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.     

Use of allele-specific sequencing primers is an efficient alternative to PCR subcloning of low-copy nuclear genes

Direct Sanger sequencing of polymerase chain reaction (PCR)-amplified nuclear genes leads to polymorphic sequences when allelic variation is present. To overcome this problem, most researchers subclone the PCR products to separate alleles. An alternative is to directly sequence the separate alleles using allele-specific primers. We tested two methods to enhance the specificity of allele-specific primers for use in direct sequencing: using short primers and amplification refractory mutation system (ARMS) technique. By shortening the allele-specific primer to 15-13 nucleotides, the single mismatch in the ultimate base of the primer is enough to hinder the amplification of the nontarget allele in direct sequencing and recover only the targeted allele at high accuracy. The deliberate addition of a second mismatch, as implemented in the ARMS technique, was less successful and seems better suited for allele-specific amplification in regular PCR rather than in direct sequencing.     

Accumulation of cholesterol precursors and plant sterols in human stenotic aortic valves

The pathogenesis of aortic valve stenosis (AS) is characterized by the accumulation of LDL-derived cholesterol in the diseased valves. Since LDL particles also contain plant sterols, we investigated whether plant sterols accumulate in aortic valve lesions. Serum samples were collected from 82 patients with severe AS and from 12 control subjects. Aortic valves were obtained from a subpopulation of 21 AS patients undergoing valve surgery and from 10 controls. Serum and valvular total cholesterol and noncholesterol sterols were measured by gas-liquid chromatography. Noncholesterol sterols, including both cholesterol precursors and sterols reflecting cholesterol absorption, were detected in serum samples and aortic valves. The higher the ratios to cholesterol of the cholesterol precursors and absorption markers in serum, the higher their ratios in the stenotic aortic valves (r=0.74, P<0.001 for lathosterol and r=0.88, P<0.001 for campesterol). The valvular ratio to cholesterol of lathosterol correlated negatively with the aortic valve area (r= -0.47, P=0.045), suggesting attenuation of cholesterol synthesis with increasing severity of AS. The higher the absorption of cholesterol, the higher the plant sterol contents in stenotic aortic valves. These findings suggest that local accumulation of plant sterols and cholesterol precursors may participate in the pathobiology of aortic valve disease.     

Survival of male Tengmalm’s owls increases with cover of old forest in their territory

The loss and fragmentation of forest habitats have been considered to pose a worldwide threat to the viability of forest-dwelling animals, especially to species that occupy old forests. We investigated whether the annual survival of sedentary male Tengmalm’s owls Aegolius funereus was associated with the cover of old coniferous forests in Finland. Survival and recapture probabilities varied annually with density changes in populations of the main prey (Microtus voles). When this variation was controlled for, and relationships between survival and proportions of the three different forest age classes (old-growth, middle-aged, and young) were modeled separately, the old-growth model was the most parsimonious. Survival increased with the cover of old forest, although the extent of old forest within owl territories was relatively small (mean ∼12%, range 2–37%). This association, however, varied among years and appeared especially in years of increasing vole abundance. At such times, old forests may sustain high populations of bank voles Clethrionomys glareolus, shrews and small passerines. In addition, old forests may serve as refuges against large avian predator species, such as Ural owls Strix uralensis and goshawks Accipiter gentilis. Our results suggest that changes in habitat quality created by agriculture and forestry may have the potential to reduce adult survival, an essential component of fitness and population viability.     

All JNKs can kill, but nuclear localization is critical for neuronal death

JNKs are implicated in a range of brain pathologies and receive considerable attention as potential therapeutic targets. However, JNKs also regulate physiological and homeostatic processes. An attractive hypothesis from the drug development perspective is that distinct JNK isoforms mediate "physiological" and "pathological" responses. However, this lacks experimental evaluation. Here we investigate the isoforms, subcellular pools, and c-Jun/ATF2 targets of JNK in death of central nervous system neurons following withdrawal of trophic support. We use gene knockouts, gene silencing, subcellularly targeted dominant negative constructs, and pharmacological inhibitors. Combined small interfering RNA knockdown of all JNKs 1, 2, and 3, provides substantial neuroprotection. In contrast, knockdown or knock-out of individual JNKs or two JNKs together does not protect. This explains why the evidence for JNK in neuronal death has to date been largely pharmacological. Complete knockdown of c-Jun and ATF2 using small interfering RNA also fails to protect, casting doubt on c-Jun as a critical effector of JNK in neuronal death. Nonetheless, the death requires nuclear but not cytosolic JNK activity as nuclear dominant negative inhibitors of JNK protect, whereas cytosolic inhibitors only block physiological JNK function. Thus any one of the three JNKs is capable of mediating apoptosis and inhibition of nuclear JNK is protective.     

Complete assignments of the (1)H and (13)C chemical shifts and J(H,H) coupling constants in NMR spectra of D-glucopyranose and all D-glucopyranosyl-D-glucopyranosides

Complete assignment of the (1)H and (13)C NMR spectra of all possible d-glucopyranosyl-d-glucopyranosides was performed and the (1)H chemical shifts and proton-proton coupling constants were refined by computational spectral analyses (using PERCH NMR software) until full agreement between the calculated and experimental spectra was achieved. To support the experimental results, the (1)H and (13)C chemical shifts and the spin-spin coupling constants between the non-hydroxyl protons of alpha- and beta-d-glucopyranose (1a and 1b) were calculated with density functional theory (DFT) methods at the B3LYP/pcJ-2//B3LYP/6-31G(d,p) level of theory. The effects of different glycosidic linkage types and positions on the glucose ring conformations and on the alpha/beta-ratio of the reducing end hydroxyl groups were investigated. Conformational analyses were also performed for anomerically pure forms of methyl d-glucopyranosides (13a and 13b) and fully protected derivatives such as 1,2,3,4,6-penta-O-acetyl-d-glucopyranoses (14a and 14b).     

Phase transitions and antiferromagnetism in copper(II) hexanoates: a new tetranuclear type of copper carboxylate paddle-wheel association

A series of compounds of formula [{Cu₂(OOCC_mH_2m + 1)₄(urea)}₂] (m=5-11) have been characterized. X-ray structure analysis for the hexanoate compound reveals a new type of tetranuclear dicopper(II) tetracarboxylate, where the central coordination sphere in [{Cu₂(OOCC₅H₁₁)₄(urea)}₂] is composed of two dinuclear dicopper tetracarboxylates, connected via two inter-dinuclear Cu-O coordination bonds at a distance 2.222(2) Å through the apical positions of two dimers. Urea molecules (Cu-O 2.114(2) Å) occupy both outside apical positions of the resulting tetranuclear units. A strong antiferromagnetic behaviour has been shown for [{Cu₂(OOCC₅H₁₁)₄(urea)}₂] (−2J=261.4(4) cm⁻¹), and compared with related isolated dinuclear and polymeric hexanoate compounds [Cu₂(OOCC₅H₁₁)₄(urea)₂], [Cu₂(OOCC₅H₁₁)₄]_n, respectively. Only small differences in the magnetic susceptibility have been found, while EPR spectroscopy showed significantly different results for all three hexanoate compounds, also with the dicopper tetracarboxylate central core and square-pyramidal CuO₄O chromophores. A solid-to-solid phase transition for [{Cu₂(OOCC₅H₁₁)₄(urea)}₂] was observed by magnetic measurement and analysed for the whole series [{Cu₂(OOCC_mH_2m + 1)₄(urea)}₂] by TG, DTA, and variable temperature XRD studies.     

In situ delipidation of low-density lipoproteins in capillary electrochromatography yields apolipoprotein B-100-coated surfaces for interaction studies

An electrochromatographic method was developed for the in situ delipidation of intact low-density lipoprotein (LDL) particles immobilized on the inner wall of a 50-μm inner diameter silica capillary. In this method, the immobilized LDL particles were delipidated with nonionic surfactant Nonidet P-40 at pH 7.4 and 25 °C, resulting in an apolipoprotein B-100 (apoB-100)-coated capillary surface. The mobility of the electroosmotic flow marker dimethyl sulfoxide gave information about the surface charge, and the retention factors of β-estradiol, testosterone, and progesterone were informative of the surface hydrophobicity. The calculated distribution coefficients of the steroids produced specific information about the affinity interactions of the steroids, with capillary surfaces coated either with intact LDL particles or with apoB-100. Delipidation with Nonidet P-40 resulted in a strong decrease in the hydrophobicity of the LDL coating. Atomic force microscopy images confirmed the loss of lipids from the LDL particles and the presence of apoB-100 protein coating. The in situ delipidation of LDL particles in capillaries represents a novel approach for the isolation of immobilized apoB-100 and for the determination of its pI value. The technique requires extremely low quantities of LDL particles, and it is simple and fast.     

Habitat characteristics and macroinvertebrate assemblages in boreal forest streams: relations to catchment silvicultural activities

We compared the stream habitat characteristics and macroinvertebrate assemblages of boreal headwater streams in both the Finnish and the Russian parts of a single river basin, the Koitajoki River. Over the last 50 years, the Finnish side of the catchment has been managed using modern forestry techniques, whereas Russian side has remained nearly unexploited and is near to its natural state. Differences in silvicultural activities were observed to contribute to differences in habitat structure. The channel habitats were in fairly natural state in the Russian reference streams, whereas the impacted Finnish sites were cleared and straightened. In comparison with the impacted channels, the abundance of coarse woody debris (CWD) was 10–100-fold higher in the reference streams. Implications on the forestry-induced deterioration of water quality were also observed. On the contrary, only small differences in macroinvertebrate assemblages were detected. Despite the lower amount of retentive structures (CWD), significantly higher relative abundance of shredders was observed in the forestry-impacted streams. Otherwise the zoobenthic communities were quite similar in the two subcatchments. We suggest that several mechanisms may explain this similarity: (1) community structure is controlled by naturally acidic conditions, (2) the adverse impacts of forestry on habitat structure and water quality of streams may be compensated by increased input of deciduous litter and organic compounds from drained, structurally young riparian forests and (3) macroinvertebrate species have flexible feeding habits and may thus readily adapt to changing conditions.