Restoration of β-galactosidase to Escherichia coli M15. Complementation studies

Carboxymethylated beta-galactosidase from Escherichia coli was dissociated at 100 degrees C to form carboxymethylated fragments A and B. The mol.wts. of carboxymethylated fragments A and B were determined by gel filtration to be 64300 and 22400 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of carboxymethylated fragments A and B that had been pretreated with 2-mercaptoethanol and sodium dodecyl sulphate yielded mol.wts. of 64000 and 22100 respectively. Carboxymethylated fragments A and B had arginine as their C-terminal amino acid. When a crude extract of E. coli M15 was filtered through a column of Sepharose 6B, it was found that carboxymethylated fragment B could restore beta-galactosidase activity when added to fractions having mol.wts. estimated to be 123000, 262000 and 506000. These fractions are referred to as ;complementable fractions'. Similarly, it was found that carboxymethylated fragment A could restore enzyme activity to tractions having mol.wts. estimated to be 63000, 253000 and 506000. Estimates of the molecular weights of the beta-galactosidase activity obtained by restoration with carboxymethylated fragments A and B were made by filtering the active enzyme through another column of Sepharose 6B. The enzyme obtained by complementation with carboxymethylated fragment B, i.e. the complemented enzyme, had mol.wt. 525000, and that obtained with carboxymethylated fragment A had mol.wts. of 525000, 646000 and 2000000. The latter finding suggests that multiple forms of complemented beta-galactosidase can exist.     

Standardized Aronia melanocarpa extract regulates redox status in patients receiving hemodialysis with anemia

Molecular and Cellular Biochemistry - The aim of our study was to investigate the effects of one-month consumption of polyphenol-rich standardized Aronia melanocarpa extract (SAE) on redox status...     

Generation of Amyloid-β Is Reduced by the Interaction of Calreticulin with Amyloid Precursor Protein,Presenilin and Nicastrin

Dysregulation of the proteolytic processing of amyloid precursor protein by γ-secretase and the ensuing generation of amyloid-β is associated with the pathogenesis of Alzheimer''s disease. Thus, the identification of amyloid precursor protein binding proteins involved in regulating processing of amyloid precursor protein by the γ-secretase complex is essential for understanding the mechanisms underlying the molecular pathology of the disease. We identified calreticulin as novel amyloid precursor protein interaction partner that binds to the γ-secretase cleavage site within amyloid precursor protein and showed that this Ca²⁺- and N-glycan-independent interaction is mediated by amino acids 330–344 in the C-terminal C-domain of calreticulin. Co-immunoprecipitation confirmed that calreticulin is not only associated with amyloid precursor protein but also with the γ-secretase complex members presenilin and nicastrin. Calreticulin was detected at the cell surface by surface biotinylation of cells overexpressing amyloid precursor protein and was co-localized by immunostaining with amyloid precursor protein and presenilin at the cell surface of hippocampal neurons. The P-domain of calreticulin located between the N-terminal N-domain and the C-domain interacts with presenilin, the catalytic subunit of the γ-secretase complex. The P- and C-domains also interact with nicastrin, another functionally important subunit of this complex. Transfection of amyloid precursor protein overexpressing cells with full-length calreticulin leads to a decrease in amyloid-β₄₂ levels in culture supernatants, while transfection with the P-domain increases amyloid-β₄₀ levels. Similarly, application of the recombinant P- or C-domains and of a synthetic calreticulin peptide comprising amino acid 330–344 to amyloid precursor protein overexpressing cells result in elevated amyloid-β₄₀ and amyloid-β₄₂ levels, respectively. These findings indicate that the interaction of calreticulin with amyloid precursor protein and the γ-secretase complex regulates the proteolytic processing of amyloid precursor protein by the γ-secretase complex, pointing to calreticulin as a potential target for therapy in Alzheimer''s disease.     

Improving Education in Medical Statistics: Implementing a Blended Learning Model in the Existing Curriculum

Background

Although recent studies report on the benefits of blended learning in improving medical student education, there is still no empirical evidence on the relative effectiveness of blended over traditional learning approaches in medical statistics. We implemented blended along with on-site (i.e. face-to-face) learning to further assess the potential value of web-based learning in medical statistics.

Methods

This was a prospective study conducted with third year medical undergraduate students attending the Faculty of Medicine, University of Belgrade, who passed (440 of 545) the final exam of the obligatory introductory statistics course during 2013–14. Student statistics achievements were stratified based on the two methods of education delivery: blended learning and on-site learning. Blended learning included a combination of face-to-face and distance learning methodologies integrated into a single course.

Results

Mean exam scores for the blended learning student group were higher than for the on-site student group for both final statistics score (89.36±6.60 vs. 86.06±8.48; p = 0.001) and knowledge test score (7.88±1.30 vs. 7.51±1.36; p = 0.023) with a medium effect size. There were no differences in sex or study duration between the groups. Current grade point average (GPA) was higher in the blended group. In a multivariable regression model, current GPA and knowledge test scores were associated with the final statistics score after adjusting for study duration and learning modality (p<0.001).

Conclusion

This study provides empirical evidence to support educator decisions to implement different learning environments for teaching medical statistics to undergraduate medical students. Blended and on-site training formats led to similar knowledge acquisition; however, students with higher GPA preferred the technology assisted learning format. Implementation of blended learning approaches can be considered an attractive, cost-effective, and efficient alternative to traditional classroom training in medical statistics.     

Genetic diversity and morphological variability in the Balkan endemic Campanula secundiflora s.l. (Campanulaceae)

We reconstructed the relationships among populations of Campanula secundiflora s.l. and closely related and geographically close populations of C. austroadriatica and C. versicolor. Based on analyses of microsatellite DNA data, the investigated populations have high overall genetic diversity and abundant allelic variation over seven investigated loci. Bayesian model‐based clustering identified four clearly differentiated genetic groups of populations. The genetic differentiation was reflected by morphological differentiation, allowing us to propose a new taxonomic treatment of the constituents of C. secundiflora s.l. The populations distributed in south‐western Serbia and north‐eastern Montenegro were included in C. secundiflora. A new species, C ampanula montenegrina sp. nov. , distributed in the continental part of Montenegro and the northern part of Albania is described. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180 , 64–88.     

Conjunctival melanoma copy number alterations and correlation with mutation status,tumor features,and clinical outcome

Relatively little is known about the genetic aberrations of conjunctival melanomas (CoM) and their correlation with clinical and histomorphological features as well as prognosis. The aim of this large collaborative multicenter study was to determine potential key biomarkers for metastatic risk and any druggable targets for high metastatic risk CoM. Using Affymetrix single nucleotide polymorphism genotyping arrays on 59 CoM, we detected frequent amplifications on chromosome (chr) 6p and deletions on 7q, and characterized mutation‐specific copy number alterations. Deletions on chr 10q11.21‐26.2, a region harboring the tumor suppressor genes, PDCD4, SUFU, NEURL1, PTEN, RASSF4, DMBT1, and C10orf90 and C10orf99, significantly correlated with metastasis (Fisher's exact, p ≤ 0.04), lymphatic invasion (Fisher's exact, p ≤ 0.02), increasing tumor thickness (Mann–Whitney, p ≤ 0.02), and BRAF mutation (Fisher's exact, p ≤ 0.05). This enhanced insight into CoM biology is a step toward identifying patients at risk of metastasis and potential therapeutic targets for systemic disease.     

The impact of aerobic and anaerobic training regimes on blood pressure in normotensive and hypertensive rats: focus on redox changes

Molecular and Cellular Biochemistry - Melatonin is a crucial neurohormone synthesized in the pineal gland that influences the physiology of animals. The molecular mechanism of norepinephrine...