Prevention of farnesylation of c-Ha-Ras protein enhances synergistically the cytotoxic action of doxorubicin in cycling but not in quiescent cells

Ras, the product of a proto-oncogene, is a GTP-hydrolyzing enzyme found mutated in approximately 50% of human cancers. "Gain of function" mutations of Ras lead to an escape of transformed cells from cell-cycle control, rendering them independent to stimulation by growth factors, giving them almost unlimited proliferation capacity. The cytosolic precursor isoform of Ras is biologically inactive. After several post-translational modifications, Ras is anchored to the plasma membrane and, thereby, the protein becomes activated. The finding that lipid modifications of Ras protein, particularly farnesylation, are essential for its signal transduction activity, gave rise to the concept that blocking farnesyl protein transferase (FPTase), the enzyme catalyzing the first step in the Ras modification cascade, would prevent proper membrane anchoring and provide an improved approach in the cure of tumors harboring Ras mutations. In the present study we used transformed rat cells overexpressing a temperature-sensitive p53 protein, adopting wt conformation at 32 degrees C and mutant conformation at 37 degrees C. We treated the cells growing at 32 or 37 degrees C with doxorubicin alone, or in combination with inhibitors of FPTase. Combined treatment was more efficient and the same inhibition of cell proliferation was reached at lower DOX concentrations. The treatment strongly affected the growth rate of tumor cells but only negligibly of normal cells. However, the inhibitors of FPTase prevented the membrane anchoring in both situations. These results show two striking advantages of the combined treatment: the desired cytostatic effect on tumor cells at lower drug concentrations and clearly reduced adverse effects on quiescent cells.     

Similarity networks of protein binding sites

An increasing attention has been dedicated to the characterization of complex networks within the protein world. This work is reporting how we uncovered networked structures that reflected the structural similarities among protein binding sites. First, a 211 binding sites dataset has been compiled by removing the redundant proteins in the Protein Ligand Database (PLD) (http://www-mitchell.ch.cam.ac.uk/pld/). Using a clique detection algorithm we have performed all-against-all binding site comparisons among the 211 available ones. Within the set of nodes representing each binding site an edge was added whenever a pair of binding sites had a similarity higher than a threshold value. The generated similarity networks revealed that many nodes had few links and only few were highly connected, but due to the limited data available it was not possible to definitively prove a scale-free architecture. Within the same dataset, the binding site similarity networks were compared with the networks of sequence and fold similarity networks. In the protein world, indications were found that structure is better conserved than sequence, but on its own, sequence was better conserved than the subset of functional residues forming the binding site. Because a binding site is strongly linked with protein function, the identification of protein binding site similarity networks could accelerate the functional annotation of newly identified genes. In view of this we have discussed several potential applications of binding site similarity networks, such as the construction of novel binding site classification databases, as well as the implications for protein molecular design in general and computational chemogenomics in particular.     

Albumin endocytosis via megalin in astrocytes is caveola- and Dab-1 dependent and is required for the synthesis of the neurotrophic factor oleic acid

The synthesis and release of the neurotrophic factor oleic acid requires internalization of albumin into the astrocyte, which is mediated by megalin. In this study, we show that the binding and internalization of albumin involve its interaction with megalin, caveolin-1, caveolin-2 and cavin, but not with clathrin in astrocytes from primary culture. Electron microscopy analyses revealed albumin-gold complexes localized in caveolae, but not in clathrin-coated vesicles. Neither chlorpromazine nor silencing clathrin expression modified albumin uptake. Silencing caveolin-1 strongly reduced the binding and internalization of albumin and the distribution of megalin in the plasma membrane. However, silencing caveolin-2 only decreased albumin internalization, suggesting that caveolin-1 is responsible for megalin recruitment to the caveolae and that caveolin-2 participates in caveolae internalization. In most tissues, the cytosolic adaptor protein disabled (Dab)-2 connects megalin to clathrin, astrocytes lack Dab-2; instead, they express Dab-1, which interacts with caveolin-1 and megalin and is required for albumin internalization. The transcytosis of albumin in astrocytes, including the passage through the endoplasmic reticulum, which is a compulsory step for oleic acid synthesis, was confirmed by electron microscopy analyses. Thus, whereas silencing clathrin did not modify the synthesis and release of oleic acid, the knock-down of caveolin-1, caveolin-2 and Dab-1 strongly reduced the synthesis and release of this neurotrophic factor. In conclusion, caveola-mediated endocytosis of albumin requires megalin and the adaptor protein Dab-1 in cultured astrocytes. Albumin endocytosis may be a key step in brain development because it stimulates the synthesis of oleic acid, which in turn promotes neuronal differentiation.     

CELL CYCLE AND CELL MORTALITY OF ALEXANDRIUM MINUTUM (DINOPHYCEAE) UNDER SMALL‐SCALE TURBULENCE CONDITIONS1

Decreased net population growth rates and cellular abundances have been observed in dinoflagellate species exposed to small‐scale turbulence. Here, we investigated whether these effects were caused by alterations in the cell cycle and/or by cell mortality and, in turn, whether these two mechanisms depended on the duration of exposure to turbulence. The study was conducted on the toxic dinoflagellate Alexandrium minutum Halim, with the same experimental design and setup used in previous studies to allow direct comparison among results. A combination of microscopy and Coulter Counter measurements allowed us to detect cell mortality, based on the biovolume of broken cells and thecae. The turbulence applied during the exponential growth phase caused an immediate transitory arrest in the G2/M phase, but significant mortality did not occur. This finding suggests that high intensities of small‐scale turbulence can alter the cell division, likely affecting the correct chromosome segregation during the dinomitosis. When shaking persisted for >4 d, mortality signals and presence of anomalously swollen cells appeared, hinting at the activation of mechanisms that induce programmed cell death. Our study suggests that the sensitivity of dinoflagellates to turbulence may drive these organisms to find the most favorable (calm) conditions to complete their division cycle.     

Comparison of two histological techniques for age determination in small cetaceans

Age estimation in odontocetes is based on counts of growth layer groups (GLGs) deposited in recording structures such as teeth. Generally, tooth sections are obtained using a cryostat microtome. However, some researchers prefer obtaining thin sections using a traditional paraffin microtome. Little information is available on the application of this technique to dolphin teeth. Our main aim was to investigate if the paraffin technique can be a viable alternative. We considered whether estimated age would be affected by preparation technique, staining method, and section thickness, while controlling for effects of species, body length, and sex. We also analyzed whether the staining method would affect readability of GLGs and age reading variability. Teeth from 86 individuals (representing seven species) were used, but not all were prepared using both techniques because sufficient teeth were not available in all cases. Although the staining method had significant effects on the estimated age using both techniques, the variability of GLG counts was small and appeared to be similar for both techniques. Using Mayer's hematoxylin stained sections of 8 μm thickness, good agreement of ages was obtained from both techniques, with more preparations classified as "good quality" for the paraffin technique. Mayer's hematoxylin provided the best contrast of the GLGs when using the paraffin technique. We conclude that the paraffin technique is viable and represents a cost-effective alternative to a cryostat microtome when preparing cetacean teeth for age determination.     

Structural characterization of the alpha-hemolysin monomer from Staphylococcus aureus

Alpha-hemolysin from Staphylococcus aureus is secreted as a water-soluble monomer and assembles on membranes to oligomerize into a homo-heptameric, water-filled pore. These pores lead to lysis and cell death. Although the structure of the heptameric pore is solved by means of X-ray crystallography, structures of intermediate states-from the soluble monomer to all potential "pre-pore" structures-are yet unknown. Here, we propose a model of the monomeric alpha-hemolysin in solution based on molecular modeling, verified by small angle X-ray scattering data. This structure reveals details of the monomeric conformation of the alpha-hemolysin, for example inherent flexibility, along with definite differences in comparison to the structures used as templates.