188 research outputs found
Modest phenotypic improvements in ASA-deficient mice with only one UDP-galactose:ceramide-galactosyltransferase gene
BACKGROUND: Arylsulfatase A (ASA)-deficient mice are a model for the lysosomal storage disorder metachromatic leukodystrophy. This lipidosis is characterised by the lysosomal accumulation of the sphingolipid sulfatide. Storage of this lipid is associated with progressive demyelination. We have mated ASA-deficient mice with mice heterozygous for a non-functional allele of UDP-galactose:ceramide-galactosyltransferase (CGT). This deficiency is known to lead to a decreased synthesis of galactosylceramide and sulfatide, which should reduce sulfatide storage and improve pathology in ASA-deficient mice. RESULTS: ASA-/- CGT+/- mice, however, showed no detectable decrease in sulfatide storage. Neuronal degeneration of cells in the spiral ganglion of the inner ear, however, was decreased. Behavioural tests showed small but clear improvements of the phenotype in ASA-/- CGT+/- mice. CONCLUSION: Thus the reduction of galactosylceramide and sulfatide biosynthesis by genetic means overall causes modest improvements of pathology
Π’Π΅Ρ Π½ΠΎΠ»ΠΎΠ³ΠΈΡ ΡΠΈΠ½ΡΠ΅Π·Π° ΠΈ ΠΎΡΠΈΡΡΠΊΠΈ Π³Π»ΠΈΠΊΠΎΠ»ΠΈΠ΄Π°
ΠΠ°Π½Π½Π°Ρ ΡΠ°Π±ΠΎΡΠ° ΠΏΠΎΡΠ²ΡΡΠ΅Π½Π° ΡΠ΅Ρ
Π½ΠΎΠ»ΠΎΠ³ΠΈΠΈ ΠΏΠΎΠ»ΡΡΠ΅Π½ΠΈΡ ΠΈ ΠΎΡΠΈΡΡΠΊΠΈ Π³Π»ΠΈΠΊΠΎΠ»ΠΈΠ΄Π°, ΠΊΠ°ΠΊ ΠΌΠΎΠ½ΠΎΠΌΠ΅ΡΠ° Π΄Π»Ρ Π±ΠΈΠΎΡΠ°Π·Π»Π°Π³Π°Π΅ΠΌΡΡ
ΠΏΠΎΠ»ΠΈΠΌΠ΅ΡΠΎΠ². ΠΡΠ½ΠΎΠ²Π½ΡΠ΅ ΠΏΠΎΡΠ΅ΡΠΈ ΠΏΡΠΎΠ΄ΡΠΊΡΠ° ΠΏΡΠΎΠΈΡΡ
ΠΎΠ΄ΡΡ Π½Π° ΡΡΠ°Π΄ΠΈΠΈ ΠΏΠΎΠ»ΡΡΠ΅Π½ΠΈΡ ΠΈ ΠΎΡΠΈΡΡΠΊΠΈ ΠΌΠΎΠ½ΠΎΠΌΠ΅ΡΠ°. ΠΠΎΡΠ΅ΡΠΈ ΡΠΎΡΡΠ°Π²Π»ΡΡΡ ΠΏΠΎΡΡΠ΄ΠΊΠ° 50-60 %.
Π¦Π΅Π»ΡΡ Π΄Π°Π½Π½ΠΎΠΉ ΡΠ°Π±ΠΎΡΡ ΡΠ²Π»ΡΠ΅ΡΡΡ Π²ΡΠ±ΠΎΡ ΠΎΠΏΡΠΈΠΌΠ°Π»ΡΠ½ΠΎΠ³ΠΎ ΠΏΡΡΠΈ ΠΈ ΠΎΡΠΈΡΡΠΊΠΈ Π³Π»ΠΈΠΊΠΎΠ»ΠΈΠ΄Π°.
Π Π΄Π°Π½Π½ΠΎΠΉ ΡΠ°Π±ΠΎΡΠ΅ ΠΏΡΠΎΠ²Π΅Π΄ΡΠ½ ΠΈ ΠΏΡΠ΅Π΄ΡΡΠ°Π²Π»Π΅Π½ Π²ΡΠ΅ΡΡΠΎΡΠΎΠ½Π½ΠΈΠΉ Π»ΠΈΡΠ΅ΡΠ°ΡΡΡΠ½ΡΠΉ ΠΎΠ±Π·ΠΎΡ ΠΏΠΎ ΠΌΠ΅ΡΠΎΠ΄Π°ΠΌ ΠΏΠΎΠ»ΡΡΠ΅Π½ΠΈΡ Π³Π»ΠΈΠΊΠΎΠ»Π΅Π²ΠΎΠΉ ΠΊΠΈΡΠ»ΠΎΡΡ, Π³Π»ΠΈΠΊΠΎΠ»ΠΈΠ΄Π°, ΠΎΡΠΈΡΡΠΊΠΈ ΠΈ ΠΏΠΎΠ»ΠΈΠΌΠ΅ΡΠΈΠ·Π°ΡΠΈΠΈ Π³Π»ΠΈΠΊΠΎΠ»ΠΈΠ΄Π°. Π‘ΡΠ°Π²Π½ΠΈΠ²Π°Π»ΠΈΡΡ ΡΠ°Π·Π»ΠΈΡΠ½ΡΠ΅ ΠΊΠ°ΡΠ°Π»ΠΈΠ·Π°ΡΠΎΡΡ Π½Π° ΡΡΠ°Π΄ΠΈΡΡ
ΠΏΠΎΠ»ΠΈΠΊΠΎΠ½Π΄Π΅Π½ΡΠ°ΡΠΈΠΈ, Π΄Π΅ΠΏΠΎΠ»ΠΈΠΌΠ΅ΡΠΈΠ·Π°ΡΠΈΠΈ ΠΈ ΠΏΠΎΠ»ΠΈΠΌΠ΅ΡΠΈΠ·Π°ΡΠΈΠΈ Π³Π»ΠΈΠΊΠΎΠ»ΠΈΠ΄Π°.
Π ΡΠ°Π±ΠΎΡΠ΅ ΠΎΠΏΠΈΡΠ°Π½Ρ Ρ
Π°ΡΠ°ΠΊΡΠ΅ΡΠΈΡΡΠΈΠΊΠΈ ΡΡΡΡΡ, ΠΎΠΏΠΈΡΠ°Π½Ρ ΡΠΏΠΎΡΠΎΠ±Ρ ΠΏΠΎΠ»ΡΡΠ΅Π½ΠΈΡ, ΠΎΡΠΈΡΡΠΊΠΈ ΠΈ ΠΏΠΎΠ»ΠΈΠΌΠ΅ΡΠΈΠ·Π°ΡΠΈΠΈ Π³Π»ΠΈΠΊΠΎΠ»ΠΈΠ΄Π°. ΠΠ·Π»ΠΎΠΆΠ΅Π½Ρ ΠΌΠ΅ΡΠΎΠ΄ΠΈΠΊΠΈ Π°Π½Π°Π»ΠΈΠ·Π° Π³Π»ΠΈΠΊΠΎΠ»ΠΈΠ΄Π°.This paper is devoted to the technology of production and purification of glycolide as a monomer for biodegradable polymers. The main product losses occur at the stage of monomer production and purification. Losses are about 50-60%.
The purpose of this work is to choose the optimal path and purification of glycolide.
In this paper, we conducted and presented a comprehensive literature review on methods for producing glycolic acid, glycolide, and purification and polymerization of glycolide. Different catalysts were compared at the stages of glycolide polycondensation, depolymerization, and polymerization.
The work describes the characteristics of the raw materials, describes the methods of production, purification, and polymerization of glycolide
Depletion of Kinesin 5B Affects Lysosomal Distribution and Stability and Induces Peri-Nuclear Accumulation of Autophagosomes in Cancer Cells
Background: Enhanced lysosomal trafficking is associated with metastatic cancer. In an attempt to discover cancer relevant
lysosomal motor proteins, we compared the lysosomal proteomes from parental MCF-7 breast cancer cells with those from
highly invasive MCF-7 cells that express an active form of the ErbB2 (DN-ErbB2).
Methodology/Principal Findings: Mass spectrometry analysis identified kinesin heavy chain protein KIF5B as the only
microtubule motor associated with the lysosomes in MCF-7 cells, and ectopic DN-ErbB2 enhanced its lysosomal association.
KIF5B associated with lysosomes also in HeLa cervix carcinoma cells as analyzed by subcellular fractionation. The depletion
of KIF5B triggered peripheral aggregations of lysosomes followed by lysosomal destabilization, and cell death in HeLa cells.
Lysosomal exocytosis in response to plasma membrane damage as well as fluid phase endocytosis functioned, however,
normally in these cells. Both HeLa and MCF-7 cells appeared to express similar levels of the KIF5B isoform but the death
phenotype was weaker in KIF5B-depleted MCF-7 cells. Surprisingly, KIF5B depletion inhibited the rapamycin-induced
accumulation of autophagosomes in MCF-7 cells. In KIF5B-depleted cells the autophagosomes formed and accumulated in
the close proximity to the Golgi apparatus, whereas in the control cells they appeared uniformly distributed in the
cytoplasm.
Conclusions/Significance: Our data identify KIF5B as a cancer relevant lysosomal motor protein with additional functions in
autophagosome formatio
Novel Patient Cell-Based HTS Assay for Identification of Small Molecules for a Lysosomal Storage Disease
Small molecules have been identified as potential therapeutic agents for lysosomal storage diseases (LSDs), inherited metabolic disorders caused by defects in proteins that result in lysosome dysfunctional. Some small molecules function assisting the folding of mutant misfolded lysosomal enzymes that are otherwise degraded in ER-associated degradation. The ultimate result is the enhancement of the residual enzymatic activity of the deficient enzyme. Most of the high throughput screening (HTS) assays developed to identify these molecules are single-target biochemical assays. Here we describe a cell-based assay using patient cell lines to identify small molecules that enhance the residual arylsulfatase A (ASA) activity found in patients with metachromatic leukodystrophy (MLD), a progressive neurodegenerative LSD. In order to generate sufficient cell lines for a large scale HTS, primary cultured fibroblasts from MLD patients were transformed using SV40 large T antigen. These SV40 transformed (SV40t) cells showed to conserve biochemical characteristics of the primary cells. Using a specific colorimetric substrate para-nitrocatechol sulfate (pNCS), detectable ASA residual activity were observed in primary and SV40t fibroblasts from a MLD patient (ASA-I179S) cultured in multi-well plates. A robust fluorescence ASA assay was developed in high-density 1,536-well plates using the traditional colorimetric pNCS substrate, whose product (pNC) acts as βplate fluorescence quencherβ in white solid-bottom plates. The quantitative cell-based HTS assay for ASA generated strong statistical parameters when tested against a diverse small molecule collection. This cell-based assay approach can be used for several other LSDs and genetic disorders, especially those that rely on colorimetric substrates which traditionally present low sensitivity for assay-miniaturization. In addition, the quantitative cell-based HTS assay here developed using patient cells creates an opportunity to identify therapeutic small molecules in a disease-cellular environment where potentially disrupted pathways are exposed and available as targets
A Rapid and Sensitive Method for Measuring NAcetylglucosaminidase Activity in Cultured Cells
A rapid and sensitive method to quantitatively assess N-acetylglucosaminidase (NAG) activity in cultured cells is highly
desirable for both basic research and clinical studies. NAG activity is deficient in cells from patients with
Mucopolysaccharidosis type IIIB (MPS IIIB) due to mutations in NAGLU, the gene that encodes NAG. Currently available
techniques for measuring NAG activity in patient-derived cell lines include chromogenic and fluorogenic assays and provide
a biochemical method for the diagnosis of MPS IIIB. However, standard protocols require large amounts of cells, cell
disruption by sonication or freeze-thawing, and normalization to the cellular protein content, resulting in an error-prone
procedure that is material- and time-consuming and that produces highly variable results. Here we report a new procedure
for measuring NAG activity in cultured cells. This procedure is based on the use of the fluorogenic NAG substrate, 4-
Methylumbelliferyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (MUG), in a one-step cell assay that does not require cell
disruption or post-assay normalization and that employs a low number of cells in 96-well plate format. We show that the
NAG one-step cell assay greatly discriminates between wild-type and MPS IIIB patient-derived fibroblasts, thus providing a
rapid method for the detection of deficiencies in NAG activity. We also show that the assay is sensitive to changes in NAG
activity due to increases in NAGLU expression achieved by either overexpressing the transcription factor EB (TFEB), a master
regulator of lysosomal function, or by inducing TFEB activation chemically. Because of its small format, rapidity, sensitivity
and reproducibility, the NAG one-step cell assay is suitable for multiple procedures, including the high-throughput
screening of chemical libraries to identify modulators of NAG expression, folding and activity, and the investigation of
candidate molecules and constructs for applications in enzyme replacement therapy, gene therapy, and combination
therapies
Different Origins of Gamma Rhythm and High-Gamma Activity in Macaque Visual Cortex
High-gamma (80β200 Hz) activity can be dissociated from gamma rhythms in
the monkey cortex, and appears largely to reflect spiking activity in the
vicinity of the electrode
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