Altered mitochondrial apparent affinity for ADP and impaired function of mitochondrial creatine kinase in gluteus medius of patients with hip osteoarthritis

The cellular energy metabolism in human musculus gluteus medius (MGM) under normal conditions and hip osteoarthritis (OA) was explored. The functions of oxidative phosphorylation and energy transport systems were analyzed in permeabilized (skinned) muscle fibers by oxygraphy, in relation to myosin heavy chain (MHC) isoform distribution profile analyzed by SDS-PAGE, and to creatine kinase (CK) and adenylate kinase (AK) activities measured spectrophotometrically in the intact muscle. The results revealed high apparent Km for ADP in regulation of respiration that decreased after addition of creatine in MGM of traumatic patients (controls). OA was associated with increased sensitivity of mitochondrial respiration to ADP, decreased total activities of AK and CK with major reduction in mi-CK fraction, and attenuated effect of creatine on apparent Km for ADP compared with control group. It also included a complete loss of type II fibers in a subgroup of patients with the severest disease grade. It is concluded that energy metabolism in MGM cells is organized into functional complexes of mitochondria and ATPases. It is suggested that because of degenerative remodeling occurring during development of OA, these complexes become structurally and functionally impaired, which results in increased access of exogenous ADP to mitochondria and dysfunction of CK-phosphotransfer system.     

Connections between climatic variables and the growth and needle dynamics of Scots pine (Pinus sylvestris L.) in Estonia and Lapland

The relationships between climatic variables and Scots pine (Pinus sylvestris L.) growth and needle dynamics were studied in three stands in Estonia and in four stands located near the northern timberline in Lapland. The trees sampled in Estonia had low correlations with the analysed climatic variables (air temperature, precipitation and indices of atmospheric circulation). Moreover, the weak cross-correlation of the time-series of the Estonian sample trees indicated that Scots pine is affected mainly by local factors in that region. In Lapland, however, height increment and needle production correlated strongly among trees within a stand (mean r=0.45 and 0.46, respectively) and between stands (r=0.32 and 0.37). Radial increment also showed a high inter-correlation among the trees within a stand in Lapland (r=0.45). Both height increment and needle production were strongly influenced by the temperature regime of the previous summer in Lapland (mean r=0.64 and 0.64, respectively). Radial increment was correlated with the mean July temperature of the current year (mean r=0.29). The correlations between the indices of atmospheric circulation and tree attributes were weak, while the strongest correlation was between the Ponta Delgada NAO index (PD–NAO) and height increment and needle production in Lapland. Height increment, needle production and radial increment have increased since the 1990s in the trees growing in Lapland. This may indicate a positive effect of climate warming on tree growth in Lapland. In Estonia, where climatic conditions do not limit tree growth, the climate warming seems not to directly influence the growth and needle dynamics of Scots pine.     

S4(13)-PV cell-penetrating peptide induces physical and morphological changes in membrane-mimetic lipid systems and cell membranes: implications for cell internalization

The present work aims to gain insights into the role of peptide-lipid interactions in the mechanisms of cellular internalization and endosomal escape of the S4(13)-PV cell-penetrating peptide, which has been successfully used in our laboratory as a nucleic acid delivery system. A S4(13)-PV analogue, S4(13)-PVscr, displaying a scrambled amino acid sequence, deficient cell internalization and drug delivery inability, was used in this study for comparative purposes. Differential scanning calorimetry, fluorescence polarization and X-ray diffraction at small and wide angles techniques showed that both peptides interacted with anionic membranes composed of phosphatidylglycerol or a mixture of this lipid with phosphatidylethanolamine, increasing the lipid order, shifting the phase transition to higher temperatures and raising the correlation length between the bilayers. However, S4(13)-PVscr, in contrast to the wild-type peptide, did not promote lipid domain segregation and induced the formation of an inverted hexagonal lipid phase instead of a cubic phase in the lipid systems assayed. Electron microscopy showed that, as opposed to S4(13)-PVscr, the wild-type peptide induced the formation of a non-lamellar organization in membranes of HeLa cells. We concluded that lateral phase separation and destabilization of membrane lamellar structure without compromising membrane integrity are on the basis of the lipid-driven and receptor-independent mechanism of cell entry of S4(13)-PV peptide. Overall, our results can contribute to a better understanding of the role of peptide-lipid interactions in the mechanisms of cell-penetrating peptide membrane translocation, helping in the future design of more efficient cell-penetrating peptide-based drug delivery systems.     

Global Gene Expression Profiling Reveals SPINK1 as a Potential Hepatocellular Carcinoma Marker

Background

Liver cirrhosis is the most important risk factor for hepatocellular carcinoma (HCC) but the role of liver disease aetiology in cancer development remains under-explored. We investigated global gene expression profiles from HCC arising in different liver diseases to test whether HCC development is driven by expression of common or different genes, which could provide new diagnostic markers or therapeutic targets.

Methodology and Principal Findings

Global gene expression profiling was performed for 4 normal (control) livers as well as 8 background liver and 7 HCC from 3 patients with hereditary haemochromatosis (HH) undergoing surgery. In order to investigate different disease phenotypes causing HCC, the data were compared with public microarray repositories for gene expression in normal liver, hepatitis C virus (HCV) cirrhosis, HCV-related HCC (HCV-HCC), hepatitis B virus (HBV) cirrhosis and HBV-related HCC (HBV-HCC). Principal component analysis and differential gene expression analysis were carried out using R Bioconductor. Liver disease-specific and shared gene lists were created and genes identified as highly expressed in hereditary haemochromatosis HCC (HH-HCC) were validated using quantitative RT-PCR. Selected genes were investigated further using immunohistochemistry in 86 HCC arising in liver disorders with varied aetiology. Using a 2-fold cut-off, 9 genes were highly expressed in all HCC, 11 in HH-HCC, 270 in HBV-HCC and 9 in HCV-HCC. Six genes identified by microarray as highly expressed in HH-HCC were confirmed by RT qPCR. Serine peptidase inhibitor, Kazal type 1 (SPINK1) mRNA was very highly expressed in HH-HCC (median fold change 2291, p = 0.0072) and was detected by immunohistochemistry in 91% of HH-HCC, 0% of HH-related cirrhotic or dysplastic nodules and 79% of mixed-aetiology HCC.

Conclusion

HCC, arising from diverse backgrounds, uniformly over-express a small set of genes. SPINK1, a secretory trypsin inhibitor, demonstrated potential as a diagnostic HCC marker and should be evaluated in future studies.     

Synthesis and Biological Activity of Bimorpholine and its Carbanucleoside

A new enantiomerically pure carbacyclic nucleoside analogue with bimorpholine as a nonaromatic nucleobase was synthesized. The nucleoside analogue and bimorpholine were tested for cytotoxicity using an MTT assay and the xCELLigence System. Both assays revealed that compound 3 was highly cytotoxic at a 50 μM concentration while the cytotoxic effect of compound 1 was much less prominent. No antiretroviral activity was detected for this compound. In contrast, it acted as a potent inhibitor of hepatitis C virus (HCV) replication. Most likely this effect originates largely from the cytotoxicity of the compound; however, it is possible that a specific mechanism of HCV inhibition also exists.     

The conservation of ground layer lichen communities in alvar grasslands and the relevance of substitution habitats

Semi-natural calcareous grasslands (alvars) are biodiversity hotspots in Northern Europe, particularly for herb layer plants. In the last century, traditional management has ceased, and the area of grasslands has declined due to extensive encroachment. We were interested in the drivers of ground layer (alias terricolous or epigeic) lichen communities. Our survey consisted of 86 habitat fragments in western Estonia, covering four types of historic alvar grasslands and three types of alvar-like habitats. We found that the ground lichen communities were primarily soil-type-specific, but were also affected by historic disturbances and land use change. In contrast to knowledge about herb layer communities, for which shrub encroachment has been shown to be main driver, the increased density of the herb layer and the reduced diversity of microhabitats were major drivers for the ground layer lichen community. These drivers caused a decrease in species richness, but only within the species of conservation value, and also led to a shift in the composition of lichen growth form from the dominance of squamulose and crustose towards fruticose lichens. We conclude that the traditional practice of restoring alvars by cutting shrubs is insufficient to maintain ground layer lichen biodiversity. Alvar maintenance practices should include grazing, which creates various small-scale ground disturbances and increases microhabitat heterogeneity. Alvar-like habitats originating from large-scale historic disturbances appeared to be suitable for calcicolous epigeic lichens, and can therefore be considered to be temporary substitution habitats, i.e. refugia for the regional species pool.     

Cholesterol-rich low density lipoproteins are also more oxidized

Cardiovascular diseases are accompanied by active oxygen species and organic free radical generation. The aim of this study was to examine the possibility of using oxidized low-density lipoprotein (oxLDL) as a new diagnostic biomarker. Epidemiological study in populations of Estonia (782 subjects) and Russia (1433 subjects) was carried out in 2007-2009. The screening procedure included standard epidemiological methods. Oxidative stress was assessed by measuring the level of oxLDL using immunoassay method. Positive correlation between the levels of oxLDL and LDL cholesterol was indicated in blood of patients from estonian (r = 0.61; P < 0.05) and russian (r = 0.56; P < 0.05) populations. In russian population oxLDL/HDL cholesterol ratio was higher in the groups with highest risk of atherosclerosis development or manifest coronary artery disease (CAD). Cholesterol-rich low density lipoproteins are also more oxidized. Estimation of oxLDL/HDL ratio may be used as an independent biochemical marker for atherosclerosis.     

Direct translocation as major cellular uptake for CADY self-assembling peptide-based nanoparticles

Cell penetrating peptides constitute a potent approach to overcome the limitations of in vivo siRNA delivery. We recently proposed a peptide-based nanoparticle system, CADY, for efficient delivery of siRNA into numerous cell lines. CADY is a secondary amphipathic peptide that forms stable complexes with siRNA thereby improving both their cellular uptake and biological response. With the aim of understanding the cellular uptake mechanism of CADY:siRNA complexes, we have combined biochemical, confocal and electron microscopy approaches. In the present work, we provide evidence that the major route for CADY:siRNA cellular uptake involves direct translocation through the membrane but not the endosomal pathway. We have demonstrated that CADY:siRNA complexes do not colocalize with most endosomal markers and remain fully active in the presence of inhibitors of the endosomal pathway. Moreover, neither electrostatic interactions with cell surface heparan sulphates nor membrane potential are essential for CADY:siRNA cell entry. In contrast, we have shown that CADY:siRNA complexes clearly induce a transient cell membrane permeabilization, which is rapidly restored by cell membrane fluidity. Therefore, we propose that direct translocation is the major gate for cell entry of CADY:siRNA complexes. Membrane perturbation and uptake are driven mainly by the ability of CADY to interact with phospholipids within the cell membrane, followed by rapid localization of the complex in the cytoplasm, without affecting cell integrity or viability.     

Polymorphisms in genes involved in the NF-κB signalling pathway are associated with bone mineral density, geometry and turnover in men

Introduction

In this study, we aimed to investigate the association between single nucleotide polymorphisms (SNPs) within two genes involved in the NF-κB cascade (GPR177 and MAP3K14) and bone mineral density (BMD) assessed at different skeletal sites, radial geometric parameters and bone turnover.

Methods

Ten GPR177 SNPs previously associated with BMD with genome-wide significance and twelve tag SNPs (r²≥0.8) within MAP3K14 (±10 kb) were genotyped in 2359 men aged 40–79 years recruited from 8 centres for participation in the European Male Aging Study (EMAS). Measurement of bone turnover markers (PINP and CTX-I) in the serum and quantitative ultrasound (QUS) at the calcaneus were performed in all centres. Dual energy X-ray absorptiometry (DXA), at the lumbar spine and hip, and peripheral quantitative computed tomography (pQCT), at the distal and midshaft radius, were performed in a subsample (2 centres). Linear regression was used to test for association between the SNPs and bone measures under an additive genetic model adjusting for study centre.

Results

We validated the associations between SNPs in GPR177 and BMD_a previously reported and also observed evidence of pleiotrophic effects on density and geometry. Rs2772300 in GPR177 was associated with increased total hip and LS BMD_a, increased total and cortical vBMD at the radius and increased cortical area, thickness and stress strain index. We also found evidence of association with BMD_a, vBMD, geometric parameters and CTX-I for SNPs in MAP3K14. None of the GPR177 and MAP3K14 SNPs were associated with calcaneal estimated BMD measured by QUS.

Conclusion

Our findings suggest that SNPs in GPR177 and MAP3K14 involved in the NF-κB signalling pathway influence bone mineral density, geometry and turnover in a population-based cohort of middle aged and elderly men. This adds to the understanding of the role of genetic variation in this pathway in determining bone health.