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1.
Neurotoxicology ; 95: 181-192, 2023 03.
Article in English | MEDLINE | ID: mdl-36775208

ABSTRACT

The environment, containing pollutants, toxins, and transition metals (copper, iron, manganese, and zinc), plays a critical role in neurodegenerative disease development. Copper occupational exposure increases Parkinson's disease (PD) risk. Previously, we determined the mechanisms by which copper induces dopaminergic cell death in vitro. The copper transporter protein 1 (Ctr1) overexpression led to intracellular glutathione depletion potentiating caspase-3 mediated cell death; oxidative stress was primarily cytosolic, and Nrf2 was upregulated mediating an antioxidant response; and protein ubiquitination, AMPK-Ulk1 signaling, p62, and Atg5-dependent autophagy were increased as a protective mechanism. However, the effect of chronic copper exposure on the neurodegenerative process has not been explored in vivo. We aimed to elucidate whether prolonged copper treatment reproduces PD features and mechanisms during aging. Throughout 40 weeks, C57BL/6J male mice were treated with copper at 0, 100, 250, and 500 ppm in the drinking water. Chronic copper exposure altered motor function and induced dopaminergic neuronal loss, astrocytosis, and microgliosis in a dose-dependent manner. α-Synuclein accumulation and aggregation were increased in response to copper, and the proteasome and autophagy alterations, previously observed in vitro, were confirmed in vivo, where protein ubiquitination, AMPK phosphorylation, and the autophagy marker LC3-II were also increased by copper exposure. Finally, nitrosative stress was induced by copper in a concentration-dependent fashion, as evidenced by increased protein nitration. To our knowledge, this is the first study combining chronic copper exposure and aging, which may represent an in vivo model of non-genetic PD and help to assess potential prophylactic and therapeutic approaches. DATA AVAILABILITY: The data underlying this article are available in the article.


Subject(s)
Neurodegenerative Diseases , Parkinson Disease , Mice , Animals , Male , Copper/toxicity , Copper/metabolism , Neurodegenerative Diseases/metabolism , AMP-Activated Protein Kinases/metabolism , Mice, Inbred C57BL , alpha-Synuclein/metabolism , Parkinson Disease/metabolism , Dopaminergic Neurons , Aging
2.
Int J Mol Sci ; 24(2)2023 Jan 09.
Article in English | MEDLINE | ID: mdl-36674772

ABSTRACT

Biometals are all metal ions that are essential for all living organisms. About 40% of all enzymes with known structures require biometals to function correctly. The main target of damage by biometals is the central nervous system (CNS). Biometal dysregulation (metal deficiency or overload) is related to pathological processes. Chronic occupational and environmental exposure to biometals, including iron and copper, is related to an increased risk of developing Parkinson's disease (PD). Indeed, biometals have been shown to induce a dopaminergic neuronal loss in the substantia nigra. Although the etiology of PD is still unknown, oxidative stress dysregulation, mitochondrial dysfunction, and inhibition of both the ubiquitin-proteasome system (UPS) and autophagy are related to dopaminergic neuronal death. Herein, we addressed the involvement of redox-active biometals, iron, and copper, as oxidative stress and neuronal death inducers, as well as the current metal chelation-based therapy in PD.


Subject(s)
Parkinson Disease , Trace Elements , Humans , Parkinson Disease/pathology , Copper , Metals , Iron , Oxidative Stress , Oxidation-Reduction , Dopaminergic Neurons/pathology , Chelating Agents/pharmacology , Chelating Agents/therapeutic use
4.
Biotechnol Lett ; 39(8): 1149-1157, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28470625

ABSTRACT

OBJECTIVE: To generate an immunogenic chimeric protein containing the Entamoeba histolytica LC3 fragment fused to the retrograde delivery domains of exotoxin A of Pseudomonas aeruginosa and KDEL3 for use as an effective vaccine. RESULTS: A codon-optimized synthetic gene encoding the PEΔIII-LC3-KDEL3 fusion construct was designed for expression in Pichia pastoris. This transgene was subcloned into the plasmid pPIC9 for methanol-inducible expression. After transformation and selection of positive-transformed clones by PCR, the expression of the recombinant protein PEΔIII-LC3-KDEL3 was elicited. SDS-PAGE, protein glycosylation staining and western blot assays demonstrated a 67 kDa protein in the medium culture supernatant. The recombinant protein was detected with a polyclonal anti-6X His tag antibody and a polyclonal E. histolytica-specific antibody. A specific antibody response was induced in hamsters after immunization with this protein. CONCLUSIONS: We report for the first time the design and expression of the recombinant E. histolytica LC3 protein fused to PEΔIII and KDEL3, with potential application as an immunogen.


Subject(s)
ADP Ribose Transferases/genetics , Bacterial Toxins/genetics , Entamoeba histolytica/genetics , Exotoxins/genetics , Recombinant Fusion Proteins/genetics , Vaccines , Virulence Factors/genetics , ADP Ribose Transferases/immunology , Animals , Bacterial Toxins/immunology , Entamoeba histolytica/immunology , Exotoxins/immunology , Pichia/genetics , Recombinant Fusion Proteins/immunology , Virulence Factors/immunology , Pseudomonas aeruginosa Exotoxin A
5.
Biotechnol Lett ; 37(4): 779-85, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25515795

ABSTRACT

Fusokines are proteins formed by the fusion of two cytokines. They have greater bioavailability and therapeutic potential than individual cytokines or a combination of different cytokines. Interferon-gamma-inducible protein 10 (CXCL10) and lymphotactin (XCL1) are members of the chemotactic family of cytokines, which induce tumor regression by eliciting immune-system cell chemotaxis. We engineered a replication-deficient adenoviral system expressing CXCL10/XCL1 fusokine (Ad FIL) and assessed its chemotactic response in vitro and in vivo. The CXCL10/XCL1 fusokine elicited a greater chemotactic effect in IL-2 stimulated lymphocytes than individual or combined cytokines in vitro. CXCL10/XCL1 fusokine biological activity was demonstrated in vivo by intratumoral chemoattraction of CXCR3+ cells. Thus, this novel CXCL10/XCL1 fusokine may represent a potential tool for gene therapy treatment of cancer and other illnesses that require triggering immune-system cell recruitment.


Subject(s)
Chemokine CXCL10/metabolism , Chemokines, C/metabolism , Chemotaxis , Genetic Vectors , Lymphocytes/physiology , Mastadenovirus/genetics , Animals , Cell Line , Chemokine CXCL10/genetics , Chemokines, C/genetics , Humans , Lymphocytes/drug effects , Mice, Inbred C57BL
6.
Biotechnol Lett ; 31(11): 1795-800, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19618273

ABSTRACT

Chemokines are members of the super family of cytokines necessary for leukocyte recruitment in tissues and lymphoid organs. The interferon-gamma inducible protein-10 (IP-10) chemo-attracts CXCR3-expressing cells, such as activated T lymphocytes and monocytes. We have genetically engineered a strain of Lactococcus lactis to secrete a biologically active murine IP-10 that interacts with human CXCR3, its homolog receptor, and chemo-attracts human CD3+ T lymphocytes.


Subject(s)
CD3 Complex/metabolism , Chemotactic Factors/pharmacology , Lactococcus lactis/metabolism , Lymphocytes/drug effects , Receptors, Cytokine/metabolism , Amino Acid Sequence , Animals , Chemotaxis/drug effects , Humans , Lymphocytes/cytology , Mice , Molecular Sequence Data , Receptors, Cytokine/chemistry , Sequence Alignment
7.
Biotechnol Lett ; 31(2): 215-20, 2009 Feb.
Article in English | MEDLINE | ID: mdl-18923910

ABSTRACT

Lymphotactin-XCL1 is a chemokine produced mainly by activated CD8+ T-cells and directs migration of CD4+ and CD8+ lymphocytes and natural killer (NK) cells. We expressed human lymphotactin (LTN) by the lactic-acid bacterium Lactococcus lactis. Biological activity of LTN was confirmed by chemo-attraction of human T-cells by chemotaxis demonstrating, for the first time, how this chemokine secreted by a food-grade prokaryote retains biological activity and chemoattracts T lymphocytes. This strain thus represents a feasible well-tolerated vector to deliver active LTN at a mucosal level.


Subject(s)
Chemokines, C/biosynthesis , Chemokines, C/pharmacology , Chemotaxis/physiology , Lactococcus lactis/metabolism , Lymphocytes/drug effects , Lymphocytes/physiology , Protein Engineering/methods , Cells, Cultured , Chemokines, C/genetics , Chemotaxis/drug effects , Humans , Lactococcus lactis/genetics , Recombinant Proteins/administration & dosage , Recombinant Proteins/metabolism
8.
J Med Microbiol ; 53(Pt 5): 427-433, 2004 May.
Article in English | MEDLINE | ID: mdl-15096553

ABSTRACT

Human papillomavirus type 16 (HPV-16) is the major causative agent of cervical cancer. To date, vaccine strategies against HPV-16 are based on the ability of the E7 oncoprotein to elicit an immune response against this virus. In this study, the use of an inducible or a constitutive system to produce the HPV-16 E7 protein in Lactococcus lactis, a non-pathogenic and non-invasive Gram-positive bacterium, was compared. The highest E7 production was obtained with the inducible system. When mice were immunized intranasally with recombinant lactococci expressing either inducible or constitutive E7, an antigen-specific cellular response (i.e. secretion of IL2 and IFN-gamma cytokines) was evoked and was substantially higher in mice receiving L. lactis expressing E7 with the inducible system. As bacterial antigen location may influence the immune response, recombinant L. lactis strains that produced E7 in three cellular locations, intracellular, secreted or cell-wall-anchored were evaluated. The highest immune response was elicited by administration of L. lactis producing an inducible cell-wall-anchored form of E7 protein. These promising results represent a step towards the development of a new, safe mucosal vector to treat HPV-related cervical cancer.


Subject(s)
Cancer Vaccines/immunology , Immunity, Cellular , Lactococcus lactis/immunology , Oncogene Proteins, Viral/immunology , Papillomaviridae/immunology , Administration, Intranasal , Animals , Cancer Vaccines/administration & dosage , Cancer Vaccines/genetics , Cell Wall/metabolism , Female , Humans , Immunization , Interferon-gamma/metabolism , Interleukin-2/metabolism , Lactococcus lactis/genetics , Mice , Mice, Inbred C57BL , Oncogene Proteins, Viral/genetics , Papillomavirus E7 Proteins , Papillomavirus Infections/immunology , Papillomavirus Infections/prevention & control , Promoter Regions, Genetic , Recombinant Proteins/administration & dosage , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Uterine Cervical Neoplasms/immunology , Uterine Cervical Neoplasms/prevention & control , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology
9.
Cancer Res ; 63(24): 8664-9, 2003 Dec 15.
Article in English | MEDLINE | ID: mdl-14695178

ABSTRACT

The p53 tumor suppressor ensures maintenance of genome integrity by initiating either apoptosis or cell cycle arrest in response to DNA damage. Deletion of either mdm2 or mdm4 genes, which encode p53 inhibitors, results in embryonic lethality. The lethal phenotypes are rescued in the absence of p53, which indicates that increased activity of p53 is the cause of lethality in the mdm2- and mdm4-null embryos. Here we show that mdm2-null embryos die because of apoptosis initiated at 3.5 days postcoitum (dpc). Partial rescue of mdm2-null embryos by deletion of bax allows survival to 6.5 dpc and alters the mechanism of death from apoptosis to cell cycle arrest, indicating that bax is a critical component of the p53 pathway in early embryogenesis. The death of mdm4-null embryos is due to p53-initiated cell cycle arrest at 7.5 dpc. Deletion of p21(p21(waf1/cip1)), a p53 downstream target partially responsible for cell cycle arrest, does not rescue this phenotype; however, deletion of p21 alters the mechanism of cell death from lack of proliferation to apoptosis. Thus, in both examples, deletion of a p53 downstream target gene allows p53 to redirect its efforts, highlighting a high degree of plasticity in p53 function.


Subject(s)
Apoptosis/genetics , Genes, p53/physiology , Nuclear Proteins , Proto-Oncogene Proteins c-bcl-2 , Proto-Oncogene Proteins/genetics , Animals , Crosses, Genetic , Embryo, Mammalian , Female , Gene Deletion , Male , Mice , Proto-Oncogene Proteins/deficiency , Proto-Oncogene Proteins c-mdm2 , Ubiquitin-Protein Ligases , bcl-2-Associated X Protein
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