Suppression of oxidative phosphorylation confers resistance against bevacizumab in experimental glioma

Although bevacizumab initially shows high response rates in gliomas and other tumours, therapy resistance usually develops later. Because anti‐angiogenic agents are supposed to induce hypoxia, we asked whether rendering glioma cells independent of oxidative phosphorylation modulates their sensitivity against hypoxia and bevacizumab. LNT‐229 glioma cells without functional mitochondria (rho⁰) and control (rho⁺) cells were generated. LNT‐229 rho⁰‐cells displayed reduced expression of oxidative phosphorylation‐related genes and diminished oxygen consumption. Conversely, glycolysis was up‐regulated in these cells, as shown by increased lactate production and stronger expression of glucose transporter‐1 and lactate dehydrogenase‐A. However, hypoxia‐induced cell death in vitro was nearly completely abolished in the LNT‐229 rho⁰‐cells, these cells were more sensitive towards glucose restriction and the treatment with the glycolysis inhibitor 2‐deoxy‐D‐glucose. In an orthotopic mouse xenograft experiment, bevacizumab induced hypoxia as reflected by elevated Hypoxia‐inducible factor 1‐alpha staining in both, rho⁺‐ and rho⁰‐tumours. However, it prolonged survival only in the mice bearing rho⁺‐tumours (74 days vs. 105 days, p  = 0.024 log‐rank test) and had no effect on survival in mice carrying LNT‐229 rho⁰‐tumours (75 days vs. 70 days, p  = 0.52 log‐rank test). Interestingly, inhibition of glycolysis in vivo with 2‐deoxy‐D‐glucose re‐established sensitivity of rho⁰‐tumours against bevacizumab (98 days vs. 80 days, p  = 0.0001). In summary, ablation of oxidative phosphorylation in glioma cells leads to a more glycolytic and hypoxia‐resistant phenotype and is sufficient to induce bevacizumab‐refractory tumours. These results add to increasing evidence that a switch towards glycolysis is one mechanism how tumour cells may evade anti‐angiogenic treatments and suggest anti‐glycolytic strategies as promising approaches to overcome bevacizumab resistance.

     

Contrast sensitivity and appearance in briefly presented illusory figures.

We examined the contributions of brightness enhancement, illusory figure formation and figural completion to changes in contrast sensitivity in contour gaps. The brightness on the border of a Kanizsa-square and an outline square was measured as the point of subjective equality with the background (PSE) for small line targets. Increment and decrement thresholds were measured at the same location. We found that contrast thresholds were lower than in a control condition without inducers, and that the threshold reduction was independent of the contrast polarity of the inducers. This reduction cannot be explained by a simple summation of stimulus contrast and induced brightness. In a second experiment the inducers that define the contour of the Kanizsa and the outline square were changed so that the figure was no longer closed, keeping the local stimulus surround constant. Thresholds were equally reduced for all conditions, independently of whether the figure was completed or not, or whether an illusory contour was perceived or not. The results suggest that the reduction of contrast threshold in contour gaps is independent of the brightness perceived in these gaps and of the formation of an illusory figure. Processes that cause contrast threshold reduction in contour gaps also seem to operate independently of figural completion.     

Age-dependent changes of the ion content and the circadian leaf movement period in thePhaseolus pulvinus

The circadian movement of the lamina of primary leaves ofPhaseolus coccineus L. depends on circadian changes of the K⁺, Cl^- and (depending on the Cl^- availability) malate content in the swelling and shrinking motor cells of the laminar pulvinus. After sowing in soil, the laminar pulvinus develops within about 26 days. When the leaves emerge from the soil (about 6 days after sowing) and the pulvinus starts with the diurnal movement (about 9 days after sowing) the pulvinar dimensions are about half of those of the mature pulvinus. The anatomical structure, however, is basically the same as in the developed pulvinus. In soil-grown plants, the K⁺, Cl^- and malate content as well as the period length of the circadian leaf movement rhythm change in the developing pulvinus. In the embryo of the dry seed, the Cl^- content is low (0.03 mmol g^-1 DW), the K⁺ content, however, 22-fold higher than the Cl^- content. When the leaves emerge from the soil, the pulvinar K⁺ and Cl^- content is the same as in the whole embryo of the dry seed. In the developing pulvinus the K⁺ content increases by a factor of 2 and the Cl^- content by a factor of 41 in the mature pulvinus. The pulvinar malate content increases between the 6th and 10th day from about 40 to 180Μmol g^-1 DW, then decreases until the 17th day and remains thereafter on a low level (around 80 Μmol g^-1 DW). These results indicate that the Cl^- availability increases in the developing pulvinus with age. It explains furthermore why in young leaves malate was found as counterion to K⁺ in the osmotic leaf movement motor, in older ones, however, Cl^-. The circadian leaf movement starts 9 days after sowing. The period length decreases during the development of the pulvinus from 31.3 to 28.6 h in leaves of intact soil-grown plants. In leaves which were cut from the plants and immersed with their petioles in distilled water, the age dependent decrease of the period length is also found. However, the period lengths are shorter by more than 1 h than in the leaves of intact plants. The increasing Cl^- availability in the developing pulvinus does not seem to be the cause for the age dependent shortening of the period length, because the period length in 22 days old Cl^- deprived pulvini is the same as in 22 days old pulvini with a high Cl^- content.     

NACOB presentation to ASB Young Scientist Award: Postdoctoral. The impact of boundary conditions and mesh size on the accuracy of cancellous bone tissue modulus determination using large-scale finite-element modeling. North American Congress on Biomechanics.

The apparent properties of cancellous bone are determined by a combination of both hard tissue properties and microstructural organization. A method is desired to extract the underlying hard tissue properties from simple mechanical tests, free from the complications of microstructure. It has been suggested that microCT voxel-based large-scale finite element models could be employed to accomplish this goal (van Rietbergen et al., 1995, Journal of Biomechanics, 28, 69-81). This approach has recently been implemented and it is becoming increasingly popular as finite element models increase in size and sophistication (Fyhrie et al., 1997, Proceedings of the 43rd Annual Meeting of the Orthopaedic Research Society, San Francisco, CA, p. 815; van Rietbergen et al., 1997, Proceedings of the 43rd Annual Meeting of the Orthopaedic Research Society, San Francisco, CA, p. 62). However, no direct quantitative measurements of the accuracy of this method applied to porous structures such as cancellous bone have been made. This project demonstrates the feasibility of this approach by quantifying its best-case accuracy in determining the trabecular hard tissue modulus of analogues fabricated of a material with known material properties determined independently by direct testing. In addition we were able to assess the impact of mesh size and boundary conditions on accuracy. We found that the assumption of a frictionless boundary condition in the parallel plate compression loading configuration was a significant source of error that could be overcome with the use of rigid end-caps similar to those used by Keaveny et al. (1997 Journal of Orthopaedic Research, 15(1), 101-110). In conclusion, we found that this approach is an effective method for determining the average trabecular hard tissue properties of human cancellous bone with an expected practical accuracy level better than 5%.     

Effect of different levels of intracellular cAMP on the in vitro maturation of cattle oocytes and their subsequent development following in vitro fertilization

Serum, gonadotrophins, growth factors, and steroid hormones stimulate the in vitro maturation (IVM) of competent oocytes, acting, directly or indirectly, upon the adenylate cyclase pathway to produce the intracellular messenger, cAMP. The intracellular levels of cAMP in cattle cumulus‐oocyte complexes (COC) were manipulated by adding to the collection and maturation media invasive adenylate cyclase (iAC), a toxin produced by the bacterium, Bordetella pertussis. High concentrations of iAC (1 or 5 μg/ml) in the maturation medium inhibited the resumption of meiosis, while low concentrations (0.1 or 0.01 μg/ml) resulted in high rates of maturation to the MII stage (92.6 ± 2.5 and 98.5 ± 1.4% respectively). The same low concentrations of iAC in the maturation medium resulted in rates of development to the blastocyst stage 8 days post insemination (30.1 ± 4.2 and 45.1 ± 3.9%, respectively), which were either not different, or significantly better, than those obtained after IVM in medium supplemented only with serum and gonadotrophins (36.1 ± 2.9%). Finally, the addition of 0.1 μg/ml iAC and 0.5 mM 3‐isobutyl 1‐methylxanthine (IBMX) in the collection medium significantly improved the blastocyst rate when IVM was performed in control medium or medium supplemented with 0.01 μg/ml iAC (31.9 ± 5.5 vs. 12.1 ± 1.6 and 45.5 ± 2.9 vs. 19.1 ± 2.3% respectively). It is concluded that the maintenance of an optimal intracellular concentration of cAMP before and during IVM ensures a high developmental competence of bovine oocytes matured in medium without serum and hormones. Mol. Reprod. Dev. 54:86–91,1999. © 1999 Wiley‐Liss, Inc.     

The glycoprotein-hormones activin A and inhibin A interfere with dendritic cell maturation

Background  

Pregnancy represents an exclusive situation in which the immune and the endocrine system cooperate to prevent rejection of the embryo by the maternal immune system. While immature dendritic cells (iDC) in the early pregnancy decidua presumably contribute to the establishment of peripheral tolerance, glycoprotein-hormones of the transforming growth factor beta (TGF-beta) family including activin A (ActA) and inhibin A (InA) are candidates that could direct the differentiation of DCs into a tolerance-inducing phenotype.     

Carbohydrate availability affects growth and metabolism in peach fruit

Along with sucrose, sorbitol represents the main photosynthetic product and form of translocated carbon in peach. This study aimed at determining whether peach fruit carbohydrate metabolism is affected by changes in source–sink balance , and specifically whether sorbitol or sucrose availability regulates fruit enzyme activities and growth. In various trials, different levels of assimilate availability to growing fruits were induced in vivo by varying crop load of entire trees, leaf : fruit ratio (L:F) of fruiting shoots, or by interrupting the phloem stream (girdling) to individual fruits. In vitro, fruit tissue was incubated in presence/absence of sorbitol and sucrose. Relative growth rate (RGR), enzyme activities and carbohydrates were measured at different fruit growth stages of various peach cultivars in different years. At stage III, high crop load induced higher acid invertase (AI, EC 3.2.1.26) activities and hexose : sucrose ratios. Both sorbitol and sucrose contents were proportional to L:F, while sorbitol dehydrogenase (SDH, EC 1.1.1.14) activity was the only enzyme activity directly related to L:F in both fruit growth stages. Girdling reduced fruit RGR and all major carbohydrates after 4 days and SDH activity already after 48 h, but it did not affect sucrose synthase (SS, EC 2.4.1.13), AI and neutral invertase (NI, EC 3.2.1.27). Fruit incubation in sorbitol for 24 h induced higher SDH activities than in buffer alone. In general, assimilate availability affected both sorbitol and sucrose metabolism in peach fruit, and sorbitol may function as a signal for modulating SDH activity. Under highly competitive conditions, AI activity may be enhanced by assimilate depletion, providing a mechanism to increase fruit sink strength by increasing hexose concentrations.     

Fine structure of the ventral nerve centre and interspecific identification of individual neurons in the enigmatic Chaetognatha

The enigmatic arrow worms (Chaetognatha) are marine carnivores and among the most abundant planktonic organisms. Their phylogenetic position has been heavily debated for a long time. Most recent molecular studies still provide a diverging picture and suggest arrow worms to be some kind of basal protostomes. In an effort to understand the organization of the nervous system in this clade for a broad comparison with other Metazoa we analysed the ultrastructure of the ventral nerve centre in Spadella cephaloptera by transmission electron microscopy. We were able to identify six different types of neurons in the bilateral somata clusters by means of the cytoplasmic composition (regarding the structure of the neurite and soma including the shape and eu-/heterochromatin ratio within the nucleus) as well as the size and position of these neurons. Furthermore, our study provides new insights into the neuropil composition of the ventral nerve centre and several other fine structural features. Our second goal was to examine if individually identifiable neurons are present in the ventral nerve centres of four chaetognath species, Sagitta setosa, Sagitta enflata, Pterosagitta draco, and Spadella cephaloptera. For that purpose, we processed whole mount specimens of these species for immunolocalization of RFamide-related neuropeptides and analysed them with confocal laser-scanning microscopy. Our experiments provide evidence for the interspecific homology of individual neurons in the ventral nerve centres of these four chaetognath species suggesting that the potential to generate serially arranged neurons with individual identities is part of their ground pattern.     

Vascular remodelling of an arterio-venous blood vessel network during solid tumour growth

We formulate a theoretical model to analyze the vascular remodelling process of an arterio-venous vessel network during solid tumour growth. The model incorporates a hierarchically organized initial vasculature comprising arteries, veins and capillaries, and involves sprouting angiogenesis, vessel cooption, dilation and regression as well as tumour cell proliferation and death. The emerging tumour vasculature is non-hierarchical, compartmentalized into well-characterized zones and transports efficiently an injected drug-bolus. It displays a complex geometry with necrotic zones and “hot spots” of increased vascular density and blood flow of varying size. The corresponding cluster size distribution is algebraic, reminiscent of a self-organized critical state. The intra-tumour vascular-density fluctuations correlate with pressure drops in the initial vasculature suggesting a physical mechanism underlying hot spot formation.     

The caudal regeneration blastema is an accumulation of rapidly proliferating stem cells in the flatworm Macrostomum lignano

Background  

Macrostomum lignano is a small free-living flatworm capable of regenerating all body parts posterior of the pharynx and anterior to the brain. We quantified the cellular composition of the caudal-most body region, the tail plate, and investigated regeneration of the tail plate in vivo and in semithin sections labeled with bromodeoxyuridine, a marker for stem cells (neoblasts) in S-phase.