Lifestyle impacts on the aging‐associated expression of biomarkers of DNA damage and telomere dysfunction in human blood

Cellular aging is characterized by telomere shortening, which can lead to uncapping of chromosome ends (telomere dysfunction) and activation of DNA damage responses. There is some evidence that DNA damage accumulates during human aging and that lifestyle factors contribute to the accumulation of DNA damage. Recent studies have identified a set of serum markers that are induced by telomere dysfunction and DNA damage, and these markers showed an increased expression in blood during human aging. Here, we investigated the influence of lifestyle factors (such as exercise, smoking, body mass) on the aging‐associated expression of serum markers of DNA damage (CRAMP, EF‐1α, stathmin, n‐acetyl‐glucosaminidase and chitinase) in comparison with other described markers of cellular aging (p16^INK4a upregulation and telomere shortening) in human peripheral blood. The study shows that lifestyle factors have an age‐independent impact on the expression level of biomarkers of DNA damage. Smoking and increased body mass indices were associated with elevated levels of biomarkers of DNA damage independent of the age of the individuals. In contrast, exercise was associated with an age‐independent reduction in the expression of biomarkers of DNA damage in human blood. The expression of biomarkers of DNA damage correlated positively with p16^INK4a expression and negatively with telomere length in peripheral blood T‐lymphocytes. Together, these data provide experimental evidence that both aging and lifestyle impact on the accumulation of DNA damage during human aging.     

Mechanisms of cardiac fibrosis induced by urokinase plasminogen activator

Human hearts with end-stage failure and fibrosis have macrophage accumulation and elevated plasminogen activator activity. However, the mechanisms that link macrophage accumulation and plasminogen activator activity with cardiac fibrosis are unclear. We previously reported that mice with macrophage-targeted overexpression of urokinase plasminogen activator (SR-uPA+/o mice) develop cardiac macrophage accumulation by 5 weeks of age and cardiac fibrosis by 15 weeks. We used SR-uPA+/o mice to investigate mechanisms through which macrophage-expressed uPA causes cardiac macrophage accumulation and fibrosis. We hypothesized that: 1) macrophage accumulation and cardiac fibrosis in SR-uPA+/o mice are dependent on localization of uPA by the uPA receptor (uPAR); 2) activation of plasminogen by uPA and subsequent activation of transforming growth factor-beta1 (TGF-beta1) and matrix metalloproteinase (MMP)-2 and -9 by plasmin are critical pathways through which uPA-expressing macrophages accumulate in the heart and cause fibrosis; and 3) uPA-induced cardiac fibrosis can be attenuated by treatment with verapamil. To test these hypotheses, we bred the SR-uPA+/o transgene into mice deficient in either uPAR or plasminogen and measured cardiac macrophage accumulation and fibrosis. We also measured cardiac TGF-beta1 protein (total and active), Smad2 phosphorylation, and MMP activity after the onset of macrophage accumulation but before the onset of cardiac fibrosis. Finally, we treated mice with verapamil. Our studies revealed that plasminogen is necessary for uPA-induced cardiac fibrosis and macrophage accumulation but uPAR is not. We did not detect plasmin-mediated activation of TGF-beta1, MMP-2, or MMP-9 in hearts of SR-uPA+/o mice. However, verapamil treatment significantly attenuated both cardiac fibrosis and macrophage accumulation.     

Characterization of a recombinant C-type lectin, rCEL-IV, expressed in Escherichia coli cells using a synthetic gene

The body fluid of marine invertebrate Cucumaria echinata (Holothuroidea) contains four Ca2+-dependent galactose-specific lectins. One of these lectins, CEL-IV, is composed of a C-type carbohydrate-recognition domain homotetramer. CEL-IV exhibits higher specificity for alpha-galactosides than for beta-galactosides, while other C. echinata lectins show preferential binding of beta-galactosides. We constructed an artificial synthetic gene for recombinant CEL-IV (rCEL-IV) based on the amino acid sequence previously determined from the purified protein. rCEL-IV was expressed in Escherichia coli cells as inclusion bodies. After the refolding process, most of rCEL-IV spontaneously formed a homotetramer structure having interchain disulfide bonds. The secondary structure of rCEL-IV was similar to that of the native one, as judged by the comparison of the far UV-circular dichroism spectra of rCEL-IV and native CEL-IV (nCEL-IV). Carbohydrate-binding specificity of rCEL-IV was confirmed to be similar to that of nCEL-IV from the results of the binding-inhibition assay using liposomes composed of rabbit erythrocyte lipids. Crystals of rCEL-IV were obtained in a few days by the sitting drop vapor diffusion method. These results indicate that rCEL-IV achieved essentially correct three-dimensional structure, including the carbohydrate-binding sites, and it would be very useful for further study on the carbohydrate-recognition mechanism by mutational and X-ray crystallographic analyses.     

Effect of assumed stiffness and mass density on the impact response of the human chest using a three-dimensional FE model of the human body

The mass density, Young's modulus (E), tangent modulus (Et), and yield stress (sigma y) of the human ribs, sternum, internal organs, and muscles play important roles when determining impact responses of the chest associated with pendulum impact. A series of parametric studies was conducted using a commercially available three-dimensional finite element (FE) model, Total HUman Model for Safety (THUMS) of the whole human body, to determine the effect of changing these material properties on the predicted impact force, chest deflection, and the number of rib fractures and fractured ribs. Results from this parametric study indicate that the initial chest apparent stiffness was mainly influenced by the stiffness and mass density of the superficial muscles covering the torso. The number of rib fractures and fractured ribs was primarily determined by the stiffness of the ribcage. Similarly, the stiffness of the ribcage and internal organs contributed to the maximum chest deflection in frontal impact, while the maximum chest deflection for lateral impact was mainly affected by the stiffness of the ribcage. Additionally, the total mass of the whole chest had a moderately effect on the number of rib fractures.     

Differential analysis of D-beta-Asp-containing proteins found in normal and infrared irradiated rabbit lens

Although proteins are generally composed of l-alpha-amino acids, d-beta-aspartic acid (Asp)-containing proteins have been reported in various elderly tissues. Our previous study detected several d-beta-Asp-containing proteins in a rabbit lens derived from epithelial cell line by Western blot analysis of a 2D-gel using a polyclonal antibody that is highly specific for d-beta-Asp-containing proteins. The identity of each spot was subsequently determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Ms-Fit online database searching algorithm. In this study, we discovered novel d-beta-Asp-containing proteins from rabbit lens. The results indicate that beta-crystallin A3, beta-crystallin A4, beta-crystallin B1, beta-crystallin B2, beta-crystallin B3, gamma-crystallin C, gamma-crystallin D, and lambda-crystallin in rabbit lens contain d-beta-Asp residues. Furthermore, the occurrence of d-beta-Asp residues increases with infrared ray (IR) irradiation. Additionally, some d-beta-Asp-containing proteins only appear after IR irradiation. One such protein is the alpha-enolase, which shows homology to tau-crystallin.     

Key adhesin gene in community-acquired methicillin-resistant Staphylococcus aureus

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) possessing the Panton-Valentine leukocidin (PVL) gene (luk(PV)) is associated with skin and soft tissue infections, osteomyelitis, and necrotizing pneumonia. There are geographically two types of CA-MRSA: one (sequence type ST30) that is worldwide (pandemic) and the other (sequence types, e.g., ST1, ST8 or ST80) that is continent-specific. The pandemic type, but not continent-specific type, possessed the bone sialoprotein-adhesin gene (bbp), which was associated with osteomyelitis. No recent hospital-acquired MRSA had the bbp gene, while past PVL-positive nosocomial outbreak-derived strains did possess it. The collagen-adhesin gene (cna) was associated with pandemic CA-MRSA, though with positive cases even in continent-specific CA-MRSA and PVL-negative Japanese region-specific CA-MRSA. Thus, the pandemic type is characterized by the combination of luk(PV) and bbp (and cna) genes. A specific real-time PCR assay for the bbp gene was developed, and dual assay for bbp and luk(PV) in one test tube became possible.     

Dicer and positive charge of proteins decrease the stability of RNA containing the AU-rich element of GM-CSF

AU-rich elements (AREs) in the 3'-untranslated region of mRNAs promote rapid decay of the mRNAs for certain cytokines, including that encoding granulocyte-macrophage colony-stimulating factor (GM-CSF). We show that an RNA molecule based on the ARE of GM-CSF mRNA is cleaved between U and A residues in the presence of bovine serum albumin of which cleavage effect is attenuated by acetylation. Furthermore, the expression of RNA molecule containing the ARE of GM-CSF mRNA in human cell lines was increased by inhibition of histone deacetylase activity and attenuation of Dicer expression. These findings suggest that degradation of mRNAs containing an ARE might be regulated by positive charge of polypeptides and Dicer.     

Climatic and intertrophic effects detected in 10-year population dynamics of biological control of the arrowhead scale by two parasitoids in southwestern Japan

Relative effects of weather and three-trophic interactions were studied for a classical biological control system consisting of the arrowhead scale Unaspis yanonensis, known formerly as a serious pest of the Satsuma mandarin orange Citrus unshiu, and its two introduced parasitoids, Coccobius fulvus and Aphytis yanonensis. Yearly population responses of the three insect species on a per-tree basis for up to 10 years at two orange groves were analyzed by general linear models, with a backward stepwise procedure, to select among abiotic (summer/winter temperature and rainfall) and biotic (densities of the three insect species and orange bearing in the previous years) independent variables. Temperature positively affected the arrowhead scale and the two parasitoids. A negative correlation of rainfall was detected for all three insect species. Mandarin fruiting showed negative delayed density dependence, thereby supporting the observed alternate bearing phenomenon in mandarins, presumably due to physiological imbalance triggered by climatic factors. The arrowhead scale was negatively correlated with fruit production in the preceding years, possibly due to reduced resistance in subsequent years of mast fruiting. We found a negative correlation of the arrowhead scale with Coccobius only in a single grove and none with Aphytis. Thus, it appears that bottom-up forces may be more important than top-down control by the parasitoids in the post-transient phase of this system.     

Dispersal of Camellia japonica seeds by Apodemus speciosus revealed by maternity analysis of plants and behavioral observation of animal vectors

Seed dispersal determines a plant’s reproductive success, range expansion, and population genetic structures. Camellia japonica, a common evergreen tree in Japan, has been the subject of recent genetic studies of population structure, but its mode of seed dispersal has been assumed, without detailed study, to be barochory. The morphological and physiological features of C. japonica seeds, which are large and nutritious, suggest zoochorous dispersal, however. We compared actual distances between mother trees and seedlings with distances attributable to gravity dispersion only, to test the zoochory hypothesis of C. japonica. The animals that transport the seeds for caching were identified experimentally. We also examined the extent to which seed dispersal is affected by the behavior of animal vectors. Seed dispersal by Apodemus speciosus was confirmed by taking photographs of animals that were consuming seeds experimentally deposited on the ground. Camellia seeds hoarded by the rodents under the litter or soil were protected from drying. On the basis of microsatellite analysis of maternal tissue from the seed coat, the mother trees of 28 seedlings were identified. Maternity analysis revealed the average seed-dispersal distance from mother trees was 5.8 m±6.0 SD, a distance greater than initial dispersal by gravity alone. These results indicate that C. japonica is a zoochorous species dispersed by A. speciosus. Fifty percent of the seed dispersal occurred from mature evergreen forests to dwarf bamboo thickets. This directional seed dispersal would contribute to range expansion of C. japonica. Home range sizes of A. speciosus were 0.85 ha at most and covered with different types of vegetation, from evergreen forests to grassland. This low specificity of their microhabitat use might enhance seed dispersal to different types of vegetation.All animal experiments complied with Japanese laws.