Histone H1-mediated transfection: role of calcium in the cellular uptake and intracellular fate of H1-DNA complexes

Previously, we have shown that the transgene expression in the endothelial cell line ECV 304 strongly depends on the presence of low concentrations of Ca2+. However, it remained unclear, which transfection steps are controlled by Ca2+ ions. In the present study, we constructed transfection complexes of digoxigenin-labelled DNA and FITC-labelled histone H1. We monitored the pathway of these complexes with the use of anti-digoxigenin and anti-cathepsin B antibodies and immunofluorescence microscopy. Double labelling of DNA and cathepsin B permitted the localization of transfection complexes into endosomes/lysosomes which suggests an uptake of transfection complexes via endocytosis. It was also found that the uptake of transfection complexes by the cells was independent of the presence or absence of Ca2+ ions in the transfection medium. On the other hand, the presence of Ca2+ in the transfection medium dramatically changed the composition of the transfection complexes inside the endosome/lysosome compartment, which resulted in a strong reduction of H1 binding to DNA. Presence of Ca2+ in the postincubation medium for 24 h resulted in release of the transfection complexes with reduced H1 content from the endosomes/lysosomes into the cytosol. In the absence of Ca2+ the transfection complexes practically disappeared. These results allow us to come to the following conclusions: Ca2+ ions control the reorganization of the transfection complexes in endosomes/lysosomes and their release into the cytosol, which is an important prerequisite for transgene expression, whereas uptake of transfection complexes by the cells is not dependent on Ca2+.     

The topology of early- and late-replicating chromatin in differentially decondensed chromosomes

In this study we used a novel technique to reveal both longitudinal and transverse differentiation within mammalian mitotic chromosomes. Structural changes in chromosomes that we term ‘differential decondensation’ were produced in cells that were first incubated in hypotonic medium (15% Hanks’ solution), then adapted to normotonic conditions and thereafter exposed to a second short hypotonic shock. Such a double hypotonic treatment (DHT) is not critical for cell viability, but considerably elongates the G2 phase of the cell cycle. Giemsa staining of differentially decondensed chromosomes corresponds to standard G-banding, but does not need the standard post-fixation treatment. Using ‘dynamic’ BrdU banding, we show that such ‘differential’ staining is a result of differential resistance of the R- and G-bands to DHT. Thus, early-replicating foci, markers of R-bands, are localized in the peripheral chromatin halo, whereas late-replicating foci, corresponding to G-bands, remain associated with the axial regions of chromatids. Remarkably, despite these major changes in the structure of the chromosomal bands, the replication foci still preserve their discrete structure.     

Brain volume dynamics in multiple sclerosis. A case-control study

ABSTRACT

Objectives: In this study, we aimed to explore the extent and clinical relevance of brain volume dynamics in relapsing remitting multiple sclerosis (RRMS).

Methods: Sixty-three patients with RRMS with a disease duration of about 5 years (36 women, mean age 39.9 ± 9.4 years; mean EDSS1.4 ± 1.2, mean relapse rate 0.98 ± 1.17) and 50 healthy control individuals (24 women, mean age 39.1 ± 10.2 years) were recruited and imaged on a MRI scanner by using post-gadolinium high-resolution3D T1W sequences. Cross-sectional and longitudinal volumetric data were obtained by using SIENA(X) and FIRST software.

Results: Patients showed significantly lower subcortical volumes compared to healthy controls. Interestingly, the educational level predicted the rate of right thalamus atrophy. The mean annualized percentage of brain volume change (aPBVC) was ?0.92% (±1.64%) and was presented in higher rates during the first five years after MS diagnosis.

Conclusion: Brain atrophy mainly involved subcortical grey matter structures and was more conspicuous during the first years of MS diagnosis. The buffering role of education in atrophy was also corroborated by this study.     

Potential role of hepatic progenitor cells expression in cases of chronic hepatitis C and their relation to response to therapy: a clinicopathologic study.

BACKGROUND: This study investigates the correlation of hepatic progenitor cells (HPC) expression with treatment response in patients with chronic hepatitis C. DESIGN: The study comprised 77 liver biopsies with chronic hepatitis C (HCV). All patients were PCR-HCV (+) and received antiviral therapy with interferon or pegylated interferon alpha-2b and ribavirin. Twenty-nine patients were assigned as responders (group A), 29 as nonresponders (group B) and 19 as relapsers (group C). Ten normal liver biopsies were used as controls. Liver paraffin sections were subjected (a) to immunohistochemistry using antibodies for cytokeratins 19 (CK19) and 7 (CK7), alpha-fetoprotein (AFP), leukocyte common antigen (LCA) and CD34 antigen (b) to in situ hybridization for AFP mRNA and (c) to immunohistochemistry+in situ hybridization. Results were expressed as % of positive cells following morphometric analysis. RESULTS: HPC expression was present in all 87 specimens. In the control biopsies, rare HPC were detected. In the CH cases and according to AFP mRNA expression, the grade for % HPC expression was: group B: 53.2+/-2.6> group C: 48.37+/-1.8> group A: 31.4+/-1.6 (group A vs B P<0.01, group A vs C P<0.01, group B vs C P>0.05. Double stain revealed that HPC coexpressed CK19/AFP mRNA, CK7/AFP mRNa and AFP protein/AFP mRNA. HPC-percentages were directly correlated with total HAI score (P<0.01), fibrosis stage (P<0.01), and transaminase values (P<0.05). CONCLUSIONS: This study demonstrates that in cases of chronic hepatitis C, the significant association of HPC expression with the severity of disease and more specifically with the response to treatment implies that HPC development and proliferation may provide additional prognostic information and predict prognosis in such cases.     

Effect of Long-Term Hydroxytyrosol Administration on Body Weight,Fat Mass and Urine Metabolomics: A Randomized Double-Blind Prospective Human Study

Hydroxytyrosol (HT) is a natural antioxidant found in olive products and characterized by well-documented beneficial effects on human health. Several research studies are ongoing that aim to investigate its potency and molecular mechanism of action. The present study aimed to investigate the potential effect of HT on human obesity through a randomized double-blind prospective design. HT in two different doses (15 and 5 mg/day) and a placebo capsule was administered to 29 women with overweight/obesity for six months and their weight and fat mass were monitored at three time points (baseline, 4, 12 and 24 weeks). Statistically significant weight and visceral fat mass loss (%weight loss: p = 0.012, %visceral fat loss: p = 0.006) were observed in the group receiving the maximum HT dosage versus placebo after 4 weeks of the intervention, with attenuation of these findings at 12 and 24 weeks of the study. Urine samples were collected during the intervention and analyzed via liquid chromatography–high-resolution mass spectrometry for untargeted metabolomic purposes and comparisons between study groups were performed. HT administration was safe and well-tolerated. To the best of our knowledge, this is the first human cohort investigating the effects of HT on obesity for a prolonged study period.