Isolation and characterization of 21 novel polymorphic microsatellite loci in the Chinese soft-shelled turtle Pelodiscus sinensis

Twenty-one novel polymorphic microsatellite loci were isolated from the Chinese soft-shelled turtle, Pelodiscus sinensis (Trionychidae). This is a commonly consumed Asian species, especially in China, where it is reared in large numbers on farms. We screened 34 unrelated individuals from Wuhu of Anhui province in China and detected high levels of polymorphism for all 21 loci, with the number of alleles/locus ranging from 5 to 23 (mean 15.67). The expected and observed heterozygosities ranged from 0.421 to 0.946 and from 0.324 to 0.941, respectively. Hence, these microsatellites could facilitate studies on genetic diversity and population structure and marker-assisted breeding of this vulnerable species.     

Body adiposity in later life and the incidence of dementia: the health in men study

Objective

To determine if adiposity in later life increases dementia hazard.

Methods

Cohort study of 12,047 men aged 65–84 years living in Perth, Australia. Adiposity exposures were baseline body mass index (BMI), waist circumference (WC) and waist-to-hip ratio (WHR). We used the Western Australian Data Linkage System (WADLS) to establish the presence of new cases of dementia between 1996 and 2009 according to the International Classification of Diseases (ICD). Crude and adjusted hazard ratio (HR, 95% confidence interval, 95%CI) of dementia for each adiposity marker was calculated using Cox regression models. Other measured factors included age, marital status, education, alcohol use, smoking, diet, physical activity, and prevalent hypertension, diabetes, dyslipidaemia and cardiovascular disease.

Results

Compared with men with BMI<25, participants with BMI between 25–30 had lower adjusted HR of dementia (HR = 0.82, 95% CI = 0.70–0.95). The HR of dementia for men with BMI≥30 was comparable to men with BMI<25 (HR = 0.82, 95%CI = 0.67–1.01). Waist circumference showed no obvious association with dementia hazard. Men with WHR≥0.9 had lower adjusted HR of dementia than men with WHR <0.9 (HR = 0.82, 95%CI = 0.69–0.98). We found a “J” shape association between measures of obesity and the hazard of dementia, with the nadir of risk being in the overweight range of BMI and about 1 for WHR.

Conclusions

Higher adiposity is not associated with incident dementia in this Australian cohort of older men. Overweight men and those with WHR≥0.9 have lower hazard of dementia than men with normal weight and with WHR<0.9.     

Cell-type-specific regulation of raft-associated Akt signaling

20S-protopanaxadiol (aPPD) is a metabolite of ginseng saponins, which is reported to be pro-apoptotic in some cells but anti-apoptotic in neuronal cells by regulating Akt signaling. Owing to its cholesterol-like structure, we hypothesized that aPPD may regulate Akt signaling by interacting with lipid rafts. Here, we compared Akt signaling in glioblastoma U87MG and neuroblastoma Neuro-2a cells treated with aPPD. aPPD did not change Akt activity in the total plasma membranes of each cell type, but drastically altered the activity of raft-associated Akt. Strikingly, Akt activity was decreased in the rafts of U87MG cells but increased in N2a cells by aPPD through regulating raft-associated dephosphorylation. The bidirectional regulation of raft-associated Akt signaling by aPPD enhanced the chemotoxicity of Paclitaxel or Vinblastine in U87MG cells but attenuated the excitotoxicity of N-methyl--aspartate in N2a cells. Our results demonstrated that the activity of raft-associated but not total membrane Akt determines its cellular functions. Lipid rafts differ in different types of cells, which allows for the possibility of cell-type-specific targeting for which aPPD might prove to be a useful agent.     

Isolation and biochemical characterization of two lipases from a metagenomic library of China Holstein cow rumen

Two novel lipase genes RlipE1 and RlipE2 which encoded 361- and 265-amino acid peptides, respectively, were recovered from a metagenomic library of the rumen microbiota of Chinese Holstein cows. A BLAST search revealed a high similarity (90%) between RlipE2 and a carboxylesterase from Thermosinus carboxydivorans Nor1, while there was a low similarity (below 50%) between RlipE1 and other lipases. Phylogenetic analysis indicated that RlipE2 clustered with the lipolytic enzymes from family V while RlipE1 clustered with six other putative bacterial lipases which might constitute a new subfamily. The recombinant lipases were thermally unstable and retained 60% activity over a pH range of 6.5-8.5. Substrate specificity assay indicated that both enzymes had higher hydrolytic activity toward laurate (C₁₂), palmitate (C₁₆) and stearate (C₁₈). The novel phylogenetic affiliation and high specificity of both enzymes for long-chain fatty acid make them interesting targets for manipulation of rumen lipid metabolism.     

Uniform Action Potential Repolarization within the Sarcolemma of In Situ Ventricular Cardiomyocytes

Previous studies have speculated, based on indirect evidence, that the action potential at the transverse (t)-tubules is longer than at the surface membrane in mammalian ventricular cardiomyocytes. To date, no technique has enabled recording of electrical activity selectively at the t-tubules to directly examine this hypothesis. We used confocal line-scan imaging in conjunction with the fast response voltage-sensitive dyes ANNINE-6 and ANNINE-6plus to resolve action potential-related changes in fractional dye fluorescence (ΔF/F) at the t-tubule and surface membranes of in situ mouse ventricular cardiomyocytes. Peak ΔF/F during action potential phase 0 depolarization averaged −21% for both dyes. The shape and time course of optical action potentials measured with the water-soluble ANNINE-6plus were indistinguishable from those of action potentials recorded with intracellular microelectrodes in the absence of the dye. In contrast, optical action potentials measured with the water-insoluble ANNINE-6 were significantly prolonged compared to the electrical recordings obtained from dye-free hearts, suggesting electrophysiological effects of ANNINE-6 and/or its solvents. With either dye, the kinetics of action potential-dependent changes in ΔF/F during repolarization were found to be similar at the t-tubular and surface membranes. This study provides what to our knowledge are the first direct measurements of t-tubule electrical activity in ventricular cardiomyocytes, which support the concept that action potential duration is uniform throughout the sarcolemma of individual cells.     

Ezrin Induces Long-Range Interdomain Allostery in the Scaffolding Protein NHERF1

Scaffolding proteins are molecular switches that control diverse signaling events. The scaffolding protein Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) assembles macromolecular signaling complexes and regulates the macromolecular assembly, localization, and intracellular trafficking of a number of membrane ion transport proteins, receptors, and adhesion/antiadhesion proteins. NHERF1 begins with two modular protein-protein interaction domains—PDZ1 and PDZ2—and ends with a C-terminal (CT) domain. This CT domain binds to ezrin, which, in turn, interacts with cytosekeletal actin. Remarkably, ezrin binding to NHERF1 increases the binding capabilities of both PDZ domains. Here, we use deuterium labeling and contrast variation neutron-scattering experiments to determine the conformational changes in NHERF1 when it forms a complex with ezrin. Upon binding to ezrin, NHERF1 undergoes significant conformational changes in the region linking PDZ2 and its CT ezrin-binding domain, as well as in the region linking PDZ1 and PDZ2, involving very long range interactions over 120 Å. The results provide a structural explanation, at mesoscopic scales, of the allosteric control of NHERF1 by ezrin as it assembles protein complexes. Because of the essential roles of NHERF1 and ezrin in intracellular trafficking in epithelial cells, we hypothesize that this long-range allosteric regulation of NHERF1 by ezrin enables the membrane-cytoskeleton to assemble protein complexes that control cross-talk and regulate the strength and duration of signaling.     

Effects of nitrogen mineralization on paddy rice yield under low nitrogen input conditions in irrigated rice-based multiple cropping with intensive cropping of vegetables in southwest China

The farm household responsibility system (FHRS) was adopted in Chinese rural areas during the economic reform in the early 1980s. Since then, many farm households have increased cropping intensity by using large quantities of nitrogen (N) fertilizers in their responsible fields to increase agricultural income. However, intensive cropping systems with low N input are still common in remote places of the southwestern region of China. Maintenance and improvement of soil quality in intensive cropping systems is critical for sustaining agricultural productivity and environmental quality for future generations. The effects of intensive cropping of vegetables on paddy rice (Oryza sativa L.) yield using small quantities of N fertilizers through N mineralization of paddy soil in irrigated rice-based multiple cropping systems were studied in 15 paddy fields in Sichuan Province, China for 3 years. Intensification of vegetable cropping with negative N balance and removal of vegetable crop residues has greatly decreased total N (TN) contents in paddy soil leading to low levels of effective cumulated soil temperature and thickness of plow layer. As a result, the N mineralization in paddy field during paddy rice growing period was decreased. In addition to the low levels of chemical fertilizer N input and residual mineral N input, the lower level of N mineralization in paddy fields and low N recovery efficiency decreased the amount of N accumulated in aboveground biomass of paddy rice at maturity, resulting in limited rice yields. The amount of mineralized N only correlated with TN (partial correlation analysis). Therefore, in paddy fields with very low N input, the N mineralization in paddy soil during the paddy rice-growing period was the major limiting factor affecting the total yield increases. In addition, a decline in soil fertility can be determined using TN as an indicator. To improve paddy rice yield and avoid soil deterioration, the development and adoption of rational soil management programs are needed. These include input of plant residues, conscientious soil tillage for the maintenance of soil temperature and thickness of the plow layer, and the split application of fertilizer for the improvement of N recovery efficiency.     

ATR and H2AX Cooperate in Maintaining Genome Stability under Replication Stress

Chromosomal abnormalities are frequently caused by problems encountered during DNA replication. Although the ATR-Chk1 pathway has previously been implicated in preventing the collapse of stalled replication forks into double-strand breaks (DSB), the importance of the response to fork collapse in ATR-deficient cells has not been well characterized. Herein, we demonstrate that, upon stalled replication, ATR deficiency leads to the phosphorylation of H2AX by ATM and DNA-PKcs and to the focal accumulation of Rad51, a marker of homologous recombination and fork restart. Because H2AX has been shown to play a facilitative role in homologous recombination, we hypothesized that H2AX participates in Rad51-mediated suppression of DSBs generated in the absence of ATR. Consistent with this model, increased Rad51 focal accumulation in ATR-deficient cells is largely dependent on H2AX, and dual deficiencies in ATR and H2AX lead to synergistic increases in chromatid breaks and translocations. Importantly, the ATM and DNA-PK phosphorylation site on H2AX (Ser¹³⁹) is required for genome stabilization in the absence of ATR; therefore, phosphorylation of H2AX by ATM and DNA-PKcs plays a pivotal role in suppressing DSBs during DNA synthesis in instances of ATR pathway failure. These results imply that ATR-dependent fork stabilization and H2AX/ATM/DNA-PKcs-dependent restart pathways cooperatively suppress double-strand breaks as a layered response network when replication stalls.Genome maintenance prevents mutations that lead to cancer and age-related diseases. A major challenge in preserving genome integrity occurs in the simple act of DNA replication, in which failures at numerous levels can occur. Besides the mis-incorporation of nucleotides, it is during this phase of the cell cycle that the relatively stable double-stranded nature of DNA is temporarily suspended at the replication fork, a structure that is susceptible to collapse into DSBs.² Replication fork stability is maintained by a variety of mechanisms, including activation of the ATR-dependent checkpoint pathway.The ATR pathway is activated upon the generation and recognition of extended stretches of single-stranded DNA at stalled replication forks (1-4). Genome maintenance functions for ATR and orthologs in yeast were first indicated by increased chromatid breaks in ATR^-/- cultured cells (5) and by the “cut” phenotype observed in Mec1 (Saccharomyces cerevisiae) and Rad3 (Schizosaccharomyces pombe) mutants (6-9). Importantly, subsequent studies in S. cerevisiae demonstrated that mutation of Mec1 or the downstream checkpoint kinase Rad53 led to increased chromosome breaks at regions of the genome that are inherently difficult to replicate (10), and a decreased ability to reinitiate replication fork progression following DNA damage or deoxyribonucleotide depletion (11-14).In vertebrates, similar replication fork stabilizing functions have been demonstrated for ATR and the downstream protein kinase Chk1 (15-20). Several possible mechanisms have been put forward to explain how ATR-Chk1 and orthologous pathways in yeast maintain replication fork stability, including maintenance of replicative polymerases (α, δ, and ε) at forks (17, 21), regulation of branch migrating helicases, such as Blm (22-25), and regulation of homologous recombination, either positively or negatively (26-29).Consistent with the role of the ATR-dependent checkpoint in replication fork stability, common fragile sites, located in late-replicating regions of the genome, are significantly more unstable (5-10-fold) in the absence of ATR or Chk1 (19, 20). Because these sites are favored regions of instability in oncogene-transformed cells and preneoplastic lesions (30, 31), it is possible that the increased tumor incidence observed in ATR haploinsufficient mice (5, 32) may be related to subtle increases in genomic instability. Together, these studies indicate that maintenance of replication fork stability may contribute to tumor suppression.It is important to note that prevention of fork collapse represents an early response to problems occurring during DNA replication. In the event of fork collapse into DSBs, homologous recombination (HR) has also been demonstrated to play a key role in genome stability during S phase by catalyzing recombination between sister chromatids as a means to re-establish replication forks (33). Importantly, a facilitator of homologous recombination, H2AX, has been shown to be phosphorylated under conditions that cause replication fork collapse (18, 34).Phosphorylation of H2AX occurs predominantly upon DSB formation (34-38) and has been reported to require ATM, DNA-PKcs, or ATR, depending on the context (37-42). Although H2AX is not essential for HR, studies have demonstrated that H2AX mutation leads to deficiencies in HR (43, 44), and suppresses events associated with homologous recombination, such as the focal accumulation of Rad51, BRCA1, BRCA2, ubiquitinated-FANCD2, and Ubc13-mediated chromatin ubiquitination (43, 45-51). Therefore, through its contribution to HR, it is possible that H2AX plays an important role in replication fork stability as part of a salvage pathway to reinitiate replication following collapse.If ATR prevents the collapse of stalled replication forks into DSBs, and H2AX facilitates HR-mediated restart, the combined deficiency in ATR and H2AX would be expected to dramatically enhance the accumulation of DSBs upon replication fork stalling. Herein, we utilize both partial and complete elimination of ATR and H2AX to demonstrate that these genes work cooperatively in non-redundant pathways to suppress DSBs during S phase. As discussed, these studies imply that the various components of replication fork protection and regeneration cooperate to maintain replication fork stability. Given the large number of genes involved in each of these processes, it is possible that combined deficiencies in these pathways may be relatively frequent in humans and may synergistically influence the onset of age-related diseases and cancer.     

Receptor-associated Protein Interacts with Amyloid-�� Peptide and Promotes Its Cellular Uptake

Brain amyloid-β (Aβ) peptide accumulation and aggregation are critical events in the pathogenesis of Alzheimer disease. Increasing evidence has demonstrated that LRP1 is involved in Alzheimer disease pathogenesis. The physiological ligands of LRP1, including apoE, play significant roles in the cellular clearance of Aβ. The receptor-associated protein (RAP) is a specialized chaperone for members of the low density lipoprotein receptor family. RAP shares structural and receptor-binding properties with apoE. Here, we show that RAP binds to both Aβ40 and Aβ42 in a concentration-dependent manner and forms complexes with them. Fluorescence-activated cell sorter analysis showed that RAP significantly enhances the cellular internalization of Aβ in different cell types, including brain vascular smooth muscle, neuroblastoma, glioblastoma, and Chinese hamster ovary cells. This effect of RAP was confirmed by fluorescence microscopy and enzyme-linked immunosorbent assay. RAP binds to both LRP1 and heparin; however, the ability of RAP to enhance Aβ cellular uptake was blocked by heparin and heparinase treatment but not by LRP1 deficiency. Furthermore, the effects of RAP were significantly decreased in heparan sulfate proteoglycan-deficient Chinese hamster ovary cells. Our findings reveal that RAP is a novel Aβ-binding protein that promotes cellular internalization of Aβ.