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Diazepinomicin is a dibenzodiazepine alkaloid with an unusual structure among the known microbial metabolites discovered so far. Diazepinomicin was isolated from the marine sponge-associated strain Micromonospora sp. RV115 and was identified by spectroscopic analysis and by comparison to literature data. In addition to its interesting preclinical broad-spectrum antitumor potential, we report here new antioxidant and anti-protease activities for this compound. Using the ferric reducing antioxidant power (FRAP) assay, a strong antioxidant potential of diazepinomicin was demonstrated. Moreover, diazepinomicin showed a significant antioxidant and protective capacity from genomic damage induced by the reactive oxygen species hydrogen peroxide in human kidney (HK-2) and human promyelocytic (HL-60) cell lines. Additionally, diazepinomicin inhibited the proteases rhodesain and cathepsin L at an IC50 of 70–90 μM. It also showed antiparasitic activity against trypomastigote forms of Trypanosoma brucei with an IC50 of 13.5 μM. These results showed unprecedented antioxidant and anti-protease activities of diazepinomicin, thus further highlighting its potential as a future drug candidate.
Evaluation of a pathophysiological role of the interleukin-6-type cytokine oncostatin M (OSM) for human diseases has been complicated by the fact that mouse models of diseases targeting either OSM or the OSM receptor (OSMR) complex cannot fully reflect the human situation. This is due to earlier findings that human OSM utilizes two receptor complexes, glycoprotein 130 (gp130)/leukemia inhibitory factor receptor (LIFR) (type I) and gp130/OSMR (type II), both with wide expression profiles. Murine OSM on the other hand only binds to the gp130/OSMR (type II) receptor complex with high affinity. Here, we characterize the receptor usage for rat OSM. Using different experimental approaches (knock-down of the OSMR expression by RNA interference, blocking of the LIFR by LIF-05, an antagonistic LIF variant and stably transfected Ba/F3 cells) we can clearly show that rat OSM surprisingly utilizes both, the type I and type II receptor complex, therefore mimicking the human situation. Furthermore, it displays cross-species activities and stimulates cells of human as well as murine origin. Its signaling capacities closely mimic those of human OSM in cell types of different origin in the way that strong activation of the Jak/STAT, the MAP kinase as well as the PI3K/Akt pathways can be observed. Therefore, rat disease models would allow evaluation of the relevance of OSM for human biology.
Aberrations in gene expression are a hallmark of cancer cells. Differential tumor-specific transcript levels of single genes or whole sets of genes may be critical for the neoplastic phenotype and important for therapeutic considerations or useful as biomarkers. As an approach to filter out such relevant expression differences from the plethora of changes noted in global expression profiling studies, we searched for changes of gene expression levels that are conserved. Transcriptomes from massive parallel sequencing of different types of melanoma from medaka were generated and compared to microarray datasets from zebrafish and human melanoma. This revealed molecular conservation at various levels between fish models and human tumors providing a useful strategy for identifying expression signatures strongly associated with disease phenotypes and uncovering new melanoma molecules.
Diversity of Nonribosomal Peptide Synthetase Genes in the Microbial Metagenomes of Marine Sponges
(2012)
Genomic mining revealed one major nonribosomal peptide synthetase (NRPS) phylogenetic cluster in 12 marine sponge species, one ascidian, an actinobacterial isolate and seawater. Phylogenetic analysis predicts its taxonomic affiliation to the actinomycetes and hydroxy-phenyl-glycine as a likely substrate. Additionally, a phylogenetically distinct NRPS gene cluster was discovered in the microbial metagenome of the sponge Aplysina aerophoba, which shows highest similarities to NRPS genes that were previously assigned, by ways of single cell genomics, to a Chloroflexi sponge symbiont. Genomic mining studies such as the one presented here for NRPS genes, contribute to on-going efforts to characterize the genomic potential of sponge-associated microbiota for secondary metabolite biosynthesis.
Background: Combination of oncolytic vaccinia virus therapy with conventional chemotherapy has shown promise for tumor therapy. However, side effects of chemotherapy including thrombocytopenia, still remain problematic. Methods: Here, we describe a novel approach to optimize combination therapy of oncolytic virus and chemotherapy utilizing virus-encoding hyper-IL-6, GLV-1h90, to reduce chemotherapy-associated side effects. Results: We showed that the hyper-IL-6 cytokine was successfully produced by GLV-1h90 and was functional both in cell culture as well as in tumor-bearing animals, in which the cytokine-producing vaccinia virus strain was well tolerated. When combined with the chemotherapeutic mitomycin C, the anti-tumor effect of the oncolytic virotherapy was significantly enhanced. Moreover, hyper-IL-6 expression greatly reduced the time interval during which the mice suffered from chemotherapy-induced thrombocytopenia. Conclusion: Therefore, future clinical application would benefit from careful investigation of additional cytokine treatment to reduce chemotherapy-induced side effects.
Both human herpes viruses and Chlamydia are highly prevalent in the human population and are detected together in different human disorders. Here, we demonstrate that co-infection with human herpes virus 6 (HHV6) interferes with the developmental cycle of C. trachomatis and induces persistence. Induction of chlamydial persistence by HHV6 is independent of productive virus infection, but requires the interaction and uptake of the virus by the host cell. On the other hand, viral uptake is strongly promoted under co-infection conditions. Host cell glutathione reductase activity was suppressed by HHV6 causing NADPH accumulation, decreased formation of reduced glutathione and increased oxidative stress. Prevention of oxidative stress restored infectivity of Chlamydia after HHV6-induced persistence. We show that co-infection with Herpes simplex virus 1 or human Cytomegalovirus also induces chlamydial persistence by a similar mechanism suggesting that Chlamydia -human herpes virus co-infections are evolutionary shaped interactions with a thus far unrecognized broad significance.
Myc is a global transcriptional regulator and one of the most frequently overexpressed oncoproteins in human tumors. It is well established that activation of Myc leads to enhanced cell proliferation but can also lead to increased apoptosis. The use of animal models expressing deregulated levels of Myc has helped to both elucidate its function in normal cells and give insight into how Myc initiates and maintains tumorigenesis. Analyses of the medaka (Oryzias latipes) genome uncovered the unexpected presence of two Myc gene copies in this teleost species. Comparison of these Myc versions to other vertebrate species revealed that one gene, myc17, differs by the loss of some conserved regulatory protein motifs present in all other known Myc genes. To investigate how such differences might affect the basic biological functions of Myc, we generated a tamoxifeninducible in vivo model utilizing a natural, fish-specific Myc gene. Using this model we show that, when activated, Myc17 leads to increased proliferation and to apoptosis in a dose-dependent manner, similar to human Myc. We have also shown that long-term Myc17 activation triggers liver hyperplasia in adult fish, allowing this newly established transgenic medaka model to be used to study the transition from hyperplasia to liver cancer and to identify Myc-induced tumorigenesis modifiers.
Molecular characterization of antimicrobial peptide genes of the carpenter ant Camponotus floridanus
(2012)
The production of antimicrobial peptides (AMPs) is a major defense mechanism against pathogen infestation and of particular importance for insects relying exclusively on an innate immune system. Here, we report on the characterization of three AMPs from the carpenter ant Camponotus floridanus. Due to sequence similarities and amino acid composition these peptides can be classified into the cysteine-rich (e.g. defensin) and glycine-rich (e.g. hymenoptaecin) AMP groups, respectively. The gene and cDNA sequences of these AMPs were established and their expression was shown to be induced by microbial challenge. We characterized two different defensin genes. The defensin-2 gene has a single intron, whereas the defensin-1 gene has two introns. The deduced amino acid sequence of the C. floridanus defensins is very similar to other known ant defensins with the exception of a short C-terminal extension of defensin-1. The hymenoptaecin gene has a single intron and a very peculiar domain structure. The corresponding precursor protein consists of a signal- and a pro-sequence followed by a hymenoptaecin-like domain and six directly repeated hymenoptaecin domains. Each of the hymenoptaecin domains is flanked by an EAEP-spacer sequence and a RR-site known to be a proteolytic processing site. Thus, proteolytic processing of the multipeptide precursor may generate several mature AMPs leading to an amplification of the immune response. Bioinformatical analyses revealed the presence of hymenoptaecin genes with similar multipeptide precursor structure in genomes of other ant species suggesting an evolutionary conserved important role of this gene in ant immunity.
Plant communities in the European Alps are assumed to be highly affected by climate change since temperature rise in this region is above the global average. It is predicted that higher temperatures will lead to advanced snowmelt dates and that the number of extreme weather events will increase. The aims of this study were to determine the impacts of extreme climatic events on flower phenology and to assess whether those impacts differed between lower and higher altitudes. In 2010 an experiment simulating advanced and delayed snowmelt as well as drought event was conducted along an altitudinal transect ca. every 250m (600-2000 m a.s.l.) in the Berchtesgaden National Park, Germany. The study showed that flower phenology is strongly affected by altitude; however there were few effects of the manipulative treatments on flowering. The effects of advanced snowmelt were significantly greater at higher than at lower sites, but no significant difference was found between both altitudinal bands for the other treatments. The response of flower phenology to temperature declined through the season and the length of flowering duration was not significantly influenced by treatments. The stronger effect of advanced snowmelt at higher altitudes might be a response to differences in treatment intensity across the gradient. Consequently, shifts in the date of snowmelt due to global warming may affect species more at higher than at lower altitudes since changes may be more pronounced at higher altitudes. Our data indicate a rather low risk of drought events on flowering phenology in the Bavarian Alps.
It is of interest to define bacterial toxin biochemical properties to use them as molecular-syringe devices in order to deliver enzymatic activities into host cells. Binary toxins of the AB7/8-type are among the most potent and specialized bacterial protein toxins. The B subunits oligomerize to form a pore that binds with high affinity host cell receptors and the enzymatic A subunit. This allows the endocytosis of the complex and subsequent injection of the A subunit into the cytosol of the host cells. Here we report that the addition of an N-terminal His6-tag to different proteins increased their binding affinity to the protective antigen (PA) PA63-channels, irrespective if they are related (C2I) or unrelated (gpJ, EDIN) to the AB7/8-family of toxins. His6-EDIN exhibited voltage-dependent increase of the stability constant for binding by a factor of about 25 when the trans-side corresponding to the cell interior was set to 270 mV. Surprisingly, the C. botulinum toxin C2II-channel did not share this feature of PA63. Cell-based experiments demonstrated that addition of an N-terminal His6-tag promoted also intoxication of endothelial cells by C2I or EDIN via PA63. Our results revealed that addition of His6-tags to several factors increase their binding properties to PA63 and enhance the property to intoxicate cells.