A new simple preparation method for NaK-ATPase-rich membrane fragments

A method for the isolation of highly active membrane bound NaK-ATPase without detergents in quantity from the electric organ of the electric eel (Electrophorus electricus) is described. This method consists of the homogenation of electric organ with an isotonic solution containing sucrose, histidine, EDTA, and arginine, and of the separation of the higher active membrane fraction from the microsomal fraction by density gradient centrifugation. The enzyme has a specific activity of about 20 μmol Pi/min/mg at 37°C, and 13 μmol Pi/min/mg at 30°C. Although it is not as pure as the detergent-treated enzyme preparation based on the level of phosphorylated protein, ouabain binding, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, its enzyme activity is comparable to that of the purified enzymes. This preparation is very stable and is able to change its medium by Sephadex chromatography without any loss of enzyme activity and protein content. This preparation is also expected to keep the original characteristics as well as the enzyme in the tissue.     

DNA strand scission by neocarzinostatin: molecular recognition process responsible for site-specificity

A series of hexanucleotides possessing A-T, G-C, inosine (I)-C and 2-aminoadenine (ANH2)-T base pairs at 5'-side of the target thymine were prepared and their selectivity for C-5' and C4' oxidation in the NCS-mediated degradation was investigated. Quantitative product analysis indicated that preferential C5' oxidation of deoxyribose moiety of the target T occurs at -5'-AT- and 5'-IT- sites, whereas C5' and C4' oxidation occurs competitively at T of -5'-GT- and -5'-ANH2T- sites. Based on the experimental results, an intercalation model that permits competitive hydrogen abstraction from C5' and C4' of deoxyribose moiety has been proposed.     

A Disintegrin and Metalloprotease 10 (ADAM10) Is Indispensable for Maintenance of the Muscle Satellite Cell Pool

Satellite cells (SCs) are muscle-specific stem cells that are essential for the regeneration of damaged muscles. Although SCs have a robust capacity to regenerate myofibers, the number of SCs decreases with aging, leading to insufficient recovery after muscle injury. We herein show that ADAM10 (a disintegrin and metalloprotease 10), a membrane-bound proteolytic enzyme with a critical role in Notch processing (S2 cleavage), is essential for the maintenance of SC quiescence. We generated mutant mice in which ADAM10 in SCs can be conditionally abrogated by tamoxifen injection. Tamoxifen-treated mutant mice did not show any apparent defects and grew normally under unchallenged conditions. However, these mice showed a nearly complete loss of muscle regeneration after chemically induced muscle injury. In situ hybridization and flow cytometric analyses revealed that the mutant mice had significantly less SCs compared with wild type controls. Of note, we found that inactivation of ADAM10 in SCs severely compromised Notch signaling and led to dysregulated myogenic differentiation, ultimately resulting in deprivation of the SC pool in vivo. Taken together, the present findings underscore the role of ADAM10 as an indispensable component of Notch signaling in SCs and for maintaining the SC pool.     

Environmental perturbations influence telomere dynamics in long-lived birds in their natural habitat

Telomeres are regarded as markers of biological or cellular ageing because they shorten with the degree of stress exposure. Accordingly, telomere lengths should show different rates of change when animals are faced with different intensities of environmental challenges. However, a relationship between telomere length and the environment has not yet been tested within a natural setting. Here, we report longitudinal telomere dynamics in free-living, black-tailed gulls (Larus crassirostris) through the recapture of birds of a known age over 2–5 consecutive years. The rate of change in telomere lengths differed with respect to year but not sex or age. The years when gulls showed stable telomere lengths or increases in telomere lengths (from 2009 to 2010) and decreases in telomere lengths (from 2010 to 2011) were characterized by El Niño and the Great Japan Earthquake, respectively. Both events are suspected to have had long-lasting effects on food availability and/or weather conditions. Thus, our findings that telomere dynamics in long-lived birds are influenced by dramatic changes in environmental conditions highlight the importance of environmental fluctuations in affecting stress and lifespan.     

Assessment of reproductive potential in multiple-spawning fish with indeterminate fecundity: a case study of yellow sea bream Dentex hypselosomus in the East China Sea

This study examined the spawning season, spawning frequency and batch fecundity of yellow sea bream Dentex hypselosomus in the East China Sea to reassess the previously reported reproductive characteristics of the species. Time-course sampling showed that this species had a diurnal ovarian maturation rhythm. Late tertiary yolk-stage oocytes appeared 2 days before spawning, starting the process of germinal vesicle movement and breakdown. On the day of spawning, ovulation and subsequent spawning occurred in the early morning (0400–0800 hours). Postovulatory follicles disappeared from the ovaries within c. 24 h of ovulation. Seasonal changes in the ovarian conditions indicated that this species spawned more or less throughout the year, with the peak ranging from spring to autumn. The compositions of the developing oocytes and degenerating postovulatory follicles in the ovaries suggested that most females spawned repeatedly over 2 to 3 consecutive days during the peak of the spawning season. Somatic body condition did not have a significant effect on batch fecundity, but there was a significant relationship between batch fecundity and fork length according to spawning status. Females spawning on consecutive days were more fecund than those spawning every other day. The findings show that this species has much greater reproductive potential than previously estimated.     

Early Staphylococcal Biofilm Formation on Solid Orthopaedic Implant Materials: In Vitro Study

Biofilms forming on the surface of biomaterials can cause intractable implant-related infections. Bacterial adherence and early biofilm formation are influenced by the type of biomaterial used and the physical characteristics of implant surface. In this in vitro research, we evaluated the ability of Staphylococcus epidermidis, the main pathogen in implant-related infections, to form biofilms on the surface of the solid orthopaedic biomaterials, oxidized zirconium-niobium alloy, cobalt-chromium-molybdenum alloy (Co-Cr-Mo), titanium alloy (Ti-6Al-4V), commercially pure titanium (cp-Ti) and stainless steel. A bacterial suspension of Staphylococcus epidermidis strain RP62A (ATCC35984) was added to the surface of specimens and incubated. The stained biofilms were imaged with a digital optical microscope and the biofilm coverage rate (BCR) was calculated. The total amount of biofilm was determined with the crystal violet assay and the number of viable cells in the biofilm was counted using the plate count method. The BCR of all the biomaterials rose in proportion to culture duration. After culturing for 2–4 hours, the BCR was similar for all materials. However, after culturing for 6 hours, the BCR for Co-Cr-Mo alloy was significantly lower than for Ti-6Al-4V, cp-Ti and stainless steel (P<0.05). The absorbance value determined in the crystal violet assay and the number of viable cells on Co-Cr-Mo were not significantly lower than for the other materials (P>0.05). These results suggest that surface properties, such as hydrophobicity or the low surface free energy of Co-Cr-Mo, may have some influence in inhibiting or delaying the two-dimensional expansion of biofilm on surfaces with a similar degree of smoothness.     

Role of long-chain acyl-coenzyme A synthetases in the regulation of arachidonic acid metabolism in interleukin 1β-stimulated rat fibroblasts

Acyl coenzyme A synthetase long-chain family members (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their acyl-CoAs and play an important role in fatty acid metabolism. Here we show the role of ACSL isozymes in interleukin (IL)-1β-induced arachidonic acid (AA) metabolism in rat fibroblastic 3Y1 cells. Treatment of 3Y1 cells with triacsin C, an ACSL inhibitor, markedly enhanced the IL-1β-induced prostaglandin (PG) biosynthesis. Small interfering RNA-mediated knockdown of endogenous Acsl4 expression increased significantly the release of AA metabolites, including PGE₂, PGD₂, and PGF_2α, compared with replicated control cells, whereas knockdown of Acsl1 expression reduced the IL-1β-induced release of AA metabolites. Experiments with double knockdown of Acsl4 and intracellular phospholipase A₂ (PLA₂) isozymes revealed that cytosolic PLA₂α, but not calcium-independent PLA₂s, is involved in the Acsl4 knockdown-enhanced PG biosynthesis. Electrospray ionization mass spectrometry of cellular phospholipids bearing AA showed that the levels of some, if not all, phosphatidylcholine (PC) and phosphatidylinositol species in Acsl4 knockdown cells were decreased after IL-1β stimulation, while those in control cells were not so obviously decreased. In Acsl1 knockdown cells, the levels of some AA-bearing PC species were reduced even in the unstimulated condition. Collectively, these results suggest that Acsl isozymes play distinct roles in the control of AA remodeling in rat fibroblasts: Acsl4 acts as the first step of enzyme for AA remodeling following IL-1β stimulation, and Acsl1 is involved in the maintenance of some AA-containing PC species.