Calcium binding to tandem repeats of EGF-like modules. Expression and characterization of the EGF-like modules of human Notch-1 implicated in receptor-ligand interactions.

The Ca(2+)-binding epidermal growth factor (cbEGF)-like module is a structural component of numerous diverse proteins and occurs almost exclusively within repeated motifs. Notch-1, a fundamental receptor for cell fate decisions, contains 36 extracellular EGF modules in tandem, of which 21 are potentially Ca(2+)-binding. We report the Ca(2+)-binding properties of EGF11-12 and EGF10-13 from human Notch-1 (hNEGF11-12 and hNEGF10-13), modules previously shown to support Ca(2+)-dependent interactions with the ligands Delta and Serrate. Ca2+ titrations in the presence of chromophoric chelators, 5,5''-Br2BAPTA and 5-NBAPTA, gave two binding constants for hNEGF11-12, Kd1 = 3.4 x 10(-5) M and Kd2 > 2.5 x 10(-4) M. The high-affinity site was found to be localized to hNEGF12. Titration of hNEGF10-13 gave three binding constants, Kd1 = 3.1 x 10(-6) M, Kd2 = 1.6 x 10(-4) M, and Kd3 > 2.5 x 10(-4) M, demonstrating that assembly of EGF modules in tandem can increase Ca2+ affinity. The highest affinity sites in hNEGF11-12 and hNEGF10-13 had 10 to 100-fold higher affinity than reported for EGF32-33 and EGF25-31, respectively, from fibrillin-1, a connective tissue protein with 43 cbEGF modules. A model of hNEGF11-12 based on fibrillin-1 EGF32-33 demonstrates electronegative potential that could contribute to the higher affinity of the Ca(2+)-binding site in hNEGF12. These data demonstrate that the Ca2+ affinity of cbEGF repeats can be highly variable among different classes of cbEGF containing proteins.     

Correction of the biochemical defect in porphobilinogen deaminase deficient cells by non-viral gene delivery

Porphobilinogen deaminase (PBGD), the third enzyme in the biosynthesis of heme, is deficient in acute intermittent porphyria (AIP). AIP is a genetic disease characterized by neurovisceral and psychiatric disturbances. Despite a palliative treatment, it may still be lethal. An initial step towards gene therapy was recently taken by showing that PBGD could be expressed to correct the enzyme deficiency in AIP fibroblasts. The aim of the present study was to investigate whether the biochemical defect can be corrected by using non-viral gene delivery. The biochemical defect in human and mouse PBGD deficient fibroblasts was demonstrated by analyzing synthesis of the heme precursor, protoporphyrin (PP), after addition of 5-aminolevulinic acid (ALA). Human AIP fibroblasts synthesized 21% and mouse PBGD deficient fibroblasts only 11% of the PP amount synthesized in respective control cells. Gene delivery increased the PBGD activity 88–200 fold in human AIP fibroblasts and synthesis of PP was increased from 21–152% of normal after ALA incubation. Similar results were obtained in mouse PBGD deficient cells, although the PP levels were several-fold lower as compared to human cells. HPLC analysis confirmed that PP was the main porphyrin intermediate that was formed. Addition of porphobilinogen (PBG) resulted in 3–7 fold lower synthesis of PP as compared to ALA addition. These results show that non-viral gene delivery of plasmids encoding PBGD results in a high expression of functional PBGD shown by induced synthesis of PP in PBGD deficient cells after supplementation of ALA and PBG.     

Can small‐scale experiments predict ecosystem responses? An example from peatlands

Oligotrophic, Sphagnum -dominated peatlands have been regarded as long-term stable ecosystems that function as carbon sinks. As a result of environmental perturbations, particularly anthropogenic N deposition, this view is now increasingly questioned. We examined whether small-scale field experiments can predict the direction and magnitude of ecosystem responses to increased N supply. We, therefore, compared data from a 10-year field experiment (involving deposition of 2, 15 and 30 kg N ha⁻¹ year⁻¹) with data from a gradient associated with increased N deposition (2, 8 and 12 kg N ha⁻¹ year⁻¹). We chose to compare: (1) the physiological response of Sphagnum balticum , measured in the form of N accumulation as free amino acids (N_AA); and (2) changes in the total Sphagnum cover, the cover of S. balticum , and vascular plant cover. In all cases we found a highly significant correlation between the two data sets. We attribute the high correspondence between the two data sets to the key function of the dominant group of organisms, the Sphagna, that monopolize N availability and control the water balance, creating an environment hostile to vascular plants. Thus the key role of Sphagna as ecosystem engineers seems to supersede the role of other, scale-dependent processes. We also conclude that N_AA is a sensitive indicator that can be used to signal the slow and gradual shift from Sphagnum to vascular plant dominance.     

Sequential Salinomycin Treatment Results in Resistance Formation through Clonal Selection of Epithelial-Like Tumor Cells

Acquiring therapy resistance is one of the major obstacles in the treatment of patients with cancer. The discovery of the cancer stem cell (CSC)–specific drug salinomycin raised hope for improved treatment options by targeting therapy-refractory CSCs and mesenchymal cancer cells. However, the occurrence of an acquired salinomycin resistance in tumor cells remains elusive. To study the formation of salinomycin resistance, mesenchymal breast cancer cells were sequentially treated with salinomycin in an in vitro cell culture assay, and the resulting differences in gene expression and salinomycin susceptibility were analyzed. We demonstrated that long-term salinomycin treatment of mesenchymal cancer cells resulted in salinomycin-resistant cells with elevated levels of epithelial markers, such as E-cadherin and miR-200c, a decreased migratory capability, and a higher susceptibility to the classic chemotherapeutic drug doxorubicin. The formation of salinomycin resistance through the acquisition of epithelial traits was further validated by inducing mesenchymal-epithelial transition through an overexpression of miR-200c. The transition from a mesenchymal to a more epithelial-like phenotype of salinomycin-treated tumor cells was moreover confirmed in vivo, using syngeneic and, for the first time, transgenic mouse tumor models. These results suggest that the acquisition of salinomycin resistance through the clonal selection of epithelial-like cancer cells could become exploited for improved cancer therapies by antagonizing the tumor-progressive effects of epithelial-mesenchymal transition.     

Some effects of processing on the molecular structure and morphology of thermoplastic starch

Hydroxypropylated and oxidised potato starch (HONPS) was used together with glycerol and water to produce thermoplastic starch. The amount of glycerol was kept constant at 22 parts by weight per 100 parts of dry starch. The thermoplastic starch was converted into films/sheets using three different processing techniques; casting, compression moulding and film blowing. The last two methods represent typical thermoplastic conversion techniques requiring elevated processing temperatures. By means of size-exclusion chromatography, it was found that compression moulding and film blowing led to some degradation of high-molecular weight amylopectin as well as of high-molecular weight amylose-like molecules. The degradation was significantly less pronounced for the cast films. The morphology of the specimens was quite complex and phase separations on different levels were identified. In the cast films and, to a lesser extent, in the compression-moulded specimens, a fine network structure could be distinguished. Such a structure could however not be ascertained in the film-blown material and this is discussed in terms of the thermo-mechanical treatment of the starch materials.     

Association Between Obesity,Flow Rate of Whole Saliva,and Dental Caries in Adolescents

In a cross‐sectional study design, we test the hypothesis whether childhood obesity is associated with reduced flow rate of stimulated whole saliva and dental caries. Obese adolescents (n = 65) with a mean age of 14.5 years and normal weight subjects (n = 65) with a mean age of 14.2 years were clinically examined with respect to dental caries, visible plaque accumulation (visible plaque index (VPI%)), gingival inflammation in terms of bleeding on probing (BOP%) as well as answered a questionnaire concerning medical history, medication, oral hygiene habits, smoking habits, and sociodemographic background. The flow rate of stimulated whole saliva (ml/min) was determined. BMI was calculated and adjusted for age and gender (BMI‐sds). The obese subjects exhibited higher number of decayed surfaces (DS), 0.7 vs. 0.1 (P = 0.008) and lower flow rate of stimulated whole saliva 1.2 vs. 2.0 ml/min (P < 0.001). Of obese patients, 17 subjects had VPI% >25 and 21 had BOP% >25, both compared to only 5 subjects of the normal weight with P values of 0.005 and <0.001, respectively. In a multivariate logistic regression model BMI‐sds was significantly associated with the flow rate of stimulated whole saliva less than the median value 1.5 ml/min (P < 0.001; odds ratio (OR) 1.36) as well as with DS (DS >0) (P = 0.002; OR 1.31) and the associations were not found to be confounded by any of the studied variables. The results indicate that childhood obesity is associated with reduced flow rate of stimulated whole saliva and dental caries and further strengthens obesity's negative effect on children's oral health.