Combining metabolic pathway analysis with Evolutionary Game Theory: explaining the occurrence of low-yield pathways by an analytic optimization approach

Elementary-mode analysis is a powerful method for detecting all potential pathways in a metabolic network and computing the associated molar yields. Metabolic pathways can be interpreted as different strategies of organisms. Thus, methods from Evolutionary Game Theory can be employed. In Flux Balance Analysis (FBA), it is usually assumed that molar yields of relevant products (such as biomass or ATP) have been maximized during evolution. This has been questioned on game-theoretical grounds. In particular, in situations that can be characterized as a Prisoner's Dilemma, maximization of flux is not in line with maximization of yield. Under other conditions (that is, for other parameter values of maximal velocities), a Harmony game can result, where the above two maximization criteria give the same result. Here, we analyse the optimal situations under varying conditions. In particular, we consider the case where the cell can allocate a certain amount of protein on several enzymes in a varying distribution and model this by a linear programming problem in which not only the rates but also the maximal velocities are variable. It turns out that in the case of low or moderate synthesis costs for the enzymes of the high-yield pathway, maximizing pathway flux is in line with maximizing molar yield while in the case of high costs, it is not. This may explain the observation that many cells such as striated muscle cells, tumour cells, activated lymphocytes and several yeasts do not reallocate protein away from glycolytic enzymes towards TCA cycle and respiratory chain enzymes, in spite of the higher efficiency of respiration. This provides a straightforward explanation of the Warburg effect in tumour cells.     

Blockade of tumor necrosis factor-induced Bid cleavage by caspase-resistant Rb

Tumor necrosis factor-alpha (TNF) activates caspase-8 to cleave effector caspases or Bid, resulting in type-1 or type-2 apoptosis, respectively. We show here that TNF also induces caspase-8-dependent C-terminal cleavage of the retinoblastoma protein (Rb). Interestingly, fibroblasts from Rb(MI/MI) mice, in which the C-terminal caspase cleavage site is mutated, exhibit a defect in Bid cleavage despite caspase-8 activation. Recent results suggest that TNF receptor endocytosis is required for the activation of caspase-8. Consistent with this notion, inhibition of V-ATPase, which plays an essential role in acidification and degradation of endosomes, specifically restores Bid cleavage in Rb(MI/MI) cells. Inhibition of V-ATPase sensitizes Rb(MI/MI) but not wild-type fibroblasts to TNF-induced apoptosis and stimulates inflammation-associated colonic apoptosis in Rb(MI/MI) but not wild-type mice. These results suggest that Rb cleavage is required for Bid cleavage in TNF-induced type-2 apoptosis, and this requirement can be supplanted by the inhibition of V-ATPase.     

Detritus as a potential food source for protozoans: utilization of fine particulate plant detritus by a heterotrophic flagellate, <Emphasis Type="Italic">Chilomonas paramecium</Emphasis>, and a ciliate, <Emphasis Type="Italic">Tetrahymena pyriformis</Emphasis>

We investigated the direct utilization of fine particulate detritus (dried and homogenized plant material in the size range of bacteria) as a food source for protozoans using axenic cultures of the cryptomonad, heterotrophic flagellate, Chilomonas paramecium, and the hymenostome ciliate, Tetrahymena pyriformis. When fed media containing only particulate detritus, these species revealed growth rates similar to those reported for field populations. The growth rates of Chilomonas fed exclusively particulate detritus were similar to those obtained on a bacterial diet. Considering the high percentage of detritus particles in the size range of bacteria in lakes, our results imply that direct utilization of detritus by protozoans may form an additional pathway of carbon in aquatic food webs that has generally been overlooked.     

A role for complexes of survival of motor neurons (SMN) protein with gemins and profilin in neurite-like cytoplasmic extensions of cultured nerve cells

Spinal muscular atrophy (SMA) is caused by reduced levels of SMN (survival of motor neurons protein) and consequent loss of motor neurons. SMN is involved in snRNP transport and nuclear RNA splicing, but axonal transport of SMN has also been shown to occur in motor neurons. SMN also binds to the small actin-binding protein, profilin. We now show that SMN and profilin II co-localise in the cytoplasm of differentiating rat PC12 cells and in neurite-like extensions, especially at their growth cones. Many components of known SMN complexes were also found in these extensions, including gemin2 (SIP-1), gemin6, gemin7 and unrip (unr-interacting protein). Coilin p80 and Sm core protein immunoreactivity, however, were seen only in the nucleus. SMN is known to associate with beta-actin mRNA and specific hnRNPs in axons and in neurite extensions of cultured nerve cells, and SMN also stimulates neurite outgrowth in cultures. Our results are therefore consistent with SMN complexes, rather than SMN alone, being involved in the transport of actin mRNPs along the axon as in the transport of snRNPs into the nucleus by similar SMN complexes. Antisense knockdown of profilin I and II isoforms inhibited neurite outgrowth of PC12 cells and caused accumulation of SMN and its associated proteins in cytoplasmic aggregates. BIAcore studies demonstrated a high affinity interaction of SMN with profilin IIa, the isoform present in developing neurons. Pathogenic missense mutations in SMN, or deletion of exons 5 and 7, prevented this interaction. The interaction is functional in that SMN can modulate actin polymerisation in vitro by reducing the inhibitory effect of profilin IIa. This suggests that reduced SMN in SMA might cause axonal pathfinding defects by disturbing the normal regulation of microfilament growth by profilins.     

Dose-dependent effects of UVB-induced skin carcinogenesis in hairless p53 knockout mice

Exposure to (solar) UVB radiation gives rise to mutations in the p53 tumor suppressor gene that appear to contribute to the earliest steps in the molecular cascade towards human and murine skin cancer. To examine in more detail the role of p53, we studied UVB-induced carcinogenesis in hairless p53 knock-out mice. The early onset of lymphomas as well as early wasting of mice interfered with the development of skin tumors in p53 null-mice. The induction of skin tumors in the hairless p53+/- mice was accomplished by daily exposure to two different UV-doses of approximately 450 J/m2 and 900 J/m2 from F40 lamps corresponding to a fraction of about 0.4 and 0.8 of the minimal edemal dose. Marked differences in skin carcinogenesis were observed between the p53+/- mice and their wild type littermates. Firstly, at 900 J/m2, tumors developed significantly faster in the heterozygotes than in wild types, whereas at 450 J/m2 there was hardly any difference, suggesting that only at higher damage levels loss of one functional p53 allele is important. Secondly, a large portion (25%) of skin tumors in the heterozygotes were of a more malignant, poorly differentiated variety of squamous cell carcinomas, i.e. spindle cell carcinomas, a tumor type that was rarely observed in daily UV exposed wild type hairless mice. Thirdly, the p53 mutation spectrum in skin tumors in heterozygotes is quite different from that in wild types. Together these results support the notion that a point mutation in the p53 gene impacts skin carcinogenesis quite differently than allelic loss: the former is generally selected for in early stages of skin tumors in wild type mice, whereas the latter enhances tumor development only at high exposure levels (where apoptosis becomes more prevalent) and appears to increase progression (to a higher grade of malignancy) of skin tumors.     

PAK5 kinase is an inhibitor of MARK/Par-1, which leads to stable microtubules and dynamic actin

MARK/Par-1 is a kinase involved in development of embryonic polarity. In neurons, MARK phosphorylates tau protein and causes its detachment from microtubules, the tracks of axonal transport. Because the target sites of MARK on tau occur at an early stage of Alzheimer neurodegeneration, we searched for interaction partners of MARK. Here we report that MARK2 is negatively regulated by PAK5, a neuronal member of the p21-activated kinase family. PAK5 suppresses the activity of MARK2 toward its target, tau protein. The inhibition requires the binding between the PAK5 and MARK2 catalytic domains, but does not require phosphorylation. In transfected Chinese hamster ovary (CHO) cells both kinases show a vesicular distribution with partial colocalization on endosomes containing AP-1/2. Although MARK2 transfected alone destabilizes microtubules and stabilizes actin stress fibers, PAK5 keeps microtubules stable through the down-regulation of MARK2 but destabilizes the F-actin network so that stress fibers and focal adhesions disappear and cells develop filopodia. The results point to an inverse relationship between actin- and microtubule-related signaling by the PAK5 and MARK2 pathways that affect both cytoskeletal networks.     

High-resolution analysis of chromosomal imbalances using the Affymetrix 10K SNP genotyping chip

Array-based comparative genome hybridization is a powerful tool for detecting chromosomal imbalances at high resolution. However, the design and setup of such arrays are time consuming and expensive and thus worthwhile only when large numbers of arrays will be processed. To provide a feasible solution, we have developed an algorithm that renders the publicly available Affymetrix 10K SNP genotyping chip useful for high-resolution analysis of chromosomal imbalances. We have used our newly developed algorithm to analyze data from Affymetrix 10K chips that were hybridized with DNA probes from a variety of different sources, such as primary tumors, cell lines, and blood from patients with unbalanced translocations. In summary, we were able to (i) demonstrate the capability of our method by reproduction of published and unpublished data obtained with alternative methods and (ii) identify novel imbalances that were not shown before.     

Evaluation of ultrasound velocity measurements for estimating protease activities using casein as substrate

Ultrasonic resonator technology (URT) was compared with the well established UV–Vis/ninhydrin assay to estimate protease activities in defined buffer systems. Hydrolysis of casein was measured using subtilisin, trypsin, halophilic protease from Haloferax mediterranei and Bacillus lentus alkaline protease. Sensitivity, reproducibility, working range as well as the limit of detection and the limit of quantification were comparable for both methods. Salt concentrations (0.5 M NaCl) interfered with the URT method. The quantification of protease activity by URT was possible when the product concentration measured by the UV–Vis/ninhydrin assay was correlated to the corresponding ultrasonic velocity signals.