Differentiation-dependent glycosylation of gp190, an oncofetal crypt cell antigen expressed by Caco-2 cells

gp190 is a glycoprotein expressed on the cell surface of several human colon carcinoma cells in culture, on epithelial cells of fetal colon, but not on the normal mucosa of adult colon; thus it is referred to as an oncofetal crypt cell antigen. We report the characterisation of O[emsp4 ]-linked glycans carried by gp190 synthesised by [³H]glucosamine-labelled Caco-2 cells at the confluence (undifferentiated cells) and at three weeks of postconfluence (differentiated cells). By using a specific monoclonal antibody, gp190 was isolated and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The mobility of gp190 from differentiated cells was found to be lower than that from undifferentiated cells, suggesting a more extensive glycosylation process in the former glycoprotein. The major results of the glycan characterisation have been as follows: (i) gp190 carries mainly, if not exclusively, O-linked glycans with the core-2 structure; (ii) the elongation with N-acetyllactosamine units of the Gal1,4GlcNAc1,6(Gal1,3)GalNAc tetrasaccharide predominates in gp190 synthesised by differentiated cells, whereas the direct 2,3sialylation of the tetrasaccharide is prevalent in gp190 synthesised by undifferentiated cells. The increment in the core-2 1,6GlcNAc-transferase activity under the Caco-2 differentiation process may be relevant in producing the larger occurrence of polylactosaminoglycans in gp190 from differentiated cells. Since no change in the activity of the 2,3sialyltransferases upon cell differentiation was observed, we suggest that the lower 2,3sialylation in gp190 synthesised by polarised cells might be due to a changed transit-rate through the distal Golgi apparatus.     

Occurrence of Fusaproliferin and Beauvericin in Fusarium-Contaminated Livestock Feed in Iowa

Fusarium fungal contaminants and related mycotoxins were investigated in eight maize feed samples submitted to the Iowa State University Veterinary Diagnostic Laboratory. Fusarium moniliforme, F. proliferatum, and F. subglutinans were isolated from seven, eight, and five samples, respectively. These strains belonged to mating populations A, D, and E of the teleomorph Gibberella fujikuroi. Fusaproliferin was detected at concentrations of 0.1 to 30 μg/g in four samples, and beauvericin was detected (0.1 to 3.0 μg/g) in five samples. Fumonisins were detected in all eight samples (1.1 to 14 μg/g). Ten of 11 strains of F. proliferatum and all 12 strains of F. subglutinans isolated from the samples produced fusaproliferin in culture on whole maize kernels (4 to 350 and 100 to 1,000 μg/g, respectively). Nine F. proliferatum strains also produced beauvericin in culture (85 to 350 μg/g), but none of the F. subglutinans strains produced beauvericin. Fumonisin B₁ was produced by all nine F. moniliforme strains (50 to 2,000 μg/g) and by 10 of the F. proliferatum strains (1,000 to 2,000 μg/g). This is the first report of the natural occurrence of fusaproliferin outside Italy and of the natural occurrence of beauvericin in North America.     

Calpain-3 Impairs Cell Proliferation and Stimulates Oxidative Stress-Mediated Cell Death in Melanoma Cells

Calpain-3 is an intracellular cysteine protease, belonging to Calpain superfamily and predominantly expressed in skeletal muscle. In human melanoma cell lines and biopsies, we previously identified two novel splicing variants (hMp78 and hMp84) of Calpain-3 gene (CAPN3), which have a significant lower expression in vertical growth phase melanomas and, even lower, in metastases, compared to benign nevi. In the present study, in order to investigate the pathophysiological role played by the longer Calpain-3 variant, hMp84, in melanoma cells, we over-expressed it in A375 and HT-144 cells. In A375 cells, the enforced expression of hMp84 induces p53 stabilization, and modulates the expression of a few p53- and oxidative stress-related genes. Consistently, hMp84 increases the intracellular production of ROS (Reactive Oxygen Species), which lead to oxidative modification of phospholipids (formation of F₂-isoprostanes) and DNA damage. Such events culminate in an adverse cell fate, as indicated by the decrease of cell proliferation and by cell death. To a different extent, either the antioxidant N-acetyl-cysteine or the p53 inhibitor, Pifithrin-α, recover cell viability and decrease ROS formation. Similarly to A375 cells, hMp84 over-expression causes inhibition of cell proliferation, cell death, and increase of both ROS levels and F₂-isoprostanes also in HT-144 cells. However, in these cells no p53 accumulation occurs. In both cell lines, no significant change of cell proliferation and cell damage is observed in cells over-expressing the mutant hMp84^C42S devoid of its enzymatic activity, suggesting that the catalytic activity of hMp84 is required for its detrimental effects. Since a more aggressive phenotype is expected to benefit from down-regulation of mechanisms impairing cell growth and survival, we envisage that Calpain-3 down-regulation can be regarded as a novel mechanism contributing to melanoma progression.     

Interaction between Calpain-1 and HSP90: New Insights into the Regulation of Localization and Activity of the Protease

Here we demonstrate that heat shock protein 90 (HSP90) interacts with calpain-1, but not with calpain-2, and forms a discrete complex in which the protease maintains its catalytic activity, although with a lower affinity for Ca²⁺. Equilibrium gel distribution experiments show that this complex is composed by an equal number of molecules of each protein partner. Moreover, in resting cells, cytosolic calpain-1 is completely associated with HSP90. Since calpain-1, in association with HSP90, retains its proteolytic activity, and the chaperone is displaced by calpastatin also in the absence of Ca²⁺, the catalytic cleft of the protease is not involved in this association. Thus, calpain-1 can form two distinct complexes depending on the availability of calpastatin in the cytosol. The occurrence of a complex between HSP90 and calpain-1, in which the protease is still activable, can prevent the complete inhibition of the protease even in the presence of high calpastatin levels. We also demonstrate that in basal cell conditions HSP90 and calpain-1, but not calpain-2, are inserted in the multi-protein N-Methyl-D-Aspartate receptor (NMDAR) complex. The amount of calpain-1 at the NMDAR cluster is not modified in conditions of increased [Ca²⁺]_i, and this resident protease is involved in the processing of NMDAR components. Finally, the amount of calpain-1 associated with NMDAR cluster is independent from Ca²⁺-mediated translocation. Our findings show that HSP90 plays an important role in maintaining a given and proper amount of calpain-1 at the functional sites.     

Functional Organization of the Action Observation Network in Autism: A Graph Theory Approach

Background

The ability to recognize, understand and interpret other’s actions and emotions has been linked to the mirror system or action-observation-network (AON). Although variations in these abilities are prevalent in the neuro-typical population, persons diagnosed with autism spectrum disorders (ASD) have deficits in the social domain and exhibit alterations in this neural network.

Method

Here, we examined functional network properties of the AON using graph theory measures and region-to-region functional connectivity analyses of resting-state fMRI-data from adolescents and young adults with ASD and typical controls (TC).

Results

Overall, our graph theory analyses provided convergent evidence that the network integrity of the AON is altered in ASD, and that reductions in network efficiency relate to reductions in overall network density (i.e., decreased overall connection strength). Compared to TC, individuals with ASD showed significant reductions in network efficiency and increased shortest path lengths and centrality. Importantly, when adjusting for overall differences in network density between ASD and TC groups, participants with ASD continued to display reductions in network integrity, suggesting that also network-level organizational properties of the AON are altered in ASD.

Conclusion

While differences in empirical connectivity contributed to reductions in network integrity, graph theoretical analyses provided indications that also changes in the high-level network organization reduced integrity of the AON.     

IgG absorption by Santa Ines lambs fed Holstein bovine colostrum or Santa Ines ovine colostrum

The objective of this study was to evaluate the immunoglobulin G (IgG) absorption by Santa Ines lambs under two colostrum management systems usually used by producers. Twenty-seven Santa Ines newborn lambs received two meals of 250 ml of bovine colostrum from Holstein cows (BC group) or ovine colostrum from Santa Ines ewes (OC group) at 0 and 6 h of life. Pools of BC and OC were analyzed by radial immunodiffusion to quantify IgG. Results are expressed as least-square means and standard errors of mean (means ± s.e.m.). The concentration of IgG in bovine and ovine pools averaged 115.7 ± 20.5 and 48.1 ± 5.0 mg/ml, respectively, levels of concentration found in similar regular colostrum managements. The efficiency of IgG absorption was evaluated under two aspects, maximum apparent efficiency of absorption and total apparent efficiency of absorption (AEAmax and AEAtotal, respectively). The AEAmax was calculated taking into account the mass of IgG ingested just in the first meal of colostrum at birth and the serum IgG concentration at 6 h while the AEAtotal took into account the serum IgG concentration at 24 h of life that reflects the first colostrum offered at birth and the second meal at 6 h. The IgG and apparent efficiency of absorption results were transformed into the square root and log base 10, respectively, and were presented as geometric least-square means. In BC, lower (P < 0.05) AEAmax and AEAtotal were verified (14.2% and 15.6%, respectively), in relation to OC (23.6% and 24.4%, respectively). Serum IgG concentrations at 24 h were significantly higher (P < 0.05) in BC (31.4 mg/ml, respectively) compared with OC (22.2 mg/ml, respectively). The results in this study confirm that there is a limitation to the process of IgG absorption by the enterocytes of newborn lambs, which determined a nonlinear behavior of passive immunity acquisition. Similar values of AEAmax and AEAtotal for the two sources of colostrum reveal that the process of IgG absorption from the first and second meals during the first 6 h of life did not change and indicates that the ingestion of a second feeding of quality colostrum can enhance the acquisition of immune protection of newborn lambs.     

Assessing the functional turnover of species assemblages with tailored dissimilarity matrices

It is often suggested that community functional diversity is an appropriate predictive measure of ecosystem functioning, particularly if relevant species traits for the ecological property of interest are carefully selected. However, methods for selecting traits are often based on expert knowledge or on theoretical models of ecosystem functioning, but usually do not include explicitly developed quantitative procedures. Here we propose to construct a so‐called ‘tailored dissimilarity matrix’ between species assemblages to emphasize their functional turnover in response to some user‐defined ecological property. First, a subset of community weighted mean trait values (CWM) is selected by stepwise regression on the ecological process of interest. The selected CWM values are then replaced by the residuals of the least‐squares regressions of each single CWM on the ecological process of interest and pairwise Euclidean distances between the residual values at each sampling site are calculated. We illustrate the advantages of the tailored approach using two distinct plant and bee communities under contrasting fire regimes in temperate forests of southern Switzerland. Our results demonstrated that, unlike for the original CWM values, the tailored approach optimized the degree of functional differentiation among bee and plant species assemblages, i.e. the species functional turnover, with respect to different fire regimes.     

Improving indicator species analysis by combining groups of sites

Indicator species are species that are used as ecological indicators of community or habitat types, environmental conditions, or environmental changes. In order to determine indicator species, the characteristic to be predicted is represented in the form of a classification of the sites, which is compared to the patterns of distribution of the species found at the sites. Indicator species analysis should take into account the fact that species have different niche breadths: if a species is related to the conditions prevailing in two or more groups of sites, an indicator species analysis undertaken on individual groups of sites may fail to reveal this association. In this paper, we suggest improving indicator species analysis by considering all possible combinations of groups of sites and selecting the combination for which the species can be best used as indicator. When using a correlation index, such as the point‐biserial correlation, the method yields the combination where the difference between the observed and expected abundance/frequency of the species is the largest. When an indicator value index (IndVal) is used, the method provides the set of site‐groups that best matches the observed distribution pattern of the species. We illustrate the advantages of the method in three different examples. Consideration of combinations of groups of sites provides an extra flexibility to qualitatively model the habitat preferences of the species of interest. The method also allows users to cross multiple classifications of the same sites, increasing the amount of information resulting from the analysis. When applied to community types, it allows one to distinguish those species that characterize individual types from those that characterize the relationships between them. This distinction is useful to determine the number of types that maximizes the number of indicator species.