Enhanced immunodetection of cell-free synthesized erythropoietin under denaturing conditions

A monoclonal antibody produced by hydridoma cell line, ATCC HB8209, was used to detect and purify erythropoietin synthesized in a cell-free system. The antibody was raised against the N-terminal 20 residues of erythropoietin. It retained anti-erythropoietin activity in 6 M urea in which most of the cell-free synthesized erythropoietin became soluble and gave an enhanced activity of the antibody.     

Glymphatic distribution of CSF-derived apoE into brain is isoform specific and suppressed during sleep deprivation

Background

Apolipoprotein E (apoE) is a major carrier of cholesterol and essential for synaptic plasticity. In brain, it’s expressed by many cells but highly expressed by the choroid plexus and the predominant apolipoprotein in cerebrospinal fluid (CSF). The role of apoE in the CSF is unclear. Recently, the glymphatic system was described as a clearance system whereby CSF and ISF (interstitial fluid) is exchanged via the peri-arterial space and convective flow of ISF clearance is mediated by aquaporin 4 (AQP4), a water channel. We reasoned that this system also serves to distribute essential molecules in CSF into brain. The aim was to establish whether apoE in CSF, secreted by the choroid plexus, is distributed into brain, and whether this distribution pattern was altered by sleep deprivation.

Methods

We used fluorescently labeled lipidated apoE isoforms, lenti-apoE3 delivered to the choroid plexus, immunohistochemistry to map apoE brain distribution, immunolabeled cells and proteins in brain, Western blot analysis and ELISA to determine apoE levels and radiolabeled molecules to quantify CSF inflow into brain and brain clearance in mice. Data were statistically analyzed using ANOVA or Student’s t- test.

Results

We show that the glymphatic fluid transporting system contributes to the delivery of choroid plexus/CSF-derived human apoE to neurons. CSF-delivered human apoE entered brain via the perivascular space of penetrating arteries and flows radially around arteries, but not veins, in an isoform specific manner (apoE2?>?apoE3?>?apoE4). Flow of apoE around arteries was facilitated by AQP4, a characteristic feature of the glymphatic system. ApoE3, delivered by lentivirus to the choroid plexus and ependymal layer but not to the parenchymal cells, was present in the CSF, penetrating arteries and neurons. The inflow of CSF, which contains apoE, into brain and its clearance from the interstitium were severely suppressed by sleep deprivation compared to the sleep state.

Conclusions

Thus, choroid plexus/CSF provides an additional source of apoE and the glymphatic fluid transporting system delivers it to brain via the periarterial space. By implication, failure in this essential physiological role of the glymphatic fluid flow and ISF clearance may also contribute to apoE isoform-specific disorders in the long term.

     

Establishment of Transfer Functions of the Pollen-Climatic Factors in Northern China and the Quantitative Climatic Reconstruction at DJ Core

The 2.4 meter-long core was extracted from the Diaojiao lake (41º18′N, 112º21′E) at the foot of the northern part of Daqingshan Mts. Pollen analysis from collections subsampled in the laboratory at 2 cm intervals, revealed plentiful pollen and spores from over 10 arboreal genera, including Pinus, Betula, Picea, Abies, Carpinus, Quercus, Ulmus and more than 20 non-arboreal genera, mainly of Artemisia, Labiatae, Nitraria, Polygonaceae, Ranunculus, Thalictrum, Umbel- liferae, Caryophyllaceae and Cyperaceae. Fern spores, aquatic pollen and algae were also observed in some parts of the core. The transfer functions were established by the stepwise regression analysis using the climatic factors and 13 pollen taxa. The different Fl and F2 value were used as the thresh- old value of F test (i. e. used for selecting and deleting factors). Each regressed equation was obtained from 70 times of calculations with a step-wise increase of 0.1 for Fl and F2 and those having the smallest regression deviation and the largest multiple correlation coefficient were the final four transfer functions. Substituting the pre-factor obtained from the stratigraphic sampled into the regression equations, the estimates of temperature and precipitation in January and in July, and annual mean temperature values could be calculated. Some climatic stages were inferred from total pollen influx and pollen percentage from the core using a transfer function: humid-cool (from 10 000 to 7 800 a BP), arid-cold (9 200 to 7 900 a BP), arid-warm (7 900 to 7 100 a BP), humid-warm (7 100 to 4 400 a BP), arid-warm (4 400 to 3 000 a BP), arid-cold (3 000 to 2 100 a BP). The highest annual mean temperature during Holocene was ca. 4 ℃ higher and the lowest was ca. 2 ℃ lower than the present temperature. Annual precipitation was 250 mm higher and 300 mm lower than the present.     

Biflavonoids are superior to monoflavonoids in inhibiting amyloid-β toxicity and fibrillogenesis via accumulation of nontoxic oligomer-like structures

Polymerization of monomeric amyloid-β peptides (Aβ) into soluble oligomers and insoluble fibrils is one of the major pathways triggering the pathogenesis of Alzheimer's disease (AD). Using small molecules to prevent the polymerization of Aβ peptides can, therefore, be an effective therapeutic strategy for AD. In this study, we investigate the effects of mono- and biflavonoids in Aβ42-induced toxicity and fibrillogenesis and find that the biflavonoid taiwaniaflavone (TF) effectively and specifically inhibits Aβ toxicity and fibrillogenesis. Compared to TF, the monoflavonoid apigenin (AP) is less effective and less specific. Our data show that differential effects of the mono- and biflavonoids in Aβ fibrillogenesis correlate with their varying cytoprotective efficacies. We also find that other biflavonoids, namely, 2',8'-biapigenin, amentoflavone, and sumaflavone, can also effectively inhibit Aβ toxicity and fibrillogenesis, implying that the participation of two monoflavonoids in a single biflavonoid molecule enhances their activity. Biflavonoids, while strongly inhibiting Aβ fibrillogenesis, accumulate nontoxic Aβ oligomeric structures, suggesting that these are off-pathway oligomers. Moreover, TF abrogates the toxicity of preformed Aβ oligomers and fibrils, indicating that TF and other biflavonoids may also reduce the toxicity of toxic Aβ species. Altogether, our data clearly show that biflavonoids, possibly because of the possession of two Aβ binders separated by an appropriate size linker, are likely to be promising therapeutics for suppressing Aβ toxicity.     

A Durable Alternative for Proton‐Exchange Membranes: Sulfonated Poly(Benzoxazole Thioether Sulfone)s

To develop a durable proton‐exchange membrane (PEM) for fuel‐cell applications, a series of sulfonated poly(benzoxazole thioether sulfone)s ( SPTESBOs) are designed and synthesized, with anticipated good dimensional stability (via acid–base cross linking), improved oxidative stability against free radicals (via incorporation of thioether groups), and enhanced inherent stability (via elimination of unstable end groups) of the backbone. The structures and the degree of sulfonation of the copolymers are characterized using Fourier‐transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy (¹H NMR and ¹⁹F NMR). The electrochemical stabilities of the monomers are examined using cyclic voltammetry in a typical three‐electrode cell configuration. The physicochemical properties of the membranes vital to fuel‐cell performance are also carefully evaluated under conditions relevant to fuel‐cell operation, including chemical and thermal stability, proton conductivity, solubility in different solvents, water uptake, and swelling ratio. The new membranes exhibit low dimensional change at 25°C to 90°C and excellent thermal stability up to 250°C. Upon elimination of unstable end groups, the co‐polymers display enhanced chemical resistance and oxidative stability in Fenton's test. Further, the SPTESBO‐HFB‐60 (HFB‐60=hexafluorobenzene, 60 mol% sulfone) membrane displays comparable fuel‐cell performance to that of an NRE 212 membrane at 80°C under fully humidified condition, suggesting that the new membranes have the potential to be more durable but less expensive for fuel‐cell applications.     

Atherosclerosis,Oxidation and Endometriosis

Endometriosis affects younger women of childbearing age. Atherosclerosis is considered as a disease of the old and increases with the ageing process. Both diseases are characterized by the increased presence of activated macrophages and associated increases in growth promoting activity and the production of inflammatory cytokines. In this review, we propose that oxidative stress and the presence of forms of oxidized low-density lipoprotein (LDL) might contribute to both Atherosclerosis and Endometriosis.     

Quantitative Förster resonance energy transfer analysis for kinetic determinations of SUMO-specific protease

Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research, and it is a very powerful tool for elucidating protein interactions in either dynamic or steady state. SUMOylation (the process of SUMO [small ubiquitin-like modifier] conjugation to substrates) is an important posttranslational protein modification with critical roles in multiple biological processes. Conjugating SUMO to substrates requires an enzymatic cascade. Sentrin/SUMO-specific proteases (SENPs) act as an endopeptidase to process the pre-SUMO or as an isopeptidase to deconjugate SUMO from its substrate. To fully understand the roles of SENPs in the SUMOylation cycle, it is critical to understand their kinetics. Here, we report a novel development of a quantitative FRET-based protease assay for SENP1 kinetic parameter determination. The assay is based on the quantitative analysis of the FRET signal from the total fluorescent signal at acceptor emission wavelength, which consists of three components: donor (CyPet–SUMO1) emission, acceptor (YPet) emission, and FRET signal during the digestion process. Subsequently, we developed novel theoretical and experimental procedures to determine the kinetic parameters, k_cat, K_M, and catalytic efficiency (k_cat/K_M) of catalytic domain SENP1 toward pre-SUMO1. Importantly, the general principles of this quantitative FRET-based protease kinetic determination can be applied to other proteases.     

BcMF11, a novel non-coding RNA gene from Brassica campestris, is required for pollen development and male fertility

Key message

BcMF11 as a non-coding RNA gene has an essential role in pollen development, and might be useful for regulating the pollen fertility of crops by antisense RNA technology.

Abstract

We previously identified a 828-bp full-length cDNA of BcMF11, a novel pollen-specific non-coding mRNA-like gene from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino). However, little information is known about the function of BcMF11 in pollen development. To investigate its exact biological roles in pollen development, the BcMF11 cDNA was antisense inhibited in transgenic Chinese cabbage under the control of a tapetum-specific promoter BcA9 and a constitutive promoter CaMV 35S. Antisense RNA transgenic plants displayed decreasing expression of BcMF11 and showed distinct morphological defects. Pollen germination test in vitro and in vivo of the transgenic plants suggested that inhibition of BcMF11 decreased pollen germination efficiency and delayed the pollen tubes’ extension in the style. Under scanning electron microscopy, many shrunken and collapsed pollen grains were detected in the antisense BcMF11 transgenic Chinese cabbage. Further cytological observation revealed abnormal pollen development process in transgenic plants, including delayed degradation of tapetum, asynchronous separation of microspore, and aborted development of pollen grain. These results suggest that BcMF11, as a non-coding RNA, plays an essential role in pollen development and male fertility.