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1.
Eur J Clin Microbiol Infect Dis ; 43(2): 355-371, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38099986

ABSTRACT

PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.


Subject(s)
Toxoplasma , Pregnancy , Female , Humans , Toxoplasma/genetics , Genotype , Multiplex Polymerase Chain Reaction , High-Throughput Nucleotide Sequencing , DNA, Protozoan/genetics , Genetic Variation , Polymorphism, Restriction Fragment Length
2.
Food Waterborne Parasitol ; 27: e00156, 2022 Jun.
Article in English | MEDLINE | ID: mdl-35498549

ABSTRACT

The International Trichinella Reference Centre (ITRC) is the official laboratory of the International Commission on Trichinellosis, of the World Organization for Animal Health and of the European Union Reference Laboratory for Parasites. The ITRC was established in 1988 as a repository of Trichinella strains and a source of reference materials and information for international scientific research. To date, more than 8000 Trichinella isolates collected throughout the world have been identified at the species or genotype level by the ITRC staff and the information has been stored in a freely accessible database providing the largest collection of data available for scientists involved in the systematics and epidemiology of this parasite. This paper presents a summary of the data collected over 33 years of activity and describes the database functionalities. It finally advocates the potential of the database to improve knowledge of the epidemiology and taxonomy of Trichinella, which in turn may help the international surveillance of Trichinella species.

3.
FEBS Lett ; 481(2): 152-8, 2000 Sep 15.
Article in English | MEDLINE | ID: mdl-10996315

ABSTRACT

Growth arrest specific (gas) 1 gene product is expressed in non-transformed fibroblasts in response to stimuli driving cells into Go phase. Gas1 has been demonstrated to inhibit cell proliferation when over-expressed in proliferating fibroblasts. This activity depends on a function of the p53 protein independent of its transactivating ability. To better define the pathway leading from Gas1, which is located on the plasma membrane, to p53, we have undertaken a detailed characterization of its topology. We demonstrate that the protein undergoes cotranslational modifications in the endoplasmic reticulum, consisting of signal peptide cleavage, N-linked glycosylation and glycosyl-phosphatidylinositol anchor addition. Immunoelectron microscopy shows that, in its mature form, Gas1 is randomly distributed over the outer leaflet of the plasma membrane and that upon antibody-induced clustering it relocalizes to caveolae.


Subject(s)
Cell Membrane/metabolism , Glycosylphosphatidylinositols/metabolism , Membrane Glycoproteins/metabolism , Saccharomyces cerevisiae Proteins , 3T3 Cells , Animals , COS Cells , Cell Cycle Proteins , Cell Division , Consensus Sequence/physiology , Endoplasmic Reticulum/metabolism , GPI-Linked Proteins , Glutaral , Humans , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/genetics , Membrane Glycoproteins/ultrastructure , Membrane Proteins , Mice , Microscopy, Immunoelectron , Palmitic Acid/metabolism , Phosphatidylinositol Diacylglycerol-Lyase , Precipitin Tests , Protein Binding , Protein Sorting Signals/physiology , Tissue Fixation , Transfection , Type C Phospholipases/metabolism
4.
FEBS Lett ; 481(2): 159-63, 2000 Sep 15.
Article in English | MEDLINE | ID: mdl-10996316

ABSTRACT

The product of the growth arrest specific gene, gas1, is a membrane-associated protein which activates a p53-dependent growth suppression signalling pathway. We have shown that Gas1 is linked to the plasma membrane through a glycosyl-phosphatidylinositol (GPI) anchor. Several GPI-anchored protein have been identified as part of receptor complexes either as co-receptors or as membrane bound ligands. In this report, we characterize the Gas1 domains required for its growth suppression function and demonstrate the dispensability of Gas1 GPI anchor.


Subject(s)
Cell Division , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Mutation , Saccharomyces cerevisiae Proteins , 3T3 Cells , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Animals , COS Cells , Glycosylphosphatidylinositols/genetics , Glycosylphosphatidylinositols/metabolism , Humans , Membrane Glycoproteins/administration & dosage , Membrane Glycoproteins/genetics , Mice , Microinjections , Protein Sorting Signals/genetics , Protein Sorting Signals/physiology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence Deletion/genetics , Solubility , Transfection
5.
Genomics ; 57(1): 120-9, 1999 Apr 01.
Article in English | MEDLINE | ID: mdl-10191091

ABSTRACT

The serum deprivation response gene (SDPR, alias sdr) has been previously isolated for its high mRNA expression in serum-starved cells compared to contact-inhibited NIH3T3 cells; such regulation is not observed in single-oncogene transformed NIH3T3 cells after serum starvation. More recently Sdpr has been identified as a substrate of protein kinase C (PKC): this interaction determines the compartimentalization of PKC to caveolae, a plasma membrane invagination of which Sdpr is a major component. Lack of Sdpr-PKC interaction in transformed cells has been proposed to be involved in the alteration of PKC subcellular localization and substrate specificity. Here we report the cloning of the human SDPR homologue (HGMW-approved symbol SDPR) and its mapping to 2q32-q33 in the human genome. In analogy with the murine system, SDPR mRNA expression is increased when human fibroblasts are serum starved, it becomes down-regulated during synchronous cell-cycle reentry, but it is not induced in cells arrested by contact inhibition. Analysis of SDPR expression in human tissues reveals a near ubiquitous expression, with highest levels found in heart and lung. We show that human SDPR encodes PS-p68, a previously characterized phosphatidylserine-binding protein purified from human platelets. Accordingly, recombinant Sdpr is able to specifically bind phosphatidylserine in the absence of Ca2+. SDPR is homologous to two genes in the databank, one of which, srbc, is similarly regulated during growth arrest and encodes a phosphatidylserine-binding protein that is a substrate of PKC.


Subject(s)
Carrier Proteins/genetics , Carrier Proteins/metabolism , Chromosomes, Human, Pair 2 , Phosphatidylserines/metabolism , Amino Acid Sequence , Blotting, Northern , Cell Cycle/physiology , Cloning, Molecular , Fibroblasts/metabolism , Fluorescent Antibody Technique , Humans , In Situ Hybridization, Fluorescence , Liver/metabolism , Models, Genetic , Molecular Sequence Data , Phosphate-Binding Proteins , Recombinant Proteins , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Time Factors , Tissue Distribution , Transfection
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