No abstract available.
Aged ; Cytidine Deaminase/*genetics/metabolism ; Dasatinib/therapeutic use ; Disease Progression ; Drug Resistance, Neoplasm ; Fusion Proteins, bcr-abl/genetics/metabolism ; Humans ; Imatinib Mesylate/*therapeutic use ; In Situ Hybridization, Fluorescence ; Karyotype ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/*drug therapy ; Male ; Microfilament Proteins/*genetics/metabolism ; Oncogene Proteins/*genetics/metabolism ; Protein Kinase Inhibitors/*therapeutic use

Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease-causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical application of such approaches. Recently, a base editor (BE) system built on cytidine (C) deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine (T) efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high-fidelity version of base editor 2 (HF2-BE2), and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.
APOBEC-1 Deaminase ; genetics ; metabolism ; Animals ; Bacterial Proteins ; genetics ; metabolism ; Base Sequence ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cytidine ; genetics ; metabolism ; Embryo Transfer ; Embryo, Mammalian ; Endonucleases ; genetics ; metabolism ; Gene Editing ; methods ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Mice ; Mice, Inbred C57BL ; Microinjections ; Plasmids ; chemistry ; metabolism ; Point Mutation ; RNA, Guide ; genetics ; metabolism ; Thymidine ; genetics ; metabolism ; Zygote ; growth & development ; metabolism ; transplantation

APOBEC3 is a class of cytidine deaminase, which is considered as a new member of the innate immune system, and APOBEC3G belongs to this family. The research about APOBEC3G is a new direction of innate immune defense mechanism against virus. APOBEC3G has the restrictive activity on many viral replications, which deaminates dC to dU in the viral genome and then induces extensive hypermutation. APOBEC3G can also interrupt viral replication at several phases such as reverse transcription, replication, nucleocapsid and so on by non-deamination mechanisms. However, virus can encode viral proteins to counteract the restriction activity of APOBEC3G. Elucidation of the antagonistic interaction between APOBEC3G and the virus will be contributed to development of new antiviral drugs in the future.
APOBEC-3G Deaminase ; Animals ; Cytidine Deaminase ; genetics ; metabolism ; DNA Replication ; Deamination ; HIV-1 ; physiology ; Hepacivirus ; genetics ; physiology ; Hepatitis B virus ; genetics ; physiology ; Humans ; Paramyxoviridae ; genetics ; physiology ; Retroviridae ; physiology ; Virus Replication ; vif Gene Products, Human Immunodeficiency Virus ; metabolism

OBJECTIVETo investigate the association between rs185983011 single-nucleotide polymorphisms (SNP) of apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G (APOBEC3G) and the susceptibility to chronic hepatitis B.

METHODSThe blood samples were collected from 186 healthy subjects and 159 patients with chronic hepatitis B. The rs185983011 SNP was detected and genotyped by sequencing with Sanger's method to analyze the relationship between rs185983011 SNP and chronic hepatitis B.

RESULTSOnly C/C and C/T genotypes of the alleles of rs185983011 SNP were found in the tested subjects, and the C/C genotype was predominant (97.7%). The distribution frequencies of rs185983011 SNP genotypes and alleles showed no significant difference between healthy subjects and patients with chronic hepatitis B (P>0.05).

CONCLUSIONThe predominant genotype of rs185983011 SNP of APOBEC3G is C/C in the tested subjects, and rs185983011 SNP does not appear to associate with the susceptibility to chronic hepatitis B.

APOBEC-3G Deaminase ; Adult ; Alleles ; Case-Control Studies ; Cytidine Deaminase ; genetics ; Female ; Genetic Predisposition to Disease ; Genotype ; Hepatitis B, Chronic ; genetics ; Humans ; Male ; Polymorphism, Single Nucleotide ; Young Adult

BACKGROUNDThe cytosine arabinoside (Ara-C)-based chemotherapy is the major remedial measure for acute myeloid leukemia (AML). Deoxycytidine kinase (DCK) and cytidine deaminase (CDA) are the key enzymes in the metabolism of Ara-C. Many single nucleotide polymorphisms (SNPs) and haplotypes of DCK and CDA, which contribute to susceptibility to Ara-C, have been identified in Africans and Europeans. However, there has been no report about the relation among three SNPs in DCK (rs115543896, rs72552079, and rs111454937) and two SNPs in CDA (rs2072671 and rs60369023), and their clinical response to Ara-C for a Chinese population. In this study, we aimed to investigate whether these five SNPs are associated with the therapeutic outcomes of Ara-C-based chemotherapy regimens in patients with AML.

METHODSA total of 151 Chinese patients with AML were enrolled in our study. SNPs genotyping were performed using the MassARRAY system by means of the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) method.

RESULTSThe results illustrated that DCKrs111454937 AA genotype was more frequent in patients with higher platelet count, and A allele frequency was significantly higher in the group £40 years, lower white blood cell (WBC) count patients group and the group with platelet counts > 60'10(9)/L. Meanwhile, both DCKrs72552079 TC (OR = 1.225, 95%CI = 1.225 - 9.851, P = 0.0192) and CDArs60369023 GA (OR = 9.851, 95%CI = 1.31 - 77.93, P = 0.0263) significantly improved Ara-C-based chemotherapy response. While DCKrs11554389 AA (OR = 0.147, 95%CI = 0.027 - 0.801, P = 0.0267) was associated with the decrease of Ara-C-based chemotherapy response.

CONCLUSIONIt is evident that the DCK and CDA polymorphisms might be the important markers for the AML patients' therapy outcomes in a Chinese population.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Cytarabine ; therapeutic use ; Cytidine Deaminase ; genetics ; Deoxycytidine Kinase ; genetics ; Female ; Gene Frequency ; genetics ; Humans ; Leukemia, Myeloid, Acute ; drug therapy ; genetics ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; genetics ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Treatment Outcome ; Young Adult

Activation-induced cytidine deaminase (AID) is required for the generation of antibody diversity through initiating both somatic hypermutation (SHM) and class switch recombination. A few research groups have successfully used the feature of AID for generating mutant libraries in directed evolution of target proteins in B cells in vitro. B cells, cultured in suspension, are not convenient for transfection and cloning. In this study, we established an AID-based mutant accumulation and sorting system in adherent human cells. Mouse AID gene was first transfected into the human non-small cell lung carcinoma H1299 cells, and a stable cell clone (H1299-AID) was selected. Afterwards, anti-hTNF-α scFv (ATscFv) was transfected into H1299-AID cells and ATscFv was displayed on the surface of H1299-AID cells. By 4-round amplification/flow cytometric sorting for cells with the highest affinities to hTNF-alpha, two ATscFv mutant gene clones were isolated. Compared with the wild type ATscFv, the two mutants were much more efficient in neutralizing cytotoxicity of hTNF-alpha. The results indicate that directed evolution by somatic hypermutation can be carried out in adherent non-B cells, which makes directed evolution in mammalian cells easier and more efficient.
Animals ; Antibody Affinity ; Cells, Cultured ; Cytidine Deaminase ; genetics ; metabolism ; HEK293 Cells ; Humans ; Immunoglobulin Variable Region ; genetics ; immunology ; Mice ; Mutation ; Single-Chain Antibodies ; chemistry ; genetics ; immunology ; Somatic Hypermutation, Immunoglobulin ; genetics ; immunology ; Tumor Necrosis Factor-alpha ; immunology

OBJECTIVETo study the expression level and intracellular localization of APOBEC3G in peripheral blood mononuclear cells (PBMCs) and liver tissues of chronic HBV patients.

METHODSThe expression level and intracellular localization of APOBEC3G in PBMCs and liver tissues were detected using the western blot and confocal laser scanning microscope (CLSM).

RESULTSWestern-blot showed that the expression level of APOBEC3G in PBMCs of healthy controls was very low. The relative expression levels of APOBEC3G in PBMC of patients with chronic hepatitis B, chronic severe hepatitis, liver cirrhosis, or liver cancer were 4.12+/-0.21, 4.07+/-0.28, 4.16+/-0.36 or 4.21+/-0.39 respectively, which were higher than that in the healthy controls. However, there was no significant difference in APOBEC3G expression among different chronic HBV patients (q = 0.931, 0.744, 1.675, 1.675, 2.606 or 0.931, respectively, all P values more than 0.05). In addition, there was no significant difference on APOBEC3G in liver tissues between chronic hepatitis B patients and hepatocellular carcinoma patients (4.40+/-0.34 vs 4.34+/-0.43, q = 0.588, P more than 0.05). CLSM indicated that the localization of APOBEC3G protein was in cytoplasm of PBMCs and hepatocytes.

CONCLUSIONAPOBEC3G is upregulated in the PBMCs of chronic hepatitis B patients.

APOBEC-3G Deaminase ; Blotting, Western ; Case-Control Studies ; Cytidine Deaminase ; genetics ; metabolism ; Cytoplasm ; metabolism ; Hepatitis B, Chronic ; metabolism ; pathology ; virology ; Humans ; Leukocytes, Mononuclear ; metabolism ; Liver ; metabolism ; pathology ; Liver Cirrhosis ; metabolism ; pathology ; virology ; Liver Neoplasms ; metabolism ; pathology ; virology ; Microscopy, Confocal ; methods ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo assay the expression of cytidine deaminase (CDA), ribonucleotide reductase subunit 1 (RRM1), phosphatase and tensin homologue deleted from chromosome 10 (PTEN), excision repair cross-complementation group 1 (ERCC1), deoxycytidine kinase (dCK) and RRM1(-)37A/C polymorphism, which have been shown relevant to gemcitabine resistance in two human gemcitabine-resistant non-small cell lung cancer cell lines A549/Gem and NCI-H460/Gem, so as to make clear how do they vary during the course of acquiring resistance to gemcitabine.

METHODSThe human gemcitabine-resistant non-small cell lung cancer cell lines A549/Gem and NCI-H460/Gem were established in our Department by repeated clinical serum peak concentration and gradually increasing doses. Real-time fluorescent quantitative PCR was used to examine the expression of CDA, RRM1, PTEN, ERCC1, dCK and RRM1(-)37A/C polymorphism in those cell lines at different time points during their induction process.

RESULTSThe resistance indexes of A549/Gem and NCI-H460/Gem cells reached 163.228 and 181.684, and then remained stable at 115.297 and 129.783, respectively. The expression of CDA, RRM1, PTEN and ERCC1 varied along with the changing gemcitabine resistance indexes, but expression of dCK did not change apparently. The wild type promoter was able to amplify the genomic DNA in different induction stages of A549/Gem and NCI-H460/Gem cells, but allelotype did not, indicating that the gene type of A549/Gem, NCI-H460/Gem and their parental cells remaining still wild type.

CONCLUSIONCompared with their parental cells, the expressions of CDA, RRM1, PTEN and ERCC1 in human gemcitabine-resistant non-small cell lung cancer cell lines A549/Gem and NCI-H460/Gem rise, the expression of dCK changes inapparently, therefore, their gene type are remaining wild type.

Antimetabolites, Antineoplastic ; pharmacology ; Carcinoma, Large Cell ; genetics ; metabolism ; pathology ; Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cytidine Deaminase ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Deoxycytidine Kinase ; genetics ; metabolism ; Drug Resistance, Neoplasm ; Endonucleases ; genetics ; metabolism ; Humans ; Lung Neoplasms ; genetics ; metabolism ; pathology ; PTEN Phosphohydrolase ; genetics ; metabolism ; Polymorphism, Single Nucleotide ; Promoter Regions, Genetic ; RNA, Messenger ; metabolism ; Tumor Suppressor Proteins ; genetics ; metabolism

Human immunodeficiency virus type 1 (HIV-1) viral infectivity factor (Vif), one of the accessory proteins, which is a small basic phosphoprotein, is essential for viral replication and pathogenesis. The best well-characterized function of Vif is its ability to neutralize the host cell antiviral factor, apolipoprotein B mRNA editing enzyme catalytic polypeptide like 3G (APOBEC3G), which makes the viral particles more infective. In addition, Vif can regulate the reverse transcription and the advanced stage of replication of the virus particle, as well as induce the termination of cell cycle at G2 stage and so on. The designed drug aimed directly at Vif can efficiently block the maturation and infectivity of HIV-1. In this review, the structure, function and especially the related inhibitors of Vif are reviewed.
APOBEC-3G Deaminase ; Amino Acid Sequence ; Anti-HIV Agents ; pharmacology ; Cytidine Deaminase ; metabolism ; Ethylenediamines ; pharmacology ; HIV-1 ; physiology ; Humans ; Reverse Transcription ; Virus Replication ; vif Gene Products, Human Immunodeficiency Virus ; antagonists & inhibitors ; genetics ; metabolism ; physiology

OBJECTIVETo observe the inhibitory effect of a replication-defective hepatitis B virus (HBV) vector plasmid expressing A3C on HBV replication in vitro.

METHODSThe HBV vector plasmisd pCH-LJ3-A3C and pCH-LJ3-hrGFP expressing A3C and hrGFP were constructed using PCR and gene recombination technique. The two recombinant plasmids were separately cotranfected into HepG2 cells along with the wild-type HBV plasmid pCH-3093. The HBV DNA in the cell cytoplasmic lysates and in the cell culture supernatant was extracted for Southern blotting, and the nucleocapsid-associated HBV DNA were amplified by PCR, cloned and sequenced.

RESULTSpCH-LJ3-A3C showed obvious inhibitory effect on HBV DNA in the cytoplasmic lysates and cell culture supernatant, causing a reduction of the HBV DNA by 31% and 40%, respectively. The pCH-LJ3-A3C plasmid was capable of editing the HBV DNA. Among the 50 sequenced clones, 36 clones had G-A mutations, with a total of 982 such mutations.

CONCLUSIONpCH-LJ3-A3C can inhibit the replication of HBV primarily by editing HBV DNA. The pCH-LJ3-A3C plasmid may serve as a new antiviral agent against human HBV infection.

APOBEC-3G Deaminase ; Antiviral Agents ; pharmacology ; Base Sequence ; Cytidine Deaminase ; genetics ; metabolism ; Genetic Vectors ; genetics ; Hep G2 Cells ; Hepatitis B virus ; genetics ; physiology ; Humans ; Molecular Sequence Data ; Plasmids ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Virus Replication ; genetics