Molecular Chaperone GRP94 Binds to the Fanconi Anemia Group C Protein and Regulates Its Intracellular Expression

Blood ◽  
1998 ◽  
Vol 91 (11) ◽  
pp. 4379-4386 ◽  
Author(s):  
Taizo Hoshino ◽  
Jianxiang Wang ◽  
Marcel P. Devetten ◽  
Nobuhisa Iwata ◽  
Sachiko Kajigaya ◽  
...  
Keyword(s):  
Fanconi Anemia ◽  
Molecular Chaperone ◽  
B Lymphocyte ◽  
Complementation Group ◽  
Splicing Mutation ◽  
Cytoplasmic Proteins ◽  
Intracellular Expression ◽  
Anemia Group

Abstract The FAC protein encoded by the gene defective in Fanconi anemia (FA) complementation group C binds to at least three ubiquitous cytoplasmic proteins in vitro. We used here the complete coding sequence ofFAC in a yeast two-hybrid screen to identify interacting proteins. The molecular chaperone GRP94 was isolated twice from a B-lymphocyte cDNA library. Binding was confirmed by coimmunoprecipitation of FAC and GRP94 from cytosolic, but not nuclear, lysates of transfected COS-1 cells, as well as from mouse liver cytoplasmic extracts. Deletion mutants of FAC showed that residues 103-308 were required for interaction with GRP94, and a natural splicing mutation within the IVS-4 of FAC that removes residues 111-148 failed to bind GRP94. Ribozyme-mediated inactivation of GRP94 in the rat NRK cell line led to significantly reduced levels of immunoreactive FAC and concomitant hypersensitivity to mitomycin C, similar to the cellular phenotype of FA. Our results demonstrate that GRP94 interacts with FAC both in vitro and in vivo and regulates its intracellular level in a cell culture model. In addition, the pathogenicity of the IVS-4 splicing mutation in the FAC gene may be mediated in part by its inability to bind to GRP94.

Molecular Chaperone GRP94 Binds to the Fanconi Anemia Group C Protein and Regulates Its Intracellular Expression

Blood ◽  
1998 ◽  
Vol 91 (11) ◽  
pp. 4379-4386 ◽  
Author(s):  
Taizo Hoshino ◽  
Jianxiang Wang ◽  
Marcel P. Devetten ◽  
Nobuhisa Iwata ◽  
Sachiko Kajigaya ◽  
...  
Keyword(s):  
Fanconi Anemia ◽  
Molecular Chaperone ◽  
B Lymphocyte ◽  
Complementation Group ◽  
Splicing Mutation ◽  
Cytoplasmic Proteins ◽  
Intracellular Expression ◽  
Anemia Group

The FAC protein encoded by the gene defective in Fanconi anemia (FA) complementation group C binds to at least three ubiquitous cytoplasmic proteins in vitro. We used here the complete coding sequence ofFAC in a yeast two-hybrid screen to identify interacting proteins. The molecular chaperone GRP94 was isolated twice from a B-lymphocyte cDNA library. Binding was confirmed by coimmunoprecipitation of FAC and GRP94 from cytosolic, but not nuclear, lysates of transfected COS-1 cells, as well as from mouse liver cytoplasmic extracts. Deletion mutants of FAC showed that residues 103-308 were required for interaction with GRP94, and a natural splicing mutation within the IVS-4 of FAC that removes residues 111-148 failed to bind GRP94. Ribozyme-mediated inactivation of GRP94 in the rat NRK cell line led to significantly reduced levels of immunoreactive FAC and concomitant hypersensitivity to mitomycin C, similar to the cellular phenotype of FA. Our results demonstrate that GRP94 interacts with FAC both in vitro and in vivo and regulates its intracellular level in a cell culture model. In addition, the pathogenicity of the IVS-4 splicing mutation in the FAC gene may be mediated in part by its inability to bind to GRP94.

Defective hematopoiesis and hepatic steatosis in mice with combined deficiencies of the genes encoding Fancc and Cu/Zn superoxide dismutase

Blood ◽  
2001 ◽  
Vol 98 (4) ◽  
pp. 1003-1011 ◽  
Author(s):  
Suzana Hadjur ◽  
Karen Ung ◽  
Louis Wadsworth ◽  
James Dimmick ◽  
Evica Rajcan-Separovic ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Fanconi Anemia ◽  
Redox State ◽  
Complementation Group ◽  
Normal Numbers ◽  
Cell Surface Antigens ◽  
Genes Encoding ◽  
B Lymphoid

Several lines of evidence point to an abnormality in the response of Fanconi anemia cells to reactive oxygen species. To investigate the potential pathologic consequences of an in vivo alteration of redox state in mice lacking one of the Fanconi anemia genes, animals were generated having combined deficiencies of the cytosolic Cu/Zn superoxide dismutase (Sod1) and Fanconi anemia complementation group C (Fancc) genes. Interestingly, hepatocytes of Fancc−/−Sod1−/−mice exhibited a zonal pattern of microvesicular steatosis, possibly as a result of oxidative stress-induced injury to hepatocyte membranes. Consistent with this idea, freshly explantedFancc−/−Sod1−/−hepatocytes demonstrated increased spontaneous production of superoxide in vitro. The second phenotypic feature ofFancc−/− Sod1−/−mice was that of bone marrow hypocellularity accompanied by significant decreases in peripheral blood erythrocyte and leukocyte numbers as compared with wild-type controls. Although flow cytometry analysis with monoclonal antibodies against cell surface antigens revealed normal numbers of primitive hematopoietic progenitor populations inFancc−/−Sod1−/−marrow, lineage-positive progenitor numbers were significantly reduced in these mice. Furthermore, the in vitro clonogenic growth ofFancc−/−Sod1−/−erythroid, myeloid, and early B-lymphoid colonies in semisolid media was profoundly compromised. These results suggested that the altered redox state likely present inFancc−/− Sod1−/−hematopoietic progenitors was responsible for an impairment of cell proliferation or survival.

Continuous in vivo infusion of interferon-gamma (IFN-γ) preferentially reduces myeloid progenitor numbers and enhances engraftment of syngeneic wild-type cells in Fancc-/- mice

Blood ◽  
2004 ◽  
Vol 104 (4) ◽  
pp. 1204-1209 ◽  
Author(s):  
Xiaxin Li ◽  
Yanzhu Yang ◽  
Jin Yuan ◽  
Ping Hong ◽  
Brian Freie ◽  
...  
Keyword(s):  
Stem Cells ◽  
Fanconi Anemia ◽  
Cancer Susceptibility ◽  
Phase 1 ◽  
Interferon Γ ◽  
Wild Type ◽  
Ifn Γ ◽  
Anemia Group

AbstractFanconi anemia (FA) is characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of many FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. Previous studies in FA murine models and in a phase 1 clinical trial suggest that myelopreparation is required for significant engraftment of exogenous, genetically corrected stem cells. Since myeloid progenitors from Fancc-/- mice and human Fanconi anemia group C protein (FANCC) patients have increased apoptosis in response to interferon γ (IFN-γ) in vitro, we hypothesized that IFN-γ may be useful as a nongenotoxic, myelopreparative conditioning agent. To test this hypothesis, IFN-γ was administered as a continuous infusion to Fancc-/- and wild-type (WT) mice for 1 week. Primitive and mature myeloid lineages were preferentially reduced in IFN-γ-treated Fancc-/- mice. Further, IFN-γ conditioning of Fancc-/- recipients was sufficient as a myelopreparative regimen to allow consistent engraftment of isogenic WT repopulating stem cells. Collectively, these data demonstrate that Fancc-/- hematopoietic cell populations have increased hypersensitivity to IFN-γ in vivo and that IFN-γ conditioning may be useful as a nongenotoxic strategy for myelopreparation in this disorder. (Blood. 2004;104:1204-1209)

scholarly journals The Effect of Oxidized Dopamine on the Structure and Molecular Chaperone Function of the Small Heat-Shock Proteins, αB-Crystallin and Hsp27

2021 ◽  
Vol 22 (7) ◽  
pp. 3700
Author(s):  
Junna Hayashi ◽  
Jennifer Ton ◽  
Sparsh Negi ◽  
Daniel E. K. M. Stephens ◽  
Dean L. Pountney ◽  
...  
Keyword(s):  
Heat Shock ◽  
Protein Aggregation ◽  
Molecular Chaperone ◽  
Cross Linking ◽  
Αb Crystallin ◽  
The Cross ◽  
Shock Proteins ◽  
And Function

Oxidation of the neurotransmitter, dopamine (DA), is a pathological hallmark of Parkinson’s disease (PD). Oxidized DA forms adducts with proteins which can alter their functionality. αB-crystallin and Hsp27 are intracellular, small heat-shock molecular chaperone proteins (sHsps) which form the first line of defense to prevent protein aggregation under conditions of cellular stress. In vitro, the effects of oxidized DA on the structure and function of αB-crystallin and Hsp27 were investigated. Oxidized DA promoted the cross-linking of αB-crystallin and Hsp27 to form well-defined dimer, trimer, tetramer, etc., species, as monitored by SDS-PAGE. Lysine residues were involved in the cross-links. The secondary structure of the sHsps was not altered significantly upon cross-linking with oxidized DA but their oligomeric size was increased. When modified with a molar equivalent of DA, sHsp chaperone functionality was largely retained in preventing both amorphous and amyloid fibrillar aggregation, including fibril formation of mutant (A53T) α-synuclein, a protein whose aggregation is associated with autosomal PD. In the main, higher levels of sHsp modification with DA led to a reduction in chaperone effectiveness. In vivo, DA is sequestered into acidic vesicles to prevent its oxidation and, intracellularly, oxidation is minimized by mM levels of the antioxidant, glutathione. In vitro, acidic pH and glutathione prevented the formation of oxidized DA-induced cross-linking of the sHsps. Oxidized DA-modified αB-crystallin and Hsp27 were not cytotoxic. In a cellular context, retention of significant chaperone functionality by mildly oxidized DA-modified sHsps would contribute to proteostasis by preventing protein aggregation (particularly of α-synuclein) that is associated with PD.

Fetal liver pro-B and pre-B lymphocyte clones: expression of lymphoid-specific genes, surface markers, growth requirements, colonization of the bone marrow, and generation of B lymphocytes in vivo and in vitro

1992 ◽  
Vol 12 (2) ◽  
pp. 518-530
Author(s):  
R Palacios ◽  
J Samaridis
Keyword(s):  
B Cell ◽  
B Lymphocytes ◽  
Stromal Cells ◽  
B Lymphocyte ◽  
Germ Line ◽  
Fetal Liver ◽  
Rag 1 ◽  
B Cell Precursors

We describe here the development and characterization of the FLS4.1 stromal line derived from 15-day fetal liver of BALB/c embryos and defined culture conditions that efficiently support the cloning and long-term growth of nontransformed B-220+ 14-day fetal liver cells at two stages of B-cell development, namely, pro-B lymphocytes (immunoglobulin [Ig] genes in germ line configuration) and pre-B cells (JH-rearranged genes with both light-chain Ig genes in the germ line state). All B-cell precursor clones require recombinant interleukin-7 (rIL-7) and FLS4.1 stromal cells for continuous growth in culture, but pro-B lymphocyte clones can also proliferate in rIL-3. None proliferate in rIL-1, rIL-2, rIL-4, rIL-5, rIL-6, or leukemia inhibitory factor. FLS4.1 stromal cells synthesize mRNA for Steel factor but not for IL-1 to IL-7; all pro-B and pre-B clones express c-Kit, the receptor for Steel factor, and a c-Kit-specific antibody inhibits the enhanced proliferative response of fetal liver B-220+ B-cell precursors supported by FLS4.1 stromal cells and exogenous rIL-7 but does not affect that promoted by rIL-7 alone. Northern (RNA) blot analysis of the expression of the MB-1, lambda 5, Vpre-B, c mu, RAG-1, and RAG-2 genes in pro-B and pre-B clones show that transcription of the MB-1 gene precedes IgH gene rearrangement and RNA synthesis from c mu, RAG-1, RAG-2, lambda 5, and Vpre-B genes. All clones at the pre-B-cell stage synthesize mRNA for c mu, RAG-1, and RAG-2 genes; transcription of the lambda 5 and Vpre-B genes seems to start after D-to-JH rearrangement in B-cell precursors, indicating that the proteins encoded by either gene are not required for B-cell progenitors to undergo D-to-JH gene rearrangement. These findings mark transcription of the MB-1 gene as one of the earliest molecular events in commitment to develop along the B-lymphocyte pathway. Indeed, both pro-B and pre-B clones can generate in vitro and in vivo B lymphocytes but not T lymphocytes; moreover, these clones do not express the CD3-gamma T-cell-specific gene, nor do they have rearranged gamma, delta, or beta T-cell antigen receptor genes.

scholarly journals Safety of measles, mumps, and rubella vaccine in egg allergy:in vivo and in vitro management

2020 ◽  
Author(s):  
Stefania Magistà ◽  
Marcello Albanesi ◽  
Nada Chaoul ◽  
Danilo Di Bona ◽  
Elisabetta Di Leo ◽  
...  
Keyword(s):  
Lymphocyte Proliferation ◽  
B Lymphocyte ◽  
Ex Vivo ◽  
Diagnostic Strategy ◽  
Egg Allergy ◽  
Lymphocyte Proliferation Assay ◽  
Proliferation Assay ◽  
Mmr Vaccine

Abstract Background Egg allergy is the second most prevalent form of food allergy in childhood. In spite of the evidence accumulated, inoculating egg allergy children with attenuated vaccines grown on chick embryo cell cultures, such as the measles, mumps, and rubella (MMR) vaccine, is regarded (erroneously) as potentially dangerous or even anaphylactogenic, by many. An issue perceived as particularly conflicting also by Health Professionals.Case presentation A 15-year-old boy, with a history of severe egg allergy in early infancy, who was still sensitized to egg allergens, including baked egg, had never received MMR vaccination, in fear of possible anaphylaxis, in spite of the fact that this vaccination is mandatory in the first year of life, in Italy. Because of that, he was not allowed to attend school, longer, and was referred to us in order to assess the potential risk of MMR vaccination. Upon thorough allergologic workup, sensitization to MMR vaccine components was excluded by an in vivo approach, consisting in skin prick tests, intradermal tests, and subcutaneous injection test, corroborated by vaccine-specific B-lymphocyte proliferation assay, ex vivo. T-cell proliferation in response to MMR vaccine was also excluded. Eventually, the boy was inoculated with MMR vaccine and was readmitted to school.Conclusions The diagnostic strategy adopted appears feasible and easy-to-perform and may be adopted in controversial cases (as the one reported), characterized by previous severe allergic reactions to egg. The B-lymphocyte proliferation assay we developed may represent a useful and reliable tool not only in research but also in clinical practice.

scholarly journals B lymphocyte binding to E- and P-selectins is mediated through the de novo expression of carbohydrates on in vitro and in vivo activated human B cells.

1994 ◽  
Vol 94 (4) ◽  
pp. 1585-1596 ◽  
Author(s):  
A A Postigo ◽  
M Marazuela ◽  
F Sánchez-Madrid ◽  
M O de Landázuri
Keyword(s):  
B Cells ◽  
B Lymphocyte ◽  
De Novo ◽  
Human B Cells

scholarly journals In vitro immunization approach to generate specific murine monoclonal IgG antibodies

2020 ◽  
Author(s):  
Sophia Michelchen ◽  
Burkhard Micheel ◽  
Katja Hanack
Keyword(s):  
Monoclonal Antibodies ◽  
Legionella Pneumophila ◽  
B Lymphocyte ◽  
Laboratory Animals ◽  
Igg Antibody ◽  
Simplified Approach ◽  
Humoral Immune ◽  
Viral Coat

AbstractGenerating monoclonal antibodies to date is a time intense process requiring immunization of laboratory animals. The transfer of the humoral immune response into in vitro settings shortens this process and circumvents the necessity of animal immunization. However, orchestrating the complex interplay of immune cells in vitro is very challenging. We aimed for a simplified approach focusing on the protagonist of antibody production: the B lymphocyte. We activated purified murine B lymphocytes in vitro with combinations of antigen and stimuli. Within ten days of culture we induced specific IgM and IgG antibody responses against a viral coat protein. Permanently antibody-producing hybridomas were generated. Furthermore we used this method to induce a specific antibody response against Legionella pneumophila. We thus established an effective protocol to generate monoclonal antibodies in vitro. By overcoming the necessity of in vivo immunization it may be the first step towards a universal strategy to generate antibodies from various species.

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