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Terrestrial actinomycetes are noteworthy producers of a multitude of antibiotics, however the marine representatives are much less studied in this regard. In this study, 90 actinomycetes were isolated from 11 different species of marine sponges that had been collected from offshore Ras Mohamed (Egypt) and from Rovinj (Croatia). Phylogenetic characterization of the isolates based on 16S rRNA gene sequencing supported their assignment to 18 different actinomycete genera representing seven different suborders. Fourteen putatively novel species were identified based on sequence similarity values below 98.2% to other strains in the NCBI database. A putative new genus related to Rubrobacter was isolated on M1 agar that had been amended with sponge extract, thus highlighting the need for innovative cultivation protocols. Testing for anti-infective activities was performed against clinically relevant, Gram-positive (Enterococcus faecalis, Staphylococcus aureus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria, fungi (Candida albicans) and human parasites (Leishmania major, Trypanosoma brucei). Bioactivities against these pathogens were documented for 10 actinomycete isolates. These results show a high diversity of actinomycetes associated with marine sponges as well as highlight their potential to produce anti-infective agents.
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.
Die Funktionalität β1- und β2-adrenerger Rezeptoren wird durch Polymorphismen in ihrer kodierenden Region moduliert. Wir haben uns die Technik des Fluoreszenz-Resonanz- Energie-Transfers (FRET) zu Nutze gemacht, um den Einfluss der am häufigsten vorkommenden Polymorphismen (Ser49Gly und Gly389Arg im β1AR, Arg16Gly und Gln27Glu im β2AR) auf die Rezeptorkonformation nach Aktivierung zu untersuchen. Dafür wurden FRET-Sensoren für die beiden βAR-Subtypen mit einem gelb-fluoreszierenden Protein (YFP) sowie einem cyan-fluoreszierenden Protein (CFP oder Cerulean) in der dritten intrazellulären Schleife bzw. am C-Terminus verwendet. Nach Stimulierung der βARSensoren konnte die Aktivierung der polymorphen Rezeptorvarianten in lebenden Zellen in Echtzeit untersucht werden. Dabei behielten die FRET-Sensoren sowohl die Bindungsaffinitäten der nativen Rezeptoren als auch eine intakte Funktionalität hinsichtlich der Bildung von sekundären Botenstoffen. Der Vergleich der Aktivierungskinetiken der verschieden polymorphen Varianten des β1AR und β2AR ergab keine signifikanten Unterschiede nach einer einmaligen Stimulation. Es zeigte sich jedoch, dass Rezeptorpolymorphismen die Aktivierungskinetik vorstimulierter βAR erheblich beeinflussen. So konnten wir im Vergleich zur ersten Aktivierung eine schnellere Aktivierung der Gly16-Varianten des β2AR sowie des Gly49Arg389-β1AR feststellen, während die Arg16-β2AR-Variante und der Ser49Gly389-β1AR dagegen bei einer wiederholten Stimulation langsamer aktiviert wurden. Diese Ergebnisse lassen auf ein "Rezeptorgedächtnis" schließen, das spezifisch für bestimmte polymorphe Rezeptorvarianten ist und eine βAR-Subtyp-spezische Ausprägung zeigt. Die Ausbildung der unterschiedlichen Aktivierungskinetiken hing von der Interaktion des Rezeptors mit löslichen intrazellulären Faktoren ab und bedurfte einer Phosphorylierung intrazellulärer Serin- und Threonin-Reste durch G-Protein-gekoppelte Rezeptorkinasen. Die Interaktion mit löslichen intrazellulären Faktoren scheint für den β1AR weniger stark ausgeprägt zu sein als für den β2AR. Die cAMP-Produktion war für die schneller werdenden, “hyperfunktionellen” Gly16-β2ARVarianten signifikant um mehr als 50% höher im Vergleich zur “hypofunktionellen” Arg16- Variante. Die unterschiedliche Funktionalität spiegelte sich im Therapieausgang bei Tokoysepatientinnen wider, dessen Erfolg mit dem Arg16Gly Polymorphismus verknüpft war. Die Daten implizieren eine intrinsische, polymorphismusabhängige Eigenschaft der βAR, die die Aktivierungskinetik der Rezeptoren bei wiederholten Stimulationen determiniert. Diese könnte auch für die zwischen Individuen variierende Ansprechbarkeit auf β-Agonisten und β-Blocker mitverantwortlich sein.
Chlamydia trachomatis is an obligate intracellular pathogenic bacterium that has been refractory to genetic manipulations. Although the genomes of several strains have been sequenced, very little information is available on the gene structure of these bacteria. We used deep sequencing to define the transcriptome of purified elementary bodies (EB) and reticulate bodies (RB) of C. trachomatis L2b, respectively. Using an RNAseq approach, we have mapped 363 transcriptional start sites (TSS) of annotated genes. Semiquantitative analysis of mapped cDNA reads revealed differences in the RNA levels of 84 genes isolated from EB and RB, respectively. We have identified and in part confirmed 42 genome- and 1 plasmid-derived novel non-coding RNAs. The genome encoded non-coding RNA, ctrR0332 was one of the most abundantly and differentially expressed RNA in EB and RB, implying an important role in the developmental cycle of C. trachomatis. The detailed map of TSS in a thus far unprecedented resolution as a complement to the genome sequence will help to understand the organization, control and function of genes of this important pathogen.
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.
A gene (Imsod) encoding superoxide dismutase (SOD; EC 1.15.1.1) of the facultative intracellular pathogen, Listeria monocytogenes, was cloned by functional complementation of an SOD-deficient Escherichia coli mutant. The nucleotide sequence was determined and the deduced amino acid (aa) sequence (202 aa) showed close similarity to manganese-containing SOD's from other organisms. Subunits of the recombinant L. monocytogenes SOD (re-SOD) and of both E. coli SODs formed enzymatically active hybrid enzymes in vivo. DNA/DNA-hybridization experiments showed that this type of recombinant re-sod gene is conserved within the genus Listeria.
N-MYC is a member of the human MYC proto-oncogene family, which comprises three transcription factors (C-, N- and L-MYC) that function in multiple biological processes. Deregulated expression of MYC proteins is linked to tumour initiation, maintenance and progression. For example, a large fraction of neuroblastoma displays high N-MYC levels due to an amplification of the N-MYC encoding gene. MYCN-amplified neuroblastoma depend on high N-MYC protein levels, which are maintained by Aurora-A kinase. Aurora-A interaction with N-MYC interferes with degradation of N-MYC via the E3 ubiquitin ligase SCFFBXW7. However, the underlying mechanism of Aurora-A-mediated stabilisation of N-MYC remains to be elucidated.
To identify novel N-MYC interacting proteins, which could be involved in N-MYC stabilisation by Aurora-A, a proteomic analysis of purified N-MYC protein complexes was conducted. Since two alanine mutations in MBI of N-MYC, T58A and S62A (N-MYC mut), disable Aurora-A-mediated stabilisation of N-MYC, N-MYC protein complexes from cells expressing either N-MYC wt or mut were analysed. Proteomic analysis revealed that N-MYC interacts with two deubiquitinating enzymes, USP7 and USP11, which catalyse the removal of ubiquitin chains from target proteins, preventing recognition by the proteasome and subsequent degradation. Although N-MYC interaction with USP7 and USP11 was confirmed in subsequent immunoprecipitation experiments, neither USP7, nor USP11 was shown to be involved in the regulation of N-MYC stability. Besides USP7/11, proteomic analyses identified numerous additional N-MYC interacting proteins that were not described to interact with MYC transcription factors previously. Interestingly, many of the identified N-MYC interaction partners displayed a preference for the interaction with N-MYC wt, suggesting a MBI-dependent interaction. Among these were several proteins, which are involved in three-dimensional organisation of chromatin domains and transcriptional elongation by POL II. Not only the interaction of N-MYC with proteins functioning in elongation, such as the DSIF component SPT5 and the PAF1C components CDC73 and CTR9, was validated in immunoprecipitation experiments, but also with the POL III transcription factor TFIIIC and topoisomerases TOP2A/B. ChIP-sequencing analysis of N-MYC and TFIIIC subunit 5 (TFIIIC5) revealed a large number of joint binding sites in POL II promoters and intergenic regions, which are characterised by the presence of a specific motif that is highly similar to the CTCF motif. Additionally, N-MYC was shown to interact with the ring-shaped cohesin complex that is known to bind to CTCF motifs and to assist the insulator protein CTCF. Importantly, individual ChIP experiments demonstrated that N-MYC, TFIIIC5 and cohesin subunit RAD21 occupy joint binding sites comprising a CTCF motif.
Collectively, the results indicate that N-MYC functions in two biological processes that have not been linked to MYC biology previously. Furthermore, the identification of joint binding sites of N-MYC, TFIIIC and cohesin and the confirmation of their interaction with each other suggests a novel function of MYC transcription factors in three-dimensional organisation of chromatin.
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.
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.
A cloned cytolytic determinant from the genome of Bacillus cereus GP-4 has been characterized at the molecular Ievel. Nucleotide sequence determination revealed the presence of two open reading frames. 8oth open reading frames were found by deletion and complementation analysis to be necessary for expression of the hemolytic phenotype by Bacillus subtilis and Escherichia coli hosts. The 5' open reading frame was found to be nearly identical to a recently reported phospholipase C gene derived from a mutant B. cereus strain which overexpresses the respective protein, and it conferred a lecithinase-positive phenotype to the B. subtilis host. The 3' open reading frame encoded a sphingomyelinase. The two tandemly encoded activities, phospholipase C and sphingomyelinase, constitute a biologically functional cytolytic determinant of B. cereus termed cereolysin AB.
To foster sustainable environmentally friendly behavior in children it is important to provide an effective form of environmental education. In this context we studied three important factors: Attitude towards nature, environmental knowledge and advanced expert knowledge.
Concerning attitude towards nature our first question was: “Is it possible to affect primary school children’s environmental values during a one-day visit at a wildlife park?”
As a control, the program was also conducted in schools, leading to two different learning settings- wildlife park and school.
Regarding environmental knowledge, in our second question we wanted to know, if our modified teaching approach “guided learning at workstations” (G) combining instructional and constructivist elements would lead to good cognitive learning results of primary school children. Additionally, we compared it to a stronger teacher-centered (T) as well as to a stronger student-centered (S) approach.
The third question we asked was “Is it possible to convey fascinating expert knowledge on a more advanced subject to primary school children using conceptual change theory?” After gathering primary school children’s preconceptions, we defined different groups due to the heterogeneity of their pre-existing conceptions and the change in conceptions. Based on this research we designed a program along with an instrument to measure the impact of the conceptual change teaching method.
After years of building a strong cooperation between the section Didactics of Biology at the Julius-Maximilians University Würzburg, the nearby schools and the wildlife park “Wild-Park Klaushof” near Bad Kissingen in northern Bavaria it was time to evaluate the environmental education programs prepared and applied by undergraduate university students. As a model species we chose the European wildcat (Felis silvestris silvestris) which represents endangered wildlife in Europe and the need for human interaction for the sake of preserving a species by restoring or recreating the habitat conditions needed while maintaining current infrastructure. Drawing from our own as well as teachers’ and university students’ experiences, we built, implemented and evaluated a hands-on program following several workstations between the wildcat enclosure and the wildlife park’s green classroom.
The content of our intervention was presented as a problem-oriented lesson, where children were confronted with the need for human interaction in order to preserve the European wildcat. Not only on a theoretical basis, but very specific to their hometowns they were told where and when nature conservation groups met or where to donate money.
692 Bavarian third grade primary school children in 35 classes participated in the one-day intervention that took place between the months of april, 2014 and november, 2015 in the wildlife park or in their respective classrooms. The ages varied between 8 and 11 years with the mean age being 8.88 ± 0.56 years old. 48.6 % of them were boys, 51.4 % were girls.
(1) To measure primary school children’s environmental attitudes a questionnaire on two major environmental values- preservation and utilization of nature- was administered in a pre, post- and retention test design. It was possible to affect primary school children’s environmental preservation values during our one-day program. This result could be found not only at the wildlife park but unexpectedly also in school, where we educated classes for control purposes. We also found this impact consistent in all used teaching approaches and were surprised to see the preservation values change in a way we did not expect from higher tendency towards preservation of nature to a lower one.
We presume that children of this age group reflected on the contents of our intervention. This had an influence on their own values towards preservation which led to a more realistic marking behavior in the questionnaire. We therefore conclude that it is possible to affect primary school children’s environmental values with a one-day program on environmental content.
(2) We were interested in conveying environmental knowledge about the European wildcat; its morphology, ecology and behavior. We designed and applied a knowledge questionnaire also in a pre-, post- and retention test design, to find out, whether different forms of instruction made a difference in learning success of primary school children.
We used two approaches with a teacher in the role of a didactic leader- our modified guided approach (G) as well as a stronger teacher-centered one (T) with a higher focus on instruction. The third approach was presented as a strong student-centered learning at workstations (S) without a didactic leader we also called “free learning at workstations”.
Overall, all children’s knowledge scores changed significantly from pre- to post-test and from pre- to retention test, indicating learning success. Differences could only be found between the posttest values of both approaches with a didactic leader (G, T) in comparison to the strong student-centered (S) form.
It appears that these primary school children gained knowledge at the out of school learning setting regardless of the used teaching approach.
On the subject of short-term differences, we discuss, that the difference in learning success might have been consistent from post to retention test if a consolidation phase had been added in the days following the program as should be common practice after a visit to an out-of- school learning setting but was not part of our intervention.
When comparing both approaches with a didactic leader (G, T), we prefer our modified guided learning at workstations (G) since constructivist phases can be implemented without losses concerning learning success. Moreover, the (at least temporary) presence of a teacher in the role of a didactic leader ensures maintained discipline and counteracts off-task behavior.
To make sure, different emotional states did not factor in our program, we measured children’s situational emotions directly after the morning intervention using a short scale that evaluated interest, wellbeing and boredom. We found, that these emotions remained consistent over both learning settings as well as different forms of instruction. While interest and wellbeing remained constantly high, boredom values remained low.
We take this as a sign of high quality designing and conducting the intervention.
(3) In the afternoon of the one-day intervention, children were given the opportunity to investigate the wildcat further, this time using the conceptual change theory in combination with a more complex and fascinating content: cats’ vision in dusk and dawn.
Children were confronted with their preconceptions which had been sampled prior to the study and turned into three distinctive topics reflected in a special questionnaire.
In a pre-, post and retention test design we included the most common alternative conceptions, the scientifically correct conceptions as well as other preconceptions.
We gathered a high heterogeneity of preconceptions and defined three groups based on conceptual change literature: “Conceptual change”, “Synthetic Models” and “Conceptual Growth”. In addition to these we identified two more groups after our data analysis: “Knowledge” and “Non-addressed Concepts”.
We found that instruction according to the conceptual change theory did not work with primary school children in our intervention. The conceptual change from the addressed alternative conceptions as well as from other preconceptions towards the scientifically correct conceptions was successfully achieved only on occasion.
In our case and depending on the topic only one third to one fourth of the children actually held the addressed conception while the rest was not targeted by the instruction. Moreover, we conclude children holding other conceptions were rather confused than educated by the confrontation. We assume that children of this age group may be overchallenged by the conceptual change method.
The homology screening approach has been used to clone a new member of the guanine-nucleotidebinding-protein-coupled receptor superfamily from guinea pig uterus. The cloned cDNA encodes a 399-amino-acid protein and shows the highest amino acid similarity to members of the bombesin receptor family; 52% and 47% similarity to the gastrin-releasing-peptide (GRP) receptor and the neuromedin-B receptor, respectively. Bindingexperiments with the stably transfected LLC-PK<sub>1</sub> cell line expressing the new receptor protein confmned the bombesin-like nature of the cloned receptor. The relative order ofligand affinity, GRP = neuromedin C >> neuromedin B, suggests that the cloned cDNA represents the GRP subtype rather than the neuromedin-B subtype of bombesin receptors. Northern-blot analysis of mRNA species from several guinea-pig tissues showed that the mRNA for the new bombesin receptor subtype is expressed mainly in uteri of pregnant animals.
A cDNA encoding guinea-pig uterine substance P (SP) receptor has been isolated using the homology screening approach. Northern blot analysis reveals that the corresponding mRNA, of approx. 4.8 kb, is expressed in all tissues tested, but predominantly in the uteri of non-pregnant animals; during pregnancy its expression is reduced. The guinea-pig SP receptor was expressed in COS-7 cells and demonstrated relative Iigand affinity in the order: SP >> neurokinin A > neurokinin B.
CATIO~IC drugs of different types and structures (antihistaminics, antiarrhythmics, sedatives, opiates, cytostatics and antibiotics, for example) are excreted in mammals by epithelial cells of the renal proximal tubules and by hepatocytes in the liver<sup>1-4</sup>. In the proximal tubules, two functionally disparate transport systems are involved which are localized in the basolateral and luminal plasma membrane and are different from the previously identified neuronal monoamine transporters and A TP-dependent multidrug exporting proteins<sup>1-3,5-12</sup>. Here we report the isolation of a complementary DNA from rat kidney that encodes a 556-amino-acid membrane protein, OCT1, which has the functional characteristics of organic cation uptake over the basolateral membrane of renal proximal tubules and of organic cation uptake into hepatocytes. OCTl is not homologous to any other known protein and is found in kidney, liver and intestine. As OCTl translocates hydrophobic and hydrophilic organic cations of different structures, it is considered to be a new prolotype of polyspecific transporters that are important for drug elimination.
Mammalian Sun1 belongs to an evolutionarily conserved family of inner nuclear membrane proteins, which are known as SUN domain proteins. SUN domain proteins interact with KASH domain partners to form bridging complexes, so-called LINC complexes, that physically connect the nuclear interior to the cytoskeleton. LINC complexes are critical for nuclear integrity and play fundamental roles in nuclear positioning, shaping and movement. The mammalian genome codes for at least five different SUN domain proteins used for the formation of a number of different LINC complexes. Recently, we reported on the identification of everal Sun1 isoforms, which tremendously enlarges the alternatives to form functional LINC complexes. We now confirmed that Sun1 actually exists in at least seven distinct splice variants. Besides that, we observed that expression of individual Sun1 isoforms remarkably depends on the cell type, suggesting a cell type-specific adaption of Sun1 dependent LINC complexes to specific cellular and physiological requirements.