Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance

Growth factors and mitogens use the Ras/Raf/MEK/ERK signaling cascade to transmit signals from their receptors to regulate gene expression and prevent apoptosis. Some components of these pathways are mutated or aberrantly expressed in human cancer (e.g., Ras, B-Raf). Mutations also occur at genes encoding upstream receptors (e.g., EGFR and Flt-3) and chimeric chromosomal translocations (e.g., BCR-ABL) which transmit their signals through these cascades. Even in the absence of obvious genetic mutations, this pathway has been reported to be activated in over 50% of acute myelogenous leukemia and acute lymphocytic leukemia and is also frequently activated in other cancer types (e.g., breast and prostate cancers). Importantly, this increased expression is associated with a poor prognosis. The Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt pathways interact with each other to regulate growth and in some cases tumorigenesis. For example, in some cells, PTEN mutation may contribute to suppression of the Raf/MEK/ERK cascade due to the ability of activated Akt to phosphorylate and inactivate different Rafs. Although both of these pathways are commonly thought to have anti-apoptotic and drug resistance effects on cells, they display different cell lineage specific effects. For example, Raf/MEK/ERK is usually associated with proliferation and drug resistance of hematopoietic cells, while activation of the Raf/MEK/ERK cascade is suppressed in some prostate cancer cell lines which have mutations at PTEN and express high levels of activated Akt. Furthermore the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt pathways also interact with the p53 pathway. Some of these interactions can result in controlling the activity and subcellular localization of Bim, Bak, Bax, Puma and Noxa. Raf/MEK/ERK may promote cell cycle arrest in prostate cells and this may be regulated by p53 as restoration of wild-type p53 in p53 deficient prostate cancer cells results in their enhanced sensitivity to chemotherapeutic drugs and increased expression of Raf/MEK/ERK pathway. Thus in advanced prostate cancer, it may be advantageous to induce Raf/MEK/ERK expression to promote cell cycle arrest, while in hematopoietic cancers it may be beneficial to inhibit Raf/MEK/ERK induced proliferation and drug resistance. Thus the Raf/MEK/ERK pathway has different effects on growth, prevention of apoptosis, cell cycle arrest and induction of drug resistance in cells of various lineages which may be due to the presence of functional p53 and PTEN and the expression of lineage specific factors.     

Rana/Pol III: a family of SINE-like sequences in the genomes of western Palearctic water frogs.

The highly repetitive Rana/Pol III family consists of short, tandemly arrayed sequences, scattered throughout the genomes of Palearctic green water frogs. The repeat unit is about 250 bp in length and is a composite element: it contains a SINE-like retroposon with a tRNA structure, flanked by two short direct repeats, and the occurrence of two internal repeats gives evidence that an additional transposition event may have inserted a segment within the already transposed element. Rana/Pol III family is present in the genomes of Rana lessonae, R. ridibunda, and their hybrid form R. esculenta, as well as in R. shqiperica. R. epeirotica, R. cretensis, and the Italian taxon. These sequences are also present in the Iberian R. perezi, although less abundant, but appear to be lacking in the north African species R. saharica. The distribution of Rana/Pol III in the genomes of Palearctic green frogs is in agreement with the phyletic history based on genetic data. The evolutionary pattern proposed for the genus Rana enables us to suppose that the hybridogenetic mechanism is one of the factors accounting for the possible horizontal transfer of Rana/Pol III elements from the central-north Europe species to R. perezi.     

A conformational study in solution of pro-somatostatin fragments by NMR and computational methods

The results of a conformational study by nuclear magnetic spectroscopy and computational methods on a series of point-mutated synthetic peptides, containing 14 amino acid residues and mimicking the region containing the Arg-Lys dibasic cleavage site of pro-somatostatin, have confirmed the possible role of a well defined secondary structure in the recognition phenomenon by processing enzymes. The importance of the residues located near the Arg-Lys dibasic site in the C-terminal region of the pro-hormone for the cleavage of the precursor into somatostatin-14 has been confirmed. The present structural analysis indicates the occurrence of two β-turns in the 4–7 and 11–14 regions, flanking the cleavage site, for all the peptides recognized as substrates by the processing enzyme. Interestingly, in the point-mutated analogue not processed by the enzyme and containing the replacement of proline by alanine in position 5 the first β-turn is displaced by one residue and involves the Ala⁵-Arg⁸ segment. This observation may explain the lack of recognition by the maturation enzyme. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Chiral recognition by the copper(II) complex of 6-deoxy-6-N-(2-methylaminopyridine)-β-cyclodextrin

A modified β-cyclodextrin bearing a 2-aminomethylpyridine binding site for copper(II) (6-deoxy-6-[N-(2-methylamino)pyridine)]-β-cyclodextrin, CDampy was synthesized by C₆-monofunctionalization. The acid-base properties of the new ligand in aqueous solution were investigated by potentiometry and calorimetry, and its conformations as a function of pH were studied by NMR and circular dichroism (c.d.). The formation of binary copper(II) complexes was studied by potentiometry, EPR, and c.d. The copper(II) complex was used as chiral selector for the HPLC enantiomeric separation of underivatized aromatic amino acids. Enantioselectivity in the overall stability constants of the ternary complexes with D- or L-Trp was detected by potentiometry, whereas the complexes of the Ala enantiomers did not show any difference in stability. These results were consistent with a preferred cis coordination of the amino group of the ligand and of the amino acid in the ternary complexes (“cis effect”), which leads to the inclusion of the aromatic side chain of D-Trp, but not of that of L-Trp. In Trp-containing ternary complexes, the two enantiomers showed differences in the fluorescence lifetime distribution, consistent with only one conformer of D-Trp and two conformers of L-Trp, and the latter were found to be more accessible to fluorescence quenching by acrylamide and KI. Chirality 9:341–349, 1997. © 1997 Wiley-Liss, Inc.     

Predicting the actigraphy-based acrophase using the Morningness–Eveningness Questionnaire (MEQ) in college students of North Italy

Actigraphy is the reference objective method to measure circadian rhythmicity. One simpler subjective approach to assess the circadian typology is the Morningness–Eveningness Questionnaire (MEQ) by Horne and Ostberg. In this study, we compared the MEQ score against the actigraphy-based circadian parameters MESOR, amplitude and acrophase in a sample of 54 students of the University of Milan in Northern Italy. MEQ and the acrophase resulted strongly and inversely associated (r = ?0.84, p < 0.0001), and their relationship exhibited a clear-cut linear trend. We thus used linear regression to develop an equation enabling us to predict the value of the acrophase from the MEQ score. The parameters of the regression model were precisely estimated, with the slope of the regression line being significantly different from 0 (p < 0.0001). The best-fit linear equation was: acrophase (min) = 1238.7–5.49·MEQ, indicating that each additional point in the MEQ score corresponded to a shortening of the acrophase of approximately 5 min. The coefficient of determination, R², was 0.70. The residuals were evenly distributed and did not show any systematic pattern, thus indicating that the linear model yielded a good, balanced prediction of the acrophase throughout the range of the MEQ score. In particular, the model was able to accurately predict the mean values of the acrophase in the three chronotypes (Morning-, Neither-, and Evening-types) in which the study subjects were categorized. Both the confidence and prediction limits associated to the regression line were calculated, thus providing an assessment of the uncertainty associated with the prediction of the model. In particular, the size of the two-sided prediction limits for the acrophase was about ±100 min in the midrange of the MEQ score. Finally, k-fold cross-validation showed that both the model’s predictive ability on new data and the model’s stability to changes in the data set used for parameter estimation were good. In conclusion, the actigraphy-based acrophase can be predicted using the MEQ score in a population of college students of North Italy.     

Rainfall reduction impacts rhizosphere biogeochemistry in eucalypts grown in a deep Ferralsol in Brazil

Plant and Soil - Comparing root functioning under contrasting rainfall regimes can help assessing the capacity of plant species to cope with more intense and frequent drought predicted under...     

Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events

Recently, certain lots of heparin have been associated with an acute, rapid onset of serious side effects indicative of an allergic-type reaction. To identify potential causes for this sudden rise in side effects, we examined lots of heparin that correlated with adverse events using orthogonal high-resolution analytical techniques. Through detailed structural analysis, the contaminant was found to contain a disaccharide repeat unit of glucuronic acid linked beta1-->3 to a beta-N-acetylgalactosamine. The disaccharide unit has an unusual sulfation pattern and is sulfated at the 2-O and 3-O positions of the glucuronic acid as well as at the 4-O and 6-O positions of the galactosamine. Given the nature of this contaminant, traditional screening tests cannot differentiate between affected and unaffected lots. Our analysis suggests effective screening methods that can be used to determine whether or not heparin lots contain the contaminant reported here.     

Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast

Background  

The ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT) aiming to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv) format represents a powerful reagent to allow in in vitro and in vivo detection of CD expression in GDEPT/ADEPT studies.