scholarly journals Novel subtype of mucopolysaccharidosis caused by arylsulfatase K (ARSK) deficiency

2021 ◽  
pp. jmedgenet-2021-108061
Author(s):  
Sarah Verheyen ◽  
Jasmin Blatterer ◽  
Michael R Speicher ◽  
Gandham SriLakshmi Bhavani ◽  
Geert-Jan Boons ◽  
...  
Keyword(s):  
Dermatan Sulfate ◽  
Reversed Phase ◽  
Site Directed Mutagenesis ◽  
Facial Features ◽  
Liquid Chromatography Mass Spectrometry ◽  
Human Phenotype ◽  
Protein Levels ◽  
Ht1080 Cells ◽  
Enzyme Defects ◽  
Functional Consequences

BackgroundMucopolysaccharidoses (MPS) are monogenic metabolic disorders that significantly affect the skeleton. Eleven enzyme defects in the lysosomal degradation of glycosaminoglycans (GAGs) have been assigned to the known MPS subtypes (I–IX). Arylsulfatase K (ARSK) is a recently characterised lysosomal hydrolase involved in GAG degradation that removes the 2-O-sulfate group from 2-sulfoglucuronate. Knockout of Arsk in mice was consistent with mild storage pathology, but no human phenotype has yet been described.MethodsIn this study, we report four affected individuals of two unrelated consanguineous families with homozygous variants c.250C>T, p.(Arg84Cys) and c.560T>A, p.(Leu187Ter) in ARSK, respectively. Functional consequences of the two ARSK variants were assessed by mutation-specific ARSK constructs derived by site-directed mutagenesis, which were ectopically expressed in HT1080 cells. Urinary GAG excretion was analysed by dimethylene blue and electrophoresis, as well as liquid chromatography/mass spectrometry (LC-MS)/MS analysis.ResultsThe phenotypes of the affected individuals include MPS features, such as short stature, coarse facial features and dysostosis multiplex. Reverse phenotyping in two of the four individuals revealed additional cardiac and ophthalmological abnormalities. Mild elevation of dermatan sulfate was detected in the two subjects investigated by LC-MS/MS. Human HT1080 cells expressing the ARSK-Leu187Ter construct exhibited absent protein levels by western blot, and cells with the ARSK-Arg84Cys construct showed markedly reduced enzyme activity in an ARSK-specific enzymatic assay against 2-O-sulfoglucuronate-containing disaccharides as analysed by C18-reversed-phase chromatography followed by MS.ConclusionOur work provides a detailed clinical and molecular characterisation of a novel subtype of mucopolysaccharidosis, which we suggest to designate subtype X.

scholarly journals FBXW7 mutations reduce binding of NOTCH1, leading to cleaved NOTCH1 accumulation and target gene activation in CLL

Blood ◽  
2019 ◽  
Vol 133 (8) ◽  
pp. 830-839 ◽  
Author(s):  
Viola Close ◽  
William Close ◽  
Sabrina Julia Kugler ◽  
Michaela Reichenzeller ◽  
Deyan Yordanov Yosifov ◽  
...  
Keyword(s):  
Target Gene ◽  
Hypoxia Inducible Factor ◽  
Gene Activation ◽  
Lymphocytic Leukemia ◽  
Negative Regulator ◽  
Missense Mutations ◽  
Protein Levels ◽  
In Silico Modeling ◽  
Functional Consequences ◽  
Wd40 Domain

Abstract NOTCH1 is mutated in 10% of chronic lymphocytic leukemia (CLL) patients and is associated with poor outcome. However, NOTCH1 activation is identified in approximately one-half of CLL cases even in the absence of NOTCH1 mutations. Hence, there appear to be additional factors responsible for the impairment of NOTCH1 degradation. E3-ubiquitin ligase F-box and WD40 repeat domain containing-7 (FBXW7), a negative regulator of NOTCH1, is mutated in 2% to 6% of CLL patients. The functional consequences of these mutations in CLL are unknown. We found heterozygous FBXW7 mutations in 36 of 905 (4%) untreated CLL patients. The majority were missense mutations (78%) that mostly affected the WD40 substrate binding domain; 10% of mutations occurred in the first exon of the α-isoform. To identify target proteins of FBXW7 in CLL, we truncated the WD40 domain in CLL cell line HG-3 via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9). Homozygous truncation of FBXW7 resulted in an increase of activated NOTCH1 intracellular domain (NICD) and c-MYC protein levels as well as elevated hypoxia-inducible factor 1-α activity. In silico modeling predicted that novel mutations G423V and W425C in the FBXW7-WD40 domain change the binding of protein substrates. This differential binding was confirmed via coimmunoprecipitation of overexpressed FBXW7 and NOTCH1. In primary CLL cells harboring FBXW7 mutations, activated NICD levels were increased and remained stable upon translation inhibition. FBXW7 mutations coincided with an increase in NOTCH1 target gene expression and explain a proportion of patients characterized by dysregulated NOTCH1 signaling.

Effect of microgravity on the expression of mitochondrial enzymes in rat cardiac and skeletal muscles

1998 ◽  
Vol 84 (2) ◽  
pp. 593-598 ◽  
Author(s):  
Michael K. Connor ◽  
David A. Hood
Keyword(s):  
Skeletal Muscle ◽  
Enzyme Activity ◽  
Skeletal Muscles ◽  
Male Rats ◽  
Mitochondrial Enzymes ◽  
Mrna Levels ◽  
Triceps Brachii ◽  
Protein Levels ◽  
Functional Consequences ◽  
Microgravity Conditions

Connor, Michael K., and David A. Hood. Effect of microgravity on the expression of mitochondrial enzymes in rat cardiac and skeletal muscles. J. Appl. Physiol. 84(2): 593–598, 1998.—The purpose of this study was to examine the expression of nuclear and mitochondrial genes in cardiac and skeletal muscle (triceps brachii) in response to short-duration microgravity exposure. Six adult male rats were exposed to microgravity for 6 days and were compared with six ground-based control animals. We observed a significant 32% increase in heart malate dehydrogenase (MDH) enzyme activity, which was accompanied by a 62% elevation in heart MDH mRNA levels after microgravity exposure. Despite modest elevations in the mRNAs encoding subunits III, IV, and VIc as well as a 2.2-fold higher subunit IV protein content after exposure to microgravity, heart cytochrome c oxidase (CytOx) enzyme activity remained unchanged. In skeletal muscle, MDH expression was unaffected by microgravity, but CytOx activity was significantly reduced 41% by microgravity, whereas subunit III, IV, and VIc mRNA levels and subunit IV protein levels were unaltered. Thus tissue-specific (i.e., heart vs. skeletal muscle) differences exist in the regulation of nuclear-encoded mitochondrial proteins in response to microgravity. In addition, the expression of nuclear-encoded proteins such as CytOx subunit IV and expression of MDH are differentially regulated within a tissue. Our data also illustrate that the heart undergoes previously unidentified mitochondrial adaptations in response to short-term microgravity conditions more dramatic than those evident in skeletal muscle. Further studies evaluating the functional consequences of these adaptations in the heart, as well as those designed to measure protein turnover, are warranted in response to microgravity.

On-line reversed phase liquid chromatography-mass spectrometry

1979 ◽  
Vol 51 (14) ◽  
pp. 2324-2328 ◽  
Author(s):  
B. L. Karger ◽  
D. P. Kirby ◽  
Paul. Vouros ◽  
R. L. Foltz ◽  
B. Hidy
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Reversed Phase ◽  
Liquid Chromatography Mass Spectrometry ◽  
Reversed Phase Liquid Chromatography ◽  
Chromatography Mass Spectrometry ◽  
On Line ◽  
Phase Liquid

Targeted analysis of phenolic compounds by LC-MS v1

2021 ◽  
Author(s):  
Bashar Amer ◽  
Ramu Kakumanu ◽  
yangtian not provided ◽  
Aymerick Eudes ◽  
Edward EK Baidoo
Keyword(s):  
Phenolic Compounds ◽  
Plant Biomass ◽  
Reversed Phase ◽  
Industrial Applications ◽  
Liquid Chromatography Mass Spectrometry ◽  
Reversed Phase Liquid Chromatography ◽  
Sample Matrix ◽  
Targeted Analysis ◽  
Challenges And Opportunities ◽  
Phase Liquid

Cell-wall-bound (CWB) aromatics such as ferulate and p-coumarate play important physiological roles in plant development and response to stresses. Their presence also poses some challenges and opportunities during processing of plant biomass in various agro-industrial applications. To this end, we have developed a robust high-throughput reversed-phase liquid chromatography mass spectrometry method for quantifying CWB phenolic compounds. The method showed excellent linearity (R2 = ≥0.999) and intraday retention time repeatability (≤ 0.31 %RSD) for ferulate and p-coumarate. The limits of detection and quantitation for these analytes were ≤ 39 nM and 130 nM, respectively. Furthermore, there was very little effect of the CWB sample matrix on the retention times of the analytes and analyte percent recoveries from the CWB sample matrix was ≥83.91%.

scholarly journals Upregulation of the Sarco-Endoplasmic Reticulum Calcium ATPase 1 Truncated Isoform Plays a Pathogenic Role in Alzheimer’s Disease

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1539 ◽  
Author(s):  
Bussiere ◽  
Oulès ◽  
Mary ◽  
Vaillant-Beuchot ◽  
Martin ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Neuroblastoma Cells ◽  
Amyloid Β ◽  
Aβ Oligomers ◽  
Human Neuroblastoma ◽  
Endoplasmic Reticulum Calcium ◽  
Protein Levels ◽  
Functional Consequences ◽  
Β Amyloid

Dysregulation of the Endoplasmic Reticulum (ER) Ca2+ homeostasis and subsequent ER stress activation occur in Alzheimer Disease (AD). We studied the contribution of the human truncated isoform of the sarco-endoplasmic reticulum Ca2+ ATPase 1 (S1T) to AD. We examined S1T expression in human AD-affected brains and its functional consequences in cellular and transgenic mice AD models. S1T expression is increased in sporadic AD brains and correlates with amyloid β (Aβ) and ER stress chaperone protein levels. Increased S1T expression was also observed in human neuroblastoma cells expressing Swedish-mutated β-amyloid precursor protein (βAPP) or treated with Aβ oligomers. Lentiviral overexpression of S1T enhances in return the production of APP C-terminal fragments and Aβ through specific increases of β-secretase expression and activity, and triggers neuroinflammation. We describe a molecular interplay between S1T-dependent ER Ca2+ leak, ER stress and βAPP-derived fragments that could contribute to AD setting and/or progression.

scholarly journals Determination of 7B3 Residues in Cotton Plants by Liquid Chromatography/Mass Spectrometry

2009 ◽  
Vol 92 (1) ◽  
pp. 302-306 ◽  
Author(s):  
Xiao-Jing Yan ◽  
Xiao-Mei Liang ◽  
Yan-Jun Xu ◽  
Shu-Hui Jin ◽  
Dao-Quan Wang
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Limit Of Detection ◽  
Reversed Phase ◽  
Liquid Chromatography Mass Spectrometry ◽  
Chromatography Mass Spectrometry ◽  
Relative Standard ◽  
Limit Of Quantitation ◽  
Cotton Plants

Abstract A method was developed for the determination of 7B3 (12-propyloxyimino-1,15-pentadecanlactam), a novel macrolactam fungicide, by liquid chromatography/mass spectrometry (LC/MS) with positive electrospray ionization (ESI+). The method used a reversed-phase C18 column and acetonitrilewater (60 + 40, v/v) mobile phase. The quick, easy, cheap, effective, rugged, and safe method was used for extraction of 7B3 from cotton plants, which involved the extraction of 10 g homogenized sample with 10 mL acetonitrile, followed by the addition of 4 g anhydrous MgSO4 and 1.0 g NaCl. After centrifugation, 1 mL of the buffered acetonitrile extract was transferred into a tube containing 50 mg primary secondary amine sorbent and 100 mg anhydrous MgSO4. After shaking and centrifugation, the final extract was transferred to an autosampler vial for concurrent analysis by LC/MS. The results of 7B3 determined by LC/MS in the selective ion monitoring mode were linear, and the matrix effect of the method was evaluated. The average recoveries of 7B3 fortified at different levels were within 84.1100.2, and the relative standard deviations were <7.5 for all samples analyzed. The method limit of detection and the limit of quantitation values were 0.03 and 0.1 mg/kg, respectively. The proposed method was successfully applied to determine 7B3 residues in practical samples. This method is sensitive, accurate, reliable, simple, and safe.

scholarly journals Molecular Analysis of the ABCA4 Gene Mutations in Patients with Stargardt Disease Using Human Hair Follicles

2020 ◽  
Vol 21 (10) ◽  
pp. 3430
Author(s):  
Aneta Ścieżyńska ◽  
Marta Soszyńska ◽  
Michał Komorowski ◽  
Anna Podgórska ◽  
Natalia Krześniak ◽  
...  
Keyword(s):  
Molecular Analysis ◽  
Splice Site ◽  
Human Hair ◽  
Gene Mutations ◽  
Hair Follicles ◽  
Cdna Sequencing ◽  
Stargardt Disease ◽  
Protein Levels ◽  
Abca4 Gene ◽  
Functional Consequences

ABCA4 gene mutations are the cause of a spectrum of ABCA4 retinopathies, and the most common juvenile macular degeneration is called Stargardt disease. ABCA4 has previously been observed almost exclusively in the retina. Therefore, studying the functional consequences of ABCA4 variants has required advanced molecular analysis techniques. The aim of the present study was to evaluate whether human hair follicles may be used for molecular analysis of the ABCA4 gene splice-site variants in patients with ABCA4 retinopathies. We assessed ABCA4 expression in hair follicles and skin at mRNA and protein levels by means of real-time PCR and Western blot analyses, respectively. We performed cDNA sequencing to reveal the presence of full-length ABCA4 transcripts and analyzed ABCA4 transcripts from three patients with Stargardt disease carrying different splice-site ABCA4 variants: c.5312+1G>A, c.5312+2T>G and c.5836-3C>A. cDNA analysis revealed that c.5312+1G>A, c.5312+2T>G variants led to the skipping of exon 37, and the c.5836-3C>A variant resulted in the insertion of 30 nucleotides into the transcript. Our results strongly argue for the use of hair follicles as a model for the molecular analysis of the pathogenicity of ABCA4 variants in patients with ABCA4 retinopathies.

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