Sandhoff disease (SD) is a neurodegenerative lysosomal β-hexosaminidase (Hex) deficiency involving excessive accumulation of undegraded substrates, including terminal GlcNAc-oligosaccharides and GM2 ...ganglioside. Microglia-mediated neuroinflammation contributes to the pathogenesis and progression of SD. Our previous study demonstrated that MIP-1α, a putative pathogenic factor for SD, is up-regulated in microglial cells derived from SD model mice (SD-Mg) through activation of Akt and JNK.
In this study, we first demonstrated that prostaglandin E2 (PGE2), which is one of the lipid mediators derived from arachidonic acid and is known to suppress activation of microglia, reduced the aberrant MIP-1α production by SD-Mg to the same level as by WT-Mg. PGE2 also attenuated the activation of Akt and JNK. The inhibition of MIP-1α production and the activation of Akt and JNK occurred through the EP2 and 4/cAMP/PKA signaling pathway in the murine microglia derived from SD model mice.
We propose that PGE2 plays a role as a negative regulator of MIP-1α production in the pathogenesis of SD, and that PGE2-EP2 and 4/cAMP/PKA signaling could be a target pathway for therapy for SD.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Sandhoff disease (SD) is a lysosomal disease caused by a mutation of the
HEXB
gene associated with excessive accumulation of GM2 ganglioside (GM2) in lysosomes and neurological manifestations. ...Production of autoantibodies against the accumulated gangliosides has been reported to be involved in the progressive pathogenesis of GM2 gangliosidosis, although the underlying mechanism has not been fully elucidated. The thymus is the key organ in the acquired immune system including the development of autoantibodies. We showed here that thymic involution and an increase in cell death in the organ occur in SD model mice at a late stage of the pathogenesis. Dramatic increases in the populations of Annexin-V
+
cells and terminal deoxynucletidyl transferase dUTP nick end labeling (TUNEL)
+
cells were observed throughout the thymuses of 15-week old SD mice. Enhanced caspase-3/7 activation, but not that of caspase-1/4, -6 ,-8, or −9, was also demonstrated. Furthermore, the serum level of corticosterone, a potent inducer of apoptosis of thymocytes, was elevated during the same period of apoptosis. Our studies suggested that an increase in endocrine corticosterone may be one of the causes that accelerate the apoptosis of thymocytes leading to thymic involution in GM2 gangliosidosis, and thus can be used as a disease marker for evaluation of the thymic condition and disease progression.
Human β-hexosaminidase A (HexA) is a heterodimeric glycoprotein composed of α- and β-subunits that degrades GM2 gangliosides in lysosomes. GM2 gangliosidosis is a lysosomal storage disease in which ...an inherited deficiency of HexA causes the accumulation of GM2 gangliosides. In order to prepare a large amount of HexA for a treatment based on enzyme replacement therapy (ERT), recombinant HexA was produced in the methylotrophic yeast Ogataea minuta instead of in mammalian cells, which are commonly used to produce recombinant enzymes for ERT. The problem of antigenicity due to differences in N-glycan structures between mammalian and yeast glycoproteins was potentially resolved by using α-1,6-mannosyltransferase-deficient (och1Δ) yeast as the host. Genes encoding the α- and β-subunits of HexA were integrated into the yeast cell, and the heterodimer was expressed together with its isozymes HexS (αα) and HexB (ββ). A total of 57 mg of β-hexosaminidase isozymes, of which 13 mg was HexA (αβ), was produced per liter of medium. HexA was purified with immobilized metal affinity column for the His tag attached to the β-subunit. The purified HexA was treated with α-mannosidase to expose mannose-6-phosphate (M6P) residues on the N-glycans. The specific activities of HexA and M6P-exposed HexA (M6PHexA) for the artificial substrate 4MU-GlcNAc were 1.2 ± 0.1 and 1.7 ± 0.3 mmol/h/mg, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern suggested a C-terminal truncation in the β-subunit of the recombinant protein. M6PHexA was incorporated dose dependently into GM2 gangliosidosis patient-derived fibroblasts via M6P receptors on the cell surface, and degradation of accumulated GM2 ganglioside was observed.
We propose a novel method for solving the quadratic assignment problems. First, we realize the conventional tabu search on a neural network, and modify it to a chaotic version. Our novel method ...includes both effects of chaotic dynamics and tabu search. We compare the performance of the novel chaotic search with the conventional tabu search and an exponential tabu search whose memory effect for tabu (forbidding previous moves) decays exponentially. We show that the exponential tabu search has higher performance than the conventional tabu search, and further that the novel method with a chaotic neural network exhibits the best performance. We also propose a controlling method of the chaotic neural network for realizing easy and robust applications of our method. Then, better performance can be realized without manual parameter setting for various problems.
In cellulo crystallization is a developing technique to provide crystals for protein structure determination, particularly for proteins that are difficult to prepare by in vitro crystallization. This ...method has a key advantage: it requires neither a protein purification step nor a crystallization step. However, there is still no systematic strategy for improving the technique of in cellulo crystallization because the process occurs spontaneously. Here we report a protocol to produce and extract in cellulo crystals of human lysosomal neuraminidase-1 (NEU1) in human cultured cells. Overexpression of NEU1 protein by the retransfection of cells pretransfected with neu1-overexpressing plasmid improved the efficiency of NEU1 crystallization. Microscopic analysis revealed that NEU1 proteins were not crystallized in the lysosome but in the endoplasmic reticulum (ER). Screening of the buffer conditions used to extract crystals from cells further improved the crystal yield. The optimal pH was 7.0, which corresponds to the pH in the ER. Use of a high-yield flask with a large surface area also yielded more crystals. These optimizations enabled us to execute a serial femtosecond crystallography experiment with a sufficient number of crystals to generate a complete data set. Optimization of the in cellulo crystallization method was thus shown to be possible.
Sandhoff disease (SD) is a lysosomal β-hexosaminidase (Hex) deficiency involving excessive accumulation of undegraded substrates, including GM2 ganglioside, and progressive neurodegeneration. ...Macrophage inflammatory protein-1α (MIP-1α) is a crucial factor for microglia-mediated neuroinflammation in the onset or progression of SD. However, the transmitter-mediated production of MIP-1α in SD is still poorly understood.
Extracellular nucleotides, including uridine diphosphate (UDP), leaked by either injured or damaged neuronal cells activate microglia to trigger chemotaxis, phagocytosis, macropinocytosis, and cytokine production.
In this study, we demonstrated that UDP enhanced the production of MIP-1α by microglia derived from SD mice (SD-Mg), but not that from wild-type mice (WT-Mg). The UDP-induced MIP-1α production was mediated by the activation of P2Y6 receptor, ERK, and JNK. We also found the amount of dimeric P2Y6 receptor protein to have increased in SD-Mg in comparison to WT-Mg. In addition, we demonstrated that the disruption of lipid rafts enhanced the effect of UDP on MIP-1α production and the disordered maintenance of the lipid rafts in SD-Mg. Thus, the accumulation of undegraded substrates might cause the enhanced effect of UDP in SD-Mg through the increased expression of the dimeric P2Y6 receptors and the disordered maintenance of the lipid rafts. These findings provide new insights into the pathogenic mechanism and therapeutic strategies for SD.
Phytochemical study on a non-medicinal part of a plant material for herbal medicine, the roots of Schisandra chinensis, was conducted to isolate five new triterpenes, schinensins A–D (1–4) and ...3-O-methylchangnanic acid (5), together with 21 known compounds including 10 triterpenes, one sterol, two sesquiterpenes, seven lignans, and one flavonoid. The structures of new triterpenes (1–5) were assigned on the basis of spectroscopic analyses aided with TDDFT ECD calculations. Schinensin A (1) was a dinortriterpene possessing 28-norschiartane skeleton, while schinensins B–D (2–4) were assigned as 3,4:9,10-disecocycloartane, 3,4-secocycloartane, and cycloartane triterpenes, respectively. In an evaluation of antiproliferative activities against human cancer cell lines, some triterpenes exhibited significant activities against human breast carcinoma MCF-7 cells as compared to the other cell lines (A549, HeLa, and RPMI8226).
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•The roots of Schisandra chinensis are a non-medicinal part.•Phytochemical investigation of the roots gave 26 compounds including 15 triterpenes.•Schinensin A was assigned as a new 28-norschiartane type dinortriterpene.•Some triterpene exhibited significant antiproliferative activity against MCF-7 cells.•A diversity of specialized metabolites of Shisandra chinensis roots was demonstrated.
Formohyperins A–F, previously undescribed meroterpenes, and grandone, a prenylated benzoylphloroglucinol being considered to be one of their biogenetic precursors, were isolated from the flowers of a ...Hypericaceous plant, Hypericum formosanum Maxim. Detailed spectroscopic analyses showed that formohyperins A–D were meroterpenes with an enolized 3-phenylpropane-1,3-dione moiety. Formohyperins E and F were elucidated as meroterpenes having a 4-benzoyl-5-hydroxycyclopent-4-ene-1,3-dione moiety. Formohyperins A–C and E were optically active, and their absolute configurations were deduced by comparison of the experimental and TDDFT calculated ECD spectra. In contrast, formohyperin D was concluded to be a racemate. Formohyperins A–F and grandone were found to show inhibitory activities against LPS-stimulated IL-1β production from murine microglial cells with EC50 values of 13.2, 6.6, 8.5, 24.3, 4.1, 10.9, and 3.0 μM, respectively.
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•Formohyperins A–F were isolated from the flowers of Hypericum formosanum Maxim.•Their structures were elucidated by spectroscopic analyses and ECD calculations.•Formohyperins A–F are meroterpenes biogenetically related to prenylated benzoylphloroglucinols.•Formohyperins A–F showed inhibitory activity against IL-1β release from LPS-stimulated murine microglial cells.
Gastric adenocarcinomas comprise one of the common types of cancers in Asian countries including Japan. Comprehensive protein profiling of paired surgical specimens of primary gastric adenocarcinomas ...and nontumor mucosae derived from Japanese patients was carried out by means of two-dimensional gel electrophoresis (2D-EP) and liquid chromatography-electrospray ionic tandem mass spectrometry (LC-ESI-MS) to establish gastric cancer-specific proteins as putative clinical biomarkers and molecular targets for chemotherapy.
Relatively common alterations in protein expression were revealed in the tumor tissues. Increases in manganese dismutase and nonhistone chromosomal protein HMG-1 (HMG-1) were observed, while decreases in carbonic anhydrases I and II, glutatione-S-transferase and foveolin precursor (gastrokine-1) (FOV), an 18-kDa stomach-specific protein with putative tumor suppressor activity, were detected. RT-PCR analysis also revealed significant down-regulation of FOV mRNA expression in tumor tissues.
A possible pathological role for down-regulation of FOV in gastric carcinogenesis was demonstrated. Evaluation of the specific decreases in gene and protein expression of FOV in patients may be utilized as clinical biomarkers for effective diagnosis and assessment of gastric cancer.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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