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71.
Thiyagaragan?M.?Achariyar Baoman?Li Weiguo?Peng Philip?B.?Verghese Yang?Shi Evan?McConnell Abdellatif?Benraiss Tristan?Kasper Wei?Song Takahiro?Takano David?M.?Holtzman Maiken?Nedergaard Rashid?DeaneEmail author 《Molecular neurodegeneration》2016,11(1):74
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.72.
MOTIVATION: In contrast with conventional PCR using a pair of specific primers, some applications utilize a single unique primer in combination with a common primer, thereby relying solely on the former for specificity. These applications include rapid amplification of cDNA ends (RACE), adaptor-tagged competitive PCR (ATAC-PCR), PCR-mediated genome walking and so forth. Since the primers designed by conventional methods often fail to work in these applications, an improved strategy is required, particularly, for a large-scale analysis. RESULTS: Based on the structure of 'off-target' products in the ATAC-PCR, we reasoned that the practical determinant of the specificity of primers may not be the uniqueness of entire sequence but that of the shortest 3'-end subsequence that exceeds a threshold of duplex stability. We termed such a subsequence as a 'specificity-determining subsequence' (SDSS) and developed a simple algorithm to predict the performance of the primer: the algorithm identifies the SDSS of each primer and examines its uniqueness in the target genome. The primers designed using this algorithm worked much better than those designed using a conventional method in both ATAC-PCR and 5'-RACE experiments. Thus, the algorithm will be generally useful for improving various PCR-based applications. 相似文献
73.
Sakano S Hasegawa Y Murata Y Ito T Genda E Iwata H Ishiguro N Seo H 《Biochemical and biophysical research communications》2002,293(2):680-685
Basic fibroblast growth factor (bFGF) is reported to stimulate repair of fracture and bony defects in in vivo animal studies. However, most studies performed in vitro demonstrate inhibitory effect of bFGF on cartilage and bone differentiation. To understand the discrepancy observed in in vivo and in vitro studies, we evaluated the effect of bFGF on chondro-osteogenesis initiated by bone matrix powder (MP). MP was implanted in the murine hamstring muscles with or without administration of bFGF. Injection of 1 microg of bFGF markedly reduced the size of heterotopic bone induced by MP, as detected by X-ray. Injection of 10 microg of bFGF completely inhibited ossification and only fibrous tissues were observed at the site of MP implantation. The expressions of alkaline phosphatase and osteocalcin mRNAs, markers for bone differentiation, were completely suppressed by 10 microg of bFGF. These results demonstrate the inhibitory effect of bFGF on endochondral ossification in vivo, implicating a precaution for its use in musculo-skeletal disorders. 相似文献
74.
Most delta-endotoxins produced by Bacillus thuringiensis require proteolytic processing in order to become active. The in vitro and in vivo activation processes of Cry39A, a delta-endotoxin that is highly toxic to Anopheles stephensi, were investigated. Cry39A with a molecular mass of 72 kDa was processed in vitro into a 60 kDa fragment by trypsin and gut extract from A. stephensi larvae. N-terminal amino acid sequencing of the 60 kDa fragment revealed that trypsin and the protease(s) in the gut extract cleaved Cry39A between Arg(61) and Gly(62). In contrast, 40 and 25 kDa polypeptides were generated in vivo by intramolecular cleavage of the 60 kDa fragment in A. stephensi larvae. Further, a co-precipitation assay was used to investigate the binding property of the activated Cry39A to A. stephensi BBMV. Cry39A bound to A. stephensi BBMV specifically and did not compete with the Cry4Aa toxin. This indicated that the binding molecule(s) for Cry39A might differ from those for Cry4A. In addition, Cry39A preferentially bound to the Triton X-100-insoluble membrane fraction. 相似文献
75.
Nathan D. Mathewson Orr Ashenberg Itay Tirosh Simon Gritsch Elizabeth M. Perez Sascha Marx Livnat Jerby-Arnon Rony Chanoch-Myers Toshiro Hara Alyssa R. Richman Yoshinaga Ito Jason Pyrdol Mirco Friedrich Kathrin Schumann Michael J. Poitras Prafulla C. Gokhale L. Nicolas Gonzalez Castro Marni E. Shore Kai W. Wucherpfennig 《Cell》2021,184(5):1281-1298.e26
76.
Yasuro Fuse Takahiro Okamoto Kazuhide Hayakawa Hajime Karatani Etsu Yamada 《Limnology》2016,17(3):207-221
Humic substances extracted and purified from bottom sediments of northern Lake Biwa, Japan, in November 2012 and 2013 were characterized using elemental analysis, Fourier-transform infrared spectroscopy, hydrogen-1 nuclear magnetic resonance (1H NMR) analysis, and pyrolysis gas chromatography/mass spectrometry (Py-GC/MS). The infrared (IR) bands in the spectra of humic acids showed the presence of amide linkages and polysaccharides. Results of 1H NMR analysis showed that the humic acids contained approximately twice the number of aliphatic protons as those in the Japanese soil standards used for comparison. Results of the Py-GC/MS analysis, which evaluates pyrolysis temperature dependency of the amount of pyrolysis products, showed that the generation of pyrolysis products in humic acids also differed from that in Japanese soil standards but was similar to that of phytoplankton in Lake Biwa. This analysis method is the first to provide extensive information about the chemical structure of humic substances; conventional Py-GC/MS provides limited information for a single temperature. Data suggest that humic acids in lake sediments are related to chemical characteristics of phytoplankton. Results shed new light on the origins of humic substances in deep-water-lake sediments and provide insights into material recycling in such sediments. 相似文献
77.
Six amylose‐2‐acetyl‐3,6‐bis(phenylcarbamate) (AAPC) samples ranging in weight‐average molar mass Mw from 1.8 × 104 g mol?1 to 1.1 × 106 g mol?1 have been prepared from enzymatically synthesized amylose samples. Static light scattering, small‐angle X‐ray scattering, sedimentation equilibrium, and viscosity measurements were made for the samples in 1,4‐dioxane (DIOX), 2‐ethoxyethanol (2EE), and 2‐butanone (MEK) all at 25°C to determine particle scattering functions, z‐average radii of gyration, intrinsic viscosities, as well as Mw. The data were analyzed in terms of the wormlike cylinder model mainly to yield the helix pitch per residue h and the Kuhn segment length λ?1, which corresponds to twice of the persistence length. The latter parameters (λ?1) in 2EE (11 nm) and MEK (12 nm) are quite smaller than those for amylose tris(phenylcarbamate) (ATPC) in the same solvent (16 nm in 2EE and 18 nm in MEK) whereas those for AAPC (21 nm) and ATPC (22 nm) in DIOX are essentially the same as each other. This indicates that the chain stiffness of AAPC is more strongly influenced by the solvents since the number of intramolecular H‐bonds of AAPC is more changeable than that for ATPC. © 2012 Wiley Periodicals, Inc. Biopolymers 97:1010–1017, 2012. 相似文献
78.
Dani Permana Kosuke Minamihata Tsuneyuki Tatsuke Jae M. Lee Takahiro Kusakabe Masahiro Goto Noriho Kamiya 《Biotechnology journal》2019,14(6)
The polymerization of proteins can create newly active and large bio‐macromolecular assemblies that exhibit unique functionalities depending on the properties of the building block proteins and the protein units in polymers. Herein, the first enzymatic polymerization of horseradish peroxidase (HRP) is reported. Recombinant HRPs fused with a tyrosine‐tag (Y‐tag) through a flexible linker at the N‐ and/or C‐termini are expressed in silkworm, Bombyx mori. Trametes sp. laccase (TL) is used to activate the tyrosine of Y‐tagged HRPs with molecular O2 to form a tyrosyl‐free radical, which initiates the tyrosine coupling reaction between the HRP units. A covalent dityrosine linkage is also formed through a HRP‐catalyzed self‐crosslinking reaction in the presence of H2O2. The addition of H2O2 in the self‐polymerization of Y‐tagged HRPs results in lower activity of the HRP polymers, whereas TL provides site‐selectivity, mild reaction conditions and maintains the activity of the polymeric products. The cocrosslinking of Y‐tagged HRPs and HRP‐protein G (Y‐HRP‐pG) units catalyzed by TL shows a higher signal in enzyme‐linked immunosorbent assay (ELISA) than the genetically pG‐fused HRP, Y‐HRP‐pG, and its polymers. This new enzymatic polymerization of HRP promises to provide highly active and functionalized polymers for biomedical applications and diagnostics probes. 相似文献
79.
Immunodiagnostic and molecular approaches for the detection of taeniid cestode infections 总被引:3,自引:0,他引:3
This article summarizes the most recent advances in techniques and applications for the detection of taeniid cestode-infected persons or animals. In addition, the use of molecular approaches for strain identification and control of parasite transmission is discussed. 相似文献
80.