the synthesis of cymantrene(CpMn(CO)₃)–CPP conjugates with biological activity on different cancer cell lines. However, the precise mechanism of cytotoxicity remained elusive in these studies. To investigate the role of the metal center and the linker between the CpM(CO)₃ moiety and the peptide, a number of derivatives with manganese replaced by rhenium and the keto linker originally used substituted by a methylene group were prepared and fully characterized by ¹H NMR spectroscopy, infrared spectroscopy, electrospray ionization mass spectrometry, and elemental analysis as well as X-ray structure determination. The organometal–peptide conjugates as well as carboxyfluorescein-labeled derivatives thereof were prepared by solid-phase peptide synthesis, purified by high-performance liquid chromatography, and analyzed by mass spectrometry. Fluorescence microscopy studies of MCF-7 human breast cancer cells revealed an efficient cellular uptake and pronounced nuclear localization of the bioconjugates with the methylene linker compared with systems with the keto group. In addition, the latter also showed a higher cytotoxicity. In contrast, the variation of the metal center from manganese to rhenium had a negligible effect. The structure–activity relationships determined in the present work will aid in the further tuning of the biological activity of organometal–peptide conjugates.