Reduction of 8-hydroxyguanine in human leukocyte DNA by physical exercise

We investigated the effect of physical exercise on the level of 8-hydroxyguanine (8-OH-Gua), a form of oxidative DNA damage, and its repair activity in human peripheral leukocytes. Whole blood samples were collected by venipuncture from 21 healthy male volunteers (10 trained athletes and 13 untrained men), aged 19-50 years, both before and after physical exercise. Trained athletes showed a lower level of 8-OH-Gua (2.4 ± 0.5/10⁶ Gua, p = 0.0032) before exercise when compared to that of untrained men (6.2 ± 3.5). The mean levels of 8-OH-Gua of untrained subjects decreased significantly (p = 0.0057) from 6.2 ± 3.5/10⁶ Gua (mean ± SD/10⁶ Gua) to 3.3 ± 1.4/10⁶ Gua after physical exercise. On the other hand, the mean levels of repair activity of untrained subjects significantly increased after exercise (p = 0.0093) from 0.037 ± 0.024 (mean DNA cleavage ratio ± SD) to 0.056 ± 0.036. In the trained athletes 8-OH-Gua level and its repair activity were not changed before and after the exercise. We also observed inter-individual differences in 8-OH-Gua levels and its repair activities. These results suggest that physical exercise causes both rapid and long-range reduction of oxidative DNA damage in human leukocytes, with individually different efficiencies.     

ATP-dependent aggregation of single-stranded DNA by a bacterial SMC homodimer.

SMC (structural maintenance of chromosomes) proteins are putative ATPases that are highly conserved among Bacteria, Archaea and Eucarya. Eukaryotic SMC proteins are implicated in a diverse range of chromosome dynamics including chromosome condensation, dosage compensation and recombinational repair. In eukaryotes, two different SMC proteins form a heterodimer, which in turn acts as the core component of a large protein complex. Despite recent progress, no ATP-dependent activity has been found in individual SMC subunits. We report here the first biochemical characterization of a bacterial SMC protein from Bacillus subtilis. Unlike eukaryotic versions, the B.subtilis SMC protein (BsSMC) is a simple homodimer with no associated subunits. It binds preferentially to single-stranded DNA (ssDNA) and has a ssDNA-stimulated ATPase activity. In the presence of ATP, BsSMC forms large nucleoprotein aggregates in a ssDNA-specific manner. Proteolytic cleavage of BsSMC is changed upon binding to ATP and ssDNA. The energy-dependent aggregation of ssDNA might represent a primitive type of chromosome condensation that occurs during segregation of bacterial chromosomes.     

Non-synonymous FGD3 Variant as Positional Candidate for Disproportional Tall Stature Accounting for a Carcass Weight QTL (CW-3) and Skeletal Dysplasia in Japanese Black Cattle

Recessive skeletal dysplasia, characterized by joint- and/or hip bone-enlargement, was mapped within the critical region for a major quantitative trait locus (QTL) influencing carcass weight; previously named CW-3 in Japanese Black cattle. The risk allele was on the same chromosome as the Q allele that increases carcass weight. Phenotypic characterization revealed that the risk allele causes disproportional tall stature and bone size that increases carcass weight in heterozygous individuals but causes disproportionately narrow chest width in homozygotes. A non-synonymous variant of FGD3 was identified as a positional candidate quantitative trait nucleotide (QTN) and the corresponding mutant protein showed reduced activity as a guanine nucleotide exchange factor for Cdc42. FGD3 is expressed in the growth plate cartilage of femurs from bovine and mouse. Thus, loss of FDG3 activity may lead to subsequent loss of Cdc42 function. This would be consistent with the columnar disorganization of proliferating chondrocytes in chondrocyte-specific inactivated Cdc42 mutant mice. This is the first report showing association of FGD3 with skeletal dysplasia.     

Creutzfeldt-Jakob Disease with a prion protein gene codon 180 mutation presenting asymmetric cortical high-intensity on magnetic resonance imaging

ABSTRACT. Here we report a genetically confirmed case of Creutzfeldt-Jakob disease with a prion protein gene codon 180 mutation presenting atypical magnetic resonance imaging findings. The present case exhibited an acute onset and lateralized neurologic signs, and progressive cognitive impairment. No myoclonus or periodic synchronous discharges on electroencephalography were observed. Diffusion-weighted images revealed areas of high signal intensity in the right frontal and temporal cortices at onset that extended to the whole cortex and basal ganglia of the right cerebral hemisphere at 3 months. Although the cerebrospinal fluid (CSF) was initially negative for neuron specific enolase, tau protein, 14–3–3 protein, and abnormal prion protein, the CSF was positive for these brain-derived proteins at 3 months after onset.     

Macrophage Receptor with Collagenous Structure (MARCO) Is Processed by either Macropinocytosis or Endocytosis-Autophagy Pathway

The Macrophage Receptor with COllagenous structure (MARCO) protein is a plasma membrane receptor for un-opsonized or environmental particles on phagocytic cells. Here, we show that MARCO was internalized either by ruffling of plasma membrane followed by macropinocytosis or by endocytosis followed by fusion with autophagosome in CHO-K1 cells stably transfected with GFP-MARCO. The macropinocytic process generated large vesicles when the plasma membrane subsided. The endocytosis/autophagosome (amphisome) generated small fluorescent puncta which were visible in the presence of glutamine, chloroquine, bafilomycin, ammonia, and other amines. The small puncta, but not the large vesicles, co-localized with LC3B and lysosomes. The LC3-II/LC3-I ratio increased in the presence of glutamine, ammonia, and chloroquine in various cells. The small puncta trafficked between the peri-nuclear region and the distal ends of cells back and forth at rates of up to 2–3 μm/sec; tubulin, but not actin, regulated the trafficking of the small puncta. Besides phagocytosis MARCO, an adhesive plasma membrane receptor, may play a role in incorporation of various extracellular materials into the cell via both macropinocytic and endocytic pathways.     

Cultured human corneal epithelial stem/progenitor cells derived from the corneal limbus

Stem/progenitor cells of the human corneal epithelium are present in the human corneal limbus, and several corneal epithelial stem/progenitor cell markers have been reported. Recently, the neurotrophin family receptors were reported to be useful markers of corneal epithelial stem/progenitor cells. Therefore, we examined an enzymatic separation method for obtaining corneal epithelial stem/progenitor cells and measuring the change in the expression of low-affinity neurotrophin receptor p75 (p75^NTR), a receptor belonging to the neurotrophin family. As a result, it was found that our separation method preserved cell viability. Furthermore, p75^NTR was mainly observed in epithelial basal cells as were the corneal epithelial stem/progenitor markers p63 and integrin β1. p75^NTR was also observed in the cultured cells, but its frequency decreased with passage. In conclusion, we propose that our culture method will enable the culture of corneal stem cells and that it is a useful tool for elucidating the molecular basis of the niche that is necessary for the maintenance of epithelial stem cells in the corneal limbus. Furthermore, we conclude that p75^NTR is a useful cell marker for evaluating the characteristics of stem/progenitor cells in culture.     

Islands as refugia of <Emphasis Type="Italic">Trifolium repens</Emphasis> genetic diversity

Island populations are often thought to be more susceptible to the loss of genetic diversity as a consequence of limited population size and genetic drift, greater susceptibility to detrimental stochastic events and low levels of immigration. However the geographic isolation of islands may create refuges for native crop species whose genetic diversity is threatened from the genetic erosion occurring in mainland areas as a result of crop-wild gene flow and genetic swamping. Many UK islands remain uncharacterised in terms of plant genetic diversity. In this study we compared the genetic diversity of mainland populations and landraces of Trifolium repens with wild populations collected from the islands surrounding the UK, including the island of Hirta in the St Kildan archipelago. Individuals from St Kilda represent a unique conservation resource, with populations both highly differentiated from UK mainland populations and genetically distinct from cultivated varieties, whilst able to retain diversity through limited human influence on the islands. In contrast, there is relative genetic similarity of wild UK populations to cultivated forms highlighted in mainland populations, but with geographic barriers preventing complete homogenisation of the mainland UK genepool. We underline the need for conservation priorities to include common species that are threatened by gene flow from cultivation, and draw attention to the potential of islands to preserve natural levels of genetic diversity.     

The obligate alkaliphile <Emphasis Type="Italic">Bacillus clarkii</Emphasis> K24-1U retains extruded protons at the beginning of respiration

Alkaliphiles grow under alkaline conditions that might be disadvantageous for the transmembrane pH gradient (ΔpH, outside acidic). In this study, the behaviors of extruded protons by the respiration of obligate alkaliphilic Bacillus clarkii K24-1U were investigated by comparison with those of neutralophilic Bacillus subtilis IAM 1026. Although whole-cell suspensions of both Bacillus species consumed oxygen immediately after the addition of air, there were lag times before the suspensions were acidified. Under alkaline conditions, the lag time for B. clarkii significantly increased, whereas that for B. subtilis decreased. In the presence of valinomycin or ETH-157, which disrupts the membrane electrical potential (Δψ), the cell suspensions of both Bacillus species acidified immediately after the addition of air. Artificial electroneutral antiporters (nigericin and monensin) that eliminate the ΔpH exhibited no significant effect on the lag times of the two Bacillus species except that monensin increased the lag times of B. clarkii. The inhibition of ATPase and the Na⁺ channel also exhibited little effects on the lag times. The increased lag time for B. clarkii may represent the Δψ-dependent proton retention on the outer surface of the cytoplasmic membrane to generate a sufficient ΔpH under alkaline conditions.     

Sofalcone, a gastric mucosa protective agent, increases vascular endothelial growth factor via the Nrf2-heme-oxygenase-1 dependent pathway in gastric epithelial cells

Sofalcone, 2′-carboxymethoxy-4,4-bis(3-methyl-2-butenyloxy)chalcone, is an anti-ulcer agent that is classified as a gastric mucosa protective agent. Recent studies indicate heat shock proteins such as HSP32, also known as heme-oxygenase-1(HO-1), play important roles in protecting gastrointestinal tissues from several stresses. We have previously reported that sofalcone increases the expression of HO-1 in adipocytes and pre-adipocytes, although the effect of sofalcone on HO-1 induction in gastrointestinal tissues is not clear. In the current study, we investigated the effects of sofalcone on the expression of HO-1 and its functional role in rat gastric epithelial (RGM-1) cells. We found that sofalcone increased HO-1 expression in RGM-1 cells in both time- and concentration-dependent manners. The HO-1 induction was associated with the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in RGM-1 cells. We also observed that sofalcone increased vascular endothelial growth factor (VEGF) production in the culture medium. Treatment of RGM-1 cells with an HO-1 inhibitor (tin-protoporphyrin), or HO-1 siRNA inhibited sofalcone-induced VEGF production, suggesting that the effect of sofalcone on VEGF expression is mediated by the HO-1 pathway. These results suggest that the gastroprotective effects of sofalcone are partly exerted via Nrf2-HO-1 activation followed by VEGF production.