scholarly journals Assessment of galactose-1-phosphate uridyltransferase activity in cells and tissues

2021 ◽  
Vol 8 (2) ◽  
pp. e149
Author(s):  
Megan L. Brophy ◽  
John E. Murphy ◽  
Robert D. Bell
Keyword(s):  
Cell Culture ◽  
Autosomal Recessive ◽  
Genetic Disorders ◽  
Unmet Need ◽  
Novel Therapies ◽  
Animal Tissues ◽  
Galactose Metabolism ◽  
Pathophysiological Mechanisms ◽  
Leloir Pathway ◽  
In Cells

Galactosemias are a family of autosomal recessive genetic disorders resulting from impaired enzymes of the Leloir pathway of galactose metabolism including galactokinase, galactose uridyltransferase, and UDP-galactose 4-epimerase that are critical for conversion of galactose into glucose-6-phosphate. To better understand pathophysiological mechanisms involved in galactosemia and develop novel therapies to address the unmet need in patients, it is important to develop reliable assays to measure the activity of the Leloir pathway enzymes. Here we describe in-depth methods for indirectly measuring Galacose-1-Phosphate Uridyltransferase activity in cell culture and animal tissues.

scholarly journals Solving a case of allelic dropout in the GNPTAB gene: implications in the molecular diagnosis of mucolipidosis type III alpha/beta

2016 ◽  
Vol 29 (10) ◽  
Author(s):  
Maria Francisca Coutinho ◽  
Marisa Encarnação ◽  
Francisco Laranjeira ◽  
Lúcia Lacerda ◽  
Maria João Prata ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Molecular Diagnosis ◽  
Autosomal Recessive ◽  
Genetic Disorders ◽  
Causative Mutation ◽  
Lysosomal Storage ◽  
Allelic Dropout ◽  
Type Iii ◽  
Alpha Beta ◽  
Mucolipidosis Type

AbstractWhile being well known that the diagnosis of many genetic disorders relies on a combination of clinical suspicion and confirmatory genetic testing, not rarely, however, genetic testing needs much perseverance and cunning strategies to identify the causative mutation(s). Here we present a case of a thorny molecular diagnosis of mucolipidosis type III alpha/beta, which is an autosomal recessive lysosomal storage disorder, caused by a defect in the

Practice of Genetics in Clinical Medicine

2010 ◽  
Author(s):  
Bruce R Korf ◽  
Carlos Gallego
Keyword(s):  
Genetic Testing ◽  
Autosomal Recessive ◽  
Clinical Medicine ◽  
Maternal Inheritance ◽  
Genetic Disorders ◽  
Gene Mutations ◽  
Basic Principles ◽  
Direct To Consumer ◽  
Risk Assessment And Prevention ◽  
Traditional Area

This review provides a general overview of the genetic approach in medical practice, discusses the principles of genetic testing, including interpretation of genetic tests and direct-to-consumer genomic testing, and looks at genetic counseling and approaches to treatment. The internist should become familiar with genetic disorders such as those associated with mutations in single genes or changes in chromosome number or structure. This is the traditional area of focus for medical geneticists and is likely to remain so. The internist should be familiar with basic principles of care for individuals with the more common of these conditions and needs to recognize clues that suggest the presence of these disorders, especially in family history. The section on genetics of common disorders focuses on pharmacogenetics, risk assessment, and prevention. Figures illustrate commonly used standard pedigree symbols and examples of autosomal recessive, autosomal dominant, X-linked recessive, and maternal inheritance. Tables offer different forms of genetic testing and types of gene mutations at genome and DNA levels. This review contains 2 figures, 2 tables, and 83 references.

scholarly journals From Interconnection between Genes and Microenvironment to Novel Immunotherapeutic Approaches in Upper Gastro-Intestinal Cancers—A Multidisciplinary Perspective

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2105
Author(s):  
Giulia Accordino ◽  
Sara Lettieri ◽  
Chandra Bortolotto ◽  
Silvia Benvenuti ◽  
Anna Gallotti ◽  
...  
Keyword(s):  
Unmet Need ◽  
Predictive Biomarkers ◽  
Standard Of Care ◽  
Integrated Approach ◽  
Point Of View ◽  
Novel Therapies ◽  
Car T ◽  
Treatment Data ◽  
Multidisciplinary Perspective

Despite the progress during the last decade, patients with advanced gastric and esophageal cancers still have poor prognosis. Finding optimal therapeutic strategies represents an unmet need in this field. Several prognostic and predictive factors have been evaluated and may guide clinicians in choosing a tailored treatment. Data from large studies investigating the role of immunotherapy in gastrointestinal cancers are promising but further investigations are necessary to better select those patients who can mostly benefit from these novel therapies. This review will focus on the treatment of metastatic esophageal and gastric cancer. We will review the standard of care and the role of novel therapies such as immunotherapies and CAR-T. Moreover, we will focus on the analysis of potential predictive biomarkers such as Modify as: Microsatellite Instability (MSI) and PD-L1, which may lead to treatment personalization and improved treatment outcomes. A multidisciplinary point of view is mandatory to generate an integrated approach to properly exploit these novel antiproliferative agents.

scholarly journals Towards Enhanced Galactose Utilization by Lactococcus lactis

2010 ◽  
Vol 76 (21) ◽  
pp. 7048-7060 ◽  
Author(s):  
Ana R. Neves ◽  
Wietske A. Pool ◽  
Ana Solopova ◽  
Jan Kok ◽  
Helena Santos ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Poor Quality ◽  
Genetically Engineered ◽  
Phosphotransferase System ◽  
Galactose Metabolism ◽  
Gene Encoding ◽  
Leloir Pathway ◽  
Lactose Fermentation ◽  
Consumption Rates ◽  
Galactose Utilization

ABSTRACT Accumulation of galactose in dairy products due to partial lactose fermentation by lactic acid bacteria yields poor-quality products and precludes their consumption by individuals suffering from galactosemia. This study aimed at extending our knowledge of galactose metabolism in Lactococcus lactis, with the final goal of tailoring strains for enhanced galactose consumption. We used directed genetically engineered strains to examine galactose utilization in strain NZ9000 via the chromosomal Leloir pathway (gal genes) or the plasmid-encoded tagatose 6-phosphate (Tag6P) pathway (lac genes). Galactokinase (GalK), but not galactose permease (GalP), is essential for growth on galactose. This finding led to the discovery of an alternative route, comprising a galactose phosphotransferase system (PTS) and a phosphatase, for galactose dissimilation in NZ9000. Introduction of the Tag6P pathway in a galPMK mutant restored the ability to metabolize galactose but did not sustain growth on this sugar. The latter strain was used to prove that lacFE, encoding the lactose PTS, is necessary for galactose metabolism, thus implicating this transporter in galactose uptake. Both PTS transporters have a low affinity for galactose, while GalP displays a high affinity for the sugar. Furthermore, the GalP/Leloir route supported the highest galactose consumption rate. To further increase this rate, we overexpressed galPMKT, but this led to a substantial accumulation of α-galactose 1-phosphate and α-glucose 1-phosphate, pointing to a bottleneck at the level of α-phosphoglucomutase. Overexpression of a gene encoding α-phosphoglucomutase alone or in combination with gal genes yielded strains with galactose consumption rates enhanced up to 50% relative to that of NZ9000. Approaches to further improve galactose metabolism are discussed.

scholarly journals Galactose Metabolism Plays a Crucial Role in Biofilm Formation by Bacillus subtilis

mBio ◽  
2012 ◽  
Vol 3 (4) ◽  
Author(s):  
Yunrong Chai ◽  
Pascale B. Beauregard ◽  
Hera Vlamakis ◽  
Richard Losick ◽  
Roberto Kolter
Keyword(s):  
Bacillus Subtilis ◽  
Biofilm Formation ◽  
Carbon Sources ◽  
Galactose Metabolism ◽  
Content Type ◽  
Leloir Pathway ◽  
The Matrix ◽  
Living Organisms ◽  
Galactose Metabolites ◽  
Biofilm Matrix

ABSTRACTGalactose is a common monosaccharide that can be utilized by all living organisms via the activities of three main enzymes that make up the Leloir pathway: GalK, GalT, and GalE. InBacillus subtilis, the absence of GalE causes sensitivity to exogenous galactose, leading to rapid cell lysis. This effect can be attributed to the accumulation of toxic galactose metabolites, since thegalEmutant is blocked in the final step of galactose catabolism. In a screen for suppressor mutants restoring viability to agalEnull mutant in the presence of galactose, we identified mutations insinR, which is the major biofilm repressor gene. These mutations caused an increase in the production of the exopolysaccharide (EPS) component of the biofilm matrix. We propose that UDP-galactose is the toxic galactose metabolite and that it is used in the synthesis of EPS. Thus, EPS production can function as a shunt mechanism for this toxic molecule. Additionally, we demonstrated that galactose metabolism genes play an essential role inB. subtilisbiofilm formation and that the expressions of both thegalandepsgenes are interrelated. Finally, we propose thatB. subtilisand other members of theBacillusgenus may have evolved to utilize naturally occurring polymers of galactose, such as galactan, as carbon sources.IMPORTANCEBacteria switch from unicellular to multicellular states by producing extracellular matrices that contain exopolysaccharides. In such aggregates, known as biofilms, bacteria are more resistant to antibiotics. This makes biofilms a serious problem in clinical settings. The resilience of biofilms makes them very useful in industrial settings. Thus, understanding the production of biofilm matrices is an important problem in microbiology. In studying the synthesis of the biofilm matrix ofBacillus subtilis, we provide further understanding of a long-standing microbiological observation that certain mutants defective in the utilization of galactose became sensitive to it. In this work, we show that the toxicity observed before was because cells were grown under conditions that were not propitious to produce the exopolysaccharide component of the matrix. When cells are grown under conditions that favor matrix production, the toxicity of galactose is relieved. This allowed us to demonstrate that galactose metabolism is essential for the synthesis of the extracellular matrix.

scholarly journals Tau aggregates are RNA-protein assemblies that mis-localize multiple nuclear speckle components

2021 ◽  
Author(s):  
Evan Lester ◽  
Felicia K. Ooi ◽  
Nadine Bakkar ◽  
Jacob Ayers ◽  
Amanda L. Woerman ◽  
...  
Keyword(s):  
Cell Culture ◽  
Frontotemporal Dementia ◽  
Corticobasal Degeneration ◽  
Mrna Splicing ◽  
Protein Assemblies ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Tau Aggregation ◽  
In Cells

AbstractTau aggregates contribute to neurodegenerative diseases including frontotemporal dementia and Alzheimer’s disease (AD). Although RNA promotes tau aggregation in vitro, whether tau aggregates in cells contain RNA is unknown. We demonstrate in cell culture and mouse brains that both cytosolic and nuclear tau aggregates contain RNA, with enrichment for snRNAs and snoRNAs. Nuclear tau aggregates colocalize with and alter the composition, dynamics, and organization of nuclear speckles, which are membraneless organelles involved in pre-mRNA splicing. Moreover, several nuclear speckle components, including SRRM2, mislocalize to cytosolic tau aggregates in cells, mouse brains, and patient brains with AD, frontotemporal dementia (FTD), and corticobasal degeneration (CBD). Consistent with these alterations we observe the presence of tau aggregates is sufficient to alter pre-mRNA splicing. This work identifies tau alteration of nuclear speckles as a feature of tau aggregation that may contribute to the pathology of tau aggregates.

scholarly journals The Metabolic Chemical Reporter Ac46AzGal Could Incorporate Intracellular Protein Modification in the Form of UDP-6AzGlc Mediated by OGT and Enzymes in the Leloir Pathway

2021 ◽  
Vol 9 ◽  
Author(s):  
Jiajia Wang ◽  
Biao Dou ◽  
Lu Zheng ◽  
Wei Cao ◽  
Peiyu Dong ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Protein Modification ◽  
Time Dependent ◽  
Dependent Manner ◽  
Intracellular Protein ◽  
Galactose Metabolism ◽  
Naturally Occurring ◽  
Leloir Pathway ◽  
Chemical Reporter ◽  
Complex Glycans

Galactose is a naturally occurring monosaccharide used to build complex glycans that has not been targeted for labeling as a metabolic reporter. Here, we characterize the cellular modification of proteins by using Ac46AzGal in a dose- and time-dependent manner. It is noted that a vast majority of this labeling of Ac46AzGal occurs intracellularly in a range of mammalian cells. We also provided evidence that this labeling is dependent on not only the enzymes of OGT responsible for O-GlcNAcylation but also the enzymes of GALT and GALE in the Leloir pathway. Notably, we discover that Ac46AzGal is not the direct substrate of OGT, and the labeling results may attribute to UDP-6AzGlc after epimerization of UDP-6AzGal via GALE. Together, these discoveries support the conclusion that Ac46AzGal as an analogue of galactose could metabolically label intracellular O-glycosylation modification, raising the possibility of characterization with impaired functions of the galactose metabolism in the Leloir pathway under certain conditions, such as galactosemias.

scholarly journals Restoration of LDL receptor function in cells from patients with autosomal recessive hypercholesterolemia by retroviral expression of ARH1

2002 ◽  
Vol 110 (11) ◽  
pp. 1695-1702 ◽  
Author(s):  
Emily R. Eden ◽  
Dilipkumar D. Patel ◽  
Xi-Ming Sun ◽  
Jemima J. Burden ◽  
Michael Themis ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Ldl Receptor ◽  
Receptor Function ◽  
Autosomal Recessive Hypercholesterolemia ◽  
In Cells

scholarly journals Leader of the Capsid Protein in Feline Calicivirus Promotes Replication of Norwalk Virus in Cell Culture

2008 ◽  
Vol 82 (19) ◽  
pp. 9306-9317 ◽  
Author(s):  
Kyeong-Ok Chang ◽  
David W. George ◽  
John B. Patton ◽  
Kim Y. Green ◽  
Stanislav V. Sosnovtsev
Keyword(s):  
Cell Culture ◽  
Capsid Protein ◽  
Recombinant Plasmid ◽  
Fluorescent Protein ◽  
Feline Calicivirus ◽  
Norwalk Virus ◽  
Human Noroviruses ◽  
Infected Cells ◽  
Green Fluorescent ◽  
In Cells

ABSTRACT The inability to grow human noroviruses in cell culture has greatly impeded the studies of their pathogenesis and immunity. Vesiviruses, in the family Caliciviridae, grow efficiently in cell culture and encode a unique protein in the subgenomic region designated as leader of the capsid protein (LC). We hypothesized that LC might be associated with the efficient replication of vesiviruses in cell culture and promote the replication of human norovirus in cells. To test this hypothesis, a recombinant plasmid was engineered in which the LC region of feline calicivirus (FCV) was placed under the control of the cytomegalovirus promoter (pCI-LC) so that the LC protein could be provided in trans to replicating calicivirus genomes bearing a reporter gene. We constructed pNV-GFP, a recombinant plasmid containing a full-length NV genome with a green fluorescent protein (GFP) in the place of VP1. The transfection of pNV-GFP in MVA-T7-infected cells produced few GFP-positive cells detected by fluorescence microscopy and flow cytometry analysis. When pNV-GFP was cotransfected with pCI-LC in MVA-T7-infected cells, we observed an increase in the number of GFP-positive cells (ca. 3% of the whole-cell population). Using this cotransfection method with mutagenesis study, we identified potential cis-acting elements at the start of subgenomic RNA and the 3′ end of NV genome for the virus replication. We conclude that LC may be a viral factor which promotes the replication of NV in cells, which could provide a clue to growing the fastidious human noroviruses in cell culture.

Sign in / Sign up

Export Citation Format

Share Document