Glycosylation-induced conformational modification positively regulates receptor-receptor association: a study with an aberrant epidermal growth factor receptor (EGFRvIII/DeltaEGFR) expressed in cancer cells

The epidermal growth factor receptor (EGFR) is a multisited and multifunctional transmembrane glycoprotein with intrinsic tyrosine kinase activity. Upon ligand binding, the monomeric receptor undergoes dimerization resulting in kinase activation. The consequences of kinase stimulation are the phosphorylation of its own tyrosine residues (autophosphorylation) followed by association with and activation of signal transducers. Deregulation of signaling resulting from aberrant expression of the EGFR has been implicated in a number of neoplasms including breast, brain, and skin tumors. A mutant epidermal growth factor (EGF) receptor missing 267 amino acids from the exoplasmic domain is common in human glioblastomas. The truncated receptor (EGFRvIII/DeltaEGFR) lacks EGF binding activity; however, the kinase is constitutively active, and cells expressing the receptor are tumorigenic. Our studies revealed that the high kinase activity of the DeltaEGFR is due to self-dimerization, and contrary to earlier reports, the kinase activity per molecule of the dimeric DeltaEGFR is comparable to that of the EGF-stimulated wild-type receptor. Furthermore, the phosphorylation patterns of both receptors are similar as determined by interaction with a conformation-specific antibody and by phosphopeptide analysis. This eliminates the possibility that the defective down-regulation of the DeltaEGFR is due to its altered phosphorylation pattern as has been suggested previously. Interestingly, the receptor-receptor self-association is highly dependent on a conformation induced by N-linked glycosylation. We have identified four potential sites that might participate in self-dimerization; these sites are located in a domain that plays an important role in EGFR functioning.     

High-performance liquid chromatography/mass spectrometry characterization of Ki4B-Ras in PSN-1 cells treated with the prenyltransferase inhibitor L-778,123

Cellular transformation by Ras oncoproteins requires the posttranslation modification of farnesylation in a reaction catalyzed by farnesyl protein transferase (FPTase). Thus, inhibitors of FPTase have been developed as potential anticancer agents. However, recent studies with selective inhibitors of FPTase have shown that Ki4B-Ras retains its ability to transform cells by undergoing alternative prenylation by the related geranylgeranyl protein transferase I (GGPTase-I) in human tumor cells. We have developed a high-performance liquid chromatography/mass spectrometry assay for the detection and quantitation of the different processing states of Ki4B-Ras isolated from PSN-1 cells (a human pancreatic cell line with an activating Gly12 to Arg mutation) treated with the prenyltransferase inhibitor, L-778,123. Recently tested in the clinic, L-778,123 is a potent inhibitor of FPTase (in vitro IC50 = 2 nM) with some activity against GGPTase-I (in vitro IC50 = 98 nM). We find primarily farnesylated-Ki4B-Ras in vehicle-treated PSN-1 cells, a mixture of farnesylated- and geranylgeranylated-Ki4B-Ras in cells treated with nanomolar concentrations of L-778,123, and a mixture of unprocessed, farnesylated, and geranylgeranylated-Ki4B-Ras in cells treated with micromolar concentrations of compound. Of importance, this technique does not require metabolic labeling and may be used as a pharmacodynamic assay for Ki4B-Ras processing in mouse models.     

Maintenance of NF-kappaB activation in T-lymphocytes and a naive T-cell population in autoimmune-prone (NZB/NZW)F(1) mice by feeding a food-restricted diet enriched with n-3 fatty acids

We have previously shown that feeding a fish oil (FO) supplemented diet in combination with 40% food restriction (FO/FR) has a greater impact on extending life span in lupus-prone (NZB x NZW)F1 mice than either FO ad libitum (FO/AL) or corn oil food restricted (CO/FR) alone. Lupus disease is associated with increased Th-2 (i.e., IL-6 and IL-10) cytokine production and reduced IL-2 production and NF-kappaB activation. We hypothesized that the mechanism of action by which FO/FR increases life span may involve alterations in T-lymphocyte signaling and subsequent cytokine production. To test this hypothesis, we isolated and then stimulated splenic T-lymphocytes ex vivo with anti-CD3 and -CD28 monoclonal antibodies. We report here that CO/FR and FO/FR and to a lesser extent FO/AL offset disease-associated losses in Th-1 cytokine production, CD69 expression, and NF-kappaB activation in splenic T-lymphocytes activated ex vivo. Similarly, CO/FR and FO/FR prevented the disease-dependent rise in Th-2 cytokine production ex vivo and CD69 expression in vivo. In essence, the T-lymphocyte phenotype in the old CO/FR and FO/FR groups was identical to that in the young disease-free mice. Taken together, the data suggest that both CO/FR and FO/FR increase life span, in part, by maintaining a youthful immune phenotype in autoimmune-prone mice. However, FO/FR appears to represent a more potent dietary strategy in delaying disease-associated immune dysregulation than CO/FR.     

Cystic fibrosis: low frequency of DF508 mutation in 2 population samples from Rio de Janeiro, Brazil

Blood samples from 44 unrelated cystic fibrosis (CF) patients from Rio de Janeiro, Brazil, were analyzed for the 8 European CF mutations. Six homozygous and 15 heterozygous carriers of the DF508 mutation were found, corresponding to 47.7% of CF patients (allele frequency 0.3068). The G542X and G551D mutations were also observed with allele frequencies of 0.0227 and 0.0114, respectively. An analysis of the DF508 mutation in 291 randomly chosen, healthy individuals was performed, and only 3 heterozygous carriers were identified. These results show that the frequency of the DF508 allele in Rio de Janeiro is much lower than the world average; this may be due to the extremely heterogeneous ethnic admixture of the study population. By combining the results of these 2 different samples (CF patients and random population) and admixture data from Rio de Janeiro, we can estimate the CF incidence in this population to be 1:3542 individuals. However, taking into account the Rio de Janeiro ethnic admixture, we can find an estimate of 1:6902 individuals.     

Regional heterogeneities in the production of uric acid from adenosine in the bivascularly perfused rat liver

The heterogeneity of the liver parenchyma in relation to uric acid production from adenosine was investigated using the bivascularly perfused rat liver in the anterograde and retrograde modes. Adenosine was infused in livers from fed rats during 20 min at four different concentrations (20, 50, 100 and 200 M) according to four experimental protocols as follows: (A) anterograde perfusion, with adenosine infusion into the portal vein; (B) anterograde perfusion, with adenosine in the hepatic artery, (C) retrograde perfusion, with adenosine in the hepatic vein; (D) retrograde perfusion, with adenosine in the hepatic artery. With protocols A, B, and D uric acid production from adenosine was always characterized by initial bursts followed by progressive decreases toward smaller steady-states. With protocol C the initial burst was present only when 200 M adenosine was infused. The initial bursts in uric acid production were accompanied by simultaneous increases in the ratio of uric acid production/adenosine uptake rate. These initial bursts are thus representing increments in the production of uric acid that are not corresponded by similar increments in the metabolic uptake rates of adenosine. Global analysis of uric acid production revealed that the final steady-state rates were approximately equal for all infusion rates with protocols A, B and C, but smaller with protocol D. This difference, however, can be explained in terms of the differences in accessible cellular spaces, which are much smaller when protocol D is employed. When the analysis was performed in terms of the extra amounts of uric acid produced during the infusion of adenosine, where the initial bursts are also taken into account, different dose-response curves were found for each experimental protocol. These differences cannot be explained in terms of the accessible cell spaces and they are likely to reflect regional heterogeneities. From the various dose-response curves and from the known characteristics of the microcirculation of the rat liver it can be concluded that the initial bursts in uric acid production are generated in periportal hepatocytes. The reason for this heterogeneity could be related to the metabolic effects of adenosine, especially to oxygen uptake inhibition, which is likely to produce changes in the ATP/AMP ratios.     

Edge effect and species–area relationships in the gall-forming insect fauna of natural forest patches in the Brazilian Pantanal

The aim of this study was to (i) measure differences in species richness between edge habitats versus interior habitats, or more precisely the edge effect, and (ii) test the species–area relationship for gall-forming insects in natural forest patches in a Brazilian floodplain (Pantanal of Mato Grosso do Sul). These patches are regionally known as capões, basically composed of woody vegetation. Twenty-seven patches were surveyed. In each patch two transects were conducted for gall sampling. One transect encircled the patches while the other was conducted in the interior of the patch, totaling 54h of sampling. Host plant and galling insect species composition differed quite characteristically between the edge and the interior of patches, but galling insect richness did not. When insect gall richness was expressed as the ratio between insect gall and host plant richness (gall per plant ratio), a weak species–area relationship was found. Our results suggest that the number of galling insects per individual plant is not affected by the size of the patch. Despite these results, the natural forest patches found in this region seem well suited for long-term studies addressing species–area relationships. With regard to herbivorous insects, these studies should be combined with research on host plant dynamics during flooding and dry seasons.     

Induction of baroresistance by hydrogen peroxide, ethanol and cold-shock in Saccharomyces cerevisiae

The acquisition of tolerance to high hydrostatic pressure of 220 MPa (HHP) in response to a 0.4 mM hydrogen peroxide, 6% ethanol and cold-shock (10 degrees C) pretreatment for different lengths of times was studied in the yeast Saccharomyces cerevisiae. The protection conferred by these different treatments was similar ( approximately 3 log cycles) and time-dependent. Analysis of the induction of the most pressure up-regulated genes under these conditions was investigated by RT-PCR. Our results revealed that the cell stress response to HHP shares common features with hydrogen peroxide and ethanol stresses, but differs in some way to cold-shock.     

Two novel muramidases from skin mucosa of rainbow trout (Oncorhynchus mykiss)

Two novel antibacterial muramidases were purified to homogeneity from skin exudates of rainbow trout (Oncorhynchus mykiss). Unusually, one has an acidic isoelectric point and it is the first anionic muramidase to be reported for fish. Its molecular mass is 14,268 Da, as determined by mass spectrometry. The other muramidase is cationic with a mass of 14,252 Da. Partial N-terminal amino acid sequencing and peptide mapping strongly point to it being a c-type lysozyme, the first to be purified and characterised from skin of a salmonid. Its optimum pH ranges from 4.5 to 5.5 and its optimum temperature, at pH 5.0, is 33-49 degrees C, although it still exhibits activity at 5 degrees C. It is strongly bactericidal to the Gram-(+) bacterium Planococcus citreus, with a minimum bactericidal concentration of 100 U ml(-1), but is neither chitinolytic nor haemolytic. These two muramidases probably contribute to epithelial defence of the fish against microbes, either alone or in synergism with antibacterial peptides.