Absence of endogenous phospholipid transfer protein impairs ABCA1-dependent efflux of cholesterol from macrophage foam cells

In vitro experiments have demonstrated that exogenous phospholipid transfer protein (PLTP), i.e. purified PLTP added to macrophage cultures, influences ABCA1-mediated cholesterol efflux from macrophages to HDL. To investigate whether PLTP produced by the macrophages (i.e., endogenous PLTP) is also part of this process, we used peritoneal macrophages derived from PLTP-knockout (KO) and wild-type (WT) mice. The macrophages were transformed to foam cells by cholesterol loading, and this resulted in the upregulation of ABCA1. Such macrophage foam cells from PLTP-KO mice released less cholesterol to lipid-free apolipoprotein A-I (apoA-I) and to HDL than did the corresponding WT foam cells. Also, when plasma from either WT or PLTP-KO mice was used as an acceptor, cholesterol efflux from PLTP-KO foam cells was less efficient than that from WT foam cells. After cAMP treatment, which upregulated the expression of ABCA1, cholesterol efflux from PLTP-KO foam cells to apoA-I increased markedly and reached a level similar to that observed in cAMP-treated WT foam cells, restoring the decreased cholesterol efflux associated with PLTP deficiency. These results indicate that endogenous PLTP produced by macrophages contributes to the optimal function of the ABCA1-mediated cholesterol efflux-promoting machinery in these cells. Whether macrophage PLTP acts at the plasma membrane or intracellularly or shuttles between these compartments needs further study.     

Grosmannia and Leptographium spp. associated with conifer-infesting bark beetles in Finland and Russia, including Leptographium taigense sp. nov.

Species of Grosmannia with Leptographium anamorphs include important forest pathogens and agents of blue stain in timber. They are commonly found in association with forest pests, such as bark beetles. During a survey of ophiostomatoid fungi in eastern parts of Finland and neighboring Russia, species belonging to the genus Grosmannia were isolated from 12 different bark beetle species infesting Picea abies and Pinus sylvestris, the most economically important conifers in the region. Identification of these fungi was based on morphology, DNA sequence comparisons for three gene regions and phylogenetic analyses. A total of ten taxa were identified. These belonged to six different species complexes in Grosmannia. The phylogenetic analyses provided an opportunity to redefine the G. galeiformis-, L. procerum-, L. lundbergii-, G. piceiperda-, G. olivacea- and G. penicillata-complexes, and to consider the species emerging from the survey within the context of these complexes. The species included G. galeiformis, G. olivacea, L. chlamydatum, L. lundbergii, L. truncatum and a novel taxon, described here as L. taigense sp. nov. In addition, species closely related to G. cucullata, G. olivaceapini comb. nov., G. piceiperda and L. procerum were isolated but their identity could not be resolved. The overall results indicate that the diversity of Grosmannia species in the boreal forests remains poorly understood and that further studies are needed to clarify the status of several species or species complexes.     

High-throughput karyotyping of human pluripotent stem cells

Genomic integrity of human pluripotent stem cell (hPSC) lines requires routine monitoring. We report here that novel karyotyping assay, utilizing bead-bound bacterial artificial chromosome probes, provides a fast and easy tool for detection of chromosomal abnormalities in hPSC lines. The analysis can be performed from low amounts of DNA isolated from whole cell pools with simple data analysis interface. The method enables routine screening of stem cell lines in a cost-efficient high-throughput manner.     

Expression analysis of immune response genes in fish epithelial cells following ranavirus infection

Ranaviruses (family Iridoviridae) are a growing threat to fish and amphibian populations worldwide. The immune response to ranavirus infection has been studied in amphibians, but little is known about the responses elicited in piscine hosts. In this study, the immune response and apoptosis induced by ranaviruses were investigated in fish epithelial cells. Epithelioma papulosum cyprini (EPC) cells were infected with four different viral isolates: epizootic haematopoietic necrosis virus (EHNV), frog virus 3 (FV3), European catfish virus (ECV) and doctor fish virus (DFV). Quantitative real-time PCR (qPCR) assays were developed to measure the mRNA expression of immune response genes during ranavirus infection. The target genes included tumour necrosis factor α (TNF-α), interleukin-1β (IL-1β), β2-microglobulin (β2M), interleukin-10 (IL-10) and transforming growth factor β (TGF-β). All ranaviruses elicited changes in immune gene expression. EHNV and FV3 caused a strong pro-inflammatory response with an increase in the expression of both IL-1β and TNF-α, whereas ECV and DFV evoked transient up-regulation of regulatory cytokine TGF-β. Additionally, all viral isolates induced increased β2M expression as well as apoptosis in the EPC cells. Our results indicate that epithelial cells can serve as an in vitro model for studying the mechanisms of immune response in the piscine host in the first stages of ranavirus infection.     

Somatic progenitor cell vulnerability to mitochondrial DNA mutagenesis underlies progeroid phenotypes in Polg mutator mice

Somatic stem cell (SSC) dysfunction is typical for different progeroid phenotypes in mice with genomic DNA repair defects. MtDNA mutagenesis in mice with defective Polg exonuclease activity also leads to progeroid symptoms, by an unknown mechanism. We found that Polg-Mutator mice had neural (NSC) and hematopoietic progenitor (HPC) dysfunction already from embryogenesis. NSC self-renewal was decreased in vitro, and quiescent NSC amounts were reduced in vivo. HPCs showed abnormal lineage differentiation leading to anemia and lymphopenia. N-acetyl-L-cysteine treatment rescued both NSC and HPC abnormalities, suggesting that subtle ROS/redox changes, induced by mtDNA mutagenesis, modulate SSC function. Our results show that mtDNA mutagenesis affected SSC function early but manifested as respiratory chain deficiency in nondividing tissues in old age. Deletor mice, having mtDNA deletions in postmitotic cells and no progeria, had normal SSCs. We propose that SSC compartment is sensitive to mtDNA mutagenesis, and that mitochondrial dysfunction in SSCs can underlie progeroid manifestations.     

Mannose inhibits hyaluronan synthesis by down-regulation of the cellular pool of UDP-N-acetylhexosamines

We found that d-mannose dose-dependently decreases hyaluronan synthesis in cultured epidermal keratinocytes to approximately 50%, whereas glucose, galactose, and fructose up to 20 mm concentration had no effect. The full inhibition occurred within 3 h following introduction of mannose and did not involve down-regulation of hyaluronan synthase (Has1-3) mRNA. Following introduction of mannose, there was an approximately 50% reduction in the cellular concentration of UDP-N-acetylhexosamines (UDP-HexNAc, i.e. UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine). On the other hand, 2 mm glucosamine in the culture medium increased UDP-HexNAc content, stimulated hyaluronan secretion, and negated the effect of mannose, supporting the notion that the inhibition by mannose on hyaluronan synthesis was because of down-regulated UDP-HexNAc content. The content of UDP-glucuronic acid, the other building block for hyaluronan synthesis, was not reduced by mannose but declined from 39 to 14% of controls by 0.2-1.0 mm 4-methylumbelliferone, another compound that inhibits hyaluronan synthesis. Applying 4-methylumbelliferone and mannose together produced the expected reductions in both UDP sugars but no additive reduction in hyaluronan production, indicating that the concentration of each substrate alone can limit hyaluronan synthesis. Mannose is a potentially useful tool in studies on hyaluronan-dependent cell functions, as demonstrated by reduced rates of keratinocyte proliferation and migration, functions known to depend on hyaluronan synthesis.     

Bifidobacterium microbiota and parameters of immune function in elderly subjects

Faecal and serum samples were collected over a period of 6 months from 55 institutionalized elderly subjects, who were enrolled in a double-blind placebo-controlled study. Participants were randomized in one of the three treatment groups: intervention (two probiotic Bifidobacterium longum strains: 2C and 46), placebo and commercial control (Bifidobacterium lactis Bb-12). The faecal Bifidobacterium microbiota was characterized by genus and species-specific PCR. Serum levels of the cytokines IL-10, tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1 were determined by enzyme-linked immunosorbent assay. Each participant harboured on average approximately three different bifidobacterial species. The most frequently detected species were B. longum, Bifidobacterium adolescentis and Bifidobacterium bifidum. Depending on the treatment, the intervention resulted in specific changes in the levels of certain Bifidobacterium species, and positive correlations were found between the different species. Negative correlations were observed between the levels of Bifidobacterium species and the pro-inflammatory cytokine TNF-alpha and the regulatory cytokine IL-10. The presence of faecal B. longum and Bifidobacterium animalis correlated with reduced serum IL-10. The anti-inflammatory TGF-beta1 levels were increased over time in all three groups, and the presence of Bifidobacterium breve correlated with higher serum TGF-beta1 levels. This indicates that modulation of the faecal Bifidobacterium microbiota may provide a means of influencing inflammatory responses.