The PI3K inhibitor pictilisib and the multikinase inhibitors pazopanib and sorafenib have an impact on Rac1 level and migration of medulloblastoma in vitro

Metastatic disease is the leading cause of death in children suffering from medulloblastoma and a major treatment challenge. The evidence of leptomeningeal dissemination defines the most aggressive tumours and is associated with increased mortality; thus, inhibition of migration as a factor involved in the process of metastatic disease is fundamental for the treatment and prevention of metastatic dissemination. Targeting the small Rho GTPases Rac1 has been shown to effectively impair medulloblastoma cell migration in vitro. Yet clinically applicable selective Rac1 inhibitors are still lacking. In view of the pertinent oncogenic role of the PI3K signalling cascade and tyrosine kinase‐mediated signalling pathways in medulloblastoma, we explored clinically available targeted therapeutics to this effect. Here, we show that Rac1 is expressed in both the cytoplasm and nucleus in the medulloblastoma cell lines Daoy and MEB‐Med‐8A representative of two high risk medulloblastoma entities. We demonstrate that activated Rac1 is subject to substantial downmodulation following administration of the clinically available inhibitor of the PI3K pathway Pictilisib (GDC‐0941) and the multityrosine kinase inhibitors Pazopanib and Sorafenib. The application of those drugs was associated with reduced mobility of the medulloblastoma cells and alterations of the actin skeleton. Of note, PI3K inhibition reveals the strongest anti‐migratory effect in Daoy cells. Thus, our in vitro observations provide new insights into different strategies of blocking Rac1 and inhibiting migration in medulloblastoma employing clinically available agents paving the way for confirmatory studies in in vivo models.     

Protonephridial organs in Myzostoma cirriferum (Myzostomida)

Myzostoma cirriferum Leuckart, 1836 possesses five paired, serially arranged, blindending nephridial organs which are described for the first time. Ultrastructural investigations reveal that each nephridium is composed of three terminal cells and one tubular cell that forms the emission tubule. The central lumen of the individual terminal cells contains six to nine flagella, each of which is surrounded regularly by cytoplasmic rods arranged in parallel. Weir-like fenestrations in the peripheral wall of the terminal cells make up the connection between the central lumina and the extracellular space around the nephridial organ. The canal of the emission tubule possesses cilia, microvilli and cytoplasmic structures, suggesting involvement of this cell with active transport and storage. It opens into the cuticle at the ventral surface of the animal.     

Simultaneous fitting of real-time PCR data with efficiency of amplification modeled as Gaussian function of target fluorescence

Background  

In real-time PCR, it is necessary to consider the efficiency of amplification (EA) of amplicons in order to determine initial target levels properly. EAs can be deduced from standard curves, but these involve extra effort and cost and may yield invalid EAs. Alternatively, EA can be extracted from individual fluorescence curves. Unfortunately, this is not reliable enough.     

Social Organization of Lepilemur ruficaudatus

We characterize the spatial organization of red-tailed sportive lemurs (Lepilemur ruficaudatus) as a key aspect of their social organization and social system. Sportive lemurs are small (<1000 g), nocturnal and folivorous primates endemic to Madagascar. We studied a population of 57 individually-marked individuals in Kirindy Forest, western Madagascar, between 1995 and 2001. We radio-tracked 20 males and 26 females of the marked population to obtain detailed information on the size and location of their home ranges. Census data and morphometric measurements provided complementary data sets. Males and females occupied small (<1 ha) home-ranges. Long-term records from 9 individuals revealed home-range stability over several years. In 4 cases home ranges overlapped extensively with that of one member of the opposite sex; in 2 cases, a spatial association of 1 male and 2 females occurred. However, home ranges overlapped very little with neighboring individuals of both sexes. During the study period, spatially associated individuals used on average 5.6 sleeping trees within 117 days, but they spent on average only about every fourth night together. The data suggest that home ranges in red-tailed sportive lemurs are exclusively used by pairs or trios and that the modal social organization of red-tailed sportive lemurs is pair-living.     

Incidence of Klebsiella Species in Surface Waters and Their Expression of Virulence Factors

To investigate the occurrence of different Klebsiella spp. in aquatic environments, a total of 208 samples of natural surface waters was examined. From half (53%) of these samples, 123 Klebsiella strains were isolated, the most common species being Klebsiella pneumoniae. A comparison of these isolates to a group of 207 clinical K. pneumoniae isolates demonstrated that water isolates of K. pneumoniae, unlike those of K. oxytoca and K. planticola, are as capable as clinical isolates of expressing putative virulence factors such as serum resistance and capsular polysaccharides, pili, and siderophores.     

Glutamine transport by rat basolateral membrane vesicles

Glutamine, a neutral amino acid, is unlike most amino acids, has two amine moieties which underlies its importance as a nitrogen transporter and a carrier of ammonia from the periphery to visceral organs. The gastrointestinal tract utilizes glutamine as a respiratory substrate. The intestinal tract receives glutamine from the luminal side and from the arterial side through the basolateral membranes of the enterocyte. This study characterizes the transport of glutamine by basolateral membrane vesicles of the rat. Basolateral membranes were prepared by a well validated technique of separation on a percoll density gradient. Membrane preparations were enriched with Na+/K+-ATPase and showed no 'overshoot' phenomena with glucose under sodium-gradient conditions. Glutamine uptake represented transport into the intravesicular space as evident by an osmolality study. Glutamine uptake was temperature sensitive and driven by an inwardly directed sodium gradient as evident by transient accumulation of glutamine above the equilibrium values. Kinetics of glutamine uptake under both sodium and potassium gradients at glutamine concentrations between 0.01 and 0.6 mM showed saturable processes with Vmax of 0.39 +/- 0.008 and 0.34 +/- 0.05 nmol/mg protein per 15 s for both sodium-dependent and sodium-independent processes, respectively. Km values were 0.2 +/- 0.01 and 0.55 +/- 0.01 mM, respectively. pH optimum for glutamine uptake was 7.5. Imposition of negative membrane potential by valinomycin and anion substitution studies enhanced the sodium-dependent uptake of glutamine suggesting an electrogenic process, whereas the sodium-independent uptake was not enhanced suggesting an electroneutral process. Other neutral amino acids inhibited the initial uptake of glutamine under both sodium-dependent and sodium-independent conditions. We conclude that glutamine uptake by basolateral membranes occurs by carrier-mediated sodium-dependent and sodium-independent processes. Both processes exhibit saturation kinetics and are inhibited by neutral amino acids. The sodium-dependent pathway is electrogenic whereas the sodium-independent pathway is electroneutral.     

Über die RSN-Synthese bei Saccharomyces-Zellen nach photodynamischer Behandlung und nach Röntgenbestrahlung

Zusammenfassung Die RNS-Synthese inSaccharomyces- Zellen wird bis zu 10 Std nach photodynamischer Behandlung mit Thiopyronin und vergleichsweise auch nach Röntgenbestrahlung untersucht. Während die RNS-Synthese selbst nach Einwirkung hoher Röntgendosen bis zu 2 Std nach Bestrahlung in allen Zellen nur geringfügig gehemmt ist, dann aber in nicht mehr teilungsfähigen Zellen stark inhibiert wird, erfolgt die Blockierung der RNS-Synthese nach photodynamischer Behandlung in nicht mehr teilungsfähigen Zellen sofort, während die überlebenden, teilungsfähigen Zellen offensichtlich in ihrem Stoffwechsel nicht beeinträchtigt sind. Die Ergebnisse werden als weitere Bestätigung der Tatsache gewertet, daß die photodynamische Wirkung in Hefezellen nicht wie nach Röntgenbestrahlung hauptsächlich durch eine DNS-Schädigung verursacht wird, sondern wahrscheinlich durch sofortige Inaktivierung plasmatischer Strukturen.

---

Synthesis of RNA inSaccharomyces after photodynamic treatment or irradiation with X-rays

Summary RNA synthesis inSaccharomyces has been investigated up to 10 h after X-irradiation or photodynamic treatment with thiopyronine and visible light. Even with high doses of X-rays RNA synthesis is only very slightly inhibited in all cells up to about 2 h after irradiation. Then RNA synthesis decreases strongly in cells which cannot divide. On the contrary, after photodynamic treatment the inhibition of RNA synthesis in cells which lost their colony-forming ability begins immediately. In cells not inactivated in colony-forming ability, RNA synthesis shows nearly the same extent as control cells.These results give further supports that inactivation by photodynamic action in yeast cells is not caused by DNA damage — which is the case after irradiation with X-rays — but by inactivating plasmatic structures.

---

---

Herrn Prof. Dr. Dr. h. c. mult. B. Rajewsky zum 80. Geburtstag gewidmet.

---

Herrn Prof. Dr. W. Pohlit danken wir herzlich für intensive Diskussion der vorliegenden Ergebnisse.